Xenomonitoramento molecular para avaliação da infecção vetorial por Wuchereria bancrofti em áreas endêmicas de filariose linfática na Região Metropolitana do Recife/PE / Molecular xenomonitoring for the evaluation of vector infection rate by Wuchereria bancrofti in endemic areas for lymphatic filariasis in Metropolitan Region of Recife/PE

A filariose linfática (FL) ou bancroftiana é uma doença parasitária causada por Wuchereria bancrofti, um verme filarial transmitido no Brasil pelo mosquito Culex quinquefasciatus. De acordo com a Organização Mundial de Saúde (OMS) esta doença afeta 120 milhões de pessoas em 58 países. Portanto, para enfrentar a FL, a OMS lançou um programa global para eliminá-la até 2020 e o Brasil tornou signatário dessa proposta criando o Plano Nacional de Eliminação da Filariose Linfática (PNEFL). Atualmente, a Região Metropolitana do Recife (RMR) é uma área de importante transmissibilidade e, assim, foi preconizado o Tratamento Coletivo (TC) da população com o medicamento Dietilcarbamazina (DEC) e o controle vetorial para reduzir a transmissão da doença. Como ferramenta complementar, desde a vigilância até a verificação da eliminação, o xenomonitoramento molecular (baseado na PCR para detecção de W. bancrofti em mosquitos) é um importante método não invasivo para monitorar indiretamente se a transmissão de larvas de W. bancrofti está ocorrendo na população humana. A fim de verificar a taxa de infecção vetorial no mosquito C. quinquefasciatus pela W. bancrofti foram coletadas 43.981 fêmeas do mosquito em doze localidades na RMR. Além disso, foi desenvolvido um novo protocolo (PCR duplex) para o diagnóstico de infecção vetorial e o número ideal de fêmeas por pools foi estabelecido. Os resultados mostraram que Linha do Tiro (Recife), uma área com alto índice de microfilaremia na população humana, apresentou status de transmissão durante o TC com uma taxa de infecção vetorial de 0,80 por cento, diferente das outras localidades com transmissão reduzida não foram detectados pools positivos. Portanto, observa-se que onde o TC é conduzido a taxa de infecção vetorial tende a ser reduzida. O xenomonitoramento molecular é um indicador importante para avaliação da eficiência das estratégias do PGEFL implantado em áreas endêmicas, até que ocorra a certificação da interrupção do ciclo de transmissão da filariose.

Canine heartworm disease in Porto Velho: first record, distribution map and occurrence of positive mosquitoes / Dirofilariose canina em Porto Velho: primeiro registro, mapa de distribuicao e ocorrencia de mosquitos positivos

The aim of this study was to make the first report on canine heartworm disease in the state of Rondônia and confirm its transmission in this state. Blood samples were randomly collected from 727 dogs in the city of Porto Velho. The samples were analyzed to search for microfilariae and circulating antigens, using three different techniques: optical microscopy on thick blood smears stained with Giemsa; immunochromatography; and PCR. Mosquitoes were collected inside and outside the homes of all the cases of positive dogs and were tested using PCR to search for DNA of Dirofilaria immitis. Ninety-three blood samples out of 727 (12.8%) were positive according to the immunoassay technique and none according to the thick smear method. Among the 93 positive dogs, 89 (95.7%) were born in Porto Velho. No difference in the frequency of infection was observed between dogs raised indoors and in the yard. PCR on the mosquitoes resulted in only one positive pool. This result shows that the transmission of canine heartworm disease is occurring in the city of Porto Velho and that there is moderate prevalence among the dogs. The techniques of immunochromatography and PCR were more effective for detecting canine heartworm than thick blood smears. The confirmation of canine heartworm disease transmission in Porto Velho places this disease in the ranking for differential diagnosis of pulmonary nodules in humans in Rondônia.

