Norovirus gastroenteritis outbreaks in military units: a systematic review.

INTRODUCTION: Norovirus gastroenteritis is one of the most frequent causes of personnel unavailability in military units, being associated with significant morbidity and degradation of their operational effectiveness. The disease is usually mild but can be severe and life-threatening in young and healthy soldiers, who are prone to dehydration due to intensive daily activity. Despite its impact, the full extent of the norovirus gastroenteritis burden in military forces remains unclear. This systematic review aims to evaluate the impact and ascertain clinical and epidemiological features of norovirus outbreaks that have occurred in the military forces. METHODS: The systematic review followed the Preferred Reporting Items for Systemic Reviews and Meta-Analysis (PRISMA) guidelines and used three databases: PubMed, Scopus, and LILACs. Papers published up to 1 September 2019 were included without restrictions if they reported one or more outbreaks in the military forces on active duty, either on national territories or deployed overseas. RESULTS: A total of 343 papers were retrieved from the literature search. After inclusion/exclusion criteria a total of 39 eligible papers were considered. From 1988 (first reported outbreak in the military) to 2018 more than 101 norovirus outbreaks have been reported in the military, accounting for at least 24 332 cases. Secondary transmission was emphasised as the main route of norovirus transmission in the military forces, with eating outside the military setting an important route for the primary cases. CONCLUSIONS: The present review highlights that norovirus gastroenteritis has been a burden to military troops both in combat and on peacekeeping operations. Norovirus disease has been shown to exact a substantial toll on mission readiness and operational effectiveness. It is noteworthy that the impact of norovirus outbreaks among military units is underestimated because the literature review retrieved information from the armed forces from only nine countries.

Simultaneous norovirus outbreak in three Portuguese army bases in the Lisbon region, December 2017.

INTRODUCTION: Norovirus outbreaks frequently occur in communities and institutional settings acquiring a particular significance in armed forces where prompt reporting is critical. Here we describe the epidemiological, clinical and laboratorial investigation of a multicentre gastroenteritis outbreak that was detected simultaneously in three Portuguese army units with a common food supplier, Lisbon region, between 5 and 6 December 2017. METHODS: Questionnaires were distributed to all soldiers stationed in the three affected army units, and stool specimens were collected from soldiers with acute gastrointestinal illness. Stool specimens were tested for common enteropathogenic bacteria by standard methods and screened for a panel of enteric viruses using a multiplex real-time PCR assay. Food samples were also collected for microbiological analysis. Positive stool specimens for norovirus were further genotyped. RESULTS: The three simultaneous acute gastroenteritis outbreaks affected a 31 (3.5%) soldiers from a total of 874 stationed at the three units and lasted for 2 days. No secondary cases were reported. Stool specimens (N=11) were negative for all studied enteropathogenic agents but tested positive for norovirus. The recombinant norovirus GII.P16-GII.4 Sydney was identified in all positive samples with 100% identity. CONCLUSIONS: The results are suggestive of a common source of infection plausibly related to the food supplying chain. Although centralisation of food supplying in the army has economic advantages, it may contribute to the multifocal occurrence of outbreaks. A rapid intervention is key in the mitigation of outbreak consequences and in reducing secondary transmission.

Effects of peanut drying and blanching on Salmonella spp.

In order to evaluate the behavior of Salmonella during peanut drying and blanching, a study was conducted with Runner type peanuts. Samples of raw in-shell or unblanched peanuts were inoculated by spraying with a pool of five Salmonella serotypes isolated from the peanut supply chain (Miami, Muenster, Yoruba, Javiana and Glostrup). The in-shell peanuts were submitted to drying at 35 and 40 °C up to 18 h. After this time, the Salmonella counts went down ca. 2.0 log MPN/g at 35 and 40 °C. According to the Weibull model the time needed to achieve Salmonella 3-log reduction (T3d) and 5-log reduction (T5d) on the in-shell peanuts would be ca. 49 and 117 h at 35 °C and 35 and 79 h at 40 °C, respectively. The results showed that there was no statistical difference (p > .05) between either of the temperatures employed in the process. The blanching process was performed in two steps: pre-roasting (step 1) and skin removal (step 2). Reduction of up to 2.1 log MPN/g was observed after blanching at 100 °C/15 min plus 15 s of air impact. The skin removal process did not result in recontamination of the final sample. The Weibull model predicted 3- and 5-log reductions of Salmonella in 37.0 and 68.9 min for blanching at 95 °C, and in 39.1 and 114.9 min at 100 °C. The results demonstrated that drying and blanching processes did not generate large reductions of Salmonella in the peanut samples. Thus, the product resulting from these steps may be a possible source of cross-contamination for the processing plant and the final product.

