Bloodstream Infections Caused by Magnusiomyces capitatus and Magnusiomyces clavatus: Epidemiological, Clinical, and Microbiological Features of Two Emerging Yeast Species.

Magnusiomyces clavatus and Magnusiomyces capitatus are emerging yeasts with intrinsic resistance to many commonly used antifungal agents. Identification is difficult, and determination of susceptibility patterns with commercial and reference methods is equally challenging. For this reason, few data on invasive infections by Magnusiomyces spp. are available. Our objectives were to determine the epidemiology and susceptibility of Magnusiomyces isolates from bloodstream infections (BSI) isolated in Germany and Austria from 2001 to 2020. In seven institutions, a total of 34 Magnusiomyces BSI were identified. Identification was done by internal transcribed spacer (ITS) sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Antifungal susceptibility was determined by EUCAST broth microdilution and gradient tests. Of the 34 isolates, M. clavatus was more common (n = 24) than M. capitatus (n = 10). BSI by Magnusiomyces spp. were more common in men (62%) and mostly occurred in patients with hemato-oncological malignancies (79%). The highest in vitro antifungal activity against M. clavatus/M. capitatus was observed for voriconazole (MIC50, 0.03/0.125 mg/L), followed by posaconazole (MIC50, 0.125/0.25 mg/L). M. clavatus isolates showed overall lower MICs than M. capitatus. With the exception of amphotericin B, low essential agreement between gradient test and microdilution was recorded for all antifungals (0 to 70%). Both species showed distinct morphologic traits on ChromAgar Orientation medium and Columbia blood agar, which can be used for differentiation if no MALDI-TOF MS or molecular identification is available. In conclusion, most BSI were caused by M. clavatus. The lowest MICs were recorded for voriconazole. Gradient tests demonstrated unacceptably low agreement and should preferably not be used for susceptibility testing of Magnusiomyces spp.

Candidalysin is a fungal peptide toxin critical for mucosal infection.

Cytolytic proteins and peptide toxins are classical virulence factors of several bacterial pathogens which disrupt epithelial barrier function, damage cells and activate or modulate host immune responses. Such toxins have not been identified previously in human pathogenic fungi. Here we identify the first, to our knowledge, fungal cytolytic peptide toxin in the opportunistic pathogen Candida albicans. This secreted toxin directly damages epithelial membranes, triggers a danger response signalling pathway and activates epithelial immunity. Membrane permeabilization is enhanced by a positive charge at the carboxy terminus of the peptide, which triggers an inward current concomitant with calcium influx. C. albicans strains lacking this toxin do not activate or damage epithelial cells and are avirulent in animal models of mucosal infection. We propose the name 'Candidalysin' for this cytolytic peptide toxin; a newly identified, critical molecular determinant of epithelial damage and host recognition of the clinically important fungus, C. albicans.

Proteogenomics analysis of CUG codon translation in the human pathogen Candida albicans.

BACKGROUND: Yeasts of the CTG-clade lineage, which includes the human-infecting Candida albicans, Candida parapsilosis and Candida tropicalis species, are characterized by an altered genetic code. Instead of translating CUG codons as leucine, as happens in most eukaryotes, these yeasts, whose ancestors are thought to have lost the relevant leucine-tRNA gene, translate CUG codons as serine using a serine-tRNA with a mutated anticodon, [Formula: see text]. Previously reported experiments have suggested that 3-5% of the CTG-clade CUG codons are mistranslated as leucine due to mischarging of the [Formula: see text]. The mistranslation was suggested to result in variable surface proteins explaining fast host adaptation and pathogenicity. RESULTS: In this study, we reassess this potential mistranslation by high-resolution mass spectrometry-based proteogenomics of multiple CTG-clade yeasts, including various C. albicans strains, isolated from colonized and from infected human body sites, and C. albicans grown in yeast and hyphal forms. Our data do not support a bias towards CUG codon mistranslation as leucine. Instead, our data suggest that (i) CUG codons are mistranslated at a frequency corresponding to the normal extent of ribosomal mistranslation with no preference for specific amino acids, (ii) CUG codons are as unambiguous (or ambiguous) as the related CUU leucine and UCC serine codons, (iii) tRNA anticodon loop variation across the CTG-clade yeasts does not result in any difference of the mistranslation level, and (iv) CUG codon unambiguity is independent of C. albicans' strain pathogenicity or growth form. CONCLUSIONS: Our findings imply that C. albicans does not decode CUG ambiguously. This suggests that the proposed misleucylation of the [Formula: see text] might be as prevalent as every other misacylation or mistranslation event and, if at all, be just one of many reasons causing phenotypic diversity.

