Method and kit for identifying drug-resistant fungal pathogen

Abstract

Provided is a method for identifying a drug-resistant fungal pathogen. The method includes: detecting a single nucleotide polymorphism genotype on XYR1 gene and SNQ2 gene of a fungal pathogen. Further provided is a kit for identifying drug-resistant fungal pathogen. The kit includes: a lysis buffer for breaking down the fungal pathogen; and a primer for detecting XYR1 gene and / or SNQ2 gene of a fungal pathogen.

Description

TITLEMETHOD AND KIT FOR IDENTIFYING DRUG-RESISTANT FUNGAL PATHOGENCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 550,611, filed on February 7th, 2024. The content of the application is incorporated herein by reference.BACKGROUND OF THE INVENTION1. FIELD OF THE INVENTION

[0002] The present application hereby incorporates by reference the entire contents of the text file named “NHR-P0029-PUS-Sequencing Listing. xml” in XML format. The text file containing the Sequencing Listing of the present application was created on Jan. 22, 2025 and is 45,519 bytes in size.

[0003] The present relates to a method and a kit for identifying drug-resistant fungal pathogen, and particularly to a method and a kit for identifying drug-resistant Candida tropicalis with clade 4 genotype.2. DESCRIPTION OF THE PRIOR ART

[0004] Approximately 2.5 million deaths are related to fungal infections annually, posing serious public health problems, and drug-resistant pathogenic fungi are increasing due to the extensive use of drugs. The number of fungal infection cases is underestimated since the difficulty of diagnosis. The estimated cost of fungal infections in the United States was approximately USD 11.5 billion in 2019, of which medical costs accounted for USD 7.5 billion.

[0005] Candida species (spp.) are typically harmless eukaryotic commensal yeasts found in various environmental, human, and other mammalian sources. Within humans, Candida spp. play a vital role as a part of the microflora present on the skin, oral, and vaginal mucosa, as well as within the gastrointestinal tract. However, in cases of severely compromised immunity, Candida spp. may transform into opportunistic pathogens, leading to illness and even lethality. The drug-resistant clone of Candida spp. is spreading in many countries. Currently, whole genome sequencing or conventional biochemical identification is commonly used to identify the predominant drug-resistant genotype of Candida spp. However, these methods are time and cost consuming.

[0006] In view of the foregoing, there is an unmet need in the art to develop a rapid detection method to identify the predominant drug-resistant genotype of Candida spp. Therefore, this information may help healthcare professionals to determine appropriate antifungal drugs for the treatment of fungal infections.SUMMARY OF THE INVENTION

[0007] In view of the foregoing, the present disclosure provides a method for identifying a drug-resistant fungal pathogen. The method includes: detecting a single nucleotide polymorphism genotype on XYR1 gene and SNQ2 gene of a fungal pathogen.

[0008] The present disclosure further provides a kit for identifying a drug-resistant fungal pathogen. The kit includes: a lysis buffer for breaking down a fungal pathogen; and a primer for detecting XYR1 gene and / or SNQ2 gene of the fungal pathogen.

[0009] The objectives of the present disclosure will become readily understandable to those of ordinary skill in the art after reading the following detailed description of the embodiments that are illustrated in the various figures and drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0011] FIG. 1 is a schematic representation of the two XYR1 genes according to an embodiment of the present disclosure.

[0012] FIG. 2 is a box plot illustrating the distance of intragenotype and intergenotype for two XYR1 genes according to an embodiment of the present disclosure (the box represents the interquartile range, with the lines extending the range of the data; the points outside the range are mild outliers, with values greater than 1.5x the upper bound of the interquartile range; and “**” indicates p value < 0.01).

[0013] FIG. 3 A is an image illustrating amplification of target genes by multiplex polymerase chain reaction (PCR) according to an embodiment of the present disclosure (the template for the multiplex PCR is ATCC750; and the nine amplicons are divided into three groups based on sizeof PCR product, including Group 1 (blue, -750 bp): ICL1, MDR1, SAPT2, SAPT4, ZWFla, and SNQ2,' Group 2 (green, -1 kb): CTRG 05978 and GTRG 05993; and Group 3 (orange, -1.5 kb): ERG1 ).

[0014] FIG. 3B is an image illustrating results of multiplex PCR at two annealing temperatures, 60°C (lanes 1 and 2) and 61 °C (lanes 3 and 4) according to an embodiment of the present disclosure (negative controls are shown in lanes 2 and 4).

[0015] FIG. 4 is a bar chart showing the percentage of reads for nine amplicons across seven isolates according to an embodiment of the present disclosure (percentage are calculated using Samtools to determine the identity of individual reads).

[0016] FIG. 5A is a box plot illustrating distribution of read percentages for six MLST target genes (SAPT2, SAPT4, XYR1, ZWF1, ERG11, SNQ2) across 96 samples according to an embodiment of the present disclosure (the boxplot represents the interquartile range for read percentages of each gene, with the median marked by a line inside the box; and whiskers extend to the minimum and maximum values of the data, and individual outliers are plotted).

