Rotavirus Diagnosis Research Articles

An outbreak of diarrhea in a neonatal medium care unit caused by a novel strain of rotavirus: investigation using both epidemiologic and microbiological methods.

In December 1999, an outbreak of diarrhea was reported in a general hospital neonatal medium care unit (NMCU) caused by a novel strain of rotavirus with genotype P[6], G9. An investigation was conducted to determine risk factors for illness among neonates. Rotavirus diagnosis was by latex agglutination and typing by reverse transcriptase polymerase chain reaction. A case-control study was performed using data collected from medical records on exposures in a 3-day period before illness (cases) or a random 3-day period (controls). Environmental swabs were tested for rotavirus. Antenatal blood samples from mothers and blood samples provided by hospital staff were analyzed for rotavirus antibodies. Fifty-six cases of rotaviral illness were confirmed by latex agglutination. Forty-seven of these were among 118 neonates exposed to the NMCU (attack rate, 40%). There was a 4-week period with no clinical cases in the course of the outbreak. Increased frequency (> or = 15 times in 3 days) of ungloved nasogastric feeding was a significant risk factor (adjusted odds ratio, 8.79), controlling for birth weight and gestational age. Environmental sampling showed persistence of the virus on ward surfaces despite cleaning. None of 24 NMCU staff members had high levels of antibodies against P[6], G9. Three (8%) of 38 mothers had high antibody levels; 2 had infants who became ill. The outbreak ended with a 7-day ward closure, disinfection, and introduction of gloved nasogastric feeding. Case-control studies can be successful in identiffying risk factors for nosocomial outbreaks of diarrhea. High levels of rotavirus antibodies in mothers may not protect infants. The environment may be the most important reservoir of rotavirus during outbreaks.

Evaluation of the ImmunoCardSTAT Rotavirus Assay For On-site Detection of Bovine Rotavirus

Diarrhea is the most important disease in neonatal dairy and beef calves. Significant economic loss occurs due to increased morbidity and mortality, treatment costs and reduced growth rates. Rotaviruses usually cause diarrhea during the first three weeks of life. Clinical signs can be quite variable and are similar to those caused by other neonatal bovine enteropathogens such as corona virus and Eschericia coli.
 Diagnosis of rotavirus in calves is based on the identification of viral particles, antigens or nucleic acids in feces. Tests include direct or immune electron microscopy, immunofluorescence, antigen capture enzyme- linked immunosorbant assay (ELISA), latex agglutination and polymerase chain reaction (RT-PCR). All of these tests are useful for diagnosing rotavirus infections, but in general are relegated to use by diagnostic laboratories. Development of an on-site diagnostic assay for bovine rota virus would be a useful tool for veterinarians and cattle producers in making management decisions regarding neonatal bovine diarrhea.
 The ImmunoCardSTAT Rotavirus (ICS-RV) Assay (Meridian Diagnostics, Cincinnati, OH) is a patient-side assay designed to detect group A rotavirus in humans. This assay is based upon rapid immunomigration (RIM) technology. This technology allows for packaging of all reagents into a single-step assay that can then be used patient-side (similar to a "home pregnancy test"). The objective of this study was to evaluate the ICS-RV assay's usefulness as an on-site diagnostic test for bovine rotavirus.

Culture amplification in human colon adenocarcinoma cell line (CaCo-2) combined with an ELISA as a supplementary assay for accurate diagnosis of rotavirus

Culture amplification in colon adenocarcinoma cell line (CaCo-2) combined with enzyme immunoassay (Pathfinder ELISA) was developed as a supplementary tool for rotavirus diagnosis. One hundred and thirty stools in which results by polyacrylamide gel electrophoresis (PAGE) were in agreement with those obtained by ELISA were amplified in the CaCo-2 cell line. After the first passage 100% specimens were revealed as positive by ELISA. This result was confirmed by PAGE and direct electron microscopy (EM) which increased the rates of rotavirus detection up to 100% after the third and fifth cell passages, respectively. All of the amplified negative stools were confirmed as negative. Among discordant results, three of the eight specimens positive by ELISA but negative by PAGE were confirmed as true positive after the third cell passage. False positive ELISA results could be discarded when the samples were culture amplified and retested by the same ELISA. Using the CaCo-2 amplification-ELISA as supplementary assay, sensitivity and specificity were 1.000 and 0.953 for ELISA and 0.917 and 1.000 for PAGE, respectively. The combined CaCo-2 cell line amplification-immunoassay method proved to be suitable both to evaluate increase in sensitivity of newly developed rotavirus assays and for rotaviral amplification before antigen assays.

