Polymerase chain reaction for the evaluation of Schistosoma mansoni infection in two low endemicity areas of Minas Gerais, Brazil

This study aimed to evaluate the occurrence of schistosomiasis in areas with low endemicity using polymerase chain reaction (PCR) as a diagnostic method. We analysed faecal samples from 219 individuals residing in Piau and Coronel Pacheco, state of Minas Gerais, Brazil, using a single faecal sample from each individual and two slides of the Kato-Katz technique as a gold standard. Fifteen out of the 219 samples were positive with both methods of diagnosis. One sample was diagnosed as positive by the Kato-Katz technique only and 61 were diagnosed only by PCR. The positivity rates were 7.3% with the Kato-Katz method and 34.7% with PCR. When both techniques were assumed to have 100% specificity and positive individuals were identified by both methods, the sensitivity of the Kato-Katz method was 20.8% and the PCR sensitivity was 98.7%. The Kappa index between the two techniques was 0.234, suggesting weak agreement. The assessment of a single faecal sample by PCR detected more cases of infection than the analysis of one sample with two slides using the Kato-Katz technique, suggesting that PCR can be a useful diagnostic tool, particularly in areas with low endemicity.

In vitro effects of amodiaquine on paired Schistosoma mansoni adult worms at concentrations of less than 5 µg/mL

In this study, the in vitro effects of amodiaquine (AQ) monotherapy on the egg output of paired adult Schistosoma mansoni worms and their survival during in vitro culture were assessed. In addition, the gross morphological alterations of male and female worms caused by AQ were visually observed under a dissecting microscope. AQ significantly reduced the daily egg output of paired adult S. mansoni worms following incubation for 14 days at 1-5 µg/mL, but not at 0.5 µg/mL, compared with the control group. AQ also reduced the survival of male and female worms at concentrations of 2 and 5 µg/mL, respectively. Moreover, exposure to 5 µg/mL AQ caused severe swelling and/or localisation of black content in the body of all male and female worms within one or two days of incubation; subsequently, shrinkage in the male worms and elongation in the female worms were observed. The initial morphological alterations caused by AQ occurred along the intestinal tract of the male and female worms. To our knowledge, this is the first study to report not only the efficacy of AQ at concentrations lower than 5 µg/mL on paired adult S. mansoni worms, but also the effects of AQ on the intestinal tracts of worms in in vitro culture.

Schistosoma mansoni in a low-prevalence area in Brazil: the importance of additional methods for the diagnosis of hard-to-detect individual carriers by low-cost immunological assays

Schistosomiasis diagnosis is based on the detection of eggs in the faeces, which is laborious and lacks sensitivity, especially for patients with a low parasite burden. Immunological assays for specific antibody detection are available, but they usually demonstrate low sensitivity and/or specificity. In this study, two simple immunological assays were evaluated for the detection of soluble Schistosoma mansoni adult worm preparation (SWAP) and egg-specific IgGs. These studies have not yet been evaluated for patients with low parasite burdens. Residents of an endemic area in Brazil donated sera and faecal samples for our study. The patients were initially diagnosed by a rigorous Kato-Katz analysis of 18 thick smears from four different stool samples. The ELISA-SWAP was successful for human diagnosis with 90% sensitivity and specificity, confirming the Kato-Katz diagnosis with nearly perfect agreement, as seen by the Kappa index (0.85). Although the ELISA-soluble S. mansoni egg antigen was 85% sensitive, it exhibited low specificity (80%; Kappa index: 0.75) and was more susceptible to cross-reactivity. We believe that immunological assays should be used in conjunction with Kato-Katz analysis as a supplementary tool for the diagnosis of schistosomiasis for patients with low infection burdens, which are usually hard to detect.

