Antifungal Susceptibility Testing Research Articles

Epidemiology of cryptococcal meningitis and fluconazole heteroresistance in Cryptococcus neoformans isolates from a teaching hospital in southwestern China.

Cryptococcal meningitis (CM), a common and serious opportunistic infection mostly caused by Cryptococcus neoformans, is primarily treated with fluconazole. Nevertheless, Cryptococcus neoformans strains that undergo repeated exposure to azoles can gradually acquire heteroresistance to fluconazole. The management of this specific CM infection poses a substantial challenge. Determining a globally accepted definition for fluconazole heteroresistance and developing effective and prompt methods for identifying heteroresistance is of utmost importance. We collected data on the clinical and epidemiological characteristics of patients diagnosed with CM. All the available Cryptococcus neoformans strains isolated from these patients were collected and subjected to antifungal susceptibility testing and evaluation of fluconazole heteroresistance. AIDS was present in 40.5% of the patients, whereas 24.1% did not have any underlying diseases. Patients with chronic diseases or impaired immune systems are susceptible to infection by Cryptococcus neoformans, a fungus that frequently (39.6%, 19/48) shows heteroresistance to fluconazole, as confirmed by population analysis profile (PAP).IMPORTANCEFluconazole heteroresistance poses a significant threat to the efficacy of fluconazole in treating cryptococcal meningitis (CM). Unfortunately, the standard broth microdilution method often misses the subtle percentages of subpopulations exhibiting heteroresistance. While the population analysis profile (PAP) method is esteemed as the gold standard, its time-consuming and labor-intensive nature makes it impractical for routine clinical use. In contrast, the Kirby-Bauer (KB) disk diffusion method offers a simple and effective screening solution. Our study highlights the value of KB over PAP and minimum inhibitory concentration (MIC) by demonstrating that when adjusting the inoculum concentration to 1.0 McFarland and subjecting samples to a 72-hour incubation period at 35°C, the KB method closely mirrors the outcomes of the PAP approach in detecting fluconazole heteroresistance. This optimization of the KB method not only enhances assay efficiency but also provides a blueprint for developing a timely and effective strategy for identifying heteroresistance.

Epidemiology of Candidemia in Mashhad, Northeast Iran: A Prospective Multicenter Study (2019–2021)

Candidemia is a major cause of morbidity and mortality in health care settings, and its epidemiology is changing. In the last two decades, the proportion of non-albicans Candida (NAC) yeasts in candidemia has increased. These yeasts more often display resistance to common antifungals. In many western countries, candidemia is mainly caused by susceptible C. albicans, while in resource-limited countries, including Iran, the candidemia species distribution is studied less often. Here, we investigated the species distribution, resistance levels, and characteristics of patients with candidemia in five hospitals in Mashhad (northeast Iran) for two years (2019–2021). Yeast isolates from blood were identified with MALDI-TOF MS and subjected to antifungal susceptibility testing (AFST) using the broth microdilution method, while molecular genotyping was applied to Candida parapsilosis isolates. In total, 160 yeast isolates were recovered from 160 patients, of which the majority were adults (60%). Candidemia was almost equally detected in men (48%) and women (52%). Almost half of patients (n = 67, 49%) were from intensive care units (ICUs). C. parapsilosis (n = 58, 36%) was the most common causative agent, surpassing C. albicans (n = 52, 33%). The all-cause mortality rate was 53%, with C. albicans candidemia displaying the lowest mortality with 39%, in contrast to a mortality rate of 59% for NAC candidemia. With microbroth AFST, nearly all tested isolates were found to be susceptible, except for one C. albicans isolate that was resistant to anidulafungin. By applying short tandem repeat (STR) genotyping to C. parapsilosis, multiple clusters were found. To summarize, candidemia in Mashhad, Iran, from 2019 to 2021, is characterized by common yeast species, in particular C. parapsilosis, for which STR typing indicates potential nosocomial transmission. The overall mortality is high, while resistance rates were found to be low, suggesting that the high mortality is linked to limited diagnostic options and insufficient medical care, including the restricted use of echinocandins as the first treatment option.

Current therapy and advancements in the treatment of equine fungal keratitis.

