Evaluation of the CHROMagar Candida Plus medium for presumptive identification of yeasts and MALDI-TOF MS identification.

Rapid and accurate yeasts species identification in clinical laboratories is important for appropriate and timely antifungal treatment. We evaluate the performance of the new medium CHROMagar™ Candida Plus for presumptive identification of yeasts species and MALDI-TOF identification. We identify 303 strains belonging to 60 clinically relevant yeasts species by using the new medium. Presumptive identification was correct at the Candida albicans complex, Candida tropicalis and Pichia kudriavzevii (Candida krusei) species. However, although this medium was able to identify all Candida auris and Candida glabrata strains, other species were misidentified as C. auris or C. glabrata. A total of 215 strains were identified by using MALDI-TOF and evaluated two incubation temperatures (30°C and 37°C) and two incubation times (24 h and 72 h). Most strains (94%; 202/215) were correctly identified at the species (n:190) or complex level (n:12) at both temperatures and incubation times. However, we observed that the time of incubation (24 h vs. 72 h) affects the score values when yeasts are incubated at 37°C, but does not affect score values when yeasts are incubated at 30°C. In conclusion, the new medium has a good performance in the presumptive identification of the C. albicans complex, C. tropicalis and P. kudriavzevii (C. krusei). In addition, this medium is useful for the screening of C. auris and C. glabrata isolates, but identification should be confirmed by other more specific techniques, like MALDI-TOF.

Genotypic Diversity and Antifungal Susceptibility of Clinical Isolates of Cryptococcus Gattii Species Complex from Argentina.

The aim of this study was to determine the genotypic diversity of 22 Cryptococcus gattii species complex clinical isolates from Argentina and to place these genotypes within the diversity of clinical, veterinary and environmental isolates from Latin America. Mating type and antifungal susceptibility of the isolates were also determined. By URA5-RFLP, nine isolates were identified as molecular type VGI, 10 as VGII, one as VGIII and two as VGIV. Multilocus sequence typing (MSLT), following the International Society for Human and Animal Mycology (ISHAM) consensus MLST scheme, was used to determine the genotypic diversity. Our results suggest that, in Argentina, VGI isolates have low genetic diversity, while VGII isolates have high genetic diversity. Both isolates identified as VGIV by URA5-RFLP were genotyped by MLST as belonging to the currently named VGVI clade. From all isolates, eight sequence types (STs) were unique for Argentina, while five STs have been reported already in other countries, being of high interest the genotypes ST20 and ST7 since they belong to the subtypes VGIIa and VGIIb, respectively, which are associated with hypervirulent strains responsible for outbreaks in North America. To note, geographical analysis showed that some genotypes may be associated with some regions in Argentina. Most isolates were MATα, but we are reporting one isolate MATa for the first time in the country. Antifungal susceptibility tests showed that itraconazole, voriconazole and posaconazole had high activity against all isolates, while amphotericin B, fluconazole and 5-fluorocytosine were the least active drugs against all studied isolates.

Effectiveness of the National Quality Control Program in Mycology of Argentina and performance evaluation of participating laboratories.

The National Quality Control Program in Mycology (PNCCM) of Argentina was established in 1996 to improve the quality of the mycological diagnosis, to help establish and to set up standardized procedures and continuous training of laboratory staff. The aim of this study was to assess the effectiveness of the PNCCM in the 1996-2018 period. Data from the National Mycology Laboratory Network (NMLN) and PNCCM database was used to estimate the increase in the number of controlled laboratories and jurisdictions, the percentage of participation, the improvement in the quality of results and the adherence to the program. Satisfaction surveys were performed to assess user satisfaction. The number of controlled laboratories increased from 29 to 146; participation increased from 49% to 93% and general adherence was 72% in the evaluated period (1996-2018). Improvement in the quality of the results was 15% for low complexity samples; 7% for intermediate complexity samples and 14% for the identification of high complexity strains. Up to 84% of the users consider the PNCCM to be "very good" and 16% "satisfactory". These results show the importance of the PNCCM, which is widely accepted by mycological diagnostic laboratories from Argentina.

Frequency and geographical distribution of genotypes and mating types of Cryptococcus neoformans and Cryptococcus gattii species complexes in Argentina.

