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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE 1
WSLH: Laboratory Update
Tuberculosis SummitVerona, WIApril 24, 2014
Julie Tans-Kersten, MS, BS-MT (ASCP)Tuberculosis Laboratory Program CoordinatorWisconsin State Laboratory of [email protected](608) 263-5364
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Laboratory UpdateObjectives
• Background• Specimen Collection• Specimen Preparation and
Transport• Testing performed at WSLH• Reporting and Interpretation of
Results, Expected turn-around times
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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Mycobacteriology Testing at WSLH• WSLH serves as a public health
laboratory for the Wisconsin State Department of Public Health and Wisconsin local public health agencies.
• WSLH serves as a primary diagnostic facility and reference laboratory for clinicians and private mycobacteriology laboratories located throughout Wisconsin
• Full-service mycobacteriology laboratory
• Biosafety Level-3 facility
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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Submission of Patient Specimens to WSLH for Mycobacteriology Testing
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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.
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Sputum
• Recently discharged material from the bronchial tree, with minimal amounts of oral or nasal material
• Expectorated: from deep productive cough
• Indications for sputum collection– To establish an initial diagnosis of TB– To monitor the infectiousness of the patient– To determine the effectiveness of treatment
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Sputum Quality• Specimens are thick and contain
mucoid or mucopurulent material• Ideally, 3–5 ml in volume, although
smaller quantities are acceptable if the quality is satisfactory
• Poor quality specimens are thin and watery. Saliva and nasal secretions are unacceptable
• Laboratory requisition form should indicate when a specimen is induced to avoid the specimen being labeled as “unacceptable” quality
Clinical and Laboratory Standards Institute. Laboratory detection and identification of mycobacteria; approved guideline. CLSI Document M48-A. Wayne, PA: CLSI; 2008.
http://www.stoptb.org/wg/gli/assets/documents/29_specimen_condition_transport.doc 7
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Sputum Quality
Thick, Mucopurulent
Hemoptysis
Watery (induced?)
Salivary
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Sputum Collection
• 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
• Optimally, sputum should be collected before the initiation of drug therapy
Centers for Disease Control and Prevention. Guidelines for Preventing the Transmission of Mycobacterium tuberculosis in Health-Care Settings, MMWR 2005:54, RR-17
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Sputum Collection
• Provide supervised sputum collection for at least the first sputum specimen, until the patient demonstrated the ability to properly collect the specimen.
• Use respiratory precautions when collecting sputum specimens
• All mycobacteria specimens are collected into a sealed leak proof container
• Label the specimen with patient name, collection date/time and specimen type.
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Storage and Transport of Sputum Specimens
• Collection sites should refrigerate samples that cannot be transported immediately to reduce growth of contaminating organisms
• Specimens should be delivered to the laboratory as soon as possible, within 24 hours of collection is optimal (avoid batching)
• Recommended: include a cold pack with specimen transport materials
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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Submission of Patient Specimens to WSLH for Mycobacteriology Testing
Insulated mailer with labels
Absorbent pad
Cold pack
Sterile plastic conical tube with label
Sealable biohazard specimen transport bag
Instruction sheet
WSLH Respiratory Collection Kit #8
Order: 1-800-862-1088
Kits are free
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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Submission of Patient Specimens to WSLH for Mycobacteriology Testing
Requisition Form A
Order: 1-800-862-1088
Preprinted with account number
Submit one form with each specimen.