[0017] FIG. 5B is bar chart illustrating sequencing success rates for six MLST genes under four single nucleotide polymorphism (SNP) filter thresholds using multiplex PCR-MinlON sequencing across 96 C. tropicalis isolates according to an embodiment of the present disclosure (bars represent percentage of accuracy compared to available sequencing results in the art using bcltools; and the four SNP filter thresholds include: Filter 1 : minimum 40% reads for all MLST genes, except XYR1 (20%). Filter 2: minimum 30% reads for all MLST except AI7?7(15%). Filter 3 : minimum 25% reads for all MLST gens, except XYR1 (10%). Filter 4: minimum 10% reads for all MLST genes).

[0018] FIG. 5C is bar chart illustrating optimization of read counts for accurate assembly in multiplex PCR-MinlON sequencing according to an embodiment of the present disclosure (accuracy rates for identifying six MLST gene fragments in 96 C. tropicalis isolates under varying read count thresholds (5,000, 7,000, 10,000, 12,000) with a consistent 10% SNP filter threshold is applied).

[0019] Left panel of FIG. 6 is an allele discrimination plot for CTRG 05978 displaying alleles as GA / GA homozygous (lower right red cluster), GA / AA heterozygous (middle green cluster),and AA / AA homozygous (upper left blue cluster) according to an embodiment of the present disclosure (the middle green cluster strains correspond to the CTRG 05978 type IV genotype (GA / AA), and 6 strains are located in the middle green cluster, including YM140156, YM180233, YM180351, YM180473, YM180681, and YM180969).

[0020] Right panel of FIG. 6 is an allele discrimination plot for SNQ2 A2977G, presenting alleles as G / G homozygous (lower right red cluster), G / A heterozygous (middle green cluster), and A / A homozygous (upper left blue cluster) according to an embodiment of the present disclosure (eight strains exhibit mutations in SNQ2 mutation; and by combining the results of CTRG 05978 and SNQ2 genotypes using the TaqMan Genotyping Assay, the identification of CTRG_05978 type IV and SNQ2 A2977G becomes straightforward).

[0021] FIG. 7 is a schematic workflow illustrating diagnosis and management of the clade 4 C. tropicalis by detecting CTRG 05978 SNP and SNQ2 N2977G mutation according to an embodiment of the present disclosure (the SNPs are marked by red arrows; and yeasts in red refers predominant drug-resistant clade 4 genotype and in other colors represents different genotypes).DETAILED DESCRIPTION

[0022] The following embodiments are provided to illustrate the present disclosure in detail. A person having ordinary skills in the art can easily understand the advantages and effects of the present disclosure after reading the disclosure of this specification, and also can implement or apply in other different embodiments. Therefore, it is possible to modify and / or alter the following embodiments for carrying out this disclosure without contravening its scope for different aspects and applications, and any element or method within the scope of the present disclosure disclosed herein can combine with any other element or method disclosed in any embodiments of the present disclosure.

[0023] In order that the present disclosure may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of the present disclosure.

[0024] As used herein, the singular forms “a,” “an,” and “the” include plural referents, unless expressly and unequivocally limited to one referent. The term “or” is used interchangeably withthe term “and / or” unless the context clearly indicates otherwise.

[0025] As used herein, the term “comprising,” “comprises” “include,” “including,” “have,” “having,” “contain,” “containing,” and any other variations thereof are intended to cover a non-exclusive inclusion. For example, when describing an object “comprises” a limitation, unless otherwise specified, it may additionally include other ingredients, elements, components, structures, regions, parts, allele, locus, steps, or connections, etc., and should not exclude other limitations.

[0026] In at least one embodiment of the present disclosure, the single nucleotide polymorphism genotype on the XYR1 gene may include a sequence of ATRA, and R is A or G. In some embodiments, the sequence of ATRA may be a first 4-mer of the XYR1 gene.

[0027] In at least one embodiment of the present disclosure, the single nucleotide polymorphism genotype on the SNQ2 gene may include a nucleotide A at position 2977 substituted with a nucleotide G.

[0028] In at least one embodiment of the present disclosure, the fungal pathogen may be a non-albicans Candida species. In some embodiments, the non-albicans Candida species is at least one selected from the group consisting of Candida tropicalis. Candida parapsilosis. Candida glabrata, Candida auris. Candida gtiilliermondii. and Candida krusei. In some embodiments, the non-albicans Candida species may be Candida tropicalis. In some embodiments, the Candida tropicalis may include a clade 4 genotype.

[0029] In at least one embodiment of the present disclosure, the drug-resistant fungal pathogen may have a drug resistance against a drug comprising azole.

[0030] In at least one embodiment of the present disclosure, the single nucleotide polymorphism genotype on XYR1 gene and SNQ2 gene of the fungal pathogen may be detected by high-throughput identification. In some embodiments, the high-throughput identification may include combining a multiplex polymerase chain reaction approach and a MinlON nanopore sequencing approach.

[0031] In at least one embodiment of the present disclosure, the single nucleotide polymorphism genotype on XYR1 gene and SNQ2 gene of the fungal pathogen may be detectedby a TaqMan genotyping assay.

[0032] In at least one embodiment of the present disclosure, the kit may further include a DNA polymerase; and a PCR buffer comprising Mg2+.

