Comparison of different PCR amplification targets for molecular diagnosis of Strongyloides stercoralis.

Molecular techniques are an alternative for the diagnosis of strongyloidiasis, produced by Strongyloides stercoralis. However, it is necessary to determine the best amplification target for the populations of this parasite present in a geographical area and standardize a polymerase chain reaction (PCR) protocol for its detection. The objectives of this work were the comparison of different PCR targets for molecular detection of S. stercoralis and the standardization of a PCR protocol for the selected target with the best diagnostic results. DNA extraction was performed from parasite larvae by saline precipitation. Three amplification targets of the genes encoding ribosomal RNA 18S (18S rDNA) and 5.8S (5.8S rDNA) and cytochrome oxidase 1 (COX1) of S. stercoralis were compared, and the PCR reaction conditions for the best target were standardized (concentration of reagents and template DNA, hybridization temperature, and number of cycles). The analytical sensitivity and specificity of the technique were determined. DNA extraction by saline precipitation made it possible to obtain DNA of high purity and integrity. The ideal target was the 5.8S rDNA, since the 18S rDNA yielded non-reproducible results and COX1 never amplified under any condition tested. The optimal conditions for the 5.8S rDNA-PCR were: 1.5 mM MgCl2, 100 µM dNTPs, 0.4 µM primers, and 0.75 U DNA polymerase, using 35 cycles and a hybridization temperature of 60 °C. The analytical sensitivity of the PCR was 1 attogram of DNA, and the specificity was 100%. Consequently, the 5.8S rDNA was shown to be highly sensitive and specific for the detection of S. stercoralis DNA.

Comparison between merthiolate-iodine-formalin and Kato-Katz methods for the diagnosis of human helminth infections in resource-limited settings.

Diagnosis of intestinal parasites through examination of fresh faecal samples is hampered by its unpleasantness and the urgent need to detect all parasitic forms. In this paper, we compared the standard Kato-Katz (KK) technique with a traditional fixation method, the merthiolate-iodine-formalin (MIF) method. Two hundred and twenty-seven faecal samples from individuals living in a rural setting in Venezuela with high to moderate prevalences of Ascaris lumbricoides (Al), Trichuris trichiura (Tt) and hookworm infections were examined. The 'gold standard' used here was derived from the combination of the outcomes from both methods. KK performed better at detecting Tt, and showed higher sensitivity and negative predictive value for both Tt and Al, probably due to a higher capacity of KK to detect low parasite loads. Both methods showed an almost perfect agreement using the Kappa index. MIF provided a higher median of parasitic loads for low and total egg counts for the three helminths. Differentiating fertile from infertile eggs of Al did not affect the results; infertile eggs were present only at low and intermediate parasitic loads, but absent at high loads. KK was not able to detect high loads of any of the helminths. MIF allowed for the detection of other helminths, such as Strongyloides stercoralis, and protozoan infections, for which KK is not specific. In conclusion, MIF is a simple and inexpensive technique that performs competitively with KK in both laboratory and field work on intestinal helminths, particularly in resource-limited settings.

Polymerase chain reaction for the amplification of the 121-bp repetitive sequence of Schistosoma mansoni: a highly sensitive potential diagnostic tool for areas of low endemicity.

Schistosomiasis is a disease caused by parasitic flatworms of the genus Schistosoma, whose diagnosis has limitations, such as the low sensitivity and specificity of parasitological and immunological methods, respectively. In the present study an alternative molecular technique requiring previous standardization was carried out using the polymerase chain reaction (PCR) for the amplification of a 121-bp highly repetitive sequence for Schistosoma mansoni. DNA was extracted from eggs of S. mansoni by salting out. Different conditions were standardized for the PCR technique, including the concentration of reagents and the DNA template, annealing temperature and number of cycles, followed by the determination of the analytical sensitivity and specificity of the technique. Furthermore, the standardized PCR technique was employed in DNA extracted, using Chelex®100, from samples of sera of patients with an immunodiagnosis of schistosomiasis. The optimal conditions for the PCR were 2.5 mm MgCl2, 150 mm deoxynucleoside triphosphates (dNTPs), 0.4 µm primers, 0.75 U DNA polymerase, using 35 cycles and an annealing temperature of 63°C. The analytical sensitivity of the PCR was 10 attograms of DNA and the specificity was 100%. The DNA sequence was successfully detected in the sera of two patients, demonstrating schistosomiasis transmission, although low, in the community studied. The standardized PCR technique, using smaller amounts of reagents than in the original protocol, is highly sensitive and specific for the detection of DNA from S. mansoni and could be an important tool for diagnosis in areas of low endemicity.

