Molecular subtyping of clinical isolates of <Emphasis Type="Italic">Candida albicans</Emphasis> and identification of <Emphasis Type="Italic">Candida dubliniensis</Emphasis>in Malaysia

The genotypes of 221 recent isolates of Candida albicans from various clinical specimens of 213 patients admitted to the University Malaya Medical Centre, Malaysia was determined based on the amplification of a transposable intron region in the 25 S rRNA gene. The analyses of 178 C. albicansisolated from nonsterile clinical specimens showed that they could be classified into three genotypes: genotype A (138 isolates), genotype B (38 isolates) and genotype C (2 isolates). The genotyping of 43 clinical isolates from sterile specimens showed that they belonged to genotype A (29 isolates), genotype B (10 isolates), genotype C (2 isolates) and genotype D (2 isolates). The overall distribution of C. albicans genotypes in sterile and nonsterile specimens appeared similar, with genotype A being the most predominant type. This study reported the identification of C. dubliniensis (genotype D) in 2 HIV-negative patients with systemic candidiasis, which were missed by the routine mycological procedure. The study demonstrated the genetic diversity of clinical isolates of C. albicans in Malaysia.     

Molecular typing of clinical Candida strains using random amplified polymorphic DNA and contour‐clamped homogenous electric fields electrophoresis

Aims: This report describes an investigation into the genetic profiles of 38 Candida albicans and 19 Candida glabrata strains collected from a dental hospital of Monastir (Tunisia) and the Laboratory of Parasitology, Farhat Hached Hospital of Sousse (Tunisia), using two typing methods: random amplified polymorphic DNA (RAPD) and contour‐clamped homogenous electric fields (CHEF). Methods and Results: The two methods (RAPD and CHEF electrophoresis) were able to identify clonal‐related isolates from different patients. RAPD method using two primers (CA1 and CA2) exhibited the highest discriminatory power by discriminating 22 genotypes for C. albicans with CA1 oligonucleotides and 19 genotypes with CA2 primer. For C. glabrata, 17 genotypes were obtained when both primers CA1 and CA2 were combined. The CHEF karyotyping of C. albicans has discriminated only 17 different karyotypes. Conclusion: The genotype of each isolate and genotypic difference among C. albicans and C. glabrata isolates were patient specific and not associated with the site of infection, geographic origin or date of isolation. Significance and Impact of the Study: Identification of relatedness between Candida species using molecular approaches with high discriminatory power is important in determining adequate measures for interruption of transmission of this yeast.     

The synergy of honokiol and fluconazole against clinical isolates of azole‐resistant Candida albicans

Aims: To evaluate the interaction of fluconazole (FLC) and honokiol (HNK) in vitro and vivo against azole‐resistant (azole‐R) clinical isolates of Candida albicans. Methods and Results: A checkerboard microdilution method was used to study the in vitro interaction of FLC and HNK in 24 azole‐R clinical isolates of C. albicans. In vivo antifungal activity was performed to further analyse the interaction between FLC and HNK. In the in vitro study, synergism was observed in all 24 FLC‐resistant strains tested as determined by fractional inhibitory concentration index (FICI), and in 22 strains by ΔE models. No antagonistic activity was observed in any of the strains tested. These positive interactions were also confirmed by using the time‐killing test for the selected strain C. albicans YL371, which shows strong susceptible to the combination of HNK and FLC. In the in vivo study, the mice with candidiasis were treated successfully by a combination therapy of HNK with FLC, the results showed a decrease of the colony forming unit in infected and treated animals compared to the controls, at the conditions of the treatment used in this study. Conclusions: Synergistic activity of HNK and FLC against clinical isolates of FLC‐resistant C. albicans was observed in vitro and in vivo. Significance and Impact of the Study: This report might provide a potential therapeutic method to overcome the problem of drug‐resistance in C. albicans.     

