Rotavirus In Fecal Specimens Research Articles

Prevalence of Rotavirus among Iraqi children with diarrhea in Diyala province

Background: Rotavirus is the most common cause of acute gastroenteritis in children below five years.
 Objectives: The aim of this study to evaluate the prevalence of rotavirus among children with diarrhea, the efficacy of Immuno Chromatography Assay (ICA) and Enzyme-linked Immunosorbent assay (ELISA) and evaluate the relationship between the demographic agent and clinical presentation associated with rotavirus.
 Material and Methods: A cross-sectional study was conducted at AL-Batool Teaching Hospital in Baqubah, from the first of April 2019 to 31 of March 2020, on (300) children with diarrhea below five years of age. The patients included (n=161, 53.6%) males and (n=139, 46.3%) females. The patient’s age, gender, living area, season, mode of feeding, source of water supply and sterilization, were collected through a questionnaire prepared for this study. The Rapid Test and ELISA are used for the detection of rotavirus in fecal specimens.
 Results: The rotavirus antigen was detected in (47.33%) stool specimens by the ELISA test and (59%) by ICA from (300) children with diarrhea and high infection in the age group between 13-24 months at the rate (49.40%). The highest infection rate in spring was (56.00%) and lower in summer (33.33%). The infection males more than females (42.24% vs. 39.57%). High significant infection among children living in rural areas (55.68%) compared to those living in urban areas (35.48%). According to the clinical features, the results showed the rate of rotavirus infection in children with fever was (54.90%), vomiting (42.03%), those suffering from severe dehydration (48.85) some dehydration 78 (46.15%), however, rotavirus infection was significant with fever only.

Prevalence of Rotavirus among Iraqi children with diarrhea in Diyala province

Background: Rotavirus is the most common cause of acute gastroenteritis in children below five years.
 Objectives: The aim of this study to evaluate the prevalence of rotavirus among children with diarrhea, the efficacy of Rotavirus Rapid Test Device (RTD)and ELISA and evaluate the relationship between the demographic agent and clinical presentation associated with rotavirus.
 Material and Methods: A cross-sectional study was conducted at AL-Batool Teaching Hospital in Baqubah, from the first of April 2019 to 31 of March 2020, on (300) children with diarrhea below five years of age. The patients included (n=161, 53.6%) males and (n=139, 46.3%) females. The patient’s age, gender, living area, season, mode of feeding, source of water supply and sterilization, were collected through a questionnaire prepared for this study. The Rapid Test and ELISA are used for the detection of rotavirus in fecal specimens.
 Results: The rotavirus antigen was detected in (47.33%) stool specimens by the ELISA test and (59%) by Rotavirus Rapid Test Device (RTD) from (300) children with diarrhea and high infection in the age group between 13-24 months at the rate (49.40%). The highest infection rate in spring was (56.00%) and lower in summer (33.33%). The infection males more than females (42.24% vs. 39.57%). High significant infection among children living in rural areas (55.68%) compared to those living in urban areas (35.48%). According to the clinical features, the results showed the rate of rotavirus infection in children with fever was (54.90%), vomiting (42.03%), those suffering from severe dehydration (48.85) some dehydration 78 (46.15%), however, rotavirus infection was significant with fever only.

Development of a new enzyme immunoassay for improved detection of rotavirus in fecal specimens of vaccinated infants