Sensitization against Fungi in Patients with Airway Allergies over 20 Years in Germany.

BACKGROUND: Fungal spores are ubiquitous allergens. Severe forms of asthma are particularly highly associated with fungal sensitization. National and international asthma guidelines recommend the implementation of allergen immunotherapy if indicated. Thus, detection and treatment of relevant allergies are key components of primary care of these patients. OBJECTIVES: The aims of the study were (i) to investigate trends in the prevalence of sensitization to twelve fungi in central Germany over the last 20 years and (ii) to dissect specific sensitization patterns among the 3 most important fungi: Aspergillus, Alternaria, and Cladosporium. METHODS: This single-center study evaluated skin prick test (SPT) results of 3,358 patients with suspected airway allergies over a period of 20 years (1998-2017). RESULTS: While 19.2% of all study patients had positive test results to at least 1 of the 3 fungi (Alternaria, Aspergillus, or Cladosporium) in the first study decade, this rate increased to 22.5% in the second decade. Slight increases in sensitization rates to almost all fungi were observed over the 20-year period. In the last decade, polysensitization to Alternaria, Aspergillus, and Cladosporium increased significantly. Sensitization to fungi is age-dependent and peaks in the age-group of 21-40 years during the second decade. CONCLUSION: Fungi are relevant allergens for perennial and seasonal allergy symptoms. We currently recommend including Aspergillus, Alternaria, and Cladosporium in the standard series of SPTs for airway allergies.

Molecular Typing of Candida glabrata.

The yeast Candida glabrata has emerged, second only to Candida albicans, to be one of the most frequently isolated fungi in clinical specimen from human. Its frequent resistance towards azole antifungal drugs and the high capacity to form biofilms on indwelling catheters of individual isolates render it an often difficult to treat pathogen. Hence, there is a notably increasing scientific and clinical interest in this species. This has led to the development of a variety of molecular tools for genetic modification, strain collections, and last but not least different approaches to analyse the population structure among isolates of different geographical and clinical contexts. Often, these are used to study correlations (or the absence thereof) with different pathogenicity, virulence, or drug resistance traits. Three molecular methods have been used to type within the C. glabrata population on a genetic level by multiple studies: multi-locus sequence typing, microsatellite length polymorphisms, and clustering of whole-genome sequencing data, and these are subject of this review.

High diversity of Candida glabrata in a tertiary hospital-Mwanza, Tanzania.

Candida glabrata is a genetically diverse human pathogenic yeast, whose subpopulations have been documented to vary geographically. Here, we report MLST genotypes and antifungal drug susceptibility of C. glabrata isolates from Africa. Among 47 mostly urogenital isolates, we found 13 sequence types, amounting to a 27% genetic population difference. More than half of the isolates were of novel sequence types. ST18 was most predominant and had reduced susceptibility to fluconazole. There was clear segregation of STs between urine and vaginal specimen. In Tanzania, the C. glabrata population is genetically diverse, and divergent from those seen in other countries.

Virulence and susceptibility patterns of clinical Candida spp. isolates from a tertiary hospital, Tanzania.

