Rapid Identification and Drug Susceptibility Testing of Mycobacterium tuberculosis: Standard Operating Procedure for Non-Commercial Assays: Part 2: Nitrate Reductase Assay v1.3.12

Medical fitness

In accordance with the 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 about 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 workflow

• Procedures

• Waste management

• Importance of laboratory results for patient management

• Importance of laboratory results for the national TB program

• 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 the 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 the 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 the inner glove with disinfectant before touching anything else in the lab

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.

Preparation of the media

1. Homogenized whole eggs

   Fresh hens' eggs, not more than 7 days old, are cleaned by scrubbing thoroughly with a hand brush in warm water and plain alkaline soap. Let the eggs soak for 30 min in the soap solution. Rinse eggs thoroughly in running water and soak them in 70% ethanol for 15 min. Before handling the clean dry eggs scrub the hands and wash them. Crack the eggs with a sterile forceps into a sterile flask and beat them with a sterile egg whisk or with a sterile blender.

2. Complete medium

   The following ingredients are aseptically pooled in a large, sterile flask, and mixed well:

   • Mineral salt solution, 600 ml

   • Malachite green solution, 20 ml

   • Homogenized eggs (20-25 eggs, depending on size), 1000 ml

   Divide medium in three different portions: Nitrate control, PNB control, and drug-containing medium.

   Nitrate control: Potassium nitrate stock 0.2 g/ml - add 5 ml of stock to 1000 ml of medium.

   PNB control: PNB stock 0.2 g/ml - add 2.5 ml of stock to 1,000 ml of medium.

3. Drug-containing medium

   Divide this portion again in two parts and add drugs to medium as per given in Table 2 as well as add potassium nitrate.

   The complete egg medium is distributed in 6-8 ml volumes in sterile 28 ml McCartney bottles or in 20 ml volumes in 20 × 150 mm screw-capped test tubes and the tops are securely fastened. Inspissate the medium within 15 min of distribution to prevent sedimentation of the heavier ingredients.

4. Coagulation of medium

   Before loading, heat the inspissator to 80°C to quicken the build-up of the temperature. Place the bottles in a slanted position in the inspissator and coagulate the medium for 45 min at 80-85°C (since the medium has been prepared with sterile precautions this heating is to solidify the medium, not to sterilize it). Heating for a second or third time has a detrimental effect on the quality of the medium. The quality of egg media deteriorates when coagulation is done at too high a temperature or for too long. Discoloration of the coagulated medium may be due to excessive temperature. The appearance of little holes or bubbles on the surface of the medium also indicates faulty coagulation procedures. Poor quality media should be discarded.

Sterility check

After inspissation, the whole media batch or a representative sample of culture bottles should be incubated at 35 - 37°C for 24 h as a check of sterility.

Storage

The L-J medium should be dated and stored in the refrigerator and can be kept for several weeks if the caps are tightly closed to prevent drying out of the medium. For optimal isolation from specimens, L-J medium should not be older than 4 weeks.

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).

For samples requiring decontamination process the specimen Process the specimen with sodium hydroxide-N-acetyl-l-cysteine (NaOH-NALC) modified Petroff's method.

Reagents required

• 4% NaOH

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

• NALC

• Anhydrous disodium hydrogen phosphate (Na ₂ HPO ₄ )

• Mono-potassium dihydrogen phosphate (KH ₂ PO ₄ )

• DW

Procedure for decontamination of sputum using sodium hydroxide-N-acetyl-l-cystein: Modified Petroff's method

1. Preparation of NaOH-NALC

   • The mucolytic agent, NALC is used for rapid digestion of sputum and this enables the decontaminating agents like NaOH, to be used at a lower concentration (in sputum) of 1%

   • The NaOH and sodium citrate may be mixed as given in Table 3, 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. (Note: Acetyl-cysteine loses activity rapidly in solution, so should be made fresh daily. Sodium citrate is included in the digestant mixture to bind the heavy metal ions that might be present in the specimen and could inactivate the specimen and could inactivate the acetyl-cysteine.)

   For preparation of NaOH-NALC solution refer to Table 3.

