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.

Five-Year National Surveillance of Invasive Candidiasis: Species Distribution and Azole Susceptibility from the China Hospital Invasive Fungal Surveillance Net (CHIF-NET) Study.

Data on the epidemiology of invasive candidiasis (IC) and the antifungal susceptibility of Candida isolates in China are still limited. Here we report on surveillance for IC from the China Hospital Invasive Fungal Surveillance Net (CHIF-NET) study. Sixty-five tertiary hospitals collected 8,829 Candida isolates from 1 August 2009 to 31 July 2014. Matrix-assisted laser desorption ionization-time of flight mass spectrometry supplemented by ribosomal DNA sequencing was used to define the species, and the fluconazole and voriconazole susceptibilities were determined by the Clinical and Laboratory Standards Institute disk diffusion method. A total of 32 Candida species were identified. Candida albicans was the most common species (44.9%), followed by the C. parapsilosis complex (20.0%), C. tropicalis (17.2%), and the C. glabrata complex (10.8%), with other species comprising <3% of isolates. However, in candidemia, the proportion of cases caused by C. albicans was only 32.3%. C. albicans and C. parapsilosis complex isolates were susceptible to fluconazole and voriconazole (<6% resistance), while fluconazole and azole cross-resistance rates were high in C. tropicalis (13.3% and 12.9%, respectively), C. glabrata complex (18.7% and 14%, respectively), and uncommon Candida species (44.1% and 10.3%, respectively) isolates. Moreover, from years 1 to 5 of the study, there was a significant increase in the rates of resistance to fluconazole among C. glabrata complex isolates (12.2% to 24.0%) and to both fluconazole (5.7% to 21.0%) and voriconazole (5.7% to 21.4%) among C. tropicalis isolates (P < 0.01 for all comparisons). Geographic variations in the causative species and susceptibilities were noted. Our findings indicate that antifungal resistance has become noteworthy in China, and enhanced surveillance is warranted.

First case report of endocarditis caused by haematobacter massiliensis in China.

BACKGROUND: Haematobacter massiliensis, a rare species of fastidious Gram-negative, non-motile, non-sporing, non-fermentative, pleomorphic, aerobic bacilli, has rarely been documented as the cause of infectious endocarditis in literature. Here we report the first case of infectious endocarditis (IE) caused by H. massiliensis in China. CASE PRESENTATION: A 44-year-old woman presented to the infectious department of Peking Union Medical College Hospital (Beijing) in August 2013, with a 7-week history of fevers, chills, sore throat, muscular soreness, occasional joint pain, and cough. The organism obtained by blood culture, identified as H. massiliensis by 16S rRNA gene sequencing, was finally implicated as the cause of infectious endocarditis. The patient was cured with amoxicillin/clavulanate combined with amikacin for 6 weeks. CONCLUSION: This is the first case report in China, of the isolation of H. massiliensis from the bloodstream of a patient with endocarditis. The microbiology and clinical study of the organism will help us understand it better in future clinical practice.

Identification and Antifungal Susceptibility Profile of Candida guilliermondii and Candida fermentati from a Multicenter Study in China.

With molecular sequencing as a gold standard, the Vitek MS, Bruker Biotyper MS, and Vitek-2 Compact systems correctly identified 92.7%, 97.0%, and 15.2% of 164 Candida guillermondii isolates, respectively, and none of 8 C. fermentati isolates. All of the isolates showed high susceptibility to echinocandins, but some C. guilliermondii isolates showed low azole susceptibility.

Identification and Antifungal Susceptibility Profiles of Candida haemulonii Species Complex Clinical Isolates from a Multicenter Study in China.

