Cat. no. TB100 TB MODS Kit 100 tests/kit

Transcription

1 TB MODS KIT Cat. no. TB100 TB MODS Kit 100 tests/kit Each kit contains: Z29A Middlebrook 7H9 Broth + OADC, 5ml with white cap Z29B Deionized Water, 2.5ml (green dot with imprinted N) Z29C Middlebrook 7H9 Broth + OADC, 3ml (5 red dot with imprinted R, 5 blue dot with imprinted I) Z29D Middlebrook 7H9 Broth + OADC, 5ml with blue cap Yellow dot on lid and vial Pink dot on lid and vial Orange dot on lid and vial Z29I Isoniazid Tablets, 5 per vial (blue dot with imprinted I) Z29R Rifampicin Tablets, 5 per vial (red dot with imprinted R) Z29N NAPTA Tablets, 5 per vial (green dot with imprinted N) Z29E Silicone Safety Lid (autoclaved) P52000 Tissue Culture Plate, 24-well R55800 Orientation Card 100 vials 5 vials 10 vials 40 vials 10 vials 15 vials 15 vials 1 vial 1 vial 1 vial 25 lids 25 trays 1 card INTENDED USE Hardy Diagnostics TB MODS (Microscopic Observation Drug Susceptibility Assay) Kit is recommended for use as an accelerated culture and direct drug susceptibility test (DST) method for detection of drug-susceptible, monoresistant, and multi-drug resistant tuberculosis (MDR-TB). SUMMARY One-third of the world's population is currently infected with Mycobacterium tuberculosis. 5-10% of people who are infected with the TB bacillus become ill or infectious at some time during their life. Patients co-infected with both HIV and TB are much more likely to develop active TB disease. Almost every country in the world has reported strains of M. tuberculosis that are resistant to at least one of the anti-tb drugs. Drug-resistant TB can develop as the result of inconsistent or partial treatment of the infection, or if the wrong treatment regimens are prescribed. These drug-resistant strains can then be transmitted to others. A strain is designated as multi-drug resistant (MDR) when the organism is resistant to both isoniazid (INH) and rifampicin (RIF), the two most potent and widely used anti-tb drugs. (1) Monoresistant strains (e.g. resistant to either INH or RIF but not both) can also occur. In 2008, the World Health Organization reported the current prevalence of INH resistance as 10.3% for new cases and 27.7% for previously treated cases. (2) In some instances rifampicin monoresistant strains have been linked to patients who are incarcerated, infected with HIV, or with poor intestinal absorption of TB medications. In addition, patients infected with rifampicin monoresistant strains are more likely to have extrapulmonary infection. Treatment of rifampicin monoresistant strains is challenging because alternate drugs can be difficult to administer, are associated with numerous adverse effects, and patient compliance is difficult to obtain. (3) MDR strains that are also resistant to any fluoroquinolones and at least one of the three following injectable drugs: capreomycin, kanamycin, or amikacin, are termed extensively drug-resistant (XDR). In 2008, the World Health Organization reported that strains of XDR M. tuberculosis were found in at least 45 countries. (4) km TB MODS Kit Page 1 of 16

