Rapid Identification and Drug Susceptibility Testing of Mycobacterium tuberculosis: Standard Operating Procedure for Non-Commercial Assays: Part 1: Microscopic Observation Drug Susceptibility Assay v2.4.12

Medical fitness

In accordance with national laws and practices, arrangements should be made for appropriate health surveillance of TB laboratory workers:

• Before enrolment in the TB laboratory

• At regular intervals thereafter, annually or bi-annually

• After any biohazard incident

• In case of onset of TB symptoms

All cases of disease or death identified in accordance with national laws and/or practice as resulting from occupational exposure to biological agents shall be notified to the competent authority.

Education and training

Personnel are required to be knowledgeable of the procedures in this SOP. Documentation of training and familiarization with this SOP can be found in the training file for each employee.

The laboratory staff shall confirm (i.e., documentation in the training file of familiarization with the SOP) that they can properly perform the procedure before commencing work. Education and training must be given on the following topics:

• Potential risks to health (symptoms of TB disease and transmission)

• Precautions to be taken to minimize aerosol formation and prevent exposure

• Hygiene requirements

• Wearing and use of protective equipment and clothing

• Handling of potentially infectious materials

• Laboratory design, including airflow conditions

• Use of biological safety cabinets (BSC) (operation, identification of malfunctions, maintenance)

• Use of autoclaves, incubators (operation, identification of malfunctions, maintenance)

• Prevention of incidents and steps to be taken by workers in the case of incidents (biohazard incidents, chemical, electrical and fire hazards)

• Good laboratory practice and good microbiological techniques

• Organization of work flow and procedures

• Waste management

• Importance of laboratory results for patient management

• Importance of laboratory results for the national TB programme

• Training shall be given before a staff member takes up his/her post

• Repeat training periodically, preferably every year

Biological safety cabinets

• Switch ON the safety cabinets for at least 30 min before use. Note that the reading on the mini gauge pressure is satisfactory.

• Wear double pair of gloves, every time you work inside the cabinet.

• Biosafety cabinets need to be cleaned with 5% phenolic or 1% hypochlorite solution before work.

• Keep disposal bin/vessel with 5% phenolic or 5% hypochlorite disinfect inside the cabinet at right side corner.

• Wipes of Gauge-cloth soaked in 5% phenolic or 5% hypochlorite, should be readily available inside the cabinet.

• Arrange all un-infected material required towards left side.

• All the processed samples need to be arranged right side.

• Do not process more than six specimens at a time, inside the cabinet.

• After completion of work, wipe off the surface with 5% phenolic solution, and discard all wipes in biohazard bags, or in disposal container meant for infectious materials.

• Discard off the outer glove, too, inside the bio-safety cabinet.

• Wipe off inner glove with disinfectant before touching anything else in the laboratory.

Waste disposal and handling

All infectious waste should be discarded in the bio-safety disposal bin. All infectious solid waste-wipes, swabs, plastic, paper towels, gauze pads, gloves, etc., should be placed inside the double autoclave bags, sealed with autoclave tape and sterilized at 121°C for 30 min in the autoclave.

Liquid waste, in the steel discarding bins, should be disinfected in 5% phenol for at least 1 h, before sealing the caps and autoclaved at 121°C for 30 min.

Accidents and spillages

1. Spills inside biological safety cabinets

   All workers using the bio-safety cabinets should keep absorbent materials (gauge cloth/adsorbent sheet) and 5% phenol within the cabinet.

   • Alert all people in lab of immediate area of in the event of spill

   • Spread 5% phenol soaked wipe immediately, while the BSC continues to operate. Wait for 15-20 min

   • Use paper towels to wipe up the spill, working from the edges into the center

   • Decontaminate equipment: Items that are not readily or easily surface decontaminated should be carefully placed into autoclave bags and removed for further treatment (e.g., decontamination by autoclaving)

   • Contaminated gloves and clothes (sleeves are most likely to be contaminated); remove and decontaminate the lab coat by autoclaving or soaking in decontaminant

2. Spills outside containment room in the biological safety cabinets

   Spills on equipment (such as vortex, centrifuge, incubator, refrigerator etc), laboratory benches, walls, or floors:

   • Immediately indicate to all personnel working in the lab, and evacuate for 1 h to allow dissipation of aerosols created by the spill (negative air pressure system would clear the aerosols)

   • Leave the BSC operating and cultures inside cabinet

   • Leave the containment facility following exit procedures

   • Close laboratory doors and post-warning signs to prevent others from entering the laboratory

