Etiological and epidemiological features of acute meningitis or encephalitis in China: a nationwide active surveillance study.

BACKGROUND: Acute meningitis or encephalitis (AME) results from a neurological infection causing high case fatality and severe sequelae. AME lacked comprehensive surveillance in China. METHODS: Nation-wide surveillance of all-age patients with AME syndromes was conducted in 144 sentinel hospitals of 29 provinces in China. Eleven AME-causative viral and bacterial pathogens were tested with multiple diagnostic methods. FINDINGS: Between 2009 and 2018, 20,454 AME patients were recruited for tests. Based on 9,079 patients with all-four-virus tested, 28.43% (95% CI: 27.50%‒29.36%) of them had at least one virus-positive detection. Enterovirus was the most frequently determined virus in children <18 years, herpes simplex virus and Japanese encephalitis virus were the most frequently determined in 18-59 and ≥60 years age groups, respectively. Based on 6,802 patients with all-seven-bacteria tested, 4.43% (95% CI: 3.94%‒4.91%) had at least one bacteria-positive detection, Streptococcus pneumoniae and Neisseria meningitidis were the leading bacterium in children aged <5 years and 5-17 years, respectively. Staphylococcus aureus was the most frequently detected in adults aged 18-59 and ≥60 years. The pathogen spectrum also differed statistically significantly between northern and southern China. Joinpoint analysis revealed age-specific positive rates, with enterovirus, herpes simplex virus and mumps virus peaking at 3-6 years old, while Japanese encephalitis virus peaked in the ≥60 years old. As age increased, the positive rate for Streptococcus pneumoniae and Escherichia coli statistically significantly decreased, while for Staphylococcus aureus and Streptococcus suis it increased. INTERPRETATION: The current findings allow enhanced identification of the predominant AME-related pathogen candidates for diagnosis in clinical practice and more targeted application of prevention and control measures in China, and a possible reassessment of vaccination strategy. FUNDING: China Mega-Project on Infectious Disease Prevention and the National Natural Science Funds.

Resistance of poliovirus 1 and enterovirus A71 against alcohol and other disinfectants.

Poliovirus 1 (PV 1) is the standard virus used in tests to support claims of virucidal property in commercial hand sanitizers and disinfectants in China. Classified within the same genus as poliovirus, enterovirus A71 (EV A71), which causes hand-foot-mouth disease among children, has caused numerous outbreaks in China and other countries. Hand hygiene and surface cleaning are critical to prevent and control this disease and many other infectious diseases. This study compared the efficacies of 17 self-made alcohol-based hand sanitizers and 10 commercially available disinfectants (4 high-level, 4 intermediate-level, 2 low-level) against these two viruses. The results showed that by itself, ethanol needed to reach a concentration of 75 % to meet the inactivation requirement of 4-log reduction in average TCID50 against PV 1. Nine out of 13 laboratory-formulated alcohol-based hand sanitizers reached the 4-log inactivation requirement against PV 1 after 4.5 min, while the remaining four sanitizers did not. Unexpectedly, none of the tested ethanol-based sanitizers inactivated EV A71 by 4-log. For the commercially available disinfectants, all four high-level and one intermediate-level disinfectants passed the inactivation requirements against both PV 1 and EV A71, while two intermediate-level disinfectants met the inactivation requirement against PV 1 but failed against EV A71. The last intermediate-level and both low-level disinfectants did not meet the requirement for either PV 1 or EV A71. Therefore, PV 1 is more susceptible to inactivation by many common alcohol-based and non-alcohol-based disinfectants than EV A71. Therefore, the adoption of EV A71 as the standard test virus would elevate the disinfectant requirement standard and provide better protection for the public. Based on these results, seven new alcohol-based hand sanitizer recipes were formulated and found to be effective against both PV 1 and EV A71, with two candidates reaching the required 4-log virus reduction efficacy within 1 min.

Etiological and epidemiological features of acute respiratory infections in China.

