Genetic relatedness among azole-resistant Candida tropicalis clinical strains in Taiwan from 2014 to 2018.

To monitor trends in the distribution of yeast species and the susceptibilities of these species to commonly prescribed antifungal drugs, we conduct the Taiwan Surveillance of Antimicrobial Resistance of Yeasts (TSARY) every 4 years. We found that 25 of 294 Candida tropicalis isolates from TSARY 2014 and 31 of 314 C. tropicalis isolates from TSARY 2018 were resistant to fluconazole. We determined the genetic relatedness among fluconazole-resistant C. tropicalis isolates by multilocus sequence typing (MLST). Among 174 C. tropicalis isolates, including all 56 fluconazole-resistant, all 26 susceptible-dose dependent and 92 selected fluconazole-susceptible isolates, 59 diploid sequence types (DSTs) were identified. We found that 22 of the 25 fluconazole-resistant C. tropicalis from TSARY 2014 and 29 of the 31 fluconazole-resistant C. tropicalis from TSARY 2018 were genetically related and belonged to the same cluster (clade 4). A combination of mutation and overexpression of ERG11, encoding the target of azole drugs, was the major mechanism contributing to drug resistance. Approximately two-thirds of reviewed patients infected or colonised by fluconazole-resistant C. tropicalis were azole-naïve. Furthermore, there was no evidence of patient-to-patient transmission. Because the clade 4 fluconazole-resistant C. tropicalis strain persists in Taiwan, it is important to identify the source of azole-resistant C. tropicalis to prevent the spread of this resistant strain.

Mixed yeast infections in Taiwan.

Clinically significant yeast isolates were collected via Taiwan Surveillance of Antimicrobial Resistance of Yeasts (TSARY) in 2014, and mixed infections were investigated. Among 44 out of 1092 specimens containing multiple species, 17, 11, 5, 3, and 8 were from urine, sputum, blood, ascites, and 6 others, respectively. There predominant combinations of mixed infection were 14 Candida albicans/Candida glabrata, 13 C. albicans/Candida tropicalis, and 9 C. glabrata/C. tropicalis. Furthermore, we also detected fluconazole resistant isolates Candida norvegensis and Candida krusei. Hence, it is important to accurately identify the species with different drug susceptibilities when they are in the same specimen.

The survey of tinea capitis and scalp dermatophyte carriage in nursing home residents.

Tinea capitis is a contagious dermatophyte infection of scalp and associated hairs. On the other hand, asymptomatic carriage is a status of positive dermatophyte scalp culture, but without signs or symptoms of tinea capitis, and no evidence of hair shaft invasion confirmed by direct microscopy. Tinea capitis and asymptomatic carriage mostly occur in children, but adult females are becoming another population in recent decades. In this study, we focused on the prevalence and related fungi of tinea capitis and asymptomatic carriage in elderly by the shampoo brush method, as well as the source of transmission, in 10 nursing home residents. Two hundred and thirteen residents were screened, and 186 isolates were identified, of which only three were dermatophytes (1.4%). The scalp dermatophyte isolates were identified as Trichophyton rubrum by morphological characters and sequences comparisons in all three cases. After revisiting, these cases were proved to be asymptomatic carriers by negative microscopic and culture examination; however, two cases were found to have concurrent tinea pedis and onychomycosis, which were identified as T. rubrum and Trichophyton interdigitale. The source of the T. rubrum scalp carriage may come from tinea elsewhere on the body of the same subject or from other people in the same institute. Finding and treating the source of carriage, as well as treating scalp carriage patients according to the colony counts, may help prevent disease spreading.

Prevalence of and risk factors for methicillin-resistant Staphylococcus aureus colonization among human immunodeficient virus-infected outpatients in Taiwan: oral Candida colonization as a comparator.

