Current status of pathogen handling in European laboratories: focus on viral inactivation process.

For handling safely infectious agents, European laboratories must comply with specific EC Directives, national regulations and recommendations from the World Health Organization (WHO). To prevent laboratory acquired infections (LAIs) and pathogens dissemination, a key biosafety rule requires that any infectious material (clinical specimens or research samples) manipulated outside a biosafety cabinet (BSC) must be inactivated unless the lack of infectivity is proven. This inactivation process is a crucial step for biosafety and must be guided by a rigorous experimental qualification and validation procedure. However, for diagnostic or research laboratories, this process is not harmonized with common standard operation procedures (SOPs) but based on individual risk assessment and general international guidelines which can pose problems in emergency situations such as major outbreaks or pandemics. This review focuses on viral inactivation method, outlining the current regulatory framework, its limitations and a number of ways in which biosafety can be improved.

Comparing the Performance of Three Models Incorporating Weather Data to Forecast Dengue Epidemics in Reunion Island, 2018-2019.

We developed mathematical models to analyze a large dengue virus (DENV) epidemic in Reunion Island in 2018-2019. Our models captured major drivers of uncertainty including the complex relationship between climate and DENV transmission, temperature trends, and underreporting. Early assessment correctly concluded that persistence of DENV transmission during the austral winter 2018 was likely and that the second epidemic wave would be larger than the first one. From November 2018, the detection probability was estimated at 10%-20% and, for this range of values, our projections were found to be remarkably accurate. Overall, we estimated that 8% and 18% of the population were infected during the first and second wave, respectively. Out of the 3 models considered, the best-fitting one was calibrated to laboratory entomological data, and accounted for temperature but not precipitation. This study showcases the contribution of modeling to strengthen risk assessments and planning of national and local authorities.

Deep Proteomics Network and Machine Learning Analysis of Human Cerebrospinal Fluid in Japanese Encephalitis Virus Infection.

Japanese encephalitis virus is a leading cause of neurological infection in the Asia-Pacific region with no means of detection in more remote areas. We aimed to test the hypothesis of a Japanese encephalitis (JE) protein signature in human cerebrospinal fluid (CSF) that could be harnessed in a rapid diagnostic test (RDT), contribute to understanding the host response and predict outcome during infection. Liquid chromatography and tandem mass spectrometry (LC-MS/MS), using extensive offline fractionation and tandem mass tag labeling (TMT), enabled comparison of the deep CSF proteome in JE vs other confirmed neurological infections (non-JE). Verification was performed using data-independent acquisition (DIA) LC-MS/MS. 5,070 proteins were identified, including 4,805 human proteins and 265 pathogen proteins. Feature selection and predictive modeling using TMT analysis of 147 patient samples enabled the development of a nine-protein JE diagnostic signature. This was tested using DIA analysis of an independent group of 16 patient samples, demonstrating 82% accuracy. Ultimately, validation in a larger group of patients and different locations could help refine the list to 2-3 proteins for an RDT. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD034789 and 10.6019/PXD034789.

SARS-CoV-2 IgG seroprevalence surveys in blood donors before the vaccination campaign, France 2020-2021.

We conducted a cross-sectional study for SARS-CoV-2 anti-S1 IgG prevalence in French blood donors (n = 32605), from March-2020 to January-2021. A mathematical model combined seroprevalence with a daily number of hospital admissions to estimate the probability of hospitalization upon infection and determine the number of infections while correcting for antibody decay. There was an overall seroprevalence increase over the study period and we estimate that ∼15% of the French population had been infected by SARS-CoV-2 by January-2021. The infection/hospitalization ratio increased with age, from 0.31% (18-30yo) to 4.5% (61-70yo). Half of the IgG-S1 positive individuals had no detectable antibodies 4 to 5 months after infection. The seroprevalence in group O donors (7.43%) was lower (p = 0.003) than in A, B, and AB donors (8.90%). We conclude, based on seroprevalence data and mathematical modeling, that a large proportion of the French population was unprotected against severe disease prior to the vaccination campaign.

In vitro activity of therapeutic antibodies against SARS-CoV-2 Omicron BA.1, BA.2 and BA.5.

