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Clostridioides difficile sequence type (ST) 35 has been found in humans and animals worldwide. However, its genomic epidemiology and clonal transmission have not been explored in detail. In this study, 176 C. difficile ST35 isolates from six countries were sequenced. Genomic diversity, clonal transmission and epidemiological data were analyzed. Sporulation and virulence capacities were measured. Four ribotypes (RT) were identified including RT046 (97.2%), RT656 (1.1%), RT427 (0.6%), and RT AI-78 (1.1%). Phylogenetic analysis of 176 ST35 genomes, along with 50 publicly available genomes, revealed two distinctive lineages without time-, region-, or source-dependent distribution. However, the distribution of antimicrobial resistance genes differed significantly between the two lineages. Nosocomial and communal transmission occurred in humans with the isolates differed by ≤ two core-genome single-nucleotide polymorphism (cgSNPs) and clonal circulation was found in pigs with the isolates differed by ≤ four cgSNPs. Notably, interspecies clonal transmission was identified among three patients with community acquired C. difficile infection and pigs with epidemiological links, differed by ≤ nine cgSNPs. Toxin B (TcdB) concentrations were significantly higher in human isolates compared to pig isolates, and ST35 isolates exhibited stronger sporulation capacities than other STs. Our study provided new genomic insights and epidemiological evidence of C. difficile ST35 intraspecies and interspecies clonal transmission, which can also be facilitated by its strong sporulation capacity.

ABSTRACT Group B Streptococcus (GBS) remains the leading cause of serious neonatal infection. Prenatal screening for vaginal and rectal GBS colonization is necessary in late pregnancy. Our objective was to compare the performance of four methods including the Xpert Xpress GBS, standard broth enrichment culture, real-time quantitative PCR (qPCR), and direct culture, for detecting of GBS in rectovaginal specimens. Among 939 pregnant women at 35–37 weeks of gestation recruited in the study, 168 (17.9%) were determined to be GBS-colonized by broth enrichment culture, the gold standard. The sensitivity and specificity of the Xpert Xpress GBS were 94.6% and 95.9%, respectively. The hands-on time and running time of the Xpert Xpress GBS were 1 minute and less than 1 hour, respectively. As a random-access, integrated device, the Xpert Xpress GBS test efficiently and accurately screens rectovaginal swabs for GBS colonization in pregnant women. IMPORTANCE This was the first study evaluating the performance of the Xpert Xpress group B Streptococcus (GBS) test using rectovaginal swabs from Chinese pregnant women. Compared to the other three assays, the Xpert Xpress GBS test demonstrated high sensitivity and specificity when screening 939 pregnant women for GBS in rectovaginal specimens. Additionally, its reduced time to obtain results makes it valuable for the rapid detection of GBS.

The World Health Organization has identified high-priority target product profiles for new TB diagnostics which include rapid biomarker-based, non-sputum-based diagnostic testing, using an easily accessible sample. The Cepheid 3-gene Host Response Fingerstick Blood Prototype Test (MTB-HR) quantifies relative mRNA levels of a 3-gene signature (GBP5, DUSP3, and KLF2) from a whole-blood sample on the GeneXpert platform. The objective of the present study was to evaluate the performance of the MTB-HR to distinguish between active tuberculosis (ATB), latent Mycobacterium tuberculosis infection (LTBI), other pulmonary diseases, and healthy volunteers at a tertiary care centre. Among 653 participants enrolled in this study, 192 were diagnosed as having ATB, and the remaining 461 were classified as non-ATB, including 137 cases of LTBI, 224 cases of other pulmonary diseases, and 100 healthy volunteers. The corresponding AUCs of the MTB-HR in distinguishing untreated ATB from non-ATB, LTBI, other pulmonary diseases, and healthy volunteers were 0.814 (95% CI, 0.760-0.868, sensitivity 76.1%, specificity 71.6%), 0.739 (95% CI, 0.667-0.812, sensitivity 59.7%, specificity 78.1%), 0.825 (95% CI, 0.770-0.880, sensitivity 82.1%, specificity 65.6%), 0.892 (95% CI, 0.839-0.945, sensitivity 76.1%, specificity 88.0%), respectively. When only samples with TAT of less than 1 h were included, the AUC of the MTB-HR in distinguishing untreated ATB from non-ATB was largest, 0.920 (95% CI, 0.822-1.000, sensitivity 81.3%, specificity 87.7%). In conclusion, the MTB-HR assay shows potential as a rapid, blood-based screening and triage test for ATB, especially for untreated ATB, with the advantage of increased diagnostic yield since blood is more readily available.

ABSTRACTBackground: Posterior oropharyngeal saliva (POS) is increasingly recognized as an alternative specimen for detecting respiratory pathogens. The accuracy of Xpert® MTB/RIF Ultra (X-Ultra), when performed on POS obtained from patients with paucibacillary pulmonary tuberculosis (TB) is unclear. Methods: We consecutively recruited adults with symptoms suggestive of pulmonary TB who were negative by both smear microscopy and Xpert MTB/RIF (X-Classic). Each participant was required to provide one bronchoalveolar lavage fluid (BALF) and one POS specimen, respectively. Diagnostic performances of X-Ultra and X-Classic on POS were compared against clinical and mycobacterial reference standards. Findings: 686 participants meeting inclusion criteria were consecutively enrolled into the study. The overall diagnostic sensitivities of X-Ultra and X-Classic on POS samples were 78.9% [95% confidence interval (CI): 72.8–83.8] and 56.4% (95% CI: 49.7–62.9), respectively; the specificities were 96.6% (95% CI: 94.3–98.1) for X-Ultra and 97.6 (95CI: 95.5–98.8) for X-Classic in POS specimens. Notably, the sensitivity of X-Ultra on POS was as sensitive as X-Classic on BALF against microbiological reference standard (78.9% VS 73.1%). Against clinical diagnosis as a reference standard, the sensitivities of X-Ultra and X-Classic on POS were 55.9% (95% CI: 50.5–61.2; 193/345) and 40.0% (95% CI: 34.8–45.4; 138/345), respectively. The risk of negative results with POS was dramatically increased with decreasing bacterial loads. Conclusions: The testing of POS using X-Ultra shows promise as a tool to identify patients with paucibacillary TB. Considering that bronchoscopy is a semi-invasive procedure, POS testing ahead of bronchoscopy, may decrease the need for bronchoscopic procedures, and the cost of care.

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) has been a major threat to human health due to its increased morbidity and mortality in clinical settings. Carbapenemase genes are less frequently found in CRPA compared with carbapenem-resistant Enterobacterales, of which carbapenemase producers are common. In this study, we identified 11 blaKPC-2-harbouring P. aeruginosa isolates from 139 carbapenemase-insensitive P. aeruginosa isolates collected between 2010 and 2021 in a tertiary hospital in China. Nine isolates belonged to ST697, while the other two were ST463. The antibiotic susceptibility testing showed that all the isolates were multidrug resistant, including resistance to imipenem, meropenem, ceftazidime, and tigecycline. Patients with Klebsiella pneumoniae carbapenemase-2 (KPC-2)-producing P. aeruginosa infections were mostly associated with complicated diseases and prolonged hospital stay, with 30% deterioration. The whole-genome sequencing analysis showed that these isolates carried multiple antibiotic resistance genes and virulence genes, and the KPC-2 genetic elements were highly related in ST697 isolates. The complete sequencing of ST697 isolate SE5416 showed that the harbouring of blaKPC-2 resulted from complex transposition and homologous recombination of an IncpRBL16 plasmid and other mobile elements. The Galleria mellonella infection model experiment showed that these KPC-2-producing P. aeruginosa–infected larvae had low survival rates and high virulence. The present study revealed the shifting of CRPA from ST697 to ST463 in East China; ST463 had higher drug resistance, posing greater challenges for clinical management.

