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The identification of Candida species isolated from clinical specimens of immunocompromised patients with PCR and determination of antifungal resistance genes with RFLP and sequencing analysis

Year 2012, Volume: 39 Issue: 2, 242 - 250, 01.06.2012
https://doi.org/10.5798/diclemedj.0921.2012.02.0135

Abstract

References

  • Hazen KC, Howell SA. Candida, Cryptococcus, and other yeasts of medical importance, “Murray PR, Baron EJ, Jorgensen JH, Pfaller MA, Yolken RH (eds): Manual of Cilinical Microbiology, 8th edn.” ASM Press,Washington, 2003:1693-710.
  • Kurtz MB, Cortelyou MW, Kirsch DR. Integrative transfor- mation of Candida albicans, using a cloned Candida ADE2 gene. Mol Cell Biol 1986;6(1):142–9.
  • Chang HC, Leaw SN, Huang AH, Wu TL, Chang TC. Rapid identification of yeasts in positive blood cultures by a mul- tiplex PCR method. J Clin Microbiol 2001;39(10):3466-71.
  • Luo G, Mitchell TG. Rapid identification of pathogenic fungi directly from cultures by using multiplex PCR., J Clin Mi- crobiol 2002;40(8):2860-5.
  • Romeo O, Racco C, Criseo G. Amplification of the hyphal wall protein 1 gene to distinguish Candida albicans from Candida dubliniensis. J Clin Microbiol 2006;44(7):2590-2.
  • Sanglard D, Bile J. Current understanding of the modes of action and resistance mechanisms to conventional and emerging antifungal agents for treatment of Candida of in- fections, “Calderone RA (ed): Candida and Candidiasis, 1th edn” kitabında ASM Press, Washington, 2002:349-83.
  • Whelan WL, Partridge RM, Magee PT. Heterozygosity and segregation in Candida albicans. Mol Gen Genet 1980; 180(1):107–13.
  • Sanglard D, Ischer F, Bille J. Role of ATP-binding-cassette transporter genes in high-frequency acquisition of resis- tance to azole antifungals in Candida glabrata. Antimicrob Agents Chemother 2001;45(4):1174-83.
  • Arıkan S. Fungal hastalıklarda genomik ve proteomiklerin yeri ve önemi. ANKEM Derg 2009;23(Ek 2): 53-6.
  • Clinical and Laboratory Standards Institute (CLSI): Ref- erence method for broth dilution antifungal susceptibil- ity testing of yeasts, Approved Standard 3rd edn, CLSI Document M27-A3, Clinical and Laboratory Standards Institute,Wayne, PA 2008;28(14).
  • Xu Y, Chen L, Li C. Susceptibility of clinical isolates of Candida species to fluconazole and detection of Candida albicans ERG11 mutations. J Antimicrob Chemother 2008;61(4):798-804.
  • Alpar R. Spor Bilimlerinde Uygulamalı İstatistik, 2. Baskı, s.219-55, Nobel Tıp Kitabevi, İstanbul (2001).
  • Günalp A. Gen ve Moleküler Biyoloji, 1st edn, , Hacettepe Üniversitesi Yayınları, Ankara, 1965;1:226-63.
  • Hawthorne DC, Mortimer RK. Chromosome mapping in Saccharomyces centromere-linked genes. Genetics 1960;45(8):1085-110.
  • Shin JH, Nolte FS, Morrison CJ. Rapid identification of Candida species in blood cultures by a clinically useful PCR method. J Clin Microbiol 1997;35(6):1454-9.
  • Ausubel FM, Brent R, Kingston RE et al. Short Protocols in Molecular Biology, 3rd edn, John Wiley&Sons, New York, 1995:642-50.
  • Einsele H, Hebart H, Roller G, Loffler J, Rothenhofer I, Muller CA. Detection and identification of fungal patho- gens in blood by using molecular probes. J Clin Microbiol 1997;35(6):1353-60.
  • Maaroufi Y, Heymans C, De Bruyne JM, Duchateau V, Ro- driguez-Villalobos H. Rapid detection of Candida albicans in clinical blood samples by using a TaqMan-based PCR assay. J Clin Microbiol 2003;41(7):3293-8.
  • Li YL, Leaw SN, Chen JH, Chang HC, Chang TC. Rap- id identification of yeasts commonly found in positive blood cultures by amplification of the internal transcribed spacer regions 1 and 2. Eur J Clin Microbiol Infect Dis 2003;22(11):693-6.
  • Skladny H, Buchheidt D, Baust C, Krieg-Schneider F, Sei- farth W, Leib-Mosch C. Specific detection of Aspergillus species in blood and bronchoalveolar lavage samples of immunocompromised patients by two-step PCR., J Clin Microbiol 1999;37(12):3865-71.
  • Hayette MP, Vaira D, Susin F, Boland P, Christiaens G, Melin P. Detection of Aspergillus species DNA by PCR in bronchoalveolar lavage fluid. J Clin Microbiol 2001;39(6):2338-40.
  • Whıte TC, Marr KA, Bowden RA. Clinical, cellular, and molecular factors that contribute to antifungal drug resis- tance. Clin Microbiol Rev 1998;11(2):382-402 .
  • Haitao Ji, Zhang W, Zhou Y, Zhang M, Zhu J, Song Y. A three-dimensional model of lanosterol 14α-demethylase of Candida albicans and its interaction with azole antifungals. J Med Chem 2000;43(13):2493-505.
  • Favre B, Didmon M, Ryder NS. Multiple amino acid substitutions in lanosterol 14α-demethylase contribute to azole resistance in Candida albicans. Microbiology 1999;145(10):2715-25.
  • Marichal P, Koymans S, Willemsens S, Bellens D, Ver- hasselt P, Luyten W, Borgers M,Ramaekers FC, Odds FC and Vanden Bossche H. Contribution of mutations in the- 111cytochrome P450 14α-demethylase (Erg 11p, Cyp 51p) to azole resistance in Candida albicans. Microbiology 1999;145: 2701-13.
  • Löffler J, Kelly SL, Hebart H, Schumacher U, Lass- Flörl C, Einsele H. Molecular analysis of cyp 51 from flucon- azole resistant Candida albicans strains. FEMS Microbiol Lett1997;151:263-8.

