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 ~  Abstract
 ~  Materials and Me...
 ~  BACTEC PZA susce...
 ~  Results
 ~  Discussion
 ~  References

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Year : 2004  |  Volume : 22  |  Issue : 3  |  Page : 166-168
 

Comparison of pyrazinamide drug susceptibility of m.tuberculosis by radiometric bactec and enzymatic pyrazinamidase assay


Department of Microbiology, P.D Hinduja National Hospital and Medical Research Centre, Mumbai - 400 016, India

Date of Submission10-Jun-2003
Date of Acceptance03-Oct-2003

Correspondence Address:
Department of Microbiology, P.D Hinduja National Hospital and Medical Research Centre, Mumbai - 400 016, India

 ~ Abstract 

The aim of this study was to perform pyrazinamide (PZA) susceptibility testing of M.tuberculosis by enzymatic PZA assay and compare the results with radiometric BACTEC 460 TB system and LJ proportion method. One hundred and thirty clinical isolates of M.tuberculosis were included in the study. Of the 130 clinical isolates tested, five were resistant and 124 were sensitive by both methods thus giving overall sensitivity and specificity of 83.33% and 100% respectively. Concordance was found in 129 out of 130 strains tested by all three methods. Pyrazinamidase assay can be used as an alternative to BACTEC and LJ proportion method.

How to cite this article:
Krishnamurthy A, Almeida D, Rodrigues C, Mehta A. Comparison of pyrazinamide drug susceptibility of m.tuberculosis by radiometric bactec and enzymatic pyrazinamidase assay. Indian J Med Microbiol 2004;22:166-8


How to cite this URL:
Krishnamurthy A, Almeida D, Rodrigues C, Mehta A. Comparison of pyrazinamide drug susceptibility of m.tuberculosis by radiometric bactec and enzymatic pyrazinamidase assay. Indian J Med Microbiol [serial online] 2004 [cited 2019 Dec 11];22:166-8. Available from: http://www.ijmm.org/text.asp?2004/22/3/166/11210


Tuberculosis remains a major public health problem, claiming three million deaths annually in developing countries. The emergence of drug resistant M. tuberculosis strains (resistant to both rifampicin and isoniazid) compounded with human immunodeficiency virus coinfection has compromised our ability to control tuberculosis.[1] Pyrazinamide (PZA) is an important first line anti-tuberculosis drug. Along with isoniazid, rifampicin, ethambutol, PZA is part of the currently used short course treatment regimens, also called directly observed therapy, short course (DOTS) recommended by World Health Organization (WHO). It acts in the extracellular microenvironment and kills at least 95% of bacilli during the first two weeks of treatment. Following its addition to anti-tuberculosis drug regimens, the length of a course of treatment has been reduced to six months, thus aiding compliance and decreasing the risk of development of MDR-TB.[2] The resistance of clinical isolates of M. tuberculosis to PZA correlates with the absence of an amidase activity, pyrazinamidase. This enzyme transforms PZA into pyrazinoic acid (POA) which is active against M. tuberculosis.[3] PZA is not active under normal culture conditions but it is active in acid medium.[1] Strains of M. tuberculosis which are susceptible to PZA have pyrazinamidase that hydrolyses PZA to POA, whereas, resistant strains of M. tuberculosis do not possess this enzyme and are not able to convert PZA to POA. Therefore, pyrazinamidase activity test was originally proposed as a taxonomic test and used in some laboratories as a PZA susceptibility test.[4] Conventional culture based techniques for susceptibility testing takes several weeks to complete whereas both radiometric and nonradiometric liquid culture systems have significantly reduced turn around times for obtaining results.[5] The BACTEC radiometric system for mycobacterial culture, identification and susceptibility testing includes a test to assay for PZA susceptibility.[6]
This study aimed to perform PZA susceptibility testing by enzymatic pyrazinamidase assay and compare it with semi automated, radiometric BACTEC 460 TB system and the conventional LJ proportion method.

 ~ Materials and Methods Top

Cultures
One hundred and thirty clinical isolates of M. tuberculosis grown on LJ slants were used for evaluating the performance of pyrazinamidase assay. PZA drug susceptibility testing was performed using LJ Proportion method, BACTEC 460 TB system and Wayne's pyrazinamidase assay. BACTEC method of susceptibility testing was used as the gold standard for the comparison of the other two methods.

 ~ BACTEC PZA susceptibility testing Top

All strains underwent the BACTEC PZA susceptibility testing as per the recommendations of the manufacturer. Actively growing culture in BACTEC 12 B vials supplemented with polyoxyethylene stearate (POES) were appropriately inoculated into two BACTEC PZA test medium vials, one containing PZA 100mg/mL and POES and the other used as control with POES only. Daily readings of the growth index (GI) in both vials were done till it reached 200 and results were interpreted by comparing the GI in drug vial to GI in control vial. If GI difference between the two vials was less than 9% it was considered susceptible, if more than 9% it was resistant and between 9-11% was considered borderline. [7]
PZA Susceptibility by LJ Proportion method
PZA is active only in acidic condition, hence susceptibility testing requires special media with acidic pH. The pH of LJ medium was adjusted to 4.85+ 0.05, as this has been found to support the growth of majority of strains from British, south Indian and Chinese patients.[8] Using LJ proportion method, one loopful of 10-[2] mg/mL and 10-[4] mg/mL dilution was inoculated on the LJ slants containing the drug and acidic drug free control slope. The slopes were incubated at 37C for six weeks.[8]
Wayne's Pyrazinamidase assay
It was performed using the enzymatic deamidation of PZA to pyrazinoic acid and ammonia as described by Wayne's method.[9] The procedure and interpretation are described in brief in [Figure - 1].
Interpretation
A pink band which forms in the subsurface agar and diffuses in the medium, indicates enzymatic hydrolysis of PZA to free pyrazinoic acid.
Positive = Pink band in agar (Sensitive)
Negative = No pink band in agar (Resistant)

 ~ Results Top

The results are summarised in [Table - 1] and [Table - 2]. In [Table - 1], concordance was observed in 129 of the 130 strains tested for pyrazinamide susceptibility. Out of 130 strains, five were resistant and 124 were sensitive by both methods. Similar results were obtained when compared with LJ proportion method. Considering TB-BACTEC to be the recommended method,[10] the sensitivity and specificity of pyrazinamidase assay is shown in [Table - 2].