Clinical microbiology has possessed a marvellous past, an important present and a bright future. Western medicine modernization started with the discovery of bacterial pathogens, and from then, clinical bacteriology became a cornerstone of diagnostics. Today, clinical microbiology uses standard techniques including Gram stain morphology, in vitro culture, antigen and antibody assays, and molecular biology both to establish a diagnosis and monitor the progression of microbial infections. Clinical microbiology has played a critical role in pathogen detection and characterization for emerging infectious diseases as evidenced by the ongoing COVID-19 pandemic. Revolutionary changes are on the way in clinical microbiology with the application of “-omic” techniques, including transcriptomics and metabolomics, and optimization of clinical practice configurations to improve outcomes of patients with infectious diseases.

Abstract Background The current COVID-19 pandemic and the previous SARS/MERS outbreaks of 2003 and 2012 have resulted in a series of major global public health crises. We argue that in the interest of developing effective and safe vaccines and drugs and to better understand coronaviruses and associated disease mechenisms it is necessary to integrate the large and exponentially growing body of heterogeneous coronavirus data. Ontologies play an important role in standard-based knowledge and data representation, integration, sharing, and analysis. Accordingly, we initiated the development of the community-based Coronavirus Infectious Disease Ontology (CIDO) in early 2020. Results As an Open Biomedical Ontology (OBO) library ontology, CIDO is open source and interoperable with other existing OBO ontologies. CIDO is aligned with the Basic Formal Ontology and Viral Infectious Disease Ontology. CIDO has imported terms from over 30 OBO ontologies. For example, CIDO imports all SARS-CoV-2 protein terms from the Protein Ontology, COVID-19-related phenotype terms from the Human Phenotype Ontology, and over 100 COVID-19 terms for vaccines (both authorized and in clinical trial) from the Vaccine Ontology. CIDO systematically represents variants of SARS-CoV-2 viruses and over 300 amino acid substitutions therein, along with over 300 diagnostic kits and methods. CIDO also describes hundreds of host-coronavirus protein-protein interactions (PPIs) and the drugs that target proteins in these PPIs. CIDO has been used to model COVID-19 related phenomena in areas such as epidemiology. The scope of CIDO was evaluated by visual analysis supported by a summarization network method. CIDO has been used in various applications such as term standardization, inference, natural language processing (NLP) and clinical data integration. We have applied the amino acid variant knowledge present in CIDO to analyze differences between SARS-CoV-2 Delta and Omicron variants. CIDO's integrative host-coronavirus PPIs and drug-target knowledge has also been used to support drug repurposing for COVID-19 treatment. Conclusion CIDO represents entities and relations in the domain of coronavirus diseases with a special focus on COVID-19. It supports shared knowledge representation, data and metadata standardization and integration, and has been used in a range of applications.

Carbapenem-resistant (CR) Klebsiella oxytoca complex can be associated with high mortality, emerging as a new threat to the public health. K. oxytoca complex is phylogenetically close to K. pneumoniae, one of most common species associated with multidrug resistance in Enterobacterale. The latest research showed that K. oxytoca is a complex of six species. Currently, the epidemiological and genomic characteristics of CR K. oxytoca complex in China are still unclear. Here, we conducted a multi-center study on 25 CR K. oxytoca complex collected from five representative regions in China. These isolates were, respectively, recovered from respiratory tract (12 cases, 48.0%), abdominal cavity (5 cases, 20.0%), blood (4 cases, 16.0%), urine tract (3 cases, 12.0%) and skin or soft tissue (1 cases, 4.0%). Among them, 32.0% (8/25) of patients infected with K. oxytoca complex had a poor prognosis. In this study, three K. oxytoca complex species were detected, namely K. michiganensis, K. oxytoca and K. pasteurii, among which K. michiganensis was the most common. Three carbapenemase genes were identified, including blaNDM-1 (10, 38.5%), blaKPC-2 (9, 34.6%) and blaIMP (6 blaIMP-4 and 1 blaIMP-8; 7, 26.9%). Subsequent multilocus sequence typing identified various sequence types (STs), among which ST43, ST92 and ST145 were relatively common. Different from the clonal dissemination of high-risk carbapenem-resistant K. pneumoniae strains, our research revealed a polyclonal dissemination characteristic of CR K. oxytoca complex in China. S1-nuclease PFGE and Southern blot experiment showed that carbapenemase genes were encoded in plasmids of different sizes. Two blaNDM-harboring plasmids were subsequently sequenced, and were characterized to be IncX3 and IncC incompatibility groups, respectively. This is the first multi-center study of CR K. oxytoca complex in China, which improved our understanding of the prevalence and antimicrobial resistance characteristics of CR K. oxytoca complex in China.

ABSTRACT Early detection of microbial pathogens causing respiratory tract infection plays a crucial role in clinical management. The BioCode Respiratory Pathogen Panel (BioCode RPP) utilizes reverse transcriptase PCR (RT-PCR) in combination with barcoded magnetic beads to amplify, detect, and identify respiratory pathogens. This panel qualitatively detects and identifies 14 viruses, including influenza virus A with H1 pdm09, H1, and H3 subtyping; influenza B; respiratory syncytial virus (RSV); human metapneumovirus; parainfluenza virus 1; parainfluenza virus 2; parainfluenza virus 3; parainfluenza virus 4; coronavirus (229E, NL63, OC43, and HKU1); adenovirus; and human rhinovirus/enterovirus, and 3 bacteria, including Chlamydia pneumoniae, Mycoplasma pneumoniae, and Bordetella pertussis. Reproducibility, which was assessed with contrived specimens containing 12 targets at 3 clinical sites, with 2 operators at each site for 5 days, was 99.4% for Flu A H3 and Flu B, 98.9% for RSV, and 100% for the remaining 9 targets assayed. A multicenter clinical trial evaluated the performance of the BioCode RPP with 2,647 nasopharyngeal swab specimens from 5 geographically distinct sites and revealed comparable performance between the BioCode RPP and FilmArray Respiratory Panel (FA-RP). Specifically, the positive percent agreements (PPAs) for various pathogens ranged between 80.8% and 100% compared with the FA-RP (1.7 and 2.0). Negative percent agreement ranged from 98.4% to 100% for BioCode RPP. The BioCode RPP also offers scalable automated testing capability of up to 96 specimens in a single run with total sample-to-result time under 5 h. The invalid rate of the BioCode RPP on initial testing was 1.0% (26/2,649). IMPORTANCE Early detection of microbial pathogens causing respiratory tract infection plays a crucial role in clinical management. The BioCode Respiratory Pathogen Panel (BioCode RPP) is a high-throughput test that utilizes RT-PCR in combination with barcoded magnetic beads to amplify, detect, and identify 17 respiratory pathogens, including 14 viruses and 3 bacteria. This study summarizes data generated from a multicenter clinical trial evaluating the performance of the BioCode RPP on 2,647 nasopharyngeal swab specimens from five geographically distinct sites.

The vortex dynamics of a supersonic flow past an impulsively started circular cylinder are studied numerically. A well-developed high-order finite difference method is adopted to solve the two-dimensional Navier–Stokes equations. In particular, Ma = 1.1 and Re = 2.2 × 104 are selected to report the vortex evolution process. The results confirm that the α-, β-, sub-α-, and sub-β-phenomena observed in incompressible flow in the separated region do exist in the supersonic regime. In addition, the influence of Mach number and Reynolds number on the vortex is also studied, and the vortex changes are compared at Ma = 1.1, 1.7 and Re = 1 × 104, 2 × 104, 2.5 × 104, 3 × 104, 5 × 104, and 1 × 105.

