Special Issue May 2019 Vol. 19
ISSN: 1755-6783
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©Annals of Tropical Medicine & Public Health-Special Issues
Table of Content May 2019 Vol. 19
Title Page Comparing the techniques of laminectomy SP2000-19
Development of X-ray application in cancer
treatment and the use of medical imaging techniques
SP2001-19
Correlation of FNAC lymph node cytology with CD4
count in HIV seropositive adults
SP2002-19
RnBeads 2.0: Comprehensive analysis of DNA
methylation data
SP2003-19
Safety disposal of electrophoresis gels and PCR
contaminate with ethidium bromide and alternative
methods
SP2004-19
In-vitro chick chorioallantoic membrane study of
chitosan capped 5-fluorouracil conjugated gold
nanoparticles
SP2005-19
Dental ergonomics: Awareness and practices among
dental undergraduates in Saudi Arabia
SP2006-19
Application: Coating of nanoparticles to minimizing
harmful environmental pollution by chemical
fungicide
SP2007-19
Genetic detection of hepatitis B virus by PCR
technique among children under 7 years in Hilla city,
Iraq
SP2008-19
Comparison of APGAR Score in neonates born after
elective spinal versus general anaesthesia
SP2009-19
Frequency of norovirus in Egyptian children with
gastroenteritis and confection with rotavirus
SP2010-19
A descriptive cross-sectional survey among
International Islamic University Malaysia’s student
on e-waste generation and public health problem in
Malaysia
SP2011-19
Resistance of Trypanosoma congolense forest
isolates to isometamedium chloride and diminazene
aceturate in West Democratic Republic of Congo
SP2012-19
Hemophagocytic lymphohistiocytosis in a Nigerian
child: A review of the literature
SP2013-19
Changing trends of lower respiratory tract
pathogens: A five year study
SP2014-19
Pathogens in milk and product diary: Study in Europe SPe168-19
i
©Annals of Tropical Medicine & Public Health-Special Issues
Salih (2019): Laminectomy techniques May 2019 Vol.19
SP2000-19 ©Annals of Tropical Medicine & Public Health
COMPARING THE TECHNIQUES OF LAMINECTOMY
ABDULNASER ABDULQADER SALIH1
1. DEPARTMENT OF NEUROLOGICAL SURGERY IN TIKRIT UNIVERSITY COLLAGE OF
MEDICINE
Email: [email protected]
Abstract
CL - shortcut for Classical Laminectomy - is the standard lumbar spine stenosis (LSS) treatment and
succeeds in (60: 87 %) of patients. Conversely, it is related with high difficulty rates such as back pain, spinal
instability and muscular atrophy. The goal in our study is to compare the clinical results of Transspinous-Split
Laminectomy TSSL, which reduces paraspinal muscle destruction, and protects the alignment of vertebrates. The
data consists of 50 patients which divided to group A with 25 patients who treatment via transspinous split
laminectomy and group B with 25 patients who treatment via classic laminectomy. The two groups were
administered by the same surgeon and the process technique is chosen randomly. The sample does not contain
pregnant patients and does not have any infectious diseases, malignant infections, previous treatment of spinal
fractures or disc herniation, and they have previous injury in the treatment of iatrogenic, spondylosis, spondylosis or
radiation-documented instability. The incision length and amount of hemorrhage were matched to group A and
group B. The length of the incision was shorter in group A. Avoided circumference dissecting muscles in group A
reduces the amount of hemorrhage. Paraspinal muscle atrophy (PMA) rating was lower in group A. The Minimal
muscle atrophy is scheduled during dissection of minimal muscular atrophy in TSSL technique. The reduction of the
values of postoperative Oswestry pain grade of patients compared to another group clinically verifies that adequate
decompression has been alleviated. The smaller the size of the incision, the less bleeding during the operation and
the injury of the paraspinal muscles results in less pain after the surgery and thus recovering faster. The minimally
invasive metod (TSSL) is a good alternative to the conventional of the traditional spinal laminectomy.
Key words: laminectomy, TSSL, back pain, lumbar spine stenosis, Oswestry pain grade
How to cite this article: Salih AA (2019): Comparing of techniques of laminectomy, Ann
Trop Med & Pub Health-Special issue; 19: SP2000-19
1. Introduction
Laminectomy or the decompression surgery removes the skeletal roof covering the spinal cord and nerves to provide
more gaps for them to move easily. Stenosis or narrowing of the spinal canal can produce chronic pain, numbness,
tingling, and muscle faintness in the arms or legs as shown in figure 1(1).
Salih (2019): Laminectomy techniques May 2019 Vol.19
SP2000-19 ©Annals of Tropical Medicine & Public Health
Is the removal of the entire bone lamina, part of the enlarged facet joints, and the thick ligaments above the spinal cord
and nerves.
Laminectomy
Types of decompression surgery
Figure 1 Spinal stenosis is a narrowing of the spinal canal
The stenosis (narrowing) is frequently produced by osteoporosis associated with age, inflated joints, and thick
ligaments. Stress relief may be suggested if your symptoms do not recover with physical therapy or medications.
Surgery requires hospitalization from 1 to 3 days and the recovery period takes from 5 to 6 weeks (2).
Decompression can be achieved on the spine wherever starting from the spine of the neck (cervical) to the lower
back (lumbar). The technique is performed through a surgical incision in the back (posterior). The lamina is the bone
that forms the back of the spinal canal and makes the surface above the spinal cord. Eliminating the lamina and other
soft tissues gives more room for nerves and allows removal of bone tingling. Depending on how narrow (extent of
stenosis), there may be one vertebra (one level) or more (multi-level)(3).
Laminotomy Is the removal of a small part of the lamina and ligaments, frequently on one side. Using this technique the normal
support of the lamina is left in place, which reduces the chance of spinal instability after surgery. Sometimes, an
internal endoscope can be used, allowing for a slighter, less invasive incision.
Laminaplasty Is the extension of the spinal canal via by cutting the lamina on one side and fluctuation them open like a door. It is
used only in the cervical area.
In various situations, the spinal fusion can be merged at the same time to help stabilize the parts of the spine treated
with the laminectomy. Fusion uses a combination of bone bait, screws, and rods to connect two separate vertebrae
together in one new section of bone. Joint integration prevents spinal stenosis from repeating and can help eliminate
the pain of an unstable spine (4).
Foraminotomy Is the removal of the bone around the neural puncture - the space between the vertebrae where the nerve root emerges
from the spinal canal. This technique is used when the dissolution of the disk causes the collapse of the high puncture,
leading to a pinched nerve. This can be done using a laminectomy or laminotomy.
Salih (2019): Laminectomy techniques May 2019 Vol.19
SP2000-19 ©Annals of Tropical Medicine & Public Health
not improved with physical therapy or medication
diagnostic tests (MRI, CT, myelogram) that show stenosis in
the central canal or lateral recess
leg pain worse than back pain
difficulty walking or standing that affects your quality of life
The surgery of decompression spinal contraction is optional, except for occasional cases in the tail syndrome or a
rapidly developing neurological deficit. The doctor may advocate treatment options, but only you can decide if
surgery is right for you. The patients have to be sure of check out all the risks and benefits before making a decision
of surgery. The decompression does not treat spinal stenosis and does not remove arthritis. It only releases some of
the symptoms. Regrettably, symptoms may repeat with the continued degenerative process that leads to stenosis (5).
significant pain, weakness, or numbness in your leg or foot
It may be a candidate to decompression if you have
Salih (2019): Laminectomy techniques May 2019 Vol.19
SP2000-19 ©Annals of Tropical Medicine & Public Health
The orthopedic or neurologist surgeon can achieve spinal surgery. Numerous spine surgeons will concentrate in
multifaceted spinal surgery. Patient have to ask surgeon about training, particularly if patient’s condition is
complicated or you have undergone more than one spinal surgery (6).
CL - shortcut for Classical Laminectomy - is the standard lumbar spine stenosis (LSS) treatment and succeeds in
(60: 87 %) of patients. Conversely, it is related with high difficulty rates such as back pain, spinal instability and
muscular atrophy. The long hospital rest and recovery time, and high blood loss. The minimal invasive operation
techniques began to develop after the operation microscopes are used. In 2002, Shiraishi described the technique of
vertebral lamination of the cervical spine that protected the paraspinal muscles. Furthermore in 2005; it is improved
to be used in the lumbar spine by Kota Watanabe. After 10 years, relevant publications supported the effectiveness
of this surgical technique (7).
The goal in our study is to compare the clinical results of Transspinous-Split Laminectomy TSSL, which reduces
paraspinal muscle destruction, and protects the alignment of vertebrates.
2. Methods
The data consists of 50 patients which divided to group A with 25 patients who treatment via transspinous split
laminectomy and group B with 25 patients who treatment via classic laminectomy. The two groups were
administered by the same surgeon and the process technique is chosen randomly. The sample does not contain
pregnant patients and does not have any infectious diseases, malignant infections, previous treatment of spinal
fractures or disc herniation, and they have previous injury in the treatment of iatrogenic, spondylosis, spondylosis or
radiation-documented instability.
The mean age of group A patients was 55.9± 1.25 and for group B was 53.23± 2.13. The gender of sample was 26
female and 24 male. Group A have treatment by TSSL which performed of 10 male and 15 female patients. Group B
have treatment by CL which performed of 14 male and 9 female patients. the symptoms mean duration were 5.10
years. Patients for group A and B had pain at lower back and leg. Group A have 15 patients with numbness and
group B have 10 patients only. Group A have 8 patients had claudication and group B had 11 patients claudication
as shown in figure 2.
Salih (2019): Laminectomy techniques May 2019 Vol.19
SP2000-19 ©Annals of Tropical Medicine & Public Health
Figure 2 Postoperative early period CT image
Figure 3 Postoperative MRI and CT images
Patients were assessed by oswestry scale, narcoticscale, and preoperative. Muscle atrophy was measured prior to
preoperative and postoperative surgery by MIR images. Patients' responses about walking in the oswestry scale were
analyzed using the assumption process of I=0, II=1, III=3, IV=4, V=5.
May 2019 Vol. 9
Muscle atrophy had assessed by determining two dimensions of the muscles prior to preoperative and postoperative
neurosurgery using weighted T2 images at the intervertebral disc level. In multi-level decompression is considered
and the average values are estimated. The rate of muscular atrophy was calculated using the following formula
Precentage of Atrophy Rating = (1 − 𝑇𝑜𝑡𝑎𝑙 𝑝𝑜𝑠𝑡𝑜𝑝𝑒𝑟𝑎𝑡𝑖𝑣𝑒 𝑎𝑟𝑒𝑎
𝑇𝑜𝑡𝑎𝑙 𝑝𝑟𝑒𝑜𝑝𝑒𝑟𝑎𝑡𝑖𝑣𝑒 𝑎𝑟𝑒𝑎
) × 100
After the effects of general anesthesia, patients placed on the operating table in the prone position, with semi flex
waist. The level of spinous operations has been distinguished with a portable endoscopy arm c. The operating
microscope was used at all stages of the surgery. The skin was passed under the uncover spinous operations. The
Spinous processes operations were divided into two sections with osteotomi process applied by high speed motor
and retractor incision was used to enlarge the surgery area. The Spinous processes and the muscles surrounding the
medulla connect to them and keep it. Data were analyzed using a statistical program (SPSS 14) and P <0.05 for
statistical significance.
3. Results
Group A have 13 patients (52%) with one level of decompression and 12 patients (48%) with multilevel. Group B
have 11 patients (44%) with one level of decompression and 14 patients (56%) with multilevel of decompression.
The units of measurement were measured of lengths of incision in centimeter, operating times in hours, amount of
bleeding in (cc), hospital stay in day, oswestry patients scores and grade, pain scores, anesthesia scores, VAS scores,
walk distances and tumor paraspinal musclesatrophy (PMA) before and after surgery for each group as shown in
table 1
.
SP2000-19 ©Annals of Tropical Medicine & Public Health
Salih (2019): Laminectomy techniques May 2019 Vol.19
SP2000-19 ©Annals of Tropical Medicine & Public Health
Incision Length 3.72 ± 1.10 4.32 ± 1.95
Blooding 235.32 ± 188.15 263.12 ± 191.35
Before 63.50% ± 13.21 58.51% ± 17.25
Oswestry grade
After 19.50% ± 13.89 42.34% ± 18.54
Before 6.65 ± 1.52 5.32 ± 1.82
Pain grade
After 0.95 ± 1.63 2.42 ± 1.92
Before 2.74 ± 0.27 4.78 ± 0.57
Narcotic grade
After 1.02 ± 0.57 1.13 ± 0.24
Before 9.12 ± 1.61 8.24 ± 1.95
VAS
After 2.24 ± 1.75 4.87 ± 1.11
Before 3.52 ± 1.75 3.41 ± 1.32
Distance of walk
After 0.80 ± 0.52 2.82 ± 1.15
PMA 9.56% ± 1.85 38.52% ± 13.89
Table 1 Statistics
Items Group A Group B
Time of operation 2.31 ± 0.15 2.11 ± 0.55
Days for stay at hospital 2.15 ± 1.10 2.65 ± 2.12
The incision length and amount of hemorrhage were matched to group A and group B. The length of the incision
was shorter in group A. Avoided circumference dissecting muscles in group A reduces the amount of hemorrhage.
Paraspinal muscle atrophy (PMA)rating was lower in group A. the incision lengths, amounts of bleeding and
degrees of muscular atrophy assessment using Mann Whitney were tested and statistically better results were found
in group A (P = 0.001). The Minimal muscle atrophyis scheduled during dissection of minimal muscular atrophy in
TSSL technique. The main cause of postoperative back pain is loss of muscle function, which is better maintained in
TSSL technique.
Salih (2019): Laminectomy techniques May 2019 Vol.19
SP2000-19 ©Annals of Tropical Medicine & Public Health
4. Discussion
The lumbar spine stenosis (LSS) is the greatest familiar degenerative disorder in the lumbar spine which affects
about 1 in every 1000 people over age 55. The spread of the disease increases with age. The standard of lumbar
spine stenosis (LSS) for surgical treatment is pressure relief. The technique of surgical used is the removal of the
traditional spinal laminectomy to remove the pressure from the neurotransmitter described by Mexter and Pierre
from 70 years (8).
Corresponding to the results of 7-10 years of surgery, 26% of patients were re-operated and 34% of patients
presented with low back pain and hip after operation. Standard laminectomy involves the removal of a wide
laminectomy and partial or complete facial removal (9).
While this classic surgery offers sufficient nerve decompression, the posterior column, which has a significant role
in stabilizing the spine, is absolutely detached with the process. Removing the spinous process and lamina process
causes loss of tissue, formation of dead space, skin contour and cosmetic surgery problems, simplifies local wound
complications such as infection, and causes increased neural root scar adhesions. This in turn may increase pain and
lead to a syndrome of failure again in the coming period (10).
Since there is no an intersegmental such as the other muscles of paraspinal, and it was isolated only by the medial
branch of the dorsal ramus. The multifidus muscle is most susceptible to injury during backbone surgery. Lateral
deflection of the muscle dimension during the operation and subsequent compression of the medial part of the dorsal
branch can lead to the removal of muscle adenoid surrounding the uterus and postoperative muscular atrophy(11).
Line with others researchers, in their analysis of 18 patients, showed [sub laminar chimney pressure (CSD)]
technique were a way that minimized muscle damage with good neurological results in 2006. Published, they
reported that stripping the surrounding muscles of the marrow of a vertebra led to remove muscular embolism and
revascularization. Muscle contraction disrupts the local blood flow and increases oxidation, inflammatory cell
infiltration and increase in the nuclear factor KB associated with COX-2 expression the progressive edema of
muscle fibers. Postoperative injury pain is minimized using the new CSD procedure, as it does not involve muscle
stripping and muscles of paraspinal. Importantly, with this technique and early recovery, short hospital stay, a
decrease in complications related to hospital survival such as obstructive and urinary tract infections were also
observed(12).
Shetty and others involved in their report using LSPSL technology pointed out that this method provides a suitable
surgical area. They also observed that postoperative recovery was faster with minimal ligaments in the middle of the
midline and muscle damage (13).
5. Conclusion
In our report, it was shown that the minimally invasive common technique provide adequate expansion of the spinal
canal diameter by postoperative management of MIR and CT.
May 2019 Vol. 9
SP2000-19 ©Annals of Tropical Medicine & Public Health
Consistency, the reduction of the values of postoperative Oswestry pain grade of patients compared to another group
clinically verifies that adequate decompression has been alleviated. The smaller the size of the incision, the less
bleeding during the operation and the injury of the paraspinal muscles results in less pain after the surgery and thus
recovering faster
In the end of this report, as a minimally invasive method (TSSL) is a good alternative to the conventional of the
traditional spinal laminectomy. The disadvantage of this study is the low number of patients. At last, as a minimally
invasive technique; TSSL is a good alternative to conventional laminectomy. The problem of this study is that the
sample does not expand (only 50 patients). To reach a high resolution result, the sample could be increased to
include different situations and situations with different age groups.
6. References
1. Robot-Assisted Decompressive Laminectomy Planning Based on 3D Medical Image. Yu Sun, Zhongliang Jiang,. 1, s.l. :
IEEE, 2018 , Vol. 12.
2. Clinical Comparison Between Patients Operated for Unilateral Radiculopathy via a Contralateral ( Facet-Sparing ) and
Ipsilateral Side Approach. Ahmet OGRENCI, Orkun Koban. 12, s.l. : Sedat Dalbayrak, 2017, Vol. 22.
3. Clinical Outcomes of Patients over 75 Years of Age with Degenerative Spondylolisthesis Following Bilateral Decompression
via Unilateral Approach. Mustafa Kemal Ilik, Mustafa Golen,. 1, s.l. : Turkish neurosurgery, 2017, Vol. 2.
4. Feng Chang, Ting Zhang,.Comparison of the Minimally Invasive and Conventional Open Surgery Approach in the Treatment
of Lumbar Stenosis: A Systematic Review and a Meta-Analysis. Singapore : Annals of the Academy of Medicine, , 2017.
5. Decompression surgery for lumbar spinal canal stenosis in octogenarians; a single center experience of 121 consecutive
patients. Alexander Antoniadis, Nils Harry-Bert Ulrich. 2, s.l. : British journal of neurosurgery, 2017, Vol. 15.
6. Lumbar Microlaminectomy vs Traditional Laminectomy. Langevin, J M. s.l. : Journal of neurosurgery. Spine, 2017.
7. How safe is minimally invasive pedicle screw placement for treatment of thoracolumbar spine fractures? Timo Michael
Heintel, Stefan Dannigkeit,. s.l. : European Spine Journal, 2016.
8. Microsurgical unilateral laminotomy for decompression of lumbar spinal stenosis: long-term results and predictive factors.
Karsten Schöller, Thomas Steingrüber,. s.l. : Acta Neurochirurgica, 2016.
9. Comparative Analysis of Inpatient and Outpatient Interspinous Process Device Placement for Lumbar Spinal Stenosis. Alicia
Ortega, J. Manuel Sarmiento. 3, s.l. : Journal of neurological surgery, 2015, Vol. 2.
May 2019 Vol. 9
SP2000-19 ©Annals of Tropical Medicine & Public Health
10. Comparing cost-effectiveness of X-Stop with minimally invasive decompression in lumbar spinal stenosis: a randomized
controlled trial. Greger Lønne, Lars G. Johnsen,. Spine : s.n., 2015.
11. Does Obesity Affect Outcomes After Decompressive Surgery for Lumbar Spinal Stenosis? A Multicenter, Observational,
Registry-Based Study. Charalampis Giannadakis, Ulf S. Nerland,. s.l. : World neurosurgery, 2015.
12. Minimally Invasive Surgery for Intradural extramedullary Spinal Tumors : A Comprehensive Review with Illustrative
Clinical Cases. Martin H Pham, Ki-eun Chang,. 2016.
13. Minimally invasive removal of thoracic and lumbar spinal tumors using a nonexpandable tubular retractor. André Nzokou,
A G Weil, Daniel Shedid. Spine : Journal of neurosurgery, 2013.
Abbas (2019): X-ray application May 2019 Vol. 19
©Annals of Tropical Medicine & Public Health SP2001-19
Development of X-ray applications in cancer treatment and the use of medical
imaging techniques
Marwa H. Abbas
Department of dentistry, Al-Esra'a University College, Iraq
Email: [email protected]
Summary:
Role of radiotherapy in palliative care of cancer patients Radiotherapy plays an important role in
palliative care of cancer patients. This type of therapy accounts for about 30 to 50% of all radiation
delivered in a medical ward. The goal is to improve the patient's quality of life by providing rapid relief
of symptoms which depends on each specific localization. The cause of the symptoms must be
carefully identified (agreement between radiological and clinical findings) in order to establish the
precise target for radiation. Radiation dose and fractionation should be adapted to the patient's general
status and expected duration of life in order to limit the duration of treatment and secondary effects.
Key words: X-ray, radiology, prognosis, bone disease
How to cite this article: Abbas MH (2019): Development of X-ray application in cancer treatment
and the use of medical imaging techniques, Ann Trop Med & Pub Health-Special Issue; 19:
SP2001-19
Introduction
Painful bone metastases (20% of palliative care radiation protocols) can be irradiated in one or more
sessions with even analgesic effect with different results for prevention of fractures and cord
compression. Cord compression is a therapeutic emergency. The chances of recovering a motor deficit
after radiotherapy depend mainly on the rate of installation and the degree of deficit at the time of
treatment.
Patients with multiple brain metastases can be treated with 20-30 Gy delivered in 5-10 fractions on the
entire encephalon. More aggressive and technically more complex treatment (metastasectomy with
postoperative radiation, radiosurgery) can be reserved for selected patients. Radiotherapy can also be
effective for palliative care of patients with thoracic gold pelvic cancer.
The irradiation plays a major role in taking palliative load of patients with non-cancer curable [1].
Overall, the external irradiations carried out for palliative purposes represent 30% to 50% of activity of
a radiotherapy department, bone metastases painful alone constituting 20% of this activity. The purpose
Abbas (2019): X-ray application May 2019 Vol. 19
©Annals of Tropical Medicine & Public Health SP2001-19
of these treatments is to relieve and prevent symptoms, improve the quality of life or even increase
survival in some case. Because of their potentially myelo-toxic effects they must be fully integrated
into the in charge of the patients, especially not to risk compromising chemotherapy active. Moreover,
"time being precious when life is short "[2], the total duration of radiotherapy (or spreading) must be
adapted to the general condition of the patient and overall prognosis of the condition to limit maximum
unnecessary travel for patients whose life expectancy is short. Overall, a patient in desperate state never
benefits from palliative irradiation.
Reminder: fractionation of the dose in radiotherapy, the dose unit is gray (Gy), expressing the amount
of energy absorbed by the tissues irradiated. Apart from palliative situations or certain total body
irradiation protocols before transplant marrow, irradiations are not delivered in single dose but
according to a fractionation that is classically 5 weekly sessions of 1.8 to 2 Gy each. This fractionation
of the dose increases the effect of radiotherapy on the tumor while reducing its effects on the tissues
healthy.
In a palliative situation, it is possible to increase size of the fractions so as to shorten the total duration
of the treatment (spreading) while getting faster the desired analgesic or decompressive effect. This is
possible to the extent that the total doses required are much lower than during curative radiation. This
last element, associated with reduced life expectancy patients, limits the risk of complications late
radiotherapy. Schematically, it is therefore possible to deliver an irradiation with a spreading even
shorter than:
The life expectancy of the patient is short;
The target volume does not contain healthy tissue at risk acute complications (small
intestine, brain ...);
The total dose delivered is low;
The size of the fields is reduced.
Bone metastases:
Lesions with fracture risk should benefit of a first surgical treatment. The irradiations of bone
metastases account for 20% of the activity of a radiotherapy service. Split doses or doses unique are
comparable in terms of analgesic response objective.
External radiotherapy:
Overview 50% of patients with breast cancer, lung or prostate will develop metastases bones at one
time or another from the evolution of their disease. Conversely, 80% of bone metastases come from
breast cancer, lung cancer or prostate. Other cancers more rarely involved are those of the kidney,
thyroid and bladder. Without exception (including kidney cancers), bone metastases are in multiple
rule. Treatment of bone metastases includes two shutters:
Abbas (2019): X-ray application May 2019 Vol. 19
©Annals of Tropical Medicine & Public Health SP2001-19
the general treatment of the cancerous disease (chemotherapy, hormone therapy) or its
consequences bones (bisphosphonates);
the local treatment of bone metastases (surgery, radiotherapy).
Radiation therapy is a palliative treatment with aim to improve the quality of life of patients. Indeed, it
has the potential of being able to regress lesions tumors developed in bones and soft tissues adjacent
and thereby to obtain an analgesic effect, prolong the ambulatory state and reduce the risk of fracture
and spinal cord compression.
Several conditions must be met before starting radiotherapy of bone metastases:
1. Establish that the identified bone lesion is a metastasis. In the majority of cases, the diagnosis is
obvious. However, there are some difficult cases especially when the metastasis is unique and it occurs
more than 2 years after primary treatment of the primary tumor. He is then essential to confirm the
metastatic nature of the lesion by a biopsy or a puncture at the fine needle.
2. Make sure there is no indication of osteosynthesis prior to irradiation. Bone consolidation requiring
several months to be effective, it is better to use a primary surgery when the fracture risks are high.
Various criteria are used to evaluate in practice the fracture risk.
3. Integrate radiotherapy into the therapeutic plan general of the cancerous disease. Preservation of
capital medulla is different depending on whether it is a myeloma multiple for which myelotoxic
chemotherapy is considered or for carcinoma prostate for which androgenosuppressive hormone
therapy will constitute the bulk of systemic treatment.
4. Look for other bone metastases (scintigraphy bone), with little or no symptoms, but it is desirable to
irradiate at the same time because potentially fracturing in a relatively short (lytic metastases of long
bones or acetabulum for example).
The mechanisms by which radiotherapy allows to obtain an analgesic effect are poorly known. Indeed,
there is no clear correlation between the analgesic effect obtained on the one hand and the
radiosensitivity of the tumor or the dose delivered on the other hand. When the analgesic effect is rapid,
occurring in the first days after irradiation, one can invoke a reduction or a stop of the secretion of
chemical mediators of pain. The effects analgesics obtained later, in the weeks following the
irradiation, are rather in favor of a reduction tumor volume or bone recalcification for very late effects.
The average life expectancy of patients with bone metastases was evaluated at 29.3 months for prostate
cancers, 22.6 months for breast cancer and 3.3 months for bronchopulmonary cancers. However, some
subgroups of patients have a better prognosis. This is particularly the case of patients with exclusively
bone metastases secondary to carcinoma mammary (45% survival at 5 years with survival average of
52 months) or prostatic carcinoma hormone-sensitive (survival 43 months). The main criteria
correlated with life expectancy are as follows:
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©Annals of Tropical Medicine & Public Health SP2001-19
number of metastatic sites;
duration of the free interval between the first treatment primary cancer and the emergence
of metastases;
general condition of the patient reflected by the assessment of
the performance index (WHO or Karnofsky scale) (KPS));
Expected sensitivity of the neoplastic disease to a systemic treatment.
Overall, radiotherapy of bone metastases provides an analgesic effect in 70 to 80% of case. This
analgesic effect is:
Complete in 30 to 60% of the cases according to the series;
Delayed compared to the start of treatment; we can estimate that 50% to 60% of patients are calmed
in 2 weeks and 20% in 1 month or more.
Durable when it is complete (more than 1 year at 70% of the patients totally calmed);
Influenced by the nature of the primary tumor: the effect analgesic is more frequently obtained, more
complete and more durable when it comes to localizations of myeloma multiple or metastases of breast
cancer or prostate only when it comes to cancer metastasis thyroid, bronchial or renal;
Not influenced by the anatomical site of metastasis bone. Obtaining an analgesic effect (especially if
it is complete and durable) after irradiation of a first metastasis is a criterion of effectiveness during
irradiation another bone metastatic location. By against, the analgesic effect is delayed compared to the
beginning irradiation and allopathic analgesic treatment must be maintained the first sessions.
