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Türkiye'nin Doğu Anadolu bölgesinden izole edilen M. tuberculosis izolatlarının inhouse PCR yöntemi ile moleküler epidemiyolojik tiplendirilmesi

Year 2022, Volume: 47 Issue: 1, 191 - 198, 31.03.2022
https://doi.org/10.17826/cumj.997873

Abstract

Amaç: Örneklerin mikroskopi ve kültür sonuçlarının karşılaştırılması, izolatların ilaç direnç oranlarının belirlenmesi, suşlar arasındaki epidemiyolojik ilişkilerin ERIC-PCR, RAPD-PCR, OUT-PCR ile ev içi PCR tekniğine dayalı olarak değerlendirilmesi amaçlandı.
Gereç ve Yöntem: 2010 numunenin direkt mikroskopi ve kültür sonuçları analiz edildi. İlaç duyarlılık sonuçları TULSA'dan elde edildi. İzolatların tiplendirilmesi, bir devlet üniversitesinin tıp fakültesinin mikrobiyoloji laboratuvarında, in house PCR yöntemi ile gerçekleştirilmiştir.
Bulgular: Pozitif örneklerin %2.68'inde Aside dirençli basil (ARB) pozitif + kültür pozitifliği, yaymada %2.93'ünde ARB pozitif ve kültür negatif, yaymada %1.34'ünde ARB negatif ve kültür pozitifliği vardı. Kültür pozitif olan 33 izolatta primer antitüberküloz (anti-TB) ilaçlara direnç görülmezken, bir veya daha fazla primer anti-TB ilaca direnç %9.09'da gözlendi. Tek ilaç direnci %3.03, Isoniazid (INH) ve INH kritik ilaçlara direnç %6.06 idi. İzolatlar ERIC-PCR ile 3 gruba, OUT-PCR için 5 grup ve RAPD-PCR ile 6 gruba ayrıldı.
Sonuç: Çalışılan numune oranının düşük olması, tedavi sürecinde alınan numunelerin negatif olması nedeniyle pozitiflik oranları düşüktü. ERIC-PCR, RAPD-PCR ve OUT-PCR gibi moleküler teknikler, kökenler arasındaki farklılıkları, benzerlikleri değerlendirmede Mycobacterium tuberculosis'in (MTB) epidemiyolojik tiplendirilmesi için kolay, hızlı ve ucuz yöntemlerdir.

