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.

The first outbreak of Candida haemulonii fungemia is described. The seven isolates from the blood of four neonates were identified by DNA sequencing of the ribosomal DNA. They were all resistant to amphotericin B, fluconazole, and itraconazole. This report highlights the emergence of C. haemulonii as an opportunistic pathogen in immunocompromised patients.

Basidiobolus ranarum is a known cause of subcutaneous zygomycosis. Recently, its etiologic role in gastrointestinal infections has been increasingly recognized. While the clinical presentation of the subcutaneous disease is quite characteristic and the disease is easy to diagnose, gastrointestinal basidiobolomycosis poses diagnostic difficulties; its clinical presentation is nonspecific, there are no identifiable risk factors, and all age groups are susceptible. The case of gastrointestinal basidiobolomycosis described in the present report occurred in a 41-year-old Indian male who had a history of repair of a left inguinal hernia 2 years earlier and who is native to the southern part of India, where the subcutaneous form of the disease is indigenous. Diagnosis is based on the isolation of B. ranarum from cultures of urine and demonstration of broad, sparsely septate hyphal elements in histopathologic sections of the colon, with characteristic eosinophilic infiltration and the Splendore-Hoeppli phenomenon. The titers of both immunoglobulin G (IgG) and IgM antibodies to locally produced antigen of the fungus were elevated. The patient failed to respond to 8 weeks of amphotericin B therapy, and the isolate was later found to be resistant to amphotericin B, itraconazole, fluconazole, and flucytosine but susceptible to ketoconazole and miconazole. One other noteworthy feature of the fungus was that the patient's serum showed raised levels of Th2-type cytokines (interleukins 4 and 10) and tumor necrosis factor alpha. The present report underscores the need to consider gastrointestinal basidiobolomycosis in the differential diagnosis of inflammatory bowel diseases and suggests that, perhaps, more time should be invested in developing standardized serologic reagents that can be used as part of a less invasive means of diagnosis of the disease. CASE REPORTA 41-year-old, Indian male was admitted to Mubarak AlKabeer Hospital, Kuwait, in December 1999 with a history of abdominal pain of 20 days' duration. On examination, he was febrile (38.8°C) and his abdomen was tender and distended, particularly at the right hypochondrium and the right iliac fossa, where a palpable, nodular mass (9 by 5 cm) was located. The patient's history was not remarkable, except that he had a history of repair of a left inguinal hernia 2 years earlier. He was nondiabetic and nonneutropenic but had persistently low hemoglobin levels (7.7 to 8.7 g/dl). Ultrasound of the abdomen and pelvis confirmed the presence of a thick mass in the right iliac fossa and showed a marked thickening of the ascending colon and cecum and an increased echogenicity of the renal cortex, suggesting bilateral compromised function. Moreover, his prostate was markedly enlarged. There was fecal impaction in the colon. His blood urea nitrogen, serum creatinine, and uric acid levels were within the normal ranges. Examination of his urine showed many leukocytes and red blood cells, besides elevated levels of protein and bilirubin. On 11 January 2000, a right hemicolectomy was per...

Background: Candidemia is a major infectious complication of seriously immunocompromised patients. In the absence of specific signs and symptoms, there is a need to evolve an appropriate diagnostic approach. A number of methods based on the detection of Candida mannan, nucleic acid and (1,3)-beta-D-glucan (BDG) have been used with varying specificities and sensitivities. In this retrospective study, attention has been focused to evaluate the usefulness of two or more disease markers in the diagnosis of candidemia.

