Multiplex Semi-nested RT-PCR Research Articles

Long-term impact of rotavirus vaccination on all-cause and rotavirus-specific gastroenteritis and strain distribution in Central Kenya: An 11-year interrupted time-series analysis

Background: Kenya introduced a monovalent rotavirus vaccine administered orally at 6 and 10 weeks of age into her National Immunization Program in July 2014. The study evaluated the long-term impact of the vaccine on hospitalization for all-cause and rotavirus-specific acute gastroenteritis (AGE) and strain epidemiology in Kenya. Methods: Data on all-cause and rotavirus-specific AGE and strain distribution were derived from an eleven-year hospital-based surveillance of AGE among children aged <5 years at Kiambu County Teaching and Referral Hospital (KCTRH) in Central Kenya between 2009 and 2020. Fecal samples were screened for group A rotavirus using ELISA and genotyped using multiplex semi-nested RT-PCR. Trends in all-cause and rotavirus-related AGE and strain distribution were compared between the pre-vaccine (July 2009–June 2014), early post-vaccine (July 2014–June 2016) and late post-vaccine (February 2019–October 2020) periods. Results: Rotavirus-specific AGE was detected at 27.5% (429/1546, 95% CI: 25.5–30.1%) in the pre-vaccine period; 13.8% (91/658, 95% CI: 11.3–16.6%) in the early post-vaccine period (July 2014–June 2016); and 12.0% (229/1916, 95% CI: 10.6–13.5%) in the late post-vaccine period (February 2019–October 2020). This amounted to a decline of 49.8% (95% CI: 34.6%–63.7%) in rotavirus-specific AGE in the early post-vaccine period and 53.4% (95% CI: 41.5–70.3%) in the late post-vaccine period when compared to the pre-vaccine period. All-cause AGE hospitalizations declined by 40.2% (95% CI: 30.8%–50.2%) and 75.3% (95% CI: 65.9–83.1%) in the early post-vaccine and late post-vaccine periods, respectively, when compared to the pre-vaccine period. G3P [8] was the predominant strain in the late post-vaccine period, replacing G1P[8] which had predominated in the pre-vaccine and early post-vaccine periods. Additionally, we detected considerable proportions of uncommon strains G3P[6] (4.8%) and G12P[6] (3.5%) in the post-vaccine era. Conclusion: Rotavirus vaccination has resulted in a significant decline in all-cause and rotavirus-specific AGE, and thus, provides strong evidence for public health policy makers in Kenya to support the sustained use of the rotavirus vaccine in routine immunization. However, the shift in strain dominance and age distribution of rotavirus AGE in the post-vaccine era underscores the need for continued surveillance to assess any possible vaccine-induced selective pressure that could diminish the vaccine effectiveness over time.

Effect of rotavirus vaccine implementation on the prevalence of coinfections with enteric viruses in Norway.

Acute gastroenteritis (AGE) is a common illness in both adults and children worldwide and is caused by several microorganisms including viruses, bacteria, and parasites. Rotavirus (RV), which is the main cause of AGE, can occur as a mixed infection with other viruses. The aim of this study is to assess the molecular epidemiology of viral enteric viruses and assess RV coinfections with other enteric viruses and their influence on disease severity before and after RV vaccine introduction in children under 5 years of age. A total of 600 samples collected from children hospitalized for AGE in five large hospitals in Norway, and were analyzed for viral gastroenteritis agents by enzyme immunoassay and quantitative real-time polymerase chain reaction (qRT-PCR). Positive results confirmed either by Sanger sequencing or genotyped by multiplex semi-nested RT-PCR. In total, 243 of the 300 (81%) samples, collected from the prevaccine cohort, were positive for at least one of the four viruses tested in this study. RV was most frequently identified in 82.6% of the samples. In the postvaccine cohort, 114 of the 300 (38%) samples were positive for at least one of the viruses tested. RV found in 36.5% of the samples. Coinfections found less frequently in the postvaccine cohort. Among circulating enteric viruses in Norway, RV is the most important cause of viral gastrointestinal infection. As expected, there were fewer RV positive and fewer coinfections after RV vaccine implementation. The results provide valuable data that can aid in further evaluation of the vaccine impact.