The replacement of the Omicron BA.1 variant of SARS-CoV-2 by the BA.2 and the rapid growth of the BA.5 sub lineage, which have both different sets of mutations in the spike glycoprotein, alters the spectrum of activity of therapeutic antibodies currently licensed in the European Union. Using clinical strains of the Omicron BA.2 and BA.5 variants, we compared the neutralising power of monoclonal antibodies against the Omicron BA.1, BA.2 and BA.5 variants, using an ancestral strain (lineage B.1, D614G) and a Delta variant strain as reference. Sotrovimab/Vir-7831 is less active against BA.2 than against BA.1 (fold change reduction ~ 1,4) and even less active against BA.5 (fold change reduction ~ 2.7). Within the Evusheld /AZD7442 cocktail, Cilgavimab/AZD1061 is more active against BA.2 and BA.5 than against BA.1 (fold change increase ~ 32), whilst the very low activity of Tixagevimab/AZD8895 against BA.1 is not enhanced against BA.2 nor BA.5. In total, compared to BA.1, the activity of the Evusheld/AZD7442 is significantly improved against BA.2 while BA.5 is intermediate but closer to BA.2.

Immunoglobulin M seroneutralization for improved confirmation of Japanese encephalitis virus infection in a flavivirus-endemic area.

BACKGROUND: The mainstay of diagnostic confirmation of acute Japanese encephalitis (JE) involves detection of anti-JE virus (JEV) immunoglobulin M (IgM) by enzyme-linked immunosorbent assay (ELISA). Limitations in the specificity of this test are increasingly apparent with the introduction of JEV vaccinations and the endemicity of other cross-reactive flaviviruses. Virus neutralization testing (VNT) is considered the gold standard, but it is challenging to implement and interpret. We performed a pilot study to assess IgG depletion prior to VNT for detection of anti-JEV IgM neutralizing antibodies (IgM-VNT) as compared with standard VNT. METHODS: We evaluated IgM-VNT in paired sera from anti-JEV IgM ELISA-positive patients (JE n=35) and negative controls of healthy flavivirus-naïve (n=10) as well as confirmed dengue (n=12) and Zika virus (n=4) patient sera. IgM-VNT was subsequently performed on single sera from additional JE patients (n=76). RESULTS: Anti-JEV IgG was detectable in admission serum of 58% of JE patients. The positive, negative and overall percentage agreement of IgM-VNT as compared with standard VNT was 100%. A total of 12/14 (86%) patient samples were unclassified by VNT and, with sufficient sample available for IgG depletion and IgG ELISA confirming depletion, were classified by IgM-VNT. IgM-VNT enabled JE case classification in 72/76 (95%) patients for whom only a single sample was available. CONCLUSIONS: The novel approach has been readily adapted for high-throughput testing of single patient samples and it holds promise for incorporation into algorithms for use in reference centres.

COVID-19 convalescent plasma: Evolving strategies for serological screening in France.

Quantitation of anti-SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) neutralizing antibodies (Nabs) is a key parameter in determining the effective dose for treatment with COVID-19 convalescent plasma (CCP). Interpretation of results from clinical trials conducted worldwide requires comparison of Nabs titres obtained from different methods. As virus neutralization tests (VNTs) are not standardized scalable or commercially available, strategies based on intensity of ELISA (Enzyme Linked Immunosorbent Assay) or chemiluminescent binding serological tests were implemented to allow comparisons and establish criteria for determining 'high-titres' of anti-SARS-CoV-2 antibodies (Abs). To this end, the FDA (Food and Drug Administration) has proposed criteria to define high-titre plasmas using different serological assays, including the one used in France for the CCP SARS-CoV-2 Abs screening (Euroimmun anti-S1 IgG). A retrospective study revealed that when using the FDA criteria (ELISA signal-to-cut-off [S/C ratio] ≥3.5), 91% of CCP had Nabs titres ≥40 as assessed with an in-house VNT. French strategy to ensure sufficient stocks of CCP of increasing titre has evolved over time. Recently, we improved our strategy by collecting only plasma from vaccinated convalescent donors as we confirmed that the mean IgG antibody level (ELISA S/C ratio) was significantly higher in plasma from vaccinated convalescent donors compared to donations from unvaccinated convalescent donors: 9.31 (CI 95%: 8.46-10.16) versus 3.22 (CI 95%: 3.05-3.39) (p < 0.001).