   • Thoroughly wash hands and other apparently contaminated areas with soap and water. Put on clean disposable gloves

   • If personal clothing is contaminated, remove all outer clothing and place it in the autoclave or container for autoclaving. Put on clean garments

   • Upon returning to the laboratory wear the N95 mask, fresh lab coat and double pair gloves to start decontamination, cover the spill area with paper towels soaked in 5% Phenol solution or 1:10 dilution of 20% bleach (freshly prepared), or 70% ethanol solution (do not pour decontamination solution directly onto the spill in order to avoid additional release of aerosols)

   • Let stand for 20 min then wipe up with paper towels

   • Wipe up the spill with the soaked paper towels and place the used towels in an autoclave bag and autoclave

   • Place gloves and paper towels in autoclave bag and autoclave

   • Spill inside the centrifuge bucket/tube: Always use the aerosol containment cups for centrifuging. Always open the centrifuge buckets inside the bio-safety cabinet. Autoclave the buckets

   • Wash hands and other apparently contaminated areas again with soap and water

3. Don't

   • Eating, drinking, smoking, applying cosmetics, use of mobile phones, or applying contact lenses in the TB laboratory

   • Do not allow unauthorized personnel to enter the TB laboratory

   • Mouth pipetting

   • Crowding of lab with material that is not required inside

Sample

• Clinical sample for direct MODS.

• Pure cultures of acid - fast bacilli grown on solid media/liquid medium, for indirect MODS.

Equipment and materials

• BSC, class II, annually certified

• Inverted light microscope

• Incubator set at 37°C

• Autoclave

• Weighing balance

• Refrigerator/freezer

• Vortex

• Micropipettes (1000 μl, 200 μl, and 20 μl)

Reagents and solutions preparation

• Sodium hydroxide (NaOH) (Merck, MB1M61-321)

• Tri-sodium citrate (Na ₃ C ₆ H ₅ O ₇·2H ₂ O) (Fisher Scientific, 14005)

• N-acetyl-l-cysteine (NALC) (C ₅ H ₉ NO ₃ S) (Central Drug House, 011011)

• Potassium dihydrogen phosphate (KH ₂ PO ₄ ) (Merck, 1048729025)

• Di-sodium hydrogen phosphate anhydrous (Na ₂ HPO ₄ ) (Merck, MG7M571666)

• Di-methyl sulfoxide (Sigma, 472301)

• Glycerol Reagent Grade (Merck, 356350)

• Tween-80 (Merck, SI9S590405).

Drugs

• RIF (Sigma, ref. R7382)

• INH (Sigma, ref. I3377).

Media

• Middlebrook 7H9 broth (ref. 271310-500 g-Becton Dickinson) to be supplemented with glycerol, OADC (ref. 211886 - 10× 20 ml - Becton Dickinson) and PANTA (ref. B4345114 BD Diagnostic Systems).

Other consumables

• Sterile micropipette tips

• Sterile microcentrifuge tubes

• Screw cap glass tubes

• Sterile plastic tubes

• 24-well plates

• Storage vials

• Millipore filters, 0.2 μm filters

• Parafilm or Zip lock bags

• 50 ml polypropylene centrifuge tubes.

NaOH-NALC solution preparation

4% NaOH and 2.9% Na citrate solution with 0.5% NALC (decontamination/processing solution)

• 4% Sodium hydroxide

• 2.9% Sodium citrate dehydrate or 2.6% sodium citrate anhydrous

• NALC

The sodium hydroxide and sodium citrate may be mixed as given in Table 1, sterilized and stored in sterile screw cap bottle for use. After NALC has been added, the prepared volume of digestant must be used within 24 h, as NALC loses mucolytic activity on standing for long. For preparation of NaOH-NALC solution [Table 1].

Table 1 Preparation of sodium hydroxide-N-acetyl-l-cysteine buffer

Preparation of 0.067 M phosphate buffer (pH 6.8)

1. Disodium phosphate: Dissolve 9.47 g of anhydrous Na ₂·HPO ₄ in 1 l of distilled water (DW).

2. Mono-potassium phosphate: Dissolve 9.07 g of KH ₂ PO ₄ in 1 l of DW.

   • Mix 950 ml of solution A with 950 ml of solution B and stir; keep back 50 ml of each solution to adjust pH if necessary

   • If final buffer requires pH adjustments, add solution (A) to raise the pH or solution (B) to lower it

Final 7H9-OADC and 7H9-OADC-PANTA medium preparation

1. 7H9-OAD

   • OADC: Enrichment supplement (oleic acid, albumin, dextrose, and catalase): Commercial preparation comes ready for use. OADC should be used at 10% in 7H9 medium.