Objectives: The number of lung transplants is increasing year by year in China and globally. With the widespread use of donation after brainstem death (DBD) donor lungs and donation after circulatory death (DCD) donor lungs, donor-derived infection (DDI) poses a major challenge in lung transplantation. Using donor lungs infected or colonized with carbapenem-resistant Enterobacteriaceae (CRE) may have serious implications in lung-transplant recipients. Currently, traditional microbial culture along with antimicrobial susceptibility testing cannot fully meet the need for rapid and accurate diagnosis of CRE infection in a donor before organ harvest. Methods: The Xpert Carba-R device (Cepheid, Sunnyvale, CA) was used to detect and differentiate Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-β-lactamase (NDM), Verona integron-encoded metallo-β-lactamase (VIM), active-on-imipenem (IMP), and OXA-48 carbapenemase genotypes in bronchial lavage fluid from donor lungs before organ harvest. Positive detection of 1 or more of these genotypes indicated a potentially CRE-infected donor lung, and these organs were removed from the lung transplantation cohort. Donor lungs negative for all KPC, NDM, VIM, IMP, and OXA-48 genotypes determined by the Xpert Carba-R device were used for lung transplantation. The incidence of CRE-associated DDI and infection-related complications were compared in the Xpert Carba-R screening group and an historic control group. Results: In this study, 21 donor lungs were tested with the Xpert Carba-R device to detect and differentiate carbapenemase genotypes. Among them, 4 were positive for 1 or more carbapenemase genotypes and were discarded, and the remaining 17 donor lungs showing no carbapenemase gene presence were used for lung transplantation. No CRE-associated DDI occurred in these 17 lung-transplant recipients. Conclusions: Rapid and accurate detection of the carbapenemase gene in donor lungs at the point of care before transplantation using the Xpert Carba-R device reduced the risk of CRE-associated DDI in lung-transplant recipients.

Objectives: Rapid and accurate screening for carbapenemase-producing organism (CPOs) in hospitalized patients is critical for infection control and prevention. The Xpert Carba-R assay is designed for rapid detection of CPOs, but 1 assay is usually conducted for only 1 sample. We evaluated a pooling strategy for CPO screening using the Xpert Carba-R assay. Methods: Swab sets containing 2 swabs were collected from 415 unique patients at Peking University People’s Hospital. One swab was used for the pooling test, in which 5 swabs from different patients were mixed in 1 sample treatment solution. The prevalence of CPOs in the hospital (5.3%) predicted that 5:1 pooling was most economical. As the reference method, the other swab was tested by culture using sequencing. Results: Of 415 samples, 383 were CPO negative using the pooling test strategy and 31 were positive. All samples that were negative by pooling were negative by culture and sequencing. Among the 31 positive samples identified by the pooling strategy, 26 were positive by culture and sequencing (including 24 samples with 1 targeted gene and 2 samples with double targeted genes, 1 NDM+/IMP+ and 1 VIM+/IMP+), and 5 were negative. Overall, 198 tests were conducted in the study, and 217 were saved compared with testing individually. The efficiency of the pooling strategy was 215%. The overall sensitivity was 1 (95% CI, 0.840–1), the specificity was 0.987 (95% CI, 0.968–0.995), the accuracy was 0.987 (95% CI, 0.970–0.996), positive predictive value was 0.838 (95% CI, 0.655–0.939), and the negative predictive value was 1 (95% CI, 0.988–1). Conclusions: The pooling strategy using the Xpert Carba-R assay showed good potential in screening CPO with good sensitivity and a significantly lower cost.

ABSTRACT The objective of this study was to compare the performances of BioFire Respiratory Panel 2 (RP2) plus, quantitative real-time PCR (qPCR), and culture for the detection of Bordetella pertussis in nasopharyngeal swab (NPS) specimens. Consecutive NPS specimens were collected from patients with clinically suspected pertussis from 1 March 1 to 31 July 2018 in Shenzhen Children’s Hospital. All the specimens were tested in parallel by RP2 plus, qPCR, and culture methods. A total of 464 children were enrolled in this study. The positive pertussis rates of culture, RP2 plus, and qPCR were 23.1%, 39.0%, and 38.4%, respectively. Compared to the combined reference standard, the sensitivity, specificity, positive predictive value, and negative predictive values were, respectively, 56.6% (95% confidence interval [CI], 49.2 to 63.7%), 100% (98.3 to 100%), 100% (95.7 to 100%), and 77.0% (72.2 to 81.2%) for culture, 89.9% (84.5 to 93.7%), 96.0% (92.8 to 97.9%), 93.9% (89.1 to 96.8%), and 93.3% (89.5 to 95.8%) for RP2 plus, and 86.8% (80.9 to 91.1%), 94.9% (91.4 to 97.1%), 92.1% (86.9 to 95.5%), and 91.3% (87.2 to 94.2%) for qPCR. The most prevalent codetected pathogen was human rhinovirus/enterovirus (n = 99, 52.4%), followed by parainfluenza virus (n =32, 16.9%) and respiratory syncytial virus (n = 29, 15.3%), in children with B. pertussis present, which was consistent with the top three pathogens previously found in children with B. pertussis absent. Turnaround times for RP2 plus, qPCR, and culture were 2 h, 8 h, and 120 h, respectively. RP2 plus quickly and accurately detected B. pertussis, providing valuable information for an early clinical diagnosis and optimal choice of therapy. IMPORTANCE In recent years, there have been some epidemic or local outbreaks of pertussis in countries with high vaccination rates. One of the crucial factors in controlling pertussis is early diagnosis, which is based on specific laboratory measurements, including culture, serological tests, and PCR assays. Compared to culture and serological tests, PCR is more suitable for clinical application, with a fast detection speed of several hours independent of the disease stage and individual vaccination status. BioFire Respiratory Panel 2 plus, a multiplex PCR assay for simultaneously detecting 22 respiratory pathogens, facilitates the quick detection of Bordetella pertussis and coinfecting respiratory pathogens. It also provides valuable information for an early clinical diagnosis and optimal choice of therapy for children with clinically suspected pertussis.

Abstract The aim of this study was to carry out a case control study comparing the HPV genome in patients with oral cavity squamous cell carcinoma (OC-SCC) to normal patients using metagenomic shotgun sequencing. We recruited 50 OC-SCC cases which were then matched with a control patient by age, gender, race, smoking status and alcohol status. DNA was extracted from oral wash samples from all patients and whole genome shotgun sequencing performed. The raw sequence data was cleaned, reads aligned with the human genome (GRCH38), nonhuman reads identified and then HPV genotypes identified using HPViewer. In the 50 patients with OC-SCC, the most common subsite was tongue in 26 (52%). All patients were treated with primary resection and neck dissection. All but 2 tumors were negative on p16 immunohistochemistry. There were no statistically significant differences between the cases and controls in terms of gender, age, race/ethnicity, alcohol drinking, and cigarette smoking. There was no statistically significant difference between the cancer samples and control samples in the nonhuman DNA reads (medians 4,228,072 vs. 5,719,715, P value = 0.324). HPV was detected in 5 cases (10%) of OC-SCC (genotypes 10, 16, 98) but only 1 tumor sample (genotype 16) yielded a high number of reads to suggest a role in the etiology of OC-SCC. HPV was detected in 4 control patients (genotypes 16, 22, 76, 200) but all had only 1–2 HPV reads per human genome. Genotypes of HPV are rarely found in patients with oral cancer.

