Training Workshop Islamabad 24th August Warren Jones

Transcription

1 Training Workshop Islamabad 24th August 2016 Warren Jones

2 SPUTUM COLLECTION AND LABORATORY ESSENTIALS

3 TB TB Bacilli SPREAD THROUGH THE AIR Probability TB Will Be Transmitted - Infectiousness of person with TB - Environment in which exposure occurred - Duration of exposure -Virulence of the organism Sources:

4 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)

5 TB PATHOGENESIS Inactive - 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 TB Active - Intestinal bypass or gastrectomy - Chronic malabsorption syndromes - Low body weight (10% or more below the ideal)

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7 Mycobacterium tuberculosis-morphology Slender, straight or slightly curved bacillus, non-motile, non-encapsulated and does not form spores Acid fast bacillus (AFB) Aerobic Slow growing- divides every hr. Resistant to drying and chemical disinfectants Sensitive to heat (Pasteurization) and UV light

8 Thin section transmission electron micrograph of Mycobacterium tuberculosis

9 close-up of a Mycobacterium tuberculosis culture revealing this organism s colonial morphology

10 TB COMMON SITES OF TB DISEASE Drug-Resistant TB - 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 Lymphatic Pleura Central nervous system system Bones and joints Genitourinary systems Disseminated (miliary TB)

11 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 Arrow points to cavity in patient's right upper lobe.

12 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 12

13 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. 13

14 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 14

15 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 15

16 Specimen Collection II Health centre staff instructed on proper specimen collection and transportation procedures Staff properly instruct patients on specimen collection 16

17 Sputum Collection PPE required by technician/nurse collecting N95 Mask Gloves Laboratory coat Infectious waste bin and bags

18 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) 18

19 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 19

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22 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 22

23 23 Form Example

24 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 24

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27 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 27

28 Transportation Regulations Your country s specific rules for transportation of patient specimens must be followed International Air Transport Association (IATA): 28

29 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 29

30 Assessing Quality of Sputum Specimens Saliva Muco-purulent Bloody Mucoid Photo kindly provided by Ms. Akiko Fujiki 30

31 Assessing Quality of Sputum Specimens Saliva Bloody Muco-purulent Mucoid Photo kindly provided by Ms. Akiko Fujiki 31

32 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 32

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34 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.

35 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

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37 AFB Microscopy Staining Techniques

38 REAGENT

39 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.

40 STEP 3,4 : Rinse the slide with water. Flood the slide with 0.5% Acid Alcohol and allow to decolorize for 2 minutes.

41 STEP 6: Flood each slide with Potassium Permanganate (MSDS) and allow to quench for 1 minute.

42 ZIEHL-NEELSEN ACID-FAST STAINS

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44 FLUORESCENCE STAINS

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49 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)

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52 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

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56 M. intracellulare non-tuberculosis mycobacteria NTM

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64 Immunochromatographic Identification

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71 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.

72 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

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75 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.

76 MYCOBACTERIA M.tuberculosis Complex (MTBC) Non-Tuberculous Mycobacteria (NTM) FOUND IN: HUMANS DOMESTIC ANIMALS WILD ANIMALS FOUND IN: THE ENVIRONMENT DUST SOIL WATER (most common contaminate in the

77 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.

78 METHODOLOGY (ALL ASSAYS USE SAME TEST PROTOCOL)

79 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)

80 1. DNA PREPARATION: Crude boil method: 2. Centrifugation 3. heat inactivation

81 Centrifuge and heat block

82 2. PCR: Master Mix preparation Extracted DNA addition Amplification (denaturation, annealing, extension)

83 Thermocycler

84 3. HYBRIDIZATION: Amplicon denaturation Reverse hybridization to strip Strepavidin/AP conjugation Staining to detect banding pattern

85 DEVELOPED STRIPS MTBDRplus CM/AS

86 SCHEMATIC EXAMPLES OF POSSIBLE RESULTS FOR MTBDRplus ASSAY

87 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

88 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 resistance inha = Low level Isoniazid resistance katg = High level Isoniazid resistance

89 MB SUITE USAGE RULES: STRICT COMPLIANCE WITH UNI-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

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91 GenXpert

92 Introduction. 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

93 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.

94 How the GeneXpert works. 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.

95 Models of GeneXpert: GeneXpert module GX-I GX-II GX-IV GX-XVI GX-48 & infinity

96 GeneXpert Models Cont.. 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

97 GeneXpert components. GeneXpert System includes: Modules : - Thermal and optical system Cartridge : - Self-Contained Disposable Computer System : - Laptop/Desktop Computer Software Barcode scanner Other Accessories Printer

98 Benefits of Molecular Diagnostics Accurate Results Fast Easy to Use!

99 Xpert MTB/Rif Assay kit.

100 GeneXpert module. Syringe Motor Mother Board I-CORE Syringe Motor

101 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.

102 Parts of a GeneXpert cartridge Rxtn tube Cartridge lid 11 Rxtin chambers Reaction chambers Body Reaction tube Syringe drive Valve body Foot/Base Ultrasonic horn

103 Samples for use on GeneXpert 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).

104 Design Specifications. Near-patient No wet interface between instrument and cartridge to eliminate carry-over Easy to perform Controls are in built, hence no need for external positive or negative controls Safe to perform Rapid Automated data reduction and results interpretation As sensitive as culture Exploits proven SmartCycler technology (Incorporate same solid-state thermal cycler/fluorometer module) As specific as culture Detection of Rifampicin resistance Integrated ultrasonic horn for rapid lysis of spores. Robust Encoded software driven motors for valve movement and integral hydraulic drives Room temperature storage Total internal control of reagent system meaning no separate external positive or negative controls required. Advanced micro fluidics technologies to enable complex sample prep processing protocols.

105 Sample Processing: Fresh sputum 2. Shake then incubate for 10 min at Room Temp 1. Add 2:1 Buffer to Sample 4. Transfer 2ml or more into the cartridge 3. Shake then incubate for a further 5 min Once cartridge is ready, test should be launched within 30 minutes!! 5.Begin Test

106 Sample Processing: Sediments Once cartridge is ready, test should be launched within 30 minutes!!

107 Running the MTB/RIF test 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

108 Summarized xpert process. (VIDEO)

109 Xpert Controls 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

110 Interpretation of Results 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 Low Very Low >28

111 Limitations. 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.

112 Precautions!! 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.

113 Reasons to repeat an Xpert assay 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.

114 Common Causes of Errors 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

115 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

116 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?

117 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?

118 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

119 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?

120 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?