Emergence of the novel sixth Candida auris Clade VI in Bangladesh

ABSTRACT 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. IMPORTANCE Candida 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.


Patient information
Ten C. auris isolates, representing eight ICU patients and two NICU patients, were described in this study.Patients were admitted with various underlying health complica tions; none had candidemia (File S1).Among the patients, only one patient (101022) from the ICU had a previous travel history to the United Arab Emirates within 1 year of enrollment.Overall, 40% (four patients from ICU) developed nosocomial colonization with C. auris, and none received any antifungal treatment since patients either expired or were discharged by the time the test result was released (Table 1).

WGS SNP calling and phylogeny analysis: emergence of a novel clade
Whole genome-based SNP calling was performed to distinguish all BD isolates in different clades, incorporating a total of 25 C. auris isolates that represent the known five clades (File S2).Around 92.68% (SD ± 1.17) reads of BD isolates mapped with the reference B11205, with an average GC content of 44.41% (SD ± 0.15; File S3).Phyloge netic analysis revealed that BD-ICU isolates (sample IDs 101019, 101008, 101009, 101022, 101077, 101082, 101101, and 101087) clustered closely with Clade I with a maximum SNP difference of 163 SNPs (Fig. 1; File S4).Notably, these isolates differed by only ≤17 SNPs from each other except isolates 101019 and 101022.The isolate (101022) collected from the ICU patient who traveled to UAE was separated by only 18 SNPs from the UAE_B14000 isolate.Isolate 101019 had the highest SNP differences with other Clade I BD-ICU isolates (87-102 SNPs).Within the Clade I clusters, Clade I BD isolates had closer resemblance to UK_15B5 isolate (51-86 SNPs) than those from India (108-155 SNPs), Pakistan (109-148 SNPs), and New Jersey, USA (124-163).Interestingly, the BD-NICU isolates (102096 and 102170) clustered separately from all known clade isolates (Fig. 1).These two isolates differed by only 68 SNPs and were genetically distinct from all the clades by at least 42,447 single-nucleotide polymorphisms and, hence, formed a novel clade, Clade VI (7).South American Clade (Clade IV) was the closest neighbor (>42,447 SNPs) for Clade VI, whereas the Iranian Clade (Clade V) was the most distant neighbor (>254,068 SNPs).To pinpoint the origin of this novel clade, we inquired the neonates' parents about any international travel history, but none had any in 2020 or before.The two neonates were born in different hospitals, but when the condition of perinatal asphyxia worsened, they were transferred to the NICU of study hospital.It was unknown whether their parents were suffering from fungal colonization or infections; or whether these neonates acquired C. auris colonization during birth or from the previous hospitals.To investigate the emergence of this novel clade, phenotypic differences between the Clade I and Clade VI isolates were observed and are described in

Molecular-resistance mechanisms
All the BD isolates were screened for mutations associated with antifungal resistance.For fluconazole and other triazole resistance, ERG11, CDR1, TAC1B, and MRR1A gene mutations were screened in several positions leading to increased minimum inhibi tion concentration (MIC) values.All BD-ICU isolates of Clade I harbored ERG11_Y132F substitution.No mutations were detected in CDR1, TAC1B, and MRR1A genes.No known FKS1 gene mutation was observed in BD-ICU isolates which is reflected by susceptibility to caspofungin in antifungal susceptability testing results (Table 3).FCY1_S70R mutation which confers 5-flucytocine resistance was detected in all BD-ICU isolates by genomic analysis.No mutations in ERG3 and ERG6 genes were detected which could explain the amphotericin B resistance in isolate 101022.Surprisingly, Clade VI isolates from BD-NICU patients had no known mutations in any of the drug-resistance genes (Table 3).

Antifungal susceptibility testing
Antifungal susceptibility testing (AFST) was performed on all BD C. auris isolates against fluconazole, voriconazole, caspofungin, and amphotericin B (Table 3).Based on U.S.   4).Mutational analysis on the resistance profile aligned with the AFST results except for isolate 101022 and 102170; no known mutation was found that confer amphotericin B resistance.