Human immuodeficency virus (HIV)-infected patients receiving highly active antiretroviral therapy (HAART) and community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) have increased in recent years in Taiwan. This study was undertaken to determine the prevalence of and risk factors for nasal and oral S. aureus and MRSA colonization among contemporary HIV-infected populations. Clinical variables for S. aureus and MRSA colonization among HIV-infected outpatients from three hospitals were analyzed and compared with those for oral Candida colonization. Genetic characteristics of MRSA isolates were analyzed. A total of 714 patients were screened for nasal S. aureus colonization, and a subset of 457 patients were also screened for oral S. aureus colonization. Of all patients, 79.4% were receiving HAART, and their mean CD4 count was 472 cells/mm3. The colonization rates in the oral cavity, nasal cavity, and at either site were 18.8%, 31.7%, and 36.8%, respectively, for S. aureus, and 3.1%, 4.4%, and 5.5%, respectively, for MRSA. These rates were all much lower than the previously reported rate of oral Candida colonization (52.4%). By multivariate analysis, a suppressed viral load (<200 copies/mL) protected against oral S. aureus, MRSA, and Candida colonization, and recent use of antibacterial agents protected against oral and nasal S. aureus colonization. Recent incarceration increased the risk of nasal MRSA colonization, while recent hospitalization, tuberculosis, older age, and intravenous drug use increased the risk of oral Candida colonization. Candida spp. did not augment S. aureus or MRSA colonization in the oral cavity. Most of the 41 MRSA isolates recovered belonged to the SCCmec IV/pvl-negative (51.2%) and VT/pvl-positive (26.8%) ST59 local prevalent CA-MRSA clones. Distinct carriage rates demonstrated here suggested that mucosal immunity against colonization might differ in terms of microbes and sites. A decreased risk in oral carriage of MRSA and Candida might be a benefit of HAART.

Fruits as the vehicle of drug resistant pathogenic yeasts.

OBJECTIVE: We investigated the diversity and drug susceptibility of pathogenic yeasts on fruit surfaces. METHOD: Fruits were purchased from supermarkets and washed with buffer. The pellets were re-suspended in medium after centrifugation. The cell suspensions were plated onto CHROMagar Candida medium. Yeasts were identified by ribosomal DNA sequencing and their drug susceptibilities were determined by broth microdilution assay. RESULTS: Of 184 isolates, comprised of 55 species, from 22 different types of fruits, 29 species, including Candida famata, Candida fermentati, Candida guilliermondii, Candida intermedia, Candida krusei, Candida orthopsilosis, Candida parapsilosis, Candida pelliculosa, Candida tropicalis, and others have been reported to cause diseases in humans. In addition to C. krusei, intrinsically resistant to fluconazole, all Rhodotorula and Rhodosporidium species were resistant to fluconazole. One each of C. tropicalis isolate was belonged to diploid sequence type (DST)149 and DST225, genotypes also detected in isolates from humans. Furthermore, the DST225 isolate was less susceptible to azole drugs. The susceptibilities to azole drugs for clinical and agricultural usage were associated to each other. CONCLUSION: It is important to be aware of the existence of pathogenic yeasts, especially drug-resistant ones, on the fruit surfaces, a potential route for pathogenic yeasts to be transmitted to humans.

The distribution and drug susceptibilities of clinical Candida species in TSARY 2014.

The species distribution and drug susceptibilities of 1106 Candida isolates collected in Taiwan Surveillance of Antimicrobial Resistance of Yeasts (TSARY) in 2014 were determined. Candida albicans is still the dominant species, accounting for 35.9%, followed by 28.3% C. glabrata, 26.6% C. tropicalis, 5.2% C. parapsilosis, 1.0% C. krusei, and 3.0% of 13 other species. Interestingly, the prevalence of candidemia caused by C. glabrata in the present study is significantly higher than that in previous three surveys (39/220 vs. 54/471, P=0.025). We found that 31 (2.8%), 24 (2.2%), 1 (0.09%), and 0 isolates were resistant to fluconazole, voriconazole, anidulafungin, and amphotericin B, respectively. There is a significant increase in fluconazole (P=0.00002) and voriconazole (P=0.00006) resistant rates when compared to the isolates collected in 2010. Importantly, all the 24 voriconazole resistant isolates identified were also resistant to fluconazole. Hence, cross-resistance among azole-type drugs is an emerging issue for managing fungal infections.

