Edward F. Silva

Prevalence of intestinal parasites in children from public daycare centers in the city of Belo Horizonte, Minas Gerais, Brazil

The objective of this study was to verify the occurrence of intestinal parasites in 3 to 6-year-old children from daycare centers maintained by the municipal government of Belo Horizonte, Minas Gerais, Brazil. Coproparasitological tests performed in 472 children have shown that 24.6% of them had some type of parasites, 6.6% of the children having more than one type. Among protozoa, Entamoeba coli (14.0%) and G. duodenalis (9.5%) were the most prevalent, whereas Ascaris lumbricoides (3.0%) and Trichuris trichiura (1.1%) were the most frequent among the helminths. Thus, we can observe that intestinal parasites still represent a serious public health problem in Belo Horizonte, especially among children and in areas where the socioeconomic conditions are less favorable.

An improved method to distinguish Entamoeba histolytica and Entamoeba dispar

A 482 base pair gene fragment from samples of amoebae E. histolytica and E. dispar was amplified by PCR. The amplification products of fragments from the 2 species of amoebae presented differences in mobility in non-denaturing polyacrylamide gel, probably due to sequence-dependent conformational alterations in the DNA fragments. The method described here permits E. histolytica and E. dispar to be distinguished with greater sensitivity and rapidity.

A single step duplex PCR to distinguish Entamoeba histolytica from Entamoeba dispar

In this study, a single-step duplex polymerase chain reaction procedure was developed for rapid, specific and sensitive identification of Entamoeba histolytica and for its diagnostic differentiation from E. dispar. Specific oligonucleotide primers were combined for the amplification of a cysteine proteinase 5 gene target sequence of 242 bp, present only in E. histolytica. Additionally, another oligonucleotide primer pair for both the E. histolytica and E. dispar actin gene target of 300 bp was designed to amplify only from amoebae DNA. The PCR developed was specific and efficiently identified and differentiated these parasites from each other in either cultured parasites or from stool material.

Arbitrarily primed PCR fingerprinting of RNA and DNA in Entamoeba histolytica.

Differences were detected in the gene expression of strains of E. histolytica using RNA (RAP-PCR) and DNA fingerprinting (RAPD). Analysis of the electrophoretic profiles of the gels revealed some polymorphic markers that could be used in the individual characterization of the strains. The 260 bands generated by using five different primers for RAP-PCR, as well as RAPD, were employed in the construction of dendograms. The dendogram obtained based on the RAPD products permitted the distinction of symptomatic and asymptomatic isolates, as well the correlation between the polymorphism exhibited and the virulence of the strains. The dendogram obtained for the RAP-PCR products did not show a correlation with the virulence of the strains but revealed a high degree of intraspecific transcriptional variability that could be related to other biological features, whether or not these are involved in the pathogenesis of amebiasis.

Pathogenicity of Entamoeba dispar under xenic and monoxenic cultivation compared to a virulent E. histolytica

Two xenic isolates and cloned cultures of Entamoeba dispar were submitted to monoxenization using Crithidia fasciculata as the associated organism. Growth in monoxenic cultivation and ability of xenic and monoxenic trophozoites to destroy VERO cells and produce lesions in hamster livers were compared to those of a virulent E. histolytica. Parental and cloned E. dispar under monoxenic cultivation showed a remarkable lower growth than the monoxenic E. histolytica and were avirulent in both in vivo and in vitro tests. When xenically cultured, trophozoites of E. dispar showed a moderate lytic activity against VERO cells (1.5 to 41.8% of destruction) but caused severe hepatic lesions in hamsters as those caused by the virulent E. histolytica (29 to 100% in prevalence and 0.86 to 4.00 in lesion degree). Although E. dispar has not been associated with invasive disease in men, the ability of xenic trophozoites to produce prominent tissue damage in experimental conditions has indicated that some strains have a considerable pathogenic potential when in presence of bacteria.

Molecular characterization of Brazilian human Giardia duodenalis isolates using isoenzyme and random amplified polymorphic DNA analysis.

