Antifungal susceptibility profiles and drug resistance mechanisms of clinical Candida duobushaemulonii isolates from China.

Candida duobushaemulonii, type II Candida haemulonii complex, is closely related to Candida auris and capable of causing invasive and non-invasive infections in humans. Eleven strains of C. duobushaemulonii were collected from China Hospital Invasive Fungal Surveillance Net (CHIF-NET) and identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), VITEK 2 Yeast Identification Card (YST), and internal transcribed spacer (ITS) sequencing. Whole genome sequencing of C. duobushaemulonii was done to determine their genotypes. Furthermore, C. duobushaemulonii strains were tested by Sensititre YeastOne™ and Clinical and Laboratory Institute (CLSI) broth microdilution panel for antifungal susceptibility. Three C. duobushaemulonii could not be identified by VITEK 2. All 11 isolates had high minimum inhibitory concentrations (MICs) to amphotericin B more than 2 µg/ml. One isolate showed a high MIC value of ≥64 µg/ml to 5-flucytosine. All isolates were wild type (WT) for triazoles and echinocandins. FUR1 variation may result in C. duobushaemulonii with high MIC to 5-flucytosine. Candida duobushaemulonii mainly infects patients with weakened immunity, and the amphotericin B resistance of these isolates might represent a challenge to clinical treatment.

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.

Species distribution and antifungal susceptibilities of clinical isolates of Penicillium and Talaromyces species in China.

Morphologically identified Penicillium (n = 103) and Talaromyces marneffei (n = 8) isolates were collected from various clinical sources between 2016 and 2017 at a medical centre in Beijing, China. Identification to species level was confirmed by sequencing of the internal transcribed spacer (ITS) region, ß-tubulin gene (benA) and RNA polymerase II second largest subunit (RPB2) gene. Of the 111 isolates, 56 (50.5%) were identified as Penicillium spp. and 55 (49.5%) as Talaromyces spp. Eleven species of Penicillium were detected, of which Penicillium oxalicum was the commonest, accounting for 51.8% (29/56), followed by Penicillium rubens (10.7%; 6/56) and Penicillium citrinum (10.7%; 6/56). Among the 55 Talaromyces isolates, nine species were identified, with Talaromyces funiculosus (36.4%; 20/55), Talaromyces stollii (27.3%; 15/55) and Talaromyces marneffei (14.5%; 8/55) being the most common. Of note, 89.3% (50/56) of the Penicillium isolates and 98.2% (54/55) of the Talaromyces isolates exhibited growth at 37°C. The isolates were mainly recovered from patients with pulmonary disorders (56.8%; 63/111), autoimmune disease (12.6%; 14/111) and AIDS (5.4%; 6/111). The azoles and amphotericin B exhibited potent activity against T. marneffei, while various levels of activity were observed against Penicillium and other Talaromyces species The echinocandins had the lowest MECs (MEC90, ≤0.12 mg/L) against most Penicillium and Talaromyces species, with the exception of T. marneffei whose MEC90 (4 mg/L) was five or more dilutions higher than that of the other species tested. These data on the species distribution and antifungal susceptibility expand the current clinical knowledge of Penicillium and Talaromyces species.

Evaluation of Autof MS 1000 and Vitek MS MALDI-TOF MS System in Identification of Closely-Related Yeasts Causing Invasive Fungal Diseases.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been accepted as a rapid, accurate, and less labor-intensive method in the identification of microorganisms in clinical laboratories. However, there is limited data on systematic evaluation of its effectiveness in the identification of phylogenetically closely-related yeast species. In this study, we evaluated two commercially available MALDI-TOF systems, Autof MS 1000 and Vitek MS, for the identification of yeasts within closely-related species complexes. A total of 1,228 yeast isolates, representing 14 different species of five species complexes, including 479 of Candida parapsilosis complex, 323 of Candida albicans complex, 95 of Candida glabrata complex, 16 of Candida haemulonii complex (including two Candida auris), and 315 of Cryptococcus neoformans complex, collected under the National China Hospital Invasive Fungal Surveillance Net (CHIF-NET) program, were studied. Autof MS 1000 and Vitek MS systems correctly identified 99.2% and 89.2% of the isolates, with major error rate of 0.4% versus 1.6%, and minor error rate of 0.1% versus 3.5%, respectively. The proportion of isolates accurately identified by Autof MS 1000 and Vitek MS per each yeast complex, respectively, was as follows; C. albicans complex, 99.4% vs 96.3%; C. parapsilosis complex, 99.0% vs 79.1%; C glabrata complex, 98.9% vs 94.7%; C. haemulonii complex, 100% vs 93.8%; and C. neoformans, 99.4% vs 95.2%. Overall, Autof MS 1000 exhibited good capacity in yeast identification while Vitek MS had lower identification accuracy, especially in the identification of less common species within phylogenetically closely-related species complexes.

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.

In vitro Activity of a New Fourth-Generation Cephalosporin, Cefoselis, Against Clinically Important Bacterial Pathogens in China.

The objective of this study was to systematically evaluate the in vitro activity of cefoselis and other comparators against common bacterial pathogens collected from 18 hospitals across China. Minimum inhibitory concentrations (MICs) were determined by the broth microdilution method following Clinical and Laboratory Standards Institute (CLSI) guidelines. Cefoselis showed poor activity against extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis, with susceptibility rates of < 10% each, while the susceptibility rates of this antibiotic against non-ESBL-producing strains of these organisms were 100%, 94.3%, and 97.0%, respectively. Cefoselis exhibited susceptibility rates of 56.7-83.3% against other tested Enterobacteriaceae isolates. For Acinetobacter baumannii and Pseudomonas aeruginosa isolates, the susceptibility rates to cefoselis were 18.7% and 73.3%, respectively. All methicillin-resistant Staphylococcus aureus (MRSA) strains were resistant to cefoselis, while all methicillin-sensitive S. aureus (MSSA) strains were susceptible to this antibiotic. In conclusion, cefoselis showed good activity against non-ESBL-producing E. coli, K. pneumoniae, and P. mirabilis, MSSA, and was also potent against Enterobacteriaceae, P. aeruginosa, and Streptococcus.

