Rapid and reliable inactivation protocols for the diagnostics of emerging viruses: The example of SARS-CoV-2 and monkeypox virus.

The emergence and sustained transmission of novel pathogens are exerting an increasing demand on the diagnostics sector worldwide, as seen with the ongoing severe acute respiratory coronavirus 2 (SARS-CoV-2) pandemic and the more recent public health concern of monkeypox virus (MPXV) since May 2022. Appropriate and reliable viral inactivation measures are needed to ensure the safety of personnel handling these infectious samples. In the present study, seven commercialized diagnosis buffers, heat (56°C and 60°C), and sodium dodecyl sulfate detergent (2.0%, 1.0%, and 0.5% final concentrations) were tested against infectious SARS-CoV-2 and MPXV culture isolates on Vero cell culture. Cytopathic effects were observed up to 7 days postinoculation and viral load evolution was measured by semiquantitative polymerase chain reaction. The World Health Organization recommends an infectious titer reduction of at least 4 log10 . As such, the data show efficacious SARS-CoV-2 inactivation by all investigated methods, with >6.0 log10 reduction. MPXV inactivation was also validated with all investigated methods with 6.9 log10 reductions, although some commercial buffers required a longer incubation period to yield complete inactivation. These results are valuable for facilities, notably those without biosafety level-3 capabilities, that need to implement rapid and reliable protocols common against both SARS-CoV-2 and MPXV.

Determinants of protection against SARS-CoV-2 Omicron BA.1 and Delta infections in fully vaccinated outpatients.

We aimed to evaluate the association between the humoral and cellular immune responses and symptomatic SARS-CoV-2 infection with Delta or Omicron BA.1 variants in fully vaccinated outpatients. Anti-receptor binding domain (RBD) IgG levels and interferon-gamma (IFN-γ) release were evaluated at PCR-diagnosis of SARS-CoV-2 in 636 samples from negative and positive patients during Delta and Omicron BA.1 periods. Median levels of anti-RBD IgG in positive patients were significantly lower than in negative patients for both variants (p < 0.05). The frequency of Omicron BA.1 infection in patients with anti-RBD IgG concentrations ≥1000 binding antibody units (BAU)/mL was 51.0% and decreased to 34.4% in patients with concentrations ≥3000 BAU/mL. For Delta infection, the frequency of infection was significantly lower when applying the same anti-RBD IgG thresholds (13.3% and 5.3% respectively, p < 0.05). In addition, individuals in the hybrid immunity group had a 4.5 times lower risk of Delta infection compared to the homologous vaccination group (aOR = 0.22, 95% CI: [0.05-0.64]. No significant decrease in the risk of Omicron BA.1 infection was observed in the hybrid group compared to the homologous group, but the risk decreased within the hybrid group as anti-RBD IgG titers increased (aOR = 0.08, 95% CI: [0.01-0.41], p = 0.008). IFN-γ release post-SARS-CoV-2 peptide stimulation was not different between samples from patients infected (either with Delta or Omicron BA.1 variant) or not (p > 0.05). Our results show that high circulating levels of anti-RBD IgG and hybrid immunity were independently associated with a lower risk of symptomatic SARS-CoV-2 infection in outpatients with differences according to the infecting variant (www.clinicaltrials.gov; ID NCT05060939).

Two-step strategy for the identification of SARS-CoV-2 variant of concern 202012/01 and other variants with spike deletion H69-V70, France, August to December 2020.

We report the strategy leading to the first detection of variant of concern 202012/01 (VOC) in France (21 December 2020). First, the spike (S) deletion H69-V70 (ΔH69/ΔV70), identified in certain SARS-CoV-2 variants including VOC, is screened for. This deletion is associated with a S-gene target failure (SGTF) in the three-target RT-PCR assay (TaqPath kit). Subsequently, SGTF samples are whole genome sequenced. This approach revealed mutations co-occurring with ΔH69/ΔV70 including S:N501Y in the VOC.