2 The majority of diagnosed TB cases occur in developing countries where resources are scarce. In these settings, the diagnosis of tuberculosis may be limited to performing microscopic examination of sputum smears, for which the sensitivity of detection is low (estimated at only 50% sensitive). (4,5) The Hardy Diagnostics TB MODS Kit offers an accelerated, cost effective, liquid culturebased diagnostic tool that can aid in the diagnosis of susceptible, monoresistant and multi-drug resistant tuberculosis. Growth of TB is obtained faster than with conventional testing and drug susceptibility results are obtained concurrently. The MODS method has been reported to have a sensitivity of 97.8% and specificity of 99.6%. (5) The kit contains all of the necessary media, supplements, and antibiotics in an easy-to-use format. A flexible silicone lid used to seal the trays containing patient specimens, during incubation and examination under the microscope, provides an increased level of safety. The lid can be easily pierced with a syringe if subculture is necessary. A second kit, Hardy Diagnostics TB DeCon Red (Cat. no. Z30), contains all of the reagents necessary for required digestion and decontamination of patient sputum specimens. The Microscopic Observation Drug Susceptibility Assay for Tuberculosis (MODS method) was developed by researchers from the Universidad Peruana Cayetano Heredia, Peru, John Hopkins University, and Imperial College, London. The MODS technology has been in use since 2000 and is a core component of the Peruvian National TB Control policy. The MODS test system is based on three principles: 1) growth of Mycobacterium tuberculosis in liquid media is faster than on solid media, 2) the morphology of M. tuberculosis in liquid culture is characteristic and easy to recognize since M. tuberculosis demonstrates visually observable "cording" of the bacterial cells, and 3) reliable direct drug susceptibility testing is performed simultaneously without the need for subculture. (5) The MODS procedure utilizes sterile, plastic 24-well culture plates. After processing the patient sputum specimen, the plate wells are filled with supplement-enriched Middlebrook 7H9 broth and decontaminated sputum (four wells per sample). Two of the plate wells also have antibiotic solutions (rifampicin and isoniazid) added to them at the time of inoculation. The plate wells are permanently sealed with a silicone sealing lid and incubated. Periodically during incubation the plate wells are examined using an inverted light microscope. Positive results are identified by growth with cord formation (which is characteristic of M. tuberculosis) in the drug-free control wells. Nontuberculous mycobacteria are recognized by their lack of cording. M. chelonae (the only nontuberculous mycobacteria that forms cords but is rarely isolated from sputum) demonstrates rapid overgrowth by day five of incubation and thus is readily distinguishable. If growth occurs in the drug-free wells and in the wells containing either isoniazid or rifampicin, the organism is drug-resistant. If an isolate is identified by the MODS method as being multi-drug resistant, further drug susceptibility testing of extended first and second line agents is recommended to guide appropriate therapy. In one study, 99.5% of positive MODS cultures were identified within two weeks, whereas only 34% of Lowenstein-Jensen (LJ) cultures of the same samples were found to be positive within the same time period. (4) In 2006, Moore et al. compared the results of MODS testing with two reference methods: automated mycobacterial culture and culture on Lowenstein-Jensen medium. They found that the sensitivity of detection was 97.8% for MODS culture, 89% for automated mycobacterial culture, and 84% for Lowenstein- Jensen culture. The median time to positivity was 7 days, 13 days, and 26 days, respectively. Agreement between MODS and the reference standard for susceptibility test results was 100% for rifampicin, 97% for isoniazid, and 99% for rifampicin and isoniazid (combined results for multi-drug resistance). (5) The medium used in the MODS procedure is Middlebrook 7H9 Broth Base. The 7H9 basal medium contains glycerol, biotin and sodium citrate. Glycerol provides an abundant source of carbon and energy for the tubercle organisms. Biotin helps stimulate the revival of damaged organisms as well as being involved in a variety of carboxylation and decarboxylation reactions. Sodium citrate, when converted to citric acid, holds inorganic cations in solution. The basal medium of 7H9 broth is supplemented with Middlebrook OADC Enrichment. The supplementation provides nutrients necessary for mycobacterial growth. OADC Enrichment contains the following required additives: albumin to protect the tubercle bacilli against toxic agents; oleic acid, a fatty acid utilized in the metabolism of the organism; sodium chloride to maintain osmotic equilibrium; catalase to destroy any toxic peroxides in the medium; and dextrose as an energy source. The antibiotics NAPTA, INH (Isoniazid), and RIF (Rifampicin) are provided in easy-to-use tablet form and are packaged in vials which have a desiccant included in the vial lid. NAPTA (an antibiotic mixture containing Nalidixic acid, Azlocillin, Polymyxin B, Trimethoprim and Amphotericin B) is added to the supplemented 7H9 broth to suppress the growth of contaminants. INH and RIF are the two most potent anti-tb drugs. The final working concentration of the anti-tb drugs in the plate wells is 0.4µg/ml for INH and 1µg/ml for RIF. Growth in either of the wells containing these concentrations of drugs indicates resistance. Growth in both INH and RIF containing wells indicates the isolate is MDR. Hardy Diagnostics TB DeCon Red Kit (sold separately Cat. no. Z30) is recommended for the digestion and decontamination of clinical sputum specimens suspected to contain mycobacteria, especially Mycobacterium tuberculosis. Sodium hydroxide (NaOH), in the TB Base Digestant Red, acts as an emulsifier and a decontaminant, breaking down mucoid material and inhibiting the growth of contaminants. A ph indicator is used in the TB Base Digestant Red to insure that the proper ph is maintained. TB Base Digestant Red will remain pink during the decontamination steps, and turn clear after the solution has been neutralized by the addition of 1M hydrochloric acid (HCl). N-Acetyl-L-Cysteine (NALC), when combined with NaOH, facilitates decontamination by further digesting mucopurulent specimens which allows the NaOH to penetrate. Sodium citrate, in the TB Base Digestant Red, aids in liquefaction by km TB MODS Kit Page 2 of 16