DISCUSSION
C. auris, first detected in Bangladesh in 2019, has emerged as a global multidrug-resistant healthcare-associated pathogen (29).The WGS-based approach revealed the circulation  The genome sequencing approach separated the clades of C. auris by tens of thousands of SNP differences (7).The ~42,447 SNPs difference of BD-NICU isolates (102096 and 102170) indicate the emergence of the new clade, Clade VI, in Bangladesh.Though tracing was not possible to identify the source of this nosocomial infection in neonates, it is evident that Clade VI is circulating in Bangladesh.By the time of writing this paper, a preprint published by a group of researchers from Singapore claimed the presence of a new clade separated by >36,000 SNPs from all known five clades of C. auris (31).These isolates, namely, isolate A (LIMS ID F3485, Biosample SAMN36753178), isolate B (LIMS ID F1580, Biosample SAMN36753179), isolate C (LIMS ID F0083, Biosample SAMN36753180), and 102096 from our study (SRR24877248) were distinct by at least 70 SNPs (31).Isolate C of the novel clade was collected from the blood of a Bangladeshi patient in 2018, who was airlifted directly to Singapore General Hospital for treatment and was initially classified as Clade IV (30).The presence of candidemia in this patient indicates the undetected circulation of novel C. auris clade in Bangladesh and a later transfer to Singapore in 2018 before the first detection of C. auris in Bangladesh in 2019  Clade I BD-ICU isolates showed the general trend of azole resistance for both mutational (genotypic) and AFST (phenotypic) results against fluconazole and voricona zole (7).Surprisingly, these isolates showed inconsistency for 5-flucytosine resistance pattern except for isolate 101019.A similar observation was reported from a 29-year-old woman residing in New Jersey, who was diagnosed with severe short bowel syndrome (20).She underwent a multi-visceral transplantation surgery, and during her post-opera tive period of 72 days, the patient developed refractory fungal peritonitis.Nineteen C. auris isolates were obtained from the patient at various time intervals.While the initial 12 isolates collected within the initial 33 days demonstrated sensitivity to 5-flucytosine, the subsequent seven isolates obtained between days 33 and 72 exhibited resistance to this antifungal agent.Genomic analysis of these isolates revealed the presence of the FCY1_S70R mutation in all 19 isolates.The authors argued FCY1_S70R mutation to be inconsequential in describing 5-flucytosine resistance.The findings in our study reflected the discrepancy between FCY1_S70R mutation and resistance pattern for Clade I isolates, strengthening the argument of inconsequential behavior of FCY1_S70R mutation in describing the 5-flucytosine resistance.Two BD-NICU isolates representing Clade VI exhibited no resistance to the antifungals tested (except amphotericin B for isolate 102170) and harbored no known mutation conferring resistance.Active clinical surveillance, monitoring of clinical symptoms, and extensive epidemiological investiga tion will accelerate understanding of the significance of this newly discovered sixth clade of C. auris.

Limitations
Our study presents the WGS-based approach to detect the circulation of C. auris clades for the first time in Bangladesh.The isolates sequenced in this study were collected from the archived samples in 2021 as part of regular surveillance to monitor infections and colonization associated with ICU/NICU admitted patients.Tracing the contacts of C. auris-positive cases was not performed at that time.The small sample size in this study may not be the representative picture of C. auris circulation in Bangladesh.Including a large sample size would increase the chances of capturing other undetected clades circulating in Bangladesh.Long-read sequencing would ensure the recovery of the nearly full genome and the comprehensive genomic study on the antifungal resistance profile.Overall, our study demonstrates the necessity of C. auris genomic surveillance to understand the transmission dynamics, pathobiology, and containment of this urgent antimicrobial-resistant threat in Bangladesh.

Conclusion
We report the circulation of Clade I C. auris in Bangladesh along with the emergence of the novel Clade VI detected patients in the ICU and NICU.Bangladesh remains the hub of this novel clade since a Clade VI isolate was also found in the blood of a Bangladeshi patient airlifted in Singapore in 2018.It is critical that we enforce nation-wide genomic surveillance to better understand the emergence and transmission of this novel clade in Bangladesh.