Bloodstream infections due to Candida species are important complications in severely ill hospitalized patients. This study presents data on species distribution and antifungal susceptibility profiles of Candida bloodstream isolates obtained from Kuwait during a 10-year period. All the bloodstream isolates were identified to species level by the germ tube test and carbohydrate assimilation profile using the VITEK 2 yeast identification system. Using E-test strips for amphotericin B, fluconazole, 5-flucytosine and voriconazole, MICs were determined on RPMI agar supplemented with 2 % glucose. The MIC breakpoints for resistance were based on Clinical and Laboratory Standards Institute criteria or those published by reference laboratories, and were as follows: amphotericin B, >1 mg ml . In all, 607 bloodstream yeast isolates were obtained over the past 10 years in Kuwait. Candida albicans was the predominant species (39.5 %), followed by Candida parapsilosis (30.6 %), Candida tropicalis (12.4 %), Candida glabrata (5.6 %) and Candida krusei (1.6 %). All C. albicans, C. tropicalis and C. glabrata isolates were susceptible to amphotericin B. Of 186 isolates of C. parapsilosis tested, only four (2 %) exhibited an MIC for amphotericin B of >1 mg ml "1 . Resistance to fluconazole was observed in nine (3.8 %) C. albicans isolates, two (5.8 %) C. glabrata isolates and four (40 %) C. krusei isolates. Resistance to 5-flucytosine was observed in two (0.8 %) C. albicans isolates, seven (9.3 %) C. tropicalis isolates, three (1.6 %) C. parapsilosis isolates and all ten (100 %) C. krusei isolates. All the isolates of C. albicans, C. tropicalis, C. parapsilosis, C. glabrata and C. krusei were susceptible to voriconazole, including those resistant to fluconazole. Although amphotericin B and fluconazole are widely used in clinical practice in Kuwait, resistance to these drugs remained low. INTRODUCTIONCandida species are one of the major causes of nosocomial bloodstream infections worldwide (Jarvis, 1995;. Despite the availability of an expanded antifungal armamentarium, the mortality associated with invasive Candida infections remains high, ranging between 19 and 49 % (Blot et al., 2002;Alonso-Valle et al., 2003;Gudlaugsson et al., 2003;Morgan, 2005). The incidence and associated mortality due to candidaemia can be influenced by several factors including characteristics of the population at risk, standard of the healthcare facilities available, distribution of Candida species and prevalence of resistance (Hobson, 2003;Eggimann et al., 2003). Hence, epidemiological information available for one centre or geographical region may not be applicable to others (Hobson, 2003). The increased isolation rates of non-albicans Candida species and a gradual shift in the antifungal susceptibility profile, especially against azole antifungal agents, have underlined the need to monitor laboratory data for possible emergence of resistance and to select the most appropriate antifungal agent for therapy (Sanglard & Odds, 2002;Eggimann et al., 2003). C...

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Candidal adhesion has been implicated as the initial step in the pathogenesis of oral candidiasis and cell surface hydrophobicity (CSH) has been implicated in adhesion to mucosal surfaces. Candida dubliniensis is an opportunistic pathogen associated with recurrent oral candidiasis. Chlorhexidine gluconate is by far the commonest antiseptic mouth wash prescribed in dentistry. At dosage intervals the intraoral concentration of this antiseptic fluctuates considerably and reaches sub-therapeutic levels due to the dynamics of the oral cavity. Hence, the organisms undergo only a limited exposure to the antiseptic during treatment. The impact of this antiseptic following such exposure on CSH of C. dubliniensis isolates has not been investigated. Hence, the main objective of this study was to investigate the effect of brief exposure to sub-therapeutic concentrations of chlorhexidine gluconate on the CSH of C. dubliniensis isolates. Twelve oral isolates of C. dubliniensis were briefly exposed to three sub-therapeutic concentrations of 0.005%, 0.0025% and 0.00125% chlorhexidine gluconate for 30 min. Following subsequent removal of the drug, the CSH of the isolates was determined by a biphasic aqueous-hydrocarbon assay. Compared with the controls, exposure to 0.005% and 0.0025% chlorhexidine gluconate suppressed the relative CSH of the total sample tested by 44.49% (P < 0.001) and 21.82% (P < 0.018), respectively, with all isolates being significantly affected. Although exposure to 0.00125% of chlorhexidine gluconate did not elicit a significant suppression on the total sample tested (7.01%; P > 0.05), four isolates of the group were significantly affected. These findings imply that exposure to sub-therapeutic concentrations of chlorhexidine gluconate may suppress CSH of C. dublinienis isolates, thereby reducing its pathogenicity and highlights further the pharmacodynamics of chlorhexidine gluconate.

Recent molecular studies have led to the recognition of three distinct species within the Candida parapsilosis complex, namely Candida parapsilosis, Candida orthopsilosis and Candida metapsilosis. As currently available yeast identification systems fail to differentiate these species, there is a paucity of information on their occurrence in different geographical regions. This study describes a simple PCR-based protocol for rapid discrimination among C. parapsilosis, C. orthopsilosis and C. metapsilosis strains by using primers derived from unique sequences within the internally transcribed spacer 1 (ITS1)–5.8 rRNA–ITS2 region. Retrospective analysis of 114 C. parapsilosis-complex isolates recovered from clinical specimens in Kuwait identified 109 as C. parapsilosis, five as C. orthopsilosis and none as C. metapsilosis. The results were further validated by PCR-RFLP patterns of the secondary alcohol dehydrogenase gene fragment. DNA sequencing of the ITS region and the D1/D2 regions of the 28S rRNA gene confirmed the species-specific identification of all five C. orthopsilosis strains. The amplicon length of the intergenic spacer between the 28S and 5S rRNA genes (IGS1) was also species-specific, and PCR-RFLP analyses of the IGS1 region identified two distinct genotypes among the five C. orthopsilosis strains, which corresponded with the ITS region sequence data. The three bloodstream C. orthopsilosis strains were confined to a single genotype. Among 81 randomly selected C. parapsilosis strains, two genotypes were detected by IGS1 region analyses, indicating limited genotypic heterogeneity among C. parapsilosis sensu stricto strains. As far as is known, this is the first report on the identification of C. orthopsilosis from a bloodstream infection in the Arabian Gulf region.