Equine fungal keratitis represents a substantial portion of keratitis cases in horses, with fungal involvement identified in approximately half of all infectious keratitis cases. Despite its prevalence, more comprehensive retrospective analyses are needed to better understand this condition. Outcomes vary, with approximately two-thirds of cases achieving complete healing with retained vision, although enucleation is often necessary. Predominant pathogens include Aspergillus and Fusarium, with yeast reported in a minority of cases. Resistance to common antifungal agents among filamentous fungi poses a significant challenge. Advances in diagnostics, including repeat culture and antifungal susceptibility testing, as well as the incorporation of PCR technology, hold promise for improving detection and guiding treatment decisions. Newer antifungals, combination therapies, and innovative modalities such as photodynamic therapy offer hope for improved outcomes. Continued research efforts are essential to further elucidate the epidemiology, pathogenesis, and optimal management strategies for this condition.

Twenty Years in EUCAST Anti-Fungal Susceptibility Testing: Progress & Remaining Challenges.

Since its inception in 2002, the EUCAST Antifungal Susceptibility Testing Subcommittee (AFST) has developed and refined susceptibility testing methods for yeast, moulds and dermatophytes, and established epidemiological cut-off values and breakpoints for antifungals. For yeast, three challenges have been addressed. Interpretation of trailing growth in fluconazole susceptibility testing, which has been proven without impact on efficacy if below the 50% endpoint. Variability in rezafungin MIC testing due to laboratory conditions, which has been solved by the addition of Tween 20 to the growth medium in E.Def 7.4. And third, interpretation of MICs for rare yeast with no breakpoints, where recommendations have been established for MIC-based clinical advice. For moulds, refinements include the validation of spectrophotometer reading for A. fumigatus to facilitate objective MIC determination, and for dermatophytes the establishment of a microdilution method with automated reading and a selective medium to minimise the risk of contaminations. Recent initiatives involve development and validation of agar-based screening assays for detection of potential azole and echinocandin resistance in A. fumigatus and Aspergillus species, respectively, and of terbinafine resistance in Trichophyton species. Moreover, the development of a EUCAST guidance document for molecular resistance testing represents an advancement, particularly for identifying target gene alterations associated with resistance. In summary, EUCAST AFST continues to play a pivotal role in standardizing AFST and facilitating accurate interpretation of susceptibility data for clinical decision-making. Adoption of EUCAST breakpoints for commercial test methods, however, requires thorough validation to ensure concordance with EUCAST reference testing species-specific MIC distributions.

Hospitalized COVID-19 Patients with Urinary Tract Infection in Iran: Candida Species Distribution and Antifungal Susceptibility Patterns

Candida species, typically part of the human skin and mucous membrane flora, can cause opportunistic fungal infections, notably urinary tract infections (UTIs), which are on the rise among hospitalized COVID-19 patients. The lack of understanding of UTIs in this population, coupled with the emergence of multidrug-resistant strains, poses significant challenges for effective treatment and further investigations. In this study, urine samples were collected from 70 COVID-19 patients with UTIs in sterile containers for microbiology examination. After microscopic observation, the isolates were identified both by phenotypic and molecular techniques such as multiplex PCR. Antifungal susceptibility testing (AFST) against fluconazole (Flu), itraconazole (Itr), and amphotericin B (AMB) was performed according to CLSI M27/S4 standard methods, with the frequency of isolates including Candida albicans (n = 20, 51.3%), Candida tropicalis (n = 15, 38.4%), Nakaseomyces glabrata (previously Candida glabrata) (n = 2, 5.1%), Pichia kudriavzevii (previously Candida krusei), and Candida parapsilosis (n = 1, 2.5%). All isolates of C. albicans, C. tropicalis, C. glabrata, and C. parapsilosis were sensitive to amphotericin B, while C. kruzei was resistant to AMB. Around 70% of C. albicans isolates were sensitive to Flu; 20% of C. tropicalis were resistant to itraconazole, while 33% were resistant to fluconazole. C. albicans and C. tropicalis were the main causes of candiduria in infected cases and both Flu and AMB showed good results in AFST in these species. Performing drug susceptibility testing for clinical isolates of Candida spp. provided guidance for appropriate management and control, and timely antifungal treatment.

