Hepatitis E virus in Italy: molecular analysis of travel-related and autochthonous cases

Human hepatitis E virus (HEV) is considered an emerging pathogen in industrialized countries. The aim of the present study was to contribute to the body of knowledge available on the molecular epidemiology of acute hepatitis E in Italy. Three sets of HEV-specific primers targeting the ORF1 and ORF2 were used to examine serum samples collected from acute hepatitis patients positive for anti-HEV IgG and/or IgM, between 2007 and 2010. Seventeen patients (39.5 %) tested HEV RNA-positive: 12 infections, due to genotype 1, were associated with travel to endemic areas (Bangladesh, India and Pakistan), while five infections, due to genotype 3, were presumably autochthonous. Risk factors identified in this group included exposure to raw seafood, pork liver sausages and wild boar. Results from the present study confirm that human HEV infection in Italy is caused by different genotypes, depending on whether the infection is travel-related or autochthonous

Simultaneous control of DNA and RNA processing efficiency using a nucleic acid calibration set.

PCR-based detection techniques enables reliable and sensitive nucleic acid target detection. However, quantitative determination methods often fail to control for the efficiency of nucleic acid extraction, reverse transcription, and PCR amplification. This problem is even more prominent when working with clinical samples due to target sequence loss during nucleic acid processing or the co-purification of PCR inhibitors (1,2). Handling processes are often assumed to approach 100% efficiency in the laboratory, even if practical experience shows that this efficiency can be much lower. This inability to ensure accuracy can lead to significant error in uncalibrated DNA sample quantitation. The additional need for reverse transcription of RNA may further increase the quantitative error rate, as yet another enzymatic process is involved. Nucleic acid controls have been developed based upon known sequences to calibrate either DNA or RNA handling; DNA calibrators have been used to control for the amplification of target sequences using realtime PCR methods (3–8), while RNA calibrators have been developed to test reverse transcription and amplification efficiencies (9–11). A nonpathogenic viral particle carrying a sequence for use as an external positive control of extraction and amplification has also been described (12). Unfortunately, most of the established processing controls are only suitable for limited applications (i.e., either DNA or RNA detection). Cross-contamination of biological samples or minute detection from natural sources reveals the need for completely synthetic sequences, with no homology to sequences in the nucleic acid databases. It is, therefore, beneficial to design an internal, synthetic calibration system that can control for both DNA and RNA processing steps in a single tube. This set includes both RNA and DNA targets with identical primer binding sites and, thus, primer binding efficiency, but easily distinguishable sequence characteristics, allowing for simultaneous detection, quantitation, and calibration of nucleic acid processing efficiency. A 150-bp randomly generated nucleic acid sequence was developed for use as a short control (SC). A GCrich 75-bp sequence was inserted in the middle of the 150-bp sequence to generate a 225-bp sequence, long control (LC). Besides size, the two sequences were designed to have easily distinguishable probe binding sites with a predicted product melting temperature difference of 4°C. Calibrator sequences have been published as GenBank® accession nos. EF143258 (DNA control, LC) and EF143257 (RNA control, SC). Simultaneous control of DNA and RNA processing efficiency using a nucleic acid calibration se

Autochthonous acute hepatitis E: treatment with sofosbuvir and ribavirin

Introduction: Hepatitis E virus (HEV) is an emerging cause of autochthonous-acute-hepatitis and acute-on-chronic-liver-failure in western countries. Treatment is not routinely used, despite ribavirin has a good antiviral effect. In vitro sofosbuvir inhibits HEV replication, but clinical data are lacking. Case report: We report a case of acute-on-chronic-liver-failure due to HEV treated with sofosbuvir and ribavirin. The treatment was capable of rapidly inducing both HCV and HEV viral suppression. Conclusion: In conclusion, although more data are required before firm conclusions could be drawn, the combination of sofosbuvir and ribavirin in not immunosuppressed patients with acute hepatitis E may be able to clear HEV infection

household transmission and disease transmissibility of a large hav outbreak in lazio italy 2016 2017

Abstract A major outbreak of Hepatitis A Virus (HAV) has swept through Europe between mid-2016 and 2017, mainly within the community of men who have sex with men (MSM). Over the same period, about 1,000 outbreak-related cases of acute Hepatitis A (AHA) were recorded in Lazio region, Italy. We calibrated a Bayesian model to reconstruct likely transmission events within all 44 households where multiple infections were recorded, representing a total of 103 cases from the HAV outbreak in Lazio. Based on information on the observed times of symptom onset, we estimated for the probability distribution function of the HAV generation time and used it to compute the effective and instantaneous reproduction numbers for the considered outbreak from the overall epidemic curve (N = 998 cases). We estimated a mean generation time of 30.2 days (95%CI: 25.2-33.0) and an effective reproduction number of about 1.63 (95% CI: 1.35-1.94). Transmissibility peaked in January 2017, shortly before targeted awareness and vaccination campaigns were put in place by health authorities; however, transmission remained above the epidemic threshold until June 2017. Within households, children (0-15) and young adults (16-30) infected preferentially individuals of the same age class, whereas transmission within older age groups was substantially homogeneous. These results suggest that the implemented interventions were able to slow down HAV transmission, but not to bring it rapidly to a halt. According to our estimates of the HAV transmissibility, about 50% of the at-risk persons should be immunized to prevent similar outbreaks in the future. Our results also indicate spillover from community transmission to household members, suggesting the opportunity of vaccinating household contacts of cases to prevent further spread of the epidemics