Schistogram changes after administration of antischistosomal drugs in mice at the early phase of Schistosoma mansoni infection

Mice infected with Schistosoma mansoni were treated with oxamniquine, praziquantel, artesunate at the pre-patent phase, aiming at observing schistogram alterations. Half of the animals were perfused five days post-treatment for counting and classification of immature worms, based on pre-established morphological criteria (schistogram); the remaining animals were evaluated 42 or 100 days after infection and perfusion of the portal-system was performed for collection and counting of adult worms and oogram. It was observed that oxamniquine and artesunate treatment administered at the pre-postural phase causes significant reduction in the number of immature and adult worms. However, there was little reduction with praziquantel when used at the dose of 400 mg/kg for treatments administered 14, 15, 21 or 23 days post-infection. Artesunate was responsible for significant alterations in development of young worms, as well as for a higher number of worms presenting intestinal damages. Immature adult worms were detected in mice treated with artesunate or oxamniquine at the pre-patent phase of infection and recovered by perfusion 100 days after infection. Schistogram proved to be a very useful tool for experimental evaluation of the activity of antischistosomal drugs and a good model to identify the most sensitive stages to drugs.

Cytokines as determinants of resistance and pathology in human Schistosoma mansoni infection

The role of different cytokines in the peripheral blood mononuclear cell (PBMC) proliferative response and in in vitro granuloma formation was evaluated in a cross-sectional study with patients with the different clinical forms and phases of Schistosoma mansoni infection, as well as a group of individuals "naturally" resistant to infection named normal endemic (NE). The blockage of IL-4 and IL-5 using anti-IL-4 and anti-IL-5 antibodies significantly reduced the PBMC proliferative response to soluble egg (SEA) and adult worm (SWAP) antigens in acute (ACT), chronic intestinal (INT) and hepatosplenic (HS) patients. Similar results were obtained in the in vitro granuloma formation. Blockage of IL-10 had no significant effect on either assay using PBMC from ACT or HS. In contrast, the addition of anti-IL-10 antibodies to PBMC cultures from INT patients significantly increased the proliferative response to SEA and SWAP as well as the in vitro granuloma formation. Interestingly, association of anti-IL-4 and anti-IL-10 antibodies did not increase the PBMC proliferative response of these patients, suggesting that IL-10 may act by modulating IL-4 and IL-5 secretion. Addition of recombinant IL-10 decreased the proliferative response to undetectable levels when PBMC from patients with the different clinical forms were used. Analysis of IFN-g in the supernatants showed that PBMC from INT patients secreted low levels of IFN-g upon antigenic stimulation. In contrast, PBMC from NE secreted high levels of IFN-g. These data suggest that IL-10 is an important cytokine in regulating the immune response and possibly controlling morbidity in human schistosomiasis mansoni, and that the production of IFN-g may be associated with resistance to infection.

Collagen arrangement in hepatic granuloma in mice infected with Schistosoma mansoni: dependence on fiber radiation centers

The collagen structure of isolated and in situ liver granuloma from Swiss Webster mice infected with Schistosoma mansoni was sequentially and three-dimensionally analyzed during different times of infection (early acute, acute, transitional acute-chronic, and chronic phases) by laser scanning confocal microscopy and electron scanning variable vacuum microscopy. The initial granuloma structure is characterized by vascular collagen residues and by anchorage points (or fiber radiation centers), from where collagenous fibers are angularly shed and self-assembled. During the exudative-productive stage, the self-assembly of these fibers minimizes energy and mass through continuous tension and focal compression. The curvature or angles between collagen fibers probably depends on the fibroblastic or myofibroblastic organization of stress fibers. Gradually, the loose unstable lattice of the exudative-productive stage transforms into a highly packed and stable architecture as a result of progressive compactness. The three-dimensional architecture of granulomas provides increased tissue integrity, efficient distribution of soluble compounds and a haptotactic background to the cells.