References

  • World Health Organization. Global Tuberculosis Report, 2020. Geneva, WHO, 2020.
  • T.C. Ministry of Health General Directorate of Public Health Tuberculosis Department. TB War in Turkey 2018 Report. Ankara, Ministry of Health, 2018.
  • T.C. Ministry of Health General Directorate of Public Health Tuberculosis Department, TB war in Turkey 2019 Report. Ankara, Ministry of Health, 2020.
  • Dheda K, Ruhwald M, Theron G, Peter J, Yam WC. Point-of-care diagnosis of tuberculosis: past, present and future. Respirology. 2013;18:217-32.
  • Perkins MD, Cunningham J. Facing the crisis: improving the diagnosis of tuberculosis in the HIV era. J Infect Dis. 2007;15:15-27.
  • Cattamanchi A, Davis JL, Pai M, Huang L, Hopewell PC, Steingart KR. Does bleach processing increase the accuracy of sputum smear microscopy for diagnosing pulmonary tuberculosis? J Clin Microbiol. 2010;48:2433-9.
  • Alp A. Current status in laboratory diagnosis of tuberculosis. Hacettepe Tıp Dergisi. 2011;42:28-33.
  • Meehan CJ, Goig GA, Kohl TA, Verboven L, Dippenaar A, Ezewudo M et al. Whole genome sequencing of Mycobacterium tuberculosis: current standards and open issues. Nat Rev Microbiol. 2019;17:533-45.
  • Igarashi Y, Chikamatsu K, Aono A, Yi L, Yamada H, Takaki A et al. Laboratory evaluation of the Anyplex II MTB/MDR and MTB/XDR tests based on multiplex real-time PCR and melting-temperature analysis to identify Mycobacterium tuberculosis and drug resistance. Diagn Microbiol Infect Dis. 2017;89:276-81.
  • Ruiz P, Causse M, Vaquero M, Gutierrez JB, Casal M. Evaluation of a new automated Abbott RealTime MTB RIF/INH assay for qualitative detection of rifampicin/isoniazid resistance in pulmonary and extra-pulmonary clinical samples of Mycobacterium tuberculosis. Infect Drug Resist. 2017;6:463-7.
  • World Health Organization. Technical Report on Critical Concentrations for Drug Susceptibility Testing of Medicines Used in the Treatment of Drug-Resistant Tuberculosis . Geneva, WHO, 2018.
  • Centers for Disease Control and Prevention (CDC). Multiple misdiagnoses of tuberculosis resulting from laboratory error--Wisconsin, 1996. MMWR Morb Mortal Wkly Rep.1997;29:797-801.
  • Burman WJ, Reves RR. Review of false-positive cultures for Mycobacterium tuberculosis and recommendations for avoiding unnecessary treatment. Clin Infect Dis. 2000;31:1390-5.
  • Cook VJ, Stark G, Roscoe DL, Kwong A, Elwood RK. Investigation of suspected laboratory cross-contamination: interpretation of single smear-negative, positive cultures for Mycobacterium tuberculosis. Clin Microbiol Infect. 2006;12:1042-5.
  • Fitzpatrick L, Braden C, Cronin W, English J, Campbell E, Valway S et al. Investigation of Laboratory cross-contamination of Mycobacterium tuberculosis cultures. Clin Infect Dis. 2004;15;38:52-4.
  • Carroll NM, Richardson M, van Helden PD. Criteria for identification of cross-contamination of cultures of Mycobacterium tuberculosis in routine microbiology laboratories. J Clin Microbiol. 2003;41:269-70.
  • Jain A, Dixit P. Multidrug-resistant to extensively drug resistant tuberculosis: what is next? J Biosci. 2008;33:605-16.
  • Dündar D, Sönmez-Tamer G. Resistance rates of Mycobacterium tuberculosis complex isolates to primary antituberculous drugs. Klimik. 2009;22:52-4.
  • Kanduma E, McHugh TD, Gillespie SH. Molecular methods for Mycobacterium tuberculosis strain typing: a users guide. J Appl Microbiol. 2003;94:781-91.
  • Durmaz R, Zozio T, Gunal S, Allix C, Fauville-Dufaux M, Rastogi N. Population-based molecular epidemiological study of tuberculosis in Malatya, Turkey. J Clin Microbiol. 2007;45:4027-35.
  • N. Fomukong, M. Beggs, E.I. Hajj, K. Prabhakar, P.R. Narayanam. Differences tuberculosis strains: low and high copy number of IS6110. Tuber Lung Dis. 1997;78:109-16.