Rapid vertical agarose: silver stain detection of rotavirus.

Human rotaviruses (HRV) are the most important etiologic agents of acquired diarrhea in infants and young children worldwide. Therefore, the early diagnosis is essential for effective patient management and infection control. We have developed a rapid, simple technique for the diagnosis of rotavirus based on the sensitive detection of rotavirus double-stranded RNA genome segments separated in vertical agarose gels and developed by silver staining (AGE-SS). This method also has the ability to detect differences in the electrophoretic mobility of RNA bands among group C rotaviruses, reovirus, and group A rotaviruses. The results indicate that this assay is as sensitive and specific as the polyacrylamide gel electrophoresis and silver stain method (PAGE/SS) and it could be applied on large scale for the screening of stool suspected of rotaviral diarrhea. This assay does not need sophisticated equipment and the cost per sample is minimal compared with other available assays.

The epidemiology of rotavirus diarrhea in the United States: surveillance and estimates of disease burden.

The decision to develop rotavirus vaccines was predicated on the extensive burden of rotavirus disease among children worldwide. US reports on nationwide hospitalizations (1979-1992) and deaths (1968-1991) due to diarrhea and weekly reports of rotavirus infection by 74 laboratories were reviewed to estimate the burden of rotavirus disease, identify epidemiologic trends, and consider methods for evaluating an immunization program when a vaccine becomes available. From 1968 to 1985, diarrhea-related deaths among US children <5 years old declined from 1100 to 300/year. This decline was associated with the disappearance of winter peaks for diarrhea-related deaths previously associated with rotavirus infection among children 4-23 months old. From 1979 to 1992, however, hospitalizations for diarrhea averaged 186,000/year and retained their winter peaks, which have been linked to rotavirus infections. Each year an estimated 54,000-55,000 US children are hospitalized for diarrhea, but <40 die with rotavirus. A rotavirus vaccine program will require improved surveillance, including the timely collection of data from sentinel hospitals, in which a diagnosis of rotavirus can be established or ruled out for all children hospitalized for diarrhea.

WC3 reassortant vaccines in children.

Bovine rotavirus strain WC3 (P7[5], G6) administered at the 12th passage level was well tolerated clinically in infants and efficiently induced serum virus neutralizing antibody (VNA) with bovine rotavirus G6 specificity. The protective efficacy of WC3 vaccine against all rotavirus disease was inconsistent, varying in four separate trials from 76% to 0%; some selective protection against severe disease was seen in all trials. WC3 reassortants containing the gene for an individual human rotavirus VP7 (G) or VP4 (P) surface antigen were also well tolerated, but preferentially induced VNA to the WC3 parent. Efficacy trials of human G1 VP7 reassortant WI79-9 (P7[5], G1) consistently led to > 60% protection against all rotavirus disease. A quadrivalent WC3 reassortant vaccine was developed to contain four separate monovalent reassortants expressing human rotaviruses surface proteins G1, G2, G3, and P1A [8] respectively. In a multicenter trial including 439 infants, this vaccine induced 67.1% protection against all rotavirus disease (defined as positive for rotavirus antigen by ELISA only [p = < 0.001]) and 72.6% protection when the standard for rotavirus diagnosis was a positive test of stool for both rotavirus antigen by ELISA and rotavirus RNA by electropherotype analysis (p = < 0.001). In this trial, episodes of the most severe rotavirus disease (clinical severity score > 16.0 eight cases) occurred only in placebo recipients.