Indications: choice of dose and fractionation in 1998, the American College of Radiology
recommended the use of 3 regimens: 20 Gy in 5 fractions, 30 Gy in 10 fractions or 35 Gy in 14
fractions [3]. It suggests taking more account of hope of patients' lives by focusing on treatments short
for patients whose life expectancy is short.
Even Nielsen, who defends the principle of irradiation as a single dose, has reservation for this type of
treatment for unselected patients [4]. Single dose irradiations bone metastases are indicated when the
objective is to obtain a rapid analgesic effect. It is indeed effective treatment of painful bone
metastases, particularly simple and not very constraining for the patient, finally easily achievable for
the services of radiotherapy and inexpensive. The doses usually delivered range from 6 to 10 Gy
depending on the size of the fields and the organs at risk contained in the target volume. It is the
treatment of choice of metastases of non-bearing bones, poor lytic metastases of the carrier bones and
metastases Bones occurring in Patients with Hope life very reduced. Fractional dose irradiations should
be favored in patients with a relatively long life expectancy long when the goal is to get in addition to
the effect analgesic, i.e. tumor decompression (infiltration of the medullary canal, the paravertebral
muscles, a hole of conjugation ...) is a bone reconstruction (Lytic metastasis of the bones bearing). The
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irradiations carried out after bone healing surgery are also performed in split mode. The most protocols
employees deliver 30 Gy / 10 fractions or 20 Gy / 5 fractions.
Diffuse metastatic osteosis in treatment failure is good indications of irradiation hemoglobin when the
hemogram of the patient allows it. These irradiations are most often performed in single dose (6 Gy on
the upper hemi-body, 8 Gy on the lower hemi-body). Arcangeli [5] published an analysis of the dose
response relationship in the field of analgesic radiotherapy of metastases bone. In this study, high doses
appeared increase the response rates but the methodology employee was questionable. It was indeed a
retrospective study conducted in a single institution, with a significant bias due to the fact that high
doses were delivered to patients with a good prognosis. Ben-Josef [6] also found a dose-response
relationship after a compilatory study of 5 randomized trials published.
The short-term analgesic efficacy of radiotherapy bone metastasis is established but there are persistent
divergences on the level of dose to be delivered and on the fractionation use. Phase III studies already
carried out did not show superiority of a treatment regimen compared to others [7-10]. As a result, wide
varieties of protocols are used. The most common deliver 8 Gy in 1 fraction, 20 Gy in 5 fractions or 30
Gy in 10 fractions; depending on the choice in practice various criteria such as the habits of the
institution, the convictions of the responsible practitioner, the workload of the processing units and the
means financial services. The tests that have been published have concerned for patients with a life
expectancy short, of the order of 3 to 4 months, either did not take counts the life expectancy of
patients in the criteria inclusion. Most studies done on radiotherapy bone metastases have focused on
the analgesic effect obtained during the 3 months following the treatment. In total, almost all of these
tests have confirmed that a single fraction of 6 to 10 Gy offered in the short term a rate of clinical
responses similar to that obtained by fractional irradiations. On the other hand, it appears from these
studies that long-term results single-dose radiotherapy is not established since there is no reliable data
available. Attempts involving randomized patients also showed that the re-irradiations were more
frequent in primotravity patients by a single dose but they have not studied precisely the reasons of
these re-irradiations. He can the effect is earlier or more intense recurrences of the syndrome after
single dose irradiation. It is also possible, as suggested by Steenland [11], that the first treatment with a
single fraction has conditioned a higher rate of early re-irradiations and this independently of the
intensity of the pain. The results published are in favor of reducing the number of pathological fractures
after fractional irradiation compared to single dose irradiations.
After irradiation, cells shown to be tumors were replaced by calcifying fibrous tissue gradually. An
irradiated lytic metastasis would be able to fill itself by direct osteogenesis without prior chondrogenic
phase, whereas a pathological fracture would require going through a chondrogenesis phase to be able
to consolidate. Chondrogenesis would be sensitive to radiotherapy, unlike osteogenesis. For this
reason, radiotherapy would reduce the consolidation of pathological fractures while it would have no
effect on the healing of lytic lesions not fractured. Radiologically there are signs of remineralization
lytic bone metastases after radiotherapy in 65 to 85% of cases. The essential benefit is obtained after
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early radiotherapy of lyric lesions vertebral, pelvic or other weight-supporting bones from the body. No
reduction in the number of spinal cord compression has not been highlighted.
Hemi-corporal irradiation:
These irradiations were mainly used in analgesic treatment of multiple bone locations myeloma, breast
and prostate cancer. This treatment is indicated when these cancers are linked with systemic treatment
and that the patient keeps a hemogram satisfactory (the granular elements must be greater than 1,800 /
mm 3 and platelets at 100,000 / mm 3).
The previous bone irradiations do not constitute a contraindication. The irradiated volumes are as a
rule: for the upper hemi-body, extended from the angle from the jaw to the umbilicus: a single dose of
6 Gy or fractional irradiation of 12-24 Gy / 4-8 fractions; for the lower hemi-body, extended from the
umbilicus up to the knees: a single dose of 8 Gy or an irradiation fractionated from 15-30 Gy / 5-10
fractions.
The "middle" hemi-body, extended from the cervical spine to the upper third of the femurs, is more
rarely irradiated because the risk of bone marrow hypoplasia is high. The analgesic results published
are satisfactory. Thus, we obtain 77% of answers, 21% of which were answered completely. The
analgesic effect is obtained quickly, in 48 hours in 50% of responders and in 1 week in 80% of them,
sometimes after a brief period exacerbation of pain syndrome due to edema radiation-induced. The
immediate tolerance to these irradiations is globally very good. Acute side effects are mostly the fact of
the superior hemi-corporal irradiations. They may cause nausea and vomiting, fever and hypotension
and frequently lead to the suggestion of establishment of a venous route (alkalization), a premedication
with atropine, anti-emetic and corticosteroids, or else a medical observation during a few hours with
blood pressure monitoring. Immediate tolerance of hemi-bodily radiation lower is excellent. They can
be realized outpatient and require no precaution special. However, a few days later diarrhea occurs.
Late complications are rare. Some pneumopathies have been reported. In fact, only one monitoring of
the hemogram is necessary. Irradiation on the other half-body or chemotherapy can be performed 3 to 4
weeks later if the hemogram permits.
Metabolic radiotherapy
Although theoretically out of the frame of this review, metabolic irradiation is also a treatment
effective analgesic bone metastases. Irradiations metabolites by Strontium® or Quadramet constitute a
good alternative to external radiotherapy in case of multiple pain metastases, especially if they are
osteoconductive and the patient is still in good condition. Strontium chloride 89 (Metastro transmitter β
pure) is indicated for metastases of prostate cancer while the Samarium 153-EDTMP (Quadramet,
transmitter β and γ) is indicated for any tumor metastatic bone. Metabolic radiotherapy proves effective
in management of prostatic painful bone metastases, with 60 to 65% analgesic effect and a reduction
taking analgesics as well as the development of new painful sites.
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The average response time is 6 months (Sr 89), 4.5 month (Sm 153).
The effect is all the more clear and prolonged bone is moderate. This treatment has low hematological
toxicity (granular line and platelets). It can however to appear a discrete and transitory worsening of the
pains (10 to 20%). The time of appearance is variable: long for the Metastron (up to 1 month), faster
for Strontium (1 week). Metabolic radiotherapy can be renewed within a minimum of 12 weeks.
Medullary compression:
Spinal cord compressions are therapeutic emergencies and the degree of recovery from the motor
deficit depends mainly on the speed of installation of the deficit and early management of treatment.
Metastatic spinal cord compressions represent a therapeutic emergency. Spinal pain previous
neurological symptomatology in more than 90% of the cases.
Intramedullary metastases are exceptional (1% of cases). Almost all spinal cord compressions
metastatic (99%) corresponds to invasion tumor of epidural space by 3 mechanisms:
Hematogenous bone spread responsible for a epidural invasion of contiguity (85% of cases);
Invasion of the para-vertebral spaces, in particular from the lymph nodes, gaining then the epidural
space after invasion of the bone tissue or by infiltration of the conjugation hole (10 to 15% of case);
Hematogenous dissemination directly localizing in the vessels of the epidural space (1 to 5% of
cases).
Bone metastases reaching much more frequently the vertebral body as the pedicle or blades, it is the
anterior part of the epidural space which is the more often invaded. These earlier epidurites are
associated with more or less destruction extension of the vertebral body opposite. Direct compression
of the marrow by the tumor tissue is accompanied by hemodynamic disturbances that may at any time
cause irreversible acute ischemia.
The posterior surgical approach limits considerably the possibilities of excision of these previous
lesions and makes run the risk of intraoperative neurological aggravation by lesion of posterior
vascular elements while the anterior vessels are already deficient made of the tumor. Surgery is
reserved for patients with doubted diagnosis, spinal instability, a compressive lesion obvious and
extirpable or a compression in an already irradiated territory. Traditionally, this is of a posterior
laminectomy allowing decompression of the spinal canal or more rarely of a posterior stabilization
(even a reconstruction posterior vertebral). Mortality rates and morbidity is 3 to 14% and 5 to 30% [12,
13]. A Postoperative irradiation is recommended at the dose of 20-30 Gy / 5-10 fractions [14]. A
prospective study [15] involving 209 patients mainly affected by localizations of mammary origin,
bronchopulmonary, prostatic and myeloma and treated with external radiotherapy associated with
corticosteroids, emphasized the importance of early maturity of care about the chances of recovery
from walking. Functional recovery is significantly influenced by the slow appearance of deficit signs.
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Thus, a study of 98 cases [16] showed that, if the delay in setting up the disorders was long, the rate of
recovery was high. In case of rapid installation of motor disorders, only 10% of patients were improved
by irradiation.
The same author describes in a non-randomized trial [17] 3 fractionation schemes in patients with bone
marrow compression (49% of patients non-ambulatory): 30 Gy / 10 fractions, 37.5 Gy / 15 fractions, 40
Gy / 20 fractions, splitting being function the workload of the processing machines.
An outpatient activity was included in 19 to 24% of case, without difference between the 3 splits,
leading the authors to adopt the most splitting short (30 Gy / 10 fractions).
For altered patients (index WHO = 2, reduced life expectancy), Maranzano used an irradiation
hypofractionated of 8 Gy in 1 fraction in at least 53 patients paraparetic, repeated at 1 week interval in
case of response or stability. He reported 67% improvement in pain and 63% reduction motor deficit
without any radiation myelopathy [18].
When spinal cord compression occurs in territory already irradiated, reirradiation is at risk significant
amounts of radiation myelitis [19, 20]. In theory, a surgical solution must be preferred. In practice,
intervention is only exceptionally its own risks and the general condition often poor patients. The
utility of a reirradiation must then to be discussed on a case-by-case basis according to cumulative dose
and expectancy of the patient's life [21].
Brain metastases:
Panencephalic irradiation is recommended for symptomatic patients with metastases multiple. A
radiosurgical association or radiosurgery can be offered to selected patients whose prognosis is more
favorable. The brain is the 4 e metastatic site after the skeleton, lungs and liver [22]. Twenty-five
percent of patientsmetastatic will develop brain localizations secondary.
The median survival is 4 months longer (median 5-6 months) in patients treated with irradiation and
corticosteroids versus 1 month for patients treated with corticosteroids only [23]. The Radiation
Therapy Oncology Group analyzed the factors influencing the duration and quality of life of patients
treated for brain metastases and identified 3 prognostic groups. Median survival the highest (7.1
months) was for patients with KPS> 70, age <65 years, primary tumor controlled and absent other
metastatic locations. The adverse group had a median survival of 2.3 months [24].
Currently, an irradiation of 30 Gy in 10 fractions on the whole brain is the most common pattern used
in patients with metastases multiple cerebral In case of significant alteration of the condition In general,
an irradiation of 20 Gy in 5 fractions can be proposed [25].
For patients with isolated brain metastases, the radio-surgical association (metastasectomy then
postoperative pan-encephalic irradiation of 30 Gy / 10 fractions) appears to increase the median of
survival in the German study of Schackert (13 months vs.8 months) in patients in good general
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condition and whose disease systemic was controlled [26]. Other studies have reported comparable
results with obtaining a profit significant in terms of survival, local control cerebral and quality of life
[27, 28].
Radiosurgery is a radiotherapy technique under stereotactic conditions to deliver a high dose of
radiation in a single session in a small intracerebral target volume with high precision [29].
The results obtained with this technique are similar to those of surgery in selected patients [30]. The
selection criteria usually used are the following: general state conserved (KPS = 70), less than 4 brain
metastases on MRI [31], and size <30 mm.
Chest tumors:
Bronchopulmonary Cancers:
Compressive, painful or haemorrhagic symptoms to a thoracic tumor constitute good indications of
radiotherapy. Endobronchial brachytherapy high dose rate may be an alternative external radiotherapy
in case of bronchial obstruction or tracheal.
Symptoms related to pleuro-pericardial effusion or an aero-digestive fistula is not indications
radiotherapy.
Thoracic irradiations performed for palliative purposes concern a large number of patients. Indeed,
several situations can lead to this indication:
Bronchial cancers initially too advanced or occurring in patients who are in poor general
condition to benefit from a surgical or radiochemotherapeutic project for curative purposes;
Bronchial cancers recurring locally;
Intrathoracic metastases of other cancers.
Again, many tests have been performed to try to shorten the duration of external beam radiotherapy in
these patients whose life expectancy is short. The Medical Research Council conducted two
randomized trials [32, 33] comparing an irradiation delivering 17 Gy in 2 fractions and 1 week at
irradiation performed with a more conventional fractionation (30-39 Gy / 10-13 fractions). The
palliation of the main symptoms (including cough and hemoptysis) was identical in the different groups
treated both in terms response rate than duration of the palliation, without significant change in
survival. Conversely, a test Canadian randomized patient [34] comparing a single session of 10 Gy at
an irradiation delivering 20 Gy in 5 fractions and 1 week reported better palliative effect (cough, pain
thoracic, gene in daily activity and quality of global life) in the split arm. In addition, survival was also
significantly longer in the arm split (6 months vs. 4.2 months) for the subgroup patients in good general
and non-metastatic condition. Endoluminal brachytherapy with high dose rate is also an effective
palliative treatment, especially in the treatment of endotracheal recurrences or endobronchial infections
occurring in irradiated territory and hemoptysis or dyspnea. This method is to circulate for a few
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minutes a source of high-dose iridium in the light bronchial, over the entire length of the tumor
segment.
Applications are most often performed under anesthesia general, at the rate of an application of 5 to 15
Gy weekly for 1 to 5 weeks depending on the dose already received by the patient, the oncological
situation and condition general of the patient. The retrospective study of MD Anderson Hospital [35]
involving 175 patients reported a rate 78% objective response and significant improvement median
survival of responder patients (7 months vs. 3 months). Comparable results have been reported by a
British prospective study [36].
A randomized British trial [37] compared external irradiation (30 Gy / 10 fractions) at one application
unique endobronchial brachytherapy delivering 15 Gy. The conclusions of this study were in favor of
external beam radiotherapy which allowed for palliation more frequent and lasting symptoms than
brachytherapy as well as a modest but statistically of patient survival, at the price however, a higher
rate of radiation-induced dysphagia.
Note that the hemoptysis rate was similar in both arms (7%). Currently, the majority of authors agree to
reserve very concentrated irradiations (1 or 2 sessions from 8 to 10 Gy) to patients in poor general
condition, including life expectancy is very low. In total, patients in good general condition treated for
a non-metastatic disease seem to benefit from external irradiation spread as regards the improvement of
symptoms as survival time. Conversely, schemas using one or two strong fractions seem more adapted
to the symptomatic management of patients whose general condition is impaired and the prognosis
unfavorable short term. Radiation-induced esophagitis is the main complication acute occurring from
doses of 30 to 40 Gy. Smooth diet associated with a local treatment with anesthetics contact (gel or
syrup of Xylocaine) is often effective for dysphagia minor. For radio-mucositis more severe, general
treatment by major analgesics and corticosteroids as well as the installation of a probe may be
necessary. These dysphagia are transient, regressing a few days to a few weeks depending on their
severity [32].
For asymptomatic patients at the thoracic level, Falk et al did not found any benefit of immediate
irradiation compared to an irradiation deferred [38]. Survival rates were similar and only 42% of
patients in the group with "delayed irradiation" finally received palliative irradiation. This study
confirms the primary role of chemotherapy for patients asymptomatic in good general condition.
Superior cellar syndrome A superior vena cava syndrome due to bronchial cancer small cells or
untreated malignant lymphoma is a preferred indication of chemotherapy.
Radiation therapy is indicated if the SCS is due either to non-small cell lung cancer, either to a small
cell lung cancer or lymphoma in a situation of recurrence or failure of chemotherapy. The placement of
an endocaval vascular prosthesis represents a fast and efficient way to lift compression and can be a
complement or alternative to radiotherapy.
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Three-quarters of the superior vena cava compressions are due to bronchopulmonary cancers, of which
40% to small cell cancers. About 5% of bronchopulmonary carcinomas are complicated by Upper Cave
Syndrome (SCS). Lymphomas are responsible approximately 10% of SCS cases. The clinical
symptomatology depends to a large extent on the speed of cave obstruction, slow obstructions allowing
the development of a collateral circulation that partially drains blood flow and thereby reduces the
importance of symptoms related to obstruction of the vein cellar.
The prognosis is not favorable in the absence of treatment with a median survival of 6 weeks.
Treatment aggressive and responsive symptoms by at least partially restoring a flow in the vena cava
and to obtain an overall survival rate at 1 year estimated at 25%.
In the case of small cell lung carcinomas not pretreated, chemotherapy is currently advised in first
intention whenever possible. Indeed, the results of chemotherapy are comparable to those of
radiotherapy, while having the advantage to treat distant metastases early and avoid irradiation of a
large cardiopulmonary volume. The regression SCS is fast (7 to 10 days) and is obtained in 43 to 100%
of cases depending on the series. A randomized study comparing chemotherapy with or without
radiotherapy confirmed the absence of benefit of radiotherapy in this situation [39]. Radiotherapy is
however formally indicated when the SCS occurs in the part of a bronchial carcinoma to small cells
chemoresistant from the outset or in local recidivism. The same attitude is recommended in case of
SCS secondary to lymphoma. Unlike small cell cancers, radiotherapy plays a vital role in the initial
care SCS due to non-small bronchial cancers cells.
It has been proposed to start the irradiation with 3 fractions from 3 to 4 Gy in order to obtain a fast
decompressive effect, with resumption of radiotherapy according to a fractionation conventional [40].
In a small series, Egelmeers [41] reported a response rate of 76% in group of patients irradiated for
SCS due to carcinoma bronchial non-small cell, half of responders remaining under control until death.
The obstruction of the superior vena cava can also be treated by percutaneous insertion of a prosthesis
expansive metallic endocave (stent). So a Japanese study [42] compared the pose of a stent to one
mediastinal radiotherapy or chemotherapy (10 patients).
The clinical response between stent and radiotherapy was identical (78 and 80%), as the median
survival time (145 and 146 days). Previous treatment did not change median survival and clinical
response rate. Other studies [43] confirmed the good results of this technique which allows obtaining a
quick lifting of the obstacle and may be supplemented by subsequent radiotherapy. Esophageal cancers
The importance of initial dysphagia is a factor essential prognosis of oesophageal cancers. Lifting the
dysphagia is the main goal of the mediastinal irradiation, possibly associated with an endoscopic
désobstruction. Esophageal cancers remain of poor prognosis with a 5-year survival rate of about 5% at
all stages confused. The main symptom is dysphagia causing malnutrition, impairment of general
condition and loss weight. It is also frequently painful. Lift dysphagia is the primary goal of treatment
symptomatic. It has been shown that a dose of at least 50 Gy delivered in 25 fractions and 5 weeks
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increased survival rates overall and free interval without dysphagia. The patients very dysphagic do not
benefit from an increase of the dose beyond 50 Gy and a désobstruction mechanical is necessary.
Thus the retrospective study of Caspers [44] showed a radiotherapy dose equivalent to 50 Gy / 25
fractions / 5 weeks improved dysphagia in 70.5% of case. In this study, 50% of patients maintained the
benefit of this irradiation until their death. Severity dysphagia before treatment influenced survival
overall. A randomized Indian study compared a treatment endoscopic (esophageal dilatation and / or
prosthesis oesophageal) followed by external irradiation by oesophageal prosthesis alone [45]. For the
group of patients without initial esophageal fistula, the 1-year survival was better in the combined arm
(17% vs. 2.3%). In case of fistula present before treatment, radiotherapy did not improve median
survival but the size of this subgroup was too small to conclude to the inefficiency of radiotherapy.
In case of tumor recurrence or progressive continuation responsible for dysphagia, an endoscopic
treatment should be considered. Esophageal endoluminal brachytherapy with high dose rate has also
been used in patients whose life expectancy was less than 3 months or already treated by external
irradiation. The technique was similar to that used in the endo-luminal bronchial carcinomas. A single
fraction of 12 to 15 Gy proved effective on dysphagia, allowing obtaining response rates of 42% to
90% and a free interval of dysphagia ranging from 3.3 to 5 months [46].
A dose escalation of 2 fractions of 8 Gy or 3 fractions of 6 Gy [47] improved dysphagia and local
control but at the cost of complications mainly stenoses and fistulas.
Pelvic tumors:
Digestive or urinary obstructions should be benefit of a derivation before irradiation. Loco-regional
evolution of pelvic tumors may result in various clinical presentations. Thus, tumors centro-pelvic are
rather the cause of compressions or invasion of the bladder, rectum or of the vagina while latero-pelvic
tumors associate more or less completely limb lymphedema inferior, ipsilateral radiculalgia and
uretero-hydronephrosis. In case of obstruction of the urinary tract (obstacle to level of the
ureterovesical junction or bladder neck), the installation of a suprapubic catheter or a ureteral catheter
is necessary before any irradiation [48].
For rectal cancers, it has been shown that irradiation exclusive allowed to reduce syndromes rectal in
50 to 75% of cases, rectorrhagia in 2/3 of cases and pelvic pain in near the half of the cases. However,
a dose effect has been obviously, a minimum dose of 45 Gy delivered in 4 to 5 weeks being necessary.
Below this dose, the chances of being effective on the symptoms are limited (<15%). These palliative
effects are unfortunately transient, less than 15% of patients remain relieved at 1 year [49]. In order to
reduce the total duration of treatment, hypofractionated irradiations have been proposed. For example,
Swedish teams have established that preoperative, 25 Gy delivered in 5 fractions and 1 week were
equivalent to 45 Gy delivered in 25 fractions and 5 weeks [50].
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For bladder cancer, palliative radiotherapy significantly improves symptoms such as dysuria, bladder
irritation and hematuria. A randomized study from the Medical Research Council [51] compared 2
different fractions (21 Gy / 3 fractions / 2 weeks vs. 35 Gy / 10 fractions / 12 days) in the setting
symptomatic load of 500 cancer patients bladder. The symptoms were reduced in 50 - 53% at the end
of irradiation and in 64 to 71% of patients at 3 months. There was no difference in effectiveness or a
difference in survival (median of 7.5 months) between the 2 arms. Other concentrated irradiation
schemes have been successfully proposed, for example 17 Gy issued in 2 fractions and 3 days [52]. In
terms of palliation, this scheme resulted in a better reduction of hematuria and pains that more
conventional irradiation (45 Gy / 15 fractions) but at the price of a reduction of survival that proved
significantly more short in the hypofractionated arm (9.77 months vs. 14.47 months).
For prostate cancer, the conclusions are similar. Radiation therapy improves the symptoms associated
with invasion of pelvic structures [53]. In total, palliative radiation therapy for pelvic cancers is an
effective, highly concentrated radiation protocol (1 to 3 sessions of 7 to 10 Gy) being reserved for
patients in poor general condition whose life expectancy is short.
Various irradiations:
For haemostatic radiotherapy of uterine cancers, Metrorrhagia is frequently indicative of cervical
cancer. Present at diagnosis in 70 to 80% of cases, they can be abundant and even represent a
therapeutic emergency. Irradiation allows rapid tumor reduction and Hemostasis by occlusion vessels.
In a retrospective study of 20 patients with metrorrhagia since 6.25 months in average and worsening in
the 5 last days preceding the consultation, not giving in to a dressing vaginal compress, Biswalet a [54],
have reported of haemostasis in all patients after 3 irradiation sessions. These results were similar to
those found by Kraiphibul [55].
Pancreatic cancers:
Pancreatic cancer is rarely diagnosed at early stage surgery. A derivation is necessary in case of an
inoperable tumor responsible for jaundice compression. The method choice of extrahepatic biliary
obstruction is represented by the endoscopic placement of prosthesis. This technique allows lifting the
obstacle in 80 to 90% of cases [56]. The way transhepatic is used in case of failure of the endoscopic
approach. Surgery is also possible by bypass biliary or digestive. The results of these different methods
on jaundice regression are equivalent to short term [57].
Inoperable, locally advanced cancers of the pancreas can benefit from a concomitant association
radiotherapy and chemotherapy if the general condition of the patients allows it. Studies have shown
that this association could increase the quality and duration of survival (9 to 10 months vs.5 to 6
months) compared to a treatment by external radiotherapy alone [58-60] or chemotherapy only [61].
In patients in poor general condition, radiotherapy palliative is relatively little used because the doses
necessary to achieve a palliative effect are relatively elevated while organs located in the party upper
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abdomen have poor tolerance to irradiation. So, in a randomized trial, local control was obtained in
only 20% of patients at 2 years after an exclusive irradiation of 60 Gy [62].
Splenomegaly:
In myeloproliferative syndromes (especially in chronic myeloid leukemias and splenomegaly myeloid),
splenomegaly sometimes monstrous, occupying a part of the abdomen and possibly of the pelvis often
sign aggravation of the hematological disease. Palliative irradiation low dose is effective, allowing a
reduction in symptoms (gravity, pain, fever, sweating, slimming) in 60% of cases, splenic volume in 42
to 81% of cases and transfusion needs in 32 to 60% of cases [63, 64]. The irradiation field includes the
entire spleen. The total dose is 5 to 10 Gy delivered per daily fraction of 0.5 Gy. The size of the fields
is adapted to the regression of splenic volume during irradiation.
Side effects of radiotherapy:
The acute side effects of radiotherapy are correlated with the size of the fractions and the total dose
delivered. The usual protocols used (6 to 8 Gy in 1 fraction, 20 Gy in 5 fractions, 30 Gy in 10 fractions)
are in generally very well tolerated. Nevertheless, side effects may occur:
A cutaneous erythema, especially frequent at the level of folds and zones tangent to the beams,
resolving in 2 at 3 weeks;
Nausea and vomiting during irradiation abdominoplasty, especially when part of the stomach is
included in the fields (utility of anti-HT3 issued before each meeting);
Diarrhea after abdominal or pelvic irradiation including a significant intestinal volume;
Dysphonia and dysphagia during irradiation cervical or thoracic, including all or one part of the
pharyngolaryngeal sector or the esophagus. The doses delivered relatively low and especially the
expectation reduced life of these patients limit the risks occurrence of late complications. Only myelitis
radiation may be a problem during re-irradiation vertebral (especially where there is a risk of fields) in
patients with an expectation of life of several months.
Conclusion:
Palliative radiation therapy is an important part of the activity of a radiotherapy service and each
treatment must be adapted for the patient to draw a real benefit. Thus, we will favor a hypofractionated
irradiation or as a single dose for patients in terminal phase, which will be delivered quickly and will
not be less effective; transport and manipulation will be limited in these frail patients and the costs
treatment will be reduced [65]. More splitting conventional or a dose increase may in certain situations
(patient in good general condition, illness poorly evolved) improve local control rates and overall
survival. Innovative techniques (radiotherapy conformational, radiosurgery, brachytherapy dose rate)
have their place in this type of irradiation.
