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SPUTUM COLLECTION AND LABORATORY ESSENTIALS
TB
TB Bacilli SPREAD THROUGH THE AIR
- Infectiousness of person with TB
- Environment in which exposure occurred
- Duration of exposure
-Virulence of the organism
Probability TB Will Be Transmitted
Sources: www.gwc.maricopa.edu/...samples/greenman.htm
TB
TRANSMISSION AND PATHOGENESIS
- Spread by droplet nuclei
- Expelled when person with infectious TB coughs, sneezes, speaks, or sings
- Close contacts at highest risk of becoming infected
- Transmission occurs from person with infectious TB disease (not latent TB infection)
TB
PATHOGENESIS - 10% of infected persons with normal immune systems develop TB at some point in life
- HIV strongest risk factor for development of TB if infected
- Risk of developing TB disease 7% to 10% each year
- Certain medical conditions increase risk that TB infection will progress to TB disease
- Cancer of the head and neck
- Hematologic and reticuloendothelial diseases
- End-stage renal disease
- Intestinal bypass or gastrectomy
- Chronic malabsorption syndromes
- Low body weight (10% or more below the ideal)
Inactive
TB Active
• Slender, straight or slightly curved bacillus, non-motile, non-encapsulated and does not form spores
• Acid fast bacillus (AFB) • Aerobic • Slow growing- divides every 18-24 hr. • Resistant to drying and chemical disinfectants • Sensitive to heat (Pasteurization) and UV light
Mycobacterium tuberculosis-Morphology
Thin section transmission electron micrograph of Mycobacterium tuberculosis
close-up of a Mycobacterium tuberculosis culture revealing this organism’s colonial morphology
TB
COMMON SITES OF TB DISEASE
- Drug-resistant TB transmitted same way as drug-susceptible TB
- Drug resistance is divided into two types:
*Primary resistance develops in persons initially infected with resistant organisms
*Secondary resistance (acquired resistance) develops during TB therapy
Lungs Pleura Central nervous system
Lymphatic system Genitourinary systems
Bones and joints Disseminated (miliary TB)
Drug-Resistant TB
Chest Radiograph
•Abnormalities often seen in apical
or posterior segments of upper lobe or superior segments of lower lobe
•May have unusual appearance in
HIV-positive persons
•Cannot confirm diagnosis of TB
Arrow points to cavity in patient's right upper lobe.
Quality Test Results Require Quality Specimens…
• It is important to remember that…………
– To obtain the most accurate results from TB culture procedures
– Patient specimens must be• Collected with the utmost care
• Promptly transported to the laboratory
• Analyzed for quality before testing
• Processed as soon as possible after arrival
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Collection Strategy
STRATEGY
• Collection of three sputum specimens is recommended at the following times:
– Three early morning fasted specimens
– Collected on three consecutive days
– If not possible, one day rest between collections is acceptable, but not two.
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Containers for Sputum Collection
•Strong, unbreakable•Leak proof, screw-capped with a water-tight seal
•Sterile
•Up to 50 ml capacity•Translucent or clear material
•Single-use •Easily-labeled walls•50ml conical tubes ideal since they can be used for processing
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Specimen Collection I
• Collect sputum in sterile, single use, plastic containers without fixatives (wax containers must not be used)
• Clearly label container with the coded individual ID number, or bar-code and date of collection
• Complete specimen request form
• Instruct patient on proper collection
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Specimen Collection II
• Health centre staff instructed on proper specimen collection and transportation procedures
• Staff properly instruct patients on specimen collection
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Sputum Collection
PPE required by technician/nurse collecting
• N95 Mask
• Gloves
• Laboratory coat
• Infectious waste bin and bags
Specimen Collection III∙Never collect sputum in the laboratory∙Dedicate a well-ventilated or open air
area at a distance from other people
∙Instruct patients to rinse mouth with clean / bottled water before collecting specimen to remove food particles and decrease normal flora
∙Skip if no clean water is available
∙Collection should avoid areas of possible contamination (e.g., tap water due to the presence of environmental mycobacteria)
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Specimen Collection IV
• Patient Instructions• Rinse mouth with clean distilled
or bottled water (Not from water coolers)
• Open container but do not touch inside container or cap
• Take 3–4 deep breaths, holding breath for 3-5 seconds before exhaling
• Cough after the last exhalation• Empty sputum into container
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Sputum Collection V
• Complete specimen request form
• Package sputum for transport to Lab– One sputum per sealable plastic bag
