Epidemiology and Clinical Characteristics of Seasonal Human Coronaviruses in Children Hospitalized in Hebei Province, China Before and During the COVID-19 Pandemic.

Background: This study aimed to assess the impact of the COVID-19 pandemic on the prevalence and clinical characteristics of seasonal human coronavirus (HCoV) infections among children hospitalized in Hebei, China. Methods: We examined nasopharyngeal aspirate (NPA) specimens for seasonal HCoVs from January 2018 to December 2021, at the Children's Hospital of Hebei Province. We used a GeXP-based multiplex reverse transcription PCR assay for the detection of 11 common respiratory viruses (including seasonal HCoVs), chlamydia, and Mycoplasma pneumoniae. The demographic and clinical characteristics of children who tested positive for seasonal HCoVs were recorded and analyzed. Results: A total of 377 (1.96%) of the 19,248 specimens from 2018 to 2019 and 263 (1.96%) of the 13,426 specimens from 2020 to 2021 exhibited seasonal HCoVs. Compared to 2018 and 2019, the positive rate of seasonal HCoVs was lower from January to July of 2020 and increased beginning in August 2020, peaking in the autumn and winter. In 2020-2021, nasal blockage and swollen adenoids were detected more frequently in children who tested positive for seasonal HCoVs. During 2018-2019, however, the duration of fever was significantly longer, and cough and dyspnea were more prominent among children who had fallen ill. In addition, seasonal HCoV-positive patients in 2018-2019 were more likely to experience complications, had a higher risk of severe community-acquired pneumonia (CAP), and had a tendency to require a longer hospital stay than patients in 2020-2021. Conclusion: According to our findings, there were significant changes in the epidemiology of seasonal HCoVs in Hebei, China during the COVID-19 pandemic, and children infected with seasonal HCoVs usually experienced milder clinical symptoms during the pandemic than before it.

Simultaneous detection of 9 respiratory pathogens using a newly developed multiplex real-time PCR panel based on an automatic molecular detection and analysis system.

Timely identification of respiratory pathogens guides specific treatment, reduces hospital costs and minimizes the excessive use of antibiotics. A new multiplex real-time PCR panel was developed based on an automatic molecular detection and analysis system (AutoMolec system), consisting of three separate internally controlled assays. Mycoplasma pneumoniae, Chlamydia pneumoniae, adenovirus, human metapneumovirus, influenza B virus, respiratory syncytial virus and human parainfluenza virus 1-3 may be directly detected in original samples. The system's clinical performance was evaluated by comparison with an approved commercial kit, using 517 clinical samples. The limit of detection of the AutoMolec mRT-PCR panel ranged from 4 × 10-4 ∼3.3 TCID50/mL and no cross-reaction with common respiratory pathogens was observed. The AutoMolec mRT-PCR panel had 99.09% sensitivity and 100.0% specificity and overall detection consistency was 99.61%, making it comparable to that of the commercial kit. Therefore, the AutoMolec mRT-PCR panel has great potential for routine screening of respiratory infection in China.

Clinical Study of Human Coronavirus NL63, OC43, 229E, HKU1 Infentions in Hospitalized Children from 2015 to 2020.

Objective: This study aims to analyze the clinical characteristics of hospitalized children infected with HCoV-NL63, OC43, 229E, HKU1 and provide the basis for disease diagnosis and treatment. Methods: A retrospective analysis was conducted on clinical manifestations, imaging data, and treatment measures of hospitalized children with positive HCoV-NL63, OC43, 229E, HKU1 from 2015 to 2020. Results: A total of 1062 children aged 33 days to 12 years were analyzed, including 879 (82.77%) between 33 days to three years. Lower respiratory tract infections were the most common in 698 children positive for HCoVs (65.72%). The incidences of runny nose, cough, pharyngeal hyperemia, and fine crackles in the mild case group (n = 894, 84.18%) were significantly higher than in the severe case group, and the differences were statistically significant (P < 0.01). The incidences of gasp, stridor, and convulsions, the proportion of underlying diseases, such as congenital heart disease, laryngomalacia, and general developmental disorders, anemia, and abnormal liver function, and mixed infections in the severe group (n = 168, 15.82%) were significantly higher than in the mild group, and the differences were statistically significant (P < 0.01 or P < 0.05). Imaging manifestations differed. Pleural effusion and atelectasis occurred in the severe cases. After treatment, patients fully recovered or improved and were discharged from the hospital. There were no deaths. Conclusion: HCoV-NL63, OC43, 229E, HKU1 infection is most common in children under three years old, and the infection site is mainly the lower respiratory tract. The main clinical manifestations include fever, cough, and runny nose. Inspiratory three concave signs, respiratory failure, and heart failure occurred in the severe cases, with pleural effusion and atelectasis possibly occurring at the same time. Severe cases should be identified early so that they may be given comprehensive treatment in time to improve the prognosis.

A highly sensitive 1-tube nested real-time RT-PCR assay using LNA-modified primers for detection of respiratory syncytial virus.

