The first established microsatellite markers to distinguish Candida orthopsilosis isolates and detection of a nosocomial outbreak in China.

The infection proportion of Candida orthopsilosis, a member of the C. parapsilosis complex, has increased globally in recent years, and nosocomial outbreaks have been reported in several countries. This study aimed to establish microsatellite loci-based typing method that was able to effectively distinguish among C. orthopsilosis isolates. Three reference C. orthopsilosis genome sequences were analyzed to identify repeat loci. DNA sequences containing over eight bi- or more nucleotide repeats were selected. A total of 51 loci were initially identified, and locus-specific primers were designed and tested with 20 epidemiologically unrelated isolates. Four loci with excellent reproducibility, specificity, and resolution for molecular typing purposes were identified, and the combined discriminatory power (DP, based on 20 epidemiologically unrelated isolates) of these four loci was 1.0. Reproducibility was demonstrated by consistently testing three strains each in triplicate, and stability, demonstrated by testing 10 successive passages. Then, we collected 48 C. orthopsilosis non-duplicate clinical isolates from the China Hospital Invasive Fungal Surveillance Net study to compare the DP of the microsatellite-based typing with internal transcribed spacer (ITS) and amplified fragment length polymorphism (AFLP) typing analyses, using ATCC 96139 as a reference strain. These 49 isolates were subdivided into 12 microsatellite types (COMT1-12), six AFLP types, and three ITS types, while all the isolates with the same COMT belonged to consistent AFLP and ITS type, demonstrating the high DP of our microsatellite-type method. According to our results, COMT12 was found to be the predominant type in China, and COMT5 was the second largest and responsible for causing a nosocomial outbreak. This microsatellite-type method is a valuable tool for the differentiation of C. orthopsilosis and could be vital for epidemiological studies to determine strain relatedness and monitor transmission.

Evolution of loss of heterozygosity patterns in hybrid genomes of Candida yeast pathogens.

BACKGROUND: Hybrids are chimeric organisms with highly plastic heterozygous genomes that may confer unique traits enabling the adaptation to new environments. However, most evolutionary theory frameworks predict that the high levels of genetic heterozygosity present in hybrids from divergent parents are likely to result in numerous deleterious epistatic interactions. Under this scenario, selection is expected to favor recombination events resulting in loss of heterozygosity (LOH) affecting genes involved in such negative interactions. Nevertheless, it is so far unknown whether this phenomenon actually drives genomic evolution in natural populations of hybrids. To determine the balance between selection and drift in the evolution of LOH patterns in natural yeast hybrids, we analyzed the genomic sequences from fifty-five hybrid strains of the pathogenic yeasts Candida orthopsilosis and Candida metapsilosis, which derived from at least six distinct natural hybridization events. RESULTS: We found that, although LOH patterns in independent hybrid clades share some level of convergence that would not be expected from random occurrence, there is an apparent lack of strong functional selection. Moreover, while mitosis is associated with a limited number of inter-homeologous chromosome recombinations in these genomes, induced DNA breaks seem to increase the LOH rate. We also found that LOH does not accumulate linearly with time in these hybrids. Furthermore, some C. orthopsilosis hybrids present LOH patterns compatible with footprints of meiotic recombination. These meiotic-like patterns are at odds with a lack of evidence of sexual recombination and with our inability to experimentally induce sporulation in these hybrids. CONCLUSIONS: Our results suggest that genetic drift is the prevailing force shaping LOH patterns in these hybrid genomes. Moreover, the observed LOH patterns suggest that these are likely not the result of continuous accumulation of sporadic events-as expected by mitotic repair of rare chromosomal breaks-but rather of acute episodes involving many LOH events in a short period of time.

Molecular Investigation of the Fatal Bloodstream Candida orthopsilosis Infection Case following Gastrectomy.

