Detection Of Rotavirus In Stools Research Articles

Evaluation of Enzyme Immunoassay Method versus Real-time PCR for Diagnosis of Rotavirus Infection in Acute Infantile Diarrhea

Background: Acute infectious gastroenteritis is a common cause of fatality between children in the developing countries which is usually due to viral etiology. Rotavirus is a ds-RNA (60-80nm), non-enveloped virus with a segmented genome. Group (A) of the virus is an important human pathogen that accounts for (90%) of the isolates. An easy, rapid, non-expensive and sensitive method is needed to detect this virus for clinical controlling. The objective of this study is to evaluate Enzyme immunoassay technique versus Quantitative real-time PCR in the diagnosis of infection with Rotavirus in the children with acute diarrhea. Methodology: This study was conducted on (75) infants and young children, from The Pediatric Department at Tanta University Hospitals in the period from December 2019 to March 2020 and were diagnosed according to history and clinical examination using Vesikari scoring system for acute severe gastroenteritis. Also, 10 healthy infants and children were taken as a control group. Stool samples were obtained from the patients and the controls. These specimens were tested with ELISA and Quantitative real-time PCR for detection of Rotavirus in stool. Results: The study revealed that 62 patients (82.6 %) were positive by ELISA and 74 cases (98.6 %) were positive with real time RT-PCR. Additionally, all the control group gave negative results by the two techniques. Conclusion: Enzyme immunoassay is an accurate and suitable method as a routine diagnostic measure for Rotavirus that can run a large number of samples. But, it is expensive when used for a single sample. Quantitative real time PCR was more sensitive and specific measure that can detect Rotavirus RNA in too minimal amounts in stools.

Multicenter Trial of a Combination Probiotic for Children with Gastroenteritis

BackgroundGastroenteritis accounts for approximately 1.7 million visits to the emergency department (ED) by children in the United States every year. Data to determine whether the use of probiotics improves outcomes in these children are lacking.MethodsWe conducted a randomized, double-blind trial involving 886 children 3 to 48 months of age with gastroenteritis who presented to six pediatric EDs in Canada. Participants received a 5-day course of a combination probiotic product containing Lactobacillus rhamnosus R0011 and L. helveticus R0052, at a dose of 4.0×109 colony-forming units twice daily or placebo. The primary outcome was moderate-to-severe gastroenteritis, which was defined according to a post-enrollment modified Vesikari scale symptom score of 9 or higher (scores range from 0 to 20, with higher scores indicating more severe disease). Secondary outcomes included the duration of diarrhea and vomiting, the percentage of children who had unscheduled physician visits, and the presence or absence of adverse events.ResultsModerate-to-severe gastroenteritis within 14 days after enrollment occurred in 108 of 414 participants (26.1%) who were assigned to probiotics and 102 of 413 participants (24.7%) who were assigned to placebo (odds ratio, 1.06; 95% confidence interval [CI], 0.77 to 1.46; P=0.72). After adjustment for trial site, age, detection of rotavirus in stool, and frequency of diarrhea and vomiting before enrollment, trial-group assignment did not predict moderate-to-severe gastroenteritis (odds ratio, 1.06; 95% CI, 0.76 to 1.49; P=0.74). There were no significant differences between the probiotic group and the placebo group in the median duration of diarrhea (52.5 hours [interquartile range, 18.3 to 95.8] and 55.5 hours [interquartile range, 20.2 to 102.3], respectively; P=0.31) or vomiting (17.7 hours [interquartile range, 0 to 58.6] and 18.7 hours [interquartile range, 0 to 51.6], P=0.18), the percentages of participants with unscheduled visits to a health care provider (30.2% and 26.6%; odds ratio, 1.19; 95% CI, 0.87 to 1.62; P=0.27), and the percentage of participants who reported an adverse event (34.8% and 38.7%; odds ratio, 0.83; 95% CI, 0.62 to 1.11; P=0.21).ConclusionsIn children who presented to the emergency department with gastroenteritis, twice-daily administration of a combined L. rhamnosus–L. helveticus probiotic did not prevent the development of moderate-to-severe gastroenteritis within 14 days after enrollment. (Funded by the Canadian Institutes of Health Research and others; PROGUT ClinicalTrials.gov number, NCT01853124.)