Human Papillomavirus in Head and Neck Cancer

Human papillomavirus (HPV) is currently considered to be a major etiologic factor, in addition to tobacco and alcohol, for oropharyngeal cancer (OPC) development. HPV positive OPCs are epidemiologically distinct from HPV negative ones, and are characterized by younger age at onset, male predominance, and strong association with sexual behaviors. HPV16 is the most prevalent types in oral cavity cancer (OCC), moreover the prevalence of beta, and gamma HPV types is higher than that of alpha HPV in oral cavity

mRNA- and Adenovirus-Based Vaccines against SARS-CoV-2 in HIV-Positive People

About two years have passed since the identification of SARS-CoV-2 in China. The rapid spread of this virus all over the world and its high transmissibility and pathogenicity in humans have resulted in a global pandemic. The negative impact of COVID-19 on health, society and the economy at the global level has pushed researchers and pharmaceutical companies to develop effective vaccines to fight SARS-CoV-2. Thanks to this collaborative effort, the first COVID-19 vaccine was developed in less than a year. Since then, several COVID-19 vaccines have been validated for use by the World Health Organization. Among these, mRNA- (BNT162b2 and mRNA1273) and adenovirus-based (ChAdOx1) vaccines were developed through the use of novel technologies. While all three of these vaccines have shown effectiveness against the COVID-19 disease and their immunogenicity was characterized in clinical trials in the general population, data on their efficacy and immunogenicity in people living with HIV (PLWH) are limited. In this review, we provide a description of the characteristics of mRNA- and adenovirus-based vaccines and of the immune response elicited in the general population by vaccination. Then we describe the use of these vaccines and their efficacy and immunogenicity in people living with HIV and we conclude with a discussion regarding some open questions concerning the use of mRNA- and adenovirus-based COVID-19 vaccines in PLWH

Human Papillomavirus Oral Infection: Review of Methodological Aspects and Epidemiology

Oral infection by Human Papillomavirus (HPV) has recently gained great attention because of its involvement in the development of a subset of head and neck squamous cell carcinoma. The role of specific Alpha-HPVs in this regard has been well established, whereas the contribution of other genera is under investigation. Despite their traditional classification as “cutaneous” types, Beta and Gamma HPVs are frequently detected in oral samples. Due to the lack of a standardized protocol, a large variety of methodologies have been used for oral sample collection, DNA extraction, HPV detection and genotyping. Laboratory procedures influence the evaluation of oral HPV prevalence, which largely varies also according to the population characteristics, e.g., age, gender, sexual behavior, Human Immunodeficiency Virus (HIV) status. Nevertheless, oral infection by Beta and Gamma HPVs seems to be even more common than Alpha-HPVs. The latter is 5–7% in the general population, and increases up to 30% approximately in HIV-infected men who have sex with men. Despite major advances in the evaluation of oral HPV prevalence, its natural history is still little understood, especially for Beta and Gamma HPVs. The latest technologies, such as Next Generation Sequencing (NGS), can be exploited to gain new insights into oral HPV, and to improve the identification of novel HPV types

Hepatitis C Virus Genetic Variability, Human Immune Response, and Genome Polymorphisms: Which Is the Interplay?

Hepatitis C virus (HCV) infection is the main cause of chronic hepatitis, affecting an estimated 150 million people worldwide. Initial exposure to HCV is most often followed by chronic hepatitis, with only a minority of individuals spontaneously clearing the virus. The induction of sustained and broadly directed HCV-specific CD4⁺ and CD8⁺ T cell responses, together with neutralizing antibodies (nAb), and specific genetic polymorphism have been associated with spontaneous resolution of the infection. However, due to its high variability, HCV is able to overwhelm the host immune response through the rapid acquisition of mutations in the epitopes targeted by T cells and neutralizing antibodies. In this context, immune-mediated pressure represents the main force in driving HCV evolution. This review summarizes the data on HCV diversity and the current state of knowledge about the contributions of antibodies, T cells, and host genetic polymorphism in driving HCV evolution in vivo

Pre-analytic phase in molecular biology: criticism and non-compliance management

Introduction: During workflow in Laboratories the most delicate and important step is pre-analytic sample treatment because it involves more than one operator of the same structure and often different health services. In fact, the biological materials used for the diagnosis should be collected, sent and properly treated before the analytic phase. Correct methods for collecting and handling biological materials, including guidelines to users of laboratory services, improve performance of Laboratory testing activity. In the pre-analytic phase the operators check sample integrity, and prepare the sample for the subsequent analytic phase: in all these steps monitoring and control of “non- compliance” is crucial. Methods: During 2007-2008 we created a “non- compliance” check-list, to monitor errors which occurred in different sectors of the preanalytic phase, particularly in the nucleic acid extraction step. These “non-compliances” are analysed to identify and to remove errors, adopting preventive and corrective proceedings. Since 2008 we have been using DNA/RNA internal controls synthesized in our Laboratory. They can be amplified by the same primers and recognized by different probes. Results: Examination of the “non compliance” check-list for molecular biology investigations shows that the percentage of urine repeat samples decreased from 17% to 2% and the percentage of stool repeat samples from 27% to 2%. Regarding use of internal controls, they allow the assessment of inhibitory factors that can prevent gene amplification. Conclusions: Monitoring “non-compliance” cases and dividing them by typology allow us identifying the most frequent causes of incorrect sample handling, as a non optimal procedure of pre-treatment, thus improving the pre-analytic phase. Therefore by monitoring the preanalytic phase we can prevent the introduction of confounding factors that may negatively influence the accuracy of results and their interpretation. Before proceeding to gene amplification, biological samples must be properly purified to eliminate lipids, proteins, polysaccharides and other potential inhibitors of DNA polymerase