Serological Detection of Rotavirus Among Children with Diarrhea in Relation to Different Environmental Conditions

Rotaviruses are the single most important etiologic agents of severe diarrhea of infants and young children worldwide. In the present study, serological detection of rotavirus was done using an enzyme linked immunosorbent assay (ELISA), on 247 stool specimens. These were collected from children with acute diarrhea attending the outpatient clinic of Alexandria University Children's Hospital at El-Shatby, from October 2005 to April 2007. Rotavirus was detected in 33.6% of the collected samples; no specific age group or sex predilection was observed. It was presented with a marked seasonal peak during autumn and winter (58.3% and 40.5%, respectively). Rotavirus was found to be infecting most commonly under-weight children (46.9 %) resulting into fluid loss and severe dehydration (80%). Rotavirus acute gastroenteritis was found to be associated with fever (38.8%), vomiting (39.9%), watery stools, and long duration of diarrheal episodes lasting from one up to six days. The appearance of convulsions among rotavirus-positive cases even in the absence of fever (84.6%) was an important finding. Exclusive formula-fed infants appeared to exhibit the highest disease incidence (50%) while exclusive breast-fed infants had a lower incidence level (35.2%) of the disease. The virus was found to be significantly affecting children living in rural areas of Egypt (43.8%) rather than urban ones (26.1%). Environmental factors that were shown to affect the disease incidence include: the presence of impurities in water (41.6%), broken pipes (58.1%) and water tanks (58.7%) at the residence place. On the other hand, neither the kind of water source nor the presence of a sewage-disposal network was significantly related to the disease. Therefore, the study recommended to screen for rotavirus in children with diarrhea in order to avoid the use of unnecessary medications. In addition, encouragement of breast feeding practices and improvement of environmental conditions are important means of prevention of rotavirus infection.

Bull High Inst Public Health Vol.38 No. 3 [2008]   the world. (1)Data presented at the 6 th International Rotavirus Symposium in Mexico City, indicated that the annual number of deaths due to rotavirus infection may be as high as 608,000 deaths per year, 82% of which occur in developing countries. (2)The high disease burden motivated major efforts to develop a suitable rotavirus vaccine.However the vaccine efficacy is being challenged by the extensive strain diversity.This made rotavirus a prime target for global estimation of the prevalence and type distribution of the virus strains all over the world. (3,4)taviruses are classified as a genus in the family Reoviridae.The rotavirus genus currently has five species (Rotavirus A to Rotavirus E), with two possible additional species (Rotavirus F and Rotavirus G) based on serological identification of common antigens.Most human infections are caused by group A rotaviruses, although groups B and C rotaviruses have occasionally been associated with human illness. (5)taviruses are non enveloped, medium-sized viruses ranging from 60 to classifying rotaviruses into G (VP7) and P (VP4) serotypes. (6,7)e predominant mode of rotavirus transmission is fecal-oral.The infectious particles are shed in high concentrations in the stools of infected children up to one week after infection or for more than 30 days in immunocompromised patients. (8)read through respiratory secretions, person-to-person contact, or contaminated environmental surfaces has been also speculated. (9,10)nimal-to-human transmission does not appear to be common, although human rotavirus strains that possess a high degree of genetic homology with animal strains have been identified. (10)taviruses can survive for weeks in potable and recreational waters and for at least four hours on human hands.The viruses are relatively resistant to commonly used hard-surface disinfectants and hygienic hand-wash agents. (11)In addition, their massive excretion begins with the first day of diarrhea. (12)Although they are found in wastewater and can also be concentrated by shellfish; rotaviruses have not been linked with infectious disease following seafood consumption. (13)In case of improper wastewater management, these large numbers of viruses are usually carried to the surface and ground waters and consequently become a source of infection to those who are drinking, using and in contact with contaminated waters. (14)wage sludge is a complex mixture of consequently, viruses become environmental pollutants. (15)lids-associated viruses in wastewater effluents are discharged into aquatic environments and accumulate in the sediments where they persist longer than in the surface water. (16)As a matter of Bull High Inst Public Health Vol.38 No.3 [2008]   fact, sediments act as a reservoir from which viruses are re-suspended in the water by several natural or artificial phenomena. (17,18)cordingly, humans are exposed to enteric viruses through one or more of the various transmission routes such as eating of fresh vegetables grown in contaminated land, bathing in sewage-polluted recreational waters and drinking contaminated water. (

Aim of the work:
The aim of the present work was to study the occurrence of rotavirus infection as a cause of diarrhea among children under 5 years of age in relation to different environmental conditions.