Sm14 gene expression in different stages of the Schistosoma mansoni life cycle and immunolocalization of the Sm14 protein within the adult worm

Sm14 is a 14-kDa vaccine candidate antigen from Schistosoma mansoni that seems to be involved in cytoplasmic trafficking of fatty acids. Although schistosomes have a high requirement for lipids, they are not able to synthesize fatty acids and sterols de novo. Thus, they must acquire host lipids. In order to determine whether Sm14 is present in different stages of the life cycle of the parasite, we performed RT-PCR. Sm14 mRNA was identified in all stages of the life cycle studied, mainly schistosomulum, adult worm and egg. Additionally, we used a rabbit anti-Sm14 polyclonal antibody in an indirect immunofluorescence assay to localize Sm14 in adult worm sections. The basal lamella of the tegument and the gut epithelium were strongly labeled. These tissues have a high flow of and demand for lipids, a finding that supports the putative role of Sm14 as an intracellular transporter of fatty acids from host cells.

Observations on the miraxonal attraction exercised by sexually immature or adult Biomphalaria glabrata infected or not by Schistosoma mansoni

OBJECTIVE: The influence of age and the presence of secondary sporocysts in the miraxonal attraction exercised by Biomphalaria glabrata on miracidia of Schistosoma mansoni of the BH strain were studied. MATERIAL AND METHOD: A glass apparatus containing two compartments joined by a tube and previously tested in other experiments, was used. Specimens of B. glabrata or its snail conditioned water (SCW) selected before the first oviposition (sexually immature), after the first oviposition (adult), with or without secondary sporocysts, were used to attract the miracidia. RESULTS: It was noted that snails or their SCW containing secondary sporocysts lost the ability to attract miracidia. The sexual maturity of the snail did not influence miraxonal attraction.

Stability of Schistosoma mansoni progeny to antischistosomal drugs

The susceptibility of the MAP Brazilian strain (F1 to F5 progenies) of S. mansoni to four antischistosomal drugs has been reported in a previous study. In the present investigation, progeny F14 of the same strain, was tested for stability to the same 4 drugs. A new medication, Oltipraz (35,972 RP), was added to the study. Five groups of 12 mice infected with cercariae by tail immersion were treated with hycanthone, oxamniquine, niridazole, praziquantel and Oltipraz. An untreated group was used as control. Schistosomal activity was assessed by the localization of worms in the portal vein system, by oogram changes, and percentage of parasite reduction. The stability of the susceptibility of progeny F14 did not change in relation to generations F1 to F5; the progeny was resistant to hycanthone and oxamniquine; but sensitive to niridazole, praziquantel and Oltipraz. We emphasize the importance of the phenomenon of resistance of the worm in view of the fact that oxamniquine has been widely used in Brazilian areas where mansonic schistosomiasis is endemic.

Experimental infection with Schistosoma mansoni of Biomphalaria straminea from different parts of the Northeast of Brazil

In the western part of the State of Bahia Biomphalaria straminea and B. glabrata both occur, but in the majority of cases they do not share the same habitat. In the State of Ceará, however, B. straminea is the sole snail host of Schistosoma mansoni. In this survey, no naturally infected B. straminea was found among snails collected from Bahia and Ceará, evidently because of the very low infection rates. The susceptibility of laboratory-reared specimens to infection with a Puerto Rican strain of S. mansoni was then tested experimentally. In general, the snails showed very low susceptibility. The infection rates were 1.1% among snails from Redenção (Ceará); 2.3% in those from Pentecoste (Ceará); 2.9% in snails from São Desidério (Bahia), while they were very high among an albino strain (NIH) of B. glabrata used as control. Another group of B. straminea from São Desidério was exposed to a Bahian strain of S. mansoni and the infection rate was still very low (3.6%) Apparently, the very low susceptibility of B. straminea, despite high snail density, is correlated with moderate infection rates with S. mansoni among humans, as shown by the results of stool examinations conducted by SUCAM in the municipalities of Redenção and Pentecoste, in Ceará.

