Simultaneous Detection of 13 Key Bacterial Respiratory Pathogens by Combination of Multiplex PCR and Capillary Electrophoresis

Objective Lower respiratory tract infections continue to pose a significant threat to human health. It is important to accurately and rapidly detect respiratory bacteria. To compensate for the limits of current respiratory bacteria detection methods, we developed a combination of multiplex polymerase chain reaction （PCR） and capillary electrophoresis （MPCE） assay to detect thirteen bacterial pathogens responsible for lower respiratory tract infections, including Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catorrholis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Mycoplasma pneumoniae, Legionella spp., Bordetella pertussis, Mycobacterium tuberculosis complex, Corynebactefium diphthefiae, and Streptococcus pyogenes. Methods Three multiplex PCR reactions were built, and the products were analyzed by capillary electrophoresis using the high-throughput DNA analyzer. The specificity of the MPCE assay was examined and the detection limit was evaluated using DNA samples from each bacterial strain and the simulative samples of each strain. This assay was further evaluated using 152 clinical specimens and compared with real-time PCR reactions. For this assay, three nested-multiplex-PCRs were used to detect these clinical specimens. Results The detection limits of the MPCE assay for the 13 pathogens were very low and ranged from 10-7 to 10-2 ng/μL. Furthermore, analysis of the 252 clinical specimens yielded a specificity ranging from 96.5%-100.0%, and a sensitivity of 100.0% for the 13 pathogens. Conclusion This study revealed that the MPCE with high specificity and sensitivity. This assay survey of respiratory pathogens. assay is a rapid, reliable, and high-throughput method has great potential in the molecular epidemiological.

Location of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus in Tissues of Natural Cases

[ Objective] The aim of this study was to provide a theoretical basis for the prevention and treatment of highly pathogenic porcine reproductive and respiratory syndrome （HP-PRRS）. [Method] Antigen location and histopathological observation in natural cases infected by highly pathogenic porcine reproductive and respiratory syndrome virus （HP-PRRSV） were analyzed by immunohistochemistry and H. E. staining. [Result] The virus antigen mainly existed in epithelial calls, and also a few in mecrophages, lymphocytes and brain nerve cells. [ Conclusion] The cell and tissue tropism of HP-PRRSV strain in natural cases is different from that of previous strains.

Evaluation of the Pathogenicity of a Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Variant in Piglets

Since May 2006,a highly pathogenic porcine reproductive and respiratory syndrome virus （HP-PRRSV） variant characterized by 30 amino acids deletion within its NSP2-coding region emerged and caused extensive economic losses to China＇s pig industry.To investigate the in vivo pathogenicity and immune responses of the newly emerging PRRSV,3 groups of 60-d-old conventional piglets were inoculated intranasally with a representative strain of the HP-PRRSV variant HuN4 with 3 different infection doses （3×103-3×105 TCID50）.The results revealed that the virus variant caused severe disease in piglets and the significant clinical characteristics consisted of persistently high fever （41.0-41.9oC） and high morbidity and mortality （60-100%）,the marked clinical signs of PRRS and severe histopathogenic damages in multiple organs.It induced rapid and intense humoral immune responses and seroconversion was detected in most infected pigs at 7 d post-infection （DPI）.The virus vigorously replicated in vivo and the highest virus average titer was 9.7 log copies mL-1 serum at 7 DPI.Elevated levels of IFN-g and IL-10 cytokine production in serum in this study were also observed.Taken together,our results demonstrated that the HP-PRRSV variant HuN4 strain is highly pathogenic for piglets and suitable to be a reference strain of highly virulent PRRSV for evaluating the efficacy of the new vaccines.