Despite the increased burden of human immunodeficiency virus (HIV) and other comobidities in developing countries, information regarding antifungal susceptibility patterns of Candida spp. and their virulence potential are still limited. Here, we report the virulence and antifungal susceptibility patterns of Candida spp. from varieties spectrum of candidiasis in a tertiary hospital, Tanzania. The study was conducted from March to December 2017. Candida spp. from clinical samples were characterized. Antifungal susceptibility patterns based on EUCAST guidelines and virulence activities (phospholipase, protease, hemolysin, and coagulase activity) were determined. A total of 399 Candida spp. isolates were obtained, of these, 278, 51 and 47 were C. albicans, C. tropicalis, and C. glabrata, respectively. Phospholipase 193/268, protease 32/51 and coagulase 25/47 were the most frequently detected virulence activities in C. albicans, C. tropicalis, and C. glabrata, respectively. Protease and phospholipase were frequently detected virulence activities from C. albicans from blood and esophageal brushes. The median zone diameter of protease activities was significantly larger among C. tropicalis than C. albicans. C. albicans, and C. tropicalis isolates were 100% sensitive to caspofungin. The proportions of C. albicans isolate resistant to fluconazole, voriconazole and posaconazole were 3.1, 3.6%, and 1.8%, respectively. In conclusion, the majority of Candida spp. isolates were sensitive to fluconazole. There are different phenotypes of C. albicans, C. glabrata and C. tropicalis based on susceptibility and virulence activities patterns, necessitating further molecular characterizations to place them in global perspective. Routine antifungal susceptibility testing to guide clinical therapy should be encouraged in developing countries.

Molecular Tools for the Detection and Deduction of Azole Antifungal Drug Resistance Phenotypes in Aspergillus Species.

The incidence of azole resistance in Aspergillus species has increased over the past years, most importantly for Aspergillus fumigatus. This is partially attributable to the global spread of only a few resistance alleles through the environment. Secondary resistance is a significant clinical concern, as invasive aspergillosis with drug-susceptible strains is already difficult to treat, and exclusion of azole-based antifungals from prophylaxis or first-line treatment of invasive aspergillosis in high-risk patients would dramatically limit drug choices, thus increasing mortality rates for immunocompromised patients. Management options for invasive aspergillosis caused by azole-resistant A. fumigatus strains were recently reevaluated by an international expert panel, which concluded that drug resistance testing of cultured isolates is highly indicated when antifungal therapy is intended. In geographical regions with a high environmental prevalence of azole-resistant strains, initial therapy should be guided by such analyses. More environmental and clinical screening studies are therefore needed to generate the local epidemiologic data if such measures are to be implemented on a sound basis. Here we propose a first workflow for evaluating isolates from screening studies, and we compile the MIC values correlating with individual amino acid substitutions in the products of cyp51 genes for interpretation of DNA sequencing data, especially in the absence of cultured isolates.

Progressive Dispersion of Azole Resistance in Aspergillus fumigatus: Fatal Invasive Aspergillosis in a Patient with Acute Myeloid Leukemia Infected with an A. fumigatus Strain with a cyp51A TR46 Y121F M172I T289A Allele.

Patients with hematologic malignancies as well as allogeneic hematopoietic stem cell transplantation (HSCT) patients are at high risk for invasive aspergillosis. Here, we report a culture- and autopsy-proven fatal invasive aspergillosis in an allogeneic HSTC patient which he developed despite posaconazole prophylaxis. The agent was determined to be an azole-resistant Aspergillus fumigatus strain bearing the cyp51A mutation combination TR46 Y121F M172I T289A. At increasing frequency, the azole resistance of A. fumigatus is being reported globally, limiting treatment options and complicating regimens.

Prevalence of azole-resistant Aspergillus fumigatus in the environment of Thailand.

