The yeast genus Tardiomyces gen. nov. with one new species and two new combinations.

PURPOSE: Rare yeasts species are increasingly reported as causative agents of invasive human infection. Proper identification and antifungal therapy are essential to manage these infections. Candida blankii is one of these emerging pathogens and is known for its reduced susceptibility to multiple antifungals. METHODS: To obtain more insight into the characteristics of this species, 26 isolates reported as C. blankii were investigated using genetic and phenotypical approaches. RESULTS: Among the 26 isolates, seven recovered either from blood, sputum, urine, or the oral cavity, displayed substantial genetic and some phenotypical differences compared to the other isolates, which were confirmed as C. blankii. We consider these seven strains to represent a novel species, Tardiomyces depauwii. Phylogenomics assigned C. blankii, C. digboiensis, and the novel species in a distinct branch within the order Dipodascales, for which the novel genus Tardiomyces is erected. The new combinations Tardiomyces blankii and Tardiomyces digboiensis are introduced. Differences with related, strictly environmental genera Sugiyamaella, Crinitomyces, and Diddensiella are enumerated. All three Tardiomyces species share the rare ability to grow up to 42 °C, display slower growth in nutrient-poor media, and show a reduced susceptibility to azoles and echinocandins. Characteristics of T. depauwii include high MIC values with voriconazole and a unique protein pattern. CONCLUSION: We propose the novel yeast species Tardiomyces depauwii and the transfer of C. blankii and C. digboiensis to the novel Tardiomyces genus.

Evaluation of the Vitek 2 system for antifungal susceptibility testing of Candida auris using a representative international panel of clinical isolates: overestimation of amphotericin B resistance and underestimation of fluconazole resistance.

Although the Vitek 2 system is broadly used for antifungal susceptibility testing of Candida spp., its performance against Candida auris has been assessed using limited number of isolates recovered from restricted geographic areas. We therefore compared Vitek 2 system with the reference Clinical and Laboratory Standards Institute (CLSI) broth microdilution method using an international collection of 100 C. auris isolates belonging to different clades. The agreement ±1 twofold dilution between the two methods and the categorical agreement (CA) based on the Centers for Disease Control and Prevention's (CDC's) tentative resistance breakpoints and Vitek 2-specific wild-type upper limit values (WT-ULVs) were determined. The CLSI-Vitek 2 agreement was poor for 5-flucytosine (0%), fluconazole (16%), and amphotericin B (29%), and moderate for voriconazole (61%), micafungin (67%), and caspofungin (81%). Significant interpretation errors were recorded using the CDC breakpoints for amphotericin B (31% CA, 69% major errors; MaEs) and fluconazole (69% CA, 31% very major errors; VmEs), but not for echinocandins (99% CA, 1% MaEs for both micafungin and caspofungin) for which the Vitek 2 allowed correct categorization of echinocandin-resistant FKS1 mutant isolates. Discrepancies were reduced when the Vitek 2 WT-ULV of 16 mg/L for amphotericin B (98% CA, 2% MaEs) and of 4 mg/L for fluconazole (96% CA, 1% MaEs, 3% VmEs) were used. In conclusion, the Vitek 2 system performed well for echinocandin susceptibility testing of C .auris. Resistance to fluconazole was underestimated whereas resistance to amphotericin B was overestimated using the CDC breakpoints of ≥32 and ≥2 mg/L, respectively. Vitek 2 minimun inhibitory concentrations (MICs) >4 mg/L indicated resistance to fluconazole and Vitek 2 MICs ≤16 mg/L indicated non-resistance to amphotericin B.

Genotyping and clonal origin of Sporothrix brasiliensis in human sporotrichosis cases in Argentina.

Sporothrix brasiliensis is considered a highly virulent emerging pathogen that causes sporotrichosis in humans, mainly after zoonotic transmission from infected cats. The epidemic of this zoonosis that originated from Brazil has spread in the last decades, generating hyperendemic regions in Latin America. We present two cases of human sporotrichosis causes by S. brasiliensis in Buenos Aires, Argentina, with good clinical response to differing treatments after contact with sick cats. Using Short tandem repeat (STR) genotyping, the two S. brasiliensis cases appear to be introduced from Brazil and likely originate from the same source.