Evolutionary accumulation of FKS1 mutations from clinical echinocandin-resistant Candida auris

Introduction: Drug resistance to echinocandins, first-line drugs used to treat Candida auris infection, is rapidly emerging. However, the accumulation of mutations in genes other than FKS1 (before an isolate develops to resistance via FKS1 mutations), remains poorly understood. Methods Four clinical cases and 29 isolates associated with the incremental process of echinocandin resistance were collected and analyzed using antifungal drug susceptibility testing and genome sequencing to assess the evolution of echinocandin resistance. Findings: Six echinocandin minimum inhibitory concentration (MIC)-elevated C. auris strains and seven resistant strains were isolated from the urinary system of patients receiving echinocandin treatment. Meanwhile, phylogenetic analyses illustrated that the echinocandin-resistant strains were closely related to other strains in the same patient. Genomic data revealed that the echinocandin-resistant strains had FKS1 mutations. Furthermore, three categories (ECN-S/E/R) of non-synonymous mutant SNP genes (such as RBR3, IFF6, MKC1, MPH1, RAD2, and MYO1) in C. auris appeared to be associated with the three-stage-evolutionary model of echinocandin resistance in C. glabrata: cell wall stress, drug adaptation, and genetic escape (FKS mutation). Interpretation: Echinocandin-resistant C. auris undergoes spatial and temporal phase changes closely related to echinocandin exposure, particularly in the urinary system. These findings suggest that FKS1 mutations mediate an evolutionary accumulation of echinocandin resistance followed by modulation of chromosome remodeling and DNA repair processes that ultimately lead to FKS1 hot spot mutations and the development of drug resistance. This study provides an in-depth exploration of the molecular pathways involved in the evolution of Candida auris echinocandin resistance.

Comparative study of antifungal susceptibility testing methods for clinical Candida albicans isolates

PurposeCandida albicans is the second most common cause of candidemia in Malaysia. The Clinical and Laboratory Standards Institute (CLSI) broth microdilution method is the gold standard for determining its minimum inhibitory concentration (MIC); however, it is laborious and time-consuming. This study was conducted to evaluate the usefulness of alternative methods, namely Sensititre YeastOne (SYO), VITEK 2 system, and E-test for determining the MIC of clinical C. albicans isolates. Materials and methodsThe susceptibilities of 95 C. albicans isolates were compared between SYO, VITEK 2 system, and E-test with CLSI broth microdilution method. The categorical agreement (CA), essential agreement (EA), very major errors (VME), major errors (ME) and minor errors (MiE) were calculated. ResultsOur finding showed the CA varied for SYO from 96.8% to 100%, while the EA ranged from 91.6% to 100%. The SYO method showed 1.1% of VME and ME, and up to 3.2% of MiE. Next, the CA and EA ranges for the VITEK 2 system were 97.8%–100% and 23.2%–100%, respectively. In the VITEK 2 technique, 1.1% of VME were found. For the E-test, the CA varied from 83.2% to 100% while the EA ranged from 64.2% to 98.9%. The E-test method showed 1.1% of VME and up to 16.8% of MiE. ConclusionsIn conclusion, SYO and VITEK 2 (except flucytosine) could be potential alternatives to the CLSI broth microdilution method in determining the MIC of C. albicans.

Susceptibility Testing of Environmental and Clinical Aspergillus sydowii Demonstrates Potent Activity of Various Antifungals

The genus Aspergillus consists of a vast number of medically and environmentally relevant species. Aspergillus species classified in series Versicolores are ubiquitous in the environment and include the opportunistic pathogen Aspergillus sydowii, which is associated with onychomycosis and superficial skin infections. Despite frequent clinical reports of A. sydowii and related series Versicolores species, antifungal susceptibility data are scarce, hampering optimal treatment choices and subsequent patient outcomes. Here, we employed antifungal susceptibility testing (AFST) based on microbroth dilution on a set of 155 series Versicolores strains using the common antifungals amphotericin B, itraconazole, voriconazole, posaconazole, isavuconazole and micafungin with the addition of luliconazole and olorofim. All strains were identified using partial calmodulin gene sequencing, with 145 being A. sydowii, seven A. creber and three A. versicolor, using the latest taxonomic insights. Overall, tested antifungals were potent against the entire strain collection. In comparison to A. fumigatus, azole and amphotericin B MICs were slightly elevated for some strains. AFST with luliconazole and olorofim, here reported for the first time, displayed the highest in vitro activity, making these antifungals interesting alternative drugs but clinical studies are warranted for future therapeutic use.