The aim of this work was to know the frequency and geographical distribution of genotypes and mating types of Cryptococcus neoformans and Cryptococcus gattii species complexes isolated from human infections in Argentina during the period from April 2009 to April 2011. A multicenter study was conducted, in which 372 isolates were obtained from 61 laboratories throughout the country. Of those, 98.8% of the isolates belonged to the C. neoformans species complex and 1.1% to the C. gattii species complex. Genotype VNI (MATα) was the most frequently isolated (n=326, 87.6%), followed by VNII (MATα) (n=22, 5.9%), the recently described VNII-VNIV (aADα) hybrid (n=14, 3.8%), VNIV (MATα) (n=4, 1.1%), VNIII (αADa) hybrid (n=1, 0.3%), and VNIII (αADα) hybrid (n=1, 0.3%). The Argentine Central region showed the greatest number of cases and genotype diversity. Interestingly, a relative high frequency was observed in genotype VNII (MATα) in the Cuyo, Northeast and Northwest regions and, also in VNII-VNIV (aADα) hybrids in the Northwest region. C. gattii species complex was isolated at a low rate; 3 VGI (MATα) and 1 VGII (MATα) isolates were obtained from the Northwest and Central regions. In conclusion, this study shows that genotype frequencies seem to vary among regions in Argentina and reveals a relatively high frequency of rare hybrids in the Northwest region. Further regional clinical and environmental studies may help to elucidate if those variations in frequencies are associated with the existence of regional ecological niches or any other regional factors.

Reidentification and antifungal susceptibility profile of Candida guilliermondii and Candida famata clinical isolates from a culture collection in Argentina.

The aim of this work was to reidentify strains previously identified as Candida guilliermondii and Candida famata by conventional phenotypic methods conserved in a culture collection from Argentina using ribosomal DNA sequencing, ACT1 gene sequencing, and matrix-assisted laser desorption ionization - time of flight mass spectrometry (MALDI-TOF MS). In addition, we performed antifungal susceptibility tests of eight antifungal drugs commonly used in clinical treatment. We identified 68 isolates belonging to the Candida guilliermondii species complex (59 C. guilliermondii, 8 C. fermentati, and 1 Candida carpophila), 16 isolates belonging to the Candida famata species complex (8 C. famata, 6 Debaryomyces nepalensis, 1 Debaryomyces fabryi, and 1 Debaryomyces tyrocola). Although sequencing of ITS region was able to identify C. guilliermondii and D. nepalensis isolates, sequencing of ACT1 gene seems to be the most appropriate technique for differentiation between C. fermentati and C. carpophila and between members of the C. famata species complex others than D. nepalensis. MALDI-TOF MS has a good potential for the identification of these yeasts, particularly in clinical laboratories since is a rapid and easy to perform technique. Here, we report the first isolation of D. tyrocola from a human patient and the first isolation of D. nepalensis from lungs and blood of human patients. Finally, correct identification and determination of antifungal susceptibility of those closely related species could be a useful tool for clinicians to choose the most effective antifungal treatment.

Development and validation of an extended database for yeast identification by MALDI-TOF MS in Argentina.

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) has revolutionized the identification of microorganisms in clinical laboratories because it is rapid, relatively simple to use, accurate, and can be used for a wide number of microorganisms. Several studies have demonstrated the utility of this technique in the identification of yeasts; however, its performance is usually improved by the extension of the database. Here we developed an in-house database of 143 strains belonging to 42 yeast species in the MALDI Biotyper platform, and we validated the extended database with 388 regional strains and 15 reference strains belonging to 55 yeast species. We also performed an intra- and interlaboratory study to assess reproducibility and analyzed the use of the cutoff values of 1.700 and 2.000 to correctly identify at species level. The creation of an in-house database that extended the manufacturer's database was successful in view of no incorrect identification was introduced. The best performance was observed by using the extended database and a cutoff value of 1.700 with a sensitivity of .94 and specificity of .96. A reproducibility study showed utility to detect deviations and could be used for external quality control. The extended database was able to differentiate closely related species and it has potential in distinguishing the molecular genotypes of Cryptococcus neoformans and Cryptococcus gattii.

National production of certified reference fungal cultures.