   • *You can add the OADC after autoclave of the 7H9 medium and cooling down. Add 100 ml OADC to 900 ml of 7H9 medium

2. 7H9-OADC-PANTA

   • PANTA: Antibiotic supplement used to minimize contamination of MODS culture by oral flora micro-organisms not killed during the decontamination process. The BBL MGIT PANTA vial contains a lyophilized mixture of antimicrobial agents

   • Reconstitute a lyophilized vial of BBL MGIT PANTA with 3 ml of sterile DW [Table 2].Final media preparation should only be performed on the day of using 7H9 medium, OADC and PANTA

Table 2 Polymyxin, amphotericin, nalidixic acid, trimethoprim, azlocillin concentration after reconstitution

Set out

• For every sample: 1 Tube with 4.5 ml 7H9 medium +0.5 ml OADC for every sample/or 5 ml 7H9-OADC already prepared [Figure 1]

• For the medium control (MC) column: 1 Tube with 4.5 ml 7H9 medium for every plate +0.5 ml OADC/or 5 ml 7H9-OADC already prepared

• For the quality control (QC): 2 Tubes with 4.5 ml 7H9 medium +0.5 ml OADC/or 5 ml 7H9-OADC already prepared (1 for H ₃₇ Rv and 1 for MDR strain)

• For drug solution preparation - 2 tubes/sterile micro centrifuge tube (MCT's) with 990 μl 7H9-OADC [Table 3].

• Reconstitute PANTA in 3 ml sterile DW and add 0.1 ml to each sample tube and to the MC tubes (7H9-OADC-PANTA: Total volume = 5.1 ml).

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Figure 1

Preparation of medium tubes

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The final volume of 7H9-OADC and 7H9-OADC-PANTA required will vary according to the number of plates being set-up. (Note: Complete medium 7H9-OADC-PANTA is used for samples and MCs. Use 7H9-OADC without PANTA for QCs and for antibiotic solution preparation.)

For preparation of drug stock and drug dilutions

• Take 990 μl of 7H9-OADC in each of the two micro-centrifuge tubes (MCT-1 and MCT-2)

• Add 10 μl of INH stock (0.4 mg/ml) solution to MCT-1 (total volume 1000 μl); mix well. This gives working concentration (4 μg/ml) of INH.

• Add 10 μl on RIF stock (1 mg/ml) solution to MCT-2 (total volume 1000 μl); mix well. This gives working concentration (10 μg/ml) of RIF.

• The tubes are ready for further use [Tables 3 and 4].

Table 3 Preparation of drug dilutions to be used in medium

Table 4 Preparation of drug stock

Quality control

Well characterized TB strains: One fully susceptible strain (H₃₇Rv standard strain) and one MDR strain (if possible) are run as QC. For each batch of MODS plate being set-up 1 batch of QC is run. If the controls do not show the expected pattern, the results of samples plated on that day are not valid.

1. Requirements

   • Susceptible control strain

   • MDR control strain

   • 7H9-OADC, 5 ml

   • 10% sterile Tween 80, 40 μl

   • Sterile DW, 10 ml

2. Procedure

   • Mix 10 ml sterile DW and 40 μl of 10% sterile Tween 80 in a sterile tube (final Tween 80 concentration = 0.04%)

   • Using a sterile loop, harvest several colonies of mycobacteria and place in a sterile tube containing 100 μl water-Tween 80 solution and sterile glass beads (do not scrape the medium)

   • Cap tube tightly and vortex for 2 - 3 min; (till there are no visible clumps)

   • Let it stand for 5 min

   • Open tube and add 3 ml of water-Tween 80; cap tightly and vortex again for 20 s (till suspension has uniform turbidity). Let it stand for 30 min

   • Transfer the supernatant to another sterile tube using a pipette

   • Adjust turbidity to McFarland Scale 1 (approximately 3 × 10⁸ CFU/ml) with 0.04% water-Tween 80 solution

   • The growth suspension can be used up to 4 weeks (keep at 2-8°C)

3. Preparation of inoculum

   • Mix 5 μl of each McFarland 1 (McF 1) control strain suspension with 5 ml of 7H9-OADC medium. This is the QC suspension ready for plating. The culture suspension for both susceptible strain and MDR strains is prepared separately for plating, as shown in Figure 2

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Figure 2

Preparation of inoculum tubes for direct microscopic observation drug susceptibility assay

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Indirect MODS from culture medium

Pure culture of acid - fast bacilli grown on solid medium is used for indirect MODS. Confirm the growth as M. tuberculosis using MPT-64, PNB or any other standard methods being practiced in the local laboratory.

