Genotypic heterogeneity and molecular basis of 5-flucytosine resistance amongCandida dubliniensisisolates recovered from clinical specimens in Kuwait

Ahmad, Suhail; Khan, Zia U.; Joseph, Leena; Asadzadeh, Mohammad; Theyyathel, A.

doi:10.3109/13693786.2011.597446

Cited by 15 publications

(29 citation statements)

References 29 publications

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“…dubliniensis isolates from Kuwait [21], we found 68 ITSH1 and 25 ITSH4 isolates and five additional haplotypes (labeled as ITSH5 to ITSH9) but failed to detect any ITSH2 or ITSH3 isolates, described previously by Gee et al [18]. The significance of these haplotypes among clinical C .…”

Section: Introductionsupporting

confidence: 46%

Population structure and molecular genetic characterization of 5-flucytosine-susceptible and -resistant clinical Candida dubliniensis isolates from Kuwait

Asadzadeh¹,

Ahmad²,

Al‐Sweih³

et al. 2017

PLoS ONE

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Candida dubliniensis and Candida albicans are two closely related species. Although C. dubliniensis is less pathogenic, it has a higher propensity to develop resistance to fluconazole and some strains exhibit intrinsic resistance to 5-flucytosine (5-FC). All 5-FC-resistant isolates from Kuwait were previously shown to belong to one of seven internal transcribed spacer (ITS) region of rDNA-based haplotypes. This study performed fingerprinting of C. dubliniensis isolates by multilocus sequence typing (MLST) to determine population structure of 5-FC-resistant and -susceptible strains and compared the results with data from a global collection of isolates. Fifty-two C. dubliniensis isolates previously analyzed and 58 additional isolates mostly collected during 2010–2013 and characterized by phenotypic and molecular methods were used. ITS-based haplotypes were identified by haplotype-specific PCR and/or by PCR-DNA sequencing of rDNA. Population structure was determined by 8-loci-based MLST. E-test was used to determine susceptibility to 5-FC, fluconazole, voriconazole and amphotericin B. Five ITS haplotypes (ITSH) were detected among 110 C. dubliniensis isolates. The ITSH1 was most common (n = 80 isolates) followed by ITSH4 (n = 25 isolates). Two isolates each belonged to ITSH5 and ITSH8 while one isolate belonged to ITSH7. MLST identified 16 diploid sequence types (DSTs) including six new DSTs. DST11 (n = 52) and DST14 (n = 25) were dominant genotypes and were confined (together with DST21) to Middle-Eastern countries. Other DSTs (excluding some new DSTs) had a wider global distribution as they were identified from various other countries. Only ITSH4 isolates (n = 25) belonged to DST14, were resistant to 5-FC and contained S29L mutation in CdFCA1. ITSH5, ITSH7 and ITSH8 isolates belonged to different DSTs. Thus, clinical C. dubliniensis isolates in Kuwait exhibited limited genotypic heterogeneity and most isolates belonged to region-specific DSTs. All 5-FC-resistant C. dubliniensis isolates belonged to ITSH4 and MLST-based DST14 genotype. Placement of some isolates into additional ITS haplotypes is also supported by MLST data.

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Section: Introductionsupporting

confidence: 46%

Population structure and molecular genetic characterization of 5-flucytosine-susceptible and -resistant clinical Candida dubliniensis isolates from Kuwait

Asadzadeh¹,

Ahmad²,

Al‐Sweih³

et al. 2017

PLoS ONE

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“…In a previous study, we developed a thermal duplex PCR assay with two pairs of species-specific primers for detection and differentiation of strains of C. albicans and C. dubli niensis, and the amplicons were detected by agarose gel electrophoresis [30]. However, when the same primers were used in real-time PCR, the difference in T m values between C. albicans and C. dubliniensis strains was small which was not suitable for routine use as the ITS region sequences vary among clinical C. albicans and C. dublini ensis strains [5,7,31]. Thus, other sets of primers were tested, and the primers used in this study yielded a T m difference of nearly 4 ° C between reference strains of C. albicans and C. dubliniensis which was most suitable for accurate screening of a large number of samples.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, the difference in T m values for C. albicans and C. dubliniensis strains in these reports was either small (approx. 0.5 ° C) or negligible which may lead to misidentification of some isolates since ITS region sequences vary among clinical C. albicans and C. dubliniensis isolates [5,7,31]. The other method is a nested PCR involving a first round of amplification with pan-Candida primers followed by real-time PCR amplification with 8 different species-specific primer pairs for the identification of 8 different Candida species including C. albicans and C. dubliniensis [36].…”

Section: Discussionmentioning

confidence: 99%

Rapid and Accurate Identification of Candida albicans and Candida dubliniensis by Real-Time PCR and Melting Curve Analysis

