Prevalence and Genetic Diversity of Adenovirus 40/41, Astrovirus, and Sapovirus in Children with Acute Gastroenteritis in Kansas City 2011-2016.

BACKGROUND: Most U.S. acute gastroenteritis (AGE) episodes in children are attributed to norovirus, whereas very little information is available on adenovirus 40/41 (AdV40/41), astrovirus or sapovirus. The New Vaccine Surveillance Network (NVSN) conducted prospective, active, population-based AGE surveillance in young children. METHODS: We tested and typed stool specimens collected between December 2011 to June 2016 from one NVSN site in Kansas City for the three viruses, and calculated hospitalization and emergency department (ED) detection rate. RESULTS: Of 3,205 collected specimens, 2,453 (76.5%) were from AGE patients (339 inpatients and 2,114 ED patients) and 752 (23.5%) were from healthy controls (HC). In AGE patients, astrovirus was detected in 94 (3.8%), sapovirus in 252 (10.3%) and AdV40/41 in 101 (4.5%) of 2249 patients. In HC, astrovirus was detected in 13 (1.7%) and sapovirus in 15 (2.0%) specimens. Astrovirus type 1 (37.7%) and genogroup I sapoviruses (59.3%) were most prevalent.Hospitalization rates were 5 (AdV40/41), 4 (astrovirus) and 8 (sapovirus) per 100,000 children <11 years old, whereas ED rates were 2.4 (AdV40/41), 1.9 (astrovirus) and 5.3 (sapovirus) per 1000 children <5 years old. CONCLUSIONS: Overall, AdV40/41, astrovirus, and sapovirus were detected in 18.6% of AGE in a large pediatric hospital in Kansas City.

Timing and genotype distribution of symptomatic and asymptomatic sapovirus infections and re-infections in a Nicaraguan birth cohort.

OBJECTIVES: To characterize the timing and genotype distribution of symptomatic and asymptomatic sapovirus infections and re-infections in a Nicaraguan birth cohort. METHODS: Infants (N = 444) were enrolled at 10-14 days of life and observed weekly until 2 years of age. Stool samples were collected for each acute gastroenteritis (AGE) episode, and routine stool samples were collected monthly. Stool samples were tested for sapovirus using RT-qPCR, and positive samples were genotyped. RESULTS: A total of 348 children completed 2 years of AGE weekly surveillance; 93 (26.7%) of them experienced sapovirus AGE. Most infections occurred after 5 months of age and mainly during the second year of life (62.4%, 58/93) and early in the rainy season. Sapovirus screening in all stools from a subset of 67 children who consistently provided samples showed sapovirus infections in 91 of 330 (27.6%) AGE episodes and in 39 of 1350 (2.9%) routine stools. In this subset, the median age at the first sapovirus AGE was 11.2 months (95% CI, 9.3-15.9 months); 38 of 67 (57%) children experienced re-infections, 19 symptomatic and 19 asymptomatic. On average, sapovirus re-infections were reported 7.2 months after symptomatic and 5.3 months after asymptomatic infections. Genogroup GI (64%, 69/108) was the most common detected. Sapovirus GI.1 was more frequently detected in AGE stool samples than in routine stool samples (47.2%, 43/91 vs. 25.6%, 10/39; p 0.005), and re-infection with the same genotype was uncommon. DISCUSSION: The first sapovirus infections occurred at approximately 11 months of age, whereas the median time to symptomatic re-infection was 7.2 months. Re-infections with the same sapovirus genotype were rare during 2 years of life suggesting genotype-specific protection after natural infection.

Norovirus Infection in Young Nicaraguan Children Induces Durable and Genotype-Specific Antibody Immunity.