Candida albicans in the oral cavities of pets: biofilm formation, putative virulence, antifungal resistance profiles and classification of the isolates.

The study aimed to investigate Candida albicans presence, antifungal resistance, biofilm formation, putative virulence genes, and molecular characterization in oral samples of dogs and cats. A total of 239 oral samples were collected from cats and dogs of various breeds and ages at Erciyes University, Faculty of Veterinary Medicine Clinics, between May 2017 and April 2018. Among 216 isolates obtained, 15 (6.95%) were identified as C. albicans, while 8 (3.7%) were non-albicans Candida species. Antifungal susceptibility testing revealed sensitivities to caspofungin, fluconazole, and flucytosine in varying proportions. Molecular analysis indicated the presence of fluconazole and caspofungin resistance genes in all C. albicans isolates. Additionally, virulence genes ALS1, HWP1, and HSP90 showed variable presence. Biofilm formation varied among isolates, with 46.7% strong, 33.3% moderate, and 20% weak producers. PCA analysis categorized isolates into two main clusters, with some dog isolates grouped separately. The findings underscore the significance of oral care and protective measures in pets due to C. albicans prevalence, biofilm formation, virulence factors, and antifungal resistance in their oral cavity, thereby aiding clinical diagnosis and treatment in veterinary medicine.

Detection and characterisation of a sixth Candida auris clade in Singapore: a genomic and phenotypic study

The emerging fungal pathogen Candida auris poses a serious threat to global public health due to its worldwide distribution, multidrug resistance, high transmissibility, propensity to cause outbreaks, and high mortality. We aimed to characterise three unusual Cauris isolates detected in Singapore, and to determine whether they constitute a novel clade distinct from all previously known Cauris clades (I-V). In this genotypic and phenotypic study, we characterised three Cauris clinical isolates, which were cultured from epidemiologically unlinked inpatients at a large tertiary hospital in Singapore. The index isolate was detected in April, 2023. We performed whole-genome sequencing (WGS) and obtained hybrid assemblies of these Cauris isolates. The complete genomes were compared with representative genomes of all known Cauris clades. To provide a global context, 3651international WGS data from the National Center for Biotechnology Information (NCBI) database were included in a high-resolution single nucleotide polymorphism (SNP) analysis. Antifungal susceptibility testing was done and antifungal resistance genes, mating-type locus, and chromosomal rearrangements were characterised from the WGS data of the three investigated isolates. We further implemented Bayesian logistic regression models to classify isolates into known clades and simulate the automatic detection of isolates belonging to novel clades as their WGS data became available. The three investigated isolates were separated by at least 37 000SNPs (range 37 000-236 900) from all existing Cauris clades. These isolates had opposite mating-type allele and different chromosomal rearrangements when compared with their closest clade IV relatives. The isolates were susceptible to all tested antifungals. Therefore, we propose that these isolates represent a new clade of Cauris, clade VI. Furthermore, an independent WGS dataset from Bangladesh, accessed via the NCBI Sequence Read Archive, was found to belong to this new clade. As a proof-of-concept, our Bayesian logistic regression model was able to flag these outlier genomes as a potential new clade. The discovery of a new Cauris clade in Singapore and Bangladesh in the Indomalayan zone, showing a close relationship to cladeIV members most commonly found in South America, highlights the unknown genetic diversity and origin of Cauris, particularly in under-resourced regions. Active surveillance in clinical settings, along with effective sequencing strategies and downstream analysis, will be essential in the identification of novel strains, tracking of transmission, and containment of adverse clinical effects of Cauris infections. Duke-NUS Academic Medical Center Nurturing Clinician Researcher Scheme, and the Genedant-GIS Innovation Program.

Clinical and microbiological characteristics of a hospital outbreak of Candida auris in a referral hospital in Lima, Peru.