Model-based assessment of Chikungunya and O'nyong-nyong virus circulation in Mali in a serological cross-reactivity context.

Serological surveys are essential to quantify immunity in a population but serological cross-reactivity often impairs estimates of the seroprevalence. Here, we show that modeling helps addressing this key challenge by considering the important cross-reactivity between Chikungunya (CHIKV) and O'nyong-nyong virus (ONNV) as a case study. We develop a statistical model to assess the epidemiology of these viruses in Mali. We additionally calibrate the model with paired virus neutralization titers in the French West Indies, a region with known CHIKV circulation but no ONNV. In Mali, the model estimate of ONNV and CHIKV prevalence is 30% and 13%, respectively, versus 27% and 2% in non-adjusted estimates. While a CHIKV infection induces an ONNV response in 80% of cases, an ONNV infection leads to a cross-reactive CHIKV response in only 22% of cases. Our study shows the importance of conducting serological assays on multiple cross-reactive pathogens to estimate levels of virus circulation.

ZIKA Virus Neutralizing Antibody Kinetics in Antenatally Exposed Infants.

BACKGROUND: Zika virus (ZIKV) is associated with severe congenital abnormalities and laboratory diagnosis of antenatal infection is difficult. Here we evaluated ZIKV neutralizing antibody (nAb) kinetics in infants born to mothers with PCR-confirmed ZIKV infection during pregnancy. METHODS: Neonates (n = 98) had serum specimens tested repeatedly for ZIKV nAb over the first 2 years of life using virus neutralization test (VNT). ZIKV neonatal infection was confirmed by RT-PCR in blood or urine and/or presence of ZIKV IgM antibodies, and results were correlated with infant clinical features. RESULTS: Postnatal laboratory evidence of ZIKV vertical transmission was obtained for 60.2% of children, while 32.7% exhibited clinical abnormalities. Congenital abnormalities were found in 37.3% of children with confirmed ZIKV infection and 31.0% of children without confirmed infection (P = .734). All but 1 child displayed a physiologic decline in ZIKV nAb, reflecting maternal antibody decay, despite an early ZIKV-IgM response in one-third of infants. CONCLUSIONS: Infants with antenatal ZIKV exposure do not develop ZIKV nAb despite an early IgM response. Therefore, ZIKV VNT in children is not useful for diagnosis of congenital infection. In light of these findings, it remains to be determined if children infected in utero are potentially susceptible to reinfection.

Lower prevalence of antibodies neutralizing SARS-CoV-2 in group O French blood donors.

We investigated the distribution of antibodies neutralizing SARS-CoV-2 according to age, sex or blood group in French blood donors. In 464 samples collected before the emergence of SARS-CoV-2 (2017 and 2018), our virus neutralization assay had a 100% specificity. It was used to test 998 samples collected from blood donors during the last week of March or the first week of April 2020. As expected at this stage of the outbreak, the prevalence was low (2.7%) and, importantly, criteria for blood donation imply that the vast majority of seropositives had asymptomatic or pauci-symptomatic SARS-CoV-2 infections. Seroprevalence values did not differ significantly among age groups (but were slightly higher in donors <30yo and ≥60yo), and between males and females (2.82% vs 2.69%), unlike what has been observed regarding hospitalizations admission to ICU and death rates in France. By contrast, we observed that the proportion of seropositives was significantly lower in group O donors (1.32% vs 3.86% in other donors, p = 0.014). We conclude that virus infection seems to occur with a similar incidence in men and women among French blood donors, but that blood group O persons are less at risk of being infected and not only of suffering from severe clinical presentations, as previously suggested.

Heat Inactivation of Different Types of SARS-CoV-2 Samples: What Protocols for Biosafety, Molecular Detection and Serological Diagnostics?