Clostridioides difficile sequence type (ST) 37 (ribotype 017) is one of the most prevalent genotypes circulating in China. However, its genomic evolution and virulence determinants were rarely explored. Whole-genome sequencing, phylogeographic and phylogenetic analyses were conducted for C. difficile ST37 isolates. The 325 ST37 genomes from six continents, including North America (n = 66), South America (n = 4), Oceania (n = 7), Africa (n = 9), Europe (n = 138) and Asia (n = 101), were clustered into six major lineages, with region-dependent distributions, harbouring an array of antibiotic-resistance genes. The ST37 strains from China were divided into four distinct sublineages, showing five importation times and international sources. Isolates associated with severe infections exhibited significantly higher toxin productions, tcdB mRNA levels, and sporulation capacities (P

BackgroundFatty acids, especially polyunsaturated fatty acid (PUFA), are found abundantly in the brain and are fundamental for a fetus's growth. The fatty acid profiles of mothers and fetuses may be affected by maternal prepregnancy body mass index (pre-BMI), thus affecting fetal growth and development.MethodsA total of 103 mother-fetus pairs were divided into overweight/obese (OW, n = 26), normal weight (NW, n = 60), and underweight (UW, n = 17) groups according to pre-BMI. Fatty acid profiles in maternal and umbilical cord plasma were analyzed by gas chromatography.ResultsThe infant birth BMI z-score of the OW group was higher than that of the NW and UW groups (p < 0.05). The OW mothers had significantly higher plasma n-6 PUFA and n-6/n-3, but lower docosahexaenoic acid (DHA) and n-3 PUFA (p < 0.05). In cord plasma, the proportions of DHA and n-3 PUFA were lower in the OW group (p < 0.05), whereas the n-6/n-3 ratio was higher in the OW group (p < 0.05). The pre-BMI was negatively correlated with cord plasma DHA in all subjects (r = −0.303, p = 0.002), and the same negative correlation can be observed in the OW group (r = −0.561, p = 0.004), but not in the NW and UW groups (p > 0.05). The pre-BMI was positively correlated with cord plasma n-6/n-3 in all subjects (r = 0.325, p = 0.001), and the same positive correlation can be found in the OW group (r = 0.558, p = 0.004), but not in NW and UW groups (p > 0.05).ConclusionsMaternal pre-BMI was associated with the maternal-fetal plasma fatty acid profiles, whereas the adverse fatty acid profiles are more noticeable in the prepregnancy OW mothers.

ABSTRACTSerology detection is recognized for its sensitivity in convalescent patients with COVID-19, in comparison with nucleic acid amplification tests (NAATs). This article aimed to evaluate the diagnostic accuracy of serologic methods for COVID-19 based on assay design and post-symptom-onset intervals. Two authors independently searched PubMed, Cochrane library, Ovid, EBSCO for case–control, longitudinal and cohort studies that determined the diagnostic accuracy of serology tests in comparison with NAATs in COVID-19 cases and used QUADAS-2 for quality assessment. Pooled accuracy was analysed using INLA method. A total of 27 studies were included in this meta-analysis, with 4 cohort, 16 case–control and 7 longitudinal studies and 4565 participants. Serology tests had the lowest sensitivity at 0–7 days after symptom onset and the highest at >14 days. TAB had a better sensitivity than IgG or IgM only. Using combined nucleocapsid (N) and spike(S) protein had a better sensitivity compared to N or S protein only. Lateral flow immunoassay (LFIA) had a lower sensitivity than enzyme-linked immunoassay (ELISA) and chemiluminescent immunoassay (CLIA). Serology tests will play an important role in the clinical diagnosis for later stage COVID-19 patients. ELISA tests, detecting TAB or targeting combined N and S proteins had a higher diagnostic sensitivity compared to other methods.

ABSTRACTThe three unprecedented outbreaks of emerging human coronavirus (HCoV) infections at the beginning of the twenty-first century have highlighted the necessity for readily available, accurate and fast diagnostic testing methods. The laboratory diagnostic methods for human coronavirus infections have evolved substantially, with the development of novel assays as well as the availability of updated tests for emerging ones. Newer laboratory methods are fast, highly sensitive and specific, and are gradually replacing the conventional gold standards. This presentation reviews the current laboratory methods available for testing coronaviruses by focusing on the coronavirus disease 2019 (COVID-19) outbreak going on in Wuhan. Viral pneumonias typically do not result in the production of purulent sputum. Thus, a nasopharyngeal swab is usually the collection method used to obtain a specimen for testing. Nasopharyngeal specimens may miss some infections; a deeper specimen may need to be obtained by bronchoscopy. Alternatively, repeated testing can be used because over time, the likelihood of the SARS-CoV-2 being present in the nasopharynx increases. Several integrated, random-access, point-of-care molecular devices are currently under development for fast and accurate diagnosis of SARS-CoV-2 infections. These assays are simple, fast and safe and can be used in the local hospitals and clinics bearing the burden of identifying and treating patients.

BackgroundWe aimed to evaluate the clinical performance of the GeneXpert® (Xpert) CT/NG assay for the detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) using urine and cervical swabs collected from patients in China.MethodsThis study was conducted from September 2016 to September 2018 in three Chinese urban hospitals. The results from the Xpert CT/NG test were compared to those from the Roche cobas® 4800 CT/NG test. Discordant results were confirmed by DNA sequence analysis.ResultsIn this study, 619 first void urine (FVU) specimens and 1,042 cervical swab specimens were included in the final dataset. There were no statistical differences between the results of the two tests for the detection of CT/NG in urine samples (p > 0.05), while a statistical difference was found in cervical swabs (p

Sepsis is a common and often treacherous medical emergency with a high mortality and long-term complications in survivors. Though antibiotic therapy can reduce death rate of sepsis significantly, it impairs gut microbiota (GM), which play imperative roles in human health. In this study, we compared the therapeutic effects of antibiotics, probiotics, and Chinese medicine QRD on the survival rates of septic model and observed the GM characteristics of experimental rats via 16S rRNA gene amplicon sequencing. The 72 h survival rates of septic rat demonstrated the significant therapeutic effects in the three groups treated with antibiotics (AT), Chinses medicine QRD (QT), and probiotics (PT), which were elevated from the survival rate of 26.67% for the sepsis control group (ST) to 100.0% for AT, 88.24% for QT, and 58.33% for PT. The original characteristics of GM identified in the sham operation controls (SC) were relatively similar to those in PT and QT; nevertheless, the AT rats were shown dramatically decreased in the GM diversity. In addition, the septic rats in AT were revealed the higher abundances of Escherichia Shigella, Proteus, Morganella, Enterococcus, and Lysinibacillus, but the lower those of Parabacteroides, Alistipes, Desulfovibrio, Bacteroides, Helicobacter, Mucispirillum, Oscillibacter, Lachnospiraceae, and Ruminiclostridium 9, when compared to the PT and QT rats. By contrast, the GM of PT and QT rats shared similar diversity and structure. Our findings indicated that QRD increased the survival rates without impairment of the GM characteristics, which provides novel insights into the role of Chinese medicine in therapy and long-term recovery of sepsis.

