RESUMEN

To evaluate the prevalence of enteric viruses and their possible association with diarrhea, 244 stool samples were collected from HIV-infected and uninfected patients with or without diarrhea (subgroups I-a, Ib, II-a, and II-b, respectively). Subjects were screened by polyacrylamide gel electrophoresis, latex agglutination, and enzyme immunoassays for rotaviruses, adenoviruses, picobirnaviruses, and astroviruses. Enteric viruses were found significantly more often in specimens from HIV patients (20%) than in specimens from uninfected HIV patients (0%) (p < 0.05). Picobirnavirus was detected in 14.63% of 82 HIV-infected patients with diarrhea, but it was detected neither in those without diarrhea (0%) (p < 0.05) nor in the groups of uninfected HIV subjects (0%) (p < 0.05). Nor could astrovirus (subgroups I-a [4.00%] versus subgroup I-b [5.26%],p > 0.05) or enteric adenovirus (subgroup I-a [1.22%] versus subgroup I-b [0%], p > 0.05) be linked to the diarrhea disorder in HIV-infected patients. Rotaviruses were not detected in any of the clinical subgroups studied. Enteric viruses were detected in 15 of 93 (16.13%) of the HIV-infected patients with CD4+ T cell count <200/microl and 3 of 19 (15.79%) of those HIV-infected individuals with a CD4+ T cell count 200-499/microl, showing no significant difference (p > 0.05). According to our data, unusual enteric viruses such as picobirnavirus, astrovirus, and enteric adenovirus occur in HIV-infected population in Córdoba, Argentina. However, only picobirnaviruses could be significantly associated with diarrhea in these patients.

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Diarrhea due to enteric pathogens is an important complication of advanced HIV infection. Picobirnaviruses are agents recently linked with human enteritis. In total, 197 fecal samples collected from HIV-infected and noninfected patients with and without diarrhea were investigated for the presence of rotavirus and picobirnavirus by polyacrylamide gel electrophoresis. Picobirnavirus was detected in 8.8% of 57 HIV-infected patients with diarrhea, but it was detected in neither those without diarrhea (p<.018) nor in the group of subjects uninfected with HIV (p<.022). All genomic electropherotypes of picobirnavirus strains had a wide pattern. Picobirnavirus genome segments varied in size between 2.4 and 2.7 and 1.6 and 1.9 kbp for the slow and fast migrating bands, respectively. Rotaviruses were not detected in any of the clinical groups studied. Two methods for the extraction of nucleic acid-phenol/chloroform and guanidinium thiocynate (GTC)/silica-were compared. Detection of picobirnavirus by polyacrylamide gel electrophoresis was 2.5 times more sensitive following guanidinium thiocynate RNA extraction. This investigation offers preliminary results about the circulation of picobirnavirus in HIV-infected patients in Córdoba, Argentina.

PIP: In 1988, a new group of viruses containing a bisegmented double-stranded RNA genome was described and named "picobirnavirus" (PBV). Viruses with similar properties have subsequently been found in fecal specimens collected from HIV-infected and noninfected patients with gastrointestinal symptoms in several countries. The present study used polyacrylamide gel electrophoresis (PAGE) to examine fecal specimens from 197 HIV infected and noninfected adults, with and without diarrhea, from Cordoba, Argentina, for rotavirus and PBV. PBVs were detected in the stools of 5 HIV-infected patients with diarrhea (8.8%), but in none of the other subgroups (HIV-positive patients without diarrhea, HIV-negative patients with diarrhea, HIV-negative patients without diarrhea). 3 of the 5 stool samples positive for PBV were also positive for intestinal parasites (mixed infection), but these parasites were found with equal frequency in HIV-infected patients without diarrhea. Rotaviruses were not detected in any of the subgroups. PBV genome segments varied in size between 2.4-2.7 and 1.6-1.9 kbp for the slow and fast migrating bands, respectively. PBV detection by the PAGE technique was 2.5 times more sensitive after guanidinium thiocyanate RNA extraction. Further research is required to determine the duration of excretion of PBVs in HIV-infected patients with diarrhea and understand the immune response to infection.

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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.

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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.