[0033] In some embodiments, among the Candida spp. found in humans, Candida albicans is the most frequently isolated in hospital settings resulting in nosocomial infections. However, there has been a shift toward the more-treatment-resistant non-albicans Candida spp., such as Candida tropi calis. Candida parapsi losis. Candida glabrata, and Candida krusei. The prevalence of these species may vary based on the geographical location. Candida tropicalis (C. tropicalis) stands out among the non-albicans Candida species as the second most or even the most prevalent pathogenic yeast in some regions, such as Asia and Latin America.

[0034] In some embodiments, molecular epidemiological investigation of C. tropicalis may be instrumental in identifying epidemic genotype, potential modes of transmission, genetic composition of drug-resistant strains, biological habitats, and population structure thereof. The multilocus sequence typing (MLST) has been developed to characterize population structures within a species and distinguish geographical origins. The combinations of alleles at different loci yield unique diploid sequence types (DSTs), enabling differentiation and classification of genetic relatedness among strains. The present MLST approach for C. tropicalis utilizes six genes, including ICL1, MDR1, SAPT2, SAPT4, XYR1, and ZWFla. Moreover, MLST has been extensively employed to assess the clonality of C. tropicalis and ascertain various traits, such as fluconazole susceptibility, hospital-based origins, and anatomical sources. Finally, the allelic profiles of isolates may be compared by utilizing the online MLST database to explore diverse geographical origins, drug resistance transmission, and genetic variation patterns.

[0035] In some embodiments, MLST has been also exerted to uncover that fluconazole-resistant C. tropicalis strains (isolates) share the same genotype, clade 4, underscoring the utility of MLST typing as a method for distinguishing drug-resistant C. tropicalis strains. Although MLST offers the easy application in determining the genetic type of C. tropicalis, the need for rapid identification and more comprehensive characterization of drug-resistant strains (isolates) remains vital in the realms of diagnosis and antifungal stewardship. Therefore, the present disclosure may elucidate the genotypes of one of XYR1 genes, CTRG 05978, and SNQ2 which is situated 25 kb upstream from CTRG 05978 and has been linked to drug susceptibility in Nakaseomyces glabratus (Candida glabrala) and Candida aurisare the potential markers enabling the rapid identification of azole-resistant C. tropicalis, contributing to the prevention of further dissemination of the predominant clade 4 strains (isolates).EXAMPLES

[0036] Exemplary embodiments of the present disclosure are further described in the following examples, which should not be construed to limit the scope of the present disclosure.Materials and MethodsC. tropicalis strains isolation

[0037] The methods for isolating C. tropicalis from the environment are described in Taiwan surveillance of Antimicrobial Resistance of Yeasts (TSARY) studies (Yang, Y.L., Lin, C.C., Chang, T.P., Lauderdale, T.L., Chen, H.T., Lee, C.F., Hsieh, C.W., Chen, P.C. and Lo, H.J. (2012) Comparison of human and soil Candida tropicalis isolates with reduced susceptibility to fluconazole. PloS one, 7, e34609; and Zhou, Z.L., Lin, C.C., Chu, W.L., Yang, YL. and Lo, H.J. (2016) The distribution and drug susceptibilities of clinical Candida species in TSARY 2014. Diagnostic microbiology and infectious disease, 86, 399-404). All yeast strains are identified by the sequences of the rDNA (Leaw, S.N., Chang, H.C., Barton, R., Bouchara, J.P. and Chang, T.C. (2007) Identification of medically important Candida and non-Candida yeast species by an oligonucleotide array. Journal of clinical microbiology, 45, 2220-2229). In order to make sure the representative population of C. tropicalis, at least 10 strains, each of 11 most common clades including 37 strains having whole-genome data, are analyzed. A total of 120 C. tropicalis strains, including ATCC750 and 119 from TSARY (Table 5) are analyzed initially to identify potential markers. To verify, 161 strains including all clade 4 whole-genome data that may be found from the NCBI database along with 10 other clades containing the most DST members are further analyzed.DNA extraction and MLST analysis of C. tropicalis strains

[0038] Genomic DNA of each C. tropicalis strain (isolate) is extracted using a Yeast Genomic DNA Kit (Geneaid) according to the manufacturer’s instructions, and DNA is quantified using the Qubit fluorometer (ThermoFisher Scientific). The DNA fragments of six genes, ICL1, MDR1,SAPT2, SAPT4, XYRL and ZWFla, are amplified and sequenced as described in previous studies (Yang, Y.L., Lin, C.C., Chang, T.P., Lauderdale, T.L., Chen, H.T., Lee, C.F., Hsieh, C.W., Chen, P.C. and Lo, H.J. (2012) Comparison of human and soil Candida tropicalis isolates with reduced susceptibility to fluconazole. PloS one, 7, e34609; and Zhou, Z.L., Tseng, K.Y, Chen, Y.Z., Tsai, D.J., Wu, C.J., Chen, Y.C., Peng, H.L., Yang, Y.L., Hsieh, L.Y, Chen, C.H. et al. (2022) Genetic relatedness among azole-resistant Candida tropicalis clinical strains in Taiwan from 2014 to 2018. International journal of antimicrobial agents, 59, 106592). The primers used are listed below (Table 1). The resultant sequences are aligned with BioNumerics 3.0 (Applied Maths, Kortrijk, Belgium) and compared with those in the database of C. tropicalis (http: / / pubmlst.org / website) to determine the level of sequence identities (DST).Table 1. Primers used in the present disclosure (“*” indicates position of translation initiation site of ATG as +1; “F” indicates forward primer; and “R” indicates reverse primer).Whole genome sequencing