There are significant challenges to the development of a pediatric norovirus vaccine, mainly due to the antigenic diversity among strains infecting young children. Characterizing human norovirus serotypes and understanding norovirus immunity in naïve children would provide key information for designing rational vaccine platforms. In this study, 26 Nicaraguan children experiencing their first norovirus acute gastroenteritis (AGE) episode during the first 18 months of life were investigated. We used a surrogate neutralization assay that measured antibodies blocking the binding of 13 different norovirus virus-like particles (VLPs) to histo-blood group antigens (HBGAs) in pre- and post-infection sera. To assess for asymptomatic norovirus infections, stools from asymptomatic children were collected monthly, screened for norovirus by RT-qPCR and genotyped by sequencing. Seroconversion of an HBGA-blocking antibody matched the infecting genotype in 25 (96%) of the 26 children. A subset of 13 (50%) and 4 (15%) of the 26 children experienced monotypic GII and GI seroconversion, respectively, strongly suggesting a type-specific response in naïve children, and 9 (35%) showed multitypic seroconversion. The most frequent pairing in multitypic seroconversion (8/12) were GII.4 Sydney and GII.12 noroviruses, both co-circulating at the time. Blocking antibody titers to these two genotypes did not correlate with each other, suggesting multiple exposure rather than cross-reactivity between genotypes. In addition, GII titers remained consistent for at least 19 months post-infection, demonstrating durable immunity. In conclusion, the first natural norovirus gastroenteritis episodes in these young children were dominated by a limited number of genotypes and induced responses of antibodies blocking binding of norovirus VLPs in a genotype-specific manner, suggesting that an effective pediatric norovirus vaccine likely needs to be multivalent and include globally dominant genotypes. The duration of protection from natural infections provides optimism for pediatric norovirus vaccines administered early in life.

Preexisting Heterotypic Ligand-blocking Antibody Does Not Protect Against Genogroup II Norovirus Episodes in Young Children.

A birth cohort design was used to understand whether heterotypic ligand-blocking norovirus antibodies provide cross-protection within the GII genogroup. We found that almost one-half of children who experienced a norovirus GII episode had preexisting antibodies heterotypic to the infecting genotype; therefore, these antibodies did not provide cross-protection.

Gut Microbiome Changes Occurring with Norovirus Infection and Recovery in Infants Enrolled in a Longitudinal Birth Cohort in Leon, Nicaragua.

Noroviruses are associated with one fifth of diarrheal illnesses globally and are not yet preventable with vaccines. Little is known about the effects of norovirus infection on infant gut microbiome health, which has a demonstrated role in protecting hosts from pathogens and a possible role in oral vaccine performance. In this study, we characterized infant gut microbiome changes occurring with norovirus-associated acute gastroenteritis (AGE) and the extent of recovery. Metagenomic sequencing was performed on the stools of five infants participating in a longitudinal birth cohort study conducted in León, Nicaragua. Taxonomic and functional diversities of gut microbiomes were profiled at time points before, during, and after norovirus infection. Initially, the gut microbiomes resembled those of breastfeeding infants, rich in probiotic species. When disturbed by AGE, Gammaproteobacteria dominated, particularly Pseudomonas species. Alpha diversity increased but the genes involved in carbohydrate metabolism and glycan biosynthesis decreased. After the symptoms subsided, the gut microbiomes rebounded with their taxonomic and functional communities resembling those of the pre-infection microbiomes. In this study, during disruptive norovirus-associated AGE, the gut microbiome was temporarily altered, returning to a pre-infection composition a median of 58 days later. Our study provides new insights for developing probiotic treatments and furthering our understanding of the role that episodes of AGE have in shaping the infant gut microbiome, their long-term outcomes, and implications for oral vaccine effectiveness.

First Episodes of Norovirus and Sapovirus Gastroenteritis Protect Against Subsequent Episodes in a Nicaraguan Birth Cohort.