Genotyping of Rotaviruses in River Nile in Giza, Egypt

In 2013, WHO estimated the annual diarrheal mortality rate among children less than five years in Egypt was 24 deaths per 100.000, 2% was due to rotavirus infection. Eight water samples were collected monthly from the Nile water stream passing through Giza over 12 months during June 2016 to May 2017. Totally, ninety-six water samples were collected and concentrated for the detection of rotavirus group A (RV-A) using RT-PCR. Multiplex semi-nested RT-PCR was performed to identify the genotypes P and G of RV-A. The detection rate of RV-A was 18.75% (18/96), whereas the rate of rotavirus genotypes G and P were 61% (11/18) and 50% (9/18), respectively. Rotaviruses G1P[8] and G1P[4] were the most common genotypes identified in our survey. In addition, the seasonal distribution findings demonstrated that the highest detection rate was 37.5% in the winter season, followed by 20.8%, 12.5% and 4% in spring, autumn and summer, respectively. Multiplex semi-nested RT-PCR is a useful method for rapid detection and genotyping of RV-A in surface water samples.

Unexpected predominance of rotavirus G9P[8] strain in Tunisian adult diarrheal patients.

Introduction. Group A Rotavirus (RVA) is known to be a major cause of acute gastroenteritis (AGE) in children but its role as a potential pathogen in immunocompetent adults is probably underestimated.Aim. To compare RVA infections in patients from different age groups.Methodology. Fecal samples were collected from patients aged from birth to 65 years, hospitalized or consulting for AGE between 2015 and 2017. All samples were screened by RT-PCR for the detection of VP6 gene specific of RVA. RVA-positive samples were VP7 and VP4 genotyped using multiplex semi-nested RT-PCR. Full-length VP7 gene of G9-positive strains were sequenced and submitted for phylogenetic analysis.Results. Of 1371 stool specimens collected from children (<5 years; n=454), older children (5 to <15 years; n=316) and adults (15-65 years; n=601), 165 (12.0 %) were RVA-positive. RVA detection rates were significantly higher in children and adults than in older children (15.8 % and 12.1 Vs 6.3 %, respectively; P<0.001). While RVA infections were mostly detected during the coldest months in children, they were observed all year-round in patients aged >5 years. Although G1P[8] remained the most prevalent combination (41.7 %) detected in children, G9P[8] strains widely predominated in adults (58.1 %), followed by G2P[4] (12.9 %). All characterized G9 strains clustered in the modern lineage III.Conclusion. RVA play an important role in AGE not only in children but also in adults. The findings of a wide G9 predominance in patients >5 years highlights the need for continuing surveillance in both pediatric and mature populations.

Influence of early use of antimicrobial on the health and performance of Holstein calves in the first month of life

ABSTRACT: The early use of antimicrobial therapy has been introduced in many farms to prevent diarrhea and respiratory disease in young calves; however, there is controversy about whether this practice has a beneficial effect on the health of these animals. This study evaluated the influence of the early use of antimicrobials on the health and performance of neonatal Holstein calves. Twenty-six Holstein calves were screened and divided into two groups, according to the administration (ATB+), or not (ATB-) of tulathromycin (2.5mg/kg, subcutaneously) within the first 12 hours of life. Calves were evaluated by general clinical examination, fecal score, respiratory score, and external palpation of the umbilical region, besides fecal output of dry matter. Anemia was determined by using an automatic system and, also, using a commercial kit for iron dosage. Diarrhea was diagnosed by a centrifuge-flotation technique using a sugar solution (Cryptosporidium) and multiplex semi-nested RT-PCR (rotavirus/coronavirus). The performance of the calves was estimated by Daily Weight Gain (DWG). The young dairy calves were evaluated within 12 hours of birth (≤12h) and at 3-5th (D3-5), 7-9th (D7-9), 13-15th (D13-15), 20-23rd (D20-23), and 27-30th (D27-30) days of life. No difference was noted between the ATB+ and ATB- groups concerning heart rate, respiratory frequency, and rectal temperature. Erythrogram showed a higher frequency of anemia in ATB- group (P=0.016) at the D3-5 check-up; lower values of serum iron were also observed simultaneously (P=0.051). Thirteen cases of respiratory disease were detected during this study; however, no significant difference was observed between the groups in this regard. The frequency of diarrhea (fecal score 2-3) was high in both groups, peaking at D13-D15. No differences were noted between the groups regarding the frequency of diarrhea when considering the dry fecal matter. The predominant etiological agent for diarrhea was Cryptosporidium spp.. The DWG was similar between groups, with maximum weight reduction on D13-15. The administration of tulathromycin in prophylactic dose (2.5mg/kg) at birth decreased the frequency of anemia but did not influence weight gain or the prevalence of diarrhea.

