Identification of a measles variant displaying mutations impacting molecular diagnostics, Geneva, Switzerland, 2023.

Real-time PCR is one of the most widely used techniques to diagnose measles cases. Here we report measles virus variants with three genetic mutations in the reverse primer annealing site of a widely used PCR. The mutations result in a slight loss of the PCR sensitivity. Variants bearing the three mutations presently circulate in different countries since at least the end of 2021. Our findings highlight the usefulness of molecular surveillance in monitoring if oligonucleotides in diagnostic tests remain adequate.

Adapting response to a measles outbreak in a context of high vaccination and breakthrough cases: an example from Vaud, Switzerland, January to March 2024.

A measles outbreak with 51 cases occurred in the canton of Vaud, Switzerland, between January and March 2024. The outbreak was triggered by an imported case, and 37 (72.5%) subsequent cases were previously vaccinated individuals. Epidemiological investigations showed that vaccinated measles cases were symptomatic and infectious. In a highly vaccinated population, it is important to raise awareness among healthcare professionals to suspect and test for measles virus when an outbreak is declared, irrespective of the vaccination status of the patients.

Normal tissue content impact on the GBM molecular classification.

Molecular classification of glioblastoma has enabled a deeper understanding of the disease. The four-subtype model (including Proneural, Classical, Mesenchymal and Neural) has been replaced by a model that discards the Neural subtype, found to be associated with samples with a high content of normal tissue. These samples can be misclassified preventing biological and clinical insights into the different tumor subtypes from coming to light. In this work, we present a model that tackles both the molecular classification of samples and discrimination of those with a high content of normal cells. We performed a transcriptomic in silico analysis on glioblastoma (GBM) samples (n = 810) and tested different criteria to optimize the number of genes needed for molecular classification. We used gene expression of normal brain samples (n = 555) to design an additional gene signature to detect samples with a high normal tissue content. Microdissection samples of different structures within GBM (n = 122) have been used to validate the final model. Finally, the model was tested in a cohort of 43 patients and confirmed by histology. Based on the expression of 20 genes, our model is able to discriminate samples with a high content of normal tissue and to classify the remaining ones. We have shown that taking into consideration normal cells can prevent errors in the classification and the subsequent misinterpretation of the results. Moreover, considering only samples with a low content of normal cells, we found an association between the complexity of the samples and survival for the three molecular subtypes.

The size, number, and distribution of nerve endings around and within the human epiglottis, focusing on tracheal intubation maneuvers.

The aim of this study was to examine the distribution of nerve endings in the mucosa, submucosa, and cartilage of the epiglottis and the vallecula area and to quantify them. The findings could inform the choice of laryngoscope blades for intubation procedures. Fourteen neck slices from seven unembalmed, cryopreserved human cadavers were analyzed. The slices were stained, and cross and longitudinal sections were obtained from each. The nerve endings and cartilage were identified. The primary metrics recorded were the number, area, and circumference of nerve endings located in the mucosa and submucosa of the pharyngeal and laryngeal sides of the epiglottis, epiglottis cartilage, and epiglottic vallecula zone. The length and thickness of the epiglottis and cartilage were also measured. The elastic cartilage of the epiglottis was primarily continuous; however, it contained several fragments. It was covered with dense collagen fibers and surrounded by adipose cells from the pharyngeal and laryngeal submucosa. Nerve endings were found within the submucosa of pharyngeal and laryngeal epiglottis and epiglottic vallecula. There were significantly more nerve endings on the posterior surface of the epiglottis than on the anterior surface. The epiglottic cartilage was twice the length of the epiglottis. The study demonstrated that the distribution of nerve endings in the epiglottis differed significantly between the posterior and anterior sides; there were considerably more in the former. The findings have implications for tracheal intubation and laryngoscope blade selection and design.

Microscopy of structures surrounding typical acupoints used in clinical practice and electron microscopic evaluation of acupuncture needles.