Candida orthopsilosis represents a closely related cryptic genospecies of Candida parapsilosis complex-misidentified in routine diagnostic assays. This is emerging in settings where central venous catheters, invasive medical interventions, and echinocandin treatments are most likely to be used. A 59-year-old, non-neutropenic male patient, was admitted to an intensive care unit (ICU) due to respiratory distress syndrome, following a partial gastrectomy. As a result of duodenal stump leakage, re-laparotomy was required, abdominal drains were provided and central line catheters were exchanged. Multiple isolates of Candida orthopsilosis drawn from consecutive blood cultures were identified, despite ongoing echinocandin therapy and confirmed in vitro echinocandins susceptibility of the isolated strain. Species identification was verified via ITS region sequencing. Herein, we report the well-documented-per clinical data and relevant laboratory diagnosis-first case of a bloodstream infection caused by Candida orthopsilosis in Poland.

Characterization, antifungal susceptibility and virulence of Candida parapsilosis complex isolates in a tertiary hospital in Cantabria, Northern Spain.

INTRODUCTION: The correct identification of the species within the Candida parapsilosis complex has become relevant due to the resistance of Candida metapsilosis to antifungals. We describe the characteristics of the Candida parapsilosis complex isolates, with respect to antifungal resistance and biofilm formation. METHODS: We perform a descriptive cross-sectional study in 30 strains, collected in a tertiary hospital. All strains, were identified by Vitek2, Vitek-MS™ systems and by ITS sequencing. The antifungal susceptibility profile was obtained with Sensititre™ panels, while biomass production and metabolic activity were quantified by means of crystal violet and XTT reduction assay, respectively. RESULTS: There was a 100% correlation between Vitek-MS™ and ITS sequencing. All isolates were susceptible to the nine antifungals tested. The metabolic activity and biomass production tests did not show any difference among the subtypes. CONCLUSIONS: The Vitek-MS™ system provides acceptable identification. We did not find significant differences neither in azole resistance nor in biofilm formation.

Characterization, antifungal susceptibility and virulence of Candida parapsilosis complex isolates in a tertiary hospital in Cantabria, Northern Spain / Caracterización, sensibilidad antifúngica y virulencia de aislamientos del complejo Candida parapsilosis en un hospital terciario en Cantabria, norte de España

Introduction: The correct identification of the species within the Candida parapsilosis complex has become relevant due to the resistance of Candida metapsilosis to antifungals. We describe the characteristics of the Candida parapsilosis complex isolates, with respect to antifungal resistance and biofilm formation. Methods: We perform a descriptive cross-sectional study in 30 strains, collected in a tertiary hospital. All strains, were identified by Vitek2, Vitek-MS™ systems and by ITS sequencing. The antifungal susceptibility profile was obtained with Sensititre™ panels, while biomass production and metabolic activity were quantified by means of crystal violet and XTT reduction assay, respectively. Results: There was a 100% correlation between Vitek-MS™ and ITS sequencing. All isolates were susceptible to the nine antifungals tested. The metabolic activity and biomass production tests did not show any difference among the subtypes. Conclusions: The Vitek-MS™ system provides acceptable identification. We did not find significant differences neither in azole resistance nor in biofilm formation.(AU)

Introducción: La correcta identificación del complejo Candida parasilopsis es relevante debido a la resistencia antifúngica de Candida metasilopsis. Describimos las características de aislados del complejo Candida parapsilosis respecto a la resistencia antifúngica y formación de biopelícula. Métodos: Se realiza un estudio descriptivo transversal de 30 cepas recolectadas en un hospital terciario. Todas se identificaron por los sistemas Vitek2, MALDI-TOF MS Vitek-MSTM y por secuenciación de las regiones ITS. La sensibilidad antifúngica se realizó con paneles SensititreTM. Para la producción de biomasa y la actividad metabólica se emplearon la medición de cristal violeta y el ensayo de reducción de XTT, respectivamente. Resultados: Hubo una correlación del 100% entre Vitek-MSTM y la secuencia de ITS. Todos los aislados fueron sensibles a los 9 antifúngicos evaluados. Los ensayos de actividad metabólica y producción de biomasa no arrojaron diferencias entre los subtipos. Conclusiones: El sistema Vitek-MSTM proporciona una identificación aceptable. No encontramos diferencias significativas ni en la resistencia a azoles ni en la formación de biopelículas.(AU)

Plasmid-Based CRISPR-Cas9 Editing in Multiple Candida Species.