The identification of Candida species isolated from clinical specimens of immunocompromised patients with PCR and determination of antifungal resistance genes with RFLP and sequencing analysis

Year 2012, Volume: 39 Issue: 2, 242 - 250, 01.06.2012
https://doi.org/10.5798/diclemedj.0921.2012.02.0135

Abstract

Amaç: Bu çalışmanın amacı immun sistemi baskılı hastalardan elde edilen klinik örneklerden izole edilen Candida türlerinde PCR tekniğinin ve RFLP ve sekans analizi ile antifungal direnç genlerinin araştırılmasıdır. Gereç ve yöntem: Çalışmada immün sistemi baskılı 200 hastadan alınan klinik örnekler (96 bronkoalveoler lavaj, 56 biyopsi-apse, 8 kan, 15 periton diyaliz sıvısı, 15 plevra sıvısı, 5 beyin omurilik ve 5 perikard sıvısı) geleneksel ve moleküler yöntemler ile incelenmiş ve sonuçlar değerlendirilmiştir. Örneklerden mantar DNA\'sı saptandıktan sonra elde edilen ürünün, multipleks PCR yardımı ile tür düzeyinde tanısı yapılmış, antifungal direnci E-test, direnç genleri ise RFLP analizi ile saptanmıştır. Bulgular: İkiyüz örneğin 30\'unda (%15) kültür yöntemleri ile pozitif sonuç alınmış [20 Candida albicans (%67), beş Candida parapsilosis (%17), beş Candida tropicalis (%17)], 170 (%85) örnekte üreme olmamıştır. Genel primerler ile yapılan PCR testi sonucunda kültürleri olumlu bulunan 30 örneğin tümünün mantar DNA\'sı içerdiği belirlenmiştir. İzole edilen suşların biri E-test yöntemiyle flukanozole dirençli, ikisi flukanozole doza bağlı duyarlı olarak saptanırken 27\'si duyarlı olarak bulunmuştur. Seçilen uygun primerler ile BamHI ve SalI enzimleri kullanılarak RFLP uygulanmış, yapılan analizler sonucunda dirençli bir C.albicans suşunda 600 bp\'lik bant görülmüştür. Candida dubliniensis (950 bp) ve Candida krusei (360 bp) ile yapılan multipleks PCR optimizasyonunda başarı sağlanmıştır. Suşların ERG genine uygun primer çiftleri ile PCR\'ları yapıldıktan sonra yapılan sekans analizi sonucunda dirençli C.albicans suşu referans gen ile karşılaştırıldığında D132E, E216D mutasyonları belirlenmiştir. Sonuç: Moleküler test yöntemleri özellikle immün sistemi baskılanmış hasta populasyonunun kısa sürede doğru tedavisine olanak sağladıkları için yaşamsal açıdan önemlidir.

