Evaluation of Extraction Methods to Detect Noroviruses in Ready-to-Eat Raw Milk Minas Artisanal Cheese.

This study aimed to assess two homogenization methods to recover norovirus from Minas artisanal cheese (MAC) made with raw bovine milk obtained from four microregions of the Minas Gerais state, Brazil, with different ripening times and geographical and abiotic characteristics. For this purpose, 33 fiscal samples were artificially contaminated with norovirus GI and GII, and Mengovirus (MgV), used as an internal process control (IPC). TRIzol® reagent and Proteinase K homogenization methods were evaluated for all samples were then subjected to RNA extraction using viral magnetic beads and RT-qPCR Taqman® for viral detection/quantification. Proteinase K method showed better efficiency results for both norovirus GI and GII, with means recovery efficiency of 45.7% (95% CI 34.3-57.2%) and 41.4% (95% CI 29.1-53.6%), respectively, when compared to TRIzol method (16.6% GI, 95% CI 8.4-24.9%, and 12.3% GII, 95% CI 7.0-17.6%). The limits of detection for norovirus GI and GII for this method were 101GC/g and 103GC/g, respectively, independent of cheese origin. MgV was detected and revealed in 100% success rate in all types of cheese, with mean recovery efficiency of 25.6% for Proteinase K, and 3.8% for the TRIzol method. According to cheese origin, Triangulo Mineiro MAC had the highest mean recovery rates for the three viral targets surveyed (89% GI, 87% GII, and 51% MgV), while Serro MAC showed the lowest rates (p < 0.001). Those results indicate that the proteinase K adapted method is suitable for norovirus GI and GII detection in MAC and corroborated MgV as an applicable IPC to be used during the process.

Acute gastroenteritis outbreak associated with multiple and rare norovirus genotypes after storm events in Santa Catarina, Brazil.

Norovirus is a major cause of acute diarrheal disease (ADD) outbreaks worldwide. In the present study, we investigated an ADD outbreak caused by norovirus in several municipalities of Santa Catarina state during the summer season, southern Brazil in 2023. As of the 10th epidemiological week of 2023, approximately 87 000 ADD cases were reported, with the capital, Florianópolis, recording the highest number of cases throughout the weeks. By using RT-qPCR and sequencing, we detected 10 different genotypes, from both genogroups (G) I and II. Some rare genotypes were also identified. Additionally, rotavirus and human adenovirus were sporadically detected among the ADD cases. Several features of the outbreak suggest that sewage-contaminated water could played a role in the surge of ADD cases. Storm events in Santa Catarina state that preceded the outbreak likely increased the discharge of contaminated wastewater and stormwater into water bodies, such as rivers and beaches during a high touristic season in the state. Climate change-induced extreme weather events, including intensified rainfall and frequent floods, can disturb healthcare and sanitation systems. Implementing public policies for effective sanitation, particularly during peak times, is crucial to maintain environmental equilibrium and counter marine pollution.

Rotavirus A during the COVID-19 Pandemic in Brazil, 2020-2022: Emergence of G6P[8] Genotype.

Rotavirus A (RVA) remains a leading cause of acute gastroenteritis (AGE) hospitalizations in children worldwide. During the COVID-19 pandemic, a reduction in vaccination coverage in Brazil and elsewhere was observed, and some reports have demonstrated a reduction in AGE notifications during the pandemic. This study aims to investigate the diversity and prevalence of RVA genotypes in children and adults presenting with AGE symptoms in Brazil during the COVID-19 pandemic between 2020 and 2022. RVA was screened using RT-qPCR; then, G and P genotypes were characterized using one-step multiplex RT-PCR. A total of 2173 samples were investigated over the three-year period, and we detected RVA in 7.7% of samples (n = 167), being 15.5% in 2020, 0.5% in 2021, and 13.8% in 2022. Higher RVA prevalence was observed in the Northeastern region (19.3%) compared to the Southeastern (6.1%) and Southern regions (5.5%). The most affected age group was children aged between 0 and 6 months old; however, this was not statistically significant. Genotyping and phylogenetic analysis identified the emergence of G6P[8] during the period; moreover, it was detected in 10.6% of samples in 2020 and in 83.5% in 2022. In contrast, the prevalence of G3P[8], the previous dominant genotype, decreased from 72.3% in 2020 to 11.3% in 2022. We also identified unusual strains, such as G3P[9] and G9P[4], being sporadically detected during the period. This is the first report on the molecular epidemiology and surveillance of RVA during the COVID-19 pandemic period in Brazil. Our study provides evidence for the importance of maintaining high and sustainable levels of vaccine coverage to protect against RVA disease. Furthermore, it highlights the need to maintain nationwide surveillance in order to monitor future trends and changes in the epidemiology of RVA in Brazil.

