Viral and Bacterial Etiology of Acute Febrile Respiratory Syndrome among Patients in Qinghai, China.

OBJECTIVE: This study was conducted to investigate the viral and bacterial etiology and epidemiology of patients with acute febrile respiratory syndrome (AFRS) in Qinghai using a commercial routine multiplex-ligation-nucleic acid amplification test (NAT)-based assay. METHODS: A total of 445 nasopharyngeal swabs specimens from patients with AFRS were analyzed using the RespiFinderSmart22kit (PathoFinder BV, Netherlands) and the LightCycler 480 real-time PCR system. RESULTS: Among the 225 (225/445, 51%) positive specimens, 329 positive pathogens were detected, including 298 (90.58%) viruses and 31 (9%) bacteria. The most commonly detected pathogens were influenza virus (IFV; 37.39%; 123/329), adenovirus (AdV; 17.02%; 56/329), human coronaviruses (HCoVs; 10.94%; 36/329), rhinovirus/enterovirus (RV/EV; 10.03%; 33/329), parainfluenza viruses (PIVs; 8.51%; 28/329), and Mycoplasma pneumoniae (M. pneu; 8.51%; 28/329), respectively. Among the co-infected cases (17.53%; 78/445), IFV/AdV and IFV/M. pneu were the most common co-infections. Most of the respiratory viruses were detected in summer and fall. CONCLUSION: In our study, IFV-A was the most common respiratory pathogen among 22 detected pathogens, followed by AdV, HCoV, RV/EV, PIV, and M. pneu. Bacteria appeared less frequently than viruses, and co-infection was the most common phenomenon among viral pathogens. Pathogens were distributed among different age groups and respiratory viruses were generally active in July, September, and November. Enhanced surveillance and early detection can be useful in the diagnosis, treatment, and prevention of AFRS, as well as for guiding the development of appropriate public health strategies.

Detection of six common human paramyxoviruses in patients with acute febrile respiratory symptoms using a novel multiplex real-time RT-PCR assay.

Human metapneumovirus (hMPV), respiratory syncytial virus type A (RSV-A), RSV-B, and human parainfluenza viruses 1, 2, and 3 (HPIV-1, HPIV-2, and HPIV-3) are common respiratory paramyxoviruses. Here, we developed a two-tube triplex one-step real-time reverse-transcription polymerase chain reaction (real-time RT-PCR) and evaluated its performance using clinical samples. The data showed that this novel assay was 100% consistent with the monoplex real-time RT-PCR assay (in-house), which was superior to the commercial routine multiplex-ligation-NAT-based assay. Meanwhile, the clinical nasopharyngeal swabs of 471 patients with the acute febrile respiratory syndrome (AFRS) were analyzed using the established method. The results showed that 52 (11.7%) cases were positive for paramyxovirus. Among them, HPIVs and RSV-A had the highest detection rate. The age and seasonal distribution of human paramyxovirus infection were analyzed. In conclusion, we developed a novel multiplex real-time RT-PCR assay for the rapid detection of six common human paramyxoviruses, which were dominant in patients with AFRS in Qinghai.

Detection and Identification of Six Foodborne Bacteria by Two-tube Multiplex Real Time PCR and Melting Curve Analysis.

OBJECTIVE: This study is aimed to develop a two-tube melting curve-based multiplex real time PCR assay (MCMRT-PCR) for the simultaneous detection of six common foodborne pathogenic bacteria (diarrhoeagenic Escherichia coli, Salmonella, and Shigella in tube 1, Staphylococcus aureus, Vibrio parahaemolyticus, and Listeria monocytogenes in tube 2). METHODS: A two-tube MCMRT-PCR assay was performed on 7900HT Fast Real-Time PCR System (Applied Biosystems, USA). Amplification by PCR was optimized to obtain high efficiency. The sensitivity and specificity of assays were investigated. RESULTS: The detection limit of optimized MCMRT-PCR assay was 3.9×102 CFU/mL for S. aureus, 4.4×102 CFU/mL for L. monocytogenes, 3.0×102 CFU/mL for Salmonella, 2.5×102 CFU/mL for Shigella, 2.1×102 CFU/mL for V. parahaemolyticus, and 1.2×102 CFU/mL for E. coli. The feasibility of MCMRT-PCR was further evaluated using artificially contaminated milk, the sensitivity was at the level of 105 CFU/mL. CONCLUSION: A two-tube MCMRT-PCR assay using six primer sets was developed for detection of multiple pathogens. Our findings demonstrates that the proposed two-tube assay is reliable, useful and rapid for simultaneous detection of six foodborne pathogenic bacteria with an intended application in provincial Centers for Diseases Control and Prevention (CDC).

[Establishment and primary application of a novel resequencing pathogen microarray-based assay for detecting pathogens in patients with unexplained diarrhea].

In this study, a novel resequencing pathogen microarray (RPM)-based multi-pathogen detection assay was developed to simultaneously detect 14 rotaviruses, 7 caliciviruses, 8 astroviruses, 28 enteroviruses, and 16 rare diarrhea viruses in patients with diarrhea syndrome. The specificity of the assay was examined using confirmed virus-positive specimens, and the sensitivity was evaluated by serial ten-fold dilutions of in vitro transcribed RNA. RPM assay could detect and differentiate virus types/subtypes at 20-2000 copies/microL. The detection threshold of RPM was determined by adjusting the reference concentration, and the detection steps were optimized to type Enterovirus. The nucleic acids of 10 stool samples from patients with unexplained diarrhea were screened, and 6 of them showed positive results. The RPM results were further verified by singleplex PCR followed by sequencing, and no difference was found between the two assays. In conclusion, we have established a high-throughput RPM assay with high specificity and sensitivity, which demonstrates a great potential for the identification of pathogens in patients with unexplained diarrhea and the management of emerging epidemic.