3 binding heavy metals, thus stabilizing NALC and allowing it to work properly. Phosphate Buffer lowers the specific gravity of the specimen and gently neutralizes the ph of the specimen after decontamination. FORMULA Ingredients per liter of deionized water:* Middlebrook 7H9 Broth: Disodium Phosphate Monopotassium Phosphate L-Glutamic Acid Ammonium Sulfate Sodium Citrate Magnesium Sulfate Ferric Ammonium Citrate Zinc Sulfate Copper Sulfate Pyridoxine Calcium Chloride Biotin Glycerol OADC Enrichment: Bovine Albumin Dextrose Sodium Chloride Beef Catalase Oleic Acid Isoniazid Tablet Isoniazid Rifampicin Tablet Rifampicin NAPTA Tablet Nalidixic Acid Amphotericin B Polymyxin B Trimethoprim Azlocillin 2.5gm 1.0gm 0.5gm 0.5gm 0.1gm 50.0mg 40.0mg 1.0mg 1.0mg 1.0mg 0.5mg 0.5mg 2.0ml 5.0gm 2.0gm 0.85gm 4.0mg 50.0mg 4µg/ml 10µg/ml 800µg/ml 200µg/ml 2000U/ml 200µg/ml 200µg/ml Final ph 6.8 +/- 0.2 at 25 degrees C. * Adjusted and/or supplemented as required to meet performance criteria. STORAGE AND SHELF LIFE Storage: Upon receipt store at 2-8 C. away from direct light. Product should not be used if there are any signs of deterioration, contamination, discoloration, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing. The expiration date applies to the product in its intact packaging when stored as directed km TB MODS Kit Page 3 of 16

4 This product has the following shelf life from the date of manufacture: 320 Days: TB100 TB MODS Kit Refer to the document "Storage" on the Hardy Diagnostics Technical Document website for more information. PRECAUTIONS This product may contain components of animal origin. Certified knowledge of the origin and/or sanitary state of the animals does not guarantee the absence of transmissible pathogenic agents. Therefore, it is recommended that these products be treated as potentially infectious and handled observing the usual universal blood precautions. Do not ingest, inhale, or allow to come into contact with skin. This product is for in vitro diagnostic use only and is to be used only by adequately trained and qualified laboratory personnel. Observe approved biohazard precautions and aseptic techniques. All laboratory specimens should be considered infectious and handled according to "standard precautions". The "Guideline for Isolation Precautions" is available from the Centers of Disease Control and Prevention at For additional information regarding specific precautions for the prevention of the transmission of all infectious agents from laboratory instruments and materials, and for recommendations for the management of exposure to infectious disease, refer to the Clinical and Laboratory Standards Institute (CLSI) document M29: Protection of Laboratory Workers from Occupationally Acquired Infections--Approved Guideline. Note: Minimum recommended safety requirements include the use of a Biosafety Level 2 (P2) laboratory facility. This includes a well-maintained class II biological safety cabinet in which any recirculated air passes through a HEPA filter. Laboratory space used for TB processing and testing should located in a separate area of the laboratory with well-sealed windows and lockable doors to prevent entry of personnel and creation of air turbulence while specimens are being processed. Furniture should be solid and resist deterioration when disinfectants are applied. Laboratory staff must be trained in biosecurity procedures and the importance of biosafety in the laboratory. Adequate protective clothing including the use of gowns and gloves is mandatory. It is recommended that respiratory protection be used at all times (N95 respirators, Cat. no. USP10206). MODS plates should be handled carefully and properly disposed. Sterilize all biohazard waste before disposal. Refer to the document "Precautions When Using Media" on the Hardy Diagnostics Technical Document website for more information. Refer to the SDS Search instructions on the Hardy Diagnostics website for more information. PROCEDURE Specimen Collection: Collect approximately 5 to 10ml of a sputum specimen in a 50ml screw-cap centrifuge tube (Cat. no. 6289) or graduated disposable sputum cup (Cat. no ). If a larger volume of sputum is collected, the specimen should be separated into 5 to 10ml volumes. Avoid contamination of the specimen with saliva or nasal secretions. Transport specimens to the laboratory as soon as possible. The specimen should be refrigerated if processing will be delayed. Method of Use: All solutions should be prepared in a class II biological safety cabinet before processing specimens and should be prepared using aseptic technique. The directions for performing the test procedures are based on runs containing 20 specimens plus controls when using the 100 test kit and 10 specimens plus controls when using the 50 test kit. Note: Blue capped vials (kit component Z29D) have different color dots on the lid and label. Each vial labeled with colored dots has a special use in the kit. The white capped vials without colored dots (kit component Z29A) will be used for processed patient specimens. A. Antibiotic solutions (prepared fresh each day of testing) Allow antibiotic tablets to come to room temperature before opening the vials km TB MODS Kit Page 4 of 16