Candida haemulonii complex (Candida haemulonii, Candida haemulonii var. vulnera, and Candida duobushaemulonii) consists of emerging pathogens. Thirty-one isolates from 14 hospitals in China were studied for their species classification and antifungal susceptibilities. Performances of molecular (i.e., ribosomal DNA [rDNA] internal transcribed spacer [ITS] sequencing, D1/D2 sequencing, and ITS sequencer-based capillary gel electrophoresis [SCGE]) and phenotypic identification methods in species identification were compared. Twenty-six (83.9%) of 31 isolates were identified as C. haemulonii and 5 isolates were identified as C. duobushaemulonii by ITS sequencing as the reference method; results obtained by D1/D2 sequencing and ITS SCGE were concordant with those obtained by ITS sequencing for all (100%) of the isolates. All 31 isolates were identified as C. haemulonii by the Vitek matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system (bioMérieux, France), whereas the Bruker MS system (Bruker Daltoniks, Germany) correctly provided species identification for 77.4% and 100% of isolates using cutoff scores for species of ≥2.0 and ≥1.70, respectively. The Vitek 2 compact (bioMérieux) only identified 9 (29%) of 31 isolates. All isolates showed high MICs for amphotericin B (range, 2 to >8 µg/ml) and fluconazole (≥128 µg/ml) but low MICs (≤0.5 µg/ml) for the echinocandins. Our results reinforce the need for MALDI-TOF MS and/or molecular differentiation of species within the C. haemulonii complex. The multiresistant antifungal susceptibility profile of these isolates represents a challenge to therapy.

Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) Analysis for the Identification of Pathogenic Microorganisms: A Review.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used in the field of clinical microbiology since 2010. Compared with the traditional technique of biochemical identification, MALDI-TOF MS has many advantages, including convenience, speed, accuracy, and low cost. The accuracy and speed of identification using MALDI-TOF MS have been increasing with the development of sample preparation, database enrichment, and algorithm optimization. MALDI-TOF MS has shown promising results in identifying cultured colonies and rapidly detecting samples. MALDI-TOF MS has critical research applications for the rapid detection of highly virulent and drug-resistant pathogens. Here we present a scientific review that evaluates the performance of MALDI-TOF MS in identifying clinical pathogenic microorganisms. MALDI-TOF MS is a promising tool in identifying clinical microorganisms, although some aspects still require improvement.

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.

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.

The tcdA-negative and tcdB-positive Clostridium difficile ST81 clone exhibits a high level of resistance to fluoroquinolones: a multi-centre study in Beijing, China.

Clostridium difficile infection (CDI) is the leading cause of antibiotic-associated diarrhoea worldwide. In order to gain a better understanding about the molecular epidemiology of C. difficile in Beijing, China, molecular typing, antimicrobial susceptibility testing and drug resistance gene sequencing were performed on 174 strains of C. difficile collected from four large tertiary hospitals in Beijing. In total, 31 sequence types (STs) were identified among the 174 strains. ST81 was found to be the most prevalent (26.4%, 46/174), followed by ST2 (16.7%, 29/174) and ST54 (9.8%, 17/174). All isolates were susceptible to metronidazole and vancomycin. The test strains displayed resistance rates of 97.1%, 44.3% and 44.3% for ciprofloxacin, levofloxacin and moxifloxacin, respectively. ST81 isolates displayed a drug resistance rate of 97.8% for levofloxacin and moxifloxacin, which was significantly higher than ST2 (0%), ST54 (17.6%) and ST42 (0%) isolates (P<0.05). An amino acid mutation (T82I) was identified in GyrA, and the total mutation rate of the C. difficile strains was 40.8% (71/174). The mutation rate of ST81 isolates was 95.7% (44/46). Three amino acid mutations (D426N, S366A and D426V) were identified in GyrB, and the total mutation rate of GyrB was 39.1%. A double-site mutation in GyrB (S366A+D426V) was identified in all ST81 (n=46) isolates. In conclusion, the C. difficile ST81 clone showed a high level of resistance to fluoroquinolones in Beijing, highlighting the need for nationwide surveillance of CDI.

Trichosporon dohaense, a rare pathogen of human invasive infections, and literature review.