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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Submission of Patient Specimens to WSLH for Mycobacteriology TestingCode Test Description
MM00250
Mycobacteria (AFB) Smear and Culture
MM00253
Mycobacteria Isolate Identification
MM00202
Mycobacterium avium complex (MAC) susceptibility
MM00204
Mycobacterium tuberculosis susceptibility 1st line drugs
MM00207
Mycobacteria rapid grower susceptibility
MM00256
Mycobacterium tuberculosis PCR
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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Submission of Patient Specimens to WSLH for Mycobacteriology Testing• Wrap specimen in absorbent material• Place in zipper portion of biohazard bag
and zip closed• Place requisition form in rear pouch of
biohazard bag• Place bags and cold pack in insulated
mailing container and seal with tape• Label mailer with WSLH address and
UN3373 (“Biological Substance Category B”) label
• Arrange for pre-paid Dunham Express Pick-up:
1-800-236-7127 (WSLH account 7271)
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Mycobacteriology Testing Performed at WSLH
• Smear Microscopy• PCR for Direct Detection• Culture• Identification• Drug Susceptibility Testing
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
AFB Smear Microscopy
• Small amount of processed and concentrated patient specimen is placed on a microscope slide and stained acid-fast organisms
• Rapid and inexpensive screening tool• Positive AFB smear results provide a
first indication of mycobacterial infection and potential TB disease
• Must be accompanied by additional testing including culture for confirmatory diagnosis
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
AFB Smear Microscopy: Interpreting Results
WSLH Report Graded Scale Qualitative Scale
Interpretation
Negative Negative Negative Potentially infectious
1-9 AFB per 100 fields
1+ Positive (rare)
Low-level infectious
1-9 AFB per 10 fields
2+ Positive (few) Moderately
infectious1-9 AFB per field
3+ Positive (moderate)
>9 AFB per field
4+ Positive (many)
Highly infectious
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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 sensitivity
– High bacterial load 5,000-10,000 AFB /mL is required for detection
– Misses >45% of U.S. TB cases• Limited specificity
– All mycobacteria are acid fast– Does not provide species identification– Local prevalence of MTB and NTM determine the
predictive values of a positive smear for MTB20
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
AFB Smear Microscopy Results Guide Decisions
• Clinical management– Patient therapy may be initiated for TB based on smear
result and clinical presentation– Changes in smear status important for monitoring
response to therapy
• Public health interventions– Smear status and grade useful for identifying the most
infectious cases – Contact investigations prioritized based on smear result– Decisions regarding respiratory isolation based on
smear result
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
PCR for Direct Detection
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
WSLH TB/MAC PCR• Detection of M. tuberculosis complex and M.
avium complex (MAC) directly from patient specimens
• Healthy People 2020 Goal: Identify new TB patients within 48 hours – Respiratory isolation– Start therapy
• Identify smear positive MAC patients– Release from isolation– Alter therapy decisions
• Presumptive rapid results for 59% of smear positive patients
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
WSLH TB/MAC PCR Specimen Requirements
Test Specimen Type
Smear Result
TB PCR
Respiratory and non-respiratory
Smear positive and smear negative
MAC PCR
Respiratory only
Smear positive only
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Patient Criteria for Fee-Exempt TB/MAC PCR Testing
• Patient must have signs and symptoms of pulmonary TB
• Patient must be reported to the local or state public health department as a suspect TB case
• Patient must be in respiratory isolation• Patient must not have been diagnosed with TB
or a non-tuberculous mycobacterial infection within the last 12 months
• Patient must have received ≤7 days of anti-mycobacterial therapy or no such treatment within the last 12 months
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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Interpretation of PCR Results
WSLH Lab Report Interpretation
“Mycobacterium tuberculosis complex DNA detected”
Positive for TB
“Mycobacterium avium complex DNA detected”
Positive for MAC
“No Mycobacterium tuberculosis complex DNA detected”
Negative for TB
“No Mycobacterium avium complex DNA detected”
Negative for MAC
“Inhibitory substances that prevent nucleic acid amplification were detected”
Test is of no diagnostic help
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Culture for Mycobacteria
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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Culture for Mycobacteria
• Detects viable mycobacteria from patient specimens
• Most sensitive method for detecting mycobacteria (“Gold standard”)
• Slowest Method– Average time to detection for TB = 15 days– Range for detection of TB: 8-30 days
• Smear and Culture always performed together
• Broth and solid media used to grow mycobacteria
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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Automated system that uses a fluorescent method for detection of oxygen consumption
Mycobacteria Growth Indicator Tube (MGIT)
Solid media plate
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Identification of Mycobacteria
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Identification of Mycobacteria at WSLH
• Multifaceted approach– Colony morphology and pigment– High performance liquid chromatography
(HPLC)– Matrix-Assisted Laser Desorption Ionization
Time of Flight (MALDI-TOF)– DNA Probes (M. tuberculosis complex, M.
avium complex, M. gordonae, M. kansasii)– DNA sequencing
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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Interpretation of Culture Results
Test Result Interpretation
“Negative for mycobacteria”
No mycobacteria detected during 6 weeks of incubation
“Isolated: M. tuberculosis complex”
Confirmation of TB by culture
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
• The laboratory identification of MTBC is the most important finding in the clinical mycobacteriology laboratory. The finding of this species has vital epidemiologic and public health consequences.– MTBC is not found in the environment– Isolation almost always signifies disease
• MTBC culture is important for conventional drug susceptibility testing
Significance of MTBC Culture Results
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
• Progress of TB treatment is measured by culture conversion
• Recommend 2 negative cultures by the end of 2 months (intensive phase) to document culture conversion.
