Lab Talk: What a Lab Talk: What a Max Salfinger, MD, FIDSA, FAAM Executive Director, Advanced Diagnostic Laboratories Laboratory Director, Mycobacteriology & Pharmacokinetics National Jewish Health Lisa True RN, MS MDR Nurse Coordinator/Program Liaison TB Control Branch Center for Infectious Diseases California Department of Public Health Lana Kay Tyer RN, MS TB Nurse Consultant Washington Department of Health Laboratory slides curtesy of Julie Tans Kersten, TB Controller, Wisconsin and APHL Essentials for the Mycobacteriology Laboratory: Promoting Quality Practices 2 WELCOME 3 www.tbcontrollers.org 1
This is a talk about TB laboratory diagnostic tests!! 4 If you find lab reports confusing, raise your hand 5 If you already understand TB lab tests, you can leave 6 www.tbcontrollers.org 2
Just kidding, please stick around to help the rest of us! 7 We hope this will be fun and informative. And cover the objectives so you get your CEUs! 8 Objectives Understand laboratory tests available for the diagnosis of tuberculosis (TB) and antimicrobial susceptibility (AST) studies to assure the best patient outcomes. Have fun! Know who to ask when lab information doesn t make sense! www.tbcontrollers.org 3
Day in the Life of TB Nurse Lab Report Patient 1-Positive Smear 11 Clinical Information Elderly woman US born History of COPD Admitted for shortness of breath www.tbcontrollers.org 4
What a Nurse Thinks 13 Day in the Life of Microbiologist SMEAR MICROSCOPY 15 www.tbcontrollers.org 5
Smear Microscopy Procedure Smear: A small amount of primary patient specimen (direct or processed) is placed on a slide for the purpose of microscopic examination. The smear is stained by a special procedure to make AFB in the specimen more visible. Acid Fast Bacilli (AFB): resist decolorization with acid alcohol due to the lipid rich mycolic acids in the cell wall thereby retaining the primary stain Microscopic examination of the smears detects acid fast organisms such as Mycobacterium tuberculosis and nontuberculous mycobacteria (NTM) 16 AFB Smear Microscopy Auramine-O (fluorescent) Kinyoun or Ziehl-Neelsen (carbol fuchsin) 17 Reporting smear results Report an approximation of the number of AFB viewed on the slide using a semiquantitative scale Report smear results within 24 hours of specimen receipt Smear positive results are considered critical values and should be reported to the health care provider and public health department as soon as results are known. 18 www.tbcontrollers.org 6
AFB Smear Microscopy Advantages and Limitations Advantage: Smear microscopy is a rapid, convenient and inexpensive test Limitations: Must be accompanied by additional testing including culture for confirmatory diagnosis Limited sensitivity High bacterial load 5,000 10,000 AFB /ml is required for detection Misses >45% of U.S. TB cases Microscopists should prepare and read a minimum of 15 smears per week to maintain technical proficiency 19 More Limitations of AFB Smear Microscopy Does not distinguish between viable and dead organisms Follow up specimens from patients on treatment may be smear positive yet culture negative Limited specificity All mycobacteria are acid fast (also Nocardia and others) Does not provide species identification Local prevalence of MTB and NTM determine the predictive values of a positive smear for MTB 20 20 What a nurse hears www.tbcontrollers.org 7
SPECIMEN COLLECTION 22 Specimen Quality is Important The results of tests, as they affect patient diagnosis and treatment, are directly related to the quality of the specimen collected and delivered to the laboratory. For initial diagnosis of TB: Collect a series of three sputum specimens, 8 24 hours apart, at least one of which is an early morning specimen* APHL: Essentials of the Mycobacteriology Laboratory: Promoting Quality Practices *Centers for Disease Control and Prevention. Guidelines for Preventing the Transmission of Mycobacterium tuberculosis in Health Care Settings, MMWR 2005:54, RR 17 23 Sputum Quality Specimens are thick and contain mucopurulent material from a deep cough. Ideally, 3 5 ml in volume, although smaller quantities are acceptable if the quality is satisfactory.* Poor quality specimens are thin and watery (unless induced). Saliva and nasal secretions are unacceptable. Check with your lab for specimen requirements. APHL: Essentials of the Mycobacteriology Laboratory: Promoting Quality Practices *Clinical and Laboratory Standards Institute. Laboratory detection and identification of mycobacteria; approved guideline. CLSI Document M48 A. Wayne, PA: CLSI; 2008. 24 www.tbcontrollers.org 8