Molecular typing and antifungal susceptibility profile of Candida krusei bloodstream isolates from Türkiye.

Candida krusei also known as Pichia kudriavzevii is a potentially multidrug-resistant yeast because it is intrinsically resistant to fluconazole and develops acquired resistance to echinocandins and polyenes. Here, we aim to provide a better understanding of the epidemiology and transmission modes of C. krusei infections by comparing invasive bloodstream (n = 35) and non-invasive vaginal (n = 20) C. krusei isolates. The genetic relatedness of the isolates was assessed using a newly described short tandem repeat (STR) analysis and their sensitivity to eight antifungal compounds was evaluated by antifungal susceptibility testing using the CLSI microbroth dilution method. All C. krusei isolates revealed unique STR genotypes, indicating the absence of clonal transmission in the study group. Furthermore, no drug-resistant or non-wild-type isolates were identified. Our findings demonstrated high resolution of STR genotyping for the detection and simultaneous genetic analysis of multiple C. krusei strains in clinical samples and excellent in vitro activity of common antifungal agents against invasive strains.

An Autochthonous Susceptible Candida auris Clade I Otomycosis Case in Iran.

Candida auris is a newly emerging multidrug-resistant fungal pathogen considered to be a serious global health threat. Due to diagnostic challenges, there is no precise estimate for the prevalence rate of this pathogen in Iran. Since 2019, only six culture-proven C. auris cases have been reported from Iran, of which, five belonged to clade V and one to clade I. Herein, we report a case of otomycosis due to C. auris from 2017 in a 78-year-old man with diabetes mellitus type II without an epidemiological link to other cases or travel history. Short tandem repeat genotyping and whole genome sequencing (WGS) analysis revealed that this isolate belonged to clade I of C. auris (South Asian Clade). The WGS single nucleotide polymorphism calling demonstrated that the C. auris isolate from 2017 is not related to a previously reported clade I isolate from Iran. The presence of this retrospectively recognized clade I isolate also suggests an early introduction from other regions or an autochthonous presence. Although the majority of reported C. auris isolates worldwide are resistant to fluconazole and, to a lesser extent, to echinocandins and amphotericin B, the reported clade I isolate from Iran was susceptible to all antifungal drugs.

The Fourier-transform infrared spectroscopy-based method as a new typing tool for Candida parapsilosis clinical isolates.

The Fourier-transform infrared spectroscopy-based IR Biotyper is a straightforward typing tool for bacterial species, but its use with Candida species is limited. We applied IR Biotyper to Candida parapsilosis, a common cause of nosocomial bloodstream infection (BSI), which is aggravated by the intra-hospital spread of fluconazole-resistant isolates. Of 59 C. parapsilosis isolates studied, n = 56 (48 fluconazole-resistant and 8 fluconazole-susceptible) and n = 3 (2 fluconazole-resistant and 1 fluconazole-susceptible) isolates, respectively, had been recovered from BSI episodes in 2 spatially distant Italian hospitals. The latter isolates served as an outgroup. Of fluconazole-resistant isolates, n = 40 (including one outgroup) harbored the Y132F mutation alone and n = 10 (including one outgroup) harbored both Y132F and R398I mutations in the ERG11-encoded azole-target enzyme. Using a microsatellite typing method, which relies on the amplification of genomic short tandem repeats (STR), two major clusters were obtained based on the mutation(s) (Y132F or Y132F/R398I) present in the isolates. Regarding IR Biotyper, each isolate was analyzed in quintuplicate using an automatic (i.e., proposed by the manufacturer's software) or tentative (i.e., proposed by us) cutoff value. In the first case, four clusters were identified, with clusters I and II formed by Y132F or Y132F/R398I isolates, respectively. In the second case, six subclusters (derived by the split of clusters I and II) were identified. This allowed to separate the outgroup isolates from other isolates and to increase the IR Biotyper typeability. The agreement of IR Biotyper with STR ranged from 47% to 74%, depending on type of cutoff value used in the analysis. IMPORTANCE Establishing relatedness between clinical isolates of Candida parapsilosis is important for implementing rapid measures to control and prevent nosocomial transmission of this Candida species. We evaluated the FTIR-based IR Biotyper, a new typing method in the Candida field, using a collection of fluconazole-resistant C. parapsilosis isolates supposed to be genetically related due to the presence of the Y132F mutation. We showed that IR Biotyper was discriminatory but not as much as the STR method, which is still considered the method of choice. Further studies on larger series of C. parapsilosis isolates or closely related Candida species will be necessary to confirm and/or extend the results from this study.

