Hepatitis A in Latin America: The current scenario.

This review aims to gather and disseminate updated information regarding hepatitis A virus (HAV) in Latin America (LA) in the last 11 years, including seroprevalence, post-vaccination studies, virus detection in aqueous matrices and food samples, and outbreak reports. Only 24 seroprevalence studies were published between 2012 and 2023 with 55%-100% reported prevalences of anti-HAV IgG. Among the 25 LA countries, only eight of them have introduced HAV vaccines into their immunisation programs. Outbreaks of hepatitis A occurred between 2017-2019, mainly affecting men who have sex with men in Argentina, Brazil and Chile, probably as a consequence of the abrupt decline of young adults' immunity. This could be due to that young adult have never been infected in childhood (due to socio-health improvements) and are above the cut-off ages to be included when the vaccination programs were introduced. Although scarce, studies focused on environmental and food HAV surveillance have shown viral presence in these samples. Surface waters presented HAV detections between 1.2% and 86.7%, and untreated wastewaters between 2.8% and 70.9%. Genotypes found in all cases were IA and IC. The only wastewater-based epidemiology study showed to be a useful tool as a complement of traditional epidemiological surveillance. Only four LA countries have looked for HAV in food samples, with genome detection rates between 9% and 33%. Latin American HAV circulation scenario is changing. In countries where socioeconomic and sanitary conditions have not improved, the virus persists with high endemicity and the access to the vaccine should be re-evaluated by local governments. In countries where access to clean water, better sanitary conditions and HAV immunisation programs have been implemented, the number of cases among young adults seems to be increasing, alerting health authorities.

Indicator and pathogenic virus removal in bench scale soil aquifer treatment.

The demonstration of enteric virus removal for indirect potable reuse of advanced purified water is necessary to ensure safe water reclamation practices. This study evaluated the efficacy of soil treatment in reducing concentrations of Pepper Mild Mottle Virus (PMMoV), Hepatitis A (HAV), and Norovirus (NoV) gene markers through bench scale unsaturated soil columns. Three different infiltration rates were evaluated to determine their impact on viral gene marker removal. The concentrations of viral markers in the column influent and effluent samples were measured through RNA extraction and then RT-qPCR, and the log reduction values (LRVs) were calculated to quantify the effectiveness of removal across the columns. The LRVs achieved for PMMoV were 2.80 ± 0.36, 2.91 ± 0.48, and 2.72 ± 0.32 for infiltration rates of 4.9 mm/h, 9.4 mm/h, and 14.0 mm/h, respectively. A one-way ANOVA indicated no statistically significant differences in LRVs among the various infiltration rates (p-value = 0.329). All samples measured for HAV were below the detection limit both in the influent and effluent of the soil columns. While NoV GI and GII markers were measurable in the soil column influent, they were removed to below the detection limit in the effluent. The use of half the Limit-of-Detection (LoD) for effluent values enabled the estimation of log removals, which were calculated as 1.42 ± 0.07, 1.64 ± 0.29, and 1.74 ± 0.18 for NoV GI and 1.14 ± 0.19, 1.58 ± 0.21, and 1.87 ± 0.41 for NoV GII at infiltration rates of 4.9 mm/h, 9.4 mm/h, and 14.0 mm/h. This highlights the efficacy of soil treatment in reducing virus gene marker concentrations at various infiltration rates, and that spreading basins employed for reclaimed water recharge to ground water aquifers are an effective method for reducing the presence of viral contaminants in indirect potable reuse systems.

Ongoing outbreak of hepatitis A associated with sexual transmission among men who have sex with men, Portugal, October 2023 to April 2024.

An outbreak of hepatitis A is ongoing in Portugal, with 71 confirmed cases from 7 October 2023 to 24 April 2024. Most cases are male, aged 18-44 years, with many identifying as men who have sex with men (MSM) and reported as suspected sexual transmission. Phylogenetic analysis identified the subgenotype IA, VRD 521-2016 strain, last observed in an MSM-associated multi-country outbreak in 2016 to 2018. We wish to alert colleagues in other countries to investigate potential similar spread.

Whole-Genome Sequencing-Based Confirmatory Methods on RT-qPCR Results for the Detection of Foodborne Viruses in Frozen Berries.