Nationwide prospective surveillance of all-age patients with acute respiratory infections was conducted in China between 2009‒2019. Here we report the etiological and epidemiological features of the 231,107 eligible patients enrolled in this analysis. Children <5 years old and school-age children have the highest viral positivity rate (46.9%) and bacterial positivity rate (30.9%). Influenza virus, respiratory syncytial virus and human rhinovirus are the three leading viral pathogens with proportions of 28.5%, 16.8% and 16.7%, and Streptococcus pneumoniae, Mycoplasma pneumoniae and Klebsiella pneumoniae are the three leading bacterial pathogens (29.9%, 18.6% and 15.8%). Negative interactions between viruses and positive interactions between viral and bacterial pathogens are common. A Join-Point analysis reveals the age-specific positivity rate and how this varied for individual pathogens. These data indicate that differential priorities for diagnosis, prevention and control should be highlighted in terms of acute respiratory tract infection patients' demography, geographic locations and season of illness in China.

Etiological, epidemiological, and clinical features of acute diarrhea in China.

National-based prospective surveillance of all-age patients with acute diarrhea was conducted in China between 2009‒2018. Here we report the etiological, epidemiological, and clinical features of the 152,792 eligible patients enrolled in this analysis. Rotavirus A and norovirus are the two leading viral pathogens detected in the patients, followed by adenovirus and astrovirus. Diarrheagenic Escherichia coli and nontyphoidal Salmonella are the two leading bacterial pathogens, followed by Shigella and Vibrio parahaemolyticus. Patients aged <5 years had higher overall positive rate of viral pathogens, while bacterial pathogens were more common in patients aged 18‒45 years. A joinpoint analysis revealed the age-specific positivity rate and how this varied for individual pathogens. Our findings fill crucial gaps of how the distributions of enteropathogens change across China in patients with diarrhea. This allows enhanced identification of the predominant diarrheal pathogen candidates for diagnosis in clinical practice and more targeted application of prevention and control measures.

Development and Evaluation of a Loop-Mediated Isothermal Amplification Assay for Rapid and Specific Identification of Carbapenem-Resistant Acinetobacter baumannii Strains Harboring blaOXA-23, and the Epidemiological Survey of Clinical Isolates.

Acinetobacter baumannii is an important nosocomial pathogen in hospital-acquired infections, and carbapenem resistance has been increasingly observed worldwide. Oxacillinase production by blaOXA-23 is a predominant and prevalent carbapenem resistance mechanism of A. baumannii, especially in China. Rapid and specific detection of blaOXA-23 may offer valuable insight for administration of directed antimicrobial therapy. In this study, we aimed to develop a loop-mediated isothermal amplification (LAMP)-based method for identifying carbapenem-resistant A. baumannii (CRAB) harboring the blaOXA-23 gene. High-specificity primers for screening blaOXA-23 were designed and synthesized, and the LAMP reactions were performed. Clinical A. baumannii strains isolated from the Former 307th Hospital of People's Liberation Army were used to determine the sensitivity and specificity of this method compared with those of phenotypic antimicrobial susceptibility testing and the traditional PCR method. Multilocus sequence typing (MLST) was performed to investigate the epidemiology of the A. baumannii bacterial population. Compared with antimicrobial susceptibility testing, the sensitivity and specificity of LAMP in detecting blaOXA-23 were 88.4% and 97.7%, respectively. However, the LAMP method is much simpler and less time-consuming (within 60 minutes) than conventional PCR and phenotypic susceptibility testing. The 113 isolates could be clustered into 30 sequence types, and most strains (83/113) belonged to clonal complex (CC) 92, which is also the dominant CC in China. The LAMP-based method detected blaOXA-23 in a simpler manner and could provide rapid results for identifying CRAB. Consequently, blaOXA-23 may serve as a surrogate marker for the presence of CRAB in patients with serious infections in clinical practice.

Microwave-assisted, rapid synthesis of 2-vinylquinolines and evaluation of their antimalarial activity.

A rapid and efficient synthesis of 2-vinylquinolines via trifluoromethanesulfonamidemediated olefination of 2-methylquinoline and aldehyde under microwave irradiation is reported. Biological evaluation of these scaffolds demonstrates that 2-vinylquinolines 3x - 3z possess excellent antimalarial activities against chloroquine-resistant Dd2 strain of Plasmodium falciparum (IC50 < 100 nM).

Establishment of the molecular beacon-loop-mediated isothermal amplification method for the rapid detection of Porphyromonas gingivalis.

