Construction of an internal RT-PCR standard control for the detection of human caliciviruses in stool.

RT-PCR is the most sensitive assay for the detection of human caliciviruses (HuCV) in stool and environmental samples. However, false negative results are commonly obtained due to the presence of RT-PCR inhibitors. In order to exclude such false negative results, an internal control (IC) was developed for the assay by cloning a 319 nt sequence of the Norwalk virus (NV) polymerase containing a 156 nt cDNA insert. The RT-PCR assay was carried out using RNA derived from the constructed plasmid and a primer set previously described for calicivirus detection, resulting in a 475 nt product. Distinct bands of the internal control and the viral specific RT-PCR products (319 nt) were obtained when the internal control was added to the samples. Similar results were also obtained when both the control RNA and viral RNA were seeded into stool samples from asymptomatic volunteers, or when the internal control was included into positive samples. Since the primer set used in the assays can detect a wide range of strains in both norovirus and sapovirus genera, this internal control should have a broad application for the diagnosis of human caliciviruses diagnosis in both clinical and environmental samples.

Prevalence and genetic diversity of human caliciviruses (HuCVs) in Mexican children.

Human caliciviruses (HuCVs) contain two genera: "Norwalk-like viruses" (NLVs) and "Sapporo-like viruses" (SLVs). The importance of the two genera as a cause of acute gastroenteritis of infants and children remains unknown. Beginning in 1989, a birth cohort of children in Mexico was enrolled and monitored for acute gastroenteritis. A subset of 115 diarrhea stool specimens from 76 children and 66 non-diarrhea stool specimens from 64 children was examined for HuCVs by RT-PCR by using a primer pair (p289/290) that detects both NLVs and SLVs. Twenty-two (19%) of the 115 diarrhea stool specimens and 5 (7%) of 66 non-diarrhea stool specimens produced RT-PCR products of expected size (319 bp for NLVs and 331 bp for SLVs). Twenty of the twenty-seven strains were cloned and sequenced. Pairwise sequence analysis showed that 9 (60%) and 6 (40%) of the 15 strains from the diarrhea stools were NLVs and SLVs, respectively. The same proportions of NLVs (60%) and SLVs (40%) were observed in the non-diarrhea stools. Strains in the NLV genus could be further divided into four clusters: Lordsdale, MxV, and HV and one potentially new cluster. Strains in the SLV genus could be divided into three clusters: Sapporo/82, Lon/92, and a potentially new cluster. Strains from the Lordsdale cluster were the most common among these children. The findings of both genera and multiple clusters of HuCVs co-circulating and the identification of new strains of HuCVs in the population justify the need for future studies of HuCVs in infants and children.

[Detection of Norwalk and Mexico viruses, two human Caliciviruses in stools of Chilean children]. / Detección de virus Norwalk y México, dos calicivirus humanos en deposiciones de niños chilenos.

BACKGROUND: Human calciviruses (HuCVs) cause diarrhea outbreaks associated with consumption of contaminated food and water. Seroepidemiological studies in developing countries, suggest that HuCVs can cause acute gastroenteritis in children. AIM: To study the presence of Norwalk (NV) and Mexico (MX) virus, two HuCVs, in stools of Chilean children from different settings. SUBJECTS AND METHODS: ELISA tests for NV and MX were performed in 677 stool samples for children aged 0 to 132 years old, with acute diarrhea occurring in day care centers or consulting in outpatient clinics or emergency rooms. We also studied eight samples from children involved in a diarrhea outbreak that occurred in a rural community in 1992. A subset of samples was tested with polymerase chain reactions using different primers. RESULTS: Only one sample from a child with acute diarrhea occurring in a day care center was positive for HuCV by polymerase chain reaction. Three samples from the outbreak were positive by the latter method and by ELISA. The HuCV obtained from the day care center was genetically different from other known HuCV. CONCLUSIONS: Despite the high seroprevalence, NV and MX viruses were detected in a very low proportion of Chilean children stools.