[0039] The high molecular weight DNAs are used to construct a multiplexing Nanopore sequencing library using the Oxford Nanopore Technologies (ONT) Ligation Sequencing Kit (SQK-LSK109) and Native Barcoding Expansion Kit (EXP-NBD104), following ONT instructions. A standard 72-hour sequencing script is executed on MinKNOW; raw reads are collected then base called and demultiplexed using the standalone application, Guppy.Investigation of XYR1 by Nanopore

[0040] The raw reads of 17 C. tropicalis strains (isolates) are aligned to the XYR1 gene (XM 002546469.1) using minimap2 (Li 2018) to produce reads containing XYR1. The XYR / -containing reads are then de novo assembled using SMARTdenovo (version 1.0) (Liu et al. 2021) to form contigs.

[0041] The assembled Al7 / / -containing contigs are further polished with Medaka (version 1.5.0) using the raw reads and rearranged to maintain the same orientation as the C. tropicalis MYA-3404 genomic scaffold supercont3.11 (NW_003020040.1). The sequence analyses for the 17 Al7 / ? / -containing contigs reveal a similar schematic representation of the two XYR1 genes compared to the MYA-3404 strain.Multiple sequence alignments and evolutionary distances

[0042] After designing the primer sequences for sequencing the partial sequences of CTRG 05978 and CTRG 05993, 120 strains of C. tropicalis, including ATCC750, and 119 from clinical sources, are analyzed. The XYR1 DNA sequence is aligned using ClustalW multiple alignment in MEGA 10. Pairwise genetic distances are separated into intraspecific and interspecific parameters and calculated to characterize both intra-and interspecific variation within and between CTRG 05978 coding DNA sequence (CDS) and CTRG 05993 CDS by using MEGA10. The statistical significance (p-value), Wilcoxon Rank Sum, and Signed Rank Tests are determined by R software version 4.1.2 (Wilcox. test package).TaqMan SNP genotyping

[0043] The TaqMan SNP genotyping assay is performed using TaqMan Genotyping Master Mix (Applied Biosystems) and the ABI QuantStudioTM 6 Flex System (Applied Biosystems), following the manufacturer’s instructions. Thermal cycle conditions in CTRG 05978 are as follows: 1 cycle of 95 °C for 10 min, 40 cycles of 95 °C for 15 sec, 54 °C for 20 sec and 60 °C for 1 min, and 1 cycle of 60 °C for 1 min; and in SNQ2 are as follows: 1 cycle of 95 °C for 10 min, 40 cycles of 95 °C for 15 sec, 60 °C for 1 min, and 1 cycle of 60 °C for 1 min.Statistical analysis

[0044] SPSS software for Windows (version 12.0) is used to analyze the data. The null hypothesis that the frequencies of the MLST genome type and CTRG 05978 start codon region type are equal is investigated by the chi-square (%2) test. The chi-squared or Fisher’s exact test with 1 -tailed correction is applied for categorical variables. Logistic regression is applied to assess the independent effects of factors with values less than 0.05 in univariate analysis. A p-value less than 0.05 is considered significant.Multiplex polymerase chain reaction

[0045] Nine target gene fragments (i.e., ICL1, MDR1, SAPT2, SAPT4, XYR1, ZWFla, ERG11, and SNQ2) are amplified using multiplex PCR with the KOD -Multi & Epi-™ DNA polymerase (Toyobo, Osaka, Japan) in a 50-pl reaction. The reaction mixture comprises 25 pl of 2* PCRbuffer, including dNTPs (dATP, dGTP, dCTP, and dTTP) and Mg2+at a final concentration of 2.0 mM. Additionally, the mixture also contained 10 pl of primer mix, 1 pl of KOD -Multi & Epienzyme, anti-KOD DNA polymerase antibodies (to neutralize the polymerase and inhibit its 3'— >5' exonuclease activities), 5 pl of yeast lysate (in 0.02 N NaOH), and 14 pl of nuclease-free water. The primer mix comprises primers for ICL1, MDR1, SAPT2, SAPT4, XYR1, ZWFla, ERG11, and SNQ2 at concentrations of 0.2 pM, 0.1 pM, 0.2 pM, 0.15 pM, 0.15 pM, 0.2 pM, 0.15 pM, and 0.1 pM, respectively. Primer sequences are detailed in Table 2. The PCR protocol is as follows: initial denaturation at 94°C for 2 min, followed by 35 cycles of denaturation at 98°C for 10 sec, annealing at 60°C for 30 sec, extension at 68°C for 1 min and 45 sec, and a final extension at 68°C for 10 min.Table 2. The oligo and probes used in the present disclosure (“*” indicates position numbers indicate location of gene fragments according to the location of the ATG translation initiation site for each gene, designated as +1; “F” indicates forward primer; and “R” indicates reverse primer).Analysis of multiplex polymerase chain reaction

[0046] The Rapid Barcoding Kit 96 (SQK-RBK114.96) is used for rapid barcoding of the 96 samples. Each sample is mixed with 5 pl of multiplex PCR product, 2.5 pl of nuclease-free water, and 2.5 pl of a rapid barcode. The mixture is incubated at 30°C for 2 min, followed by 80°C for 2 min. All 96 barcoded DNA samples are pooled, and 120 pl of the pooled DNA is mixed with an equal volume of solid phase reversible immobilization (SPRI) beads. After 5 min of incubation with a hula mixer, the barcoded DNA is cleaned twice with 240 pl of freshly prepared 80% ethanol and eluted with 15 pl of elution buffer (EB). An aliquot of 800 ng of barcoded DNA is adjusted to a total volume of 11 pl with EB. One microliter of diluted rapid adaptor F is added to the barcoded DNA, and the mixture is incubated at room temperature for 5 min. BSA priming mix is added for optimal sequencing performance.