Whole genome sequencing analysis demonstrates therapy-induced echinocandin resistance in Candida auris isolates.

Candida auris is an emerging, multidrug-resistant yeast, causing outbreaks in healthcare facilities. Echinocandins are the antifungal drugs of choice to treat candidiasis, as they cause few side effects and resistance is rarely found. Previously, immunocompromised patients from Kuwait with C. auris colonisation or infection were treated with echinocandins, and within days to months, resistance was reported in urine isolates. To determine whether the development of echinocandin resistance was due to independent introductions of resistant strains or resulted from intra-patient resistance development, whole genome sequencing (WGS) single-nucleotide polymorphism (SNP) analysis was performed on susceptible (n = 26) and echinocandin-resistant (n = 6) isolates from seven patients. WGS SNP analysis identified three distinct clusters differing 17-127 SNPs from two patients, and the remaining isolates from five patients, respectively. Sequential isolates within patients had a maximum of 11 SNP differences over a time period of 1-10 months. The majority of isolates with reduced susceptibility displayed unique FKS1 substitutions including a novel FKS1M690V substitution, and nearly all were genetically related, ranging from only three to six SNP differences compared to susceptible isolates from the same patient. Resistant isolates from three patients shared the common FKS1S639F substitution; however, WGS analysis did not suggest a common source. These findings strongly indicate that echinocandin resistance is induced during antifungal treatment. Future studies should determine whether such echinocandin-resistant strains are capable of long-term colonisation, cause subsequent breakthrough candidiasis, have a propensity to cross-infect other patients, or remain viable for longer time periods in the hospital environment.

Genotyping and susceptibility testing uncovers large azole-resistant Candida tropicalis clade in Alexandria, Egypt.

OBJECTIVES: Candida tropicalis is an emerging medically relevant Candida species. The yeast primarily causes opportunistic infections in intensive care units and is highly prevalent in tropical countries. The genetic diversity within this species is high, and nosocomial transmission has been reported. C. tropicalis genotyping of isolates from low- and middle-income countries is underrepresented when compared with that from high-income countries. Also, in Egypt, only limited genotyping has been conducted for C. tropicalis isolates, while antifungal resistance seems to increase, especially against azoles. METHODS: Antifungal susceptibility testing was performed on 64 C. tropicalis isolates from ICU patients collected from multiple hospitals in Alexandria, Egypt. Genotyping by means of short tandem repeat (STR) and whole genome sequencing (WGS) single nucleotide polymorphism (SNP) analysis was performed. RESULTS: Using antifungal susceptibility testing, fluconazole resistance was observed in 24 isolates (38%), of which 23 harboured an ERG11 G464S substitution, previously shown to cause resistance in Candida albicans. STR genotyping showed that these 23 isolates were related, forming a distinct resistant clade. WGS SNP analysis subsequently confirmed this genetic relationship, although isolates within this clade differed in at least 429 SNPs, suggesting that these were independently introduced. CONCLUSION: Overall, STR and WGS SNP analysis of this collection indicates limited C. tropicalis nosocomial transmission in Alexandria, while the presence of this large azole-resistant C. tropicalis clade within this city hampers the treatment of intensive care unit patients.

Application of Novel Short Tandem Repeat Typing for Wickerhamomyces anomalus Reveals Simultaneous Outbreaks within a Single Hospital.