Detection of <Emphasis Type="Italic">Candida albicans</Emphasis> by Mass Spectrometric Fingerprinting

Candida albicans is one of the most frequent causes of fungal infections in humans. Significant correlation between candiduria and invasive candidiasis has previously been described. The existing diagnostic methods are often time-consuming, cost-intensive and lack in sensitivity and specificity. In this study, the profile of low-molecular weight volatile compounds in the headspace of C. albicans-urine suspensions of four different fungal cell concentrations compared to nutrient media and urine without C. albicans was determined using proton-transfer reaction mass spectrometry (PTR-MS). At fungal counts of ≥1.5 × 10⁵ colony forming units (CFU)/ml signals at 45, 47 and 73 atomic mass units (amu) highly significantly increased. At fungal counts of <1.5 × 10⁵ CFU/ml signals at 47 and 73 amu also increased, but only at 45 amu a statistically significant increase was seen. Time course alterations of signal intensities dependent on different cell concentrations and after addition of Sabouraud nutrient solution were analysed. Recommendations for measurement conditions are given. Our study is the first to describe headspace profiling of C. albicans-urine suspensions of different fungal cell concentrations. PTR-MS represents a promising approach to rapid, highly sensitive and non-invasive clinical diagnostics allowing qualitative and quantitative analysis.     

Evaluation of the V404I and V509M amino acid substitutions of <Emphasis Type="Italic">ERG11</Emphasis> gene in <Emphasis Type="Italic">Candida albicans</Emphasis> isolates by pyrosequencing

The molecular mechanisms underlying fluconazole resistance in C. albicans involve mutations and the overexpression of the ERG11 gene and membrane transport proteins. We examined the relationship between the reduced fluconazole susceptibility of C. albicans and mutations of V404I and V509M in the ERG11 gene in 182 C. albicans clinical isolates using the Pyrosequencing™ method. DNAs from these clinical isolates with different levels of in-vitro fluconazole susceptibility — one resistant, five susceptible dose-dependent (SDD), four trailer, and 172 susceptible — were analyzed. None of the fluconazole-susceptible, SDD, trailer or resistant isolates had mutations of V404I or V509M. Our results showed that no correlation can be found between the V404I or V509M mutation and fluconazole susceptibility in C. albicans.     

Prevalence of <Emphasis Type="Italic">emm</Emphasis> types of Group A streptococci recovered from school children and hospital patients in Bangalore City,India

A total of sixty non repetitive isolates of group A streptococci (GAS) recovered from throat cultures of asymptomatic school children (n = 36), school children with clinical pharyngitis (n = 9) and hospital patients with clinical pharyngitis (n = 4) as well as diverse clinical samples from invasive GAS disease (n = 11) were subjected to emm typing. A total of 35 emm types including 3 new subtypes were identified among them. Types emm 12 (16.6%) and emm 71 (6.6%) were the most prevalent. The distribution of emm types identified among school children were similar to those encountered among invasive isolates but was significantly different from those reported elsewhere in India. Our study confirms the high diversity among Indian GAS isolates as well as significant regional differences among the distribution of emm types.     

Inhibitory effect of extracts from Brazilian medicinal plants on the adhesion of <Emphasis Type="Italic">Candida albicans</Emphasis> to buccal epithelial cells

Plants are known to produce a plethora of secondary metabolites which are recognized as a useful source of new drugs or drug leads. Extracts and fractions of Schinus terebinthifolius Raddi (Anacardiaceae), Piper regnellii C.D.C. (Piperaceae), Rumex acetosa L. (Polygonaceae), and Punica granatum L. (Punicaceae) were assessed for their antifungal activity against eight clinical isolates of C. albicans. They were also evaluated for their effect on the adhesion of these C. albicans isolates to buccal epithelial cells (BECs). The ethyl acetate fraction from the leaves of S. terebinthifolius showed promising activity, inhibiting the growth of three C. albicans isolates at 7.8 μg ml⁻¹ and significantly inhibiting their adhesion to BEC at 15 μg ml⁻¹ . In addition, this fraction did not show cytotoxic activity against murine macrophages. The results show the potential of the plant extracts studied as a source of new antifungal compounds. Further studies are necessary for isolation and characterization of the active compounds of these plants.     