Detección de virus Norwalk y México, dos calicivirus humanos en deposiciones de niños chilenos / Detection of Norwalk and Mexico virus, 2 human calciviruses in feces of chilean children

Background: Human calciviruses (HuCVs) cause diarrhea outbreaks associated with consumption of contaminated food and water. Seroepidemiological studies in developing countries, suggest that HuCVs can cause acute gastroenteritis in children. Aim: To study the presence of Norwalk (NV) and Mexico (MX) virus, two HuCVs, in stools of Chilean children from different settings. Subjects and methods: ELISA tests for NV and MX were performed in 677 stool samples for children aged 0 to 132 years old, with acute diarrhea occurring in day care centers or consulting in outpatient clinics or emergency rooms. We also studied eight samples from children involved in a diarrhea outbreak that occurred in a rural community in 1992. A subset of samples was tested with polymerase chain reactions using different primers. Results: Only one sample from a child with acute diarrhea occurring in a day care center was positive for HuCV by polymerase chain reaction. Three samples from the outbreak were positive by the latter method and by ELISA. The HuCV obtained from the day care center was genetically different from other known HuCV. Conclusions: Despite the high seroprevalence, NV and MX viruses were detected in a very low proportion of Chilean children stools

Evolución temporal del patrón de aislamiento de enteropatógenos en diarrea aguda / Temporal evolution of patterns isolation of enteropathogens in acute diarrhoea

Se revisan los datos existentes en el Hospital Infantil de México "Federico Gómez" en relación con los agentes bacterianos causantes de diarreas agudas de pacientes que acuden al Servicio de Hidratación Oral, considerando los más importantes: Shigella, Salmonella, Escherichia coli enteropatógena y Campylobacter. Se comparan estos datos con los que fueron obtenidos entre 1993 y 1997 en el mismo servicio hospitalario. Se estudia la relación de Shigella flexneri con S. sonnei constatando el creciente predominio de S. sonnei. Se reporta la resistencia a antibióticos (ampicilina y bactrim) de Shigella desde 1960, comparando estos hallazgos con los resultados obtenidos en los últimos años. El aumento de cepas resistentes es evidente (AU)

Evolución temporal del patrón de aislamiento de enteropatógenos en diarrea aguda / Temporal evolution of pattern's isolation of enteropathogens in acute diarrhoea

Se revisan los datos existentes en el Hospital Infantil de México "Federico Gómez" en relación con los agentes bacterianos causantes de diarreas agudas de pacientes que acuden al Servicio de Hidratación Oral, considerando los más importantes: Shigella, Salmonella, Escherichia coli enteropatógena y Campylobacter. Se comparan estos datos con los que fueron obtenidos entre 1993 y 1997 en el mismo servicio hospitalario. Se estudia la relación de Shigella flexneri con S. sonnei constatando el creciente predominio de S. sonnei. Se reporta la resistencia a antibióticos (ampicilina y bactrim) de Shigella desde 1960, comparando estos hallazgos con los resultados obtenidos en los últimos años. El aumento de cepas resistentes es evidente

Prevalence of enteric viruses in human immunodeficiency virus seropositive patients in Venezuela.

The prevalence of enteric viruses associated with gastroenteritis was determined in 125 stool samples from patients infected with the human immunodeficiency virus (HIV), with or without diarrhea. Diagnostic assays included enzyme immunoassays for the identification of rotavirus, adenovirus, and Norwalk virus; polyacrylamide gel electrophoresis for atypical rotaviruses and picobirnaviruses and polymerase chain reaction for astrovirus. Enteric viruses were detected in 6.4% (8 of 125) of the stools collected: five (4.0%) samples positive for adenoviruses, and three (2.3%) samples positive for picobirnaviruses were detected. No rotavirus, astrovirus, or Norwalk virus were observed. Only one of the viruses identified (adenovirus) was found in a sample from a patient with diarrhea. Viruses were detected in 10% of the patients with AIDS, 14% of the symptomatic patients, and none of the asymptomatic persons. These results do not support a major role for enteric viruses in the diarrhea suffered by HIV-infected patients.