• If cultures are still positive after 4 months, the patient is deemed to have failed treatment (patient management must be re-assessed)
• Patients with MDR-TB may be kept under airborne precautions until culture conversion is documented
Culture Results for Patient Management
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Susceptibility Testing
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Drug Susceptibility Testing of M. tuberculosis complex isolates• Automatically performed for all
new culture-confirmed TB patients (no need to order)
• For “conventional” culture-based susceptibility testing– Need culture growth– Need pure growth
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
WSLH TB First Line drugs
• MGIT 960 broth system– INH (0.2 ug/ml)– INH (1.0 ug/ml)– rifampin (1.0 ug/ml)– ethambutol (5.0 ug/ml)– PZA (100 ug/ml)
• Repeat testing if resistant
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
CDC TB Drug Susceptibility Testing
TB First-Line Drugs TB Second-line Drugs
•INH•Rifampin•Ethambutol•PZA
•Streptomycin•Rifabutin•Ciprofloxacin•Kanamycin•Ethionamide•Capreomycin•PAS•Ofloxacin•Amikacin
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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Interpretation of Drug Susceptibility Test Results
Result Interpretation
Susceptible Strain is likely to show responsiveness to the drug
Resistant Strain is unlikely to show responsiveness to the drug
Indeterminate Test is of no help in prediction of responsiveness to the drug
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
CDC Molecular Detection of Drug Resistance (MDDR) Program
• For rapid detection of drug resistance from cultures or smear positive patient specimens
• CDC performs sequencing to detect mutations that confer resistance– First-line drugs, fluoroquinolones, injectables
• Turn-around time is 2-3 days• Requires CDC approval for submission
– Patient must have risk factors for drug resistance• Since 2010, we have rapidly detected all of
our MDR-TB patients using this program
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
CDC MDDR Results
Gene (region) examined
Result Interpretation
rpoB (RRDR) No mutation Rifampin susceptible
inhA (promoter)
No mutationINH resistant
katG (ser315 codon)
Mutation: Ser315Thr
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Expected Turn-around TimesReporting Results
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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Summary of WSLH Turn-Around-Times
Test Expect Report
Smear 24 hours
PCR 24-48 hours
Culture M. tuberculosis complex usually grows within 15 days.
6 weeks for final report
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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Turn-Around Times for Drug Susceptibility Testing
Test Expect Report
Molecular Detection (CDC)
2-3 days from date of receipt at CDC
TB first line conventional (WSLH)
Average 30 days (range 16-98 days)
TB second line conventional (CDC)
4-5 weeks from date of receipt at CDC
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Testing Turn-around Times
Primary specimen
PCR24-48 hours
Culture7-21 daysSmear positive
respiratory
Identification0-2 days
TB first line drugs4-20 days
TB second-line drugs3-4 weeks
MDDR2-3
days
Conventional (C
ulture Based):
4-10 weeks
Molecular: 4-6 days
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
WSLH Reporting of Laboratory Results
• All requested test results are reported to the submitter
• All reportable results are electronically transferred into the Wisconsin Electronic Disease Surveillance System (WEDSS).
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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WSLH Reporting of Critical
Laboratory ResultsCritical Value Notification by
telephone and faxPositive smear result Submitter and TB Program
Positive PCR result Submitter and TB Program
New positive cultures with M. tuberculosis complex isolated
Submitter and TB Program
Resistant TB first-line drug results (conventional or molecular)
Submitter and TB Program
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Summary• Quality specimens are important for quality results
• Transport specimens ASAP using Dunham Express with cold pack
• Expect smear results in 24 hours
• Fee-exempt PCR testing is available for detection of MTBC and MAC directly from patient specimens
• Request CDC molecular detection of drug resistance for TB patients with risk factors for drug resistance
• Culture-based “conventional” testing for identification and susceptibility testing requires more time but is still considered the gold standard
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
For More Information• Julie Tans-KerstenWisconsin State Lab of Hygiene(608) 263-5364Fax: (608) [email protected]
• TB (Mycobacteriology) Lab: (608) 262-1618
• Lorna Will, Philip Wegner, Pa VangWI State TB Program608-261-6319