Objectives: Ongoing acquisition of antimicrobial resistance genes has made Morganella morganii a new clinical treatment challenge. Understanding the molecular epidemiology of M. morganii will contribute to clinical treatment and prevention.Methods: We undertook a 6-year clinical molecular epidemiological investigation of M. morganii from three tertiary hospitals in China since 2014. Antimicrobial susceptibility testing was performed using a VITEK-2 system. All isolates were screened for β-lactam and plasmid-mediated quinolone resistance genes by PCR. Isolates carrying carbapenem-resistant genes were subjected to whole-genome sequencing (WGS). The variation and evolution of these mobile genetic elements (MGEs) were then systematically analyzed.Results: Among all M. morganii isolates (n = 335), forty (11.9%) were recognized as multidrug resistant strains. qnrD1, aac(6′)-Ib-cr, blaTEM–104, and blaCTX–M–162 were the top four most prevalent resistance genes. Notably, phylogenomic and population structure analysis suggested clade 1 (rhierBAPS SC3 and SC5) associated with multiple resistance genes seemed to be widely spread. WGS showed a blaOXA–181-carrying IncX3 plasmid and a Proteus genomic island 2 variant carrying blaCTX–M–3, aac(6′)-Ib-cr coexisted in the same multidrug resistant strain zy_m28. Additionally, a blaIMP–1-carrying IncP-1β type plasmid was found in the strain nx_m63.Conclusion: This study indicates a clade of M. morganii is prone to acquire resistance genes, and multidrug resistant M. morganii are increasing by harboring a variety of MGEs including two newly discovered ones in the species. We should be vigilant that M. morganii may bring more extensive and challenging antimicrobial resistance issue.

Clostridioides difficile sequence type 2 (ST2) has been increasingly recognized as one of the major genotypes in China, while the genomic characteristics and biological phenotypes of Chinese ST2 strains remain to be determined. We used whole-genome sequencing and phylogenetic analysis to investigate the genomic features of 182 ST2 strains, isolated between 2011 and 2017. PCR ribotyping (RT) was performed, and antibiotic resistance, toxin concentration, and sporulation capacity were measured. The core genome Maximum-likelihood phylogenetic analysis showed that ST2 strains were distinctly segregated into two genetically diverse lineages [L1 (67.0% from Northern America) and L2], while L2 further divided into two sub-lineages, SL2a and SL2b (73.5% from China). The 36 virulence-related genes were widely distributed in ST2 genomes, but in which only 11 antibiotic resistance-associated genes were dispersedly found. Among the 25 SL2b sequenced isolates, RT014 (40.0%, n = 10) and RT020 (28.0%, n = 7) were two main genotypes with no significant difference on antibiotic resistance (χ2 = 0.024–2.667, P > 0.05). A non-synonymous amino acid substitution was found in tcdB (Y1975D) which was specific to SL2b. Although there was no significant difference in sporulation capacity between the two lineages, the average toxin B concentration (5.11 ± 3.20 ng/μL) in SL2b was significantly lower in comparison to those in L1 (10.49 ± 15.82 ng/μL) and SL2a (13.92 ± 2.39 ng/μL) (χ2 = 12.30, P < 0.05). This study described the genomic characteristics of C. difficile ST2, with many virulence loci and few antibiotic resistance elements. The Chinese ST2 strains with the mutation in codon 1975 of the tcdB gene clustering in SL2b circulating in China express low toxin B, which may be associated with mild or moderate C. difficile infection.

Abstract Background The merging of two divergent genomes during hybridization can result in the remodeling of parental gene expression in hybrids. A molecular basis underling expression change in hybrid is regulatory divergence, which may change with the parental genetic divergence. However, there still no unanimous conclusion for this hypothesis. Results Three species of Camellia with a range of genetic divergence and their F1 hybrids were used to study the effect of parental genetic divergence on gene expression and regulatory patterns in hybrids by RNA-sequencing and allelic expression analysis. We found that though the proportion of differentially expressed genes (DEGs) between the hybrids and their parents did not increase, a greater proportion of DEGs would be non-additively (especially transgressively) expressed in the hybrids as genomes between the parents become more divergent. In addition, the proportion of genes with significant evidence of cis-regulatory divergence increased, whereas with trans-regulatory divergence decreased with parental genetic divergence. Conclusions The discordance within hybrid would intensify as the parents become more divergent, manifesting as more DEGs would be non-additively expressed. Trans-regulatory divergence contributed more to the additively inherited genes than cis, however, its contribution to expression difference would be weakened as cis mutations accumulated over time; and this might be an important reason for that the more divergent the parents are, the greater proportion of DEGs would be non-additively expressed in hybrid.

We isolated a New Delhi metallo-β-lactamase 5 (NDM-5)–producing Klebsiella pneumoniae sequence type (ST) 258 strain in southwest China during 2017. The blaNDM-5 gene was acquired by horizontal plasmid transfer from NDM-5–producing Escherichia coli. We identified genomic characteristics in ST258 strains that differed from those of global K. pneumoniae carbapenemase–producing strains.

ABSTRACTMolecular epidemiology of Clostridium difficile infection (CDI) has been extensively studied in North America and Europe; however, limited data on CDI are available in the Asia-Pacific region. A multicentre retrospective study was conducted in this region. C. difficile isolates were subjected to multilocus sequence typing (ST) and antimicrobial susceptibility testing. Totally, 394 isolates were collected from Hangzhou, Hong Kong, China; Busan, South Korea; Fukuoka, Japan; Singapore; Perth, Sydney, Australia; New York, the United States. C. difficile isolates included 337 toxin A-positive/B-positive/binary toxin-negative (A+B+CDT-), 48 A-B+CDT-, and nine A+B+CDT+. Distribution of dominant STs varied geographically with ST17 in Fukuoka (18.6%), Busan (56.0%), ST2 in Sydney (20.4%), Perth (25.8%). The antimicrobial resistance patterns were significantly different among the eight sites (χ2 = 325.64, p < 0.001). Five major clonal complexes correlated with unique antimicrobial resistances. Healthcare-associated (HA) CDI was mainly from older patients with more frequent antimicrobial use and higher A-B+ positive rates. Higher resistance to gatifloxacin, tetracycline, and erythromycin were observed in HA-CDI patients (χ2 = 4.76-7.89, p = 0.005-0.029). In conclusion, multiple C. difficile genotypes with varied antimicrobial resistance patterns have been circulating in the Asia-Pacific region. A-B+ isolates from older patients with prior antimicrobial use were correlated with HA-CDI.

Salmonella spp. are a major cause of foodborne illness throughout the world. Traditional serotyping by antisera agglutination has been used as a standard identification method for many years but newer nucleic acid-based tests have become available that may provide advantages in workflow and test turnaround time. In this study, we evaluated the Luminex® xMAP® Salmonella Serotyping Assay (SSA), a multiplex nucleic acid test capable of identifying 85% of the most common Salmonella serotypes, in comparison to the traditional serum agglutination test (SAT) on 4 standard strains and 255 isolates from human (224), environmental, and food (31) samples. Of the total of 259 isolates, 256 could be typed by the SSA. Of these, 197 (77.0%) were fully typed and 59 (23.0%) were partially typed. By SAT, 246 of the 259 isolates (95%) were successfully typed. Sixty isolates had discrepant results between SAT and SSA and were resolved using whole genome sequencing (WGS). By SAT, 80.0% (48/60) of the isolates were consistent with WGS while by SSA 91.7% (55/60) were partially consistent with WGS. By serovar, all 30 serovars except one tested were fully or partially typable. The workflow comparison showed that SSA provided advantages over SAT with a hands-on time (HOT) of 3.5 min and total turnaround time (TAT) of 6 h, as compared to 1 h HOT and 2–6 days TAT for SAT. Overall, this study showed that molecular serotyping is promising as a rapid method for Salmonella serotyping with good accuracy for typing most common Salmonella serovars circulating in China.

