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.
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Method Article
The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis
In This Article
Summary
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
- Prepare stock solutions
- Phosphate buffer stock
- 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
- Adjust pH to 6.8 ± 0.2: add sodium phosphate dibasic solution to raise pH; add potassium phosphate monobasic solution to lower pH
- Autoclave at 121-124°C for 15 minutes to sterilize
- Plate a 100μl aliquot of the phosphate buffer solution on nutri....
- Phosphate buffer stock
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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.......
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Acknowledgements
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 contrib....
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Materials
| 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; d–s 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 Scientific | DF0713-17-9 | 500gr/bottle; culture media base |
| Casitone (pancreatic digest casein) | Reagent | Fisher Scientific | DF0259‐17‐9 | 500gr/bottle; culture media base |
| Glycerol (glycerin) lyophilized | Reagent | Sigma-Aldrich | G‐33‐500 | 500ml/bottle; culture media base |
| PANTA (Antibiotic mixture lyophilized BD) | Reagent | Fisher Scientific | B4345114 | 6 bottles/pack; antibiotic media supplement |
| OADC (Middlebrook OADC enrichment BD) | Reagent | Fisher Scientific | B11886 | 10 x 20ml/pack; nutritional media supplement |
| Dimethyl sulphoxide (Hibri-Max) | Reagent | Sigma-Aldrich | D-2650 | 100ml/bottle; to prepare rifampicin stock |
| Antibiotic stocks: isoniazid | Reagent | Sigma-Aldrich | I-3377 | 50gr/bottle; direct susceptibility testing |
| Antibiotic stocks: rifampicin | Reagent | Sigma-Aldrich | R-3501 | 1gr/bottle; direct susceptibility testing |
| Sodium hydroxide (pellets) | Reagent | Sigma-Aldrich | 221465 | 500gr/bottle; sputum decontamination |
| Sodium citrate (trisodium salt dihydrate) | Reagent | Sigma-Aldrich | S-4641 | 500gr/bottle; sputum decontamination |
| N-acetyl-L-cysteine | Reagent | Sigma-Aldrich | A-7250 | 50gr/bottle; sputum decontamination |
| Potassium Phosphate Monobasic crystal. KH2PO4 | Reagent | Sigma-Aldrich | P0662 | 500gr/bottle; sputum decontamination |
| Sodium Phosphate Dibasic, anhydrous. Na2HPO4 | Reagent | Sigma-Aldrich | S0876 | 500gr/bottle; sputum decontamination |
| Sodium hypochlorite | Reagent | household bleach | to discard contaminated waste | |
| 15ml centrifuge tubes (polypropylene 15ml Falcon 35‐2096) | Consumable | Fisher Scientific | 14‐959‐49B | 500ea/case; for sputum decontamination and concentration |
| 24 well plates (Plates Tissue 24 wells BD Falcon 35‐3047) | Consumable | Fisher Scientific | 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 international | 47729-583 | 500 tubes/case; to store aliquots of prepared broth |
| Screw cap microcentrifuge tubes (1.5ml) | Consumable | Fisher Scientific | 05‐669‐22 | 1000ea/case; to store aliquots of antibiotic stocks |
| 0.22μm filters (aqueous solvents) Syringe filter Millex blue | Consumable | Fisher Scientific | SLGL 025 OS | 50 units/case; to filter antibiotic stocks |
| 0.22μm filters (organic solvents) Syringe filter Millex yellow | Consumable | Fisher Scientific | SLGV 033 RS | 50 units/case; to filter antibiotic stocks |
| Disposable Pasteur pipettes borosilicate glass 9″ | Consumable | Fisher Scientific | 13‐678‐20C | 720ea/case; to mix PANTA with media mix |
| Aerosol barrier tips 1000‐1300μl | Consumable | Fisher Scientific | 02‐707‐51 | 1000ea/pk; to dispense media into plate |
| USA Scientific Tips One 1‐200μl yellow tips | Consumable | Fisher Scientific | 1111‐0006 | 1000 tips/bag; to dilute antibiotic stocks |
References
- Arias, M. Clinical evaluation of the microscopic-observation drug-susceptibility assay for detection of tuberculosis. Clin Infect Dis. 44, 674-674 (2007).
- Caviedes, L.
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