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.

Burkholderia pseudomallei Detection among Hospitalized Patients, Sarawak.

Burkholderia pseudomallei infections are prevalent in Southeast Asia and northern Australia and often misdiagnosed. Diagnostics are often neither sensitive nor rapid, contributing up to 50% mortality rate. In this 2018 pilot study, we enrolled 100 patients aged 6 months-79 years from Kapit Hospital in Sarawak, Malaysia, with symptoms of B. pseudomallei infection. We used three different methods for the detection of B. pseudomallei: a real-time polymerase chain reaction (PCR) assay, a rapid lateral flow immunoassay, and the standard-of-care bacterial culture-the gold standard. Among the 100 participants, 24 (24%) were positive for B. pseudomallei by one or more of the detection methods. Comparing the two individual diagnostic methods against the gold standard-bacterial culture-of any positive test, there was low sensitivity for each test (25-44%) but high specificity (93-98%). It seems clear that more sensitive diagnostics or a sensitive screening diagnostic followed by specific confirmatory diagnostic is needed for this disease.

High Prevalence of Viral Infections Among Hospitalized Pneumonia Patients in Equatorial Sarawak, Malaysia.

BACKGROUND: Although pneumonia is a known cause of morbidity and mortality in Sarawak, Malaysia, the etiology and epidemiology of pneumonia are not well described in this equatorial region. Routine clinical diagnostics for pneumonia etiology at government hospitals in Sarawak had historically involved only bacterial diagnostics. Viral diagnostics were only obtained through outside consultations. METHODS: From June 15, 2017 to May 14, 2018, we collected nasopharyngeal swabs from 600 patients of all ages older than 1 month hospitalized with pneumonia at Sibu and Kapit Hospitals. Specimens were examined at our collaborating institutions with a panel of molecular assays for viral pathogens including influenza A (IAV), IBV, ICV, and IDV, human adenovirus (AdV), human enterovirus (EV), human coronavirus (CoV), respiratory syncytial virus subtype A (RSV-A) or RSV-B, and parainfluenza virus (PIV) types 1-4. RESULTS: Of 599 samples examined, 288 (48%) had molecular evidence of 1 or more respiratory viruses. Overall, the most prevalent virus detected was RSV-A (14.2%) followed by AdV (10.4%) and IAV (10.4%), then RSV-B (6.2%), EV (4.2%), IBV (2.2%), PIV-3 (1.7%), CoV (1.0%), PIV-1 (1.0%), PIV-4 (0.7%), and PIV-2 (0.2%). No specimens were confirmed positive for ICV or IDV. CONCLUSIONS: The high prevalence of viruses detected in this study suggest that respiratory viruses may be responsible for considerable morbidity in equatorial regions such as Sarawak. Access to viral diagnostics are very necessary for medical staff to determine appropriate pneumonia treatments.

Surveillance for respiratory syncytial virus and parainfluenza virus among patients hospitalized with pneumonia in Sarawak, Malaysia.

BACKGROUND: Respiratory syncytial virus (RSV) and parainfluenza virus (PIV) are frequent causes of pneumonia and death among children at Sibu and Kapit Hospitals in Sarawak, Malaysia. OBJECTIVES: To determine the prevalence and risk factors for RSV subtypes A and B and PIV types 1-4 among patients hospitalized with pneumonia. METHODS: In a cross-sectional, pilot study nasopharyngeal swabs were studied with real-time reverse transcription polymerase chain reaction assays. Concurrently, we helped Sibu and Kapit Hospitals adapt their first molecular diagnostics for RSV and PIV. RESULTS: Of 129 specimens collected (June to July 2017), 39 tested positive for RSV-A (30.2%), two were positive for RSV B (1.6%), one was positive for PIV-3 (0.8%) and one was positive for PIV-4 (0.8%). No samples were positive for PIV-1 or PIV-2. Of the 39 RSV-A positive specimens, 46.2% were collected from children under one year of age and only 5.1% were from patients over the age of 18. A multivariable analysis found the odds of children <1 year of age testing positive for RSV-A were 32.7 (95% CI: 3.9, 276.2) times larger than >18 years of age, and the odds of patients hospitalized at Kapit Hospital testing positive for RSV-A were 3.2 (95% CI: 1.3, 7.8) times larger than patients hospitalized at Sibu Hospital. CONCLUSION: This study found an unusually high prevalence of RSV-A among pneumonia patients admitted to the two hospitals. Subsequently, Sibu Hospital adapted the molecular assays with the goal of providing more directed care for such pneumonia patients.