Occurrence of azole-resistant Aspergillus fumigatus (ARAF) in the environment is an emerging problem worldwide, likely impacting on patient treatment. Several resistance mutations are thought to have initially arisen through triazole-based fungicide use in agriculture and subsequently being propagated in a similar manner. Here we investigated the prevalence of ARAF in the environment of Thailand and characterized their susceptibility profiles toward clinically used azole compounds along with underlying resistance mutations. Three hundred and eight soil samples were collected and analyzed, out of which 3.25% (n = 10) were positive for ARAF. All isolates obtained were resistant to itraconazole (MIC ≥ 8 µg/ml), two showed additional increased MIC values toward posaconazole (MIC = 0.5 µg/ml), and one other toward voriconazole (MIC = 2 µg/ml). Sequencing of the respective cyp51A genes revealed that eight of the isolates carried the TR34/L98H allele and those two with elevated MIC values to posaconazole the G54R substitution. Although a clear correlation between the use of triazole-based fungicides and isolation of ARAF strains from agricultural lands could not be established for Thailand, but this study clearly demonstrates the spread of globally observed ARAF strains to the environment of South East Asia.

Diversity and Antifungal Drug Susceptibility of Cryptococcus Isolates in Thailand.

Yeasts of the Cryptococcus species complex are the causative agent of cryptococcosis, especially in human immunodeficiency virus (HIV) positive individuals. Cerebral or disseminated cryptococcosis has a very high mortality rate worldwide, including in Thailand. Additionally, an increasing rate of antifungal drug resistant cryptococcal isolates has been reported in several neighboring countries, complicating therapeutic approaches. To understand the situation of this infection in Thailand, we retrospectively investigated the molecular epidemiology and antifungal drug resistance in a collection of 74 clinical, 52 environmental and two veterinary isolates using the URA5-RFLP for typing and the EUCAST guideline for susceptibility testing. Where no EUCAST breakpoints (AMB and 5FC) were available, CLSI epidemiologic cutoff values were used for interpretation. Cryptococcal molecular type diversity showed most isolates were C. grubii, molecular type VNI. One clinical isolate was C. deuterogattii (mol. type VGII) and another C. grubii (mol. type VNII). One strain from environment was classified as C. grubii (mol. type VNII). No resistant strains were detected in this retrospective study for either of the antimycotics tested; however, monitoring of the epidemiology of Cryptococcus species in infected patients in Thailand needs to be continued to detect emergence of resistance.

Prevalence of pregnancy-relevant infections in a rural setting of Ghana.

BACKGROUND: Although infectious diseases still account for a high burden of morbidity and mortality in sub-Saharan Africa, simultaneous investigations on multiple infections affecting maternal and child health are missing. METHODS: We conducted a cross-sectional, single-centre pilot study in a rural area of Ghana to assess the infectiological profile during pregnancy. Screening of 180 expectant mothers was done by vaginal swabs and serology to detect the most common pregnancy-relevant infections. They were also interviewed for potential risk factors, outcome of previous pregnancies, and socio-economic aspects. RESULTS: We found a high prevalence of infections caused by hepatitis B virus (16.7% HBs antigen positive). In contrast, infections caused by hepatitis C virus (1.1% anti-HCV) and HIV (0.6%) were rare. Maternal malaria was frequent (10.6%), despite increasing acceptance of intermittent preventive treatment during pregnancy (IPTp). Group B streptococci were present in 10.6% of all pregnant women. Absence of antibodies against varicella zoster virus in 43.2%, Toxoplasma gondii in 26.8%, parvovirus B19 in 20.0%, and rubella virus in 15.7% makes a significant proportion of pregnant women susceptible for acquiring primary infections. Whereas all study participants had specific IgG antibodies against human cytomegalovirus, infections with Listeria, Brucella, or Neisseria gonorrhoeae as well as active syphilis were absent. CONCLUSIONS: Our pilot study in a rural community in Ghana indicates an urgent need for action in dealing at least with high-prevalent pregnancy-relevant infections, such as hepatitis B, malaria and those caused by group B streptococci. In addition, the resulting prevalence rates of various other infections may offer guidance for health officials to prioritize possible future intervention schemes.