G6P[8] Rotavirus a Possessing a Wa-like VP3 Gene from a Child with Acute Gastroenteritis Living in the Northwest Amazon Region.

The introduction of rotavirus A (RVA) vaccines has considerably reduced the RVA-associated mortality among children under 5 years of age worldwide. The ability of RVA to reassort gives rise to different combinations of surface proteins G (glycoprotein, VP7) and P (protease sensitive, VP4) RVA types infecting children. During the epidemiological surveillance of RVA in the Northwest Amazon region, an unusual rotavirus genotype G6P[8] was detected in feces of a 2-year-old child with acute gastroenteritis (AGE) that had been vaccinated with one dose of Rotarix® (RV1). The G6P[8] sample had a DS-1-like constellation with a Wa-like VP3 gene mono-reassortment similar to equine-like G3P[8] that has been frequently detected in Brazil previously. The results presented here reinforce the evolutionary dynamics of RVA and the importance of constant molecular surveillance.

Norovirus Epidemiology and Genotype Circulation during the COVID-19 Pandemic in Brazil, 2019-2022.

Norovirus stands out as a leading cause of acute gastroenteritis (AGE) worldwide, affecting all age groups. In the present study, we investigated fecal samples from medically attended AGE patients received from nine Brazilian states, from 2019 to 2022, including the COVID-19 pandemic period. Norovirus GI and GII were detected and quantified using RT-qPCR, and norovirus-positive samples underwent genotyping through sequencing the ORF1/2 junction region. During the four-year period, norovirus prevalence was 37.2%, varying from 20.1% in 2020 to 55.4% in 2021. GII genotypes dominated, being detected in 92.9% of samples. GII-infected patients had significantly higher viral concentrations compared to GI-infected patients (median of 3.8 × 107 GC/g and 6.7 × 105 GC/g, respectively); and patients aged >12-24 months showed a higher median viral load (8 × 107 GC/g) compared to other age groups. Norovirus sequencing revealed 20 genotypes by phylogenetic analysis of RdRp and VP1 partial regions. GII.4 Sydney[P16] was the dominant genotype (57.3%), especially in 2019 and 2021, followed by GII.2[P16] (14.8%) and GII.6[P7] (6.3%). The intergenogroup recombinant genotype, GIX.1[GII.P15], was detected in five samples. Our study is the first to explore norovirus epidemiology and genotype distribution in Brazil during COVID-19, and contributes to understanding the epidemiological dynamics of norovirus and highlighting the importance of continuing to follow norovirus surveillance programs in Brazil.

Human enteric adenovirus F40/41 as a major cause of acute gastroenteritis in children in Brazil, 2018 to 2020.

Human adenovirus (HAdV) types F40/41 have long been recognized as major viral agents of acute gastroenteritis (AGE) in children. Despite this, studies on HAdV molecular epidemiology are sparse, and their real impact is likely under-estimated. Thus, our goal was to investigate HAdV incidence, enteric and non-enteric types circulation, co-detections with rotavirus and norovirus and DNA shedding in stool samples from inpatients and outpatients from eleven Brazilian states. During the three-year study, 1012 AGE stool samples were analysed by TaqMan-based qPCR, to detect and quantify HAdV. Positive samples were genotyped by partial sequencing of the hexon gene followed by phylogenetic analysis. Co-detections were accessed by screening for rotavirus and norovirus. Overall, we detected HAdV in 24.5% of single-detected samples (n = 248), with a prevalence of type F41 (35.8%). We observed a higher incidence in children between 6 to 24 months, without marked seasonality. Additionally, we observed a statistically higher median viral load among single-detections between enteric and non-enteric types and a significantly lower HAdV viral load compared to rotavirus and norovirus in co-detections (p < 0.0001). Our study contributes to the knowledge of HAdV epidemiology and reinforces the need for the inclusion of enteric types F40/41 in molecular surveillance programs.