5 1. Anti-TB Drugs Select one vial with a blue dot with imprinted I and one vial with a red dot with imprinted R, each contain 3ml of 7H9 + OADC Broth (kit component Z29C). a. INH Using sterile forceps, remove one INH tablet and place it in the vial labeled with the blue dot with imprinted I. b. RIF Using sterile forceps, remove one RIF tablet and place it in the vial labeled with the red dot with imprinted R. 2. NAPTA - Using sterile forceps, remove one NAPTA tablet and place it in a vial containing 2.5ml of sterile Deionized (Distilled) Water labeled with a green dot with imprinted N (kit component Z29B). B. 7H9 Broth + OADC + NAPTA (for patient testing and negative control wells) 1. For each patient specimen, remove a white capped vial containing 5ml of 7H9 + OADC Broth (kit component Z29A). Label the vials with patient names or numbers corresponding to the sputum specimens. 2. Remove two (50 test kit) or three (100 test kit) blue capped vials that are labeled with a pink dot. These vials will be used in the negative control wells. 3. Add 0.1ml of the prepared NAPTA solution to each vial listed in step 1 and step 2. C. 7H9 Broth + OADC (for positive control wells and drug-free wells) 1. Remove one blue-capped vial of 7H9 + OADC Broth labeled with an orange dot (kit component Z29D) to use for each positive control that will be used in the test. Label the vials with the names of the control organisms. 2. Also remove one (50 test kit) or two (100 test kit) blue capped vials of 7H9 + OADC Broth labeled with a yellow dot (kit component Z29D) to be used in the drug-free control wells. Processing sputum specimens: For best results, the following procedure is recommended for the decontamination and digestion of sputum specimens and uses the Hardy Diagnostics TB DeCon Red Kit (Cat. no. Z30). ***Perform all procedures within a biological safety cabinet and wear appropriate Personal Protective Equipment (PPE). (See Precautions Section)*** 1. Prepare digestant solution daily by dissolving 250mg of NALC Reagent (Cat. no. Z30C) in a 50ml bottle of TB Base Digestant Red (Cat. no. X46). Do not vortex. The resulting solution should be pink. Prepare only what can be used in 24 hours. One bottle of X46 is sufficient for 5 to 10 sputum samples. 2. Transfer 5ml to 10ml of sputum specimen into a graduated 50ml, aerosol-proof, screw-capped polypropylene centrifuge tube (Cat. no. 6289). If more than 10ml of specimen is submitted, select 10ml of the most purulent, bloody or mucoid portion km TB MODS Kit Page 5 of 16

