SARS-CoV-2 RNAemia and Disease Severity in COVID-19 Patients.

OBJECTIVE: The clinical implications of SARS-CoV-2 RNA viremia in blood (RNAemia) remain uncertain despite gaining more prognostic implications for coronavirus disease 2019 (COVID-19). However, the clinical relevance of SARS-CoV-2 RNAemia has not been well documented. METHODS: We conducted a cohort study on 95 confirmed COVID-19 patients and explored the prospects with evidence of SARS-CoV-2 RNAemia in association with various clinical characteristics. We performed reverse transcription-polymerase chain reaction and studied the risk factors of SARS-CoV-2 RNAemia using logistic regression analysis. RESULTS: The presence of SARS-CoV-2 RNAemia in critical or fatal cases was the highest (66.7%), followed by severe (12.5%) and mild to moderate (1.7%) in admission samples. SARS-CoV-2 viral RNAemia was detected on admission and 1st week samples; however, RNAemia was not detected on the samples collected on the second week post-symptom onset. Multiple regression analysis showed that the severity of the disease was an independent predictor of RNAemia (p < 0.021), and the Kaplan-Meier survival curve estimated an increased mortality rate in SARS-CoV-2 RNAemia cases (p < 0.001). CONCLUSIONS: Our study demonstrated that SARS-CoV-2 RNAemia is a predictive risk factor for clinical severity in COVID-19 patients. Hence, we showed that blood RNAemia might be a critical marker for disease severity and mortality.

Diagnostic Sensitivity of Saliva and Other Respiratory Tract Samples of SARS-CoV-2 Variants in Patients with COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to emerge during the ongoing coronavirus disease 2019 (COVID-19) pandemic. Contrasting studies on the omicron variant have demonstrated higher viral loads in different clinical specimens, which is consistent with its high transmissibility. We investigated the viral load in clinical specimens that were infected with the SARS-CoV-2 wild-type, delta, and omicron variants, and we analyzed the diagnostic accuracy of upper and lower respiratory specimens for these variants. We performed nested reverse transcription (RT)-polymerase chain reaction (PCR), targeting the spike gene and sequencing for variant classification. RT-PCR was performed using upper and lower respiratory specimens, including saliva from 78 COVID-19 patients (wild-type, delta, and omicron variants). A comparison of the sensitivity and specificity, using the area under the receiver operating characteristic curve (AUC) values from the N gene, showed that the omicron variant saliva samples had a higher sensitivity (AUC = 1.000) than did the delta (AUC = 0.875) and the wild-type (AUC = 0.878) variant samples. The sensitivity of the omicron saliva samples was greater than that of the wild-type nasopharynx and sputum samples (P < 0.001). The viral loads of the saliva samples containing the wild-type, delta, and omicron variants were 8.18 × 105, 2.77 × 106, and 5.69 × 105, respectively, which did not differ significantly (P = 0.610). Statistically significant differences were not observed in the saliva viral loads between vaccinated and nonvaccinated patients who were infected with the omicron variant (P = 0.120). In conclusion, omicron saliva samples had higher sensitivity than did wild-type and delta samples, and the viral load did not significantly differ between vaccinated and nonvaccinated patients. Further research is necessary to elucidate the mechanisms underlying the sensitivity differences. IMPORTANCE Owing to the vast heterogeneity of the studies focused on the correlation between the SARS-CoV-2 omicron variant and COVID-19, accurate comparisons of the specificity and sensitivity of samples and associated outcomes are still inconclusive. Moreover, limited information is available on the leading causes of infection and the factors that are associated with the conditions that underlie the spread of infection. Although several studies have contributed important knowledge regarding infectious specimens, the impact of saliva samples remains unknown. This study showed that the sensitivity of the omicron variant saliva samples was higher than that of the wild-type nasopharyngeal and sputum samples. Moreover, neither vaccinated nor nonvaccinated patients who were infected with the omicron variant showed any significant differences in SARS-CoV-2 viral loads. Hence, this study is an important step toward understanding how saliva sample results are correlated with other specimen results, regardless of the vaccination status of patients who are infected with the SARS-CoV-2 omicron variant.