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Sumana et al (2019): FNAC lymph node in HIV+ adults May 2019 vol. 19
©Annals of Tropical Medicine & Public Health SP2002-19
Correlation of FNAC lymph node cytology with CD4 count in HIV seropositive adults”
Mukherjee Sumana1, Mukhopadhyay Keya
1 , Bhattacharya Pranab
1
1. Department of Pathology, School Of Tropical Medicine, Kolkata
Corresponding Author:
Sumana Mukherjee, BH-62, Sector-2, Salt Lake, Kolkata
Phone numbers +919830945575
E-mail: [email protected]
Abstract:
Context: Lymphoid tissues are common targets of HIV infection.FNAC is the initial investigation of choice in
these cases.
Aims: To evaluate the usefulness of FNAC in HIV positive lymphadenopathy in our center..
Methods and Material: FNAC was performed in 153 HIVpositive patients presenting with lymphadenopathy.
Smears were stained with Giemsa, ZN and PAS/Grocotts/PAP according to cytological findings.
Statistical analysis: The data was analysed using the T Test
Results: Tuberculous lymphadenitis was the most common diagnosis (44%).Smear positivity was found in 29%
cases. Necrotizing granulomas and smear positivity was significantly higher in cases with CD4 count<200.
Reactive hyperplasia was significantly higher in the CD4> 200 category.
Conclusions: FNAC is very useful and gives specific diagnosis in most cases of HIV lymphadenopathy. Lower
CD4 count significantly increases the smear positivity for AFB.
Key-words: FNAC, lymph node, HIV, CD4 count
How to cite this article: Sumana M, Keya M, Pranab B (2019): Correlation of FNAC lymph node cytology
with CD4 count in HIV seropositive adults, Ann Trop Med & Pub Health-Special issue; 19: 2002-19.
Key Messages: FNAC should be the chosen diagnostic method in HIV lymphadenopathy
because it avoids unnecessary biopsy, saves time, is cost effective, safer for the operator and
has yields mostly specific diagnosis.
Sumana et al (2019): FNAC lymph node in HIV+ adults May 2019 vol. 19
©Annals of Tropical Medicine & Public Health SP2002-19
Introduction:
Lymphoid tissues are commonly targeted in HIV infections [1]
. HIV positive individuals thus commonly present
with enlarged lymph nodes. The degree of lymphadenopathy may range from progressive generalized to
transient.The commonest infection is tuberculosis and extra pulmonary involvement is common [2]
. Occurrence
of extra pulmonary tuberculosis has increased specially in those who are severely immunodeficient [3]
.
FNAC is the initial investigation of choice in these cases. Though FNAC may not clearly demarcate all
pathologies, it is useful in diagnosis of specific infections and involves lesser risk to the performer than biopsies
[4].
We tried to note the FNAC findings of all HIV positive patients sent to our department with lymphadenopathy
and corelated it with CD4 counts. We aim to evaluate the usefulness of FNAC in HIV positive
lymphadenopathy in our center
.
Materials and Methods:
Subjects: This is a cross sectional observational study of HIV infected subjects diagnosed in an ICTC unit in a
tertiary medical center. FNAC was performed on patients presenting with lymphadenopathy.
Sample size: 153 HIV positive adults with lymphadenopathy.
Inclusion criteria: Subjects above 18 years, seropositive for HIV, lymph node size at least 1 cm.
Exclusion criteria: Retroperitoneal or non-palpable nodes, inadequate material.
Data collection: Data collected included age, sex, site of lymph node enlargement, whether on ART therapy,
clinical examination of nodes, CD4 cell count by flow cytometry and cytological features. All the smears from
the aspirates were stained with Giemsa stain and ZN stain. PAS / Grocotts / PAP stains and culture were used
depending on cytological findings.
The following categories were used to record cytological data. The groups were (1) reactive hyperplasia, (2)
necrotizing granulomatous with or without AFB, (3) necrotizing only with or without AFB, (4) other specific
diagnosis like histoplasma, Cryptococcus, suspected lymphoma or metastasis, (5) inconclusive.
Statistical analysis: The data was analysed using the T test. P-values were calculated. A p-value of < 0.005 was
considered significant.
Results:
There were 108 males (70.5%) and 45 females (29.5%). The age range was 20 to 58 years. The commonest site
was cervical (40%) followed by axillary (25%) and inguinal (5%). Some patients (30%) presented with multiple
site involvement, commonly cervical and axillary. Most nodes (55%) were discrete, non-tender. Matted nodes
constituted 25% cases, while abscess or sinus formation was seen in 20% cases (Table 1). 112 cases were on
ART, while 41 were ART naïve.
Sumana et al (2019): FNAC lymph node in HIV+ adults May 2019 vol. 19
©Annals of Tropical Medicine & Public Health SP2002-19
Reactive hyperplasia accounted for 35% of diagnosis. There were 29% cases positive for AFB (figure 1). Only
necrotizing pattern (figure 2) were noticed in 35% cases among the AFB positiveswhile most smear positives
showed necrotizing granulomas (figure 3). Necrotizing granulomatous comprised 36%. Tuberculosis was
diagnosed when there was AFB positivity irrespective of cytology and/or presence of caseation necrosis with
epithelioid granulomas. Tuberculosis was diagnosed in 44% cases. Fungal infection comprised of 2 cases,
proven by culture. Lymphomas comprised 3.2% cases. Out of the 5 cases of lymphoma, biopsy confirmed NHL
in 4 cases and 1 was florid reactive hyperplasia. Most lymphomas and fungal infections (figure 4) were in CD4
count < 200 group (Table 2).
The number of reactive hyperplasias was significantly higher in CD4 > 200 group (p=0.00). While necrotizing
granulomas and AFB positivity was significantly higher in CD4 < 200 group (p=0.0066). Similarly, only
necrosis with AFB was significantly higherin CD4 < 200 group. There was no significant difference among
cases with necrosis and no AFB in the 2 CD4 groups (p=0.00). Cases with granulomas but no necrosis or AFB
were considered inconclusive at FNAC.
Discussion:
Male predominance has been established in most studies5,6
and the higher age limit varies up to 65 years7,8,9
In
our study , however, we did not get any case above 60 years of age. Agravatet al8 showed incidence of
lymphadenopathy decreased with increasing age.
Cervical nodes were most commonin our study like Neelima et al and others 9,
10,
11
Satyanarayana et al 4 found
axillary nodes most commonly. Liatjos et al12
and Naser S et al13
had categorized cytological findings in our line.
Similar to our findings, most workers found tuberculous lymphadenitis as the most common
diagnosis.1,5,8,9,11
Chronic granulomatous lymphadenitis without caseous necrosis and no AFB or fungi, which we
categorized as inconclusive 6,14
on FNAC comprised 12 of our cases (7.8%) is in between the 19% recorded by
Satyanarayana et al4 and the single case reported by Neelima et al.
10
CD4 count < 200 is considered advanced stage while counts 200-500 and >500 are early and intermediate
stages.15
We considered two groups based on similar cut off value.
Our study corroborates with other workers 10
that fungal infections and lymphomas are common in the low CD4
categories. Kumar Guru et al 6 also found highest CD4 counts in reactive hyperplasias like our study. We found
that metastasis has also been reported by some corroborating with our findings.15
We may have missed many
opportunistic infections like viruses and toxoplasma in this cytomorphological study. Diagnostic accuracy could
be increased by using appropriate immunofluorescence kits.
Conclusion
FNAC is very useful and gives specific diagnosis in most cases of HIV lymphadenitis. Lower CD4 count
significantly increases smear positivity for AFB and fungi and may help in considering segregation of these
patients.
Sumana et al (2019): FNAC lymph node in HIV+ adults May 2019 vol. 19
©Annals of Tropical Medicine & Public Health SP2002-19
Source(s) of support: Nil
Presentation at a meeting: Not Applicable
Conflicting Interest (If present, give more details): Nil
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Table 1: Site distribution of lymph nodes and clinical features among the cases
Lynph node Number Percentage
Site
Cervical 61 40%
Axillary 38 25%
Inguinal 8 5%
Combined 46 30%
Clinical Features
Discrete 84 55%
Matted 38 25%
Abscess or Sinus 31 20%
Table 2: Correlation of cytological features of lymph nodes with CD4 count
Cytological Feature CD4 > 200 CD4 < 200
Reactive Hyperplasia 53 5
Necrotizing granulomatous without AFB 24 --
Necrotizing granulomatous with AFB 11 20
Necrotizing without AFB / fungal stain positivity 4 2
Necrotizing with AFB 2 11
Lymphoma 1 4
Metastasis 2 --
Histoplasma -- 1
Cryptococcus -- 1
Inconclusive 10 2
Total Cases 107 46
Sumana et al (2019): FNAC lymph node in HIV+ adults May 2019 vol. 19
©Annals of Tropical Medicine & Public Health SP2002-19
Figure 1: AFB positivity, ZN stain, x 1000
Figure 2: Necrotizing inflammation, Giemsa, x100
Figure 3: Granuloma, Giemsa, x400
Figure 4: Histoplasma, GMS stain, x 400
Lafta & Gangadhar (2019): RnBeads 2.0 May 2019 vol. 19
©Annals of Tropical Medicine & Public Health SP2003-19
RnBeads 2.0: Comprehensive Analysis of DNA Methylation Data
Methaq Hadi lafta1*
, Lekshmi Gangadhar2
1*Lecturer in ministry of education, Iraq,ORCID ID: 0000-0002-9978-6829
2Department of Nanotechnology, Noorul Islam Center for Higher Education, India, ORCID
ID: 0000-0002-1627-1230
ABSTRACT
DNA methylation is a widely investigated epigenetic mark with important roles in development and disease. High-
throughput assays enable genome-scale DNA methylation analysis in large numbers of samples. Here, we describe a
new version of our RnBeads software – an R/Bioconductor package that implements start-to-finish analysis
workflows for Infinium microarrays and various types of bisulfite sequencing. RnBeads 2.0 (https://rnbeads.org/)
provides additional data types and analysis methods, new functionality for interpreting DNA methylation
differences, improved usability with a novel graphical user interface, and better use of computational resources. We
demonstrate RnBeads 2.0 in four re-runnable use cases focusing on cell differentiation and cancer.
Keywords: Bisulfite sequencing, Computational epigenetic, DNA methylation, Epigenome-wide
association studies, Epigenotyping microarrays
How to cite this article: Lefta MH, Gangadhar L (2019): RnBeads 2.0: Comprehensive analysis of DNA
methylation data, Ann Trop Med & Pub Heatlh-Special Issue; 19: 2003-19
BACKGROUND
DNA methylation at CpG dinucleotides is a widely studied epigenetic marker that is involved in the regulation of
cell state and relevant for a broad range of diseases. Changes in DNA methylation at promoters and enhancers have
been associated with cell differentiation, developmental processes, cancer development, and regulation of the
immune system. The vast majority of current assays for DNA methylation profiling use bisulfite treatment to
selectively convert unmethylated cytosines (including 5-formyl-cytosine and 5-carboxy-cytosine) into uracil (which
is subsequently replaced by thymine), while methylated cytosines (including 5-hydroxy-cytosine) remain
unconverted. Bisulfite conversion thus transforms DNA methylation information into DNA sequence information
that can be read by next-generation sequencing or DNA microarrays [1].
Whole-genome bisulfite sequencing (WGBS) constitutes the current gold standard for DNA methylation profiling,
given its genome-wide coverage and single-base pair resolution [2]. However, WGBS requires deep sequencing of
the entire genome (which is a significant cost factor), while shallow sequencing leads to poor sensitivity for
detecting small differences in DNA methylation [3]. Reduced representation bisulfite sequencing (RRBS) offers a
cost-effective alternative for profiling large sets of patient samples, by focusing the sequencing on a subset of the
genome enriched using restriction enzymes. RRBS is particularly useful for studying DNA methylation
Lafta & Gangadhar (2019): RnBeads 2.0 May 2019 vol. 19
©Annals of Tropical Medicine & Public Health SP2003-19
heterogeneity, which profits from deep sequencing coverage and from analyzing many samples using a sequencing-
based assay [4]. Target-capture bisulfite sequencing enables the analysis of a defined set of genomic regions in large
numbers of samples at low cost per sample, but with high setup cost. Finally, the microarray-based Infinium DNA
methylation assays, including the MethylationEPIC BeadChip (EPIC) and its predecessor, the
HumanMethylation450 Bead Chip (450k)-facilitate standardized, high-throughput DNA methylation profiling of a
pre-defined subset of CpGs in large sample cohorts [5].
These assays have enabled DNA methylation mapping for a large number of cell types and following the concept of
epigenome-wide association studies (EWAS), for various diseases with a suspected role of epigenetic regulation [6].
The resulting datasets typically comprise DNA methylation profiles as well as sample annotations such as tissue or
cell type, phenotypic data (donor age, sex, etc.) and sample grouping (case versus control, treated versus untreated,
etc.). The primary goal for the bioinformatic analysis of such datasets is to identify characteristic and reliable DNA
methylation patterns, and to associate them with relevant annotation data. The various software packages exist that
support individual steps of the DNA methylation analysis are summarized as a feature table in Additional file 1:
Table S1. Many users would benefit from an integrative analysis tool that provides extensive, easy-to-understand
analysis reports and requires minimal configuration and no detailed bioinformatic knowledge of the various analysis
steps.
METHODOLOGY
We have previously developed the RnBeads software package as a start-to-finish pipeline for DNA methylation
analysis in accordance with established standards and practices [7]. Initially released in 2012 and published in 2014,
RnBeads has become a popular and widely used tool for DNA methylation analysis (200–300 downloads per month
from Bioconductor) [8]. Here, we present a new, substantially extended version of RnBeads, providing an up-to-
date, user-friendly, feature-rich, and readily scalable workflow for the bioinformatic analysis of DNA methylation
datasets. The new RnBeads 2.0 software package addresses feedback and feature requests from the tool’s active user
community, implements new analysis methods, introduces a graphical user interface, and improves computational
efficiency. With these advances, RnBeads provides state-of-the-art support for DNA methylation data analysis in an
easy-to-use way, with high flexibility and performance.
RnBeads includes modules for data import, quality control, filtering and normalization (“preprocessing”), export of
processed data (“tracks and tables”), covariate inference (e.g., predicting epigenetic age and cell type heterogeneity
from DNA methylation data), exploratory analysis (e.g., dimension reduction, global distribution of DNA
methylation levels, hierarchical clustering), and differential DNA methylation analysis (Fig. 1). Each analysis
module generates an HTML report that combines method descriptions, results tables, and publication-grade plots.
These reports provide the user with a comprehensive and readily sharable summary of the dataset.
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Fig. 1 Differential DNA methylation analysis
Overview of the RnBeads analysis workflow and new features added in RnBeads 2.0. Conceptual drawing of the
RnBeads workflow for DNA methylation analysis, listing key features (right) for each of the RnBeads analysis
modules (center), with newly added features indicated in bold red text. Tab, tabular (e.g., comma-separated) files;
idat, Infinium signal intensity files; geo, download from the GEO data repository
Of the various features that we introduced into RnBeads since the original publication in 2014, we specifically
highlight the following four areas:
1. New data types and cross-platform analysis: RnBeads now supports EPIC microarrays and enables
seamless data integration across different DNA methylation assays (e.g., EPIC, 450k, and 27k microarrays
as well as WGBS and RRBS), which facilitates DNA methylation meta-analyses that combine several data
sources into a single set of results.
2. Extended analysis and inference methods: We added new functionality for handling incomplete data and
missing values, for detecting genetic evidence of sample contamination or low data quality, for quantifying
DNA methylation heterogeneity, and for DNA methylation-based inference of phenotypic information. We
incorporated the LUMP algorithm, which estimates immune cell content of tumors and other heterogeneous
tissue samples, and epigenetic age prediction for both Infinium microarray and bisulfite sequencing data.
These predictions are useful not only for inferring missing donor annotations, but also for detecting
deviations indicative of accelerated aging or evidence of sample mix-ups. Additional new features include
the identification of genomic regions characterized by differential DNA methylation variability and
genomic region enrichment analysis using the LOLA tool.
3. New user-friendly interface: We provide a graphical user interface for RnBeads that facilitates the
configuration and execution of DNA methylation analyses. Together with RnBeads’ interactive and self-
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explanatory HTML reports, this new interface makes RnBeads analyses more readily accessible for users
with limited R/Bioconductor knowledge.
4. Improved computational efficiency: Using parallelization and automatic distribution of RnBeads
analyses across a high-performance computing (HPC) cluster, we were able to process datasets comprising
hundreds of RRBS/WGBS profiles and thousands of microarray-based profiles in a single analysis run.
RESULTS AND DISCUSSION
To illustrate the practical utility of these new RnBeads features, we present four use cases: (i) DNA methylation in
human peripheral blood samples, (ii) cell type-specific DNA methylation in human hematopoiesis, (iii) DNA
methylation heterogeneity in cancer samples, and (iv) cross-platform DNA methylation analysis. Detailed re-
runnable versions of these analyses, including configurations and results, are available for visualization and
download from the RnBeads website (https://rnbeads.org/methylomes.html). These pre-configured analyses and pre-
calculated reports also provide a good starting point for learning about the use of RnBeads, thereby complementing
the tutorials provided on the RnBeads website (https://rnbeads.org/tutorial.html), and for configuring custom
analyses that integrate newly generated datasets with publicly available reference data.
Use case 1: Analyzing DNA methylation in a large cohort of peripheral blood samples
To illustrate the use of RnBeads for analyzing DNA methylation microarray data in a large cohort, we obtained
Infinium 450k profiles for peripheral blood samples of 732 healthy individuals. We also included reference profiles
for sorted blood cell types, in order to account for inter-individual differences in the frequency of different cell
types. First, we used RnBeads to infer donor age and sex for each sample, thereby filling in a handful of missing
annotations with imputed values, while also checking for potential sample mix-ups among those samples that have
donor age and sex documented as part of their annotation as in Fig. 2 a, b. Second, we performed reference-based
estimation of immune cell composition as implemented in RnBeads, and we found that the inferred immune cell
content as well as other annotations are indeed associated with important principal components of the DNA
methylation dataset as in Fig. 2 c, d. Our results emphasize the need to correct for these covariates when identifying
CpGs and genomic regions that are associated with the annotation(s) of primary interest. Third, we compared
chronological age with the fraction of CD4+ T cells inferred from the DNA methylation data using sorted blood cell
types as reference as in Fig. 2e and observed a negative correlation, consistent with the known age-related shift
toward myeloid (instead of lymphoid) hematopoiesis. In summary, this use case illustrates the prediction of age, sex,
and cell composition based on DNA methylation data, and it provides a framework for microarray-based
epigenome-wide association studies.
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Fig. 2Infinium 450k profiles for peripheral blood samples
Analysis of a large DNA methylation dataset of blood samples profiled using Infinium 450k.
A) Scatterplot showing the correlation between epigenetic age predicted from DNA methylation and reported
chronological age for 729 healthy donors (three individuals were excluded because no chronological age
was reported).
B) Positioning of the samples in two-dimensional space for sex prediction
C) Statistical association between principal components (columns) and sample annotations (rows). Significant
associations with p values below 0.01 are marked by filled circles, while non-significant values are
represented as empty circles.
D) Principal component analysis for 792 blood-based DNA methylation profiles, comprising 732 peripheral
blood samples and 60 sorted blood cell populations, using the same principal components as in panel c.
Immune cell content was estimated using the LUMP algorithm. E Scatterplot showing the negative
correlation between chronological age and the estimated fraction of CD4+ T cells
Use case 2: Dissecting the DNA methylation landscape of human hematopoiesis
The efforts of the International Human Epigenome Consortium and its contributing projects have resulted in large
sets of publicly available WGBS data for various cell types. To demonstrate RnBeads’ ability to process such large
reference collections, we analyzed a DNA methylome dataset comprising 195 WGBS profiles and 26,238,599
unique CpG sites (after the preprocessing step) for various hematopoietic cell types as in Fig. 3a, which was
originally established by the BLUEPRINT project. Focusing on pre-defined genomic region sets including the
Ensemble Regulatory Build, we observed the expected distribution of DNA methylation, with high levels of DNA
methylation in genome-wide tiling regions, slightly lower levels at enhancers and transcription factor binding sites,
and much lower levels (and a bimodal distribution) of DNA methylation at gene promoters and transcription start
sites as shown in Fig. 3b. The DNA methylation profiles clustered according to cellular lineage (lymphoid vs.
myeloid cells), cell maturation stage (naive vs. effector/memory cells), and cell type as shown in Fig. 3c. Comparing
two myeloid cell types (monocytes and neutrophils), RnBeads identified decreased DNA methylation levels in
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monocytes at a subset of putative regulatory regions as in Fig. 3d. LOLA analysis for enrichment of genomic region
sets (a new feature we introduced in RnBeads 2.0 to facilitate biological interpretation) identified characteristic
enrichment for cell type-specific regulatory regions (including monocyte-specific open chromatin and its associated
histone modifications) and for the binding sites of important hematopoietic transcription factors such as CEBPB and
SPI-1/PU.1. In summary, this use case demonstrates the scalability of RnBeads to large DNA methylation datasets
(which involves the distribution of analysis jobs across an HPC cluster for efficient parallelized calculation), region-
based analysis of DNA methylation, and biological interpretation by region set enrichment analysis.
Fig. 3 Unique CpG sitesfor various hematopoietic cell types
Genome-wide analysis of DNA methylation in hematopoietic cells profiled using WGBS.
A) Overview of cell types and sample numbers in the BLUEPRINT WGBS dataset (August 2016
release), which was analyzed with RnBeads.
B) Distribution of DNA methylation levels for different types of genomic region sets.
C) t-SNE dimension reduction based on Euclidean distances of mean DNA methylation in putative
regulatory regions.
Cell types are color-coded as in panel a. d Density scatter plots showing differential DNA methylation levels
between monocytes (N = 20) and neutrophils (N = 10). Point density is indicated by blue shading. The 0.1% of
regions in the most sparsely populated areas of the plot are shown as individual points. The 500 highest-ranking
hypomethylated regions in monocytes compared to neutrophils are indicated in purple. E Log-odds ratios of the
LOLA enrichment analysis for the 500 regions highlighted in panel d. The top 20 most enriched categories of the
LOLA Core and Extended databases are shown. Differently colored bars represent different types of genomic region
data (e.g., peaks for histone marks or transcription factor binding sites) as Mf, macrophage; GM, lymphoblastoid
cell; Mo, monocyte; REMC, Roadmap Epigenomics Mapping Consortium.
Use case 3: Quantifying DNA methylation heterogeneity in a childhood cancer cohort
Epigenetic heterogeneity has recently emerged as a key property of tumor samples [9]. To demonstrate the utility of
RnBeads for cancer research, we re-analyzed 188 recently published RRBS profiles of Ewing sarcoma tumors, cell
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lines, and mesenchymal stem cells [10]. Ewing sarcoma is a pediatric bone cancer characterized by low genetic
heterogeneity but striking changes in the epigenome [11]. Data processing and quality control resulted in 2,217,186
unique CpG sites that were covered by at least five sequencing reads in more than 50% of the samples. Based on
these CpGs, we aggregated DNA methylation values in each sample across annotated genomic regions, including
putative regulatory elements defined in the Ensembl Regulatory Build [12]. Principal component analysis showed
the expected separation between tumors, cell lines, and mesenchymal stem cells, with higher sample-to-sample
heterogeneity among the tumors and cell lines compared to mesenchymal stem cells (Fig. 4a). We compared the
primary tumors with the cell lines using the differential DNA methylation module of RnBeads, and we found that
most of the differentially methylated regions were hypermethylated in the cell lines (Fig. 4b). We also observed
increased variance in the cell lines (Fig. 4c). LOLA analysis detected markedly different enrichments among
differentially methylated regions (DMRs) and differentially variable regions (DVRs), indicating that the two
measures provide complementary information about the DNA methylation landscape (Fig. 4d–f). Regions
hypermethylated in Ewing sarcoma cell lines were enriched for DNase hypersensitive sites in various healthy tissue
samples (Fig. 4d), consistent with the widespread hypermethylation and silencing of non-essential regulatory regions
in cell lines. In contrast, hypervariable regions were enriched for transcription factor binding and histone
modifications in cancer cell lines and embryonic stem cells (Fig. 4f), indicative of increased regulatory plasticity of
the Ewing sarcoma cell lines compared to the primary tumors. In summary, this use case describes the analysis of an
RRBS-based dataset (which benefits from region-based analysis due to fluctuations in single-CpG coverage), and it
demonstrates the utility of RRBS and RnBeads for investigating DNA methylation heterogeneity in tumor samples.
Fig. 4. Component analysis for investigating DNA methylation heterogeneity in tumor samples.
Dissection of DNA methylation heterogeneity in Ewing sarcoma samples profiled using RRBS is as follows.
A. Principal component analysis of an RRBS dataset for Ewing sarcoma tumors, cell lines, and mesenchymal
stem cells, based on DNA methylation values aggregated across Ensembl Regulatory Build regions.
B. Density scatter plot comparing the aggregated DNA methylation levels between Ewing sarcoma tumors
(N = 140) and Ewing sarcoma cell lines (N = 16). Marked in purple are the most highly ranking
differentially methylated regions up to an automatically selected rank cut off.
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C. Density scatter plot comparing DNA methylation variance between Ewing sarcoma tumors and cell lines.
Significant differentially variable regions are marked in brown.
D. Enrichment (log-odds ratios) based on LOLA analysis for the differentially methylated regions shown in
panel b and in panel e. Differently colored bars represent different types of genomic region data.
E. Density scatter plot comparing the log-ratios between DNA methylation levels and variance in Ewing
sarcoma tumors and cell lines.
F. Enrichment (log-odds ratios) based on LOLA analysis for differentially variable regions shown in panel c
and in panel e. ESC, embryonic stem cell; CGIs, CpG islands.
Use case 4: Analyzing DNA methylation data across different assay platforms
Several generations of Infinium DNA methylation microarrays have been used over the years, and it can be
necessary to combine multiple datasets in an integrative analysis. RnBeads now provides dedicated methods for
cross-platform analysis, making it possible to combine RnBeads data objects across the different versions of the
Infinium microarray (27k, 450k, EPIC) and with bisulfite sequencing data (RRBS, WGBS). To demonstrate this
feature, we analyzed a benchmarking dataset comprising three different assay platforms: Infinium 450k microarrays,
Infinium EPIC microarrays, and WGBS [13]. All three datasets were loaded and preprocessed separately using
RnBeads, which resulted in data objects with 443,053 (450k), 801,716 (EPIC), and 25,918,426 (WGBS) unique
CpG sites, respectively. Applying the RnBeads method for combining datasets with the option of including only
CpGs covered by all three platforms, these objects were merged into a combined dataset comprising 408,621 shared
CpGs. This combined dataset was processed using the RnBeads analysis modules.
We observed differences in the global distribution of DNA methylation levels between assays (Fig. 5a).
Nevertheless, the principal component analysis showed that the biological differences between samples dominated
over the technical differences between platforms (Fig. 5b). Focusing specifically on the comparison between a
prostate cancer cell line (LNCaP) and prostate epithelial cells (PrECs), we observed the highest correlation between
replicates for the same assay in the same cell type (Pearson’s r = 0.9979, Fig. 5c). Nevertheless, the correlation
between different assays in the same cell type (Pearson’s r = 0.9655, Fig. 5d) was still high and much stronger than
the correlation between different cell types for the same assay (Pearson’s r = 0.6471, Fig. 5e). In summary, this use
case highlights the feasibility and practical utility of cross-platform analysis of DNA methylation using RnBeads\.