Candida auris, a multidrug-resistant fungal pathogen, has received considerable attention owing to its recent surge, especially in South America, which coincides with the ongoing global COVID-19 pandemic. Understanding the clinical and microbiological characteristics of outbreaks is crucial for their effective management and control. This retrospective observational study aimed to characterize a C. auris outbreak at a Peruvian referral hospital between January 2021 and July 2023. Data were collected from hospitalized patients with positive C. auris culture results. Microbiological data and antifungal susceptibility test results were analysed. Additionally, infection prevention and control measures have been described. Statistical analysis was used to compare the characteristics between the infected and colonized patients. Thirty-three patients were identified, mostly male (66.7%), with a median age of 53 years. Among them, 18 (54.5%) were colonized, and 15 (45.5%) were infected. Fungemia was the predominant presentation (80%), with notable cases of fungemia in tuberculosis patients with long-stay devices for parenteral anti-tuberculosis therapy. Seventy-five percent of the isolates exhibited fluconazole resistance. Echinocandins were the primary treatment, preventing fungemia recurrence within 30 days. Infected patients had significantly longer hospital stays than colonized patients (100 vs. 45 days; p = .023). Hospital mortality rates were 46.7% and 25% in the infected and fungemia patients, respectively. Simultaneous outbreaks of multidrug-resistant bacteria were documented. This study underscores the severity of a C. auris outbreak at a referral hospital in Peru, highlighting its significant impact on patient outcomes and healthcare resources. The high prevalence of fluconazole-resistant isolates, leading to prolonged hospital stay and high mortality rates, particularly in cases of fungemia, underscores the critical need for effective infection prevention and control strategies.

Candiduria: Analysis of Changing Trends and Antifungal Dynamics in Acute Healthcare Settings with Special Reference to Rare Candida Species

Background: Candiduria is a common phenomenon among hospitalized patients, particularly those in intensive care units (ICUs), wherein multiple predisposing factors such as comorbidities, indwelling urinary catheters, and antimicrobial exposure contribute to its prevalence. Objective: This study aims to investigate the epidemiological trends and antifungal susceptibility patterns of Candida spp. isolated from urine samples of patients at a tertiary care center in Western Maharashtra, India. Methods: Of the 7,642 urine samples received between 01 June 2022 to 31 May 2023, 142 samples (9%) displayed the growth of Candida species. Upon retesting samples from these patients, only 70 true positive results were identified. All the isolates were detected via VITEK MS (bioMerieux, France), while antifungal susceptibility testing (AFST) was conducted using the VITEK 2 Compact with an AST-YS08 card. Results: The most common isolate was Candida tropicalis (50%), followed by Candida albicans (25.7%). Rare Candida species (e.g., Candida ciferrii, Candida kefyr, Candida metapsilosis, Candida utilis) collectively contributed to 7.1% of all Candida species. The results of the AFST revealed a high sensitivity to amphotericin B and echinocandins. Fluconazole resistance was observed in 6% of the isolates. Candida ciferrii emonstrated a distinct pattern, indicating a statistically significant association (p = 0.015) when the patient had two or more comorbidities and resistance to azoles, which was statistically significant (p-values for fluconazole (p = 0.015), voriconazole (p = 0.004), and flucytosine (p = 0.002)) when compared with other Candida species. Conclusion: Resistance trends in India have been changing with increased fluconazole-resistant Candida strains being frequently isolated, emphasizing the importance of customized therapeutic strategies based on culture and sensitivity reports.

Asymptomatic colonization of stray dogs and domestic cats with Trichophyton mentagrophytes II* in Northern Iran

BackgroundDespite changes in the epidemiology of dermatophyte infections, the incidence of fungal infections associated with Trichophyton species still remains high among dogs and cats. The objective of the present study was to isolate and characterize dermatophytes from dogs and cats in Iran. Method:From December 2022 to May 2023, skin and hair samples were collected from symptomatic and asymptomatic cats and dogs in Mazandaran, a northern province of Iran. The samples were then inoculated into Mycosel™ Agar. Dermatophyte isolates were identified by sequencing the internal transcribed spacer region. Antifungal susceptibility tests were conducted using the Clinical and Laboratory Standards Institute (CLSI-M38-A3). ResultOf the 250 samples collected (from 200 dogs and 50 cats), 20 (from 19 dogs and one cat) (8.0 %) were positive for dermatophyte growth. Based on sequence and phylogenetic analysis, all isolates belonged to T. mentagrophytes II*. Of these positive samples, 14 (70.0 %), 3 (15.0 %), 2 (10.0 %), and 1 (2.0 %) were isolated from asymptomatic stray dogs, symptomatic stray dogs, symptomatic domestic dogs, and symptomatic cats, respectively. Luliconazole and terbinafine displayed potent activity against all T. mentagrophytes isolates, with Minimum inhibitory concentration (MIC) values of 0.016 µg/ml. Miconazole and griseofulvin demonstrated higher MIC (1 and 8 µg/ml). ConclusionThe present study indicated that T. mentagrophytes II* asymptomatic carriage is frequent in stray dogs in Iran. The potential risk to public health needs to be evaluated However, T. mentagrophytes genotype VIII, considered as an endemic and emerging human pathogenic clone in several countries, was not detected during the present survey.

