Zia U. Khan

Seminested PCR for Diagnosis of Candidemia: Comparison with Culture, Antigen Detection, and Biochemical Methods for Species Identification

ABSTRACT 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.

Comparative evaluation of (1, 3)-β-D-glucan, mannan and anti-mannan antibodies, and Candida species-specific snPCR in patients with candidemia

BackgroundCandidemia 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.MethodsDiagnostic usefulness of Platelia Candida Ag for the detection of mannan, Platelia Candida Ab for the detection of anti-mannan antibodies, Fungitell for the detection of BDG, and of a semi-nested PCR (snPCR) for the detection Candida species-specific DNA have been retrospectively evaluated using 32 sera from 27 patients with culture-proven candidemia, 51 sera from 39 patients with clinically suspected candidemia, sera of 10 women with C. albicans vaginitis, and sera of 16 healthy controls.ResultsUsing cut-off values recommended by the manufacturers, the sensitivity of the assays for candidemia patients were as follows: Candida snPCR 88%, BDG 47%, mannan 41%, anti-mannan antibodies 47%, respectively. snPCR detected 5 patients who had candidemia due to more than one Candida species. The sensitivities of the combined tests were as follows: Candida mannan and anti-mannan antibodies 75%, and Candida mannan and BDG 56%. Addition of snPCR data improved the sensitivity further to 88%, thus adding 10 sera that were negative by BDG and/or mannan. In clinically suspected, blood culture negative patients; the positivities of the tests were as follows: Candida DNA was positive in 53%, BDG in 29%, mannan in 16%, and anti-mannan antibodies in 29%. The combined detection of mannan and BDG, and mannan, BDG and Candida DNA enhanced the positivity to 36% and 54%, respectively. None of the sera from Candida vaginitis patients and healthy subjects were positive for Candida DNA and mannan.ConclusionThe observations made in this study reinforce the diagnostic value of snPCR in the sensitive and specific diagnosis of candidemia and detection of more than one Candida species in a given patient. Additionally, in the absence of a positive blood culture, snPCR detected Candida DNA in sera of more than half of the clinically suspected patients. While detection of BDG, mannan and anti-mannan antibodies singly or in combination could help enhancing sensitivity and eliminating false positive tests, a more extensive evaluation of these assays in sequentially collected serum samples is required to assess their value in the early diagnosis of candidemia.

Tobacco Agar, a New Medium for Differentiating Candida dubliniensis from Candida albicans

ABSTRACT Isolates of Candida dubliniensis may be misidentified as Candida albicans in microbiological laboratories if only the germ tube and/or the chlamydospore test is used for identification to the species level. In this study, we have evaluated the efficacy of tobacco agar for the differentiation of C. dubliniensis from C. albicans. On this medium at 28°C, all 30 C. dubliniensis isolates produced yellowish-brown colonies with hyphal fringes and abundant chlamydospores, whereas 54 C. albicans isolates formed smooth, white-to-cream-colored colonies with no chlamydospore production. This medium provides a simple tool for presumptive differentiation of C. dubliniensis from C. albicans.

Rapid molecular differentiation and genotypic heterogeneity among Candida parapsilosis and Candida orthopsilosis strains isolated from clinical specimens in Kuwait

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.

Outbreak of Fungemia among Neonates Caused by Candida haemulonii Resistant to Amphotericin B, Itraconazole, and Fluconazole

ABSTRACT 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.

Prevalence of Candida dubliniensis among germ tube-positive Candida isolates in a maternity hospital in Kuwait

In this study, 1644 germ tube‐positive Candida isolates from a maternity hospital was prospectively examined for the prevalence of Candida dubliniensis. Candida species were isolated from different clinical specimens, but majority (>90%) of them came from high vaginal swabs and urine specimens. The phenotypic and molecular identification methods for C. dubliniensis included production of rough colonies and chlamydospores on simplified sunflower seed agar, determination of assimilation profile by Vitek 2 yeast identification system, specific amplification of rDNA of internally transcribed spacer (ITS)‐2 region by semi‐nested PCR and direct DNA sequencing of the ITS‐1 and ITS‐2 regions. Three germ tube‐positive Candida isolates were identified as C. dubliniensis with an overall prevalence of 0.2%. Of these, two came from urine specimens and one from a vaginal swab. None of the C. dubliniensis isolates showed resistance against fluconazole, voriconazole and amphotericin B. The study reinforces the usefulness of sunflower seed agar in presumptive identification of C. dubliniensis and confirms the prevailing view that this species forms only a minor constituent of Candida species occurring in vagina or other anatomic sites of non‐HIV/AIDS‐infected individuals.