Isolate selection
From August to September 2021, as part of ongoing hospital surveillance, we identified a total of 60 C. auris isolates and archived these isolates at the International Center for Diarrhoeal Disease Research, Bangladesh (icddr,b) Mycology Laboratory.Isolates were cultured from skin swab samples from both axilla and groin for screening the patients admitted in the ICU and NICU of two tertiary-care hospitals (one public and one private).In addition to the on-admission screening sample, subsequent follow-up samples were collected-after 72 hours of admission and before shifting from ICU-from patients to identify nosocomial colonization (32).Isolates from ICU patients were termed as BD-ICU, and isolates from NICU patients were termed BD-NICU throughout the paper.

DNA extraction and whole-genome sequencing
Ten C. auris isolates were randomly selected and retrieved from −80°C storage and grown on CHROMagar Candida Plus media for presumptive identification of C. auris.Pure C. auris colonies were transferred from CHROMagar plate to Sabouraud dextrose agar (SDA) plate and incubated at 37°C for 72 hours.Genomic DNA of the C. auris isolates was extracted using the Qiagen DNAeasy Blood and Tissue Kit, and the Illumina DNA Prep kit (Illumina Inc., San Diego, California, USA) was used for the construction of DNA libraries.Sequencing was performed on Illumina NextSeq 550 platform with 2 × 150 bp cycles at icddr,b Genome Center.

Variant calling and phylogenetic analysis
To investigate the origin and possible route of transmission of sequenced C. auris isolates from Bangladesh, phylogenetic analysis was performed using the whole-genome sequences.A total of 25 isolates representative of C. auris Clades I-V were also inclu ded in this analysis (File S2).Quality control, SNP calling, and phylogenetic analysis were performed using the reference-based pipeline, MycoSNP, v1.4 (https://github.com/CDCgov/mycosnp-nf) with default parameters and using assembly B11205 (accession no.GCA_016772135.1) generated from an isolate from India (33).In brief, the MycoSNP workflow masks repetitive regions and indexes the reference genome in preparation for read alignment and SNP calling.Paired-end reads were trimmed and filtered for low-quality data.Samples, with at least 20× coverage and a % GC content between 42% and 47.5%, were used for alignment to the reference genome using the BWA (34) and pre-processed for downstream SNP analysis using GATK (35).GATK HaplotypeCal ler function is used for haplotype calling the processed BAM files from each isolate.The ploidy parameter for C. auris is set to haploid.HaplotypeCaller uses the PairHMM (pair hidden Markov models) algorithm and performs pairwise alignment.Then, the resulting individual gVCF files are merged into a single multi-sample gVCF file by CombineGVCFs function.Next, the GenotypeGVCFs function performs SNP calling from this single gVCF file which results in a VCF file with all samples genotyped.GATK's VariantFiltration function was used to filter SNP sites based on the QualityByDepth (QD), FisherStrand score (FS), and RMSMappingQuality (MQ) parameters using filtering expression: "QD < 2.0 || FS > 60.0 || MQ < 40.0." Additional customized SNP filters were applied based on parameters such as genotype quality (GQ), allele depth (AD), and depth (DP) using the following criterion: GQ ≥ 50, AD ≥ 80%, and DP ≥ 10.The MycoSNP workflow recovered all SNP alleles from the VCF files using a Python script "vcfSnpsToFasta.py"(https://github.com/broadinstitute/broad-fungalgroup/tree/master/scripts/SNPs) to create an SNP alignment in multi-fasta format (33).
To construct maximum likelihood (ML) phylogenetic trees from SNP alignment, RAxML v8.2.12 (36) was utilized with the generalized time-reversible model and a gamma distribution (GTRGAMMA) to account for site-specific rate variation.Support for the ML phylogenetic trees was evaluated through 1,000 bootstrap replicates of the alignment.The resulting trees were visualized using iTol v5.5 (37).
The C. auris isolates from this study were also tested for susceptibility to 5-flucytosine by the disk diffusion method outlined by CLSI-M60 guidelines (43).The assays were performed using fungal suspension adjusted to match the turbidity of a 0.5 McFarland standard.The antifungal disk for Flucytosine 10 µg was purchased from Liofilchem, Italy.Mueller Hinton II Agar (BD), SDA (BD), and Roswell Park Memorial Institute Medium 1640 (RPMI-1640; Sigma-Aldrich) were used for the preparation of media of the C. auris isolates.The reference and lot number of corresponding agar and media are provided in File S6.The disks were placed on to the inoculum media, and the plates were incubated at 35°C (±2°C) for 18-24 hours.After 24 hours, the plates were examined, and the zones of inhibition diameter around the discs were measured in millimeters (mm).The inhibition zone diameters (mm) were interpreted as susceptible (S), intermediate (I), and resistant (R) based on established clinical breakpoints provided by CLSI.Susceptibility to 5-flucytosine was also tested via automated VITEK2 system using an AST-YS09 card as per the manufacturer's instructions.The time, and temperature of incubation was similar to disk diffusion assay (18−24 hours at 35°C ± 2°C), but the results were expressed as MICs.The MIC calling range was 1 to 64 mg/L for 5-flucytosine.Since the VITEK2 system lacks species-specific MICs for C. auris, the species was modified to different Candida spp. to obtain MICs for C. auris isolates.Notably, the MIC values remained consistent irrespective of the species assigned when retrieving MIC data for C. auris across all drugs.As Candida duobushaemulonii is closely related to C. auris and belongs to the Candida haemulonii complex, analysis was based on MICs retrieved from this species (44,45).The findings and conclusions of this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control (CDC).
breakpoints, all BD-ICU isolates were highly resistant to fluconazole (MIC 256 mg/L) and voriconazole (MIC 32 mg/L).Isolate 101022 collected from patient with recent travel history to the United Arab Emirates additionally showed resistance to amphotericin B (MIC 2 mg/L) along with fluconazole and voriconazole.No resistance was observed for caspofungin in BD-ICU isolates.On the contrary, BD-NICU isolates repre senting Clade VI were susceptible to three major antifungal drug classes.However, BD-NICU isolate 102170 demonstrated resistance to amphotericin B (MIC 2 mg/L).Only isolate 101019 of Clade I harboring FCY1_S70R mutation showed resistance against 5flucytosine in both disk diffusion and automated VITEK2 methods, while other Clade I isolates showed sensitivity in both methods even though these harbor the same mutation (Table