A Road Less Traveled: E-test Method for Antifungal Susceptibility Testing in Trichophyton mentagrophyte Isolates Among Patients Presenting With Dermatophytosis at a Tertiary Healthcare Center in North India.

Introduction Dermatophytosis is a common infection of the skin, hair, and nails caused by dermatophytes, a group of filamentous fungi capable of digesting and obtaining nutrients from keratin. Dermatophytes comprise three important genera: Epidermophyton, Microsporum,and Trichophyton. This study aimed to analyze the antifungal susceptibility patterns of Trichophyton mentagrophytes isolates using the epsilometer test (E-test) method. Material and methods This prospective observational study was conducted on clinically suspected cases of dermatophytosis. All samples, including skin scrapings, hair, and nails, were subjected to potassium hydroxide (KOH) examination followed by fungal culture. The Trichophyton mentagrophytes isolates were then subjected to antifungal susceptibility testing using the E-test method for the two most prescribed antifungals: itraconazole and fluconazole. Results In this study, one-third of the patients who tested positive for dermatophytosis belonged to the same family, with spouses being the most commonly affected. Tinea corporis was the most common clinical presentation, with Trichophyton mentagrophytes identified as the most common etiological agent. Itraconazole was more effective than fluconazole. Conclusion The current study demonstrated that antifungal susceptibility testing of dermatophytes using the E-test is easier and can be applied in routine laboratories as a screening method, serving as an alternative to broth microdilution.

Clinical spectrum, phenotypic and molecular characterization, and antifungal susceptibility of an emerging human pathogen, Acrophialophora, from India.

Acrophialophora is implicated in superficial and invasive infections, especially in immunosuppressed individuals. The present study was undertaken to provide clinical, microbiological, phylogenetic, and antifungal susceptibility testing (AFST) profile of Acrophialophora isolated from India. All the isolates identified as Acrophialophora species at the National Culture Collection for Pathogenic Fungi, Chandigarh, India were revived. Phenotypic and molecular characterization was performed, followed by temperature studies, scanning electron microscopy (SEM), and AFST. We also performed systematic review of all the cases of Acrophialophora species reported till date. A total of nine isolates identified as Acrophialophora species were identified by molecular method as A. fusispora (n=8) and A. levis (n=1), from brain abscess (n=4), respiratory tract (n=3), and corneal scraping (n=2). All patients but two had predisposing factors/co-morbidities. Acrophialophora was identified as mere colonizer in one. Temperature studies and SEM divulged variation between both species. Sequencing of the internal transcribed spacer ribosomal DNA and beta-tubulin loci could distinguish species, while the LSU ribosomal DNA locus could not. AFST showed the lowest minimum inhibitory concentrations (MICs) for triazoles and the highest for echinocandins. Systematic literature review revealed 16 cases (11 studies), with ocular infections, pulmonary and central nervous system infections, and A. fusispora was common species. All the patients except three responded well. High MICs were noted for fluconazole, micafungin, and caspofungin. This is the first study delineating clinical, phenotypic, and genotypic characteristics of Acrophialophora species from India. The study highlights microscopic differences between both species and emphasizes the role of molecular methods in precise identification. Triazoles appear to be the most effective antifungals for managing patients.

Emergence of the novel sixth Candida auris Clade VI in Bangladesh.

