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ABSTRACTCandida parapsilosis is known to cause severe and persistent outbreaks in clinical settings. Patients infected with multidrug-resistant C. parapsilosis (MDR Cp) isolates were identified in a large Turkish hospital from 2017–2020. We subsequently identified three additional patients infected with MDR Cp isolates in 2022 from the same hospital and two echinocandin-resistant (ECR) isolates from a single patient in another hospital. The increasing number of MDR and ECR isolates contradicts the general principle that the severe fitness cost associated with these phenotypes could prevent their dominance in clinical settings. Here, we employed a multidimensional approach to systematically assess the fitness costs of MDR and ECR C. parapsilosis isolates. Whole-genome sequencing revealed a novel MDR genotype infecting two patients in 2022. Despite severe in vitro defects, the levels and tolerances of the biofilms of our ECR and MDR isolates were generally comparable to those of susceptible wild-type isolates. Surprisingly, the MDR and ECR isolates showed major alterations in their cell wall components, and some of the MDR isolates consistently displayed increased tolerance to the fungicidal activities of primary human neutrophils and were more immunoevasive during exposure to primary human macrophages. Our systemic infection mouse model showed that MDR and ECR C. parapsilosis isolates had comparable fungal burden in most organs relative to susceptible isolates. Overall, we observed a notable increase in the genotypic diversity and frequency of MDR isolates and identified MDR and ECR isolates potentially capable of causing persistent outbreaks in the future.

Although rare, trans-kingdom infection features an interesting infection biology concept, in which highly versatile pathogenic attributes allow successful infections in evolutionarily highly divergent species. Corynespora cassiicola is a phytopathogenic fungus and occasionally causes human infections. Herein, we report a phaeohyphomycosis case caused by C. cassiicola. Given that sporadic reports may contribute to a lack of awareness of the transmission route, clinical manifestations, and diagnostic and clinical management, we systematically reviewed the cases reported thus far. Nine patients were identified and included in the pooled analysis, 88.9% (8/9) of whom were reported after 2010. All patients were from Asian, African, and Latin American countries, among whom 77.8% (7/9) were farmers or lived in areas with active agriculture. Exposed body parts were the major affected infection area, and clinical manifestations were mainly non-specific inflammatory reactions. Although biochemical and morphological examinations confirmed the presence of fungal infection, molecular analysis was used for the final diagnosis, with 77.8% (7/9) being identified by internal transcribed spacer sequencing. Whereas voriconazole, terbinafine, and AmB, either alone or in combination, resulted in successful infection resolution in most cases (5/9; 55.5%), those suffering from invasive facial infections and CARD9 deficiency showed poor outcomes. Our patient is the third case of invasive facial infection caused by C. cassiicola and was successfully treated with intravenous LAmB followed by oral voriconazole combined with topical antifungal irrigation. Molecular identification of fungus and prompt antifungal treatment is pivotal in the clinical success of patients suspected to have phaeohyphomycosis. Moreover, as evidenced by our data, itraconazole treatment is not recommended.

ABSTRACTRecent epidemiological studies documented an alarming increase in the prevalence of echinocandin-resistant (ECR) Candida glabrata blood isolates. ECR isolates are known to arise from a minor subpopulation of a clonal population, termed echinocandin persisters. Although it is believed that isolates with a higher echinocandin persistence (ECP) are more likely to develop ECR, the implication of ECP needs to be better understood. Moreover, replacing laborious and time-consuming traditional approaches to determine ECP levels with rapid, convenient, and reliable tools is imperative to advance our understanding of this emerging concept in clinical practice. Herein, using extensive ex vivo and in vivo systemic infection models, we showed that high ECP isolates are less effectively cleared by micafungin treatment and exclusively give rise to ECR colonies. Additionally, we developed a flow cytometry-based tool that takes advantage of a SYTOX-based assay for the stratification of ECP levels. Once challenged with various collections of echinocandin-susceptible blood isolates, our assay reliably differentiated ECP levels in vitro and predicted ECP levels in real time under ex vivo and in vivo conditions when compared to traditional methods relying on colony-forming unit counting. Given the high and low ECP predictive values of 92.3% and 82.3%, respectively, our assay showed a high agreement with traditional approach. Collectively, our study supports the concept of ECP level determination in clinical settings and provides a robust tool scalable for high-throughput settings. Application of this tool facilitates the interrogation of mutant and drug libraries to further our understanding of persister biology and designing anti-persister therapeutics.IMPORTANCECandida glabrata is a prevalent fungal pathogen able to replicate inside macrophages and rapidly develop resistance against frontline antifungal echinocandins. Multiple studies have shown that echinocandin resistance is fueled by the survival of a small subpopulation of susceptible cells surviving lethal concentrations of echinocandins. Importantly, bacterial pathogens that exhibit high antibiotic persistence also impose a high burden and generate more antibiotic-resistant colonies. Nonetheless, the implications of echinocandin persistence (ECP) among the clinical isolates of C. glabrata have not been defined. Additionally, ECP level determination relies on a laborious and time-consuming method, which is prone to high variation. By exploiting in vivo systemic infection and ex vivo models, we showed that C. glabrata isolates with a higher ECP are associated with a higher burden and more likely develop echinocandin resistance upon micafungin treatment. Additionally, we developed an assay that reliably determines ECP levels in real time. Therefore, our study identified C. glabrata isolates displaying high ECP levels as important entities and provided a reliable and convenient tool for measuring echinocandin persistence, which is extendable to other fungal and bacterial pathogens.

