全部 标题 作者
关键词 摘要

OALib Journal期刊
ISSN: 2333-9721
费用:99美元

查看量下载量

相关文章

更多...

Assessment of Status of rpoB Gene in FNAC Samples of Tuberculous Lymphadenitis by Real-Time PCR

DOI: 10.1155/2012/834836

Full-Text   Cite this paper   Add to My Lib

Abstract:

Introduction. Multidrug resistance tuberculosis (MDR TB), the combined resistance of Mycobacterium tuberculosis to isoniazid (INH) and rifampin (RFM) is a major public health problem in India as it ranks second among the MDR-TB high burden countries worldwide. WHO recommends RFM resistance as a “surrogate marker” for detecting MDR. FNAC is the most widely used noninvasive investigative technique for TB lymphadenitis. Real-time polymerase chain reaction, an extremely versatile technique can be used for the timely detection and treatment of MDR TB by assessing RFM resistance status in the FNAC samples of TB lymphadenitis. Aim. To assess the status of rpoB gene by real-time PCR in FNAC samples of TB lymphadenitis. Materials and Methods. Thirty FNAC samples from patients with persistent LAP or appearance of new LAP after 5 months or more of Anti Tubercular Treatment were assessed for status of rpoB gene by Real-Time PCR using probe covering the “hot spot resistance” region of the rpoB gene. Result. By using probe covering codons 531 and 526 of rpoB gene, we could detect 17 of 30 (56.7%) rifampin resistant isolate. The PCR could detect Mtb DNA in 100% of cases. Conclusion. Use of molecular methods like Real-Time PCR for detection of MDR-TB in FNAC samples is time saving, logical and economical approach over the culture based method. 1. Introduction Multidrug resistant tuberculosis (MDR-TB) is a major public health problem in India as it ranks second among the MDR-TB high-burden countries worldwide [1]. MDR-TB is defined as the combined resistance of Mycobacterium tuberculosis (Mtb) to isoniazid (INH) and rifampin (RFM). However, resistance to RFM, the first-line antituberculosis drug is considered to be more critical since it usually occurs in combination with other drugs specially INH. Hence, WHO has recommended RFM resistance as a “surrogate marker” for detecting MDR [2]. Ninety-six percent of RFM-resistant Mtb strains possess genetic alterations within an 81?bp “rifampin resistance-determining region” (RRDR) in the rpoB gene [3, 4], corresponding to codons 507 to 533. Real-time polymerase chain reaction (PCR) is a rapid and reliable method that enables both the amplification and the detection of mutations by using fluorescently labelled DNA probes [5]. The assessment of RFM resistance in cases of TB lymphadenitis (LAP), the most frequent (30–52%) cause of LAP in developing countries [6], is important for the timely detection and efficient treatment of such cases. Fine needle aspiration cytology (FNAC), a widely practised noninvasive, safe, simple, and

