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An outbreak of SARS-CoV-2 Alpha variant (Pango lineage B.1.1.7) was detected at a primary school (School X) in Lansingerland, the Netherlands, in December 2020. The outbreak was studied retrospectively, and population-based screening was used to assess the extent of virus circulation and decelerate transmission. Cases were SARS-CoV-2 laboratory confirmed and were residents of Lansingerland (November 16th 2020 until February 22th 2021), or had an epidemiological link with School X or neighbouring schools. The SARS-CoV-2 variant was determined using variant PCR or whole genome sequencing. A questionnaire primarily assessed clinical symptoms. A total of 77 Alpha variant cases were found with an epidemiological link to School X, 16 Alpha variant cases linked to the neighbouring schools, and 146 Alpha variant cases among residents of Lansingerland without a link to the schools. The mean number of self-reported symptoms was not significantly different among Alpha variant infected individuals compared to non-Alpha infected individuals. The secondary attack rate (SAR) among Alpha variant exposed individuals in households was 52% higher compared to non-Alpha variant exposed individuals (p = 0.010), with the mean household age, and mean number of children and adults per household as confounders. Sequence analysis of 60 Alpha variant sequences obtained from cases confirmed virus transmission between School X and neighbouring schools, and showed that multiple introductions of the Alpha variant had already taken place in Lansingerland at the time of the study. The alpha variant caused a large outbreak at both locations of School X, and subsequently spread to neighbouring schools, and households. Population-based screening (together with other public health measures) nearly stopped transmission of the outbreak strain, but did not prevent variant replacement in the Lansingerland municipality. [ABSTRACT FROM AUTHOR]

Background: Performance of the SD Biosensor saliva antigen rapid test was evaluated at a large designated testing site in non-hospitalized patients, with or without symptoms. Method: All eligible people over 18 years of age presenting for a booked appointment at the designated SARS-CoV-2 testing site were approached for inclusion and enrolled following verbal informed consent. One nasopharyngeal swab was taken to carry out the default antigen rapid test from which the results were reported back to the patient and one saliva sample was self-taken according to verbal instruction on site. This was used for the saliva antigen rapid test, the RT-PCR and for virus culture. Sensitivity of the saliva antigen rapid test was analyzed in two ways: i, compared to saliva RT-PCR; and ii, compared to virus culture of the saliva samples. Study participants were also asked to fill in a short questionnaire stating age, sex, date of symptom onset. Recommended time of ≥30mins since last meal, drink or cigarette if applicable was also recorded. The study was carried out in February-March 2021 for 4 weeks. Results: We could include 789 people with complete records and results. Compared to saliva RT-PCR, overall sensitivity and specificity of the saliva antigen rapid test was 66.1% and 99.6% which increased to 88.6% with Ct ≤30 cutoff. Analysis by days post onset did not result in higher sensitivities because the large majority of people were in the very early phase of disease ie <3 days post onset. When breaking down the data for symptomatic and asymptomatic individuals, sensitivity ranged from 69.2% to 50% respectively, however the total number of RT-PCR positive asymptomatic participants was very low (n = 5). Importantly, almost all culture positive samples were detected by the rapid test. Conclusion: Overall, the potential benefits of saliva antigen rapid test, could outweigh the lower sensitivity compared to nasopharyngeal antigen rapid test in a comprehensive testing strategy, especially for home/self-testing and in vulnerable populations like elderly, disabled or children where in intrusive testing is either not possible or causes unnecessary stress. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) is world-wide a major cause of liver related morbidity and mortality. No vaccine is available to prevent HCV infection. To design an effective vaccine, understanding immunity against HCV is necessary. The memory B cell repertoire was characterized from an intravenous drug user who spontaneously cleared HCV infection 25 years ago. CD27+IgG+ memory B cells were immortalized using BCL6 and Bcl-xL. These immortalized B cells were used to study antibody-mediated immunity against the HCV E1E2 glycoproteins. Five E1E2 broadly reactive antibodies were isolated: 3 antibodies showed potent neutralization of genotype 1 to 4 using HCV pseudotyped particles, whereas the other 2 antibodies neutralized genotype 1, 2 and 3 or 1 and 2 only. All antibodies recognized non-linear epitopes on E2. Finally, except for antibody AT12-011, which recognized an epitope consisting of antigenic domain C /AR2 and AR5, all other four antibodies recognized epitope II and domain B. These data show that a subject, who spontaneously cleared HCV infection 25 years ago, still has circulating memory B cells that are able to secrete broadly neutralizing antibodies. Presence of such memory B cells strengthens the argument for undertaking the development of an HCV vaccine. [ABSTRACT FROM AUTHOR]