ABSTRACT Small colony variants are relatively common among some bacterial species and are associated with poor prognosis and recalcitrant infections. Similarly, Candida glabrata—a major intracellular fungal pathogen—produces small and slow-growing respiratory-deficient colonies, termed “petite.” Despite reports of clinical petite C. glabrata strains, our understanding of petite behavior in the host remains obscure. Moreover, controversies exist regarding in-host petite fitness and its clinical relevance. Herein, we employed whole-genome sequencing (WGS), dual-RNAseq, and extensive ex vivo and in vivo studies to fill this knowledge gap. WGS identified multiple petite-specific mutations in nuclear and mitochondrially encoded genes. Consistent with dual-RNAseq data, petite C. glabrata cells did not replicate inside host macrophages and were outcompeted by their non-petite parents in macrophages and in gut colonization and systemic infection mouse models. The intracellular petites showed hallmarks of drug tolerance and were relatively insensitive to the fungicidal activity of echinocandin drugs. Petite-infected macrophages exhibited a pro-inflammatory and type I IFN-skewed transcriptional program. Interrogation of international C. glabrata blood isolates (n = 1000) showed that petite prevalence varies by country, albeit at an overall low prevalence (0%–3.5%). Collectively, our study sheds new light on the genetic basis, drug susceptibility, clinical prevalence, and host-pathogen responses of a clinically overlooked phenotype in a major fungal pathogen. Importance Candida glabrata is a major fungal pathogen, which is able to lose mitochondria and form small and slow-growing colonies, called “petite.” This attenuated growth rate has created controversies and questioned the clinical importance of petiteness. Herein, we have employed multiple omics technologies and in vivo mouse models to critically assess the clinical importance of petite phenotype. Our WGS identifies multiple genes potentially underpinning petite phenotype. Interestingly, petite C. glabrata cells engulfed by macrophages are dormant and, therefore, are not killed by the frontline antifungal drugs. Interestingly, macrophages infected with petite cells mount distinct transcriptomic responses. Consistent with our ex vivo observations, mitochondrial-proficient parental strains outcompete petites during systemic and gut colonization. Retrospective examination of C. glabrata isolates identified petite prevalence a rare entity, which can significantly vary from country to country. Collectively, our study overcomes the existing controversies and provides novel insights regarding the clinical relevance of petite C. glabrata isolates.

Arastehfar et al. show that macrophage-engulfed cells of fungal pathogen Candida glabrata exhibit multidrug tolerance and increased survival in the presence of cidal antifungal drugs, forming a reservoir from which drug-resistant mutants emerge

Patients presenting with severe COVID-19 are predisposed to acquire secondary fungal infections such as COVID-19-associated candidemia (CAC), which are associated with poor clinical outcomes despite antifungal treatment. The extreme burden imposed on clinical facilities during the COVID-19 pandemic has provided a permissive environment for the emergence of clonal outbreaks of multiple Candida species, including C. auris and C. parapsilosis. Here we report the largest clonal CAC outbreak to date caused by fluconazole resistant (FLZR) and echinocandin tolerant (ECT) C. parapsilosis. Sixty C. parapsilosis strains were obtained from 57 patients at a tertiary care hospital in Brazil, 90% of them were FLZR and ECT. Although only 35.8% of FLZR isolates contained an ERG11 mutation, all of them contained the TAC1L518F mutation and significantly overexpressed CDR1. Introduction of TAC1L518F into a susceptible background increased the MIC of fluconazole and voriconazole 8-fold and resulted in significant basal overexpression of CDR1. Additionally, FLZR isolates exclusively harboured E1939G outside of Fks1 hotspot-2, which did not confer echinocandin resistance, but significantly increased ECT. Multilocus microsatellite typing showed that 51/60 (85%) of the FLZR isolates belonged to the same cluster, while the susceptible isolates each represented a distinct lineage. Finally, biofilm production in FLZR isolates was significantly lower than in susceptible counterparts Suggesting that it may not be an outbreak determinant. In summary, we show that TAC1L518F and FKS1E1393G confer FLZR and ECT, respectively, in CAC-associated C. parapsilosis. Our study underscores the importance of antifungal stewardship and effective infection control strategies to mitigate clonal C. parapsilosis outbreaks.