BACKGROUND: Trichosporon dohaense is a rare fungal species that has not been described in human invasive infections. PATIENTS AND METHODS: In this study, we investigated two T. dohaense isolates from patients with invasive infections in two hospitals in China, as part of the China Hospital Invasive Fungal Surveillance Net (CHIF-NET) program. Both patients were under immunocompromised conditions. RESULTS: On chromogenic agar, T. dohaense isolates were dark blue, similar to the color of Candida. tropicalis, but the characteristic moist colony appearance was quite different from that of T. asahii. The two isolates were misidentified as T. asahii and T. inkin by the VITEK 2 YST system. The rDNA internal transcribed spacer (ITS) region and the D1/D2 domain sequences of the two T. dohaense isolates were 100% identical to T. dohaense type strain CBS10761T. The sequence of the intergenic spacer region-1 also clearly distinguished the species. Of the three matrix-assisted laser desorption/ionization time-of-flight mass spectrometry systems, Bruker Biotyper and Autobio MS correctly identified the two isolates to species level, whereas Vitek MS systems misidentified them as T. ovoides or T. asteroides. Echinocandins exhibited no in vitro activities against the two T. dohaense isolates. In addition, the isolates exhibited intermediate susceptibility to fluconazole (with minimal inhibitory concentrations [MICs] of 8 and 16 µg/mL) and itraconazole, voriconazole, and posaconazole (MICs of 0.25-1 µg/mL). T. dohaense demonstrated susceptibility to amphotericin B with MIC of 1 µg/mL. The MICs of fluconazole and voriconazole in our study were higher than the MIC50 of 62 for T. asahii isolates (4 and 0.064 µg/mL) in the CHIF-NET program. CONCLUSION: This case study points to a possible emergence of T. dohaense as an opportunistic human invasive fungal pathogen, and the reduced susceptibility should be noted.

Five-year China Hospital Invasive Fungal Surveillance Net (CHIF-NET) study of invasive fungal infections caused by noncandidal yeasts: species distribution and azole susceptibility.

PURPOSE: In this study, we report results from a 5-year surveillance for noncandidal yeast species causing invasive infections from 65 hospitals in China. MATERIALS AND METHODS: Species identification was carried out by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) supplemented by rDNA sequencing, and fluconazole and voriconazole susceptibilities of yeasts were determined by Clinical and Laboratory Standards Institute (CLSI) disk diffusion methods. RESULTS: Overall, 884 noncandidal isolates belonging to 38 species were collected. Cryptococcus neoformans was the most common (75.6%), which also comprised 96.5% of the isolates from cerebrospinal fluid (CSF) and 62.6% from blood, followed by Trichosporon asahii (6.9%) and Rhodotorula mucilaginosa (5.1%). Fluconazole susceptibility and resistant rates were 74.1% and 9.7% for C. neoformans and 81.0% and 5.2% for T. asahii. Voriconazole exhibited good activity in comparison to these two species (99.5% and 98.3% of the isolates, were susceptible). However, 100% of the R. mucilaginosa isolates were resistant to both azoles. Other noncandidal yeast species showed reduced susceptibility to fluconazole (53.3%) but most were susceptible to voriconazole (94.3%). Over the 5 years, a decrease in the proportion of fluconazole-susceptible isolates was observed for C. neoformans (90%-67%, P<0.001) and other noncandidal yeast species (91%-66%, P<0.001). Moreover, the prevalence of azole-resistant R. mucilaginosa increased from 1% to 7% (P<0.001). CONCLUSION: The shift in azole susceptibilities in mainland China calls for continued surveillance for noncandidal yeasts.

High in vitro activity of fidaxomicin against Clostridium difficile isolates from a university teaching hospital in China.

BACKGROUND: Clostridium difficile infection (CDI) is a significant cause of morbidity and mortality in both the acute care setting and the wider healthcare system. The purpose of this study was to evaluate the in vitro activity of fidaxomicin against C. difficile isolates from a university teaching hospital in China. METHODS: One hundred and one C. difficile isolates were collected and analyzed for toxin genes by multiplex PCR. The toxin gene positive strains were also typed by multilocus sequence typing (MLST) and PCR-ribotyping. The MICs of the isolates were determined against fidaxomicin, metronidazole, vancomycin, tigecycline and moxifloxacin, by the agar dilution method. RESULTS: All the 101 isolates exhibited low MICs to fidaxomicin (0.032-1 mg/L), metronidazole (0.125-1 mg/L), vancomycin (0.25-2 mg/L) and tigecycline (0.016-0.5 mg/L). Tigecycline showed the lowest geometric mean MIC value (0.041 mg/L), followed by fidaxomicin (0.227 mg/L), metronidazole (0.345 mg/L), and vancomycin (0.579 mg/L). About 35% of the strains (n = 35) were resistant to moxifloxacin, and the resistance rate to moxifloxacin for A-B+CDT- isolates (85.0%) was much higher than that of A+B+CDT- (15.7%) and A-B-CDT- (29.2%) isolates (P < 0.001). The MIC values of fidaxomicin, metronidazole, vancomycin and moxifloxacin against the 3 ST1 isolates were higher than for other STs. All the 28 moxifloxacin-resistant toxigenic isolates carried a mutation either in gyrA or/and gyrB. CONCLUSION: Fidaxomicin exhibited high antimicrobial activity against all C. difficile isolates tested, which shows promise as a new drug for treating Chinese CDI patients.