BackgroundGroup A rotavirus is the most common cause of acute diarrhea in young children worldwide. A simple and rapid enzyme immunoassay (EIA) has been commonly used to detect rotavirus infection and evaluate rotavirus vaccines. Currently licensed commercial EIA kits have low sensitivity. A more sensitive detection of rotavirus can improve rotavirus diagnostics and vaccine efficacy studies. ObjectiveA biotin-avidin based sandwich EIA was developed and compared with commercial EIA kits for improved detection of viral shedding in fecal samples from infants who received human rotavirus vaccine Rotarix in Mexico. Study designA monoclonal antibody (mAb: 1D4) specific to human rotavirus group antigen VP6 was prepared and used to develop a biotin-avidin based sandwich EIA. This EIA was employed to test 128 fecal samples from vaccinated infants, in comparison with two commercial EIA kits using RT-PCR as a reference. ResultsA new biotin-avidin based sandwich EIA showed specific reaction to group A rotaviruses, but not to other enteric viruses. This new EIA had a detection rate of 36.7% for rotavirus antigen shedding in fecal specimens, which was two times higher (16.4%, 18.0%) than those from two commercial EIA kits. ConclusionThe new EIA had specificity and higher sensitivity than commercial kits. This new EIA has the potential to detect rotavirus at lower concentration in clinical specimens and thus should be further evaluated as a more sensitive kit for use in diagnostics and vaccine efficacy and effectiveness studies.

Detection of Rotavirus in children with acute gastroenteritis in Zagazig University Hospitals in Egypt.

Introduction:Rotavirus is the major cause of acute gastroenteritis (AGE) in infants and young children all over the world. The objective of the study was to compare different methods for detecting rotavirus and to assess the burden of rotavirus as a causative agent for AGE in children younger than five.Methods:This case control study included 65 children with AGE and 35 healthy control children. They were chosen from the Pediatric Department of Zagazig University Hospitals from October 2014 to March 2015. Stool samples were obtained and assayed for rotavirus by the immunochromatography test (ICT), enzyme linked immunosorbent assay (ELISA) and quantitative real time RT-PCR (qr RT-PCR).Results:Fifty out of the 65 patients (76.9%) were positive for qr RT-PCR. Forty-five (69.2%) and 44 (67.7%) were positive for ICT and ELISA, respectively. There was a significant association between the severity of the disease as determined by the Vesikari score and rotavirus infection.Conclusion:This study demonstrated that ICT is a useful method for the rapid screening of group A rotavirus in fecal specimens, because it is rapid, inexpensive, easy to perform, and requires very little equipment. In addition, this study highlights the substantial health burden of rotavirus AGE among children less than five.

Rapid and sensitive electrochemiluminescence detection of rotavirus by magnetic primer based reverse transcription-polymerase chain reaction

A novel method for detection of rotavirus has been developed by integrating magnetic primer based reverse transcription-polymerase chain reaction (RT-PCR) with electrochemiluminescence (ECL) detection. This is realized by accomplishing RT of rotavirus RNA in traditional way and performing PCR of the resulting cDNA fragment on the surface of magnetic particles (MPs). In order to implement PCR on MPs and achieve rapid ECL detection, forward and reverse primers are bounded to MPs and tris-(2,2′-bipyridyl) ruthenium (TBR), respectively. After RT-PCR amplification, the TBR labels are directly enriched onto the surface of MPs. Then the MPs–TBR complexes can be loaded on the electrode surface and analyzed by magnetic ECL platform without any post-modification or post-incubation process. So some laborious manual operations can be avoided to achieve rapid yet sensitive detection. In this study, rotavirus in fecal specimens was successfully detected within 1.5h. Experimental results showed that the detection limit of the assay was 0.2pgμL−1 of rotavirus. The ECL intensity was linearly with the concentration from 0.2pgμL−1 to 400pgμL−1. What's more, the specificity of this method was confirmed by detecting other fecal specimens of patients with nonrotavirus-associated gastroenteritis. We anticipate that the proposed magnetic primer based RT-PCR with ECL detection strategy will find numerous applications in food safety field and clinical diagnosis.

Comparison of the Rapid Methods for Screening of Group A Rotavirus in Stool Samples

In this study, the sensitivity and specificity of three immunochromatography (IC) test kits for rapid detection of group A rotavirus were compared and evaluated with stool samples collected from children, who suffered from acute gastroenteritis during February to June, 2009 in Japan. A total of 86 stool samples were tested and compared with a reference RT-PCR method. The sensitivity among IP-Rota V, Dipstick Eiken ROTA and ROTA-ADENO test kits were 97.2, 95.8 and 88.7%, while the specificity were 100, 93.3 and 100%, respectively. It was demonstrated that the IC kits evaluated in this study could be used as an alternative method for the rapid screening of group A rotavirus in fecal specimens, especially during acute gastroenteritis outbreak season.