 ~ Discussion Top

Until the development of BACTEC PZA susceptibility test method, susceptibility testing of M. tuberculosis was performed by either conventional LJ method or enzymatic pyrazinamidase assay. This usually meant a delay of several weeks (4-5 weeks) by LJ method and 7 days for pyrazinamidase assay. Conventional PZA susceptibility testing of M. tuberculosis requires growth of the organism and may lead to uninterpretable results due to poor growth in acidified environment.[11] The BACTEC susceptibility testing for PZA is rapid, as it is available within 5 days. In our study, 1 strain was classified as resistant by BACTEC while from pyrazinamidase assay it was sensitive. This strain was re-tested by LJ proportion method and was found to be resistant. Compared with BACTEC we observed sensitivity, specificity and efficiency of PZA assay to be 83.33%, 100% and 99.23% respectively. Our overall correlation was similar to the study conducted by Miller et al in which an overall correlation of 98.2-100% was observed.[6]
To conclude, a good correlation was found between the three PZA susceptibility testing methods, employing radiometric, LJ proportion and pyrazinamidase activity. So far, no study has examined a large number of PZA susceptible and resistant clinical isolates of M.tuberculosis by means of comparison of BACTEC assay and conventional method of detection of pyrazinamidase. In a study performed by Miller et al[6] PZA susceptibility by BACTEC was compared with pyrazinamidase assay (Wayne's method) in which a correlation of 98.2% and 100% was observed for susceptible and resistant strains. Radiometric BACTEC method is commonly used method for PZA susceptibility testing in developed countries. However, pyrazinamidase assay can be used as an alternative to BACTEC especially in areas where BACTEC is not available. 

 ~ References Top

1.Scorpio A, Lindholm-Levy P, Heifets L, Gilman R, Siddiqi S, Cynamon M, Zhang Y. Characterization of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis. Antimicrob Agents Chemother 1997;41:540-543.  Back to cited text no. 1    
2.Davies AP, Billington OJ, McHugh TD, Mitchison DA, Gillespie SH. Comparison of phenotypic and genotypic methods for pyrazinamde susceptibility testing with Mycobacterium tuberculosis. J Clin Microbiol 2000;38:3686-3688.  Back to cited text no. 2    
3.Raynaud C, Laneelle M, Ryan HS, Draper P, Laneelle G, Daffe M. Mechanisms of pyrazinamide resistance in mycobacteria: importance of lack of uptake in addition to lack of pyrazinamidase activity. Microbiol 1999;145:1359-1367.  Back to cited text no. 3    
4.Heifets LB, Iseman MD. Radiometric method for testing susceptibility of mycobacteria to pyrazinamide in 7H12 broth. J Clin Microbiol 1985;21:200-204.  Back to cited text no. 4    
5.Eltringham IJ, Wilson SM, Drobniewski FA. Evaluation of a bacteriophage-based assay (Phage Amplified Biologically Assay) as a rapid screen for resistance to isoniazid, ethambutol, streptomycin, pyrazinamide and ciprofloxacin among clinical isolates of Mycobacterium tuberculosis. J Clin Microbiol 1999;37:3528-3532.  Back to cited text no. 5    
6.Miller MA, Thibert L, Desjardins F, Siddiqi SH, Dascal A. Testing of susceptibility of Mycobacterium tuberculosis to pyrazinamide: Comparison of BACTEC method with Pyrazinamidase assay. J Clin Microbiol 1995;33:2468-2470.  Back to cited text no. 6    
7.Siddiqi S. 1996. BACTEC 460 TB System, Product and Procedure manual. Revision E. Becton Dickinson Microbiology Systems, Sparks, Md.  Back to cited text no. 7    
8.Canetti G, Fox W, Khomenko A, Mahler HT, Menon NK, Mitchison DA, Rist N, Smelev NA. Advances in techniques of testing, mycobacterial drug sensitivity, and the use of sensitivity tests in Tuberculosis Control Programmes. Bull World Health Organ 1969;41:21-43.  Back to cited text no. 8  [PUBMED]  
9.Kent PT, Kubica GP. Public health mycobacteriology. A guide for the level III laboratory. Centers for Disease Control, Atlanta, Ga. 1985;71-120.  Back to cited text no. 9    
10.Keihn T E, Cynamon MH, Inderlied CB, Roberts GD, Siddiqi SH, Wallace RJ, Warren NG. NCCLS. Susceptibility testing of mycobacteria, nocardia and other aerobic actinomycetes; tentative standard, 2nd Edn. NCCLS document M24-T24, NCCLS, 1995;15(16); Wayne, Pa.  Back to cited text no. 10    
11.Miller MA, Thibert L, Desjardins F, Siddiqi SH, Dascal A. Growth inhibition of Mycobacterium tuberculosis by Polyoxyethylene stearate present in the BACTEC Pyrazinamide susceptibility test. J Clin Microbiol 1996;34:84-86.  Back to cited text no. 11    
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