– Place request form in outside pouch on bag
• Log each specimen on the specimen transportation log
• Store specimens in the refrigerator until picked up
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23Form Example
Transporting specimens• Specimens are transported in a containment system
– Primary containment• Collection container with screw cap top
– Secondary containment• Specimen container in a sealable, biohazard bag • Place requisition in outside pouch of biohazard bag
– Tertiary containment• Specimens in bags are placed in transport box
• Use transport boxes (Styrofoam with fibreboard, plastic, or metal) and ensure lid is securely fastened
• Package specimens per box, pack specimens vertically to avoid leaking. • Use cold chain transport and keep specimens protected from light• Do not use dry ice Use water ice for cold chain• Temperature 4-8°C
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Critical Collection and Transport Issues
• Containers should be clearly labeled on the side, not the cap• Identification number on each specimen container
corresponds to the identification number on the specimen log• Request forms must be separated from specimen container• Specimen log should include the requested data for each
patient• Number of specimen containers in the box corresponds to the
number of entries on the specimen log• Date shipped and the name of the health centre are included
on the specimen log
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Transportation Regulations
• Your country’s specific rules for transportation of patient specimens must be followed
• International Air Transport Association (IATA): www.iata.org
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Quality Assessment of Sputum Specimens
• Characteristics of a good sputum specimen– Mucoid or mucopurulent appearance
– Minimum amounts of saliva
– Optimal volume: 5ml-10ml
– Minimum volume: 0.5 ml
Record characteristics on form
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Assessing Quality of Sputum Specimens
Photo kindly provided by Ms. Akiko Fujiki
Saliva Muco-purulent Bloody Mucoid
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Assessing Quality of Sputum Specimens
Saliva Muco-purulent
Bloody Mucoid
Photo kindly provided by Ms. Akiko Fujiki31
Summary points• Sputum specimens may be collected using three consecutive early morning
collections strategy
• Acceptable containers for sputum collection include those having 50ml capacity, are single use, sturdy, screw-cap, and translucent
• At a minimum, specimens should be labelled with the individual code number or identifier, or bar-code, institution, and date collected
• Good quality sputum specimens are those having a minimum volume of 0.5 ml, and contain solid or purulent particles
• • Specimens should be submitted using cold chain transport, and should reach the
laboratory within 4-5 days of collection
• When transport delays are unavoidable, specimens should be refrigerated
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Timelines
• Time from collection to processing is CRITICAL. Must be as soon as possible.
• More than an hour needs cold chain and cold ice-packs
• Do NOT pack in dry-ice.• Limit is 7 days provided cold chain ensured,
this is for remote refugee camp settings.Can use transport thermometers for packing 4-8°C.
Other Methods
• Induced with nebulizer. Be sure to mark induced specimen (For children 3 years and above)
• Gastric Aspirate (most intrusive procedure) and these specimens MUST be pH neutralised directly after collection. Need to liaise with the medical staff
AFB Microscopy Staining Techniques
REAGENT
STEP 2:
Flood the slide with Auramine Rhodamine stain and allow to stain for 15 minutes.Be sure that the stain stays on the smear. Do NOT heat. Do NOT use paper strips.
STEP 3,4 : Rinse the slide with water. Flood the slide with 0.5% Acid Alcohol and allow to decolorize for 2 minutes.
STEP 6: Flood each slide with Potassium Permanganate (MSDS) and allow to quench for 1 minute.
ZIEHL-NEELSEN ACID-FAST STAINS
FLUORESCENCE STAINS
Smear Testing
Advantages
• Inexpensive
• Quick turnaround
• Indicates TB disease
• Positive for the most infectious patients
• High specificity
• Can be used to monitor treatment outcomes.
Disadvantages
• Low Sensitivity 20-25%
• Does not indicate viable organisms
• Further testing required for diagnosis (Depending on other factors)
Culture testing
Advantages• Can grow the organism• Shows viable bacilli• Can perform DST
testing• Can positively identify
MTB• High sensitivity can
detect 10 bacilli• Can monitor treatment
outcomes
Disadvantages• Needs expensive
infrastructure• Slow turnaround 1-8
weeks
M. intracellularenon-tuberculosis mycobacteriaNTM
Immunochromatographic Identification
Molecular Xpert
Advantages
• Quick turnaround time
• Indicates RIF resistance (If conditions met)
• No expensive infrastructure required.
• Sensitivity 60-90% of culture
• High specificity
Disadvantages
• Cannot determine viable organisms
• Cannot monitor treatment
• Cannot provide further information regarding DST results.
