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.

Rapid two-stage amplification in a single tube for simultaneous detection of norovirus GII and group a rotavirus.

The most prevalent viruses currently causing diarrhea are norovirus and rotavirus, and rapid and sensitive detection methods are essential for the early diagnosis of disease. The purpose of this study was to establish a sensitive single-tube two-stage nucleic acid amplification method-reverse transcription recombinase-assisted PCR (RT-RAP)-for simultaneous detection of norovirus GII and group A Rotavirus, with the first stage consisting of isothermal reverse transcription recombinase-aided amplification (RT-RAA) and the second stage consisting of qPCR (quantitative PCR). RT-RAP is more sensitive than either RT-RAA or qRT-PCR (quantitative RT-PCR) alone. And the addition of a barrier that can be disassembled after heating enabled the detection of samples within 1 h in a single closed tube. Sensitivity was 10 copies/reaction of norovirus (Novs) GII and group A rotavirus (RVA). In parallel, two hundred fecal specimens were used to evaluate the method and compare it with a commercial fluorescent quantitative RT-PCR. The data showed kappa values of 0.957 and 0.98 (p < 0.05) for detecting Novs GII and RVA by the two methods, indicating the potential of the newly established assay to be applied to clinical and laboratory testing.

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.

Development of a duplex recombinase-aided amplification assay for direct detection of Mycoplasma pneumoniae and Chlamydia trachomatis in clinical samples.

BACKGROUND: Pneumonia caused by Mycoplasma pneumoniae is common in the elderly and children, and pneumonia caused by Chlamydia trachomatis is prevalent in newborns. This study aimed to establish a rapid, sensitive, and simple method for the direct detection of M. pneumoniae and C. trachomatis in clinical samples without DNA extraction. METHODS: We established a duplex recombinase-aided amplification (RAA) assay with the RNAseP gene as an internal control for detecting the P1 gene of M. pneumoniae and the ORF8 gene of C. trachomatis, respectively. The results were obtained at 39 °C within 15-20 min. A total of 130 clinical samples suspected of M. pneumoniae or C. trachomatis infection were collected and tested by duplex RAA and PCR. DNA extracted via a commercial kit or treated with a nucleic acid-releasing agent was used and compared, respectively. Standard recombinant plasmids were used to test the sensitivity of the duplex RAA assay. In addition, other similar common pathogens were used to verify the specificity of the duplex RAA assay. RESULTS: The sensitivity of the duplex RAA assay for detecting M. pneumoniae and C. trachomatis was 10 copies/µL using recombinant plasmids. Compared with PCR, the sensitivity and specificity of duplex RAA assays for M. pneumoniae and C. trachomatis was 100% using clinical DNA samples extracted using a commercial kit and a nucleic acid-releasing agent, and the Kappa value was 1. CONCLUSION: The advantages of this duplex RAA assay include high sensitivity and specificity, short duration, and simple extraction steps, with potential for use in the on-site detection of M. pneumoniae and C. trachomatis in resource-limited settings.

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 one-tube nested quantitative real-time PCR assay for specific detection of Bordetella pertussis using the LNA technique.

OBJECTIVES: Bordetella pertussis is a highly contagious respiratory agent and is the causative pathogen of pertussis, which primarily affects children. Current diagnostic techniques for this pathogen have a variety of limitations including a long culture time, low bacterial load, and lack of specificity. METHODS: This article reports the development of a one-tube nested quantitative real-time PCR assay using the locked nucleic acid (LNA) technique (LNA-OTN-q-PCR), targeting the BP485 gene and using a simple inexpensive extraction method. A total of 130 clinical samples from patients with clinically suspected pertussis, collected from the Children's Hospital of Hebei, China, were tested by LNA-OTN-q-PCR assay. RT-PCR and two-step semi-nested PCR assays were performed in parallel for comparison. RESULTS: Only strains of B. pertussis were identified as positive, whereas all of the remaining strains were appropriately identified as negative by the LNA-OTN-q-PCR assay. A single copy per reaction can be detected by the LNA-OTN-q-PCR assay. Additionally, the sensitivity of this method was 100 times that of the RT-PCR assay (100 copies per reaction). Sixty-three of the 130 clinical samples were detected positive by LNA-OTN-q-PCR assay; in contrast, RT-PCR was able to detect only 41 positive samples. Following this, all 63 samples were positively identified by two-step semi-nested PCR. Compared with the two-step semi-nested PCR assay, both the specificity and sensitivity of the LNA-OTN-q-PCR assay using purified DNA and crude extract were 100%. CONCLUSIONS: This assay was able to detect B. pertussis infection with high sensitivity and specificity. This test shows great potential as a promising technique to detect B. pertussis in both clinical laboratories and public health settings.