Therapeutical evaluation of different dose regimens of praziquantel in schistosomiasis mansoni, based on the quantitative oogram technique

A clinical trial involving 80 patients of both sexes, from ages 15 to 55, with chronic intestinal or hepatointestinal schistosomiasis mansoni, was carried out to evaluate the therapeutical efficacy of different dose regimens of praziquantel. The patients were randomly allocated into four groups with an equal number of cases and were then treated with one of the following dosages: 60 mg/kg for 1 day; 60 mg/kg daily for 2 days; 60 mg/kg daily for 3 days; and 30 mg/kg daily for 6 days. The assessment of parasitological cure was based on the quantitative oogram technique through rectal mucosa biopsies which were undertaken prior to, as well as, 1,2,4 and 6 months post-treatment. Concurrently, stool examinations according to the qualitative Hoffman, Pons & Janer (HPJ) and the quantitative Kato-Katz (K-K) methods were also performed. The best tolerability was observed with 30 mg/kg daily for 6 days whereas the highest incidence of side-effects (mainly dizziness and nausea) was found with 60 mg/kg daily for 3 days. No serious adverse drug reaction has occurred. The achieved cure rates were: 25% with 60 mg/kg for 1 day; 60% with 60 mg/kg daily for 2 days; 89.5% with 60 mg/kg daily for 3 days; and 90% with 30 mg/kg daily for 6 days. At the same time there has been a downfall of 64%, 73%, 87% and 84% respectively, in the median number of viable S. mansoni ova per gram of tissue. Thus, a very clear direct correlation between dose and effect could be seen. The corresponding cure rates according to stool examinations by HPJ were 39%, 80%, 100% and 95%; by K-K 89%, 100%, 100% and 100%. This discrepancy in results amongst the three parasitological methods is certainly due to their unequal accuracy. In fact, when the number of viable eggs per gram of tissue fell below 5,000 the difference in the percentage of false negative findings between HPJ (28%) and K-K (80%) became significative. When this number dropped to less than 2,000 the percentage of false negative results obtained with HPJ (49%) turned significant in relation to the oogram as well. In conclusion, it has been proven that praziquantel is a highly efficacious agent against S. mansoni infections. If administered at a total dose of 180 mg/kg divided into either 3 or 6 days, it yields a 90% cure rate. Possibly, one could reach 100% by increasing the total dose to 240 mg/kg. Furthermore, it was confirmed that the quantitative oogram technique is the most reliable parasitological method when evaluating the efficacy of new drugs in schistosomiasis mansoni.

Efficacy of oxamniquine, praziquantel and a combination of both drugs in schistosomiasis mansoni in Brazil

A randomized clinical trial was carried out to compare the efficacy of a low-dosage combination of oxamniquine (7.5 mg/kg) plus praziquantel (20 mg/kg) against either agent, oxamniquine (15 mg/kg) or praziquantel (40 mg/kg) alone, in the treatment of schistosomiasis mansoni in the Brazilian north-east. The drugs were randomly administered per os to 91 patients. Six and twelve months after treatment 89% of those admitted to the trial were reexamined by Kato-Katz method (ten slides) and MIF technique (one gram of stool) The achieved cure rates, as defined by absence of S. mansoni eggs in the faeces of individual patients at all points during the parasitological follow-up, were 81.8%, 81.2% and 67.6% for praziquantel, oxamniquine and the combination respectively. The reduction of eggs excretion in non cured patients six months after therapy ranged from 93.8-96.8% with praziquantel, 32.5-97% with oxamniquine and 76.9-99.5% with the combination. It is concluded that, at the used dosages, the three therapeutical regimens give similar and satisfactory results in the treatment of uncomplicated S. mansoni infection in Brazil.