Background: Atopic dermatitis (AD) is a common cutaneous disease, associated with imbalances in the skin microbiota.Objective: To explore the characteristics of the cutaneous microbiota and its dynamic changes during clinical treatment.Methods: Cutaneous swab samples were collected from 51 AD patients before treatment, and 40 AD patients remained after a 2-week treatment with mometasone and mupirocin.Results: AD patients exhibited significant enrichments of Prevotella and Desulfovibrio as well as obvious reductions of Corynebacterium, Streptococcus and Parabacteroides. Based on the proportion of Staphylococcus aureus, the AD patients were further classified into S. aureus-predominant group (AD.S) and S. aureus-non-dominant (AD.ND) group. The AD.S group exhibited lower skin microbial diversity and higher atopic dermatitis (SCORAD) index. In the AD.S group, the cutaneous microbial diversity significantly increased from 2.9 ± 0.8 to 3.7 ± 1.0, while the relative abundance of S. aureus decreased from 42.5 ± 20.7 to 10.3 ± 28.4 after treatment. In contrast, no significant skin microbiota changes were detected in the AD.ND group.Conclusions: AD patients with predominant S. aureus had higher disease severity and lower microbiota diversity compared to patients in the AD.ND group. Mometasone and mupirocin therapy had significant effects on skin microbiota in AD.S patients, but had a paradoxical response in the AD.ND patients.

KPC-producing Klebsiella pneumoniae (KPC-KP) is the most widely spread carbapenem-resistant Enterobacteriaceae (CRE) in China. Avibactam is a novel non-β-lactam β-lactamase inhibitor which is highly active against KPC. Recently, ceftazidime-avibactam (CAZ-AVI) was approved for clinical treatment in China. Here we conducted a retrospective study to examine the antimicrobial susceptibility of CAZ-AVI prior to its usage in China, and evaluated the potential to develop resistance in KPC-KP. CAZ-AVI MICs were tested in 347 KPC-KP isolates collected from patients with no prior treatment with this combination from six medical centers in China. Almost all isolates (n = 346; 99.7%) were CAZ-AVI-susceptible, with only 12 (3.5%) which showed reduced susceptibility (MIC ≥ 4/4 μg/ml) or resistance. The 12 isolates belong to ST11 and half of them carry virulence genes. In comparison to susceptible isolates, these isolates demonstrated higher blaKPC–2 copy numbers and expressions, and demonstrated higher frequency of developing CAZ-AVI resistance.

ObjectiveThe emergence of carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-hvKp) strains poses a significant public threat, and effective antimicrobial therapy is urgently needed. Recent studies indicated that apramycin is a potent antibiotic with good activity against a range of multi-drug resistant pathogens. In this study, we evaluated the in vitro activity of apramycin against clinical CR-hvKp along with carbapenem-resistant non-hvKp (CR-non-hvKp) isolates.MethodsBroth microdilution method was used to evaluate the in vitro activities of apramycin, gentamicin, amikacin, imipenem, meropenem, doripenem, ertapenem and other comparator “last-resort” antimicrobial agents, including ceftazidime-avibactam, colistin and tigecycline, against eighty-four CR-hvKp and forty CR-non-hvKp isolates collected from three Chinese hospitals. Multilocus Sequence typing (MLST), molecular capsule typing (wzi sequencing) and antimicrobial resistance genes were examined by PCR and Sanger sequencing. Pulsed-field gel electrophoresis and next generation sequencing were conducted on selected isolates.ResultsAmong the 84 CR-hvKp isolates, 97.6, 100, 97.6, and 100% were resistant to imipenem, meropenem, doripenem and ertapenem, respectively. Apramycin demonstrated an MIC50/MIC90 of 4/8 μg/mL against the CR-hvKp isolates. In contrast, the MIC50/MIC90 for amikacin and gentamicin were >64/>64 μg/mL. All CR-hvKp isolates were susceptible to ceftazidime-avibactam, colistin and tigecycline with the MIC50/MIC90 values of 0.5/1, 0.25/0.5, 1/1, respectively. For CR-non-hvKp, The MIC50/90 values for apramycin, gentamicin and amikacin were 2/8, >64/>64, and >64/>64 μg/mL, respectively. There were no statistical significance in the resistance rates of antimicrobial agents between CR-hvKp and CR-non-hvKp groups (p > 0.05). Genetic analysis revealed that all CR-hvKp isolates harbored blaKPC–2, and 94% (n = 79) belong to the ST11 high-risk clone. 93.6% (44/47) of amikacin or gentamicin resistant strains carried 16S rRNA methyltransferases gene rmtB.ConclusionApramycin demonstrated potent in vitro activity against CR-hvKp isolates, including those were resistant to amikacin or gentamicin. Further studies are needed to evaluate the applicability of apramycin to be used as a therapeutic antibiotic against CR-hvKp infections.

Little is known regarding differences in the gut microbiomes of rheumatoid arthritis (RA) patients and healthy cohorts in China. This study aimed to identify differences in the fecal microbiomes of 66 Chinese patients with RA and 60 healthy Chinese controls. The V3-V4 variable regions of bacterial 16S rRNA genes were sequenced with the Illumina system to define the bacterial composition. The alpha-diversity index of the microbiome of the RA patients was significantly lower than that of the control group. The bacterial genera Bacteroides (p = 0.02202) and Escherichia-Shigella (p = 0.03137) were more abundant in RA patients. In contrast, Lactobacillus (p = 0.000014), Alloprevotella (p = 0.0000008615), Enterobacter (p = 0.000005759), and Odoribacter (p = 0.0000166) were less abundant in the RA group than in the control group. Spearman correlation analysis of blood physiological measures of RA showed that bacterial genera such as Dorea and Ruminococcus were positively correlated with RF-IgA and anti-CCP antibodies. Furthermore, Alloprevotella and Parabacteroides were positively correlated with the erythrocyte sedimentation rate, and Prevotella-2 and Alloprevotella were positively correlated with C-reactive protein, both biomarkers of inflammation. These findings suggest that the gut microbiota may contribute to RA development via interactions with the host immune system.

Enterobacter cloacae has recently emerged as one of the most common carbapenem-resistant Enterobacteriaceae. The emergence and spread of metallo-β-lactamase-producing E. cloacae have posed an immediate threat globally. Here, we investigated the molecular characteristics of 84 carbapenem-resistant Enterobacter cloacae (CREL) collected from three tertiary hospitals in China between 2012 and 2016. Species identification and antimicrobial susceptibility testing were performed using a VITEK-2 system. Carbapenems, polymyxins B, and tigecycline were tested by broth microdilution method. The carbapenem in activation method (CIM) and cefoxitin three-dimensional test were used to detect carbapenemase and AmpC β-lactamase, respectively. Isolates were screened for β-lactam resistance genes by PCR, and expression of ompC and ompF was determined by qRT-PCR. Genetic relatedness was performed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST), while selected isolates were subjected to whole-genome sequencing. Among the 84 CREL isolates, 50 (59.5%) were detected as carbapenemase producers. NDM-1 was the dominant carbapenemase (80.0%), followed by IMP-26 (8.0%) and IMP-4 (6.0%). Notably, we identified the first NDM-1 and IMP-1 co-producing E. cloacae, carrying plasmids of several incompatibility (Inc) groups, including IncHI2, IncHI2A, and IncN. Most isolates showed decreased expression of ompC and/or ompF, and contained a broad distribution of ESBLs and AmpC β-lactamases. These findings suggested that different molecular mechanisms, including carbapenemase, ESBL and/or AmpC plus loss of porins, have contributed to carbapenem resistance. The blaNDM−1-harboring plasmids contained highly conserved gene environment around blaNDM−1 (blaNDM−1-bleMBL-trpF-dsbD-cutA1-groES-groEL), which could be associated with the potential dissemination of blaNDM−1. IMP-type MBL was located within a variety of integrons and usually contained various gene cassettes encoding multidrug resistance. These isolates produced 54 different pulsotypes, and were classified into 42 STs by MLST. Nineteen blaNDM−1-positive E. cloacae isolates obtained from Ningxia had the same pulsotype (PFGE type 1), belonging to ST78 within clonal complex 74 (CC74). The plasmid-based replicon typing indicated that IncX3 plasmids mediated the dissemination of blaNDM−1 among these homologous strains. This is the first report on the outbreak of NDM-1-producing E. cloacae ST78 with contribution of IncX3 plasmids in Northwestern China. There's an immediate need to intensify surveillance attentively to prevent and control the further spread of NDM-1 in China.