Acid proteinase,phospholipase, and biofilm production of <Emphasis Type="Italic">Candida</Emphasis> species isolated from blood cultures

Three virulence factors comprising proteinase, phospholipase, and biofilm among 68 Candida albicans and 31 non-albicans Candida strains (11 C. tropicalis, 8 C. parapsilosis, 6 C. glabrata, 4 C. guillermondii, 2 C. krusei) isolated from blood cultures were analyzed. In total, 61 (89.7%) C. albicans strains were detected as proteinase positive whereas eight (25.8%) non-albicans Candida strains were proteinase positive (P < 0.05). Phospholipase production was detected in 41 (60.3%) C. albicans strains. All non-albicans Candida strains were phospholipase negative. Biofilm production was determined by both visual and spectrophotometric methods. Eight (11.8%) of C. albicans strains and 13 (41.93%) of 31 non-albicans Candida strains were biofilm positive with two of the methods (P < 0.05). According to our results, we may suggest that detection of hydrolytic enzyme and biofilm production abilities of the Candida isolates in clinical mycology laboratories may warn the clinican for a possible hematogenous infection.     

Frequency of <Emphasis Type="Italic">Candida</Emphasis> spp. in the Oral Cavity of Brazilian HIV-Positive Patients and Correlation with CD4 Cell Counts and Viral Load

The aim of this study was to evaluate the prevalence of Candida spp., and particularly C. dubliniensis, among oral isolates from Brazilian HIV-positive patients correlating these results with CD4 cell counts and viral load. Forty-five individuals (23 female and 22 male) diagnosed as HIV-positive by ELISA and Western-blot, under anti-retroviral therapy for at least 1 year and without oral candidosis signals were included in the study. The control group was constituted by 45 healthy individuals, matched to the test group in relation to age, gender, and oral conditions. Oral rinses were collected and the identification was performed by phenotypic tests. The existence of C. dubliniensis among the isolates was analyzed using a validated multiplex PCR assay. Candida spp. were detected at significantly higher number in the oral cavity of HIV-positive patients in relation to the controls (P = 0.0008). C. albicans was the most frequently isolated species in both groups. In the HIV group, C. glabrata, C. lipolytica, C. krusei, C. guilliermondii, and C. parapsilosis were also identified. In the control group, we additionally identified C. tropicalis and C. dubliniensis. Two isolates (1.9%, 2/108) from control individuals were identified as C. dubliniensis and this species was not verified in the HIV group. Candida spp. counts were statistically lower (P = 0.0230) in the oral cavity of patients with low viral load (<400 copies/mm3). Candida spp. counts did not differ statistically among groups with different levels of CD4 cells counts (P = 0.1068).     

Identification of <Emphasis Type="Italic">Candida</Emphasis> Species Isolated from Bovine Mastitic Milk and Their In Vitro Hemolytic Activity in Western Turkey

In this study, identification of 207 Candida isolates, previously isolated from mastitic bovine quarter milk samples at the level of genus, was made using API 20 C AUX system. The most frequently isolated species were Candida krusei (34.8%), followed by Candida rugosa (16.4%), Candida kefyr (12.6%), Candida albicans (10.1%), and Candida tropicalis (9.2%). Less common isolates were Candida zeylanoides (5.8%), Candida parapsilosis (4.3%), Candida guilliermondii (3.4%), Candida famata (1.9%), and Candida glabrata (1.5%). Additionally, in vitro hemolytic activity of all Candida strains were also examined in the present study. C. krusei (72 isolates), C. kefyr (26), C. albicans (21), C. tropicalis (19), C. zeylanoides (12), and C. glabrata (3) demonstrated both alpha and beta hemolysis at 48-h postinoculation. Only alpha hemolysis was detected in C. rugosa (34), C. guilliermondii (7), and C. famata (4), while C. parapsilosis (9) did not show any hemolytic activity after incubation for 72 h. Statistically significant difference (P < 0.001) was determined between the beta-hemolytic activities of Candida strains. The hemolytic activities of C. zeylanoides, C. albicans and C. kefyr were higher than other strains. This is the first study to describe variable hemolysis types exhibited by different Candida strains isolated from bovine mastitic milk in Turkey.     

