The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis

Summary

The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, low-tech tool for high-performance detection of tuberculosis (TB) and multidrug-resistant tuberculosis (MDRTB). This video describes the MODS liquid media culture method.

Abstract

Patients with active pulmonary tuberculosis (TB) infect 10-15 other persons per year, making diagnosing active TB essential to both curing the patient and preventing new infections. Furthermore, the emergence of multidrug resistant tuberculosis (MDRTB) means that detection of drug resistance is necessary for stopping the spread of drug-resistant strains. The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, low-tech tool for high-performance detection of TB and MDRTB. The MODS assay is based on three principles: 1) mycobacterium tuberculosis (MTB) grows faster in liquid media than on solid media 2) microscopic MTB growth can be detected earlier in liquid media than waiting for the macroscopic appearance of colonies on solid media, and that growth is characteristic of MTB, allowing it to be distinguished from atypical mycobacteria or fungal or bacterial contamination 3) the drugs isoniazid and rifampicin can be incorporated into the MODS assay to allow for simultaneous direct detection of MDRTB, obviating the need for subculture to perform an indirect drug susceptibility test. Competing current diagnostics are hampered by low sensitivity with sputum smear, long delays until diagnosis with solid media culture, prohibitively high cost with existing liquid media culture methods, and the need to do subculture for indirect drug susceptibility testing to detect MDRTB. In contrast, the non-proprietary MODS method has a high sensitivity for TB and MDRTB, is a relatively rapid culture method, provides simultaneous drug susceptibility testing for MDRTB, and is accessible to resource-limited settings at just under $3 for testing for TB and MDRTB.

Protocol

1. Prepare stock solutions
   1. Phosphate buffer stock
      1. Mix 950ml of sodium dibasic solution (9.47g of sodium phosphate dibasic dissolved in 1000ml distilled water) with 950ml of potassium phosphate monobasic solution (9.07g of potassium phosphate monobasic dissolved in 1000ml distilled water) and stir; keep back 50ml of each solution to adjust pH if necessary
      2. Adjust pH to 6.8 ± 0.2: add sodium phosphate dibasic solution to raise pH; add potassium phosphate monobasic solution to .......

Discussion

The MODS assay is targeted at resource-limited settings.  For the first time, MODS brings the ability for rapid liquid culture detection of tuberculosis and multidrug resistant tuberculosis to resource-limited settings at just under $3 per test.  MODS is a non-proprietary, iterative methodology, and the MODS community is always interested in improvements that other laboratories have managed to make.

A recurrent concern is the biosafety of liquid media culture of tuberculosis because.......

Materials

Material Name	Type	Company	Catalogue Number	Comment
Name	Company	Catalog Number	Comments
Refrigerator/ freezer	Equipment			to store pre-prepared broth and antibiotic stocks
Vortex	Equipment			to aid sputum decontamination
Centrifuge	Equipment			for sputum concentration; capable of reaching 3000 g; does not need to be refrigerated, but MUST be biosafe (buckets can be sealed)
Incubator (37 degree C)	Equipment			for culture; need not be CO2 enriched
Inverted light microscope	Microscope			to read MODS plates
Autoclave	Equipment			to sterilize media, PBS and used plates
Balance	Equipment			to weigh isoniazid, rifampicin and NALC
Middlebrook 7H9 broth (Difco)	Reagent	Fisher	DF0713-17-9	500gr/bottle; culture media base
Casitone (pancreatic digest casein)	Reagent	Fisher	DF0259‐17‐9	500gr/bottle; culture media base
Glycerol (glycerin) lyophilized	Reagent	Sigma	G‐33‐500	500ml/bottle; culture media base
PANTA (Antibiotic mixture lyophilized BD)	Reagent	Fisher	B4345114	6 bottles/pack; antibiotic media supplement
OADC (Middlebrook OADC enrichment BD)	Reagent	Fisher	B11886	10 x 20ml/pack; nutritional media supplement
Dimethyl sulphoxide (Hibri-Max)	Reagent	Sigma	D-2650	100ml/bottle; to prepare rifampicin stock
Antibiotic stocks: isoniazid	Reagent	Sigma	I-3377	50gr/bottle; direct susceptibility testing
Antibiotic stocks: rifampicin	Reagent	Sigma 99	R-3501	1gr/bottle; direct susceptibility testing
Sodium hydroxide (pellets)	Reagent	Sigma	221465	500gr/bottle; sputum decontamination
Sodium citrate (trisodium salt dihydrate)	Reagent	Sigma	S-4641	500gr/bottle; sputum decontamination
N-acetyl-L-cysteine	Reagent	Sigma	A-7250	50gr/bottle; sputum decontamination
Potassium Phosphate Monobasic crystal. KH2PO4	Reagent	Sigma	P0662	500gr/bottle; sputum decontamination
Sodium Phosphate Dibasic, anhydrous. Na2HPO4	Reagent	Sigma	S0876	500gr/bottle; sputum decontamination
Sodium hypochlorite	Reagent	household bleach		to discard contaminated waste
15ml centrifuge tubes (polypropylene 15ml Falcon 35‐2096)	Consumable	Fisher	14‐959‐49B	500ea/case; for sputum decontamination and concentration
24 well plates (Plates Tissue 24 wells BD Falcon 35‐3047)	Consumable	Fisher	08-772-1	50 plates/case; for culture and reading
Sealable polythene bags 6 X 6 “ (ziplock)	Consumable			for biosecurity to contain 24 well plate
Glass tubes with lid (16 x 100mm and 18 x 145mm)	Consumable	VWR	47729-583	500 tubes/case; to store aliquots of prepared broth
Screw cap microcentrifuge tubes (1.5ml)	Consumable	Fisher	05‐669‐22	1000ea/case; to store aliquots of antibiotic stocks
0.22μm filters (aqueous solvents) Syringe filter Millex blue	Consumable	Fisher	SLGL 025 OS	50 units/case; to filter antibiotic stocks
0.22μm filters (organic solvents) Syringe filter Millex yellow	Consumable	Fisher	SLGV 033 RS	50 units/case; to filter antibiotic stocks
Disposable Pasteur pipettes borosilicate glass 9″	Consumable	Fisher	13‐678‐20C	720ea/case; to mix PANTA with media mix
Aerosol barrier tips 1000‐1300μl	Consumable	Fisher	02‐707‐51	1000ea/pk; to dispense media into plate
USA Scientific Tips One 1‐200μl yellow tips	Consumable	Fisher	1111‐0006	1000 tips/bag; to dilute antibiotic stocks