6 3. Add an equal volume of digestant solution to the sputum. Avoid touching the lip of the specimen container with reagent bottles. If the specimen is bloody, do not add more than 8ml of digestant solution. The sample should be pink after the addition of the digestant solution. If the solution turns clear add digestant solution until the sample is pink. 4. Tighten caps firmly. Mix the centrifuge tube for no more than 30 seconds on a vortex mixer within the biological safety cabinet. 5. Allow the centrifuge tube to stand at room temperature (15-30 degrees C.) for 15 minutes, to digest and decontaminate the specimen. If the specimen is extremely thick, it can be in contact with the digestion reagent for up to 20 minutes. Do not allow the specimen to be in contact with the digestion reagent longer than 15 minutes if non-mucoid or 20 minutes if mucoid. 6. Open centrifuge tube and fill tube to within 2cm of the top of the tube, with Phosphate Buffer (Cat. no. U192). Avoid touching the lip of the specimen container with the reagent bottle. Recap the tube tightly, and invert several times until solution is mixed. 7. Centrifuge at least 15 to 20 minutes at 3000xg. 8. Under a biological safety cabinet, carefully decant the supernatant into a splash proof container containing a cold sterilant (e.g. a 10% bleach solution sodium hypochlorite). Wipe the lip of the centrifuge tube with disinfectant. Do not allow the disinfectant to enter the tube. 9. Using a small tipped dropper pipet (e.g., transfer pipet, Cat. no. 2121S), titrate with 1M HCl, dropwise, until the color of the sediment changes from red to a persistent yellow. Bloody specimens may remain pink due to residual hemoglobin and can be assumed to be at the proper ph as long as no more than 8ml of digestant solution was used. 10. For each specimen, remove 2ml from a previously labeled white capped 7H9 + OADC + NAPTA vial and add it to the sediment in the centrifuge tube. Set vial aside for use in Step #12 below. 11. Mix sediment well with a transfer pipet (Cat. no. 2121S) and remove 1ml of resuspended sediment to a previously labeled microcentrifuge (Cat. no ) tube as a back-up. Store the back-up sample at 2-8 degrees C. or freeze. 12. Add remaining sediment back to the corresponding (previously used in step 10 above) white capped 7H9 + OADC + NAPTA vial. This tube will be used to inoculate one column of the test wells in the 24-well plate. 13. When all of the specimens have been processed, they can be added to the tissue culture plate (see next section). Adding Antibiotics and Specimens to the Plate: The prepared antibiotic solutions (vials with blue and red dots) and 7H9 + OADC Broth (yellow dot) vials that will be added to the drug-free wells) should be added to the plates in the biological safety cabinet. All processed patient specimens must be added to the plate while working in the biological safety cabinet. Note: The 24-well plate that will be used for the positive controls can also be used initially to aliquot the 7H9 + OADC Broth (yellow dot), INH (blue dot with imprinted I) and RIF (red dot with imprinted R) solutions if a multi-channel pipettor will be used to dispense the material to the 24-well plates (see steps 2, 3, and 4 below). Do not add the positive controls to the plate used for the broth and antibiotics until all patient samples have been added to the plates. 1. Orient the plate so that the letters A, B, C, D are on the left vertical edge and the numbers 1 through 6 are located on the horizontal axis. Post the Orientation card inside the biological safety cabinet for easy reference in setting up the plates. 2. Label the front edge of the 24-well plate with specimen numbers with a waterproof marking pen. 3. Add 100µl of 7H9 + OADC (yellow dot) to rows A and B km TB MODS Kit Page 6 of 16

7 4. Add 100µl of INH solution (blue dot with imprinted I) to row C. 5. Add 100µl of RIF solution (red dot with imprinted R) to row D. 6. Add 900µl of processed sputum specimens (from step 13 above) to the columns. Columns 1, 2, 4, 5, and 6 will each contain a different patient specimen. Column 3 is used as the negative control and it will not contain a specimen. Do not add any specimens to column 3 of each plate. 7. Add 900µl of 7H9 + OADC + NAPTA (pink dot) to column 3. Place silicone sealing lid on each plate and carefully make sure that the lid is sealed firmly on the plate by pressing lightly over each well. Incubate the plates aerobically at 37 degrees C. A CO 2 incubator is not required. Positive Controls - preparation of stock cultures Note: All procedures required to prepare the positive controls must be done within a class II biological safety cabinet. Perform this work after all samples for the day have been plated. Two positive control strains must be run each day specimens are processed; one fully susceptible strain and one MDR strain. If laboratories prefer to avoid handling of MDR strains then two separate strains can be used, one isoniazid monoresistant and the other rifampicin monoresistant. Positive controls test the medium and the quality of the antibiotic solutions used on the day of sample processing. If positive controls do not grow in the expected pattern, the results of samples plated on the same day are not valid. Preparation of Positive Controls and Suspensions: 1. Reconstitute commercially prepared mycobacteria control strains using the manufacturer s instructions. 2. Plate each strain to a separate Petri dish containing Middlebrook 7H11 Agar (Cat. no. W35). 3. Incubate at 37 degrees C. for days. 4. Using a sterile loop, harvest several colonies of mycobacteria and place in TB diluting fluid (Cat. no. R54) containing beads (if not available, mix 10ml of sterile distilled water and 40µl of 10% sterile Tween 80 in a sterile tube. The final concentration of Tween is 0.04%). Cap the tube tightly and vortex for 2-3 minutes; let stand for 5 minutes. 5. Open tube and add contents to a tube (3ml) of Water with Tween (Cat. no. V03); cap tightly and vortex again for 20 seconds. Let stand for 30 minutes. 6. Transfer the supernatant to another sterile tube using a transfer pipette. 7. Adjust the turbidity to a McFarland Standard #1 (Cat. no. ML1) using Water with Tween (Cat. no. V03). 8. The McFarland #1 control suspension can be kept tightly sealed at 2-8 degrees C. for repeated use for up to 15 days. 9. The remaining growth on the Middlebrook 7H11 plate can be replated every four to six weeks to maintain a continuous supply. Alternatively, growth from the plate can be harvested and frozen at -70 degrees C. for long-term storage. Positive Controls - day of use: To minimize the risk of cross-contamination, the positive controls are set up in a separate plate after all plates with samples have been sealed and placed in the incubator km TB MODS Kit Page 7 of 16

