Investigación de blastocystis spp, giardia spp y cryptosporidium spp en aguas de consumo en una comunidad de Caracas- Venezuela: Reporte preliminar / Surveillance of blastocystis spp, giardia spp and cryptosporidium spp in human consumption water in a community of Caracas- Venezuela

Uno de los mecanismos de transmisión de protozoarios intestinales, es el consumo de agua contaminada con quistes y ooquistes, cuya eliminación por cloración o filtración no resulta eficaz. En una comunidad de Caracas, se evaluó la posible contaminación del agua de consumo, con Blastocystis spp, Giardia spp y Cryptosporidium spp. El sedimento obtenido mediante filtración y separación inmunomagnética de 15 muestras de agua, se examinó microscópicamente al fresco, con coloraciones, inmunofluorescencia y cultivo en medio de Boeck- Drbohlav modificado (BDM). Se recopiló información sobre las condiciones de suministro, almacenamiento y consumo del agua, además del procedimiento utilizado para el lavado de frutas y vegetales. El único parásito observado fue Blastocystis spp (60%), mediante examen directo/cultivo (33%). Se observó un mayor consumo de agua filtrada que hervida (p= 0,001). Predominó el uso del agua de chorro para el lavado de vegetales y frutas, más que con agua y vinagre (p= 0,011). Se observó una mayor proporción de averías en los sistemas de recolección de aguas servidas (78,6%), más que en los sistemas de aguas blancas (28,6%, p= 0,011). El hallazgo de Blastocystis spp en el agua, se correlaciona con la prevalencia del parásito en habitantes de este sector. Destaca el papel del agua en la transmisión de Blastocystis spp, por lo cual se recomienda filtrarla y hervirla para prevenir la infección con este parásito.

Many intestinal protozoa are transmitted by contaminated water with cysts and oocysts and methods for their elimination as filtration or chlorination are not completely effective. We evaluated a possible consumption water contamination with Blastocystis spp, Giardia spp and Cryptosporidium spp in a community located in Caracas, Venezuela. The pellet obtained by immunomagnetic separation filtration of 15 water samples were examined by microscopic observation (direct examination), stain techniques, immunofluorescence and culture in Drbohlav Boeck-modified medium (BDM). We also collected information about consuming habits, water supply, storage and washing procedures of vegetables and fruits at assessed homes. The only parasite detected was Blastocystis spp (60%), by direct examination/culture (33%). A higher consumption of boiled filtered water (p = 0.001) was observed. The use of tub water for washing vegetables and fruits was predominant, instead of using water and vinegar (p = 0.011). We observed a higher proportion of nonfunctioning sewage collection (78.6%), rather than white water systems (28.6%, p = 0.011). Finding Blastocystis spp in water samples correlates with prevalence of this parasite in residents of this sector. The role of water in Blastocystis spp transmission is significant, so we recommend filtering and boiling it to prevent infection with this parasite.

In vivo killing of Giardia trophozoites harbouring bacterial endosymbionts by intestinal Paneth cells: an ultrastructural study.

To date Paneth cells have not previously been reported to kill Giardia trophozoites and other protozoa in vivo. Here we report the first evidence for in vivo killing of Giardia trophozoites by intestinal Paneth cells. Transmission electron microscopic (TEM) examination of duodenal specimens taken from naturally infected mice revealed that only Giardia trophozoites harbouring peripheral bacterial endosymbionts were destroyed and lysed in the vicinity of the activated Paneth cells. Additionally, intestinal epithelium was more affected by Giardia harbouring bacterial endosymbionts than Giardia with no endosymbionts. Our findings imply that the bacterial endosymbionts within Giardia trophozoites have a role in both host protective and pathological mechanisms, probably through altering the trophozoite antigencity. These observations might shed light on the diversity in infectivity and host specificity of Giardia species.

Diarrea aguda: tratamiento nutricional / Acute diarrhea: nutrtional treatment

El tratamiento de la diarrea aguda consistirá básicamente en rehidratación oral si existiera deshidratación, realimentación precoz y excepcionalmente farmacológico. La rehidratación debe durar 4-6 horas, que se prolonga a 8-12 horas si la deshidratación es hipernatrémica, pasando posteriormente a la fase de mantenimiento. Las soluciones de rehidratación oral son las recomendadas, usándose en países en vías de desarrollo la solución de la OMS por las pérdidas importantes de sodio en las heces y soluciones con menor contenido de sodio en los países industrializados al ser las pérdidas de sodio menores. La realimentación debe ser lo mas precoz y equilibrada posible, recoméndandose la lactancia materna si es la forma de alimentación o la fórmula sin diluir si realiza lactancia artificial. No es aconsejable sistemáticamente las fórmulas sin lactosa. El uso de probióticos mejora el cuadro. No se precisa tratamiento farmacológico y los antibióticos sólo están indicados en pacientes inmunodeprimidos, cólera, lactantes menores de 3 meses con coprocultivos bacterianos positivos, enfermedad sistémica, infección por amebas, giardias, clostridium difficile y shigella que permanece sintomática (AU)

RNA dependent RNA polymerase activity associated with the double-stranded RNA virus of Giardia lamblia.