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Con el objeto de determinar el perfil local de infección por rotavirus humanos (RVH), se llevó a cabo en la ciudad de Córdoba, Argentina, entre enero de 1979-dic. 1989, un estudio retrospectivo de la circulación de RVH. Se analizaron un total de 1839 muestras de materias fecales, obtenidas de niños menores de 4 años de edad, con diagnóstico clínico de gastroenteritis aguda. El análisis de la frecuencia y circulación de rotavirus se realizó a través de la detección y seguimiento de los diferentes electroferotipos virales utilizando presencia de rotavirus grupo A (frecuencia global: 13,9 por ciento). El análisis del ácido nucléico viral permitió identificar 16 variedades de electroferotipos: 11 de tipo largo (82,2 por ciento) y 5 del tipo corto (18,8 por ciento). Entre las variedades detectadas, las predominantes fueron: L79-5 (31,4 por ciento), L84-8 (22,1 por ciento), S79-7 (11,9 por ciento) y S86-10 (4,4 por ciento), persistiendo en la comunidad entre 4 y 6 años. Anualmente se observó la circulación de más de una variante electroferotípica. Los resultados obtenidos proveen información sobre los diferentes electroferotipos de RVH que han circulado en la ciudad de Córdoba, la aparición secuencial de estos electroferotipos y la persistencia de ellos dentro de la comunidad. La sistematización en la clasificación de los electroferotipos permite, de ahora en más, determinar la continuidad de la circulación de variantes electroferotípicas, las recombinaciones de segmentos del genoma viral entre cepas circulantes y la identificación de fuentes primarias de infección (AU)

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Con el objeto de determinar el perfil local de infección por rotavirus humanos (RVH), se llevó a cabo en la ciudad de Córdoba, Argentina, entre enero de 1979-dic. 1989, un estudio retrospectivo de la circulación de RVH. Se analizaron un total de 1839 muestras de materias fecales, obtenidas de niños menores de 4 años de edad, con diagnóstico clínico de gastroenteritis aguda. El análisis de la frecuencia y circulación de rotavirus se realizó a través de la detección y seguimiento de los diferentes electroferotipos virales utilizando presencia de rotavirus grupo A (frecuencia global: 13,9 por ciento). El análisis del ácido nucléico viral permitió identificar 16 variedades de electroferotipos: 11 de tipo largo (82,2 por ciento) y 5 del tipo corto (18,8 por ciento). Entre las variedades detectadas, las predominantes fueron: L79-5 (31,4 por ciento), L84-8 (22,1 por ciento), S79-7 (11,9 por ciento) y S86-10 (4,4 por ciento), persistiendo en la comunidad entre 4 y 6 años. Anualmente se observó la circulación de más de una variante electroferotípica. Los resultados obtenidos proveen información sobre los diferentes electroferotipos de RVH que han circulado en la ciudad de Córdoba, la aparición secuencial de estos electroferotipos y la persistencia de ellos dentro de la comunidad. La sistematización en la clasificación de los electroferotipos permite, de ahora en más, determinar la continuidad de la circulación de variantes electroferotípicas, las recombinaciones de segmentos del genoma viral entre cepas circulantes y la identificación de fuentes primarias de infección

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A neutralization enzyme-linked immunosorbent (Nt-ELISA) assay for determination of protective immunity to measles virus was developed and evaluated. This procedure uses the same initial steps as performed to determine antibody titers by seroneutralization (Nt) test. However, a reduction in virus infectivity by neutralizing antibody was determined by quantitation of viral antigen using ELISA. The serum dilution that resulted in neutralization of 50% of infectious virus could be determined from the absorbance values. To be able to screen a large number of specimens, the conditions of the Nt-ELISA test were adjusted such that negative sera for measles antibodies and the positive ones were clearly distinguished on the basis of a single dilution (1:4). This test showed similar sensitivity (88.3%) and equal specificity as the Nt test when screening 136 serum samples from normal subjects. The estimation of protective antibody titers by Nt and Nt-ELISA methods was strongly correlated (correlation coefficient = 0.91). Thus, the measles Nt-ELISA test is rapid, reproducible, sensitive, and specific for detection of protective measles antibodies.

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A retrospective survey was carried out to determine the occurrence and circulation of different human rotavirus electropherotypes. Stool specimens were collected from children under four years old admitted to pediatric health services of Cordoba city, Argentina, from January 1979 to December 1989. Polyacrylamide gel electrophoresis followed by silver staining was employed for rotavirus detection. Of 1839 faecal samples 228 (13.9%) were positive for rotavirus. Examination of the electrophoretic patterns allowed us to identify 16 different electropherotypes (EPT), eleven with a long pattern (82.2%) and 5 with a short one (18.8%). The L 79-5 (31.4%), L 84-8 (22.1%), S 79-7 (11.9%) and S 86-10 (4.4%) were the predominant EPT, persisting between 4 and 6 years in the community. A cocirculation of more than one electropherotype was observed annually. The results provide information concerning the number of different electropherotypes of human rotavirus in the community, the sequential pattern of electropherotype appearance and the length of time for which an electropherotype may persist.

