Stacy White, PhD May 12, TB for Community Providers. Phoenix, Arizona
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1 Role of the Laboratory in TB Diagnosis Stacy White, PhD May 12, 2015 TB for Community Providers May 12, 2015 Phoenix, Arizona EXCELLENCE EXPERTISE INNOVATION Stacy White, PhD has the following disclosures to make: No conflict of interests No relevant financial relationships with any commercial companies pertaining to this educational activity 1
2 Tuberculosis Testing and Laboratory Updates Arizona State Public Health Laboratory Stacy White, PhD Arizona Department of Health Services Bureau of State Laboratory Services Mycobacteriology/Molecular Methods and Research Overview Description of laboratory services at Arizona State Laboratory (ASL) Specimen collection and transport Testing workflow Tests available 2
3 Arizona State Laboratory Diagnostic and Reference laboratory Hours of Operation: Monday Friday, 8:00am -5:00pm AZDHS Website ndex.htm Guide to Laboratory Services Laboratory Submission form Arizona State Laboratory TB Services Process Diagnostic Specimens AFB Microscopy AFB Culture and Identification TB Drug Susceptibility testing Broth method (rapid) Agar Proportion method 3
4 Specimen - Sources Respiratory Non-respiratory - Accuracy of laboratory testing dependent on quality of specimen - Factors: - Collection - Storage - Transportation - Labeling Specimen Collection Specimen collection kits and mailing containers are provided by ASL Contact by mail, phone, or fax Arizona Department of Health Services Bureau of State Laboratory Services ATTN: Receiving Section 250 N. 17 th Ave Phoenix, AZ Fax: (602) Phone: (602)
5 Example of Collection Kit Supplied by ASL Inner metal screw capped container, outer screw capped cardboard container, place submission form around the outside of the inner metal container. Label the specimen collection container with patient name and collection date. Specimen Collection Collect aseptically, or bypass contamination as much as possible Avoid contamination with tap water (NTM may be present) Collect prior to therapy if possible No swabs, fixatives, preservatives 5
6 Specimen Collection Specimen collection containers requirements Sterile Leak-proof Disposable Non-breakable Appropriately labeled!!! Laboratory Submission Form Laboratory Submission Form 6
7 Laboratory Submission Form Complete all highlighted (required) fields AFB Specimen Transport Transport in as short as a time as possible to avoid overgrowth of contaminating bacteria Specimens that cannot be transported to the lab immediately should be refrigerated Reference the Guide to Laboratory Services CDC recommendation: Specimen received at laboratory within 24 hours of specimen collection. 7
8 Sputum Most common specimen for isolation Thick and mucopurulent- deep within lungs Desired minimal volume = 3mL (5-10mL preferred) Initial diagnosis: Three specimens, 8-24 hours apart with at least one from early morning Optimally collected before treatment starts Do not pool specimens Typical Specimen Workflow at ASL Specimen Arrives at State Lab 24hrs AFB Smear AFB Culture Positive Negative Positive Negative NAAT Identification 3-8 weeks Drug Susceptibility (MTB) 2-3 weeks 8
9 Specimen Processing sterile site = no digestion or decontamination (CSF, synovial fluid, etc.) Non-sterile site (sputum) Digestion and decontamination (and/or concentration) to prevent overgrowth of normal flora Many different ways: NALC-NaOH (most common) Oxalic Acid Cetylpyridinium chloride AFB Smear Microscopy Quick and inexpensive 24 hour TAT Low sensitivity Results dependent of specimen quality Limited specimen- culture priority Not specific for MTBC Does not distinguish between live and dead bacilli culture negative/smear positive 400X 9
10 Reporting Smear Results Fluorescent Microscopy- CDC Scale 250X (30 fields) 450X (70 fields) Report as 0 AFB/smear 0 AFB/Smear Negative for AFB 1-2/30 fields 1-2/70 fields Report exact count order repeat specimen 1-9/10 fields 2-18/50 fields /field 4-36/10 fields /field 4-36/field 3+ >90/field >36/field 4+ What Can AFB Smear Results Tell Us? Positive smear indicative of infectiousness provides strong inferential diagnosis of tuberculosis initiation of therapy impact decision for respiratory isolation* can delegate whether laboratory performs NAAT Smear grade bacterial load correlates with disease presentation monitoring of therapy (changes in smear status) prioritization of contact investigations 10
11 ASL NAAT FDA cleared Cepheid GeneXpert MTB/RIF Molecular beacons Detects MTBC as well as possible mutations associated with rifampin resistance often associated with isoniazid resistance (together = MDR) processed sputum samples do not test on pediatric patients actual testing time is less than 2 hours Per FDA: Patients can be removed from respiratory isolation following one or two negative Xpert MTB/RIF results Nucleic Acid Amplification Tests Pros rapid direct detection (growth not required) very sensitive very specific AFB smear rapid but insensitive/nonspecific Cons expensive cross contamination live/dead cells? 11
12 ASL NAAT TAT: hours from specimen receipt Performed on smear positive samples (EPI approval otherwise) Sensitivity decreases for smear negative specimens Smear Positive >99% vs 76% (culture positive) Does not replace the need for culture confirmation - phenotypic confirmation -DST - genotyping ASL Reporting of NAAT Results Result MTB Not Detected MTB Detected/rpoB gene mutation NOT Detected MTB Detected/rpoB gene mutation DETECTED* Action Continue with culture for isolation; notify TB Control Continue with culture for isolation; notify submitter and TB Control; send to CDC if requested Continue with culture for isolation; notify submitter and TB Control; send to CDC for MDDR * Not all mutations confer resistance 12