Effectiveness of Pentavalent Rotavirus Vaccine in a Large Urban Population in the United States

The goal was to assess the effectiveness of complete (3-dose) or partial (1- or 2-dose) immunization with pentavalent rotavirus vaccine (RV5) against rotavirus acute gastroenteritis (AGE) in US clinical practice. A case-control evaluation was conducted in February through June 2008 at an emergency department in Houston, Texas. Case patients with rotavirus AGE (N = 90) were identified through testing for rotavirus in fecal specimens obtained from 205 children 15 days through 23 months of age presenting with AGE. Control groups included rotavirus-negative AGE patients (N = 115), concurrently enrolled patients with acute respiratory infection (ARI) (N = 228), and up to 10 age- and zip code-matched children sampled from the Houston-Harris County Immunization Registry (HHCIR) for each case patient >8 months of age. Immunization data were obtained from parent records, health care providers, and/or the HHCIR. Vaccine effectiveness was calculated as 1 minus odds of RV5 vaccination for case patients versus control patients, after adjustment for age at presentation and birth date. The vaccine effectiveness of a complete RV5 series was 89% (95% confidence interval [CI]: 70%-96%) and 85% (95% CI: 55%-95%) with rotavirus-negative AGE and ARI control patients, respectively. Immunization data were available for 44% of case patients (n = 40) from the HHCIR; the estimated 3-dose vaccine effectiveness with these HHCIR control patients was 82% (95% CI: 19%-96%). A complete RV5 series conferred 100% protection (95% CI: 71%-100%) against severe rotavirus disease requiring hospitalization and 96% protection (95% CI: 72%-99%) against disease requiring intravenous hydration. Vaccine effectiveness of 1 and 2 doses against hospitalization and emergency department visits was 69% (95% CI: 13%-89%) and 81% (95% CI: 13%-96%), respectively, using rotavirus-negative AGE and ARI control groups combined. In this setting, a complete series of RV5 was highly effective against severe rotavirus AGE. Partial immunization also conferred substantial protection.

Molecular epidemiology of rotaviruses among healthy calves in Japan: Isolation of a novel bovine rotavirus bearing new P and G genotypes

A total of 171 fecal specimens collected from healthy calves on a beef farm in Gifu Prefecture, Japan in 2006–2007 were examined for group A rotaviruses by RT-semi-nested PCR targeting the coding region for VP8*. Nine specimens were positive for rotavirus. G and P genotyping indicated that one strain was G10P[11]-like and six strains were considered to be the same unknown G and P genotypes. Among these six untypeable strains, one strain, AzuK-1, was adapted to cell culture and analyzed. Sequence and phylogenetic analyses of the full lengths of VP4 and VP7 genes revealed that AzuK-1 strain is a novel bovine rotavirus bearing new G21 and P[29] genotypes as confirmed by the RCWG. Furthermore, we detected G21P[29] rotaviruses in fecal specimens collected from healthy calves in Hokkaido, Japan during the period from 1997 to 1998. These findings suggest that novel G21P[29] rotaviruses have been widely prevalent among cattle for over 10 years in Japan.

Comparison of electron microscopy, enzyme-linked immunosorbent assay and latex agglutination for the detection of bovine rotavirus in faeces.

