Draft Genome Sequence of Candida saopaulonensis from a Very Premature Infant with Sepsis.

The rare fungus Candida saopaulonensis has never been reported to be associated with human infection. We report the draft genome sequence of the first clinical isolate of C. saopaulonensis, which was isolated from a very premature infant with sepsis. This is the first genome assembly reaching the near-complete chromosomal level with structural annotation for this species, opening up avenues for exploring evolutionary patterns and genetic mechanisms of pathogenesis.

Emergence of multiple fluconazole-resistant Candida parapsilosis sensu stricto clones with persistence and transmission in China.

OBJECTIVES: We explored the epidemiological and molecular characteristics of Candida parapsilosis sensu stricto isolates in China, and their mechanisms of azole resistance. METHODS: Azole susceptibilities of 2318 non-duplicate isolates were determined using CLSI broth microdilution. Isolates were genotyped by a microsatellite typing method. Molecular resistance mechanisms were also studied and functionally validated by CRISPR/Cas9-based genetic alterations. RESULTS: Fluconazole resistance occurred in 2.4% (n = 56) of isolates, and these isolates showed a higher frequency of distribution in ICU inpatients compared with susceptible isolates (48.2%, n = 27/56 versus 27.8%, 613/2208; P = 0.019). Microsatellite-genotyping analysis yielded 29 genotypes among 56 fluconazole-resistant isolates, of which 10 genotypes, including 37 isolates, belonged to clusters, persisting and transmitting in Chinese hospitals for 1-29 months. Clusters harbouring Erg11Y132F (5/10; 50%) were predominant in China. Among these, the second most dominant cluster MT07, including seven isolates, characteristically harbouring Erg11Y132F and Mrr1Q625K, lent its carriage to being one of the strongest associations with cross-resistance and high MICs of fluconazole (>256 mg/L) and voriconazole (2-8 mg/L), causing transmission across two hospitals. Among mutations tested, Mrr1Q625K led to the highest-level increase of fluconazole MIC (32-fold), while mutations located within or near the predicted transcription factor domain of Tac1 (D440Y, T492M and L518F) conferred cross-resistance to azoles. CONCLUSIONS: This study is the first Chinese report of persistence and transmissions of multiple fluconazole-resistant C. parapsilosis sensu stricto clones harbouring Erg11Y132F, and the first demonstration of the mutations Erg11G307A, Mrr1Q625K, Tac1L263S, Tac1D440Y and Tac1T492M as conferring resistance to azoles.

Developing Two Rapid Protein Extraction Methods Using Focused-Ultrasonication and Zirconia-Silica Beads for Filamentous Fungi Identification by MALDI-TOF MS.

Filamentous fungi identification by Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been challenging due to the lack of simple and rapid protein extraction methods and insufficient species coverage in the database. In this study, we created two rapid protein extraction methods for filamentous fungi: a one-step zirconia-silica beads method (ZSB) and a focused-ultrasonication method (FUS). The identification accuracy of two methods were evaluated with the VITEK MS, as well as number of spectra peaks and signal-to-noise ratio (S/N) with M-Discover 100 MALDI-TOF MS compared to the routine method. The better method was applied to build a filamentous fungi in-house spectra library for the M-Discover 100 MS, and then another one and routine method were performed in parallel to verify the accuracy and commonality of the in-house library. Using the two optimized methods, the dedicated operating time before MALDI-TOF MS analysis was reduced from 30 min to 7 (ZSB) or 5 (FUS) min per sample, with only a few seconds added for each additional strain. And both two methods identified isolates from most mold types equal to or better than the routine method, and the total correct identification rate using VITEK MS was 79.67, 76.42, and 76.42%, respectively. On the other hand, the two rapid methods generally achieved higher maximum and minimum S/N ratios with these isolates tested as compared to the routine method. Besides, the ZSB method produced overall mean of maximum and minimum S/N ratio higher than that by FUS. An in-house library of M-Discover MS was successfully built from 135 isolates from 42 species belonging to 18 genera using the ZSB method. Analysis of 467 isolates resulted in 97.22% correctly identified isolates to the species level by the ZSB method versus 95.50% by the routine method. The two novel methods are time- and cost-effective and allow efficient identification of filamentous fungi while providing a simplified procedure to build an in-house library. Thus, more clinical laboratories may consider adopting MALDI-TOF MS for filamentous fungi identification in the future.

Clinical and Microbiological Characterization of Invasive Pulmonary Aspergillosis Caused by Aspergillus lentulus in China.