BACKGROUND: Norovirus and sapovirus cause a large burden of acute gastroenteritis (AGE) in young children. We assessed protection conferred by norovirus and sapovirus AGE episodes against future episodes. METHODS: Between June 2017 and July 2018, we recruited 444 newborns in León, Nicaragua. Weekly household surveys identified AGE episodes over 36 months, and AGE stools were tested by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) for norovirus genogroup (G)I/GII and sapovirus. We used recurrent-event Cox models and negative control methods to estimate protection conferred by first episodes, controlling for observed and unobserved risk factors, respectively. RESULTS: Sapovirus episodes conferred a 69% reduced hazard of subsequent episodes using the negative control method. Norovirus GI (hazard ratio [HR] = 0.67; 95% confidence interval [CI] = 0.31, 1.3) and GII (HR = 0.20; 95% CI = 0.04, 0.44) episodes also appeared highly protective. Protection against norovirus GII was enhanced following two episodes. CONCLUSIONS: Evidence of natural immunity in early childhood provides optimism for the future success of pediatric norovirus and sapovirus vaccines.

Secretor Status Strongly Influences the Incidence of Symptomatic Norovirus Infection in a Genotype-Dependent Manner in a Nicaraguan Birth Cohort.

BACKGROUND: The role of histo-blood group on the burden and severity of norovirus gastroenteritis in young infants has not been well documented. METHODS: Norovirus gastroenteritis was assessed in 443 Nicaraguan children followed from birth until 3 years of age. Stool samples were tested for norovirus by reverse-transcription quantitative polymerase chain reaction (RT-qPCR), and histo-blood group antigens (HBGAs) were determined by phenotyping of saliva and blood. Hazard ratios and predictors of norovirus acute gastroenteritis (AGE) outcome stratified by HBGA were estimated using Cox proportional hazards models. RESULTS: Of 1353 AGE episodes experienced by children, 229 (17%) tested positive for norovirus with an overall incidence of 21.9/100 child-years. Secretor children were infected as early as 2 months of age and had a higher incidence of norovirus GII compared to nonsecretor children (15.4 vs 4.1/100 child-years, P = .006). Furthermore, all GII.4 AGE episodes occurred in secretor children. Children infected with GI (adjusted odds ratio [aOR], 0.09 [95% confidence interval {CI}, .02-.33]) or non-GII.4 viruses (aOR, 0.2 [95% CI, .07-.6]) were less likely to have severe AGE compared to GII.4-infected children. CONCLUSIONS: Secretor status in children strongly influences the incidence of symptomatic norovirus infection in a genogroup or genotype-dependent manner and provides evidence that clinical severity in children depends on norovirus genotypes.

Global distribution of sporadic sapovirus infections: A systematic review and meta-analysis.

Acute gastroenteritis (AGE), characterized by diarrhea and vomiting, is an important cause of global mortality, accounting for 9% of all deaths in children under five years of age. Since the reduction of rotavirus in countries that have included rotavirus vaccines in their national immunization programs, other viruses such as norovirus and sapovirus have emerged as more common causes of AGE. Due to widespread use of real-time RT-PCR testing, sapovirus has been increasingly reported as the etiologic agent in both AGE outbreaks and sporadic AGE cases. We aimed to assess the role of sapovirus as a cause of endemic AGE worldwide by conducting a systematic review of published studies that used molecular diagnostics to assess the prevalence of sapovirus among individuals with AGE symptoms. Of 106 articles included, the pooled sapovirus prevalence was 3.4%, with highest prevalence among children <5 years of age (4.4%) and among individuals in community settings (7.1%). Compared to studies that used conventional RT-PCR, RT-qPCR assays had a higher pooled prevalence (5.6%). Among individuals without AGE symptoms, the pooled sapovirus prevalence was 2.7%. These results highlight the relative contribution of sapovirus to cases of AGE, especially in community settings and among children <5 years of age.

Risk Factors and Clinical Profile of Sapovirus-associated Acute Gastroenteritis in Early Childhood: A Nicaraguan Birth Cohort Study.