Fluconazole-resistant Candida parapsilosis (FLZR-CP) outbreaks are a growing public health concern and have been reported in numerous countries. Patients infected with FLZR-CP isolates show fluconazole therapeutic failure and have a significantly increased mortality rate. Because fluconazole is the most widely used antifungal agent in most regions with outbreaks, it is paramount to restore its antifungal activity. Milbemycin oxim (MOX), a well-known canine endectocide, is a potent efflux pump inhibitor that significantly potentiates the activity of fluconazole against FLZR C. glabrata and C. albicans. However, the FLZ-MOX combination has not been tested against FLZR-CP isolates, nor is it known whether MOX may also potentiate the activity of echinocandins, a different class of antifungal drugs. Furthermore, the extent of involvement of efflux pumps CDR1 and MDR1 and ergosterol biosynthesis enzyme ERG11 and their link with gain-of-function (GOF) mutations in their transcription regulators (TAC1, MRR1, and UPC2) are poorly characterized among FLZR-CP isolates. We analyzed 25 C. parapsilosis isolates collected from outbreaks in Turkey and Brazil by determining the expression levels of CDR1, MDR1, and ERG11, examining the presence of potential GOF mutations in their transcriptional regulators, and assessing the antifungal activity of FLZ-MOX and micafungin-MOX against FLZR and multidrug-resistant (MDR) C. parapsilosis isolates. ERG11 was found to be universally induced by fluconazole in all isolates, while expression of MDR1 was unchanged. Whereas mutations in MRR1 and UPC2 were not detected, CDR1 was overexpressed in three Brazilian FLZR-CP isolates, which also carried a novel TAC1L518F mutation. Of these three isolates, one showed increased basal expression of CDR1, while the other two overexpressed CDR1 only in the presence of fluconazole. Interestingly, MOX showed promising antifungal activity against FLZR isolates, reducing the FLZ MIC 8- to 32-fold. However, the MOX and micafungin combination did not exert activity against an MDR C. parapsilosis isolate. Collectively, our study documents that the mechanisms underpinning FLZR are region specific, where ERG11 mutations were the sole mechanism of FLZR in Turkish FLZR-CP isolates, while simultaneous overexpression of CDR1 was observed in some Brazilian counterparts. Moreover, MOX and fluconazole showed potent synergistic activity, while the MOX-micafungin combination showed no synergy.

Abstract Background The clinical profiles and outcomes of cryptococcal meningitis have been shown to vary depending on the underlying condition. The aim of this study was to investigate clinical characteristics and outcomes in patients with and without type II diabetes mellitus. Methods A retrospective study was performed. Clinical data of HIV-negative cryptococcal meningitis patients with type II diabetes mellitus (n = 26) and without type II diabetes mellitus (n = 52) referring to the Jiangxi Chest Hospital between January 2012 to December 2018 were analyzed. The data were analyzed using chi square, none-parametric tests, and logistic regression. P-values

Abstract Background Despite being associated with a high mortality and economic burden, data regarding candidemia are scant in Algeria. The aim of this study was to unveil the epidemiology of candidemia in Algeria, evaluate the antifungal susceptibility pattern of causative agents and understand the molecular mechanisms of antifungal resistance where applicable. Furthermore, by performing environmental screening and microsatellite typing we sought to identify the source of infection. Methods We performed a retrospective epidemiological-based surveillance study and collected available blood yeast isolates recovered from the seven hospitals in Algiers. To identify the source of infection, we performed environmental screening from the hands of healthcare workers (HCWs) and high touch areas. Species identification was performed by API Auxa-Color and MALDI-TOF MS and ITS sequencing was performed for species not reliably identified by MALDI-TOF MS. Antifungal susceptibility testing followed CLSI M27-A3/S4 and included all blood and environmental yeast isolates. ERG11 sequencing was performed for azole-resistant Candida isolates. Microsatellite typing was performed for blood and environmental Candida species, where applicable. Results Candida tropicalis (19/66) was the main cause of candidemia in these seven hospitals, followed by Candida parapsilosis (18/66), Candida albicans (18/66), and Candida glabrata (7/66). The overall mortality rate was 68.6% (35/51) and was 81.2% for C. tropicalis-infected patients (13/16). Fluconazole was the main antifungal drug used (12/51); 41% of the patients (21/51) did not receive any systemic treatment. Candida parapsilosis was isolated mainly from the hands of HCWs (7/28), and various yeasts were collected from high-touch areas (11/47), including Naganishia albida, C. parapsilosis and C. glabrata. Typing data revealed interhospital transmission on two occasions for C. parapsilosis and C. glabrata, and the same clone of C. parapsilosis infected two patients within the same hospital. Resistance was only noted for C. tropicalis against azoles (6/19) and fluconazole-resistant C. tropicalis isolates (≥8 μg/ml) (6/19) contained a novel P56S (5/6) amino acid substitution and a previously reported one (V234F; 1/6) in Erg11p. Conclusions Collectively, our data suggest an urgent need for antifungal stewardship and infection control strategies to improve the clinical outcome of Algerian patients with candidemia. The high prevalence of C. tropicalis joined by fluconazole-resistance may hamper the therapeutic efficacy of fluconazole, the frontline antifungal drug used in Algeria.

Wickerhamomyces anomalus is an emerging pathogen, which has been associated with clonal outbreaks and poor clinical outcomes. Despite being an important emerging yeasts species, our understanding concerning the microbiological and clinical characteristics of infections due to this species is limited. Herein, we are reporting a retrospective analysis of fungemia patients with W. anomalus from a 2,100-bed hospital in Shanghai during 2014–2016. Moreover, we conducted an extensive literature review to gain a deeper clinical and microbiological insights. Detailed clinical data were recorded. Antifungal susceptibility testing (AFST) followed CLSI M27-A3, and isolates were identified using MALDI-TOF MS. In total, 13 patients were identified with a mortality rate of 38.5% (5/13). Central venous catheter (CVC), broad-spectrum antibiotic therapy, total parenteral nutrition (TPN), surgery, and mechanical ventilation were the most frequently observed risk factors. Eight patients (61.5%) experienced mixed bacterial/Candida bloodstream infections, and four patients developed mixed candidemia (MC). W. anomalus isolates showed high minimum inhibitory concentrations (MICs) against all azoles tested and flucytosine, while AMB showed the highest in vitro activity. Azoles were used for 84.6% (11/13) of the cases, while 36.4% (4/11) of them died. When combining with the AFST data and the literature review, our study highlights the poor efficacy of azoles and optimal efficacy of AMB and LAMB against infections caused by W. anomalus. In conclusion, our study highlights the emerging threat of W. anomalus affecting both neonates and adults. Furthermore, our results advocate the use of AMB formulations rather than azoles among patients infected with W. anomalus. Future studies are warranted to reach a definitive consensus regarding the utility of echinocandins among such patients.