Sodium Choleate (NaCho) Effects on <Emphasis Type="Italic">Candida albicans</Emphasis>: Implications for Its Role as a Gastrointestinal Tract Inhabitant

Candida albicans at times resides in the intestinal tract, where it experiences exposure to bile salts suggesting a study of the effects of crude bile salts in the form of sodium choleate (NaCho) on C. albicans growth, expression of virulent phenotypes, and adaptation to physiological challenges in vitro. Growth and phenotype alteration was examined by challenging clinical isolates of C. albicans with a wide range of NaCho concentrations by using conventional microbial physiology methods. Our results showed that (1) NaCho did not inhibit growth of yeast cells, up to 40 mg/ml; (2) NaCho powerfully stimulated the hypha formation; (3) NaCho at 2.5 and 5 mg/ml significantly induced CDR1p and biofilm formation, but these effects decreased at higher NaCho concentrations; (4) loss of cell integrity with exposure to 56°C for 15 min, was exacerbated by increasing levels of NaCho; (5) NaCho protected yeast from hydrogen peroxide damage in a dose-dependent manner; and (6) catalase activity was increased by NaCho exposure.     

Detection of <Emphasis Type="Italic">Candida</Emphasis><Emphasis Type="Italic">dubliniensis</Emphasis> in Venezuela

Over the past decades there has been a significant increase in fungal infections caused by Candida species, and continues to be common in immunocompromised individuals infected with the human immunodeficiency virus (HIV). Although Candida albicans remains the fungal species most frequently isolated as an opportunistic oral pathogen, other non-albicans are often identified in this cohort of patients, including C. dubliniensis. This yeast is closely related to and shares many phenotypic characteristics with C. albicans. Colonies of these two species appear morphologically identical when not grown on special media. The shared phenotypic characteristics of C. dubliniensis and C. albicans suggest that many C. dubliniensis isolates may have been misidentified as C. albicans in the past. The present studies aim is to recover and identify C. dubliniensis, and presumptive clinical C. albicans, from the oral cavities of HIV-seropositive individuals, comparing conventional media to obtain a simple, low-cost and reliable identification system for C. dubliniensis. A total of 16 isolates (3,98%) had been obtained from 402 HIV infected individuals with recurrent oropharyngitis and were identified as C. dubliniensis. Out of these C. dubliniensis isolates 19% were resistant, with MICs above 64 μg/ml to fluconazole. This constitutes, to the authors knowledge the first recovery of this organism in Venezuela.     

Detection of invasive Candida albicans infection using a specific 99mTc-labeled monoclonal antibody for the C. albicans germ tube

Accurate diagnosis is critical for effective treatment of the invasive infection by Candida albicans. Here, we investigated whether a ^99m technetium (Tc)-labeled Fab’ fragment of the monoclonal antibody specific for the C. albicans germ tube could specifically identify an invasive C. albicans infection. The germ tube of C. albicans was used as an immunogen to obtain monoclonal antibodies and the Fab’ fragment of MAb03.2 C1–C2 with highest affinity and specificity was labeled with ^99mTc. In vitro binding assays showed that the labeled Fab’ preferentially bound to the germ tubes of C. albicans (4.23 ± 0.17 × 10² Bq per 1 × 10⁷ cells). These values were significantly higher than those for blastospores of C. albicans, blastospores of heat-killed C. albicans, Aspergillus fumigatus, Staphylococcus aureus, and Escherichia coli (P < 0.05). By using in vivo biodistribution and planar imaging with single photon emission computed tomography, we demonstrated a significant specific accumulation of radioactivity in C. albicans-infected tissues. In summary, ^99mTc-MAb03.2 C1–C2 Fab’ is able to specifically accumulate in C. albicans-infected tissues, but not in tissue infected with A. fumigatus or bacteria or in a sterile inflammation. This study provides a new and specific radiopharmaceutical for the diagnosis of invasive C. albicans infections.     

