TB NAAT testing at the Los Angeles County Public Health Laboratory Hector Rivas Public Health Microbiology Supervisor II Los Angeles County Public Health Laboratory hrivas@ph.lacounty.gov April 2012 1
Los Angeles County Population approx. 10 million 4,057 square miles of land area 8 Service Planning Areas (SPAs) 11 public health clinics and 3 county hospitals that care for TB patients 2011- LAC reported 30% of the cases in California (n = 681) 2
Number of TB cases reported to CDPH TB Control Branch 1200 1000 1025 950 930 906 885 Number of cases 800 600 400 815 792 706 674 681 200 0 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 Year 3
TB diagnostics Goal = Detect TB as soon as possible in primary specimen and identify drug resistance Culture is still the gold standard Rapid diagnostic methods have advanced detection and identification of TB complex NAAT has high PPV value (>95%) for AFB smear-positive specimens in settings in which NTM are common NAAT is useful for confirming TB AFB in smear-negative, culture-positive specimens 4
LACPHL Workload 2008 2009 2010 2011 # of individual patients whose cultures were processed for identification of mycobacteria 3768 3363 4207 5914 # of individual patients with MTB complex initially identified 308 221 228 232 # of individual patients for whom NAAT confirmed M. tuberculosis complex 143 145 105 137 # of individual patients with Molecular Beacon testing (in-house & reference specimens/cultures) 91 100 228 305 5
Culture Confirmed Pulmonary TB Cases at LA County (2011) Smear Result No. % Positive 310 67.2 Negative 140 30.4 Missing 11 2.4 Total 461 100.0 Reported to TB Control Program 6
TB NAAT performed at LACPHL Amplified M. tuberculosis Direct Test (MTD) Molecular Beacon Test Target Amplification and Detection TMA/HPA* assay used to detect M. tuberculosis rrna Real-time PCR used for detection of mutations associated with isoniazid and rifampin resistance Acceptable Specimens Respiratory specimens (induced, expectorated, BAL, BA, TA) Smear positive/negative Respiratory specimens Smear positive (2+) Culture isolates *Transcription-mediated amplification/hybridization protection 7
LACPHL NAAT algorithm NALC-NaOH pulmonary specimen* Fluorescent AFB smear First-time Positive Smear Negative Reflex MTD MTD upon request Positive Negative Molecular Beacon Test *Culture also simultaneously performed 8
Incorporation of MTD at LACPHL MTD was FDA-approved in 1995 for smear-positive specimens and revised approval for smear-negative specimens in 1999 In 2003, LACPHL instituted the MTD test Reasons why test chosen: FDA-approved and reagents provided as a kit Easier validation procedure compared to home-brew test High sensitivity and specificity Required basic equipment already in use at the laboratory 9
Principle of TMA-HPA GenProbe 10
MTD vs. PCR TMA PCR Target DNA or RNA DNA Enzymes RNA Polymerase and DNA Polymerase Reverse Transcriptase Thermal Conditions Isothermal Thermal Cycling Amplicon Product RNA DNA 11
Test Results Interpretation Results: 500,000 RLU positive for M. tuberculosis complex rrna < 30,000 RLU negative for M. tuberculosis complex rrna 30,000 to 499,999 RLU probable M. tuberculosis complex rrna positive; repeat to verify results: Repeat 30,000 RLU positive for M. tuberculosis complex rrna Repeat < 30,000 RLU negative for M. tuberculosis complex rrna 12
Test Results Reporting Qualitative Results: M. tuberculosis complex rrna detected M. tuberculosis complex rrna not detected Result Inconclusive due to presence of specimen inhibitors. Testing of an additional specimen is recommended. Culture is always recommended 13
FDA package insert performance using patient diagnosis as endpoint Smear Positive Patients LACPHL performance using culture as endpoint Smear Positive Patients MTD Test Internal Performance Analysis 2003 # of patients Sensitivity Specificity PPV NPV 39 96% 100% 100% 87.5% 35 95.6% 100% 100% 96.2% 14
2000 2003 2005 2009 2012 15
Physician guidelines Patients must meet the following criteria: High to moderate clinical suspicion of disease There is NO documentation of 1 or more of the following items: NAAT (+) Culture (+) AFB Smear 2x (+) and NAAT 2x (-) before 7 days of anti-tb treatment at time of testing The patient has NOT received 7 days of anti-tb treatment for current episode The patient has NOT received anti-tb treatment within the past 12 months For low clinical suspicion, only smear positive samples will be tested 16
FA Smear Result MTD Result TB NAAT Clinician Guidelines Inhibit. Test Inhibit. Detected Interpretation Positive Positive -Presume Patient has TB while awaiting culture Positive Negative Yes No Yes -Use clinical judgment to begin anti-tb treatment or -Presume MOTT if 2 or more specimens are NAAT negative -NAAT test of no value -Use clinical judgment pend. culture Negative Positive -Use clinical judgment to begin anti-tb treatment or -Presume TB if 2 or more specimens are NAAT positive Negative Negative -Use clinical judgment to begin antitb treatment pend. culture Based on CDC guidelines (2009) 17
Advantages of MTD FDA approved to test directly test smear positive and negative pulmonary specimens High sensitivity/specificity Commercially available Uses common lab equipment such as heat block, sonicator, water bath, luminometer Easier validation compared to home-brew Results same day External technical support 18
Disadvantages of MTD Detects viable and non-viable organisms False negative results may occur due to low number of TB or TB in the presence other mycobacteria M. celatum and M. terrae-like organisms will cross-react if present at high concentrations Test efficacy has not been determined for other sources extrapulmonary Labor intensive and low-throughput Need to test within 7 days of treatment No internal control Not approved for culture isolates Contamination risk 19
Future NAAT Testing: IS6110/rpoB RT-PCR and PSQ Multiplex real-time PCR: IS6110 and rpob Two- step detection tool for TB High sensitivity and specificity IS6110 is a 1.3 kb insertion sequence used in molecular epidemiology as genetic marker to track spread of TB Conserved target with 1-20+ copies Present in 99.9 % TB complex strains PSQ used to detect mutations in genes associated with resistance New targets can be added on as methods are developed Halse et al. 2010. JCM.48(4):1182 8. 20
Acknowledgements Los Angeles County Department of Public Health Mycobacteriology/Mycology Unit J. Michael Janda, PhD, D(ABMM) Nicole M. Green, PhD, D(ABMM) Jonas Petterson, PhD TB Control Program Wadsworth Center, NY State DOH Kimberlee Musser, PhD Tanya Halse CDPHL MDL Ed Desmond, PhD, D (ABMM) Grace Lin, MS 21
IS6110 IS A SENSITIVE, MULTICOPY PCR TARGET Tuberculosis and the Tubercule Bacillus, 2005 ASM Press 22