Laboratuvarımızda Konvansi̇yonel ve Moleküler Yöntemlerle Tüberküloz Tanısı: 4 Yıllık Bir Değerlendirme
Year 2024,
, 31 - 36, 01.03.2024
Cihadiye Elif Öztürk
,
Betül Dönmez
,
Pınar Yıldız
,
Emel Çalışkan
,
Gulfidan Uzan Çakmak
Abstract
Amaç: Bu çalışmanın amacı, tüberküloz tanısında direkt mikroskopi, kültür ve Polimeraz Zincir Reaksiyonu (PCR) yöntemlerini karşılaştırmak ve son 4 yılın antibiyotik direnç profilini konvansiyonel ve moleküler yöntemlerle karşılaştırmalı olarak sunmaktır.
Gereç ve Yöntemler: Tüm örneklere Ehrlich-Ziehl-Neelsen (EZN) boyama, Mycobacterium kültürü ve PCR testleri yapıldı. EZN boyama ile aside dirençli basil (ARB) saptanan örneklere direkt hızlı direnç testi yapıldı.
Bulgular: Çalışmaya 968 örnek dahil edildi. Bunların 81’inde (%8) kültür, 78’inde (%8) PCR ve 39’unda (%46) EZN pozitif bulundu. Hem solunum hem de diğer örneklerde aynı gün yapılan PCR kültür ile çok iyi uyum gösterirken, EZN boyaması orta düzeyde uyum gösterdi. Hızlı direnç testinin kültürde saptanmayan rifampisin direncini saptadığı, INH durumunda ise kültür antibiyogramı ile hızlı direnç testinin tam uyumlu olduğu görülmüştür. Hızlı direnç testinin EZN boyaması pozitif olan her hastaya uygulanması, direncin çok erken tespit edilmesini sağlamıştır.
Sonuç: Şüpheli klinik örneklerde tüberküloz ve direncin hızlı tanısında PCR testlerinin yararlı olduğu sonucuna varılmıştır.
References
- Migliori GB, Matteelli A, Cirillo D, Pai M. Diagnosis of multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis: current standards and chal¬lenges. Can J Infect Dis MedM icrobiol 2008; 19:169- 72.
- Nyendak MR, Lewinsohn DA, Lewinsohn DM. New diagnostic methods for tuberculosis. Curr Opin Infect Dis 2009; 22:174-82. http://dx.doi.org/10.1097/QCO.0b013e3283262fe9.
- Mathew P, Kuo YH, Vazirani B, Eng RH, Weinstein MP. Are three sputum acid-fast bacillus smears neces¬sary for discontinuing tuberculosis isolation? J Clin Microbiol 2002; 40: 3482-4.
- Andersen AB, Thybo S, Faussett PG, Stoker NG. Polymerase chain reaction for detection of Mycobacterium tuberculosis in sputum. Eur J Clin Microbiol İnfect Dis 1993;12: 922-7.
- Kikuchi Y, Oka S, Kimura S, Shimada K. Clinical application of the polymerase chain reaction for a rapid diagnosis of Mycobacterium tuberculosis infection. Intern Med 1992;31(8): 1016-22.
- Özdemir Ö. Tüberkülozda Tanı Yöntemleri. Türkiye Klinikleri Tıp Bilimleri Dergisi Tüberküloz Özel Sayısı 1994;14(6): 420-4.
- Karslıgil T, Alagöz SG, Zer Y, Sağlam M, Çay M, Buğur Ş. Comparison of PCR and MGIT 960 florometric methods in the detection of Mycobacterium tuberculosis .Gaziantep Med J 2015;21(4):248-251.
- Nolte FS, Metchock B, Mc Gowan JE, Edwards A, Okwumabua O, Thurmond C. et al. Direct detection of Mycobacterium tuberculosis in Sputum by polymerase chain reaction and DNA hybridization. J Clin Microbiol 1993; 31(7): 1777-82.
- Hashimato T, Suzuki K, Amitani R, Kuze F. Rapid detection of Mycobacterium tuberculosis in sputa by the amplification of IS6110. Intern Med 1995; 34(7): 605-10.