Standard precautions to minimize the risk of SARS-CoV-2 transmission implies that infected cell cultures and clinical specimens may undergo some sort of inactivation to reduce or abolish infectivity. We evaluated three heat inactivation protocols (56 °C-30 min, 60 °C-60 min and 92 °C-15 min) on SARS-CoV-2 using (i) infected cell culture supernatant, (ii) virus-spiked human sera (iii) and nasopharyngeal samples according to the recommendations of the European norm NF EN 14476-A2. Regardless of the protocol and the type of samples, a 4 Log10 TCID50 reduction was observed. However, samples containing viral loads > 6 Log10 TCID50 were still infectious after 56 °C-30 min and 60 °C-60 min, although infectivity was < 10 TCID50. The protocols 56 °C-30 min and 60 °C-60 min had little influence on the RNA copies detection, whereas 92 °C-15 min drastically reduced the limit of detection, which suggests that this protocol should be avoided for inactivation ahead of molecular diagnostics. Lastly, 56 °C-30 min treatment of serum specimens had a negligible influence on the results of IgG detection using a commercial ELISA test, whereas a drastic decrease in neutralizing titers was observed.

Evaluation of Chemical Protocols for Inactivating SARS-CoV-2 Infectious Samples.

Clinical samples collected in coronavirus disease 19 (COVID-19), patients are commonly manipulated in biosafety level 2 laboratories for molecular diagnostic purposes. Here, we tested French norm NF-EN-14476+A2 derived from European standard EN-14885 to assess the risk of manipulating infectious viruses prior to RNA extraction. SARS-CoV-2 cell-culture supernatant and nasopharyngeal samples (virus-spiked samples and clinical samples collected in COVID-19 patients) were used to measure the reduction of infectivity after 10 minute contact with lysis buffer containing various detergents and chaotropic agents. A total of thirteen protocols were evaluated. Two commercially available formulations showed the ability to reduce infectivity by at least 6 log 10, whereas others proved less effective.

Prolonged Infectivity of SARS-CoV-2 in Fomites.

We spotted severe acute respiratory syndrome coronavirus 2 on polystyrene plastic, aluminum, and glass for 96 hours with and without bovine serum albumin (3 g/L). We observed a steady infectivity (<1 log10 drop) on plastic, a 3.5 log10 decrease on glass, and a 6 log10 drop on aluminum. The presence of proteins noticeably prolonged infectivity.

Zika Virus Circulation in Mali.

The circulation of Zika virus (ZIKV) in Mali has not been clearly characterized. Therefore, we conducted a serologic survey of 793 asymptomatic volunteers >15 years of age (2016), and 637 blood donors (2013) to assess the seroprevalence of ZIKV infection in 2 ecoclimatic regions of Mali, tropical savannah and warm semiarid region, using ELISA and seroneutralization assays. The overall seroprevalence was ≈12% and increased with age, with no statistical difference between male and female participants. In the warm semiarid study sites we detected immunological markers of an outbreak that occurred in the late 1990s in 18% (95% CI 13%-23%) of participants. In tropical savannah sites, we estimated a low rate of endemic transmission, with 2.5% (95% CI 2.0%-3.1%) of population infected by ZIKV annually. These data demonstrate the circulation of ZIKV in Mali and provide evidence of a previously unidentified outbreak that occurred in the late 1990s.

Low Zika Virus Seroprevalence in Vientiane, Laos, 2003-2015.

Zika virus (ZIKV) has been presumed to be endemic in Southeast Asia (SEA), with a low rate of human infections. Although the first ZIKV evidence was obtained in the 1950s through serosurveys, the first laboratory-confirmed case was only detected in 2010 in Cambodia. The epidemiology of ZIKV in SEA remains uncertain because of the scarcity of available data. From 2016, subsequent to the large outbreaks in the Pacific and Latin America, several Asian countries started reporting increasing numbers of confirmed ZIKV patients, but no global epidemiological assessment is available to date. Here, with the aim of providing information on ZIKV circulation and population immunity, we conducted a seroprevalence study among blood donors in Vientiane, Laos. Sera from 359 asymptomatic consenting adult donors in 2003-2004 and 687 in 2015 were screened for anti-ZIKV IgG using NS1 ELISA assay (Euroimmun, Luebeck, Germany). Positive and equivocal samples were confirmed for anti-ZIKV-neutralizing antibodies by virus neutralization tests. Our findings suggest that ZIKV has been circulating in Vientiane over at least the last decade. Zika virus seroprevalence observed in the studied blood donors was low, 4.5% in 2003-2004 with an increase in 2015 to 9.9% (P = 0.002), possibly reflecting the increase of ZIKV incident cases reported over this period. We did not observe any significant difference in seroprevalence according to gender. With a low herd immunity in the Vientiane population, ZIKV represents a risk for future large-scale outbreaks. Implementation of a nationwide ZIKV surveillance network and epidemiological studies throughout the country is needed.