Molecular epidemiology of Clostridium difficile in two tertiary care hospitals in Shandong Province, China.

PURPOSE: The incidence and severity of Clostridium difficile infection (CDI) have markedly increased over the past decade. However, there is very limited epidemiological data on CDI in China so far, specifically no data in Shandong Province. The aim of this study was to evaluate diagnostic algorithm for CDI and to gain data on molecular epidemiology of CDI in the Shandong Province of China. MATERIALS AND METHODS: Nonrepetitive unformed fecal specimens (n=504) were investigated by the glutamate dehydrogenase (GDH), C. difficile toxin A&B (CDAB) tests and toxigenic culture. Furthermore, 85 isolates were characterized by toxin gene detection, multilocus sequence typing, ribotyping and antimicrobial susceptibility testing. RESULTS: The algorithm of combining GDH and CDAB tests could define diagnosis of 54.2% CDI cases and excluded 90% of non-CDI. Further adding the toxigenic culture to the algorithm enhanced the detection sensitivity to 100%. Toxigenic strains comprised 84.7% of isolates, including A+B+CDT- (71.8%, 61/85), A-B+CDT- (11.8%, 10/85) and A+B+CDT+ (1.2%, 1/85) isolates. RT046/ST35 (13.9%, 10/72), RT014/ST2 (12.5%, 9/72) and RT017/ST37 (12.5%, 9/72) were the more common genotypes among toxigenic C. difficile strains. The clinical severity score of A-B+CDT- toxin genes genotype (3.50±0.85) was significantly higher than the A+B+CDT- type (2.59±0.93) (P<0.05). RT046/ST35 isolates were highly prevalent and had high clinical severity scores (3.80±0.92). Variations in resistance from different sequence types (STs) were observed. Toxigenic strains showed higher resistance rates to erythromycin, clindamycin and ciprofloxacin compared to nontoxigenic strains (P<0.05). CONCLUSION: The epidemiology of C. difficile in Shandong Province differed from other regions in China. Comprehensive optimized diagnosis strategy and continuous surveillance should be established and applied in order to curb the spread of toxigenic C. difficile strains, especially for hospitalized patients.

Use of matrix-assisted laser desorption ionization-time of flight mass spectrometry to identify MLST clade 4 Clostridium difficile isolates.

Clostridium difficile is the leading cause of health care-associated infections. Previous studies suggest that C. difficile MLST clade 4 strains with higher drug resistance rates constitute the major clone spreading in China. Thus development of a rapid and accurate typing method for these strains is needed to monitor the epidemiology of this clone and to guide clinical treatment. A total of 160 non-duplicate C. difficile isolates recovered from three large teaching hospitals in Beijing were studied. All the 41 clade 4 C. difficile isolates clustered together on the PCA dendrogram. Spectra peak statistics revealed that five markers (2691.43Da, 2704.91Da, 2711.93Da, 3247.27Da and 3290.76Da) can easily and reliably distinguish between clade 4 and non-clade 4 isolates, with area under the curve (AUC) values of 0.991, 0.997, 0.973, 1 and 1, respectively. In conclusion, MALDI-TOF MS is a very simple and accurate method for identifying C. difficile MLST clade 4 strains.

Moraxella catarrhalis Macrolide-Resistant Isolates Are Highly Concentrated in Two MLST Clonal Complexes -CCN10 and CC363.