- Kocagöz T, Yılmaz E, Özkara Ş, Kocagöz S, Hayran M, Sachedeva M, Chambers HF. Detection of Mycobacterium tuberculosis in sputum samples by polymerase chain reaction using a simplified procedure. J Clin Microbiol 1993; 31(6): 1435-8.
- Ozturk CE, Balbay OA, Kaya D, Ceyhan I, Bulut I, Sahin I. The resistance to major antituberculous drugs of Mycobacterium tuberculosis strains isolated from the respiratory system specimens of tuberculosis patients in Duzce, Turkey. Jpn J Infect Dis 2005;58(1):47-9.
- Zhuang Y, Wu X, Zhang X, Li G. Detection of Mycobacterium tuberculosis in sputum specimens of pulmoner tuberculosis by DNA amplification. Wei Sheng Wu Hsueh Pao 1992; 32(5): 364-9.
- Chin DP, Yajko DM, Hadley WK, Sanders CA, Nassos P S, Madej JJ, Hopewell PC. Clinical utility of a commerical test based on the polymerase chain reaction for detecting Mycobacterium tuberculosis in respiratory specimens. Am J Respir Crit Care Med 1995; 151: 1872-7.
- Barnard M, Albert H, Coetzee G, O’Brien R, Bosman ME. Rapid molecular screening for multidrug-resistant tuberculosis in a high-volume public health laboratory in South Africa, Am J Respir Crit Care Med 2008;177(7):787-92. http://dx.doi.org/10.1164/rccm.200709-1436OC PMid:18202343.
- Abdulmajed O, Koç AN, Gültekin A , Atalay MA , Kılıç H. Klinik Örneklerden Mikobakteri Türlerinin İzolasyonunda Klasik Tanı Yöntemlerin Karşılaştırılması ve Primer antitüberküloz İlaçlara Duyarlılıklarının Belirlenmesi. Dergi Park 2012;21: 127 – 135.
- Alp A. Tüberkülozun laboratuvar tanısında güncel durum. Hacettepe Tıp Derg 2011; 42(1): 28-33.
- Grange JM (1990) Tuberculosis: Topley and Wilson’s Principle of bacteriology, virology and immunity, vol II, 8th edn. Butler and Tanner Ltd, London, pp 93–118.
- Rozales FP, Machado ABMP, De Paris F, Zavascki AP , Barth AL. PCR to detect Mycobacterium tuberculosis in respiratory tract samples: evaluation of clinical data. Epidemiol Infect. 2014;142(7):1517-23.
- Parsons LM, Somoskövi A, Gutierrez C, Lee E, Paramasivan CN, Abimiku A, et al. Laboratory diagnosis of tuberculosis in resource-poor countries: Challenges and opportunities. Clin Microbiol Rev. 2011; 24(2):314-50. doi: 10.1128/CMR.00059-10.
- Cheng VCC, Yew WW, Yuen KY. Molecular diagnostics in tuberculosis. Eur J Clin Microbiol Infect Dis 2005; 24:711-20.
- Greco S, Girardi E, Navarra A, Saltini C. Current evidence on diagnostic accuracy of commercially based nucleic acid amplification tests for the diagnosis of pulmonary tuberculosis. Thorax 2006; 61:783-90.
- Sarmiento OL, Weigle KA, Alexander J, Weber DJ, Miller WC. Assessment by meta-analysis of PCR for diagnosis of smear-negative pulmonary tuberculosis. J Clin Microbiol 2003; 41: 3233-40.
- Greco S, Rulli M, Girardi E, Piersimoni C, Saltini C. Diagnostic accuracy of in-house PCR for pulmonary tuberculosis in smear-positive patients: meta-analysis and metaregression. J Clin Microbiol 2009; 47: 569-76.
