Comparison of a colorimetric assay for Pyrazinamide susceptibility testing using Nicotinamide for Mycobacterium tuberculosis with Manual Mycobacterium Growth Indicator Tube (MGIT) assay
6. BRIEF RESUME OF THE STUDY
6.1 NEED FOR STUDY
Tuberculosis is a major global health problem with respect to morbidity and mortality.1 The situation becomes worse with it’s association to HIV and emergence of Multi-drug resistant(MDR) tuberculosis and Extensively drug resistant(XDR) tuberculosis strains.2
Pyrazinamide is an important first line drug in short course therapy and also to treat MDR tuberculosis as the 2nd line drug.3It acts in the acidic extracellular microenvironment found in acute inflammation and kills at least 95% of bacilli during the first 2 weeks of treatment.4 So, timely detection of sensitivity to pyrazinamide becomes essential for proper management of the patient.
Pyrazinamide is pyrazine analogue of nicotinamide which gets converted to pyrazinoic acid by pyrazinamidase at an acidic pH. Pyrazinamidase converts Nicotinamide to nicotinic acid too but at physiological pH.without hampering the growth of the bacilli.5 Similar studies has been done with varying sensitivity and specificity for Pyrazinamide but none of the test assays are considered gold standard.6,7,8
The nitrate reductase assay (colorimetric test) is a proved technique for the sensitivity testing for first line drugs.9,10,11 The test is simple, rapid and does not require sophisticated instruments and costly reagents. In comparison the reagents used in Manual MGIT is expensive as well as it requires expensive growth supplement. The liquid culture contamination rate is also high.12 Hence this study will be undertaken to compare the results of nitrate reductase assay with that of in-use Manual MGIT assay.
6.2 REVIEW OF LITERATURE
Tuberculosis is one of the most important public health problem worldwide.1 The emergence of multidrug-resistant strains of Mycobacterium tuberculosis (resistant to isoniazid and rifampicin) has hindered the control of this disease. At present resistance to Pyrazinamide, one of the important drugs in short course regimen and also used in MDR-Tuberculosis as the 2nd line drug is a great concern. For this reason, rapid diagnosis of tuberculosis drug resistance is a priority to avoid the spread of resistant strains.13
There are various diagnostic techniques available for detection of tuberculosis drug resistance. The conventional proportion method using the egg based/ agar based media is considered as the gold standard. But its use is restricted as it requires several weeks to give results. The radiometric BACTEC 460 TB method has been proven to be both sensitive and rapid for detecting mycobacteria and rapid method for susceptibility testing. But the major drawbacks of the system is known to be related for the disposal of the radioactive substrate.13,14 Mycobacteria growth indicator tube(MGIT) a non radiometric technique is at present, has been reported as a sensitive and rapid method for the growth and detection of mycobacteria from clinical specimens. The MGIT contains a modified Middlebrook 7H9 broth in conjunction with a fluorescence quenching-based oxygen sensor(silicon rubber impregnated with ruthenium pentahydrate).The studies have reported that the MGIT can also be used for AST, but those evaluations were carried out mostly with the first line drugs and few studies on 2nd line drugs. As the BACTEC 960 automated instrument is expensive, a manual MGIT has been introduced for the purpose of Mycobacterial growth detection .Here a hand-held UV device is used for the fluorescence detection which is less expensive and practical for routine use in resource poor settings. Other Commercial tests (ETest) and molecular tools (INNO-LiPA) are also expensive and impractical to be routinely used .13
Studies have shown that manual MGIT also has higher recovery rate, easy to perform, rapid turn- around time and able to give accurate results in case of antimycobacterial drug sensitivity test for first line and second line drugs used in tuberculosis treatment.14 But still using MGIT in resource poor country is impractical because of the expensive reagents.
In such settings there are alternative colorimetric assays such as nitrate reductase assays (NRA), Resazurin micro-titre plate assay (REMA) etc.13 Nitrate reductase assay is a colorimetric method and it is easy to perform. Many studies have shown that nitrate reductase assay is comparable with manual MGIT for the first line drugs like INH and Rifampicin.9,15 But studies comparing the sensitivity testing for 2nd line drugs and also for Pyrazinamide are scanty. As this method can be employed in resource poor settings, hence the study was undertaken to compare the Pyrazinamide sensitivity testing using NRA with that of manual MGIT.