One small step for a yeast--microevolution within macrophages renders Candida glabrata hypervirulent due to a single point mutation.

Candida glabrata is one of the most common causes of candidemia, a life-threatening, systemic fungal infection, and is surpassed in frequency only by Candida albicans. Major factors contributing to the success of this opportunistic pathogen include its ability to readily acquire resistance to antifungals and to colonize and adapt to many different niches in the human body. Here we addressed the flexibility and adaptability of C. glabrata during interaction with macrophages with a serial passage approach. Continuous co-incubation of C. glabrata with a murine macrophage cell line for over six months resulted in a striking alteration in fungal morphology: The growth form changed from typical spherical yeasts to pseudohyphae-like structures - a phenotype which was stable over several generations without any selective pressure. Transmission electron microscopy and FACS analyses showed that the filamentous-like morphology was accompanied by changes in cell wall architecture. This altered growth form permitted faster escape from macrophages and increased damage of macrophages. In addition, the evolved strain (Evo) showed transiently increased virulence in a systemic mouse infection model, which correlated with increased organ-specific fungal burden and inflammatory response (TNFα and IL-6) in the brain. Similarly, the Evo mutant significantly increased TNFα production in the brain on day 2, which is mirrored in macrophages confronted with the Evo mutant, but not with the parental wild type. Whole genome sequencing of the Evo strain, genetic analyses, targeted gene disruption and a reverse microevolution experiment revealed a single nucleotide exchange in the chitin synthase-encoding CHS2 gene as the sole basis for this phenotypic alteration. A targeted CHS2 mutant with the same SNP showed similar phenotypes as the Evo strain under all experimental conditions tested. These results indicate that microevolutionary processes in host-simulative conditions can elicit adaptations of C. glabrata to distinct host niches and even lead to hypervirulent strains.

Aspergillus fumigatus carrying TR34/L98H resistance allele causing complicated suppurative otitis media in Tanzania: Call for improved diagnosis of fungi in sub-Saharan Africa.

BACKGROUND: Suppurative otitis media (SOM) is a major public health concern worldwide and is associated with increased morbidity. Cases of fungal suppurative otitis media were studied to establish the effect of fungi in otitis media. METHODS: Ear swabs from 410 patients were collected aseptically using sterile cotton swabs from discharging ear through perforated tympanic membrane. Swabs were subjected to microscopic and culture investigations. The species of fungal growing on Sabouraud's agar were identified using MALDI-TOF MS. For moulds broth micro dilution method following EUCAST guidelines was employed to determine susceptibility patterns against itraconazole, voriconazole and posaconazole. RESULTS: A total of 44 (10.74 %) cases with positive fungal culture growth were studied. The median age of patients with fungal infection was 29.5 (IQR 16-43) years. Of 44 patients; 35 (79.6 %) had pure growth of one type of fungal. Candida albicans was the most common fungus isolated (n = 13; 29.6 %) followed by Aspergillus versicolor (n = 8; 18.2 %). A total of 7 (15.9 %) patients had disease complication at time of enrollment; of them 6 (13.6 %) had hearing loss. On follow up 7 (15.9 %) had poor treatment outcome. All five Aspergillus fumigatus strains resistant itraconazole with reduced susceptibility to voriconazole and posaconazole carried carrying TR34/L98H resistance allele. In addition, all Penicillium citrinum isolates were resistant to voriconazole while all Penicillium sumatrense were resistant to both itraconazole and voriconazole. There were non-significant association of poor treatment outcome and female gender, being HIV positive and being infected with moulds. CONCLUSION: Fungal infections play a significant role in SOM pathology in our setting. Diagnosis of fungal infections in developing countries should be improved so that appropriate management can be initiated on time to prevent associated complications.