Zebrafish Egg Infection Model for Studying Candida albicans Adhesion Factors.

Disseminated candidiasis is associated with 30-40% mortality in severely immunocompromised patients. Among the causal agents, Candida albicans is the dominant one. Various animal models have been developed for investigating gene functions in C. albicans. Zebrafish injection models have increasingly been applied in elucidating C. albicans pathogenesis because of the conserved immunity, prolific fecundity of the zebrafish and the low costs of care systems. In this study, we established a simple, noninvasive zebrafish egg bath infection model, defined its optimal conditions, and evaluated the model with various C. albicans mutant strains. The deletion of SAP6 did not have significant effect on the virulence. By contrast, the deletion of BCR1, CPH1, EFG1, or TEC1 significantly reduced the virulence under current conditions. Furthermore, all embryos survived when co-incubated with bcr1/bcr1, cph1/cph1 efg1/efg1, efg1/efg1, or tec1/tec1 mutant cells. The results indicated that our novel zebrafish model is time-saving and cost effective.

Cph1p negatively regulates MDR1 involved in drug resistance in Candida albicans.

The cph1/cph1 efg1/efg1 double mutant in Candida albicans is defective in filamentous growth and is avirulent in a mouse model. We previously reported that Efg1p but not Cph1p is involved in drug resistance by negatively regulating ERG3 in C. albicans. In the current study, we have found that overexpression of CPH1 in Saccharomyces cerevisiae increases susceptibility to the antifungal drug fluconazole. Furthermore, in C. albicans, null mutation of CPH1 increased the expression of MDR1 as well as decreased susceptibility to fluconazole and voriconazole but not to amphotericin B. These findings indicate that although Efg1p and Cph1p may have the same effects on virulence, they have opposite effects on drug resistance in C. albicans.

Ndt80p is involved in L-sorbose utilization through regulating SOU1 in Candida albicans.

Ndt80p, a known transcriptional factor, regulates various targets involved in stress responses, filamentous growth, and virulence in Candida albicans. Potential targets of Ndt80p have been identified at the transcriptional level. The present study was conducted to identify genes regulated by Ndt80p from the protein level. We found that the levels of Ahp1p, Fma1p, Hsp21p, Rfa2p, Snz1p, Sod1p, Sou1p, Trp99p, orf19.251, orf19.1862, and orf19.5620, were affected by the null mutation of NDT80 by two-dimensional polyacrylamide gel-electrophoresis analysis. Among the 11 proteins, all but Sou1p and Rfa2p are suggested to be involved in known functions of Ndt80p. Here, we demonstrate that Ndt80p plays a role in l-sorbose utilization through regulating SOU1 in C. albicans.

Yeast oropharyngeal colonization in human immunodeficiency virus-infected patients in central taiwan.

A prospective, cross-sectional study was conducted at a medical center in central Taiwan to understand the prevalence, associated factors, and microbiologic features for oropharyngeal yeast colonization in human immunodeficiency virus-infected outpatients. Oral yeast colonization was detected in 127 (45 %) patients, including 21 (16.5 %) colonized by more than one species. Of the 154 isolates, Candida albicans was the most common species (114, 74 %), followed by Candida dubliniensis (10, 6.5 %), Candida glabrata (10, 6.5 %), Candida tropicalis (7, 4.5 %), and 13 others. We found that receiving antituberculous drug (p = 0.046) or atazanavir (p = 0.045) was two predictors for patients colonized by non-C. albicans species (p = 0.005) and risking mixed yeast colonization (p = 0.009). Even though our data showed that clinical antifungal drugs remained effective in vitro against the colonizing yeasts, the increased mixed yeast colonization indicates a potential issue for controlling mixed infections in hospital settings.