Norwalk virus infection in Venezuela.

The presence of antibodies against Norwalk virus (NV) was studied in sera from different Venezuelan populations, using an enzyme immuno-assay (EIA) based on recombinant NV protein. Antibodies to NV were found in 47%-53% of urban subjects from Caracas, 83% of rural subjects from the west of the country, and 73%-93% of Amerindian subjects. The prevalences found in the rural and Amerindian groups were significantly higher than that in the urban group. Although about 50% of the children studied were seropositive for NV by the age of 5 years, only four (0.4%) of 1120 faecal samples from children with diarrhoea which were tested for the presence of NV antigen by sandwich EIA were found positive. An increase of at least 4-fold in the titre of anti-NV IgA was found in three (5%) of 61 pairs of sera taken during and 1 month after an acute episode of diarrhoea not due to rotavirus. NV was therefore not a predominant aetiological cause of gastro-enteritis in young children in Venezuela between 1993 and 1995, although it can be the cause of diarrhoea in infants.

Contaminación de los alimentos por virus: un problema de salud pública poco comprendido / Viral contamination of food products: a poorly understood public health problem

En todas partes del mundo han surgido epidemias de enfermedades transmitidas por los alimentos (ETA) sobre las que no existe suficiente información para guiar las acciones de las instituciones de salud pública. El presente estudio se hizo con objeto de contribuir a la diseminación de información sobre esas enfermedades, sus agentes etiológicos y su epidemiología y control. Se utilizaron datos de 61 estudios, entre ellos revisiones, descripciones de brotes y sistematización de datos. De los resultados obtenidos se pudo concluir que hay un gran problema de subregistro y falta de datos sobre estas enfermedades en los diversos países, pero los virus constituyen la segunda causa más importante de ETA en los Estados Unidos de América. Dos agentes, el virus Norwalk y el de la hepatitis A, ocuparon el quinto y sexto lugares, respectivamente, entre las causas principales de ETA, aunque el primero ocupó el primer puesto en 1982 y el segundo lugar como causa principal de enfermedades de transmisión hídrica durante el período de 1986 a 1988. A pesar de la escasez de datos al respecto, los rotavirus, poliovirus, virus de la hepatitis E, astrovirus y pequeños virus gastroentéricos también tienen importancia como agentes de ETA. En el artículo se discute también la importancia de las zoonosis víricas, especialmente de las fiebres hemorrágicas transmitidas por excretas de roedores y las encefalitis víricas transmitidas por garrapatas (fiebre difásica de la leche). Asimismo se presenta la polémica sobre la enfermedad de las vacas locas y su posible transmisión por los alimentos, además de los cuidados alimentarios relacionados con el sida y otras infecciones víricas. Por último, se describen los procedimientos de prevención y control de las ETA víricas

Throughout the world there have been several epidemics of food-borne diseases (FBD) about which there is lack of sufficient information for public health institutions to take appropriate measures. This study was conducted for the purpose of contributing to the dissemination of information on these diseases and their etiologic agents, epidemiology, and control. The study was based on data from 61 sources, including review articles, reports of outbreaks, and databases. Results reveal considerable underregistration and lack of data on FBD throughout the various countries, with viruses being the second most important cause of FBD in the United States of America. Two agents, Norwalk virus and hepatitis A virus, were the fifth and sixth most frequent causes, respectively, although the former was the single most frequent cause of FBD in 1982 and the second most frequent cause of water-borne diseases during the period from 1986 to 1988. Despite the scarcity of information on the problem, rotavirus, poliovirus, hepatitis E virus, astrovirus, and small gastroenteric viruses are also important causes of FBD. We also discuss the importance of viral zoonoses, especially hemorrhagic fevers transmitted by contact with rodent feces and tick-borne viral encephalitides (Lassa fever). There is discussion of the controversial mad cow disease and its potential transmission through food products, as well as of dietary aspects of the management of AIDS and other viral infections. Finally, measures for the prevention and control of FBD are described.