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A retrospective survey was carried out to determine the occurrence and circulation of different human rotavirus electropherotypes. Stool specimens were collected from children under four years old admitted to pediatric health services of Cordoba city, Argentina, from January 1979 to December 1989. Polyacrylamide gel electrophoresis followed by silver staining was employed for rotavirus detection. Of 1839 faecal samples 228 (13.9

) were positive for rotavirus. Examination of the electrophoretic patterns allowed us to identify 16 different electropherotypes (EPT), eleven with a long pattern (82.2

) and 5 with a short one (18.8

). The L 79-5 (31.4

), L 84-8 (22.1

), S 79-7 (11.9

) and S 86-10 (4.4

) were the predominant EPT, persisting between 4 and 6 years in the community. A cocirculation of more than one electropherotype was observed annually. The results provide information concerning the number of different electropherotypes of human rotavirus in the community, the sequential pattern of electropherotype appearance and the length of time for which an electropherotype may persist.

RESUMEN

Two rapid procedures modifying the polyacrylamide gel electrophoresis (PAGE) standard technique for the diagnosis of acute gastroenteritis caused by rotavirus infections are described. These assays permit the detection of both group A and non-group-A rotaviruses, and can be employed in most microbiology laboratories. The cost per sample is minimal compared with that of enzyme immune assays (ELISA) or the agglutination tests available commercially.

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Out of 317 human gastroenteritis cases studied between August 1988-August 1989, two atypical antigenically distinct rotaviruses (pararotaviruses) were detected in faecal samples among 19 rotaviruses shedding children from Misiones province, North-Eastern Argentina. A 1 3/4 year old girl a 3 years old boy, both with vomiting and normal temperature, shed these atypical rotaviruses. Their morphology by electron microscopy was identical to other rotaviruses; they contained 11 double-stranded RNA segments detected by polyacrylamide gel electrophoresis and failed to react with the antibody directed against the rotavirus group specific antigen (Rotazyme II ELISA). The electrophoretic migration of these RNAs (electropherotype) in polyacrylamide gels did not coincide with the typical pattern of distinct size classes observed in most human rotaviruses reported, instead, they appeared to be related to patterns of rotaviruses group C.

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This article presents the results obtained in the search for the hepatitis B antigen by the counter-immunoelectrophoresis technique in sera of more than 50.000 persons from Córdoba, Argentina. Groups of voluntary blood donors, general population, 20 years old army recruits, patients, with acute hepatitis and patients admitted to a hospital due to other diseases were included in this study. In the apparenly healthy population the positivity of the test ranged between 0.33 to 0.53 percent; the highest rate was found among the recruits. In the group of patients with acute hepatitis, 38.53% of the sera were positive. No statistically significant differences were observed in the monthly distribution of the hepatitis B antigen during the period of study. These results are compared with those found by other workers in different countries. This study shows the importance of the early detection of the hepatitis B antigen in blood donors, to reduce the incidence of hepatitis transmitted by transfusions. The importance of establishing centers for the standardization and control of reagents, as well as for the training of laboratory personnel is emphasized.

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This article presents the results obtained in the search for the hepatitis B antigen by the counter-immunoelectrophoresis technique in sera of more than 50.000 persons from Córdoba, Argentina. Groups of voluntary blood donors, general population, 20 years old army recruits, patients, with acute hepatitis and patients admitted to a hospital due to other diseases were included in this study. In the apparenly healthy population the positivity of the test ranged between 0.33 to 0.53 percent; the highest rate was found among the recruits. In the group of patients with acute hepatitis, 38.53

of the sera were positive. No statistically significant differences were observed in the monthly distribution of the hepatitis B antigen during the period of study. These results are compared with those found by other workers in different countries. This study shows the importance of the early detection of the hepatitis B antigen in blood donors, to reduce the incidence of hepatitis transmitted by transfusions. The importance of establishing centers for the standardization and control of reagents, as well as for the training of laboratory personnel is emphasized.

RESUMEN

This article presents the results obtained in the search for the hepatitis B antigen by the counter-immunoelectrophoresis technique in sera of more than 50.000 persons from Córdoba, Argentina. Groups of voluntary blood donors, general population, 20 years old army recruits, patients, with acute hepatitis and patients admitted to a hospital due to other diseases were included in this study. In the apparenly healthy population the positivity of the test ranged between 0.33 to 0.53 percent; the highest rate was found among the recruits. In the group of patients with acute hepatitis, 38.53

of the sera were positive. No statistically significant differences were observed in the monthly distribution of the hepatitis B antigen during the period of study. These results are compared with those found by other workers in different countries. This study shows the importance of the early detection of the hepatitis B antigen in blood donors, to reduce the incidence of hepatitis transmitted by transfusions. The importance of establishing centers for the standardization and control of reagents, as well as for the training of laboratory personnel is emphasized.