Nosocomial acute gastroenteritis outbreak caused by an equine-like G3P[8] DS-1-like rotavirus and GII.4 Sydney[P16] norovirus at a pediatric hospital in Rio de Janeiro, Brazil, 2019.

Worldwide, rotavirus (RVA) and norovirus are considered major etiological agents of acute gastroenteritis (AGE) in pediatric population admitted to hospitals. This study describes the investigation of nosocomial infections caused by emergent RVA and norovirus strains reported at a pediatric hospital in southern Brazil in May 2019. This outbreak affected 30 people among children and adults. Nine stool samples (eight children and one nurse) were obtained and analyzed by RT-qPCR to detect and quantify RVA and norovirus. Positive samples were genotyped by sequencing and subjected to phylogenetic analysis. We detected RVA in 44.4% (4/9) and norovirus in 55.5% (5/9) at high viral loads, ranging from 3.5 × 107 to 6.1 × 107 and 3.2 × 102 to 3.2 × 109 genome copies/g of stool, respectively. Co-infections were not observed. RVA VP4 and VP7 gene sequencing in combination with polyacrylamide gel electrophoresis identified the circulation of equine-like G3P[8] DS-1-like, and the partial sequencing of the other nine genes revealed that strains possessed I2-R2-C2-M2-A2-N1-T2-E2-H2 genotype background. The emergent recombinant norovirus variant, GII.4 Sydney[P16], was identified by ORF1-2 sequencing. Active surveillance and effective prevention measures should be constantly reinforced to avoid the spread of nosocomial viral infections into hospitals, which could severely affect pediatric patients admitted with underlying health conditions.

Epidemiology of enteric virus infections in children living in the Amazon region.

OBJECTIVES: To verify the frequency of viruses causing acute gastroenteritis (AGE) in association with the histo-blood group antigen (HBGA) and Rotarix™ vaccination coverage in children from the Amazon region. DESIGN: Fecal and saliva samples were collected from children with AGE (n = 485) and acute respiratory infection (ARI) (n = 249) clinical symptoms. Rotavirus A (RVA), norovirus, human adenovirus (HAdV), and sapovirus (SaV) were verified in feces by molecular detection. Saliva samples were used for HBGA phenotyping/FUT3 genotyping. Blood group types, clinical aspects and Rotarix™ RVA vaccination data were recorded. RESULTS: Norovirus remained the most prevalently detected cause of AGE (38%, 184/485 and ARI 21.3%, 53/249). High HAdV frequencies were observed in AGE children (28.6%, 139/485) and ARI children (37.3%, 93/249). RVA was the third most prevalent virus causing AGE (22.7%, 110/485 and ARI 19.3%, 48/249) and a low RV1 coverage (61%, 448/734) was verified. The SaV frequencies were lower (7.2%, 35/485 for AGE and 6.8%, 17/249 for ARI). Secretor children were HBGA susceptible to HAdV infection (OR 1.5, 95% CI 1.0-2.3; P = 0.04) but not to RVA, norovirus or SaV infection. CONCLUSIONS: Norovirus could be considered the main etiological agent of AGE. No association was verified for HBGA susceptibility to RVA, norovirus and SaV. Secretor children showed a slight susceptibility to HAdV infection and the Le (a-b-) heterogeneous SNPs on the FUT3 gene.

SARS-CoV-2 RNA detection in stool samples from acute gastroenteritis cases, Brazil.