O objetivo deste estudo foi de registrar pela primeira vez a dirofilariose canina no estado de Rondônia e confirmar sua transmissão neste estado. Amostras de sangue de 727 cães foram colhidas aleatoriamente na cidade de Porto Velho. As amostras foram analisadas em busca de microfilárias e antígenos circulantes usando três técnicas diferentes: microscopia ótica de gota espessa corada com Giemsa e imunocromatografia de fluxo lateral e PCR. Mosquitos foram colhidos no domicilio e peridomicílio de todos os casos de cães positivos, estes mosquitos foram testados pela PCR na detecção de DNA de Dirofilaria immitis. Noventa e três das 727 amostras de sangue foram positivas na técnica de imunoensaio (12,8%). Nenhuma amostra foi positiva na gota espessa. Entre os 93 cães positivos, 89 (95,7%) foram nascidos em Porto Velho. Nenhuma diferença na frequencia de infecção foi observada entre cães criados dentro da casa ou no quintal. O PCR dos mosquitos resultou em apenas um pool positivo. Este resultado mostra que a transmissão de dirofilariose canina está ocorrendo na cidade de Porto Velho e a frequência que ocorre nos cães é considerada moderada. As técnicas de imunocromatografia e PCR são mais eficazes na detecção de dirofilariose comparadas a gota espessa. A confirmação de transmissão de dirofilariose canina em Porto Velho, coloca esta doença no ranking de diagnóstico diferencial de nódulos pulmonares em seres humanos em Rondônia.

Survey of Bancroftian filariasis infection in humans and Culex mosquitoes in the western Brazilian Amazon region: implications for transmission and control

Introduction The aim of this work was to identify possible lymphatic filariasis foci in the western Brazilian Amazonian that could be established from the reports of Rachou in the 1950s. The study was conducted in three cities of the western Brazilian Amazon region - Porto Velho and Guajará-Mirim (State of Rondônia) and Humaitá (State of Amazonas). Methods For human infection evaluation thick blood smear stained with Giemsa was used to analyze samples collected from 10pm to 1am. Polymerase chain reaction (PCR) was used to examine mosquito vectors for the presence of Wuchereria bancrofti DNA. Humans were randomly sampled from night schools students and from inhabitants in neighborhoods lacking sanitation. Mosquitoes were collected from residences only. Results A total 2,709 night students enrolled in the Program for Education of Young Adults (EJA), and 935 people registered in the residences near the schools were examined, being 641 from Porto Velho, 214 from Guajará-Mirim and 80 from Humaitá. No individual examined was positive for the presence of microfilariae in the blood stream. A total of 7,860 female Culex quinquefasciatus specimens examined were negative by PCR. Conclusions This survey including human and mosquito examinations indicates that the western Amazon region of Brazil is not a focus of Bancroftian filariasis infection or transmission. Therefore, there is no need to be included in the Brazilian lymphatic filariasis control program. .

Molecular detection of Schistosoma japonicum in infected snails and mouse faeces using a real-time PCR assay with FRET hybridisation probes

A real-time polymerase chain reaction (PCR) assay with fluorescence resonance energy transfer (FRET) hybridisation probes combined with melting curve analysis was developed to detect Schistosoma japonicum in experimentally infected snails and in faecal samples of infected mice. This procedure is based on melting curve analysis of a hybrid between an amplicon from the S. japonicum internal transcribed spacer region 2 sequence, which is a 192-bp S. japonicum-specific sequence, and fluorophore-labelled specific probes. Real-time FRET PCR could detect as little as a single cercaria artificially introduced into a pool of 10 non-infected snails and a single egg inoculated in 100 mg of non-infected mouse faeces. All S. japonicum-infected snails and all faecal samples from infected mice were positive. Non-infected snails, non-infected mouse faeces and genomic DNA from other parasites were negative. This assay is rapid and has potential for epidemiological S. japonicum surveys in snails, intermediate hosts and faecal samples of final hosts.

Faecal examination and PCR to detect Strongyloides venezuelensis in experimentally infected Lewis rats

More sensitive methodologies are necessary to improve strongyloidiasis diagnosis. This study compared the sensitivities of the McMaster modified technique and polymerase chain reaction (PCR) assays, both performed in faecal samples. Lewis rats were subcutaneously infected with 4,000, 400 or 40 infective third-stage larvae, considered as high, moderate or low infection, respectively. Seven days later, they were euthanized to count adult nematodes recovered from the small intestine. Stool samples were used to count the number of eggs per gram (EPG) of faeces and to detect parasite DNA by PCR performed with a species and a genus primer pair. The sensitivity of these assays depended upon parasite burden and the primer specificity. All assays presented 100 percent sensitivity at the highest parasite load. In the moderate infection, EPG and PCR with the genus primer maintained 100 percent specificity, whereas PCR sensitivity with the species primer decreased to 77.7 percent. In low infection, the sensitivity was 60 percent for EPG, 0 percent for PCR with the species primer and 90 percent for PCR done with the genus primer. Together, these results suggest that PCR with a genus primer can be a very sensitive methodology to detect Strongyloides venezuelensisin faeces of Lewis rats infected with very low parasite burden.

