PART 2: Culture andDrug Susceptible Testing (DST)

Heidi Behm, RN, MPHTB Nurse Consultant/TB Controller, Public Health Division

Oregon Health Authority

Importance of TB Culture

• Recognized as the gold standard for diagnosis of TB disease• Increases TB case identification• Used for species identification, drug-susceptibility testing, genotyping• In patients with mixed populations, allows for isolation of pure MTBC

culture, important for accurate drug susceptibility testing• Used to monitor patient response to treatment―For drug-susceptible TB patient, failure to convert to negative culture

following 2 months of treatment may indicate need to extend treatment. Conversion per RVCT is one negative culture but some clinical guidelines recommend 2 consecutively negative cultures to document conversion.―A persistent positive culture after 4 months of treatment may represent

treatment failure

Mycobacterium tuberculosis complex (MTBC)

M. tuberculosisM. africanumM. bovis (cattle)M. canettii (rare)M. orygis (herd animals)M. microti (rodents)M. caprae (sheep/goats)M. pinnipedii (seals/sea lions)M. suricattae (meerkats)M. mungi (mongoose)

Source: NIAID image 18139

Human pathogens

3

Types of Media used to grow TB

Solid Media• Egg-based• Lowenstein Jensen (LJ)

• Agar-based• Middlebrook 7H10 and 7H11

Liquid Media• Becton Dickinson BACTEC MGITTM

• bioMerieux BacT/ALERTÒ

• Thermo Scientific VersaTREKTM

PositiveNegative

MGIT tube image source: Grace Lin, CDPH/MDL

Sample TB Culture Result

• Negative culture result reported 6-8 weeks after inoculation• Positive result reported as soon

as growth identified• Update provided once

identification made (MTBC or Non-tuberculous mycobacteria [NTM])

GeneXPERT MTB/RIF

• Is a NAAT, a molecular test.

• Detects MTB AND mutations associated with Rifampin resistance.

• Smear positive specimen -Sensitivity for detection MTB approximately 97%.• Smear negative specimen- Sensitivity for detection MTB approximately 55%.

• Usually if RIF resistant, also INH resistant = multidrug resistant.• GeneXpert may report silent, clinically insignificant, mutations as RIF drug

resistance. Call your public health lab or state TB program if you get a RIF resistant result! Needs to be confirmed by CDC MDDR.

• Note: Does not detect INH resistance which is most common.

Conventional growth-based drug susceptibility testing (DST)

• Conventional growth-based DST = phenotypic, conventional, or culture-based drug susceptibility testing

• A culture is needed to setup growth-based DST. A culture is not required for molecular tests.

• When there is drug resistance, there may be DST results from multiple methods and laboratories. Discordant results may occur and are challenging to interpret!

Reliability of growth-based DST by drug

• Reliable: isoniazid (INH), rifampin (RIF), fluoroquinolones, amikacin (AK), capreomycin (CM), kanamycin (KM)

• Less reliable or no data: ethambutol (EMB) (more often tests susceptible by MGIT 960 compared to agar proportion), pyrazinamide (PZA) (more often falsely resistant), streptomycin (SM), oral second-line drugs, third-line drugs

Polling Question 3

A specimen that is PCR positive for TB means live Mycobacterium tuberculosis organisms must be present in the patient.

a. Trueb. Falsec. Don’t Know

Drug Susceptible Testing: liquid broth systems

• MGIT 960 most common U.S.• FDA approved INH, RIF, EMB, PZA, SM• Results in 4-14 days after test is set-up (culture must be grown first)• Other: VersaTREK, Sensititre, MODs

Drug Susceptible Testing: agar proportion method

• Solid media• Reference or gold standard in U.S.• A cell suspension is prepared from a culture and inoculated onto each quadrant of

an agar plate. Each quadrant contains a specific drug at its critical concentration or no drug as a control. Plates are incubated for 21 days before colony counts are taken.• The isolate is resistant if number of colonies in the drug quadrant is equal to or more

than 1% of that in the control quadrant.• Indirect DST refers to testing on positive culture growth, while direct DST is done on

AFB smear-positive sediments.

Drug susceptibility tests: review

• Liquid broth systems- MGIT most common in U.S.- Result typically available 4-14 days

after culture+ MTB- PZA liquid broth only

MGIT INDIRECT

• Agar proportion method- Phenotypic, “gold standard”- Result typically available 21 days after

susceptibility culture is set up, usually 4 weeks after culture + MTB

- INH/RIF/EMB and second line drugs- Inoculate a drug containing media with culture

from the original specimen and observe growth (indirect method)

Molecular methods to detect M. tuberculosis DNA and drug resistance mutations

• Understanding of drug resistance at the genetic level is variable and incomplete. 100% sensitivity for detecting all drug resistance is not possible currently.

• Traditional DST is gold standard.

• GeneXpert – RIF only (rpoB), if mutations detected MUST verify. Send to CDC MDDR service

• Pyrosequencing (CDC MDDR service, CA, NY and others)

• Sanger sequencing (CDC MDDR service)

CDC MDDR Service

• Molecular Detection of Drug Resistance Laboratory User Guide

• Requires approval TBLab@cdc.gov• Must be NAAT + or culture+ MTB typically to be accepted• Pyrosequencing – INH and RIF• Sanger Sequencing – if RIF resistant or upon request.

Second line drugs• Times to request CDC MDDR: patient failing treatment,

patient has taken treatment for TB disease or LTBI previously, GeneXpert is RIF resistant, specimen is contaminated with other species, patient very ill or infant involved, large contact investigation, other reason to suspect drug resistance

Breakout Practice Instructions: PART 2

• Small group facilitated discussion• Video cam on for introductions at the

start• Use Learner Packet to record

discussion points• If audio problems or technical

assistance needed, select blue “LEAVE” button, bottom right, then “LEAVE BREAKOUT”üTake note of breakout room number