CRISPR-Cas9 technology radically changed the approach to genetic manipulation of both medically and industrially relevant Candida species, as attested by the ever-increasing number of applications to the study of pathogenesis, drug resistance, gene expression, and host pathogen interaction and drug discovery. Here, we describe the use of plasmid-based systems for high efficiency CRISPR-Cas9 gene editing into any strain of four non-albicans Candida species, namely, Candida parapsilosis, Candida orthopsilosis, Candida metapsilosis, and Candida tropicalis. The plasmids pCP-tRNA and pCT-tRNA contain all the elements necessary for expressing the CRISPR-Cas9 machinery, and they can be used in combination with a repair template for disrupting gene function by insertion of a premature stop codon or by gene deletion. The plasmids are easily lost in the absence of selection, allowing scarless gene editing and minimizing detrimental effects of prolonged Cas9 expression.

Evaluation of MALDI-TOF MS for Identification of Species in the Candida parapsilosis Complex from Candidiasis Cases.

BACKGROUND: Phenotypically identified Candida parapsilosis is actually a complex of 3 member species named Candida parapsilosis sensu stricto (CPSS), Candida orthopsilosis (CO), and Candida metapsilosis (CM), which can be identified only by molecular methods and automated methods such as MALDI-TOF mass spectrometry (MS). This study was undertaken to evaluate the VITEK MS, which uses the principle of MALDI-TOF MS for the identification of member species of C. parapsilosis complex (CPC). METHODS: In this cross-sectional study, 126 presumptively identified and stocked isolates of CPC were included. Definite identification to species level was done by VITEK MS and PCR as the gold standard method. Clinico-demographic characters and risk factors were analyzed. Antifungal susceptibility testing was performed for fluconazole and voriconazole. RESULTS: Twelve isolates were not identified as CPC either by VITEK MS or PCR and hence were excluded from the analysis. Out of 114 CPC isolates, 89 (78.1%), 18 (15.8%), and 7 (6.1%) isolates were identified as CPSS, CO, and CM, respectively, by VITEK MS. PCR identified 84 (79.2%), 15 (14.2%), and 7 (6.6%) isolates as CPSS, CO, and CM, respectively. However, PCR did not detect 8 isolates of CPSS detected by VITEK MS. VITEK MS showed 95.3% agreement in species identification and showed a kappa coefficient of 0.87, which is almost perfect agreement. Predominant isolations of all 3 species were from blood. Resistance was observed more in CPSS for both the azoles. CONCLUSION: MALDI-TOF MS can be used as a rapid, reliable, cost-effective method to identify the species of CPC.

Hexane extract from Spiranthera odoratissima A. St.-hil. leaves: chemical composition and its bioactive potential against Candida pathogenic species, Leishmania amazonensis and Xylella fastidiosa.

Spiranthera odoratissima A. St.-Hil. (Rutaceae) has been popularly used against abdominal pain and rheumatism. This study aimed at extracting hexane from S. odoratissima (HE-SO) leaves to identify and quantify its volatile compounds by GC-MS and GC-FID and to evaluate its antifungal, antileishmanial and antibacterial activities in vitro. HE-SO exhibited antileishmanial activity against promastigote forms of Leishmania (Leishmania) amazonensis (IC50 = 38.16 µg/mL) and was moderately active against Xylella fastidiosa (MIC = 100 µg/mL). HE-SO also showed remarkable antifungal potential against six strains of Candida species, i. e., C. albicans, C. glabrata, C. parapsilosis, C. krusei, C. tropicalis and C. orthopsilosis. The lowest MIC values were between 31.25 and 250 µg/mL. Spathulenol (20.2%), τ-cadinol (11.7%), α-cadinol (9.4%), caryophyllene oxide (9.2%) and isoaromadendrene epoxide (8.2%) were the major components identified in HE-SO. Therefore, results showed that HE-SO has promising antileishmanial and antifungal actions.