Molecular Detection and Genotyping of Group A Rotavirus by Multiplex Semi-Nested RT-PCR in Sewage Water and Sludge

Molecular Detection and Genotyping of Group A Rotavirus by Multiplex Semi-Nested RT-PCR in Sewage Water and Sludge

Multiplex Semi-Nested RT-PCR for Genotyping of Rotaviruses Group A in Giza Tap Water, Egypt

Multiplex Semi-Nested RT-PCR for Genotyping of Rotaviruses Group A in Giza Tap Water, Egypt

Genetic diversity of rotavirus strains in children with diarrhea in Lagos, Nigeria

ObjectiveTo describe the genetic diversity of rotavirus strains circulating in Lagos, Nigeria. MethodsA total of 302 stool samples were obtained from diarrheic children on admission to four hospitals in Lagos and screened for rotavirus antigen by enzyme immunoassay, while rotavirus VP4 and VP7 genotypes were determined by multiplex semi-nested RT-PCR using recognized primers and methods. ResultsOverall, the globally important genotypes, G1, G2, G3, G4 and G9, associated with P[4], P[6] and P[8] were detected. A rotavirus carrying G12 specificity was also detected at low frequency. The different rotavirus strains found in this study were G[2]P[6], G[2]P[8], G[1]P[8], G[1]P[6], G[3]P[6], G[9]P[6], G[2]P[4], G[4]P[4], G[12]P[8] and some mixed infections. ConclusionsThe study highlights the wide diversity of rotavirus strains and the potential emergence of unusual rotavirus in this region. It is therefore important to continue the epidemiological studies to monitor rotavirus strains associated with gastroenteritis in hospitals before and after the introduction of rotavirus vaccine.

Natural transovarial transmission of dengue virus 4 in Aedes aegypti from Cuiabá, State of Mato Grosso, Brazil.

Dengue is the most prevalent arboviral disease in tropical areas. In Mato Grosso, outbreaks are reported every year, but studies on dengue in this state are scarce. Natural transovarial infection of Aedes aegypti by a flavivirus was investigated in the Jardim Industriário neighborhood of Cuiabá, Mato Grosso. Eggs were collected with ovitraps during the dry, intermediate, and rainy seasons of 2012. After the eggs hatched and the larvae developed to adulthood, mosquitoes (n = 758) were identified and allocated to pools of 1-10 specimens according to the collection location, sex, and climatic period. After RNA extraction, multiplex semi-nested RT-PCR was performed to detect the four dengue virus (DENV) serotypes, yellow fever virus, West Nile virus and Saint Louis encephalitis virus. DENV-4 was the only flavivirus detected, and it was found in 8/50 pools (16.0%). Three of the positive pools contained females, and five contained males. Their nucleotide sequences presented 96-100% similarity with DENV-4 genotype II strains from Manaus, Amazonas. The minimum infection rate was 10.5 per 1000 specimens, and the maximum likelihood estimator of the infection rate was 11.6 (95% confidence interval: 4.8; 23.3). This study provides the first evidence of natural transovarial infection by DENV-4 in Ae. Aegypti in Mato Grosso, suggesting that this type of infection might serve as a mechanism of virus maintenance during interepidemic periods in Cuiabá, a city where dengue epidemics are reported every year. These results emphasize the need for efficient vector population control measures to prevent arbovirus outbreaks in the state.

Assessment of gastroenteric viruses from wastewater directly discharged into Uruguay River, Uruguay.

