Detection and identification of Candida species associated with Candida vaginitis by real-time PCR and pyrosequencing

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Abstract

Real-time polymerase chain reaction (PCR) is currently considered the most sensitive method to detect low abundance DNA of pathogens in clinical samples. Furthermore, obtaining DNA sequence is the ‘gold standard’ of precise molecular detection. Here we combine species-specific real-time PCR and pyrosequencing to rapidly amplify and sequence ribosomal DNA from Candida albicans, Candida glabrata, Candida parapsilosis, and Candida tropicalis, which are commonly associated with candida vaginitis (CV). A standard curve was developed from plasmids containing the target DNA for each of the Candida species. A minimum real-time PCR and pyrosequencing detection limit of 100 copies per reaction was achieved. The combined technique was applied to the identification of the four Candida species in DNA extracts from vaginal samples. The results from 231 samples were compared with conventional PCR methods of identification. The results of both methods agreed on all but two samples, which were determined by both methods to contain C. albicans, but real-time PCR and pyrosequencing identified a second species that went undetected by conventional PCR. This is the first application of real-time PCR and pyrosequencing to DNA from vaginal samples for identification of four Candida species associated with CV, without the need for time-consuming culture methods.

Introduction

Vaginal candidiasis causes 20–25% of infectious vaginitis cases, second only to the 40–50% of cases caused by bacterial vaginosis [1]. Candida vaginitis (CV) is marked by pruritis, soreness, a change in discharge, dyspareunia, vulvar erythema, edema, and fissures [1], [2]. The condition is rare before puberty, but by the age of 25, nearly one-half of all women will have had at least one clinician-diagnosed episode of CV. Overall, it is estimated that 75% of all women will experience an episode of CV in their lifetime [1], [3]. Among the Candida species causing infections, Candida albicans, Candida glabrata, Candida parapsilosis, and Candida tropicalis account for 80–90% of fungal isolates encountered worldwide [4], [5]. Although C. albicans is implicated in 85–95% of all cases of CV [1], [6], the widespread use of azole antifungal drugs is postulated to have promoted the shifting of vaginal colonization and selection of more naturally resistant species, such as C. glabrata [7], [8], [9], [10]. The 2004 guidelines for the treatment of candidiasis put forth by P.G. Pappas, et al. state that knowledge of the infecting species is highly predictive of likely drug susceptibility and that this information should be used as a guide for selecting therapy [11]. Therefore, rapid and specific identification of Candida species will facilitate appropriate antifungal selection and improve patient care.

Commonly, Candida in vaginal samples is identified by microscopic examination of a wet mount with potassium hydroxide (KOH, amine test). This technique detects budding yeast cells in only 50–70% of women with CV [12], [13] and may fail to detect non-albicans species [14]. Alternatively, C. albicans and C. tropicalis can be distinguished by growth on chromogenic agar medium and other Candida spp. can be identified by enzymatic tests. However, each of these tests requires isolated organisms to be grown on solid medium for 24–48 h before they can be performed or interpreted [15], [16]. In addition, the ‘gold standard’ for definitive biochemical identification requires analysis of assimilation and fermentation, taking up to 30 days to complete [16].

In recent years, numerous DNA-based techniques have been developed to improve the identification of Candida species. PCR amplification of Candida target DNA [17], [18], [19], [20], [21], [22] is particularly promising because of its simplicity, specificity, and sensitivity. However, these strategies require post-amplification analyses and are considered to have lower sensitivity than real-time PCR techniques that directly monitor amplification via fluorescent probes [23]. Real-time PCR strategies have been developed to identify Candida species [24], [25], [26], [27], but these methods were designed and optimized for detection of Candida in blood or blood culture. Strategies for the detection of Candida species in DNA extracted from vaginal samples, especially without time-consuming culture, are lacking. In addition, current DNA-based Candida detection methods do not take into account the fact that DNA sequencing is generally accepted as the most precise method for discriminating among closely related species.

The purpose of this study was to develop a rapid method for the detection of four Candida species associated with CV in DNA extracted directly from vaginal samples. The detection method combines the sensitivity and specificity of real-time PCR with the unambiguous species identification and validation capability of DNA sequencing by pyrosequencing, an established bioluminometric technique that employs a cascade of coupled enzymatic reactions to monitor DNA synthesis [28].

Section snippets

Clinical samples and DNA extraction

A total of 231 vaginal samples from female subjects were tested. The subjects' symptoms, HIV status, and clinician diagnoses were not disclosed. Patient care providers collected specimens from a vaginal sampling using a Cellmatics swab (BD, Sparks, MD), which was then placed in 2 ml of its accompanying transport medium. Upon receipt, swabs were immediately processed for PCR analysis. Established procedures for SDS/proteinase K lysis and phenol/chloroform DNA extraction [29] from 470 μl of swab

Analysis of real-time PCR conditions

To confirm amplification quality, the real-time PCR products generated from 5×106 copies of the positive control plasmid, 10 ng of DNA extracted from an isolate purchased from ATCC, and 0.5 μg of DNA extracted from a positive vaginal sample (confirmed by conventional PCR) of each Candida species were subjected to agarose gel electrophoresis (Fig. 1). The product generated from each template type was a single band of the expected size and lacked the formation of any primer dimers. This indicates

Discussion and conclusions

The development of a rapid, sensitive, and unambiguous method for identification of Candida species for aiding diagnosis and treatment of CV is critical mainly because of species-specific differences in susceptibility to antifungal drugs. To meet this need, the real-time PCR and pyrosequencing method described here is rapid, taking only approximately 2 h to complete (10 min for real-time PCR preparation, 75 min real-time PCR run-time, 15 min pyrosequencing preparation, and 20 min minimum for

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