Characterization of a synthetic peptide mimicking trypsin-cleavage site of rotavirus VP4

A synthetic peptide corresponding to the trypsin cleavage site on the 84 k protein of bovine rotavirus was synthesized (VP4-peptide). This synthetic peptide could be cleaved by trypsin and therefore possessed the enzyme binding site present on the authentic protein. Further proof that this peptide mimicks the authentic trypsin cleavage site was the specific reaction of anti-peptide serum with the 84 k protein. The reaction of anti-peptide serum with infectious virus neutralized infectivity thereby supporting the biological importance of this site. Another interesting characteristic of this peptide was its ability to bind to the nucleocapsid protein resulting in a laddering effect on the nucleocapsid monomer (45 k), dimer (90 k) and trimer (135 k) [Gorzilia et al., J. Gen. Virol., 66, 1889–1900 (1985); Sabara et al., J. Virol., 53, 58–66 (1985); Sabara et al., J. Gen. Virol., 67, 201–212 (1986)]. Definitive proof of binding was provided by the fact that the increments in the ladder corresponded to the molecular weight of the synthetic peptide and that anti-peptide serum specifically reacted with the ladder formations. The laddering of the nucleocapsid could be eliminated by incubation with trypsin thus further supporting the formation of a synthetic peptide-nucleocapsid complex. Due to the ability of the peptide to bind to trypsin and to the nucleocapsid protein its biological activity was investigated. It appeared that increasing concentrations of the peptide reduced the rate of virus plaque formation, thereby suggesting that virus replication was inhibited. These results illustrate two features of this synthetic peptide which warrant further investigation; (1) its capacity to mimic an enzyme cleavage site and, (2) its ability to complex tightly to another protein. In protection-challenge experiments performed using a murine model, animals immunized with VP4-peptide provided protection passively, to neonates suckling on the immune dams, against a virulent rotavirus. The potential applications of this peptide in rotavirus diagnosis, therapy and synthetic peptides based vaccine is discussed.

Rotaviral Diarrhea

Rotavirus has been identified as the most common cause of foal diarrhea outbreaks in major breeding areas worldwide. This article reviews the latest information on the practical diagnosis, prevention, and control of rotavirus on horse farms. Until an effective vaccine is developed, certain farm management methods and disinfection techniques have proved effective in containing rotavirus outbreaks. Research involving electropherotyping techniques is furthering our knowledge of the different rotavirus serotypes, which will aid in epidemiologic studies and vaccine development.

Comparison of a Commercial Visible Reaction Disc Enzyme Immunoassay to a Commercial Spectrophotometric Bead Enzyme Immunoassay and Electron Microscopy for the Diagnosis of Rotavirus in Bovines

contain a group-specific (gs) antigen located in the inner capsid of the virus. This gs antigen is the basis for techniques such as enzyme immunoassays for the diagnosis of rotavirus infections. Initially, rotaviruses were identified by electron microscopy and by isolation in cell culture. Recently, commercial immunoassay kits developed for identification of human rotaviruses have been utilized for diagnosis of animal rotaviruses. This article presents data comparing a commercial visible reaction disc enzyme immunoassay (VRDEIA) with a commercial spectrophotometric bead enzyme immunoassay (SPB-EIA) and electron microscopy (EM). Fecal specimens and/or intestinal contents from 78 cattle with a history of diarrhea were tested for the presence of rotavirus antigens. The majority of the specimens were from dairy breeds. Samples for the enzyme immunoassays were stored undiluted at -70 C. Specimens were thawed and tested by both immunoassays on the same day. Samples for electron microscopy were diluted with sterile distilled water (1:1 ratio) and stored at -70 C. For the VRD-EIA, a the fecal specimens and intestinal contents were thawed and mixed, and 0.1 g of the sample was transferred to a dilution cup containing 1 measured dropperful of phosphate-buffered saline (0.01 M). The sample was then tested as described in the package insert. A positive sign (+) of any color intensity on the reaction device indicated the presence of rotavirus antigen, whereas a negative sign (-) indicated the absence (or undetectable quantity) of rotavirus antigen. Tests with no sign were considered invalid and were repeated. For the SPB-EIA, a the fecal specimens or intestinal contents were thawed, mixed, and diluted to an approximate 10% suspension using the sample diluent provided. A 200µ1 aliquot of each diluted sample and each control was tested as described in the package insert. The color reactions were visually compared to a color development chart provided in

