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.
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 liquids can be spilt or aerosolized. We believe that the MODS assay is more biosafe than any assay that involved indirect drug susceptibility testing because indirect drug susceptibility testing involves the manipulation of highly concentrated solutions of mycobacteria with the attendant risks of spillage and aerosolization; in contrast, MODS simply involves the inoculation of a sputum sample into a plate, after which the plate is sealed within a plastic bag and never again opened. This is supported by data from Korea (Kim 2007) which showed that the occupational risk to laboratory workers who plated out sputum samples without doing drug-susceptibility testing was no greater than that in workers doing sputum smear microscopy; in contrast, those doing drug-susceptibility testing had much higher occupational risk for tuberculosis.
We strongly recommend that none of these procedures be undertaken without proper precautions for laboratory workers. This includes N-95 masks for personal respiratory protection, a Class 2 biological safety cabinet with exhausted air filtered through HEPA filters, and a lock on the laboratory door to stop turbulence of airflow while samples are being manipulated.
Standard operating procedures for processing extrapulmonary samples, a photo library of mycobacterium tubercuclosis and other mycobacteria and bacterial and fungal contamination, a recommended quality assurance strategy, a procedure for accreditation of laboratories to start using MODS, and an FAQ sheet are available at modsperu.org
We would like to acknowledge Sean Fitzwater and Carmen Giannina Luna Colombo for the tuberculosis growth time-lapse video segment. We thank Marty Roper for her thorough and excellent feedback during the editing and co-authoring the User Guide, from which the current protocol was taken mostly verbatim. Production of this video was funded by the NIH/ Fogarty International Center http://www.fic.nih.gov/ David A.J. Moore contributed as a Wellcome Trust Clinical Research Fellow in Tropical Medicine and Reader in Infectious Diseases at Imperial College London (Fellowship award number 078067/Z/05). Mark F. Brady contributed as an NIH/Fogarty International Center Research Fellow.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
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 |