Improved DNA‐FISH for cytometric detection of Candida spp

Aims: We developed improved methods for DNA‐based fluorescence in situ hybridization (FISH) for rapid detection of Candida spp. and Candida albicans via flow cytometry. Methods and Results: Two previously reported C. albicans‐targeted DNA probes were evaluated against whole cells of C. albicans and related Candida species using a rapid, high‐temperature hybridization protocol. One probe (CalB2208) was shown for the first time to be suitable as a FISH probe. Although cell labelling for both probes was relatively bright, we were able to substantially improve our results by altering fixation and hybridization conditions. For fixation, a 60 : 40 mixture of 10% buffered formalin and ethanol was most effective. Probe intensity was improved as much as ten‐fold through the use of unlabelled helper probes, and buffer containing 0·9 mol l^?1 NaCl plus 10% formamide yielded the best hybridizations for both probe/helper cocktails. Although optimal labelling occurred with longer hybridizations, we found that C. albicans could be completely differentiated from the nontarget yeast Rhodotorula glutinis after only 15 min using the brightest probe (Calb‐1249) and that a formal washing step was not required. Specificities of probe/helper cocktails under optimal conditions were determined using a panel of target and nontarget cell types, including four strains of Candida dubliniensis. Calb‐1249 cross‐reacted slightly with Candida parapsilosis and strongly with both Candida tropicalis and C. dubliniensis. In contrast, we found that CalB2208 was exclusive for C. albicans. The molecular basis of this specificity was confirmed by DNA sequencing. Conclusions: We describe DNA probe‐based approaches for rapid and bright labelling of Candida spp. and for specific labelling of C. albicans without cross‐reaction with C. dubliniensis. Our work improves upon previously described methods. Significance and Impact of the Study: The methods described here for rapid FISH‐based detection of Candida spp. may have applications in both clinical and food microbiology.     

Evaluation of Antifungal Susceptibility Testing with Microdilution and Etest Methods of <Emphasis Type="Italic">Candida</Emphasis> Blood Isolates

Candida species that show an increasing number of clinical and/or microbiological resistance to several antifungals and are the most common agents of invasive fungal infections. The aim of this study was to investigate the in vitro susceptibility of Candida blood isolates to antifungal agents (amphotericin B, fluconazole, itraconazole, and voriconazole) by comparative use of the CLSI reference microdilution method and Etest. Four hundred Candida blood isolates (215 Candida albicans, 185 non-albicans Candida strains) were included in the study. The broth microdilution test was performed according to the CLSI M27 A2 document. Etest was carried out according to the manufacturer’s instructions. The MIC results obtained with reference microdilution were compared with those obtained with the Etest by using percent and categorical agreements. According to MIK₉₀ values, voriconazole was the most active and itraconazole was the least active drug in vitro against all Candida species. Other than voriconazole, statistically significant differences were found when the susceptibility of Candida albicans and non-albicans Candida spp. to amphotericin B, fluconazole, and itraconazole were compared. These antifungal agents were found to be more active to C. albicans. Among the non-albicans Candida species, the lowest MIC values were obtained for Candida parapsilosis isolates. When the standard method was compared with Etest, the total agreement was higher for C. albicans than for non-albicans species, especially for fluconazole and voriconazole. In view of the findings, it was concluded that itraconazole showed the lowest activity against all Candida species. Etest could be an alternative method in assessing the in vitro antifungal susceptibility of Candida spp., but it is more convenient to use the microdilution method for studying in vitro susceptibility of non-albicans species, in particular for those possessing high MIC values against azoles.     