Beware of covert enemies: Candida orthopsilosis malignant otitis externa with base of the skull osteomyelitis, a case report and review of literature.

BACKGROUND: Malignant otitis externa (MOE) is a serious infection of the external auditory canal that is frequently associated with skull base osteomyelitis (SBO) as well as secondary neurological sequelae. Patients with poorly controlled diabetes mellitus or immunosuppression are at increased risk of developing such critical infection for multiple local and systemic factors. While most cases are secondary to bacterial infections particularlyPseudomonas aeruginosa, fungal infections are also occasionally encountered, often associated with delayed diagnosis and high morbidity and mortality. CASE REPORT: We report a case of a 63 years old man with uncontrolled diabetes mellitus who presented with symptoms and signs of MOE, supported by radiological assessments. The patient was treated presumptively with a prolonged course of antibiotics without clinical improvement, coupled with progression of radiological findings and significant disease extension. Reassessment with biopsies and tissue cultures from external auditory meatus, tempo-mandibular bone, as well as base of the skull grew Candida orthopsilosis. The patient received induction treatment with high dose liposomal amphotericin followed by fluconazole to control disease progression and complications. CONCLUSION: Candida MOE with secondary skull base osteomyelitis is rare and difficult to diagnose with no clear guidance on assessment and management. Clinicians should be aware of the unusual presentations where microbiological and histopathological evaluations are essential for proper management.

Prevalence and Antifungal Susceptibility of Candida parapsilosis Species Complex in Eastern China: A 15-Year Retrospective Study by ECIFIG.

Candida parapsilosis complex is one of the most common non-albicans Candida species that cause candidemia, especially invasive candidiasis. The purpose of this study was to evaluate the antifungal susceptibilities of both colonized and invasive clinical C. parapsilosis complex isolates to 10 drugs: amphotericin (AMB), anidulafungin (AFG), caspofungin (CAS), micafungin (MFG), fluconazole (FLZ), voriconazole (VRZ), itraconazole (ITZ), posaconazole (POZ), 5-flucytosine (FCY), and isaconazole (ISA). In total, 884 C. parapsilosis species complex isolates were gathered between January 2005 and December 2020. C. parapsilosis, Candida metapsilosis, and Candida orthopsilosis accounted for 86.3, 8.1, and 5.5% of the cryptic species, respectively. The resistance/non-wild-type rate of bloodstream C. parapsilosis to the drugs was 3.5%, of C. metapsilosis to AFG and CAS was 7.7%, and of C. orthopsilosis to FLZ and VRZ was 15% and to CAS, MFG, and POZ was 5%. The geometric mean (GM) minimum inhibitory concentrations (MICs) of non-bloodstream C. parapsilosis for CAS (0.555 mg/L), MFG (0.853 mg/L), FLZ (0.816 mg/L), VRZ (0.017 mg/L), ITZ (0.076 mg/L), and POZ (0.042 mg/L) were significantly higher than those of bloodstream C. parapsilosis, for which the GM MICs were 0.464, 0.745, 0.704, 0.015, 0.061, and 0.033 mg/L, respectively (P < 0.05). The MIC distribution of the bloodstream C. parapsilosis strains collected from 2019 to 2020 for VRZ, POZ, and ITZ were 0.018, 0.040, and 0.073 mg/L, significantly higher than those from 2005 to 2018, which were 0.013, 0.028, and 0.052 mg/L (P < 0.05). Additionally, MIC distributions of C. parapsilosis with FLZ and the distributions of C. orthopsilosis with ITZ and POZ might be higher than those in Clinical and Laboratory Standards Institute studies. Furthermore, a total of 143 C. parapsilosis complex isolates showed great susceptibility to ISA. Overall, antifungal treatment of the non-bloodstream C. parapsilosis complex isolates should be managed and improved. The clinicians are suggested to pay more attention on azoles usage for the C. parapsilosis complex isolates. In addition, establishing the epidemiological cutoff values (ECVs) for azoles used in Eastern China may offer better guidance for clinical treatments. Although ISA acts on the same target as other azoles, it may be used as an alternative therapy for cases caused by FLZ- or VRZ-resistant C. parapsilosis complex strains.

