Multi-locus sequence typing and phylogenetics of Cryptococcus neoformans AD hybrids.

Hybrid AD strains of the human pathogenic Cryptococcus neoformans species complex have been reported from many parts of the world. However, their origin, diversity, and evolution are incompletely understood. In this study, we analyzed 102 AD hybrid strains representing 21 countries on five continents. For each strain, we obtained its mating type and its allelic sequences at each of the seven loci that have been used for genotyping haploid serotypes A and D strains of the species complex by the Cryptococcus research community. Our results showed that most AD hybrids exhibited loss of heterozygosity at one or more of the seven analyzed loci. Phylogenetic and population genetic analyses of the allelic sequences revealed multiple origins of the hybrids within each continent, dating back to one million years ago in Africa and up to the present in other continents. We found evidence for clonal reproduction and long-distance dispersal of these hybrids in nature. Comparisons with the global haploid serotypes A and D strains identified new alleles and new haploid multi-locus genotypes in AD hybrids, consistent with the presence of yet-to-be discovered genetic diversity in haploid populations of this species complex in nature. Together, our results indicate that AD hybrids can be effectively genotyped using the same multi-locus sequencing type approach as that established for serotypes A and D strains. Our comparisons of the AD hybrids among each other as well as with the global haploid serotypes A and D strains revealed novel genetic diversity as well as evidence for multiple origins and dynamic evolution of these hybrids in nature.

Assessment of caspofungin susceptibility of Candida glabrata by the Etest®, CLSI, and EUCAST methods, and detection of FKS1 and FKS2 mutations.

Candida glabrata has emerged as a major pathogen in invasive candidiasis in recent years. Currently, guidelines for invasive candidiasis treatment recommend fluconazole or an echinocandin as the first-line therapy. Nevertheless, the resistance of Candida glabrata to echinocandin is an emerging problem and has been partly associated with mutations in the FKS1 and FKS2 genes. The Etest® is an appropriate method for determining antifungal susceptibility in emergency routine diagnosis. In this work, we evaluated the reliability of the Etest® in comparison with the two reference broth microdilution methods, Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST), to assess the caspofungin resistance of 193 isolates of Candida glabrata. The interpretation of minimum inhibitory concentration (MIC) values was also discussed according to different breakpoints. Moreover, FKS1 and FKS2 mutations were investigated for isolates with high MICs. Our results showed that the MIC50 value was similar to the MIC90 value for each method. The Etest® method showed the lowest MIC values, whereas EUCAST presented the highest. Categorical agreement between the Etest® and CLSI methods was 100 % and 36 % using the breakpoints proposed by Arendrup et al. (Antimicrob Agents Chemother 56(7):3965-3968, 2012) and Pfaller et al. (Int J Antimicrob Agents 38(1):65-69, 2011), respectively. Two isolates showed high MIC values with the three methods and both presented FKS2 mutations. A novel FKS2 mutation was also reported for one isolate. Future epidemiological studies should also evaluate the reliability of the Etest® to detect echinocandin resistance, as it remains a routine method.

Taxonomy and antifungal susceptibility of clinically important Rasamsonia species.

In recent years, Geosmithia argillacea has been increasingly reported in humans and animals and can be considered an emerging pathogen. The taxonomy of Geosmithia was recently studied, and Geosmithia argillacea and related species were transferred to the new genus Rasamsonia. The diversity among a set of Rasamsonia argillacea strains, including 28 clinical strains, was studied, and antifungal susceptibility profiles were generated. Data obtained from morphological studies and from phylogenetic analyses of internal transcribed spacer (ITS) and partial ß-tubulin and calmodulin sequences revealed the presence of four species in the Rasamsonia argillacea complex, two of which are newly described here: R. piperina sp. nov. and R. aegroticola sp. nov. In contrast to other related genera, all Rasamsonia species can be identified with ITS sequences. A retrospective identification was performed on recently reported clinical isolates from animal or human patients. Susceptibility tests showed that the antifungal susceptibility profiles of the four members of the R. argillacea complex are similar, and caspofungin showed significant activity in vitro, followed by amphotericin B and posaconazole. Voriconazole was the least active of the antifungals tested. The phenotypically similar species R. brevistipitata and R. cylindrospora had different antifungal susceptibility profiles, and this indicates that correct species identification is important to help guide appropriate antifungal therapy.