References

  • Hazen KC, Howell SA. Candida, Cryptococcus, and other yeasts of medical importance, “Murray PR, Baron EJ, Jorgensen JH, Pfaller MA, Yolken RH (eds): Manual of Cilinical Microbiology, 8th edn.” ASM Press,Washington, 2003:1693-710.
  • Kurtz MB, Cortelyou MW, Kirsch DR. Integrative transfor- mation of Candida albicans, using a cloned Candida ADE2 gene. Mol Cell Biol 1986;6(1):142–9.
  • Chang HC, Leaw SN, Huang AH, Wu TL, Chang TC. Rapid identification of yeasts in positive blood cultures by a mul- tiplex PCR method. J Clin Microbiol 2001;39(10):3466-71.
  • Luo G, Mitchell TG. Rapid identification of pathogenic fungi directly from cultures by using multiplex PCR., J Clin Mi- crobiol 2002;40(8):2860-5.
  • Romeo O, Racco C, Criseo G. Amplification of the hyphal wall protein 1 gene to distinguish Candida albicans from Candida dubliniensis. J Clin Microbiol 2006;44(7):2590-2.
  • Sanglard D, Bile J. Current understanding of the modes of action and resistance mechanisms to conventional and emerging antifungal agents for treatment of Candida of in- fections, “Calderone RA (ed): Candida and Candidiasis, 1th edn” kitabında ASM Press, Washington, 2002:349-83.
  • Whelan WL, Partridge RM, Magee PT. Heterozygosity and segregation in Candida albicans. Mol Gen Genet 1980; 180(1):107–13.
  • Sanglard D, Ischer F, Bille J. Role of ATP-binding-cassette transporter genes in high-frequency acquisition of resis- tance to azole antifungals in Candida glabrata. Antimicrob Agents Chemother 2001;45(4):1174-83.
  • Arıkan S. Fungal hastalıklarda genomik ve proteomiklerin yeri ve önemi. ANKEM Derg 2009;23(Ek 2): 53-6.
  • Clinical and Laboratory Standards Institute (CLSI): Ref- erence method for broth dilution antifungal susceptibil- ity testing of yeasts, Approved Standard 3rd edn, CLSI Document M27-A3, Clinical and Laboratory Standards Institute,Wayne, PA 2008;28(14).
  • Xu Y, Chen L, Li C. Susceptibility of clinical isolates of Candida species to fluconazole and detection of Candida albicans ERG11 mutations. J Antimicrob Chemother 2008;61(4):798-804.
  • Alpar R. Spor Bilimlerinde Uygulamalı İstatistik, 2. Baskı, s.219-55, Nobel Tıp Kitabevi, İstanbul (2001).
  • Günalp A. Gen ve Moleküler Biyoloji, 1st edn, , Hacettepe Üniversitesi Yayınları, Ankara, 1965;1:226-63.
  • Hawthorne DC, Mortimer RK. Chromosome mapping in Saccharomyces centromere-linked genes. Genetics 1960;45(8):1085-110.
  • Shin JH, Nolte FS, Morrison CJ. Rapid identification of Candida species in blood cultures by a clinically useful PCR method. J Clin Microbiol 1997;35(6):1454-9.
  • Ausubel FM, Brent R, Kingston RE et al. Short Protocols in Molecular Biology, 3rd edn, John Wiley&Sons, New York, 1995:642-50.
  • Einsele H, Hebart H, Roller G, Loffler J, Rothenhofer I, Muller CA. Detection and identification of fungal patho- gens in blood by using molecular probes. J Clin Microbiol 1997;35(6):1353-60.
  • Maaroufi Y, Heymans C, De Bruyne JM, Duchateau V, Ro- driguez-Villalobos H. Rapid detection of Candida albicans in clinical blood samples by using a TaqMan-based PCR assay. J Clin Microbiol 2003;41(7):3293-8.
  • Li YL, Leaw SN, Chen JH, Chang HC, Chang TC. Rap- id identification of yeasts commonly found in positive blood cultures by amplification of the internal transcribed spacer regions 1 and 2. Eur J Clin Microbiol Infect Dis 2003;22(11):693-6.
  • Skladny H, Buchheidt D, Baust C, Krieg-Schneider F, Sei- farth W, Leib-Mosch C. Specific detection of Aspergillus species in blood and bronchoalveolar lavage samples of immunocompromised patients by two-step PCR., J Clin Microbiol 1999;37(12):3865-71.
  • Hayette MP, Vaira D, Susin F, Boland P, Christiaens G, Melin P. Detection of Aspergillus species DNA by PCR in bronchoalveolar lavage fluid. J Clin Microbiol 2001;39(6):2338-40.
  • Whıte TC, Marr KA, Bowden RA. Clinical, cellular, and molecular factors that contribute to antifungal drug resis- tance. Clin Microbiol Rev 1998;11(2):382-402 .
  • Haitao Ji, Zhang W, Zhou Y, Zhang M, Zhu J, Song Y. A three-dimensional model of lanosterol 14α-demethylase of Candida albicans and its interaction with azole antifungals. J Med Chem 2000;43(13):2493-505.
  • Favre B, Didmon M, Ryder NS. Multiple amino acid substitutions in lanosterol 14α-demethylase contribute to azole resistance in Candida albicans. Microbiology 1999;145(10):2715-25.
  • Marichal P, Koymans S, Willemsens S, Bellens D, Ver- hasselt P, Luyten W, Borgers M,Ramaekers FC, Odds FC and Vanden Bossche H. Contribution of mutations in the- 111cytochrome P450 14α-demethylase (Erg 11p, Cyp 51p) to azole resistance in Candida albicans. Microbiology 1999;145: 2701-13.
  • Löffler J, Kelly SL, Hebart H, Schumacher U, Lass- Flörl C, Einsele H. Molecular analysis of cyp 51 from flucon- azole resistant Candida albicans strains. FEMS Microbiol Lett1997;151:263-8.
There are 26 citations in total.