Country-wide surveillance of norovirus outbreaks in the Portuguese Army, 2015-2017.

INTRODUCTION: Gastrointestinal infections are among the most common foodborne and waterborne diseases in military populations, with direct implications in operational efficiency and force readiness. Through the surveillance system of reportable acute gastrointestinal illness in the Portuguese Army, four norovirus outbreaks were identified between October 2015 and October 2017 in mainland Portugal and the Azores archipelago. The present study documents the epidemiological, clinical and laboratory investigations of these norovirus outbreaks. METHODS: Cases were investigated and epidemiological questionnaires were distributed to all soldiers in each military setting where the outbreaks occurred. Stool samples from soldiers with acute gastroenteritis illness were collected and screened for common enteropathogenic agents. Food and water samples served on the settings were also collected for microbiological investigation. Norovirus-positive samples were further characterised by sequence analysis using a public automated genotyping tool. RESULTS: The four outbreaks affected a total of 99 soldiers among the 618 stationed on base units and in a military exercise. A total of 27 soldiers provided a stool sample, of which 20 were positive for norovirus by real-time PCR. Phylogenetic analysis showed that the noroviruses involved were all genogroup II, namely GII.17, GII.Pe-GII.4 Sydney 2012, GII.P2-GII.2 and GII.P16-GII.2. Of note, 30 soldiers had to receive treatment at the military hospital due to severity of symptoms. CONCLUSION: In this short, two-year surveillance period, a total of four norovirus gastroenteritis outbreaks were detected in the Portuguese Army which caused a considerable morbidity, showing once again the impact of norovirus on Army effectiveness and force readiness.

Serologic evidence for Schmallenberg virus circulation at high altitude, Central Portugal, 2015-2016.

A few years after the initial 2011 large scale Schmallenberg virus (SBV) epidemic that affected Europe, a subsequent decrease in new SBV infections was observed presumably associated with natural substitution of previously exposed animals. In the present work, a 2-year prospective serosurvey was performed to evaluate SBV circulation in a population of sheep living at high altitude in the central region of Portugal and with restricted movement. Blood from a representative sample of 168 autochthonous sheep was collected in 2015 and again in 2016, and tested for the presence of anti-SBV IgG by ELISA. Of the 2015 sample collection, seven animals tested positive for anti-SBV IgG, corresponding to a seroprevalence of 4.2% while of the 2016 sample collection, 10 presented SBV antibodies, showing a seroprevalence of 6.0% (p = 0.619). Results show that SBV is endemic in sheep of central Portugal, even in herds at high altitude locations. When comparing anti-SBV seroprevalences of 2015/2016 found in this study, to one detected in 2014 in the same region, a steep decrease could be observed (p < 0.001). This is in accordance with what has been documented in Western European countries, where a decrease in the number of SBV-infected sheep has been found, a fact which may pose a new threat for SBV re-emergence.

Salmonella, Escherichia coli and Enterobacteriaceae in the peanut supply chain: From farm to table.

Due to recent foodborne outbreaks, peanuts have been considered a potential risk for Salmonella transmission. For this reason, the aim of this study was to determine the prevalence and contamination load of Salmonella, Escherichia coli and Enterobacteriaceae throughout the peanut supply chain in Brazil. Samples of peanuts and peanut-containing processed products from post-harvest (n=129), secondary processing (n=185) and retail market (n=100) were analyzed. The results showed high Enterobacteriaceae counts in the post-harvest samples. At the end of the secondary processing, 16% of the samples remained contaminated by this group of microorganisms. Six peanut samples from primary production and one sample of peanut butter were tested positive for E. coli while Salmonella was detected in nine samples (2.2%): six from post-harvest, two from the initial stage of the secondary processing and one from retail. The Salmonella counts ranged between 0.004 and 0.092MPN/g and five serotypes were identified (Muenster, Miami, Javiana, Oranienburg, Glostrup). The results demonstrated a high prevalence of Enterobacteriaceae and low prevalence of E. coli throughout the peanut supply chain. Furthermore, it was verified that peanuts may become contaminated by Salmonella during different stages of the supply chain, especially at post-harvest.

