ABSTRACT
PURPOSE: The purpose of this study is to evaluate the frequency of viral and bacterial respiratory pathogens detected by molecular methods in sputum samples of patients hospitalized for COVID-19 and to evaluate its impact on mortality and unfavorable outcomes (in-hospital death or mechanical ventilation). PATIENTS AND METHODS: The prospective cohort included patients with diagnosis of COVID-19 hospitalized at Hospital Nacional Hipólito Unanue. Sociodemographic and clinical data were collected from clinical records. Sputum samples were analyzed with the Biofire Filmarray Pneumonia plus® respiratory panel. Crude and adjusted associations with unfavorable outcomes were evaluated using logistic regression models. RESULTS: Ninety-three patients who were able to collect sputum samples were recruited between September 8 and December 28, 2020. The median age was 61.7 years (IQR 52.3-69-8) and 66 (71%) were male. The most frequent symptoms were dyspnea, cough, fever, and general malaise found in 80 (86%), 76 (82%), 45 (48%), and 34 (37%) patients, respectively. Fifty-three percent of patients had comorbidities. Seventy-six (82%) patients received antibiotics prior to admission and 29 (31%) developed unfavorable outcome. Coinfection was evidenced in 38 (40.86%) cases. The most frequently found bacteria were Staphylococcus aureus, Streptococcus agalactiae, Haemophilus influenzae and Klebsiella pneumoniae in 11 (11.83%), 10 (10.75%), 10 (10.75%), and 8 (8.6%) cases, respectively. Streptococcus pneumoniae was found in one case (1.08%). We neither identify atypical bacteria nor influenza virus. No association was found between the presence of viral or bacterial microorganisms and development of unfavorable outcomes (OR 1.63; 95% CI 0.45-5.82). CONCLUSION: A high frequency of respiratory pathogens was detected by molecular methods in patients with COVID-19 pneumonia but were not associated with unfavorable outcomes. No atypical agents or influenza virus were found. The high use antibiotics before admission is a concern. Our data suggest that the use of drug therapy against atypical bacteria and viruses would not be justified in patients hospitalized for COVID-19.
ABSTRACT
Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a major threat to public health. Rapid molecular testing for convenient and timely diagnosis of SARS-CoV-2 infections represents a challenge that could help to control the current pandemic and prevent future outbreaks. We aimed to develop and validate a multiplex and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay using lyophilized LAMP reagents for sensitive and rapid detection of SARS-CoV-2. LAMP primers were designed for a set of gene targets identified by a genome-wide comparison of viruses. Primer sets that showed optimal features were combined into a multiplex RT-LAMP assay. Analytical validation included assessment of the limit of detection (LoD), intra- and inter-assay precision, and cross-reaction with other respiratory pathogens. Clinical performance compared to that of real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) was assessed using 278 clinical RNA samples isolated from swabs collected from individuals tested for COVID-19. The RT-LAMP assay targeting the RNA-dependent RNA polymerase (RdRp), membrane (M), and ORF1ab genes achieved a comparable LoD (0.65 PFU/mL, CT=34.12) to RT-qPCR and was 10-fold more sensitive than RT-qPCR at detecting viral RNA in clinical samples. Cross-reactivity to other respiratory pathogens was not observed. The multiplex RT-LAMP assay demonstrated a strong robustness and acceptable intra- and inter-assay precision (mean coefficient of variation, 4.75% and 8.30%). Diagnostic sensitivity and specificity values were 100.0% (95% CI: 97.4-100.0%) and 98.6% (95% CI: 94.9-99.8%), respectively, showing high consistency (Cohen's kappa, 0.986; 95% CI: 0.966-1.000; p<0.0001) compared to RT-qPCR. The novel one-step multiplex RT-LAMP assay is storable at room temperature and showed similar diagnostic accuracy to conventional RT-qPCR, while being faster (<45 min), simpler, and cheaper. The new assay could allow reliable and early diagnosis of SARS-CoV-2 infections in primary health care. It may aid large-scale testing in resource-limited settings, especially if it is integrated into a point-of-care diagnostic device.