Background: Recent studies from multiple countries have shown a high prevalence of coronavirus disease 2019 (COVID-19)-associated pulmonary aspergillosis (CAPA) among severely ill patients. Despite providing valuable insight into the clinical management of CAPA, large-scale prospective studies are limited. Here, we report on one of the largest multicenter epidemiological studies to explore the clinical features and prevalence of COVID-19-associated pulmonary mold infections (CAPMIs) among mechanically ventilated patients.Methods: Bronchoalveolar lavage (BAL) and serum samples were collected for culture, galactomannan (GM), and β-D-glucan (BDG) testing. Patients were classified as probable CAPMI based on the presence of host factors, radiological findings, and mycological criteria.Results: During the study period, 302 COVID-19 patients were admitted to intensive care units (ICUs), among whom 105 were mechanically ventilated for ≥4 days. Probable CAPMI was observed among 38% of patients (40/105), among whom BAL culture of 29 patients turned positive for molds, while galactomannan testing on BAL (GM index ≥1) and serum (GM index >0.5) samples were positive for 60% (24/40) and 37.5% (15/39) of patients, respectively. Aspergillus (22/29; 75.8%) and Fusarium (6/29; 20.6%) constituted 96.5% of the molds isolated. Diaporthe foeniculina was isolated from a COVID-19 patient. None of the patients who presented with CAPMI were treated with antifungal drugs.Conclusion: Despite being prevalent, the absence of appropriate antifungal treatment highlights that CAPMI is a neglected complication among mechanically ventilated COVID-19 patients admitted to ICUs. CAPMI can be caused by species other than Aspergillus.

As the second leading etiological agent of candidemia in Turkey and the cause of severe fluconazole-non-susceptible (FNS) clonal outbreaks, Candida parapsilosis emerged as a major health threat at Ege University Hospital (EUH). Evaluation of microbiological and pertinent clinical profiles of candidemia patients due to C. parapsilosis in EUH in 2019–2020. Candida parapsilosis isolates were collected from blood samples and identified by sequencing internal transcribed spacer ribosomal DNA. Antifungal susceptibility testing was performed in accordance with CLSI M60 protocol and ERG11 and HS1/HS2-FKS1 were sequenced to explore the fluconazole and echinocandin resistance, respectively. Isolates were typed using a multilocus microsatellite typing assay. Relevant clinical data were obtained for patients recruited in the current study. FNS C. parapsilosis isolates were recovered from 53% of the patients admitted to EUH in 2019–2020. Y132F was the most frequent mutation in Erg11. All patients infected with C. parapsilosis isolates carrying Y132F, who received fluconazole showed therapeutic failure and significantly had a higher mortality than those infected with other FNS and susceptible isolates (50% vs. 16.1%). All isolates carrying Y132F grouped into one major cluster and mainly recovered from patients admitted to chest diseases and pediatric surgery wards. The unprecedented increase in the number of Y132F C. parapsilosis, which corresponded with increased rates of fluconazole therapeutic failure and mortality, is worrisome and highlights the urgency for strict infection control strategies, antifungal stewardship, and environmental screening in EUH.

Recurrent vulvovaginal candidiasis (RVVC) is one of the most prevalent fungal infections in humans, especially in developing countries; however, it is underestimated and regarded as an easy-to-treat condition. RVVC may be caused by dysbiosis of the microbiome and other host-, pathogen-, and antifungal drug-related factors. Although multiple studies on host-related factors affecting the outcome have been conducted, such studies on Candida-derived factors and their association with RVVC are lacking. Thus, fluconazole-tolerant (FLZT) isolates may cause fluconazole therapeutic failure (FTF), but this concept has not been assessed in the context of Candida-associated vaginitis. Iran is among the countries with the highest burden of RVVC; however, comprehensive studies detailing the clinical and microbiological features of this complication are scarce. Therefore, we conducted a 1-year prospective study with the aim to determine the RVVC burden among women referred to a gynecology hospital in Tehran, the association of the previous exposure to clotrimazole and fluconazole with the emergence of FLZT and fluconazole-resistant (FLZR) Candida isolates, and the relevance of these phenotypes to FTF. The results indicated that about 53% of the patients (43/81) experienced RVVC. Candida albicans and C. glabrata constituted approximately 90% of the yeast isolates (72 patients). Except for one FLZT C. tropicalis isolate, FLZR and FLZT phenotypes were detected exclusively in patients with RVVC; among them, 27.9% (12/43) harbored FLZR strains. C. albicans constituted 81.2% of FLZR (13/16) and 100% of the FLZT (13/13) isolates, respectively, and both phenotypes were likely responsible for FTF, which was also observed among patients with RVVC infected with fluconazole-susceptible isolates. Thus, FTF could be due to host-, drug-, and pathogen-related characteristics. Our study indicates that FLZT and FLZR isolates may arise following the exposure to over-the-counter (OTC) topical azole (clotrimazole) and that both phenotypes can cause FTF. Therefore, the widespread use of OTC azoles can influence fluconazole therapeutic success, highlighting the necessity of controlling the use of weak topical antifungals among Iranian women.