Respiratory syncytial virus (RSV) causes serious respiratory tract infection worldwide. The relatively low RSV load makes it difficult to detect in frail, elderly, and severely immune-compromised patients. In the present study, we developed a locked nucleic acid--based 1-tube nested real-time RT-PCR (OTNRT-PCR) assay with the advantages of extremely high sensitivity, facile operability, and less likelihood of cross-contamination. The sensitivity, specificity, and clinical performance of the OTNRT-PCR assay were compared in parallel with a conventional TaqMan probe-based real-time PCR (qRT-PCR) assay and a traditional 2-step nested RT-PCR assay. The limit of detection of the OTNRT-PCR assay was 1.02 × 10-1 TCID50/mL, equivalent to the traditional 2-step nested RT-PCR assay and 25-fold lower than the qRT-PCR assay. Of 616 nasopharyngeal aspirates tested, 143 RSV-negative samples by qRT-PCR were confirmed as positive by sequencing the OTNRT-PCR products. We therefore conclude that OTNRT-PCR is more sensitive than qRT-PCR for detection of RSV in clinical samples.

A multiplex one-tube nested real time RT-PCR assay for simultaneous detection of respiratory syncytial virus, human rhinovirus and human metapneumovirus.

BACKGROUND: Respiratory syncytial virus (RSV), human Rhinovirus (HRV) and human Metapneumo Virus (HMPV) are important viral pathogens causing acute respiratory tract infections in the hospitalized patients. Sensitive and accurate detection of RSV, HRV and HMPV is necessary for clinical diagnosis and treatment. RESULTS: A locked nucleic acid (LNA)-based multiplex closed one-tube nested real-time RT-PCR (mOTNRT-PCR) assay was developed for simultaneous detection of RSV, HRV and HMPV. The sensitivity, specificity, reproducibility and clinical performance of mOTNRT-PCR were evaluated and compared with individual real time PCR (RT-qPCR) assay using clinical samples. The analytical sensitivity of mOTNRT-PCR assay was 5 copies/reaction for RSV, HRV and HMPV, respectively, and no cross-reaction with other common respiratory viruses was observed. The coefficients of variation (CV) of intra-assay and inter-assay were between 0.51 to 3.67%. Of 398 nasopharyngeal aspirates samples tested, 109 (27.39%), 150 (37.69%) and 44 (11.06%) were positive for RSV, HRV and HMPV, respectively, whereas 95 (23.87%), 137 (34.42%) and 38 (9.55%) were positive for RSV, HRV and HMPV, respectively, by individual RT-qPCR assay. Thirty three samples that were positive by mOTNRT-PCR but negative by RT-qPCR were confirmed as true positives by sequencing using reported traditional two-step nested PCR assay. CONCLUSION: mOTNRT-PCR assay reveals extremely higher sensitivity than that of RT-qPCR assay for detecting RSV, HRV and HMPV in clinical settings.

Adenovirus associated with acute diarrhea: a case-control study.

BACKGROUND: Diarrhea is a major source of morbidity and mortality among young children in low-income and middle-income countries. Human adenoviruses (HAdV), particular HAdV species F (40, 41) has been recognized as important causal pathogens, however limited data exist on molecular epidemiology of other HAdV associated with acute gastroenteritis. METHODS: In the present preliminary study, we performed a case-control study involving 273 children who presented diarrheal disease and 361 healthy children matched control in Children's hospital of Hebei Province (China) to investigate the relationship between non-enteric HAdV and diarrhea. HAdV were detected and quantified using quantitative real-time PCR (qPCR) and serotyped by sequencing and phylogenetic analysis. Odds ratio (OR) was used to assess the risk factor of HAdV. RESULTS: HAdV were detected in 79 (28.94%) of 273 children with diarrhea including 7 different serotypes (HAdV 40, 41, 3, 2,1,5 and 57) with serotypes 40, 41 and 3 being the most dominant and in 26 (7.20%) of 361 healthy children containing 9 serotypes (HAdV 40, 41, 3, 2,1,5,57,6 and 31). A majority (91.14%) of HAdV positives occurred in diarrhea children and 65.38% in controls< 3 years of age. No significant difference in the viral load was found between case and control groups or between Ad41-positive patients and healthy controls. In addition to HAdV 40 and 41, HAdV 3 was also associated with diarrhea (OR = 17.301, adjusted OR = 9.205, p < 0.001). CONCLUSIONS: Our results demonstrate a high diversity of HAdV present among diarrhea and healthy children and implicate that non-enteric HAdV3 may lead to diarrhea.

A triplex quantitative real-time PCR assay for differential detection of human adenovirus serotypes 2, 3 and 7.