Giardia lamblia, a parasitic protozoan, can contain a double-stranded RNA (dsRNA) virus, GLV (1). We have identified an RNA polymerase activity present specifically in cultures of GLV infected cells. This RNA polymerase activity is present in crude whole cell lysates as well as in lysates from GLV particles purified from the culture medium. The RNA polymerase has many characteristics common to other RNA polymerases (e.g. it requires divalent cations and all four ribonucleoside triphosphates), yet it is not inhibited by RNA polymerase inhibitors such as alpha-amanitin or rifampicin. The RNA polymerase activity synthesizes RNAs corresponding to one strand of the GLV genome, although under the present experimental conditions, the RNA products of the reaction are not full length viral RNAs. The in vitro products of the RNA polymerase reaction co-sediment through sucrose gradients with viral particles; and purified GLV viral particles have RNA polymerase activity. The RNA polymerase activities within and outside of infected cells closely parallel the amount of virus present during the course of viral infection. The similarities between the RNA polymerase of GLV and the polymerase associated with the dsRNA virus system of yeast are discussed.

A single-stranded RNA copy of the Giardia lamblia virus double-stranded RNA genome is present in the infected Giardia lamblia.

An isolate of Giardia lamblia infected with the double-stranded RNA virus (GLV) has two major species of RNA that are not present in an uninfected isolate. One of these species is the previously characterized double-stranded RNA genome of GLV (1). The second species of RNA appears to be a full length copy of one strand of the double-stranded RNA genome. This full length single-stranded RNA is not present in viral particles isolated from the growth medium. The cellular concentration of the single-stranded RNA changes during exponential and stationary phases of cell growth in a fashion consistent with a viral replicative intermediate or mRNA. The single-stranded species does not appear to be polyadenylated.

Uptake of bacteria by trophozoites of Giardia duodenalis (Say).

Rod-like bacteria were found in the cytoplasm of trophozoites of Giardia duodenalis (Say) in domestic rats (Rattus rattus). These structures were always in phagocytic vacuoles without signs of bacteria digestion or degradation of the trophozoite cytoplasm. The uptake of the bacteria was observed from their attachment to the trophozoite membrane until their total incorporation by phagocytosis.

Giardiasis / Giardiasis

Una vez que los quistes de Giardia son expulsados del huesped mueren rapidamente si son deshidratados, pero pueden sobrevivir hasta dos meses en aguas frias con temperaturas de 8 C. Por tanto, las giardiasis se transmiten por la ingestion de heces o aguas contaminadas. Generalmente la infeccion produce diarrea, en las formas subaguda o cronica se pueden desarrollar signos y sintomas adicionales de malestar intestinal

Antibodies to the Giardia lamblia double-stranded RNA virus major protein can block the viral infection.

The double-stranded RNA virus-like particles, found among several independent isolates and cloned strains of Giardia lamblia, have previously been reported to be spheres of 35 nm with a genome of 7 kilobase pairs and a major protein of 100 kDa. The virus is capable of infecting certain virus-free isolates of G. lamblia. Antisera raised in mice against the intact virus did not react with the double-stranded RNA, but reacted strongly with the 100 kDa protein in Western blots. Preincubation of the virus with antisera abolished viral infectivity, whereas the antisera against double-stranded RNA showed only a weak blocking effect. Inclusion of the antiviral sera in the cultures of virus-infected G. lamblia at 10(3)-fold dilution resulted in elimination of the virus from the protozoa. Apparently, the 100 kDa protein is necessary for the initiation of viral infection and possibly subsequent assembly or replication of viral progeny particles.

Identification of Giardia lamblia isolates susceptible and resistant to infection by the double-stranded RNA virus.

The presence or absence of the Giardia lamblia double-stranded RNA virus (GLV) was surveyed among 38 axenic isolates of G. lamblia derived from both humans and animals. Of the 28 isolates lacking the virus, 19 could readily be infected by the virus. The remaining 9 isolates proved to be resistant to GLV infection even when the ratio between virus to parasite reached as high as 10(6) to 1. Evidence is also presented indicating that there are at least two "Portland 1" strains being used by the current scientific community, one containing the virus and the other lacking the virus.

Purification and characterization of the Giardia lamblia double-stranded RNA virus.