Wickerhamomyces anomalus, previously known as Candida pelliculosa, occasionally causes candidemia in humans, primarily infecting neonates, and infants. The mortality rate of these invasive infections is high, and isolates with a reduced susceptibility to fluconazole have been reported. W. anomalus outbreaks are regularly reported in healthcare facilities, especially in neonatal intensive care units (NICUs). In order to rapidly genotype isolates with a high-resolution, we developed and applied a short tandem repeat (STR) typing scheme for W. anomalus. Six STR markers were selected and amplified in two multiplex PCRs, M3 and M6, respectively. In total, 90 W. anomalus isolates were typed, leading to the identification of 38 different genotypes. Four large clusters were found, unveiling simultaneous outbreak events spread across multiple units within the same hospital. STR typing results of 11 isolates were compared to whole-genome sequencing (WGS) single nucleotide polymorphism (SNP) calling, and the identified genotypic relationships were highly concordant. We performed antifungal susceptibility testing of these isolates, and a reduced susceptibility to fluconazole was found for two (2.3%) isolates. ERG11 genes of these two isolates were examined using WGS data, which revealed a novel I469L substitution in one isolate. By constructing a homology model for W. anomalus ERG11p, the substitution was found in close proximity to the fluconazole binding site. In summary, we showed multiple W. anomalus outbreak events by applying a novel STR genotyping scheme.

Overestimation of Amphotericin B Resistance in Candida auris with Sensititre YeastOne Antifungal Susceptibility Testing: a Need for Adjustment for Correct Interpretation.

Significant variation in minimal inhibitory concentrations (MIC) has been reported for amphotericin B (AMB) and C. auris, depending on the antifungal susceptibility testing (AFST) method. Although the Sensititre YeastOne (SYO) is widely used in routine laboratory testing, data regarding its performance for the AFST of C. auris are scarce. We tested AMB against 65 C. auris clinical isolates with the SYO and the reference methodology by the Clinical and Laboratory Standards Institute (CLSI). The essential agreement (EA, ±1 dilution) between the two methods and the categorical agreement (CA) based on the Centers for Disease Control and Prevention (CDC)'s tentative breakpoint of MIC ≥ 2 mg/L were determined. The SYO wild type upper limit value (WT-UL) was determined using the ECOFFinder. The modal (range) CLSI growth inhibitory MIC was lower than the SYO colorimetric MIC [1(0.25-1) versus 2(1-8) mg/L, respectively]). The CLSI-colorimetric SYO EA was 29% and the CA was 11% (89% major errors; MaE). MaE were reduced when the SYO WT-UL of 8 mg/L was used (0% MaE). Alternatively, the use of SYO growth inhibition endpoints of MIC-1 (75% growth inhibition) or MIC-2 (50% growth inhibition) resulted in 88% CA with 12% MaE and 97% CA with 3% MaE, respectively. In conclusion, SYO overestimated AMB resistance in C. auris isolates when colorimetric MICs, as per SYO instructions and the CDC breakpoint of 2 mg/L, were used. This can be improved either by using the method-specific WT-UL MIC of 8 mg/L for colorimetric MICs or by determining growth inhibition MIC endpoints, regardless of the color. IMPORTANCE Candida auris is an emerging and frequently multidrug-resistant fungal pathogen that accounts for life-threatening invasive infections and nosocomial outbreaks worldwide. Reliable AF is important for the choice of the optimal treatment. Commercial methods are frequently used without prior vigorous assessment. Resistance to AMB was over-reported with the commercial colorimetric method Sensititre YeastOne (SYO). SYO produced MICs that were 1 to 2 twofold dilutions higher than those of the reference CLSI method, resulting in 89% MaE. MaE were reduced using a SYO-specific colorimetric wild type upper limit MIC value of 8 mg/L (0%) or a 50% growth inhibition endpoint (3%).

High-Throughput Microsatellite Markers Development for Genetic Characterization of Emerging Sporothrix Species.