Evaluation of Molecular Epidemiology, Clinical Characteristics, Antifungal Susceptibility Profiles, and Molecular Mechanisms of Antifungal Resistance of Iranian Candida parapsilosis Species Complex Blood Isolates.

Clonal expansion of fluconazole resistant (FLZ-R) Candida parapsilosis isolates is increasingly being identified in many countries, while there is no study exploring the antifungal susceptibility pattern, genetic diversity, and clinical information for Iranian C. parapsilosis blood isolates. Candida parapsilosis species complex blood isolates (n = 98) were recovered from nine hospitals located in three major cities, identified by MALDI-TOF MS, and their genetic relatedness was examined by AFLP fingerprinting. Antifungal susceptibility testing followed CLSI-M27-A3 and ERG11, MRR1 and hotspots 1/2 (HS1/2) of FKS1 were sequenced to assess the azole and echinocandin resistance mechanisms, respectively. Ninety-four C. parapsilosis and four Candida orthopsilosis isolates were identified from 90 patients. Only 43 patients received systemic antifungal drugs with fluconazole as the main antifungal used. The overall mortality rate was 46.6% (42/90) and death mostly occurred for those receiving systemic antifungals (25/43) relative to those not treated (17/47). Although, antifungal-resistance was rare, one isolate was multidrug-resistant (FLZ = 16 µg/ml and micafungin = 8 µg/ml) and the infected patient showed therapeutic failure to FLZ prophylaxis. Mutations causing azole and echinocandin resistance were not found in the genes studied. AFLP revealed five genotypes (G) and G1 was the main one (59/94; 62.7%). Clinical outcome was significantly associated with city (P = 0.02, α <0.05) and Mashhad was significantly associated with mortality (P = 0.03, α <0.05). Overall, we found a low level of antifungal resistance for Iranian C. parapsilosis blood isolates, but the noted MDR strain can potentially become the source of future infections and challenge the antifungal therapy in antifungal-naïve patients. AFLP typing results warrants confirmation using other resolutive typing methods.

The Transcriptional Aftermath in Two Independently Formed Hybrids of the Opportunistic Pathogen Candida orthopsilosis.

Interspecific hybridization can drive evolutionary adaptation to novel environments. The Saccharomycotina clade of budding yeasts includes many hybrid lineages, and hybridization has been proposed as a source for new pathogenic species. Candida orthopsilosis is an emerging opportunistic pathogen for which most clinical isolates are hybrids, each derived from one of at least four independent crosses between the same two parental lineages. To gain insight into the transcriptomic aftermath of hybridization in these pathogens, we analyzed allele-specific gene expression in two independently formed hybrid strains and in a homozygous strain representative of one parental lineage. Our results show that the effect of hybridization on overall gene expression is rather limited, affecting ∼4% of the genes studied. However, we identified a larger effect in terms of imbalanced allelic expression, affecting ∼9.5% of the heterozygous genes in the hybrids. This effect was larger in the hybrid with more extensive loss of heterozygosity, which may indicate a tendency to avoid loss of heterozygosity in these genes. Consistently, the number of shared genes with allele-specific expression in the two independently formed hybrids was higher than random expectation, suggesting selective retention. Some of the imbalanced genes have functions related to pathogenicity, including zinc transport and superoxide dismutase activities. While it remains unclear whether the observed imbalanced genes play a role in virulence, our results suggest that differences in allele-specific expression may add an additional layer of phenotypic plasticity to traits related to virulence in C. orthopsilosis hybrids.IMPORTANCE How new pathogens emerge is an important question that remains largely unanswered. Some emerging yeast pathogens are hybrids originated through the crossing of two different species, but how hybridization contributes to higher virulence is unclear. Here, we show that hybrids selectively retain gene regulation plasticity inherited from the two parents and that this plasticity affects genes involved in virulence.

