A pan-coronavirus RT-PCR assay for rapid viral screening of animal, human, and environmental specimens.

We examined a collection of 386 animal, 451 human, and 109 archived bioaerosol samples with a new pan-species coronavirus molecular assay. Thirty-eight (4.02%) of 946 specimens yielded evidence of human or animal coronaviruses. Our findings demonstrate the utility of employing the pan-CoV RT-PCR assay in detecting varied coronavirus among human, animal, and environmental specimens. This RT-PCR assay might be employed as a screening diagnostic for early detection of coronaviruses incursions or prepandemic coronavirus emergence in animal or human populations.

Panspecies molecular assays detect viral pathogens missed by real-time PCR/reverse-transcriptase PCR among pneumonia patients, Sarawak, Malaysia.

BACKGROUND: In a year-long pneumonia etiology study conducted June 2017 to May 2018 in Sarawak, Malaysia, 599 patients' nasopharyngeal swab specimens were studied with real-time polymerase chain reaction (rPCR)/ reverse-transcription (rRT-PCR) assays for respiratory pathogens known to contribute to the high burden of lower respiratory tract infections. The study team sought to compare real-time assay results with panspecies conventional molecular diagnostics to compare sensitivities and learn if novel viruses had been missed. METHODS: Specimens were studied for evidence of adenovirus (AdV), enterovirus (EV) and coronavirus (CoV) with panspecies gel-based nested PCR/RT-PCR assays. Gene sequences of specimens positive by panspecies assays were sequenced and studied with the NCBI Basic Local Alignment Search Tool software. RESULTS: There was considerable discordance between real-time and conventional molecular methods. The real-time AdV assay found a positivity of 10.4%; however, the AdV panspecies assay detected a positivity of 12.4% and the conventional AdV-Hexon assay detected a positivity of 19.6%. The CoV and EV panspecies assays similarly detected more positive specimens than the real-time assays, with a positivity of 7.8% by the CoV panspecies assay versus 4.2% by rRT-PCR, and 8.0% by the EV panspecies assay versus 1.0% by rRT-PCR. We were not able to ascertain virus viability in this setting. While most discordance was likely due to assay sensitivity for previously described human viruses, two novel, possible zoonotic AdV were detected. CONCLUSIONS: The observed differences in the two modes of amplification suggest that where a problem with sensitivity is suspected, real-time assay results might be supplemented with panspecies conventional PCR/RT-PCR assays.

A RT-PCR assay for the detection of coronaviruses from four genera.

BACKGROUND: During the past two decades, three novel coronaviruses (CoVs) have emerged to cause international human epidemics with severe morbidity. CoVs have also emerged to cause severe epidemics in animals. A better understanding of the natural hosts and genetic diversity of CoVs are needed to help mitigate these threats. OBJECTIVE: To design and evaluate a molecular diagnostic tool for detection and identification of all currently recognized and potentially future emergent CoVs from the Orthocoronavirinae subfamily. STUDY DESIGN AND RESULTS: We designed a semi-nested, reverse transcription RT-PCR assay based upon 38 published genome sequences of human and animal CoVs. We evaluated this assay with 14 human and animal CoVs and 11 other non-CoV respiratory viruses. Through sequencing the assay's target amplicon, the assay correctly identified each of the CoVs; no cross-reactivity with 11 common respiratory viruses was observed. The limits of detection ranged from 4 to 4 × 102 copies/reaction, depending on the CoV species tested. To assess the assay's clinical performance, we tested a large panel of previously studied specimens: 192 human respiratory specimens from pneumonia patients, 5 clinical specimens from COVID-19 patients, 81 poultry oral secretion specimens, 109 pig slurry specimens, and 31 aerosol samples from a live bird market. The amplicons of all RT-PCR-positive samples were confirmed by Sanger sequencing. Our assay performed well with all tested specimens across all sample types. CONCLUSIONS: This assay can be used for detection and identification of all previously recognized CoVs, including SARS-CoV-2, and potentially any emergent CoVs in the Orthocoronavirinae subfamily.

Virus detections among patients with severe acute respiratory illness, Northern Vietnam.

Severe acute respiratory illness (SARI) is a major cause of death and morbidity in low- and middle-income countries, however, the etiologic agents are often undetermined due to the lack of molecular diagnostics in hospitals and clinics. To examine evidence for select viral infections among patients with SARI in northern Vietnam, we studied 348 nasopharyngeal samples from military and civilian patients admitted to 4 hospitals in the greater Hanoi area from 2017-2019. Initial screening for human respiratory viral pathogens was performed in Hanoi, Vietnam at the National Institute of Hygiene and Epidemiology (NIHE) or the Military Institute of Preventative Medicine (MIPM), and an aliquot was shipped to Duke-NUS Medical School in Singapore for validation. Patient demographics were recorded and used to epidemiologically describe the infections. Among military and civilian cases of SARI, 184 (52.9%) tested positive for one or more respiratory viruses. Influenza A virus was the most prevalent virus detected (64.7%), followed by influenza B virus (29.3%), enterovirus (3.8%), adenovirus (1.1%), and coronavirus (1.1%). Risk factor analyses demonstrated an increased risk of influenza A virus detection among military hospital patients (adjusted OR, 2.0; 95% CI, 1.2-3.2), and an increased risk of influenza B virus detection among patients enrolled in year 2017 (adjusted OR, 7.9; 95% CI, 2.7-22.9). As influenza A and B viruses were commonly associated with SARI and are treatable, SARI patients entering these hospitals would benefit if the hospitals were able to adapt onsite molecular diagnostics.

Bioaerosol Sampling to Detect Avian Influenza Virus in Hanoi's Largest Live Poultry Market.

BACKGROUND: Newly emergent and virulent strains of H7N9 avian influenza virus are rapidly spreading in China and threaten to invade Vietnam. We sought to introduce aerosol sampling for avian influenza viruses in Vietnam. METHODS: During October 2017, National Institute for Occupational Safety and Health 2-stage aerosol samplers were assembled on a tripod and run for 4 hours. Concomitantly, up to 20 oropharyngeal (OP) swab samples were collected from chickens and ducks distanced at 0.2-1.5 m from each sampler. RESULTS: The 3 weeks of sampling yielded 30 aerosol samples that were 90% positive for influenza A, by quantitative reverse-transcription polymerase chain reaction, and 116 OP swab sample pools (5 samples per pool) that were 47% positive. Egg cultures yielded 1 influenza A virus (not H5 or H7) from aerosol and 25 influenza A viruses from OP swab sample pools (5 were H5 positive). The association between positive sample types (over time and position) was strong, with 91.7% of positive OP pooled swab samples confirmed by positive aerosol samples and 81% of influenza A positive aerosol samples confirmed by positive OP swab samples. CONCLUSIONS: We posit that aerosol sampling might be used for early warning screening of poultry markets for novel influenza virus detection, such as H7N9. Markets with positive aerosol samples might be followed up with more focused individual bird or cage swabbing, and back-tracing could be performed later to locate specific farms harboring novel virus. Culling birds in such farms could reduce highly pathogenic avian influenza virus spread among poultry and humans.