Molecular Line Probe Assay
Advantages• Quick turnaround 3-4
hours.• Indicates INH and RIF
resistance• Gives identification of
MTB • Has high
sensitivity/specificity for MTB and resistance
Disadvantages• Expensive infrastructure• Can only be used directly
on strong positive smear specimens and culture positive isolate
• Not approved for smear negative cases
• Cannot provide further DST information
• Cannot monitor treatment
Line Probe Assay
• Requires laboratory with infrastructure the same as for culture.
• Needs at least two separate rooms with one being positive air-pressure
• Best used in conjunction with level II or III mycobacteria laboratories.
FOUND IN: THE ENVIRONMENTDUSTSOILWATER (most common contaminate in the laboratory)
MYCOBACTERIA
M.tuberculosis Complex (MTBC)
Non-Tuberculous Mycobacteria (NTM)
FOUND IN:HUMANSDOMESTIC ANIMALSWILD ANIMALS
MTBDRplus ASSAY:
1. QC tool to validate DST results of M.tuberculosis cultures for all programs
2. Rapid diagnostic tool to detect high/low level Isoniazid and/or Rifampicin resistance in MTB strains directly from PROCESSED SEDIMENT or from CULTURE.
3. Diagnostic tool to detect MDR TB strains in contaminated DST cultures.
METHODOLOGY(ALL ASSAYS USE SAME TEST PROTOCOL)
Sources of Nucleic Acids:
MTBDRplus assay:culture (ex broth or solid media) and smear-positive processed sediments
CM/AS assays:Culture only (ex broth or solid media)
1. DNA PREPARATION:
Crude boil method:2. Centrifugation3. heat inactivation
Centrifuge and heat block
2. PCR:• Master Mix preparation
• Extracted DNA addition
•Amplification (denaturation, annealing, extension)
Thermocycler
3. HYBRIDIZATION:
• Amplicon denaturation
• Reverse hybridization to strip
• Strepavidin/AP conjugation
• Staining to detect banding pattern
DEVELOPED STRIPS
MTBDRplus
CM/AS
SCHEMATIC EXAMPLES OF POSSIBLE RESULTS FOR MTBDRplus ASSAY
4. EVALUATION: MTBDRplus
• Strips pasted onto evaluation sheet to document test results• Valid test results require all internal control bands (CC, AC, TUB, gene locus) to be present
Interpretation of the MTBDRplus banding pattern:
•only bands with colour intensity ≥ Amplification Control to be considered • all wild type probe bands must be present if strain to be considered drug susceptible• any missing or weakly-stained wild type bands indicates the following drug resistances:
rpoB = Rifampicin resistanceinhA = Low level Isoniazid resistancekatG = High level Isoniazid resistance
MB SUITE USAGE RULES:
• STRICT COMPLIANCE WITHUNI-DIRECTIONAL WORK FLOW (ROOM 1 TO ROOM 3 ONLY)
• CORRECT USE OF PPE AT ALL TIMES (Personal Protection Equipment)• STRICT COMPLIANCE WITH HANDWASHING AND WORKFLOW PROCEDURES
• NO ADMITTANCE TO NON-LAB STAFF (INCLUDING CLEANERS)
• WEEKLY CLEANING/MAINTENANCE OF MB ROOMS TO BE DONE BY LAB STAFF ONLY WITH DEDICATED EQUIPMENT
GenXpert
➢ The GeneXpert Dx System automates and integrates preparation, nucleic acid amplification, and
detection of the target sequence in simple or complex samples using real-time Polymerase Chain
Reaction(PCR).
➢ The system is suited for in vitro diagnostic applications that require hands-off processing of patient
samples (specimens) and provides both summarized and detailed test results data in tabular and
graphic formats.
➢ Detection of Mycobacterium tuberculosis (MTB) and Rifampicin resistant Mycobacterium tuberculosis.
➢ Detects - rpoB gene responsible for the resistance to Rifampicin for the wild type of Mycobacterium
tuberculosis
Introduction.
GenXpert
• Best used in Provincial/DistrictHospitals
• HIV clinics
• Areas of high TB burden with high MDRTB rates
• Health facilities without the infrastructure for culture testing.
• Detects DNA sequences specific for Mycobacterium
Tuberculosis and Rifampicin resistance by PCR
• Based on Nucleic Acid Amplification Test (NAAT). The
Xpert® MTB/RIF; -
• purifies.
• concentrates.
• amplifies (by real-time PCR) and
• identifies targeted nucleic acid sequences in the
Mycobacterium tuberculosis genome.
How the GeneXpert works.