Utility of two PCR-RFLP-based techniques for identification of Candida parapsilosis complex blood isolates.

BACKGROUND: Candida parapsilosis is the second or third most frequently isolated Candida species related to nosocomial infections, even overtaking Candida albicans in some hospitals. C. parapsilosis constitutes a complex of closely related species: Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis. Accurate detection of these species is of importance, as the incidence of C. orthopsilosis has been reported to surpass that of Candida krusei. OBJECTIVE: To evaluate the diagnostic utility of two PCR-RFLP methods targeting the SADH and FKS1 genes and to determine the prevalence of cryptic species in 96 bloodstream isolates of C. parapsilosis from 93 patients. METHODS: Restriction patterns of the SADH and FKS1 genes were analysed, and sequencing of the D1/D2 regions of the ribosomal RNA was used to evaluate the reliability of both PCR-RFLP methods. RESULTS: In our study, 77 C. parapsilosis sensu stricto, 13 C. orthopsilosis and five C. metapsilosis were identified by sequencing. Both PCR-RFLP methods demonstrated strong agreement with D1/D2 sequencing in the identification of C. parapsilosis and C. orthopsilosis, while both methods were unable to identify the C. metapsilosis isolates. Moreover, unexpected restriction patterns were observed for two isolates on SADH PCR-RFLP and for four isolates on FKS1 PCR-RFLP. Mixed bloodstream infections of C. parapsilosis sensu stricto and C. orthopsilosis were detected for three patients, for which differential growth characteristics were observed. CONCLUSION: The molecular method chosen for identification could have an impact on determination of the real prevalence of C. metapsilosis in candidaemia, and mixed fungaemias can remain undetected.

CORT0C04210 is required for Candida orthopsilosis adhesion to human buccal cells.

Candida orthopsilosis is a human fungal pathogen belonging to the Candida parapsilosis sensu lato species complex. C. orthopsilosis annotated genome harbors 3 putative agglutinin-like sequence (ALS) genes named CORT0B00800, CORT0C04210 and CORT0C04220. The aim of this study was to investigate the role played by CORT0C04210 (CoALS4210) in the virulence and pathogenicity of this opportunistic yeast. Heterozygous and null mutant strains lacking one or both copies of CoALS4210 were obtained using the SAT1-flipper cassette strategy and were characterized in in vitro, ex vivo and in vivo models. While no differences between the mutant and the wild-type strains were observed in in vitro growth or in the ability to undergo morphogenesis, the CoALS4210 null mutant showed an impaired adhesion to human buccal epithelial cells compared to heterozygous and wild type strains. When the pathogenicity of CoALS4210 mutant and wild type strains was evaluated in a murine model of systemic candidiasis, no statistically significant differences were observed in fungal burden of target organs. Since gene disruption could alter chromatin structure and influence transcriptional regulation of other genes, two independent CRISPR/Cas9 edited mutant strains were generated in the same genetic background used to create the deleted strains. CoALS4210-edited strains were tested for their in vitro growing ability, and compared with the deleted strain for adhesion ability to human buccal epithelial cells. The results obtained confirmed a reduction in the adhesion ability of C. orthopsilosis edited strains to buccal cells. These findings provide the first evidence that CRISPR/Cas9 can be successfully used in C. orthopsilosis and demonstrate that CoALS4210 plays a direct role in the adhesion of C. orthopsilosis to human buccal cells but is not primarily involved in the onset of disseminated candidiasis.

Evaluation of Exoenzyme Activities, Biofilm Formation, and Co-hemolytic Effect in Clinical Isolates of Candida parapsilosis Species Complex.

