BackgroundCandida albicans is the most pathogenic Candida species but shares many phenotypic features with Candida dubliniensis and may, therefore, be misidentified in clinical microbiology laboratories. Candidemia cases due to C. dubliniensis are increasingly being reported in recent years. Accurate identification is warranted since mortality rates are highest for C. albicans infections, however, C. dubliniensis has the propensity to develop resistance against azoles more easily. We developed a duplex PCR assay for rapid detection and differentiation of C. albicans from C. dubliniensis for resource-poor settings equipped with basic PCR technology and compared its performance with three phenotypic methods.MethodsDuplex PCR was performed on 122 germ tube positive and 12 germ tube negative isolates of Candida species previously identified by assimilation profiles on Vitek 2 ID-YST system. Typical morphologic characteristics on simplified sunflower seed agar (SSA), and reaction with a commercial (Bichro-Dubli) latex agglutination test were also performed. The assay was further applied on 239 clinical yeast and yeast-like fungi and results were confirmed by DNA sequencing of internal transcribed spacer (ITS) region of rDNA.ResultsThe results of duplex PCR assay for 122 germ tube positive and 12 germ tube negative isolates of Candida species were comparable to their identification by Vitek 2 ID-YST system, colony characteristics on SSA and latex agglutination test. Application of duplex PCR also correctly identified all 148 C. albicans and 50 C. dubliniensis strains among 239 yeast-like fungi.ConclusionsThe data show that both, duplex PCR and Bichro-Dubli are reliable tests for rapid (within few hours) identification of clinical yeast isolates as C. dubliniensis or C. albicans. However, duplex PCR may be applied directly on clinical yeast isolates for their identification as C. dubliniensis or C. albicans as it does not require prior testing for germ tube formation or latex Candida agglutination.

There is a paucity of information about genotypic heterogeneity among Candida dubliniensis isolates recovered from different geographic regions. This study explored genotypic heterogeneity among 103 C. dubliniensis strains obtained over a six-year period from clinical specimens in Kuwait. Genotype assignment was based on amplification with genotype-specific primers and sequencing of rDNA. Susceptibility to 5-flucytosine was determined by means of the Etest. DNA sequencing of cytosine deaminase was performed to determine the molecular basis of resistance to 5-flucytosine. DNA sequencing of rDNA identified seven different genotypes, i.e., 68 (66%) isolates were found to belong to genotype 1, 25 to genotype 4, six to genotype 5 and one each to genotypes 6-9. Strains of genotype 2 or genotype 3 were not detected. All isolates of genotype 4 but none of other genotypes were resistant to 5-flucytosine and the resistant strains all contained S29L mutation. Isolates of all other genotypes contained wild-type codon 29 in cytosine deaminase. A simple, PCR-RFLP-based method has been developed to facilitate rapid detection of S29L mutation in cytosine deaminase. A noteworthy observation of our study is the identification of five new genotypes of C. dubliniensis isolates, recovered from oral/respiratory specimens from patients of Middle Eastern origin. Furthermore, all 5-flucytosine resistant C. dubliniensis isolates in Kuwait belonged to genotype 4 only.

The aim of this study was to detect Aspergillus fumigatus-specific DNA by nested PCR (nPCR) in serum and bronchoalveolar lavage (BAL) specimens of experimentally infected rats and compare the results with (1-3)-beta-D-glucan (BDG) and galactomannan (GM) detection. Sixty Wistar rats, immunosuppressed with an intraperitoneal injection of cyclophosphamide (70 mg kg(-1)) were infected with 1 x 10(6)A. fumigatus conidia. The rats were killed on days 1, 3, 5, 7 and 9 postinfection in groups of six each and their BAL, blood and lungs were cultured. The A. fumigatus-specific DNA, BDG and GM in serum and BAL were detected by nPCR, Fungitell kit and Aspergillus Platelia kit respectively. Base line values were obtained by using sera from six healthy rats. Except the lungs, blood and BAL specimens of all the infected rats were negative for A. fumigatus culture. The BDG, GM and nPCR positivity in serum specimens was 80%, 77% and 63% respectively. The sensitivity of GM and nPCR tests in BAL specimens was 77% and 70% respectively. The data suggest that BDG and GM appear early in the course of infection, and have similar kinetics (r = 0.483, P = 0.007). Hence, their combined detection could be useful in the early diagnosis of invasive aspergillosis.

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Fusarium infections are increasingly being encountered in immunocompromised patients. Fusarium solani accounts for nearly half of these infections. A specific nested PCR (nPCR) assay has been developed by using DNA isolated from several Fusarium species and other common fungi. Furthermore, DNA samples isolated from bronchoalveolar lavage (BAL) and serum samples from mice infected intravenously with F. solani conidia and sacrificed on every third day post infection were used for the evaluation of the established nPCR protocol. The lung homogenate, BAL and blood from infected animals were also cultured. The nPCR assay was specific for F. solani and detected 450 fg of DNA corresponding roughly to 11 F. solani cells. Cultures of lung homogenate of infected animals up to day 16 yielded F. solani with decreasing fungal load and were negative thereafter. The nPCR positivity in BAL was 100% concordant with lung tissue culture results. Although detection of F. solani DNA in serum was less sensitive than in BAL, it remained positive for longer duration. Our data from an experimental mouse model show that detection of DNA in BAL and to a lesser extent in serum by nPCR offers a sensitive and specific diagnostic approach to invasive F. solani infection.