Molecular and morphological characterization of heterorhabditid entomopathogenic nematodes from the tropical rainforest in Brazil

Despite massive losses of primary forest, the Amazonian rainforest remains an extremely rich source of biodiversity. In recent years, entomopathogenic nematodes (EPNs) have been isolated from soil in various parts of the world and used successfully as biological control agents against numerous insect pests. Therefore, a sampling in the rainforest of Monte Negro, Rondônia, Brazil was conducted with the aim of discovering new strains and/or species of EPNs for future development as biological control agents. From 156 soil samples taken at nine collecting sites, 19 isolates were obtained, all of them belonging to the genus Heterorhabditis. Four strains were subjected to detailed morphological and molecular evaluation. Based on morphometrics and internal transcribed spacer (ITS) sequence data, the strains LPP1, LPP2 and LPP4 were identified as Heterorhabditis indica, whereas LPP7 was considered Heterorhabditis baujardi. Comparative analysis of the ITS1 sequence of H. indica and H. baujardi isolates showed a polymorphic site for the restriction enzyme Tth 111 that could be used to distinguish the two species. Consequently, strains LPP1, LPP2, LPP3, LPP4, and LPP9 were identified as H. indica, whereas LPP5, LPP7, LPP8 and LPP10 were identified as H. baujardi.

Detection of Onchocerca volvulus (Nematoda: Onchocercidae) infection in vectors from Amazonian Brazil following mass Mectizan™ distribution

Detection of Onchocerca volvulus in Simulium populations is of primary importance in the assessment of the effectiveness of onchocerciasis control programs. In Brazil, the main focus of onchocerciasis is in the Amazon region, in a Yanomami reserve. The main onchocerciasis control strategy in Brazil is the semi-annually mass distribution of the microfilaricide ivermectin. In accordance with the control strategy for the disease, polymerase chain reaction (PCR) was applied in pools of simuliids from the area to detect the helminth infection in the vectors, as recommended by the Onchocerciasis Elimination Program for the Americas and the World Health Organization. Systematic sampling was performed monthly from September 1998 to October 1999, and a total of 4942 blackflies were collected from two sites (2576 from Balawaú and 2366 from Toototobi). The molecular methodology was found to be highly sensitive and specific for the detection of infected and/or infective blackflies in pools of 50 blackflies. The results from the material collected under field conditions showed that after the sixth cycle of distribution of ivermectin, the prevalence of infected blackflies with O. volvulus had decreased from 8.6 to 0.3 percent in Balawaú and from 4 to 0.1 percent in Toototobi.

Development of a real time polymerase chain reaction for quantitation of Schistosoma mansoni DNA

This report describes the development of a SYBR Green I based real time polymerase chain reaction (PCR) protocol for detection on the ABI Prism 7000 instrument. Primers targeting the gene encoding the SSU rRNA were designed to amplify with high specificity DNA from Schistosoma mansoni, in a real time quantitative PCR system. The limit of detection of parasite DNA for the system was 10 fg of purified genomic DNA, that means less than the equivalent to one parasite cell (genome ~580 fg DNA). The efficiency was 0.99 and the correlation coefficient (R²) was 0.97. When different copy numbers of the target amplicon were used as standards, the assay could detect at least 10 copies of the specific target. The primers used were designed to amplify a 106 bp DNA fragment (Tm 83°C). The assay was highly specific for S. mansoni, and did not recognize DNA from closely related non-schistosome trematodes. The real time PCR allowed for accurate quantification of S. mansoni DNA and no time-consuming post-PCR detection of amplification products by gel electrophoresis was required. The assay is potentially able to quantify S. mansoni DNA (and indirectly parasite burden) in a number of samples, such as snail tissue, serum and feces from patients, and cercaria infested water. Thus, these PCR protocols have potential to be used as tools for monitoring of schistosome transmission and quantitative diagnosis of human infection.

Molecular approaches for the detection of Schistosoma mansoni: possible applications in the detection of snail infection, monitoring of transmission sites, and diagnosis of human infection

The detection of specific DNA sequences by polymerase chain reaction (PCR) has proved extremely valuable for the analysis of genetic disorders and the diagnosis of a variety of infectious disease pathogens. However, the application to the detection of Schistosoma mansoni is rare, despite a recommendation of the World Health Organization that a major focus of research on schistosomiasis should be on the development and evaluation of new strategies and tools for control of the disease. In this context, a few studies were published for the detection of the parasite in snails, monitoring of cercariae in water bodies, and diagnosis of human infection. The present minireview describes sensitive and specific PCR based systems to detect S. mansoni, indicating possible applications in the detection of snail infection, monitoring of transmission sites, and diagnosis of human infection.