An emerging issue of mixed yeast cultures.

BACKGROUND: Different yeast species have different susceptibilities to commonly prescribed antifungal drugs. Thus, it is important to accurately determine the species of pathogenic yeasts, especially when more than one species are in a specimen. METHODS: Clinically significant yeast isolates were collected via the Taiwan Surveillance of Antimicrobial Resistance of Yeasts from July to September in 2010. The identifications of isolates were assessed in the core laboratory at the National Health Research Institutes. RESULTS: Of the 1127 isolates recovered, 1088 were of Candida genus, accounting for 96.53% of the total isolates, followed by Cryptococcus (15, 1.33%), Trichosporon (12, 1.06%), Kodamaea (4, 0.35%), Pichia (4, 0.35%), and three others. In all, 38 out of 1116 (3.4%) specimens had mixed yeast cultures. One ascites specimen had three species, Candida albicans, Candida glabrata, and Candida tropicalis. In the remaining 37 specimens, 16 had a combination of C. albicans and C. glabrata, eight C. albicans and C. tropicalis, five C. glabrata and C. tropicalis, three Candida krusei and C. tropicalis, and five with different combinations. CONCLUSION: The high prevalence of cultures with mixed yeasts may be an emerging issue. Thus, to determine mixed yeast cultures in the same specimen, we highly recommend CHROMagar Candida medium to culture yeast isolates directly from the specimen.

Candida albicans ENO1 null mutants exhibit altered drug susceptibility, hyphal formation, and virulence.

We previously showed that the expression of ENO1 (enolase) in the fungal pathogen Candida albicans is critical for cell growth. In this study, we investigate the contribution of the ENO1 gene to virulence. We conducted our functional study of ENO1 in C. albicans by constructing an eno1/eno1 null mutant strain in which both ENO1 alleles in the genome were knockouted with the SAT1 flipper cassette that contains the nourseothricin-resistance marker. Although the null mutant failed to grow on synthetic media containing glucose, it was capable of growth on media containing yeast extract, peptone, and non-fermentable carbon sources. The null mutant was more susceptible to certain antifungal drugs. It also exhibited defective hyphal formation, and was avirulent in BALB/c mice.

Distribution and drug susceptibilities of Candida species causing candidemia from a medical center in central Taiwan.

Invasive fungal infections have increased significantly in the past few decades because of the increase in high-risk populations. To investigate the distribution and drug susceptibilities of such infections, we analyzed all 152 Candida isolates causing candidemia from 2004 to 2006 at the China Medical University Hospital, a medical center in central Taiwan. Candida albicans was the most common species, accounting for 52.6% of the isolates, followed by C. tropicalis (19.7%), C. parapsilosis (14.5%), C. glabrata (8.6%), C. guilliermondii (3.9%), and C. pelliculosa (0.7%). All isolates were susceptible to amphotericin B, anidulafungin, micafungin, and voriconazole according to minimum inhibitory concentrations (MICs) after a 24-h incubation; 0.7%, 6.6%, and 7.9% of isolates were resistant to amphotericin B, fluconazole, and voriconazole, respectively, after 48-h incubation. Both C. albicans and C. parapsilosis had high degrees of agreement for azoles between 24- and 48-h incubation periods, whereas C. glabrata (38.5-46.2%) and C. tropicalis (56.7-63.3%) did not. The majority of the isolates with high azole MICs displayed a trailing growth phenotype. Hence, the MICs of different drugs after 24-h incubation may be considered for prognosis of candidemia.

Genotype analysis based on intergenic spacer 1 sequences of Trichosporon asahii collected in Taiwan.