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Fig. 5 Global distribution of DNA methylation levels
Cross-platform data integration using RnBeads is as follows.
A. Distribution of DNA methylation levels for the same samples profiled across different assay platforms.
B. Principal component analysis of the assay comparison dataset. Point shapes and colors depict assay
platforms and cell types, respectively.
C. Density scatter plots comparing replicates of prostate epithelial cells profiled using the EPIC assay (panel
c); prostate epithelial cells profiled using the EPIC assay and WGBS (panel d); and prostate epithelial cells
as well as a prostate cancer cell line profiled with the EPIC assay (panel e).
Point density is indicated by blue shading. The 0.1% of CpGs in the most sparsely populated areas of the plot are
shown as individual points. All plots are based on CpGs that were covered by all three assay platforms. Pearson
correlation coefficients are shown below each diagram as NAF, non-malignant tissue-associated fibroblasts; CAF,
cancer-associated fibroblasts; PrEC, prostate epithelial cells; LNCaP, prostate cancer cell line.
Comparison to other software tools for DNA methylation analysis
To assess the computational efficiency of RnBeads, we compared its performance to that of other software packages
for DNA methylation analysis, separately for DNA methylation microarray data, RRBS data, and WGBS data (see
the “Methods” section for details and Additional file 2: Table S2 for tool configurations) [14]. Given that the
different tools provide vastly different feature sets, we considered three scenarios: (i) data import only, (ii) core
modules and (iii) comprehensive analysis with more features activated (Additional file 3: Figure S1). RnBeads was
the only tool that supported both microarray-based and bisulfite sequencing-based analysis. For microarray-based
analysis, the low-level data processing packages minfi, methylumi, and wateRmelon were faster than ChAMP and
RnBeads (which need to prepare the dataset for their more extensive downstream analyses) [15]. Compared to
ChAMP, RnBeads was more memory-efficient and faster in the comprehensive setting. For bisulfite sequencing-
based analysis, RnBeads showed better performance than methylKit on the WGBS dataset in the core module
setting, but somewhat longer runtime and higher memory usage on the RRBS dataset. These differences can be
attributed to the reformatting into memory-efficient data structures that RnBeads performs during data import [16].
In summary, the runtime performance of RnBeads was similar to that of other tools with more limited functionality,
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suggesting that the choice of the most suitable tool for DNA methylation analysis depends mainly on the desired
features and analysis modes. To assist with an informed selection, we thus surveyed a broad range of tools for DNA
methylation analysis and assembled a detailed feature table based on the tool documentations (Additional file 1:
Table S1). RnBeads emerged from this comparison as the software that implements the most comprehensive
workflow for analyzing DNA methylation data, while also providing a user-friendly interface and extensive options
for reporting and reproducibility.
CONCLUSION
RnBeads is an integrated software package for the analysis and interpretation of DNA methylation data. Due to its
modular design and optional graphical user interface, the software is well suited for both beginners and experts in
the field of DNA methylation analysis. Interactive reports provide a comprehensive overview of DNA methylation
datasets while also fostering reproducibility (by documenting parameter settings) and robustness (by making it easy
to evaluate different parameter settings). RnBeads 2.0 implements various new features that arose from
technological advances and valuable user feedback. For example, support for the Infinium EPIC assay and for cross-
platform data integration broaden the scope of RnBeads analyses; new and improved methods for the DNA
methylation-based prediction of age and sex are useful for large cohort studies; a graphical user interface makes
many RnBeads features more easily accessible; the analysis of DNA methylation variability adds a new dimension
to epigenome-wide association studies; and LOLA-based region set enrichment analysis facilitates the biological
interpretation of DNA methylation differences. RnBeads also establishes a convenient way of interfacing with
reference epigenome datasets and integrating them into custom analyses, as demonstrated by the integration of
sorted blood cell profiles in our first use case. We processed several such datasets and provide re-runnable analyses
on the website (https://rnbeads.org/methylomes.html). The presented use cases provide concrete examples for
RnBeads analysis of DNA methylation and illustrate some of the tool’s key features. Typical applications of
RnBeads include epigenome-wide association studies, reference epigenome analysis, investigation of cancer
heterogeneity, and epigenetic biomarker development.
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2. Makambi, K. (2003). Weighted inverse chi-square method for correlated significance tests. Journal of Applied
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6. Adler, D., Gl¨aser, C., Nenadic, O., Oehlschl¨agel, J. and Zucchini, W. (2012) ff: memoryefficient storage of large
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Lafta & Shamran (2019): Electrophoresis gels and PCR contamination May 2019 vol. 19
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Safety Disposal of Electrophoresis Gels and PCR contaminate with
Ethidium Bromide and alternative methods.
Methaq Hadi lafta¹ Amal Raqib Shamran²
Iraqi Education Ministry, Qadisiyah ¹
University of Babylon /collage of science for weman²
Abstract:
Electrophoresis gel is commonly used in molecular biology laboratories for the identification of nucleic acid
(DNA or RNA) and proteins. This gel will typically be agarose-based. This electrophoresis process utilized an
organic fluorescence dye or inorganic such as Silver (which is an EPA regulated material) to stain DNA or
proteins. Waste of the DNA identification process must be managed and disposed in a manner to protect public
health and the environment. Purpose: To ensure safe, prudent disposal as well as reduce the amount of
Hazardous Waste material. This can be accomplished by choosing less toxic materials and work practical
methods that minimize thquantity of waste generated as well as the toxicity of the waste material itself. In cases
where safer materials or work practices cannot be employed, waste collection methods and regulatory agency
requirements are to be followed. Background: There are a number of different protocols and alternative dyes
used in the preparation of electrophoresis gels. Gels can be cast with or without dyes. The DNA /proteins can
be stained by adding the dye to the sample before electrophoresis; the gel can be placed in a dye solution after
electrophoresis has been completed. Waste Management: Waste disposal requirements will vary on the dye
used and the methodology used to stain the cells.
The electrophoretic waste of the DNA or proteins product identification process must be managed and disposed
in a manner that is consistent with the commitment it has to protect public health and the environment. The
following provides Lab managers/generators with the proper procedures for managing and disposing
electrophoresis-associated gel wastes.
Key words: PCR, electrophoresis, ethidium bromide, contaminate, waste management
How to cite this article: Lafta MH, Shamran AR (2019): Safety Disposal of Electrophoresis Gels and PCR
contaminate with Ethidium Bromide and alternative methods, Ann Trop Med & Pub Health-Special Issue;
19: 2004-19.
MUTAGENIC DYES
Ethidium Bromide, Acridine Orange, SYBR Green I, SYBR Green II, SYBR Gold, GelStar. These dyes have
been determined to have mutagenic properties. The gel that have been cast with these dyes in them, unwanted
dye stock solutions, and all contaminated debris must be collected for disposal by the HWMU. Gels that have
undergone Electrophoresis and staining, and then have been destained.
Where all excess dye has been washed out the gel (the only dye left in the gel is a trace amount contained in
DNA /protein sample material) can be discarded in the trash. Contaminated “non-sharp” lab debris (e.g.,
gloves, towels, tubes, etc.) should be collected and disposed of the HWMU.
The buffer solutions that have been run through the approved filter should be checked under an appropriate light
source for complete removal of
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©Annals of Tropical Medicine & Public Health SP2004-19
the dyes, and if it passes (does not fluoresce), the liquid can be disposed of
down the drain with a copious amount of water as long as it contains no other materials that would cause it to be
regulated as a Hazardous Waste.
Waste Management of Materials for Disposal through the Hazardous Waste Management Unit.
What is hazardous waste?
■Chemicals (room 0114)
■'Risk waste': Blood, cells, viruses and bacteria, sharp or pointed objects (room 0116)
■Biological waste: Dead experimental animals, etc. (room 0116 / freezer room in the waste area)
■Radioactive waste (room 0115)
Chemicals
The safety data sheet will tell you whether your waste is to be classified as hazardous waste. In addition
it will provide information about the necessary protective equipment and about any protective devices that must
be used.
All waste must be packaged and labelled properly. If possible, use the original packaging if this is
suitable. Containers must be in good condition, and caps must be screwed on well. Avoid using packaging
material that can be confused with household products – for example coffee jars, soft-drink bottles and the like.
The university purchases plastic cans for hazardous waste, labels for marking waste, and polystyrene
boxes that can be used to protect glass bottles against impact. These are kept in the waste area in the basement.
If you can not find any available or have any other questions, please contact the HSE coordinator.
Each substance/substance compound must be clearly labelled with:
■contents (name of chemical and approximate composition). If the contents are not known, the
container must be labelled “Content not known".
■date
■name, department and telephone number
Chemical waste, used oil and batteries must be placed in Room 0114 in the basement of Domus
Medica.
Volatile substances must be place in the innermost part of the waste room with suction removal. The
waste must be put on the shelves, away from the floor.
Explosive chemicals must not be put in this room. Contact the HSE Coordinator for the disposal of
explosives.
The room must be kept locked at all times.
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Hazardous waste is collected by Ragn Sells AS.
Risk- and biological waste
When handling 'risk waste', adequate protective equipment and, if relevant, protective devices must be
used. Read the safety data sheet for information.
'Risk waste' and biological waste can include:
■Biological material
■Sharps and cutting equipment
■Substances that may be carcinogenic/mutagenic
■Cytostatics etc.
All waste must be packaged and labelled properly. 'Risk waste' is packaged in yellow plastic containers
that are labelled with a completed declaration form for risk waste. Yellow plastic containers and declaration
forms are available in the waste department in the basement. If you can not find any available or have any other
questions, please contact the HSE coordinator.
Yellow toxic waste containers must be placed in the basement of Domus Medica, room number 0116.
The room must be kept locked at all times.
Risk- and biological waste is collected by Ragn Sells AS.
Radioactive waste
When handling radioactive waste, adequate protective equipment and if relevant protective devices
must be used, such as closed hoods. Please read the safety data sheet for more information. All radioactive waste
needs to be clearly marked with a label for radioactive waste. These are available in the waste department. If
you can not find any available or have any other questions, please contact the HSE coordinator.
Radioactive waste, which is also 'risk waste' and biological waste, must also be packed into yellow
plastic containers and be marked with a label for radioactive waste. This also applies to waste that has
previously been radioactive but that has become inactive. Please use bequerel as a unit for activity.
All radioactive waste, as well as waste that has previously been radioactive, must be logged when it is
placed in the waste room. The log is kept inside of room 0115. If you have any problems using the log you are
required to notify the HSE coordinator.
The waste must be placed in the basement of Domus Medica, Room 0115. The room must be kept
locked at all times.
Waste described in this document must be stored in the following manner:
■32P is kept in plexiglass shielding containers
■51Cr and 125I are kept in lead containers
■35S, 3H and 14C can be stored without shielding
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©Annals of Tropical Medicine & Public Health SP2004-19
Mutagenic or Toxic product of (PCR) and Contaminated “Non-Sharp” Lab Debris (e.g., gloves, pads ,
tubes, etc.) into a plastic container 5 gallon bucket ( optimal of volume of waste generated). This container
should have a plastic bag as an inner liner. The container must be closed at all times except when immediately
adding or removing wastes from the container. Contact the Hazardous Waste Management Unit (HWMU) if you
need a 5 gallon bucket to collect your waste. Labelled the container’s which waste constituents are present in the
pail (e.g., “Hazardous Waste - Ethidium Bromide Contaminated Gels, Gloves, Paper”).
Sharp items (e.g., needles, Pasteur pipettes, Tips, etc) are to be placed into the containers or 5-gallon
pails. See below for the proper means for disposing of contaminated sharps lab debris.
For a pickup. An empty replacement pail will be provided at the time of the collection if needed. Collection and
Disposal of Chemically Contaminated Sharps
Chemically contaminated sharps (needles, Pasteur pipettes, etc.) must be collected in an approved sharps
shelter (NOT RED – use the white/translucent or yellow). It must be labelled(Hazardous Waste – Chemically
Contaminated Sharp). Any biohazard labels should be removed or completely defaced.
■ DNA and PCR products
■Kits
■ Agaros seals
■Used running buffer
Protective Equipment
■Gloves (nitrile gloves when handling ethidium bromide)
■Laboratory coat
■Safety glasses
Procedure for disposal
■ All DNA, plasmids etc. must be handled as toxic waste.
■ All pipette tips, tubes etc. that have been used for DNA work must be disposed of in yellow toxic waste
containers.
■ Agaro seals with EtBr and SYBR-safe/SYBR-green etc. are disposed of in yellow toxic waste containers.
■Contaminated glass equipment must be rinsed well before being put ready for washing.
■Kits: waste must be handled in accordance with the HSE data sheet that comes with the kit.
■Small volumes of isopropanol and ethanol waste must be collected in Eppendorf tubes and disposed of in
yellow toxic waste containers. Larger volumes must be handled as hazardous waste.
■Used running buffer must be handled as an absorption medium. Used absorption media must be disposed of in
yellow toxic waste containers. Read the safety data sheet that comes with the absorption medium.
Examples of absorption media
■ Activated carbon, activated charcoal, C3014-500G, untreated, granular, 20-60 mesh
Lafta & Shamran (2019): Electrophoresis gels and PCR contamination May 2019 vol. 19
©Annals of Tropical Medicine & Public Health SP2004-19
■Destaining bags, cat. no: 730-0997
■BondEX 50 Set (5), product code M740703
Handling GMO(Genetically Modified Organisms ) waste
■Waste from GMO must be handled as biological waste and regarded as toxic waste. This applies to both solid and liquid waste.
■Waste from GMO in class 3 and class 4 must be inactivated before it is placed in yellow toxic waste
containers.
■Inactivation takes place by autoclaving for one hour at 121 degrees, or by using Virkon 1-3% or 5%
hypochlorite.
Alternative Dyes
SYBR® Safe, GelRed, GelGreen, and EvaGreen. These dyes determined to be nonmutagenic dyes in
testing by independent licensed testing laboratories.All gels and contaminated “non-sharp” lab debris ( gloves,
towels, etc.) that are processed using this dye can be discarded in the trash.Spent running buffer solutions and
destaining solutions that contain the dyes can either be collected and disposed of through the HWMU or
collected and run through an approved filter device.
The buffer solutions that have been run through the approved filter should be checked under the
appropriate light source for complete removal of the dyes, and if it passes (does not fluoresce), the liquid can be
disposed of down the drain with a copious amount of water as long as no other materials are present that would
cause the material to be a (Hazardous Waste). The filters that have been used up and are no longer effective
must be disposed of through the HWMU.
Figure (1) show the right disposed of carcinogenic Lab waste
Lafta & Shamran (2019): Electrophoresis gels and PCR contamination May 2019 vol. 19
©Annals of Tropical Medicine & Public Health SP2004-19
References:
Armour, Margaret-Ann. Hazardous Laboratory Chemicals Disposal Guide, 3rd Edition.
Ethidium Bromide MSDS: http:/msds.ehs.cornell.edu/msds/siri/files/cfg/cfggr.html
GelRed, GelGreen Safety Report:
http://www.biotium.com/product/product_info/Newproduct/GelStains.asp
EvaGreen Safety Report:
http://www.biotium.com/product/price_and_info.asp?item=31000&Sub_Section=09A
GelStar Product Protocol:
http://www.cambrex.com/Content/Documents/Bioscience/GelStar%2818111%29.pdf
GelRed, GelGreen Ames Testing: z
www.biotium.com/product/product_info/Safety_Report/GR%20&%20GG%20safety.pd
http;//juniordentist.com
Praseetha et al (2019): Study of chitosan May 2019 vol. 19
©Annals of Tropical Medicine & Public Health SP2005-19
IN-VITRO CHICK CHORIOALLANTOIC MEMBRANE STUDY OF CHITOSAN
CAPPED 5-FLUOROURACIL CONJUGATED GOLD NANOPARTICLES
Akhila Rajan
1, P.K. Praseetha
2*, Methaq Hadi lafta
3, V.N. Ariharan
4, S. T. Gopu Kumar
5
1,2*Department of Nanotechnology, Noorul Islam Centre For Higher Education, Kumaracoil –
629180, India.
3Lecturer in Ministry of Education, Iraq.
4Inbiotics, William Hospital Campus, Nagercoil – 629001, India.
5Thampi Institute of Medical Educational and Research, William Hospital Campus,
Nagercoil – 629001, India.
E-mail:[email protected]
ABSTRACT
Introduction: Chitosan is a linear polysaccharide composed of randomly distributed β-(1→4) -
linked D-Glucosamine (DE acetylated unit) and N-acetyl-D-Glucosamine (acetylated
unit). It is made by treating the chitin shells of shrimp and other crustaceans with an
alkaline substance, like sodium hydroxide. Chitosan has a number of commercial and
possible biomedical uses. Chitosan's properties also allow it to be used in transdermal
drug delivery.
Aim: The aim of the work is to deliver therapeutic compound to the desirable site in the
treatment of diseases.
Methods: Chitosan Nanoparticles (NPs) was prepared using the ionic deletion method. 5’
Fluorouracil (5’FU) loaded Chitosan nanoparticle (5FCN) was synthesized and tested
for its Angiogenic activity on Cell lines by a chick chorioallantoic membrane (CAM)
assay.
Conclusion: The angiogenic activity of 5-FU loaded chitosan nanoparticles provided a platform
for treating cancer with biopolymer nanomaterials. The combination of nanoparticles
with Chitosan in the form of Nano composite matrices provides the high surface area
required to achieve a high loading of enzymes, drugs, and a compatible micro-
environment to facilitate stability.
Keywords: 5-fluorouracil, chick chorioallantoic membrane, Chitosan, Gold Nano particle.
How to cite this article: Rajan A, Praseetha PK, et al (2019): In-vitro chick chorioallantoic
membrane study of chitosan capped 5-fluorouracil conjugated gold
nanoparticles, Ann Trop Med & Pub Health-Special issue; 19: Sp2005-19.
Praseetha et al (2019): Study of chitosan May 2019 vol. 19
©Annals of Tropical Medicine & Public Health SP2005-19
INTRODUCTION
Recent advancements in the synthesis of various nanomaterials of different sizes, shapes and functions have
established nanotechnology as an indispensable technology for agriculture 1. Materials which show unique
properties linked to their size (ranging from 1 to 1000 nm at least in one dimension) are considered
nanoparticles and deemed under nanotechnology. Nano materials have some high value properties like high
surface-to-volume ratio, more molecules/reactive groups on the surface; prefers Nano-encapsulation and are
independent of gravity 2.
The rapid expansion of nanotechnology promises to have great benefits for society. Nanomaterial is matter at
dimensions of roughly 1-100 nm, where a unique phenomenon offers novel applications. The major advantages
of nanoparticles as a delivery system are in controlling particle size, surface properties, and release of
pharmacologically active agents in order to achieve the site-specific action of the drug at the therapeutically
optimal rate and dose regimen. Although nanoparticles offer many advantages as drug carrier systems, there are
still many limitations to be solved such as poor oral bioavailability, instability in circulation, inadequate tissue
distribution, and toxicity 3.
Chitin is found in the exoskeleton of some anthropoid’s, insects, and some fungi. Commercial sources of chitin
are the shell wastes of crab, shrimp, lobster, etc. Chitosan is usually prepared by the DE acetylation of chitin.
The conditions used for DE acetylation will determine the average molecular weight (Mw) and degree of DE
acetylation (DD). The DD of typical commercial chitosan is usually between 70 and 95%, and the Mw between
10 and 1,000 kDa.
The use of nanoparticles in the biological field has increased due to the superior properties that they possess
when compared to their bulk counterparts such as enhanced permeability and retention and the ease with which
they are taken up by the cells as demonstrated by Y. Liu et al in the use of Nano vectors for gene delivery in the
case of low density carbon nanotubes 4. Apart from this, nanoparticles own a functional surface which gives the
nanoparticles the ability to bind, adsorb and carry other compounds thus, making them suitable for drug
delivery. Besides this, the nanoparticles also protect the drug from degradation, enable a prolonged release of
the drug, improve the bioavailability of the drug, reduce the toxic side effects of the drug and offer an
appropriate form for all routes of administration 5. Of these nanoparticles, noble metal nanoparticles are
preferred due to their optical properties, non-toxicity and biocompatibility8 when compared to the other metals.
Majorly, gold nanoparticles can convert light or radio frequency into heat, thus enabling the thermal ablation of
the targeted cancer cells 6. This type of phenomenon has been observed for gold nanoparticles. Hence, the
ability to combine drug delivery and photo thermal therapy on gold nanoparticle based delivery platforms prove
it to be a system that is capable of eliminating the cancer cells 7. One major drawback that arises while using
gold nanoparticles is its agglomeration during reduction from its metal salts. It has reported the importance of
the control of nanoparticle size as the increased particle size reduces the scope for their use in drug delivery
since it is well known that particles with size ranging from 10 to 50 nm are the ones that are easily taken up by
the cells. Thus, an effective way to prevent the aggregation of gold nanoparticles is mandatory. An effective
strategy for this would be the use of a stabilizing agent or surfactant. The most promising and environment
friendly stabilizing agents are enzymes 15 and polymers. The role of a stabilizing agent/surfactant is played by a
chitosan18. Chitosan is a biopolymer that is biocompatible, biodegradable, eco-friendly, non-toxic and has NH2
Praseetha et al (2019): Study of chitosan May 2019 vol. 19
©Annals of Tropical Medicine & Public Health SP2005-19
and OH groups which act as chelating sites for drugs (5-Fluorouracil in our case) and for other molecules.
Moreover, as the combination of nanoparticles with Chitosan in the form of Nano composite matrices provide
the high surface area required to achieve a high loading of enzymes, drugs, and a compatible micro-environment
to facilitate stability as Chitosan is suitable for use as a drug delivery carrier. There are a number of reports on
the use of gold nanoparticles and Chitosan separately for drug delivery 8. A Nano composite system consisting
of both gold and Chitosan will encompass the properties of both the moieties, which makes it a powerful choice
for use in the bio-compatible, targeted delivery and sustained release of 5-Fluorouracil (5-FU).
MATERIALS AND METHODS
The chicken chorioallantoic membrane (CAM) assay is a standard assay used to evaluate the angiogenic
potential of biomaterials. Angiogenesis is the formation of new blood capillaries from existing blood vessels for
supplying nutrients to the cells that are distant from existing blood vessels. Angiogenesis is a complex process
that is mediated by the endothelial cells that line blood vessels. Angiogenesis is a regulated process involving a
number of stimulators such as fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF),
angiopoietins, activators of integrins and inhibitors such as thrombospondin, angiostatin and endostatin.
5 Days Old Fertilized Hen Eggs, Pyruvic Acid (Cat No:107360, Sigma), NaCl (Cat No.RES0926S-A, Sigma),
Parafilm (#380030, Tarsons), DMSO (#PHR1309, Sigma), D-PBS (#TL1006, Himedia), 50 ml centrifuge tubes
(# 546043 TORSON), 1.5 ml centrifuge tubes (TORSON), 1 ml serological pipette (TORSON), 10 to 1000 ul
tips (TORSON).
Equipment’s used Sterilized Sharp Forcep, 18-gauge hypodermic needle (#72-567, Harward Apparatus),
Camera (Canon), Pipettes: 2-10μl, 10-100μl, and 100-1000μl. Assay controls Negative Control (0.9% NaCl),
Positive Control (1% w/v SDS).
The chickchorio-allantoic membrane (CAM) assay for screening the effect of test samples on angiogenesis was
performed. Fertilized white leghorn chicken eggs were collected from a local hatchery at day ‘5’ and checked
for the damage. The eggs were cleaned with 70 % ethanol and incubated under conditions of constant humidity
at 37°C. On the 3rd day of incubation a small hole was drilled at the narrow end and 2-3 ml of albumin was
withdrawn with 18-gauge hypodermic needle. The window was sealed with transparent tape and again
incubated. On the 7thday of incubation a small square window was opened in the shell and sterile gel foam
(3mm×3mm×1mm) piece was implanted on top of the membrane. The vehicle control group was impregnated
with sterile normal 0.9% NaCl, standard group was impregnated with 1% SDS and the test group was
impregnated with IC50 Concentration. The eggs returned to the incubator and they were incubated undisturbed
till 24hours. After 24hours of incubation the eggs were removed from the incubator and the CAM tissues
directly beneath each sponge was resected from control and treated CAM samples. The number of vessel branch
points contained in a squire region equal to the area of each sponge was counted and findings from 3 CAM
preparations were analyzed for each treatment group. The resulting angiogenesis index is the mean of new
branch points in each set of samples.
Chitosan Coated With Gold Nanoparticles
Praseetha et al (2019): Study of chitosan May 2019 vol. 19
©Annals of Tropical Medicine & Public Health SP2005-19
100 ml Solution (2 ml acetic acid + 98 ml water) + O.34 gm. Chitosan (0.34 %) + 2 ml gold solution (5nM) +
2ml sodium borohydrate solution (50 mm). Stirred for 3 hours (room temperature). PH of the solution brought
to pH 5.0 using NaOH. The Solution is centrifuged at 3500 RPM for 15 minutes to separate Chitosan coated
with Gold Nanoparticles. The sample is dried at 50OC overnight.
5-Fluorouracil Loaded Chitosan - Gold Nanoparticles
100 ml Solution (2 ml acetic acid + 98 ml water) + O.34 gm. Chitosan (0.34 %) + 3.8 mm 5-Fluorouracil + 1.4
mm sodium polyphosphate + 0.5 % v/v Tween 80+ 2 ml gold solution (5nM) + 2ml sodium borohydrate
solution (50 mm). Stirred for 3 hours (room temperature).PH of the solution brought to pH 5.0 using NaoH. The
Solution is centrifuged at 3500 RPM for 15 minutes to separate Chitosan coated with Gold Nanoparticles. The
sample is dried at 50OC overnight.The conjugated nanoparticle is characterized by FTIR, UV, and TG/DTA.
RESULT AND DISCUSSION
FTIR Spectrum of 5flurouracil Linked Gold Coated Chitosan
The FTIR spectrum of Chitosan coated gold nanocomposite exhibits an NH2 twisting peak at ~1566.50 cm-1,
stretching at ~ 715.07 cm-1, bending at 1404.11 cm-1, a peak of NH3 + at ~1661.48 cm-1. These peaks
correspond well to the peaks of pure Chitosan except for minor differences that establish the formation of the
nanocomposite. The splitting of the NH3 + and NH2 peak increases with increasing the amount of gold in the
nanocomposite. This is because of the neutralization of the protonated amine group. An enhancement in the
intensity of the NH2 peak on increasing the amount of gold is an indication of the increase in the percentage of
gold, incorporated into the nanocomposite. The FTIR spectrum of 5-FU encapsulated nanocomposite is shown
in figure 1. All these observations show the encapsulation of 5-FU to Chitosan coated gold nanocomposite.
3500 3000 2500 2000 1500 1000
Wavenumber cm-1
Figure 1: FTIR Spectrum of 5Flurouracil linked Gold coated Chitosan.
Tra
nsm
ittan
ce [%
]
85
75
80
90
95
3249
.86
2932
.69
1651
.48
1565
.86
1556
.50
1404
.11
1247
.67
1151
.00
1069
.92
1011
.13
92
1.2
4
89
1.7
2
75
1.0
7
68
8.9
5
64
5.9
7
61
2.7
8
58
8.4
8
56
9.6
4
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©Annals of Tropical Medicine & Public Health SP2005-19
19.51% 475.9Cel
76.80uV 531.5Cel
81.62uV
463.7Cel
61.76uV -342uV.s/mg
550.6Cel
62.41uV 52.27%
495.5Cel 503.1Cel 60.00uV 58.98uV
-181uV.s/mg
TG/DTACurve Of 5flurouracil Linked Gold Coated Chitosan
Thermal Analysis (TA) techniques study the properties of materials as they change with temperature. It provides
information on properties like thermal capacity, mass changes, enthalpy, coefficient of heat expansion etc. It is
widely used in solid state chemistry to study solid state reactions, thermal degradation reactions, and phase
diagrams. The weight loss curve indicates changes in sample structure, heat stability, and other
parameters.Differential Thermal Analysis (DTA) involves exposing the material under study and an inactive
reference to similar heat cycles. Any temperature distinction amongst test and reference is recorded. In this
procedure, the heat flow to the specimen and reference is kept constant instead of the temperature. The peak
intensities are observed at 475.9 cells, 531.5Cel, 550.6 Cel.