Porphyromonas gingivalis, a clinically important oral pathogen causing periodontal disease, is difficult to culture in routine conditions. Hence, it is necessary to establish a reliable technique to detect this pathogen. Previously, our laboratory developed a new isothermal detection method, called MB-LAMP (molecular beacon-Loop-mediated isothermal amplification), which combines the advantages of LAMP and qPCR through the accurate and quantitative detection of LAMP products. This approach offers significant potential for the point-of-care detection of P. gingivalis. Here, MB-LAMP was used to detect P. gingivalis targeting a specific fragment, and the sensitivity was as high as 1.4 × 10-1 pg µL-1. The method showed no cross-reaction with 14 other bacterial pathogens. For clinical samples, this assay showed a high diagnostic sensitivity (100%) and specificity (100%), equivalent to that of real-time quantitative polymerase chain reaction (real-time qPCR). Moreover, detection with MB-LAMP was significantly faster than that with real-time qPCR, reducing the time required for clinical diagnosis. Finally, we established an absolute quantification method with MB-LAMP for P. gingivalis using pilot samples. Thus, the highly specific, sensitive, and rapid assay developed in this study makes it feasible to diagnose P. gingivalis.

Elevated MIC Values of Imidazole Drugs against Aspergillus fumigatus Isolates with TR34/L98H/S297T/F495I Mutation.

The use of azole fungicides in agriculture is believed to be one of the main reasons for the emergence of azole resistance in Aspergillus fumigatus Though widely used in agriculture, imidazole fungicides have not been linked to resistance in A. fumigatus This study showed that elevated MIC values of imidazole drugs were observed against A. fumigatus isolates with TR34/L98H/S297T/F495I mutation, but not among isolates with TR34/L98H mutation. Short-tandem-repeat (STR) typing analysis of 580 A. fumigatus isolates from 20 countries suggested that the majority of TR34/L98H/S297T/F495I strains from China were genetically different from the predominant major clade comprising most of the azole-resistant strains and the strains with the same mutation from the Netherlands and Denmark. Alignments of sterol 14α-demethylase sequences suggested that F495I in A. fumigatus was orthologous to F506I in Penicillium digitatum and F489L in Pyrenophora teres, which have been reported to be associated with imidazole resistance. In vitro antifungal susceptibility testing of different recombinants with cyp51A mutations further confirmed the association of the F495I mutation with imidazole resistance. In conclusion, this study suggested that environmental use of imidazole fungicides might confer selection pressure for the emergence of azole resistance in A. fumigatus.

Identification and Characterization of Key Charged Residues in the Cofilin Protein Involved in Azole Susceptibility, Apoptosis, and Virulence of Aspergillus fumigatus.

Through some specific amino acid residues, cofilin, a ubiquitous actin depolymerization factor, can significantly affect mitochondrial function related to drug resistance and apoptosis in Saccharomyces cerevisiae; however, this modulation in a major fungal pathogen, Aspergillus fumigatus, was still unclear. Hereby, it was found, first, that mutations on several charged residues in cofilin to alanine, D19A-R21A, E48A, and K36A, increased the formation of reactive oxygen species and induced apoptosis along with typical hallmarks, including mitochondrial membrane potential depolarization, cytochrome c release, upregulation of metacaspases, and DNA cleavage, in A. fumigatus Two of these mutations (D19A-R21A and K36A) increased acetyl coenzyme A and ATP concentrations by triggering fatty acid ß-oxidation. The upregulated acetyl coenzyme A affected the ergosterol biosynthetic pathway, leading to overexpression of cyp51A and -B, while excess ATP fueled ATP-binding cassette transporters. Besides, both of these mutations reduced the susceptibility of A. fumigatus to azole drugs and enhanced the virulence of A. fumigatus in a Galleria mellonella infection model. Taken together, novel and key charged residues in cofilin were identified to be essential modules regulating the mitochondrial function involved in azole susceptibility, apoptosis, and virulence of A. fumigatus.

Development and application of a rapid Mycobacterium tuberculosis detection technique using polymerase spiral reaction.