Accurate detection, identification, and subsequent confirmation of pathogens causing foodborne illness are essential for the prevention and investigation of foodborne outbreaks. This is particularly true when the causative agent is an enteric virus that has a very low infectious dose and is likely to be present at or near the limit of detection. In this study, whole-genome sequencing (WGS) was combined with either of two non-targeted pre-amplification methods (SPIA and SISPA) to investigate their utility as a confirmatory method for RT-qPCR positive results of foods contaminated with enteric viruses. Frozen berries (raspberries, strawberries, and blackberries) were chosen as the food matrix of interest due to their association with numerous outbreaks of foodborne illness. The hepatitis A virus (HAV) and human norovirus (HuNoV) were used as the contaminating agents. The non-targeted WGS strategy employed in this study could detect and confirm HuNoV and HAV at genomic copy numbers in the single digit range, and in a few cases, identified viruses present in samples that had been found negative by RT-qPCR analyses. However, some RT-qPCR-positive samples could not be confirmed using the WGS method, and in cases with very high Ct values, only a few viral reads and short sequences were recovered from the samples. WGS techniques show great potential for confirmation and identification of virally contaminated food items. The approaches described here should be further optimized for routine application to confirm the viral contamination in berries.

Evaluation of a new automated viral RNA extraction platform for hepatitis A virus and human norovirus in testing of berries, lettuce, and oysters.

Fruits, vegetables, and shellfish are often associated with outbreaks of illness caused particularly by human norovirus (HuNoV) and hepatitis A virus (HAV), the leading causative agents of foodborne illness worldwide. The aim of this study was to evaluate a new automated nucleic acid extraction platform (EGENE-UP EASYPREP) for enteric viruses in several at-risk food matrices and to test its limit of detection in comparison to a semi-automated method (EGENE-UP) using Boom methodology for nucleic acid extraction as suggested in the reference method ISO 15216-2:2019. Fresh and frozen raspberries, frozen blackberries, romaine lettuce and oyster digestive glands were artificially contaminated with HAV, HuNoV GII.4 or HuNoV GI.7 at 102, 103 or 104 genome copies/sample. Virus was then recovered from the food matrix using the ISO method. Viral RNA extracted from frozen berry samples by the automated system was purified on a column for additional removal of RT-qPCR inhibitors. For fresh raspberry, oysters, and romaine lettuce, the two extraction platforms were deemed equivalent. For frozen raspberry, the automated platform appeared to be more efficient for viral recovery, particularly for HAV and HuNoV GI at lower concentrations. With frozen blackberries, the two platforms may be considered equivalent for all targeted viruses. However, the automated method led to less sample-associated inhibition of the PCR, 56.5 % of samples versus 95.0 % for the semi-automated. We thus found that the automated extraction can be performed easily by users while obtaining equivalent or even superior results to the ISO 15216-2:2019 method, and therefore appears to be suitable for routine sanitary monitoring in food processing and for tracing outbreaks of illness.

Concentration of foodborne viruses eluted from fresh and frozen produce: Applicability of ultrafiltration.

Foodborne illnesses involving raw and minimally processed foods are often caused by human noroviruses (HuNoV) and hepatitis A virus (HAV). Since food is contaminated usually with small numbers of virions, these must be eluted from the food surface and then concentrated for detection. The objective of this study was to optimize an ultrafiltration (UF) concentration method for HAV and HuNoVs present on various fresh and frozen produce. The detection range of the optimized method and its applicability to different food matrices was compared to the reference method ISO 15216-1:2017. Strawberry, raspberry, blackberry, lettuce, and green onion (25 g) were contaminated with HAV, HuNoV GI.7 and HuNoV GII.4 and then recovered therefrom by elution. A commercial benchtop UF device was used for the concentration step. Viral RNA was extracted and detected by RT-qPCR. From fresh strawberries, recovery of HAV loaded at 104 genome copies per sample was 30 ± 13 %, elution time had no significant impact, and UF membrane with an 80-100 kDa cut-off in combination with Tris-glycine elution buffer at pH 9.5 was found optimal. At lower copy numbers on fresh strawberry, at least 1 log lower numbers of HuNoV were detectable by the UF method (103 vs 104 GII.4 copies/sample and 101 vs 103 GI.7 copies/sample), while HAV was detected at 101 genome copies/sample by both methods. Except on raspberry, the UF method was usually equivalent to the ISO method regardless of the virus tested. The UF method makes rapid viral concentration possible, while supporting the filtration of large volume of sample. With fewer steps and shorter analysis time than the ISO method, this method could be suitable for routine analysis of viruses throughout the food production and surveillance chain.