[0047] A pre-sequencing mix (PSM) is prepared by adding 37.5 pl of Sequencing Buffer and 25.5 pl of Library Beads (LIB) mixed immediately before use to a 12 pl DNA library. The PSM is loaded via the SpotON port into a primed Flow Cell (FLO-MINI 14) for sequencing. Basecalling and demultiplexing are performed in real-time using MinKNOW GUI implemented with GPU Gupp on a desktop PC with an NVIDIA RTX 3090 graphics card with 24 GB RAM to produce high-accuracy reads in FASTQ format. The default output set for MinKNOW is a FASTQ file containing 10,000 reads. To assemble MLST genes, ERG1R and SNQ2, the FASTQ file containing reads is polished with Medaka, and heterozygous sites are identified from the presence of variant sites between 10% to 90% in those 9 alleles.Assembly MLST gene

[0048] To evaluate the accuracy of consensus sequences generated from Multiplex PCR-MinlON sequencing data, compare the consensus sequences with available sequencing results in the art. A custom Python script is used to process Nanopore FASTQ files and analyze the impact of different single nucleotide polymorphism (SNP) filtering criteria based on the alternative allele ratio. The scripts perform sequence alignment using Minimap2, consensus calling with Medaka, and variant calling using Bcftools to generate VCF files containing SNPsand their alt / ref ratios.

[0049] The script is executed with appropriate command-line arguments to specify the input FASTQ file, reference FASTA file, and output directory. The outputs, including consensus sequences, filtered VCF files, and SNP ratio summaries, are compared to assess the effects of different SNP filtering and sequence modification criteria. The accuracy of the consensus sequences generated by the script is evaluated by aligning them to reference sequences and comparing SNP positions, base substitutions, and overall sequence concordance.ResultsXYR1 has the highest typing efficiency among six genes (i.e., ICL1, MDR1, SAPT2, SAPT4, XYR1, and ZWFla) included for MLST analysis

[0050] As the MLST method is capable of differentiating the predominant drug-resistant group, clade 4, the member with the highest typing efficiency emerges as a particularly valuable candidate for further analysis. Among the six gene fragments used for MLST, a total of 49 polymorphic sites are identified \n ICLl, 73 in MDRl, 93 in SAPT2, 106 in SAPT4, 63 in XYR1, and 49 in ZWFla, according to the C. tropicalis MLST database. XYR1 exhibits the highest typing efficiency, with the ability to distinguish 230 genotypes per polymorphism across 63 polymorphic sites (Table 3). The comprehensive analysis of XYR1 sequence, which exhibited the highest typing efficiency within the MLST analysis, is preformed subsequently.Table 3. Six Candida tropicalis fragments used for MLST.Two XYR1 genes are detected on one chromosome

[0051] Referring to FIG. 1, two XYR1 genes, CTRG 05978 and CTRG 05993 (GenBank accession number: NW_003020040.1), on chromosome VI with approximately 30 kb apart from each other in opposite orientations is found. The direction of the arrows indicates the direction of two XYR1 genes. Since the two XYR1 genes are high consensus, in order to differentiate the sequence of MLST fragments of two XYR1 genes, primer set HJL2808 / HJL3097 and HJL2808 / HJL3098 for CTRG 05978 and CTRG 05993, respectively, is employed. Seventeen different DST strains previously collected, as well as ATCC750, are analyzed, and all of them (such as MYA-3404) possess two XYR1 genes.Two XYR1 genes are likely to be ancestral duplications

[0052] Two XYR1 genes, CTRG 05978 and CTRG 05993 (GenBank accession number: NW_003020040.1), on chromosome VI with approximately 30 kb apart from each other in opposite orientations are detected (FIG. 1). A distance matrix analysis, including intra and inter-genotype distances for CTRG 05978 and CTRG 05993, is performed using MEGA X software (Kumar, S., Stecher, greater than ergenotype cohorts (median = 0.003, mean ± SD = 0.004 ± 0.005), the CTRG_05993 intergenotype cohorts (median = 0, mean ± SD = 0.002 ± 0.001), and the intragenotype cohorts (median = 0.005, mean ± SD = 0.009 ± 0.007) is generated. Notably, the largest distance is obtained from the intragenotype comparisons (0-0.022), while the distances within the CTRG 05993 intergenotype cohorts show relatively low values (0- 0.005) (FIG. 2).