BACKGROUND: Human adenovirus (HAdV) serotypes 2, 3 and 7 are more prevalent than other serotypes and have been associated with severe pneumonia in pediatric children. Molecular typing of HAdV is not routinely performed in clinical diagnostic laboratories as it is time-consuming and labor-intensive. METHODS: In the present study, we developed a triplex quantitative real-time PCR assay (tq-PCR) in a single closed tube for differential detection and quantitative analysis of HAdV serotypes 2, 3 and 7. The sensitivity, specificity, reproducibility and clinical performance of tq-PCR were evaluated. RESULTS: The analytical sensitivity of the tq-PCR was 100 copies/reaction for each of HAdV serotypes 2, 3 and 7, and no cross-reaction with other common respiratory viruses or HAdV serotypes 1,4,5,6,31,55 and 57 was observed. The coefficients of variation (CV) of intra-assay and inter-assay were between 0.6% to 3.6%. Of 138 previously-defined HAdV-positive nasopharyngeal aspirates samples tested, the detection agreement between tq-PCR and nested PCR was 96.38% (133/138). CONCLUSION: The proposed tq-PCR assay is a sensitive, specific and reproducible method and has the potential for clinical use in the rapid and differential detection and quantitation of HAdV serotypes 2, 3 and 7.

VSITA, an Improved Approach of Target Amplification in the Identification of Viral Pathogens.

OBJECTIVE: Unbiased next generation sequencing (NGS) is susceptible to interference from host or environmental sequences. Consequently, background depletion and virome enrichment techniques are usually needed for clinical samples where viral load is much lower than background sequences. METHODS: A viral Sequence Independent Targeted Amplification (VSITA) approach using a set of non-ribosomal and virus-enriched octamers (V8) was developed and compared with traditionally used random hexamers (N6). Forty-five archived clinical samples of different types were used in parallel to compare the V8 and N6 enrichment performance of viral sequences and removal performance of ribosomal sequences in the step of reverse transcription followed by quantitative PCR (qPCR). Ten sera samples from patients with fever of unknown origin and 10 feces samples from patients with diarrhea of unknown origin were used in comparison of V8 and N6 enrichment performance following NGS analysis. RESULTS: A minimum 30 hexamers matching to viral reference sequences (sense and antisense) were selected from a dataset of random 4,096 (46) hexamers (N6). Two random nucleotides were added to the 5' end of the selected hexamers, and 480 (30 × 42) octamers (V8) were obtained. In general, VSITA approach showed higher enrichment of virus-targeted cDNA and enhanced ability to remove unwanted ribosomal sequences in the majorities of 45 predefined clinical samples. Moreover, VSITA combined with NGS enabled to detect not only more viruses but also achieve more viral reads hit and higher viral genome coverage in 20 clinical samples with diarrhea or fever of unknown origin. CONCLUSION: The VSITA approach designed in this study is demonstrated to possess higher sensitivity and broader genome coverage than traditionally used random hexamers in the NGS-based identification of viral pathogens directly from clinical samples.

Development of a reverse transcription recombinase-aided amplification assay for the detection of coxsackievirus A10 and coxsackievirus A6 RNA.

Hand, foot and mouth disease (HFMD) is a serious public health problem, and coxsackievirus A6 (CVA6) and coxsackievirus A10 (CVA10) are two of the major causative pathogens, in addition to enterovirus 71 (EV71) and coxsackievirus A16 (CVA16). A simple and rapid reverse transcription recombinase-aided amplification assay (RT-RAA) was developed for the detection of CVA10 and CVA6 in this study. The analytical sensitivity for detection of CVA10 and CVA6 at 95% probability by probit regression analysis was 35 copies per reaction and 38 copies per reaction, respectively, with 100% specificity. Compared with commercial RT-qPCR assays, when testing 455 fecal specimens, the kappa value of the RT-RAA assay for CVA10 and CVA6 was 0.920 (p < 0.001) and 0.952 (p < 0.001), respectively. Moreover, four samples that were positive for CVA10 and five that were positive for CVA6 by RT-RAA but negative by RT-qPCR were further determined to be true positives. These results demonstrate that the proposed RT-RAA assays are very valuable tools for the detection of CVA10 and CVA6 and have potential for use in resource-limited settings.

Use of a rapid reverse-transcription recombinase aided amplification assay for respiratory syncytial virus detection.

In this study, a rapid reverse-transcription recombinase aided amplification (RT-RAA) assay was developed to detect respiratory syncytial virus (RSV) subgroups A and B, respectively. The reaction was performed at 39°C in less than 30min. The analytical sensitivities of RSVA and RSVB at 95% probability by probit regression analysis were 38copies per reaction and 35 copies per reaction, respectively, and no cross reactions with other related respiratory viruses were observed. The RT-RAA assay was further utilized to detect and subgroup 306 clinical specimens and the results showed that 79(25.82%, 79/306) samples were positive for RSV, of those 16(20.25%, 16/79) were identified as RSVA and 63(79.75%, 63/79) were RSVB, which is completely consistent with the results obtained by RSV RT-qPCR assay. In conclusion, the developed RAA assay will be of benefit as a faster, sensitive and specific alternative tool for detection of RSV.