Distribution and Antifungal Susceptibility of Candida Species Causing Candidemia in China: An Update From the CHIF-NET Study.

BACKGROUND: Candidemia is the most common, serious fungal infection and Candida antifungal resistance is a challenge. We report recent surveillance of candidemia in China. METHODS: The study encompassed 77 Chinese hospitals over 3 years. Identification of Candida species was by mass spectrometry and DNA sequencing. Antifungal susceptibility was determined using the Clinical and Laboratory Standards Institute broth microdilution method. RESULTS: In total, 4010 isolates were collected from candidemia patients. Although C. albicans was the most common species, non-albicans Candida species accounted for over two-thirds of isolates, predominated C. parapsilosis complex (27.1%), C. tropicalis (18.7%), and C. glabrata complex (12.0%). Most C. albicans and C. parapsilosis complex isolates were susceptible to all antifungal agents (resistance rate <5%). However, there was a decrease in voriconazole susceptibility to C. glabrata sensu stricto over the 3 years and fluconazole resistance rate in C. tropicalis tripled. Amongst less common Candida species, over one-third of C. pelliculosa isolates were coresistant to fluconazole and 5-flucytocine, and >56% of C. haemulonii isolates were multidrug resistance. CONCLUSIONS: Non-albicans Candida species are the predominant cause of candidemia in China. Azole resistance is notable amongst C. tropicalis and C. glabrata. Coresistance and multidrug resistance has emerged in less common Candida species.

Candida isolates causing refractory or recurrent oropharyngeal candidiasis in 11 hospitals in China.

Introduction: We studied the species distribution and antifungal susceptibilities of Candida isolates causing refractory or recurrent oropharyngeal candidiasis (OPC) in a multicenter study in China (2013-2016). Methods: Species identification was performed using the Bruker Biotyper (Bruker Daltonics, Germany) matrix-assisted laser desorption/ionization time of flight mass spectrometry system supplemented by internal transcribed spacer sequencing as required. Antifungal susceptibilities were determined by the Clinical and Laboratory Standards Institute document (CLSI) M27-A3 broth microdilution methodology. Results: A total of 558 non-duplicate Candida isolates comprising 10 species were obtained from 535 patients. Candida albicans was the most common species (89.6%), followed by C. glabrata (5.2%), C. tropicalis (2.9%), and C. parapsilosis (0.7%). Azoles were active against C. albicans with susceptibility rates of 96% and 95.8% for fluconazole and voriconazole, respectively. MIC50 values of C. albicans to fluconazole, voriconazole, itraconazole, and miconazole were 1, 0.03, 0.25 and 0.12 µg/mL, respectively, higher than those in previous studies of which OPC patients (corresponding MIC50 values of 0.25 , 0.015 , 0.06 , and 0.03 µg/mL). Except for itraconazole, the MIC50 and MIC90 values of 58 non-C. albicans to other azoles were two to threefold higher than C. albicans. Miconazole, amphotericin B, nystatin, and 5-flucytosine had good in vitro antifungal activity for all isolates. Conclusion: The study provides valuable data on the species distribution and antifungal susceptibility of oropharyngeal Candida isolates from geographically diverse areas of China. C. albicans remains the most common species but with increasing rates of azoles resistance.

Evaluation of VITEK MS, Clin-ToF-II MS, Autof MS 1000 and VITEK 2 ANC card for identification of Bacteroides fragilis group isolates and antimicrobial susceptibilities of these isolates in a Chinese university hospital.

BACKGROUND AND PURPOSE: Bacteroides fragilis group isolates are most frequently isolated anaerobic pathogens. This study aimed to evaluate the accuracy of VITEK MS, Clin-ToF-II MS, Autof MS 1000 and VITEK 2 ANC card on the identification of clinical B. fragilis group isolates, as well as to determine their antimicrobial susceptibilities. METHODS: A total of 138 isolates of B. fragilis group isolates were identified with the three MALDI-TOF MS systems and VITEK 2 ANC cards. 16S rRNA gene sequencing was used as the reference identification method for comparison. Antimicrobial susceptibilities were determined by agar dilution method to 19 antimicrobial agents recommended by Clinical and Laboratory Standards Institute (CLSI). RESULTS: Hundred thirty three isolates of Bacteroides spp. and 5 isolates of Parabacteroides spp. were identified by 16S rRNA sequencing. The rates of accurate identification at species level of VITEK MS, Clin-ToF-II MS, Autof MS 1000 and VITEK 2 ANC card were 94.2%, 94.2%, 98.6% and 94.9%, respectively, while that at genus level were 99.3%, 100%, 100% and 97.8%, respectively. Metronidazole and chloramphenicol were the most susceptible agents (99.3% and 92.8%, respectively), followed by meropenem, ertapenem, imipenem and piperacillin/tazobactam to which the susceptible rates ranged from 76.8% to 79.0%. The susceptible rates to carbapenems decreased 12.4-15.3% from 2010-2013 to 2014-2017. CONCLUSION: All the four systems provided high accurate rate on the identification of B. fragilis group isolates. Metronidazole showed highest activity against these isolates. Attention should be paid to the higher resistant rates to carbapenems, clindamycin, moxifloxacin and tigecycline than the other countries.