Although the general functionality and structures of acupoints have been studied, there has been little insight into their underlying morphology and physical characteristics. We describe the microanatomical structures surrounding acupoints, the electron microscopic appearance of the needles, and the physical effects of acupuncture needling on the fascia. We injected heparinized blood solution through thin needles at seven known and commonly used "sweat acupoints" in eight fresh, unembalmed, cryopreserved human cadavers to mark the needle positions, and later, during histological examination, to identify them. After the solution was injected, samples were dissected and prepared for histological examination. We examined 350 cross-sections of five different paraffin wax sections from each acupoint microscopically. Acupuncture needles were photographed and superimposed on the cross-sectioned tissues at similar magnifications. Needles were also examined under a scanning electron microscope to judge the roughness or smoothness of their surfaces. A greater conglomeration of nerve endings surrounded the acupoints than in tissues more than 1-3 cm distant from them. Nerve endings and blood vessels were in close contact with a complex network of membranes formed by interlacing collagen fibers, and were always enclosed within those collagen membranes. Nerve endings were found within hypodermis, muscles, or both. Scanning electron microscopy demonstrated the three-dimensional shapes and sizes of the needles, and the degree of roughness or smoothness of their polished external surfaces. We demonstrate a delicate arrangement of nerve endings and blood vessels enclosed within complex collagen membrane networks at acupoints within the hypodermis and muscle. This arrangement could explain why needling is an essential step in the acupuncture process that provides favorable outcomes in clinical practice.

Viral chimeras decrypt the role of enterovirus capsid proteins in viral tropism, acid sensitivity and optimal growth temperature.

Despite their genetic similarities, enteric and respiratory enteroviruses (EVs) have highly heterogeneous biophysical properties and cause a vast diversity of human pathologies. In vitro differences include acid sensitivity, optimal growth temperature and tissue tropism, which reflect a preferential in vivo replication in the respiratory or gastrointestinal tract and are thus key determinants of EV virulence. To investigate the underlying cause of these differences, we generated chimeras at the capsid-level between EV-D68 (a respiratory EV) and EV-D94 (an enteric EV). Although some chimeras were nonfunctional, EV-D94 with both the capsid and 2A protease or the capsid only of EV-D68 were both viable. Using this latter construct, we performed several functional assays, which indicated that capsid proteins determine acid sensitivity and tropism in cell lines and in respiratory, intestinal and neural tissues. Additionally, capsid genes were shown to also participate in determining the optimal growth temperature, since EV-D94 temperature adaptation relied on single mutations in VP1, while constructs with EV-D68 capsid could not adapt to higher temperatures. Finally, we demonstrate that EV-D68 maintains residual binding-capacity after acid-treatment despite a loss of infectivity. In contrast, non-structural rather than capsid proteins modulate the innate immune response in tissues. These unique biophysical insights expose another layer in the phenotypic diversity of one of world's most prevalent pathogens and could aid target selection for vaccine or antiviral development.

Hepatitis A virus adaptation to cellular shutoff is driven by dynamic adjustments of codon usage and results in the selection of populations with altered capsids.

UNLABELLED: Hepatitis A virus (HAV) has a highly biased and deoptimized codon usage compared to the host cell and fails to inhibit host protein synthesis. It has been proposed that an optimal combination of abundant and rare codons controls the translation speed required for the correct capsid folding. The artificial shutoff host protein synthesis results in the selection of variants containing mutations in the HAV capsid coding region critical for folding, stability, and function. Here, we show that these capsid mutations resulted in changes in their antigenicity; in a reduced stability to high temperature, low pH, and biliary salts; and in an increased efficacy of cell entry. In conclusion, the adaptation to cellular shutoff resulted in the selection of large-plaque-producing virus populations. IMPORTANCE: HAV has a naturally deoptimized codon usage with respect to that of its cell host and is unable to shut down the cellular translation. This fact contributes to the low replication rate of the virus, in addition to other factors such as the highly inefficient internal ribosome entry site (IRES), and explains the outstanding physical stability of this pathogen in the environment mediated by a folding-dependent highly cohesive capsid. Adaptation to artificially induced cellular transcription shutoff resulted in a redeoptimization of its capsid codon usage, instead of an optimization. These genomic changes are related to an overall change of capsid folding, which in turn induces changes in the cell entry process. Remarkably, the adaptation to cellular shutoff allowed the virus to significantly increase its RNA uncoating efficiency, resulting in the selection of large-plaque-producing populations. However, these populations produced much-debilitated virions.