NUCLEIC ACID AMPLIFICATION TESTING (NAAT) 25 Nucleic Acid Amplification Testing (NAAT) Use of molecular amplification techniques to detect MTBC DNA directly from the patient specimen Not a replacement for culture MTBC detected Sputum DNA extraction Amplification MTBC not detected 26 Nucleic Acid Amplification Testing (NAAT) Cepheid Xpert MTB/RIF (Sunnyvale, CA) Hologic Amplified MTD (San Diego, CA) Line Probe Assay Fujirebio Innogenetics INNO LiPA (Belgium/Sweden) HAIN Lifescience GenoType MTBC (Germany) Laboratory developed tests 27 www.tbcontrollers.org 9
Recommendation for Use of NAAT 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. Updated Guidelines for the Use of Nucleic Acid Amplification Tests in the Diagnosis of Tuberculosis MMWR 2009; 58 (01); 7-10 28 NAAT Limitations and Considerations Does not distinguish between live and dead organisms Not routinely used to differentiate between members of the TB complex Limited sensitivity for smear negative specimens Not indicated for use to determine bacteriologic cure or to monitor response to therapy 29 NAAT Limitations and Considerations Smear needed for interpretation of test results Culture is still the gold standard for TB diagnosis Culture is necessary for phenotypic antimicrobial susceptibility testing and genotyping 30 www.tbcontrollers.org 10
What a nurse hears Results from NAAT tests Two additional sputa collected Both were smear positive again GenXpert tests both negative for TB Tested for inhibitors, not present Patient released from airborne isolation No TB treatment, NTM identified on culture Lab Report Patient 2: Positive AFB Growth on Culture www.tbcontrollers.org 11
Clinical Information 63 year old diabetic man from the Philippines Fall at home, CXR showed infiltrates Sputa x3, all were smear negative 4 weeks later there is a report of +AFB growth on culture What a nurse thinks? MYCOBACTERIAL CULTURE 36 www.tbcontrollers.org 12
Purpose of Mycobacterial Culture Detection of Mycobacterium tuberculosis complex (MTBC) The most clinically significant mycobacterial species for public health Isolation almost always signifies disease, except in the case of laboratory cross contamination MTBC organisms are not present in the environment Detection of Nontuberculous Mycobacteria (NTM) Are opportunistic pathogens in humans and may cause significant human disease Clinicians ultimately responsible for determining the importance of a NTM Almost all of these species can be found in environmental samples 37 Culture for Mycobacteria Detects viable mycobacteria from patient specimens Most sensitive and specific method for detecting mycobacteria ( Gold standard ) Slowest Method Average time to identification of TB from smear positive specimens= 12.8 days* Average time to identification of TB from smear negative specimens = 20.2 days* Range for detection of TB: 6 81 days* Laboratory will hold cultures 6 8 weeks before reporting as negative *Wisconsin State Laboratory of Hygiene, unpublished data 38 Mycobacterial Culture Use of culture increases the number of TB cases found by 30 50% over smear alone ~10 viable bacilli/ml of sputum needed for culture compared to at least 5,000 bacilli/ml of sputum for microscopy Culture used for species identification, antimicrobial susceptibility testing (AST), and genotyping Culture also used to monitor patient response to treatment 39 www.tbcontrollers.org 13
Culture for Mycobacteria at WSLH Type of Media Incubation Broth In automated instrument Detects bacterial growth Incubated for 42 days before reported as negative BD BACTEC TM MGIT TM Solid Plate or tube Visually inspected for colony growth 40 IDENTIFICATION OF MYCOBACTERIAL SPECIES 41 Identification of Mycobacterial Species Once a culture is growing, assays must be performed to identify the organisms Most laboratories use a multifaceted approach that includes: Phenotypic characteristics: The observable physical or biochemical characteristics of an organism Genotypic characteristics: the genetic makeup of an organism http://www.healthypeople.gov/2020/data-search/search-the-data?nid=4702 42 www.tbcontrollers.org 14
