Clinical Evaluation of Nasopharyngeal, Oropharyngeal, Nasal Swabs, and Saliva for the Detection of SARS-CoV-2 by Direct RT-PCR.

Nasopharyngeal swab (NPS) and oropharyngeal swab (OPS) are the most widely used upper respiratory tract specimens for diagnosis of SARS-CoV-2 using RT-qPCR. In contrast, nasal swab (NS) and saliva (SS), recently recommended by the WHO, are rarely used, and their test accuracy is limited. The method for direct RT-PCR detection of SARS-CoV-2 does not require an RNA extraction and is faster and easier than standard RT-PCR tests with RNA extraction. This study aimed to compare the diagnostic performance of upper respiratory tract samples for SARS-CoV-2 detection using the direct RT-PCR without preliminary heat inactivation. Here we report the application and validation of direct RT-PCR SARS-CoV-2 RNA on 165 clinical specimens of NPS/OP, and 36 samples of NS/NPS and 37 saliva samples (for the latter with prior deproteinization). The overall sensitivity estimates were 95.9%, 94.2%, 88.9%, and 94.6% for NPS/OPS/NS/SS samples, respectively, and the specificity was 100% against standard RT-PCR with RNA extraction. Overall, NS and SS testing by direct RT-PCR had sufficient sensitivity to detect SARS-CoV-2. They can be acceptable alternative to NPS/OPS for rapid detection of SARS-CoV-2 infections in future.

Identification of Hotspot Mutations in the N Gene of SARS-CoV-2 in Russian Clinical Samples That May Affect the Detection by Reverse Transcription-PCR.

Sensitive and reliable diagnostic test systems based on real-time PCR are of great importance in the fight against the ongoing SARS-CoV-2 pandemic. The genetic variability of the SARS-CoV-2 virus leads to the accumulation of mutations, some of which may affect the sensitivity of modern PCR assays. The aim of this study was to search in Russian clinical samples for new mutations in SARS-CoV-2 gene N that can affect the detection by RT-PCR. In this study, the polymorphisms in the regions of the target gene N causing failed or poor detection of the target N in the RT-PCR assay on 12 selected samples were detected. Sequencing the entire N and E genes in these samples along with other 195 samples that were positive for both target regions was performed. Here, we identified a number of nonsynonymous mutations and one novel deletion in the N gene that affected the ability to detect a target in the N gene as well a few mutations in the E gene of SARS-CoV-2 that did not affect detection. Sequencing revealed that majority of the mutations in the N gene were located in the variable region between positions 193 and 235 aa, inside and nearby the phosphorylated serine-rich region of the protein N. This study highlights the importance of the further characterization of the genetic variability and evolution of gene N, the most common target for detecting SARS-CoV-2. The use of at least two targets for detecting SARS-CoV-2, including one for the E gene, will be necessary for reliable diagnostics.

Detection of S-HBsAg Mutations in Patients with Hematologic Malignancies.

Multiple studies of hepatitis B virus (HBV) genetic variability and its relationship with the disease pathogenesis are currently ongoing, stemming from growing evidence of the clinical significance of HBV mutations. It is becoming increasingly evident that patients with hematologic malignancies may be particularly prone to a higher frequency of such mutations. The present report is the first extensive study of the prevalence of escape mutations in S-HBsAg, performed using isolates from 59 patients from hospital hematology departments with diagnoses of leukemia (n = 32), lymphoma (n = 20), multiple myeloma (n = 3), and non-tumor blood diseases (n = 4). The isolates were serologically examined for the presence of HBV markers and sequenced using either next-generation sequencing (NGS) or Sanger sequencing. Occult hepatitis B was found in 5.1% of cases. Genetic analysis of the region corresponding to S-HBsAg demonstrated an exceptionally high mutation frequency in patients with leukemias (93.4%) and lymphomas (85.0%), along with the prominent mutation heterogeneity. Additionally, more than 15 mutations in one sample were found in patients with leukemias (6.3% of cases) and lymphomas (5.0% of cases). Most of the mutations were clinically significant. The study analyzes the mutation profile of HBV in different oncohematological diseases and the frequency of individual mutations. The data strongly suggest that the NGS method, capable of detecting minor populations of HBV mutations, provides a diagnostic advantage, lays the foundation for the development of screening methods, and allows for the study of the virological and pathogenetic aspects of hepatitis B.