Use of CHROMagar in the Differentiation of Common Species of <Emphasis Type="Italic">Candida</Emphasis>

CHROMagar has been reported to be useful for the rapid and accurate identification of Candida species. We tested 135 isolates of Candida species isolated from oropharyngeal candidiasis in HIV patients and found that it was useful in the presumptive identification of Candida albicans and Candida krusei. Occasional strains of C. tropicalis produced colonies with a greenish tinge making it difficult to differentiate from C. albicans.     

An Evaluation of Risk Factors in Pregnant Women with <Emphasis Type="Italic">Candida</Emphasis> Vaginitis and the Diagnostic Value of Simultaneous Vaginal and Rectal Sampling

In this study, we investigated the epidemiological characteristics of VVC among pregnant women. We conducted a prospective survey among 372 pregnant women to investigate the prevalence, clinical forms, etiological agents, and predisposing factors of VVC. In addition, we determined the relationship between vaginal and rectal flora by simultaneously obtaining one high vaginal swab and one rectal swab from each patient using sterile cotton-tipped swabs. Furthermore, we compared the recovery and identification performances of chromID Candida agar to Sabouraud dextrose agar with gentamicin and chloramphenicol. Clinically and mycologically confirmed cases of VVC were detected in 139 (37.4%) and vaginal colonization described in 42 (11.3%) of 372 pregnant women. Rectal cultures were also positive in 98 of the 139 (70.5%) VVC cases. Candida albicans and C. glabrata were identified in vaginal samples in 58.0 versus 19.0% and from rectal samples in 49.0 versus 13.5%, respectively. Increases in gestational week and gravidae were identified to be statistically significant in patients with acute VVC (AVVC) and symptomatic recurrent VVC (RVVC), and asymptomatic RVVC (P = 0.04 and P = 0.03, respectively). In the laboratory diagnosis of VVC, specifically tailored chromogenic media are reliable tools for both the recovery and rapid identification of common Candida spp., particularly C. albicans, as well as for the detection of polyfungal populations in vaginal samples (P > 0.05). In addition, rectal colonization is a common finding in cases of AVVC and symptomatic-RVVC cases and corresponds well with the presence of the same yeast species in the vagina.     

Genetic relatedness of commensal strains of <Emphasis Type="Italic">Candida albicans</Emphasis> carried in the oral cavity of patients’ dental prosthesis users in Brazil

The aim of this study is to describe the degree of yeast-colonization in diabetic and hemodialysed-users of dental prostheses. Individuals (306) were examined using an oral rinse technique in order to evaluate the incidence of yeast-carriage, and genotype of C. albicans. Yeasts were isolated from 68.4% (91/133) individual’s dental prostheses users. Dental prostheses were found to be a significant factor for the yeast colonization (P < 0.05). Overall, the intensity of carriage was higher in diabetic patients as compared with health and hemodialysed individuals (P < 0.05). The isolation rates were: C. albicans (51.7%), C. parapsilosis (20.9%), C. tropicalis (14.3%), C. glabrata (6.6%), C. krusei (3.3%), C. rugosa (1.1%), and Pichia (Pichia ohmeri, 2.2%). Ready-To-Go RAPD Analysis Beads were used and primer OPJ 6 distinguished the C. albicans isolates found in prostheses users. All the isolates were grouped into 11 RAPD profiles in four main clusters and, the average S _AB for the entire collection of 47 C.␣albicans isolates were 0.779 ± 0.178. Over 85% of isolates had a similarity level higher than or equal to 0.8 reinforcing the idea that the use of dental prostheses, independently of the host’s clinical condition, probably provides the necessary conditions for these strains to gain a growth-specific advantage over others.