Optimized nested PCR enhances biological diagnosis and phylogenetic analysis of human parvovirus B19 infections.

Diagnosis and epidemiological analysis of human parvovirus B19 (hB19V) infections are essential for disease management in severely ill patients. This study aimed to evaluate the performance of an optimized NS1-VP1u nested PCR for detection and sequencing of viruses in clinical samples using 224 clinical and five reference samples. PCR sensitivity, specificity, and positive and negative predictive values were perfect (100%). While phylogenetic analysis of a 615 bp-long fragment demonstrated that the viruses in all of the samples belonged to genotype 1, this study confirmed that this optimized PCR could detect all known hB19V with high performance.

Successful H1N1 influenza vaccination of children receiving chemotherapy for solid tumors.

BACKGROUND: The hemato-oncology community has been seriously concerned about the H1N1 pandemic of 2009. Here, we report on the evaluation of the immunogenicity and tolerability of H1N1v monovalent vaccines in young patients with cancer during this pandemic. PROCEDURE: Between December 7, 2009 and February 26, 2010, 20 children receiving chemotherapy for solid tumors at the Institute of Hematology and Pediatric Oncology of Lyon, were immunized by 2 doses of either AS03-adjuvanted or nonadjuvanted vaccine. The level of specific antibodies was assessed at D21 and D42. RESULTS: Seroconversion was observed in 13 of the 20 cases (65%), and 18 of 20 cases (90%) had protective titers after the 2 doses. Exploratory univariate analysis failed to show a significant influence of prevaccination lymphocyte counts on seroresponse rates. CONCLUSIONS: These data suggest that H1N1v monovalent vaccines were well tolerated by young cancer patients while on chemotherapy and achieved protective immune response in most cases.

SARS-CoV-2 Omicron Variant, Lineage BA.1, Is Associated with Lower Viral Load in Nasopharyngeal Samples Compared to Delta Variant.

OBJECTIVES: High viral load in upper respiratory tract specimens observed for Delta cases might contribute to its increased infectivity compared to the other variant. However, it is not yet documented if the Omicron variant's enhanced infectivity is also related to a higher viral load. Our aim was to determine if the Omicron variant's spread is also related to higher viral loads compared to the Delta variant. METHODS: Nasopharyngeal swabs, 129 (Omicron) and 85 (Delta), from Health Care Workers were collected during December 2021 at the University Hospital of Lyon, France. Cycle threshold (Ct) for the RdRp target of cobas® 6800 SARS-CoV-2 assay was used as a proxy to evaluate SARS-CoV-2 viral load. Variant identification was performed using a screening panel and confirmed by whole genome sequencing. RESULTS: Herein, we showed that the RT-PCR Ct values in Health Care Workers sampled within 5 days after symptom onset were significantly higher for Omicron cases than Delta cases (21.7 for Delta variant and 23.8 for Omicron variant, p = 0.008). This difference was also observed regarding patient with complete vaccination. CONCLUSIONS: This result supports the studies showing that the increased transmissibility of Omicron is related to other mechanisms than higher virus excretion.

Dynamics of viral shedding during ancestral or Omicron BA.1 SARS-CoV-2 infection and enhancement of pre-existing immunity during breakthrough infections.

