External quality assessment of orthohantavirus and lymphocytic choriomeningitis virus molecular detection and serology in Europe, 2021.

BackgroundRodent-borne viruses such as orthohantaviruses and arenaviruses cause considerable disease burden with regional and temporal differences in incidence and clinical awareness. Therefore, it is important to regularly evaluate laboratory diagnostic capabilities, e.g. by external quality assessments (EQA).AimWe wished to evaluate the performance and diagnostic capability of European expert laboratories to detect orthohantaviruses and lymphocytic choriomeningitis virus (LCMV) and human antibody response towards orthohantaviruses.MethodsWe conducted an EQA in 2021; molecular panels consisted of 12 samples, including different orthohantaviruses (Seoul, Dobrava-Belgrade (DOBV), Puumala (PUUV) and Hantaan orthohantavirus), LCMV and negative controls. Serological panels consisted of six human serum samples reactive to PUUV, DOBV or negative to orthohantaviruses. The EQA was sent to 25 laboratories in 20 countries.ResultsThe accuracy of molecular detection of orthohantaviruses varied (50‒67%, average 62%) among 16 participating laboratories, while LCMV samples were successfully detected in all 11 participating laboratories (91-100%, average 96%). The accuracy of serological diagnosis of acute and past orthohantavirus infections was on average 95% among 20 participating laboratories and 82% in 19 laboratories, respectively. A variety of methods was used, with predominance of in-house assays for molecular tests, and commercial assays for serological ones.ConclusionSerology, the most common tool to diagnose acute orthohantavirus infections, had a high accuracy in this EQA. The molecular detection of orthohantaviruses needs improvement while LCMV detection (performed in fewer laboratories) had 95% accuracy. Further EQAs are recommended to be performed periodically to monitor improvements and challenges in the diagnostics of rodent-borne diseases.

Comparative study between virus neutralisation testing and other serological methods detecting anti-SARS-CoV-2 antibodies in Europe, 2021.

One consequence of the ongoing coronavirus disease pandemic was the rapid development of both in-house and commercial serological assays detecting anti-SARS-CoV-2 antibodies, in an effort to reliably detect acute and past SARS-CoV-2 infections. It is crucial to evaluate the quality of these serological tests and consequently the sero-epidemiological studies that are performed with the respective tests. Here, we describe the set-up and results of a comparative study, in which a laboratory contracted by the European Centre for Disease Prevention and Control offered a centralised service to EU/EEA Member and pre-accession Member States to test representative serum specimens with known serological results, with the gold standard technique (virus neutralisation tests) to determine the presence of neutralising antibodies. Laboratories from 12 European countries shared 719 serum specimens with the contractor laboratory. We found that in-house serological tests detecting neutralising antibodies showed the highest percent agreement, both positive and negative, with the virus neutralisation test results. Despite extensive differences in virus neutralisation protocols neutralisation titres showed a strong correlation. From the commercial assays, the best positive percent agreement was found for SARS-CoV-2 IgG (sCOVG) (Siemens - Atellica IM Analyzer). Despite lower positive percent agreement of LIAISON SARS-CoV-2 TrimericS IgG kit (Diasorin Inc.), the obtained results showed relatively good correlation with neutralisation titres. The set-up of this study allowed for high comparability between laboratories and enabled laboratories that do not have the capacity or capability to perform VNTs themselves. Given the variety of in-house protocols detecting SARS-CoV-2 specific neutralising antibodies, including the virus strain, it could be of interest to select reference isolates for SARS-CoV-2 diagnostic to be made available for interested EU Member States and pre-accession countries.

Seroprevalence of Toxoplasma gondii and associated risk factors for infection in the Netherlands: third cross-sectional national study.

A third nationally representative serosurvey was performed to study the changes in Toxoplasma gondii (T. gondii) seroprevalence in the Netherlands over a 20-year time span and to identify and confirm risk factors for acquired toxoplasmosis. This cross-sectional study (conducted in 2016/2017) was designed similarly to the previous two studies (1995/1996 and 2006/2007) and included a questionnaire and serum sampling among Dutch residents. Factors associated with seropositivity for T. gondii were determined using multivariable analysis of the questionnaire-derived data. The earlier observed decrease in T. gondii seroprevalence between 1995/1996 and 2006/2007 (from 40.5% to 26.0%) did not continue into 2016/2017 (29.9%). Similarly to the previous studies, the seroprevalence increased with age and varied among regions. In all studies, higher T. gondii seropositivity was associated with increasing age, lower educational level, not living in the Southeast, and eating raw or semi-cooked pork. The incidence of congenital toxoplasmosis was estimated at 1.3/1000 (95% CI 0.9-1.8) live-born children in 2017. As the seroprevalence of T. gondii in the Netherlands did not decrease over the last decade, an increase in public health awareness is needed and prevention measures may need to be taken to achieve a further reduction in T. gondii infections in the Netherlands.