Loxapine inhibits replication of hepatitis A virus in vitro and in vivo by targeting viral protein 2C.

No antiviral drugs currently are available for treatment of infection by hepatitis A virus (HAV), a causative agent of acute hepatitis, a potentially life-threatening disease. Chemical screening of a small-compound library using nanoluciferase-expressing HAV identified loxapine succinate, a selective dopamine receptor D2 antagonist, as a potent inhibitor of HAV propagation in vitro. Loxapine succinate did not inhibit viral entry nor internal ribosome entry site (IRES)-dependent translation, but exhibited strong inhibition of viral RNA replication. Blind passage of HAV in the presence of loxapine succinate resulted in the accumulation of viruses containing mutations in the 2C-encoding region, which contributed to resistance to loxapine succinate. Analysis of molecular dynamics simulations of the interaction between 2C and loxapine suggested that loxapine binds to the N-terminal region of 2C, and that resistant mutations impede these interactions. We further demonstrated that administration of loxapine succinate to HAV-infected Ifnar1-/- mice (which lack the type I interferon receptor) results in decreases in the levels of fecal HAV RNA and of intrahepatic HAV RNA at an early stage of infection. These findings suggest that HAV protein 2C is a potential target for antivirals, and provide novel insights into the development of drugs for the treatment of hepatitis A.

Foodborne Viruses and Somatic Coliphages Occurrence in Fresh Produce at Retail from Northern Mexico.

Foodborne disease outbreaks linked to consumption of vegetables have been often attributed to human enteric viruses, such as Norovirus (NoV), Hepatitis A virus (HAV), and Rotavirus (RoV). Information about the occurrence of these viruses is scarce in many fresh-producing countries. Viral contamination detection of indicators, such as somatic coliphages, could indirectly reflect the presence of viral pathogens, being a valuable tool for better viral risk assessment in food industry. This study aimed to establish the occurrence and correlation of foodborne viruses and somatic coliphages in leafy greens in northern Mexico. A total of 320 vegetable samples were collected, resulting in 80 composite rinses, 40 of lettuce and 40 of parsley. Somatic coliphages were determined using the EPA 1602 method, while foodborne viruses (HAV, RoV, NoV GI, and GII) were determined by qPCR. The occurrence of RoV was 22.5% (9/40, mean 2.11 log gc/g) in lettuce and 20% (8/40, mean 1.91 log gc/g) in parsley. NoV and HAV were not detected in any samples. Somatic coliphages were present in all lettuce and parsley samples, with mean levels of 1.85 log PFU/100 ml and 2.28 log PFU/100 ml, respectively. Spearman analysis established the correlation of somatic coliphages and genomic copies of RoV, resulting in an r2 value of - 0.026 in lettuce and 0.349 in parsley. Although NoV or HAV were undetected in the samples, the presence of RoV is a matter of concern as leafy greens are usually eaten raw, which poses a potential risk of infection.

Meta-analysis of the prevalence of norovirus and hepatitis a virus in berries.

Norovirus (NoV) and hepatitis A virus (HAV) stand as the predominant agents associated with viral foodborne infections. Outbreaks have been documented to be caused by various types of food items, including fresh and/or frozen berries. Comprehensive data concerning crucial viral pathogens in berries remain limited and are not currently available in aggregate form. Consequently, the present study aimed to compile the existing information regarding the prevalence of NoV and HAV in this matrix. Records of foodborne viruses were systematically extracted from database repositories up to December 2022, adhering to PRISMA standards and were subjected to a multilevel random effect meta-analysis model to determine the mean occurrence rate of NoV and HAV. A high heterogeneity across studies was observed (I2 = 82 %), reflecting variations in the prevalence of sampling locations, years, berry types, and sample conditions, among other potential contributing factors. The prevalence of NoV and HAV in berries was calculated at 2.12 % (95 % CI 1.74-2.59 %), and no statistically differences were observed among the viral types or genogroup categories. However, it is important to clarify that this estimate should be taken with caution given the high heterogeneity. There was no discernible correlation between viral prevalence and any particular berry type. However, there was a temporal correlation observed with the year of sampling, revealing a significantly decreasing trend throughout the study period. A significant influence of the sample condition (fresh or frozen) was observed in relation to the prevalence of NoV GII and HAV. Overall higher viral prevalences were identified in berries originating from African countries as compared to those sourced from other continents. It was also noted that the prevalence of NoV GI was significantly higher in samples collected directly from farms compared to those obtained from retailers. The outcomes of this comprehensive meta-analysis propose that while viral contamination of berries is diminishing in more recent times, the prevalence remains substantial in certain African countries, having a significant risk for foodborne infections. It is imperative to implement intervention strategies in these regions to enhance product safety.