8 Procedure: 1. Add 5µl of each McFarland #1 control strain suspension to separate 7H9 + OADC tubes (orange dot). Mix well. 2. Label the front edge of the plate with control strain numbers. 3. Add 900µl of each positive control suspension to the four wells of a column on the positive control plate. 4. For each control strain, add 100µl aliquots from the 7H9 + OADC tubes (yellow dot) and RIF (red dot with imprinted R) and INH (blue dot with imprinted I) antibiotic working solutions as was done for the patient samples. 5. Place sealing lid on the tray, making sure that it is well sealed. Incubate aerobically at 37 degrees C. with the other plates processed the same day. INTERPRETATION OF RESULTS Start examining the drug-free wells (rows A and B) for growth on day 5. Remove the plates from the incubator. Do not remove the silicone sealing lids. Examine wells using an inverted microscope with the 10x microscope objective (100x total magnification) to search for early colony forms. For readings on day 6 and later, use the 4x objective (40x total magnification) to examine the entire contents of each well. Examination of the plates for growth: 1. Early mycobacterial growth looks like small curved commas or spirals seen on days 5-9. Colony formation usually progresses to cords, and later to more irregular, tangled growth (see images 1-5). 2. If two or more colonies are detected in each of the two drug-free wells (rows A and B), the result is "positive". When a positive result (clear cording instead of just commas ) is observed in both drug-free wells; examine the wells containing isoniazid and rifampicin on the same day (rows C and D). See section "Drug Resistance Determination" below. 3. If results are negative on day 5, continue reading the drug-free wells daily (or on alternate days depending on the laboratory's workload) until two or more mycobacterial colonies are observed in each of the two wells. 4. If no growth is observed by day 15, examine again on day 18 and day 21. If there is no growth detected on day 21, the final report is "negative". 5. Note: If only one colony forming unit (CFU) appears in either drug-free well, or in both, the result is "indeterminate". Wells containing drugs (rows C and D) should not be read if the drug-free well results are "negative" or "indeterminate". 6. Bacterial or fungal contamination will cause the wells to appear cloudy. If bacterial or fungal contamination is observed, the backup sample that was stored in the freezer should be re-processed or a new sample requested. If contamination is present, results cannot be interpreted. Growth of M. tuberculosis does not cause cloudiness in the well. Contamination is uncommon but is usually apparent by day five. 7. The negative control wells in column three are the internal controls for each plate that is processed. These wells have culture media but no patient samples. All four negative control wells in this column should not show any growth. If mycobacterial colonies are observed in any well, there has been cross-contamination. The entire plate should be discarded and back-up samples processed if available or new samples requested. A search for potential sources of cross-contamination should occur and if the source is identified, appropriate remedial action should be taken. Drug Resistance Determination: Antibiotic-containing wells should be examined only if the drug-free wells demonstrate mycobacterial growth km TB MODS Kit Page 8 of 16