BACKGROUND: Sapovirus is increasingly recognized as an important cause of acute gastroenteritis (AGE) in children. We identified risk factors and characterized the clinical profile of sapovirus AGE in a birth cohort in León, Nicaragua. METHODS: We conducted a case-control study nested within a birth cohort (n = 444). Fieldworkers conducted weekly household AGE surveillance. AGE stools were tested for sapovirus by reverse transcriptase quantitative polymerase chain reaction. For each first sapovirus episode, we selected 2 healthy age-matched controls and estimated independent risk factors of sapovirus AGE using conditional logistic regression. We compared clinical characteristics of sapovirus AGE episodes with episodes associated with other etiologies and identified co-infections with other enteric pathogens. RESULTS: From June 2017 to July 2019, we identified 63 first sapovirus AGE episodes and selected 126 controls. Having contact with an individual with AGE symptoms and vaginal delivery were independent risk factors for sapovirus AGE. All cases experienced diarrhea, lasting a median 6 days; 23% experienced vomiting. Compared with children with AGE due to another etiology, sapovirus AGE was similar in severity, with less reported fever. Most cases experienced co-infections and were more likely than controls to be infected with diarrheagenic Escherichia coli or astrovirus. CONCLUSIONS: Sapovirus was a commonly identified AGE etiology in this Central American setting, and symptoms were similar to AGE associated with other etiologies. The association between vaginal delivery and sapovirus is a novel finding. Gut microbiome composition might mediate this relationship, or vaginal delivery might be a proxy for other risk factors. Further investigation into more specific biological mechanisms is warranted.

Human Calicivirus Typing tool: A web-based tool for genotyping human norovirus and sapovirus sequences.

BACKGROUND: The family Caliciviridae consists of a genetically diverse group of RNA viruses that infect a wide range of host species including noroviruses and sapoviruses which cause acute gastroenteritis in humans. Typing of these viruses relies on sequence-based approaches, and therefore there is a need for rapid and accurate web-based typing tools. OBJECTIVE: To develop and evaluate a web-based tool for rapid and accurate genotyping of noroviruses and sapoviruses. METHODS: The Human Calicivirus Typing (HuCaT) tool uses a set of curated reference sequences that are compared to query sequences using a k-mer (DNA substring) based algorithm. Outputs include alignments and phylogenetic trees of the 12 top matching reference sequences for each query. RESULTS: The HuCaT tool was validated with a set of 1310 norovirus and 239 sapovirus sequences covering all known human norovirus and sapovirus genotypes. HuCaT tool assigned genotypes to all queries with 100 % accuracy and was much faster (17 s) than BLAST (150 s) or phylogenetic analyses approaches. CONCLUSIONS: The web-based HuCaT tool supports rapid and accurate genotyping of human noroviruses and sapoviruses.

Single-step RT-PCR assay for dual genotyping of GI and GII norovirus strains.

BACKGROUND: Noroviruses are the major cause of acute gastroenteritis (AGE) in people of all ages globally. Standardized genotyping is key for outbreak investigations and surveillance networks. OBJECTIVE: Here we describe the validation of a one-step conventional RT-PCR assay for sequence-based dual typing of GI and GII noroviruses. This polymerase (P) and capsid (C) dual typing assay uses a combination of previously published oligonucleotide primers amplifying a genomic region spanning the 3'-end of ORF1 and 5'end of ORF2 resulting in a 579 bp product for GI and 570 bp product for GII viruses. RESULTS: The limit of detection of the assay ranged from 5 to 50 copies of viral RNA per reaction for GI and GII. To validate the assay, we tested 2,663 noroviruspositive stool samples from outbreaks and sporadic cases of AGE in Bangladesh, Guatemala, Peru, and USA collected between 2010-2019, of which 2,392 (90 %) were genotyped successfully. Most of the known genotypes infecting humans (GI (n = 9) and GII (n = 23)) and P types (GI (n = 15), GII, (n = 20)) could be detected. The remaining 270 samples had low viral load (Ct > 30) by real-time RT-PCR. A panel of 166 samples positive for other enteric viruses (rotavirus, astrovirus, sapovirus, adenovirus type 40/41) tested negative. CONCLUSION: The use of broadly reactive genotyping assays greatly strengthens exchange of standardized genotype data globally to monitor trends in genotype diversity which is important for both the development of vaccines and to measure their impact.