Comparison of Latex Agglutination with Enzyme Immunoassay for Detection of Rotavirus in Children with Acute Diarrhea in Diyala Province, Iraq

Rotavirus (RV) is the most important etiological agent of diarrhea in children under 5 years old, mainly in developing countries. The laboratory diagnosis is usually based on detection of viral antigen using enzyme immunoassay or direct latex agglutination techniques. Objectives: To compare the sensitivity and specificity of enzyme immunoassay versus direct agglutination test for the detection of rotavirus in stool of children less than 5 years of age with acute diarrhea in Diyala province. Patients and methods: The present study was conducted during the period from 1/August/2012 to 30/November/2013. 120 patients with acute diarrhea were included. The patients attended Al-Batool Maternity and children Teaching Hospital and other Primary Health Care Centers in Baquba, the center of Diyala province. Seventy (58.3%) patients were males and 50 (41.7%) were females. The age range was 2 months to 5 years. Information regarding age, sex, residence, type of feeding and source of drinking water were collected. Detection of RV in stool specimens using direct agglutination test (Diaspot-USA) was performed as soon as possible after collection. The enzyme linked immunosorbant assay (ELISA) was carried out using the commercially available kit (DRG-Germany). Data were statistically analyzed and P value < 0.05 was considered significant. Results: The results showed that the detection rate of RV in stool specimens by DAT and ELISA were 70% and 93.3% respectively. The sensitivity, specificity, positive predictive value and negative predictive values of DAT was 75.56%, 66.67%, 97.22%, and 15.39% respectively, and for ELISA was 91.3%, 66.67%, 97.67%, and 33.33% respectively. Spearman's correlation analyses revealed that the age group 10-15 months was significantly affected (P= 0.046). Conclusion: Rotavirus antigen detection by EIA is a rapid, sensitive, and specific method, and could be used in large scale application for screening stool samples of patients with acute diarrhea.

Detection of Rotavirus in Stools by Means of Reversed Passive Haemagglutination (R-PHA)

Detection of Rotavirus in Stools by Means of Reversed Passive Haemagglutination (R-PHA)

An observational study of naturally acquired immunity to rotaviral diarrhea in a cohort of 363 Egyptian children. Calculation of risk for second episodes using age-specific person-years of observation.

A cohort of 363 rural children in Bilbeis, Egypt, were followed from birth from 1981 to 1983, with twice-weekly home visits made to detect diarrheal illness. Enzyme-linked immunosorbent assay was used for detection of rotavirus in stools collected during episodes of diarrhea. Rotavirus-associated diarrhea was detected once in 74 children and twice in 12 children. Using a technique not previously described, the authors calculated the age-specific incidence rates for initial episodes and second episodes of rotavirus-associated diarrhea to estimate the effectiveness of naturally acquired immunity. Assuming that the risk of exposure was the same before and after the first episode, the observed and expected numbers of second episodes of rotaviral diarrhea were equal (age-adjusted rate ratio = 1.01; 95 percent confidence interval 0.55-1.86), given the age-specific person-years at risk. The assumption of equal risk for reexposure to rotavirus appears to be invalid, however, since the children with one and two rotavirus-positive episodes appeared to be at greater risk for diarrheal illness of all causes (rate ratios of 1.42 and 1.78, respectively). The clinical illness may have been less severe in second episodes; emesis was reported more often with first rotavirus episodes than with second rotavirus episodes, and the only fatal case was in an initial episode. These data are compatible with the existence of partial immunity, since it appears that the risk of reexposure may be greater in children who experienced rotaviral gastroenteritis earlier in life. In four of seven children, rotavirus isolates from first and second episodes were of identical serotypes, indicating that even serotype-specific immunity for rotaviral diarrhea was incomplete.

Comparison of a new rapid test (TestPack Rotavirus) with standard enzyme immunoassay and electron microscopy for the detection of rotavirus in symptomatic hospitalized children

We compared a new, rapid, qualitative test for rotavirus (TestPack Rotavirus; Abbott Laboratories, North Chicago, Ill.) with another enzyme immunoassay (Pathfinder Rotavirus; Kallestad Laboratories, Inc., Austin, Tex.) and electron microscopy to determine its clinical utility in a population of symptomatic hospitalized children. In the first part of the study, 100 frozen stool samples were tested. The results after resolution with a blocking reagent showed a sensitivity of only 50% and a specificity of 88% for TestPack Rotavirus. In the second part of the study, we tested TestPack Rotavirus on 100 fresh, unfrozen samples. The results (sensitivity/specificity) were as follows: TestPack Rotavirus, 95/90%; Pathfinder Rotavirus, 84/98%; direct electron microscopy, 63/100%. Although it was not as sensitive or specific as immune electron microscopy, TestPack Rotavirus was more sensitive than direct electron microscopy or Kallestad Pathfinder Rotavirus. TestPack Rotavirus represents a rapid, qualitative method for the detection of rotavirus in stools of symptomatic children.