80.00
70.00 90.00
60.00
80.00
50.00
70.00
40.00
60.00
30.00
50.00
20.00
40.00
10.00
0.00
100.0
200.0
300.0
400.0 Temp Cel
500.0
600.0
700.0
30.00
800.0
Figure 2. TG/DTA Curve of 5Flurouracil linked Gold coated Chitosan.
UV Spectrum Of 5-Flurouracil Linked Gold Coated Chitosan
The UV-Vis spectra taken for the composite containing different weight percentages of gold is shown in figure
3. The UV-VIS spectra for the formation of gold nanoparticles was monitored and obtained. A single peak
corresponding to the surface plasmon resonance of gold was observed in the wavelength range 210-300 nm. A
peak in this range is generally attributed to the surface plasmon excitation of small spherical gold nanoparticles.
The damped nature of the peak is indicative of the small particle size. In small particles, the mean free path of
the electrons is reduced, which eventually leads to the peak dampening. The intensity of the peak increased with
concentration of gold, which implies that the amount of gold binding to Chitosan increased
.
TG
%
Praseetha et al (2019): Study of chitosan May 2019 vol. 19
©Annals of Tropical Medicine & Public Health SP2005-19
2.5
2.0
1.5
1.0
0.5
0.0
200 300 400 500 600 700
Wavelength (nm)
Figure 3. UV Spectrum of 5-Flurouracil linked Gold coated Chitosan.
Angiogenic Activity Of 5-Fluorouracil Loaded Chitosan And Gold By Chickchorio-
Allantoic Membrane (CAM) Assay
Sample A (C+G) and Sample B (C+F+G) were evaluated to check the Angiogenesis Activity on the Fertilized
Chick Eggs. The used Concentrations of the compounds and No of Branching Points resulted mentioned in the
Table 1.
Table 1. Details of Drug Treatment to respective Fertilized Eggs
Sl.No Test Compounds Concentration
1 Untreated NaCl (0.9%)
2 Standard Pyruvic Acid
3 Sample A (C+G) IC50 (uG)
4 Sample B (C+F+G) IC50 (uG)
Control
266
5-Flurouracil loaded chitosan linked Gold
Abso
rban
ce (a
.u)
Praseetha et al (2019): Study of chitosan May 2019 vol. 19
©Annals of Tropical Medicine & Public Health SP2005-19
Sample A (C+G)
Sample B (C+G+F
STD
Figure 4. Angiogenic activity of the drug under various samples.
CAM Study Of The Test Compounds Using Fertilized Hen Eggs
The Observations in Statistical data of CAM Study suggesting that on Fertilized Eggs, given Test Compound-A
(C+G)) showing a slight increase in the growth of nerve branches with the blood vessel density 26 after the
incubation of 24hours of the drug treatment. On the other hand, Test Compound-B (C+F+G)) showing a high
increase in the growth of nerve branches with the blood vessel density 43 after the incubation of 24hours of the
drug treatment compared to the Standard Drug showing 86% of Blood vessel Density. In this study, The
angiogenesis index in the chick CAM was High in the Sample B Compared to the Sample A. The networks in
CAM and the newly synthesized vessels participated actively in the circulating of blood cells. Taken together,
the observations in the present study suggest that the given Test Compounds exhibit strong angiogenic actions
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©Annals of Tropical Medicine & Public Health SP2005-19
and also may have the potential to be a useful activator of the large number of serious diseases characterized by
deregulated angiogenesis
.
Figure 5. The Angiogenic activity of Chitosan loaded gold nanoparticles.
Fertilized Hen Eggs is treated with Chitosan and gold nanocomposite under different samples, branching points
count and blood vessel density is calculated for different samples. The result is tabulated in table 2. The blood
vessel density is minimum at control and standard (86) and the blood vessel density increase in the sample B
(43.66666667).
Table 2. Details of Drug Treatment to different samples and counting of blood vessels
Concentration Unit: uG/mL Incubation: 24hrs
Test CONTROL STD Sample A Sample B
Concentration 0.9% NaCl 1% SDS IC 50 IC 50
No of Branching Points Count 1 76 168 102 121
No of Branching Points Count 2 78 161 104 125
No of Branching Points Count 3 81 164 108 120
Mean No of Branching Points 78.33333333 164.3333333 104.6666667 122
Blood Vessel Density 0 86 26.33333333 43.66666667
CONCLUSION
It was observed from the present investigation that, the angiogenic activity of 5-FU loaded chitosan
nanoparticles provided a platform for treating cancer with biopolymer nanomaterials. Chitosan is safe to use as a
carrier for the drug 5-FU, which was observed from the present investigation. This could contribute to the
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©Annals of Tropical Medicine & Public Health SP2005-19
discovery of a new method for the treatment of various cell lines that may overcome the difficulties in the
currently available procedures or therapies. The increased angiogenic activity was found even at lower
concentrations of the drug. Thus synthesized nanoparticles were more efficient in the biomedical applications in
cancer treatment for their high angiogenic activity.
ACKNOWLEDGEMENT
The authors are thankful to the chancellor, Noorul Islam Centre for Higher Education, Kumaracoil for
providing all facilities.
ETHICAL CLEARANCE
Ethical clearance is taken from advisory committee, Department of Nanotechnology, Noorul Islam
University.
SOURCE OF FUNDING
This work is a self funded project.
CONFLICT OF INTEREST
No conflicts of interest declared.
REFERENCES
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2. Yamamoto K, Hagino M, Kotaki H, Iga T. Quantitative determination of domperidone in rat plasma by high-
performance liquid chromatography with fluorescence detection. Journal of Chromatography B: Biomedical Sciences
and Applications. 1998 Dec 11;720(1-2):251-5.
3. Carmeliet P. Angiogenesis in health and disease. Nature medicine. 2003 Jun;9(6):653.
4. Ng YS, D'Amore PA. Therapeutic angiogenesis for cardiovascular disease. Trials. 2001 Dec;2(6):278.
5. Rajan A, Gopukumar ST, Praseetha PK. Journal of Global Trends in Pharmaceutical Sciences. 2013 Feb. 13-22.
6. Dutta J. Engineering of Polysaccharides via Nanotechnology. InMultifaceted Development and Application of
Biopolymers for Biology, Biomedicine and Nanotechnology 2013 Jun. 87-134.
7. Tong R, Cheng J. Anticancer polymeric nanomedicines. Journal of Macromolecular Science, Part C: Polymer Reviews.
2007 Aug 1;47(3):345-81.
8. Kim CK, Ghosh P, Pagliuca C, Zhu ZJ, Menichetti S, Rotello VM. Entrapment of hydrophobic drugs in nanoparticle
monolayers with efficient release into cancer cells. Journal of the American Chemical Society. 2009 Jan 9;131(4):1360-
1.
Zakirulla et al (2019): Dental ergonomics May 2019 vol. 19
©Annals of Tropical Medicine & Public Health SP2006-19
Dental Ergonomics: Awareness and practices among dental
undergraduates in Saudi Arabia
M Zakirulla1*
, Salem Almoammar1, Amerah Bedah Muraih
1, Amjad Ali Al-whlan
1, Ohood Saeed Al-merei
1, Rasha
Hussain Al-zahrani1, Abdulaziz Bedah Alshahrani
2
1. Department of Pediatric Dentistry & Orthodontic Sciences, College of Dentistry, King Khalid University,
Abha, Kingdom of Saudi Arabia.
2. Damman University, Damman, Kingdom of Saudi Arabia.
* Corresponding Author: Dr. M Zakirulla, Assistant Professor, Department of Pediatric Dentistry &
Orthodontic Sciences, College of Dentistry, King Khalid University, Abha, Kingdom of Saudi Arabia. E-mail:
ABSTRACT
Aim: The present study aims to assess knowledge, practice, and condition of work place regarding ergonomics
among dental students in Abha city, Saudi Arabia. Materials and Methods: A cross-sectional study was carried out
on the sample size of 156 dental students (Males 91 & Females 68) from 5th
, 6th
year and internship year of the BDS
program (Students involving clinical courses) in College of Dentistry, King Khalid University, Abha, Saudi Arabia.
The survey with close-ended, self-administered questionnaires that focused on various positions while working was
used as the data collection method. The survey data was collected and organized into Microsoft Excel spreadsheets
(Microsoft Inc., USA), and was statistically analyzed utilizing the Statistical Package for the Social Sciences version
20.0 software (IBM Inc., USA). The statistical test used here was the chi-square test and P values less than 0.05
were considered to be statistically significant (P < 0.05). Results: when students were asked about this question,
92% of students agreed with sitting dentistry, 5% practised standing dentistry, and 3% were not sure. Student’s
interest in learning and exercising the correct working posture: 93% of students said yes. Majority of students were
interested in learning the right working posture. When the students were asked about the importance of
properposture in dental clinics: 90 % of students said yes. Conclusions: Findings confirmed that there is a positive
Zakirulla et al (2019): Dental ergonomics May 2019 vol. 19
©Annals of Tropical Medicine & Public Health SP2006-19
awareness of ergonomics among dental students. There is no statistical difference between males and females in
knowledge and behaviour related to ergonomic principles among dental students.
Keywords: Ergonomics, dental students, Posture, Pain, Awareness, Saudi Arabia.
How to cite this article: Zakirulla M, Almoammar S , et al (2019): Dental Ergonomics: Awareness and
practices among dental undergraduates in Saudi Arabia, Ann Trop Med & Pub Health-Special Issue; 19:
2006-19.
INTRODUCTION
Ergonomics is derived from Greek words, “Ergo” meaning work and “Nomos” meaning natural laws or systems.
Ergonomics is an applied science which deals with devising ways and product designs to enhance efficiency and
safety atthe workplace. The goal of ergonomics is to reduce the risk of work‑related injury at workplaces.[1]
Ergonomics in dentistry is defined as a reduction in cognitive and physical stress, preventing occupational diseases,
thereby improving efficiency with better quality and greater comfort for both the practitioners and patients.[2]
Dentistry is a profession that generally produces muscular pain and soreness which are innocuous and slow to
appear. As a result, the symptomswere usually ignored until they become chronic.[3]
Improving worker productivity and occupational health and safety at workplaces are current global
concerns.[4]Musculoskeletal disorders (MSDs), also known as cumulative trauma disorders are injuries to muscles,
nerves, tendons, ligaments, joints, cartilage, and spinal discs. They often present as pains in the upper extremities,
neck, and back and shoulders, etc.[5] Poor posture at work, repetitive movements, ill‑structured job, poor
workstation design, and prolonged working time among others have been reported as risk factors for the
development of MSDs. MSDs of the upper limbs isone of the most frequent occupational problems among dental
health‑care workersand have psychological and social in addition to physical consequences. When they reach a
certain level of severity, they directly affect a person’s ability to work, causing absenteeism, and even early
retirements.[6] Dental students are also at high risk of developing these disorders,mainly owing to the deleterious
postural habits they acquired during professional training [7]. Garcia et al.[8] perceived high risk for the
Zakirulla et al (2019): Dental ergonomics May 2019 vol. 19
©Annals of Tropical Medicine & Public Health SP2006-19
development of musculoskeletal disorders in students in their final year of graduation. Dental students are also at
high risk of developing these disorders, mainly owing to the deleterious postural habits they acquired during
professional training. [6-8]
In literature, researchers have documented the varied prevalence of MSDs among dentists. Dental education can
play an important role intraining dental students, helping them to adoptadequate knowledge related to ergonomic
posture.[9]Occupational health programs and training activities are not being carriedout satisfactorily.[10] Injury
preventionand dental ergonomics education are still in its infancyin Saudi Arabia. With this in mind, this work aims
toanalyze knowledge, practice, and condition of workplace regarding ergonomics among dental students in Abha
city, Saudi Arabia.
MATERIALS AND METHODS
A cross-sectional study was carried out on the samplesize of 156dental students (Males 91 & Females 68)from 5
th,
6th
year and internship year of the BDS program (Students involving clinical courses) in College of Dentistry, King
Khalid University, Abha, Saudi Arabia. The inclusion criteria were 5th
, 6th
year and internship year of the BDS
program, whovolunteered to participate in the study. The survey with close‑ended, self‑administeredquestionnaires
that focused on various positions whileworking was used as the data collection method. Thequestionnaire was
prepared by us keeping in mind thedifferent faulty positions of students working in the dentalclinics.Written consent
was taken from the respondents,and ethical approval for performing the survey was obtained from the Scientific
Research committee of King Khalid University, College of Dentistry.The questions were designed such that they
evaluatedthe level of postural awareness by eliciting the details of the body posture of the dentist. The
questionnairewas formulated which comprised Variables included inthe study were age (only quantitative variable),
gender, classof research, knowledge, and practice of ergonomic postureand condition of work place. The other part
of the questionnaire comprised 12questions were prepared based on other studies.[11] Using the convenience
sampling, the questionnaireswere distributed to all the 185 male,and female dental students present andworking in
the student clinics of the College of Dentistry. All the students were aged between 20 years and 26 years. Among the
185 students, 159 (86%) responded.
Zakirulla et al (2019): Dental ergonomics May 2019 vol. 19
©Annals of Tropical Medicine & Public Health SP2006-19
A self-administered structured questionnaire was developed and tested among a convenience sample of 20dental
students, who were interviewed to gain feedback on the overall acceptability of the questionnaire in terms of length
and language clarity, according to their feedback the questions were corrected. Face validity was also assessed
before the start of the study. Both descriptive and analytical statistical measurements were used to describe the main
variables by SPSS 18 (IBM Corporation, Armonk, New York, USA) software. Chi‑square, ANOVA was used to
compare the qualitative and quantitative variables. The statistical significance of the coefficients in the statistical
analyses will be tested at 0.05 (< = 0.05) level.
RESULTS
Knowledge related to posture during working patient on a dental chair [Table 1]: when students were asked about
this question, their 92% of studentswere agreed with sitting dentistry, 5% were practised standing dentistry, and 3%
were not sure. Student’s interest in learning and practising thecorrect working posture:93% of students said yes.
Majority of students wereinterested in learning the right working posture. When the students were asked about the
importance of properposture in dental clinics: 90 % of students said yes.The necessity to teach correct working
posture in classes and need education regarding this:95% of studentsthought that it is necessary to teach correct
workingposture during the clinical classes, so that theywould be able to analyze themselves regarding
theproblem.Whenthey were asked the position of head and elbow while working:79% ofstudents agreed for the
straight posture of the head,and 64 % said elbow should be at shoulder level.Regarding students asked about the
lower back is supported by the back of the stool while treating a patient on dental chair: 83% of students saidyes,and
3 % of students said no. Correlation between incorrect sitting postureswill cause harm to the body and affect dental
practice in the long run: 94 % ofstudents agreed with the statement. 78% of student agreed that they experienced
neck, finger, shoulder, back, or any other muscular pain related to your work in the dental clinic [Figure 1].
Zakirulla et al (2019): Dental ergonomics May 2019 vol. 19
©Annals of Tropical Medicine & Public Health SP2006-19
Table 1: Distribution of dental students in the study according to gender and class, by number and percentage of
respondents in each group.
GENDER 5TH YEAR 6TH YEAR INTERN TOTAL P VALUE
Male 31 (34%) 28 (30%) 32 (36%) 91 (58%)
0.859 Female 26 (38%) 20 (29%) 22 (32%) 68 (42%)
Total 57 (37%) 48 (31%) 54 (32%) 156 (100%)
*P<0.05; n = Number; % = Percentage. Figure 1: Do you ever experienced neck, finger, shoulder, back, or any other muscular pain related to your work in the
dental clinic?
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©Annals of Tropical Medicine & Public Health SP2006-19
Table 2: Comparison of males and females dental students in terms of knowledge and attitude towards Ergonomics
QUESTIONS Males
(n)-91
% Females
(n)-68
% Total
159
% P value
Q1.Do you practice
Sitting dentistry 84 93 62 91 146 92
Standing dentistry 4 4 4 6 8 5 0.908
Both 3 3 2 3 5 3
Q2. Do you think that adopting the correctposture
in clinics is important?
0
Yes 81 89 63 93 144 90
No
Don’t know
10
0
11
0
5
0
7
0 15
0
10
0
0.437
Q3. Do you think incorrect workingposture will
cause harm to your body and affect your dental
practice in long run?
Yes 89 97 60 88 149 94
No
Don’t know
2
0
3
0
4
4
6
6 6
4
3
3
0.028*
Q4. Is your lower back supported by the back of the
stool?
Yes 76 84 57 84 133 83
No
Don’t know
12
3
13
3
10
1
15
1 22
4
14
3
0.747
Q5. Have you ever experienced neck, finger,
shoulder, back, or any other muscular pain related
to your work in the dental clinic?
Yes 64 70 60 88 124 78 0.026*
No
Don’t know
21
6
23
7
6
2
9
3 27
8
17
5
Q6. What is the position of your head while
working?
Straight 73 80 53 78 126 79 0.678
Bent forward and tilted 15 16 11 16 26 16
Bent forward 3 4 4 6 7 5
Q7. What is the position of your elbow while
working?
At the shoulder level 55 60 47 69 102 64
Above the shoulder level, 23 25 18 26 41 26 0.119
Below the shoulder level 13 15 3 5 16 10
Q8. What is your seat position when you are
working on the patient?
Straight, 79 87 59 87 138 87
tilted slightly forward 12 13 9 13 21 13 0.992
Q9. What correct working posturecalled as?
Ergonomics 86 95 67 98 153 96
Astronomics 3 3 1 1 4 3 0.354
Economics 2 2 0 0 2 1
Q10. Is it possible to include correct working
posture education in preclinical subject?
Yes 89 98 62 91 151 95
No
Don’t know
2
0
2
0
5
1
7
2
7
1
4
1
0.114
*P<0.05; n = Number; % = Percentage.
Zakirulla et al (2019): Dental ergonomics May 2019 vol. 19
©Annals of Tropical Medicine & Public Health SP2006-19
DISCUSSION
The right and timely application of the principles of ergonomicsat work places promotes the health, efficiency, and
well‑being ofthe workers.[12] However, the practicability of ergonomic principlesis a function of its awareness.
Against this background, thisstudy aimed at assessing the awareness and knowledge ofergonomics among dental
undergraduates in Saudi Arabia.
In the present study, a bulk of respondents (89% males; 93% females) agreed that adopting correct postures in
clinics is important a majority of respondents were aware of the fat that muscular pain is related to improper lower
back supported by the stool. Posture reflects the position that an individual maintains inspace via his bone–muscle-
skeletal system, according to a staticor dynamic balance.[13] By maintaining a good posture, thebody lowers its
energy expenditure, improves organ functioningand is protected against disturbances that could
undermineoccupational practice.[14] In our study, 98% of males and 91% of femalesstudents were interested in
learning the proper workingposture,and it should be included in the curriculum rightfrom the preclinical classes.
Learning the proper workingposture would help reduce pain and discomfort. Itwas found that the students were of
the opinion that learningthis would not only help reduce pain but also will behelpful in the long run for their future
clinical practice duringclinical postings as well as professional working places. There was no statistical significant
difference in knowledge between both males and females undergraduate dental students.
Majority of respondents agreed with the posture of head and seat backrestis straight while working on the
patient in the dental chair. Dental stool must fit correctly; it must offer neutral back, neck,and shoulder support for
optimal posture, be at the correctheight and tilt, and offer optimal arm and elbow support.[15] In another study, 60%
of students in their final year ofdental school had difficulty visualizing the oral cavity during aprocedure, because of
incorrect use of the dental stool backrest,which resulted in an inadequate working posture.[16] Garciaet al. likewise
found that 11.7% of dental students adopt amalaligned right-tilted posterior column, probably to improve
viewing.[17]
Dental professionals with poor applicationof principles of ergonomics have increased the risk for
thedevelopment of work‑musculoskeletal disorders (MSDs),which could adversely affect his performance on the
job, qualityof test result, and ultimately patient’s management and care.MSDs, also known as cumulative trauma
disorders or repetitivestress injuries,[18] are injuries to muscles, nerves, tendons,ligaments, joints, cartilage, and
spinal discs. They oftenpresent as pains in the upper extremities, neck, and back andshoulders, etc.[19] MSDs are an
Zakirulla et al (2019): Dental ergonomics May 2019 vol. 19
©Annals of Tropical Medicine & Public Health SP2006-19
increasing health problem inworkplaces, resulting in workers’ disability, loss of precioustime from work, and huge
economic and social costs.[20] Poorposture at work, repetitive movements, ill‑structured job,poor workstation
design, and prolonged working time amongothers have been reported as risk factors for the developmentof MSDs
among clinical laboratory workers.[20,21].
A poor understanding of ergonomic principlesamong dental students, a gap between thetheoretical discipline and its
clinical application will be unsuitable for the ergonomically correct dental working environment. In thissense, the
education systemmust allow students to apply their theoretical ergonomicsknowledge to their clinical practice. The
use of digitalimages reproducing ergonomic dental positioning proved to bea positive methodology in this aspect.
Early acquisition of ergonomicsknowledge among dental students can results in improved assimilation and
incorporation,preventing deleterious habit formation.[15, 22]
A key limitation of this research is that this study is questionnaire-based, knowledge and practice of
ergonomics may not be best assessed by this method. The sample size in this study is small,and furtherresearch with
a larger sample size and a more elaboratequestionnaire will help understand thedentists’ requirements. This will
enable the planningand execution of appropriate training programs toprevent ill health effects associated with
improper postures during dental practice.
REFERENCES
1. Ismaila SO. A study on ergonomics awareness in Nigeria. Aust J Basic ApplSci 2010;4:731‑4.
2. Siddiqui T, Aisha W, Owais K, Mohsin K, Farjad Z. Assessment of knowledge, practice, and work environment related
toergonomics among dental students and dental practitioners. Int J Contemp Dent Med 2016;1-5.
3. Kumar RV, Anuroopa P, Nalini MS, Savita S. Prevalence of musculoskeletal pain and assessment of ergonomics factor in
under graduate dental students: An observational study. J Health Sci Res 2014;5:1‑5.
4. Shikdar AA, Sawaqed NA. Ergonomics, and occupational health and safety in the oil industry: A managers’ response.
ComputIndEng 2004;47:223‑32.
5. Darragh AR, Harrison H, Kenny S. Effect of an ergonomics intervention on workstations of microscope workers. Am J
OccupTher 2008;62:61‑9.
6. de Carvalho MV, Soriano EP, de França Caldas A Jr., Campello RI, de Miranda HF, Cavalcanti FI, et al. Work‑related
musculoskeletal disorders among Brazilian dental students. J Dent Educ 2009;73:624‑30.
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7. Rambhad C, Pande N, Radke U. Assessment of knowledge of ergonomics among preclinical undergraduate students: A
cross-sectional study. J IntClin Dent Res Organ 2018;10:65-70.
8. Garcia PP, Pinelli C, Derceli JR, Campos JA. Musculoskeletal disorders in upper limbs in dental students: Exposure level to
risk factors. Braz J Oral Sci 2012;11:148‑53.
9. Valachi B, Valachi K. Preventing musculoskeletal disorders in clinical dentistry: Strategies to address the mechanisms
leadingto musculosketal disorders. J Am Dent Assoc 2003;134:1604-12.
10. Sartorio F, Franchgnoni F, Ferriero G, Vercelli S, Odesclchi L, Augusti D, et al. Work related musculoskeletal disorders in
dentistryprofessionals. 2. Prevention, ergonomic strategies and therapeutic programs. G Ital Med Lav Ergon 2005;27:442-8.
11. Kanaparthy A, Kanaparthy R, Boreak N. Postural awareness among dental students in Jizan, Saudi Arabia. J IntSoc Prevent
Communit Dent 2015;5:S107-11.
12. Stone R, McCloy R. Ergonomics in medicine and surgery. BMJ 2004;328:1115‑8.
13. Wilson EL, Madigan ML, Davidson BS, Nussbaum MA. Postural strategy changes with fatigue of the lumbar extensor
muscles. GaitPosture 2006: 23: 348–54.
14. Moffat M, Vickery S. Manual of maintenance and postural re-education. Porto Alegre: Artmed,2002.
15. Kee D, Karwowski W. A comparison of three observational techniques for assessing postural loads in industry. Int J
OccupSaf Ergon 2007;13:3-14.
16. Maehler P. I study of the sobrecargasposturais in academics of dentistry of the state university of the west of Parana´ -
Unioeste – Rattlesnake. Cascavel: Universidade Estadual do Oeste do Parana´ 2003.
17. Garcia PPNS, Campos JADB, Zuanon ACC. Clinical evaluation of the working positions adopted by undergraduate dental.
Pesq BrasOdontopedClinIntegr 2008: 8: 31–7.
18. Kozak A, Schedlbauer G, Peters C, Nienhaus A. Self‑reported musculoskeletal disorders of the distal upper extremities and
the neck in German veterinarians: A cross-sectional study. PLoS One 2014;9:e89362.
19. Darragh AR, Harrison H, Kenny S. Effect of an ergonomicsintervention on workstations of microscope workers. Am
JOccupTher 2008;62:61‑9.
20. Franco G. Health disorders and ergonomic concerns from the use of the microscope: A voice from the past. Am J ClinPathol
2011;135:170‑1.
21. George E. Occupational hazard for pathologists: Microscope use and musculoskeletal disorders. Am J
ClinPathol2010;133:543‑8.
22. Rising DW, Bennett BC, Hursh K, Plesh O. Reports of body pain in a dental student population. J Am Dent Assoc 2005:
136: 81–6.
Khaleel (2019): Coating of nanoparticle May 2019 Vol. 19
Application: Coating of Nanoparticles to Minimizing Harmful Environmental Pollution by Chemical Fungicide
Rana Ibrahim Khaleel
1 *
1* College of Engineering,University of Samarra, Samarra, Iraq
Abstract
Field experiment carried out for evaluation using nano-silver solution to reduce environment
pollution by fungicides. Four concentrations of AgNPs, (100 ppm, 50 ppm, 25 ppm, and 12.5 ppm)
were prepared by diluting the original stock solution with distilled water. From the concentration
was ready used in coated orang fruit compared with fungicide and uncoated fruit as control
treatment. The fruits were kept in box carton (40x40x30cm) and incubated under room conditions
for 28 days to study the formation to Green mold and Black fungal and shelf life for fruit stored.
The result showed high infection recorded in uncoated fruit compared with other treatments with
values reached (86.66 and 91.1) for Green mold and Black rot respectively, while ppm recorded
completely inhibition infections (0%) for fungal decay index. Also coating fruit with 100 and 50
ppm recorded significant different reached (3.8 and 3.9%) from other concentrations and fungicide
that, recorded high weight loss with mean reached (13.66%) after four weeks from storage.
Nanotechnology applied could play a fundamental role for this purpose and research in packaging
fresh fruit and minimize environmental pollution by chemical fungicides.
©Annals of Tropical Medicine & Public Health SP2007-19
©Annals of Tropical Medicine & Public Health SP2007-19
Khaleel (2019): Coating of nanoparticle May 2019 Vol. 9
Keywords: Silver-Nanoparticles, Potential Alternative, Orange, Fungicide.
How to cite this article: Khaleel RI (2019): Application: Coating of Nanoparticles to
Minimizing Harmful Environmental Pollution by Chemical Fungicide , Ann Trop Med & Pub Health-Special Issue; 19: 2007-19.