- Phetsuksiri B, Rudeeaneksin J, Srisungngam S, Bunchoo S, Klayut W, Nakajima C, Hamada S, Suzuki Y. Comparison of Loop-Mediated Isothermal Amplification, Microscopy, Culture, and PCR for Diagnosis of Pulmonary Tuberculosis. Jpn J Infect Dis. 2020; 22;73(4):272-277. doi: 10.7883/yoken. JJID.2019.335.
- Acharya B, Acharya A, Gautam S, Ghimire SP , Mishra G , Parajuli N, Sapkota B. Advances in diagnosis of Tuberculosis: an update into molecular diagnosis of Mycobacterium tuberculosis. Mol Biol Rep. 2020;47(5):4065-4075. doi:10.1007/s11033-020-05413-7.
- Saygan MB, Ocak F, Ceyhan, ve ark. Bölge tüberküloz laboratuarlarından gönderilen Mycobacterium tuberculosis suslarının major antitüberküloz ilaçlara duyarlılıkları, Refik Saydam Hıfzısıhha Merkez Baskanlıgı Tüberküloz Referans ve Araştırma Laboratuvarı, Sıhhiye, Ankara Mikrobiyoloji Bülteni 2007; 41: 403-9.
- Ling, DI, Zwerling AA, Pai M. GenoType MTBDR assays for the diagnosis of multidrug-resistant tuberculosis: a meta-analysis, Eur Respir J 2008; 32(5):1165-74. http://dx.doi.org/10.1183/09031936.00061808 PMid:18614561.
- Scarparo C, Ricordi P, Ruggiero G, Piccoli P. Evaluation of the fully automated BACTEC MGIT 960 system for testing susceptibility of Mycobacterium tuberculosis to pyrazinamide, streptomycin, isoniazid, rifampin, and ethambutol and comparison with the radiometric BACTEC 460TB method. J Clin Microbiol 2004; 42: 1109-14.
- Bergmann JS, Woods GL. Reliability of mycobacteria growth indicator tube for testing susceptibility of Mycobacterium tuberculosis to ethambutol and streptomycin. J Clin Microbiol 1997;35: 3325-7.
- Dorman SE, Chihota VN, Lewis J., Minty van der Meulen M, Mathema B, Beylis N et al. Genotype MTBDRplus for direct detection of Mycobacterium tuberculosis and drug resistance in gold miners in South Africa. J Clin Microbiol. 2012; 50(4): 1189–1194.
Diagnosis of Tuberculosis by Conventional and Molecular Methods in Our Laboratory: A 4-Year Assessment
Year 2024,
, 31 - 36, 01.03.2024
Cihadiye Elif Öztürk
,
Betül Dönmez
,
Pınar Yıldız
,
Emel Çalışkan
,
Gulfidan Uzan Çakmak
Abstract
Objective: The aim of this study was to compare direct microscopy, culture and Polymerase Chain Reaction (PCR) methods and to present the antibiotic resistance profile of the last 4 years comparatively by conventional and molecular methods.
Material and Method: Bacterial culture, EZN and PCR methods were applied to all samples. Direct rapid resistance test was performed for EZN positive samples.
Results: 968 patients were included in the study. Culture was positive in 81 (8%), PCR in 78 (8%) and EZN in 39 (46%) patients. PCR performed on the same day in both respiratory and other samples showed very good agreement with culture, while EZN staining showed moderate agreement. It was observed that the rapid resistance test detected rifampicin resistance which was not detected in culture, and in the case of INH, culture antibiogram and rapid resistance test were fully compatible. Application of the rapid resistance test to every patient with positive EZN staining resulted in very early detection of resistance.
Conclusion: It was concluded that PCR tests are useful in the rapid diagnosis of tuberculosis and resistance in suspicious clinical samples.
Ethical Statement
This study was supported by Düzce University Scientific Research Project numbered BAP-2019.04.01.951.
References
- Migliori GB, Matteelli A, Cirillo D, Pai M. Diagnosis of multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis: current standards and chal¬lenges. Can J Infect Dis MedM icrobiol 2008; 19:169- 72.