Hepatitis A virus genotype distribution during a decade of universal vaccination of preadolescents.

A universal vaccination program among preadolescents was implemented in Catalonia, Spain, during the period of 1999-2013 and its effectiveness has been clearly demonstrated by an overall significant attack rate reduction. However, reductions were not constant over time, and increases were again observed in 2002-2009 due to the occurrence of huge outbreaks. In the following years, in the absence of large outbreaks, the attack rate decreased again to very low levels. However, an increase of symptomatic cases in the <5 age group has recently been observed. This is an unexpected observation since children younger than 6 are mostly asymptomatic. Such a long vaccination campaign offers the opportunity to analyze not only the effectiveness of vaccination, but also the influence of the circulating genotypes on the incidence of hepatitis A among the different age groups. This study has revealed the emergence of genotype IC during a foodborne outbreak, the short-lived circulation of vaccine-escape variants isolated during an outbreak among the men-having-sex-with-men group, and the association of genotype IIIA with the increase of symptomatic cases among the very young. From a public health perspective, two conclusions may be drawn: vaccination is better at an early age, and the vaccination schedule must be complete and include all recommended vaccine doses.

Molecular basis of the behavior of hepatitis a virus exposed to high hydrostatic pressure.

Food-borne hepatitis A outbreaks may be prevented by subjecting foods at risk of virus contamination to moderate treatments of high hydrostatic pressure (HHP). A pretreatment promoting hepatitis A virus (HAV) capsid-folding changes enhances the virucidal effect of HHP, indicating that its efficacy depends on capsid conformation. HAV populations enriched in immature capsids (125S provirions) are more resistant to HHP, suggesting that mature capsids (150S virions) are more susceptible to this treatment. In addition, the monoclonal antibody (MAb) K24F2 epitope contained in the immunodominant site is a key factor for the resistance to HHP. Changes in capsid folding inducing a loss of recognition by MAb K24F2 render more susceptible conformations independently of the origin of such changes. Accordingly, codon usage-associated folding changes and changes stimulated by pH-dependent breathings, provided they confer a loss of recognition by MAb K24F2, induce a higher susceptibility to HHP. In conclusion, the resistance of HAV to HHP treatments may be explained by a low proportion of 150S particles combined with a good accessibility of the epitope contained in the immunodominant site close to the 5-fold axis.

Standardized multiplex one-step qRT-PCR for hepatitis A virus, norovirus GI and GII quantification in bivalve mollusks and water.

A quadruplex Real-Time RT-PCR assay for the simultaneous quantitative detection of hepatitis A virus (HAV), norovirus (NoV) GI and GII, and mengovirus (used as process control for determination of the virus/nucleic acid extraction efficiency) has been developed. This multiplex assay has been comparatively evaluated with the individual monoplex assays and showed to be slightly less sensitive, with average ΔCq values of 0.90, 0.28 and 0.44 for HAV, NoV GI and NoV GII, respectively, in standard curves of viral RNA, or 0.32, 0.37 and 0.51 for the same viruses respectively, in naturally-contaminated samples. These ΔCq values were mostly negligible since it represented, in the worst case scenario, a loss of 0.43 log in genome copy numbers. The quadruplex assay shows similar theoretical detection limits than the monoplex assay for NoV GII, and 10 times higher for HAV and NoV GI. However, when naturally-contaminated food and water samples were tested, these theoretical detection thresholds were often exceeded and very low genome copy numbers (below the limit of detection) could be quantified. The quadruplex assay fulfills the requirements of the method developed by the European Committee on Standardization (CEN) for virus detection in selected foodstuffs with significant advantages in labor and reagent costs.