Nicotinamide being structurally similar to pyrizinamide is being acted upon by pyrazinamidase to give nicotinic acid at physiological pH. Unlike Pyrazinamide which when acted upon by pyrazinamidase gives pyrazinoic acid at an acidic pH.This property of Nicotinamide can be utilized to see for pyrazinamide resistance.5
6.3 AIMS AND OBJECTIVES OF THE STUDY
To compare the nitrate reductase assay with the manual MGIT as a reference method to detect Pyrazinamide susceptibility to Mycobacterium tuberculosis using nicotinamide.
7. MATERIALS AND METHODS
7.1 SOURCE OF DATA
The clinical samples (both pulmonary and extra-pulmonary) positive for AFB received in the Mycobacteria laboratory, Department Of Microbiology, St. John’s Medical College will be included for the study.
A total of 87 isolates of Mycobacterium tuberculosis will be included in the study.
The strains isolated and identified as Mycobacterium tuberculosis will be subjected to drug susceptibility testing by
Manual MGIT (As per the manufacturer’s instruction)
Nitrate reductase assay(NRA)
Drug Used FOR NRA Nicotinamide ( critical concentration 500mg/l)
PROCEDURE Nitrate Reductase Assay9
LJ medium containing potassium nitrate at 1000mg/ml and Nicotinamide at 500mg/ml
Inoculum turbidity will be adjusted to Mc farland standard 1
It is then diluted to 1:10 in phosphate buffer saline and 200 µl will be inoculated to 3 control tubes
200 ul of undiluted suspension is added to Nicotinamide containing tube
Tubes will be incubated aerobically at 37oC
After 7 days 500 ul of reagent mixture containing 1 part 50% concentrated Hcl, 2 parts of 0.25% sulphanilamide, 2 parts of 0.1% n-1-napthylethylene diamine dihydrochloride will be added to 1 of the three control tubes.
If no colour developed tubes will be reincubated to repeat the procedure on 10 and 14 days
A strain is considered resistant if colour developed in nicotinamide containing tube darker than the colour appearing in growth control tube
MEDIA TO BE USED
Control tube:LJ media with potassium nitrate at 1000mg/l.
Test tube : LJ medium with potassium nitrate at 1000mg/l+ nicotinamide at a concentration of 500mg/l
Manual MGIT Pyrazinamide Susceptibility Testing
Based on the principle used for primary isolation, susceptibility is indicated by absence of fluorescence in drug containing tubes. Resistance is indicated by the presence in fluorescence when compared to growth control tube within 2 days of control tube showing fluorescence.
PZA drug critical concentration is 100 mcg/ml
PROCEDURE ( AS PER THE MANUFACTURER’S INSTRUCTION) AND INTERPRETATION
MGIT PZA uses Middlebrooks 7H9 broth at pH 5.9
Two MGIT PZA tubes, one as GC (Growth Control) and one as PZA (drug containing) tube are used and both with growth supplement
PZA at a critical concentration of 100 microlitre/ml will be used in the drug containing tube.
0.5 ml of the culture suspension will be added to the PZA tube and at 1:10 dilution to the GC tube and incubated.
Results will be interpreted once growth control tube shows fluorescence.
Resistance is considered if drug containing tube fluoresces within 2 days of the time that growth control tube fluoresces.
QUALITY CONTROL: H37RV WILL BE USED AS THE STANDARD REFERENCE STRAIN
RESULTS AND STASTICAL ANALYSIS
The results of the test will be analysed by
Agreement between the 2 tests using Cohen’s Kappa measures
Sensitivity and specificity will be calculated by chi square analysis taking MGIT as the reference method
As the microbiological data will be linked to clinical data, appropriate written consent will be taken from the patient / relatives at the time of sample collection.
7.4 Has ethical clearance been obtained from your institution
Awaiting IEC clearance
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