Post on 03-Mar-2017
transcript
BRIEF ON LABORATORY
DIAGNOSIS OF VECTOR BORNE
DISEASES
Dr. Md. Ashraf Ali S N
Department of Microbiology
2
MALARIA
RICKETTSIA
DENGUE
CHIKUNGUNYA
Dr Md Ashraf Ali
Laboratory diagnosis of Malaria1. Microscopy
Light microscopy on peripheral blood smear
Fluorescent microscopy
• ACRIDINE ORANGE staining on thin smear
• Quantitative Buffy Coat (QBC)
2. Rapid diagnostic tests (RDT)- Antigen detection
3. Molecular techniques
Dr Md Ashraf Ali
I. MICROSCOPY
1. LIGHT MICROSCOPY
• Thick and thin blood smear- GIEMSA
• Morphological forms of parasite-
demonstrated
2. FLUORESCENT MICROSCOPY
• Stain- ACRIDINE ORANGE (kawamoto
technique)
• Increases the sensitivity
Dr Md Ashraf Ali
Advantage
• Quick, saves time
• Sensitivity more than “PS for MP”.
Disadvantages
• COST: High(capillaries and equipment)
• Species identification and quantification
difficult
Quantitative Buffy Coat (QBC ®)
capillaries
Dr Md Ashraf Ali
II. RAPID DIAGNOSTIC TESTS (RDT)
Antigen detection
Target- pLDH, HRP-2, Aldolase
Formats-
1. Card
2. Dipstick
3. Casette
Low cost- Rs 100-150/- per test
Disadvantage:
low levels of parasitemia cannot be detected
1 2
3
Dr Md Ashraf Ali
III . MOLECULAR TECHNIQUES
1. Polymerase chain reaction (PCR)
Adv: • Can detect low levels of
parasitemia• Can identify species• Drug resistance.Disadv:• Expensive set up• Trained personnel Lane S: Molecular bp standard (50-bp ladder).
Lane 1: P. vivax (size: 120 bp). Lane 2: P. malariae (size: 144 bp). Lane 3: P. falciparum (size: 205 bp). Lane 4: P. ovale (size: 800 bp).
2. Loop mediated isothermal amplification ( LAMP)
Dr Md Ashraf Ali
Summary of malaria diagnostic test
TESTPeripheral
smearQBC
Rapid diag.
testPCR
LAMP
Assay
Sample to be
sent
Finger prick Blood
(EDTA)
Finger prick •finger prick,
•Blood (heparin)
Time taken
(min)30 10 20
Around
5hrs60
Sensitivity 85% 96% 93-97% 76% 98%
SpecificityAbsolutely
specific 93% 95% 100% 99%
Cost
effectiveness• Rs.100 250/- 100-150/- Ref. labs
Dr Md Ashraf Ali
LABORATORY DIAGNOSIS OF DENGUE
GENERAL
• Profound Leucopenia,
• Thrombocytopenia ( < 100,000)
Dr Md Ashraf Ali
MICROBIOLOGICAL1. SEROLOGY- MOST COMMONLY USED
i. Antigen detection
ii. Detection of antibodies
2. MOLECULAR TECHNIQUES- highly specific and sensitive
3. VIRUS ISOLATION- gold standard
Molecular detection and virus isolation done in reference
centres- NIMHANS Bengaluru, NIV Pune.