Distinct phenotype of SARS-CoV-2 Omicron BA.1 in human primary cells but no increased host range in cell lines of putative mammalian reservoir species.

SARS-CoV-2's genetic plasticity has led to several variants of concern (VOCs). Here we studied replicative capacity for seven SARS-CoV-2 isolates (B.1, Alpha, Beta, Gamma, Delta, Zeta, and Omicron BA.1) in primary reconstituted airway epithelia (HAE) and lung-derived cell lines. Furthermore, to investigate the host range of Delta and Omicron compared to ancestral SARS-CoV-2, we assessed replication in 17 cell lines from 11 non-primate mammalian species, including bats, rodents, insectivores and carnivores. Only Omicron's phenotype differed in vitro, with rapid but short replication and efficient production of infectious virus in nasal HAEs, in contrast to other VOCs, but not in lung cell lines. No increased infection efficiency for other species was observed, but Delta and Omicron infection efficiency was increased in A549 cells. Notably replication in A549 and Calu3 cells was lower than in nasal HAE. Our results suggest better adaptation of VOCs towards humans, without an extended host range, and may be relevant to the search for the putative intermediate host and reservoirs prior to the pandemic.

Total en bloc vertebrectomy and immunochemotherapy for chondrosarcoma colliding with intraosseous lymphoma.

A 59-year-old woman diagnosed with a Grade I chondrosarcoma in T7 underwent total en bloc vertebrectomy. Analysis of the surgical piece established diagnosis of a Grade 1 chondrosarcoma confined to T7. Surprisingly, an infiltration with diffuse large B-cell lymphoma was found. Systemic disease was ruled out and diagnosis was established as intracompartmental Grade 1 chondrosarcoma colliding with intraosseous extranodal diffuse large B-cell lymphoma. Resection of chondrosarcoma was considered complete and treatment with four cycles of RCHOP was indicated. Two years after surgery, the patient remains at complete metabolic response. To date, this is the first reported case of chondrosarcoma colliding with lymphoma. Although Grade 1 chondrosarcoma is typically managed with local control through complete surgical resection, the mentioned finding of the lymphoma indicated the need for systemic treatment with immunochemotherapy.

Direct Dengue Virus Genome Sequencing from Antigen NS1 Rapid Diagnostic Tests: A Proof-of-Concept with the Standard Q Dengue Duo Assay.

With nearly half of the world's population being at risk of infection, dengue virus represents a major global health issue. The use of dengue antigen rapid diagnostic tests (Ag-RDTs) represents an alternative to PCR methods for the diagnosis of acute infections since they display excellent sensitivities and specificities and can be performed outside the laboratory. The high genetic diversity of the dengue virus genome represents a challenge for vaccine development, and the progressive expansion of this virus into previously nonendemic regions justifies the implementation of a genomic surveillance program. In this proof-of-concept study, we show the feasibility of sequencing dengue virus genomes directly from positive Ag-RDT (Standard Q Dengue Duo Test assay, n = 7) cassettes stored up to 31 days at room temperature after testing. For 5 of the 7 samples, a high number of reads were obtained allowing phylogenetic analyses to be carried out to determine not only the serotypes (dengue 1, 2, 3 and 4 were detected) but also the genotypes. Furthermore, in one sample, our unbiased metagenomic next-generation sequencing approach made it possible to detect epizootic hemorrhagic disease virus sequences, an arthropod-transmitted virus in ruminants. To conclude, as such an approach requires no cold storage or freezing of samples, dengue Ag-RDTs represent a very pragmatic and robust alternative for the genomic surveillance of dengue virus.

Altered cell function and increased replication of rhinoviruses and EV-D68 in airway epithelia of asthma patients.