Evolutionary relationships and biogeography of Biomphalaria (Gastropoda: Planorbidae) with implications regarding its role as host of the human bloodfluke, Schistosoma mansoni.

The wide geographic distribution of Schistosoma mansoni, a digenetic trematode and parasite of humans, is determined by the occurrence of its intermediate hosts, freshwater snails of the genus Biomphalaria (Preston 1910). We present phylogenetic analyses of 23 species of Biomphalaria, 16 Neotropical and seven African, including the most important schistosome hosts, using partial mitochondrial ribosomal 16S and complete nuclear ribosomal ITS1 and ITS2 nucleotide sequences. A dramatically better resolution was obtained by combining the data sets as opposed to analyzing each separately, indicating that there is additive congruent signal in each data set. Neotropical species are basal, and all African species are derived, suggesting an American origin for the genus. We confirm that a proto-Biomphalaria glabrata gave rise to all African species through a trans-Atlantic colonization of Africa. In addition, genetic distances among African species are smaller compared with those among Neotropical species, indicating a more recent origin. There are two species-rich clades, one African with B. glabrata as its base, and the other Neotropical. Within the African clade, a wide-ranging tropical savannah species, B. pfeifferi, and a Nilotic species complex, have both colonized Rift Valley lakes and produced endemic lacustrine forms. Within the Neotropical clade, two newly acquired natural hosts for S. mansoni (B. straminea and B. tenagophila) are not the closest relatives of each other, suggesting two separate acquisition events. Basal to these two species-rich clades are several Neotropical lineages with large genetic distances between them, indicating multiple lineages within the genus. Interesting patterns occur regarding schistosome susceptibility: (1) the most susceptible hosts belong to a single clade, comprising B. glabrata and the African species, (2) several susceptible Neotropical species are sister groups to apparently refractory species, and (3) some basal lineages are susceptible. These patterns suggest the existence of both inherent susceptibility and resistance, but also underscore the ability of S. mansoni to adapt to and acquire previously unsusceptible species as hosts. Biomphalaria schrammi appears to be distantly related to other Biomphalaria as well as to Helisoma, and may represent a separate or intermediate lineage.

Lacto-N-fucopentaose III (Lewis x), a target of the antibody response in mice vaccinated with irradiated cercariae of Schistosoma mansoni.

Carbohydrates on soluble egg antigens are major epitopes for the antibody responses of patients and mice infected with Schistosoma mansoni. Recently, protective sera of mice vaccinated with irradiated cercariae were shown to recognize carbohydrate epitopes on schistosomal glutathione S-transferase. The present study demonstrates that carbohydrate epitopes are major targets of sera from C57BL/6J and CBA/J mice vaccinated with 15- or 50-kilorad-irradiated cercariae of S. mansoni. Antibody titers to carbohydrate epitopes increased with the number of vaccinations and were considerably higher in C57BL/6J mice than in CBA/J mice. The specificity of this anticarbohydrate response was determined by measuring antibody binding to defined oligosaccharide residues known to be present on the parasite. A predominant target of the humoral anticarbohydrate response of vaccinated mice was lacto-N-fucopentaose III, a molecule relevant for cell trafficking. We observed no binding to its nonfucosylated homolog, lacto-N-neotetraose, or to oligosaccharides present on keyhole limpet hemocyanin. The strongest antibody response to lacto-N-fucopentaose III was observed for C57BL/6J and CBA/J mice repeatedly vaccinated with 15-kilorad-irradiated cercariae, which also achieve the highest levels of protection. Immunoglobulin M was the predominant antibody class binding to lacto-N-fucopentaose III. We conclude that in the irradiated-cercariae vaccine model, C57BL/6J and CBA/J mice produce anticarbohydrate antibodies against various stages of S. mansoni and that the oligosaccharide lacto-N-fucopentaose III is one target of this response. Lacto-N-fucopentaose III and its specific antibodies may profoundly affect host resistance and parasite homing.

Isotype responses to candidate vaccine antigens in protective sera obtained from mice vaccinated with irradiated cercariae of Schistosoma mansoni.