Acute diarrhea associated viruses : An update

ResumeLes virus retrouvés dans les selles sont soit des virus cultivables (entérovirus, adénovirus autres que les types 40 et 41), soit des virus non cultivables ou “fastidieux” : rotavirus, adénovirus 40 et 41, virus de Norwalk, calcivirus, astrovirus, “petits virus” structurés ou non (SRSV et SRV). Les virus non cultivables ont été retrouvés dans de nombreux cas de diarrhées. Les virus de Norwalk, certains calicivirus, SRV et SRSV ont été retrouvés à l'origine de nombreuses épidémies, tandis que rotavirus, astrovirus et adénovirus 40 et 41 sévissent plutôt sur le mode endémique. Les méthodes diagnostiques sont soit des méthodes ouvertes, soit des méthodes spécifiques. Les méthodes ouvertes sont représentées essentiellement par la microscopie électronique, mais il faut que le virus soit intact et en quantité suffisante. Les petits virus de 20–35 nm sont plus difficiles à identifier. L'électrophorèse en gel de polyacrylamide des acides nucléiques apporte des renseignements épidémiologiques de premier ordre pour les rotavirus surtout. Les méthodes spécifiques font appel aux méthodes immunoenzymatiques ou d'agglutination de particules de latex sensibilisées. Elles sont très employées pour les rotavirus et les adénovirus (tous les types ou seulement les types 40 et 41). Les faux positifs sont possibles et en général bien évalués pour un kit donné, les faux négatifs se rencontrent surtout en cas de souches antigéniques atypiques ou lorsque la quantité de virus est trop faible. Devant une épidémie, l'examen en microscopie électronique est irremplaçable. Devant un cas isolé, la recherche de rotavirus, voire d'adénovirus 40 et 41, grâce aux kits commerciaux est le seul examen réalisable en routine s'il n'y a pas possibilité d'accéder à un microscope électronique. Cependant une minorité de selles peuvent contenir plusieurs virus potentiellement pathogènes et agissant en synergie. A l'opposé il existe de nombreux porteurs asymptomatiques.

The immunological diagnosis of rotavirus

The immunological diagnosis of rotavirus

Latex Test for Rapid Rotavirus Diagnosis in Calves

A latex agglutination test (LA) was compared with direct electron microscopy (EM) for detection of rotavirus infection in calves. A total of 375 samples from 62 calves were collected. Samples were taken when the calves were 1, 3, 5, 7, 10 and 20 days old and some scours samples were collected as well. Altogether 45/375 (12%) specimens were positive in LA and 10/375 (2.7%) were positive in EM. Samples positive in EM were also positive in LA. Out of the 62 calves studied 26/62 (42%) were positive in LA and 8/62 (13%) in EM. We found the LA very easy to perform, to be more sensitive than the EM method and probably a rather specific method for detection of rotavirus infection.

Inter-laboratory comparison of diagnosis of rotavirus infections in Scotland

Samples of 23 rotavirus positive and 17 rotavirus negative faecal specimens were distributed to 15 laboratories in Scotland which undertake rotavirus diagnosis. The results with the various techniques, electron microscopy (EM), polyacrylamide gel electrophoresis (PAGE), enzyme-linked immunoassay and latex agglutination were compared. EM and PAGE were specific and sensitive. Although the results from most commercial kits were satisfactory, there were some which were unsatisfactory. Considerable inter-laboratory variation with the same kit was also noted.