Omicron variant is circulating in the presence of a globally acquired immunity unlike the ancestral SARS-CoV-2 isolate. Herein, we investigated the normalized viral load dynamics and viral culture status in 44 fully vaccinated healthcare workers (HCWs) infected with the Omicron BA.1 variant. Viral load dynamics of 38 unvaccinated HCWs infected with the 20A variant during the first pandemic wave was also studied. We then explored the impact of Omicron infection on pre-existing immunity assessing anti-RBD IgG levels, neutralizing antibody titres against 19A, Delta and Omicron isolates, as well as IFN-γ release following cell stimulation with SARS-CoV-2 peptides. We reported that two weeks after diagnosis a greater proportion of HCWs infected with 20A (78.9%, 15/19) than with Omicron BA.1 (44.7%, 17/38; p = 0.02) were still positive by RT-qPCR. We found that Omicron breakthrough infections led to an overall enhancement of vaccine-induced humoral and cellular immunity as soon as a median [interquartile range] of 8 [7-9] days post symptom onset. Among samples with similar high viral loads, non-culturable samples exhibited higher neutralizing antibody titres and anti-RBD IgG levels than culturable samples. Additionally, Omicron infection led to an enhancement of antibodies neutralization capacity against other SARS-CoV-2 isolates. Taken together, the results suggest that Omicron BA.1 vaccine breakthrough infection is associated with a faster viral clearance than that of the ancestral SARS-CoV-2, in addition this new variant leads to a rapid enhancement of the humoral response against multiple SARS-CoV-2 variants, and of the cellular response.

Analytical Performances of the Panther Fusion System for the Detection of Respiratory Viruses in the French National Reference Centre of Lyon, France.

Respiratory infection are mainly caused by viral pathogens. During the 2017-2018 epidemic season, Panther Fusion® Respiratory kits (Influenza virus A&B (FluA&B), respiratory syncytial virus (RSV), adenovirus (ADV), metapneumovirus (MPV), rhinovirus (RV), parainfluenzae virus (PIV), were compared to the Respiratory MultiWells System r-gene. Respiratory clinical specimens were tested retrospectively (n = 268) and prospectively (n = 463). Analytical performances were determined (sensitivity -Sep-, specificity -Spe- and κ) considering concordances of ≥2 molecular testing specific to each viral target (discrepant results were verified at the National Reference Centres for Enteroviruses or Respiratory viruses, Lyon, France). After retrospective (and prospective) testing, Sep, Spe, and κ were 100% (97.7%), 100% (99%) and 100% (94%) for FluA: 100% (95.5%), 100% (99.3%) and 100% (94%) for FluB, and 100% (88.5%), 100% (98.7%) and 100% (89%) for RSV; 82.1% (41.7%), 100% (99.5%) and 86% (54%) for ADV; 94.7% (73.7%), 96.1% (98.0%) and 91% (65%) for MPV; 96.1% (94.6%), 90.2% (98.5%) and 86% (91%) for HRV; and 90% (72.7%), 100% (99.3%) and 91% (72%), respectively, for PIV. Analytical performances were above 85% for all viruses except for ADV, MPV and PIV, confirming the analytical performance of the Panther Fusion system, a high throughput system with reduced turn-around-time, when compared to non-automated systems.

Performance Assessment of SARS-CoV-2 PCR Assays Developed by WHO Referral Laboratories.

A reliable diagnostic assay is crucial to early detect new COVID-19 cases and limit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission. Since the onset of the COVID-19 pandemic, the World Health Organization has published several diagnostic molecular approaches developed by referral laboratories, including Charité (Germany), HKU (Hong Kong), China CDC (China), US CDC (United States), and Institut Pasteur, Paris (France). We aimed to compare the sensitivity and specificity of these different RT-PCR assays using SARS-CoV-2 cell culture supernatants and clinical respiratory samples. Overall, the different RT-PCR assays performed well for SARS-CoV-2 detection and were all specific except the N Charité (Germany), and N2 US CDC (United States) assays. RdRp Institut Pasteur (IP2, IP4), N China CDC, and N1 US CDC were found to be the most sensitive assays. The data presented herein are of prime importance to facilitate the equipment choice of diagnostic laboratories, as well as for the development of marketed tests.

Decontamination of Intravaginal Probes Infected by Human Papillomavirus (HPV) Using UV-C Decontamination System.