The dsRNA virus which infects some strains of Giardia lamblia has been purified and characterized with respect to its effect on growth of the parasite. Extensive purification of the virus from G. lamblia growth medium was accomplished by Millipore filtration and two successive CsCl gradient centrifugations. The purified virus possessed a single major protein species of 100,000 molecular weight. Effects of the extensively purified virus on growth of the virus-free parasite were studied. A cloned WB strain, sensitive to the viral infection, and a cloned E-9/M strain, resistant to the infection, were studied. With the WB strain, infection can occur at a ratio as low as 10 viral particles per organism. As the virus to parasite ratio increased, the rate of growth of the parasite decreased and the percentage of parasites not adhering to the culture tube wall also increased. These nonadhering cells, which differed from the nonadhering cells under normal growth conditions, were unable to divide. They contained an average number of 500,000 viral particles per cell which may be the threshold intracellular density of viral particles arresting the growth of G. lamblia. The results also suggest that the specific consequence of viral infection, even at extremely high multiplicity of infection, is not lysis of G. lamblia trophozoites but cessation of growth.

Occurrence and transfection of a Giardia virus.

The presence of virus-derived RNA was investigated in 38 axenically growing Giardia isolates from different geographic areas. The RNA virus was demonstrated in Giardia strains from humans in the U.S.A., England and the majority of strains from Poland. Two strains isolated respectively from a cat and a cavia also contained it. Giardia strains from humans in Belgium and Israel did not contain this RNA virus. Transfection of the RNA virus was accomplished from English and Polish strains, as well as from the cat isolate to isolates lacking it. Differences were observed both in sensitivity of Giardia strains to transfection and in infectivity of the RNA virus from different Giardia strains. Transfection could be carried out with sonicated Giardia extract as well as with filter sterilized medium in which Giardia strains containing RNA virus had grown. The RNA virus did not replicate in Giardia-free medium. No correlation could be demonstrated between the presence of the RNA virus in Giardia isolates and their in vitro resistance to some antiprotozoal drugs, nor with the fact that the strain originated from symptomatic or asymptomatic carriers. The presence of the RNA virus in Giardia trophozoites did not influence the isoenzyme patterns or restriction endonuclease patterns of repetitive DNA. A correlation may exist with the length of time since the isolation in axenic culture of the strain.

Discovery of a specific double-stranded RNA virus in Giardia lamblia.

Nucleic acid samples purified from trophozoites of Giardia lamblia Portland I strain contain an ethidium-stainable band that comigrates with 7.0 kilobase DNA in agarose gel electrophoresis. The band was degradable by alkali, ribonuclease A and ribonuclease T1, but the susceptibility toward the ribonucleases decreased with increasing ionic strength, suggestive of double-stranded RNA (dsRNA). This identification was confirmed by electron micrographs of the purified samples, which showed linear double-stranded structures with an estimated average length of 1.5 micron. In crude homogenates of G. lamblia, this dsRNA was protected against added ribonuclease A but disappeared upon adding sodium dodecyl sulfate or proteinase K. Differential centrifugations suggested an association of the dsRNA with the nuclear fraction, but it was freed to the 109,000 X g pelletable fraction with increasing homogenization. The dsRNA was purified by CsCl buoyant density gradient centrifugations in a distinct band with a rho value of 1.368 g ml-1. Electron microscopy revealed spherical virus-like particles (VLP) with a diameter of 33 nm. VLP of similar shape and size were also identified in the nuclei of sectioned G. lamblia trophozoites. VLP yield a major protein with an estimated molecular weight of 66,000 in sodium dodecyl sulfate polyacrylamide gel electrophoresis. VLP are abundant in the culture media of stationary-phase G. lamblia Portland I strain and are able to infect the G. lamblia WB strain, which is free of the virus. There is no sequence homology between the dsRNA and the nuclear DNA of G. lamblia and thus no apparent integration of viral genome into host DNA.

Ultrastructural observations on giardiasis in a mouse model. II. Endosymbiosis and organelle distribution in Giardia muris and Giardia lamblia.

Ultrastructural observations of Giardia muris in a mouse model revealed endosymbiotic microbes not previously reported in Giardia. Endosymbionts 240--360 nm wide, 600--1,400 nm long, and with an internal structure similar to that of bacilli were not seen entering Giardia but were found and appeared to divide within Giardia. No evidence was found of digestion of the endosymbionts by the giardia host in either the trophozoite or the cyst form. Endosymbionts were concentrated centrally around the nuclear area and were uncommon in peripheral feeding regions. The same cellular organelles seen in G. muris were found in Giardia lamblia from human jejunal biopsy material, but no endosymbionts were identified in G. lamblia trophozoites from the seven patients examined. Endosymbionts within Giardia may be found to alter trophozoite pathogenicity, metabolism, range of infectivity, antigenic surface characteristics, and host specificity, as they do in other protozoa.