[0053] The fact that the intragenotype distance is greater than the individual intergenotype distances of CTRG 05978 and CTRG 05993, suggesting that these two alleles may be ancestral duplications.The SNP of the translation initiation codon of CTRG 05978 associat with the predominant clade 4 azole-resistant C. tropicalis genotype

[0054] Due to the higher diversity of CTRG 05978, the sequence of CTRG 05978 is further analyzed. Several SNPs are in the translation initiation codon of CTRG 05978 (Table 4 and Table 5). One of these SNPs with G to A substitution in the start codon result in a loss-of-function mutation. A more comprehensive analysis of the first 60 nucleotides in thecodon sequences of CTRG 05978 across all 120 strains (isolates) is then conducted. Notably, the present disclosure reveals that approximately 82.5% (99 out of 120) of the strains (isolates) possess at least one allele of CTRG 05978 that harbors a loss-of-function mutation in Xyrl.Table 4. The sequence of the first 4-mer of CTRG 05978 and CTRG 05993.Table 5. SNPs of CTRG 05978 and SNQ2 (DST stands for diploid sequence type; WT stands for wild type; Hom stands for homozygous; and Het stands for heterozygous).

[0055] Furthermore, the first 4-mer of CTRG 05978 open reading frame may be classified into 7 different sequence types: ATAA, ATGA, ATGT, ATGW, ATRA, ATRW, and ATRW (Table 4). Interestingly, an association between the sequence of CTRG 05978 and DST by MLST is observed (Table 6 and Table 5). The majority of clade 4 strains tested, with the exception of YM180542 (DST506) and YM180921 (DST508), exhibit the genotype IV (ATRA) sequence (36 / 48 vs. 2 / 72, p < 0.001). This significant association between the sequence of CTRG_05978 and the clade 4 genotype was further confirmed by logistic regression analysis (p < 0.001; OR = 96.92) (Table 7).Table 6. The association of genotype and sequence types of CTRG 05978 among 120 C. tropicalis isolates.Table 7. Logistic regression for type IV and clade 4 (OR stands for Odds ratios; and CI stands for confidence interval).The SNP of SNQ2 is associated with the SNP of the sequence type CTRG 05978, and their combination effectively distinguish the Clade 4 genotype

[0056] Since the clade 4 being identified as the primary azole-resistant genotype, an additional investigation to determine the potential involvement of other gene(s) associated with drug resistance is embarked. The present disclosure uncovers a missense mutation I993V due to the SNP (A2977G) in SNQ2, which is situated 25 kb upstream from CTRG_05978, which has been linked to drug susceptibility in N. glabratus (C. glabrata) and Candida auris. A possible connection between the mutation in SNQ2 and the sequence profile of CTRG 05978 is suggested (Table 8 and Table 5).Table 8. The association of genotype and SNP of SNQ2 at 2977 among 120 C. tropicalis isolates.

[0057] The SNQ2 A2977G having high association with the genotype IV of CTRG 05978 (35 / 43 vs. 13 / 77, p < 0.001, at phi = 0.631) and the clade 4 genotype (36 / 43 vs. 2 / 77, p < 0.001, at phi = 0.820) is observed. The combination of the SNQ2 A2977G and the genotype IV of CTRG 05978 appears to be effective in distinguishing the clade 4 C. tropicalis strains (35 / 35 vs. 3 / 85, p < 0.001), and the relationship exhibits a notably high association (phi = 0.943).

[0058] To obtain a more comprehensive understanding of whether the SNPs of SNQ2 and CTRG 5978 may efficiently identify the clade 4 in C. tropicalis, a thorough analysis of whole-genome data from 161 C. tropicalis strains available in the NCBI database (https: / / www.ncbi.nlm.nih.gov / sra) is extended. These strains encompass geographic variations, with the majority originating from Asia (76%), followed by Oceania (15%), Europe (5%), and North America (3%).

[0059] Overall, the present disclosure reveals a significant prevalence of the SNQ2 A2977G and CTRG 05978 genotype IV among the clade 4 strains (Table 9). SNQ2 and CTRG 05978, indeed, may serve as markers for identifying clade 4 C. tropicalis (95 / 96 vs. 22 / 185, p < 0.001, at phi = 0.838).Table 9. The association of genotype and SNPs of SNQ2 and CTRG 05978 among 281 C. tropicalis isolates

[0060] Combination of SNP of CTRG 05978 and SNQ2 may effectively distinguish the clade 4 C. tropicalis strains from others, which the specificity of identification increases from 0.877 to 0.991, allowing for a better discrimination of clade 4 C. tropicalis strains (Table 10).Table 10. The sensitivity and specificity of identifying clade 4 C. tropicalis strains with SNP of CTRG 05978 and / or SNQ2.