BACKGROUND: This three-step study evaluated ultraviolet-C (UV-C) efficacy against human papillomavirus (HPV) found on vaginal ultrasound probes. METHODS: The first two steps evaluated UV-C disinfection of vaginal ultrasound probes in routine condition. During the first phase, the probe (n = 100) was sampled after a complete cleaning and disinfection protocol, i.e., cleaning with chemically impregnated wipes, followed by UV-C. During the second phase, the probe (n = 47) was sampled after cleaning and UV-C. The final step consisted of applying mixes of HPV on a dedicated, covered probe (n = 15) then sampling the cover, the probe after removal of the cover, after cleaning, and after UV-C. HPV detection was performed using CLART® HPV2 PCR (Genomica, Madrid, Spain). RESULTS: In the first phase, no probes were found to be positive for both DNA after UV-C. In the second phase, eight probes were found to be positive after cleaning (seven with human DNA and one with HPV) and negative after UV-C. In the final phase, one probe was found to be positive for HPV for each sample except after UV-C. CONCLUSIONS: Covers followed by a chemically impregnated wipe are not sufficient to ensure patient safety during vaginal ultrasound examinations. UV-C is effective in routine conditions against contaminations found on vaginal ultrasound probes, especially HPV.

The impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis.

INTRODUCTION: Bronchiolitis is a major cause of morbidity and mortality in early childhood worldwide. The presence of more than one pathogen may influence the natural history of acute bronchiolitis in infants. OBJECTIVE: To investigate the relevance of dual viral infection in infants with severe bronchiolitis hospitalized in a short-term unit compared with those in a pediatric intensive care unit (PICU). STUDY DESIGN: One hundred eighty infants <1 year old hospitalized with bronchiolitis in a short-term unit (n = 92) or admitted to the PICU (n = 88) during 2 consecutive winter seasons 2003/2004 and 2004/2005 were evaluated. Molecular biology and standard methods were used to diagnose human respiratory viruses in nasal/throat swabs and nasal aspirates. Clinical data related to host factors and viral prevalence were compared among infants requiring or not PICU support. RESULTS: A viral agent was identified in 96.1% of infants with bronchiolitis. Respiratory syncytial virus (70.6% and 73.6%, respectively in the short-term unit and PICU) and rhinovirus (18.5% and 25.3%, respectively in the short-term unit and PICU) were the main detected respiratory viruses in infants hospitalized in both units. No significant difference in viral prevalence was observed between the populations studied. From multivariate analysis, infants with coinfections were 2.7 times (95% CI: 1.2-6.2) more at risk for PICU admission than those with a single infection. Respiratory syncytial virus and rhinovirus were the viruses most frequently identified in mixed infections in infants hospitalized with bronchiolitis. CONCLUSIONS: Dual viral infection is a relevant risk factor for the admission of infants with severe bronchiolitis to the PICU.

Influenza burden in febrile infants and young children in a pediatric emergency department.

BACKGROUND: In France, epidemiologic data in children in ambulatory settings are scarce. We aimed to measure the burden of influenza in young children. METHODS: Febrile children younger than 36 months were consecutively recruited in a pediatric emergency department during the 2002 epidemic peak. Virology analysis and follow-up were systematic. RESULTS: During calendar weeks 3 to 6, 2002, 575 children were recruited; 49% were positive: A/H3N2 in 44% and B in 5%. Prevalence rate was 57% in 12- to 35-month-old children and 39% in infants younger than 12 months. The main clinical pictures were nonrespiratory in one third of them. One of 8 patients had a complication. One of 10 patients was hospitalized, and the estimated specific hospitalization rate for the study period was 237 of 100,000 in the general population among infants younger than 12 months. Forty-two percent of children (n = 110) were prescribed antibiotics and at least 34% of them were inappropriate (n = 89). Median length of disease was 8 days, and 25% of the children had not fully recovered by day 11. Only one child had been previously vaccinated of 65 with chronic conditions. Both epidemic strains were covered by the vaccine. CONCLUSIONS: Health outcomes showed that influenza disease burden in young French children is similar to that observed in North America. An active vaccination strategy would have strongly reduced the burden of influenza and lowered antibiotic use. Continuous efforts are needed to reach requirements of our influenza vaccination policy.