Identification of the Boudicca and Sinbad retrotransposons in the genome of the human blood fluke Schistosoma haematobium

Schistosomes have a comparatively large genome, estimated for Schistosoma mansoni to be about 270 megabase pairs (haploid genome). Recent findings have shown that mobile genetic elements constitute significant proportions of the genomes of S. mansoni and S. japonicum. Much less information is available on the genome of the third major human schistosome, S. haematobium. In order to investigate the possible evolutionary origins of the S. mansoni long terminal repeat retrotransposons Boudicca and Sinbad, several genomes were searched by Southern blot for the presence of these retrotransposons. These included three species of schistosomes, S. mansoni, S. japonicum, and S. haematobium, and three related platyhelminth genomes, the liver flukes Fasciola hepatica and Fascioloides magna and the planarian, Dugesia dorotocephala. In addition, Homo sapiens and three snail host genomes, Biomphalaria glabrata, Oncomelania hupensis, and Bulinus truncatus, were examined for possible indications of a horizontal origin for these retrotransposons. Southern hybridization analysis indicated that both Boudicca and Sinbad were present in the genome of S. haematobium. Furthermore, low stringency Southern hybridization analyses suggested that a Boudicca-like retrotransposon was present in the genome of B. truncatus, the snail host of S. haematobium.

Application of multiplex pcr for species discrimination using individual metacercariae of Paragonimus occurring in Thailand.

A total of 6 lung fluke species have been documented in Thailand, of which P. heterotremus is the most important, since it affects humans. Although P. westermani is found as metacercariae in the same crab species as P. heterotremus in Thailand, human infections with P. westermani have not been confirmed. To accurately discriminate between the individual metacercariae of these two species, we established a multiplex PCR method. Using this method, two products each were amplified from the metacercarial DNA samples of P. heterotremus (ca. 310 and 520 bp) and P. westermani (ca. 140 and 520 bp). In contrast, 520-bp products alone were found to be generated from the DNA samples of P. siamensis, P. bangkokensis and P. harinasutai, 3 other species of lung flukes known to occur in Thailand. Digestion of these 520-bp products with the restriction enzyme ScrFI could unequivocally discriminate species by the number and size of the produced band(s): 3 bands (ca. 60, 210 and 250 bp) for P. harinasutai, 2 bands (ca. 250 and 270 bp) for P. bangkokensis, and an uncut band (520 bp) for P. siamensis. The established multiplex PCR used in combination with restriction enzyme digestion (PCR-RFLP with ScrFI) is effective for discriminating the 5 different species of lung flukes occurring in Thailand, even at the metacercarial stage.

Towards the international collaboration for detection, surveillance and control of taeniasis/ cysticercosis and echinococcosis in Asia and the Pacific.

Both cysticercosis and echinococcosis are potentially among the most serious helminth zoonoses threatening human health worldwide. However, due to the lack of reliable tools for confirmation or identification of patients or infected animals, epidemiological data are expected to be underestimated. Conversely, sometimes, such data are over estimated due to the lack of specificity. The most important issue for doing field surveys is that they use evidence based science. In this communication, advanced immunological and molecular tools for detection of individuals infected with either metacestodes or adult tapeworms are briefly overviewed, and the applications of such tools for epidemiological surveys in Indonesia, China and other countries are introduced. As immunological tools are based on antigen-antibody responses, there may exist some cross-reactions. Therefore, immunodiagnostic tools are expected to be useful for primary screening, and should be combined with confirmation of direct parasitological evidence (morphology or DNA), and imaging techniques for cysts. As a risk factor for human cysticercosis is the presence of tapeworm carriers, detection of taeniasis cases and differentiation of the three human Taenia species (Taenia solium, T. saginata and T. asiatica) in Asia and the Pacific requires consideration. Similarly, in northwest China, Echinococcus granulosus and E. multilocularis are coendemic and differentiation of these species is required in humans and definitive hosts. It is stressed that combination of several tools for identification of the parasite and for confirmation of diseases is important for obtaining highly reliable data before consideration of control of these zoonoses. Recent projects coordinated by Asahikawa Medical College have concentrated on immunological and molecular diagnostic techniques transferable to colleagues from endemic regions of Asia and the Pacific, and on organization of two international symposia to establish a platform for further collaboration in the future.