Background: Recent studies indicate that 27% of Dutch blood donors have evidence of past infection with HEV. However, the low number of diagnosed HEV infections indicates either an asymptomatic course or under diagnosis. Objectives: We investigated whether HEV is a cause of acute hepatitis in Dutch patients and which diagnostic modality (serology or PCR) should be used for optimal detection. Study design: Serum samples were retrospectively selected from non-severely immuno-compromised patients from a university hospital population, suspected of having an infectious hepatitis. Criteria were: elevated alanine aminotransferase (ALT> 34 U/l) and request for antibody testing for CMV, EBV or Hepatitis A (HAV). Results: All samples were tested for HEV using ELISA and PCR. Ninety patients/sera were tested, of which 22% were HEV IgG positive. Only one serum was IgM positive. HEV PCR was positive in two patients: one patient was both HEV IgM and IgG positive, the other patient was only IgG positive. Both HEV RNA positive samples belonged to genotype 3. Evidence of recent infection with CMV, EBV and HAV was found in 13%, 10% and 3% respectively. Conclusions: Although our study is limited by small numbers, we conclude that HEV is a cause of acute hepatitis in hospital associated patients in The Netherlands. Moreover, in our study population the prevalence of acute HAV (3%) was almost similar to acute HEV (2%). We propose to incorporate HEV testing in panels for acute infectious hepatitis. Negative results obtained for HEV IgM in a HEV PCR positive patient, indicates that antibody testing alone may not be sufficient and argues for PCR as a primary diagnostic tool in hospital associated patients. The high percentage of HEV IgG seropositivity confirms earlier epidemiological studies. [ABSTRACT FROM AUTHOR]

Background & Aims: Telaprevir, a hepatitis C virus NS3/4A protease inhibitor has significantly improved sustained viral response rates when given in combination with pegylated interferon alfa-2a and ribavirin, compared with current standard of care in hepatitis C virus genotype 1 infected patients. In patients with a failed sustained response, the emergence of drug-resistant variants during treatment has been reported. It is unclear to what extent these variants persist in untreated patients. The aim of this study was to assess using ultra-deep pyrosequencing, whether after 4 years follow-up, the frequency of resistant variants is increased compared to pre-treatment frequencies following 14 days of telaprevir treatment. Methods: Fifteen patients from 2 previous telaprevir phase 1 clinical studies (VX04-950-101 and VX05-950-103) were included. These patients all received telaprevir monotherapy for 14 days, and 2 patients subsequently received standard of care. Variants at previously well-characterized NS3 protease positions V36, T54, R155 and A156 were assessed at baseline and after a follow-up of 4±1.2 years by ultra-deep pyrosequencing. The prevalence of resistant variants at follow-up was compared to baseline. Results: Resistance associated mutations were detectable at low frequency at baseline. In general, prevalence of resistance mutations at follow-up was not increased compared to baseline. Only one patient had a small, but statistically significant, increase in the number of V36M and T54S variants 4 years after telaprevir-dosing. Conclusion: In patients treated for 14 days with telaprevir monotherapy, ultra-deep pyrosequencing indicates that longterm persistence of resistant variants is rare. [ABSTRACT FROM AUTHOR]