Interlaboratory reproducibility of Etest amphotericin B and caspofungin yeast susceptibility testing and comparison with the CLSI method.

This study aimed to assess the interlaboratory reproducibility at four university hospital laboratories in the southeast region of France of the Etest technique for the determination of caspofungin (CAS) and amphotericin B (AMB) MICs and to compare it to the CLSI broth microdilution reference method. Consecutive clinical yeast isolates (n = 198) were included in the study. AMB and CAS MICs were read at 24 and 48 h. Interlaboratory reproducibility was estimated by using (i) an intraclass correlation coefficient (ICC), (ii) essential agreement (EA), and (iii) categorical agreement (CA). For Etest interlaboratory reproducibility for CAS, ICCs were 0.80 (95% confidence interval [CI], 0.76 to 0.84) and 0.81 (95% CI, 0.77 to 0.85) at 24 and 48 h, respectively. For AMB, the ICCs were 0.51 (95% CI, 0.43 to 0.58) and 0.69 (95% CI, 0.63 to 0.74) at 24 and 48 h, respectively. At 48 h, the between-center EAs ranged from 94.4 to 99.0% for both antifungals. For the comparison of the CLSI method and the Etest, the between-technique ICCs were 0.69 (95% CI, 0.63 to 0.74) and 0.62 (95% CI, 0.55 to 0.68) for CAS and AMB, respectively. The EAs ranged from 76.5 to 98.5% for CAS and from 90.3 to 97.4% for AMB according to the centers. CAs ranged from 87.9% to 91.4%, with four very major errors for 2 strains (1 Candida albicans strain and 1 Candida krusei strain), for CAS and from 97.5 to 99.5%, with four major errors, for AMB. In conclusion, the Etest showed a good interlaboratory reproducibility and a good correlation with the CLSI technique. It is well suited for the routine clinical laboratory and can thus be used to monitor clinical yeast isolates' in vitro susceptibilities in this setting.

A Candida albicans strain with high MIC for caspofungin and no FKS1 mutations exhibits a high chitin content and mutations in two chitinase genes.

We studied the cell wall of a Candida albicans laboratory mutant exhibiting a high minimum inhibitory concentration (MIC; 8 µg ml(-1)) for caspofungin without bearing FKS1 mutations. This strain showed a reduced level of ß 1,3 D glucan (0.43×) and a higher chitin content (2.3×) than a control strain even when grown without caspofungin. No significant over- or under-expression of chitin synthase or chitinase genes was observed. However, point mutations were detected in the chitinase 2 and 3 genes. These mutations, which may affect the enzymatic activity of the encoded protein products involved in the degradation of the chitin, could have led to an increased concentration of that component, allowing the strain to compensate for its low ß 1,3 D glucan content and the effect of caspofungin.

Comparison of the Sensititre YeastOne® dilution method with the Clinical Laboratory Standards Institute (CLSI) M27-A3 microbroth dilution reference method for determining MIC of eight antifungal agents on 102 yeast strains.

The Clinical Laboratory Standards Institute ([CLSI] formerly NCCLS) reference broth microdilution testing method (protocol M27-A3) was compared with a commercially available methods (Sensititre YeastOne(®)) by testing two quality control strains and 102 isolates of Candida sp. and Cryptococcus sp. against fluconazole, itraconazole, ketoconazole, posaconazole, voriconazole, flucytosin, amphotericin B and caspofungin. Minimal inhibitory concentrations (MIC) endpoints were determined after 24h of incubation for Sensititre YeastOne(®) and after 24 and 48 h for CLSI microdilution method. Essential agreements between methods vary from 70.6 to 92.2%. Categorical agreements vary from 94.1% for 5FC to 72.6% for AMB. Sensititre YeastOne(®) reading appears to be useful for avoiding very major errors and this confirms the interest of this method for evaluating new antifungals activity in vitro.

First report of cryptococcosis due to Cryptococcus gattii sensu stricto VGI in an Ivorian HIV negative patient.

