Frances Morgan, PhD October 21, Comprehensive Care of Patients with Tuberculosis and Their Contacts October 19 22, 2015 Wichita, KS
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1 The Laboratory s Role in Caring for Patients Diagnosed with TB Frances Morgan, PhD October 21, 2015 Comprehensive Care of Patients with Tuberculosis and Their Contacts October 19 22, 2015 Wichita, KS EXCELLENCE EXPERTISE INNOVATION Frances Morgan, 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 Testing and Monitoring for Tuberculosis: State Laboratory Roles and Perspectives October 19-22, Frances Morgan, PhD Kansas Department of Health and Environment Laboratories Deputy Director Health Section Chief fmorgan@kdheks.gov
3 Kansas Department of Health and Environment Laboratories Care of patients with tuberculosis (TB) starts with a quality assured diagnosis Kansas Department of Health and Environment Laboratories KHEL Mycobacteriology section identifies Mycobacterium species Emphasis on detection of Mycobacterium tuberculosis Patient specimens are processed and analyzed by fluorescent microscopy and liquid culture 3
4 SPECIMEN SLIDE NAAT CULTURE HPLC DST FINAL ID FINAL CULT 24 HRS 48 HRS 6 weeks 8-14 DA DA 3 WKS 6 WKS AFB FOUND NOT FOUND Hx TB MYCOBACTERIUM FOUND FINAL ID SPECIES CONFIRMATION TB complex RNA DETECTED NOT DETECTED 7H11 Plate Culture Selective non-mixed 7-10 DA M TB Complex NOT M TB Complex SUSCEPTIBILITY PRIMARY ANTIBIOTICS MYCOBACTERIUM NOT FOUND REFERRED CULTURE HRS 7-10 DA 2-3 WKS 7 Mycobacterium Mycobacteria - rod-shaped bacteria (bacilli) long, slender, straight or curved, acid fast bacilli Acid-fast bacilli (AFB) Retain stain color after an acid wash AFB tests detect the bacteria in a sample Several types of AFB that may be detected Most common and medically important ones are members of the genus Mycobacterium Mycobacterium tuberculosis is one of the most prevalent and infectious species of mycobacteria STRUCTURE COMPOSED OF HIGH MOLECULAR WEIGHT ACIDIC WAXES, MYCOLIC ACID, CORD FACTOR 4
5 Types of Specimens Sputum Pulmonary Aspirates Gastric Aspirates Body Fluids Tissues Blood Feces Other Specimen Quality Sputum specimens (3 obtained 8-24 hours apart) for AFB microscopy and mycobacterial cultures Morning sputum, more positives Adequate specimen volume (>5 ml) Sputum not saliva (epithelial cells?) Condition of specimen (ph, etc.) Appropriate specimen container Complete and accurate information Proper packaging and Prompt delivery 5
6 Collect Three Specimens Three samples are collected to increase the probability that organisms will be detected Direct Microscopy Identification M. tuberculosis is a acid fast bacterium, rod-shaped bacterium measuring 2-4 x μm They appear as bright red rods against a contrasting background The Ziehl-Neelsen stain is used to demonstrate the presence of the bacilli in a smear The technique is simple, inexpensive and detects those cases of tuberculosis who are infectious M. tuberculosis appearing as bright red bacilli (rods) in a sputum smear stained with the Ziehl-Neelsen stain 6
7 AFB Smears AFB smears are quick way to determine if an infection may be due to one of the mycobacteria, such as M. tuberculosis Requires an adequate specimen (100,000 AFB/mL) Specimen is spread thinly onto a glass slide, treated with a special stain, acid washed and examined under a microscope for "acid-fast" bacteria AFB smears can provide presumptive results within a few hours and are valuable in helping to make decisions about treatment while culture results are pending AFB Smear test is less sensitive than culture to diagnosis a mycobacterial infection AFB Smear - Negative Negative smear does not rule out AFB A negative AFB smear may mean that: 1. No infection is present 2. Symptoms are caused by something other than mycobacteria 3. Mycobacteria were not present in sufficient numbers to be seen under the microscope 7
8 AFB Smear Negative (cont.) If AFB smears are negative and there is still a strong suspicion of a mycobacterial infection, then additional samples may be collected and tested on different days A smear negative sample may still grow mycobacteria since the culture media allows low numbers of bacteria that cannot be seen in a microscopic examination to multiply and be detected AFB Smear - Positive Positive AFB smears indicate a probable mycobacterial infection Definitive diagnosis requires results from a culture Positive smear AFB present (viability?) Positive smear Mycobacterium spp., etc. 8
9 AFB Smear Positive (cont.) Smear-positive patients are 4-20 times more infectious Untreated, a smear-positive patient may infect persons/year Smear-positive patients are much more likely to die if untreated Quantification 40X Observed AFB Report 4 40 per 100 fields* per 10 fields* per field* 3+ > 40 per field* 4+ 9