Reference fungal cultures (RFCs) are essential for the internal quality control of laboratories. The production of these cultures requires standardized procedures (IRAM 14950:2016 and ISO 17034:2016 standards) carried out by a recognized and accredited laboratory. The aim of this work was to produce RFC in paper disks of autochthonous strains, characterized by two, homogeneous and stable reference methods traceable at species level. RFC were produced using 14 regional species (7 yeasts and 7 filamentous fungi) from the fungal culture collection (DMic). Paper disks were impregnated with a culture suspension, dried and packed. Homogeneity, viability, identity and purity were verified. Short- and long-term stability at different temperatures and storage times were studied. Characterization of each strain allowed to confirm its identity and to ensure its traceability at international level. Produced batches were homogeneous and stable at -20±5°C for 30 months. This method of production was adequate to produce homogeneous and stable RFC with phenotypic and genotypic characteristics correctly defined and internationally traceable. Standardized procedures were developed for the production of certified RFC that could be transferred to other microorganisms. Providing RFC that represent regional strains allows laboratories to produce more reliable results with a favorable impact on medical diagnosis, the environment or the food industry.

Candida glabrata species complex prevalence and antifungal susceptibility testing in a culture collection: First description of Candida nivariensis in Argentina.

The presence of the cryptic species belonging to the Candida glabrata complex has not been studied in Argentina. We analyzed a collection of 117 clinical isolates of C. glabrata complex belonging to a National Culture Collection of Instituto Nacional de Microbiología "Dr. Carlos G. Malbrán" from Argentina (40 isolates from blood samples, 18 from other normally sterile sites, 20 from vagina, 14 from urine, 7 from oral cavity, 3 from catheter, 1 from a stool sample and 14 isolates whose clinical origin was not recorded). The aims of this work were to determine the prevalence of the cryptic species Candida nivariensis and Candida bracarensis and to evaluate the susceptibility profile of isolates against nine antifungal drugs. Identification was carried out by using classical phenotypic tests, CHROMagar™ Candida, PCR and MALDI-TOF. The minimal inhibitory concentrations of amphotericin B, 5-fluorocytosine, fluconazole, itraconazole, voriconazole, ketoconazole, posaconazole, caspofungin and anidulafungin were determined according to the EDef 7.3 (EUCAST) reference document. Of the 117 isolates, 114 were identified as C. glabrata and three as C. nivariensis by using PCR and MALDI-TOF. There were no major differences between C. nivariensis and C. glabrata susceptibility profiles. No resistant strains were found to echinocandins. We have found that the percentage of C. nivariensis in our culture collection was 2.56. This is the first description of C. nivariensis in Argentina, and data obtained could contribute to the knowledge of the epidemiology of this cryptic species.

Amino acid substitution in Cryptococcus neoformans lanosterol 14-α-demethylase involved in fluconazole resistance in clinical isolates.

The molecular basis of fluconazole resistance in Cryptococcus neoformans has been poorly studied. A common azole resistance mechanism in Candida species is the acquisition of point mutations in the ERG11 gene encoding the enzyme lanosterol 14-α-demethylase, target of the azole class of drugs. In C. neoformans only two mutations were described in this gene. In order to evaluate other mutations that could be implicated in fluconazole resistance in C. neoformans we studied the genomic sequence of the ERG11 gene in 11 clinical isolates with minimal inhibitory concentration (MIC) values to fluconazole of ≥16µg/ml. The sequencing revealed the G1855A mutation in 3 isolates, resulting in the enzyme amino acid substitution G484S. These strains were isolated from two fluconazole-treated patients. This mutation would not intervene in the susceptibility to itraconazole and voriconazole.

First Case of Fungemia Due to Pseudozyma aphidis in a Pediatric Patient with Osteosarcoma in Latin America.

We report the first case of blood infection due to Pseudozyma aphidis in Latin America. We contribute evidence showing this organism to be a potential human pathogen, and we provide new data about its identification, drug susceptibility, and treatment outcome.

Molecular identification, genotyping, and antifungal susceptibility testing of clinically relevant Trichosporon species from Argentina.