Material and Methods:
The were also included in the questionnaire.In addition, the body weight and degree of dehydration of each case was clinically assessed. (19)ter taking the parents' consent, stool Significance was taken at 5% (0.05) level.
Values ≤ 0.05 were considered statistically significant. (20)sults and discussion: The World Health Organization (WHO) has stated that in considering routine immunization against rotavirus, countries will need sound epidemiological data to assess the burden of disease, to examine trends and seasonality, to evaluate the age patterns of incidence and to determine the serotypes of strains currently in circulation. (21)The present study was an results showed remarkable agreement with a study done by Radwan et al., (22) (23) This may be related to the difference in environmental conditions in each study.
Internationally, the same results were reported in a surveillance study done in Venezuela over five years period (1998-2002), where 33% of the stool specimens collected from children admitted to hospital with severe diarrhea were positive for rotavirus. (24)Another surveillance system done during the same period (1998-2002)   in Spain by Sànchez-Fauquier et al. (25) found that rotavirus was responsible for 31% of acute diarrhea in children less than 5 years old.Moreover, in 2002 Coluchi et al., (26)  A marked seasonal peak during the cold seasons of the year (autumn and winter), was also observed in England, (9) Finland, (28) Spain (25) and Tunisia. (29)This was also similar to a previous cohort study conducted in Bilbeis (Egypt), where the rate of rotavirus isolation predominated in the colder months (November -April). (30)wever, the seasonal peak of rotavirus infection in Egypt tends to shift over consecutive years. (22)In a previous population-based cohort study of children less than 3 years of age residing in Abu Homos in 1995 till 1996, rotavirus infection predominated during the warmer months (July -November), with a peak incidence in August. (31)oreover, rotavirus predominated from August to December during the period of 1992-1993 as published by Radwan et al. (22) These variations in the timing of the peak rotavirus activity in Egypt have been also reported in other settings all over the world.It recalls the patterns observed in less developed countries with a tropical climate, in which seasonality of rotavirus infection is unclear or nonexistent. (32,33) table (4), there was a significant association between rotavirus infection and body weight of affected children (P=0.027).
Bull High Inst Public Health Vol.38 No. 3 [2008]   Rotavirus was found to be infecting most commonly under-weight children (46.9%), while it was found in 30.3% of normalweight children.
As regards the duration of diarrhea,  (34) It is worth notice that in the present study, eleven of the rotavirus-infected children suffered from convulsions that were not associated with fever.A remark consistent with several some case reports that found evidence of rotavirus RNA by PCR technique in the cerebrospinal fluid of rotavirus-infected children who had seizures (35) and in liver and kidney sections of immunocompromised children. (36)other study used RT-PCR, immunohistochemistry, and in situ hybridization to detect rotavirus in a variety of internal organs of two children who died with severe rotavirus-associated diarrhea and who also had neurological disease. (37)e elegant study used an EIA test designed to detect rotavirus antigen in stool samples but applied it to serum and found that 22 out of 33 immunocompetent children with confirmed rotavirus gastroenteritis had rotavirus antigenemia, providing evidence that rotavirus may commonly escape the gastrointestinal tract.
The clinical significance of these findings remains unclear but is under active investigation. (38) table (7), the highest percent of rotavirus positive cases was found among infants who were exclusively formula-fed, while those who were exclusively breastfed showed a lower incidence level (50% Vs 35.2%).Infants who received both breast and formula feeding had the least percent of positive cases (22.6%) and those who received neither (children more than 2 years old) showed 25.9% of rotavirus positivity.However, this difference did not reach a statistically significant level. (P=0.062) Our results were coincident with previous studies done in Egypt (39) and Bangladesh (40) ; breast feeding was associated with a lower incidence of rotavirus diarrheal episodes, which add to the multitude of benefits that have been associated with breast feeding.Moreover, it was reported that early initiation of breastfeeding was associated with a marked reduction of the rate of diarrhea throughout the first 6 months of life, possibly because of the beneficial effects of human colostrum. (39)ble (8) shows that rotavirus infection was found to be significantly affecting  (30) in 1986.
Another study done in Pakistan in 1992 reported that most of the positive cases were collected from those living in remote rural areas of the country, where increased family size and over crowding were common factors. (42)These are significant risk factors for the mode of transmission of rotavirus due to environmental contamination and contact with the hands of infected persons. (43)e role of water in the transmission of viruses is well established.However, the epidemiological evidence of water-borne transmission of human viruses is limited to only few including rotavirus. (44)The work Knowledge of rotavirus prevalence and strains circulating in our community will aid in assessing the suitability of candidate vaccines, in order to protect against all currently circulating rotavirus strains.