Development and evaluation of a panel of multiplex one-tube nested real time PCR assay for simultaneous detection of 14 respiratory viruses in five reactions.

Multiplex real-time quantitative polymerase chain reaction (mRT-qPCR) assay is commonly used to detect respiratory viruses, however, the sensitivity is limited for most reports. A panel of locked nucleic acid based multiplex closed one-tube nested real-time PCR (mOTNRT-PCR) assay consisting of five separate internally controlled RT-qPCR assays was developed for detection of 14 respiratory viruses. The sensitivity and reproducibility of mOTNRT-PCR panel were evaluated using plasmid standards and the specificity was evaluated using clinical samples. The clinical performance of mOTNRT-PCR panel was further evaluated with 468 samples collected from patients with an acute respiratory infection and compared with individual real-time PCR (RT-qPCR) assay. The analytical sensitivities of mOTNRT-PCR panel ranged from 2 to 20 copies/reaction, and no cross-reaction with common respiratory viruses was observed. The coefficients of variation of intra-assay and inter-assay were between 0.35% and 8.29%. Totally 35 clinical samples detected by mOTNRT-PCR assay panel were missed by RT-qPCR and confirmed true positive by sequencing of nested PCR products. The mOTNRT-PCR assay panel provides a more sensitive and high-throughput method for the detection of 14 respiratory viruses.

Molecular and clinical characterization of human adenovirus associated with acute respiratory tract infection in hospitalized children.

BACKGROUND: Human adenovirus (HAdV) is a common pathogen in children that can cause acute respiratory tract infection (ARTI), but the molecular epidemiological and clinical information relating to HAdV among hospitalized children with ARTI are few reported in China. OBJECTIVES: To evaluate the epidemiological, clinical, and molecular characteristics of HAdV infections among hospitalized children with ARTI in Hebei, Northern China from June 2017 to May 2018. STUDY DESIGN: A 12-month longitudinal, retrospective study on HAdV, typed by nested polymerase chain reaction targeting the hexon gene's hypervariable region (typing was merely performed by sequencing of the hexon neutralization epitope and thus genotypes could not be identified unequivocally), associated with ARTI was performed. The epidemiological and clinical data of different types of HAdV were analyzed using statistical product and service solutions (SPSS) 21.0 software. RESULTS: HAdV was detected in 330 (3.71%) of the 8906 specimens, with most (88.48%, 292/330) HAdV-positives cases detected among children < 3 years old. HAdV were detected throughout the year with a higher prevalence in spring. 11 types were identified, with HAdV-2 (33.33%, 110/330) as the predominant type, followed by HAdV-3 (21.21%, 70/330) and HAdV-7 (13.94%, 46/330). Of the 330 HAdV-positive specimens, 247 (74.85%) were co-detected with other respiratory pathogens, most commonly rhinovirus (HRV) (58.7%, 145/247). Additionally, patients with HAdV-7 positive had longer duration of fever than HAdV-2 or -3 positive patients. CONCLUSIONS: During the study period, HAdV-2, HAdV-3 and HAdV-7 were the predominant types identified from children with ARTI in Hebei Province. Pediatric patients with HAdV-7 positive may not present more severe clinical outcome except a longer duration of fever.

Impact and clinical profiles of Mycoplasma pneumoniae co-detection in childhood community-acquired pneumonia.

BACKGROUND: Increasing number of hospitalized children with community acquired pneumonia (CAP) is co-detected with Mycoplasma pneumoniae (Mp). The clinical characteristics and impact of Mp co-detected with other bacterial and/or viral pathogens remain poorly understood. The purpose of this study was to evaluate the demographic and clinical features of CAP children with Mp mono-detection and Mp co-detection. METHODS: A total of 4148 hospitalized children with CAP were recruited from January to December 2017 at the Children's Hospital of Hebei Province, affiliated to Hebei Medical University. A variety of respiratory viruses, bacteria and Mp were detected using multiple modalities. The demographic and clinical features of CAP children with Mp mono-detection and Mp co-detection were recorded and analyzed. RESULTS: Among the 110 CAP children with Mp positive, 42 (38.18%) of them were co-detected with at least one other pathogen. Co-detection was more common among children aged ≤3 years. No significant differences were found in most clinical symptoms, complications, underlying conditions and disease severity parameters among various etiological groups, with the following exceptions. First, prolonged duration of fever, lack of appetite and runny nose were more prevalent among CAP children with Mp-virus co-detection. Second, Mp-virus (excluding HRV) co-detected patients were more likely to present with prolonged duration of fever. Third, patients co-detected with Mp-bacteria were more likely to have abnormal blood gases. Additionally, CAP children with Mp-HRV co-detection were significantly more likely to report severe runny nose compared to those with Mp mono-detection. CONCLUSION: Mp co-detection with viral and/or bacterial pathogens is common in clinical practice. However, there are no apparent differences between Mp mono-detection and Mp co-detections in terms of clinical features and disease severity.