10 Nucleic Acid Amplification Testing (NAAT) For people with signs and symptoms of an active TB infection, AFB smear results are considered together with results from NAAT for TB Definitive diagnosis requires results from a culture, results from the smear and NAAT may be helpful in deciding how to proceed with patient care For example, if there is a presumptive diagnosis of TB based on rapid test results, most physicians would treat Simultaneously detects TB and rifampicin resistance directly from sputum in < 2 hours NAAT test does not replace AFB cultures Nucleic Acid Amplification Testing (NAAT) NAAT testing has become a routine procedure in many institutions for the diagnosis of TB Because NAAT tests can rapidly and reliably detect M. tuberculosis directly in a specimen one or more weeks earlier than culture Earlier laboratory confirmation of TB can lead to earlier treatment initiation, better patient care and outcomes, greater opportunities to interrupt transmission, and improved public health interventions 10
11 AFB Smear NAAT Interpretation Positive Positive Presumptive diagnosis for TB Negative Positive Negative Positive Negative Negative NAAT is more sensitive than smear so this may occur in people with true disease; may test additional samples using NAAT; if more than one sample is positive by NAAT, this is a presumptive diagnosis for TB Questionable results for TB; an inhibitor maybepresentinthespecimenortheafb seen on the smear are not M. tuberculosis; a test for the inhibitor may be performed Symptoms probably not due to active mycobacterial infection 21 AFB Cultures AFB cultures used to diagnose 1. Active M. tuberculosis infections 2. Identify if infections are due to nontuberculous mycobacteria (NTM) 3. Determine whether TB-like symptoms are due to another cause 4. Used to help determine if TB is confined to the lungs (pulmonary disease) or has spread to organs outside the lungs (extrapulmonary disease) 5. Used to monitor the effectiveness of treatment and can help determine when a person is no longer infectious AFB culture is more sensitive than an AFB smear, but it takes longer for results to become available Centers for Disease Control and Prevention - all samples submitted for AFB testing should be cultured to ensure that any mycobacteria that are present are available for further testing 11
12 AFB Culture Negative A negative culture means 1. No active AFB infection 2. Mycobacteria not present in that sample 3. Mycobacteria present in low numbers Reason multiple samples are often collected Cultures are held for six to eight weeks before being reported as negative. The person tested may have a latent infection that caused a TB screening test to be positive but does not have active TB AFB Culture Positive Positive AFB cultures identify the particular mycobacterium causing symptoms Positive AFB smear or culture several weeks after drug treatment has started may mean that the treatment regimen is not effective and needs to be changed It also means that the person is still likely to be infectious and can pass the mycobacteria to others through coughing or sneezing Negative culture several weeks after treatment indicates that the TB infection is responding to drug treatment and that the person is no longer infectious 12
13 AFB Culture Positive If patient has a TB infection in another part of the body, a different type of sample may need to be collected and tested to identify the infection Mycobacteria grow more slowly than other types of bacteria so positive identification may take days to several weeks, while negative results (no mycobacterial growth) can take up to 6 to 8 weeks to confirm M. Tuberculosis Colonies Slow grower Rough Non-pigmented 35 C - 37 C Morphology is the final answer 13
14 HPLC Species Identification Mycobacterium positive on AFB culture Selective culture on 7H11 plate Analyzed by high performance liquid chromatography (HPLC) to identify Mycobacterium species or groups For all patients, the initial M. tuberculosis isolate should be tested for drug resistance (DST) Mycobacterium Growth Indicator Tubes (MGIT) Liquid culture (MGIT) on all specimens A fluorescent compound is embedded in silicone on the bottom of 16 x 100 mm round-bottom tubes Grown for six weeks or until positive 14
15 Streaked on 7H11 media plate Kinyoun stained Positive stains are identified Negative stains are monitored Positive MGIT HPLC - Identification Mycobacteria species contain mycolic acids in there cell walls Mycolic acids produce a profile on HPLC that varies by species Mycolic acids are extracted from unknown Mycobacteria cultures and are quantified and identified through the HPLC process All profiles produced from the HPLC are compared against an FDA approved mycolic acid profile library, then named according to similarity to that library. 15