1. Requirements

   • 7H9-OADC-PANTA, 5 ml

   • 10% sterile Tween 80, 40 μl

2. Procedure

   • Mix 10 ml sterile DW and 40 μl of 10% sterile Tween 80 in a sterile tube (final Tween 80 concentration = 0.04%)

   • Using a sterile loop, harvest several colonies of Mycobacteria and place in a sterile tube containing 100 μl water-Tween 80 solution and sterile glass beads (do not scrape the medium)

   • Cap tube tightly and vortex for 2-3 min; (till there are no visible clumps)

   • Let it stand for 5 min

   • Open tube and add 3 ml of water-Tween 80; cap tightly and vortex again for 20 s (till suspension has uniform turbidity). Let it stand for 30 min

   • Transfer the supernatant to another sterile tube using a pipette

   • Adjust turbidity to McFarland Scale 1 (approximately 3 × 10 ⁸ CFU/ml) with 0.04% water-Tween 80 solution. (Note: Preparation of McF 1 suspension involves manipulation of concentrated suspensions of mycobacteria and should only be carried out in a BSC)

3. Preparation of inoculum

   • Mix 5 μl of each McF 1 growth suspension with 5 ml of 7H9-OADC-PANTA medium. This is the final culture suspension ready for plating, as shown in Figure 3.

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Figure 3

Preparation of inoculum tubes for indirect microscopic observation drug susceptibility assay

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For direct MODS from clinical sample

Clinical sample is used. Sample decontamination is must to avoid bacterial/fungal growth in plate being set-up. Decontamination is carried out using the sodium hydroxide-N-acetyl-l-cysteine (NaOH-NALC). NALC is a mucolytic agent; it must be added to the sterile NaOH-Na citrate solution on the day of use, as activity wanes, if stored for long. The sodium citrate in the NaOH solution prevents heavy metal ions that may be present from deactivating the NALC.

1. Samples not requiring decontamination

   The following specimens usually do not need decontamination when aseptically collected into sterile containers:

   • Spinal or other internal body fluids but should be collected aseptically

   • Bone marrow aspirate if collected aseptically

   • Pus from closed cold abscesses such as fine needle aspiration cytology (FNAC) material

   • Surgically resected specimens (excluding autopsy material)

   • Material obtained from pleural, liver and lymph nodes as well as biopsies (if not fistulised).

2. Requirements for one sample

   • NaOH-NALC solution, 3-5 ml

   • 0.067 M phosphate buffer (pH 6.8), 45 ml

   • 7H9-OADC-PANTA, 5.1 ml

3. Procedure for decontamination of sputum using N-acetyl-l-cysteine sodium hydroxide (NaOH-NALC): Modified Petroff's Method

   The mucolytic agent, NALC is used for rapid digestion of sputum this enables the decontaminating agents like NaOH, to be used at a lower concentration (in sputum).

   • Transfer a maximum volume of 3-5 ml of specimen to a sterile 50 ml centrifuge tube (aerosol free and graduated).

   • Add equal volume of NALC-NaOH-Na citrate solution aseptically.

   • Mix the control for approx. 20 s on vortex mixture. Be sure to invert the tube so that NALC-NaOH comes in contact with the entire surface of the tube.

   • Allow the mixture to stand at room temperature for 15 min to decontaminate the specimen with occasional gentle shaking.

   • Neutralize the mixture by adding sterile phosphate buffer (pH 6.8) up to the 50 ml in the tube and recap it.

   • Centrifuge the tube at 3000g for 15 min.

   • Decant the supernatant.

   Sputum → NAOH/NALC decontamination → re-suspend the pellet in 2 ml 7H9-OADC-PANTA (900 μl will be plated).

4. Inoculum preparation

   Using 7H9-OADC-PANTA (from the tube containing 5.1 ml), re-suspend the sample pellet in a total volume of 2 ml in the centrifuge tube with a Pasteur pipette; mix well.

   • Remove 1 ml of sample suspension and store in a storage vial at 2-8°C as a backup.