To gain some insights into the molecular evolution of Moraxella catarrhalis macrolide resistance, PCR and sequencing analysis of the 23S rRNA gene, copB typing and multilocus sequence typing (MLST) were performed on 181 M. catarrhalis isolates. The isolates were obtained from children (n = 47) and adults (n = 134) presenting with respiratory disease in the years 2010-2014. Macrolide resistance was highly age-related, and nucleotide position alterations at A2330T could be detected in all macrolide-resistant isolates. copB 0 and copB NT (non-typable) were only found in macrolide-susceptible isolates from adults. Furthermore, copB I/III was the main type in adult or macrolide-susceptible isolates, while copB II was the most common type in children or macrolide-resistant isolates. Twenty-two different MLST clusters (sharing 7 of the 8 identical loci) were detected and only four likely primary founders (ST224, ST363, STN08, and STN10) which belong to clonal complex (CC) 224, CC363, CCN08, and CCN10, were detected, respectively. Macrolide-resistant M. catarrhalis isolates were highly concentrated in two CCs (CCN10 and CC363), which indicates some potential evolutionary advantage or co-evolution to some extent. However, further studies are needed to fully elucidate the evolution of CCN10 and CC363 in macrolide resistance.

Molecular epidemiology and azole resistance mechanism study of Candida guilliermondii from a Chinese surveillance system.

We studied the molecular epidemiology and mechanism of azole resistance of 164 C. guilliermondii isolates from a nationwide multi-center surveillance program. The isolates were identified by ITS gene sequencing, and the in vitro susceptibility to fluconazole and voriconazole was determined by broth microdilution method. The 14-α-demethylase gene ERG11 was amplified and sequenced, and microsatellite analysis was performed to study the genetic relatedness of the isolates. Amongst the 164 C. guilliermondii isolates, 15 (9.1%) and 17 (10.4%) isolates were assigned to be non-wild type (non-WT) to fluconazole and voriconazole, respectively. Sixteen sequence types (STs) were detected by comparing the amino acid sequence polymorphisms of the ERG11 gene. Fifteen isolates of STs 9, 10, 12, 13, 14, 15 and 16, were all assigned to be non-WT to fluconazole and voriconazole. By microsatellite analysis, 40 different genotypes were identified. Thirty-seven isolates from one hospital (Z1) shared the same ERG11 sequence type (ST 2), microsatellite genotype (PU40) and drug resistance pattern. In conclusion, this is the first molecular epidemiology study of C. guilliermondii in China. The rate of non-WT isolates to azoles was high and the accurate contribution of ERG11 gene mutations to azole resistance need be confirmed by further studies.

The First Two Clostridium difficile Ribotype 027/ST1 Isolates Identified in Beijing, China-an Emerging Problem or a Neglected Threat?

Clostridium difficile hyper-virulent ribotype 027 strain has become a significant concern globally, but has rarely been reported in Asian countries including China. Recently, a retrospective single-center study in Beijing, China, detected two ribotype 027 C. difficile isolates from two patients coming for outpatient visits in 2012 and 2013. We performed a systematic investigation of the two isolates (and patients). Both C. difficile isolates had the typical PCR ribotype 027 profile; were positive for tcdA, tcdB and binary toxin genes; belonged to multilocus sequence type 1 (ST1); had typical ribotype 027 deletions in the tcdC gene; and were highly-resistant to fluoroquinolones; but had a different MLVA profile and were not genetically related to any previously reported international ribotype 027 clones. A review of the patients' medical records showed that neither received appropriate antimicrobial treatment and were lost to follow-up after outpatient visits. We propose that C. difficile infections caused by ribotype 027 are probably a neglected problem in China, and the subsequent impact of unawareness of this problem is worrying. Appropriate testing assays and multi-center or national level surveillance for C. difficile infections and specifically for ribotype 027 should be introduced to provide essential data and guide future clinical practice.

Molecular Epidemiology and Antimicrobial Susceptibility of Clostridium difficile Isolates from a University Teaching Hospital in China.