Introduction
Nanoscale materials have emerged as novel antimicrobial agents owing to their high surface area to
volume ratio and the unique chemical and physical properties, which increases their contact with
microbes and their ability to permeate cells (Nejad et al., 2014). Also, nanotechnology has amplified
the effectiveness of silver particles as antimicrobial agents. Nanoparticles such as silver is known to
attack a broad range of biological processes in microorganisms including cell membrane structure
and functions (Lamsal et al., 2012). Nanoparticles also inhibit the expression of proteins associated
with ATP production, although its specific antimicrobial mechanisms are still not completely
understood. The use of nano-sized particles as antimicrobial agents has become more common as
technological advances make their production more economical. One of the potential applications of
Nanoparticles
is to manage plant disease. Since silver displays multiple modes of inhibitory action to
microorganisms (Kamran et al., 2011), it may be used for controlling various plant pathogens in a
relatively safer way compared to synthetic fungicides. Until now, . Reducing the particle size of
materials is an efficient and reliable tool for improving their biocompatibility. In fact,
nanotechnology helps in overcoming the limitations of size and can change the outlook of the world
regarding science (Gavanji et al., 2013). Furthermore, nano-materials can be modified for better
©Annals of Tropical Medicine & Public Health SP2007-19
Khaleel (2019): Coating of nanoparticle May 2019 Vol. 9
efficiency to facilitate their applications in different fields such as bioscience and medicine. This
simple size comparison gives an idea of using nanoparticles as very small probes that would allow
the elucidation of the cellular machinery without introducing too much interference (Lamsal et al.,
2012). Understanding of biological processes on the nano scale level is a strong driving force
behind development of nanotechnology (Salata et al., 2004). Current study came to investigate in
activity of silver nanoparticles as alternative of fungicides to decrease environmental pollution.
2. Methodology
AgNPs Solation Preparation
AgNPs, WA-CV-WA13B (CV)were used in the experiment that classified and using in different
manufacturing processes. Four concentration (100 ppm, 50 ppm, 25 ppm, and 12.5 ppm) were
prepared by diluting the original stock solution with distilled water and stored at 4℃ until further
use.
Fruit collection of Fungal Pollination
Citrus fruit (orange) of medium size and in good physical conditions such as being insect and
disease and damage free were purchased from local market in Samarra, Iraq. The fruits were
visually inspected for any damage or disease during collection. They were brought to the
enlivenment lab and kept in a cool room at 4oC for 24hrs to be used the next day for the experiment.
Preparation Fruit for Inoculation
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Khaleel (2019): Coating of nanoparticle May 2019 Vol. 9
The current research in cooperated a modification of this range to selected 100, 50, 25 and
12.5ppm. Fruits (oranges) washed with tap water, air-dried and then sterilized by immersion in 70%
ethanol for 1 min. The fruits were then randomly divided into six equal groups (Four nano-treatment
+ two controls). Fruits were wounded at a depth of 5.0mm with a 1.25mm diameter needle at the
equator for all the treatments and inoculated by spraying with suspension of fungi. The treated fruit
were left for an hour after spraying to stabilize the spores on the wound.
Preparation Fruit of Coating Treatment
From the concentration were prepared previously the homogenous solutions (100, 50, 25 and
12.5/ppm) used in coated fruit using a clean and soft brush. Coated fruits were allowed to dry at
ambient temperature about 30 minutes, after that transfer to package it.
Packaging Treated Fruit
The fruits were kept in box carton (40x40x30cm) and incubated under room conditions (25ºC±2
and 65-75 % RH) for 28 days to study the formation of fungal rot and shelf life for fruit stored .
There were three replicate trials of five fruits per replicate using the Completely Randomized
Design (RBD).
Fungal decay
Fungal decay in stored fruit was evaluated after 21 days under storage conditions. The percentage
of infection was determined using scale of 1-5 (Scale modified from Tzortzakis, 2006) as shown in
Table 1.
Khaleel (2019): Coating of nanoparticle May 2019 Vol. 19
©Annals of Tropical Medicine & Public Health SP2007-19
Table .1 Percentage of infection fungal decay index
Percentage infection Remarks
0 Clean
1-5 Trace
5-15 Slight
15-30 Moderate
>30 High
Shelf Life Fruit
The percentage of shelf life fruit was calculated by determining the progressive reduction in fruit
weight during storage after a period of 21 days in relation to the original fresh weight at the
beginning of storage (Tune et al., 2007).
WL (%) = WL before storage - WL after storage/ WL before storage X 100 (Eq 1)
Statistical Analysis
The experiment was laid out in the Completely Randomized Design (CRD) with three replicate.
Data collected and analysed using analysis of variance (ANOVA). Significant differences between
mean values were determined using Duncan’s Multiple Range test (P≤ 0.05). Following ANOVA
statistical analysis, which were performed using SPSS version 19- 2012 (SPSS Inc., Chicago,
USA).
3. Result and Discussion
Khaleel (2019): Coating of nanoparticle May 2019 Vol. 19
©Annals of Tropical Medicine & Public Health SP2007-19
Fungal Decay Index
The statistical analysis (ANOVA) as reported in Table.2 shows statistical different between the
treated and untreated fruit. High infection recorded in uncoated fruit compared with other treatments
with values reached (86.66 and 91.1) for Green mold and Black rot respectively. Also not
statistical difference (P ≤0.05) between 25, 50 and 100 ppm treatments which exhibited
significantly decrease in disease severity trace degree to completely inhibition infections (0%),
compared with 12.5ppm and fungicide which recorded (6.66 and 11.10) fungal decay index .
Table. 2 Effect of coating fruit with different concentrations of Silver (12.5 to 100 ppm) on the
development of mycelium growth Penicillium digitatum and Aspergillus niger(colony
diameter/cms) on the orange surface during storage for 21 days at 25oC±2 and 65-75% RH.
Treatments (ppm)
Penicillium
digitatum
Aspergilius
Niger
**Infection
12.5 6.66 b 11.10
c Slight
25 0 a 2.22
b Trace
50 0 a 0 a Trace
1000 0a 0
a Trace
Water 86.66 c 91.10
d High
Fungicides 6.66 b 11.10
c Slight
* Alphabets different in the same columns shows significant difference at (P≤0.05) between concentrations and average was calculated from three replicates.
Result the current study comes a similar to previous studies that, nanoparticles with antimicrobial
properties can be employed for safer food packaging. Nanoparticles such as titanium dioxide, zinc
oxide and magnesium oxide, as well as a combination of them, once functionalized can be efficient
in killing micro-organisms and cheaper and safer instead of metal based nanoparticles (Barik et al.
2008; Cui et al. 2010). Finding study of Sasson et al (2007) reported that, Nanotechnology has the
potential for efficient delivery of chemical and biological pesticides using nanosized preparations or
nanomaterial based agrochemical formulations. The benefits of nano material based formulations
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Khaleel (2019): Coating of nanoparticle May 2019 Vol. 9
are the improvement of efficacy due to higher surface area, higher solubility, induction of systemic
activity due to smaller particle size and higher mobility and lower toxicity due to elimination of
organic solvents in comparison to conventionally used pesticides and their formulation. Also
findings Abdelmalek and Salaheldin ,(20116) are agreement with result current study that, Silver
nanoparticle, 150 ppm, showed potent antifungal activity against the isolated fungi from fresh fruit.
Also current result come out with study (Lucimeire et al., 2014) that, coatings chitosan
nanoparticles of 110 nm showed higher antimicrobial activity against mold and yeasts, and
mesophilic and psychrotrophic bacteria.than the other treatments. Silver nanoparticles may directly
attach to and penetrate the cell membrane to kill spores, although penetration of silver nanoparticles
into microbial cell membranes is not completely understood (Morones and Elechiguerra,2005).
Shelf life Fruit
Effect different coatings on percentage of weight loss were shown in Table.3.The result showed
difference between the effects of the coating treatments on weight loss at different concentrations.
Coating fruit with 100 and 50 ppm recorded significant different (3.8 and 3.9%) from other
concentrations and fungicide after four weeks from storage. Also statistical analysis (P≤ 0.05) did
not showed significant difference between concentration 25 and 12.5ppm ( 5.42 and 5.5) which
difference from fungicide that, recorded high weight loss with mean reached (13.66%) after four
weeks from storage.
Table.3 Effect of coating fruit using nano-silver solution on weight loss (%) that storage at 25±2oC
and 65- 75 % RH for four weeks(28S) days.
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Khaleel (2019): Coating of nanoparticle May 2019 Vol. 9
Treatments
W1 W2 W3 W4 Mean
100ppm 2.9 a 3.5
a 3.9
a 4.9
b 3.8
a
50ppm 3.5b 3.5
a 4
b 4.63
a 3.9
a
25ppm 5 c 5.5
cb 5.5
c 5.70
c 5.42
b
ppm 5 c 5
b 6
d 6
d 5.5
b
Uncoated fruit 10.3 e 11.9
d 13.7
e 18.76
e 13.66
c
*Alphabets different in the same column show significant difference using Duncan’s Multiple Range test (P≤ 0.05) and average was
calculated from three replicates.
The treatment of 100ppm of silver showed significant decrease in weight loss compared with
uncoated fruit and other treatments Table. 3 Active coating using nano-silver may be helped to
improve and modify the atmosphere around the fruits, thereby preventing extremely low levels of
O2 and/or high levels of CO2, which could produce anaerobic metabolism with possibility of off-
flavour generation (Serrano et al. 2008; Valero et al. 2006 ; Valverde et al. 2005). Result current
study recorded clear increase in weight loss ranged (5.7- 6%) with treated 25 and 12.5 ppm
respectably. Current finding in agreement with report each of (Bautisty et al. 2003; Ali et al .
2011) that, coating fruit by some materalis such as chitosan conc.2-5% with crude plant extract of
custard apple leaves, papaya leaves and papaya seeds 1000-3000 did not influence the percentage
weight loss during the storage fruit but there was inhibition of fruit decay percentage. Result
current study are agreement with finding (Li and Wang. 2006 ; Li et al. 2009) that, nano-packing
technology applied on fruit and vegetables exhibited better physico-chemical , sensory
physiological and preservation properties compared to normal packaging . Also study of (Lopez-
Rubio et al. 2006 ; Jimenez et al. 2004) that, Nano encapsulation of active compounds used in
©Annals of Tropical Medicine & Public Health SP2007-19
Khaleel (2019): Coating of nanoparticle May 2019 Vol. 9
edible coating control rates and conditions and thus protect the coated items from moisture, heat or
other extreme conditions while enhancing their stability and viability.
Figure .1 Effect of coating fruit using nano silver on weight loss (%) at 25±2
oC and 65- 75%
RH storage condition for 28 days.
The experimental data by (Arnon et al. 2015; Ouzounidou et al. 2013) confirmed that -Ag coatings
markedly reduced the weight loss in those kinnow mandarin treatments. Weight loss in fresh fruits
and vegetables has not only been associated with vapor pressure at various locations, but also occurs
through respiration phenomenon. Result current study confirmed finding (Zambrano-Zaragza et al.
2013) that, Nano-emulsions and Nanoparticles can improve barrier properties and increase
functionality of coating because submicronic systems allow for more efficient control of coating
properties with better distribution and homogeneity on the coated fruit skin. Also support study
20
18
16
14
12
10
8
6
4
2
0
W1 W2 W3 W4
100ppm 50ppm 25ppm 12.5ppm Uncoated
We
igh
t lo
ss
©Annals of Tropical Medicine & Public Health SP2007-19
Khaleel (2019): Coating of nanoparticle May 2019 Vol. 9
(Hazirah and Armylisas, 2016) that reported, Nano-particles are recognized as micro additives and
submicroscopic particles which enhance the physical properties of food packaging such as edible
films and coating.
Conclusion
Application of coating nano-materials as further proved the quality of fresh fruits by decline fungal
decay index and increasing lengthen fruit shelf life. Using of the silver nanoparticles as alternative
fungicides for crop protection is emerging technology in food packaging but still need more
studies to assesses nano-silver to avoid side negative and their possible negative and toxicological
effects on environmental and human health.
Acknowledgements
Acknowledgements and Reference heading should be left justified, bold, with the first letter
capitalized but have no numbers. Text below continues as normal.
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Khamis et al (2019): Genetic detection of HBV May 2019 vol. 19
©Annals of Tropical Medicine & Public Health SP2008-19
Genetic detection of hepatitis B virus by PCR technique among children under
7 years in Hilla city, Iraq
AMAL RAQIB SHAMRAN1, Tsahel H. AL.Dulaimi
1, Ammar S.Khamis
2 RULA DHAHIR ABDULMOHSIN
1
1. Collage of science for woman
2. Collage of pharmacy University of Babylon
Abstract
Hepatitis disease is mean the inflammatory and destruction of the liver. Its commonly caused by viral
infection, Hepatitis B Virus (HBV) is one of the main resone of liver destruction, it spread when people come in
contact with the blood, open sores, or body fluids of someone who has the (HBV), 3.5% of global population is
chronic infected with HBV although the incidence of HBV infections is decreasing owing to vaccination and to a
lesser extent. Hepatitis is a serious public health problem distressing many of people worldwide. Inadequate
data is available on this issue in Iraq. This study was carry out with the aim of determining the genetic
sequencing in human and risk factors of hepatitis Bvirus (HBV) among the general population and among blood
donors. Methods: Blood samples from volunteers; have been detected the hepatitis-gen by PCR ,we see that
the 48 out of 250 children patients ( 19.2% ) among 5months to 5 years , in this study population the biggest
percentage were 45.83 % in aged 3-5 years while the second percent was 31.25 % in aged month-1year,while
it was 12.5% in age 1-3 years and 10.41% in age 5-7 years, table (1) ,in this study we see that male were 22
out of 48 ,45% while the femal were 26 out of 48 ,55%.
Key words: HBV, PCR, hepatitis, genetic detection
How to cite this article: Shamran AR, Al-Dulaimi TH, et al (2019): Genetic detection of hepatitis B virus by
PCR technique among children under 7 years in Hilla city, Iraq, Ann Trop Med & Pub Health-Special Issue; 19:
2008-19
Introduction
Hepatitis generally is disease involving the liver as target for viruses and other disease agents .The infections
that occur in the liver is called Hepatitis. There are many variety agents in addition to the virus infections that
cause liver destruction such as bacteria, fungi, drugs (1). Viruses which responsible for liver diseases include
HBV, HCV, HDV. HBV was discovered in 1963 by Dr.Blumberg at the National Institute of Health (NIH), (2).
HBV infections were recognized and reported as main reasons of significant morbidity and mortality in the
world (3). WHO reported that 2 billion people are infected with HBV and 450 millions have chronic infections
and the others peoples have risk of infected by HBV and HDV, many factors seem to associated with fibrosis
,age, daily alcohol consumption , gender (4). Many studies have reported that the risk of infection of the HBV
has increased in the countries which have high level of sexual activities, drugs use, family history of HBV
infections, vertical transmission haemolysis and exchange organs and bloods (5). This study aims of
determining the genetic sequencing in human and risk factors of HBV in the general population and among
blood donors.
Material l and methods
In this study total 250 patients children blood samples were collected from Alnoor-hospital ,during the period
Nov2017-Mar2018; then analyzed by PCR technique .
Khamis et al (2019): Genetic detection of HBV May 2019 vol. 19
©Annals of Tropical Medicine & Public Health SP2008-19
HBV DNA Detection through PCR for detection of HBV, the specific purification of the gen was amplified by
PCR using specific forward & reverse primers.
Viral DNA Extraction : DNA of the virus was isolate from the blood samples by from patients by (Qiagen
kit)according to manufacture protocol .
PCR – test,Thermal Cycling, Place the tubes wich contain (DNA sample ,master mix ,10mM
ofsenseprimer(Macrogen,Korea),5-CCGAATTCGCCACCATGCATCCTGCTGCTATGCCTCATCT-3and 10mM of
antisense primer (Macrogen, Korea), and 5-CCC-GAATTCGCCACCATGCGAACCACTGAACAAATG-GCACT-3 .
Cycling Profile
94°C to 45 second, (denaturation)
94° to 1 minute
64°C to 45 seconds (annealing)
72°C to 72 second (elongation)
For 30 cycles,
72°C to 7 minutes
Agarose Gel Electrophoresis, for visualization of the amplification products, Manufacture organisation
advocate using a 4 % NuSieve® 3:1 Plus TB buffer precast gel, 4% NuSieve, three:1 agarose gel in 1X TB buffer
contain 0.5μg/ml EthB.
Results
Screening of Cases infected with HBV was 48 out of 250 children patients ( 19.2% ) among 5 months to 5
years, in this study population, the biggest percentage were 45.83 % in those aged 3-5 years while the second
percent was 31.25 % in those aged month-1year,while it was 12.5% in age 1-3 years and 10.41% in age 5-7
years, table (1) ,in this study we see that male were 22 out of 48 ,45% while the femal were 26 out of 48 ,
55%. There is no significant different of the gender in the patients, and the same number of patients who live
in the rural and the urban (24 patient 50%), table (2).
Table 1: age group of patient population group
No.
Age group /year
Tested subject
No.
%
1 1 month-1year 15 31.25
2 1-3years 6 12.5
3 3-5years 22 45.83
4 5-7 years 5 10.41
Khamis et al (2019): Genetic detection of HBV May 2019 vol. 19
©Annals of Tropical Medicine & Public Health SP2008-19
age group of hepatitis distribution
year1 month-1
years year- 1
years years-
years years-
Figure 1: age group of hepatitis patients
Table2: general characteristic of the study of the patients’ population
Gender Inhabit
Male Female Urban Rural
No. % No. % No. % No. %
22
45.38
26
54.16
25
50
25
50
Khamis et al (2019): Genetic detection of HBV May 2019 vol. 19
©Annals of Tropical Medicine & Public Health SP2008-19
Figure 2: positive result
Discussion
All though there have been massive advances in immunization for HBV infections over three decades, the
burden of HBV remains a public health concern around the world. 2 billion people are estimated to be
affected by HBV disease, which causes a broad range of liver sickness, including intense or fulminant hepatitis,
inert administration state, reactivation, relentless hepatitis, cirrhosis and hepatocellular carcinoma (HCC). An
estimated 420 million people around the world requires ceaseless HBV contaminations; 15%-40% of them may
die due liver disease complications or HCC [3]. Symptoms of HBV disease differs around the globe, around
45% of the overall people live in risky environment, including sub-Saharan Africa, the Pacific and particularly
Asia(6). A study by Ye et al (2006) reported that a small number of case become much less infected due to
perinatal transmission from mother to infants. A number of studies reported of similar findings. The
extraordinary sexual activities in this study may explain the reason for the risk among the subjects, although
there is no statistical association between them (P>0.05). This contradicts the study of Omer and
Thewaini(1979) which reported that the males were higher than females in ratio (1.6:1) in Iraqi people as well
as Al- Mashhadani (1998) stated that males were higher than female (2:1),while Sabri reported that it was
2.5:1 . The prevalence of the virus within the urban and rural areas were identical as reported by Boisier et al
(1996) which reported that the agriculture regions have tremendous large HBV infection of around 26% while
the city has 53% (7).
References
[1] G. B.Gaeta,C. Sardaro,G.Giusti et al., “Prevalence of anti-HCV antibodies in patients with chronic liver disease and its
relation-ship to HBV and HDV infections,” Infection,vol.18,no.5,pp. 277–279, 1990.
[2] D. Lavanchy, “Hepatitis B virus epidemiology, disease burden, treatment, arid current and emerging prevention and
control measures,” Journal of Viral Hepatitis, vol.11, no.2, pp.97–107, 2004.
[3] T. L. Wright, “Introduction to chronic hepatitis B infection,” American Journal of Gastroenterology, vol.101, no.1, pp.S1–
S6, 2006.
Khamis et al (2019): Genetic detection of HBV May 2019 vol. 19
©Annals of Tropical Medicine & Public Health SP2008-19
[4] A. Sangiovanni ,G.M.Prati, P. Fasanietal., “The natural history of compensated cirrhosis due to hepatitis C virus: a 17-
year cohort study of 214 patients,”Hepatology,vol.43,no.6,pp.1303– 1310, 2006.
[5] B. Tsatsralt-Od, M. Takahashi, T. Nishizawa, K. Endo, J. Inoue, and H. Okamoto, “High prevalence of dual or triple
infection of hepatitis B, C, and delta viruses among patients with chronic liver disease in Mongolia,” Journal of Medical
Virology,vol.77, no. 4, pp. 491–499, 2005.
6) Takako Utsumi et al (2014) Molecular epidemiology of hepatitis B virus in Asia World J Med Genet. May 27, 2014; 4(2):
19-26
7) P. Boisier et al (2009) reported that Hepatitis B virus infections in general population in Madagascar evidence for
different epidemiology pattern in urban and rural areas , VOL 117,issue 1 august (1996) 133-137
8) Ye F., Yue Y., Li S., Chen T., et al. (2006). Presence of HBsAg, HBcAg and HBV DNA in ovary and ovum of the patients
with chronic hepatitis B virus infection. Am. J. Obstet. Gynecol. 194:387-392.
9) Al-Mashhadani J.I., (1998). Seroepidemiological study on HBV and HCV infections among health care workers. Ph. D.
thesis. College of Medicine. Baghdad University.
Khalaf (2019): APGAR Score in neonates May 2019 Vol. 19
©Annals of Tropical Medicine & Public Health SP2009-19
COMPARISON OF APGAR SCORE IN NEONATES BORN AFTER ELECTIVE:
SPINAL VERSUS GENARAL ANAESTHESIA.
Idrees Jameel Khalaf1
1. Department of surgery Tikrit College of medicine Tikrit, Iraq
ABSTRACT:
BACKGROUND: ten point Apgar score has been used to compare the effect of spinal anesthesia versus general
anesthesia on Apgar score of neonates born by elective caesarean section.
STUDY DESIGN: Randomized prospective study.
SETTING: This study was conducted in the Department of pediatrics-Haweejha general Hospital/Kirkuk/Iraq., between
December2017toJune2018.
SUBJECTS AND METHODS: In this study was achieved on sixteen women bestowing for Elective lower segment
caesarean section. Thirty mothers were given general anaesthesia and other 30 mothers received spinal anaesthesia. The Apgar
score was recorded at 1 minute and 5 minute interim after each delivery.
RESULTS: Out of thirty mothers, who received general anaesthesia, 25 patients (82.3%) give birth to neonates having
Apgar score ≤ 6 at one minute after birth and the remaining 5 neonates (17.7%) had Apgar score of ≥7. On the other hand out of 30 mothers who received spinal anaesthesia only 10 mothers give birth to neonate having Apgar score ≤ 6 at one minute after birth, who improved at 5 minutes interval, and their Apgar score were ≥7.It had been found that those neonates who were born under G.A were ten folds more likely to have Apgar score less than or equivalent to 6 at first
minute compared to those with spinal anaesthesia, the odds ratio=10 and 96%assurance interval of the odds ratio (2.95-
35) and p=0.00025 which is highly significant, G.A had greater risk on newborn at the first minute.
KEY WORDS: Apgar score, Spinal Anesthesia, General Anesthesia, Neonate, Elective Caesarean Section.
How to cite this article: Khalaf IJ (2019): comparison of Apgar Score in neonates born after elective spinal
versus general anaethesia, Ann Trop Med & Pub Health-Special Issue; 19: 2009-19.
INTRODUCTION: An Apgar test is usually performed for the fetus twice: once at first minute after
birth, and again at 5 minutes after birth. Infrequently, if there are apprehensions about the baby
condition and the first two scores are low, less than 7, the scoring is also performed at 10, 15and 20
minutes after delivery (1)
. Five factors are used to estimate the baby’s condition and each factor is
scored on a scale of 0 to 2, with 2 being the best score for each: Activity and muscle tone, Pulse,
Grimace response, Appearance and Respiration. Scores obtainable are between0- 10, with 10 is highest
possible score. There are several factors that affect Apgar sore counting false positive and false
Khalaf (2019): APGAR Score in neonates May 2019 Vol. 19
©Annals of Tropical Medicine & Public Health SP2009-19
(3)
negative score(2)
.
The Apgar score was mostly designed as a research implement, enabling the grouping
ofinfantsaccordingtotheirconditionatbirth.In1953,VirginiaApgar,M.D.publishedherproposal
foranewmethodofevaluationofthenewborninfant.APGARscoreisaclinicalexperimentimplementedo
na newbornoneandfiveminutesafterbirth.Itisacompositemeasureofbreathingeffort,heartrate,
muscletone,reflexes,andskincolorandisanindicatorofthenewborn'sneedformedicalconsiderationpres
entlyafterbirth.Apgarscore(AS)isusuallyusedforvaluationofnewbornsinstantlyafter
birthandconsistsoffivevariablesviz.Respiratoryefforts,heartrate,color,muscletone,andreflex
irritability. It is being used as a standardized tool for expressing the physiologic condition of
newborn at birth and also to record fetal to neonatal transition. However, Apgar score has
mainconfineslikehavingalimitedtimeframeandcountingsubjectiveconstituents.Eachoftheseis given
a score of 0,1 and 2. Thescoreisconventionallyreportedat1and5minafterbirth,A score of 0-3 at 5
min is a suggestive criteria for asphyxia insult and is a predictor of neonatal mortality.
Newbornswithascoreof≥7are measured normal .
Apgar scoring is a research instrument rather than a criteria for clinical valuation on which
to baseadministrationchoiceorprognosis.Apgarscoreat1,5 min have allow specificity for asphyxia
and subsequently poor predictive value for long term neurological squeal, score can be falsely
lowinverypreterm,maternaldrugintake,CHD,andCNSdisfigurements,hencelowscorescannotbe
alwaysparalleledtoasphyxia.Thoughitisusefulinassesscardiopulmonarystatus,tellaboutneedof
resuscitation and its effectiveness. It is assigned every 5 min until 20 min or till 2 consecutive
score are 7 or greater. The neonatal revival program (NRP) guidelines state that “Apgar scores
should not be used to dictate appropriate resuscitative actions, nor should interventions for
depressedinfantsbedelayeduntilthe1-minuteassessment.”However, an Apgar score that remains 0
beyond 10 minutes of age may be useful in determining whether additional resuscitative efforts
are indicated. The outcome of anaesthesia either spinal or general depends upon the condition of
the mother and more importantly effects on newborn. APGAR score is the best parameter to
assess the direct condition of the baby.(4,5)
This is the reason to select this
topic.Theutmostimportantcauseoffetaldistressinanyanestheticmethodisthelesseninginthe amount
of O2 available to the fetus as a result of the reduction of uteroplacental blood flow. Motherly,
placental, and fetal factors play roles in such reduction. The effect of anesthetic drugs is direct or
through the changes in the mother (6)
.The Apgar scores are taken at 01and 05 minutes after
delivery. Of the two scores, the 05 minutes score is regarded as the better predictor of survival in
infancy in the long term. Whereas the 01 minute score definitely has the value for; assessing the
effectsofdifferentdrugsgiventothemotherduringtheCesareansection.Thismethodisevenmore
interesting because it is noninvasive, (7,8)
MATERIAL AND METHODS:
.
Khalaf (2019): APGAR Score in neonates May 2019 Vol. 19
©Annals of Tropical Medicine & Public Health SP2009-19
This study was carried out in Mansoura hospital from February 2017 to July 2018, and was
performed on 60 healthy full term mothers presenting for elective lower section caesarean section
,thirty mothers were given general anaesthesia and other sixteen mothers received Spinal
anaesthesia, the Apgar scores were recorded at 1minute and 5 minute interval after each delivery.
Total sixteen mothers were encompassed and a written consent was taken from each patient.
Method for general anaesthesia: History was taken for all patients in the suite which include age,
parity, duration of pregnancy and any maternal health history, anaesthesia interrelated obstetric
history, blood pressure measurement, and airway assessment. An18gauge intravenous catheter is
employed.