Candida auris, initially identified in 2009, has rapidly become a critical concern due to its antifungal resistance and significant mortality rates in healthcare-associated outbreaks. To date, whole-genome sequencing (WGS) has identified five unique clades of C. auris, with some strains displaying resistance to all primary antifungal drug classes. In this study, we presented the first WGS analysis of C. auris from Bangladesh, describing its origins, transmission dynamics, and antifungal susceptibility testing (AFST) profile. Ten C. auris isolates collected from hospital settings in Bangladesh were initially identified by CHROMagar Candida Plus, followed by VITEK2 system, and later sequenced using Illumina NextSeq 550 system. Reference-based phylogenetic analysis and variant calling pipelines were used to classify the isolates in different clades. All isolates aligned ~90% with the Clade I C. auris B11205 reference genome. Of the 10 isolates, 8 were clustered with Clade I isolates, highlighting a South Asian lineage prevalent in Bangladesh. Remarkably, the remaining two isolates formed a distinct cluster, exhibiting >42,447 single-nucleotide polymorphism differences compared to their closest Clade IV counterparts. This significant variation corroborates the emergence of a sixth clade (Clade VI) of C. auris in Bangladesh, with potential for international transmission. AFST results showed that 80% of the C. auris isolates were resistant to fluconazole and voriconazole, whereas Clade VI isolates were susceptible to azoles, echinocandins, and pyrimidine analogue. Genomic sequencing revealed ERG11_Y132F mutation conferring azole resistance while FCY1_S70R mutation found inconsequential in describing 5-flucytosine resistance. Our study underscores the pressing need for comprehensive genomic surveillance in Bangladesh to better understand the emergence, transmission dynamics, and resistance profiles of C. auris infections. Unveiling the discovery of a sixth clade (Clade VI) accentuates the indispensable role of advanced sequencing methodologies.IMPORTANCECandida auris is a nosocomial fungal pathogen that is commonly misidentified as other Candida species. Since its emergence in 2009, this multidrug-resistant fungus has become one of the five urgent antimicrobial threats by 2019. Whole-genome sequencing (WGS) has proven to be the most accurate identification technique of C. auris which also played a crucial role in the initial discovery of this pathogen. WGS analysis of C. auris has revealed five distinct clades where isolates of each clade differ among themselves based on pathogenicity, colonization, infection mechanism, as well as other phenotypic characteristics. In Bangladesh, C. auris was first reported in 2019 from clinical samples of a large hospital in Dhaka city. To understand the origin, transmission dynamics, and antifungal-resistance profile of C. auris isolates circulating in Bangladesh, we conducted a WGS-based surveillance study on two of the largest hospital settings in Dhaka, Bangladesh.

Identification And Antifungal Susceptibility Testing Of Candida Species Isolated From Cancer Patients

Identification And Antifungal Susceptibility Testing Of Candida Species Isolated From Cancer Patients

Onychomycosis: Prevalence, Fungal Pathogens, Risk Factors, and Antifungal Susceptibility Profile among People Living with Diabetes Mellitus in Kano, Northwestern Nigeria

Abstract Context: Onychomycosis is a fungal infection of the nail unit that over time results in dystrophy and disfigurement. It affects the quality of life and increases the prevalence and severity of foot ulcers in patients with diabetes. Aim: The study investigated the prevalence of onychomycosis, the associated fungal pathogens, and their antifungal susceptibility profile in a cohort of people living with diabetes (PLWDM) in Kano, Nigeria. Settings and Designs: This cross-sectional hospital-based study recruited and enrolled 300 PLWDM from two major diabetes treatment centers in Kano. Materials and Methods: Participants were screened for thickened, brittle, crumbly, or ragged nails, and 30 met the inclusion criteria. Sociodemographic data, clinical history, and other risk factors for onychomycosis were also evaluated. Toenail and fingernail, nail clippings, and scrapings were collected and cultured on Sabouraud dextrose agar (SDA). The identification of yeasts and mold isolates was performed using gram stain, germ tube test, CHROMagar, lactophenol cotton blue (LPCB) mount, and slide cultures. Antifungal susceptibility testing was achieved by the agar well-diffusion method. Statistical Analysis Used: The data generated were analyzed using descriptive statistics and associations between variables checked by the chi-square test or Fisher’s exact test. Results: The prevalence of onychomycosis was 63.3% (19/30), and fingernails were mostly affected. Fungal pathogens recovered include dermatophytes, yeasts, and non-dermatophyte molds, with a predominance of Aspergillus spps. (7/19 (36.8%)), Trichophytonmentagrophytes (4/19 (21.1%)), Trichophytonrubrum (4/19 (21.1%)), and Candidaalbicans (2/19 (10.5%)). A preponderance of the isolates shows a good susceptibility profile to itraconazole, ketoconazole, and clotrimazole, but demonstrated high resistance to fluconazole. Age and use of oral hypoglycemic agents were some of the risk factors identified. Conclusion: The study shows a high prevalence of onychomycosis among PLWDM in Kano and calls for routine screening to minimize or eliminate all possible complications.