Introduction: Rhinovirus (RV) infections constitute one of the main triggers of asthma exacerbations and an important burden in pediatric yard. However, the mechanisms underlying this association remain poorly understood. Methods: In the present study, we compared infections of in vitro reconstituted airway epithelia originating from asthmatic versus healthy donors with representative strains of RV-A major group and minor groups, RV-C, RV-B, and the respiratory enterovirus EV-D68. Results: We found that viral replication was higher in tissues derived from asthmatic donors for all tested viruses. Viral receptor expression was comparable in non-infected tissues from both groups. After infection, ICAM1 and LDLR were upregulated, while CDHR3 was downregulated. Overall, these variations were related to viral replication levels. The presence of the CDHR3 asthma susceptibility allele (rs6967330) was not associated with increased RV-C replication. Regarding the tissue response, a significantly higher interferon (IFN) induction was demonstrated in infected tissues derived from asthmatic donors, which excludes a defect in IFN-response. Unbiased transcriptomic comparison of asthmatic versus control tissues revealed significant modifications, such as alterations of cilia structure and motility, in both infected and non-infected tissues. These observations were supported by a reduced mucociliary clearance and increased mucus secretion in non-infected tissues from asthmatic donors. Discussion: Altogether, we demonstrated an increased permissiveness and susceptibility to RV and respiratory EV infections in HAE derived from asthmatic patients, which was associated with a global alteration in epithelial cell functions. These results unveil the mechanisms underlying the pathogenesis of asthma exacerbation and suggest interesting therapeutic targets.

Analytical Sensitivity of Eight Different SARS-CoV-2 Antigen-Detecting Rapid Tests for Omicron-BA.1 Variant.

The emergence of each novel SARS-CoV-2 variant of concern (VOC) requires investigation of its potential impact on the performance of diagnostic tests in use, including antigen-detecting rapid diagnostic tests (Ag-RDTs). Although anecdotal reports have been circulating that the newly emerged Omicron-BA.1 variant is in principle detectable by Ag-RDTs, few data on sensitivity are available. We have performed (i) analytical sensitivity testing with cultured virus in eight Ag-RDTs and (ii) retrospective testing in duplicates with clinical samples from vaccinated individuals with Omicron-BA.1 (n = 59) or Delta (n = 54) breakthrough infection on seven Ag-RDTs. Overall, in our analytical study we have found heterogenicity between Ag-RDTs for detecting Omicron-BA.1. When using cultured virus, we observed a trend toward lower endpoint sensitivity for Omicron-BA.1 detection than for earlier circulating SARS-CoV-2 and the other VOCs. In our retrospective study, the detection of Delta and Omicron-BA.1 was assessed in a comparable set of stored clinical samples using seven Ag-RDTs. Four hundred ninety-seven of all 826 tests (60.17%) performed on Omicron-BA.1 samples were positive, compared to 489/756 (64.68%) for Delta samples. In the analytical study, the sensitivity for both Omicron-BA.1 and Delta between the Ag-RDTs was variable. All seven Ag-RDTs showed comparable sensitivities to detect Omicron-BA.1 and Delta in the retrospective study. IMPORTANCE Sensitivity for detecting Omicron-BA.1 shows high heterogenicity between Ag-RDTs, necessitating a careful consideration when using these tests to guide infection prevention measures. Analytical and retrospective testing is a proxy and timely solution to generate rapid performance data, but it is not a replacement for clinical evaluations, which are urgently needed. Biological and technical reasons for detection failure by some Ag-RDTs need to be further investigated.

Pathogenicity and virulence of hepatitis A virus.

Hepatitis A is an acute infection of the liver, which is mostly asymptomatic in children and increases the severity with age. Although in most patients the infection resolves completely, in a few of them it may follow a prolonged or relapsed course or even a fulminant form. The reason for these different outcomes is unknown, but it is generally accepted that host factors such as the immunological status, age and the occurrence of underlaying hepatic diseases are the main determinants of the severity. However, it cannot be ruled out that some virus traits may also contribute to the severe clinical outcomes. In this review, we will analyze which genetic determinants of the virus may determine virulence, in the context of a paradigmatic virus in terms of its genomic, molecular, replicative, and evolutionary features.

Advances for the Hepatitis A Virus Antigen Production Using a Virus Strain With Codon Frequency Optimization Adjustments in Specific Locations.