Candida tropicalis is the fourth leading cause of candidemia in Turkey. Although C. tropicalis isolates from 1997 to 2017 were characterized as fully susceptible to antifungals, the increasing global prevalence of azole-non-susceptible (ANS) C. tropicalis and the association between high fluconazole tolerance (HFT) and fluconazole therapeutic failure (FTF) prompted us to re-evaluate azole susceptibility of C. tropicalis in Turkey. In this study, 161 C. tropicalis blood isolates from seven clinical centers were identified by ITS rDNA sequencing, genotyped by multilocus microsatellite typing, and tested for susceptibility to five azoles, two echinocandins, and amphotericin B (AMB); antifungal resistance mechanisms were assessed by sequencing of ERG11 and FKS1 genes. The results indicated that C. tropicalis isolates, which belonged to 125 genotypes grouped into 11 clusters, were fully susceptible to echinocandins and AMB; however, 18.6% of them had the ANS phenotype but only two carried the ANS-conferring mutation (Y132F). HFT was recorded in 52 isolates, 10 of which were also ANS. Large proportions of patients infected with ANS and HFT isolates (89 and 40.7%, respectively) showed FTF. Patients infected with azole-susceptible or ANS isolates did not differ in mortality, which, however, was significantly lower for those infected with HFT isolates (P = 0.007). There were significant differences in mortality (P = 0.02), ANS (P = 0.012), and HFT (P = 0.007) among genotype clusters. The alarming increase in the prevalence of C. tropicalis blood isolates with ANS and HFT in Turkey and the notable FTF rate should be a matter of public health concern.

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

The genus Madurella comprising four species, M. fahalii, M. mycetomatis, M. pseudomycetomatis, and M. tropicana, represents the prevalent cause of eumycetoma worldwide. The four species are phenotypically similar and cause an invariable clinical picture, but differ markedly in their susceptibility to antifungal drugs, and epidemiological pattern. Therefore, specific identification is required for optimal management of Madurella infection and to reveal proper epidemiology of the species. In this study, a novel multiplex real-time PCR targeting the four Madurella species was developed and standardized. Evaluation of the assay using reference strains of the target and non-target species resulted in 100% specificity, high analytical reproducibility (R2 values >0.99) and a lowest detection limit of 3 pg target DNA. The accuracy of the real-time PCR was further assessed using biopsies from eumycetoma suspected patients. Unlike culture and DNA sequencing as gold standard diagnostic methods, the real-time PCR yielded accurate diagnosis with specific identification of the causative species in three hours compared to one or two weeks required for culture. The novel method reduces turnaround time as well as labor intensity and high costs associated with current reference methods.

Candida parapsilosis is the second most common cause of candidemia in some geographical areas and in children in particular. Yet, the proportion among children varies, for example, from 10.4% in Denmark to 24.7% in Tehran, Iran. As this species is also known to cause hospital outbreaks, we explored if the relatively high number of C. parapsilosis pediatric cases in Tehran could in part be explained by undiscovered clonal outbreaks. Among 56 C. parapsilosis complex isolates, 50 C. parapsilosis were genotyped by Amplified Fragment Length Polymorphism (AFLP) fingerprinting and microsatellite typing and analyzed for nucleotide polymorphisms by FKS1 and ERG11 sequencing. AFLP fingerprinting grouped Iranian isolates in two main clusters. Microsatellite typing separated the isolates into five clonal lineages, of which four were shared with Danish isolates, and with no correlation to the AFLP patterns. ERG11 and FKS1 sequencing revealed few polymorphisms in ERG11 leading to amino-acid substitutions (D133Y, Q250K, I302T, and R398I), with no influence on azole-susceptibilities. Collectively, this study demonstrated that there were no clonal outbreaks at the Iranian pediatric ward. Although possible transmission of a diverse C. parapsilosis community within the hospital cannot be ruled out, the study also emphasizes the necessity of applying appropriately discriminatory methods for outbreak investigation.

Abstract Background Candida albicans, Candida glabrata, and Candida parapsilosis are three prevalent causes of candidiasis, worldwide. These species are considered as nine medically important complex species. Limited knowledge about these newly recognized species prompted us to develop a one-step, multiplex PCR to detect and identify them in clinical settings. Methods Primers targeting Hyphal Wall Protein I gene for the C. albicans, C. dubliniensis, C. africana, Intergenic Spacer for the C. glabrata, C. nivariensis, C. bracarensis, and Intein and ITS rDNA for the C. parapsilosis, C. orthopsilosis, and C. metapsilosis were designed. Using 168 CBS reference strains and 280 clinical isolates, the specificity and reproducibility of the developed assay were evaluated. Results Our developed assay successfully identified and distinguished all the nine species. No cross-reaction with closely- and distantly-related yeast species, Aspergillus species and human DNA was observed, resulting in 100% specificity. The ambiguous results obtained by MALDI-TOF for C. albicans and C. africana were corrected by our 9-plex PCR assay. This assay identified all the cryptic complex species from two test sets from Iran and China, correctly. Conclusions Our developed multiplex assay is accurate, specific, cost/time-saving, and works without the tedious DNA extraction steps. It could be integrated into routine clinical laboratories and as a reliable identification tool and has the potential to be implemented into epidemiological studies to broaden the limited knowledge of cryptic species complexes.