The aim of this study was to assess the viral contamination of group A rotavirus (RVA), norovirus (NoV), and human astrovirus (HAstV) in sewage directly discharged into Uruguay River and to characterize RVA genotypes circulating in Uruguay. For this purpose, sewage samples (n = 96) were collected biweekly from March 2011 to February 2012 in four Uruguayan cities: Bella Unión, Salto, Paysandú, and Fray Bentos. Each sample was concentrated by ultracentrifugation method. Qualitative and quantitative RT-PCR for RVA, NoV, and HAstV were performed. A wide dissemination of gastroenteric viruses was observed in the sewage samples analyzed with 80% of positivity, being NoV (51%) the most frequently detected followed by RVA with a frequency of 49% and HAstV with 45%. Genotypes of RVA were typed using multiplex semi-nested RT-PCR as follows: P[8] (n = 15), P[4] (n = 8), P[10] (n = 1), P[11] (n = 1), G2 (n = 29), and G3 (n = 2). The viral load ranged from 10(3) to 10(7) genomic copies/liter, and they were detected roughly with the same frequency in all participant cities. A peak of RVA and HAstV detection was observed in colder months (June to September), whereas no seasonality was observed for NoV. This study demonstrates for the first time, the high degree of gastroenteric viral contamination in the country; highlighting the importance of developing these analyses as a tool to determine the viral contamination in this hydrographic boundary region used by the local populations for recreation and consumption, establishing an elevated risk of gastroenteric diseases for human health.

Molecular characterization of group A bovine rotavirus in southeastern and central-western Brazil, 2009-2010

Rotavirus is an important cause of neonatal diarrhea in humans and several animal species, including calves. A study was conducted to examine 792 fecal samples collected from calves among 65 dairy and beef herds distributed in two of Brazil's major livestock producing regions, aiming to detect the occurrence of rotavirus and perform a molecular characterization of the rotavirus according to G and P genotypes in these regions. A total of 40 (5.05%) samples tested positive for rotavirus by the polyacrylamide gel electrophoresis (PAGE) technique. The molecular characterization was performed by multiplex semi-nested RT-PCR reactions, which indicated that the associations of genotypes circulating in herds in Brazil's southeastern region were G6P[11], G10P[11], G[-]P[5] + [11], G[-]P[6] in the state of São Paulo and G6P[11], G8P[5], G11P[11], G10P[11] in the state of Minas Gerais. In the central-western region, the genotypes G6P[5] + [11], G6P[5], G8P[-], G6P[11], G [-] P[1], G[-] P[11], and G[-] P[5] were detected in the state of Goiás, while the genotypes G6P[5], G8[P11], G6[P11], G8[P1], G8[P5], G6[P1] were circulating in herds in the state of Mato Grosso do Sul. The genotypic diversity of bovine rotavirus found in each region under study underlines the importance of characterizing the circulating samples in order to devise the most effective prophylactic measures.

Multiplex semi-nested RT-PCR with exogenous internal control for simultaneous detection of bovine coronavirus and group A rotavirus

Neonatal calf diarrhea is a multi-etiology syndrome of cattle and direct detection of the two major agents of the syndrome, group A rotavirus and Bovine coronavirus (BCoV) is hampered by their fastidious growth in cell culture. This study aimed at developing a multiplex semi-nested RT-PCR for simultaneous detection of BCoV ( N gene) and group A rotavirus ( VP1 gene) with the addition of an internal control (mRNA ND5). The assay was tested in 75 bovine feces samples tested previously for rotavirus using PAGE and for BCoV using nested RT-PCR targeted to RdRp gene. Agreement with reference tests was optimal for BCoV (kappa = 0.833) and substantial for rotavirus detection (kappa = 0.648). the internal control, ND5 mRNA, was detected successfully in all reactions. Results demonstrated that this multiplex semi-nested RT-PCR was effective in the detection of BCoV and rotavirus, with high sensitivity and specificity for simultaneous detection of both viruses at a lower cost, providing an important tool for studies on the etiology of diarrhea in cattle.

Study on the epidemiological of rotavirus diarrhea in Lulong in 2008-2009

To analyze the feature of epidemiological of rotavirus diarrhea in Lulong county, Hebei province. 426 stool specimens were collected from inpatant with acute diarrhea from children less than 5 years old. Rotavirus-positive specimens were identified by ELISA kit. G/P typing assays were confirmed with multiplex seminested RT-PCR. Rotavirus was detected in 202 of 426 (47.42%) specimens. Genotyping of rotavirus showed that G3 was predominant (57.9%), followed by Gmix (16.3%), G9 (14.9% ), G1 (7.9%), G4 (1%), G2 (0.5%), P-genotyping showed that P [8], Pmix, P [4], P [9], type were found in 58.4%, 28.7%, 6.9% and 1% respectively. The most common G/P combination identified was G3P [8]. Group A rotaviruses was a major pathogen of diarrhea in Children in Lulong. G3P [8] was the predominant type in 2009, Gmix and Pmix abound, and G9 serotypes has become the second predominant after G3 strain in the region.