- Nyendak MR, Lewinsohn DA, Lewinsohn DM. New diagnostic methods for tuberculosis. Curr Opin Infect Dis 2009; 22:174-82. http://dx.doi.org/10.1097/QCO.0b013e3283262fe9.
- Mathew P, Kuo YH, Vazirani B, Eng RH, Weinstein MP. Are three sputum acid-fast bacillus smears neces¬sary for discontinuing tuberculosis isolation? J Clin Microbiol 2002; 40: 3482-4.
- Andersen AB, Thybo S, Faussett PG, Stoker NG. Polymerase chain reaction for detection of Mycobacterium tuberculosis in sputum. Eur J Clin Microbiol İnfect Dis 1993;12: 922-7.
- Kikuchi Y, Oka S, Kimura S, Shimada K. Clinical application of the polymerase chain reaction for a rapid diagnosis of Mycobacterium tuberculosis infection. Intern Med 1992;31(8): 1016-22.
- Özdemir Ö. Tüberkülozda Tanı Yöntemleri. Türkiye Klinikleri Tıp Bilimleri Dergisi Tüberküloz Özel Sayısı 1994;14(6): 420-4.
- Karslıgil T, Alagöz SG, Zer Y, Sağlam M, Çay M, Buğur Ş. Comparison of PCR and MGIT 960 florometric methods in the detection of Mycobacterium tuberculosis .Gaziantep Med J 2015;21(4):248-251.
- Nolte FS, Metchock B, Mc Gowan JE, Edwards A, Okwumabua O, Thurmond C. et al. Direct detection of Mycobacterium tuberculosis in Sputum by polymerase chain reaction and DNA hybridization. J Clin Microbiol 1993; 31(7): 1777-82.
- Hashimato T, Suzuki K, Amitani R, Kuze F. Rapid detection of Mycobacterium tuberculosis in sputa by the amplification of IS6110. Intern Med 1995; 34(7): 605-10.
- Kocagöz T, Yılmaz E, Özkara Ş, Kocagöz S, Hayran M, Sachedeva M, Chambers HF. Detection of Mycobacterium tuberculosis in sputum samples by polymerase chain reaction using a simplified procedure. J Clin Microbiol 1993; 31(6): 1435-8.
- Ozturk CE, Balbay OA, Kaya D, Ceyhan I, Bulut I, Sahin I. The resistance to major antituberculous drugs of Mycobacterium tuberculosis strains isolated from the respiratory system specimens of tuberculosis patients in Duzce, Turkey. Jpn J Infect Dis 2005;58(1):47-9.
- Zhuang Y, Wu X, Zhang X, Li G. Detection of Mycobacterium tuberculosis in sputum specimens of pulmoner tuberculosis by DNA amplification. Wei Sheng Wu Hsueh Pao 1992; 32(5): 364-9.
- Chin DP, Yajko DM, Hadley WK, Sanders CA, Nassos P S, Madej JJ, Hopewell PC. Clinical utility of a commerical test based on the polymerase chain reaction for detecting Mycobacterium tuberculosis in respiratory specimens. Am J Respir Crit Care Med 1995; 151: 1872-7.
- Barnard M, Albert H, Coetzee G, O’Brien R, Bosman ME. Rapid molecular screening for multidrug-resistant tuberculosis in a high-volume public health laboratory in South Africa, Am J Respir Crit Care Med 2008;177(7):787-92. http://dx.doi.org/10.1164/rccm.200709-1436OC PMid:18202343.
- Abdulmajed O, Koç AN, Gültekin A , Atalay MA , Kılıç H. Klinik Örneklerden Mikobakteri Türlerinin İzolasyonunda Klasik Tanı Yöntemlerin Karşılaştırılması ve Primer antitüberküloz İlaçlara Duyarlılıklarının Belirlenmesi. Dergi Park 2012;21: 127 – 135.
- Alp A. Tüberkülozun laboratuvar tanısında güncel durum. Hacettepe Tıp Derg 2011; 42(1): 28-33.
- Grange JM (1990) Tuberculosis: Topley and Wilson’s Principle of bacteriology, virology and immunity, vol II, 8th edn. Butler and Tanner Ltd, London, pp 93–118.