Dna quantities and qualities from various stages of some trematodes using optical and HAT-RAPD methods.

The aim of this experiment was to minimize DNA quantity and quality for detection by optical (spectrophotometer at 260 nm and 280 nm) and HAT-RAPD methods. Total DNA from different stages, adult, metacercaria and eggs of 6 trematode species were isolated for analysis. In this experiment, the adult trematodes were classified into 3 groups by size: small, Haplorchis taichui and Stellantchasmus falcatus; medium, Opisthorchis viverrini and Ganeo tigrinus; and large, Paramphistomum epiclitum and Fischoederius elongatus. The adult minimal DNA quantities and qualities of all specimen samples detected by optical method were 97.22,72.28, 3,167.00, 1,490.62, 21,382.66, and 27,321.77 ng; eggs were 3.92, 3.57, 3.72, 6.23, 17.53, and 14.01 ng, respectively; and metacercarial stages 50.70 and 40.98 ng in H. taichui and S. falcatus. In addition, the HAT-RAPD technique was chosen to amplify the minimal DNA qualities and quantities of all trematode specimens. Total DNA was 1-1 x 10(-12) ng; DNA templates in each dilution were used for amplification by primer OPA-09. DNA concentrations ranging between 1 x 10(-8) and 1 x 10(-11) ng were amplified with high polymorphism. Our experiment concluded that only a single specimen of each egg, metacercaria, or adult stage could be amplified with distinct bands.

Gnathostoma binucleatum (Spirurida: Gnathostomatidae) en peces dulceacuícolas de Tabasco, México / Gnathostoma binucleatum (Spirurida: Gnathostomatidae) from the freshwater fish in Tabasco, Mexico

Human gnathostomiasis is a food-born parasitic disease of relative importance in many countries in Southeast Asia. It is caused by several species of nematodes of the genus Gnathostoma. In Mexico is an emerging public health problem since 1970, when first cases were reported. Until today, larval morphometric characters that have been proposed to differentiate between the three species of Gnathostoma present in this country, are not satisfactory. Recently, the presence of advanced third-stage larvae AdvL3 (infective form for humans) in freshwater fishes from Pantanos de Centla, Tabasco. was recorded but their specific identity was not clarified . Examination of four species of freshwater fishes from the same locality revealed that three of them: Petenia splendida (n=58), Cichlasoma managuense (n=35) and Gobiomorus dormitor (n=9) were infected by 15 AdvL3 of Gnathostoma binucleatum. Specific identity was obtained comparing the internal transcribed spacer 2 (ITS2) of the ribosomal DNA with sequences reported in Genbank. This is the first record of G. binucleatum in P. splendida and G. dormitor from Tabasco and the first specific determination of the parasite in the locality.

A comparative study on mouse MHC class I sequences detected in Schistosoma japonicum recovered from BALB/c (H-2d) and C57BL/6 (H-2b) mice.

The mouse major histocompatibility complex (MHC) class I sequence was detected in all the 8-week-old Schistosoma japonicum recovered from BALB/c (H-2d) and C57BL/6 (H-2b) mice by in situ polymerase chain reaction (in situ PCR). The signals of the mouse class I MHC sequence were observed in the nuclei of the mesenchymal and reproductive cells of 8-week-old S. japonicum. Furthermore, the class I MHC sequence was detected in each DNA extracted from S. japonicum cercariae maintained in BALB/c and C57BL/6 mice by nested PCR. To prove both horizontal and vertical transmission of this sequence in schistosomes, we have used cercariae obtained from parasites maintained in BALB/c mice to infect C57BL/6 and BALB/c mice, and vice versa. The MHC sequences from adult worms were compared to the cercarial MHC and host MHC sequences. Nucleotide sequence comparisons between adult worm DNA, host (H-2d and H-2b mice) DNA and cercarial DNA used for the infection suggested that the sequence of mouse class I MHC was incorporated into schistosome adults and inherited throughout their life-cycle.