Isoenzymes and RAPD (random amplified polymorphic DNA) analysis were used to characterize three Brazilian human isolates of Giardia duodenalis and its clones. The Portland-1 strain (ATCC 30888) was included in the study as a reference pattern. Both methods divided the isolates into two main groups, one represented by the Portland-1 strain, the other constituted by the Brazilian isolates, which, in turn, were divided into 2 subgroups. The dendogram constructed with the RAPD data, using seven primers, revealed a great heterogeneity between Brazilian isolates and the Portland-1 strain. There was no relationship to the clinical characteristics of the isolates. Although a lot of similarity has been observed among Brazilian isolates and its clones, individual polymorphism was detected, which could be related to the clonal reproduction of this protozoan.

Entamoeba histolytica: cysteine proteinase activity and virulence. Focus on cysteine proteinase 5 expression levels.

Cysteine proteinase (CP) activity and CP5 mRNA levels were analyzed in eleven samples of Entamoeba histolytica isolated from patients presenting different clinical profiles. The virulence degree of the isolates, determined in hamster liver, correlated well with the clinical form of the patient and culture conditions. CP5 mRNA levels were also determined in sample freshly picked up directly from liver amoebic abscess. Differences were not observed in the levels of CP5 mRNA and CP specific activity among the cultured samples. However, different levels of CP5 mRNA were observed in trophozoite freshly isolated from hepatic amoebic lesions. These results reinforce the importance of CP5 for the virulence of amoebae and the need for studies with the parasite present in lesions to validate mechanisms involved in pathogenesis of amoebiasis.

RAPD in the analysis of isolates of Entamoeba histolytica.

Genetic variability in Entamoeba histolytica was analyzed by random amplified polymorphic DNA (RAPD) using ten arbitrary primers. Due to intrinsic characteristics of the RAPD technique only axenic samples were analyzed. since the presence of any microorganism in the cultures interfered in the DNA profile by generating RAPDs not pertaining to E. histolytica. The RAPD profiles of E. histolytica samples isolated from patients with different clinical manifestations from different regions of the Americas shared about 70% of the bands produced. These profiles were compared to those obtained for E. moshkorskii, and E. invadens. The combined data for the ten primers were used in the phenetic analysis of all the isolates studied by using the Dice similarity coefficient as the genetic distance measure between the samples. Three distinct groups could be separated by phenon line: one including E. moshkovskii samples, which shared > 90% of the RAPDs produced by the different primers; one consisting solely of E. invadens; and a third comprising samples of E. histolytica, which showed considerable intraspecific variability.

Isoenzyme profile as parameter to differentiate pathogenic strains of Entamoeba histolytica in Brazil

The isoenzyme profiles (IP) of 33 strains of Entamoeba histolytica isolated from patients and carriers of two regions in Brazil (Amazonia and Southeast) were determined. The enzymes phosphoglucomutase, glucose-phosphate isomerase, hexokinase and malic enzyme were considered. IP of the strains was correlated with culture conditions, time of maintenance in laboratory and clinical history of patients. The strains were maintained under polyxenic, monoxenic and axenic culture conditions: 27 polyxenic, 1 polyxenic and monoxenic, 1 polyxenic, monoxenic and axenic and 4 axenic only. The patients were symptomatic and asymptomatic. The symptomatic patients presented either non dysenteric (NDC) or dysenteric colitis (DC), associated or not with hepatic abscess (HA). One patient presented anal amoeboma (AM). The analysis of IP for isolates maintained in polyxenic culture showed non pathogenic IP (I) for strains from carriers and patients with NDC, while the strains isolated from patients presenting DC, HA and AM resulted in isolates II or XIX pathogenic IP. This parameter was not able to differentiate strains from carriers from symptomatic patients when these strains were found in axenic or monoxenic culture. All these strains displayed pathogenic IP (II), demonstrating the inability of this parameter to classifying for virulence since it showed identical IP for strains isolated from carriers or symptomatic patients.

Glycogen as a carbohydrate energy reserve in trophozoites of Giardia lamblia

Although there is indirect evidence to suggest that glycogen is present in G. lamblia, to date it has not been purified and identified from this organism. In this study, a high molecular weight carbohydrate was purified and characterized and its physiological role as an energetic reserve was established. The monosaccharide constituents of the carbohydrate reserve were identified as glucose by two independent methods: thin layer chromatography and an enzymatic assay. The degree of branching of the molecule was evaluated by comparing its absorbance spectrum in the presence of lugol with spectra of standard solutions of glycogen and starch under the same conditions. The results strongly suggest that glycogen is present in G. lamblia and acts as an energy reserve in trophozoites of this organism.