9 1. On the same day that both drug-free wells (rows A and B) have definite mycobacterial growth (clear cording instead of just commas ) of greater than or equal to 2 CFU, examine the wells containing isoniazid and rifampicin (rows C and D). 2. If there is any growth greater than or equal to 2 CFU in a drug-containing well, the sample is resistant to that drug (at the concentration present). No growth means the sample is sensitive to the drug. 3. If there is positive growth in both isoniazid and rifampicin containing wells, the sample is multi-drug resistant (MDR). 4. Drug-containing wells should NOT be re-examined on the days after the drug-free wells have been interpreted as positive. Scant breakthrough growth in drug-containing wells after prolonged incubation does not indicate resistance. 5. Growth of drug-resistant M. tuberculosis in drug-containing wells is usually obvious when drug-free wells are positive. Growth in the drug-containing wells may be less than the drug-free wells but this is interpreted as a "positive result" when there is greater than or equal to 2 CFU in the drug-free well. Growth in drug-containing wells should only be considered as indicating resistance if drug-free wells for the same sample have also shown growth. 6. Positive Controls: Drug-free wells should have positive mycobacterial growth (greater than or equal to 2 CFU). Absence of growth in all drugfree wells suggests that the medium does not support growth and patient specimen results are not valid. All samples plated on the same day should be retested with a new lot of medium or should be tested with a new suspension of the control. Antibiotic containing wells should be read only if the drug-free control wells are positive. The drug-susceptible control strain (H37Ra or other) should not grow in either of the drug-containing wells. Growth indicates incorrect reconstitution of the antibiotic tablets or failure to add the antibiotic solution to the wells. The drug-resistant control strain (MDR strain or two monoresistant strains) should grow in the drug-containing wells. Absence of growth indicates that the final isoniazid and/or rifampicin concentrations are too high and that the antibiotic tablets were reconstituted incorrectly or that too much of the antibiotic solution was added to the well. If positive controls do not perform as expected in antibiotic-containing wells, the susceptibility results for patient specimens plated at the same time are invalid. Discard all plates and re-process back-up samples or request new specimens. Absence of growth of control strains may also indicate that the organisms are non-viable. Consider preparing fresh control strains. LIMITATIONS Refer to the document "Limitations of Procedures and Warranty" on the Hardy Diagnostics Technical Document website for more information. 1. Rarely there is a single CFU detected in the drug-containing wells (read at the correct time) and this is interpreted as "indeterminate". 2. Best results are obtained when specimens are digested and decontaminated using reagents and solutions contained in the TB DeCon Red Kit (Cat. no. Z30). 3. Reduced drug activity may be due to incorrect storage or improper handling of antibiotic tablets km TB MODS Kit Page 9 of 16

10 MATERIALS REQUIRED BUT NOT PROVIDED Standard microbiological supplies and equipment such as Middlebrook 7H11 Agar (Cat. no. W35), McFarland Latex Standard 1 (Cat. no. ML1), Water with Tween (Cat. no. V03), TB diluting fluid (Cat. no. R54), 1M HCl, loops, swabs, applicator sticks, other culture media, centrifuge, centrifuge tubes, pipets, transfer pipets (available individually wrapped, Cat. no. 2121S or in packs of 20, Cat. no S), pipettors, sterile pipette tips, M. tuberculosis H37Ra ATCC (Cat. no. 0112P), disinfectants, incinerators, and incubators, etc., are not provided. N95 particulate respirator mask (Cat. no. USP10206). Graduated disposable specimen cup (Cat. no ). TB DeCon Red Kit (Cat. no. Z30). Used for the Clockwise from left: graduated 50ml disposable digestion and decontamination of sputum specimens. centrifuge tube (Cat. no. 6289), Water with Tween 80 (Cat. no. V03), TB Diluting Fluid (Cat. no. R54), McFarland Latex Standard 1 (Cat. no. ML1), SpeedStreaks disposable 10ul loop (Cat. no. HS10F), nylon tipped flocked swab (Cat. no. FS1HD), graduated 1ml disposable transfer pipet (Cat. no. 2121S), 1.5ml microtube (Cat. no ) km TB MODS Kit Page 10 of 16

11 Phosphate Buffer, 1L (Cat. no. U192). MODS test tube rack (Cat. no ) with Middlebrook 7H9 Broth + OADC vials (Cat. no. Z29D). Middlebrook 7H11 Agar (Cat. no. W35). Disposable 1ml serological pipet (Cat. no. 1700). Pipettors, adjustable volume (2-20µl, Cat. no. P394020; Pipet tips, sterile (1-200µl, Cat. no ; µl, Cat. no. P ; µl, Cat. no µl, Cat. no ). P ) km TB MODS Kit Page 11 of 16