Levels of (1→3)-β-D-glucan, Candida mannan and Candida DNA in serum samples of pediatric cancer patients colonized with Candida species

BackgroundSurveillance cultures may be helpful in identifying patients at increased risk of developing invasive candidiasis. However, only scant information exists on the effect of Candida colonization on serum levels of diagnostic biomarkers. This prospective surveillance study determined the extent of Candida colonization among pediatric cancer patients and its possible impact on serum levels of (1-3)-β-D-glucan (BDG), Candida mannan and Candida DNA.MethodsA total of 1075 swabs originating from oropharynx (n = 294), nostrils (n = 600), rectum (n = 28), groin (n = 50), ear (n = 54), and axilla (n = 49) of 63 pediatric cancer patients were cultured for the isolation of Candida spp. Patients yielding Candida spp. from any sites were considered as colonized. Serum samples were collected from patients at the time of first surveillance culture for detection of BDG by Fungitell kit and Candida mannan by Platelia Candida Ag. Candida DNA was detected by using panfungal primers and identification was carried out by using species-specific primers and DNA sequencing.ResultsSeventy-five (7.6%) swab cultures from 35 (55.5%) patients yielded Candida spp. These isolates included C. albicans (n = 62), C. dubliniensis (n = 8), C. glabrata and C. tropicalis (n = 2 each) and C. krusei (n = 1). Eleven patients were colonized at three or more sites. Eight of 36 serum samples from 6 colonized patients yielded BDG values higher than the currently recommended cut-off value of ≥80 pg/ml. However, none of the serum samples yielded Candida mannan levels ≥0.5 ng/ml and PCR test for Candida DNA was also negative in all the serum samples of colonized patients. During the study period, only two colonized patients subsequently developed candidemia due to C. tropicalis. Besides positive blood cultures, C. tropicalis DNA, BDG and Candida mannan were also detected in serum samples of both the patients.ConclusionsThe present study demonstrates that while mucosal colonization with Candida species in pediatric cancer patients is common, it does not give rise to diagnostically significant levels of Candida mannan or Candida DNA in serum specimens. However, BDG values may be higher than the cut-off value in some pediatric patients without clinical evidence of invasive Candida infection. The study suggests the utility of Candida mannan or Candida DNA in the diagnosis of invasive candidiasis, however, the BDG levels in pediatric cancer subjects should be interpreted with caution.

Cryptococcus randhawai sp. nov., a novel anamorphic basidiomycetous yeast isolated from tree trunk hollow of Ficus religiosa (peepal tree) from New Delhi, India.

A novel anamorphic Cryptococcus species is described, which was isolated in New Delhi (India) from decaying wood of a tree trunk hollow of Ficus religiosa. On the basis of sequence analysis of the D1/D2 domains of the 26S rRNA gene and the internally transcribed spacer (ITS)-1 and ITS-2 region sequences, the isolate belonged to the Cryptococcus albidus cluster (Filobasidiales, Tremellomycetes) and was closely related to Cryptococcus saitoi, Cryptococcus cerealis and Cryptococcus friedmannii with 98% sequence identity. Phenotypically, the species differed from C. saitoi with respect to growth temperature (up to 37oC), presence of a thin capsule, ability to grow in the absence of vitamins, and inability to assimilate citrate and ethylamine. With respect to C. friedmannii, it differed in growth temperature, ability to assimilate lactose, raffinose, l-rhamnose, myo-inositol, and inability to utilize citrate. Furthermore, our isolate also differed from C. cerealis in growth temperature, presence of capsule and inability to assimilate l-sorbose. In view of the above phenotypic differences and unique rDNA sequences, we consider that our isolate represents a new species of Cryptococcus, and therefore, a new species, Cryptococcus randhawai is proposed for this taxon. The type strain J11/2002 has been deposited in the culture collection of the Centraalbureau voor Schimmelcultures (CBS10160) and CABI Biosciences (IMI 393306).

Mucor circinelloides as a Cause of Invasive Maxillofacial Zygomycosis: an Emerging Dimorphic Pathogen with Reduced Susceptibility to Posaconazole

ABSTRACT A case of maxillofacial zygomycosis caused by Mucor circinelloides, identified by phenotypic and molecular methods and treated successfully with liposomal amphotericin B (AmBisome) and surgical debridement, is described. The isolate was resistant to posaconazole. This report underscores the importance of prior susceptibility testing of zygomycetes to guide therapy with the most effective antifungal agent for an improved prognosis.

Antifungal susceptibility of clinical Candida parapsilosis isolates in Kuwait.

This study presents data on antifungal susceptibility of 114 Candida parapsilosis isolates recovered from clinical specimens in Kuwait. Candida parapsilosis isolates originating from blood (n = 66) and other clinical specimens (n = 48) were tested by Etest against amphotericin B (AP), caspofungin (CS), 5‐flucytosine (FC), fluconazole (FL) and voriconazole (VO). The plates were incubated at 35 °C and readings for minimum inhibitory concentrations (MIC) were recorded after 24 and 48 h of incubation. The MIC ranges and MIC90 read after 48 h were as follows: 0.064–1 and 0.5 μg ml−1 for AP; 0.125–4 and 1.5 μg ml−1 for CS; 0.047 to >256 and 1 μg ml−1 for FL; 0.023 to >32 and 0.125 μg ml−1 for FC and <0.002–1 and 0.047 μg ml−1 for VO respectively. According to Clinical Laboratory Standards Institute criteria, all the isolates were susceptible to VO, and resistance against FC and FL was <2%. Eight (7%) isolates exhibited reduced susceptibility (MIC >1 μg ml−1) to CS including six isolates with MIC of ≥2 μg ml−1 at 48 h reading. The antifungal resistance among bloodstream isolates of C. parapsilosis against AP, FL, FC and VO in Kuwait is rare. This is the first report on CS susceptibility of C. parapsilosis isolates from Arabian Gulf region.