INTRODUCTION: Cryptococcus gattii species complex is endemic to tropical and subtropical regions and is described as a causative agent of cryptococcosis in immunocompetent individuals. CASE PRESENTATION: We describe the first case of cryptococcosis in a HIV-negative patient from Ivory Coast infected by Cryptococcus gattii sensu stricto VGI. Isolates were recovered from cerebrospinal fluid (CSF) prior to systemic antifungal treatment up to 42 days after detection of the presence of yeasts in the CSF. Eighteen isolates were recovered, genetic diversity and antifungal susceptibility analyses were performed. All the isolates belonged to the Cryptococcus gattii sensu stricto (B;VGI) and were identified as a new sequence type (ST) 553 by Multilocus Sequence Typing (MLST) analyses. Susceptibility testing showed that all the strains had a wild-type phenotype for fluconazole, amphotericin B and flucytosine. Treatment with fluconazole (1200mg/day) was initiated with success. CONCLUSION: This is the first case report of the presence of C. gattii sensu stricto VGI in a HIV-negative ivorian patient and the second report of the presence of species from the C. gattii complex species in this country.

Antifungal susceptibility of 205 Candida spp. isolated primarily during invasive Candidiasis and comparison of the Vitek 2 system with the CLSI broth microdilution and Etest methods.

Infections due to Candida spp. are frequent, particularly in immunocompromised and intensive care unit patients. Antifungal susceptibility tests are now required to optimize antifungal treatment given the emergence of acquired antifungal resistance in some Candida species. An antifungal susceptibility automated method, the Vitek 2 system (VK2), was evaluated. VK2 was compared to the CLSI broth microdilution reference method and the Etest procedure. For this purpose, 205 clinical isolates of Candida spp., including 11 different species, were tested for fluconazole, voriconazole, and amphotericin B susceptibility. For azoles, essential agreement ranged from 25% to 100%, depending on the method used and the Candida species tested. Categorical agreements for all of the species averaged 92.2% and ranged from 14.3 to 100%, depending on the 24-h or 48-h MIC reading by the Etest and CLSI methods and on the Candida species. Results obtained for Candida albicans showed excellent categorical and essential agreements with the two comparative methods. For Candida glabrata, the essential agreement was high with the CLSI method but low with the Etest method, and several very major errors in interpretation were observed between VK2 and the Etest method for both azoles. Low MICs of fluconazole were obtained for all of the Candida krusei isolates, but the VK2 expert software corrected all of the results obtained to resistant. Amphotericin B results showed MICs of < or = 1 mg/liter for 201 (VK2), 190 (CLSI), and 202 (Etest) isolates. The AST-YS01 Vitek 2 card system (bioMérieux) is a reliable and practical standardized automated antifungal susceptibility test. Nevertheless, more assays are needed to better evaluate C. glabrata fluconazole sensitivity.

African ST173 Cryptococcus deuterogattii strains are commonly less susceptible to fluconazole: An unclear mechanism of resistance.

OBJECTIVES: Fluconazole (FCZ), either alone or in combination, is often administered for treatment of cryptococcal meningitis, especially in sub-Saharan Africa. Its extensive use has led to the emergence of FCZ-resistant strains. The mechanisms underlying FCZ resistance are poorly documented for yeasts belonging to the Cryptococcus gattii species complex. The literature suggests that resistance could be due to mutations in and/or overexpression of the ERG11 gene (encoding the 14-α-demethylase) and efflux pumps such as MDR and AFR (two subclasses of ABC transporters). Here we highlight the presence of genotype VGII strains (Cryptococcus deuterogattii) from the Ivory Coast with a rare sequence type (ST173) associated with high FCZ minimum inhibitory concentrations (MICs) compared with strains originating from the Pacific Northwest (USA). METHODS: Mechanisms of FCZ resistance were investigated in 28 Ivorian clinical C. deuterogattii isolates recovered from three patients during their antifungal treatment and follow-up. RESULTS: The results demonstrated that: (i) these strains exhibited no mutations in the ERG11 gene; (ii) some strains had increased ERG11 and MDR1 mRNA expression, whilst AFR1 and AFR2 were not overexpressed in strains with high FCZ MICs compared with the expression levels for strains with low FCZ MICs; and (iii) exposure to FCZ in strains with high MICs induced AFR1 mRNA overexpression. CONCLUSION: This study demonstrated that the FCZ resistance mechanism commonly described in Cryptococcus neoformans was not responsible for resistance to FCZ in rare subtype strains.