Sporotrichosis is the main subcutaneous mycosis worldwide transmitted by animal or plant vectors and often escalates to outbreaks or epidemics. The current cat-transmitted sporotrichosis driven by Sporothrix brasiliensis has become a significant public health issue in South America. Transmission dynamics remain enigmatic due to the lack of development of polymorphic markers for molecular epidemiological analysis. This study used a high-throughput mining strategy to characterize simple sequence repeat (SSR) markers from Sporothrix genomes. A total of 118,140-143,912 SSR loci were identified (82,841-98,369 unique markers), with a 3651.55-3804.65 SSR/Mb density and a majority of dinucleotides motifs (GC/CG). We developed a panel of 15 highly polymorphic SSR markers suitable for genotyping S. brasiliensis, S. schenckii, and S. globosa. PCR amplification revealed 240 alleles in 180 Sporothrix isolates with excellent polymorphic information content (PIC = 0.9101), expected heterozygosity (H = 0.9159), and discriminating power (D = 0.7127), supporting the effectiveness of SSR markers in uncovering cryptic genetic diversity. A systematic population genetic study estimated three clusters, corresponding to S. brasiliensis (population 1, n = 97), S. schenckii (population 2, n = 49), and S. globosa (population 3, n = 34), with a weak signature of mixed ancestry between populations 1 and 2 or 3 and 2. Partitioning of genetic variation via AMOVA revealed highly structured populations (ΦPT = 0.539; Nm = 0.213; p < 0.0001), with approximately equivalent genetic variability within (46%) and between (54%) populations. Analysis of SSR diversity supports Rio de Janeiro (RJ) as the center of origin for contemporary S. brasiliensis infections. The recent emergence of cat-transmitted sporotrichosis in northeastern Brazil indicates an RJ-Northeast migration resulting in founder effects during the introduction of diseased animals into sporotrichosis-free areas. Our results demonstrated high cross-species transferability, reproducibility, and informativeness of SSR genetic markers, helping dissect deep and fine-scale genetic structures and guiding decision making to mitigate the harmful effects of the expansion of cat-transmitted sporotrichosis.

Genotyping and antifungal susceptibility testing of Sporothrix brasiliensis isolates from Southern Brazil.

Sporotrichosis is an implantation mycosis caused by the dimorphic fungus Sporothrix and mostly involves cutaneous and subcutaneous tissues and the lymphatic vessels. Among more than 50 different species, only Sporothrix schenckii, Sporothrix globosa and Sporothrix brasiliensis are frequently reported to cause infections in humans. Sporothrix brasiliensis is remarkably virulent and has been spreading rapidly in Brazil and other Latin American countries. In this study, we aimed to determine the genetic relatedness and antifungal susceptibility of Sporothrix strains by analysing 89 isolates from humans and cats in Curitiba, Southern Brazil. Calmodulin sequencing identified 81 S. brasiliensis and seven S. schenckii isolates. Amplified fragment length polymorphism genotyping analysis showed feline and human isolates clustering together. In vitro susceptibility testing with seven antifungals demonstrated a broad activity against all tested S. brasiliensis isolates, with no significant differences in minimal inhibitory concentration (MIC) values between feline and human isolates. Resistance was solely observed in one human isolate against itraconazole and posaconazole, with MICs of ≥16 µg/mL against both antifungals. Whole genome sequencing (WGS) analysis on this isolate and two related susceptible isolates did not reveal any unique substitutions in resistance-associated genes, including cyp51, hmg and erg6, when compared to two related susceptible isolates. The novel antifungal olorofim exhibited excellent activity against this large isolate collection, with all isolates considered as susceptible. Altogether, we indicate zoonotic transmission based on genotyping and revealed a broad activity of seven common antifungals, including olorofim, against a large S. brasiliensis isolate collection.

Short Tandem Repeat Genotyping and Antifungal Susceptibility Testing of Latin American Candida tropicalis Isolates.