Accuracy of Real-Time Polymerase Chain Reaction in COVID-19 Patients.

Coronavirus disease 2019 (COVID-19) is a mild to severe respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The diagnostic accuracy of the Centers for Disease Control and Prevention (CDC)- or World Health Organization (WHO)-recommended real-time PCR (RT-qPCR) primers in clinical practice remains unproven. We conducted a prospective study on the accuracy of RT-qPCR using an in-house-designed primer set (iNP) targeting the nucleocapsid protein as well as various recommended and commercial primers. The accuracy was assessed by culturing or seroconversion. We enrolled 12 confirmed COVID-19 patients with a total of 590 clinical samples. When a cutoff value of the cycle threshold (Ct) was set to 35, RT-qPCRs with WHO RdRp primers and CDC N1, N2, and N3 primers showed sensitivity of 42.1% to 63.2% and specificity of 90.5% to 100% in sputum, and sensitivity of 65.2% to 69.6% and specificity of 65.2% to 69.6% in nasopharyngeal samples. The sensitivity and specificity of iNP RT-qPCR in sputum and nasopharyngeal samples were 94.8%/100% and 69.6%/100%, respectively. Sputum testing had the highest sensitivity, followed by nasopharyngeal testing (P = 0.0193); self-collected saliva samples yielded better characteristics than oropharyngeal samples (P = 0.0032). Our results suggest that iNP RT-qPCR has better sensitivity and specificity than RT-PCR with WHO (P < 0.0001) or CDC (N1: P = 0.0012, N2: P = 0.0013, N3: P = 0.0012) primers. Sputum RT-qPCR analysis has the highest sensitivity, followed by nasopharyngeal, saliva, and oropharyngeal assays. Our study suggests that considerable improvement is needed for the RT-qPCR WHO and CDC primer sets for detecting SARS-CoV-2. IMPORTANCE Numerous research campaigns have addressed the vast majority of clinical and diagnostic specificity and sensitivity of various primer sets of SARS-CoV2 viral detection. Despite the impressive progress made to resolve the pandemic, there is still a need for continuous and active improvement of primers used for diagnosis in clinical practice. Our study significantly exceeds the scale of previously published research on the specificity and sensitivity of different primers comparing with different specimens and is the most comprehensive to date in terms of constant monitoring of primer sets of current usage. Henceforth, our results suggest that sputum samples sensitivity is the highest, followed by nasopharyngeal, saliva, and oropharyngeal samples. The CDC recommends the use of oropharyngeal specimens, leading to certain discrepancy between the guidelines set forth by the CDC and IDSA. We proved that the oropharyngeal samples demonstrated the lowest sensitivity for the detection of SARS-CoV-2.

Utility of Nested Reverse-Transcriptase Polymerase Chain Reaction of Clinical Specimens for Early Diagnosis of Hemorrhagic Fever with Renal Syndrome.

Hemorrhagic fever with renal syndrome (HFRS) is confirmed by the isolation of hantavirus from serum, detection of virus-specific IgM, or a four-fold change in IgG titers during the acute and convalescent periods measured using an immunofluorescence assay (IFA). However, these tests are inefficient for early diagnosis. Therefore, this study investigated the usefulness of reverse-transcriptase nested polymerase chain reaction (RT-nPCR) for early diagnosis of HFRS using clinical samples such as urine and serum. Electronic medical records of eight patients with confirmed HFRS using IFA and RT-nPCR between May 2016 and May 2020 at Chosun University Hospital were reviewed. The virus was detected in all patients using RT-nPCR targeting the large (L) segment of hantavirus during the early phase in urine and serum. Importantly, the virus was identified in urine at a time when it was not identified in serum. Additionally, the virus was detected in urine and serum for up to 1 month after initial presentation with illness, but not in saliva, using RT-nPCR. We report eight HFRS cases diagnosed using urine and serum, but not using saliva, with RT-nPCR targeting the L-segment. Hantavirus RNA detection by RT-nPCR in urine and serum may aid the rapid diagnosis of HFRS during the early phase of the disease. In particular, HFRS should not be ruled out based on negative RT-PCR results in serum, and RT-PCR should be performed using urine as well as serum during the early phase of symptoms.