The prevalence of carbapenem-resistant Enterobacteriaceae (CRE) is increasing globally, with different molecular mechanisms described. Here we studied the molecular mechanisms of carbapenem resistance, including clonal and plasmid dissemination, of 67 CRE isolates collected between 2012 and 2016 from a tertiary hospital in Eastern China, an CRE endemic region. Species identification and susceptibility testing were performed using the BD Phoenix Automated Microbiology System. Isolates were characterized by PCR (for carbapenemases, ESBLs, AmpC and porin genes), multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and conjugation transfer experiments. Selected blaKPC-2 -harboring plasmids were subjected to next-generation sequencing using the Illumina Miseq platform. Among the 67 CRE isolates, 42 Klebsiella pneumoniae, 10 Serratia marcescens, 6 Enterobacter cloacae, 2 Raoultella ornithinolytica, 2 K. oxytoca, 1 K. aerogenes, and 4 Escherichia coli isolates were identified. Six different carbapenemases were detected, including blaKPC-2 (n = 45), blaKPC-3 (n = 1), blaNDM-1 (n = 6), blaNDM-5 (n = 1), blaIMP-4 (n = 2), and blaVIM-1 (n = 2); blaOXA-48-like genes were not detected. One E. cloacae strain possessed both blaNDM-1 and blaKPC-3, while two E. cloacae isolates harbored blaNDM-1 and blaVIM-1. ESBLs (CTX-M, SHV, and TEM) and/or AmpC (CMY, DHA, and ACT/MIR) genes were also identified in 59 isolates, including 13 strains that lacked carbapenemases. Several insertions or stop codon mutations were found within porin genes of K. pneumoniae, E. coli and S. marcescens isolates, both with and without carbapenemases. The 42 K. pneumoniae isolates belonged to 12 different sequence types (ST), with ST11 being the most common, while the 6 E. cloacae isolates comprised 4 different STs. The 10 S. marcescens all shared the same PFGE pulsotype, suggestive of clonal spread. Complete plasmid sequencing and PCR screening revealed both intra-strain and inter-species spread of a common blaKPC-2-harboring plasmid in our hospital. Taken together, our study revealed extensive genetic diversity among CRE isolates form a single Chinese hospital. CRE isolates circulating in the hospital differ significantly in their species, STs, porin genes, carbapenemase genes, and their plasmid content, highlighting the complex dissemination of CRE in this endemic region.

The purpose of this study was to identify fecal bacterial microbiome changes in patients with chronic human immunodeficiency virus (HIV) infection in China. Bacterial 16S rRNA genes were amplified, sequenced (454 pyrosequencing), and clustered into operational taxonomic units using the QIIME software. Relative abundance at the phylum and genus levels were calculated. Alpha diversity was determined by Chao 1 and observed-species indices, and beta diversity was determined by double principal component analysis using the estimated phylogeny-based unweighted Unifrac distance matrices. Fecal samples of the patients with chronic HIV-infection tended to be enriched with bacteria of the phyla Firmicutes (47.20%±0.43 relative abundance) and Proteobacteria (37.21%±0.36) compared with those of the non-HIV infected controls (17.95%±0.06 and 3.81%±0.02, respectively). Members of the genus Bilophila were exclusively detected in samples of the non-HIV infected controls. Bacteroides and arabacteroides were more abundant in the chronic HIV-infected patients. Our study indicated that chronic HIV-infected patients in China have a fecal bacterial microbiome composition that is largely different from that found in non-HIV infected controls, and further study is needed to evaluate whether microbiome changes play a role in disease complications in the distal gut, including opportunistic infections.Emerging Microbes & Infections (2016) 5, e31; doi:10.1038/emi.2016.25; published online 6 April 2016Chinese patients with chronic HIV exhibit clear differences in their gut microbe populations compared with healthy controls. Infections and illness, along with the drugs used to treat them, can change the natural microbial ecosystem in the gut, which varies not just from person to person but also between ethnic groups. To investigate gut microbe diversity in the Chinese population, Hong-zhou Lu at Fudan University, Shanghai, together with Ben Li at Shanghai TargetDrug Ltd and co-workers, conducted a small-scale genetic sequencing study of microbial diversity in the feces of 13 patients with HIV and four healthy controls. The HIV-infected individuals exhibited significant differences compared with the controls, including reduced microbial diversity and higher levels of certain bacteria. Further investigations could clarify the role of this diversity in disease complications, and enhance understanding of differences between ethnic groups.

In the pediatric population, respiratory infections are the most common cause of physician visits. Although many respiratory illnesses are self-limiting viral infections that resolve with time and supportive care, it can be critical to identify the causative pathogen at an early stage of the disease in order to implement effective antimicrobial therapy and infection control. Over the last few years, diagnostics for respiratory infections have evolved substantially, with the development of novel assays and the availability of updated tests for newer strains of pathogens. Newer laboratory methods are rapid, highly sensitive and specific, and are gradually replacing the conventional gold standards, although the clinical utility of these assays is still under evaluation. This article reviews the current laboratory methods available for testing for respiratory pathogens and discusses the advantages and disadvantages of each approach.

OBJECTIVE:Necrotizing enterocolitis (NEC) is the most common surgical emergency in preterm infants, and pathogenesis associates with changes in the fecal microbiome. As fecal samples incompletely represent microbial communities in intestinal mucosa, we sought to determine the NEC tissue-specific microbiome and assess its contribution to pathogenesis. DESIGN:We amplified and sequenced the V1-V3 hypervariable region of the bacterial 16S rRNA gene extracted from intestinal tissue and corresponding fecal samples from 12 surgical patients with NEC and 14 surgical patients without NEC. Low quality and non-bacterial sequences were removed, and taxonomic assignment was made with the Ribosomal Database Project. Operational taxonomic units were clustered at 97%. We tested for differences between NEC and non-NEC samples in microbiome alpha- and beta-diversity and differential abundance of specific taxa between NEC and non-NEC samples. Additional analyses were performed to assess the contribution of other demographic and environmental confounding factors on the infant tissue and fecal microbiome. RESULTS:The fecal and tissue microbial communities were different. NEC was associated with a distinct microbiome, which was characterized by low diversity, higher abundances of Staphylococcus and Clostridium_sensu_stricto, and lower abundances of Actinomyces and Corynebacterium. Infant age and vancomycin exposure correlated with shifts in the tissue microbiome. CONCLUSION:The observed low diversity in NEC tissues suggests that NEC is associated with a bacterial bloom and a distinct mucosal bacterial community. The exact bacterial species that constitute the bloom varied by infant and were strongly influenced by age and exposure to vancomycin.