Dr Md Ashraf Ali
i. ANTIGEN DETECTION
• Sample- serum
• Time- within first 5 days of illness
• Target
o NS1 (non structural protein)
o E/M (structural)
Formats
• Immunochromatographic card test
• NS1 Ag ELISA (Early ELISA)
• NS5 based ELISA also under trialDr Md Ashraf Ali
ii. ANTIBODY DETECTION-
• SAMPLE- Serum
1. ELISA: most widely used
– IgM Antibody Capture (MAC) ELISA
• Detects recent infection
• Sensitivity- 90% and Specificity-98%
– IgG ELISA
Cost- around Rs. 200/- Dr Md Ashraf Ali
Primary V/s Secondary Infection
• Commonly encountered in endemic countries
• Can be differentiated by ratio of specific IgM and IgG
antibody response
• IgM:IgG ratio > 4 indicates Primary infection
• IgM:IgG ratio < 4 Indicates Secondary infection
( re infection)
Dr Md Ashraf Ali
• Other tests- Not routinely used for diagnosis
2. Haemagglutination-Inhibition (HI) assay
3. Neutralization test (NT)
4. Complement Fixation test (CFT)
Dr Md Ashraf Ali
DENGUE COMBO KIT
• NS1 Ag, IgM and IgG detection can be done in a
single kit
Dr Md Ashraf Ali
II. MOLECULAR METHODS
• Detection of viral RNA
• Done on ACUTE PHASE SERA
1. RT-PCR ( RT-Reverse transcriptase)
– real time RT-PCR- 90% sensitive 95% specific
– Nested PCR
2. RT-LAMP
3. NASBA (Nucleic acid sequence based amplification)
Dr Md Ashraf Ali
III. VIRUS ISOLATION
• Samples serum, whole blood(washed buffy coat) Autopsy tissues (liver, spleen)
• Time of collection – within 6days of onset of illness
• Isolation in cell line- mosquito cell line (C6-36)
• Isolation in Suckling mice ( detect antigen in brain by IFA)
after development of encephalitis
• Report- requires a weekDr Md Ashraf Ali
LABORATORY DIAGNOSIS OF CHIKUNGUNYA
Dr Md Ashraf Ali
1. Antibody detection
a) E L I S A- Widely used
b) IgM rapid test
c) Other tests- Haemagglutination Inhibition
2. Detection of viral RNA• RT-PCR
3. Virus isolation
Dr Md Ashraf Ali
ANTIBODY DETECTION
a) MAC ELISA
• Detects Chikungunya specific IgM antibodies
• Sample- serum
• Timing- after 5 days of onset of illness
• Advantage- rapid, accurate.
• Price — range 200/-
Dr Md Ashraf Ali
b) Chikungunya Rapid test
• Sensitivity: 97.1%
• Specificity: 91.1%
• Time: 10 minutes
IgM positiveDr Md Ashraf Ali
LABORATORY DIAGNOSIS OF RICKETTSIA
Dr Md Ashraf Ali
1. SEROLOGICAL TESTS
2. MOLECULAR DETECTION:
– P C R,
– real time PCR
3. ISOLATION OF RICKETTSIAE-
– Cell lines
– Shell vial technique
Reference labsDr Md Ashraf Ali
SEROLOGICAL TESTS
1. Weil- felix
2. Indirect immunofluorescent assay- gold standard
3. ELISA
4. Immunoperoxidase test
5. CFT, Microagglutination test, Haemagglutination
test
Dr Md Ashraf Ali
WEIL FELIX TEST
• Heterophile agglutination test
• Sensitivity- 33%, Specificity- 46%
Advantage
• Inexpensive test
• Acute diagnosis in resource poor settings
Disadvantage
• Not specific
Dr Md Ashraf Ali
E L I S A
• Both specific and sensitive
Disadvantage
• Diagnostic antibody titers are low, during the first
week of illness
Dr Md Ashraf Ali
Facilities available at KIMS
Vector-borne Disease Test
Dengue • NS1 ELISA
• Dengue Combo kit
• MAC ELISA
Chikungunya MAC ELISA
Japanese encephalitis MAC ELISA
Rickettsia Weil-Felix test
Malaria Peripheral blood smear
Dr Md Ashraf Ali
Statistics of vector borne diseases detected in KIMS
January 13 to December 13Name of the test done Total tested Positive
Dengue IgM 1512 555 (36.70%)
Dengue NS1 438 102 (23.83%)
Chikungunya IgM 123 44 (35.78%)
Weil-felix 45 7 (15.56%)
Japanese encephalitis 22 Nil
All the above tests for the diagnosis of vector borne diseases are done free of cost under IDSP (Integrated disease surveillance
project)Dr Md Ashraf Ali
THANK YOU
Dr Md Ashraf Ali