We described the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in stool samples from patients presenting only acute gastroenteritis (AGE) symptoms. From January to July 2020, 121 AGE stool samples were screened by quantitative reverse-transcription polymerase chain reaction. We detected SARS-CoV-2 in 27.5% of samples received during the epidemic period. No infectious viruses were observed in Vero E6 cells.

Rotavirus A in Brazil: Molecular Epidemiology and Surveillance during 2018-2019.

Rotavirus A (RVA) vaccines succeeded in lowering the burden of acute gastroenteritis (AGE) worldwide, especially preventing severe disease and mortality. In 2019, Brazil completed 13 years of RVA vaccine implementation (Rotarix™) within the National Immunization Program (NIP), and as reported elsewhere, the use of Rotarix™ in the country has reduced childhood mortality and morbidity due to AGE. Even though both marketed vaccines are widely distributed, the surveillance of RVA causing AGE and the monitoring of circulating genotypes are important tools to keep tracking the epidemiological scenario and vaccines impact. Thus, our study investigated RVA epidemiological features, viral load and G and P genotypes circulation in children and adults presenting AGE symptoms in eleven states from three out of five regions in Brazil. By using TaqMan®-based one-step RT-qPCR, we investigated a total of 1536 stool samples collected from symptomatic inpatients, emergency department visits and outpatients from January 2018 to December 2019. G and P genotypes of RVA-positive samples were genetically characterized by multiplex RT-PCR or by nearly complete fragment sequencing. We detected RVA in 12% of samples, 10.5% in 2018 and 13.7% in 2019. A marked winter/spring seasonality was observed, especially in Southern Brazil. The most affected age group was children aged >24-60 months, with a positivity rate of 18.8% (p < 0.05). Evaluating shedding, we found a statistically lower RVA viral load in stool samples collected from children aged up to six months compared to the other age groups (p < 0.05). The genotype G3P[8] was the most prevalent during the two years (83.7% in 2018 and 65.5% in 2019), and nucleotide sequencing of some strains demonstrated that they belonged to the emergent equine-like G3P[8] genotype. The dominance of an emergent genotype causing AGE reinforces the need for continuous epidemiological surveillance to assess the impact of mass RVA immunization as well as to monitor the emergence of novel genotypes.

High genetic diversity of noroviruses in children from a community-based study in Rio de Janeiro, Brazil, 2014-2018.

We report on the occurrence and diversity of noroviruses in children (younger than 5 years old of age) from a low-income urban area in Rio de Janeiro, Brazil. Sixty-one stool specimens collected from children between 1 and 4 years old with acute diarrhoeic episodes (ADE) and non-ADE were investigated. RT-qPCR and sequencing of PCR products after conventional RT-PCR analysis were performed. Noroviruses were detected in 29 (47.5%) samples: 21 (46.7%) from cases with ADE and 8 (50%) from non-ADE cases. Molecular characterization showed 10 different genotypes circulating in this community between November 2014 and April 2018.

Phenotyping of Lewis and secretor HBGA from saliva and detection of new FUT2 gene SNPs from young children from the Amazon presenting acute gastroenteritis and respiratory infection.

The Histo-blood group antigens (HBGA) are host genetic factors associated with susceptibility to rotavirus (RV) and human norovirus (HuNoV), the major etiological agents of viral acute gastroenteritis (AGE) worldwide. The FUT2 gene expressing the alpha-1, 2-L- fucosyltransferase enzyme is important for gut HBGA expression, and also provides a composition of the phenotypic profile achieved through mutations occurring in populations with different evolutionary histories; as such, it can be considered a genetic population marker. In this study, Lewis and secretor HBGA phenotyping was performed using 352 saliva samples collected from children between three months and five years old born in the Amazon (Brazil, Venezuela and English Guyana) presenting AGE or acute respiratory infection (ARI), the latter considered as control samples. The total of children phenotyped as secretors was 323, corresponding to 91.80%. From these, 207 (58.80%) had a Le (a + b+) profile. The HBGA profiles were equally found in children with AGE as well as with ARI. The rs1047781 of the FUT2 gene was not detected in DNA from saliva cells with a Le (a+b+) profile. However, mutations not yet described in the FUT2 gene were observed: missense 325A>T, 501C>T, 585C>T, 855A>T and missense substitutions 327C>T [S (Ser) > C (Cys)], 446 T>C [L(Leu) > P(Pro)], 723C>A [N(Asn) > K(Lys)], 724A>T [I(Ile) > F(Phe)], 736C>A [H(His) > N(Asn)]. The SNP distribution in the FUT2 gene of the analyzed samples was very similar to that described in Asian populations, including indigenous tribes.