Efeitos de imunopotenciadores não específicos na infecção experimental pelo Schistosoma mansoni: I. levamisole

Os efeitos do levamisole nas alterações histopatológicas, resistência do hospedeiro e quimiotaxia "in vivo" foram estudados na infecção experimental pelo Schistosoma mansoni em camundongos da linhagem C57B1/10. O tratamento profilático resultou em um aumento no número de vermes adultos obtidos pela perfusão e também em uma taxa de mortalidade maior (p < 0,05). As alterações histopatológicas (fígado e intestino) foram similares em todos os grupos. Uma redução significante da quimiotaxia "in vivo" ocorreu em camundongos controles infectados, assim como naqueles submetidos a tratamento profilático com levamisole. A atividade quimiotática atingiu os mesmos níveis dos camundongos controles normais (não-infectados e não-tratados com levamisole), quando o esquema curativo foi usado. O levamisole parece aumentar a susceptibilidade de camundongos da linhagem C57B1/10 à infecção pelo S. mansoni quando administrado antes da infecção e normaliza a atividade quimiotática, quando dado após a infecção.

Immunoelectrophoretic study on common antigens of São Lourenço da Mata and Belo Horizonte strains of Schistosoma mansoni adult worms and Biomphalaria snails

Immunoelectrophoretic studies on common antigens were carried out by using rabbits sera immunized against São Lourenço da Mata and Belo Horizonte strains of Schistosoma mansoni adult worms and antigens of Biomphalaria glabrata pigmented (Jaboatão - PE); B. glabrata albino (Belo Horizonte - MG) and B. straminea (São Lourenço da Mata, PE). Furthermore, the reverse approach was proceeded, namely, sera anti Biomphalaria snails produced in rabbits were tested against both strains of Schistosoma adult worm antigens. The analysis of the common antigens between the SLM strains of S. mansoni adult worm and B. glabrata pigmented showed 8 to 9 precipitin bands, 3 bands with B. glabrata albino and only 1 band with B. straminea crude extracts. On the other hand, the BH strain of S. mansoni adult worm antisera produced 6 to 7 bands with B. glabrata pigmented, 5 bands with B. glabrata albino and 1 band with B. straminea antigenic extract. Biomphalaria snails crude extracts were fractionated by Sephadex G-100 column and three fractions were collected from each snail strain. The fractions were tested with anti SLM and BH strains of S. mansoni adult worm sera by immunoelectrophoresis. The common antigens fractionated from Biomphalaria snails crude extracts and those found for both strains of S. mansoni adult worm mostly existed in the first fraction and they were estimated to have molecular weight over 158,000 daltons. In our laboratory, it was found a relationship between the antigenic similarities and experimental infection rates of S. mansoni towards Biomphalaria snails so that more bands were seen with increasing infection rates of S. mansoni.

Biological and morphological characteristics of Schistosoma mansoni from Ribeira Valley, State of São Paulo, Brazil: I - susceptibility of Biomphalaria tenagophila snail to sympatric S. mansoni strain

In the São Paulo State, Brazil, where the Biomphalaria tenagophila is the intermediate host, the Ribeira Valley is an important endemic schistosomiasis mansoni area. During last eleven years there has been intense control measures focusing on schistosomiasis. The efforts have been concentrated in the municipalities of Pedro de Toledo and Itariri. We determined the susceptibility of B. tenagophila to sympatric strain of S. mansoni, both recently isolated from Itariri field. In 1988, this strain was isolated and maintained in the experimental model: Swiss mice - sympatric B. tenagophila. The second generation of the worm was evaluated. The snail were divided in the three groups of 60 snails each. One group was exposed to 1 miracidium and other to 10. The third group was the control. The mortality and the shedding of cercariae were checked during 78 days. After that, the positive snails were observed until they ceased to shed cercariae. The exposed molluscs showed mortality rates of 23% and 31% and infection indexes were of 8% and 60% to 1 and 10 miracidia respectively. The mortality was of 22% in the control group. The periods of shedding cercariae in the two groups were 82 and 104 days. We can conclude that B. tenagophila is an effective intermediate host to the sympatric strain of S. mansoni sympatric strain