  • Salfinger M, Pfyffer GE. The new diagnostic mycobacteriology laboratory. Eur J Clin Microbiol Infect Dis. 1994;13:961-79.
  • Ministry of Health of the Republic of Turkey. National Standards of Microbiology. National Tuberculosis diagnostic guide (UTTR). Ankara, Aydogdu Offset, 2014.
  • Zeka AN, Tasbakan S, Cavusoglu C. Evaluation of the GeneXpert MTB/RIF assay for rapid diagnosis of tuberculosis and detection of rifampin resistance in pulmonary and extrapulmonary specimens. J Clin Microbiol. 2011;49:4138-41.
  • Ismail N, Ismail F, Omar SV, Blows L, Gardee Y, Koornhof H et al. Drug resistant tuberculosis in Africa: Current status, gaps and opportunities. Afr J Lab Med. 2018;6;7:781.
  • Pillay M, Sturm AW. Evolution of the extensively drug-resistant F15/LAM4/KZN strain of Mycobacterium tuberculosis in KwaZulu-Natal, South Africa. Clin Infect Dis. 2007;1;45:1409-14.
  • Kisa O, Tarhan G, Gunal S, Albay A, Durmaz R, Saribas Z et al. Distribution of spoligotyping defined genotypic lineages among drug-resistant Mycobacterium tuberculosis complex clinical isolates in Ankara, Turkey. PLoS One. 2012;7:e30331.
  • Özen N, Kula Atik T, Çetin Duran A. Evaluation of Mycobacterium tuberculosis culture and drug susceptibility test results and the distribution of nontuberculosis mycobacteria from the clinical specimens. Mikrobiyol Bul. 2020;54:559-74.
  • Dye C, Scheele S, Dolin P, Pathania V, Raviglione MC. Consensus statement. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO Global Surveillance and Monitoring Project. JAMA. 1999;18;282:677-86.
  • Kurtoğlu MG, Ozdemir M, Keşli R, Ozkalp B, Baysal B. Isolation rate of Mycobacterium tuberculosis complex from patients with suspected tuberculosis and identification of the strains with BACTEC™ NAP and immunochromatographic TB Ag MPT64 Rapid™ Test. Mikrobiyol Bul. 2011;45:266-73.
  • Kurtoğlu MG, Yüksekkaya Ş, Özdemir M, Baysal B. Comparison of direct preparation and culture results in mycobacteriology laboratory of an educational and research hospital. Selcuk Med J. 2011;27:2:69-72.
  • Şenoğlu S, Şahin M, Pelivanoğlu F, Şengöz G. Investigation of anti-tuberculosis drug sensitivity results in sixty-one extra-pulmonary samples by MGIT method. Med Bull Hasek. 2019;57:279-84.
  • Floodhead AH, Hasbek M, Steel C, caretaker MZ. Resistance to primary antituberculous drugs in Sivas Cumhuriyet University Medical Faculty Hospital between 2011 and 2018. ANKEM Derg. 2019;33:83-8.
  • Akşkan HE, Bostanoğlu E, Turunç T, Çolakoğlu Ş, Demiroğlu YZ, Lead E et al. Retrospectively, six-year results of the tuberculosis laboratory and rates of resistance to antimicobacterial drugs. Turk Toraks Derg. 2013;14:53-8.
  • Infallible D, Says G. Mycobacterium türlerinin alt solunum yolu örneklerinden basit besiyerleri ile izolasyonu ve maliyet/etkinlik analizi. İstanbul Kanuni Sultan Süleyman Tıp Dergisi. 2020;12:77-81.
  • Ozcholpan OO, driver S, Özkütük N, Çavuşoğlu C. Distribution of nontuberculous mycobacteria isolated from clinical specimens and identified with DNA sequence analysis. Microbiyol Bul. 2015;49:484-93.
  • Appak Ö, Türkel S, Esen N, Özkütük AA. Comparison of polymerase chain reaction-restriction enzyme analysis method and DNA sequence analysis results in the identification of non-tuberculous mycobacteria. Acta Microbiol Immunol Hung. 2018;65:515-27.
  • Velayati AA, Rahideh S, Nezhad ZD, Farnia P, Mirsaeidi m. Nontuberculous mycobacteria in Middle East: Current situation and future challenges. Int J Mycobacteriol. 2015;4:7-17.
  • Adjemian J, Daniel-Wayman S, Ricotta E, Prevots DR. Epidemiology of nontuberculous mycobacteriosis. Semin Respir Crit Care Med. 2018;39:325-35.
  • Tarhan G, January F, Ceyhan I. Comparison of ERIC-PCR, OUT- PCR and spoligotyping methods to diagnose of cross-contamination with mycobacterium tuberculosis. Advances in Biotechnology and Microbiology. 2016;1:55564. (2016).