Rapid detection of human rotavirus strains in stools by single-sandwich enzyme-linked immunosorbent assay systems using monoclonal antibodies

Using murine monoclonal antibodies (MAbs) raised against the common antigen of group A rotavirus (RV), two single-sandwich ELISA systems were developed for detection of RV in stools: one using polyclonal antibody (PAb) as capture and a MAb as detector antibody (referred to as PAb-MAb assay); and the other based on the use of two different MAbs as capture and detector antibodies (referred to as MAb-MAb assay). In each single-sandwich ELISA system, samples and peroxidase-labeled MAb were incubated sequentially (two-step method) or simultaneously (one-step method). Using the two-step procedure on purified RV, 50 pg of protein was detected in the PAb-MAb as well as in the MAb-MAb assay, whereas the one-step method detected 0.4 ng and a conventional double-sandwich ELISA detected 3.2 ng of viral protein. Titration of RV samples from stools and cell cultures showed that single-sandwich ELISA titers were, on the average, 10–100-fold higher than those obtained by electron microscopy (EM), but 10–100-fold lower than those obtained by solid-phase immune EM (SPIEM). However, when 200 stool samples previously examined by EM or SPIEM were tested by the single-sandwich ELISA systems, specificity and sensitivity of these assays were 100%, and comparable to SPIEM. No false positive results were obtained when 54 samples of meconium and 91 stools from newborns in the first five days of life were tested. The two-step procedure appeared to be somewhat preferable over the one-step method, which, although faster, gave a marked prozone with a few samples in the MAb-MAb assay. The use of MAbs in rapid single-sandwich ELISA systems for RV detection in stools appears highly convenient, due to reliable results and short test performance times.

Detection of human rotavirus by using an alkaline phosphatase-conjugated synthetic DNA probe in comparison with enzyme-linked immunoassay and polyacrylamide gel analysis

An alkaline phosphatase-conjugated synthetic oligodeoxyribonucleotide probe was compared with polyacrylamide gel electrophoresis (PAGE) detection of rotavirus RNA as well as an enzyme-linked immunosorbent assay (ELISA) for the detection of rotavirus in stools from young children with gastroenteritis. The synthetic probe did not cross-react with bacterial causative agents of diarrheal disease. Extraction of viral RNA from stool samples with a phenol-chloroform mixture was suitable for most samples. In some cases fecal pigments interfered with the reaction of the probe with viral RNA. The use of ion-exchange chromatography to further purify viral RNA removed contaminating pigments and increased the sensitivity of the probe assay. Of 260 stool specimens, 77 (30%) were positive for rotavirus when tested by PAGE analysis of rotavirus RNA. The synthetic probe identified 71 rotavirus specimens when RNA obtained by phenol-chloroform extraction followed by chromatographic purification was used (sensitivity, 91.0%; specificity, 96.7%). The ELISA results also agreed well with the electrophoretic analysis (sensitivity, 98.7%; specificity 94%) and the probe assay (sensitivity, 90%; specificity, 100%). Discordant results between the ELISA and the probe assay were examined further by electron microscopy and PAGE analysis of viral RNA. The positive and negative predictive values of the probe assay in comparison with PAGE were 92.2 and 96.1%, respectively. Rotaviruses showing both long and short RNA electrophoretic patterns were detected by the probe. The probe assay coupled with chromatographic purification of rotavirus RNA is an effective method for detecting rotavirus and compares favorably with PAGE analysis and ELISA.

Polyacrylamide gel electrophoresis and silver staining for detection of rotavirus in stools from diarrheic patients in Thailand

Detection of rotavirus by polyacrylamide gel electrophoresis in combination with silver staining and by enzyme-linked immunosorbent assay showed 96.7% identical results in tests with 1,304 stool specimens from diarrheic patients. The polyacrylamide gel electrophoresis method can be modified to reduce cost and working time. Phenol extraction of stools, however, is essential in maintaining the sensitivity of the method.