Despite the increasing occurrence of Candida orthopsilosis and Candida metapsilosis in clinical settings, little is known about their microbiological and clinical properties. Herein, we conducted a national retrospective study (2014–2019) from multiple centers in Iran. Among the 1,770 Candida isolates collected, we identified 600 Candida parapsilosis species complex isolates. Isolate identification was performed by 9-plex PCR, matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS), and rDNA sequencing, and antifungal susceptibility testing (AFST) followed CLSI M27-A3/S4; genotyping was performed by amplified fragment length polymorphism (AFLP) analysis; and clinical information was mined. Thirty-one isolates of C. orthopsilosis from various clinical sources, one mixed sample (blood) concurrently containing C. orthopsilosis and C. parapsilosis and one isolate of C. metapsilosis from a nail sample were identified. Although both 9-plex PCR and MALDI-TOF successfully identified all isolates, only 9-plex PCR could identify the agents in a mixed sample. For the C. orthopsilosis isolates, resistance (non-wild type) was noted only for itraconazole (n = 4; 12.5%). Anidulafungin and fluconazole showed the highest and voriconazole had the lowest geometric mean values. AFLP analysis showed three main and four minor genotypes. Interestingly, 90% of nail isolates clustered with 80% of the blood isolates within two clusters, and four blood isolates recovered from four patients admitted to a hospital clustered into two genotypes and showed a high degree of similarity (>99.2%), which suggests that C. orthopsilosis disseminates horizontally. Supported by our data and published case studies, C. orthopsilosis and C. metapsilosis can be linked to challenging clinical failures, and successful outcomes are not always mirrored by in vitro susceptibility. Accordingly, conducting nationwide studies may provide more comprehensive data, which is required for a better prognosis and clinical management of patients.

Occurrence of non-Candida albicans Candida (NCAC) species that are associated with elevated MIC values and therapeutic failures are increasing. As a result, timely and accurate means of identification to the species level is becoming an essential part of diagnostic practices in clinical settings. In this study, 301 clinically isolated yeast strains recovered from various anatomical sites [Blood (n = 145), other sites (n = 156)] were used to assess the accuracy and practicality of API 20C AUX and 21-plex PCR compared to MALDI-TOF MS and large subunit rDNA (LSU rDNA). MALDI-TOF MS correctly identified 98.33% of yeast isolates, 100% of top five Candida species, 95.7% of rare yeast species, while 1.3% of isolates were misidentified. API 20C AUX correctly identified 83.7% of yeast isolates, 97.2% of top five Candida species, 61.8% of rare yeast species, while 16.2% of yeast isolates were misidentified. The 21-plex PCR, accurately identified 87.3% of yeast isolates, 100% of top five Candida species, 72% of rare yeast species, but it misidentified 1.3% of rare yeast species while 9.9% of whole yeast isolates were not identified. The combination of rapidity of 21-plex PCR and comprehensiveness of API 20C AUX, led to correct identification of 92% of included yeast isolates. Due to expensiveness of MALDI-TOF MS and sequencing, this combination strategy could be the most accurate and inexpensive alternative identification strategy for developing countries. Moreover, by the advent and development of cost-effective, reliable, and rapid PCR machines that cost 130 US dollars, 21-plex could be integrated in routine laboratories of developing and resource-limited countries to specifically identify 95% causative agents of yeast-related infections in human. Databases of MALDI-TOF MS, API 20C AUX, and the number of target species identified by 21-plex require further improvement to keep up with the diverse spectrum of yeast species.

Background: Oral candidiasis (OC) has a profound effect on the life quality of immunocompromised patients, such as those undergoing chemotherapy. Objective: Systematic investigation of clinical outcome and microbiological features of yeast isolates recovered from the oral cavity of 150 Iranian patients with hematological malignancies. Design: MALDI-TOF MS, 21-plex PCR, and rDNA sequencing were used for identification. Antifungal susceptibility testing (broth microdilution, CLSI M27-A3/S4) and genotypic diversity of yeast isolates (amplified fragment length polymorphism) were assessed. Results: Nystatin treatment resulted in 70% therapeutic failure and administration of 150 mg fluconazole (FLZ) + nystatin for patients with OC relapse showed 70% clinical failure. Previous history of OC was significantly correlated with FLZ treatment requirement and nystatin failure (P = 0.005, α < 0.05). Candida albicans (80.3%) and Kluyveromyces marxianus (C. kefyr) (12.7%) were the two most prevalent yeast species isolated. FLZ and AMB exhibited the highest geometric mean values. 21-PCR showed 98.9% agreement with MALDI-TOF MS. K. marxianus isolates had the same genotype, while C. albicans isolates grouped in 15 genotypes. Conclusions: Marked rate of therapeutic failure of nystatin necessitated OC treatment with systemic antifungals. K. marxianus was the second most prevalent yeast and 21-plex PCR could be considered as an inexpensive identification tool.