A Comprehensive Sampling Study on SARS-CoV-2 Contamination of Air and Surfaces in a Large Meat Processing Plant Experiencing COVID-19 Clusters in June 2020.

OBJECTIVE: We aimed to assess SARS-CoV-2 contamination of air and surfaces to gain insight into potential occupational exposure in a large meat processing plant experiencing COVID-19 clusters. Methods: Oro-nasopharyngeal SARS-CoV-2 screening was performed in 76 workers. Environmental samples ( n = 275) including air, ventilation systems, sewage, and swabs of high-touch surfaces and workers' hands were tested for SARS-CoV-2 RNA by real-time quantitative polymerase chain reaction. Results: Twenty-seven (35.5%) of the (predominantly asymptomatic) workers tested positive with modest to low viral loads (cycle threshold ≥ 29.7). Six of 203 surface swabs, 1 of 12 personal air samples, and one of four sewage samples tested positive; other samples tested negative. Conclusions: Although one third of workers tested positive, environmental contamination was limited. Widespread SARS-CoV-2 transmission via air and surfaces was considered unlikely within this plant at the time of investigation while strict COVID-19 control measures were already implemented.

Sensitivity of Detection and Variant Typing of SARS-CoV-2 in European Laboratories.

The molecular detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is key for clinical management and surveillance. Funded by the European Centre for Disease Prevention and Control, we conducted an external quality assessment (EQA) on the molecular detection and variant typing of SARS-CoV-2 that included 59 European laboratories in 34 countries. The EQA panel consisted of 12 lyophilized inactivated samples, 10 of which were SARS-CoV-2 variants (Alpha, Beta, Gamma, Delta, Epsilon, Eta, parental B.1 strain) ranging from 2.5 to 290.0 copies/µL or pooled respiratory viruses (adenovirus, enterovirus, influenza virus A, respiratory syncytial virus, or human coronaviruses 229E and OC43). Of all participants, 72.9% identified the presence of SARS-CoV-2 RNA correctly. In samples containing 25.0 or more genome copies/µL, SARS-CoV-2 was detected by 98.3% of the participating laboratories. Laboratories applying commercial tests scored significantly better (P < 0.0001, Kruskal-Wallis test) than those using in-house assays. Both the molecular detection and the typing of the SARS-CoV-2 variants were associated with the RNA concentrations (P < 0.0001, Kruskal-Wallis test). On average, only 5 out of the 10 samples containing different SARS-CoV-2 variants at different concentrations were correctly typed. The identification of SARS-CoV-2 variants was significantly more successful among EQA participants who combined real-time reverse transcription polymerase chain reaction (RT-PCR)-based assays for mutation detection and high-throughput genomic sequencing than among those who used a single methodological approach (P = 0.0345, Kruskal-Wallis test). Our data highlight the high sensitivity of SARS-CoV-2 detection in expert laboratories as well as the importance of continuous assay development and the benefits of combining different methodologies for accurate SARS-CoV-2 variant typing.

Increase in invasive disease caused by Haemophilus influenzae b, the Netherlands, 2020 to 2021.

The incidence of most respiratory-transmitted diseases decreased during the COVID-19 pandemic as a result of containment measures. In contrast, in the Netherlands we noted an increase in invasive disease caused by Haemophilus influenzae b (Hib) (from < 0.3/100,000 before 2019 to 0.39 and 0.33/100,000 in 2020 and 2021) in vaccinated and unvaccinated age groups. We did not find a change in vaccine effectiveness against Hib invasive disease (effectiveness > 90%). We discuss factors that may have contributed to this rise.

Towards a sensitive and accurate interpretation of molecular testing for SARS-CoV-2: a rapid review of 264 studies.

