Viral Evolution and Immunology of SARS-CoV-2 in a Persistent Infection after Treatment with Rituximab.

BACKGROUND: Prolonged shedding of SARS-CoV-2 in immunocompromised patients has been described. Furthermore, an accumulation of mutations of the SARS-CoV-2 genome in these patients has been observed. METHODS: We describe the viral evolution, immunologic response and clinical course of a patient with a lymphoma in complete remission who had received therapy with rituximab and remained SARS-CoV-2 RT-qPCR positive for 161 days. RESULTS: The patient remained hospitalised for 10 days, after which he fully recovered and remained asymptomatic. A progressive increase in Ct-value, coinciding with a progressive rise in lymphocyte count, was seen from day 137 onward. Culture of a nasopharyngeal swab on day 67 showed growth of SARS-CoV-2. Whole genome sequencing (WGS) demonstrated that the virus belonged to the wildtype SARS-CoV-2 clade 20B/GR, but rapidly accumulated a high number of mutations as well as deletions in the N-terminal domain of its spike protein. CONCLUSION: SARS-CoV-2 persistence in immunocompromised individuals has important clinical implications, but halting immunosuppressive therapy might result in a favourable clinical course. The long-term shedding of viable virus necessitates customized infection prevention measures in these individuals. The observed accelerated accumulation of mutations of the SARS-CoV-2 genome in these patients might facilitate the origin of new VOCs that might subsequently spread in the general community.

Severity of COVID-19 among Hospitalized Patients: Omicron Remains a Severe Threat for Immunocompromised Hosts.

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in the general population in the context of a relatively high immunity gained through the early waves of coronavirus disease 19 (COVID-19), and vaccination campaigns. Despite this context, a significant number of patients were hospitalized, and identifying the risk factors associated with severe disease in the Omicron era is critical for targeting further preventive, and curative interventions. We retrospectively analyzed the individual medical records of 1501 SARS-CoV-2 positive hospitalized patients between 13 December 2021, and 13 February 2022, in Belgium, of which 187 (12.5%) were infected with Delta, and 1036 (69.0%) with Omicron. Unvaccinated adults showed an increased risk of moderate/severe/critical/fatal COVID-19 (crude OR 1.54; 95% CI 1.09-2.16) compared to vaccinated patients, whether infected with Omicron or Delta. In adults infected with Omicron and moderate/severe/critical/fatal COVID-19 (n = 323), immunocompromised patients showed an increased risk of in-hospital mortality related to COVID-19 (adjusted OR 2.42; 95% CI 1.39-4.22), compared to non-immunocompromised patients. The upcoming impact of the pandemic will be defined by evolving viral variants, and the immune system status of the population. The observations support that, in the context of an intrinsically less virulent variant, vaccination and underlying patient immunity remain the main drivers of severe disease.

Evaluation of the SARS-CoV-2 positivity ratio and upper respiratory tract viral load among asymptomatic individuals screened before hospitalization or surgery in Flanders, Belgium.

INTRODUCTION: The incidence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections in the Belgian community is mainly estimated based on test results of patients with coronavirus disease (COVID-19)-like symptoms. The aim of this study was to investigate the evolution of the SARS-CoV-2 reverse transcriptase polymerase chain reaction (RT-PCR) positivity ratio and distribution of viral loads within a cohort of asymptomatic patients screened prior hospitalization or surgery, stratified by age category. MATERIALS/METHODS: We retrospectively studied data on SARS-CoV-2 real-time RT-PCR detection in respiratory tract samples of asymptomatic patients screened pre-hospitalization or pre-surgery in nine Belgian hospitals located in Flanders over a 12-month period (1 April 2020-31 March 2021). RESULTS: In total, 255925 SARS-CoV-2 RT-PCR test results and 2421 positive results for which a viral load was reported, were included in this study. An unweighted overall SARS-CoV-2 real-time RT-PCR positivity ratio of 1.27% was observed with strong spatiotemporal differences. SARS-CoV-2 circulated predominantly in 80+ year old individuals across all time periods except between the first and second COVID-19 wave and in 20-30 year old individuals before the second COVID-19 wave. In contrast to the first wave, a significantly higher positivity ratio was observed for the 20-40 age group in addition to the 80+ age group compared to the other age groups during the second wave. The median viral load follows a similar temporal evolution as the positivity rate with an increase ahead of the second wave and highest viral loads observed for 80+ year old individuals. CONCLUSION: There was a high SARS-CoV-2 circulation among asymptomatic patients with a predominance and highest viral loads observed in the elderly. Moreover, ahead of the second COVID-19 wave an increase in median viral load was noted with the highest overall positivity ratio observed in 20-30 year old individuals, indicating they could have been the hidden drivers of this wave.