Correction to: Combination of ELISA screening and seroneutralisation tests to expedite Zika virus seroprevalence studies.

In the original publication of this article [1], Table 1 has some errors.

Combination of ELISA screening and seroneutralisation tests to expedite Zika virus seroprevalence studies.

Here we propose a strategy allowing implementing efficient and practicable large-scale seroepidemiological studies for Zika Virus (ZIKV). It combines screening by a commercial NS1 protein-based Zika IgG ELISA, and confirmation by a cytopathic effect-based virus neutralization test (CPE-based VNT). In post-epidemic samples from Martinique Island blood donors (a population with a dengue seroprevalence above 90%), this strategy allowed reaching specificity and sensitivity values over 98%. The CPE-based VNT consists of recording CPE directly under the optical microscope, which is easy to identify with ZIKV strain H/PF/2013 at day 5 pi. Overall, considered that CPE-based VNT is cost effective and widely automatable, the NS1 protein-based Zika IgG ELISA+CPE-based VNT combination strategy represents a convenient tool to expedite ZIKV seroprevalence studies.

Zika virus epidemiology in Bolivia: A seroprevalence study in volunteer blood donors.

BACKGROUND: Zika virus (ZIKV), was widely reported in Latin America and has been associated with neuropathologies, as microcephaly, but only few seroprevalence studies have been published to date. Our objective was to determine the seroprevalence amongst Bolivian blood donors and estimate the future potential circulation of the virus. METHODOLOGY: A ZIKV seroprevalence study was conducted between December 2016 and April 2017 in 814 asymptomatic Bolivian volunteer blood donors residing in various eco-environments corresponding to contrasting entomological activities. It was based on detection of IgG to ZIKV using NS1 ELISA screening, followed by a seroneutralisation test in case of positive or equivocal ELISA result. CONCLUSIONS/SIGNIFICANCE: Analysis revealed that ZIKV circulation occurred in tropical areas (Beni: 39%; Santa Cruz de la Sierra: 21.5%) but not in highlands (~0% in Cochabamba, La Paz, Tarija). It was modulated by Aedes aegypti activity and the virus spread was not limited by previous immunity to dengue. Cases were geo-localised in a wide range of urban areas in Santa Cruz and Trinidad. No differences in seroprevalence related to gender or age-groups could be identified. It is concluded that ZIKV has been intensely circulating in the Beni region and has still a significant potential for propagating in the area of Santa Cruz.

Biosafety and Biosecurity in European Containment Level 3 Laboratories: Focus on French Recent Progress and Essential Requirements.

Even if European Union (EU) Member States are obliged to implement EU Directives 2000/54/EC on the protection of workers from risks related to exposure to biological agents at work, national biosafety regulations and practices varied from country to country. In fact, EU legislation on biological agents and genetically modified microorganisms is often not specific enough to ensure harmonization leading to difficulties in implementation for most laboratories. In the same way, biosecurity is a relatively new concept and a few EU Member States are known to have introduced national laboratory biosecurity legislation. In France, recent regulations have reinforced biosafety/biosecurity in containment level 3 (CL-3) laboratories but they concern a specific list of pathogens with no correlation in other European Members States. The objective of this review was to summarize European biosafety/biosecurity measures concerning CL-3 facilities focusing on French specificities. Essential requirements needed to preserve efficient biosafety measures when manipulating risk group 3 biological agents are highlighted. In addition, International, European and French standards related to containment laboratory planning, operation or biosafety equipment are described to clarify optimal biosafety and biosecurity requirements.