The performance characteristics of 2 enzyme immunoassays (ELISAs) and 4 latex agglutination assays (LXs) were evaluated for the detection of bovine rotavirus in faecal specimens of young calves with diarrhoea. A total of 26 specimens from calves less than 5 months of age were examined with different commercial assays and compared with electron microscopy (EM) as the gold standard and with polyacrylamide gel electrophoresis (PAGE) for the detection of atypical, non-group A rotaviruses. In the 2nd study, EIA (Dako) and LX (Murex), the assays of choice, were used to analyse 97 further faecal specimens from calves with diarrhoea. The ELISAs proved to be the most sensitive compared with the other tests used. The EM and PAGE are 100% specific although slightly less sensitive than the commercial assays. The results show that all the commercial assays can accurately detect rotavirus in the stools of calves with gastroenteritis, although the suitability and choice of assay will depend upon the requirements of individual laboratories.

Detection of rat, porcine, and bovine group B rotavirus in fecal specimens by solid-phase enzyme immunoassay

An enzyme immunoassay that uses easily regenerated reagents was developed and evaluated for the ability to detect group B rotaviruses (GBR) in fecal specimens. Homologous rat GBR and heterologous porcine and bovine GBR were detected by this immunoassay, although a human GBR isolate was not. This immunoassay should prove useful in studies of GBR infections of animals.

Detection of animal and human group B rotaviruses in fecal specimens by polymerase chain reaction

A combined reverse transcriptase reaction-polymerase chain reaction (RT-PCR) was developed to achieve the sensitive detection of group B rotaviruses (GBR). Sequences derived from genomic segment 3 of the IDIR (intestinal disease of infant rats) strain of GBR permitted the detection of greater than or equal to 0.08 pg of purified IDIR genomic RNA (4,000 genome copies). Primers complementary to the terminal sequences of gene 11 of GBR strain ADRV (adult diarrhea rotavirus) allowed for the detection of as little as 0.008 pg of purified ADRV genomic RNA. Detection of heterologous strains of GBR was also observed with these primer pairs. IDIR gene 3 primers recognized greater than or equal to 8 pg of RNA from bovine GBR obtained from a variety of geographic locations. RNA from IDIR, but not bovine GBR, strains was detected by means of RT-PCR with ADRV gene 11 primers. Neither set of GBR primers was reactive in RT-PCR with fecal specimens containing group A rotaviruses or fecal specimens from uninfected controls. This RT-PCR assay permits the sensitive and specific detection of a variety of GBR in fecal specimens.

Removal of inhibitory substances from human fecal specimens for detection of group A rotaviruses by reverse transcriptase and polymerase chain reactions

A method was developed for the purification of rotavirus RNA from fecal extracts in order to permit the sensitive identification of group A rotavirus in fecal specimens by the polymerase chain reaction. Sequential reactions with reverse transcriptase and Taq polymerase with directed primers from rotavirus gene 6 yielded characteristic 259-base-pair fragments that were then visualized by silver stain on a polyacrylamide gel. As few as 500 genomic copies of purified rotavirus RNA could be detected in this manner. However, when the method was applied to fecal samples with added rotavirus virions, inhibition was noted in many of the fecal extracts which were tested. The inhibition could be reversed by dilution of the fecal extract, but sensitivity was also reduced by a corresponding dilutional factor. The inhibition was quantitatively removed by an added step in the extraction process that utilized chromatographic cellulose fiber powder (CF11 powder) to purify the rotavirus RNA during a series of rapid washing and elution steps. After CF11 purification, rotavirus RNA could be detected in experimental fecal samples at dilutions 1,000- to 10,000-fold beyond the detection limits of standard techniques such as enzyme immunoassay and the direct visualization of RNA following polyacrylamide gel electrophoresis. Furthermore, following purification by CF11, rotavirus RNA could be detected in all of seven enzyme-linked immunosorbent assay-positive fecal samples obtained from a child with rotavirus gastroenteritis; when CF11 purification was not performed, rotavirus RNA could be detected in only four of these samples, even after the removal of inhibitors by dilution of the extracts. Large-scale identification of rotavirus in fecal specimens may be possible by use of CF11 purification of viral RNA prior to sequential reactions with reverse transcriptase and Taq polymerase in a modified polymerase chain reaction.