Prevalence of hepatitis E virus antibodies in workers occupationally exposed to swine in Portugal.

The concept of zoonotic hepatitis E in industrialized countries has emerged with the discovery of swine strains of hepatitis E virus (HEV) genotype 3, closely related to human HEV. Different routes of zoonotic HEV transmission have been recognized, including contact with infected pigs. Workers occupationally exposed to swine (WOES) have been considered a risk group for HEV infection, but contradictory results have been reported. In the present study, we searched for anti-HEV IgG in WOES (butchers, slaughterhouse workers, veterinarians and pig farmers; n = 114) and in the general population (n = 804) in order to investigate the potential occupational risk of zoonotic HEV infection in this work group. A significantly higher (p = 0.008) anti-HEV IgG seroprevalence was found in WOES (30.7 %) when compared with the general population (19.9 %). Multivariate analysis showed that having professions with exposure to pigs for more than 16.5 years was a risk factor for being positive for anti-HEV IgG (aOR of 5.4, 95 % CI 1.9-15.6, p = 0.002). To our knowledge, this is the first study on the prevalence of anti-HEV IgG in WOES in Portugal, also showing increased probability for infection in this group.

Persistence of rubella and mumps antibodies, following changes in the recommended age for the second dose of MMR vaccine in Portugal.

In Portugal, the recommended age for the second dose of MMR (MMR2) was changed from 10-13 years to 5-6 years for those born in 1994 and afterwards. This study aimed to assess if MMR schedule and time elapsed from the last dose are associated with the concentration of rubella and mumps IgG antibodies. Three Portuguese birth cohorts (convenience samples) were selected for this study (66, 59 and 41 participants born respectively in 1990-1993, 1994-1995 and 2001-2003). Geometric mean concentrations (GMC) for mumps IgG were respectively 36, 30 and 38 RU/ml (P = 0·236) and for rubella IgG were 18, 20 and 17 IU/ml (P = 0·641). For both specific antibodies, no differences were observed with time since MMR2. Receiving MMR2 at 5-6 or 10-13 years was not associated with concentration of both antibodies. The GMC of rubella IgG was lower in males (P = 0·029). Taking into account previous evidence and the logistics needed to change vaccination schedules, it seems reasonable that sustaining very high coverage with two doses of MMR is currently the most pragmatic way to control mumps and rubella rather than any changes to the schedule.

Evidence of autochthonous hepatitis E in a Portuguese pediatric cohort, 1992-1995.

Despite the growing importance of hepatitis E virus (HEV) in industrialized countries, minimal attention has been given to autochthonous HEV infection in children. The present study screened archived sera (N = 71) from a Portuguese pediatric cohort collected in 1992-1995 for the presence of antibodies against HEV, using enzyme immunoassays and immunodot. Anti-HEV IgG was detected in two children and anti-HEV IgM in one, suggesting an acute HEV infection. This shows that HEV was circulating in the paediatric population of Portugal in the early 1990s.

Hepatitis E Virus in Sylvatic and Captive Wild Boar from Portugal.

Hepatitis E virus (HEV) is a zoonotic agent today considered a major Public Health issue in industrialized countries. HEV strains belonging to zoonotic genotype 3 are widely present in swine, being today considered important reservoirs for human disease. Unlike in swine, only scarce data are available on the circulation of HEV in wild boar. This study describes the detection and molecular characterization of HEV in livers from sylvatic wild boar hunted in Portugal and destined for consumption. Additionally, the detection of HEV in stools of a confined wild boar population also destined for consumption is also described. A total of 80 liver samples collected during the hunting season of 2011/2012 and 40 stools collected in February 2012 from a wild boar breeding farm in Portugal were tested by a nested broad-spectrum RT-PCR assay targeting open reading frame (ORF) 1. Twenty livers (25.0%) and 4 stools (10%) were positive for HEV. Phylogenetic analysis showed that all strains clustered with sequences classified as HEV genotype 3 subgenotype e. To our knowledge, this is the first report documenting the occurrence and molecular analysis of HEV in sylvatic and captive wild boar destined for human consumption in Portugal. This report demonstrates for the first time the circulation of HEV in wildlife reservoirs of Portugal adding knowledge to the epidemiology of HEV in wild boar populations.