[0061] The sensitivity (true positive rate or recall) is calculated as: sensitivity = True Positives (TP) / (True Positives (TP) + False Negatives (FN). Specificity (true negative rate) measures theproportion of actual negative cases correctly classified, and is determined as: specificity = True Negatives (TN) / (True Negatives (TN) + False Positives (FP).Multiplex PCR of nine genes is established

[0062] To better understand and manage C. tropicalis infection, rapid diagnosis alone is insufficient; efficient, cost-effective, and high-throughput methods are also required for comprehensive surveillance. A multiplex PCR approach targeting nine genes, including six genes of MLST, SNQ2, CTRG 05978, and ERG11 using 17 specific primers is employed (Table 2). These nine genes are amplified either independently or simultaneously, as demonstrated in FIG. 3A and FIG. 3B, respectively. When the nine genes are amplified independently, these genes were divided into three groups based on DNA fragment lengths (FIG. 3 A). Despite the predicted annealing temperatures of all primers exceed 61°C, multiplex PCR reveals only two major groups on the DNA electrophoresis gel instead of the expected three groups. (FIG. 3B, lane 3), with the ~1.5 Kb ERG11 fragment missing. However, when the annealing temperature is reduced to 60°C, all three groups of fragments are successfully amplified (FIG. 3B, lane 1). Importantly, whether all the nine target genes are amplified successfully and the quantity of each relative to one another requires validation using MinlON sequencing.Capacity of MinlON for molecular typing

[0063] MinlON nanopore sequencing is used to assess the quality of the multiplex PCR and to evaluate optimal primer concentrations for typing these nine genes (Table 11). The read counts of all the nine genes were analyzed across seven different strains, and the proportion of reads for eight genes (ERG11 excluded, due to copy number variation (CNV) effects) relative to the total reads for each of the seven strains is compared (FIG. 4). The results show that the optimal primer concentrations for getting the most balanced read counts are listed as flows: 0.2 pM for ICL1, SAPT2, ZWFla, 0.15 pM for MDR1, SAPT4, CTRG_05978, CTRG_05993, and E7?G77; 0.1 pM for SNQ2.Table 11. Optimization of multiplex PCR using MinlON sequencing data (six experimental conditions tested to optimize multiplex PCR reaction, including variations in primer concentrations, numbers of cycles, and annealing temperature).[0064 For MLST typing, 96 individual FASTQ output files are generated, with the total number of reads ranged from 11,211 to 169,720 per sample (combine total 8,172,043 reads and an average of 85,126 reads per sample). And the average reads depth of coverage for each target gene is as follows: ICL1 (6,061.3), MDR1 (10,594.5), SAPT2 (10,762.3), SAPT4 (15,074.4), XYR1 (12,843.1), ZWFla (4,605.9), ERG11 (10,667.7), and SNQ2 (7,709.5) (FIG. 5A). Target genes are assembled using Medaka, and variant calls were processed with Bcftools (https: / / samtools.github.io / bcftools / bcftools.html) (Danecek and McCarthy, 2017). How different SNP filtering criteria and read depths affect concordance between generated consensus sequences and available sequencing results in the art is evaluated. Initially, the accuracy of MLST gene sequences is evaluated using all reads from a single MinlON sequencing flow cell under various SNP filtering thresholds, including:

[0065] Filter 1 : MLST genes at 40% (except XYR1 at 20%); Filter 2: MLST genes at 30% (except XYR1 at 15%); Filter 3: MLST genes at 25% (except XYR1 at 10%), Filter 4: all MLST genes at 10%).

[0066] The results demonstrate that when the SNP filtering threshold is set at 10%, all MLST loci (ICL1, MDR1, SAPT2, SAPT4, XYR1, ZWFla) achieve 100% concordance with the available sequencing results in the art. However, higher SNP filtering thresholds (>10%) reduces similarity for certain loci, particularly XYR1, which exhibits only 77.08% concordance at 40% filtering threshold (FIG. 5B).

[0067] Subsequently, a 10% SNP filtering threshold is applied while varying the read counts (5000, 7000, 10,000, and 12,000) to determine the minimum number of reads required to achieve 100% accuracy. The results show that at least 10,000 reads is necessary to ensure complete concordance between the generated consensus sequences and available sequencing results in the art (FIG. 5C).

[0068] In summary, setting the SNP filtering threshold at 10% and utilizing a minimum of 10,000 reads ensures the accurate assembly of consensus sequences, achieving full concordance with available sequencing data in the art.TaqMan SNP genotyping Assay is useful in identifying CTRG 05978 type IV and SNQ2 A2977G

[0069] To identify predominant azole-resistant clade 4 C. tropicalis strains rapidly, a TaqMan SNP Genotyping Assay is established for detecting the genotype of CTRG 05978 type IV and SNQ2 A2977G. The specificity of the primers and probes is tested using purified genomic DNAs from 18 different C. tropicalis strains including ATCC 750 and 17 strains from clinical settings (Table 12). The primers used are listed in Table 13.Table 12. TaqMan Genotyping Assay enables rapid detection of the genotype IV of CTRG_05978 and the SNQ2 A2977G (*: Based on the first 4 mers of CTRG_05978, 7 types in those strains are defined: type I is ATAA; type II is ATGA; type III is ATGT; type IV is ATRA; type V is ATGW, type VI is ATRW, and type VII is ATWA; and **: CTRG 5979 detects GA and AA SNP).Table 13. Primers used in the TaqMan SNP genotyping assay (*: position of translation initiation site of ATG as +1).