Epidemiology and antifungal susceptibility testing of non-albicansCandida species colonizing mucosae of HIV-infected patients in Yaoundé (Cameroon).

Non-albicans Candida (NAC) species have emerged as potent pathogenic yeasts among HIV-infected patients. Authors evaluated the epidemiology and antifungal susceptibility testing of non-albicansCandida species colonizing Yaoundé (capital of the Republic of Cameroon, Central Africa) HIV-infected patients. The mucosal specimens were collected and submitted to the mycological diagnosis. Yeast isolates were identified by the Matrix Assisted Laser Desorption Ionisation - Time of Flight Mass Spectrometry (MALDI-TOF MS). The antifungal susceptibility testing was achieved by the CLSI-M27 protocols, and the interpretation of clinical break points (CBPs) and epidemiological cutoff values were in accordance with the CLSI-M60 and M59 recommendations. Four hundred and two patients were recruited and 1218 samples collected. The colonisation frequency was 24.1% and 304 yeasts isolated. Yeast isolates were 113 (37.2%) C. albicans, 2 (0.7%) C. africana and 172 (56.6%) NAC isolates. The NAC isolates were grouped into 13 species including C. krusei (18.1%), C. glabrata (10.9%), C. tropicalis (8.5%) and C. parapsilosis (5.9%) as the major ones. All the isolates appeared to be wild-type for amphotericin B and itraconazole. One (1/33) isolate of C. glabrata was resistant to fluconazole. C. arapsilosis isolates appeared all susceptible to fluconazole. C. tropicalis isolates presented 50% (13/26) resistance to fluconazole. The achieved results bring out new insights about epidemiology of NAC species in Cameroon. The results also highlight the resistance of NAC species to current antifungal drugs.

Cryptoccocal meningitis in Yaoundé (Cameroon) HIV infected patients: Diagnosis, frequency and Cryptococcus neoformans isolates susceptibility study to fluconazole.

Cryptococcal meningitis is a mycosis encountered especially in patients with Acquired Immunodeficiency Syndrome and is fatal in the absence of treatment. Information on epidemiology, diagnosis and susceptibility profile to antifungal drugs, are scarce in Cameroon. Authors evaluated the diagnosis possibilities of the cryptococcal meningitis in Cameroon, and studied the antifungal susceptibility of isolated strains to fluconazole, used as first line treatment of the disease in Cameroon. Between December 2009 and July 2011, 146 cerebrospinal fluids obtained from HIV patients with suspicion of meningitis were analysed. The diagnosis procedure involved macroscopic and cyto-chemical analysis, India ink test, culture on Sabouraud chloramphenicol medium and antigen latex agglutination test. Antifungal susceptibility testing of isolated strains to fluconazole was done by the E-test(®) method. The diagnosis of cryptococcal meningitis gave 28.08% positive cases. Among these patients, 80% were at stages III and IV and 20% at stage I of the HIV infection, according to the WHO previous classification. Cyto-chemical analysis showed current findings in the case of cryptococcal meningitis. India ink test and latex agglutination test exhibited very high sensitivity and specificity (>94%). Fluconazole antifungal susceptibility testing gave MICs lower than 32µg/mL to 92.7% of isolated strains and MICs greater than this value to 7.3% of isolates. These results showed that cryptococcal meningitis remains a real problem among HIV infected patients in Yaoundé. The emergence of fluconazole reduced susceptibility strains is worrying. Nevertheless, efficacy of rapid detection tests is interesting because this will help in rapid diagnosis and treatment of patients.

A longitudinal study of lung transplant recipients infected with Aspergillus: genetic polymorphism of A fumigatus.