Choice of reference assay for the detection of rotavirus in fecal specimens: electron microscopy versus enzyme immunoassay

Two previously demonstrated sensitive and specific enzyme immunoassays (EIAs) for rotavirus, one using polyclonal and monoclonal antisera (TestPack Rotavirus [TPK]; Abbott Laboratories) and the other using only monoclonal anti-rotavirus antibodies (Rotaclone [RTC]; Cambridge BioScience Corporation), were evaluated as potential reference assays for rotavirus testing in comparison with direct negative-staining electron microscopy (EM), the current laboratory standard. Two hundred and seven stool samples collected consecutively during the winter of 1989 from children with acute diarrhea admitted to a ward for infants from 0 to 2 years of age were tested by the EIAs and by EM. TPK specimens were read visually; RTC results were read spectrophotometrically. Specimens with discordant EIA and EM results were further evaluated by a fluorescent focus assay. Specimens positive by EM and those negative by EM but positive by fluorescent focus assay were considered to be positive for rotavirus. Of the 207 stools tested, 35 (17%) were positive for rotavirus by these criteria. EM had a sensitivity of only 80%. Specificities were 100% for RTC and EM and 89% for TPK. These findings indicate that EM, although very specific, is relatively insensitive compared with a highly sensitive monoclonal antibody-based EIA. An EIA with high sensitivity and specificity, such as RTC, is a more appropriate reference standard for rotavirus testing.

Detection of rotavirus by hybridization with a nonradioactive synthetic DNA probe and comparison with commercial enzyme immunoassays and silver-stained polyacrylamide gels

Three enzyme-linked immunosorbent assays (ELISAs) (rotavirus enzyme immunoassay [EIA; International Diagnostic Laboratories], Pathfinder [Kallestad Laboratories], and Rotaclone [Cambridge Bioscience, Inc.]) and hybridization of viral RNA with a nonradioactive, synthetic oligonucleotide DNA probe (SNAP; Molecular Biosystems, Inc.) were compared with silver-stained polyacrylamide gel electrophoresis (PAGE) of viral RNA for the detection of rotavirus in fecal specimens. Hybridization was performed following column purification of the viral RNA. Of 286 specimens analyzed by PAGE, SNAP, rotavirus EIA, Pathfinder, and Rotaclone, 88 were positive by PAGE. All 88 specimens were also positive by the other four assays. Nine specimens that were positive by one or more of the assays were positive by blocking ELISAs but were negative by PAGE. If these nine specimens were considered to be true positives, the final sensitivities and specificities were as follows: PAGE, 91 and 100%; SNAP, 94 and 97%; rotavirus EIA, 96 and 97%; Pathfinder, 100 and 94%; and Rotaclone, 96 and 97%, respectively.

Detection of group B rotavirus in fecal specimens by dot hybridization with a cloned cDNA probe

Cloned cDNA copies were synthesized from the genomic RNA of the IDIR strain of group B rotavirus (GBR) isolated in Baltimore, Md. These clones were screened for hybridization with heterologous GBR to identify cDNA for use in dot hybridization experiments. In multiple screening experiments, cDNA clones derived from gene segment 3 provided the most intense hybridization signals. 32P-labeled probes were produced from one of the gene 3 clones, and these were employed in dot hybridization assays. Purified preparations of bovine GBR were detected in concentrations of greater than or equal to 0.5 ng, and GBR was detected in fecal specimens obtained from five of six infected calves. Four of six human fecal specimens containing the Baltimore strain of GBR were also positive in the hybridization assay, while GBR was identified in only one of the six specimens by means of immunoelectron microscopy. A fecal specimen obtained from a patient infected with the adult diarrhea rotavirus strain of GBR was also positive in the dot hybridization assay. Fecal specimens from uninfected humans, calves, and rats, as well as specimens containing group A rotaviruses, did not hybridize with the cloned cDNA probe.