Comparison of six commercial kits for the diagnosis of rotavirus infection in man and calves

Seventy-two human and 72 bovine faecal specimens were tested for rotavirus by polyacrylamide gel electrophoresis (PAGE), four commercial enzyme-linked immunosorbent assay (ELISA) kits (Rotascreen, Wellcozyme, Rotazyme II and IDEIA) and two latex agglutination (LA) kits (RotaScreen and Wellcome). Specimens which were negative by PAGE but positive by one or more of the kits were further examined by direct and immuno-electron microscopy (DEM and IEM). If also negative by DEM and IEM the kit result was considered to be a false positive. Three kits (RotaScreen and IDEIA ELISAs and RotaScreen LA) had specificity and sensitivity greater than 90% on the human specimens but only two (RotaScreen ELISA and LA) had specificity and sensitivity over 80% on the bovine specimens. These kits can therefore be used with reasonable confidence for rotavirus diagnosis, but none of them has any advantage over PAGE other tha speed.

Cholera, rotavirus and ETEC diarrhoea: some clinico-epidemiological features

This paper analyses a few selected features from the history and clinical examination of 1258 patients with acute diarrhoea and a single laboratory diagnosis of either cholera, rotavirus, or enterotoxigenic (ETEC) Escherichia coli infection. Age distribution and seasonality in Bangladesh were also studied. The duration of illness before admission was not significantly different in the 3 groups. Cholera occurred especially in the spring and early winter. Most cholera patients were between 3 and 10 years of age. Over 37% of the patients developed severe dehydration. In about 90% of cholera cases, the stools were alkaline (pH > 7). ETEC infections were seen mostly in April–May and September–October. Infants were frequently affected but from age 25 onwards the age distribution closely followed that of cholera. Severe dehydration occurred in 8·3% of patients and was more frequent than in rotavirus cases. Stool pH was as frequently acidic as basic. Rotavirus cases were concentrated during the winter in patients under 2 years of age. They had marked vomiting, yet severe dehydration was almost absent. Cough was present in half of them. The stools were usually acidic. In spite of considerable overlap of signs and symptoms between the 3 aetiological groups, a presumptive diagnosis of cholera could be made in patients past infancy and early childhood who showed very severe dehydration. However, age-specific prevalence was strikingly different and sea-sonal variations considerable.

Rapid and reliable routine diagnosis of rotavirus using a commercial monoclonal antibody based immunoassay.

<h3>Background</h3> Pooled individual data from the landmark stroke trials of 2015 conclude that the benefit of endovascular thrombectomy for patients with intracranial arterial occlusion also extends to patients with concomitant (so-called ’tandem9) occlusions of proximal vessels. However, there is heterogeneity and debate in the management of these patients, without a clear standard of care. In particular, there is contention regarding whether the proximal or distal lesion should be treated first. We present a case control study and describe the Simultaneous Extracranial, Intracranial Management of (tandem) LESsions in Stroke (SEIMLESS) technique, an efficient approach to the acute ischemic stroke (AIS) patient who presents with tandem lesions (TLs). <h3>Methods</h3> We describe 5 patients, presenting with AIS and TLs between 2015 and 2017, who we treated with SEIMLESS. Cases were reviewed for clinical data, including arterial puncture to intracranial reperfusion, total fluoroscopy time, amount of contrast, age, and gender. Our series was matched to 5 patients treated with the standard ’sequential9 approach (angioplasty followed by thrombectomy) in the same time period by the same operator. <h3>Results</h3> Arterial access to intracranial recanalization time was significantly shorter in patients treated with SEIMLESS versus those who had angioplasty followed by thrombectomy (39.6±5.9 min vs 85.2±20.6 min; P=0.014). Patients treated ’seimlessly9 also received significantly less iodinated contrast (117±13.5 mL vs 213±48.9 mL; P=0.005) and significantly less fluoroscopy time (21.1±5.2 min vs 55.9±17.8 min; P=0.003). There was no difference in the post-procedural Thrombolysis in Cerebral Infarction score for patients treated with SEIMLESS versus the sequential method (p=0.658). <h3>Conclusion</h3> SEIMLESS is an efficient procedural method that simultaneously treats a distal intracranial occlusion and a more proximal one. Our small case controlled study finds that this technique is feasible and can lead to faster intracranial recanalization compared with the standard ’sequential9 method, utilizing less contrast and radiation in the process. Larger studies are needed to verify our findings.