As a novel risk factor, COVID-19 has led to an increase in the incidence of candidemia and an elevated mortality rate. Despite being of clinical importance, there is a lack of data regarding COVID-19-associated candidemia (CAC) among Iranian patients. Therefore, in this retrospective study, we assessed CAC epidemiology in the intensive care units (ICUs) of two COVID-19 centers in Mashhad, Iran, from early November 2020 to late January 2021. Yeast isolates from patients’ blood were identified by 21-plex polymerase chain reaction (PCR) and sequencing, then subjected to antifungal susceptibility testing according to the CLSI M27-A3 protocol. Among 1988 patients with COVID-19 admitted to ICUs, seven had fungemia (7/1988; 0.03%), among whom six had CAC. The mortality of the limited CAC cases was high and greatly exceeded that of patients with COVID-19 but without candidemia (100% (6/6) vs. 22.7% (452/1988)). In total, nine yeast isolates were collected from patients with fungemia: five Candida albicans, three C. glabrata, and one Rhodotorula mucilaginosa. Half of the patients infected with C. albicans (2/4) were refractory to both azoles and echinocandins. The high mortality of patients with CAC, despite antifungal therapy, reflects the severity of the disease in these patients and underscores the importance of rapid diagnosis and timely initiation of antifungal treatment.

Clonal outbreaks due to azole-resistant Candida parapsilosis (ARCP) isolates have been reported in numerous studies, but the environmental niche of such isolates has yet to be defined. Herein, we aimed to identify the environmental niche of ARCP isolates causing unremitting clonal outbreaks in an adult ICU from a Brazilian cancer referral center. C. parapsilosis sensu stricto isolates recovered from blood cultures, pericatheter skins, healthcare workers (HCW), and nosocomial surfaces were genotyped by multilocus microsatellite typing (MLMT). Antifungal susceptibility testing was performed by the EUCAST (European Committee for Antimicrobial Susceptibility Testing) broth microdilution reference method and ERG11 was sequenced to determine the azole resistance mechanism. Approximately 68% of isolates were fluconazole-resistant (76/112), including pericatheter skins (3/3, 100%), blood cultures (63/70, 90%), nosocomial surfaces (6/11, 54.5%), and HCW’s hands (4/28, 14.2%). MLMT revealed five clusters: the major cluster contained 88.2% of ARCP isolates (67/76) collected from blood (57/70), bed (2/2), pericatheter skin (2/3), from carts (3/7), and HCW’s hands (3/27). ARCP isolates were associated with a higher 30 day crude mortality rate (63.8%) than non-ARCP ones (20%, p = 0.008), and resisted two environmental decontamination attempts using quaternary ammonium. This study for the first time identified ARCP isolates harboring the Erg11-Y132F mutation from nosocomial surfaces and HCW’s hands, which were genetically identical to ARCP blood isolates. Therefore, it is likely that persisting clonal outbreak due to ARCP isolates was fueled by environmental sources. The resistance of Y132F ARCP isolates to disinfectants, and their potential association with a high mortality rate, warrant vigilant source control using effective environmental decontamination.

The high clinical mortality and economic burden posed by invasive fungal infections (IFIs), along with significant agricultural crop loss caused by various fungal species, has resulted in the widespread use of antifungal agents. Selective drug pressure, fungal attributes, and host- and drug-related factors have counteracted the efficacy of the limited systemic antifungal drugs and changed the epidemiological landscape of IFIs. Species belonging to Candida, Aspergillus, Cryptococcus, and Pneumocystis are among the fungal pathogens showing notable rates of antifungal resistance. Drug-resistant fungi from the environment are increasingly identified in clinical settings. Furthermore, we have a limited understanding of drug class-specific resistance mechanisms in emerging Candida species. The establishment of antifungal stewardship programs in both clinical and agricultural fields and the inclusion of species identification, antifungal susceptibility testing, and therapeutic drug monitoring practices in the clinic can minimize the emergence of drug-resistant fungi. New antifungal drugs featuring promising therapeutic profiles have great promise to treat drug-resistant fungi in the clinical setting. Mitigating antifungal tolerance, a prelude to the emergence of resistance, also requires the development of effective and fungal-specific adjuvants to be used in combination with systemic antifungals.

While being the third leading cause of candidemia worldwide, numerous studies have shown severe clonal outbreaks due to fluconazole-resistant (FLCR) Candida parapsilosis isolates associated with fluconazole therapeutic failure (FTF) with enhanced mortality. More recently, multidrug resistant (MDR) C. parapsilosis blood isolates have also been identified that are resistant to both azole and echinocandin drugs. Amphotericin B (AMB) resistance is rarely reported among C. parapsilosis isolates and proper management of bloodstream infections due to FLZR and MDR isolates requires prompt action at the time of outbreak. Therefore, using a well-established Galleria mellonella model, we assessed whether (a) laboratory-based findings on azole or echinocandin (micafungin) resistance in C. parapsilosis lead to therapeutic failure, (b) LAMB could serve as an efficient salvage treatment option, and (c) distinct mutations in ERG11 impact mortality. Our in vivo data confirm fluconazole inefficacy against FLCR C. parapsilosis isolates carrying Y132F, Y132F + K143R, Y132F + G307A, and G307A + G458S in Erg11p, while LAMB proved to be an efficacious accessible option against both FLCR and MDR C. parapsilosis isolates. Moreover, positive correlation of in vitro and in vivo data further highlights the utility of G. melonella as a reliable model to investigate azole and polyene drug efficacy.