Comparison of Illumina MiSeq and the Ion Torrent PGM and S5 platforms for whole-genome sequencing of picornaviruses and caliciviruses.

Next-generation sequencing is a powerful tool for virological surveillance. While Illumina® and Ion Torrent® sequencing platforms are used extensively for generating viral RNA genome sequences, there is limited data comparing different platforms. The Illumina MiSeq, Ion Torrent PGM and Ion Torrent S5 platforms were evaluated using a panel of sixteen specimens containing picornaviruses and human caliciviruses (noroviruses and sapoviruses). The specimens were processed, using combinations of three library preparation and five sequencing kits, to assess the quality and completeness of assembled viral genomes, and an estimation of cost per sample to generate the data was calculated. The choice of library preparation kit and sequencing platform was found to impact the breadth of genome coverage and accuracy of consensus viral genomes. The Ion Torrent S5 510 chip runs produced more reads at a lower cost per sample than the highest output Ion Torrent PGM 318 chip run, and generated the highest proportion of reads for enterovirus D68 samples. However, indels at homopolymer regions impacted the accuracy of consensus genome sequences. For lower throughput sequencing runs (i.e., Ion Torrent 510 and Illumina MiSeq Nano V2), the cost per sample was lower on the MiSeq platform, whereas with higher throughput runs (Ion Torrent 530 and Illumina MiSeq V2) there is less of a difference in the cost per sample between the two sequencing platforms ($5.47-$10.25 more per sample for an Ion Torrent 530 chip run when multiplexing 24 samples). These findings suggest that the Ion Torrent S5 and Illumina MiSeq platforms are both viable options for genomic sequencing of RNA viruses, each with specific advantages and tradeoffs.

Prevalence and genetic diversity of viral gastroenteritis viruses in children younger than 5 years of age in Guatemala, 2014-2015.

BACKGROUND: Acute diarrhea is an important cause of morbidity and mortality in children and is associated with approximately 500,000 deaths/year globally. Rotavirus and norovirus are leading causes of acute diarrhea accounting for more than half of this burden. OBJECTIVE/STUDY DESIGN: To determine the prevalence and genotype distribution of acute diarrhea caused by rotavirus, norovirus, sapovirus and astrovirus among children <5 years of age at two departments in Guatemala from January 2014 to December 2015, we tested 471 stool specimens (202 samples from hospitalized children and 269 samples from children in ambulatory clinics) by real-time reverse transcription-PCR and genotyped positive samples. RESULTS: Rotavirus was detected in 20.4%, norovirus in 18.5%, sapovirus in 7% and astrovirus in 4.2% of the samples. Co-infection of rotavirus and norovirus was found in 2.6% of the samples. Most norovirus (87.4%) and rotavirus (81.3%) infections were detected in children in the 6-12 months age group. The proportion of patients with rotavirus (34%) and norovirus (23%) was higher in hospitalized patients compared to ambulatory patients, whereas the prevalence of sapovirus and astrovirus was similar in both settings. Of the 40 genotyped norovirus strains, 62.5% were GII.4 and 15% GII.3. Sapovirus genotypes included GI.1 (15.4%), GII.2 (15.4%), GII.5 (38.5%) and GIV.1 (30.8%). CONCLUSIONS: Our data demonstrate that in 2014-2015, gastroenteritis viruses account for 50% of acute diarrhea in children younger than 5 years of age in Guatemala, highlighting the importance of continuous surveillance to guide impact of the current rotavirus vaccine and formulation of future norovirus vaccines.

Near-Complete Human Sapovirus Genome Sequences from Kenya.

We report five near-complete sapovirus genome sequences, including GI.3, GII.2, and GII.6 and two novel GII.NA (not assigned) strains. These new sequences expand the collection of human sapoviruses, allowing for a more accurate phylogenetic analysis of circulating strains and for designing broadly reactive primers for their detection and typing.

Genetic diversity of human sapovirus across the Americas.