Among 32 Trichosporon asahii isolates collected in four rounds of the Taiwan Surveillance of Antimicrobial Resistance of Yeasts (TSARY) studies, conducted in 1999, 2002, 2006, and 2010, five different intergenic spacer 1 (IGS1) genotypes were detected. Genotype 1 was the most common (43.8%), followed by genotypes 3 (28.1%), 7 (12.5%), 5 (9.4%), and 4 (6.3%). Interestingly, genotype 7 was more prevalent in Taiwan than in other areas (P = 0.01); while we did not find a significant association between IGS1 genotype and susceptibility to antifungal drugs, we did note that the majority of isolates of T. asahii were susceptible to both fluconazole and voriconazole, consistent with previous reports. A higher proportion of isolates (P = 0.05) collected in 2010 (4/12, 33.3%) had high amphotericin B MICs (≥ 2 mg/l) than those collected in the previous three TSARYs (1/21, 5%). Hence, the new data of genotypes and drug susceptibilities in the present study may contribute to the epidemiology of T. asahii.

Species distribution and drug susceptibilities of Candida isolates in TSARY 2010.

Susceptibilities to antifungal drugs of 1083 Candida isolates collected in Taiwan Surveillance of Antimicrobial Resistance of Yeasts in 2010 were determined. There were 422 (39%) C. albicans, 270 (24.9%) C. tropicalis, 258 (23.8%) C. glabrata, 87 (8%) C. parapsilosis, 18 (1.7%) C. krusei, and 28 (2.6%) of 13 other species. In the present study, we have applied species-specific clinical breakpoints for common species and epidemiological cutoff values for rare species. We found that majority of isolates were susceptible to tested drugs. A total of 15, 3, 2, and 0 isolates were not susceptible to fluconazole, voriconazole, amphotericin B, and anidulafungin, respectively. We found that three of the four fluconazole non-susceptible C. albicans isolates were resistant to voriconazole. Hence, there is an issue of cross-resistance among azole-type drugs.

Predisposing factors for oropharyngeal colonization of yeasts in human immunodeficiency virus-infected patients: a prospective cross-sectional study.

BACKGROUND: Oropharyngeal candidiasis continues to be a major opportunistic infection in human immunodeficiency virus (HIV)-infected patients. The objectives of this study were to investigate the prevalence, associated factors, and microbiologic features for oropharyngeal yeast colonization in HIV-infected patients. METHODS: From October to December 2009, consecutive HIV-infected patients older than 18 years were recruited in this study. Demographic information, underlying conditions, and clinical histories were collected. Oropharyngeal swab cultures for yeasts and antifungal drug susceptibilities of the isolates were performed. RESULTS: Of the 105 HIV-infected patients, 54 (51.4%) were colonized with yeasts, including 11 patients (20.4%) with more than one species. Among the 68 isolates, Candida albicans accounted for 73.5%, followed by Candida tropicalis (5.9%), Candida glabrata (5.9%), and Candida dubliniensis (4.4%). There were 7.5% and 6% Candida isolates resistant to fluconazole and voriconazole, respectively. All of the Candida isolates were susceptible to amphotericin B. A higher prevalence of yeast colonization was noted in patients with a CD4 cell count ≤200 cells/µL (p = 0.032). Multivariate regression analysis showed that intravenous drug use was an independent associated factor for oropharyngeal yeast colonization (odds ratio, 5.35; 95% confidence interval, 1.39-20.6; p = 0.015), as well as protease inhibitor-containing antiretroviral therapy (odds ratio, 3.59; 95% confidence interval, 1.41-9.12; p = 0.007). CONCLUSION: Despite previous studies showing that protease inhibitors decreased Candida adhesion to epithelial cells in vitro, the current study found protease inhibitor-containing antiretroviral therapy predisposed to oropharyngeal yeast colonization in HIV-infected patients.

Comparison of human and soil Candida tropicalis isolates with reduced susceptibility to fluconazole.