Candida tropicalis is emerging as one of the most common Candida species causing opportunistic infections in Latin America. Outbreak events caused by C. tropicalis were reported, and antifungal resistant isolates are on the rise. In order to investigate population genomics and look into antifungal resistance, we applied a short tandem repeat (STR) genotyping scheme and antifungal susceptibility testing (AFST) to 230 clinical and environmental C. tropicalis isolates from Latin American countries. STR genotyping identified 164 genotypes, including 11 clusters comprised of three to seven isolates, indicating outbreak events. AFST identified one isolate as anidulafungin-resistant and harboring a FKS1 S659P substitution. Moreover, we identified 24 clinical and environmental isolates with intermediate susceptibility or resistance to one or more azoles. ERG11 sequencing revealed each of these isolates harboring a Y132F and/or Y257H/N substitution. All of these isolates, except one, were clustered together in two groups of closely related STR genotypes, with each group harboring distinct ERG11 substitutions. The ancestral C. tropicalis strain of these isolates likely acquired the azole resistance-associated substitutions and subsequently spread across vast distances within Brazil. Altogether, this STR genotyping scheme for C. tropicalis proved to be useful for identifying unrecognized outbreak events and better understanding population genomics, including the spread of antifungal-resistant isolates.

Development and Application of a Short Tandem Repeat Multiplex Typing Assay for Candida tropicalis.

Candida tropicalis is a clinically important yeast that causes candidemia in humans with a high mortality rate. The yeast primarily infects immunocompromised patients, and causes outbreaks in health care facilities. Antifungal resistant isolates have been reported. We developed a short tandem repeat (STR) typing scheme for C. tropicalis to enable fast, cost-effective, and high-resolution genotyping. For the development of the typing scheme, 6 novel STR markers were selected, combined into 2 multiplex PCRs. In total, 117 C. tropicalis isolates were typed, resulting in the identification of 104 different genotypes. Subsequently, the outcome of STR typing of 10 isolates was compared to single nucleotide polymorphism (SNP) calling from whole-genome sequencing (WGS). Isolates with more than 111 SNPs were differentiated by the typing assay. Two isolates, which were identical according to SNP analysis, were separated by STR typing in 1 marker. To test specificity, the STR typing was applied to 15 related yeast species, and we found no amplification of these targets. For reproducibility testing, 2 isolates were independently typed five times, which showed identical results in each experiment. In summary, we developed a reliable and multiplex STR genotyping for C. tropicalis, which was found to correlate well to SNP calling by WGS. WGS analysis from and extensive collection of isolates is required to establish the precise resolution of this STR assay. IMPORTANCE Candida tropicalis frequently causes candidemia in immunocompromised patients. C. tropicalis infections have a high mortality rate, and the yeast is able to cause outbreaks in health care facilities. Further, antifungal resistant isolates are on the rise. Genotyping is necessary to investigate potential outbreaks. Here, we developed and applied a STR genotyping scheme in order to rapidly genotype isolates with a high-resolution. WGS SNP outcomes were highly comparable with STR typing results. Altogether, we developed a rapid, high-resolution, and specific STR genotyping scheme for C. tropicalis.

Optimization and Validation of Candida auris Short Tandem Repeat Analysis.

Candida auris is an easily transmissible yeast with resistance to different antifungal compounds. Outbreaks of C. auris are mostly observed in intensive care units. To take adequate measures during an outbreak, it is essential to understand the transmission route, which requires isolate genotyping. In 2019, a short tandem repeat (STR) genotyping analysis was developed for C. auris. To determine the discriminatory power of this method, we performed STR analysis of 171 isolates with known whole-genome sequencing (WGS) data using Illumina reads, and we compared their resolutions. We found that STR analysis separated the 171 isolates into four clades (clades I to IV), as was also seen with WGS analysis. Then, to improve the separation of isolates in clade IV, the STR assay was optimized by the addition of 2 STR markers. With this improved STR assay, a total of 32 different genotypes were identified, while all isolates with differences of >50 single-nucleotide polymorphisms (SNPs) were separated by at least 1 STR marker. Altogether, we optimized and validated the C. auris STR panel for clades I to IV and established its discriminatory power, compared to WGS SNP analysis using Illumina reads. IMPORTANCE The emerging fungal pathogen Candida auris poses a threat to public health, mainly causing outbreaks in intensive care units. Genotyping is essential for investigating potential outbreaks and preventing further spread. Previously, we developed a STR genotyping scheme for rapid and high-resolution genotyping, and WGS SNP outcomes for some isolates were compared to STR data. Here, we compared WGS SNP and STR outcomes for a larger sample cohort. Also, we optimized the resolution of this typing scheme with the addition of 2 STR markers. Altogether, we validated and optimized this rapid, reliable, and high-resolution typing scheme for C. auris.