BACKGROUND: Aspergillus infection is a well-known complication of lung transplantation and remains associated with high mortality rates. Molecular typing methods are required to elucidate the complex epidemiology of Aspergillus disease in lung transplant recipients. METHODS: Eight lung transplant recipients from one hospital were followed for A fumigatus colonization or infection. Forty-four sequential isolates from these patients were selected and typed by three molecular methods (random amplified polymorphic DNA, sequence-specific DNA primer and multi-locus enzyme electrophoresis). RESULTS: Sixteen different types were identified of which 14 were specific to 1 patient. A factorial correspondence analysis showed that variability between sequential isolates from a single patient was as high as between isolates from the other patients. Lung transplant recipients presented many different genotypes, reflecting the environmental diversity of A fumigatus. Nevertheless, throughout their follow-up, 2 of the 8 lung transplant recipients harbored a common genotype that was not replaced by others. CONCLUSIONS: These results confirm the important genetic polymorphism of the A fumigatus population. The observed genotypes were not related to the type of Aspergillus disease or anti-fungal treatment used nor to the outcome of the patient. These data confirm that all A fumigatus molecular types present the same pathogenic risk.

Clonality structure in Candida dubliniensis.

Multilocus enzyme electrophoresis was performed on 76 European strains of Candida dubliniensis. Ten of the 20 enzyme-encoding loci were polymorphic, giving rise to 10 electrophoretic types within the sample studied. Investigation of the population genetics of a subset of 36 strains from HIV-infected patients in London showed the existence of strong heterozygote deficits and excesses associated with significant linkage disequilibria between pairs of loci. These findings, together with the predominance of multilocus genotypes, strongly suggest that C. dubliniensis is mainly (if not totally) clonal. Analysis of genotypes of a larger number of strains should confirm this conclusion and improve our understanding of the epidemiology of this pathogen.

Multilocus enzyme electrophoresis analysis of Aspergillus fumigatus strains isolated from the first clinical sample from patients with invasive aspergillosis. EBGA Network. European Research Group on Biotype and Genotype of Aspergillus.

The genotypes of 50 isolates of Aspergillus fumigatus from 11 patients with invasive aspergillosis, obtained from three hospitals in different geographical areas, were determined by multilocus enzyme electrophoresis (MLEE). The study analysed the genetic polymorphism of multiple isolates from the first sample. Seven of the 14 enzymic loci studied were polymorphic, giving rise to eight different electrophoretic types. For nine of 11 patients studied, no polymorphism was observed in isolates within the first clinical sample. Analysis of genetic distance between electrophoretic types demonstrated a genetic heterogeneity within each geographical site. Moreover, some genotypes were preferentially found in a given area and this revealed a population structure within these geographical sites. Therefore, the epidemiology of A. fumigatus should be considered separately for each of these areas. The multiple discriminatory markers of MLEE seem to provide a powerful tool for increasing the understanding of the biology of this fungus.

[Kidney transplant and Candida albicans arteritis. The importance of analysing the transplant conservation liquid]. / Greffe rénale et artérite du greffon à Candida albicans. Importance de l'analyse du liquide de conservation du greffon.

INTRODUCTION: Candida arteritis can compromise the functional prognosis of the graft or even the life of the transplant recipient. The infection can be transmitted by the graft. OBSERVATION: A 46 year-old woman contracted a Candida albicans ateritis of the graft following a kidney transplant that led to a detransplantation. The yeast was probably transmitted by the graft from the donor, source of an unknown candida infection: it was found in the conservation liquid of the graft itself, and in the renal artery and vascular pedicle. Analysis of of these three elements by enzymatic electrophoresis showed that they were identical. COMMENTARIES: This case report underlines the need to establish guidelines and sanitary safety measures, notably that of systematically placing in culture the concervation solutions and alerting the transplant team if any fungi are isolated.

Genotyping and antifungal susceptibility testing of Cryptococcus neoformans isolates from Cameroonian HIV-positive adult patients.