Trichosporon species are emerging causative agents of mycoses; most are documented in immunocompromised patients. Species identification is important for epidemiological purposes in order to better define species clinical associations and to improve antifungal treatment. Here, we studied a collection of 41 Trichosporon strains recovered from hospitalized patients in Argentina. All strains were identified by sequencing the D1/D2 domain of 26S, internal transcribed spacer (ITS) regions, and intergenic spacer 1 (IGS1) region. In addition, we determined the IGS1 region genotypes of the suspected T. asahii strains. Antifungal susceptibility of all strains was investigated. Thirty-eight of the 41 strains in this study were identified as follows: 29 T. asahii, 3 T. inkin, 3 T. montevideense, 2 T. faecale, and 1 T. dermatis. The identity of the three remaining strains could not be confirmed. Strain DMic 114126 (Culture collection of the Mycology Department (DMic), National Institute of Infectious Diseases "Dr. Carlos G. Malbrán".) may represent a T. asahii subspecies or a new Trichosporon species, strain DMic 94750 was identified as T. cf. guehoae and strain DMic 114132 as T. cf. akiyoshidainum. The distribution of T. asahii genotypes was as follows: 12 genotype 3, 9 genotype 1, 4 genotype 4, 2 genotype 5, and 2 genotype 7. Amphotericin B minimal inhibitory concentrations (MICs) were ≤1 mg/l for 78% (32/41) of the strains. Fluconazole MICs were ≥2 mg/l for 90% of the strains. However, itraconazole, voriconazole, ketoconazole, and posaconazole MICs were ≤1 mg/l for 100% of the strains. Terbinafine MICs were ≤1 mg/l for 98% 40/41 of the strains.

Cryptococcus gattii in urban trees from cities in North-eastern Argentina.

In the city of Buenos Aires, Argentina, Cryptococcus gattii genotype AFLP4/VGI was found to be associated with decaying wood in hollows of different tree species. The aim of this study was to investigate the presence of C. gattii in the environment of riverside cities of the river Paraná, and to describe its serotypes and molecular types. Five hundred samples were collected in 50 parks by swabbing tree hollows. The samples were inoculated on caffeic acid agar supplemented with chloramphenicol, and incubated at 28 °C for 1 week with a daily observation. The isolates were identified by conventional methods. The serotype was determined by slide agglutination with specific antisera. Molecular typing was carried out by PCR-RFLP of the URA5 gene. Four isolates of C. gattii were recovered: Cryptococcus gattii serotype B, genotype AFLP4/VGI, isolated from Eucalyptus sp. in the city of Rosario and from Grevillea robusta in the city of La Paz; and C. gattii serotype C, genotype AFLP5/VGIII, isolated from two different Tipuana tipu trees in the city of Resistencia. Here, we report for the first time the isolation of C. gattii serotype C, genotype AFLP5/VGIII, from environmental samples in Argentina.

Comparative analyses of classical phenotypic method and ribosomal RNA gene sequencing for identification of medically relevant Candida species.

As the distribution of Candida species and their susceptibility to antifungal agents have changed, a new means of accurately and rapidly identifying these species is necessary for the successful early resolution of infection and the subsequent reduction of morbidity and mortality. The current work aimed to evaluate ribosomal RNA gene sequencing for the identification of medically relevant Candida species in comparison with a standard phenotypic method. Eighteen reference strains (RSs), 69 phenotypically identified isolates and 20 inconclusively identified isolates were examined. Internal transcribed spaces (ITSs) and D1/D2 of the 26S ribosomal RNA gene regions were used as targets for sequencing. Additionally, the sequences of the ITS regions were used to establish evolutionary relationships. The sequencing of the ITS regions was successful for 88% (94/107) of the RS and isolates, whereas 100% of the remaining 12% (13/107) of the samples were successfully analysed by sequencing the D1/D2 region. Similarly, genotypic analysis identified all of the RS and isolates, including the 20 isolates that were not phenotypically identified. Phenotypic analysis, however, misidentified 10% (7/69) of the isolates. Phylogenetic analysis allowed the confirmation of the relationships between evolutionarily close species. Currently, the use of genotypic methods is necessary for the correct identification of Candida species.

First case report of bloodstream infection due to a Candida species closely related to the novel species Candida pseudorugosa.

Candida pseudorugosa is a novel species closely related to Candida rugosa for which only one case has been reported. We report the first case of a bloodstream infection in humans caused by a Candida sp. closely related to C. pseudorugosa. We contribute evidence to show this organism as a potential human pathogen that may be misidentified by conventional methods, also pointing out its lower sensitivity to azoles and other antifungal agents.