BACKGROUND: Sapoviruses are responsible for sporadic and epidemic acute gastroenteritis worldwide. Sapovirus typing protocols have a success rate as low as 43% and relatively few complete sapovirus genome sequences are available to improve current typing protocols. OBJECTIVE/STUDY DESIGN: To increase the number of complete sapovirus genomes to better understand the molecular epidemiology of human sapovirus and to improve the success rate of current sapovirus typing methods, we used deep metagenomics shotgun sequencing to obtain the complete genomes of 68 sapovirus samples from four different countries across the Americas (Guatemala, Nicaragua, Peru and the US). RESULTS: VP1 genotyping showed that all sapovirus sequences could be grouped in the four established genogroups (GI (n = 13), GII (n = 30), GIV (n = 23), GV (n = 2)) that infect humans. They include the near-complete genome of a GI.6 virus and a recently reported novel GII.8 virus. Sequences of the complete RNA-dependent RNA polymerase gene could be grouped into three major genetic clusters or polymerase (P) types (GI.P, GII.P and GV.P) with all GIV viruses harboring a GII polymerase. One (GII.P-GII.4) of the new 68 sequences was a recombinant virus with the hotspot between the NS7 and VP1 regions. CONCLUSIONS: Analyses of this expanded database of near-complete sapovirus sequences showed several mismatches in the genotyping primers, suggesting opportunities to revisit and update current sapovirus typing methods.

Pathogen-Specific Burden of Outpatient Diarrhea in Infants in Nepal: A Multisite Prospective Case-Control Study.

BACKGROUND: Nonsevere diarrheal disease in Nepal represents a large burden of illness. Identification of the specific disease-causing pathogens will help target the appropriate control measures. METHODS: Infants aged 6 weeks to 12 months were recruited from 5 health facilities in eastern, central, and western Nepal between August 2012 and August 2013. The diarrhea arm included infants with mild or moderate diarrhea treatable in an outpatient setting; the nondiarrhea arm included healthy infants who presented for immunization visits or had a mild nondiarrheal illness. Stool samples were tested for 15 pathogens with a multiplex polymerase chain reaction (PCR) assay and real-time reverse-transcription (RT)-PCR assays for rotavirus and norovirus. Rotavirus- and norovirus-positive specimens were genotyped. We calculated attributable fractions (AFs) to estimate the pathogen-specific burden of diarrhea and adjusted for facility, age, stunting, wasting, and presence of other pathogens. RESULTS: We tested 307 diarrheal and 358 nondiarrheal specimens. Pathogens were detected more commonly in diarrheal specimens (164 of 307 [53.4%]) than in nondiarrheal specimens (113 of 358 [31.6%]) (P < .001). Rotavirus (AF, 23.9% [95% confidence interval (CI), 14.9%-32.8%]), Salmonella (AF, 12.4% [95% CI, 6.6%-17.8%]), and Campylobacter (AF, 5.6% [95% CI, 1.3%-9.8%]) contributed most to the burden of disease. In these diarrheal specimens, the most common genotypes for rotavirus were G12P[6] (27 of 82 [32.9%]) and G1P[8] (16 of 82 [19.5%]) and for norovirus were GII.4 Sydney (9 of 26 [34.6%]) and GII.7 (5 of 26 [19.2%]). CONCLUSIONS: The results of this study indicate that the introduction of a rotavirus vaccine in Nepal will likely decrease outpatient diarrheal disease burden in infants younger than 1 year, but interventions to detect and target other pathogens, such as Salmonella and Campylobacter spp, should also be considered.

Synthesis and Evaluation of Biotinylated Bivalent HistoBlood Group Antigens for Capturing Human Noroviruses.

A panel of biotinylated bivalent H-type glycans that have been reported as binding ligands for human noroviruses were synthesized using a modular synthetic strategy. These glycoconjugates were attached to streptavidin-coated magnetic beads and used to recover human norovirus from fecal samples using a magnetic bead-based assay. The biotinylated bivalent glycans synthesized for this study exhibited similar or better capturing ability when compared to commercial biotinylated glycopolymers.