Conclusion and Recommendations:
The present study confirms the huge burden of rotavirus as a major cause of 80nm in diameter with an icosahedral symmetry.They are composed of 11 double-stranded linear RNA segments surrounded by three concentric spherical protein coats.The inner coat, made of VP2, encloses the genomic RNA and another two minor proteins, VP1 and VP3.The middle layer of the mature viral particle is composed of VP6 polypeptides, which determine group specificity.The outer capsid of rotaviruses is made up of the VP7 glycoprotein and the VP4 hemagglutinin proteins.Serotype specificity is determined by the outer capsid proteins VP4 and VP7, both of which independently induce neutralizing antibodies, thus solids removed from wastewater in sewage treatment plants.It is the most hazardous by-product in wastewater treatment facilities.The type of treatment determines the concentration of pathogens and the relative risks associated with the produced sludge.Viruses are present in high numbers in raw wastewater and current water treatment practices fail to ensure the complete removal of viral pathogens; samples were taken from each child in a clean container.The samples were transferred on the same day to the laboratory where they were stored at -20°C until being tested for the presence of rotavirus using the RIDASCREEN® Rotavirus (C 0901) ELISA kit.Test kit was supplied by R-Biopharm AG (Landwehrstr.54, D-64293 Darmstadt, Germany).It is an in vitro diagnostic test developed for the qualitative determination of rotaviruses in stool samples using monoclonal antibodies against a capsid protein of gene 6 (VP6). (123)The manufacturer's instructions were closely adhered to in performing the test.Computerized tabulation was done using SPSS statistical program.The test used was paired sample -T test.
attempt to participate in the research efforts done for estimation of the occurrence of rotavirus diarrhea among children in Egypt.In addition the study examined the incidence of rotavirus diarrhea throughout the four seasons of the year, the clinical features of the disease, and the relation between breast feeding, the place of residence as well as different environmental conditions regarding the presence or absence of the infection.Rotavirus was identified in 83 (33.6%) of 247 stool specimens collected from children with acute diarrhea who attended the outpatient clinic in Alexandria University Children's Hospital at El-Shatby, over a period of 18 months starting from October 2005 till April 2007.The obtained Bull High Inst Public Health Vol.38 No.3 [2008] children living in rural areas (43.8%,P=0.003) rather than those who are living in urban areas (26.1%).Poor sanitation mainly means absence of integrated wastewater and solid waste management systems and absence of sanitary drinking water supply, which are common in Egyptian rural areas.In around 95% of Egyptian villages, wastewaters and solid wastes find their way to small canals and drains which are used in irrigation of fresh vegetables and other crops. (41)Eating of fresh vegetables which are grown in contaminated land irrigated by contaminated water without proper washing or even washed by contaminated water could be a possible source of infection with rotavirus.Also, farmers who are being in contact with contaminated land and water during day time, using the same source of water to get washed at the end of the day, then getting in contact with their children in the evening is one point to be kept in mind when considering possible sources of rotavirus infection.Table (9) demonstrates a statistically significant association between rotavirus infection and the existence of broken drinking water supply pipes (58.1%,P=0.000), presence of water storage tanks (58.7%, P=0.000) and presence of impurities in water at the residence place (41.6%, P=0.001).On the other hand, the source of water (P=0.451)and the means of sewage disposal (P=0.295) were not found to be statistically significant as regards the presence or absence of rotavirus.Contaminated drinking water is one of the most important sources of infection with rotavirus.The main causes of drinking water contamination are contaminated raw water sources (surface or ground water) the existence of broken pipes in drinking water distribution network leads to negative pressures and consequently entry of contaminants and impurities into the drinking water networks.The presence of impurities in drinking water may act as a good media for the growth of different microorganisms especially in the absence of residual disinfectant (chlorine gas) which normally occurs at the end of drinking water networks (usually in rural areas).from water-tanks and the existence of broken pipes at the residence place, are findings that fully agree with the results documented by Zaki et al., done in Pakistan was designed to examine whether or not rotaviruses were present in the influents and to evaluate the virological quality of water as delivered to the consumers in different localities of Karachi.The results showed the presence of rotavirus in 60% influent samples.It was Bull High Inst Public Health Vol.38 No.3 [2008]   also found that some of the tap-water samples collected had shown the presence of fecal coliforms, suggesting that the water source was also polluted due to either the intermittent water supply or to a leakage in the distribution pipelines or in the nearby sewage lines.Because of these factors, drinking water must be regarded as having a very significant potential as a vehicle for the transmission of rotaviruses.Therefore, the most reasonable approach for controlling the transmission of viruses through water is to recommend additional parameters to be added to the safety criteria of drinking water.(42)Nevertheless, we could not adequately account for several factors that might have affected our findings.First, the study was conducted in a selective population that may not have been representative of the entire country.Second, in our pilot study, the vast majority of the patients refused to give any data concerning their socioeconomic status (such as monthly income, number of rooms and number of people living in the house…etc.)Finally, the results of the present work underline the characterization of less common and unusual strains.
acute diarrhoea in Egyptian infants and young children.The disease was predominant in autumn and winter and was characterized by vomiting, watery diarrhea and severe dehydration.Breast-fed infants were more protected against the disease than those who were exclusively formula-fed.Residence in rural areas of the country and the quality of drinking water had an effect on the occurrence of the disease.Accordingly, we recommend rotavirus screening in children with diarrhea in order to avoid the use of unnecessary medications.In addition, encouragement of breast feeding practices, as well as, improvement of environmental conditions is recommended as means of prevention of rotavirus infection.Finally, the presence of convulsions among rotavirus-positive cases was an important finding and requires further investigation.