Molecular epidemiological typing of M. tuberculosis isolates isolated from Turkey's Eastern Anatolia with in house PCR method

Year 2022, Volume: 47 Issue: 1, 191 - 198, 31.03.2022
https://doi.org/10.17826/cumj.997873

Abstract

Purpose: The aim of this study as to compare microscopy and culture results of samples, determine drug resistance rates of the isolates, evaluate epidemiological relationship between the strains with ERIC-PCR, RAPD-PCR, OUT-PCR based on in house PCR technique.
Materials and Methods: Direct microscopy and culture results of 2010 samples were analyzed. Drug sensitivity results were obtained from TULSA. The typing of isolates based on in house PCR was carried out in the microbiology laboratory of a faculty of medicine in a state university.
Results: Of positive samples, 2.68% had Acid-resistant bacilli (ARB) positive + culture positivity, 2.93% had ARB positive and culture negative in smear, and 1.34% had ARB negative and culture positivity in smear. Resistance to primary antituberculous (anti-TB) drugs wasn’t observed in 33 culture positive isolates, whereas resistance to one or more primary anti-TB drugs was observed in 9.09%. Single drug resistance was 3.03%, resistance to Isoniazid (INH) and INH critical drugs was 6.06%. Isolates were divided 3 groups by ERIC-PCR, 5 groups for OUT-PCR and 6 by RAPD-PCR.
Conclusion: Positivity rates were low due to low rate of studied samples, negative samples taken during treatment process. Molecular techniques like ERIC-PCR, RAPD-PCR and OUT-PCR are easy, fast and inexpensive methods for the epidemiological typing of Mycobacterium tuberculosis (MTB) in evaluating distinctions, similarities between origins.