The available cell-adapted hepatitis A virus (HAV) strains show a very slow replication phenotype hampering the affordable production of antigen. A fast-growing strain characterized by the occurrence of mutations in the internal ribosome entry site (IRES), combined with changes in the codon composition has been selected in our laboratory. A characterization of the IRES activity of this fast-growing strain (HM175-HP; HP) vs. its parental strain (HM175; L0) was assessed in two cell substrates used in vaccine production (MRC-5 and Vero cells) compared with the FRhK-4 cell line in which its selection was performed. The HP-derived IRES was significantly more active than the L0-derived IRES in all cells tested and both IRES were more active in the FRhK-4 cells. The translation efficiency of the HP-derived IRES was also much higher than the L0-derived IRES, particularly, in genes with a HP codon usage background. These results correlated with a higher virus production in a shorter time for the HP strain compared to the L0 strain in any of the three cell lines tested, and of both strains in the FRhK-4 cells compared to Vero and MRC-5 cells. The addition of wortmannin resulted in the increase of infectious viruses and antigen in the supernatant of FRhK-4 infected cells, independently of the strain. Finally, the replication of both strains in a clone of FRhK-4 cells adapted to grow with synthetic sera was optimal and again the HP strain showed higher yields.

Robust innate responses to SARS-CoV-2 in children resolve faster than in adults without compromising adaptive immunity.

SARS-CoV-2 infection in children is less severe than it is in adults. We perform a longitudinal analysis of the early innate responses in children and adults with mild infection within household clusters. Children display fewer symptoms than adults do, despite similar initial viral load, and mount a robust anti-viral immune signature typical of the SARS-CoV-2 infection and characterized by early interferon gene responses; increases in cytokines, such as CXCL10 and GM-CSF; and changes in blood cell numbers. When compared with adults, the antiviral response resolves faster (within a week of symptoms), monocytes and dendritic cells are more transiently activated, and genes associated with B cell activation appear earlier in children. Nonetheless, these differences do not have major effects on the quality of SARS-CoV-2-specific antibody responses. Our findings reveal that better early control of inflammation as observed in children may be key for rapidly controlling infection and limiting the disease course.

Diagnostic accuracy of two commercial SARS-CoV-2 antigen-detecting rapid tests at the point of care in community-based testing centers.

OBJECTIVES: Determine the diagnostic accuracy of two antigen-detecting rapid diagnostic tests (Ag-RDT) for SARS-CoV-2 at the point of care and define individuals' characteristics providing best performance. METHODS: We performed a prospective, single-center, point of care validation of two Ag-RDT in comparison to RT-PCR on nasopharyngeal swabs. RESULTS: Between October 9th and 23rd, 2020, 1064 participants were enrolled. The PanbioTM Covid-19 Ag Rapid Test device (Abbott) was validated in 535 participants, with 106 positive Ag-RDT results out of 124 positive RT-PCR individuals, yielding a sensitivity of 85.5% (95% CI: 78.0-91.2). Specificity was 100.0% (95% CI: 99.1-100) in 411 RT-PCR negative individuals. The Standard Q Ag-RDT (SD Biosensor, Roche) was validated in 529 participants, with 170 positive Ag-RDT results out of 191 positive RT-PCR individuals, yielding a sensitivity of 89.0% (95%CI: 83.7-93.1). One false positive result was obtained in 338 RT-PCR negative individuals, yielding a specificity of 99.7% (95%CI: 98.4-100). For individuals presenting with fever 1-5 days post symptom onset, combined Ag-RDT sensitivity was above 95%. Lower sensitivity of 88.2% was seen on the same day of symptom development (day 0). CONCLUSIONS: We provide an independent validation of two widely available commercial Ag-RDTs, both meeting WHO criteria of ≥80% sensitivity and ≥97% specificity. Although less sensitive than RT-PCR, these assays could be beneficial due to their rapid results, ease of use, and independence from existing laboratory structures. Testing criteria focusing on patients with typical symptoms in their early symptomatic period onset could further increase diagnostic value.