Infections caused by treatment-resistant non-albicans Candida species, such as C. tropicalis, has increased, which is an emerging challenge in the management of fungal infections. Genetically related diploid sequence type (DST) strains of C. tropicalis exhibiting reduced susceptibility to fluconazole circulated widely in Taiwan. To identify the potential source of these wildly distributed DST strains, we investigated the possibility of the presence in soil of such C. tropicalis strains by pulsed field gel electrophoresis (PFGE) and DST typing methods. A total of 56 C. tropicalis isolates were recovered from 26 out of 477 soil samples. Among the 18 isolates with reduced susceptibility to fluconazole, 9 belonged to DST149 and 3 belonged to DST140. Both DSTs have been recovered from our previous studies on clinical isolates from the Taiwan Surveillance of Antimicrobial Resistance of Yeasts (TSARY) program. Furthermore, these isolates were more resistant to agricultural azoles. We have found genetically related C. tropicalis exhibiting reduced susceptibility to fluconazole from the human hosts and environmental samples. Therefore, to prevent patients from acquiring C. tropicalis with reduced susceptibility to azoles, prudent use of azoles in both clinical and agricultural settings is advocated.

Susceptibilities to amphotericin B, fluconazole and voriconazole of Trichosporon clinical isolates.

A total of 35 Trichosporon isolates were collected from the Taiwan Surveillance of Antimicrobial Resistance of Yeasts (TSARY) project from 1999 to 2006, and their identifications as well as drug susceptibilities were determined. The most frequently isolated species was T. asahii (62.9%), and the most common clinical sample that yielded Trichosporon isolates was urine (37.1%). The etiology of all seven invasive trichosporonosis was T. asahii. For the 22 T. asahii isolates, the MIC(50) and MIC(90) for amphotericin B were 0.25 and 1 µg/mL, respectively. Those for fluconazole were 2 and 4 µg/mL, respectively, and for voriconazole 0.031 and 0.063 µg/mL, respectively. When the intraclass correlation coefficients (ICCs) and agreements were calculated, we found that the MICs of fluconazole obtained from different methods were similar and the inter-method discrepancies were low. Nevertheless, no unanimous MIC of amphotericin B and voriconazole was obtained among different methods.

R432 is a key residue for the multiple functions of Ndt80p in Candida albicans.

Ndt80p is an important transcription modulator to various stress-response genes in Candida albicans, the most common human fungal pathogen in systemic infections. We found that Ndt80p directly regulated its target genes, such as YHB1, via the mid-sporulation element (MSE). Furthermore, the ndt80(R432A) allele, with a reduced capability to bind MSE, failed to complement the defects caused by null mutations of NDT80. Thus, the R432 residue in the Ndt80p DNA-binding domain is involved in all tested functions, including cell separation, drug resistance, nitric oxide inactivation, germ tube formation, hyphal growth, and virulence. Hence, the importance of the R432 residue suggests a novel approach for designing new antifungal drugs by blocking the interaction between Ndt80p and its targets.

Characterization of Candida species from different populations in Taiwan.

The opportunistic Candida species existing as part of commensal microbiota in humans are usually the etiological agents causing infections. We investigated whether isolates collected from different age groups, hospital units, and sources have distinct characteristics. A total of 913 isolates comprising 395 Candida albicans, 230 Candida tropicalis, 202 Candida glabrata, 62 Candida parapsilosis, 13 Candida krusei, and 11 of other six species were analyzed. Urine was the most common source (41.2%), followed by sputum (16.3%), blood (15.2%), and others (27.3%). Candida albicans and C. parapsilosis were more prevalent in the working group [from 19 to 65 years], whereas C. tropicalis and C. glabrata were more prevalent in the elder one (≥ 66 years). We found that the age of patients and the source of isolates affect the distribution of species. On the other hand, the drug susceptibility of isolates was associated with fungal species and whether patients were hospitalized.