In-house RT-PCR detection of hepatitis A virus in human plasma pools used for the manufacture of medicinal products.

Hepatitis A virus (HAV) is the most frequent cause of viral hepatitis worldwide and is transmitted through the fecal-oral route. However, HAV can also be transmitted by blood-derived products. This is due to the fact that viremia occurs during the asymptomatic phase of HAV infection, enabling infected blood or plasma donations to occur. Viral inactivation/removal steps are included during manufacturing of plasmaderived products. However, HAV is a small non-enveloped virus very difficult to remove with traditional viral inactivation procedures. To accomplish European guidelines for pooled human plasma (treated for virus inactivation), plasma manufacturers have been implementing HAV nucleic acid test (NAT) screening on plasma pools. In this study, we validate an in-house multiplex reverse-transcription real-time PCR (RT-PCR) assay targeting HAV RNA and an internal control with hydrolysis probes for amplicon detection. The HAV RNA test was validated by assessing limit of detection, robustness, sensitivity and specificity according to European Pharmacopoeia (Ph. Eur.) guidelines. Our assay is able to detect 100 IU/mL of all human HAV genotypes that have been described so far. The multiplex assay shows remarkable sensitivity with a 95% lower limit of detection of 5.2 IU/mL. Also, our HAV test shows good robustness, precision, and specificity. We conclude that our assay broadly meets the requirements for its purpose. The implementation of this test in the production process of plasma-derived products will increase their safety.

High-performance enrichment-based genome sequencing to support the investigation of hepatitis A virus outbreaks.

IMPORTANCE: This proof-of-concept study introduces a hybrid capture oligo panel for whole-genome sequencing of all six human pathogenic hepatitis A virus (HAV) subgenotypes, exhibiting a higher sensitivity than some conventional genotyping assays. The ability of hybrid capture to enrich multiple targets allows for a single, streamlined workflow, thus facilitating the potential harmonization of molecular surveillance of HAV with other enteric viruses. Even challenging sample matrices can be accommodated, making them suitable for broad implementation in clinical and public health laboratories. This innovative approach has significant implications for enhancing multijurisdictional outbreak investigations as well as our understanding of the global diversity and transmission dynamics of HAV.

Approaches to produce and characterize recombinant protein VP1-2A of HAV for serological rapid test application.

Studies reporting the expression of hepatitis A virus (HAV) structural proteins, specifically recombinant VP1-2A containing an immunogenic activity, use the Escherichia coli system. Recombinant HAV proteins may represent a source of less expensive antigens for application in different diagnostic platforms. However, the formation of insoluble aggregates is an obstacle to obtaining large amounts of HAV proteins in their native form. To overcome this obstacle, some approaches were applied in this study to improve purification, solubility, and protein expression levels. Critical properties were evaluated. The introduction of another insertion codon to increase the protein concentration and vector activity was observed and verified by SDS-PAGE. The expression was established with 0.4 mM IPTG for 4 h at 37 °C. The VP1 protein was partially soluble at an isoeletric point (pI) of 6.45. The majority of HAV VP1-2A proteins measured 45.19 kDa in size and had a homogeneity of 53.58%. Multi-antigen print immunoassay (MAPIA) showed antigenicity at different HAV VP1-2A concentrations, and microsphere-based immunoassays showed a specificity of 100% and a sensitivity of 84%. HAV VP1-2A was characterized using different sensitivity methods to prove its biological activity, indicating its use as a tool for the diagnosis of Hepatitis A virus infection.

Hepatitis A outbreak in a refugee shelter in Kiel, northern Germany.