Clinical Course, Antifungal Susceptibility, and Genomic Sequencing of Trichophyton indotineae

Trichophyton indotineae is an emerging dermatophyte causing outbreaks of extensive tinea infections often unresponsive to terbinafine. This species has been detected worldwide and in multiple US states, yet detailed US data on infections with T indotineae are sparse and could improve treatment practices and medical understanding of transmission. To correlate clinical features of T indotineae infections with in vitro antifungal susceptibility testing results, squalene epoxidase gene sequence variations, and isolate relatedness using whole-genome sequencing. This retrospective cohort study of patients with T indotineae infections in New York City spanned May 2022 to May 2023. Patients with confirmed T indotineae infections were recruited from 6 New York City medical centers. Improvement or resolution at the last follow-up assessment. Among 11 patients with T indotineae (6 male and 5 female patients; median [range] age, 39 [10-65] years), 2 were pregnant; 1 had lymphoma; and the remainder were immunocompetent. Nine patients reported previous travel to Bangladesh. All had widespread lesions with variable scale and inflammation, topical antifungal monotherapy failure, and diagnostic delays (range, 3-42 months). Terbinafine treatment failed in 7 patients at standard doses (250 mg daily) for prolonged duration; these patients also had isolates with amino acid substitutions at positions 393 (L393S) or 397 (F397L) in squalene epoxidase that correlated with elevated terbinafine minimum inhibitory concentrations of 0.5 μg/mL or higher. Patients who were treated with fluconazole and griseofulvin improved in 2 of 4 and 2 of 5 instances, respectively, without correlation between outcomes and antifungal minimum inhibitory concentrations. Furthermore, 5 of 7 patients treated with itraconazole cleared or had improvement at the last follow-up, and 2 of 7 were lost to follow-up or stopped treatment. Based on whole-genome sequencing analysis, US isolates formed a cluster distinct from Indian isolates. The results of this case series suggest that disease severity, diagnostic delays, and lack of response to typically used doses and durations of antifungals for tinea were common in this primarily immunocompetent patient cohort with T indotineae, consistent with published data. Itraconazole was generally effective, and the acquisition of infection was likely in Bangladesh.

Mapping the Global Spread of T. indotineae: An Update on Antifungal Resistance, Mutations, and Strategies for Effective Management.

The global spread of Trichophyton indotineae presents a pressing challenge in dermatophytosis management. This systematic review explores the current landscape of T. indotineae infections, emphasizing resistance patterns, susceptibility testing, mutational analysis, and management strategies. A literature search was conducted in November 2023 using Embase, PubMed, Scopus, and Web of Science databases. Inclusion criteria covered clinical trials, observational studies, case series, or case reports with T. indotineae diagnosis through molecular methods. Reports on resistance mechanisms, antifungal susceptibility testing, and management were used for data extraction. A total of 1148 articles were identified through the systematic search process, with 45 meeting the inclusion criteria. The global spread of T. indotineae is evident, with cases reported in numerous new countries in 2023. Tentative epidemiological cut-off values (ECOFFs) suggested by several groups provide insights into the likelihood of clinical resistance. The presence of specific mutations, particularly Phe397Leu, correlate with higher minimum inhibitory concentrations (MICs), indicating potential clinical resistance. Azole resistance has also been reported and investigated in T. indotineae, and is a growing concern. Nevertheless, itraconazole continues to be an alternative therapy. Recommendations for management include oral or combination therapies and individualized approaches based on mutational analysis and susceptibility testing. Trichophyton indotineae poses a complex clinical scenario, necessitating enhanced surveillance, improved diagnostics, and cautious antifungal use. The absence of established clinical breakpoints for dermatophytes underscores the need for further research in this challenging field.