Candida parapsilosis species complex is considered as important emerging pathogens and little is known about their pathogenicity factors and co-hemolytic activity with different bacteria species. The aim of this study was to determine in vitro exoenzyme activities, biofilm formation, and co-hemolytic effect of different bacteria species on clinical C. parapsilosis complex isolates. In total, 67 C. parapsilosis complex isolates consist of C. parapsilosis sensu stricto 63/67 and Candida orthopsilosis 4/67 were used in this study. To determine the hemolytic activity of these species, Sabouraud dextrose sheep blood agar was used. Evaluation of the CAMP-like phenomenon carried out in the presence of Staphylococcus aureus, Staphylococcus saprophyticus, Staphylococcus epidermidis, and Streptococcus agalactiae. Tube test method with ethylenediaminetetraacetic acid-rabbit plasma was used to determine coagulase activity, and biofilm formation was assessed by the tube method in assist of Sabouraud glucose broth (8%) medium. Fisher's exact tests were used for data statistical analysis. Sixty-six of 67 (98.5%) and 3/67 (4.5%) of the species showed hemolysin and coagulase activity, respectively. Fifty-five of 67 (82.1%) of species had ability for biofilm formation, and none of the samples exhibited co-hemolytic effect in the presence of four mentioned bacteria. No significant difference was found between the level of enzyme production and biofilm formation among the isolates.

Species distribution and antifungal susceptibility among clinical isolates of Candida parapsilosis complex from India.

BACKGROUND: Candida parapsilosis is recognized as a species complex: Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis are three distinct but closely related species. AIMS: To determine the species and antifungal susceptibility of members of the C. parapsilosis complex, isolated from clinical samples. METHODS: Isolates identified as C. parapsilosis complex by VITEK® 2 system were included. Antifungal susceptibility test was done using the VITEK® 2 semi-automated system. The distribution of the species in the complex was determined by multiplex PCR. RESULTS: Among the seventy-seven C. parapsilosis complex isolates, C. parapsilosis sensu stricto (57.1%) was the commonest species, followed by C. orthopsilosis (40.2%) and C. metapsilosis (2.5%). All three species were susceptible to amphotericin B, caspofungin and micafungin. Among C. parapsilosis sensu stricto isolates, 16% were resistant to fluconazole while 2.2% showed dose dependent susceptibility. Also, 18.2% of C. parapsilosis sensu stricto isolates showed dose dependent susceptibility to voriconazole. CONCLUSIONS: C. parapsilosis sensu stricto was the most commonly isolated member of the C. parapsilosis complex and it showed high resistance to fluconazole. A high prevalence of C. orthopsilosis (40.2%) was also noted.

Molecular Genotyping of Candida parapsilosis Species Complex.

BACKGROUND: The Candida parapsilosis complex species has emerged as an important cause of human disease. The molecular identification of C. parapsilosis isolates at the species level can be helpful for epidemiological studies and then for the establishment of appropriate therapies and prophylactic measures. METHODS: The present study was undertaken to analyze 13 short tandem repeat (STR) markers (7 minisatellites and 6 microsatellites) in a global set of 182 C. parapsilosis complex isolates from different origins including invasive and superficial clinical sites. RESULTS: Upon the analysis of 182 strains of C. parapsilosis complex species, 10-17 haplotypes were detected for each minisatellite marker. The combination of 7 minisatellite markers yielded 121 different genotypes with a 0.995 D value. Upon the analysis of 114 isolates (68 from invasive infections and 46 from superficial infections), 21-32 genotypes were detected for each microsatellite marker. The combination of all 13 markers yielded 96 different genotypes among 114 isolates with a high degree of discrimination (0.997 D value). The same multilocus genotype was shared by isolates recovered from some patients and from the hand of theirs correspondent healthcare worker. For another patient, the same multilocus genotype of C. metapsilosis was detected in blood and skin confirming that candidemia usually arises as an endogenous infection following prior colonization. CONCLUSIONS: These STR markers are a valuable tool for the differentiation of C. parapsilosis complex strains, to support epidemiological investigations especially studies of strain relatedness and pathways of transmission.