16 HPLC Chromatograph Drug Susceptibility Testing Set up from positive MGIT Growth tubes - done as a panel Streptomycin 1.00ug/ml Isoniazid 0.10ug/ml Rifampin 1.00ug/ml Ethambutol 5.00ug/ml Pyrazinamide 100.0ug/ml 16
17 Drug Susceptibility Testing It is crucial to identify drug resistance as early as possible to ensure effective treatment Susceptibility results promptly reported to the primary health care provider and the state or local TB control program DST results will list the antibiotics that will likely be most effective in treating the infection Isoniazid and rifampin are two drugs commonly used to treat TB Bacteria that are resistant to more than one of the primary drugs used for therapy are called multidrug-resistant TB (MDR-TB) Organisms that are resistant to multiple drugs approved for first and second lines of therapy are called extensively drug-resistant tuberculosis (XDR-TB) Drug Susceptibility Testing MDR-TB Resistant to at least isoniazid and rifampin XDR-TB Resistant to any fluoroquinolone AND resistant to at least 1 of 3 injectable 2 nd line drugs (ex: amikacin, kanamycin, or capreomycin) 17
18 Drug Susceptibility Testing Pure growths 10 days to 2 weeks to complete Rerun resistant results and send to CDC for confirmation and second line drug testing Genotyping Genotyping laboratory, in Michigan is under contract with CDC to provide genotyping services to TB programs in the United States Three genotyping methods to identify TB strains: 1. Mycobacterial interspersed repetitive unit (MIRU) analysis 2. Spoligotyping 3. IS6110-based restriction fragment length polymorphism (RFLP) analysis 18
19 Genotyping (cont.) Only for culture-confirmed TB The technique requires material from a culture Matching genotypes may indicate that TB cases are related Genotyping helps investigators understand transmission relationships between TB cases It is expected that genotypes from transmission-related TB cases will match Genotyping (cont.) CDC program for genotyping Isolate 1 specimen from every patient Spoliogotype Miru1 Miru2 19
20 TBGIMS TBGIMS TB Genotype Information Management System ubmitter Numb RVCT Number spoligotype Miru Cluster_nam ed_false_date Shipped Date Received patient_dob KS_ Mar Mar Feb KS_002 N0 25-May May Sep KS_002 N0 25-May May Jan KS_002 NO 02-May May Sep KS_002 NO 19-Oct Oct Nov KS_002 NO 01-Sep Sep Nov KS_ Mar Mar Aug KS_003 NO 03-Jun Jun Sep KS_003 NO 03-Jun Jun Sep NAC z KS_ Jan Jan Jan NAC z KS_ Mar Mar Jan KS_005 NO 19-Oct Oct Apr KS_005 NO 03-Jun Jun Nov KS_ Jan Jan Dec KS_ Jan Jan Feb KS_ Mar Mar Oct KS_005 No 01-Dec Dec Aug KS_ Aug Aug Aug KS_005 NO 09-Feb Feb Mar-62 Interferon Gamma Release Assay - IGRAS Blood tests examine immune response to TB antigens Draw blood from patient, expose white cells to TB antigens, look for signs of immune response QuantiFERON TB Gold In-Tube test (QFT-GIT) 20
21 Interferon Gamma Release Assay - IGRAS TST Blood tests Requirements to Get a High Quality Specimen to the Lab Collection Labeling Universal Forms Packaging & Shipping 21
22 Biological Substance, Category B Packing and Shipping System KDHE supplies kits Information & Instruction guide _specimen_packaging_sputum_tb_handout.pdf 5-10 ml sputum Other fluids Collection Details 22
23 Universal Forms One universal form for each specimen collected A patient may have multiple specimens collected and would have multiple universal forms Fill out universal form completely Universal Forms Key Sections 23
24 Facility Info & Specimen Source Patient Information 24
25 TB Specimen Information Universal Forms One universal form per specimen not per patient Complete all required information 25
26 Specimen Labeling Universal Form sticker Patient name Patient date of birth Date of collection Seal with Parafilm Shipping Box and Container Universal Form outside of plastic container Seal box with tape loads/diagnostic_specimen_pack aging_sputum_tb_handout.pdf 26
27 KHEL Receiving Process 27
28 KHEL Specimen Processing BSL-2 BSL-3 practices Controlled and safe Unpacked/Sorted Digested Concentrated Set up Storage 3-4 hour process Reports Preliminary Smear Negative (same day) Final Final at 6 weeks 28
29 Reports Preliminary Smear Positive (same day) NAAT (if applicable 24hrs) Identification (approx. 2 weeks) Sensitivity if TB (10 days to 2 weeks) Final If NTM final with ID TB Biochemical report and final ID (batched) Questions 29
30 30
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