The rapid detection and identification of Candida species in clinical laboratories are extremely important for the management of patients with hematogenous candidiasis. The presently available culture and biochemical methods for detection and species identification of Candida are time-consuming and lack the required sensitivity and specificity. In this study, we have established a seminested PCR (snPCR) using universal and species-specific primers for detection of Candida species in serum specimens. The universal outer primers amplified the 3 end of 5.8S ribosomal DNA (rDNA) and the 5 end of 28S rDNA, including the internally transcribed spacer 2 (ITS2), generating 350-to 410-bp fragments from the four commonly encountered Candida species, viz., C. albicans, C. tropicalis, C. glabrata, and C. parapsilosis. The species-specific primers, complementary to unique sequences within the ITS2 of each test species, amplified species-specific DNA in the reamplification step of the snPCR. The sensitivity of Candida detection by snPCR in spiked serum specimens was close to 1 organism/ml. Evaluation of snPCR for specific identification of Candida species with 76 clinical Candida isolates showed 99% concordant results with the Vitek and/or ID32C yeast identification system. Further evaluation of snPCR for detection of Candida species in sera from culture-proven (n ‫؍‬ 12), suspected (n ‫؍‬ 16), and superficially colonized (n ‫؍‬ 10) patients and healthy subjects (n ‫؍‬ 12) showed that snPCR results were consistently negative with sera from healthy individuals and colonized patients. In culture-proven candidemia patients, the snPCR results were in full agreement with blood culture results with respect to both positivity and species identity. In addition, snPCR detected candidemia due to two Candida species in five patients, compared to three by blood culture. In the category of suspected candidemia with negative blood cultures for Candida, nine patients (56%) were positive by snPCR; two of them had dual infection with C. albicans and either C. tropicalis or C. glabrata. In conclusion, the snPCR developed in this study is specific and more sensitive than culture for the detection of Candida species in serum specimens. Moreover, the improved detection of cases of candidemia caused by more than one Candida species is an additional advantage.

Candida dubliniensis is an emerging pathogen capable of causing oropharyngeal, vaginal and bloodstream infections. Although C. dubliniensis is similar to Candida albicans in several phenotypic characteristics, it differs from it with respect to epidemiology, certain virulence factors and the ability to develop resistance to fluconazole rapidly. In this study, the first seven isolations of C. dubliniensis from Kuwait are described, all originating from non-human immunodeficiency virus (HIV)-infected patients. The isolates were initially identified by the Vitek 2 yeast identification system, positive germ tube test, production of rough colonies and chlamydospores on Staib agar and by their inability to assimilate xylose, trehalose or methyl AE-D-glucoside. The species identity of the isolates was subsequently confirmed by specific amplification of rDNA targeting the internally transcribed spacer 2 (ITS2), restriction endonuclease digestion of the amplified DNA and direct DNA sequencing of the ITS2. Using the E-test method, the MICs of C. dubliniensis test isolates were in the range 0 . 125-0 . 75 ìg ml À1 for fluconazole, 0 . 002-0 . 75 ìg ml À1 for itraconazole, 0 . 006-0 . 125 ìg ml À1 for ketoconazole, 0 . 002-0 . 5 ìg ml À1 for amphotericin B and 0 . 002-0 . 016 ìg ml À1for voriconazole. Two of the isolates were resistant to 5-flucytosine (.32 ìg ml À1 ), but none against fluconazole. The study reinforces the current view that C. dubliniensis has a much wider geographical and epidemiological distribution.

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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.

A sunflower (Helianthus annuus) seed husk agar medium has been developed and evaluated for differentiation of Candida dubliniensis from Candida albicans on the basis of colony morphology and chlamydospore production. All C. dubliniensis isolates (n=40) produced rough colonies with hyphal fringes and abundant chlamydospores whereas 101 of 105 (96.2%) C. albicans isolates produced smooth colonies with no evidence of chlamydospore production. Since this medium is free from oil droplets, chlamydospores can be examined with greater clarity by Dalmau plate technique. This medium provides a simple and cost-effective tool for the presumptive differentiation of C. dubliniensis from C. albicans and is particularly suited for clinical microbiology laboratories where biochemical or molecular methods for the differentiation of these two species are not available.