Study of Norwalk virus and Mexico virus infections at Ga-Rankuwa Hospital, Ga-Rankuwa, South Africa.

Small round-structured viruses (SRSVs) or Norwalk-like viruses have been implicated as an important causative agent of gastroenteritis outbreaks. We used the relatively newly developed recombinant enzyme immunoassays (EIAs) to determine the seroprevalence of Norwalk virus (NV) and Mexico virus (MxV) in a family-based cohort and an antenatal clinic cohort at Ga-Rankuwa Hospital, Ga-Rankuwa, South Africa. High prevalences (96 to 99%) of anti-NV and anti-MxV antibodies were detected in both cohorts. We also investigated the pattern of antibody acquisition in a cohort of infants and young children without gastroenteritis and found that by 48 months of age all children had acquired adult antibody levels to both these viruses. Lastly, we tested 276 stool specimens collected from infants and young children with gastroenteritis for the presence of NV or MxV antigen by recombinant EIAs to each virus, by electron microscopy (EM), and by reverse transcription (RT)-PCR. NV and MxV antigens were present in 1.8 and 4.3% of the stool specimens, respectively, by the recombinant EIAs; 9.2% were positive for SRSVs by EM, and 25% of these SRSVs gave a positive result by RT-PCR for primer pair 35-36 directed to a region of the RNA-dependent RNA polymerase gene. The seroprevalence studies indicate a high level of exposure to these viruses in both children and adults. Although the viral antigens are not highly prevalent in diarrheal stools, it was determined by the two assays for NV and MxV that children are, nevertheless, infected early in life.

Seroprevalencia y posibles factores de riesgo para virus norwalk en Santiago de Chile / Serological prevalence and possible risk factors for norwalk virus in Santiago, Chile

El virus Norwalk ha sido reconocido como un agente frecuente de diarrea aguda en países desarrollados, especialmente como causal de brotes en adultos. La información disponible en países en desarrollo y en poblaciones pediátricas es reducida. Nuestro objetivo fue evaluar la seroprevalencia para virus Norwalk en habitantes de Santiago, e identificar posibles factores de riesgo. Se evaluaron 291 sueros obtenidos entre los años 1990-1991 de individuos menores de 21 años, estratificados por nivel socioeconómico y edad. Para cada individuo se completó un cuestionario relacionado con variables demográficas, ambientales, de hábitos recreativos y de consumo de alimentos. Los sueros fueron evaluados para anticuerpos anti-virus Norwalk mediante test de ELISA basado en partículas virales recombinantes sintéticas. La seroprevalencia global para virus Norwalk fue de 83 por ciento y el nivel de seroprevalencia fue inversamente proporcional al estrato socioeconómico evaluado (p < 0,001). La seroprevalencia aumentó con la edad (p < 0,001) observándose un aumento significativo de seroprevalencia entre los 3 (47 por ciento) y 4 años (82 por ciento) de edad (p = 0,04). La seroprevalencia fue mayor en: individuos del sexo femenino (p = 0,006) aquellos con antecedente de baño en fuentes de agua dulce (p = 0,048), aquellos que presentaban antecedentes de consumo de mariscos crudos (p = 0,0001), vegetales no cocidos (p < 0,0001), y en aquellos niños con madres con menor nivel de educación (p = 0,009). En Santiago la seroprevalencia para virus Norwalk es alta, la adquisicón de anticuerpos ocurre mayoritariamente antes de los 5 años de edad y está fuertemente asociada al nivel socioeconómico al que pertenece el individuo. El consumo de mariscos y vegetales crudos así como el contacto con fuentes de agua potencialmente contaminadas parecen ser factores significativos de riesgo para esta infección. Estos resultados deben orientar el diseño de nuevos estudios prospectivos destinados a identificar la presencia de virus en individuos pertenecientes a grupos de mayor riesgo

[Rotavirus, adenovirus, astrovirus, calicivirus and small round virus particles in feces of children with and without acute diarrhea, from 1987 to 1988, in the greater São Paulo]. / Rotavírus, adenovírus, astrovírus, calicivírus e "small round virus particles" em fezes de crianças, com e sem diarréia aguda, no período de 1987 a 1988, na grande São Paulo.