In experimental schistosomiasis, sera of mice multiply vaccinated with radiation-attenuated cercariae of Schistosoma mansoni passively transfer resistance against cercarial challenge to naive mice. To further characterize these sera, we tested their protective capacities in two mouse strains (C57BL/6J and CBA/J) and compared the antigen-specific isotype compositions of the different protective sera by means of the enzyme-linked immunosorbent assay. By using an array of purified schistosomal antigens, the patterns of antibody titers and isotypes differed for each experimental group and antigen. In the most-protective C57BL/6J sera, high levels of immunoglobulin G1 (IgG1), IgG2a, and IgG2b bound to heat shock protein 70 and the integral membrane protein Sm23, whereas recognition of these antigens by less-protective CBA/J sera was lower. Glutathione S-transferase (GST) was recognized predominantly by IgM antibodies of all vaccinated groups, and a significant portion of this response was directed against carbohydrate epitopes. Antibodies specific for triosephosphate isomerase, paramyosin, and Sm32 (hemoglobinase) were present in less-protective sera and thus seem less relevant for passive transfer of resistance. The results of this study suggest a contribution of IgG antibodies specific for heat shock protein 70 and Sm23, and possibly a contribution of GST-specific IgM antibodies, to the protective effect of sera from C57BL/6J mice vaccinated with irradiated cercariae.

Compatibility of one Brazilian and two Venezuelan strains of Schistosoma mansoni with various strains of Biomphalaria glabrata.

For evaluation of the degree of genetic heterogeneity in parasite and snail strains, the compatibility between Biomphalaria glabrata and Schistosoma mansoni from different geographical areas was studied. Venezuelan snails from Bárbula, Manuare (areas of schistosome transmission) and Tinaquillo (non-transmission area) and Brazilian BH snails were exposed to infection with miracidia of both the Venezuelan YT and SM strains and the Brazilian BH strain of S. mansoni. Snail-parasite compatibility was evaluated by quantifying the number of snails shedding cercariae during a period of 35 to 60 days post-infection. The best compatibility appeared to be between the Brazilian parasites and the Brazilian or Venezuelan snails. In contrast, the Brazilian snails appeared to be resistant to infection by the Venezuelan parasites tested. Paradoxically, the compatibility between the sympatric pair of Venezuelan parasites and Venezuelan snails appeared to be lower in comparison with the allopatric association of the Brazilian parasite and the Venezuelan snails. The results suggest an important degree of heterogeneity in the snail and parasite isolates studied and yield biological markers for both organisms.

Isoenzyme studies in one Brazilian and two Venezuelan strains of Schistosoma mansoni.

1. Enzyme polymorphism, analyzed by starch gel electrophoresis, was found to be zero for acid phosphatase, phosphoglucomutase, phosphoglucose isomerase, glucose 6-phosphate dehydrogenase, lactate dehydrogenase, malate dehydrogenase and malic enzyme, in one Brazilian and two Venezuelan strains of Schistosoma mansoni. 2. All loci studied were monomorphic within strains, but the isoenzymic patterns were, however, different among the strains. 3. Results suggest a drastic loss of the genetic variability usually found in natural populations.

Biomphalaria prona (Gastropoda: Planorbidae): a morphological and biochemical study

Two samples of Biomphalaria prona (Martens, 1873) from Lake Valencia (type locality) and seven from other Venezuelan localities were studied morphologically (shell and reproductive system) and biochemically (allozyme electrophoresis). In spite of marked differences in shell characters, all of them proved indistinguishable under the anatomic and biochemical criteria. So far B. prona has been considered an endemic species, restricted to Lake Valencia. It is now demonstrated that the extralacustrine populations refered to Biomphalaria havanensis (Pfeiffer, 1839) by several authors correspond in shell characters to an extreme variant of B. prona from the Lake and really belong to the last*mentioned species. They may be regarded as the result of a process of directional selection favoring a shell phenotype other than those making up the modal class in the Lake.

Experimental chemotherapy of Schistosoma mansoni with praziquantel and oxamniquine: differential effect of single or combined formulations of drugs on various strains and on both sexes of the parasite.