Models of GeneXpert:
GeneXpertmodule
GX-IGX-II GX-IV
GX-XVI
GX-48 & infinity
There are six different GeneXpert Dx instruments:
The GeneXpert I instrument:
Consists of one module (or one site) to process one sample.
GeneXpert II instrument:
Consists of two modules (or two sites) to process two samples.
The GeneXpert IV instrument:
Consists of up to four modules. Each module processes one sample. Up to four GeneXpert IV instruments can be connected to one computer.
The GeneXpert XVI instrument:
Consists of up to sixteen modules. Each module processes one sample.
The GeneXpert 48 instrument:
Consists of up to forty eight modules. Each module processes one sample.
The geneXpert infinty:
Consists of up to eighty modules. Each module processes one sample
GeneXpert Models Cont…..
GeneXpert System includes:
Modules :- Thermal and optical system
Cartridge :- Self-Contained Disposable
Computer System :- Laptop/Desktop ComputerSoftware
Barcode scanner
Other Accessories Printer
GeneXpert components.
Benefits of Molecular Diagnostics
Accurate Results
Fast
Easy to Use!
Xpert® MTB/Rif Assay kit.
GeneXpert module.
Syringe Motor
Mother Board
I-CORE
Syringe Motor
The geneXpert catridge;➢ The samples are prepared and
processed in the single-use, assay specific GeneXpert cartridges.
➢ You insert the sample and applicable reagents into a cartridge, and then load the cartridge into one of the available instrument modules.
Parts of a GeneXpert cartridge
Reaction chambers
Body
Reaction tube
Foot/Base
Cartridge lid
Ultrasonic horn
Syringe drive
Valve body
11 Rxtin chambers
Rxtn tube
➢ Sputum :
- Fresh sputum
- induced sputum
➢ Sputum sediments.
➢ Frozen sputum sample.
NOTE; - Use of frozen sputum sample is not part of the regular procedure on the GeneXpert; this is
not validated and generates high rates of invalids/errors (crystals will block the filters).
➢ Any other type of sample is not validated by Cepheid.
Literature regarding theses samples can be found (FIND and many other customers).
Samples for use on GeneXpert®
➢ Near-patient
➢ Easy to perform
➢ Safe to perform
➢ Rapid
➢ As sensitive as culture
➢ As specific as culture
➢ Detection of Rifampicin resistance
➢ Robust
➢ Room temperature storage
➢ Total internal control of reagent system meaning no separate external positive or negative controls required.
➢ No wet interface between instrument and cartridge to eliminate carry-over
➢ Controls are in built, hence no need for external positive or negative controls
➢ Automated data reduction and results interpretation
➢ Exploits proven SmartCycler® technology (Incorporate same “solid-state” thermal cycler/fluorometer module)
➢ Integrated ultrasonic horn for rapid lysis of spores.
➢ Encoded software driven motors for valve movement and integral hydraulic drives
➢ Advanced micro fluidics technologies to enable complex sample prep processing protocols.
Design Specifications.
Sample Processing: Fresh sputum
1. Add 2:1 Buffer to Sample
2. Shake then incubate for 10 min at Room Temp
3. Shake then incubate for a further 5 min
4. Transfer 2ml or more into the cartridge
5.Begin Test
Once cartridge is ready, test should be launched within 30 minutes!!
Sample Processing: Sediments
Once cartridge is ready, test should be launched within 30 minutes!!
1.Turn on the computer, and then turn on the GeneXpert Dx instrument.
2. On the Windows® desktop, double-click the GeneXpert Dx shortcut icon.
3. Log on to the GeneXpert Dx System software using your user name and
password.
4. In the GeneXpert Dx System window, click Create Test. The Scan Cartridge
Barcode dialog box appears.
5. Scan the barcode on the Xpert MTB/RIF cartridge. The Create Test window
appears. Using the barcode information, the software automatically fills the boxes
for the following fields: Select Assay, Reagent Lot ID, Cartridge SN, and Expiration
Date.
Running the MTB/RIF test
Summarized xpert process.
(VIDEO)
➢ Cepheid designed specific molecular methods to include internal controls that enable the system to detect specific failure modes
➢ Three elements of the molecular design were created to address all the key failure modes that could result in a false negative result
-Probe Check
-Sample Processing Controls
-Internal Control
1) Probe Check; -Probe check verifies proper
-Bead re-hydration
-PCR tube filling
-Probe integrity
-Dye or quencher instability
2) Internal control (IC) – Its components include;
-Template: Unique sequence or molecular mimic of target
-Detection probes
-Primers
What does it do?
-Verifies amplification performance of the reagent system.