A rapid and sensitive recombinase aided amplification assay to detect hepatitis B virus without DNA extraction.

BACKGROUND: Hepatitis B virus (HBV) infection is the major public health problem worldwide. In clinical practice, serological and molecular assays are the most commonly used diagnostic methods to detect HBV infection in clinical practices. METHODS: Here we present a rapid and sensitive recombinase aided amplification assay (RAA) to detect HBV at 39.0 °C for 30 min without DNA extraction from serum samples. The analytical sensitivity of RAA assay was 100 copies per reaction and showed no cross reaction with human immunodeficiency virus (HIV) and hepatitis C virus (HCV). The universality of RAA assay was validated by testing of 41 archived serum samples with predefined HBV genotypes (B, C and D). RESULTS: A total of 130 archived suspected HBV infected serum samples were detected by commercial qPCR with DNA extraction and RAA assay without DNA extraction (heat-treatment). Compared with qPCR assay as a reference, the RAA assay obtained 95.7% sensitivity and 100% specificity and a kappa value of 0.818. CONCLUSIONS: We developed a rapid, convenient, highly sensitive and specific method to detect HBV without DNA extraction in clinical samples. This RAA method of HBV detection is very suitable for clinical testing.

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 case control study on the prevalence of enterovirus in children samples and its association with diarrhea.

Some serotypes of enterovirus (EV) may lead to transient and symptomatic gastrointestinal infections while others are commensal residents of the human gut. To determine whether certain EV types are more often associated with diarrhea, we conducted a preliminary study on the prevalence of EV serotypes and common diarrhea viruses in fecal samples of diarrhea children and healthy controls. EV was tested with one step nest polymerase chain reaction and typed by direct sequencing while common causative diarrhea viruses rotavirus (RV), norovirus (NoV), adenovirus (AdV), bocavirus (HBoV), and astrovirus (AstV) were screened with multiplex PCR assays. Human Rhinovirus (HRV) and human EVs that were present in both groups were further quantified and their odds ratios (OR) were calculated. Enteric pathogens were detected in 89 (32.6%) of 273 children with diarrhea and included human EVs (51, 18.68%), HRV (32, 11.72%), RV (38, 13.92%), AdV (24, 8.79%), NoVGII (16, 8.79%), HBoV (8, 2.93%) and AstV (3, 1.09%). Potential enteric pathogens were found in 25 (6.93%) of 361 healthy controls and included human EV (59, 16.34%), HRV (8, 2.22%), RV (1, 0.28%), NoVGII (5, 1.39%), AstV (2, 0.55%), AdV (16, 4.43%) and HBoV (1, 0.28%). In addition, EV71, echovirus 3,9,14,25 and coxsackievirus A14 existed in healthy controls only, while HRV, echovirus11,18, coxsackievirus A2,4,6 and B2,4 were found in both patients and healthy controls. OR assessment confirmed a strong association of HRV (P < 0.001) and a weak one for echovirus 11 and coxsackievirus A6 with diarrhea (P > 0.05). Our results indicate the diversity of EV serotypes in diarrhea and healthy control groups varies, and the potential etiological role of HRV in diarrhea.

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.

Clinical evaluation of a new single-tube multiplex reverse transcription PCR assay for simultaneous detection of 11 respiratory viruses, Mycoplasma pneumoniae and Chlamydia in hospitalized children with acute respiratory infections.

Respiratory Pathogen 13 Detection Kit (13× kit) is able to simultaneously detect 11 respiratory viruses, Mycoplasma pneumoniae (MP) and Chlamydia in a single reaction. Using 572 Nasopharyngeal aspirates collected from hospitalized children, the clinical performance of 13× kit for detecting 11 respiratory viruses was evaluated in comparison with a routinely used 2-tube multiplex reverse transcription PCR assay (2-tube assay) at provincial Centers for Disease Control and Prevention in China. The clinical performance of 13× kit for detecting MP and Chlamydia was evaluated by commercial real-time quantitative PCR (qPCR) kits or sequencing. For tested viruses, the assay concordance was 95.98% and the kappa coefficient was 0.89. All the MP and Chlamydia positive samples detected by 13× kit were confirmed as true positives. The utilization of the 13× kit in clinical settings will be helpful for doctors to assess clinical outcome according to virus type or multiple infections, and to limit the use of antibiotics.