In the spring of 2023, three Ukrainian war refugees from a municipal community shelter and a volunteer caregiver at an affiliated daycare center in Kiel, Germany, were diagnosed with infectious jaundice attributable to a single hepatitis A virus (HAV) subgenotype IA strain. Similar HAV sequences have been observed in Germany and other European countries for several years. One refugee and the volunteer required hospitalization. Four children were asymptomatically infected but excreted high levels of HAV ribonucleic acid in the stool. The infections were probably acquired in Germany, but a source could not be determined. The outbreak was contained through vaccination, increased hygiene, and education. The existing HAV vaccination recommendation for refugee shelter staff and volunteers should be consistently implemented.

Divergent Genotype of Hepatitis A Virus in Alpacas, Bolivia, 2019.

Hepatitis A virus (HAV) is a common human pathogen found exclusively in primates. In a molecular and serologic study of 64 alpacas in Bolivia, we detected RNA of distinct HAV in ≈9% of animals and HAV antibodies in ≈64%. Complete-genome analysis suggests a long association of HAV with alpacas.

Development of a cell-free toehold switch for hepatitis A virus type I on-site detection.

Picornavirus hepatitis A virus (HAV) is a common cause of hepatitis worldwide. It is spread primarily through contaminated food and water or person-to-person contact. HAV I has been identified as the most common type of human HAV infection. Here, we have developed a cell-free toehold switch sensor for HAV I detection. We screened 10 suitable toehold switch sequences using NUPACK software, and the VP1 gene was used as the target gene. The optimal toehold switch sequence was selected by in vivo expression. The best toehold switch concentration was further found to be 20 nM in a cell-free system. 5 nM trigger RNA activated the toehold switch to generate visible green fluorescence. The minimum detection concentration decreased to 1 pM once combined with NASBA. HAV I trigger RNA could be detected accurately with excellent specificity. In addition, the cell-free toehold switch sensor was verified in HAV I entities. The successful construction of the cell-free toehold switch sensor provided a convenient, rapid, and accurate method for HAV I on-site detection, especially in developing countries, without the involvement of expensive facilities and additional professional operators.

Detection of Hepatitis A RNA, Hepatitis E RNA, Human Adenovirus F DNA, and Norovirus RNA in Fresh and Frozen Berry Products at Point of Retail in Ireland.

Soft fruits are at particular risk of contamination with enteric viruses such as Hepatitis A virus (HAV), Hepatitis E Virus (HEV), Norovirus (NoV), Human Adenovirus (HAdV) and Sapovirus (SaV). The aim of this study was to investigate, for the first time, the presence of these biological agents in ready to eat (RTE) berries at point of retail in Ireland. A sampling strategy was designed in which RTE fresh and frozen strawberries and raspberries were purchased from five retailers between May and October 2018. Reverse Transcriptase Polymerase Chain Reaction (RT-qPCR) assays for HEV RNA, Nov RNA, SaV RNA, and human Adenovirus species F DNA (HAdV-F) were performed on 239 samples (25g portions). Viral nucleic acid was present in 6.7% (n = 16) of samples tested as follows: HAV RNA (n = 5), HAdV-F DNA (n = 5), HEV RNA (n = 3) and NoV GII RNA (n = 3). Sapovirus RNA was not detected in any product. No significant differences were found between berry type, fresh/frozen status, or supermarket source. This study suggests a risk that exists across all retail outlets however only low levels of nucleic acid ranging from 0 to 16 genome copies/g were present. Although these findings may reflect non-viable/non-infectious virus the continued provision of risk mitigation advice to consumers is warranted and further work is required to ensure control measures to reduce contamination are implemented and enforced.

Hepatitis A virus subgenotypes in Latvia, 2008-2021.