Human parainfluenza virus type 4 infections: a report of 20 cases from 1998 to 2002.

BACKGROUND: Human Parainfluenza Viruses (HPIV) type 4 are responsible for respiratory infections. Unlike HPIV types 1-3, they are associated with mild infections and appear to be infrequent. Thus, they often go undetected. STUDY DESIGN: From 1998 to 2002, in 20 respiratory samples of hospitalised patient, we isolated viruses presenting a large syncytial cytopathic effect when inoculated on LLC-MK2 cells. Most of the patients (16/20) were young infants and all of them presented with respiratory infections. RESULTS: We detected 18 cases during autumn and winter, 1 case during spring and 1 during summer. We could not identify these viruses using the panel of routine assays. Samples were then analysed by specific HPIV 4 RT-PCR and IF assays. All the samples were scored positive with both methods. CONCLUSION: We conclude that HPIV 4 infections are probably underestimated. Their role in viral respiratory infections should be carefully investigated using techniques adapted to their detection and culture.

Detection of rhinovirus and enterovirus in upper respiratory tract samples using a multiplex nested PCR.

Human enteroviruses and rhinoviruses are respiratory pathogens whose role in acute respiratory infection is underestimated due to the use of diagnostic procedures with poor sensitivity. To determine the prevalence of these two pathogens in the upper respiratory tract infections, a multiplex procedure was developed that both detect and differentiate rhinoviruses and enteroviruses. This sensitive procedure allowed the detection of both pathogens from archival material (nasal swabs) collected during the previous winters and differentiated rhinoviruses from enteroviruses. This procedure can be used to determine the role of these pathogens in mild or severe upper respiratory tract infections.

Resistance of herpes simplex viruses to acyclovir: an update from a ten-year survey in France.

The widespread use of acyclovir (ACV) and the increasing number of immunocompromised patients have raised concern about an increase in ACV-resistant herpes simplex virus (HSV). ACV resistance has traditionally been a major concern for immunocompromised patients with a frequency reported between 2.5% and 10%. The aim of this study was to reassess the status of HSV resistance to ACV in immunocompetent and immunocompromised patients over a ten year period, between 2002 and 2011. This was done by retrospectively following 1425 patients. In immunocompetent patients, prevalence of resistance did not exceed 0.5% during the study period; whereas in immunocompromised patients, a significant increase was observed, rising from 3.8% between 2002 and 2006 (7/182 patients) to 15.7% between 2007 and 2011 (28/178) (p=0.0001). This sharp rise in resistance may largely be represented by allogeneic hematopoietic stem cell transplant patients, in which the prevalence of ACV resistance rose similarly from 14.3% (4/28) between 2002 and 2006 to 46.5% (26/56) between 2007 and 2011 (p=0.005). No increase in ACV resistance was detected in association with other types of immune deficiencies. Genotypic characterization of HSV UL23 thymidine kinase and UL30 DNA polymerase genes revealed 11 and 7 previously unreported substitutions, respectively. These substitutions may be related to potential polymorphisms, drug resistance, or other mutations of unclear significance.

Successful immunotherapy in life-threatening parvovirus B19 infection in a child.

We report a case of an immunocompetent child who developed parvovirus B19 infection complicated by autoinflammatory disease with myocarditis, tamponade and macrophage activation syndrome. He recovered with immunotherapy including prednisone, immunoglobulins, cyclosporin and anakinra (anti-interleukin-1). The report shows that parvovirus can provoke severe systemic inflammation with acute heart injury and that anti-interleukin-1 might be considered in such parvovirus-related inflammation.

The clinical interest of HSV1 semi-quantification in bronchoalveolar lavage.