[0070] For the CTRG_05978 genotype, all type IV strains (YM140156, YM180233, YM180351, YM180681, and YM180969) are located in the heterozygous area (green spots), while the others are located in homozygous areas (red and blue spots) (left panel of FIG. 6). In the case of the SNQ2 A2977G assay, it also specifically detected 7 mutation strains (blue and green spots) (right panel of FIG. 6). Overall, the results of TaqMan SNP Genotyping Assay are consistent with Sanger sequencing or Whole Genome Sequencing, suggesting that the aqMan SNP Genotyping Assay may be a rapid and convenient method for identifying the predominant azole-resistant clade 4 C. tropicalis genotype through detecting SNPs of CTRG 05978 and SNQ2.Summary of Examples

[0071] A method for detecting the potential marker for rapidly identifying the azole-resistant C. tropicalis with the clade 4 genotype, has been developed and tested in the examples of the present disclosure. The potential markers involve only a few nucleotide variations, which may be detected using designed capture probes targeting the SNPs of Snq2 I993V and the first 4-mer of CTRG 05978. Even though techniques such as real-time PCR applications, ELISA-like assays, or microarray analysis may also be suitable for the purpose, an easily accessible and convenient method for detection as the present disclosure are more practical for clinical settings. The TaqMan SNP genotype assay as a rapid method for identifying these SNPs in CTRG 05978 and the SNQ2 A2977G is provided in the present disclosure. Some C. tropicalis isolates of clade 4 do not belong to the CTRG 05987 type IV cluster and / or do not carry the SNQ2 A2977G mutation and one non-clade 4 strain belongs to the CTRG 05987 type IV cluster and carries the SNQ2 A2977G mutation. To increase the accuracy of rapidly identifying azole-resistant clade 4 C. tropicalis, determining whether the strains carry ERG11 mutations in addition to the genotypes of CTRG 05987 and SNQ2 identified in the present disclosure is suggested.

[0072] The SNP in CTRG_05978 and the SNQ2 A2977G are valuable tools for swiftly identifying the clade 4 genotype (FIG. 7). The present disclosure may greatly contribute to the development of targeted diagnostic resulting in a proper prescription for therapy. In conclusion, the present disclosure may present a promising approach for monitoring and managing the spread of clade 4 C. tropicalis infections, thereby offering a potential tool in mitigating significant health risks for vulnerable patient populations.

[0073] The effective management of Candida tropicalis infections necessitates rapid andscalable diagnostic solutions. To address this, the present disclosure provides two innovative approaches. First, a rapid detection marker system leverages the three selected SNP to identify dominant drug-resistant genotypes. This streamlined approach enables rapid screening, making it ideal for point-of-care testing facilities such as hospitals and clinics. Furthermore, this system is designed to optimize existing detection kits, enhancing its scalability and ease of implementation. On the other hand, the examples of the present disclosure may provide a high-throughput approach combining multiplex PCR and MinlON sequencing. This synergistic combination allows for large-scale general testing, enabling core laboratories and institutional labs to efficiently handle a high volume of samples. Together, these two approaches offer a promising strategy for monitoring and managing the spread of clade 4 C. tropicalis, addressing both the need for rapid, targeted testing and the requirement for large-scale epidemiological surveillance.

[0074] Those skilled in the art will readily observe that numerous modifications and alterations of the device and method may be made while retaining the teachings of the present disclosure. Accordingly, the above disclosure should be construed as limited only by the metes and bounds of the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A method for identifying a drug-resistant fungal pathogen, comprising: detecting a single nucleotide polymorphism genotype on XYR1 gene and SNQ2 gene of a fungal pathogen.

2. The method of claim 1, wherein the single nucleotide polymorphism genotype on the XYR1 gene comprises a sequence of ATRA, and R is A or G.

3. The method of claim 2, wherein the sequence of ATRA is a first 4-mer of the XYR1 gene.

4. The method of claim 1, wherein the single nucleotide polymorphism genotype on the SNQ2 gene comprises a nucleotide A at position 2977 substituted with a nucleotide G (A2977G).

5. The method of claim 4, wherein the single nucleotide polymorphism genotype on the XYR1 gene comprises a sequence of ATRA, and R is A or G.

6. The method of claim 5, wherein the sequence of ATRA is a first 4-mer of the XYR1 gene.

7. The method of claim 1, wherein the fungal pathogen is a non-albicans Candida species.

8. The method of claim 7, wherein the non-albicans Candida species is at least one selected from the group consisting of Candida tropicalis. Candida parapsilosis. Candida glabrata, Candida auris. Candida giiilliermondii. and Candida krusei .

9. The method of claim 8, wherein the non-albicans Candida species is Candida tropicalis.

10. The method of claim 9, wherein the Candida tropicalis comprises a clade 4 genotype.

11. The method of claim 1, wherein the drug-resistant fungal pathogen has a drug resistance against a drug comprising azole.

12. The method of claim 1, wherein the single nucleotide polymorphism genotype on XYR1 gene and SNQ2 gene of the fungal pathogen is detected by high-throughput identification, and the high-throughput identification comprises combining a multiplex polymerase chain reactionapproach and a MinlON nanopore sequencing approach.

13. The method of claim 1, wherein the single nucleotide polymorphism genotype on XYR1 gene and SNQ2 gene of the fungal pathogen is detected by a TaqMan genotyping assay.

14. A kit for identifying a drug-resistant fungal pathogen, comprising: a lysis buffer for breaking down the fungal pathogen; and a primer for detecting XYR1 gene and / or SNQ2 gene of a fungal pathogen.

15. The kit of claim 14, further comprising: a DNA polymerase; and a PCR buffer comprising Mg2+.

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