References

  • World Health Organization. Global Tuberculosis Report, 2020. Geneva, WHO, 2020.
  • T.C. Ministry of Health General Directorate of Public Health Tuberculosis Department. TB War in Turkey 2018 Report. Ankara, Ministry of Health, 2018.
  • T.C. Ministry of Health General Directorate of Public Health Tuberculosis Department, TB war in Turkey 2019 Report. Ankara, Ministry of Health, 2020.
  • Dheda K, Ruhwald M, Theron G, Peter J, Yam WC. Point-of-care diagnosis of tuberculosis: past, present and future. Respirology. 2013;18:217-32.
  • Perkins MD, Cunningham J. Facing the crisis: improving the diagnosis of tuberculosis in the HIV era. J Infect Dis. 2007;15:15-27.
  • Cattamanchi A, Davis JL, Pai M, Huang L, Hopewell PC, Steingart KR. Does bleach processing increase the accuracy of sputum smear microscopy for diagnosing pulmonary tuberculosis? J Clin Microbiol. 2010;48:2433-9.
  • Alp A. Current status in laboratory diagnosis of tuberculosis. Hacettepe Tıp Dergisi. 2011;42:28-33.
  • Meehan CJ, Goig GA, Kohl TA, Verboven L, Dippenaar A, Ezewudo M et al. Whole genome sequencing of Mycobacterium tuberculosis: current standards and open issues. Nat Rev Microbiol. 2019;17:533-45.
  • Igarashi Y, Chikamatsu K, Aono A, Yi L, Yamada H, Takaki A et al. Laboratory evaluation of the Anyplex II MTB/MDR and MTB/XDR tests based on multiplex real-time PCR and melting-temperature analysis to identify Mycobacterium tuberculosis and drug resistance. Diagn Microbiol Infect Dis. 2017;89:276-81.
  • Ruiz P, Causse M, Vaquero M, Gutierrez JB, Casal M. Evaluation of a new automated Abbott RealTime MTB RIF/INH assay for qualitative detection of rifampicin/isoniazid resistance in pulmonary and extra-pulmonary clinical samples of Mycobacterium tuberculosis. Infect Drug Resist. 2017;6:463-7.
  • World Health Organization. Technical Report on Critical Concentrations for Drug Susceptibility Testing of Medicines Used in the Treatment of Drug-Resistant Tuberculosis . Geneva, WHO, 2018.
  • Centers for Disease Control and Prevention (CDC). Multiple misdiagnoses of tuberculosis resulting from laboratory error--Wisconsin, 1996. MMWR Morb Mortal Wkly Rep.1997;29:797-801.
  • Burman WJ, Reves RR. Review of false-positive cultures for Mycobacterium tuberculosis and recommendations for avoiding unnecessary treatment. Clin Infect Dis. 2000;31:1390-5.
  • Cook VJ, Stark G, Roscoe DL, Kwong A, Elwood RK. Investigation of suspected laboratory cross-contamination: interpretation of single smear-negative, positive cultures for Mycobacterium tuberculosis. Clin Microbiol Infect. 2006;12:1042-5.
  • Fitzpatrick L, Braden C, Cronin W, English J, Campbell E, Valway S et al. Investigation of Laboratory cross-contamination of Mycobacterium tuberculosis cultures. Clin Infect Dis. 2004;15;38:52-4.
  • Carroll NM, Richardson M, van Helden PD. Criteria for identification of cross-contamination of cultures of Mycobacterium tuberculosis in routine microbiology laboratories. J Clin Microbiol. 2003;41:269-70.
  • Jain A, Dixit P. Multidrug-resistant to extensively drug resistant tuberculosis: what is next? J Biosci. 2008;33:605-16.
  • Dündar D, Sönmez-Tamer G. Resistance rates of Mycobacterium tuberculosis complex isolates to primary antituberculous drugs. Klimik. 2009;22:52-4.
  • Kanduma E, McHugh TD, Gillespie SH. Molecular methods for Mycobacterium tuberculosis strain typing: a users guide. J Appl Microbiol. 2003;94:781-91.
  • Durmaz R, Zozio T, Gunal S, Allix C, Fauville-Dufaux M, Rastogi N. Population-based molecular epidemiological study of tuberculosis in Malatya, Turkey. J Clin Microbiol. 2007;45:4027-35.
  • N. Fomukong, M. Beggs, E.I. Hajj, K. Prabhakar, P.R. Narayanam. Differences tuberculosis strains: low and high copy number of IS6110. Tuber Lung Dis. 1997;78:109-16.
  • Salfinger M, Pfyffer GE. The new diagnostic mycobacteriology laboratory. Eur J Clin Microbiol Infect Dis. 1994;13:961-79.
  • Ministry of Health of the Republic of Turkey. National Standards of Microbiology. National Tuberculosis diagnostic guide (UTTR). Ankara, Aydogdu Offset, 2014.