Analysis Mycobacteria Culture Identification Methods Method DNA Proteins Cell wall mycolic acids Enzymes AccuProbes, Line probe assays, PCR, DNA sequencing Matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF MS) High performance liquid chromatography (HPLC) Biochemical Reactions 43 Clinical Significance of MTBC and NTM Identification of MTBC is the most important finding in the laboratory and has serious clinical and public health consequences While some NTM can cause disease, not all NTM isolation is clinically significant Accurate and timely identification of mycobacteria is crucial Use a multi-faceted approach that includes a rapid identification and phenotypic assessment before issuing a final report 44 Culture Identification Results Isolated: Significance M. tuberculosis, M. bovis, M. bovis BCG, M. caprae, M. microti, M. africanum, M. canettii, M. pinnipedii, and M. mungi M. avium complex, M. gordonae, M. kansasii, M. simiae, M. terrae complex, M. xenopi M. abscessus & its subspecies, M. chelonae, M. fortuitum, M. mucogenicum M. tuberculosis complex (MTBC) Nontuberculous mycobacteria (NTM): slow growers Nontuberculous mycobacteria (NTM): rapid growers 45 www.tbcontrollers.org 15
Culture Results 46 What Nurse Thinks Lab Report Patient 3: Positive Xpert MTB/Rifampin www.tbcontrollers.org 16
Clinical Information 59 yo healthy male from Ukraine No history of previous TB disease or treatment Mild cough and fever CXR cavitation in left upper lobe Sputum smear positive x3 What a Nurse Thinks MOLECULAR DETECTION OF DRUG RESISTANCE 51 www.tbcontrollers.org 17
Molecular Detection of Drug Resistance For rapid prediction of multi drug resistant and extensively drug resistant TB Molecular detection of mutations that confer resistance to TB drugs Molecular results should be correlated with culture based results, if possible 52 Interpreting GenXpert Results Not all rpob mutations result in rifampin resistance Send isolate for additional testing Sequencing can characterize the mutation and determine whether it is a clinically significant mutation (rule out silent mutations) If the rpob mutation confers resistance, likely MDR TB Drug Rifampin INH Ethambutol PZA Kanamycin Amikacin Capreomycin Fluoroquinolone Molecular Detection of Drug Resistance Genes Sequenced rpob inha, katg embb pnca rrs, eis rrs rrs, tlya gyra www.tbcontrollers.org 18
CDC MDDR Results 55 CDC MDDR Results Locus Result Interpretation rpob (RRDR) No mutation Probably rifampin susceptible. (97% of RMP R isolates in our inhouse evaluation of 550 clinical isolates have a mutation at this locus.) 56 CDC MDDR Results Locus Result Interpretation katg (Ser315 codon) Mutation: AGC>ACC Ser315Thr Isoniazid Resistant (100% of isolates in our in house evaluation of 550 clinical isolates with this mutation are INH R). 57 www.tbcontrollers.org 19
CULTURE BASED DRUG SUSCEPTIBILITY TESTING 58 Culture-based Drug Susceptibility Testing (DST) Predicts in vivo success or failure of chemotherapy agents Guides choice of drugs provides the best chance of cure Offers insight into appropriate treatment for contacts of patients with active TB Molecular results should be correlated with culturebased results, if possible 59 Susceptibility Testing of M. tuberculosis complex isolates Drugs Tested Turn Around Time (after pure isolate obtained) TB First Line Drugs (BD MGIT ) isoniazid (INH) 0.2 ug/ml isoniazid (INH) 1.0 ug/ml rifampin 1.0 ug/ml ethambutol 5.0 ug/ml pyrazinamide 100 ug/ml 7 20 days 3 weeks TB Second Line Drugs capreomycin (10ug/ml) ethionamide (5 ug/ml) kanamycin (6 ug/ml) ofloxacin 2 ug/ml) PAS (2 ug/ml) rifabutin (0.5 ug/ml) streptomycin 2 ug/ml) streptomycin (10 ug/ml) 60 www.tbcontrollers.org 20
Challenges with Culture-based AST Drug Limitations Reference Rifampin false susceptibility may be of concern in commercial broth systems Williamson IJTLD 2012 16:216 Van Deun et al. JCM 2013; 51(8): 2633 2640 Ethambutol false susceptibility may be of concern in commercial broth systems PZA false resistance may be of concern in commercial broth systems Yakus et al., Tuberc Res Treat. 2016; Epub 2016 Jun 8 Chedore, et al. 2010. JCM 48:300 61 Recommended Turnaround Time for TB First-line AST Initial isolates of MTBC should be tested against a panel of first line drugs using a rapid commercial broth system CDC recommends that AST results should ideally be available to the submitter within 17 days of identification of MTBC 62 Culture-based AST Report 63 www.tbcontrollers.org 21
CDC Culture-based AST Report 64 What a nurse hears Resources Centers for Disease Control and Prevention. Guidelines for Preventing the Transmission of Mycobacterium tuberculosis in Health Care Settings, MMWR 2005:54, RR 17 Clinical and Laboratory Standards Institute. Laboratory detection and identification of mycobacteria; approved guideline. CLSI Document M48 A. Wayne, PA: CLSI; 2008. Essentials for the Mycobacteriology Laboratory: Promoting Quality Practices: http://forms.dev.aphl.org/aphlprograms/infectious/tuberculosis/tb Core Curriculum/Pages/default.aspx Drug Resistant Tuberculosis A Survival Guide for Clinicians, 3 rd edition, 2016. Curry International Tuberculosis Center 66 www.tbcontrollers.org 22
Questions? 67 www.tbcontrollers.org 23