Confirmation of fifth Candida auris clade by whole genome sequencing.

Candida auris has emerged globally as a multidrug-resistant pathogen causing outbreaks in health care facilities. Whole genome sequencing (WGS) analysis has identified four major clades, while earlier WGS data from a single Iranian isolate suggested the existence of a potential fifth clade. Here, we confirm the existence of this fifth clade by providing WGS data of another four Iranian isolates. These clade V isolates differed less than 100 single-nucleotide polymorphisms (SNPs) between each other, while they were separated from the other clades by more than 200,000 SNPs. Two of these isolates were resistant to fluconazole and were found to harbour mutations in the TAC1b and ERG11 genes.

Development of a Multiplex PCR Short Tandem Repeat Typing Scheme for Candida krusei.

Candida krusei is a human-pathogenic yeast that can cause candidemia with the lowest 90-day survival rate in comparison to other Candida species. Infections occur frequently in immunocompromised patients, and several C. krusei outbreaks in health care facilities have been described. Here, we developed a short tandem repeat (STR) typing scheme for C. krusei to allow the fast and cost-effective genotyping of an outbreak and compared the identified relatedness of 10 isolates to single nucleotide polymorphism (SNP) calling from whole-genome sequencing (WGS). From a selection of 14 novel STR markers, 6 were used to develop two multiplex PCRs. Additionally, three previously reported markers were selected for a third multiplex PCR. In total, 119 C. krusei isolates were typed using these nine markers, and 79 different genotypes were found. STR typing correlated well with WGS SNP typing, as isolates with the same STR genotype varied by 8 and 19 SNPs, while isolates that differed in all STR markers varied by at least tens of thousands of SNPs. The STR typing assay was found to be specific for C. krusei, stable in 100 subcloned generations, and comparable to SNP calling by WGS. In summary, this newly developed C. krusei STR typing scheme is a fast, reliable, easy-to-interpret, and cost-effective method compared to other typing methods. Moreover, the two newly developed multiplexes showed the same discriminatory power as all nine markers combined, indicating that multiplexes M3-1 and M9 are sufficient to type C. krusei.

Novel high-resolution targeted sequencing of the cervicovaginal microbiome.

BACKGROUND: The cervicovaginal microbiome (CVM) plays a significant role in women's cervical health and disease. Microbial alterations at the species level and characteristic community state types (CST) have been associated with acquisition and persistence of high-risk human papillomavirus (hrHPV) infections that may result in progression of cervical lesions to malignancy. Current sequencing methods, especially most commonly used multiplex 16S rRNA gene sequencing, struggle to fully clarify these changes because they generally fail to provide sufficient taxonomic resolution to adequately perform species-level associative studies. To improve CVM species designation, we designed a novel sequencing tool targeting microbes at the species taxonomic rank and examined its potential for profiling the CVM. RESULTS: We introduce an accessible and practical circular probe-based RNA sequencing (CiRNAseq) technology with the potential to profile and quantify the CVM. In vitro and in silico validations demonstrate that CiRNAseq can distinctively detect species in a mock mixed microbial environment, with the output data reflecting its ability to estimate microbes' abundance. Moreover, compared to 16S rRNA gene sequencing, CiRNAseq provides equivalent results but with improved sequencing sensitivity. Analyses of a cohort of cervical smears from hrHPV-negative women versus hrHPV-positive women with high-grade cervical intraepithelial neoplasia confirmed known differences in CST occurring in the CVM of women with hrHPV-induced lesions. The technique also revealed variations in microbial diversity and abundance in the CVM of hrHPV-positive women when compared to hrHPV-negative women. CONCLUSIONS: CiRNAseq is a promising tool for studying the interplay between the CVM and hrHPV in cervical carcinogenesis. This technology could provide a better understanding of cervicovaginal CST and microbial species during health and disease, prompting the discovery of biomarkers, additional to hrHPV, that can help detect high-grade cervical lesions.