-Detects degradation of enzyme(s) or other components of system
-Detects sample inhibition
Xpert® Controls
The results are interpreted by the GeneXpert DX System from
measured fluorescent signals and embedded calculation algorithms
and will be displayed in the “View Results” window. Lower Ct values
represent a higher starting concentration of DNA template; higher
Ct values represent a lower concentration of DNA template.
MTB result Ct range
➢ High <16
➢ Medium 16-22
➢ Low 22-28
➢ Very Low >28
Interpretation of Results
•The detection of MTB is dependent on the number of organisms present in the sample, reliable results are dependent on proper specimen collection, handling, and storage.
•Erroneous test results might occur from improper specimen collection, failure to follow the recommended sample collection procedure, handling or storage, technical error, sample mix-up, or an insufficient concentration of starting material.
•A positive test result does not necessarily indicate the presence of viable organisms. It is however, presumptive for the presence of MTB and Rifampicin resistance.
•Test results might be affected by antecedent or concurrent antibiotic therapy. Therefore, therapeutic success or failure cannot be assessed using this test because DNA might persist following antimicrobial therapy.
Limitations.
➢ Do not collect less than 2mL of sputum specimen.
➢ Do not accept specimens with obvious food particles or other particulates or substances that could inhibit the PCR e.g. blood, tobacco particles etc.
➢ Viable samples are as follows; -
➢ If stored at RT use before 3 days.
➢ If stored between 2–8 °C sample is good for use for 4-10 days
NB!
Specimens should be held at 2–8 °C whenever possible including during transport to the laboratory.
➢ Do not use a cartridge if it appears wet or if the lid seal appears to have been broken.
➢ Do not use a cartridge that has a damaged reaction tube.
➢ Each single-use Xpert MTB/RIF cartridge is used to process one test. Do not reuse spent cartridges.
➢ Dispose of used Xpert MTB/RIF cartridges according to your institution’s and country’s safety guidelines for hazardous material.
Precautions!!
INVALID
Result indicates that the SPC failed. The sample was not properly processed or PCR was inhibited.
ERROR
Result indicates that the Probe Check control failed and the assay was aborted possibly due to the
reaction tube being filled improperly, a reagent probe integrity problem was detected, or because
the maximum pressure limits were exceeded or there was a GeneXpert module failure.
NO RESULT
Indicates that insufficient data were collected. For example, the operator stopped a test that was in
progress.
Reasons to repeat an Xpert® assay
➢ Using a specimen with foreign particles such as food particles, fibers, blood, soot particles e.t.c
➢ Incorporating air bubbles into the specimen during transfer to cartridge.
➢ Using a specimen that is too viscous
➢ Processing a sample that is not of an adequate volume i.e. less than 2ml
➢ Using an open cartridge that has been standing for more than 30 min.
➢ Leaving a processed sample out for too long after adding buffer
➢ Using cartridges that have been knocked over/ not stored in a upright position/ leaking cartridges
➢ Stopping a test after loading the cartridge and starting the Pcr process
Common Causes of Errors
References• American Public Health Laboratories
Microscopy trainer notes
• Tina Parr David Hains Line Probe Assay 2008
• David Omar Chemjor GeneXpert Dx System 2013
• Rawee Apornsilp Photographs LJ media MTB and M. intracellulare; collection, Culture 2014
• Rawee Apornsilp Result templates 2015
• Allison Tseng BD MGIT 2007
Questions
• What to do if applicant cannot cough specimen after three attempts?
• Timeline and temperature collection to laboratory should be how long?
• Smear scanty on Dx specimen, NTM grown on that specimen rest negative. CXR shows high suspicion of MTB What actions to take?
Questions Contd.
• Two out of three specimens contaminated, what action to take?
• Xpert result positive for MTB and Rif resistance, patient from low MDR incident country. What action to take?
• Error on Xpert, but smear positive 1+. What action to take?
Questions contd.
• Result from outsourced lab shows afb growth on culture no other information. What to do?
• DST shows pan susceptible, LPA shows resistance in INH. What actions to take?
• Continued high NTM rates in culture of follow-up specimens, what actions to take?
• High contamination in all specimens from one applicant. What actions to take
Questions contd.
• Clinical strongly suggests MTB, applicant lived with MDR patient. Culture positive with resistance to Rif and INH. Xpert showed no MTB. What to suggest?
• What type of setting is most beneficial for Gen Xpert?
• What type of setting is most beneficial for LPA?
Questions contd.
• In the rain, is it suitable to collect sputum in the toilet at the clinic?
• What PPE is required for sputum collection?
• The result from the laboratory states only LPA result available. Why would this be?