Molecular characterization of accessions of Cratylia argentea (Camaratuba) using ISSR markers.

Cratylia argentea (Desv.) Kuntze (Fabaceae) is a drought-tolerant, perennial legume found primarily in Brazil, Bolivia, and Peru. The shrub is well adapted to acid soils and exhibits high productivity and nutritional value, characteristics that would favor its use as a dry season animal forage supplement in semiarid regions. In plant improvement programs, the production of elite hybrids with superior traits is generally achieved by crossing parents that exhibit the highest level of genetic divergence. Therefore, the aim of the present study was to assess genetic diversity among 13 accessions of C. argentea from the same population maintained in the active germplasm bank of Embrapa Meio-Norte using inter-simple sequence repeat (ISSR) markers. Genetic similarities between C. argentea accessions were estimated from Jaccard coefficients, and a dendrogram was constructed using the unweighted pair group method with arithmetic average (UPGMA). The set of 15 primers selected for ISSR analysis generated a total of 313 loci of which 79.23% were polymorphic. The mean number of bands per primer was 20.87, and the amplicons ranged from 280 to 3000 bp in size. Primers UBC834 and UBC827 generated the largest number of polymorphic loci and exhibited 90.91 and 100% polymorphism, respectively. The coefficients of genetic similarity among accessions varied between 0.49 and 0.73. UPGMA cluster analysis allowed the identification of four genotypic groups and demonstrated the existence of considerable variability within the collection. Potential progenitors were selected that would offer good possibilities of obtaining unusual and favorable combinations of genes in a plant breeding program.

Impairment of Interleukin-17A Expression in Canine Visceral Leishmaniosis is Correlated with Reduced Interferon-γ and Inducible Nitric Oxide Synthase Expression.

Dogs are the primary urban reservoir of Leishmania infantum and play a crucial role in the transmission of this parasite to man via sandflies. The spleen and liver are the main target organs of L. infantum infection, but few studies have evaluated the immune response to this infection in the canine liver. To identify the immunological mediators involved in resistance and/or susceptibility to canine visceral leishmaniosis (CVL), we selected 21 dogs naturally infected by L. infantum and classified as asymptomatic or symptomatic. Immunological parameters were analysed and correlations with clinical signs were determined. Symptomatic dogs showed higher numbers of parasites and less leucocyte infiltration in the liver compared with asymptomatic dogs. The progression of this disease was characterized not only by the down regulation of T helper (Th) 1-related cytokines, such as interferon (IFN)-γ and tumour necrosis factor (TNF)-α, but also by the down regulation of genes encoding interleukin (IL)-17A, inducible nitric oxide synthase (iNOS) and IL-10 in the spleen and liver in symptomatic dogs compared with asymptomatic dogs. Importantly, IL-17A gene transcription level was positively correlated with mRNA expression for iNOS and IFN-γ. Th1- and Th17-related cytokines therefore appear to play a role in restricting parasite growth via iNOS activation and decrease susceptibility of dogs to CVL.

Outbreak of Porcine Epidemic Diarrhea Virus in Portugal, 2015.

An outbreak of porcine epidemic diarrhea virus (PEDV) in the South of Portugal in January 2015 and the spread of PEDV northwards in the territory are described. Comparative analysis of the amplified sequences showed a very high (99.0%) identity with the PEDV variant most recently reported in the United States and also show complete (100%) identity to the strains recently reported in Germany, supporting the hypothesis that a unique strain is currently circulating in Europe. The origin of this PEDV variant still needs to be elucidated and further studies in the remaining European countries may contribute to the knowledge.

Evaluation of the Acute Toxicity, Cytotoxicity, and Genotoxicity of Chresta martii (Asteraceae).

