Automatic System - MGIT

GLOBAL SCENARIO  22 high burden countries (WHO ) – India 1st  Major health problem - 2.59 million cases.   India and China together account for almost 40% of the world’s TB case In India, TB kills two persons every three minutes nearly 1,000 every day Diagnosis of TB Indirect Methods Direct Methods Phenotypic Microscopy Genotypic Culture Immunological tests Phage Amplification methods Culture Methods Conventional Direct Growth observation Rapid Culture Method Colorimetric methods Automated systems Overview MGIT 1.Principles 2.Reagents 3. Inoculation 4. Unloading MGIT tubes 5. Processing MGIT tubes 6. Reporting results Principle:Fluorescence of sensor Quenched in presence of oxygen Growth occurs in tube Oxygen depleted Indicator fluoresces at 365nm UV light.  MGIT incubation is at 35-37°C  Each MGIT is monitored every 60 minutes for increasing fluorescence  A positive MGIT contains 105 to 106 colony forming units per milliliter (CFU/mL)  A negative MGIT has no fluorescence/visible signs of bacterial growth at 42 days Media  Middlebrook 7H9 broth (7ml) +OADC + PANTA  Growth supplement (OADC) is added to tubes to enable the rapid growth of Mycobacteria Oleic Acid (0.1g) Involved in metabolism of mycobacteria Albumin(50g) Protective agent by binding free fatty acids which may be toxic to Mycobacterium species Dextrose(20g) Energy Source Catalase(0.03g) Destroys toxic peroxides that may be present in media • Contamination is reduced when PANTA antibiotic mixture is added to the media Polymixin B 6000 units Amphotericin 600 µg Nalidixic acid 2400µg Trimethoprium 600µg Azlocilin 600µg Sample type Pulmonary Requirements: For Primary Culture • Falcon tubes • PPE • BSL 3 • • • • • PANTA MGIT Tubes Growth supplements Pure growth or processed specimen BACTEC Instrument Extrapulmonary DST: • • • • • • • Pure growth OADC Drugs BACTEC instrument Vortex Pipettes BHI Media Working: Reconstitute PANTA Add 0.8 ml into each MGIT tube Add 0.5 ml specimen Leave tubes for 30 minutes Incubate tubes in MGIT 960 Check MGIT 960 daily Positive flag Negative flag Unloading of tubes Positive flag tube Visual Scan Inoculate on BHI plate Subculture on LJ ZN staining Incubate at 37 degree Check at 18-24 hours & 48 hours Growth presentcontamination AFB smear Negative Reenter tube into MGIT system Positive After 3 days visually inspect the tube AFB SMEAR Still NEGATIVE BHI agar - contamination Report as contamination BHI AGAR- No contamination LJ medium- Not positive by 8 weeks Report sample as Negative Report preliminary results only after acid-fast smear evaluation Negative Flag Visually inspect Slight suspicion –AFB smear L J subculture BHI agar inoculation For Contamination: Positive AFB smear BHI + AFB showing contamination Look for another sample of same patient Not Contaminated Proceed with the new sample Contaminated/Not available Recapitulation  MGIT – Based on principle of Fluorescence  Media :Middlebrook 7H9 broth (7ml) +OADC + PANTA  Considered Positive if >4 days since incubation  Positive flag: Visual scan, BHI inoculation, LJ subculture & AFB (ZN staining)  ZN staining- Gives preliminary results  Negative flag: Suspicion go for BHI , LJ subculture, AFB(ZN staining)  Encounter contamination: Look for 2nd sample/ Decontaminate it Drug susceptibility testing  Rapid qualitative procedure for susceptibility testing of Mycobacterium tuberculosis  Treatment of TB is most commonly through a multiple drug regimen that includes: Streptomycin (STR), Isoniazid (INH), Rifampicin (RIF), Ethambutol (EMB) and Pyrazinamide (PZA)  MDR Tb- Resistance to first line drugs Isoniazid & Rifampicin  XDR Tb- MDR-TB with resistance to second line fluoroquinolones + Resistance to one of the second line aminoglycosides (e.g. kanamycin, Amikacin ,Capreomycin Principle Comparison of growth of the Mycobacterium tuberculosis isolate in a drug containing tube to a drug free tube (Growth Control GC) Overview  1 set has drug free growth control MGIT tube (GC) and one MGIT tube for each drug, as well as a bar coded tube carrier that holds the set  A known concentration of drug is added to a MGIT tube, along with the specimen  Growth of the specimen added to the drug containing tube is compared with the drug free growth control which contains the same specimen  Bacterial inoculum added to the drug-free growth control tube is one hundred fold less than the inoculum added to the drug containing tubes Growth control tube Drug containing tube  1st line Drugs reconstituted using 4 mL of sterile/deionized water  PZA must be reconstituted using 2.5 mL of sterile/deionized water  MOX must be reconstituted using 3 mL of sterile/deionized water and then diluted 1:8 with sterile/deionized water to achieve concentrations  KAN must be reconstituted using 4 mL of sterile/deionized water to achieve concentrations Procedure Label the tubes & arrange in sequence Add 0.8mL of BACTEC MGIT Supplement (SIRE and PZA) Add 0.1mL of drug MGIT Flagged Positive – Day 0 Day 1 – Tube should be incubated for 1 more day  Once a MGIT has flagged positive at or after 4 days, it must be used within 1-5 days  If a MGIT is unloaded on day 0, the tube should be reincubated for a minimum of one day (day 1) Growth control preparation : SIRE/MOX/KAN/PZA Take 0.1 ml MGIT inoculum & dilute in 9.9 ml sterile saline (.85%) Making 1:100 dilution for 1-2 day old & 1:5 for 3-5 day old Inoculate 0.5 ml of this into GC tube For PZA the concentrations vary procedure remains the same 1:10 for 1- 2 day old & 1:50 for 3-5 day old  500 µL of the organism suspension is inoculated into the drug tubes Loading of tubes  SIRE/MOX/KAN have a time in protocol of 4-13 days  PZA allows for a longer time frame of 4-21 days Results Susceptible result • Growth will be inhibited and fluorescence will be suppressed in the drug-containing tube • The drug-free growth control will grow and show increasing fluorescence • Sensitive result: Growth units are less than 100 Resistant result • Growth and its corresponding increase in fluorescence will be evident in both the drug-containing and the drug-free growth control tube • Resistant result: Growth units are more than 100 SAMPLE REPORT Errors in MGIT 200X 400X 10X • Too little inoculum • Non-viable organisms • Slow growing drug-resistant strain • Detects indications of possible contamination • Over inoculated GC tube Error in loading On repeat subculture use 3-5 days undiluted culture Quality Control • Perform DST testing on H37Rv (or equivalent fully sensitive isolate) • Each batch of reagents (drug kits and tubes) must be quality tested • If the QC fails: all results for batch must be reviewed, new reagents purchased, and testing of samples repeated Advantages  Electronic data management  Simultaneous ABST  No radioactive waste Disadvantages  False positive results due to contamination  BSL 3 is a must Recap  Comparison of growth of organism in a drug containing tube with drug free tube.  MGIT has flagged positive at or after 4 days, it must be used within 1-5 days  If a MGIT is unloaded on day 0, the tube should be re incubated for a minimum of one day (day 1)  Most drugs time protocol – 4-14 days except Pyrazinamide  Sensitive result: Growth units are less than 100  Resistant result: Growth units are more than 100  Quality Control :Perform DST testing on H37Rv (or equivalent fully sensitive isolate)