Method for general Anaesthesia: Before medication done with injection. Cimetidine 200mg i/v,
Injection Metoclopramide 10mg i/v 1 ± 2 hours prior to induction. Patients was pre oxygenated
for 3 ± 5 minutes, induction was done with, Injection Thiopentone 4mg/kg body weight i/v,
Injection Succinyl choline 1.5mg / kg i/v. After endotracheal intubation, 50% oxygen with nitrous
oxide and 0.5% halothane breathing was given each time. General anaesthesia was maintained
with non-depolarizing muscle.
Exclusion criteria: the following patients were excepted: Premature pregnancy <37weeks of
gestation, Liver, heart or kidney failure associated with gravidity, Uncontrolled metabolic
disorder (diabetes Mellitus, hypertension, thyrotoxicosis), And Multiple fetus gravidity.
Spinal anaesthesia: History was taken from patients particularly about back surgery, valvular
heart disease, hemorrhage tendency, pre-existing neurological deficits, blood pressure, pulse rate,
chest inspection was done and back examination for any deformity or back surgery at site o
injection, two 18 gauge intravenous catheters are employed, 500-1000ml of crystalloid solution
was preloaded then patient was placed in sitting position and space between 3rd
and 4th
lumbar
spine was identified and marked. After attractive all aseptic measure lumbar puncture was done
with22 gauge size spinal needle and hyperbaric Bupivacaine 0.5%, 2.5ml (12.5mg) was
administered. Directly after injection of Bupivacaine, patient was placed in supine position with
wedge under right hip for left uterine dislodgment. Monitoring was for pulse, N.I.B.P., oxygen
permeation. The following was recorded during every caesarean section under general or spinal
anaesthesia in different stages of birth: time of induction, time of incision to skin, time of incision
to uterus, time of delivering the baby. We contemplate that every fetus should deliver with less
than 10 minutes after induction of GA. In this study, Apgar score of all 60 neonates was recorded
at 1minute and 5 minutes after delivery. Birth weight was recorded and Apgar score of each baby
was compared with standard Apgar score chart.
Statistical analysis: By using statistical package for social sciences (SPSS) software for window
V.18.US , data of all cases were come in and analyzed; descriptive analytic statistic were
performed using appropriate statistical tests. Age, history was taken from patients specially about
back surgery, valvular heart disease, bleeding tendency, pre-existing neurological deficits, blood
pressure, pulse rate, chest examination was done and back examination for any deformity or back
Khalaf (2019): APGAR Score in neonates May 2019 Vol. 19
©Annals of Tropical Medicine & Public Health SP2009-19
surgery at site o injection, two 18 gauge arterial catheters are employed, 500-1000ml of
crystalloid solution was preloaded then patient was placed in sitting position and space between
3rd
and 4th
lumbar spine was identified and marked. After taking all aseptic measure lumbar
puncture was done with22 gauge size spinal needle and hyperbaric Bupivacaine 0.5%,
2.5ml(12.5mg) was administered. Immediately after injection of Bupivacaine , patient was
placed in supine and weight were expressed as (Mean± SD).
Student (t) test was used to compare these incessant variables and the mean of both groups. All
data were presented as tables, graphs or paragraph. In all statistical measures and tests P value set
at ≤0.05 to be consider as significant.
RESULTS:
Recording of Apgar score: In this study Apgar score of all 60 neonates were recorded by
neonatologist. Apgar scores were recorded at 1 minute and 5 minutes after delivery. Birth weight
of every baby was recorded. Apgar score of each baby was compared with the standard Apgar
score chart as shown below:
Apgar score (table I) of the two groups was compared by Statistical analysis performed with the
use of power calculation.
\ TABLE I: APGAR SCORE: (B= Body/ E=extremities/ C= Completely/ S= Spontaneous)
APGAR
Score
Heart
rate
Respiratory
effort
Reflex
irritability
Appearance
(color)
Muscle tone
0 Absent Absent No-response Blue or pale Flaccid
1 <100 Irregular Grimace B-pink / blue-E Good tone
2 >100 Good Cough/sneeze C-pink S-flexion
For all studied 60 mothers, the mean age of the mothers was (28.75±0.11), and the mean birth
weight of neonate was (3.1±0.16) of GA-group. While, the mean age of the mothers was
(27.91±3.12), and the mean birth weight of neonate was (31.7±0.18) of Spinal-group There was
no significant difference in age of the mothers nor had the birth weight of neonates been found in
between two groups.( P. value >0.05), table (2). Out of 30 patients who received general
anaesthesia, 25 patient (82.3%) give birth to neonate having apgar score ≤ 6 at one minute after birth and the remaining 5 neonates (17.7%) had apgar score of ≥7. At 5 minutes, twenty five neonates with low Apgar score at one minute were improved after resuscitation and showed
Apgar score of ≥7.
Table 2: Descriptive statistics of studied group.
Khalaf (2019): APGAR Score in neonates May 2019 Vol. 19
©Annals of Tropical Medicine & Public Health SP2009-19
Variables
anaesthesia Mother Age Neonates’ Birth weight
Mean ±SD(year) Ranger (year) Mean± SD(kg) Ranges (kg)
An
aes
thes
ia
GA group 28.75±0.11 18 - 37 3.1± 0.16 2.8 - 3.5
Spinal group 27.91±3.12 18 - 37 31.7 ± 0.18 2.8 - 3.5
All cases 28.6±3.75 18-37 3.07± 0.14 2.8 - 3.5
P .value 0.58 0.56
On the other hand out of 30 patients who received spinal anaesthesia, only 10 patients give birth
to neonate having Apgar score ≤ 6 at one minute after birth, who improved at 5 minute interval,
and their apgar score were ≥7. The Apgar score at 5 minutes of all 30 neonates, in spinal anaesthesia group, was ≥7.
It had been found that the infants who were born under GA were ten folds more likely to have
Apgar score less than or equal to 6 at one minute compared to those with spinal anaesthesia, the
odds ratio=10 and 96% confidence interval of the odds ratio (2.95-35) and p=0.00025 which is
highly significant, G.A had greater risk on infant at the one minute, table (3).
Table 3: Apgar score at one minute and type of anaesthesia.
Apgar score at 1min
Odd ratio
P. value Anaesthesia ≤6 ≥7 Total
GA-group Count 25 5 30
10
0.00025 %within anaesthesia 82.3% 17.7% 100%
Spinal-group Count 10 20 30
%within anaesthesia 35.5% 64.5% 100%
Total Count 35 25 60
%within anaesthesia 59.1% 40.9% 100%
DISCUSSION:
The Apgar score is a applied method of systemically assessing newborn infants directly after birth
to help identify those demanding resuscitation and to predict survival in neonatal period the 1 min.
Apgar score may signal the need for instant resuscitation, and the 5, 10, 15 and 20min.score may
indicate the probability of successfully revivifying an infant. Due to a number of factors a low
score may be including drugs given to the mother during labour, caesarean section under general
anaesthesia and immaturity(9)
. The major causes of maternal death in Pakistan are general
anesthesia. Despites the advances in anaesthetic techniques, monitoring facilities and availability
of different drugs, young women are still dying of anaesthesia related complication (10)
. In another
Khalaf (2019): APGAR Score in neonates May 2019 Vol. 19
©Annals of Tropical Medicine & Public Health SP2009-19
study observed that Apgar scores of neonates whose mothers conventional general anaesthesia
were lower than neonates whose mommies received spinal anaesthesia(11)
.
Delivery of baby by caesarean section has become gradually common, and both general and
spinal anaesthesia have certain advantages and disadvantages, but regional anaesthesia has
become the favored technique because general anaesthesia associated with higher maternal
mortality and foetal depression(12)
.
Apgar’s was between the first to report that babies delivered by Caesarean section under spinal
block were, in general, more vigorous at birth than those whose mothers had cyclopropane.
Numerous workers report a marginal enhancement in one-minute Apgar scores in infants
delivered by Caesarean section under epidural block (13,14,15,5)
,but others have found no
difference.(16,17)
.
Datta et al., observed that in absence of hypotension there is no change in Apgar scores or acid
base status with prolonged induction to delivery interval in spinal anaesthesia. Morgan describe
long skin incision to delivery time more than 8 minutes and uterine-incision-to delivery time more
than 180 seconds have been associated with foetal hypoxia and acidosis regardless of the type of
anaesthesia. The Apgar scores of neonates whose mothers received general anaesthesia were
lower than neonates whose mothers received spinal anaesthesia. (18)
.
A study other done that there is that there is statistically significant risk of fetal acidemia of
varying severity with the use of regional anaesthesia in women delivered by cesarean without
labor. Also, the umbilical artery blood PH values less than 7.10 were observed in 4% of fetuses,
among whom 1% had PH values less than 6.99. On the other hand no infant had PH values less
than 7.10 when general anaesthesia was used. They also concluded that the prevalence of low PH
values was significantly increased in those infants exposed to any of regional anesthetic
techniques compared to general anaesthesia (19,20,21)
.
In other studies for two groups of patients, one received general anaesthesia and other spinal
anaesthesia and found that no significant difference was seen in the mean 1 minute Apgar scores
in the two groups, however more neonates of the general anaesthesia group appeared depressed
soon after birth, needing free flow of oxygen and bag and mask ventilation(5)
. There are different
opinions about the ideal time at which the fetus should be delivered after induction of anaesthesia.
Barter was the first to emphasize that parturient woman should be prepped and draped before
induction of general anaesthesia(22)
. Numerous workers have mentioned that delivery is best
completed 6-8 minutes after induction of general anaesthesia as nitrous oxide could cause
neonatal depression by diffusion through the placenta (23,10)
.
A Study done at Abbasi Shaheed Hospital from March 2009 to July 2009, accomplish that there is
no significant difference between the effects of general anaesthesia and spinal anaesthesia on
Apgar score of neonates at 5 minutes interval, born after full term elective caesarean section of
healthy patients Present anaesthetic techniques, however limit the dose of intravenous agents such
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that fetal depression is usually not clinically significant with general anaesthesia and
recommended that spinal anaesthesia is safe for caesarean section of healthy patients (24)
. The
result of this study was corresponding to our result i.e. there is significant difference only at one
minute after delivery. In the other study was done at Los Angeles, California, the result of this
study was that the neonates delivered with general anaesthesia had scored significantly lower on
some of the test items than neonates delivered by spinal anaesthesia at one minute after delivery (25).
The results were in the same trend with those reported by (Haider et al.,), who found that the
general anaesthesia of 25 patients give birth to neonates having Apgar score ≤ 6 at one minute after birth and the remaining 5 neonates had Apgar score of ≥7. On the other hand out of 30 mothers who received spinal anaesthesia only 10 mothers give birth to neonate having Apgar
score ≤ 6 at one minute after birth, who improved at 5 minutes interval, and their Apgar score
were ≥7. It had been found that the neonates who were born under G.A were ten folds more likely to have Apgar score less than or equal to 6 at first minute compared to those with spinal
anaesthesia (26)
.
CONCLUSION:
From this study it is recommended that spinal anaesthesia is safe for caesarean section of healthy
patient and more safe for newborn delivery than general anaesthesia at one minute valuation of
Apgar score & more comfortable for the mother. It is preferable to do caesarean section under
spinal anaesthesia, to avoid intravenous agents which cause foetal depression at one minute after
delivery. It can be further estimated by a large studies on Apgar scores in neonates following
both elective and substitute cesarean sections.
REFERENCES:
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2. Klieyman Behrman ,Jenson Stanton .Nelson Text book of paediatrics : 18 edtion,2007;94:679.
3. American Academy of Pediatrics, Committee on Fetus and Newborn, American College of
Obstetricians and Gynecologists and Committee on Obstetric Practice. The Apgar score.
Pediatrics. 2006;117:1444–47.
4. Brown DL. Spinal, epidural, and caudal anesthesia. In: Miller RD, editor. Anaesthesia. 4th ed.
New York: Churchill Livingstone; 1995. pp. 1505-33.
5. Drowning JW, Houlton PC, Baeclay A. Extra dural anaesthesia for cesarean section: a
comparison with general anaesthesia. Br J Anaesth 1997;51:390±51:367-73.
6. Petropoulos G, Siristatidis C, Salamalekis E, Creatsas G. Spinal and epidural versus general
anesthesia for elective Cesarean section at term: effect on the acid-base status of the mother
and newborn. Journal of Maternal-Fetal and Neonatal Medicine. 2003; 13(4):260–66.
7. Skolnick AA. Apgar quartet plays perinatologist’s instruments. JAMA 1996; 276:1939-40.
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8. Khoury AD, Moretti ML, Barton JR. Fetal blood sampling in patients undergoing elective
cesarean section: a correlation with cord blood gas values obtained at delivery. Am J Obstet
Gynecol 1994; 171:679-84.
9. Klieyman Behrman ,Jenson Stanton .Nelson Text book of paediatrics : 18 edtion,2007;94:679.
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edi-torss. Care of high-risk neonate. 5th ed. Philadel-phia: W.B. Saunders; 2001. pp.45-61.
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14. Shniders M, Levinson G . Anaesthesia for obstetrics. Baltimore: Williams and Wilkins, 1979.
15. Sykes GS, Molloy PM, Johnson P, Gu W, Ashworth F, Stirrat GM, Turnbull AC. Do Apgar
scores indicate asphyxia? Lancet 1982;1:494-96.
16. Fisher J T, Mortola JP, Smith B , Fox GS, Weeks SK. Neonatal pattern of breathing
following caesarean section: epidural versus general anaesthesia. Anesthesiology 1983;
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17. Fox GS, Smith JB , Namba Y , Johnson RC. Anaesthesia for caesarean section: further
studies. American Journal ofObstetrics and Gynaecoiogy 1979;133:15-19.
18. Sultana A. Effect of Type of Anaesthesia on neonatal outcome. Annals 2004;9:552-56.
19. Roberets SW, Leveno KJ, Sidawi JE, Lucas MJ, Kelly MA. Fetal acidemia associated with
regional anaesthesia for elective cesarean delivery. Obstet Gynecol 1995; 85:79±83.
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Network. Semin Perinatol 2003;23:335-48.
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anaesthesia on Apgar score in neonates born after elective caesarean section.JLUMHS
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Khalaf (2019): APGAR Score in neonates May 2019 Vol. 19
Al-Shalah (2019): Pathogens in milk May 2019 Vol.19
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Pathogens in milk and product dairy: study in Europe
Zainab Musadaq Al-shalah1
1. Al-Qasim Green University, College of Food Science, Babylon, Iraq
Summary:
Milk and milk products contain natural microbial flora and / or addition to the origin of the diversity of
products placed on the French market. The origin of contaminations by pathogenic bacteria varies
according to the nature of the product and its mode of production and processing. The contamination of
milk and milk products by pathogenic germs can be of endogenous origin, and it then follows a
mammary excretion of the animal sick; it can also be of exogenous origin, it is then a direct contact
with infected herds or a contribution from the environment (water, personnel).
The whole process of processing and processing milk can slow down the multiplication of the germs
possibly present or, on the contrary, favor their development. For each of the main germs likely to be
found in these products, the authors describe aspects of physiology and of bacterial ecology, the
incidence in dairy products and consequences in public health. The most frequently mentioned germs
are
Mycobacteria, Brucella, Listeria monocytogenes, Staphylococcus aureus, Enterobacteria, including
Escherichia coli producing toxins and Salmonella. Currently, control of these pathogenic bacteria in
milk and derivatives requires the establishment of systems of control and surveillance that rely on
regulations now become European. The means of prevention must take into account the data now well-
known in the field of microbiology for milk and dairy products. More and more, the presence of
pathogenic bacteria in a food will have to be examined from the perspective of risk analysis for the
consumer with respect to these micro-organisms.
Keywords: Brucella , Contamination, Escherichia coli, Milk, Listeria monocytogenes ,
Mycobacteria , Prevention , Milk Products , Risk , Salmonella, Public Health, Staphylococcus aureus
How to cite this article: Al-Shalah ZM (2019): Pathogens in milk and product dairy:
study in Europe, Ann Trop Med & Pub Health; 19: SPe168-19
Salmonella:
Salmonella are Gram-negative bacteria of the type aerobic-anaerobic option belonging to the
family of Enterobacteriaceae and possessing all their characteristics biochemical. Provided
with peritrichous flagella, they are generally mobile but some serovars are motionless as S. Gallinarum
pullorum and others having lost their flagella. The genus Salmonella comprises two species :
Salmonella enterica and Salmonella bongori; the enterica species is itself subdivided in six subspecies
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defined on the basis of characters biochemical and genotypic by hybridization results DNA / DNA
(42). The subspecies thus differentiated are enterica (I), salamae (II), arizonae (IIIa), diarizonae (IIIb),
houtenae (IV) and indica (VI). The vast majority of isolated strains in humans and warm-blooded
animals belong to the subspecies enterica. Subspecies II, IIIa and IIIb are frequently found in the
commensal flora from the gut of cold-blooded animals but can be as isolated from the environment or
warm-blooded animals. The subspecies IV, VI and the bongori species are very rarely found and do not
seem to have any preferential habitat.
Each subspecies is subdivided into serovars on the base of somatic and flagella antigenic characters.
The combination of these antigenic factors makes it possible define a complete antigenic structure,
which according to the Kauffmann-White scheme characterizes a strain. The current nomenclature
grants to keep for each serovar the name that had been assigned for the first time to a strain of
identified antigenic formula; this denomination corresponds most often to an origin zoological or
geographical area for strains belonging to the first subspecies. Serovars from other subspecies are
designated only by their antigenic formula and the indication of the subspecies. To date, there are more
2,300 different serovars; new serovars are sometimes still isolated in France and recognized by the
Center National Reference Standard for Salmonella and Shigella (Institute Pasteur, Paris). Although
any isolated Salmonella is considered potentially pathogenic for humans, the determination of the
serovar makes it possible to better characterize the strain and restore it in its ecological context or
Epidemiological. Salmonella can multiply at temperatures between 5 ° C and 45 ° C with an optimum
at 35 ° C-37 ° C and at pH 4.5-9 with an optimum optimum between 6.4 and 7.5. Most salmonella can
develop in foods with water activity (Aw between 0.945 and 0.999). The potential oxidation reduction
may also be a determining factor in the growth of this microorganism.
Salmonella can be divided into three distinct groups:
- Those that are specifically adapted to humans and isolated exclusively in humans: this is the case of S.
Typhimurium, S. Paratyphi A and S. Sendai responsible for typhoid or paratyphoid fevers; serovars
specifically adapted to animal species, such as S. Gallinarum pullorum in poultry, S. Abortusovis in
sheep, S. Abortusequi in horses, S. Typhisuis and S. Choleraesuis in pigs;
- The other so-called ubiquitous serovars belonging to the third group and that can infect humans as
well which animal. They are most frequently isolated in the countries industrialized and are at the
origin of most salmonellosis human and animal. The gut of animals is the most important reservoir in
salmonella and contributes greatly to their spread in the environment where they can survive but
without multiply. The contamination of man can be done directly by contact or, most the intermediary
of contaminated food, a large number of food products being likely to be vectors. Many animal species
as well as humans can host the micro-organism in a non-apparent way as those healthy carriers
allowing even more easily this dissemination. The prevalence of contaminations by Salmonella in dairy
herds is variable according to countries and publications. In California, more than 72.7% of dairy cows
would show signs of salmonella infection, the observed serovars being S. Typhimurium, S. Dublin, S.
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Montevideo, S. Newport and S. Anatum (85). Few data are published for countries European. In France,
according to data from the serotyping of the National Center for Veterinary Studies and (CNEVA) of
Paris, salmonella of origin accounted for 18% of strains ten years ago inventories of animal origin,
while currently they corresponding to 36% of this same population (9, 10, 11, 17). At the same time,
the severity of salmonella infections requires the use of anti-infective treatments, although antibiotic
resistance of the original strains cattle has also increased.
In France and also in other European countries, S. Typhimurium is the most frequently found serovar in
cattle. According to data from the inventory of Salmonella from CNEVA Paris (2014-2015), S.
Typhimurium represents 66.6% of all isolated strains of origin bovine, whereas ten years ago serovar
Dublin was still predominant (10, 57). Currently, this last serovar appears to be increasing again in
cattle (9, 10, 11). The infected farms constitute a potential reservoir of contamination of milk and
products derived from raw milk. However, it seems that the contamination occurs more frequently
from the middle outside, from the environment or by contact with animals infected at the time of
milking as intramammary. According to the Salmonella inventory data, 443 strains were identified in
dairy products during the 2014-2015, accounting for 3.6% of the total stem from food hygiene. This
figure appears to be low, corresponds to a sharp increase in number of strains from milk and milk
products, doubtless in connection with the Decree of 30 March 2014 which Mandatory search for
Salmonella in milkshakes consumption and milk-based products when they are on the market. Table I
summarizes the evolution of the number of isolated strains and their distribution in different categories
of dairy products. The results presented depend heavily on the establishment of guidelines and
monitoring or control plans in this sector. The recent data show that serovar Typhimurium is largely
predominant and accounts for more than 35% of isolated strains, two thirds of which come from raw
milk.
Table I: Evolution of the number of isolated strains of Salmonella in milk and dairy products
Products
analyzed
Raw milk 15 44 134 94 257
Powdered
milk 9 10 6 3 0
Cheese 1 19 60 47 156
Milk products 15 13 16 25 30
Total 40 86 216 169 443
isolated strains
in hygiene
alimentary
0.9 1.3 3.2 1.9 3.6
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Is followed by serovar Montevideo with more than half of strains are isolated from cheeses (10, 63).
Although Salmonella is the leading cause of food poisoning in France, milk and products are rarely
responsible for salmonellosis Raw milk is rather infrequently contaminated and this contamination is
then most often of external origin. The pasteurized milk is usually free of all salmonella because they
are eliminated during the pasteurization. Incidents can only occur by recontamination after
pasteurization .Powders milk were responsible for several incidents; indeed, he has been shown that
some salmonella could withstand the freeze-drying heat treatment. The contamination of milk powders
may also have a external origin. Contamination of pasteurized cheeses can only be done after the
pasteurization step. The growth and survival of salmonella during the manufacturing and refining vary
according to different parameters, in particular, the acidification of the medium. A low pH variation
can reduce (pH 4.55) or contrary to promoting (pH: 4.95) the development of salmonella (68). Lactic
acid bacteria like certain Streptococcus or Lactobacillus that produce acids result in the elimination of
salmonella. During stages of manufacture and ripening, salmonella can also multiply according to the
ambient temperature. They can then persist until the moment of the market for these products.
However, for cheeses long ripening period (cooked pressed pasta), elaboration substances with
bactericidal action makes it possible to eliminate salmonella; other substances present in cheeses
marbling may also have an inhibitory action on the growth of certain strains: this is the case with
ferments according to their composition play an important role in the inhibition and elimination of
salmonella (30.) Salmonellosis can be classified in two major categories: those due to serovars strictly
humans responsible for typhoid syndromes or paratyphoid appearing in our regions as a case isolated
sporadic or restricted localized foci, and due to ubiquitous serovars of lower pathogenicity in healthy
adults, leading to clinical symptoms of food poisoning of favorable prognosis if the Host resistance is
not lowered. After a period incubation period usually from 12 to 36 hours, the table classic clinical is
that of a gastroenteritis that can be accompanied by fever, diarrhea, abdominal pain and vomiting.
Fragile populations such as subjects immunocompromised, infants and the elderly are more susceptible
to infection. The infectious dose needed to the onset of these symptoms is variable depending on the
samovar, the nature of the food and the host: it is usually estimated at 106 live bacteria but can be
above or below this value (50). During the year 2014 data from the Weekly Epidemiological Bulletin
identified 267 reported outbreaks of food-borne illness Collaborative Salmonella (TIAC) for 3,840
16.7 % were hospitalized .
Serovar Enteritidis was found in 65.5% of the outbreaks due to Salmonella. Salmonella TIACs are
more likely to occur family catering (60%) in a collective setting (35%). When the offending food
could be identified, it was the more often eggs or egg products (47.5%). Milk and dairy products were
involved in 5.5% of the cases during the year 2014; no case from these same products has declared
during the year 2015.
In France and other European countries, different types products have been involved in recent years in
episodes of food poisoning: this is raw milk, goat cheese, mozzarella, soft cow cheese, Cheddar,
Vacherin, Milk Powder, Cream and Sauce cream and ice cream. Of the milk powders, those intended
for infant feeding have recently been responsible for gastroenteritis in infants in France and in the
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United Kingdom. The survey conducted by the National Network public health network and the "Salm-
net" European network enabled trace the origin of this contamination and to identify the serovar
Anatum. Such contamination by salmonella had already been responsible for TIAC in Austria, there is
a ten years and in Australia, twenty years ago. Raw milk cheeses were responsible for two community
outbreaks of salmonellosis in the seven occurred in France between 2016 and 2017; the first was due to
serovar Paratyphi B; the second, caused by serovar Dublin, covered both France and Switzerland (24,
25). In France, the surveillance of the TIAC is carried out jointly by the National Reference Centers of
Pasteur Institute of Paris, by the National Laboratory of (LNS) receiving the reports of diagnosed cases
retransmitted by the Departmental Directorates of Affairs health and social services (DDASS). The
National Health Network The public carries out epidemiological investigations in the event of declared
outbreaks. At the same time, surveillance of zoonoses is under the control of the Directorates of
Services veterinarians from each department and the centralization of data is carried out at the level of
the Directorate-General Food. Strengthening and better organization monitoring systems have allowed
for better census of cases in recent years.
A European network has recently been set up to collect all the data from the centers of national
reference of each country. He allowed to observe Isolation frequencies of some identical serovars for
each country and also to carry out surveys epidemiological data covering several European countries
(34(. 92/46 / EEC helped to increase the number of It also seems that the implementation of the
Directive of Salmonella research in dairy products and either therefore causing the apparent increase in
the incidence of Salmonella in these product categories. Apart from the role played by the supervisory
bodies and control of food poisoning, the best way to combat the increase in the number of
salmonellosis is to set up a number of prevention measures at all levels of production. It is already
announced by the World Health Organization. Health (WHO) in 1983 (A. Zuniga Estrada, L. Mota de
la Garza and A. Lopez Merino, personal communication), with several recommended control axes
since production until consumption. These preventive measures first of all relate to the farms, where
they must allow animals to better resist infection with reducing the spread of salmonella and
maintaining a good level of hygiene through basic rules. This requires the establishment of awareness-
raising actions breeders accompanied by corrective actions in case of animals infected. Procedures on
the respect of methods appropriate cleaning and disinfection procedures have been by WHO (70). If the
use of vaccination is commonly used in the United States of America, where 45% of cows’ dairy
farmers in California are probably protected (85), this practice is little used in France. Monitoring milk
collected with regular monitoring of milk tank and tank allows a first sort that requires, in case of a
positive result, a more thorough investigation the origin of contamination in the production of the milk.
Checks are regularly carried out in milk processing workshops, whether at the level of the
pasteurization or during the manufacture and refining of cheeses. Control of the manufacturing
parameters and ripening (hygrometry, temperature, pH) as well as the place of a hazard analysis critical
control system points (HACCP: risk analysis, critical points for their control) can reduce the risks of
contamination throughout the processing chain. Finally, the consumer must be informed of the
consequences non-compliance with storage or storage instructions some products. In the United States
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of America, lately, a investigation by the Center for Disease Control and Prevention showed that most
toxi-infections were generated by the consumer himself.
Listeria monocytogenes:
The genus Listeria belongs to the phylogenetic branch of Clostridium as well as Staphylococcus,
Streptococcus, Lactococcus and Bacillus. On the basis of the DNA / DNA hybridization results and the
partial sequencing of 16S ribosomal RNA, the genus Listeria is currently divided into six species,
divided into two phylogenetic branches (55). The first includes Listeria (L) monocytogenes, L. ivanovii
(subsp. Ivanovii and subsp. iondoniensis), L. innocua, L. welshimeri, L. seeligeri. The second consists
of a single species Listeria bacteria are in the form of small bacilli of regular shape from 0.5 μm to 2
μm long and 0.4 μm to 0.5 μm in diameter, rounded at the ends and not forming neither capsule nor
spore. They are Gram positive, being able to appear in Gram stain, isolated, in V, in clusters and
sometimes even in chains.