BACKGROUND: Detecting high herpes simplex (HSV) viral load in lower respiratory tract samples is reported to be significantly associated with the severity of the illness in critical patients, particularly in patients on mechanical ventilation. It may therefore be of interest to quantify HSV in bronchoalveolar lavage (BAL). Quantitative PCR for HSV is not commonly available in clinical routine. Real-time PCR tests are, however, used commonly and provide semi-quantitative information based on the cycle threshold (Ct). OBJECTIVES: Our objectives were to determine the clinically significant threshold and to study the impact of viral load normalisation in relation to cell quantity in samples using real-time PCR. STUDY DESIGN: During the period 2011-2012, 59 HSV1 positive BAL were included. HSV viral load was determined by a quantitative real-time PCR (R-gene, Argène BioMérieux, France) and compared to a semi-quantitative real-time PCR (SmartCycler®HerpesSimplex, Cepheid, USA). Viral load normalisation was determined using a real-time PCR targeting a cellular gene (Cc r-gene kit, Argène BioMérieux, France). The significant threshold was determined versus clinical features by statistical analysis (Epiinfo Software v3.5.1 CDC). RESULTS: A viral load of 10(4) copies/ml of BAL was significantly associated with admission to the intensive care unit (p<0.001), mechanical ventilation (p<0.01) and death (p<0.01), with no influence of viral load normalisation in relation to cell quantity in the sample. This viral load was equivalent to a Ct value of 31 in the semi-quantitative technique. CONCLUSIONS: As semi-quantitative techniques are currently used in many labs, determining this Ct value could be useful for interpreting the clinical advantages of detecting HSV in BAL.

Human parechovirus infections, Lyon, France, 2008-10: evidence for severe cases.

BACKGROUND: Although data documenting the frequency and severity of human parechovirus type 3 (HPeV-3) infection in infants have been published in Canada, the USA, the UK and the Netherlands, no data from France are available. OBJECTIVES: To determine the detection frequency of HPeV in cerebrospinal fluid (CSF) samples collected from children aged <5 years hospitalized between 2008 and 2010 in the University Hospital of Lyon and to describe the clinical, virological and biological characteristics associated with HPeV infection. STUDY DESIGN: A total of 1128 CSF samples were retrospectively tested using the Parechovirus-Rgene™ real-time RT-PCR assay. Positive samples were typed by sequencing using the CDC method. Retrospective analysis of the medical charts was performed. RESULTS: Over a 3-year period, 33/1128 (2.9%) CSF samples were found to be HPeV-positive. In 2010, 9.3% of the children aged <3 months (32% in June) were detected HPeV-positive. The median age at diagnosis was 26 days (8-131 days). Most patients (86%) presented with fever or a sepsis-like syndrome. Three patients (2 with septic shock syndrome, 1 with severe respiratory distress) required hospitalization in an intensive care unit. An HPeV-3 acute infection was identified in an 11-day-old girl who died from sudden infant death syndrome. Of 29 patients genotyped, 28 were infected with HPeV-3 and one with HPeV-4. CONCLUSIONS: HPeV is a significant cause of sepsis and severe sepsis in children <3 months. Routine screening for HPeV in CSF and blood should thus be performed more extensively and could improve clinical management.

[Kidney transplantation and infection in childhood]. / Transplantation rénale et infection chez l'enfant.

Infectious risk is more important in the transplanted child than in adult because children are less often immunised against pathogens ant more exposed than adults to numerous infectious agents (virus but also bacteria including pneumococcus). The application of the standard immunisation schedule must be a permanent concern of transplantation (Tx) teams. Some vaccines that are not planned in the standard immunization schedule are particularly advised for the child and his family circle, as well as for caregivers. Immunisation response must be evaluated by a serological follow-up before Tx, in particular during the pre-Tx diagnostic work-up, then regularly after Tx. The more frequent absence of immunisation against Epstein Barr Virus (EBV) in children explains the increased frequency of post-transplant lymphoproliferative disorder at the pediatric age.