Improvement of a real-time RT-PCR assay for the detection of enterovirus RNA.

We describe an improvement of an earlier reported real-time RT-PCR assay for the detection of enterovirus RNA, based on the 5' exonuclease digestion of a dual-labeled fluorogenic probe by Taq DNA polymerase. A different extraction method, real-time RT-PCR instrument and primer set were evaluated. Our data show that the optimized assay yields a higher sensitivity and reproducibility and resulted in a significant reduced hands-on time per sample.

Distribution of HCV genotypes in Belgium from 2008 to 2015.

BACKGROUND: The knowledge of circulating HCV genotypes and subtypes in a country is crucial to guide antiviral therapy and to understand local epidemiology. Studies investigating circulating HCV genotypes and their trends have been conducted in Belgium. However they are outdated, lack nationwide representativeness or were not conducted in the general population. METHODS: In order to determine the distribution of different circulating HCV genotypes in Belgium, we conducted a multicentre study with all the 19 Belgian laboratories performing reimbursed HCV genotyping assays. Available genotype and subtype data were collected for the period from 2008 till 2015. Furthermore, a limited number of other variables were collected: some demographic characteristics from the patients and the laboratory technique used for the determination of the HCV genotype. RESULTS: For the study period, 11,033 unique records collected by the participating laboratories were used for further investigation. HCV genotype 1 was the most prevalent (53.6%) genotype in Belgium, with G1a and G1b representing 19.7% and 31.6%, respectively. Genotype 3 was the next most prevalent (22.0%). Further, genotype 4, 2, and 5 were responsible for respectively 16.1%, 6.2%, and 1.9% of HCV infections. Genotype 6 and 7 comprise the remaining <1%. Throughout the years, a stable distribution was observed for most genotypes. Only for genotype 5, a decrease as a function of the year of analysis was observed, with respectively 3.6% for 2008, 2.3% for 2009 and 1.6% for the remaining years. The overall M:F ratio was 1.59 and was mainly driven by the high M:F ratio of 3.03 for patients infected with genotype 3. Patients infected with genotype 3 are also younger (mean age 41.7 years) than patients infected with other genotypes (mean age above 50 years for all genotypes). The patients for whom a genotyping assay was performed in 2008 were younger than those from 2015. Geographical distribution demonstrates that an important number of genotyped HCV patients live outside the Belgian metropolitan cities. CONCLUSION: This national monitoring study allowed a clear and objective view of the circulating HCV genotypes in Belgium and will help health authorities in the establishment of cost effectiveness determinations before implementation of new treatment strategies. This baseline characterization of the circulating genotypes is indispensable for a continuous surveillance, especially for the investigation of the possible impact of migration from endemic regions and prior to the increasing use of highly potent direct-acting antiviral (DAA) agents.

Osteopontin synthesis and localization along the human nephron.

In normal human and rat kidneys, osteopontin (OPN) is present at the apical surface of cells in the distal nephron. After ischemic or toxic renal damage in rats, OPN is upregulated in distal tubular cells (DTC) and expressed de novo in perinuclear vesicles in proximal tubular cells (PTC). In the first phase of this study, OPN localization in ischemic human biopsies was compared with that in ischemic rat kidneys. In the second phase, cultures of PTC and DTC were used to investigate human renal OPN synthesis, secretion, and localization. OPN localization in human biopsies after renal ischemia was comparable to that in ischemic rat kidneys. Microscopic and flow cytometric detection of immunofluorescent OPN staining in tubular cell cultures demonstrated strong plasma membrane localization in DTC, whereas mainly perinuclear intracellular expression was observed in PTC. Northern blotting and reverse transcription-PCR demonstrated production of a single OPN mRNA in PTC and DTC. Detection of OPN by Western blotting and enzyme-linked immunosorbent assay demonstrated that PTC and DTC synthesized and secreted the same three molecular mass OPN forms, in comparable amounts. Finally, confocal microscopy demonstrated different staining patterns for endocytotic/lysosomal vesicles and perinuclear OPN; however, perinuclear OPN exhibited colocalization with the Golgi apparatus. In conclusion, human renal OPN localization in cell cultures demonstrated differences between PTC and DTC comparable to those observed after renal ischemia in vivo. Therefore, these cell cultures represented an excellent model for the study of human OPN synthesis, secretion, and localization in PTC versus DTC. It is reported for the first time that intracellular OPN is located in the Golgi apparatus of both PTC and DTC and that PTC and DTC are able to produce and secrete the same OPN isoforms, in comparable amounts.