Detection of rotavirus in stool specimens with monoclonal and polyclonal antibody-based assay systems.

Accurate diagnosis of rotavirus is important in both clinical and research situations. A total of 100 stool specimens from children with diarrhea were tested for rotavirus by electron microscopy. These specimens were then coded and tested for rotavirus by four procedures: a monoclonal antibody-based enzyme immunoassay (EIA) (Pathfinder; Kallestad Laboratories, Inc., Austin, Tex.), two polyclonal antibody-based EIAs (Rotazyme II; Abbott Laboratories, North Chicago, Ill.; and an EIA performed with reagents from the National Institutes of Health, Bethesda, Md. [NIH reagent EIA]), and a latex agglutination (LA) assay (Rotalex; Medical Technology Corp., Somerset, N.J.). The sensitivity of the monoclonal antibody EIA (95%) was superior to those of the polyclonal antibody EIAs (73% for Rotazyme II and 57% for the NIH reagent EIA) and the LA assay (61%). The specificity of the LA assay (98%) was slightly better than those of the other systems (88 to 96%). The positive and negative predictive values of the monoclonal antibody EIA (93 and 96%, respectively) were better than those of Rotazyme II (82 and 80%, respectively), the LA assay (96 and 76%, respectively), and the NIH reagent EIA (93 and 74%, respectively). The visual readings of the monoclonal antibody EIA correlated better with the spectrophotometric optical density readings than did the visual readings of the polyclonal antibody EIAs; however, the agreement of both with electron microscopy results was poor when 1+ or plus-minus readings were observed. The monoclonal antibody EIA is more sensitive and predictive than other rotavirus detection systems and second only to the LA assay in specificity in detecting rotavirus in stool specimens.

Rapid diagnosis of rotavirus by an enzyme-linked immunosorbent assay (ELISA) in an outbreak of neonatal diarrhea in dairy calves

An outbreak of diarrhoea affected 13 of 20 calves in a herd of 260 cows and heifers. Faeces from 10 of the 13 calves were positive or doubtful for rotavirus group-specific (gs) antigens by ELISA. Faeces from 4 cows, including the dam of one affected calf, were all negative. Cell cultures of faeces from 3 affected calves were negative for rotavirus by immunofluorescence. Supernatant fluid from a culture of one calf was positive for gs antigens by ELISA. All 13 affected calves recovered after receiving antibiotics and colostrum. One calf, vaccinated against rotavirus at birth, had slight diarrhoea but recovered. ELISA is recommended for rapid diagnosis of rotavirus in calf diarrhoea.

Rapid diagnosis of rotavirus infection by direct detection of viral nucleic acid in silver-stained polyacrylamide gels.

A rapid simple technique for the diagnosis of rotavirus has been developed based on the sensitive detection of rotavirus double-stranded RNA genome segments separated in polyacrylamide gels. The method utilizes a recently described ultrasensitive silver stain for polypeptides, which can also detect subnanogram amounts of nucleic acid. The sensitivity of the technique is comparable with that of electron microscopy or enzyme-linked immunosorbent assay.

Rotavirus viral RNA electrophoresis in hospitalized infants with diarrhea in Santiago, Chile.

Viral RNA electrophoresis technique was used to detect rotavirus in 226 children under 2 years of age with acute diarrhea, admitted to the Roberto del Río Hospital in Santiago, Chile, during the period of June 1979 through May 1980. A group of 50 children included in the aforementioned sample, admitted in winter, was compared with a control group of 25 infants without digestive pathology. In these groups, rotavirus was detected in 20 out of 50 children with diarrhea (40%) but not in the controls (0%). A positive diagnosis of rotavirus was found in 66 out of the total of 226 patients (29.2%); its monthly distribution ranged between a maximum of 83.3% (June) and a minimum of 11.1% (October).