Identification of Stachybotrys spp. by MALDI-TOF mass spectrometry.

Stachybotrys (S.) spp. are omnipresent cellulolytic molds. Some species are highly toxic owing to their ability to synthesize various secondary metabolites such as macrocyclic trichothecenes or hemolysins. The reliable identification of Stachybotrys at species level is currently limited to genome-based identification. This study aimed to establish a fast and reliable MALDI-TOF MS identification method by optimizing the pre-analytical steps for protein extraction for subsequent generation of high-quality fingerprint mass spectra. Eight reference strains of the American Type Culture Collection and the Technical University of Denmark were cultivated in triplicate (biological repetitions) for 2 days in malt extract broth. The mycelia (1.5 ml) were first washed with 75 % ethanol and an additional washing step with dimethyl sulfoxide (10 %) was added to remove unspecific low weight masses. Furthermore, mycelia were broken with roughened glass beads in formic acid (70 %) and acetonitrile. The method was successfully applied to a total of 45 isolates of Stachybotrys originating from three different habitats (indoor, feed, and food samples; n = 15 each): Twenty-seven isolates of S. chartarum and 18 isolates of S. chlorohalonata could be identified by MALDI-TOF MS. The data obtained exactly matched those obtained by genome-based identification. The mean score values for S. chartarum ranged from 2.509 to 2.739 and from 2.148 to 2.622 for S. chlorohalonata with a very good reproducibility: the relative standard deviations were between 0.3 % and 6.8 %. Thus, MALDI-TOF MS proved to be a fast and reliable alternative to identification of Stachybotrys spp. by nucleotide amplification and sequencing.

SMRT sequencing of the Campylobacter coli BfR-CA-9557 genome sequence reveals unique methylation motifs.

BACKGROUND: Campylobacter species are the most prevalent bacterial pathogen causing acute enteritis worldwide. In contrast to Campylobacter jejuni, about 5 % of Campylobacter coli strains exhibit susceptibility to restriction endonuclease digestion by DpnI cutting specifically 5'-G(m)ATC-3' motifs. This indicates significant differences in DNA methylation between both microbial species. The goal of the study was to analyze the methylome of a C. coli strain susceptible to DpnI digestion, to identify its methylation motifs and restriction modification systems (RM-systems), and compare them to related organisms like C. jejuni and Helicobacter pylori. RESULTS: Using one SMRT cell and the PacBio RS sequencing technology followed by PacBio Modification and Motif Analysis the complete genome of the DpnI susceptible strain C. coli BfR-CA-9557 was sequenced to 500-fold coverage and assembled into a single contig of 1.7 Mbp. The genome contains a CJIE1-like element prophage and is phylogenetically closer to C. coli clade 1 isolates than clade 3. 45,881 6-methylated adenines (ca. 2.7 % of genome positions) that are predominantly arranged in eight different methylation motifs and 1,788 4-methylated cytosines (ca. 0.1 %) have been detected. Only two of these motifs correspond to known restriction modification motifs. Characteristic for this methylome was the very high fraction of methylation of motifs with mostly above 99 %. CONCLUSIONS: Only five dominant methylation motifs have been identified in C. jejuni, which have been associated with known RM-systems. C. coli BFR-CA-9557 shares one (RAATTY) of these, but four ORFs could be assigned to putative Type I RM-systems, seven ORFs to Type II RM-systems and three ORFs to Type IV RM-systems. In accordance with DpnI prescreening RM-system IIP, methylation of GATC motifs was detected in C. coli BfR-CA-9557. A homologous IIP RM-system has been described for H. pylori. The remaining methylation motifs are specific for C. coli BfR-CA-9557 and have been neither detected in C. jejuni nor in H. pylori. The results of this study give us new insights into epigenetics of Campylobacteraceae and provide the groundwork to resolve the function of RM-systems in C. coli.