Analysis of Common Respiratory Infected Pathogens in 3100 Children after the Coronavirus Disease 2019 Pandemic

Objective To investigate the epidemiological features in children after the coronavirus disease 2019(COVID-19)pandemic.Methods This study collected throat swabs and serum samples from hospitalized pediatric patients of Renmin Hospital of Wuhan University,Wuhan,Hubei province,China before and after the COVID-19 pandemic.Respiratory infected pathogens[adenovirus(ADV),influenza virus A/B(Flu A/B),parainfluenza virus 1/2/3(PIV1/2/3),respiratory syncytial virus(RSV),Mycoplasma pneumoniae(MP),and Chlamydia pneumoniae(CP)]were detected.The pathogens,age,and gender were used to analyze the epidemiological features in children after the COVID-19 pandemic.Results The pathogen detection rate was significantly higher in females than in males(P<0.05),and the infection of PIV1 and MP was mainly manifested.After the COVID-19 pandemic,PIV1,PIV3,RSV,and MP had statistically different detection rates among the age groups(P<0.05),and was mainly detected in patients aged 0–6 years,0–3 years,0–3 years,and 1–6 years,respectively.When comparing before the COVID-19 pandemic,the total detection rate of common respiratory pathogens was lower(P<0.05).Except for the increase in the detection rate of PIV1 and CP,the infection rate of other pathogens had almost decreased.Conclusion The prevention and control measures for the COVID-19 pandemic effectively changed the epidemiological features of common respiratory tract infectious diseases in pediatric children.

Prevalence and Seroprevalence of Non-SARS Human Coronaviruses 229E and OC43 in East Tyrol/Austria

The recent SARS-CoV-2 pandemic renewed interest in other previously discovered human coronaviruses—the non-severe acute respiratory syndrome human coronavirus (non-SARS hCoVs) and this study is a starting point for a closer investigation of those. With the pandemic behind us we believe it to be important to also examine the current and past respiratory pathogen landscape in the patient population in our care. Therefore, 954 nasopharyngeal swabs of patients with respiratory diseases collected between October 2018 and March 2020 were tested against the pathogens Mycoplasma pneumoniae, Bordetella pertussis, Influenza A and virus, Human metapneumovirus, respiratory syncytial virus, Parainfluenza virus, human Adenovirus and Polyoma virus BK/JC. Swabs negative against these pathogens were further tested for OC43 and 229E by RT-qPCR. Human coronaviruses 229E and OC43 were proven as the causative agents in a considerable number of cases, confirmed by PCR. Overall, our results show that those two non-SARS hCoVs were responsible for 13.9% (11 of 79) of infections with flu-like symptoms of unknown etiology in the study area. In the subsequent seroprevalence study, it was shown that the seroprevalence rate of IgG antibodies against 229E and OC43 was over 50%, indicating that a big part of the population in our study area has been in contact with these non-SARS-CoVs and has built the specific humoral immune response accordingly.

mNGS-based dynamic pathogen monitoring for accurate diagnosis and treatment of severe pneumonia caused by fungal infections

Metagenomic next-generation sequencing(mNGs)has been widely applied to identify pathogens associated with infectious diseases.However,limited studies have explored the use of mNGs-based dynamic pathogen monitoring in intensive care unit patients with severe pneumonia.Here,we present a clinical case of an 86-year-old male patient with severe pneumonia caused by a fungal infection.During the clinical treatment,four mNGS analyses were performed within two consecutive weeks.Various respiratory fungal pathogens,including Candida orthopsilosis,Candida albicans,and Aspergillus fumigatus were detected by mNGS of bronchoalveolar lavage fluid(BALF).Based on conventional pathogen identification and clinical symptoms,the patient was diagnosed with severe pneumonia caused by a fungal infection.The abundance of fungal species decreased gradually in response to antifungal and empirical therapies,and the fungal infections were effectively con-trolled.In summary,our results demonstrated that mNGS could effectively identify pathogens in patients with severe pneumonia.Additionally,dynamic pathogen monitoring based on mNGS could assist in the precise diag-nosis of complex infections and may facilitate rapid induction of the most appropriate therapy.