BACKGROUND: In Latvia outbreaks of the HAV were observed between 2008 and early 2010 and again in 2017-2018. However, the risks of introducing and spreading infection still exist, as the virus spreads easily when personal hygiene is not followed. METHODS: To determine the spread of HAV subgenotypes in the territory of Latvia the VP1/P2A genomic region of HAV was amplified and sequenced for 259 case serum samples. The study carried out a molecular biological investigation and molecular epidemiological investigation. Demographic data (sex, age), disease data (hepatitis symptoms, hospitalization, vaccination) and epidemiology data (part of the outbreak, possible source of infection, recent travel) were collected. Based on the obtained sequences, the phylogenetic tree was built and analyzed for the homology and belonging to different isolated HAV clusters from other countries. RESULTS: From the obtained data, it was concluded that HAV subgenotype IA had 13 clusters and 12 sporadic cases, HAV subgenotype IB had eight clusters and 11 sporadic cases, HAV subgenotype IIIA had one cluster and nine sporadic cases. It was found that the sources of infection among the investigated cases were different, they were mostly associated with contact with a patient with HAV, travel, as well as between persons who inject drugs and men who have sex with men, and the prevalence of HAV similar sequences was observed in different years. It was concluded that patients with HAV subgenotype IA had the longest hospitalization duration and averaged 9.3 days, while patients with subgenotype IB - 7.3 days, subgenotype IIIA - 7.7 days. Analyzing the data on vaccination, it was found that mostly all were not vaccinated or had an unknown vaccination status. CONCLUSIONS: All of this has led to the conclusion that the application of molecular biological methods of the HAV and a careful analysis of epidemiological data can help to better understand the ways of spreading the infection, investigate local outbreaks, detect cases of imported infection and track the recirculation of the virus.

Molecular diagnosis of patients with hepatitis A virus infection using amplicon-based nanopore sequencing.

High-throughput sequencing is a robust tool used for identifying and tracking pathogen outbreaks. Whole-genome sequencing of hepatitis A virus (HAV) remains poor due to ultra-low viral loads, limitations of next-generation sequencing technology, and its high costs in clinical applications. This study evaluated multiplex polymerase chain reaction (PCR)-based nanopore sequencing to obtain whole-genome sequences of HAV. The HAV genomes were obtained directly from patient specimens for a rapid molecular diagnosis of viral genotypes. Serum and stool samples were collected from six patients with hepatitis A infection. Amplicon-based nanopore sequencing was performed from the clinical specimens to identify HAV genotypes by acquiring nearly complete-genome sequences. TaqMan-based quantitative PCR (qPCR) was conducted to detect and quantify multiple HAV genes. Singleplex-based nanopore sequencing demonstrated high genome coverage rates (90.4-99.5%) of HAV within 8 h, at viral RNA loads of 10 to 105 copies/µL. TaqMan qPCR showed multiplex quantification of HAV genes namely, VP0, VP3, and 3C. This study provides useful insights into rapid molecular diagnosis during hepatitis A outbreaks and may ultimately augment public health disease surveillance in the hospital and epidemiology field.

Molecular epidemiology and characterization of endemic, epidemic and imported hepatitis A virus (HAV) strains in Hungary (2003-2022).

Hepatitis A virus (HAV) is one of the most important etiological agents of acute viral hepatitis but comprehensive molecular epidemiological study with chrono-phylogeographical data are not available from Hungary.Between 2003 and 2022, a total of 8,307 HAV infections were registered officially in Hungary of which 400 (4.8%) HAV IgM antibody-positive serum samples were collected countrywide. HAV genomic RNA was successfully detected in 216/400 (54%) sera by RT-PCR subsequently confirmed by sequencing. The complete nucleotide sequences of VP1 region were determined in 32 representative HAV strains. Based on the sequence analysis, 150 (69.4%) strains were characterized as HAV sub-genotype IA and 66 (30.6%) as sub-genotype IB, respectively. Based on the combined epidemiological and molecular data, epidemic, endemic, and imported HAV strains were also characterized. The first two registered countrywide outbreaks started among men-sex-with men (MSM) in 2011 (sub-genotype IA) and 2021 (sub-genotype IB), the continuously circulating endemic/domestic HAV strain (sub-genotype IA) in East Hungary and the travel-related sub-genotype IB strains from Egypt should be highlighted. All HAV strains are deposited in the HAVNET database (https://www.rivm.nl/en/havnet).In this 20-year-long comprehensive molecular epidemiological study, we report the genetic characterization and geographic distribution of endemic, epidemic and imported HAV strains for the first time in Hungary with continuous co-circulation of sub-genotypes IA and IB HAV strains since 2003. These data provide basic information about the HAV situation in the country in an international context and can promote more effective national public health intervention strategies for the prevention of HAV transmissions and infections.