Environmental isolates of azole-resistant Aspergillus fumigatus in Germany.

Azole antifungal drug resistance in Aspergillus fumigatus is an emerging problem in several parts of the world. Here we investigated the distribution of such strains in soils from Germany. At a general positivity rate of 12%, most prevalently, we found strains with the TR34/L98H and TR46/Y121F/T289A alleles, dispersed along a corridor across northern Germany. Comparison of the distributions of resistance alleles and genotypes between environment and clinical samples suggests the presence of local clinical clusters.

Proteomic analysis of hyperadhesive Candida glabrata clinical isolates reveals a core wall proteome and differential incorporation of adhesins.

Attachment to human host tissues or abiotic medical devices is a key step in the development of infections by Candida glabrata. The genome of this pathogenic yeast codes for a large number of adhesins, but proteomic work using reference strains has shown incorporation of only few adhesins in the cell wall. By making inventories of the wall proteomes of hyperadhesive clinical isolates and reference strain CBS138 using mass spectrometry, we describe the cell wall proteome of C. glabrata and tested the hypothesis that hyperadhesive isolates display differential incorporation of adhesins. Two clinical strains (PEU382 and PEU427) were selected, which both were hyperadhesive to polystyrene and showed high surface hydrophobicity. Cell wall proteome analysis under biofilm-forming conditions identified a core proteome of about 20 proteins present in all C. glabrata strains. In addition, 12 adhesin-like wall proteins were identified in the hyperadherent strains, including six novel adhesins (Awp8-13) of which only Awp12 was also present in CBS138. We conclude that the hyperadhesive capacity of these two clinical C. glabrata isolates is correlated with increased and differential incorporation of cell wall adhesins. Future studies should elucidate the role of the identified proteins in the establishment of C. glabrata infections.

Molecular types of Cryptococcus gattii/Cryptococcus neoformans species complex from clinical and environmental sources in Nairobi, Kenya.

Cryptococcal meningitis infections cause high mortality rates among HIV-infected patients in Sub-Saharan Africa. The high incidences of cryptococcal infections may be attributed to common environmental sources which, if identified, could lead to institution of appropriate control strategies. To determine the genotypes of Cryptococcus gattii/C. neoformans- species complex from Nairobi, Kenya, 123 clinical and environmental isolates were characterised. Typing was done using orotidine monophosphate pyrophosphorylase (URA5) gene restriction fragment length polymorphism (URA5-RFLP). The majority of the isolates [105/123; 85.4%] were C. neoformans genotype (AFLPI/VNI) and 1.6% AFLP1A/VNB/VNII, whereas (13%) were C. gattii (AFLP4/VGI). This is the first report on the genotypes of C. gattii/C. neoformans species complex from clinical and environmental sources in Nairobi, Kenya and the isolation of C. gattii genotype AFLP4/VGI from the environment in Kenya.

Yeast on-target lysis (YOTL), a procedure for making auxiliary mass spectrum data sets for clinical routine identification of yeasts.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based species identification has become a reliable and fast tool for use in clinical diagnostics, including in mycology. To identify yeasts in the MALDI Biotyper system, a multistep extraction protocol, which is also used to generate the reference spectra, is recommended. Sample preparation by on-target lysis (OTL) requires significantly less hands-on time and is therefore highly desirable, but it results in too-low MALDI Biotyper log score values to allow automated species identification. To overcome this problem, we developed a procedure for generating and validating an OTL spectrum data set for the most relevant and frequently occurring yeast species in clinical specimens. The performance was evaluated against a set of OTL spectra derived during clinical routine procedures and from a set of closely related yeasts. In the diagnostic setting, the OTL procedure significantly decreased the workload but allowed species identification with high specificity and sensitivity. False identifications were not observed. The use of in-house-generated OTL reference spectra can highly accelerate MALDI-TOF MS-based yeast species identification using the MALDI Biotyper.