FIG 1
FIG 1 Phylogenetic tree based on SNPs of 35 C. auris isolates.(A) Representation of known five clades with novel Clade VI of C. auris.(B) Distances between Clade IV and novel Clade VI.All the BD isolates from our study are represented by dark blue color.Eight BD-ICU isolates cluster with reference isolate, and five Clade I isolate from different countries.Two BD-NICU isolates (102096 and 102170) cluster together distinctly from other clades forming novel Clade VI.Isolate B11243 of Clade IV has the closest SNP distances from Clade VI isolates.(C) Clade-wise heatmap of SNP distances among all C. auris isolates used in this study.

TABLE 1
Patient metadata of sequenced C. auris isolates

TABLE 2
Phenotypic differences observed between BD Clade I and Clade VI isolates of C. auris at 24, 48, and 72 hours (12,30)e I lineages and the emergence of a new Clade VI in Bangladesh.Six BD-ICU Clade I isolates (101008, 101009, 101077, 101082, 101101, and 101087) collected from adult patients, who never traveled overseas, are phylogenetically closest to each other (SNPs range 3-17), indicating the ongoing local and clonal transmission(12,30).The isolate 101022 from an ICU patient who previously traveled to UAE and died after admission was only 18 SNPs different than UAE_B14000, indicating possible Clade I transmission from UAE to Bangladesh.

TABLE 3
Results of known mutation analysis and antifungal susceptibility test for azoles, polyenes, and echinocandins

TABLE 4
Results of 5-flucytosine antifungal susceptibility test by disk diffusion and VITEK2 method 28).It is surprising to note that even though Clade I and Clade VI isolates are circulat ing in Bangladesh simultaneously, Clade VI isolates acquired no known mutation and showed no phenotypic resistance except amphotericin B (isolate 102170).It is of utmost importance to conduct nationwide genomic surveillance of prospectively collected C. auris isolates along with archived isolates stored at different hospitals in Bangladesh to understand the origin and transmission dynamics of Clade VI.
a S = susceptible, I = intermediate, and R = resistant.The categorization of resistance was based on established clinical breakpoints provided by the Clinical and Laboratory Standards Institute.b-, absent.(