Chresta martii (Asteraceae), found in the Xingó region, northeastern Brazil, is used in the treatment of gastrointestinal (GIT) and liver disorders and malaria. However, there are few studies regarding efficacy and safety of use for this species. Thus, the objective of this study was to determine in vivo acute toxicity and in vitro cytotoxicity of organic extracts of C. martii as well as in vivo genotoxicity of its semipurified fraction. Dried aerial parts of C. martii were extracted using three organic solvents (cyclohexane [ECCm], ethyl acetate [EACm], and ethanol [EECm]), and these extracts were examined for acute toxicity (50-2000 mg/kg ip or po) and cytotoxicity (50 µg/ml) in carcinogenic human cell lines (HL-60, NCIH-292, and MCF-7). The EACm, which showed evidence of toxicity (in vivo and in vitro), was fractionated on a silica column, yielding four fractions (F1-F4). The F1 was utilized for genotoxicity (50 mg/kg ip), by in vivo micronucleus (MN) assay. ECCm showed no indication of acute toxicity or occurrence of death, while the LD50 estimated for the extracts (EACm and EECm) was 500 mg/kg po and 200 mg/kg ip. The EACm (50 µg/ml) inhibited growth of tumor cells HL-60 (96.54%), NCIH-292 (73.43%), and MCF-7 (15%). The F1 fraction induced MN formation in polychromatic erythrocytes of Swiss Webster mice. Organic extracts from C. martii exhibited acute toxicity classified as mild to moderate, in addition to cytotoxicity (in vitro), while the F1 semipurified fraction induced genotoxicity (in vivo).

Verification and characterization of chromosome duplication in haploid maize.

Doubled haploid technology has been used by various private companies. However, information regarding chromosome duplication methodologies, particularly those concerning techniques used to identify duplication in cells, is limited. Thus, we analyzed and characterized artificially doubled haploids using microsatellites molecular markers, pollen viability, and flow cytometry techniques. Evaluated material was obtained using two different chromosome duplication protocols in maize seeds considered haploids, resulting from the cross between the haploid inducer line KEMS and 4 hybrids (GNS 3225, GNS 3032, GNS 3264, and DKB 393). Fourteen days after duplication, plant samples were collected and assessed by flow cytometry. Further, the plants were transplanted to a field, and samples were collected for DNA analyses using microsatellite markers. The tassels were collected during anthesis for pollen viability analyses. Haploid, diploid, and mixoploid individuals were detected using flow cytometry, demonstrating that this technique was efficient for identifying doubled haploids. The microsatellites markers were also efficient for confirming the ploidies preselected by flow cytometry and for identifying homozygous individuals. Pollen viability showed a significant difference between the evaluated ploidies when the Alexander and propionic-carmin stains were used. The viability rates between the plodies analyzed show potential for fertilization.

Study of the Gastroprotective Effect of Extracts and Semipurified Fractions of Chresta martii DC. and Identification of Its Principal Compounds.

Chresta martii (Asteraceae) is a species widely used by the population of the Xingu region of Sergipe, Brazil, in the form of a decoction (aerial parts) for the treatment of gastrointestinal diseases. The study aims to assess the gastroprotective activity of organic extracts and semipurified fractions and identify the principal compounds present in C. martii responsible for such activity. The organic extracts (cyclohexane: ECCm, ethyl acetate: EACm, and ethanol: EECm) were obtained from the dried aerial parts (500 g) of C. martii. For evaluation of the gastroprotective activity of extracts (50, 100, or 200 mg/kg; p.o.), male Swiss Webster mice (25-30 g) were used which had gastric ulcers induced by indomethacin (40 mg/kg, s.c.) or ethanol (0.2 mL/animal; p.o.). Among the extracts evaluated, EACm exhibited significant (P < 0.05) gastroprotective activity in the models used. The fractionation of EACm was performed in a silica gel column 60 eluted with the following compounds: [chloroform-F1 yield (10%)], [chloroform/ethyl acetate (1/1)-F2 yield (6%)], [ethyl acetate-F3 yield (8%)], and [ethyl/methanol acetate (1/1)-F4 yield (5%)]. Of the fractions described above, the F1 (25 mg/kg; p.o.) had greater gastroprotective activity (P < 0.05) than that displayed by ranitidine (80 mg/kg; p.o.) in the ethanol-induced ulcer model. The refractionation of F1 produced 23 subfractions and from these two yellow amorphous compounds were obtained by recrystallization, Rf: 0.46 and 0.31 (ethyl acetate : chloroform 5 : 5). The compounds isolated were characterized by nuclear magnetic resonance spectroscopy ((1)H-NMR and (13)C-NMR) and identified as flavones: chrysoeriol (yield: 0.43%) and 3',4'-dimethoxyluteolin (yield: 0.58%). Conclusion. Flavone 3',4'-dimethoxyluteolin is the principal compound present in the species C. martii and is probably responsible for gastroprotective activity observed in this species.

