WSLH: Laboratory Update Tuberculosis Summit Verona, WI April 24, 2014

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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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


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.

http://www.stoptb.org/wg/gli/assets/documents/29_specimen_condition_transport.doc 7


Sputum Quality

Thick, Mucopurulent

Hemoptysis

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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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


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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 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


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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)


Mycobacteriology Testing Performed at WSLH

• 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


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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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


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 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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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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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


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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Automated system that uses a fluorescent method for detection of oxygen consumption

Mycobacteria Growth Indicator Tube (MGIT)

Solid media plate


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


• 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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• 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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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


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


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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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


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

Result Interpretation

rpoB (RRDR) No mutation Rifampin susceptible

inhA (promoter)

No mutationINH resistant

katG (ser315 codon)

Mutation: Ser315Thr

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Expected Turn-around TimesReporting Results

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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


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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


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


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 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


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-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


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Dave

Julie TKJulie B.

Nate

Youngmi and Ana

WSLH Laboratory Team

Don

WSLH: Laboratory Update Tuberculosis Summit Verona, WI April 24, 2014

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