Asadzadeh¹,

Ahmad²,

Al‐Sweih³

et al. 2018

Med Princ Pract

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Objective: Candida albicans and Candida dubliniensis are germ tube-positive pathogenic yeast species. Accurate identification of these two species is warranted since C. albicans is a highly pathogenic species while C. dubliniensis exhibits increased adherence to buccal epithelial cells, reduced susceptibility to azoles and resistance to flucytosine. We have developed a duplex real-time PCR assay for rapid detection and differentiation between clinical C. albicans and C. dubliniensis isolates. Materials and Methods: A duplex real-time PCR assay was developed by using two species-specific primer pairs and SYBR Green dye to differentiate C. albicans and C. dubliniensis isolates via melting curve analysis of real-time PCR amplicons. Amplification products were also analyzed by agarose gel electrophoresis to confirm real-time PCR results. Results: Melting temperatures (T_m) for reference strains of C. albicans and C. dubliniensis were 86.55 and 82.75°C, respectively. No amplicon was obtained with DNA from reference strains of 8 other common Candida spp. When real-time PCR was applied on 226 clinical isolates previously identified by the Vitek 2 system and/or PCR sequencing of rDNA, T_m values for C. albicans (n = 113) and C. dubliniensis (n = 98) were 86.68 ± 0.529 and 82.616 ± 0.535°C, respectively. The results were confirmed by agarose gel electrophoresis. No amplicon was obtained from 15 isolates belonging to 9 other Candida spp. Conclusions: The real-time PCR assay described here does not require prior identification of clinical yeast isolates as C. albicans/C. dubliniensis by germ tube formation and accurately reports results within 2 h. Detection of amplicons by agarose gel electrophoresis is also suitable for resource-poor settings devoid of real-time PCR facilities.

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“…Interestingly, resistance of C. dubliniensis to fluconazole, voriconazole, and itraconazole has been documented previously [8][9][10]. Furthermore, resistance of C. dubliniensis to 5-fluorocytosine in isolates obtained from Kuwait and the Middle East region has also been documented [11,12]. Occurrence of such resistance indicates the need for possible alternative therapeutic modalities which may facilitate the management of candidiasis.…”

Section: Discussionmentioning

confidence: 99%

“…Longitudinal genotyping of C. dubliniensis isolates from patients with HIV infection has shown resistance to itraconazole, even in the absence of prior azole therapy [10]. C. dubliniensis strains resistant to 5-fluorocytosine have also been isolated in Kuwait and in the Middle East region [11,12].…”

Section: Introductionmentioning

confidence: 99%

Impact of Brief Exposure to Drugs with Antifungal Properties on the Susceptibility of Oral Candida dubliniensis Isolates to Lysozyme and Lactoferrin

Ellepola¹,

Dassanayake²,

Khan³

2018

Med Princ Pract

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Objective: Lysozyme and lactoferrin have anti-candidal activity. Candida dubliniensis is associated with oral candidiasis. Candida infections are managed with nystatin, amphotericin B, caspofungin, ketoconazole, fluconazole, and chlorhexidine. Candida species undergo a brief exposure to therapeutic agents in the mouth. There is no data on the influence of limited exposure to antimycotics on the sensitivity of C. dubliniensis to lactoferrin and lysozyme. Hence, this study observed the changes in the sensitivity of C. dubliniensis to anti-candidal action of lactoferrin and lysozyme after transitory exposure to sub-lethal concentrations of antifungals. Materials and Methods: After determination of the minimum inhibitory concentration (MIC), 20 C. dubliniensis isolates were exposed to twice the concentration of MIC of nystatin, amphotericin B, caspofungin, ketoconazole, fluconazole, and chlorhexidine for 1 h. Drugs were removed by dilution and thereafter the susceptibility of these isolates to lysozyme and lactoferrin was determined by colony-forming unit quantification assay. Results: Exposure of C. dubliniensis to nystatin, amphotericin B, caspofungin, ketoconazole, fluconazole, and chlorhexidine resulted in an increase in susceptibility to lysozyme by 9.45, 30.82, 30.04, 50.64, 55.60, and 50.18%, respectively (p < 0.05 to p < 0.001). Exposure of C. dubliniensis to nystatin, amphotericin B, caspofungin, ketoconazole, fluconazole, and chlorhexidine resulted in an increase in susceptibility to lactoferrin by 13.54, 16.43, 17.58, 19.60, 21.32, and 18.73, respectively (p < 0.05 to p < 0.001). Conclusion: Brief exposure to nystatin, amphotericin B, caspofungin, ketoconazole, fluconazole, and chlorhexidine enhances the antifungal effect of lysozyme and lactoferrin on C. dubliniensis isolates in vitro.

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Genotypic heterogeneity and molecular basis of 5-flucytosine resistance amongCandida dubliniensisisolates recovered from clinical specimens in Kuwait

Cited by 15 publications

References 29 publications

Population structure and molecular genetic characterization of 5-flucytosine-susceptible and -resistant clinical Candida dubliniensis isolates from Kuwait

Population structure and molecular genetic characterization of 5-flucytosine-susceptible and -resistant clinical Candida dubliniensis isolates from Kuwait

Rapid and Accurate Identification of <b><i>Candida albicans</i></b> and <b><i>Candida dubliniensis</i></b> by Real-Time PCR and Melting Curve Analysis

Impact of Brief Exposure to Drugs with Antifungal Properties on the Susceptibility of Oral <b><i>Candida dubliniensis</i></b> Isolates to Lysozyme and Lactoferrin

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