The evolving epidemiology of rotavirus A infection in Brazil a decade after the introduction of universal vaccination with Rotarix®.

BACKGROUND: Brazil introduced the monovalent rotavirus vaccine (Rotarix®) in 2006. This study aimed to assess the epidemiology and genotype distribution of species-A rotavirus (RVA) in Brazil, comparing the pre- and post-vaccination periods. METHODS: Laboratory-based RVA surveillance included 866 municipalities in 22 Brazilian states, over a 21-year period. A total of 16,185 children with diarrheal diseases (DD) aged up to 12 years between 1996 and 2005 (pre-vaccination period, n = 7030) and from 2006 to 2017 (post-vaccination period, n = 9155) were enrolled. RVA was detected using ELISA immune assay and/or polyacrylamide gel electrophoresis and genotyped using nested PCR and/or nucleotide sequencing. RVA-positivity and genotypes detection rates were compared in distinct periods and age groups and Rotarix vaccination status. RESULTS: RVA-positivity in pre- and post-vaccination periods was, respectively: 4-11 months bracket, 33.3% (668/2006) and 16.3% (415/2547) (p <  0.001); 12-24 months, 28.2% (607/2154) and 22.2% (680/3068) (p <  0.001); 25-48 months, 17.4% (215/1235) and 29.4% (505/1720) (p <  0.001). Genotypes distribution in the pre- and post-vaccination periods was, respectively: G1P [8]/G1P[Not Typed], 417/855 (48.8%) and 118/1835 (6.4%) (p <  0.001); G2P [4]/G2P[NT], 47/855 (5.5%) and 838/1835 (45.7%) (p <  0.001); G3P [8]/G3P[NT], 55/855 (6.4%) and 253/1835 (13.8%) (p <  0.001); G9P [8]/G9P[NT], 238/855 (27.8%) and 152/1835 (8.3%) (p <  0.001); G12P [8]/G129P[NT], 0/871 (0%) and 249/1835(13.6%) (p <  0.001). Concerning infants aged 4-11 months, RVA frequency in fully vaccinated and non-vaccinated individuals was 11.9% (125/1052) and 24.5% (58/237) (p <  0.001), respectively. In children aged 12-24 months, RVA detection rate was 18.1% (253/1395) and 29.6% (77/260) (p <  0.001), for the vaccinated and non-vaccinated individuals, respectively (p <  0.001). CONCLUSIONS: RVA infection was significantly less frequent in children aged ≤2 years with DD after implementing vaccination, mainly among vaccinated children. It was also observed a decrease of P [8] circulation and emergence of G2P[4] in 2005, and afterwards in the post-vaccine era, with spreading of G12P[8] in 2014-2015 and of G3P[8] in 2017. Continuous RVA surveillance must be carried out in this scenario.

Cynomolgus Monkeys (Macaca fascicularis) as an Experimental Infection Model for Human Group A Rotavirus.

Group A rotaviruses (RVA) are one of the most common causes of severe acute gastroenteritis in infants worldwide. Rotaviruses spread from person to person, mainly by faecal⁻oral transmission. Almost all unvaccinated children may become infected with RVA in the first two years of life. The establishment of an experimental monkey model with RVA is important to evaluate new therapeutic approaches. In this study, we demonstrated viral shedding and viraemia in juvenile⁻adult Macaca fascicularis orally inoculated with Wa RVA prototype. Nine monkeys were inoculated orally: seven animals with human RVA and two control animals with saline solution. During the study, the monkeys were clinically monitored, and faeces and blood samples were tested for RVA infection. In general, the inoculated animals developed an oligosymptomatic infection pattern. The main clinical symptoms observed were diarrhoea in two monkeys for three days, associated with a reduction in plasmatic potassium content. Viral RNA was detected in seven faecal and five sera samples from inoculated animals, suggesting virus replication. Cynomolgus monkeys are susceptible hosts for human Wa RVA infection. When inoculated orally, they presented self-limited diarrhoea associated with presence of RVA infectious particles in faeces. Thus, cynomolgus monkeys may be useful as animal models to evaluate the efficacy of new antiviral approaches.