2. Preparation of 0.067 M phosphate buffer (pH 6.8)

   For stock solutions:

   1. Disodium phosphate: Dissolve 9.47 g of anhydrous Na ₂ HPO ₄ in 1 l of DW

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

      • To prepare pH 6.8 buffer solution, mix 50 ml of (a) with 50 ml of (b) prepared above

      • Check pH

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

Table 3 Preparation of sodium hydroxide-N-acetyl-l-cysteine solution

Sample processing

• 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 approximately 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 of hand

• Dilute the mixture by adding sterile phosphate buffer (pH 6.8) upto the 50 ml mark on the tube and recap it

• Centrifuge the tube at 3000g for 15 min

• Decant the supernatant

Preparation of inoculum

• Re-suspend the pellet with 2 ml phosphate buffer (pH 6.8)

• Prepare 1:10 dilution of re-suspended pellet in 0.067 M phosphate buffer for inoculation of control tubes (add 0.2 ml of the re-suspended pellet in 0.8 ml phosphate buffer)

Inoculation of nitrate reductase assay tubes

• 0.2 ml of the 1:10 dilution is inoculated in three drugs free tubes (control tube containing potassium nitrate)

• 0.2 ml of the undiluted suspension is inoculated into the drug-containing tube

• 0.2 ml of the undiluted suspension is inoculated into the PNB containing tube

• Incubate at 37°C until 14 day

Preparation of 7H9-S

Middle brook 7H9 broth supplemented with 10% oleic acid dextrose catalase and 0.2% glycerol.

Preparation of inoculum

Confirm the growth asM. tuberculosis (MTB) using either PNB or MPT-64 rapid test or other methods being practiced in the local laboratory.

1. Inoculum from growth on solid medium

   It is very important to have fresh growth on a solid medium (21-28 days old). Older cultures may result in unreliable susceptibility test results.

   • Take a loop full of bacterial growth with a sterile loop and put it in a sterile vial with glass beads just covering the bottom of the vial with 2.5 ml of 7H9-S broth (try not to take any medium when removing growth)

   • Vortex the vial for at least 1 min to break the clumps until a fairly turbid suspension is obtained.

   • Allow it to stand for 5 min

   • Transfer the supernatant to a new sterile vial and leave it to sediment for 15 min

   • Transfer the supernatant to a new sterile vial

   • Compare the turbidity of the suspension using the scale McFarland 1.0 standard

   • Set the turbidity of inoculum as per McFarland 1.0 standard using 0.067 M phosphate buffer (pH 6.8)

2. Inoculum from a liquid medium

   It is very important to have fresh growth in liquid medium (21-28 days old). Older cultures may result in unreliable susceptibility test results.

   • Transfer 2.5 ml of the culture pellet to a sterile vial containing glass beads

   • Vortex for at least 1 min until a fairly turbid suspension is obtained

   • Allow it to stand for 5 min

   • Transfer the supernatant to a new sterile vial and leave it to sediment for 15 min

   • Transfer the s upernatant to a new sterile vial

   • Compare the turbidity of the suspension using the scale McFarland 1.0 standard

   • Set the turbidity of inoculum as per McFarland 1.0 standard using 0.067 M phosphate buffer (pH 6.8)

Dilution of the inoculum

The inoculum turbidity (from liquid or solid culture) is adjusted to a McFarland tube no. 1 and further diluted to 1:10 in 0.067 M phosphate buffer (pH 6.8).

Inoculation of nitrate reductase assay tubes

• 0.2 ml of the 1:10 dilution is inoculated in three drugs free tubes (control tubes containing potassium nitrate)

• 0.2 ml of the undiluted suspension will be inoculated into the drug-containing tube. Incubate at 37°C until 7 days. (Note: Inoculation onto PNB tube is not required in case of Indirect NRA.)

Reading of tubes

Reagents mixture: One part of 50% (v/v) concentrated HCl, two parts of 0.2% (w/v) sulfanilamide, and two parts of 0.1% (w/v) n-1-naphthylethylenediamine dihydrochloride. They will be mixed shortly before use.

After 7 days of incubation, 0.5 ml of a reagent mixture will be added to one drug free tube. If any color occurs, the corresponding antibiotic containing tube will be developed by the reagent mixture. If no color change occurs, the tube will be discarded and the other tubes will be re-incubated. The procedure will be repeated at day 10 using the second growth control tube, and if necessary, also at day 14 using the third growth control tube.