Between 1987 and 1988, 193 faecal specimens from children, with or without diarrhea, were submitted to enzyme immunoassay, polyacrylamide-gel electrophoresis and electronmicroscopy tests for virus detection. The positivity for Rotavirus, Adenovirus, Astrovirus, Calicivirus and Small Round Virus Particles (SRVP) was 11.3%, 3.1%, 2.1%, 1.0% and 4.1%, respectively, for the 97 children with acute diarrhea. Of the 96 children without diarrhea, 4.2% were positive for Rotavirus, 1.0% for Calicivirus and 7.3% for SRVP. Of 15 positive specimens for Rotavirus, 14 showed electrophoretic patterns proper to group A and 1 specimen of group C Rotavirus. The analysis of electrophorotypes demonstrated great heterogeneity of electrophoretic patterns and predominance of subgroup 2, "long". The association of virus, bacteria and parasites was present both in children with or without acute diarrhea.

Etiology of acute diarrhea among United States military personnel deployed to South America and west Africa.

A study of acute diarrhea was conducted from 1985 to 1987 among U.S. military personnel participating in routine shipboard exercises in South America and West Africa and ground troops deployed to coastal Ecuador. An enteropathogen was identified in 146 (51%) of 289 acute cases of diarrhea. Enterotoxigenic Escherichia coli, found in 50 (17%) patients with diarrhea, was the most commonly identified enteropathogen. Viral enteropathogens were also found in a high percentage of acute cases of diarrhea: rotavirus was detected in 11% of the patients and Norwalk virus infection in 10%. Most enteric pathogens were acquired in equal frequencies in South America and West Africa, except for rotavirus infection which was identified more often in West Africa and enteroaggregative E. coli infection which was identified more often in South America. Bacterial enteropathogens were frequently resistant to trimethoprim/sulfamethoxazole, but no resistance to quinolone drugs was observed, indicating that quinolone drugs have become important agents for the treatment of diarrhea in South America and West Africa.

Travelers' diarrhea in panamanian tourists in Mexico.

To determine whether residents of developing countries are unlikely to acquire travelers' diarrhea, 64 Panamanians of widely divergent socioeconomic strata were studied during a 15-day tour through Mexico. Twenty-three (36%) tourists experienced 27 episodes of travelers' diarrhea that were caused by seven different pathogens. The most commonly identified etiologic agents were rotavirus (26%), Norwalk virus (15%), and Campylobacter fetus (11%), whereas enterotoxigenic Escherichia coli was not frequently associated with travelers' diarrhea. Acquisition of travelers' diarrhea was correlated directly with high socioeconomic status. Varying levels of immunity to enteropathogens that are endemic in Panama may explain the different isolation rates of pathogens.

Nuevas luces acerca de una antigua enfermedad / New lights for an old disease

La gastroenteritis viral, mas correctamente denominada enteritis o enteropatia aguda es causa comun de enfermedad en el hombre. En los Estados Unidos es usualmente insignificante desde el punto de vista de severidad duracion y cuidado medico pero, por el contrario, en los paises en via de desarrollo es un problema importante y significativo . Se estima que es responsable de mas de 500 millones de episodios de diarrea y de 5 a 18 millones de muertes anuales. Un numero considerable de virus ha sido implicado como agentes etiologicos de la gastroenteritis. Su identificacion y caracterizacion adecuada y un claro entendimiento de la epidemiologia son indispensables para encontrar medidas preventivas, especialmente para desarrollar vacunas efectivas y disponibles para la comunidad. Teniendo en mente estas presento a discusion los mas importantes virus asociados con gastroenteritis, separandolos en tres grupos fundamentales : a. El agente Norwalk y sus relacionados; b. Los Rotavirus del hombre y de los animales y, c. Otros virus implicados como probable causa de diarrea en el hombre