12 QUALITY CONTROL The following organisms are routinely used for testing at Hardy Diagnostics: Test Organisms Mycobacterium tuberculosis H37Ra ATCC 25177** Mycobacterium tuberculosis INH resistant Clinical strain** Mycobacterium tuberculosis Rifampicin resistant Clinical strain** Inoculation Method* G G G Incubation Time Temperature Atmosphere Up to 9 days Up to 9 days Up to 9 days 35 C Aerobic 35 C Aerobic 35 C Aerobic Results Growth; colonies (with cording) visible in 5 to 9 days in drug-free wells. No growth in antibiotic containing wells. Growth; colonies (with cording) visible in 5 to 9 days in drug-free wells. Growth in well containing INH. No growth in well containing Rifampicin. Growth; colonies (with cording) visible in 5 to 9 days in drug-free wells. No growth in antibiotic well containing INH. Growth in well containing Rifampicin. * Refer to the document "Inoculation Procedures for Media QC" on the Hardy Diagnostics Technical Document website for more information ** Recommended QC strains for User Quality Control according to the CLSI document M22 when applicable. USER QUALITY CONTROL Check for signs of contamination and deterioration. Users of commercially prepared culture media may be required to perform quality control testing to demonstrate growth or a positive reaction and to demonstrate inhibition or a negative reaction (where applicable). Refer to the following documents on the Hardy Diagnostics Technical Document website for more information on QC: "Introduction to Quality Control," "Finished Product Quality Control Procedures," or "The CLSI Standard and Recommendations for User Quality Control of Media." PHYSICAL APPEARANCE Z29A, Z29C, and Z29D - Middlebrook 7H9 + OADC Broth should appear clear to slightly hazy, and colorless to light amber in color. Z29B - Deionized Water should appear clear and colorless. Z29I - Isoniazid Tablets should appear white in color. Z29R - Rifampicin Tablets should appear orange in color. Z29N - NAPTA Tablets should appear yellow in color km TB MODS Kit Page 12 of 16

13 Image 1: Mycobacterium tuberculosis (clinical isolate) Image 2: Mycobacterium tuberculosis (clinical isolate) growth visible at 5 days at 100x. growth with some cording visible at 7 days at 100x. Image 3: Mycobacterium tuberculosis (clinical isolate) Image 4: Mycobacterium tuberculosis (clinical isolate) growth with cording visible at 9 days at 100x. growth with cording visible at 21 days at 40x. Image 5: Mycobacterium tuberculosis (clinical isolate) growth with cording visible at 23 days at 40x. Photos courtesy of the MODS TB Group Universidad Peruana Cayetano Heredia Lima, Peru km TB MODS Kit Page 13 of 16

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16 REFERENCES 1. World Health Organization Tuberculosis World Health Organization Anti-tuberculosis Drug Resistance in the World, Report no. 4. World Health Organization Press, Geneva. 3. Sandman, L., Schluger, N.W., Davidow, A.L., and Bonk, S Risk Factors for Rifampicin-monoresistant Tuberculosis. A Case Control Study. Am. J. Respir. Crit. Care Med, 159: Moore, D.A.J., Mendoza, D., Gilman, R.H., et al Microscopic Observation Drug Susceptibility Assay, a Rapid, Reliable Diagnostic Test for Multidrug-Resistant Tuberculosis Suitable for Use in Resource-Poor Settings. J. Clin. Microbio.; Moore, D.A.J., Evans, C.A.W., Gilman, R.H., et al Microscopic-Observation Drug-Susceptibility Assay for the Diagnosis of TB. N. Engl. J. Med.; 355: Coronel, J., Roper, M., Caviedes, L., Moore, D MODS: A User Guide. Universidad Peruana Cayetano Heredia, Lima, Peru. 8. Versalovic, J., et al. Manual of Clinical Microbiology. American Society for Microbiology, Washington, D.C. 9. Tille, P.M., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO. 10. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C. 11. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology. J.B. Lippincott Company, Philadelphia, PA. 12. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA. ATCC is a registered trademark of the American Type Culture Collection. Tween is a registered trademark of ICI Americas, Inc km HARDY DIAGNOSTICS 1430 West McCoy Lane, Santa Maria, CA 93455, USA Phone: (805) ext Fax: (805) Website: TechService@HardyDiagnostics.com Distribution Centers: California Washington Utah Arizona Texas Ohio Florida New York North Carolina The Hardy Diagnostics manufacturing facility and quality management system is certified to ISO Copyright by Hardy Diagnostics. All rights reserved km TB MODS Kit Page 16 of 16