An improved enzyme-linked immunosorbent assay for the detection of rotavirus in faeces of neonates

A direct enzyme-linked immunosorbent assay (EIA) for the detection of rotavirus in neonatal stools was developed. Rabbit antiserum against SA 11 rotavirus was incorporated as both coating and detector antibody, and rotavirus-negative rabbit serum was applied as a coating antibody control to eliminate false positive results. Pretreatment of stools with EDTA was found to increase both the sensitivity and specificity of the assay. This effect was greatest when 0.25 M EDTA (tetrasodium salt) was included in homogenized stool suspensions before the removal of solid debris by centrifugation. By electron microscopy, this EDTA pretreatment appeared to partly uncoat human rotavirus particles in faeces. Potentially suitable solid phase supports and horseradish peroxidase substrates were evaluated in the development of the assay. Screening of stool samples revealed that repeated freezing and thawing of stools eliminated positive EIA reactions. The SA 11 coating antibody compared favourably with a reference coating antiserum prepared against human faecal rotavirus strains. This EIA showed greater sensitivity for rotavirus detection than electron microscopy of stool concentrates prepared by ultracentrifugation, on testing 143 stools from 99 neonates and children. The assay has been applied successfully to detection of rotavirus in stools of neonates containing meconium, smaller amounts of viral antigen than in older children, and lacteal antirotaviral antibody. It is likely to be particularly useful for cross-infection studies in hospital wards and neonatal nurseries.

Comparison of solid-phase immune electron microscopy by use of protein A with direct electron microscopy and enzyme-linked immunosorbent assay for detection of rotavirus in stool.

A total of 525 stool specimens collected during 1 year were examined for the presence of rotavirus by direct electron microscopy (EM), enzyme-linked immunosorbent assay (ELISA), and a solid-phase immune electron microscope method (SPIEM) utilizing protein A-coated grids for anchoring of specific viral antisera. Rotavirus was seen in 187 specimens; SPIEM detected 183 (97.8%), whereas direct EM and ELISA detected 161 (86%) and 166 (88.7%), respectively. No false-positive reactions were seen by ELISA. The sensitivity of the methods was evaluated by coded investigation of a dilution series of a positive sample, with a negative fecal specimen as diluent. SPIEM was approximately 30 times more sensitive than direct EM and 10 times more sensitive than ELISA. A study was done to compare the elapsed time for recognition of rotavirus by SPIEM and EM in 25 randomly selected positive specimens. All virus-positive specimens were detected within 2 min by SPIEM, whereas up to 9 min was required for direct EM. SPIEM with protein A is a highly sensitive method, useful for rapid detection of viruses in clinical specimens. Due to the direct visualization of virus particles by electron microscopy, there is no requirement for monospecific antisera for the method.

A DOT HYBRIDISATION ASSAY FOR DETECTION OF ROTAVIRUS

A "dot" hybridisation technique for the detection of rotavirus in stools and other biological materials is described. The assay is based on the in-situ hybridisation of labelled single-stranded RNA probes, obtained by in-vitro transcription of rotavirus particles, to heat-denatured rotavirus RNA immobilised on nitrocellulose membranes. The method is highly specific and allows for the detection of as little as 8 pg of viral RNA. Its use for the detection of rotavirus in stool suspensions and rectal swabs obtained from children with diarrhoea may facilitate epidemiological studies of rotavirus gastroenteritis.

Rapid detection of rotavirus in stool by latex agglutination: Comparison with radioimmunoassay and electron microscopy and clinical evaluation of the test

A latex agglutination test (LX) using antisera prepared against Nebraska calf diarrhea virus (NCDV) is described for the detection of rotavirus in stool of children with acute gastroenteritis. The test was compared with electron microscopy (EM) and radioimmunoassay (RIA) with 100 stools positive or negative for rotavirus. Out of 53 stools positive in RIA or EM, 49 were positive in LX and 4 were negative. Two specimens negative in EM and RIA were falsely positive in LX. The method was also tested in two clinical series with 115 stools from 101 children. Altogether 67/115 stools were positive in RIA, and 62/115 in LX. Out of 7 stools with contradictory results, 6 were negative in LX but positive in RIA, and 1 was positive in LX but negative in RIA. The results indicate that the LX is suitable for rapid screening of rotavirus gastroenteritis in clinical practice.