Monitoring of Extraction Efficiency by a Sample Process Control Virus Added Immediately Upon Sample Receipt.

When analysing food samples for enteric viruses, a sample process control virus (SPCV) must be added at the commencement of the analytical procedure, to verify that the analysis has been performed correctly. Samples can on occasion arrive at the laboratory late in the working day or week. The analyst may consequently have insufficient time to commence and complete the complex procedure, and the samples must consequently be stored. To maintain the validity of the analytical result, it will be necessary to consider storage as part of the process, and the analytical procedure as commencing on sample receipt. The aim of this study was to verify that an SPCV can be recovered after sample storage, and thus indicate the effective recovery of enteric viruses. Two types of samples (fresh and frozen raspberries) and two types of storage (refrigerated and frozen) were studied using Mengovirus vMC0 as SPCV. SPCV recovery was not significantly different (P > 0.5) regardless of sample type or duration of storage (up to 14 days at -20 °C). Accordingly, samples can be stored without a significant effect on the performance of the analysis. The results of this study should assist the analyst by demonstrating that they can verify that viruses can be extracted from food samples even if samples have been stored.

Presence of pathogenic enteric viruses in illegally imported meat and meat products to EU by international air travelers.

One hundred twenty two meat samples confiscated from passengers on flights from non-European countries at the International Airport of Bilbao (Spain) were tested for the presence of the main foodborne viral pathogens (human noroviruses genogroups I and II, hepatitis A and E viruses) during 2012 and 2013. A sample process control virus, murine norovirus, was used to evaluate the correct performance of the method. Overall, 67 samples were positive for at least one enteric viruses, 65 being positive for hepatitis E virus (53.3%), 3 for human norovirus genogroup I (2.5%) and 1 for human norovirus genogroup II (0.8%), whereas hepatitis A virus was not detected in any sample. The type of positive meat samples was diverse, but mainly was pork meat products (64.2%). The geographical origin of the positive samples was wide and diverse; samples from 15 out 19 countries tested were positive for at least one virus. However, the estimated virus load was low, ranging from 55 to 9.0 × 10(4) PDU per gram of product. The results obtained showed the potential introduction of viral agents in travelers' luggage, which constitute a neglected route of introduction and transmission.

Foods from black market at EU border as a neglected route of potential methicillin-resistant Staphylococcus aureus transmission.

The illegal entrance of foods to EU through black markets at the EU borders can constitute a neglected route of dissemination of foodborne pathogens, and in particular of methicillin-resistant Staphylococcus aureus (MRSA). In this study, we have assessed the presence of MRSA in foods sold in a black market at an EU border (the southeast part of Romania, on the border with Republic of Moldavia). We performed a search for MRSA among 200 food samples collected from 2012 to 2013. All S. aureus were studied by pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing. MRSA isolates were further characterized by multilocus sequence typing (MLST) and SCCmec typing, and tested for the presence of Panton-Valentine leukocidin (PVL) virulence factors. Overall, 32 S. aureus isolates were recovered from 16 food samples (8%). One isolate detected in a pork lard sample was MRSA (0.5%). PFGE with the restriction enzyme SmaI revealed 12 genotypes among the 32 S. aureus isolates. The MRSA isolate belonged to sequence type 398, harbored SCCmec type V, tested negative for the presence of the PVL genes and was resistant to ciprofloxacin, tetracycline and cefazolin, besides all ß-lactams. Among 31 methicillin-sensitive S. aureus (MSSA), 29% were resistant to penicillin, 9.7% to tetracycline and 3.2% to ciprofloxacin. In conclusion, in this study we report the presence of livestock-associated MRSA in foods sold in a black market at an EU border: ST398-MRSA-V. These results confirm the potential role of food in the dissemination of MRSA lineages among population, and the potential role of illegally introduced food to EU in the prevalence and evolution of MRSA clones in the community.