Norovirus GII.17 Associated with a Foodborne Acute Gastroenteritis Outbreak in Brazil, 2016.

Foodborne transmission gastroenteritis (AGE) outbreak occurred during a celebration lunch in July, 2016, Brazil. All stool samples tested were positive for noroviruses (NoV) and phylogenetic analysis revealed that strains were genetically close to GII.17 Kawasaki_2014. These findings indicated circulation of NoV GII.17 Kawasaki_2014 in the Brazilian population, associated with AGE outbreak.

G26P[19] rotavirus A strain causing acute gastroenteritis in the American continent

In Brazil, the rotavirus A genotype G26 was first identified in suckling piglets, while the P[19] genotype has not been identified in any animal species so far. This report details the genetic characterisation of a G26P[19] RVA strain detected from an eight year-old child, vaccinated with Rotarix®, hospitalised with acute diarrhoeal disease in Rio de Janeiro in 2015. Most likely, the genome constellation (I5-R1-C1-M1-A8-N1-T1-E1-H1) observed in the G26P[19] Brazilian strain was a result of interspecies transmission events between humans and pigs. In addition, a rearrangement in the NSP5 gene was observed downstream of the 3' non-coding region.

VP7 and VP8* genetic characterization of group A rotavirus genotype G12P[8]: Emergence and spreading in the Eastern Brazilian coast in 2014.

Group A rotavirus (RVA) genotype G12 is habitually associated with diarrhea disease (DD) in African children and recently its detection has increased worldwide. A total of 970 stool samples collected from individuals with DD in the Northeastern, Southeastern, and Southern Brazilian regions, Eastern coast, were analyzed and 321 (33%) were positive for RVA and of these, 241 (75%) genotyped as G12P[8]. The rate of RVA positivity was higher among children aged 5-10 years old (60%). All RVA infections observed in adults aged >21 years were G12P[8] (n = 27) showing that this genotype affected older age groups during the year of 2014 in Brazil. Phylogenetic analysis of VP7 and VP8* G12P[8] strains demonstrated an elevated similarity among Brazilian and G12-III prototypes strains circulating worldwide recently, suggesting that this lineage is associated with the global spread of the G12 genotype, considered as the 6th most prevalent human RVA genotype nowadays; while other G12 lineages remain sporadically detected and usually detected in association with other P genotypes. VP8* analysis revealed that Brazilian strains belong to P[8]-3 lineage, the single P[8] lineage presently detected in the country. No major nucleotide/amino acid disparities were observed among strains recovered from children and adults for VP7 and VP8* genes. These data are essential to support the surveillance studies, particularly in countries where the RVA vaccine was introduced in their National Immunization Program enabling identification of potential alterations in the epidemiological profile that can impact its efficacy in vaccination programs. J. Med. Virol. 89:64-70, 2017. © 2016 Wiley Periodicals, Inc.

Formação de membrana fibrótica cicatricial em paciente submetido à ceratoplastia penetrante / Fibrotic scar membrane formation in patients undergoing penetrating keratoplasty

RESUMO O surgimento de uma membrana fibrótica opacificada na córnea transplantada é pouco descrito nas literaturas nacional e mundial. O objetivo é relatar o caso de um paciente com leucoma total de olho esquerdo que foi submetido à ceratoplastia penetrante levando a formação de dupla câmara anterior devido ao surgimento de uma membrana fibrótica cicatricial. Paciente do sexo masculino, 54 anos, com leucoma total secundário a ceratite herpética, diabético há 20 anos, em uso de insulina, com retinopatia diabética não proliferativa. Realizou-se cirurgia de membranectomia com complicações pós-operatória.