Phylogenomic Placement of American Southwest-Associated Clinical and Veterinary Isolates Expands Evidence for Distinct Cryptococcus gattii VGVI.

Whole-genome sequencing has advanced our understanding of the population structure of the pathogenic species complex Cryptococcus gattii, which has allowed for the phylogenomic specification of previously described major molecular type groupings and novel lineages. Recently, isolates collected in Mexico in the 1960s were determined to be genetically distant from other known molecular types and were classified as VGVI. We sequenced four clinical isolates and one veterinary isolate collected in the southwestern United States and Argentina from 2012 to 2021. Phylogenomic analysis groups these genomes with those of the Mexican VGVI isolates, expanding VGVI into a clade and establishing this molecular type as a clinically important population. These findings also potentially expand the known Cryptococcus ecological range with a previously unrecognized endemic area.

Prevalence of Candida dubliniensis fungemia in Argentina: identification by a novel multiplex PCR and comparison of different phenotypic methods.

Candida dubliniensis is an emerging pathogen that can cause invasive disease in patients who have a variety of clinical conditions. C. dubliniensis is often misidentified as Candida albicans by clinical laboratories. In Argentina, incidence data are still scarce, and only one systemic infection has been reported. This study aims to determine the prevalence of C. dubliniensis in blood samples in Argentina, to evaluate a novel PCR multiplex as well as several phenotypic methods for the identification of this yeast, and to know the susceptibility profile of isolates against seven antifungal drugs. We have found that prevalence in Argentina appears to be lower than that reported in other countries, occurring only in 0.96% of the Candidemia cases recovered in 47 hospitals during a 1-year period. All C. dubliniensis clinical isolates included in this study were genetically identical when comparing ITS genes sequences. This is in agreement with the previous studies suggesting little genetic variation within this species. The novel multiplex PCR proved to be 100% sensitive and specific for the identification of C. dubliniensis. Therefore, we propose its use as a rapid and inexpensive method for laboratories having access to molecular techniques. Although no single phenotypic test has proved to be infallible, both colony morphology on tobacco agar, as well as abundant chlamydospore formation on both tobacco agar and on sunflower seed agar, may be used as a presumptive differentiation method in routine mycology laboratories. It has been suggested that C. dubliniensis may have higher propensity to develop azole antifungal drug resistance than C. albicans. In this study, one of the five clinical isolates of C. dubliniensis was resistant to fluconazole.

Species distribution and susceptibility profile of yeasts isolated from blood cultures: results of a multicenter active laboratory-based surveillance study in Argentina.

The Mycology Department of the Instituto Nacional de Enfermedades Infecciosas "Dr. C. Malbrán", conducted the Second National Multicenter Survey on Fungemia due to Yeasts in Argentina. The aim was to obtain updated data of the frequency of the causative species encountered and their in vitro susceptibility to seven antifungal agents. Yeast species were identified by micromorphological and biochemical studies. Antifungal susceptibility testing was performed by the reference microdilution method E.Def 7.1 of the European Committee on Antibiotic Susceptibility Testing (EUCAST). A total of 461 viable yeasts were identified. The most frequent species were: Candida albicans (38.4 %), Candida parapsilosis (26 %), Candida tropicalis (15.4 %) and Candida glabrata (4.3 %). Other uncommon species, such as Candida viswanathii (0.6 %), Candida haemulonii (0.4 %), Candida inconspicua (0.2 %) and Candida fermentati (0.2 %) were also isolated. Among the Candida spp., 5.4 % and 1.6 % were resistant to fluconazole and voriconazole, respectively. Itraconazole and caspofungin were the most efficient agents against all Candida spp. tested (MIC < 1 mg/l). For anidulafungin, 21.6 % of C. parapsilosis showed a MIC value of 4 mg/l. Fluconazole was less active against 53.1 % of Cryptococcus neoformans (MIC > 8 mg/l), 75 % of Trichosporon spp., and 100 % of Rhodotorula spp., Geotrichum candidum, Saccharomyces cerevisiae. The global percentage of mortality was 20 %. The presence of uncommon species reinforces the need for performing continuous laboratory surveillance in order to monitor possible changes, not only in the epidemiological distribution of species, but also in the resistance to antifungal drugs.