- Rozales FP, Machado ABMP, De Paris F, Zavascki AP , Barth AL. PCR to detect Mycobacterium tuberculosis in respiratory tract samples: evaluation of clinical data. Epidemiol Infect. 2014;142(7):1517-23.
- Parsons LM, Somoskövi A, Gutierrez C, Lee E, Paramasivan CN, Abimiku A, et al. Laboratory diagnosis of tuberculosis in resource-poor countries: Challenges and opportunities. Clin Microbiol Rev. 2011; 24(2):314-50. doi: 10.1128/CMR.00059-10.
- Cheng VCC, Yew WW, Yuen KY. Molecular diagnostics in tuberculosis. Eur J Clin Microbiol Infect Dis 2005; 24:711-20.
- Greco S, Girardi E, Navarra A, Saltini C. Current evidence on diagnostic accuracy of commercially based nucleic acid amplification tests for the diagnosis of pulmonary tuberculosis. Thorax 2006; 61:783-90.
- Sarmiento OL, Weigle KA, Alexander J, Weber DJ, Miller WC. Assessment by meta-analysis of PCR for diagnosis of smear-negative pulmonary tuberculosis. J Clin Microbiol 2003; 41: 3233-40.
- Greco S, Rulli M, Girardi E, Piersimoni C, Saltini C. Diagnostic accuracy of in-house PCR for pulmonary tuberculosis in smear-positive patients: meta-analysis and metaregression. J Clin Microbiol 2009; 47: 569-76.
- Phetsuksiri B, Rudeeaneksin J, Srisungngam S, Bunchoo S, Klayut W, Nakajima C, Hamada S, Suzuki Y. Comparison of Loop-Mediated Isothermal Amplification, Microscopy, Culture, and PCR for Diagnosis of Pulmonary Tuberculosis. Jpn J Infect Dis. 2020; 22;73(4):272-277. doi: 10.7883/yoken. JJID.2019.335.
- Acharya B, Acharya A, Gautam S, Ghimire SP , Mishra G , Parajuli N, Sapkota B. Advances in diagnosis of Tuberculosis: an update into molecular diagnosis of Mycobacterium tuberculosis. Mol Biol Rep. 2020;47(5):4065-4075. doi:10.1007/s11033-020-05413-7.
- Saygan MB, Ocak F, Ceyhan, ve ark. Bölge tüberküloz laboratuarlarından gönderilen Mycobacterium tuberculosis suslarının major antitüberküloz ilaçlara duyarlılıkları, Refik Saydam Hıfzısıhha Merkez Baskanlıgı Tüberküloz Referans ve Araştırma Laboratuvarı, Sıhhiye, Ankara Mikrobiyoloji Bülteni 2007; 41: 403-9.
- Ling, DI, Zwerling AA, Pai M. GenoType MTBDR assays for the diagnosis of multidrug-resistant tuberculosis: a meta-analysis, Eur Respir J 2008; 32(5):1165-74. http://dx.doi.org/10.1183/09031936.00061808 PMid:18614561.
- Scarparo C, Ricordi P, Ruggiero G, Piccoli P. Evaluation of the fully automated BACTEC MGIT 960 system for testing susceptibility of Mycobacterium tuberculosis to pyrazinamide, streptomycin, isoniazid, rifampin, and ethambutol and comparison with the radiometric BACTEC 460TB method. J Clin Microbiol 2004; 42: 1109-14.
- Bergmann JS, Woods GL. Reliability of mycobacteria growth indicator tube for testing susceptibility of Mycobacterium tuberculosis to ethambutol and streptomycin. J Clin Microbiol 1997;35: 3325-7.
- Dorman SE, Chihota VN, Lewis J., Minty van der Meulen M, Mathema B, Beylis N et al. Genotype MTBDRplus for direct detection of Mycobacterium tuberculosis and drug resistance in gold miners in South Africa. J Clin Microbiol. 2012; 50(4): 1189–1194.