[Molecular identification and in vitro antifungal susceptibility of blood isolates of the Candida parapsilosis species complex in Venezuela]. / Identificación molecular y sensibilidad a los antifúngicos de aislamientos de sangre del complejo Candida parapsilosis en Venezuela.

BACKGROUND: Candida parapsilosis is a species complex consisting of Candida parapsilosis sensu stricto, Candida orthopsilosis, and Candida metapsilosis. Studies worldwide have described its epidemiology and susceptibility to antifungal agents. AIMS: The aims of this study were to carry out the molecular identification of blood isolates belonging to the Candida parapsilosis species complex, and to determine their in vitro susceptibility to antifungals of systemic use. METHODS: A study of 86 strains of C. parapsilosis species complex collected in 2008-2011 and obtained from the Candidaemia Surveillance Network of Mycology Department of the Rafael Rangel National Institute of Hygiene, was made. Secondary alcohol-dehydrogenase gene amplification was performed using polymerase chain reaction, and the products were analysed by restriction fragments length polymorphisms using the enzyme BanI. Susceptibility tests were performed using Etest®, following the manufacturer's instructions with modifications. RESULTS: Of the 86 isolates studied, 81 (94.2%) were C. parapsilosis sensu stricto, 4 (4.6%) C. orthopsilosis, and one (1.2%) C. metapsilosis. C. parapsilosis isolates were susceptible to amphotericin B and caspofungin, showing low rates of resistance to fluconazole and voriconazole. C. orthopsilosis and C. metapsilosis were susceptible to all the antifungals tested. CONCLUSIONS: The results obtained in Venezuela provide for the first time important information about the distribution of C. parapsilosis species complex in cases of candidaemia, and support the need for continuing surveillance programs, including molecular discrimination of species and antifungal susceptibility tests, which may guide specific therapy.

Caspofungin-Non-Susceptible Candida orthopsilosis Isolated from Onychomycosis in Iran.

BACKGROUND: Although Candida albicans remains the most common fungal isolate from clinical specimens, many studies have detected a shift towards non-albicans Candida species. Despite worrying clinical pictures associated with latter species, there is little information regarding its susceptibility patterns against currently available antifungal agents, with only a small number of strains having been studied. METHODS: We evaluated the in vitro antifungal susceptibilities of clinical isolates of C. orthopsilosis already identified by two-steps PCR-RFLP and reconfirmed by sequence analysis of entire ITS rDNA region, to six antifungal drugs. RESULTS: The resulting MIC50 and MIC90 for all strains (n=18) were in increasing order, as follows: posaconazole (0.016 & 0.063 µg/ml); itraconazole (0.031 & 0.125 µg/ml); amphotericin B (0.5 & 1 µg/ml); fluconazole (0.25 & 0.5 µg/ml) and caspofungin (4 & 8 µg/ml). A uniform pattern of the MIC ranges was seen for amphotericin B, fluconazole, itraconazole, and posaconazole, while a widest range and the highest MICs were observed for caspofungin. CONCLUSION: Although we emphasis on the careful species designation of the clinical isolates of Candida, the antifungal susceptibility patterns of these clinically important organisms may have an application in clinical and epidemiological setting and deserve the implementation of local surveillance programs to monitor.

Comparison of four molecular approaches to identify Candida parapsilosis complex species

Since the description of Candida orthopsilosis and C. metapsilosis in 2005, several methods have been proposed to identify and differentiate these species from C. parapsilosis sensu stricto. Species-specific uniplex polymerase chain reaction (PCR) was performed and compared with sequencing of the D1/D2 region of the LSU 28S rDNA gene, microsatellite typing of C. parapsilosis sensu stricto, and PCR-restriction fragment length polymorphism patterns in the ITS1-5.8S-ITS2 region of the rDNA gene. There was agreement between results of testing of 98 clinical isolates with the four PCR-based methods, with 59 isolates identified as C. parapsilosis sensu stricto, 37 as C. orthopsilosis, and two as C. metapsilosis.