Évolution des souches de Rotavirus du groupe A en circulation en Tunisie sur une période de trois ans (2005–2007)

Background Rotaviruses are the most frequent agents associated with diarrhoea in children worldwide. Analysis of mobility of the 11 segments of genomic RNA by polyacrylamide gel electrophoresis (PAGE) yields a pattern which is characteristic for a particular rotavirus isolate. The group A rotaviruses can be further characterized by analysis of VP7 and VP4 genes specificities, responsible for rotavirus classification into G and P genotypes, respectively. The aim of the present study was to determine the evolution of group A Rotavirus strains circulating in Tunisia over a 3-year period (2005–2007). Material and methods A total of 1503 stool samples collected from children less than five years old, consulting or hospitalised in Tunisia for diarrhoea between 2005 and 2007, were screened for the presence of group A Rotaviruses. Rotavirus-positive specimens were further analyzed by PAGE and G/P-genotyped by multiplex semi-nested RT-PCR. Results Rotaviruses were detected in 323 stool samples over 1503 (21 %). Long electropherotypes predominated in Tunisia during the whole period of study ( N = 158 vs N = 82 short electropherotypes). VP7 genotyping showed the cocirculation of five different genotypes: G1, G2, G3, G4 and G9. VP4 typing detected four different P-genotypes: P[8], P[4], P[6] and P[11]. Rotavirus strains with G3P[8] specificity were predominating in Tunisia in 2005 and 2006, replaced by G2P[4] strains in 2007.

Relationship between electropherotypes and VP7/VP4 genotypes of group A rotaviruses detected between 2000 and 2007 in Tunisian children

Background Rotaviruses are the most frequent agents associated with diarrhoea in children worldwide. Analysis of mobility of the 11 segments of genomic RNA by polyacrylamide gel electrophoresis (PAGE) yields a pattern which is characteristic for a particular rotavirus isolate. The group A rotaviruses can be further characterized by analysis of VP7 and VP4 genes specificities, responsible for rotavirus classification into G and P genotypes, respectively. The aim of the present study was to detect a relationship between electropherotype pattern and molecular characteristics of the rotavirus strains. Material and methods Were analyzed 278 rotavirus-positive specimens by PAGE and G/P-genotyped by multiplex semi-nested RT-PCR. Pearson's correlation tests were used for statistical analysis. Results Twelve different electropherotypes were visualized, eight with a long profile (186 cases) and four with a short one (87 cases). Concerning VP7 types, G2 viral strains were found to be predominant and were detected in 91 specimens (32.7%). Strains with G1, G3, G4, G8 and G9 specificities were detected in 62 (22.3%), 82 (29.5%), 13 (4.7%), two (0.7%) and seven cases (2.5%), respectively. The results of VP4 genotyping showed a predominance of P[8] genotype which comprised half of the strains identified (139 cases, 50%). VP4 P[4], P[6] and P[11] were found in 83 (29.9%), 31 (11.1%) and 11 (4.0%) specimens, respectively. A high rate of mixed strains was also found (1.8% mixed electropherotypes, 7.6% G-mixed and 5% P-mixed strains). Electropherotype pattern of rotavirus strains was significantly correlated with VP7 genotype ( p = 0.018) and with VP4 genotype specificities ( p < 0.001).

A research on rotaviral diarrhea outbreak in Guangxi Province

To analyze epidemiological characters of an outbreak of rotavirus diarrhea in Daxing County, Guangxi Province. Rotavirus-positive specimens were identified by ELISA kit. G/P typing assays were confirmed with multiplex seminested RT-PCR. Full-length VP7 genes of 4 positive specimens were amplified and analyzed. 30 cases of Rotavirus-positive were identified from 64 specimens. The attack rate was 46.9%, and G/P typing was G1P[8]. A change of VP7 amino acid residue is at positions 68. G1P[8] rotavirus is the etiologic agents of this diarrhea outbreak. In addition, adults were included in this outbreak.