Mycobacterium tuberculosis is an age-old bacterium that is difficult to eliminate. A simple and rapid diagnostic method is of great importance to prevent the spread of M. tuberculosis. Here, we developed a low-cost rapid M. tuberculosis nucleic acid detection technique, named GenePop, which enabled the storage and transport of M. tuberculosis diagnostic reagent at ambient temperatures, without the need for professional operations or expensive instrumentation. Using a vitrification method, we vitrified heat-unstable components onto the cap of a reaction tube, and placed heat-stable components at the bottom of the reaction tube by sealing them with paraffin wax. The all-in-one detection tube, when used together with our other invention-a multi-functional sample treatment tube pre-filled with a nucleic acid-releasing agent-only required three simple steps to yield results. A comparative analysis with a commercial qPCR kit for M. tuberculosis indicated that our new technique had a concordance rate of 91.6%, showing no cross-reactivity with 11 other bacteria. The complete operation time was only 65 min. It is suitable for use in field settings or by personnel in grass-root units, and is applicable in household activities, hence can be used in developing countries.

Sensitive and Rapid Detection of the Plasmid-Encoded Colistin-Resistance Gene mcr-1 in Enterobacteriaceae Isolates by Loop-Mediated Isothermal Amplification.

The emergence of the plasmid-encoded colistin-resistance gene mcr-1 in Enterobacteriaceae represents a new threat to the treatment of infection in the clinical setting. A sensitive and rapid molecular method for detection of the mcr-1 gene in clinical isolates is needed to control the spread of this gene. In this study, we established a loop-mediated isothermal amplification (LAMP) assay for rapid detection of the mcr-1 gene. This assay was applied to cultured bacteria and spiked human stools. Real-time monitoring of turbidity and chromogenic visualization were used to assess the reaction results. The specificity and sensitivity of the primers in the LAMP reactions for detection of the mcr-1 gene were determined. All 20 clinically resistant isolates without the mcr-1 gene tested negative, indicating the high specificity of the LAMP primers. The sensitivity of LAMP, with a detection limit of 0.2 pg/µL DNA, was 10-fold greater than that of polymerase chain reaction (PCR). The assay was also conclusive when applied to human stools spiked with mcr-1-positive Escherichia coli. During clinical screening in a major hospital in Beijing, China, seven isolates were identified as positive from the 556 Enterobacteriaceae isolates. In conclusion, the LAMP assay we developed was useful for detection of the mcr-1 gene in the clinical setting.

Predicting nosocomial lower respiratory tract infections by a risk index based system.

Although belonging to one of the most common type of nosocomial infection, there was currently no simple prediction model for lower respiratory tract infections (LRTIs). This study aims to develop a risk index based system for predicting nosocomial LRTIs based on data from a large point-prevalence survey. Among the 49328 patients included, the prevalence of nosocomial LRTIs was 1.70% (95% confidence interval [CI], 1.64% to 1.76%). The areas under the receiver operating characteristic (ROC) curve for logistic regression and fisher discriminant analysis were 0.907 (95% CI, 0.897 to 0.917) and 0.902 (95% CI, 0.892 to 0.912), respectively. The constructed risk index based system also displayed excellent discrimination (area under the ROC curve: 0.905 [95% CI, 0.895 to 0.915]) to identify LRTI in internal validation. Six risk levels were generated according to the risk score distribution of study population, ranging from 0 to 5, the corresponding prevalence of nosocomial LRTIs were 0.00%, 0.39%, 3.86%, 12.38%, 28.79% and 44.83%, respectively. The sensitivity and specificity of prediction were 0.87 and 0.79, respectively, when the best cut-off point of risk score was set to 14. Our study suggested that this newly constructed risk index based system might be applied to boost more rational infection control programs in clinical settings.

Complete sequences of two novel bla NDM-1-harbouring plasmids from two Acinetobacter towneri isolates in China associated with the acquisition of Tn125.

Two novel New Delhi metallo-ß-lactamase-1 (NDM-1)-positive plasmids containing a complete composite transposon, Tn125, from two respective Acinetobacter towneri isolates were characterized. Plasmid pNDM-GJ01 (30,293 bp) isolated from A. towneri G165 did not show homology to any known plasmid structure, except for the transposon Tn125 containing bla NDM-1. A novel repB gene and two XRE-type transcriptional regulators were found in pNDM-GJ01. Plasmid pNDM-GJ02 (62,011 bp) isolated from A. towneri G295 showed the highest homology to pBJAB0715 (41% coverage, 99% nucleotide identity). In addition to the bla NDM-1-harbouring transposon Tn125, pNDM-GJ02 also had an IS26-composite transposon, which contains ISCR1 and two class 1 integrons carrying different cassette arrays. Both clinical isolates were highly resistant to ß-lactams and susceptible to tigecycline and colistin. Ten other resistance genes were detected in G295, and one other resistance gene was detected in G165. No transconjugant was obtained from any of the donors by broth and filter mating. The emergence of these two novel plasmids carrying NDM-1 in Acinetobacter spp., pNDM-GJ01 and pNDM-GJ02, suggests Tn125 mobile integration.