References

[1]  “Tuberculosis Control in the South-East Asia Region 2012,” WHO 70-71, http://www.searo.who.int/linkfiles/tuberculosis_who-tb-report-2012.pdf.
[2]  WHO/IUATLD, “Global Project on Anti-Tuberculosis Drug Resistance Surveillance (1999–2000). Drug Resistance in World,” Global Report 3, WHO/CDS/TB/2004, Geneva, Switzerland, 2004.
[3]  H. Soini and J. M. Musser, “Molecular diagnosis of mycobacteria,” Clinical Chemistry, vol. 47, no. 5, pp. 809–814, 2001.
[4]  S. Ramaswamy and J. M. Musser, “Molecular genetic basis of antimicrobial agent resistance in Mycobacterium tuberculosis: 1998 update,” Tubercle and Lung Disease, vol. 79, no. 1, pp. 3–29, 1998.
[5]  P. S. Bernard, A. Reiser, and G. H. Pritham, “Mutation detection by Fluorescent hybridization probe melting curve,” in Rapid Cycles Real-Time PCR, Methods and Applications, S. Muer, C. Wittwer, and K. Nakagawara, Eds., pp. 11–19, Springer, Heidelberg, Germany, 2001.
[6]  A. K. Gupta, M. Nayar, and M. Chandra, “Critical appraisal of fine needle aspiration cytology in tuberculous lymphadenitis,” Acta Cytologica, vol. 36, no. 3, pp. 391–394, 1992.
[7]  D. Van Sooligen, P. Hermans, W. de Haas, and J. D. van Embben, RFLP Analysis of Mycobacteria. Manual for Fingerprinting of M. tuberculosis Strains, National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands, 1995.
[8]  D. Parashar, D. S. Chauhan, V. D. Sharma, and V. M. Katoch, “Applications of real-time PCR technology to mycobacterial research,” Indian Journal of Medical Research, vol. 124, pp. 385–398, 2006.
[9]  G. Suman, K. Jamil, K. Suseela, and M. C. H. Vamsy, “Novel mutations of CYP3A4 in fine needle aspiration cytology samples of breast cancer patients and its clinical correlations,” Cancer Biomarkers, vol. 5, no. 1, pp. 33–40, 2009.
[10]  M. Espasa, J. González-Martín, F. Alcaide et al., “Direct detection in clinical samples of multiple gene mutations causing resistance of Mycobacterium tuberculosis to isoniazid and rifampicin using fluorogenic probes,” Journal of Antimicrobial Chemotherapy, vol. 55, no. 6, pp. 860–865, 2005.
[11]  M. Ruiz, M. J. Torres, A. C. Llanos, A. Arroyo, J. C. Palomares, and J. Aznar, “Direct detection of rifampin- and isoniazid-resistant Mycobacterium tuberculosis in auramine-rhodamine-positive sputum specimens by real-time PCR,” Journal of Clinical Microbiology, vol. 42, no. 4, pp. 1585–1589, 2004.
[12]  N. Lema?tre, S. Armand, A. Vachée, O. Capilliez, C. Dumoulin, and R. J. Courcol, “Comparison of the real-time PCR method and the gen-probe amplified Mycobacterium tuberculosis direct test for detection of Mycobacterium tuberculosis in pulmonary and nonpulmonary specimens,” Journal of Clinical Microbiology, vol. 42, no. 9, pp. 4307–4309, 2004, Comparative study of real-time PCR with the amplified MTD method in clinical samples.
[13]  F. Broccolo, P. Scarpellini, G. Locatelli et al., “Rapid diagnosis of mycobacterial infections and quantitation of Mycobacterium tuberculosis load by two real-time calibrated PCR assays,” Journal of Clinical Microbiology, vol. 41, no. 10, pp. 4565–4572, 2003.
[14]  D. J. Jin and C. A. Gross, “Mapping and sequencing of mutations in the Escherichia coli rpoB gene that lead to rifampicin resistance,” Journal of Molecular Biology, vol. 202, no. 1, pp. 45–58, 1988.
[15]  D. J. Jin and C. A. Gross, “Characterization of the pleiotropic phenotypes of rifampin-resistant rpoB mutants of Escherichia coli,” Journal of Bacteriology, vol. 171, no. 9, pp. 5229–5231, 1989.
[16]  N. Siddiqi, M. Shamim, S. Hussain et al., “Molecular Characterization of Multidrug-Resistant Isolates ofMycobacterium tuberculosisfrom Patients in North India,” 2002, vol. 46, no. 2, pp. 443–450.
[17]  D. L. Williams, C. Waguespack, K. Eisenach et al., “Characterization of rifampin resistance in pathogenic mycobacteria,” Antimicrobial Agents and Chemotherapy, vol. 38, no. 10, pp. 2380–2386, 1994.
[18]  C. Mani, N. Selvakumar, S. Narayanan, and P. R. Narayanan, “Mutations in the rpoB gene of multidrug-resistant Mycobacterium tuberculosis clinical isolates from India,” Journal of Clinical Microbiology, vol. 39, no. 8, pp. 2987–2990, 2001.
[19]  A. Mercy, L. Lingala, A. Srikantam, S. Jain, K. V. S. M. Rao, and P. V. R. Rao, “Clinical and geographical profiles of rpoB gene mutations inMycobacterium tuberculosisisolates from Hyderabad and Koraput in India,” Journal of Microbiology and Antimicrobials, vol. 2, no. 2, p. 13, 2010.
[20]  D. V. Gadre, U. R. Singh, K. Saxena, A. Bhatia, and V. Talwar, “Diagnosis of tubercular cervical lymphadenitis by FNAC, microscopy and culture,” Indian Journal of Tuberculosis, vol. 38, no. 1, pp. 25–27, 1991.
[21]  J. E. Clarridge, R. M. Shawar, T. M. Shinnick, and B. B. Plikaytis, “Large-scale use of polymerase chain reaction for detection of Mycobacterium tuberculosis in a routine mycobacteriology laboratory,” Journal of Clinical Microbiology, vol. 31, no. 8, pp. 2049–2056, 1993.
[22]  S. P. Pamra and G. P. Mathur, “A cooperative study of tuberculous cervical lymphadenitis,” Indian Journal of Medical Research, vol. 62, no. 11, pp. 1631–1646, 1974.

Full-Text

comments powered by Disqus

Contact Us

service@oalib.com

QQ:3279437679

WhatsApp +8615387084133

WeChat 1538708413