Cryptococcus neoformans is the most common cause of meningitis amongst adult Africans with HIV/AIDS. The widespread use of fluconazole may lead to the emergence of isolates with reduced susceptibility. We studied C. neoformans isolates from HIV-infected patients with cryptococcal meningitis. Genotyping and antifungal testing were performed to assess the genetic diversity, occurrence of mixed infections and in vitro activity of antifungal agents. Isolates were recovered from cerebrospinal fluid prior to systemic antifungal treatment. Six isolates were studied for each sample (a total of 114 isolates from 19 patients). Serotyping was performed via LAC 1 and CAP 64 gene amplification and genotyping was performed using phage M13 core, (GACA)4 and (GTG)5 primers and restriction polymorphism analysis of the URA5 gene. Susceptibilities for amphotericin B, flucytosine, fluconazole, voriconazole and posaconazole were tested by the Sensititre YeastOne® method. All strains were identified as C. neoformans var. grubii serotype A. We identified nine major genotypes. Up to two genotypes were identified in the same sample. None of the isolates were resistant to the studied drugs. However, 13 of 114 strains exhibited a reduced susceptibility to fluconazole and 13 of 114 strains exhibited a reduced susceptibility to flucytosine. No correlation was found between the genotype and susceptibility. This study confirms the prevalence of C. neoformans serotype A in Cameroon. Two genotypes may be responsible for a single episode of cryptococcosis. The possibility of mixed infection and diminished susceptibility to fluconazole or flucytosine must be considered for the management of cryptococcosis.

Typing of Saccharomyces cerevisiae clinical strains by using microsatellite sequence polymorphism.

It seems that S. cerevisiae, which was thought for about 30 years to be a nonpathogenic yeast, should now be considered an opportunistic pathogen. In this study, we estimated the discrimination ability of the microsatellite sequence amplification technique within a sample of clinical and reference S. cerevisiae strains and S. boulardii reference strains.

Genotypic identification of Candida dubliniensis isolated from HIV patients by MLEE.

Candida dubliniensis is a novel species only recently described. This emerging pathogen shares some of the phenotypic characteristics specific to C. albicans but is genetically different. In this study we typed four strains of atypical C. albicans isolated in our laboratory and compared them to 41 strains of C. albicans and 11 strains of C. dubliniensis by several phenotypic methods and by multilocus enzyme electrophoresis. Using factorial correspondence analysis, we distinguished C. dubliniensis and the atypical C. albicans strains from all strains of C. albicans. Atypical C. albicans strains were identified as C. dubliniensis.

Genetic multilocus studies of different strains of Cryptococcus neoformans: taxonomy and genetic structure.

The genotypes of 107 strains of Cryptococcus isolated from the environment or from patients from various geographical areas were determined by multilocus enzyme electrophoresis (MLEE). We analyzed the relationships between genotype structure and serotype and between genotype structure and strain origin. Twelve of the 14 enzyme-encoding loci studied were polymorphic, giving rise to 48 electrophoretic types. The genotypes of C. neoformans and C. laurentii were very similar. MLEE could not distinguish between these two pathogenic species. A correlation between the genetic multilocus structure and the origin of the sample (from the environment or patients) existed. A second analysis detected a correlation between genotype distribution and serotype. The second analysis considered three serotype groups (B, C, and A plus D plus A/D), proving that serotypes A, D, and A/D are closely related. MLEE is a useful epidemiological tool for improving our understanding of the biology of this fungus.

Genetic polymorphism of Aspergillus fumigatus in clinical samples from patients with invasive aspergillosis: investigation using multiple typing methods.

The genotypes of 52 strains of Aspergillus fumigatus isolated from 12 patients with invasive aspergillosis were investigated using three typing methods (random amplified polymorphic DNA, sequence-specific DNA polymorphism, and microsatellite polymorphism) combined with multilocus enzyme electrophoresis. Isolates were from patients hospitalized in three different geographic areas (Lyon, France; Grenoble, France; and Milan, Italy). In each case, the genetic polymorphism of several colonies (two to five) within the first respiratory clinical sample was studied. For the 52 isolates tested, random amplified polymorphic DNA identified 8 different genotypes, sequence-specific DNA polymorphism identified 9 different types, and microsatellite polymorphism identified 14 types. A combination of these results with multilocus enzyme electrophoresis study identified 25 different types within the sample studied. We identified 3 patients (of the 12 studied) who carried a single genotype; 6 patients were infected by two genotypes, 1 patient had four genotypes, while the last patient had five. A combination of typing methods provided better discrimination than the use of a single method. Typing methods revealed a population structure within each geographical site, suggesting that the epidemiology of A. fumigatus should be considered separately for each of these geographic areas. This study demonstrates the usefulness of combining several typing methods in reaching an understanding of the epidemiology of A. fumigatus and clarifies whether it is sufficient to type one isolate from each specimen to determine the strain involved in invasive aspergillosis.