Assessing antibiotic therapy effectiveness against the major bacterial pathogens in a hospital using an integrated index.

AIM: To assess the effectiveness of antibiotic therapy against five indicator bacteria in a Chinese hospital using an index-based approach. METHODS: The study population comprises 1031 patients who had one clinically significant bacterial isolate in 2008, 2010 and 2013. Drug resistance index (DRI) based on pathogens was calculated. RESULTS: The adaptive DRIs for Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus decreased, while both adaptive and fixed DRIs for Acinetobacter spp. increased from 2008 to 2013. The adaptive DRIs for Escherichia coli increased from 2008 to 2013, while the fixed DRIs exhibited a decreasing trend. CONCLUSION: DRI could be used to demonstrate the changes of antimicrobial resistance and prescribing over time as a result of evolutionary processes and governmental regulatory interference.

Combined interventions for mitigation of an influenza A (H1N1) 2009 outbreak in a physical training camp in Beijing, China.

OBJECTIVES: Many studies have suggested the effectiveness of single control measures in the containment and mitigation of pandemic influenza A (H1N1) 2009. The effects of combined interventions by multiple control measures in reducing the impact of an influenza A (H1N1) 2009 outbreak in a closed physical training camp in Beijing, China were evaluated. METHODS: Oseltamivir was prescribed for the treatment of confirmed cases and possible cases and as prophylaxis for all other participants in this training camp. Public health control measures were applied simultaneously, including the isolation of patients and possible cases, personal protection and hygiene, and social distancing measures. Symptom surveillance of all participants was initiated, and the actual attack rate was calculated. For comparison, the theoretical attack rate for this outbreak was projected using the Newton-Raphson numerical method. RESULTS: A total of 3256 persons were present at the physical training camp. During the outbreak, 405 (68.3%) possible cases and 26 (4.4%) confirmed cases were reported before the intervention and completed oseltamivir treatment; 162 (27.3%) possible cases were reported after the intervention and received part treatment and part prophylaxis. The other 2663 participants completed oseltamivir prophylaxis. Of the possible cases, 181 with fever ≥38.5°C were isolated. The actual attack rate for this outbreak of pandemic influenza A (H1N1) 2009 was 18.2%, which is much lower than the theoretical attack rate of 80% projected. CONCLUSIONS: Combined interventions of large-scale antiviral ring prophylaxis and treatment and public health control measures could be applied to reduce the magnitude of influenza A (H1N1) 2009 outbreaks in closed settings.

Development of a multiplex loop-mediated isothermal amplification method for the simultaneous detection of Salmonella spp. and Vibrio parahaemolyticus.

Rapid detection of food-borne pathogens is important in the food industry, to monitor and prevent the spread of these pathogens through contaminated food products. We therefore established a multiplex real-time loop-mediated isothermal amplification (LAMP) assay to simultaneously detect and distinguish Salmonella spp. and Vibrio parahaemolyticus DNA in a single reaction. Two target sequences, one specific for Salmonella and the other specific for Vibrio parahaemolyticus, were amplified by specific LAMP primers in the same reaction tube. After amplification at 65 °C for 60 min, the amplified products were subjected to melting curve analysis and thus could be distinguished based on the different melting temperatures (Tm values) of the two specifically amplified products. The specificity of the multiplex LAMP assay was evaluated using 19 known bacterial strains, including one V. parahaemolyticus and seven Salmonella spp. strains. The multiplex LAMP showed 100% inclusivity and exclusivity, and a detection limit similar to that of multiplex PCR. In addition, we observed and corrected preferential amplification induced by what we call LAMP selection in the multiplex LAMP reaction. In conclusion, our assay was rapid, specific, and quantitative, making it a useful tool for the food industry.