The recent global pandemic of COVID-19 has predisposed a relatively high number of patients to acute respiratory distress syndrome (ARDS), which carries a risk of developing super-infections. Candida species are major constituents of the human mycobiome and the main cause of invasive fungal infections, with a high mortality rate. Invasive yeast infections (IYIs) are increasingly recognized as s complication of severe COVID-19. Despite the marked immune dysregulation in COVID-19, no prominent defects have been reported in immune cells that are critically required for immunity to Candida. This suggests that relevant clinical factors, including prolonged ICU stays, central venous catheters, and broad-spectrum antibiotic use, may be key factors causing COVID-19 patients to develop IYIs. Although data on the comparative performance of diagnostic tools are often lacking in COVID-19 patients, a combination of serological and molecular techniques may present a promising option for the identification of IYIs. Clinical awareness and screening are needed, as IYIs are difficult to diagnose, particularly in the setting of severe COVID-19. Echinocandins and azoles are the primary antifungal used to treat IYIs, yet the therapeutic failures exerted by multidrug-resistant Candida spp. such as C. auris and C. glabrata call for the development of new antifungal drugs with novel mechanisms of action.

Human fungal pathogens are attributable to a significant economic burden and mortality worldwide. Antifungal treatments, although limited in number, play a pivotal role in decreasing mortality and morbidities posed by invasive fungal infections (IFIs). However, the recent emergence of multidrug-resistant Candida auris and Candida glabrata and acquiring invasive infections due to azole-resistant C. parapsilosis, C. tropicalis, and Aspergillus spp. in azole-naïve patients pose a serious health threat considering the limited number of systemic antifungals available to treat IFIs. Although advancing for major fungal pathogens, the understanding of fungal attributes contributing to antifungal resistance is just emerging for several clinically important MDR fungal pathogens. Further complicating the matter are the distinct differences in antifungal resistance mechanisms among various fungal species in which one or more mechanisms may contribute to the resistance phenotype. In this review, we attempt to summarize the burden of antifungal resistance for selected non-albicansCandida and clinically important Aspergillus species together with their phylogenetic placement on the tree of life. Moreover, we highlight the different molecular mechanisms between antifungal tolerance and resistance, and comprehensively discuss the molecular mechanisms of antifungal resistance in a species level.

Like severe influenza, coronavirus disease-19 (COVID-19) resulting in acute respiratory distress syndrome (ARDS) has emerged as an important disease that predisposes patients to secondary pulmonary aspergillosis, with 35 cases of COVID-19 associated pulmonary aspergillosis (CAPA) published until June 2020. The release of danger-associated molecular patterns during severe COVID-19 results in both pulmonary epithelial damage and inflammatory disease, which are predisposing risk factors for pulmonary aspergillosis. Moreover, collateral effects of host recognition pathways required for the activation of antiviral immunity may, paradoxically, contribute to a highly permissive inflammatory environment that favors fungal pathogenesis. Diagnosis of CAPA remains challenging, mainly because bronchoalveolar lavage fluid galactomannan testing and culture, which represent the most sensitive diagnostic tests for aspergillosis in the ICU, are hindered by the fact that bronchoscopies are rarely performed in COVID-19 patients due to the risk of disease transmission. Similarly, autopsies are rarely performed, which may result in an underestimation of the prevalence of CAPA. Finally, the treatment of CAPA is complicated by drug–drug interactions associated with broad spectrum azoles, renal tropism and damage caused by SARS-CoV-2, which may challenge the use of liposomal amphotericin B, as well as the emergence of azole-resistance. This clinical reality creates an urgency for new antifungal drugs currently in advanced clinical development with more promising pharmacokinetic and pharmacodynamic profiles.

Candida auris, C. haemulonii, C. duobushaemulonii, and C. pseudohaemulonii are closely related and highly multidrug resistant yeast pathogens. The high cost and low accuracy of current diagnostics may underestimate their prevalence, especially in medical resource-limited regions. In this study, we used 172 C. auris stains and its relatives and 192 other fungal strains to establish and validate a novel multiplex end-point PCR. A prospective and a retrospective clinical screenings using this assay were further performed in China and Iran respectively. We identified the first isolate of C. pseudohaemulonii in China and the first isolate of C. haemulonii in Iran from 821 clinical isolates in total, without any false positive. Animal models of C. auris and C. haemulonii were established for validation. The overall positive rates of the assay for mice blood and tissue were 28.6 and 92.9%, respectively. Compared with previously developed assays, our assay is more available and affordable to the developing countries, and may contribute to a better understanding of the epidemiology of C. auris and its relatives in these regions.

Extensive advances in technology offer a vast variety of diagnostic methods that save time and costs, but identification of fungal species causing human infections remains challenging in developing countries. Since the echinocandins, antifungals widely used to treat invasive mycoses, are still unavailable in developing countries where a considerable number of problematic fungal species are present, rapid and reliable identification is of paramount importance. Unaffordability, large footprints, lack of skilled personnel, and high costs associated with maintenance and infrastructure are the main factors precluding the establishment of high-precision technologies that can replace inexpensive yet time-consuming and inaccurate phenotypic methods. In addition, point-of-care lateral flow assay tests are available for the diagnosis of Aspergillus and Cryptococcus and are highly relevant for developing countries. An Aspergillus galactomannan lateral flow assay is also now available. Real-time PCR remains difficult to standardize and is not widespread in countries with limited resources. Isothermal and conventional PCR-based amplification assays may be alternative solutions. The combination of real-time PCR and serological assays can significantly increase diagnostic efficiency. However, this approach is too expensive for medical institutions in developing countries. Further advances in next-generation sequencing and other innovative technologies such as clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic tools may lead to efficient, alternate methods that can be used in point-of-care assays, which may supplement or replace some of the current technologies and improve the diagnostics of fungal infections in developing countries.