CDST_LT: Second line drug testing

Multidrug-resistant (MDR) TB is caused by strains of MTB resistant to at least isoniazid and rifampicin; for effective MDR patient management, optimized treatment regimens are required. These complex regimens can include a fluoroquinolone, aminoglycoside, cyclic peptide or compounds from other drug classes, and any remaining first-line susceptible drugs. 

Therefore, reliable drug susceptibility testing (DST) of these anti-TB drugs is crucial for the management of MDR-TB and for preventing the emergence of additional drug resistance in these patients.

 

Isolated cultures from TB patients are subjected to growth in the presence of a known concentration of a test drug. Growth control is also included without the addition of the drug. If the patient’s isolate grows in control but does not grow in the presence of the drug, it is considered susceptible. On the other hand, if it grows in both tubes, then it is considered to be resistant to that drug. Many methods are in use for susceptibility testing. The most commonly used method is the proportion method, where resistance is established at the 1% cut-off point for all the drugs. This means that if 1% or more of the total bacterial population is resistant to a drug, the strain is designated to be resistant to that drug.

 

Resource

Mycobacteriology Laboratory Manual    

Assessment

Question​	Answer 1​	Answer 2​	Answer 3​	Answer 4​	Correct answer​	Correct explanation​	Page id​	Part of Pre-test​	Part of Post-te
In the proportion method, resistance is established at which of the following cut-off points?	At 1% cut-off point for all the drugs	Is established by MIC	Both MIC and 1% cut-off point for all drugs	At a 1% cut-off point for some of the drugs	Answer 1	The most commonly used method is the proportion method, where resistance is established at the 1% cut-off point for all the drugs. This means that if 1% or more of the total bacterial population is resistant to a drug, the strain is designated to be resistant to that drug.

 

 

Preparation of second-line drug stock:

 

Drug preparation: Prepare the drug concentration as per national and/or internationally defined drugs critical concentration, solvent and diluents of second-line drugs for DST by MGIT.

Drugs	Critical concentrations (μg/ml)	Solvent	Diluent
Kanamycin	2.5	DW	DW
Amikacin	1.0	DW	DW
Capreomycin	2.5	DW	DW
Ofloxacin	2.0	NaOH	DW
Levofloxacin	1.5	NaOH	DW
Moxifloxacin	0.5 /1.0	1N NaOH	DW
Linezolid	1.0	DMSO	DW
Clofazimine	1.0	DMSO	DMSO
PAS	4.0	Ethanol	DW
Ethionamide	5.0	DMSO	DW

 

 

All antimicrobial agents are assayed for standard units of activity. The assay units may differ widely from the actual weight of the powder and often may differ between drug production lots. Thus, the lab must standardize the antimicrobial solutions based on the potency of an individual lot of each drug powder. 

 

The following formula is used to calculate the actual weight of the drugs to be weighed:

                    

                         Volume (mL) x Concentration (mg/mL) x dilution factor

Weight (mg) = ------------------------------------------------------------------

                                      Assay potency (mg/mg)

 

1. Weight (milligrams) is the weight of powder needed to prepare the desired volume of stock solution at the desired concentration.

2. Volume (in millilitres) is the desired volume of stock solution

3. Assay potency (micrograms per milligram) is the activity or potency specified by the manufacturer of the reference standard powder. This value usually appears on the label or the certificate of analysis.

4. Concentration (micrograms per millilitre) is the desired concentration (critical concentration) of stock solution.

5. The dilution factor is the number of times the drug added to the tube (100ml) is getting diluted by the total volume of the medium in the tube (8.3ml) = 83

 

Stock solutions of each drug are to be made 83 times higher for MGIT. Prepare a stock solution with a higher concentration than the highest concentration required in the medium. The dilution factor for MGIT is 1:83 (7mL of MGIT media + 0.8 mL of DST supplement + 0.5 mL of test inoculum /culture).

 

Example: Amikacin

 

Concentration to be tested: 1 µg/ml

Volume of the stock solution: 20 ml

Potency of the drug: (e.g.) 716 µg /mg

Dilution factor: 83

Diluent: Sterile distilled water (DW)

Amount of drug to be weighed to prepare 20 ml of stock solution:

20 x 1 x 83

----------------- = 2.32 mg

     716

 

NOTE:

 

1. As it is difficult to weigh 2.32 mg (0.00232 grams), weigh 100 times this weight, i.e. 232 mg, and dissolve in 20 ml of sterile DW, aliquot in 500µl volume and store at – 20/ -70 ^oC.

 

2. At the time of setting up DST, prepare a working stock by diluting the stock 100 times, i.e. 100 µl of stock + 9.9 ml sterile DW, and add 100µl of this working solution to the designated MGIT tube.

 

3. As the assay potency is likely to vary with varying batches, care must be taken to include the correct potency factor while preparing the stock solution.

 

Resource

 

Mycobacteriology Laboratory Manual    

Assessment

 

Question​	Answer 1​	Answer 2​	Answer 3​	Answer 4​	Correct answer​	Correct explanation​	Page id​	Part of Pre-test​	Part of Post-te
Which of these solvents is NOT used for drug preparation?	NaOH	DMSO	Ethanol	PAS	Answer 4	PAS is a drug and not a solvent. The solvents used for drug preparation are NaOH, DMSO, and Ethanol.

 

 

 

Preparation and Storage of Second-line Anti-TB Drugs used for DST

1. Freeze the stock solutions preferably at -20ºC or lower in appropriate tubes and use for up to 6 months or up to the date of original expiry, whichever comes sooner.

2.  At the time of testing, make 100 folds dilution (1:100) to achieve the desired concentration and store at -20ºC or lower in appropriate tubes and use for up to 6 months or up to the date of original expiry, whichever comes sooner.

3. Once the working solution drug is thawed, use it immediately and do not re-freeze. Discard unused portions.

4. Always pipette accurately 100uL of the drug to the designated drug labelled MGIT tube.

 Important notes for preparation of drug solutions

1. All the drugs, except Ethionamide, should be filter sterilized. Use a 0.2-micron syringe filter for sterilization of the drugs. Discard the first 20% filtered solutions, and collect the remaining solution for use.

2 In the case of self-sterilizing solutions as well as other aqueous stock solutions, all further dilutions should be made in water, using sterile distilled water and aseptic techniques.

3 If a solvent other than water is recommended, only use sufficient solvent to solubilize the antimicrobial powder and then dilute to the final stock concentration with sterile distilled water.

4 Record batch no., date of preparation and expiry date for all drugs. Store the stock solutions in small aliquots at –20° C / 70° C. Once thawed and used, discard the remainder. Refreezing may affect the potency of the drugs.

Resource

 

Mycobacteriology Laboratory Manual       

Assessment

 

Question​	Answer 1​	Answer 2​	Answer 3​	Answer 4​	Correct answer​	Correct explanation​	Page id​	Part of Pre-test​	Part of Post-te
Stock solutions are kept at which of these temperatures?	Room temperature	-20ºC	4ºC	20ºC	-20ºC	Stock solutions are preferably stored at -20ºC or lower.		Yes	Yes