While the developed world has seen a significant increase in the number of scientific articles on Clostridium difficile infection (CDI), the developing world still lags behind on this subject due to limited laboratory capacity, low awareness, and limited surveillance of this problem. As such, CDI is considered a neglected but potentially huge problem in developing countries. The major aim of this study was to systemically evaluate the utility of several molecular typing tools for CDI, including their relevance in epidemiological studies in developing countries such as China. A total of 116 non-repetitive toxigenic C. difficile isolates from Chinese patients, were studied. The isolates comprised 83 (71.6%) A+B+CDT- isolates, 27 (23.3%) A-B+CDT- isolates, and 6 (5.1%) A+B+CDT+ isolates. Typing methods evaluated included multilocus variable-number tandem-repeat analysis, PCR ribotyping, multilocus sequence typing, and sequencing of slpA and tcdC genes, which identified 113, 30, 22, 18, and 8 genotypes each and exhibited discriminatory powers of 0.999, 0.916, 0.907, 0.883, and 0.765, respectively. Compared to A+B+ strains, A-B+ strains exhibited higher prevalence of drug resistance to clindamycin, erythromycin, levofloxacin, rifampicin, rifaximin, and tetracycline. Furthermore, drug resistance rates of strains with different PCR ribotypes differed, supporting the importance of molecular typing in management and control of CDI. Based on our earlier suggestion to improve the diagnostic laboratory capacity of CDI in developing countries, setting up efficient surveillance programs complemented by relevant molecular typing methods is warranted.

Sequencer-Based Capillary Gel Electrophoresis (SCGE) Targeting the rDNA Internal Transcribed Spacer (ITS) Regions for Accurate Identification of Clinically Important Yeast Species.

Accurate species identification of Candida, Cryptococcus, Trichosporon and other yeast pathogens is important for clinical management. In the present study, we developed and evaluated a yeast species identification scheme by determining the rDNA internal transcribed spacer (ITS) region length types (LTs) using a sequencer-based capillary gel electrophoresis (SCGE) approach. A total of 156 yeast isolates encompassing 32 species were first used to establish a reference SCGE ITS LT database. Evaluation of the ITS LT database was then performed on (i) a separate set of (n = 97) clinical isolates by SCGE, and (ii) 41 isolates of 41 additional yeast species from GenBank by in silico analysis. Of 156 isolates used to build the reference database, 41 ITS LTs were identified, which correctly identified 29 of the 32 (90.6%) species, with the exception of Trichosporon asahii, Trichosporon japonicum and Trichosporon asteroides. In addition, eight of the 32 species revealed different electropherograms and were subtyped into 2-3 different ITS LTs each. Of the 97 test isolates used to evaluate the ITS LT scheme, 96 (99.0%) were correctly identified to species level, with the remaining isolate having a novel ITS LT. Of the additional 41 isolates for in silico analysis, none was misidentified by the ITS LT database except for Trichosporon mucoides whose ITS LT profile was identical to that of Trichosporon dermatis. In conclusion, yeast identification by the present SCGE ITS LT assay is a fast, reproducible and accurate alternative for the identification of clinically important yeasts with the exception of Trichosporon species.

Accurate Identification of Common Pathogenic Nocardia Species: Evaluation of a Multilocus Sequence Analysis Platform and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Species identification of Nocardia is not straightforward due to rapidly evolving taxonomy, insufficient discriminatory power of conventional phenotypic methods and also of single gene locus analysis including 16S rRNA gene sequencing. Here we evaluated the ability of a 5-locus (16S rRNA, gyrB, secA1, hsp65 and rpoB) multilocus sequence analysis (MLSA) approach as well as that of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in comparison with sequencing of the 5'-end 606 bp partial 16S rRNA gene to provide identification of 25 clinical isolates of Nocardia. The 5'-end 606 bp 16S rRNA gene sequencing successfully assigned 24 of 25 (96%) clinical isolates to species level, namely Nocardia cyriacigeorgica (n = 12, 48%), N. farcinica (n = 9, 36%), N. abscessus (n = 2, 8%) and N. otitidiscaviarum (n = 1, 4%). MLSA showed concordance with 16S rRNA gene sequencing results for the same 24 isolates. However, MLSA was able to identify the remaining isolate as N. wallacei, and clustered N. cyriacigeorgica into three subgroups. None of the clinical isolates were correctly identified to the species level by MALDI-TOF MS analysis using the manufacturer-provided database. A small "in-house" spectral database was established incorporating spectra of five clinical isolates representing the five species identified in this study. After complementation with the "in-house" database, of the remaining 20 isolates, 19 (95%) were correctly identified to species level (score ≥ 2.00) and one (an N. abscessus strain) to genus level (score ≥ 1.70 and < 2.00). In summary, MLSA showed superior discriminatory power compared with the 5'-end 606 bp partial 16S rRNA gene sequencing for species identification of Nocardia. MALDI-TOF MS can provide rapid and accurate identification but is reliant on a robust mass spectra database.