13 Updated Guidelines for the Use of NAAT in the Diagnosis of TB NAAT should be performed on at least one respiratory specimen from each patient with signs and symptoms of pulmonary TB for whom a diagnosis of TB is being considered but has not yet been established, and for whom the test result would alter case management or TB control activities. MMWR, 2009, 58:7-10 State TB control program and ASL collaborated to determine criteria for testing. How to get approval for NAAT? Contact State TB control program Approval will be based on whether the patient meets the following criteria Have a cavitary lesion seen on CXR Are in a long-term care facility Are HIV positive Are immunocompromised Are on dialysis If it will make a difference in the treatment/isolation of the patient foreign born 13
14 AFB Culture Gold standard Current recommendations: use at least two types of media to maximize the recovery of mycobacterium Liquid Solid Cultures monitored 6-8 weeks AFB Culture Liquid (broth media) Automated system (3 FDA cleared) Rapid use is recommended standard practice for mycobacteriology laboratories Solid Egg based (LJ) Agar based (7H10 and 7H11) 14
15 AFB Culture Liquid Culture Rapid Can become positive for MTB growth in days Increase recovery some fastidious mycobacteria Cannot tell if pure culture Cannot give colony morphology Solid Culture Can determine purity Mixed infections? Contamination? Colony Morphology Enumerate growth Much slower relative to liquid culture 14 vs. 25 days (average)for MTBC Identification MTBC is the most clinically significant mycobacteriaonce identified, if new case drug susceptibility patterns must be determined Some NTMs can be clinically significant; others contaminants M. gordonae M. mucogenicum M. terrae complex 15
16 Identification Conventional Colony morphology Pigmentation Consistency and texture Growth rate Rapid Growers- 3-7 days Slow Growers- MTBC 14 days (liquid) to 24 days (solid Rapid Methods HPLC MALDI-TOF DNA sequencing (16SrDNA) ASL Identification HPLC High Performance Liquid Chromatography mycolic acid analysis peak patterns inexpensive rapid (under 3 hours) 16
17 ASL Identification HPLC Advantages Rapid Liquid and solid culture Contaminated sample FDA cleared library Disadvantages Extensive technical expertise required Experience with analysis Support declining Can identify approx. 30 species (with experience) ASL Identification MALDI-TOF Matrix assisted laser desorption ionization time of flight mass spectrometry analysis of proteins (mostly ribosomal proteins) 17
18 ASL Identification MALDI-TOF Advantages inexpensive Fast Full plate (48 samples) can be read within 45 minutes Resolution: library <200 species Easy to use Disadvantages initial cost high requires fresh growth on solid media Liquid culture??? Not FDA cleared ASL Identification 16SrDNA Seq TAT 2 days Technical expertise required Labor intensive Expensive Isolated colonies Reserved for less common Mycobacterial spp isolates or unusual non mycobacteria spp. 18
19 TB Drug Susceptibility Testing Broth Systems Rapid results Testing done from culture FDA approved for first line TB drugs Isoniazid, Rifampin, Ethambutol, PZA Recommended on all initial cases of MTBC Should be repeated if there is clinical evidence of failure to respond to treatment or if cultures fail to convert to negative after 3 months of treatment. Proportion Agar Drug Susceptibility Testing 19
20 CDC Molecular Detection of Drug Resistance Service CDC Molecular Detection of Drug Resistance (MDDR) Service PCR-based DNA sequencing for drug resistance Criteria for accepting specimens High risk patients (RIF-R, MDR-TB) NAAT + High profile patients Known RIF-R Mixed or non-viable cultures CDC Molecular Detection of Drug Resistance Service Drugs and Genes offered with MDDR service (CDC) Drug Gene RIF rpob INH inha, katg FQ gyra KAN rrs, eis AMK rrs CAP rrs, tlya EMB embb PZA pnca 20
21 CDC Molecular Detection of Drug Resistance Service Does not replace conventional, phenotypic drug testing Not all mutations are not mapped out (ie cannot rule out resistance) Can be used only as a guide for treatment 48 hr TAT Example MDDR Report 21
22 How to request MDDR services? Contact State TB control program to make a request ASL will contact the CDC for approval to send the specimen ASL will arrange to ship the specimen to the CDC Preliminary report issued with molecular results Final report issued upon completion of agar proportion drug susceptibility testing National TB Genotyping Program Began in January, 2004 Goal: Genotype at least one isolate for every culturepositive case of TB Arizona State Lab actively participates in program by routinely submitting isolates to the assigned genotyping laboratory 22
23 National TB Genotyping Program Results are uploaded to a national database Results are used to Confirm suspected links Detect unsuspected transmission Detect false positive cultures What Is On the Horizon for ASPHL? Proposal for validation study for MDDR service Sensititre to replace agar proportion Replacement of HPLC- various method are being evaluated to for direct identification from broth probes real time PCR/melt curve analysis 23
24 Questions??? 24
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