Invasive aspergillosis (IA) due to Aspergillus lentulus is associated with high mortality. In this study, we investigated the clinical and microbiological characteristics of 6 fatal cases of proven or probable IA caused by A. lentulus in China. Underlying immunosuppression, prior antifungal exposure, and intensive care unit (ICU) hospitalization were important risk factors for invasive A. lentulus infection. Phenotypic differences were observed for A. lentulus isolates including slower growth, reduced sporulation, and inability to grow at 48°C, compared with Aspergillus fumigatus complex. ITS sequencing was unable to distinguish A. lentulus from A. fumigatus, but sequencing of the benA, CaM, and rod A loci enabled reliable distinction of these closely related species. Phylogenetic analysis further confirmed that the ITS region had little variation within the Aspergillus section Fumigati while the benA gene offered the highest intraspecific discrimination. Microsatellite typing results revealed that only loci on chromosomes 1, 3, 5, and 6b generated detectable amplicons for identification. All A. lentulus isolates showed in vitro resistance to multiple antifungal drugs including amphotericin B (MIC range 4 to 8 µg/ml), itraconazole (MIC 2 µg/ml), voriconazole (MIC of 4-16 µg/ml), and posaconazole (MIC of 0.5-1 µg/ml). However, MECs for the echinocandin drugs ranged from 0.03-0.25, ≤0.008-0.015, and ≤0.015-0.03 µg/ml for caspofungin, micafungin, and anidulafungin, respectively. A. lentulus is an emerging fungal pathogen in China, causing fatal disease, and clinicians as well as laboratories should be alert to their increasing presence.

Molecular Characterization of Candida parapsilosis by Microsatellite Typing and Emergence of Clonal Antifungal Drug Resistant Strains in a Multicenter Surveillance in China.

Candida parapsilosis is an important species causing invasive candidiasis (IC) in China. The present survey was a national multicenter study of the molecular epidemiology and antifungal susceptibility profiles of C. parapsilosis. Non-duplicate C. parapsilosis isolates were collected from 10 hospitals across China in the CHIF-NET program 2016-2017. Isolates were genotyped using four highly polymorphic microsatellite markers, and susceptibility profiles determined using Sensititre YeastOneTM YO10. A total of 319 C. parapsilosis from separate patients with IC were studied; 49.2, 17.9, and 10.3% isolates were from patients in surgical departments, general intensive care units (ICUs) and neonatal ICUs (NICU), respectively. C. parapsilosis showed good susceptibility to nine antifungal drugs. Microsatellite analysis identified 122 microsatellite (MT) types. Most MT types had sporadic distribution. However, we identified 32 clusters across 10 hospitals; seven clusters were caused by seven endemic genotypes involving five or more isolates in hospitals designated as H01, H02, H06, and H10. These clusters mainly affected surgical departments and ICUs, except for genotype MT42 which was seen in 22 patients from NICU (hospital H06). Of 16 fluconazole-resistant isolates, seven from hospital H02 shared the same genotype MT70, and three from hospital H04 were of genotype MT47. For 37 isolates with non-wild type MICs to 5-flucytosine, 29 were from hospital H01 (genotype MT48). Here we present the first nationwide molecular epidemiology study of C. parapsilosis in China, identified several previously unrecognized clusters, which included antifungal drug resistant isolates. These findings provide important data for control of IC in China.

Epidemiology And Antifungal Susceptibility Patterns Of Invasive Fungal Infections From 2012 To 2014 In A Teaching Hospital In Central China.

INTRODUCTION: As participants of the national China Hospital Invasive Fungal Surveillance Net program, we sought to describe the epidemiology and antifungal susceptibility patterns of yeast isolates obtained from patients with invasive fungal infection at the First Affiliated Hospital of Zhengzhou University, China. METHODS: A total of 434 yeast isolates recovered from blood and other sterile body fluids were identified to species by matrix-assisted laser desorption ionization -time of flight mass spectrometry with or without supplementation by DNA sequencing. Antifungal susceptibilities were determined by Sensititre YeastOneTM YO10 methodology. RESULTS: Candida albicans was the most common causative species (33.9% of isolates) but significantly decreased in frequency from 37.2% to 27.7% from 2012 to 2014. C. tropicalis was the next most common pathogen (25.1%), followed by C. parapsilosis complex (17.3%), C. glabrata (9%), and C. pelliculosa (6.7%), with other species comprising 8% of isolates. Caspofungin, micafungin, and anidulafungin exhibited potent in vitro activities against the majority of Candida isolates. Azoles demonstrated in vitro activities against C. albicans with a susceptibility rate of >95% and against C. parapsilosis complex, >95% isolates were susceptible. Among C. tropicalis and C. glabrata isolates, resistance rates to fluconazole and voriconazole were 11.9%, 9.1% and 7.7%, 28.2%, respectively. Of note, C. pelliculosa had a high incidence rate in newborns and high rates of resistance to fluconazole and voriconazole of 55.2% and 41.4%, respectively. CONCLUSION: The present study provided valuable local surveillance data on the epidemiology and antifungal susceptibilities of invasive yeast species, which is essential for guiding antifungal treatment protocol development.