Proforma for registration of subject for dissertation name of the candidate

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 2^nd 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 2^nd 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 2^nd 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 2^nd 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

To compare the nitrate reductase assay with the manual MGIT as a reference method to detect Pyrazinamide susceptibility to Mycobacterium tuberculosis using nicotinamide.

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 sample size was calculated based on :-

• 
  Sensitivity of new test - 85%
  
• 
  Sensitivity of reference test – 95%
  
• 
  α error - 0.05 %
  
• 
  Power of the study – 80%
  
• 
  1 or 2 sided – 2 sided
  

METHOD OF SAMPLE COLLECTION

Sample will be collected in a sterile, disposable, leak proof wide-mouthed screw capped plastic container without any preservatives.

INCLUSION CRITERIA

1. 
   Both pulmonary and extra-pulmonary samples positive for acid fast bacilli (Ziehl-Neelson staining) will be included for isolation of the strains.
   
2. 
   Actively growing(after 3-4 weeks of incubation) Mycobacterium tuberculosis strains will be studied for the Pyrazinamide susceptibility testing.
   

1. 
   Mixed cultures and contaminated cultures
   

Prospective comparative study of a new diagnostic test as compared to an in- use reference test.

SAMPLE ( PULMONARY / EXTRA-PULMONARY)

ZIEHL NEELSON STAINING

ACID FAST BACILLI POSITIVE

MODIFIED PETROFF’S METHOD FOR DECONTAMINATION

INOCULATION (2 LJ MEDIA)

GROWTH

STOCK CULTURE

1. 
   TYPICAL SERPENTINE CORD FORMATION IN
   

2. 
   NITRATE REDUCTASE TEST
   

MANUAL MGIT NRA

The strains isolated and identified as Mycobacterium tuberculosis will be subjected to drug susceptibility testing by

1. 
   Manual MGIT (As per the manufacturer’s instruction)
   
2. 
   Nitrate reductase assay(NRA)
   

1. 
   Nicotinamide ( critical concentration 500mg/l)
   

1. 
   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 37^oC
  
• 
  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 any colour (pink) is noted on the control tube, the test tube with the antibiotic will be developed with reagent mixture.
  
• 
  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
  

• 
  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
  

2. 
   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
  

• 
  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.
  

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
  

1. 
   A.KRISHNAMURTHY, D.ALMEIDA, C. RODRIGUES, A. MEHTA. Comparison of Pyrazinamide drug susceptibility of Mycobacteria tuberculosis by radiometric BACTEC and enzymatic Pyrazinamide assay. Indian Journal Of Medical Microbiology 2004, 22(3): 166-168
   
2. 
   K.ADJERS-KOSKELA , M. LEENA KATILA. Susceptibility testing with the Manual MGIT and MGIT 960 syatem provides rapid and reliable verification of MDR-tuberculosis. Journal Of Clinical Microbiology.2003, p 1235 - 1239
   
3. 
   H.SYRE , K. OVREAS, H.M.S. GREWAL. Determination of the susceptibility of Mycobacterium tuberculosis to Pyrazinamide in liquid and solid media assessed by a colorimetric nitrate reductase assay. Journal Of Antimicrobial Chemotherapy. 2010, 65: 704-712
   
4. 
   D.A. MITCHISON. The action of antitubercular drugs in short course chemotherapy. Tubercle .1985, 66:219-225
   
5. 
   N.C. MIRABEL, S.L.YZQUIERDO, D. LEMUS, M.MADRUGA, Y.MILIAN, A.MARTIN, J.C.PALOMINO. Evaluation of colorimetric methods using Nicotinamide for rapid detection of Pyrazinamide resistance in Mycobacterium tuberculosis. Journal Of Clinical Microbiology. 2010, 48(8): 2729
   
6. 
   G.E. PFYFFER, F.PALICOVA, S.RUSCH-GERDES. Testing of susceptibility of Mycobacterium tuberculosis to Pyrazinamide with non-radiometric BACTEC MGIT 960 system. Journal Of Clinical Microbiology. 2002, 40(5): 1670
   
7. 
   D.LEMUS, E. MONTORO, M.ECHEMENDIA, A. MARTIN, F. PORTEALS, J.C. PALOMINO. Nitrate reductase assay for detection of drug resistance in Mycobacterium tuberculosis; Simple and inexpensive method for low resource laboratories. Journal Of Medical Microbiology. 2006, 55:p 861-863
   
8. 
   B.SHARMA, N. PAL, B. MALHOTRA, L.VYAS, S.RISHI. Comparison and Pyrazinamide activity assay for Pyrazinamide susceptibility of Mycobacterium tuberculosis. Indian Journal Of Respiratory Medicine.2010,p 72-76
   
9. 
   A.MARTIN, S.PANEIOTOV, F.PORTEALS, S.HOFFNER, J.C. PALAMINO, K. ANGELY. The nitrate reductase assay for the rapid detection of isoniazid and rifampicin resistance in Mycobacterium tuberculosis; A systematic review and meta analysis. Journal Of Antimicrobial Chemotherapy. 2008, 62 :56-64
   
10. 
    M.GUPTA, SHAMMA, N.P. SINGH, I.R. KAUR. Evaluation of nitrate reductase assay for direct detection of drug resistance in Mycobacterium tuberculosis: Rapid and inexpensive method for low resource setting. Indian Journal Of Medical Microbiology. 2008, 28(4): 363-365
    
11. 
    P.CHEDORE, L.BERTUCCI, J. WOLFE, M. SHARMA, F. JAMIESON. Potential for erroneous results indicating resistance when using the BACTEC MGIT 960 system for testing susceptibility of Mycobacterium tuberculosis to Pyrazinamide. Journal Of Clinical Microbiology.2008,p 300-301
    
12. 
    J.A. PENA, M.J.FERRAO, C.G. HOFFMAN, J.A. BRUNDA. Growth detection failures by non radiometric BACTEC MGIT 960 mycobacterial culture system. Journal Of Clinical Microbiology. 2012, 50(6):2092
    
13. 
    J.C. PALOMINO. Non-conventional and new methods in the diagnosis of tuberculosis; feasibility and applicability in the field. European Respiratory Journal. 2005, 26:339-350
    
14. 
    A.SOMOSKOVI, C. KODMON, A. LANTOS, Z. BARTFAI. Comparison of recoveries of Mycobacterium tuberculosis using the automated BACTEC MGIT 960 system, the BACTEC 460 tb system and Lowenstein Jensen medium. Journal Of Clinical Microbiology.2000,p 2395-2397
    
15. 
    A. KRUNNER, M.D. YATES, F.A. DROBNIEWSKI. Evaluation of MGIT 960 based antimicrobial testing and determination of critical concentration of first and second line antimicrobial drugs wih drug resistant clinical strains of Mycobacterium tuberculosis. Journal Of Clinical Microbiology.2006,p 811-818
    
16. 
    B.SHARMA, N.PAL, B. MALHOTRA, L.VYAS, S.RISHI. Comparison of MGIT 960 and Pyrazinamide activity assay for Pyrazinamide susceptibility testing of Mycobacterium tuberculosis. Indian Journal Of Respiratory Medicine. 2010,p 72-76