Macrolide-resistant Mycoplasma pneumoniae (MRMP) is highly prevalent in Asia and is now being reported from Europe. Few data on MRMP are available in the United States. Using genotypic and phenotypic methods, we detected high-level MRMP in 13.2% of 91 M. pneumoniae­–positive specimens from 6 US locations.

Prompt detection and identification of fungal pathogens at the genus and species level is critical in order to provide timely antifungal therapy. Here, we highlight the vital role of molecular diagnostics in achieving a fast and definitive diagnosis of disseminated blastomycosis in a diabetic patient presenting as a brain mass initially thought to be tumoral in nature. A broad-range PCR amplification and sequencing of the fungal ribosomal RNA genes on brain biopsy tissue obtained during elective craniotomy revealed a final microbial identification of Ajellomyces dermatitidis (telemorph of Blastomyces dermatitidis).

ABSTRACT Previous work has shown that environmental and clinical isolates of Aspergillus fumigatus represent a diverse population that occupies a variety of niches, has extensive genetic diversity, and exhibits virulence heterogeneity in a number of animal models of invasive pulmonary aspergillosis (IPA). However, mechanisms explaining differences in virulence among A. fumigatus isolates remain enigmatic. Here, we report a significant difference in virulence of two common lab strains, CEA10 and AF293, in the murine triamcinolone immunosuppression model of IPA, in which we previously identified severe low oxygen microenvironments surrounding fungal lesions. Therefore, we hypothesize that the ability to thrive within these lesions of low oxygen promotes virulence of A. fumigatus in this model. To test this hypothesis, we performed in vitro fitness and in vivo virulence analyses in the triamcinolone murine model of IPA with 14 environmental and clinical isolates of A. fumigatus. Among these isolates, we observed a strong correlation between fitness in low oxygen in vitro and virulence. In further support of our hypothesis, experimental evolution of AF293, a strain that exhibits reduced fitness in low oxygen and reduced virulence in the triamcinolone model of IPA, results in a strain (EVOL20) that has increased hypoxia fitness and a corresponding increase in virulence. Thus, the ability to thrive in low oxygen correlates with virulence of A. fumigatus isolates in the context of steroid-mediated murine immunosuppression. IMPORTANCE Aspergillus fumigatus occupies multiple environmental niches, likely contributing to the genotypic and phenotypic heterogeneity among isolates. Despite reports of virulence heterogeneity, pathogenesis studies often utilize a single strain for the identification and characterization of virulence and immunity factors. Here, we describe significant variation between A. fumigatus isolates in hypoxia fitness and virulence, highlighting the advantage of including multiple strains in future studies. We also illustrate that hypoxia fitness correlates strongly with increased virulence exclusively in the nonleukopenic murine triamcinolone immunosuppression model of IPA. Through an experimental evolution experiment, we observe that chronic hypoxia exposure results in increased virulence of A. fumigatus. We describe here the first observation of a model-specific virulence phenotype correlative with in vitro fitness in hypoxia and pave the way for identification of hypoxia-mediated mechanisms of virulence in the fungal pathogen A. fumigatus.

ABSTRACT Bloodstream infections are a leading cause of morbidity and mortality. Early and targeted antimicrobial intervention is lifesaving, yet current diagnostic approaches fail to provide actionable information within a clinically viable time frame due to their reliance on blood culturing. Here, we present a novel pathogen identification (PID) platform that features the use of duplex DNA-invading γ-modified peptide nucleic acids (γPNAs) for the rapid identification of bacterial and fungal pathogens directly from blood, without culturing. The PID platform provides species-level information in under 2.5 hours while reaching single-CFU-per-milliliter sensitivity across the entire 21-pathogen panel. The clinical utility of the PID platform was demonstrated through assessment of 61 clinical specimens, which showed >95% sensitivity and >90% overall correlation to blood culture findings. This rapid γPNA-based platform promises to improve patient care by enabling the administration of a targeted first-line antimicrobial intervention. IMPORTANCE Bloodstream infections continue to be a major cause of death for hospitalized patients, despite significant improvements in both the availability of treatment options as well their application. Since early and targeted antimicrobial intervention is one of the prime determinants of patient outcome, the rapid identification of the pathogen can be lifesaving. Unfortunately, current diagnostic approaches for identifying these infections all rely on time-consuming blood culture, which precludes immediate intervention with a targeted antimicrobial. To address this, we have developed and characterized a new and comprehensive methodology, from patient specimen to result, for the rapid identification of both bacterial and fungal pathogens without the need for culturing. We anticipate broad interest in our work, given the novelty of our technical approach combined with an immense unmet need.

Substantive and significant advances have been made in the last two decades in the characterization of human immunodeficiency virus (HIV) infections using molecular techniques. These advances include the use of real-time measurements, isothermal amplification, the inclusion of internal quality assurance protocols, device miniaturization and the automation of specimen processing. The result has been a significant increase in the availability of results to a high level of accuracy and quality. Molecular assays are currently widely used for diagnostics, antiretroviral monitoring and drug resistance characterization in developed countries. Simple and cost-effective point-of-care versions are also being vigorously developed with the eventual goal of providing timely healthcare services to patients residing in remote areas and those in resource-constrained countries. In this review, we discuss the evolution of these molecular technologies, not only in the context of the virus, but also in the context of tests focused on human genomics and transcriptomics.

Plasmodium vivax is a common cause of imported malaria in the USA, second only to P. falciparum. We present a case of P. vivax malaria in a child returning from India. P. vivax was initially diagnosed by standard methodology and detected retrospectively by use of broad-range PCR and electrospray ionization mass spectrometry using a panel of primers designed to detect vector-borne pathogens. This is the first reported case of P. vivax detection using PCR and electrospray ionization mass spectrometry.Emerging Microbes & Infections (2012) 1, e48; doi:10.1038/emi.2012.49

Next generation sequencing (NGS) has supplanted traditional methods for microbiome analyses. Advantages include higher resolution and lower costs. Analyses have evolved from basic 16S rRNA surveys to metagenomic shotgun sequencing analyses. Limitations and obstacles still exist, e.g. 16S bias, bioinformatics bottlenecks, classification accuracies, and genome assembly using short reads. New tools are being developed to improve output and deal with larger amounts of data generated.

The use of molecular methods to diagnose Clostridium difficile infection (CDI) has improved diagnostic yield compared to conventional methods. However, PCR testing can detect colonization and has introduced several practical challenges pertaining to need for treatment and isolation of cases.For all new cases detected by real-time PCR, concurrent cytotoxin assay was performed and genetic characterization with MLVA (multi-locus variable number tandem repeat analysis) was done to determine relatedness. We used PCR cycle threshold (Ct) of detection as surrogate marker for bacterial burden in stool.Overall, 54 cases of CDI were detected during the study period. 42 were concurrently tested by CYT and characterized by MLVA .MLVA analysis revealed marked genetic diversity with no ongoing outbreaks; four cases were due to NAP1 strain. CYT -/PCR + cases had a higher median Ct value of detection compared to CYT+/PCR + cases (28.2 vs 22.5; p = 0.01). Among 25 strains that were genetically related, 9/11 isolates in this dominant cluster were positive by CYT compared to 4/14 in non-dominant clusters (p = 0.02).CYT-/PCR+ cases contribute to hospital based transmission. However, the risk of transmission of C. difficile from CYT +/PCR+ cases may be higher than those that are CYT-/PCR+.