Rapid identification of Streptococcus pneumoniae serotypes by cpsB gene-based sequetyping combined with multiplex PCR.

BACKGROUND/PURPOSE: Streptococcus pneumoniae is an important human pathogen that causes invasive infections in adults and children. Accurate serotyping is important to study its epidemiological distribution and to assess vaccine efficacy. METHODS: Invasive S. pneumoniae isolates (n = 300) from 27 teaching hospitals in China were studied. The Quellung reaction was used as the gold standard to identify the S. pneumoniae serotypes. Subsequently, multiplex PCR and cpsB gene-based sequetyping methods were used to identify the serotypes. RESULTS: Based on the Quellung reaction, 299 S. pneumoniae isolates were accurately identified to the serotype level and 40 different serotypes were detected. Only one strain was non-typeable, and five most common serotypes were identified: 23F (43, 14.3%), 19A (41, 13.7%), 19F (41, 13.7%), 3 (31, 10.3%), and 14 (27, 9.0%). Overall, the multiplex PCR method identified 73.3 and 20.7% of the isolates to the serotype and cluster levels, respectively, with 1.7% of the isolates misidentified. In contrast, the cpsB sequetyping method identified 59.0 and 30.3% of the isolates to the serotype and cluster levels, respectively, and 7% were misidentified. CONCLUSIONS: The cpsB gene sequetyping method combined with multiplex PCR, can greatly improve the accuracy and efficiency of serotyping, besides reducing the associated costs.

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.

Laboratory diagnosis of COVID-19 in China: A review of challenging cases and analysis.

Since the initial emergence of coronavirus disease 2019 (COVID-19) in Wuhan, Hubei province, China, a rapid spread of the disease occurred around the world, rising to become an international global health concern at pandemic level. In the face of this medical challenge threatening humans, the development of rapid and accurate methods for early screening and diagnosis of COVID-19 became crucial to containing the emerging public health threat, and prevent further spread within the population. Despite the large number of COVID-19 confirmed cases in China, some problematic cases with inconsistent laboratory testing results, were reported. Specifically, a high false-negative rate of 41% on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection by real-time reverse transcription-polymerase chain reaction (qRT-PCR) assays was observed in China. Although serological testing has been applied worldwide as a complementary method to help identify SARS-CoV-2, several limitations on its use have been reported in China. Therefore, the use of both qRT-PCR and serological testing in the diagnosis of COVID-19 in China and elsewhere, presented considerable challenges, but when used in combination, can be valuable tools in the fight against COVID-19. In this review, we give an overview of the advantages and disadvantages of different molecular techniques for SARS-CoV-2 detection that are currently used in several labs, including qRT-PCR, gene sequencing, loop-mediated isothermal amplification (LAMP), nucleic acid mass spectrometry (MS), and gene editing technique based on clustered regularly interspaced short palindromic repeats (CRISPR/Cas13) system. Then we mainly review and analyze some causes of false-negative qRT-PCR results, and how to resolve some of the diagnostic dilemma.

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.

Phosphorylcholine esterase is critical for Dolichos biflorus and Helix pomatia agglutinin binding to pneumococcal teichoic acid.

Streptococcus pneumoniae (the pneumococcus) has wall teichoic acid (WTA) and lipoteichoic acid (LTA) expressing the Forssman antigen (FA). Two lectins, Dolichos biflorus agglutinin (DBA) and Helix pomatia agglutinin (HPA), are known to bind FA. To determine the molecular structure targeted by these two lectins, different pneumococcal strains were studied for DBA/HPA binding with flow cytometry and fluorescence microscopy. Genetic experiments were used to further examine the lectins' molecular target. Twelve strains were positive for DBA binding, whereas three were negative. Super-resolution microscopy showed that DBA stained only the subcapsular area of pneumococci. The three DBA nonbinders showed no phosphorylcholine esterase (Pce) activity in vitro, whereas 10 DBA binders displayed Pce activity (the remaining two strains were DBA binders with no Pce activity in vitro). The pcegene sequence for 10 representative strains revealed two functional pce alleles, the previously recognized "allele A" and a newly discovered "allele B" (with 12 additional nucleotides). Isolates with allele B showed no Pce activity in vitro but did bind to DBA, indicating allele B Pce is functional in vivo. Genetic transfer experiments confirmed that either allele is sufficient (and necessary) for DBA binding. The three DBA nonbinders had various mutations that affected Pce function. Observations with HPA were identical to those with DBA. We show that DBA and HPA bind only to the WTA/LTA of pneumococcal isolates with a functional Pce enzyme. A newly discovered Pce variant (allele B) is functional in vivo but nonfunctional when assayed in vitro.

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.

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.

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.

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.

Invasive Infections Due to Trichosporon: Species Distribution, Genotyping, and Antifungal Susceptibilities from a Multicenter Study in China.

A total of 133 clinical Trichosporon isolates were collected in the National China Hospital Invasive Fungal Surveillance Net (CHIF-NET) program in 2009 to 2016. Accurate identification was performed by sequencing of the intergenic spacer 1 (IGS1) region. Among these isolates, Trichosporon asahii (108 isolates [81.2%]) was the leading species, followed by Trichosporon dermatis (7 isolates [5.3%]), Trichosporon asteroides (5 isolates [3.8%]), Trichosporon inkin (5 isolates [3.8%]), Trichosporon dohaense (3 isolates [2.3%]), and 1 isolate (0.7%) each of Trichosporon faecale, Trichosporon jirovecii, Trichosporon mucoides, Trichosporon coremiiforme, and Trichosporon montevideense Both the Vitek mass spectrometry (MS) (bioMérieux, Marcy l'Etoile, France) and Bruker Biotyper MS (Bruker Daltonics GmbH, Germany) platforms gave high levels (>97.5%) of correct identification when the species were present in the database. The geometric mean (GM) of amphotericin B MICs for T. asahii was 2-fold higher than that for non-asahii Trichosporon High fluconazole MICs (≥8 µg/ml) were observed for 25% of T. asahii isolates (27/108 isolates) and 16% of non-asahii Trichosporon (4/25 isolates) isolates. Itraconazole MICs were ≤0.5 µg/ml for 89.5% of the isolates. Voriconazole was the most potent antifungal agent in vitro, with a GM of 0.09 µg/ml. Genotyping of the isolates using IGS1 sequence alignment revealed that genotype 1 was most common (41.7%), followed by genotype 4 (31.5%), genotype 3 (23.1%), genotype 5 (0.9%), genotype 6 (0.9%), and genotype 7 (1.8%). Our data on species distribution, genotypes, and antifungal susceptibilities may contribute to a better understanding of the epidemiology of invasive Trichosporon infections throughout China.

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.

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.

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.