Their growth is possible between 0 ° C and 45 ° C (temperature optimum temperature: 30 ° C-37 ° C),
for pH values between 4.5 and 9.6 , up to 10% NaCl and for A w of 0.92. Between 20 ° C and 25 ° C,
they are mobile thanks to flagella implantation is peritrich.
Listeria monocytogenes can be considered as an agent food pathogen "perfect" because it is ubiquitous,
very resistant to difficult conditions (temperature, Aw, pH ...) and especially she is able to grow at
temperatures of refrigeration of food. The virulence of the strains could besides be exalted by their low
development temperature (48).
Listeria bacteria are in the form of small bacilli of regular shape from 0.5 μm to 2 μm long and 0.4 μm
to 0.5 μm in diameter, rounded at the ends and not forming neither capsule nor spore. They are Gram
positive, being able to appear in Gram stain, isolated, in V, in clusters and sometimes even in chains.
Their growth is possible between 0 ° C and 45 ° C (temperature optimum temperature: 30 ° C-37 ° C),
for pH values between 4.5 and 9.6 , up to 10% NaCl and for A w of 0.92. Between 20 ° C and 25 ° C,
they are mobile thanks to flagella implantation is peritrich , Listeria monocytogenes can be considered
as an agent food pathogen "perfect" because it is ubiquitous, very resistant to difficult conditions
(temperature, Aw, pH ...) and especially she is able to grow at temperatures of refrigeration of food.
The virulence of the strains could besides be exalted by their low development temperature (48).
Three large reservoirs at Listeria are thus identified. All first a "human" reservoir and an "animal"
reservoir, since a large frequency of healthy carriers is generally observed (6). Finally, a tank
"Environment", namely soil, vegetation, surface, wastewater, silage or food from which the man can be
contaminated. However, in in the majority of cases, the number of L monocytogenes is low (less than a
hundred or a few hundred per gram) (83).
This high incidence in food is also at the origin some major Listeriosis epidemics, the contaminations
being mainly due to of animal origin and in particular to dairy products. Thus, in France, it is estimated
that on average between 1% and 9% of raw milk samples are contaminated but the concentration is
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most often less than one bacterium / ml. Two routes of contamination are usually described
contamination by the cow (mastitis) is infrequent but the level of contamination is often high (1000 to
100,000 L. monocytogenes / ml in district milk), associated with an abnormal cell count without
clinical signs individuals ;- contamination by the environment is more frequent, but contamination
levels are also lower. The poor quality silage are therefore a source significant contamination since the
presence of L. monocytogenes in these multiplies by twenty the risk of contamination of the tank milk.
At the dairy, L monocytogenes is normally destroyed by efficient pasteurization. Contamination
possibly highlighted then result usually is a technological problem (insufficient pasteurization) or
contamination secondary.
During processing, it is estimated that 0.5% to 10% cheeses are contaminated with L. monocytogenes;
it's about mainly soft cheeses, however 75% contaminated cheeses have low levels of 1 to 100 L.
monocytogenes / g. However, some cheeses pasteurized soft can hold up to 106 / g. Contamination
may be limited to crust or extended to the dough. Indeed, Listeria generally follows the evolution of the
pH which is not homogeneous in cheese (the pH gradient can reach one or two units between the heart
and the crust). The development of Listeria is therefore more favorable under crust, thanks to the flores
of surfaces that alkalize the matrix. We can globally distinguish several types of behavior of L.
monocytogenes according to the matrix cheese (48). For some cheeses such as cheeses fresh, acidic
soft pasta (goat cheese ), Hard cheese ripening very long (parmesan, mozzarella), the matrix itself has
destructive properties. Other cheeses have inhibiting properties: pressed pastes some blues. Still others
allow growth of Listeria; these can reach levels of contamination sometimes very high in pasta soft
pasteurized (106 / g). Levels of contamination in soft pasta with raw milk are very variable, depending
on the contamination of the raw material and following the flora associated, since some flora have an
inhibitory action it can be added that L. monocytogenes has been isolated in other dairy products such
as unsweetened condensed milk, concentrated milk by ultra filtration, ice cream, or certain dried milks
(drying by the "spray" destroys L. monocytogenes, which survives but does not develop not).
Although listeriosis is a rare infection caused by virulent strains of L. monocytogenes, it has done much
talk about her in recent years because of the seriousness of the disease. This is, indeed, food-borne
infection most lethal (20% to 30% of cases). Thus, even diagnosed early, listeriosis high mortality
rates, on the one hand because the diagnosis is difficult to achieve, given the very long incubation times
variables (2-70 days) (6), on the other hand because treatments are quite random (development of
antibiotic resistance).
The infection can evolve into a mild form (episode febrile influenza type with sometimes meningeal
syndrome) but it can evolve into a more serious form like a meningitis or sepsis, leaving in 5% to 10%
of cases neurological squeal.
For example, 60% of patients during the Swiss epidemic do not have any predisposing condition). The
infectious dose is still unknown but no case human listeriosis has been linked to food containing less
than 100 L. monocytogenes / g or ml. This dose will be difficult to evaluate because it depends, among
other factors, the immune status of the host and the virulence of the strain. Since the early 1980s, the
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number of listeriosis has tendency to increase, mainly in the industrialized. Thus, if we list fifty cases
per year in France until 1970, then about 150 cases per year until 1975, the 1980s were marked by a
significant increase in the number of listeriosis cases (824 cases in 2014, 522 in 2017).
This situation is mainly due to an increase in food chains and a restoration development collective, but
also to more epidemiological surveillance organized as well as an increase in life expectancy and
number of immunocompromised individuals. Establishment of listeriosis surveillance systems in many
industrialized countries has thus made it possible to estimate the incidence of this disease. This one is
also variable from one country to another because it probably depends on everything less in part, eating
habits. So, for the year 2013, the incidence is close to ten cases / 106 inhabitants in France, while it is
estimated at around two cases / 106 inhabitants in Switzerland or Canada and seven to eight cases / 106
inhabitants in the United States of America. In France, surveillance is carried out by the National
Center reference for phage typing and molecular typing Listeria (Institut Pasteur, Paris) and by the
LNS which receives the reports of cases diagnosed by a network of biologists in the field of regulation.
Staphylococcus aureus:
Staphylococcus and Micrococcus are two genera bacteria that have long been grouped together in the
family of Micrococcaceae. This family is going to be soon overhauled, because of the remoteness
phylogenetic of these two genera. The micrococci represent a heterogeneous group of the branch of
Actinomycetes, while staphylococci form a homogeneous group connected to the branch of
Clostridium (20). The bacteria of the genus Staphylococcus are Gram cocci positive, non-sporulating,
grouped into clusters, immobile, facultative anaerobes and possessing a catalase. Thirty three species
have already been identified by DNA / DNA hybridization and new species or subspecies are regularly
described (44). The basic criterion of their classification is the coagulase production. There are three
species to positive coagulase: Staphylococcus aureus, S. intermedius, S. hyicus, and thirty coagulase
negative species.
The presence of staphylococci in food represents a risk to human health, because some strains mainly
belonging to the species S. aureus produce enterotoxins whose ingestion causes toxiinfection
Staphylococcal food .
Milk and dairy products only become toxic if they are contaminated with enterotoxin producing strains
and if favorable conditions for multiplication important bacterial and toxinogenesis can be found met.
Staphylococcus aureus is part of the flora of the skin and mucous membranes of man and animal.
Parasite usually harmless, it can cause infections (skin abscess, mastitis). Contamination of milk may
occur by through healthy or infected carriers, or by the environment. In cattle, S. aureus is isolated in
nostrils. It is found in small skin lesions and in the sleeves of milking machines. Colonization of teats
can lead to infection of the udder. It can be estimated that 25% of dairy cows can be have breast
infections. During investigations carried out in France and the United Kingdom from 2006 to 2016, the
frequency of isolation of S. aureus in infected quarters has been 20% to 40% for unseen infections, and
15 % to 30% in clinical mastitis (76). S. aureus is the most common bacterium involved in latent
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infections and chronic subclinical mastitis. Mastitis being difficult to eradicate, they represent the main
source of contamination of raw milks by S. aureus. Excretion of S. aureus in district milk varies from 0
to 1 0 4 - 1 0 5 bacteria / ml in case of subclinical mastitis and up to 1 0 8 bacteria / ml in case of
clinical mastitis. In the mixing milk, there are on average 102 to 103 S. aureus / ml. In humans, nasal
carriage concerns 20% to 50% of people. S. aureus is disseminated on the skin and hands of
temporarily or permanently. The man is considered as the main vector of contamination during the
manipulations intervening throughout the chain food. However in raw milk and milk cheeses raw,
human biotype strains remain a minority compared to strains of bovine biotype . The S. aureus strains
are not all toxinogenic. According to the numerous surveys carried out on this subject, and optimal
conditions of culture, in the laboratory, the percentage of toxigenic strains would be 30% to 60% in
sheep and goat strains, compared to only 4% in 10 % in cattle strains (21).
The temperature of milk storage and manufacturing of cheeses plays a vital role. Staphylococcus
aureus grows at temperatures included between 6 ° C and 46 ° C (optimum temperature: 37 ° C) at a
pH between 4 and 9.8 (optimum pH: 6 to 7). This species tolerate a high concentration of NaCl (up to
20%) and a Aw very reduced (0.83). Toxinogenesis is involved in conditions a little more restrictive
than those required for the growth (20).
Once formed, the enterotoxins are remarkably stable. They resist the irradiation, the enzymes
proteolytic and especially to heat. While the bacteria is destroyed during pasteurization of milk,
enterotoxins are only partially inactivated. They are not completely inactivated after 20 to 40 min at
120 ° C, according to a study with unpurified toxins at 100 ng / ml in phosphate buffer (90). More
importantly, they could form complexes with each other or with the food, preventing their detection
after heat treatment while their biological activity persists (82, 90).
Pasteurized milk is more favorable for the growth of S. aureus than raw milk because this
microorganism is a bad competitor in the presence of other bacterial flora. In the raw milk, the initial
number of S. aureus must be equal or greater than that of the concomitant flora to be able to multiply
sufficiently and produce enterotoxins .
The technological parameters of cheese making are usually favorable for the growth of S. aureus. This
occurs in the tank, then, if the pH does not drop Normally, it continues during pressing but usually not
beyond. The first twenty-four hours manufacturing therefore seem decisive. However, in case of lactic
flora, staphylococci can continue to multiply during the first weeks refining. Their number then
decreases gradually . Enterotoxins can be detected when the number of S. aureus reaches about 106 to
107 / g. In milk cheeses believed, this is exceptional because even when the level bacterial exceeds this
value, the surrounding conditions are not usually favorable for toxinogenesis. The type of cheese also
matters (temperature and pH gradient during manufacture, salt concentration, activity of the
antagonistic flora) (5, 21).
Staphylococcal food poisoning is characterized by violent and repetitive vomiting occurring 30 minutes
to 8 hours after ingestion. Disease is short lived but very challenging and spectacular complications are
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sometimes observed in ingested toxin dose and sensitivity individual. Hospitalization is reported in
14% of case on average, but mortality is exceptional (22). In France, from 1988 to 1995, staphylococci
were incriminated in more than 300 households, or 10% of TIAC reported to the health authorities.
They occupy the second row for the number of outbreaks after salmonella and reached during this
period more than 7,000 patients of which 55% in schools (2 2, 4 3). Foods responsible for food
poisoning Staphylococci are varied. Milk and dairy products are suspected in 25% of the 300
households mentioned above (22). On the one Generally, dairy products are infrequently incriminated
in original food-borne illness bacteria: they accounted for 4% of total outbreaks, France, from 1988 to
1995 (22) and, in other countries, from 0.9% to 8.3 % of households by country (32). In contrast,
staphylococci are the most common bacteria more often implicated in the toxi-infections foodstuffs due
to dairy products in France as United Kingdom (22, 56).
European Directive 92/46 / EEC of 16 June 1992 fixes the health rules for the production and placing
on the market of raw milk, heat-treated milk and products based on milk (15). Staphylococci are
presented as germs evidence of lack of hygiene, but it must be remembered that in the case of raw milk
products, the main source of contamination is bovine mastitis (5). S. aureus must be enumerated in raw
cow's milk (m = 500), cheeses fresh (m = 10), soft cheeses and marbled pasteurized milk (m = 100),
raw milk and milk cheeses thermised (m = 1000). If they contain more than 10,000 colony forming
units (CFU) per gram of S. aureus, an enterotoxin test should be performed (culture toxins isolated
strains from cheeses or toxins extracted from cheeses). However, the implementation evidence of
enterotoxins remains technically difficult despite the existence of many detection kits . Prevention of
food-borne illness Staphylococci involves setting up a program action against bovine mastitis, the
maintenance of milk refrigeration temperature and strict compliance with the rules hygiene during
handling on the farm and at the dairy, in order to to limit the number of S. aureus present in the milk.
She also requires know-how, a monitoring of parameters technologies and a choice of lactic ferments
to inhibit the growth of S. aureus as much as possible during the manufacture of raw milk cheeses (5,
20, 61).
Finally, improvements are needed at the level of declaration and investigation of toxi-infections food.
Indeed, many homes are not reported and when they are, it is not always possible to evidence of the
causative bacteria or the food in question (43, 56). In addition, most food poisoning reported are very
poorly documented. Lack of information epidemiological data, the risk due to the presence of
Staphylococci in dairy products will remain difficult to assess.
Escherichia coli:
Escherichia (E) coli form a group of motile bacilli gold still, Gram-negative, of the family of
Enterobacteriaceae. They can multiply at temperatures between 4 ° C and 46 ° C, with an optimum
growth at 37 ° C and pH between 4.6 and 9.5. E. coli that causes diarrhea, acute gastritis or colitis of
man are referred to as E. coli pathogens. Differentiation criteria based on their serotype, their virulence
and their clinical consequences have classified these pathogenic strains into four groups:
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Enteropathogenic E. coli distingites (EPEC), E. coli enterotoxigenic agents (ETEC), enteroinvasive E.
coli (EIEC) and enterohemorrhagic E. coli (EHEC).
EPECs are associated with epidemics of childhood diarrhea. They may, depending on the strain,
produce toxins or invade epithelial or intestinal cells. Clinically, the disease is characterized by fever,
vomiting, abdominal pain and severe diarrhea, accompanied by a large amount of mucus in the stool
and a little blood (49). ETECs are characterized by the production of one or two toxins, one
thermolabile (LT), the other thermostable (ST). Tea disease is characterized by watery diarrhea by
abdominal pain, discomfort and nausea. ETECs are also recognized agents of the traveler's diarrhea
(49). The infectious dose for ETEC is high: 1 0 8 - 1 0 1 0 (58).
EIECs are characterized by signs of toxemias with discomfort and fever. EIECs proliferate in tissues
epithelial cells until necrosis occurs (49). The infectious dose for this pathovar is also important: 1 0 6 -
1 0 8 (58). EHEC is responsible for hemorrhagic colitis, uremic hemolytic syndromes (HUS) and
purpura thrombocytopenia. E. coli 0157: H7 are the most often responsible for this hemorrhagic colitis.
These E. coli can produce two potent cytotoxins (VT toxins) .The infectious dose is not known with
certainty purpose it is weak (<10 / g). Escherichia coli are normal commensal of the intestine of man
and animals. It represents 80% of the flora aerobic intestinal. It is found in very large numbers in the
faecal faeces. From there, it spreads in nature: soil, waters. Its presence in the environment always
signifies a faecal contamination. A commensal bacterium, whatever its species, can acquire certain
pathogenicity factors through the contribution a new generic support (plasmid, bacteriophage,
transposons) or by the expression of genes previously silent, and thus become pathogenic (39).
Of the pathogenic bacteria that can end up in raw milk, some are usually at a very low level and are
unlikely to develop. Others are at appreciable levels and can multiply. It's the case, among others, from
E. coli that usually comes from the skin udders (77). This bacterium of fecal origin can survive on
soiled soil. Its implantation in the material milking is unusual. Some strains, fortunately rarely present,
when they are at a high level in the raw milk or in cheese, can produce gastroenteritis due to the
production of toxins. The transmission of enterohemorrhagic E. coli is essentially related to product
contamination food. Food contamination can occur during factory manufacture (followed by
multiplication possible during transport and storage), ie when meal preparation (kitchen staff). The
interpersonal contamination also exists by intermediate healthy carriers with a low dose infective.
Infectious diarrhea is a case sporadic or anademic episodes (TIAC, for example), even epidemic.
Specificities exist, on the one hand according to the regions and seasons of infection, but also
according to the age and socio-economic level of concerned and their way of (life(diets, consumption
of imported foods, fast and collective catering, travel) (39).
Seven biovars are currently identified in B. abortus, three in B. melitensis and five in B. suis (Table II).
The studies hybrid DNA / DNA and DNA / RNA compatible with the existence of a single species of
Brucella ovis and B. canis are the only two species naturally in rough phase. Bacteria of the genus
Brucella are small sticks, very frequently coccobacillary, aerobic and low glucidolytic. Their length
varies from 0.6 μm to 1.5 μm and their thickness 0.5 μm to 0.7 μm. Their morphology is very constant
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except sometimes in older cultures. The Brucella are immobile and do not form spores. No flagella, pili
or capsule. Gram-negative bacteria, Brucella do not show bipolar staining and are resistant to
discolouration by weak acids (2, 16).
Many species animals (especially ruminants, but also suidae, carnivores, equidae and rodents) can be
naturally infected by various species of Brucella (Table II). Because of the frequency and severity of
contracted human cases directly or indirectly from the animal, the brucellosis is considered a major
zoonosis.
Table II: Brucella (species and biovars) (2,69)
Species biovar Morphology
colonies
Host
Preferential
Brucella abortus 1.2.3.4.5.6.9 s Cattle
B. melitensis 1.2.3 s Sheep, goats
B. am 1 s Pigs
2 s Pigs, hares
3 s pigs
4 s Rennes
5 s Wild rodents
B. neotomae - s Neotomes *
B. ovis - R Sheep
B. canis - R Dogs
S: naturally in smooth phase R: naturally in rough phase * Neotoma lepida (desert wood rat)
In the South, the prevalence of bovine infection is now very low (for example, in France prevalence of
livestock was 0.28% in 1995) (4). In these regions of southern Europe, ovine and caprine considerably
diminished over the last ten years because of strengthening of control measures, but the prevalence
remains significant (livestock prevalence rate sheep infected in France in 1995: 2.27%) (4), in
particular in mountain areas and around the Mediterranean. Brucellosis in these species largely
explains the human cases still observed in these areas. The most common vehicle of human infection
by ingestion is raw milk, or one of its drifts, especially the cream and, in many countries, fresh cheeses,
which sometimes the cheapest source of protein and no longer available (69). Milk and dairy products
of origin cattle, sheep or goats are most frequently incriminated, but the infection can also be
contracted atfrom dairy products derived from buffaloes, camelids or yaks. Brucellosis of small
ruminants is considered nevertheless as being responsible for the majority of the cases food-borne
humans. In France, for example, the consumption of goat cheese is the first cause of unproductive
human brucellosis (54%), absorption cow's milk accounted for only 16% of these cases (80, The
generalization of the pasteurization of the milk of origin intended for consumption or processing and
level of sanitary requirements allowing the marketing raw milk products explain this situation. The
eating habits, and especially the predilection of certain populations for raw milk and its derivatives
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concur still largely to the maintenance of human brucellosis. The survival of Brucella in milk and dairy
products is related to many factors, including the type of product, the water, temperature, pH changes,
action the other bacteria present, and the duration and storage conditions of the product (38). The
results of some studies (11, 19, 65, 75, A. Zuniga Estrada, L. Mota Garza and A. Lopez Merino,
personal communication) are shown in Table III. Low concentration in medium liquid, Brucella is
easily destroyed by heat. So, the classic pasteurization, the ultra-high method temperature or a simple
prolonged boil (10 min) kill the Brucella contained in the milk (19).
Wirth Brucella, it is usually reduced in milk that has been kept for some days. Survival in ripened
fermented cheeses seems pretty short. We do not know the time of minimal fermentation necessary for
their total destruction, but it is conventionally estimated that three months are sufficient in fresh
cheeses, the survival of Brucella can be much longer, strictly lactic fermentation and of short duration
and desiccation favoring their survival. Alone past pasteurization of the milk or cream allows to
prevent the health hazard posed by these products human. Brucella are also sensitive to radiation
ionizing agents at commonly used doses, especially in colostrum. Prevention of food-borne human
brucellosis therefore goes first and foremost by the heat treatment of milk intended for direct
consumption or processing. The sale of raw milk and dairy products untreated heat must be, for its part,
closely monitored and limited to free holdings. These measures, even when accompanied by campaigns
information, are insufficient, however, to reduce significantly the incidence of human brucellosis,
numerous cases originating from direct contact with infected animals. Only countries that have
implemented animal brucellosis control programs were able to observe a consequent reduction in the
number of cases humans. The control and prevention of animal brucellosis requires the respect of a
general hygiene in the farms and the setting in place at regional level a restorative on sanitary and / or
medical measures. All of these measures cannot be really effective without a health education, training
and mobilization of professionals concerned. Finally, no measure of prophylaxis cannot be considered
without identification sustainable use of animals and flocks and strict control of their movements
(trade, transhumance).
Various general hygiene measures make it possible to limit the extension of the infection. At the farm
level, isolation animals at the time of parturition and the destruction of non-living products and foeto-
maternal appendages as well as disinfection of livestock premises are two measures essential in this
respect. Staff must also submit to a systematic disinfectant treatment of body parts and clothing that
may have been in contact with contaminated products. Given the relative inefficiency and the cost of
treatment antibiotic in livestock, elimination by slaughter of seropositive animals is one of the solutions
used in the fight against animal brucellosis. This solution implies, when setting up the program of fight,
a relatively low disease prevalence rate so that this plan is economically bearable. Such plans are
usually based on the systematic control of flocks (individual serology or regular ring-test on milk
tanker) and animal health measures positive or in clinical outbreaks, with slaughter positive animals or
total slaughter of herds infected.
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Bacteria of the genus Mycobacterium belong to the family of Mycobacteriaceae which consists of
Actynomycetales with rudimentary pseudomyelium usually presents as small bacilli, immobile,
sometimes having bulging, cuneiform or branched (0.2-0.6 μm over 1.0-10 μm). They are characterized
by their ability to maintain color despite action combined with alcohol and diluted acids: they are acid-
resistant. The optimal temperature of mycobacteria ranges from approximately 28 ° C to 45 ° C. Within
the genus Mycobacterium, there are more than 40 species recognized, including M. tuberculosis, M.
leprae and several mycobacteria called "atypical". The classification taxonomy within the genus is
based on the form of colonies, pigmentation, growth characteristics bacterial and biochemical reactivity
(Tables IV and V).
The bacteria are divided into two main groups depending on whether their growth is slow or fast. In
practice, we consider that growth is slow when it takes more than seven days incubation to obtain
visible growth.
Table IV: Cultural characteristics of the main bacteria of the genus Mycobacterium
mycobacteria Pigmentatio
n
37
°
C
42
°
C
> 7
day
s
mycobacti
n
Pyr
.
Pn
b
Tc
h
pz
a
Tb
1
em
b
M. tuberculosis - + - + - - - + - - -
M. bovis - + - + - + - - + - -
M. africanum - + - + - + - V - V -
M.
paratuberculosi
s
- + - + + + V + v Nf +
M. avium - + + + v + + + nf v +
+: positive v: variable Pyr. : pyruvate TCH: hydrazide of thiophene carboxylic acid, 2 μg /
Tb1:thiosemicarbazone, 10 μg / ml ,,, - : negative nf: not done PNB: Paranitrobenzoate, 500 μg / pza :
pyrazinamide EMB: Ethambutol, 2 μg / ml
The bacteria belong to the tuberculosis complex (M. tuberculosis, M. bovis, M. africanum, M. microti)
are mandatory pathogens. Apart from M. tuberculosis and M. leprae, several species of mycobacteria
are pathogenic for humans under certain conditions. Some species like the M. avium complex, M.
kansasii, M. malmoense and M. xenopi are more likely to cause diseases than others. The majority of
potentially pathogenic atypical mycobacteria have a slow growth. From an economic point of view, the
main diseases Mycobacterial infections in animals are due to M. bovis and M. paratuberculosis.
Mycobacterium bovis can be transmitted from animals to humans and to provoke in the latter a disease
identical to tuberculosis due to M. tuberculosis. Other mycobacteria, such as M. avium, M. marinum,
M. farcinogenes and M. silvaticum may be responsible infections in animals of very different species.
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M. bovis, agent of bovine tuberculosis, is able to infect humans and certain animal species, especially
goat, pork, dog and cat. From the point of view of veterinary medicine and public health, this germ is
the most important cause of diseases mycobacteria in animals.
The symptoms of the disease in cattle vary according to the organ reached. In case of pulmonary
infection, a dry cough appears and worsens as the disease develops. This cough is accompanied by
slimming. Infection of the uterus and / or mammary glands is responsible for a sterility and decreased
milk production. The disease tends to slow worsening. Cattle with tuberculosis are the main source of
M. bovis which is transmitted from cattle to humans mainly in two ways: by air (aerosols) and digestive
(consumption of raw milk infected). Man with pulmonary tuberculosis M. bovis is also a source of
infection for other subjects and possibly for cattle.
Table V: Biochemical characters of the main bacteria of the genus Mycobacterium
mycobacteria Nia. Nit. Cat.
20 °
C
Cat.
68 °
C
Urea 1 2 3 4 5 6
M. tuberculosis + + + - + + + +
M. bovis - - + - + + + +
M. africanum V V + - V + + +
M.
paratuberculosis
- - + + - + + +
M. avium - - + + - + + +
Nia. : niacin Cat. : catalase v: variable +: positive - : negative Nit. : nitrate reductase
Conclusion:
Other pathogenic microorganisms may be encountered in milk and milk products, among which
Yersinia enterocolitica, Campylobacter jejuni, Coxiella burnetii, Streptococcus agalactiae, Clostridium
botulinum, Bacillus cereus, toxin-producing molds and viruses. The presence and persistence of these
germs in milks and dairy products depend on their resistance to treatments raw milk (pasteurization,
acidification, curd heating, ripening conditions) and the initial level of contamination in raw
milk.Pasteurization treatments (72 ° C for 15 s) eliminate pathogenic bacteria in vegetative form, but
those in sporulated form resist ) B. cereus, C. botulinum) .
Campylobacter, very fragile germs, are found only in raw milks and insufficiently pasteurized milks
The number increased by 4-5 log instead of 3 log the foot-and-mouth disease virus is also susceptible
to pH drop. It is inactivated in 17 s at pH 6.7 at 72 ° C and in 55 s at pH 7.6 at 72 ° C .
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The survival of these pathogenic microorganisms in the dairy products is also more or less dependent
on manufacturing process. So C. burnetii can be put in evident in butter and cooked pressed cheeses
after several weeks, while in the cheeses soft and acidic dairy products, germs are very fast inactivated
(33). The FMD virus also survives longer in cooked pressed cheeses than in soft cheeses. Studies from
milk milks cow naturally infected with fever virus foot-and-mouth disease products for the production
of cheddar and camembert cheese revealed that the foot-and-mouth disease virus survives in the
cheddar after 60 days of storage but not after 120 days . In the pie chart, the virus survives 21 days at 2
° C but not 35 days. As for poisoning due to C. botulinum, in cheese technology, the parameters
(acidification, Aw, salt content) do not allow cell growth vegetative growth of C. botulinum or spore
germination (12).
Reference
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