Comparison of five enzyme immunoassays, electron microscopy, and latex agglutination for detection of rotavirus in fecal specimens

Five different enzyme immunoassays, electron microscopy, and latex agglutination (Slidex; bioMerieux) were compared for the rapid detection of human rotavirus in fecal specimens. The enzyme immunoassay using rotavirus polyclonal antiserum (Dakopatts) with simple in-house modifications was shown by the use of confirmatory tests to be the most sensitive and specific procedure.

Detection of rotavirus in faecal specimens with a monoclonal antibody enzyme-linked immunosorbent assay: Comparison with polyclonal antibody enzyme immuno-assays and a latex agglutination test

Monoclonal antibodies have been produced against the 81/36F strain of rotavirus. One of them, was chosen as diagnostic reagent: it showed high ELISA reactivity with all the bovine, human and porcine rotavirus strains tested and reacted with VP6, structural protein product known to support the common rotavirus antigen. A sandwich ELISA procedure using the chosen monoclonal as “capture and detecting” antibody was performed to detect rotavirus in faecal samples from experimentally inoculated newborn calves: it always gave a negative response with meconium and a positive response for the stool specimens which rotavirus have been isolated. This assay was compared with Enzygnost and Slidex Rota Kit tests and with a non-commercial sandwich ELISA test using polyclonal antibodies: it showed more sensitivity than the agglutination test and was as sensitive as the other two tests to detect rotavirus in routine diagnostic material. The test evaluated showed no equivocal results.

Evaluation of a new enzyme immunoassay (TESTPACK rotavirus) for the detection of rotavirus in fecal specimens

One hundred and forty fecal specimens were tested for rotavirus using two immunoassays, TESTPACK Rotavirus and Pathfinder Rotavirus. Five discordant specimens were evaluated by a blocking assay. The sensitivity, specificity, positive predictive value, and negative predictive value were 98, 100, 100, and 99% for TESTPACK and 92, 100, 100, and 96% for Pathfinder, respectively.

Comparison of nine commercial immunoassays for the detection of rotavirus in fecal specimens

One hundred fecal specimens obtained from patients with acute gastroenteritis were tested for rotavirus with nine commercial immunoassays to evaluate the sensitivity, specificity, predictive value, and diagnostic accuracy of these assays. Kits evaluated included two monoclonal antibody-based enzyme immunoassays (EIAs) (Rotaclone and Pathfinder Rotavirus), three polyclonal antibody-based EIAs (Rotavirus Immunoassay, Rotazyme II, and Wellcozyme Rotavirus), and four latex agglutination assays (Rotastat, Virogen Rotatest, Meritec-Rotavirus, and The Wellcome Latex Test). Thirty-eight of the 100 specimens were found to contain rotavirus by a reference microplate EIA. The accuracy of the reference assay was determined by RNA electrophoresis and a blocking assay on discordant specimens. The two monoclonal antibody EIAs had superior sensitivities (100%) and identified two positive specimens which were negative by the reference method but positive by the blocking assay. Among the polyclonal EIAs, all had sensitivities of greater than 90%, but specificities were variable; Rotazyme II, with a specificity of 50%, showed considerable discrepancy from other polyclonal EIAs. The latex tests had sensitivities ranging from 70 to 90% and specificities of 80 to 100%. Latex agglutination tests were more rapid than EIAs and did not require expensive equipment. The final choice of assay system will depend on the cost, speed, and accuracy requirements of the clinical laboratory.

An assessment of the sensitivity of three methods for the detection of rotavirus

A comparison was made of the sensitivity of a current commercial ELISA for detecting rotavirus in faecal specimens with the more complex, technically demanding systems of electron microscopy and polyacrylamide gel electrophoresis. Even after modification, the ELISA failed to detect 22% of specimens with particles identifiable by electron microscopy. Polyacrylamide gel electrophoresis failed to identify 2 out of 50 specimens with particles present but did distinguish 2 group C rotaviruses.