WSLH: Laboratory Update
Tuberculosis Summit
Verona, WI
April 24, 2014
Julie Tans-Kersten, MS, BS-MT (ASCP)
Tuberculosis Laboratory Program
Coordinator
Wisconsin State Laboratory of Hygiene
tanskejl@mail.slh.wisc.edu
(608) 263-5364
WISCONSIN STATE
LABORATORY OF HYGIENE
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Laboratory Update
Objectives
•
•
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•
•
Background
Specimen Collection
Specimen Preparation and Transport
Testing performed at WSLH
Reporting and Interpretation of
Results, Expected turn-around times
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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
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Submission of Patient Specimens to
WSLH for Mycobacteriology Testing
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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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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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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.
WISCONSIN STATE
http://www.stoptb.org/wg/gli/assets/documents/29_specimen_condition_transport.doc
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Sputum Quality
Thick,
Mucopurulent
Hemoptysis
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Watery
(induced?)
Salivary
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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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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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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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Submission of Patient Specimens to
WSLH for Mycobacteriology Testing
WSLH Respiratory Collection Kit #8
Order: 1-800-862-1088
Kits are free
Insulated mailer with labels
Absorbent pad
Cold pack
Sterile plastic conical tube
with label
Sealable biohazard
specimen transport bag
Instruction sheet
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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.
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Submission of Patient Specimens to
WSLH for Mycobacteriology Testing
Code
Test Description
MM00250
Mycobacteria (AFB) Smear and Culture
MM00253
Mycobacteria Isolate Identification
MM00202
Mycobacterium avium complex (MAC)
susceptibility
Mycobacterium tuberculosis susceptibility
1st line drugs
MM00204
MM00207
Mycobacteria rapid grower susceptibility
MM00256
Mycobacterium tuberculosis PCR
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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 Pickup:
1-800-236-7127 (WSLH account 7271)
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Mycobacteriology Testing Performed
at WSLH
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•
•
•
•
Smear Microscopy
PCR for Direct Detection
Culture
Identification
Drug Susceptibility Testing
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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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AFB Smear Microscopy
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AFB Smear Microscopy: Interpreting
Results
WSLH Report
Negative
Graded Scale
Qualitative
Scale
Negative
Negative
1-9 AFB per
100 fields
1+
Positive (rare)
1-9 AFB per
10 fields
1-9 AFB per
field
2+
Positive (few)
3+
Positive
(moderate)
>9 AFB per
field
4+
Positive
(many)
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Interpretation
Potentially
infectious
Low-level
infectious
Moderately
infectious
Highly
infectious
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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 MTB
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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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PCR for Direct Detection
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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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WSLH TB/MAC PCR Specimen
Requirements
Test
Specimen
Smear Result
Type
TB PCR Respiratory and Smear positive and
non-respiratory smear negative
MAC
PCR
Respiratory only Smear positive only
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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 antimycobacterial therapy or no such treatment
within the last 12 months
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Interpretation of PCR Results
WSLH Lab Report
Interpretation
“Mycobacterium tuberculosis
complex DNA detected”
Positive for TB
“Mycobacterium avium complex DNA Positive for MAC
detected”
“No Mycobacterium tuberculosis
complex DNA detected”
Negative for TB
“No Mycobacterium avium complex
DNA detected”
“Inhibitory substances that prevent
nucleic acid amplification were
detected”
Negative for MAC
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Test is of no
diagnostic help
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Culture for Mycobacteria
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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
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Mycobacteria Growth
Indicator Tube (MGIT)
Automated system that
uses a fluorescent method
for detection of oxygen
consumption
Solid media plate
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Identification of Mycobacteria
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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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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
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Significance of MTBC Culture Results
• 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
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Culture Results for Patient
Management
• 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
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Susceptibility Testing
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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
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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
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CDC TB Drug Susceptibility Testing
TB First-Line Drugs
•INH
•Rifampin
•Ethambutol
•PZA
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TB Second-line Drugs
•Streptomycin
•Rifabutin
•Ciprofloxacin
•Kanamycin
•Ethionamide
•Capreomycin
•PAS
•Ofloxacin
•Amikacin
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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
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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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CDC MDDR Results
Gene
(region)
examined
rpoB (RRDR)
Result
Interpretation
No mutation
Rifampin
susceptible
inhA
(promoter)
katG (ser315
codon)
No mutation
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INH resistant
Mutation:
Ser315Thr
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Expected Turn-around Times
Reporting Results
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Summary of WSLH Turn-AroundTimes
Test
Expect Report
Smear
24 hours
PCR
24-48 hours
Culture
M. tuberculosis complex usually grows
within 15 days.
6 weeks for final report
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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
Average 30 days (range
conventional (WSLH) 16-98 days)
TB second line
conventional (CDC)
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4-5 weeks from date of
receipt at CDC
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Testing Turn-around Times
Primary specimen
Molecular: 4-6 days
PCR
24-48 hours
Identification
0-2 days
MDDR
2-3 days
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TB first line drugs
4-20 days
TB second-line drugs
3-4 weeks
Conventional (Culture Based):
4-10 weeks
Smear positive
respiratory
Culture
7-21 days
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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WSLH Reporting of Critical
Laboratory Results
Critical Value
Notification by
telephone and fax
Positive 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
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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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For More Information
• Julie Tans-Kersten
Wisconsin State Lab of Hygiene
(608) 263-5364
Fax: (608) 890-2548
julie.tanskersten@slh.wisc.edu
• TB (Mycobacteriology) Lab: (608) 262-1618
• Lorna Will, Philip Wegner, Pa Vang
WI State TB Program
608-261-6319
WISCONSIN STATE
LABORATORY OF HYGIENE
WSLH Laboratory
Team
Nate
Dave
Julie B.
WISCONSIN STATE
LABORATORY OF HYGIENE
Don
Youngmi and Ana
Julie TK
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