Background & Aims: Since acute hepatitis C virus (HCV) infection is often asymptomatic, it is difficult to examine the rate and determinants of spontaneous clearance. Consequently, these studies are subject to bias, which can potentially lead to biased rates of viral clearance and risk estimates. We evaluated determinants of spontaneous HCV clearance among HCV seroconverters identified in a unique community-based cohort. Methods: Subjects were 106 drug users with documented dates of HCV seroconversion from the Amsterdam Cohort Study. Logistic regression was used to examine sociodemographic, behavioral, clinical, viral and host determinants, measured around acute infection, of HCV clearance. Results: The spontaneous viral clearance rate was 33.0% (95% confidence interval (CI) 24.2-42.8). In univariate analyses female sex and fever were significantly associated with spontaneous clearance. The favorable genotypes for rs12979860 (CC) and rs8099917 (TT) were associated with spontaneous clearance, although borderline significant. In multivariate analysis, females with the favorable genotype for rs12979860 (CC) had an increased odds to spontaneously clear HCV infection (adjustedOR 6.62, 95% 2.69-26.13), whereas females with the unfavorable genotype were as likely as men with the favorable and unfavorable genotype to clear HCV. Chronic Hepatitis B infection and absence of HIV coinfection around HCV seroconversion also favor HCV clearance. Conclusions: This study shows that co-infection with HIV and HBV and genetic variation in the IL28B region play an important role in spontaneous clearance of HCV. Our findings suggest a possible synergistic interaction between female sex and IL28B in spontaneous clearance of HCV. [ABSTRACT FROM AUTHOR]

In 5-40% of respiratory infections in children, the diagnostics remain negative, suggesting that the patients might be infected with a yet unknown pathogen. Virus discovery cDNA-AFLP (VIDISCA) is a virus discovery method based on recognition of restriction enzyme cleavage sites, ligation of adaptors and subsequent amplification by PCR. However, direct discovery of unknown pathogens in nasopharyngeal swabs is difficult due to the high concentration of ribosomal RNA (rRNA) that acts as competitor. In the current study we optimized VIDISCA by adjusting the reverse transcription enzymes and decreasing rRNA amplification in the reverse transcription, using hexamer oligonucleotides that do not anneal to rRNA. Residual cDNA synthesis on rRNA templates was further reduced with oligonucleotides that anneal to rRNA but can not be extended due to 39-dideoxy-C6-modification. With these modifications .90% reduction of rRNA amplification was established. Further improvement of the VIDISCA sensitivity was obtained by high throughput sequencing (VIDISCA-454). Eighteen nasopharyngeal swabs were analysed, all containing known respiratory viruses. We could identify the proper virus in the majority of samples tested (11/18). The median load in the VIDISCA-454 positive samples was 7.2 E5 viral genome copies/ml (ranging from 1.4 E3-7.7 E6). Our results show that optimization of VIDISCA and subsequent high-throughputsequencing enhances sensitivity drastically and provides the opportunity to perform virus discovery directly in patient material. [ABSTRACT FROM AUTHOR]

In 5-40% of respiratory infections in children, the diagnostics remain negative, suggesting that the patients might be infected with a yet unknown pathogen. Virus discovery cDNA-AFLP (VIDISCA) is a virus discovery method based on recognition of restriction enzyme cleavage sites, ligation of adaptors and subsequent amplification by PCR. However, direct discovery of unknown pathogens in nasopharyngeal swabs is difficult due to the high concentration of ribosomal RNA (rRNA) that acts as competitor. In the current study we optimized VIDISCA by adjusting the reverse transcription enzymes and decreasing rRNA amplification in the reverse transcription, using hexamer oligonucleotides that do not anneal to rRNA. Residual cDNA synthesis on rRNA templates was further reduced with oligonucleotides that anneal to rRNA but can not be extended due to 3'-dideoxy-C6-modification. With these modifications >90% reduction of rRNA amplification was established. Further improvement of the VIDISCA sensitivity was obtained by high throughput sequencing (VIDISCA-454). Eighteen nasopharyngeal swabs were analysed, all containing known respiratory viruses. We could identify the proper virus in the majority of samples tested (11/18). The median load in the VIDISCA-454 positive samples was 7.2 E5 viral genome copies/ml (ranging from 1.4 E3-7.7 E6). Our results show that optimization of VIDISCA and subsequent high-throughput-sequencing enhances sensitivity drastically and provides the opportunity to perform virus discovery directly in patient material.