   • Add the remaining 1 ml of sample suspension to the tube with the remaining 7H9-OADC-PANTA; mix well. This is the final sample suspension ready for plating.

For sample/culture

• Take a 24 well plate (for upto five samples).

• Mark the wells on plate: Growth control (GC), MC, INH, RIF, as shown in [Figure 4].

• Dispense 900 μl of culture/sample suspension in all 4 wells of column 1 to 6 sequentially, except column 4.

• Dispense 900 μl of MC in all 4 wells of column 4.

• Dispense 100 μl of working solution (WS) of INH in all wells of row C from MCT-1

• Dispense 100 μl of WS of RIF in all wells of row D from MCT-2.

• Add 100 μl remaining MC to rest of all the wells of row A and B to make final volume of 1 ml in each well.

• Close the plate with its lid and seal it with parafilm from all sides or zip lock bag.

• Incubate at 37°C. (Note: If more than five samples or cultures are being tested use another plate. It is advisable not to test samples/cultures on the same plate on which QC strains are being run to avoid cross-contamination.)

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Figure 4

Setting up of microscopic observation drug susceptibility assay plate for sample/culture

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For quality control

For each batch 1 set of separate QC (1 H ₃₇ Rv and 1 MDR) plate is run.

• Take a 24 well plate

• Mark the wells on plate: H ₃₇ Rv, MC, MDR, INH and RIF, as shown in Figure 5

• Dispense 900 μl of suspension of H ₃₇ Rv in all four wells of column 1

• Dispense 900 μl of suspension of MDR in all four wells of column 3

• Dispense 900 μl of MC in all 4 wells of column 2

• Dispense 100 μl of WS of INH in wells 1-3 of row C from MCT-1

• Dispense 100 μl of WS of RIF in wells 1-3 of row D from MCT-2

• Add 100 μl remaining MC to rest of all the wells of row A and B to make final volume of 1 ml in each well

• Close the plate with its lid and seal it with parafilm from all sides or place in a zip lock bag

• Incubate at 37°C

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Figure 5

Setting up of microscopic observation drug susceptibility assay plate for quality control strains

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Plate reading of GC/drug-free wells

• Start examining drug-free wells on day 5.

• Early mycobacterial growth looks like small curved commas or spirals (days 5-9).

• Confirm the growth as M. tuberculosis using MPT-64, PNB or any other standard method. Reading should be followed only if the growth is MTB (not required in case of indirect MODS).

• Colony formation usually progresses to cords, and later more irregular tangled growth.

• If two or more colonies (>2 cfu) are detected in each of the two wells, the result is positive.

• If results are negative on day 5, continue reading drug-free wells daily (or on alternate days according to laboratory workload) until >2 cfu are observed in each of the two wells.

• When a positive result is observed, read the INH and RIF containing wells on the same day.

• If no growth is observed by day 15, repeat reading on day 18 and day 21. If results are still negative on day 21 the final result is negative.

• If only 1 cfu appears in either drug-free well, or in both, the result is “indeterminate”. [Table 5]

Table 5 Reading and interpretation of drug.free wells

Reading of medium control

• All four wells in of MC should have no growth.

• If any mycobacterial colonies are observed in any well, there has been cross-contamination. The entire plate should be discarded.

• A search for potential sources of cross-contamination should occur and if the source is identified appropriate, remedial action should be taken.

Reading of quality control wells

1. Drug-free wells

   • All drug-free wells should have positive mycobacterial growth (>2cfu).

   • Absence of growth in all drug-free wells suggests that the medium does not support growth sample results are not valid. All samples plated out on the same day should be reprocessed with a new batch of medium.

   • If only one of the two positive control strains grows in drug-free wells, the strain that did not grow may not be viable. A fresh strain suspension should be used with re-testing.

2. Drug-containing wells

   • The drug-susceptible control strain should not grow in either of the drug-containing wells. Growth indicates incorrect (low) antibiotic concentrations or inadequate INH and/or RIF activity.

   • The drug-resistant control strain (1 MDR strain, or 2 mono-resistant strains) should grow in the drug-containing wells. Absence of growth indicates that the final INH and/or RIF concentrations are too high. ( Notes: Absence of control strain growth in all drug-free wells may also indicate strain non-viability. Consider use of fresh control strain preparations for re-testing.)

   • Reduced drug activity may be due to incorrect concentrations, or reduced potency related to improper handling or storage of original, undiluted, drug or drug stock solution.