Establishment of an accurate and fast detection method using molecular beacons in loop-mediated isothermal amplification assay.

This study established a constant-temperature fluorescence quantitative detection method, combining loop-mediated isothermal amplification (LAMP) with molecular beacons. The advantages of LAMP are its convenience and efficiency, as it does not require a thermocycler and results are easily visualized by the naked eye. However, a major disadvantage of current LAMP techniques is the use of indirect evaluation methods (e.g., electrophoresis, SYBR Green I dye, precipitation, hydroxynaphthol blue dye, the turbidimetric method, calcein/Mn2+ dye, and the composite probe method), which cannot distinguish between the desired products and products of nonspecific amplification, thereby leading to false positives. Use of molecular beacons avoids this problem because molecular beacons produce fluorescence signals only when binding to target DNA, thus acting as a direct indicator of amplification products. Our analyses determined the optimal conditions for molecular beacons as an evaluation tool in LAMP: beacon length of 25-45 bp, beacon concentration of 0.6-1 pmol/µL, and reaction temperature of 60-65 °C. In conclusion, we validated a novel molecular beacon loop-mediated isothermal amplification method (MB-LAMP), realizing the direct detection of LAMP product.

Metabolic shifts and structural changes in the gut microbiota upon branched-chain amino acid supplementation in middle-aged mice.

The importance of gut microbiota to health has gained extensive attention and is strongly correlated with diet. Dietary supplementation with a branched-chain amino acid-enriched mixture (BCAAem) exerts a variety of beneficial effects in mice and humans. In mice, BCAAem supplementation can promote longevity, but its influence on the gut ecosystem and the underlying mechanism remain unclear. To address this issue, BALB/C mice were fed a BCAAem-supplemented diet and their gut microbiomes were analysed by 16S rDNA sequencing. Quantitative polymerase chain reaction was performed to identify Bifidobacterium spp. in the gut, and gas chromatography-mass spectrometry was conducted for faecal-metabolite detection. The results showed that the structure of the gut microbiota changed, and BCAAem-supplementation in mice slowed the change speed of gut microbiota which is due to age. In addition, the abundance of the Akkermansia and Bifidobacterium increased in BCAAem-supplemented mice, while the ratio of Enterobacteriaceae decreased in BCAAem-supplemented mice. Moreover, 12 different metabolites, representing sugar and lipid metabolism, were altered between the supplemented and control groups. Thus, BCAAem influences the gut microbiota and gut metabolism. In addition, the BCAAem-supplemented group presented lower serum concentrations of lipopolysaccharide-binding protein. The changes are indicative of lower antigen loads in the host gut. These results suggest that dietary supplementation with BCAAem may be considered for improving health and promoting healthy aging.

Rapid Detection of Candida albicans by Polymerase Spiral Reaction Assay in Clinical Blood Samples.

Candida albicans is the most common human yeast pathogen which causes mucosal infections and invasive fungal diseases. Early detection of this pathogen is needed to guide preventative and therapeutic treatment. The aim of this study was to establish a polymerase spiral reaction (PSR) assay that rapidly and accurately detects C. albicans and to assess the clinical applicability of PSR-based diagnostic testing. Internal transcribed spacer 2 (ITS2), a region between 5.8S and 28S fungal ribosomal DNA, was used as the target sequence. Four primers were designed for amplification of ITS2 with the PSR method, which was evaluated using real time turbidity monitoring and visual detection using a pH indicator. Fourteen non-C. albicans yeast strains were negative for detection, which indicated the specificity of PSR assay was 100%. A 10-fold serial dilution of C. albicans genomic DNA was subjected to PSR and conventional polimerase chain reaction (PCR) to compare their sensitivities. The detection limit of PSR was 6.9 pg/µl within 1 h, 10-fold higher than that of PCR (69.0 pg/µl). Blood samples (n = 122) were collected from intensive care unit and hematological patients with proven or suspected C. albicans infection at two hospitals in Beijing, China. Both PSR assay and the culture method were used to analyze the samples. Of the 122 clinical samples, 34 were identified as positive by PSR. The result was consistent with those obtained by the culture method. In conclusion, a novel and effective C. albicans detection assay was developed that has a great potential for clinical screening and point-of-care testing.