ABSTRACT The emergence of opaque fibrotic membrane in transplanted cornea is little described in national and world literature. The goal is to report the case of a patient with leucoma total of left eye that was submitted to the penetrating keratoplasty leading to formation of double anterior chamber due to the emergence of a fibrotic scar membrane. Male patient, 54 years, with total herpetic keratitis secondary leucoma, diabetic for 20 years, using insulin, with non-proliferative diabetic retinopathy. Held membranectomia surgery with postoperative complications.

Epidemiology of rotavirus A diarrhea in Chókwè, Southern Mozambique, from February to September, 2011.

Acute diarrhea disease caused by Rotaviruses A (RVA) is still the leading cause of morbidity and mortality in children ≤5 years old in developing countries. An exploratory cross-sectional study was conducted between February and September, 2011 to determine the proportion of acute diarrhea caused by RVA. A total of 254 stool specimens were collected from children ≤5 years old with acute diarrhea, including outpatients (222 children) and inpatients (32 children), in three local health centers in Chókwè District, Gaza Province, South of Mozambique. RVA antigens were detected using enzyme immunoassay (EIA); the RVA G (VP7) and P (VP4) genotypes were determined by RT-PCR or analysis sequencing. Sixty (24%) out of 254 fecal specimens were positive for RVA by EIA; being 58 (97%) from children ≤2 years of age. RVA prevalence peaks in June and July (coldest and drier months) and the G[P] binary combination observed were G12P[8] (57%); G1P[8] (9%); G12P[6] (6%); and 2% for each of the following genotypes: G1P[6], G2P[6] G4P[6], and G9P[8]. Non-Typeable (NT) G and/or P genotypes were observed as follows: G12P [NT] (6%); G1P [NT], G3P[NT] and GNTP[NT] (4%). Considering the different GP combinations, G12 represented 67% of the genotypes. This is the first data showing the diversity of RVA genotypes in Mozambique highlighting the epidemiological importance of these viruses in acute diarrhea cases in children ≤2 years old. In addition, these findings will provide a baseline data before the introduction of the RVA monovalent (Rotarix(®) ) vaccine in the National Immunization Program in September 2015. J. Med. Virol. 88:1751-1758, 2016. © 2016 Wiley Periodicals, Inc.

Performance of a one-step quantitative duplex RT-PCR for detection of rotavirus A and noroviruses GII during two periods of high viral circulation.

Rotavirus A (RVA) and noroviruses (NoV) are the major viral agents of acute gastroenteritis (AGE) worldwide. In the present study, we aimed to evaluate the performance of a one-step duplex quantitative RT-PCR (dRT-qPCR) assay, established for detection and quantification of RVA and NoV genogroup II (GII) using a single DNA standard curve (SC), as well as to investigate the association between fecal viral load and optical density (OD) values, and viruses' genotyping. The results obtained by dRT-qPCR in 530 fecal samples from AGE cases were compared with methods employed for the diagnosis of those viruses as follows: enzyme immunoassay (EIA) and polyacrylamide gel electrophoresis (PAGE) for RVA; and qualitative PCR for NoV. By using dRT-qPCR, we detected RVA and NoV in 353 (66%), increasing the positivity rate by 22.5% for RVA and 11.5% NoV, comparing the number of positive samples. RVA and NoV GII were detected in a range of 5.17 × 10(3) to 6.56 × 10(9) and 3.76 × 10(3) to 9.13 × 10(10) genome copies per gram of feces, respectively. We observed a significant direct correlation between genome copies values and optical density, using dRT-qPCR and EIA assays, respectively (Spearman ρ=0.41; p<0.0001). Viruses characterization demonstrated a predominance of NoV GII.4 Sidney 2012 variant during October 2013 to February 2014, followed by the emergence of RVA genotype G12P[8] in 2014. The established assay using a single SC provides an early feedback concerning detection and quantification, with the advantage of detecting simultaneously RVA and NoV GII, reducing time and reagent costs.