First bloodstream infection due to Prototheca zopfii var. hydrocarbonea in an immunocompromised patient.

Here we describe a bloodstream infection due to P. zopfii var. hydrocarbonea in a patient with acute lymphoblastic leukemia. Identification was performed by DNA sequencing of the D1/D2 domain of 26s ribosomal DNA and by MALDI-TOF MS technique. Antifungal susceptibility tests against amphotericin B, fluconazole, itraconazole, and voriconazole showed the following MIC values, respectively: 0.25 mg/L, 128 mg/L, 0.064 mg/L, and 0.125 mg/L. The patient received amphotericin B treatment with a successful outcome.

First case report of cryptococcosis due to Cryptococcus decagattii in a pediatric patient in Argentina.

We report the first case of cryptococcosis due to Cryptococcus decagattii in an immunocompetent pediatric patient from an indigenous community in Argentina with a successful outcome. Two isolates (blood, cerebrospinal fluid) were genotyped by restriction fragment length polymorphism of the orotidine monophosphate pyrophosphorylase (URA5) gene as VGIV and identified by multi-locus sequence typing as C. decagattii. Matrix-assisted laser desorption/ionization time of flight mass spectrometry identification indicated genotype VGIII. The minimum inhibitory concentration of amphotericin B, fluconazole, itraconazole, and voriconazole was determined (cerebrospinal fluid: 0.25, 16, 0.12, and 0.12, blood: 0.25, 4, 0.12, and 0.06, respectively, all in mg/L).

Effectiveness of the National Quality Control Program in Mycology of Argentina and performance evaluation of participating laboratorios / Efectividad del Programa Nacional de Control de Calidad en Micología de Argentina y evaluación del desempeño de los laboratorios participantes

Abstract The National Quality Control Program in Mycology (PNCCM) of Argentina was establishedin 1996 to improve the quality of the mycological diagnosis, to help establish and to setup standardized procedures and continuous training of laboratory staff. The aim of this studywas to assess the effectiveness of the PNCCM in the 1996---2018 period. Data from the NationalMycology Laboratory Network (NMLN) and PNCCM database was used to estimate the increasein the number of controlled laboratories and jurisdictions, the percentage of participation, theimprovement in the quality of results and the adherence to the program. Satisfaction surveyswere performed to assess user satisfaction. The number of controlled laboratories increasedfrom 29 to 146; participation increased from 49% to 93% and general adherence was 72% inthe evaluated period (1996---2018). Improvement in the quality of the results was 15% for lowcomplexity samples; 7% for intermediate complexity samples and 14% for the identification ofhigh complexity strains. Up to 84% of the users consider the PNCCM to be ''very good'' and 16%''satisfactory''. These results show the importance of the PNCCM, which is widely accepted bymycological diagnostic laboratories from Argentina.

Resumen En 1996 se creó el Programa Nacional de Control de Calidad en Micología (PNCCM)de Argentina con el objetivo de mejorar la calidad del diagnóstico micológico, colaborar enel establecimiento de procedimientos estandarizados en aquellos laboratorios que carecen deellos y contribuir a la capacitación continua del personal.El objetivo de este estudio fue evaluar la efectividad del PNCCM en el período 1996-2018.Se utilizaron los datos de la base de la Red Nacional de Laboratorios de Micología (RNLM) ydel PNCCM para estimar el aumento en el número de laboratorios controlados y el porcentajede participación, la mejora de la calidad de los resultados y la adhesión al programa. Paraevaluar el grado de satisfacción de los usuarios, se analizaron las encuestas de satisfacción delos participantes. En el período evaluado, el número de laboratorios controlados aumentó de 29a 146, la participación aumentó de 49% a 93% y la adherencia general de los participantes fue del72%. La mejora de la calidad de los resultados de los laboratorios fue del 15% para muestras debaja complejidad, 7% para muestras de complejidad intermedia y 14% para la identificación decepas de alta complejidad. El 84% de los usuarios considera que el PNCCM es muy bueno y el 16%que es satisfactorio. Estos resultados evidencian la importancia del PNCCM, que es ampliamenteaceptado por los laboratorios que realizan diagnóstico micológico en nuestro país.