  • Zeka AN, Tasbakan S, Cavusoglu C. Evaluation of the GeneXpert MTB/RIF assay for rapid diagnosis of tuberculosis and detection of rifampin resistance in pulmonary and extrapulmonary specimens. J Clin Microbiol. 2011;49:4138-41.
  • Ismail N, Ismail F, Omar SV, Blows L, Gardee Y, Koornhof H et al. Drug resistant tuberculosis in Africa: Current status, gaps and opportunities. Afr J Lab Med. 2018;6;7:781.
  • Pillay M, Sturm AW. Evolution of the extensively drug-resistant F15/LAM4/KZN strain of Mycobacterium tuberculosis in KwaZulu-Natal, South Africa. Clin Infect Dis. 2007;1;45:1409-14.
  • Kisa O, Tarhan G, Gunal S, Albay A, Durmaz R, Saribas Z et al. Distribution of spoligotyping defined genotypic lineages among drug-resistant Mycobacterium tuberculosis complex clinical isolates in Ankara, Turkey. PLoS One. 2012;7:e30331.
  • Özen N, Kula Atik T, Çetin Duran A. Evaluation of Mycobacterium tuberculosis culture and drug susceptibility test results and the distribution of nontuberculosis mycobacteria from the clinical specimens. Mikrobiyol Bul. 2020;54:559-74.
  • Dye C, Scheele S, Dolin P, Pathania V, Raviglione MC. Consensus statement. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO Global Surveillance and Monitoring Project. JAMA. 1999;18;282:677-86.
  • Kurtoğlu MG, Ozdemir M, Keşli R, Ozkalp B, Baysal B. Isolation rate of Mycobacterium tuberculosis complex from patients with suspected tuberculosis and identification of the strains with BACTEC™ NAP and immunochromatographic TB Ag MPT64 Rapid™ Test. Mikrobiyol Bul. 2011;45:266-73.
  • Kurtoğlu MG, Yüksekkaya Ş, Özdemir M, Baysal B. Comparison of direct preparation and culture results in mycobacteriology laboratory of an educational and research hospital. Selcuk Med J. 2011;27:2:69-72.
  • Şenoğlu S, Şahin M, Pelivanoğlu F, Şengöz G. Investigation of anti-tuberculosis drug sensitivity results in sixty-one extra-pulmonary samples by MGIT method. Med Bull Hasek. 2019;57:279-84.
  • Floodhead AH, Hasbek M, Steel C, caretaker MZ. Resistance to primary antituberculous drugs in Sivas Cumhuriyet University Medical Faculty Hospital between 2011 and 2018. ANKEM Derg. 2019;33:83-8.
  • Akşkan HE, Bostanoğlu E, Turunç T, Çolakoğlu Ş, Demiroğlu YZ, Lead E et al. Retrospectively, six-year results of the tuberculosis laboratory and rates of resistance to antimicobacterial drugs. Turk Toraks Derg. 2013;14:53-8.
  • Infallible D, Says G. Mycobacterium türlerinin alt solunum yolu örneklerinden basit besiyerleri ile izolasyonu ve maliyet/etkinlik analizi. İstanbul Kanuni Sultan Süleyman Tıp Dergisi. 2020;12:77-81.
  • Ozcholpan OO, driver S, Özkütük N, Çavuşoğlu C. Distribution of nontuberculous mycobacteria isolated from clinical specimens and identified with DNA sequence analysis. Microbiyol Bul. 2015;49:484-93.
  • Appak Ö, Türkel S, Esen N, Özkütük AA. Comparison of polymerase chain reaction-restriction enzyme analysis method and DNA sequence analysis results in the identification of non-tuberculous mycobacteria. Acta Microbiol Immunol Hung. 2018;65:515-27.
  • Velayati AA, Rahideh S, Nezhad ZD, Farnia P, Mirsaeidi m. Nontuberculous mycobacteria in Middle East: Current situation and future challenges. Int J Mycobacteriol. 2015;4:7-17.
  • Adjemian J, Daniel-Wayman S, Ricotta E, Prevots DR. Epidemiology of nontuberculous mycobacteriosis. Semin Respir Crit Care Med. 2018;39:325-35.
  • Tarhan G, January F, Ceyhan I. Comparison of ERIC-PCR, OUT- PCR and spoligotyping methods to diagnose of cross-contamination with mycobacterium tuberculosis. Advances in Biotechnology and Microbiology. 2016;1:55564. (2016).
There are 40 citations in total.

Details

Primary Language English
Subjects Clinical Sciences
Journal Section Research
Authors

Elif Aydın 0000-0003-0877-453X

Gülnur Tarhan 0000-0002-1019-0798

Funda Şahin 0000-0001-8406-2011

Sami Eren This is me 0000-0002-6263-8048

Publication Date March 31, 2022
Acceptance Date January 24, 2022
Published in Issue Year 2022 Volume: 47 Issue: 1

Cite

MLA Aydın, Elif et al. “Molecular Epidemiological Typing of M. Tuberculosis Isolates Isolated from Turkey’s Eastern Anatolia With in House PCR Method”. Cukurova Medical Journal, vol. 47, no. 1, 2022, pp. 191-8, doi:10.17826/cumj.997873.