Details

Primary Language Turkish
Journal Section Research Articles
Authors

Serdar Susever This is me

Yıldız Yeğenoğlu This is me

Publication Date June 1, 2012
Submission Date March 2, 2015
Published in Issue Year 2012 Volume: 39 Issue: 2

Cite

APA Susever, S., & Yeğenoğlu, Y. (2012). The identification of Candida species isolated from clinical specimens of immunocompromised patients with PCR and determination of antifungal resistance genes with RFLP and sequencing analysis. Dicle Tıp Dergisi, 39(2), 242-250. https://doi.org/10.5798/diclemedj.0921.2012.02.0135
AMA Susever S, Yeğenoğlu Y. The identification of Candida species isolated from clinical specimens of immunocompromised patients with PCR and determination of antifungal resistance genes with RFLP and sequencing analysis. diclemedj. June 2012;39(2):242-250. doi:10.5798/diclemedj.0921.2012.02.0135
Chicago Susever, Serdar, and Yıldız Yeğenoğlu. “The Identification of Candida Species Isolated from Clinical Specimens of Immunocompromised Patients With PCR and Determination of Antifungal Resistance Genes With RFLP and Sequencing Analysis”. Dicle Tıp Dergisi 39, no. 2 (June 2012): 242-50. https://doi.org/10.5798/diclemedj.0921.2012.02.0135.
EndNote Susever S, Yeğenoğlu Y (June 1, 2012) The identification of Candida species isolated from clinical specimens of immunocompromised patients with PCR and determination of antifungal resistance genes with RFLP and sequencing analysis. Dicle Tıp Dergisi 39 2 242–250.
IEEE S. Susever and Y. Yeğenoğlu, “The identification of Candida species isolated from clinical specimens of immunocompromised patients with PCR and determination of antifungal resistance genes with RFLP and sequencing analysis”, diclemedj, vol. 39, no. 2, pp. 242–250, 2012, doi: 10.5798/diclemedj.0921.2012.02.0135.
ISNAD Susever, Serdar - Yeğenoğlu, Yıldız. “The Identification of Candida Species Isolated from Clinical Specimens of Immunocompromised Patients With PCR and Determination of Antifungal Resistance Genes With RFLP and Sequencing Analysis”. Dicle Tıp Dergisi 39/2 (June 2012), 242-250. https://doi.org/10.5798/diclemedj.0921.2012.02.0135.
JAMA Susever S, Yeğenoğlu Y. The identification of Candida species isolated from clinical specimens of immunocompromised patients with PCR and determination of antifungal resistance genes with RFLP and sequencing analysis. diclemedj. 2012;39:242–250.
MLA Susever, Serdar and Yıldız Yeğenoğlu. “The Identification of Candida Species Isolated from Clinical Specimens of Immunocompromised Patients With PCR and Determination of Antifungal Resistance Genes With RFLP and Sequencing Analysis”. Dicle Tıp Dergisi, vol. 39, no. 2, 2012, pp. 242-50, doi:10.5798/diclemedj.0921.2012.02.0135.
Vancouver Susever S, Yeğenoğlu Y. The identification of Candida species isolated from clinical specimens of immunocompromised patients with PCR and determination of antifungal resistance genes with RFLP and sequencing analysis. diclemedj. 2012;39(2):242-50.