The enterovirus protease inhibitor rupintrivir exerts cross-genotypic anti-norovirus activity and clears cells from the norovirus replicon.

Potent and safe inhibitors of norovirus replication are needed for the treatment and prophylaxis of norovirus infections. We here report that the in vitro anti-norovirus activity of the protease inhibitor rupintrivir is extended to murine noroviruses and that rupintrivir clears human cells from their Norwalk replicon after only two passages of antiviral pressure. In addition, we demonstrate that rupintrivir inhibits the human norovirus (genogroup II [GII]) protease and further explain the inhibitory effect of the molecule by means of molecular modeling on the basis of the crystal structure of the Norwalk virus protease. The combination of rupintrivir with the RNA-dependent RNA polymerase inhibitors 2'-C-methylcytidine and favipiravir (T-705) resulted in a merely additive antiviral effect. The fact that rupintrivir is active against noroviruses belonging to genogroup I (Norwalk virus), genogroup V (murine norovirus), and the recombinant 3C-like protease of a GII norovirus suggests that the drug exerts cross-genotypic anti-norovirus activity and will thus most likely be effective against the clinically relevant human norovirus strains. The design of antiviral molecules targeting the norovirus protease could be a valuable approach for the treatment and/or prophylaxis of norovirus infections.

Pet veterinarians have no increased risk of hepatitis E compared to the general population.

The discovery of autochthonous hepatitis E in industrialized countries associated to genotype 3 has changed substantially the understanding about hepatitis E virus (HEV) circulation. In these countries transmission has been associated to the consumption and direct contact to swine, a well-known reservoir for HEV. More animal reservoirs for HEV might exist since antibodies to HEV have been detected in other animal species, such as dogs and cats. Given the intimate contact between these pets and humans, the potential zoonotic HEV transmission from dogs and cats deserves to be explored. To address this issue, 493 sera from pet veterinarians (373) and matched general population (120) were tested for the presence of anti-HEV IgG using a commercial ELISA. Antibodies to HEV were found in 9.9% veterinarians and 13.3% general population. No statistically significant difference was found between these two groups showing that pet veterinarians have no increased risk to hepatitis E compared to the general population.

Antibodies to hepatitis E in Portuguese mothers and their newborns.

The high mortality of pregnant women is one of the main features of hepatitis E virus (HEV) infection in endemic countries. With the emergence of genotype 3 HEV in industrialized countries, attention should be given to pregnant women from these regions since the effect of genotype 3 HEV in pregnancy is not yet known. In this study, archived sera dating from 1993 from mothers with no history of traveling to HEV endemic countries and their newborns were tested for anti-HEV IgG antibodies. Four of the 12 maternal sera were positive for IgG anti-HEV, indicating exposure to this virus. Their newborns were also positive, showing higher anti-HEV IgG levels. Overall, these findings demonstrate not only an efficient transplacental transport of anti-HEV IgG, but also circulation of autochthonous HEV in Portugal in the early 1990s.

Inhibition of norovirus replication by the nucleoside analogue 2'-C-methylcytidine.

We here report on the activity of 2'-C-methylcytidine (2CMC) [a nucleoside polymerase inhibitor of the hepatitis C virus (HCV)] on the in vitro replication of (murine) norovirus (MNV). 2CMC inhibits (i) virus-induced CPE formation, (ii) viral RNA synthesis and (iii) infectious progeny formation with EC(50) values of ∼2µM. 2CMC acts at a time-point that coincides with the onset of viral RNA synthesis. Even following 30 passages of selective pressure no MNV-resistant virus was selected, which is in line with the high barrier to resistance of the nucleoside analogue for HCV. When combined with the broad-spectrum RNA virus inhibitor ribavirin, a marked antagonistic activity was observed indicating that these molecules should not be combined for the treatment of norovirus infections. Our results suggest that 2'-C-methyl nucleoside analogues should be further explored for the treatment and prophylaxis of norovirus infections.