The susceptibility of two Venezuelan (YT and SM) and one Brazilian (BH) strain of Schistosoma mansoni to single oral doses of praziquantel (Pz; 250 or 500 mg/kg), oxamniquine (Ox; 40, 60, or 100 mg/kg) or to low-dose combinations of both drugs (33 mg/kg Pz and 25 mg/kg Ox; 66 mg/kg Pz and 12.5 mg/kg Ox; 250 mg/kg Pz and 40 mg/kg Ox) was experimentally evaluated in mice. At lower doses of either drug, adult worms of the SM isolate were less susceptible than those of the BH and YT isolates. However, no difference in liver or intestinal egg counts (IECs) could be detected among the isolates after this treatment. At such doses, Pz was better than Ox at reducing IECs. In spite of lowered IECs, eggs continued to accumulate in the liver after Ox treatment. At higher individual doses or following treatment with low-dose combinations of both drugs, no difference in susceptibility could be detected among the parasite isolates. Under such conditions, oviposition was drastically reduced in all three isolates. We confirm that Ox preferentially kills male parasites and present for the first time evidence for the preferential killing of female worms by Pz. We propose that the synergistic effect obtained in the present study and in other investigations using low-dose combinations of both drugs may be due to the preferential cytotoxicity of each drug against a different parasite sex.

The Venezuelan experience in the control of schistosomiasis mansoni.

Schistosomiasis mansoni endemic zone of Venezuela is located in the valleys of the north central mountain region, with an extension of 15,000 km2 and inhabited by 5.1 million persons. The disease was discovered in 1906, but an organized Control Program was not established until 1943. Its basic activity has been the control of the snail vector, but prevention of man-water contact, prevention of snail infection, treatment of infected people and sanitary instruction, have also been carried out. Prevalence has diminished from 14.7% (1943-60) to 0.9% (1981-84). At present few active foci still persist, but a low transmission rate and low morbidity makes it difficult to know the exact number of infected people, which has been estimulated to be about 50,000.

Ultrastructural observations on the in vitro interaction of rat neutrophils with schistosomula of Schistosoma mansoni in the presence of antibody and/or complement.

Rat peritoneal neutrophils adhere to schistosomula of Schistosoma mansoni in vitro in the presence of antibody, complement, or both. Ultrastructural studies have demonstrated that cell adherence is not intimate, and that electron-dense secretions are not liberated onto the parasite surface in the manner described for eosinophils. Cytochemical techniques confirm that peroxidase is confined within intracellularly located neutrophil secretion granules. The metabolic burst is shown to operate during Fc-mediated interactions, but since morphological damage depends upon the presence of complement in the system, toxic oxygen products would seem not to be involved in the initiation of surface perturbation. Complement-dependent, neutrophil-mediated schistosomular damage is characterized by vesiculation of the tegumental outer membrane, an increase in density of the tegumental cytoplasm and the eventual development of focal lesions. The cells migrate laterally to push aside damaged surface tissues and then adhere intimately to the exposed musculature. Damage appears earlier when both antibody and complement are present in the system, and this correlates with higher killing efficiency. The frequently observed association of contaminant eosinophils with areas of parasite damage indicates that eosinophils and neutrophils may cooperate to effect schistosomular killing. In the presence of antibody alone, attached neutrophils exhibit intense phagocytic activity towards the antigen-antibody complex formed at the parasite surface. This phenomenon may account for the eventual detachment of cells and lack of significant parasite damage recorded in this system.

Neutrophil-mediated cytotoxicity to schistosomula of Schistosoma mansoni in vitro: studies on the kinetics of complement and/or antibody-dependent adherence and killing.

The capacity of rat peritoneal neutrophils to adhere to and kill schistosomula of Schistosoma mansoni in vitro has been investigated. Neutrophils adhere readily to schistosomula in the presence of antibody plus complement (C) and C alone (fresh normal rat serum), but not with heat-inactivated normal rat serum. However, schitosomular killing is only achieved with neutrophils and fIRS or fNRS. In the presence of hiIRS the cells detach after 6 h without producing a significant level of parasite death. The system involving neutrophils plus fIRS is the most efficient in terms of serum dilution and the rate of schistosomular killing. The complement-dependent antibody involved in this system belongs to the class IgG and occurs in rat serum at peak titres, 6-8 wk after a primary schistosome infection. Neutrophil adherence in the presence of fNRS depends upon the generation of C3b molecules at the parasite surface via the alternative pathway of C activation. Studies on the antibody alone system indicate that the lack of significant schistosomular killing might result from the absence of factors which stimulate cell migration, since if a chemokinetic agent is introduced into the assay a 30% increase in mortality is recorded. The possible participation of neutrophils in the destruction of a primary and/or challenge infection in vivo is discussed.