BackgroundSensitive molecular diagnostics and correct test interpretation are crucial for accurate COVID-19 diagnosis and thereby essential for good clinical practice. Furthermore, they are a key factor in outbreak control where active case finding in combination with isolation and contact tracing are crucial.AimWith the objective to inform the public health and laboratory responses to the pandemic, we reviewed current published knowledge on the kinetics of SARS-CoV-2 infection as assessed by RNA molecular detection in a wide range of clinical samples.MethodsWe performed an extensive search on studies published between 1 December 2019 and 15 May 2020, reporting on molecular detection and/or isolation of SARS-CoV-2 in any human laboratory specimen.ResultsWe compiled a dataset of 264 studies including 32,515 COVID-19 cases, and additionally aggregated data points (n = 2,777) from sampling of 217 adults with known infection timeline. We summarised data on SARS-CoV-2 detection in the respiratory and gastrointestinal tract, blood, oral fluid, tears, cerebrospinal fluid, peritoneal fluid, semen, vaginal fluid; where provided, we also summarised specific observations on SARS-CoV-2 detection in pregnancy, infancy, children, adolescents and immunocompromised individuals.ConclusionOptimal SARS-CoV-2 molecular testing relies on choosing the most appropriate sample type, collected with adequate sampling technique, and with the infection timeline in mind. We outlined knowledge gaps and directions for future well-documented systematic studies.

Co-detection of the measles vaccine and wild-type virus by real-time PCR: public health laboratory protocol.

In rare cases vaccination with the measles virus vaccine genotype A (MeVA) may cause a vaccine reaction with clinical signs similar to infection with wild-type measles virus (MeVwt). Rapid differentiation between MeVA and MeVwt infection is important for taking adequate public health measures. Recently, a few MeVA real-time reverse-transcription quantitative PCR methods (RT-qPCRs) were described that can distinguish between MeVA and MeVwt. However, detection of MeVA does in theory not exclude infection with MeVwt. In the present study, we established a protocol for determination of co-infections with MeVA and MeVwt. To this end, MeVA RT-qPCRs were used in combination with the routine measles virus (MeV) RT-qPCR, and the results suggested that the differences between the RT-qPCR Ct values (delta Ct, ∆Ct) could be used as criteria. Subsequently, we tested samples from vaccine-associated measles cases that were confirmed by genotyping. In addition, experimental mixtures of MeVA and MeVwt were tested in different concentrations. All tested MeVA clinical samples had ∆Ct ≤3.6. The results of experimental mixtures showed a mean ∆Ct ≤2.8 for genotype A alone and >3.2 when combined with either genotype B3 or D8. The results of a receiver operator characteristic analysis indicated that the optimum ∆Ct for use as a cut-off value was 3.5, while with ∆Ct values of 2.9 and 3.7 sensitivity and specificity were respectively 1.00. Thus, ∆Ct could be used to exclude the presence of MeVwt if MeVA is detected and ∆Ct is <2.9, while ∆Ct >3.7 were highly suggestive of co-infection and ≥2.9 ∆Ct <3.7 warranted additional confirmation, such as next-generation sequencing. This RT-qPCR-based protocol could be used for the exclusion of infection with MeVwt in cases with vaccine-associated measles reaction, crucial for the timely implementation of public health prevention and control measures.

Total HIV-1 DNA Dynamics and Influencing Factors in Long-Term ART-Treated Japanese Adults: A Retrospective Longitudinal Analysis.

BACKGROUND: Understanding HIV persistence in treated patients is an important milestone toward drug-free control. We aimed at analyzing total HIV DNA dynamics and influencing factors in Japanese patients who received more than a decade of suppressive antiretroviral treatment (ART). METHODS: A retrospective study including clinical records and 840 peripheral blood mononuclear cells samples (mean 14 samples/patient) for 59 patients (92% male) was performed. Subjects were divided into 2 groups: with and without hematological comorbidity (mainly hemophilia) plus hepatitis C virus coinfection. Total HIV DNA was measured in peripheral blood mononuclear cells by quantitative polymerase chain reaction. The dynamics, regression over time, and influence of antiretrovirals by group were estimated using a novel regression model (R software v 3.2.3). RESULTS: Total HIV DNA decreased on ART initiation, and subsequently, its dynamics varied between groups with previously undescribed fluctuations. If calculated by on-treatment, the mean total HIV DNA levels were similar. The comorbidity group had unstable levels showing different regression over time (P = 0.088/0.094 in year 1/after year 8 of ART) and significantly different treatment responses as shown by antiretroviral group switching estimates. Furthermore, curing hepatitis C virus in hemophiliacs did not significantly alter total HIV DNA levels or regression. CONCLUSIONS: Our data identified some effects of the long-term treatment on total HIV DNA levels and highlighted the partial influence of comorbidities and coinfections. Total HIV DNA monitoring contributed to therapy response estimates and HIV reservoir quantification. The results suggest that HIV DNA monitoring during ART might be useful as a persistence marker in both HIV-monoinfected patients and those with comorbidities and coinfections.