# Presentation: 240483

## Identification of M. tuberculosis and Drug Susceptibility Testing

## Identification of the M. tuberculosis Complex

- Decide whether the mycobacterium is a pathogenic species of the Mycobacterium tuberculosis complex.
- In the context of surveillance of drug  resistance only identify M. tuberculosis complex from non-tuberculosis mycobateria

## Methods to ID M. tuberculosis

- Morphology
-  Growth rate
-  Biochemical
- Genetic (Accuprobe)
-  HPLC
-  Other

## Non tuberculous Mycobacteria

- Describe mophology of each Rounyon Group and growth rate.

##  Drug Susceptibility Testing Methods

- Manual Methods- Require several weeks
- 		-Agar Proportion
- 		-L-J Proportion
- 		-L-J Resistance Ratio
- 		-Absolute Resistance
-        - E- test

## Automated Methods 

- BACTEC 460 TB
- BACTEC MGIT
- BACTEC 960 MGIT
- TREK
- BACT ALERT

## Drug Concentrations and Critical Proportions

## Drug Susceptibility Testing

- Drug resistance the result of pre-existing mutants
- Inoculum standardization most important issues in drug susceptibility testing
- Over inoculating may result in inaccurate drug testing

## The Proportion Method

- M. tuberculosis isolates are tested for susceptibility to these four drugs
- Isoniazid
- Rifampicin
- Streptomycin
- Ethambutol

## Drug Testing Procedure

- This loop will deliver 0.01 mL of inoculum
- Inoculate a sterile McCartney bottle contining1.0 mL water with 10 glass beads
- Homogenize on a vortex mixer for up to 1 minute

## Inoculating drug media

- Two serial dilutions are made from the original suspension
- A 10-2  and 10-4 dilution are used
- With the inoculating loop  inoculate one drug free colony control medium and two slopes of drug containing media
- Incubate at 370C for 4 weeks

## Drug containing media

- Appropriate concentration of drug are added to egg-based medium
- Isoniazid 0.2mg/L
- Dihydrostreptomycin sulfate 40mg/L
- Rifampicin 40 mg/L
- Ethambutol 2.0mg/L

## Drug Preparation

- Drug potencies
- Importance of weighing drugs properly
- True potency of the drug is the number of micrograms of active drug per milligram total weight of the product.
-  Not all antimicrobial drugs have been isolated in pure form.
- Desired activity (ug/mL) = (weight of drug)
- Must account for potency when weighing to get accurate final concentration

## Reading, Interpreting, and Reporting

- Examine  media after one week
- First reading of drug susceptibility is done at 4 weeks of incubation. Report any drug resistance at this time.
- Final reading is at 6 weeks.
- Resistance is reported as the % of colonies on drug-containing media in comparison to the growth on drug-free medium.

## Dilutions, Concentrations and Controls

## Slide 17

## Reading susceptibility test results

- Examine seeded media for contamination after one week.
- First reading of drug susceptibility test results is done at four weeks of incubation
-  Strains showing drug resistance can be reported as drug resistant.
-  Strains showing drug susceptibility at four weeks usually need to be incubated for two additional weeks.
- Report final reading at six weeks.

## Reporting DST Results

-  Reported results on all four drugs, list concentration and method of testing
- Interpretation for each concentration tested should be reported

## Tuberculosis Laboratory Register 

- Recording results

## Internal Quality Control for DST

- WHO/IUATLD/ NCCLS Guidelines for interpretations
- Perform controls with both known resistant and susceptible strains of M. tuberculosis
- Test each drug at low and high level resistance strains to verify drug concentration in media
-  Control strain results reflect technical performance and concerns

## Proficiency Testing 

- Exchanging samples of M. tuberculosis strains between laboratories.
- Compare results with other laboratories.
- Perform random sampling in a double-blinded manner
- Subscribe to external quality assessment programs