Evolutionary course of CsRn1 long-terminal-repeat retrotransposon and its heterogeneous integrations into the genome of the liver fluke, Clonorchis sinensis

The evolutionary course of the CsRn1 long-terminal-repeat (LTR) retrotransposon was predicted by conducting a phylogenetic analysis with its paralog LTR sequences. Based on the clustering patterns in the phylogenetic tree, multiple CsRn1 copies could be grouped into four subsets, which were shown to have different integration times. Their differential sequence divergences and heterogeneous integration patterns strongly suggested that these subsets appeared sequentially in the genome of C. sinensis. Members of recently expanding subset showed the lowest level of divergence in their LTR and reverse transcriptase gene sequences. They were also shown to be highly polymorphic among individual genomes of the trematode. The CsRn1 element exhibited a preference for repetitive, agenic chromosomal regions in terms of selecting integration targets. Our results suggested that CsRn1 might induce a considerable degree of intergenomic variation and, thereby, have influenced the evolution of the C. sinensis genome.

Divergent long-terminal-repeat retrotransposon families in the genome of Paragonimus westermani

To gain information on retrotransposons in the genome of Paragonimus westermani, PCR was carried out with degenerate primers, specific to protease and reverse transcriptase (rt) genes of long-terminal-repeat (LTR) retrotransposons. The PCR products were cloned and sequenced, after which 12 different retrotransposon-related sequences were isolated from the trematode genome. These showed various degrees of identity to the polyprotein of divergent retrotransposon families. A phylogenetic analysis demonstrated that these sequences could be classified into three different families of LTR retrotransposons, namely, Xena, Bel, and Gypsy families. Of these, two mRNA transcripts were detected by reverse transcriptase-PCR, showing that these two elements preserved their mobile activities. The genomic distributions of these two sequences were found to be highly repetitive. These results suggest that there are diverse retrotransposons including the ancient Xena family in the genome of P. westermani, which may have been involved in the evolution of the host genome.

Schistosoma japonicum strains: differentiation by RAPD and SSR-PCR.

Eight geographical isolates of Schistosoma japonicum from Taiwan and mainland China and one isolate of Schistosoma mansoni were studied by RAPD analysis using six arbitrary primers and SSR-PCR analysis using a (CA)8RY primer. The genetic distance was determined by the percentage of unshared bands. The RAPD and SSR-PCR results showed that the genetic distance between S. mansoni and S. japonicum was more than 0.900 and 0.850 respectively; the genetic distance between the eight geographical isolates of S. japonicum was 0.000 to 0.232 and 0.066 to 0.368 respectively. These results demonstrated the usefulness of RAPD and SSR-PCR for showing the differences of inter- and intra-species of Schistosoma. The results also suggest that there is genetic diversity among the different geographical strains of S. japonicum in China.

The diagnosis of human opisthorchiasis.

Opisthorchiasis viverrini is a liver fluke infection causing a serious public health problem in Thailand, Lao PDR, Cambodia, and South Vietnam because it acts as a strong promoter of cholangiocarcinoma. The diagnosis of human opisthorchiasis is based on four approaches: clinical manifestations, parasitological, molecular biological, and immunological methods. These methods have advantages and disadvantages. Clinical manifestations of the patients are practically indistinguishable from those of other liver diseases. The features of the O. viverrini eggs are, by light microscopy, difficult to differentiate from those of other minute intestinal flukes' eggs. Polymerase chain reaction (PCR) is very complicated, needs special and expensive apparatus, and is time-consuming; it is, however, highly sensitive and specific. Immunological testing is the method of choice: the techniques are applicable to both routine laboratory work and field or epidemiological studies. Of these tests, enzyme-linked immunosorbent assay (ELISA) and immunoelectrotransfer blot assay are often used for the detection of O. viverrini-specific antigens (coproantigens) and antibodies (IgM, IgG, IgA, or IgE). Monoclonal antibodies are prepared to detect coproantigens, while the crude somatic and excretory-secretory antigens from the adult worms, metacercariae, eggs, and snail intermediate hosts are prepared in order to detect antibodies in sera. To eliminate the cross reactions between parasites, the appropriate amount, type, and efficacy of antigens or antibodies preparation should be considered. In this paper, the advantages and disadvantages of the four diagnostic methods are discussed.

Resistance of Biomphalaria occidentalis from Varzea das Flores dam, Minas Gerais, to Schistosoma mansoni infection detected by low stringency polymerase chain reaction

Biomphalaria occidentalis Paraense, 1981 from Varzea das Flores dam, MG, Brazil, was exposed to infection with Schistosoma mansoni. Individual infection was performed with 140 B. occidentalis and 100 B. glabrata snails using LE and SJ strains. Two groups of B. occidentalis were killed after seven day-miracidia exposure to detect S. mansoni DNA, through the low stringency polymerase chain reaction (LS-PCR), and were negative. The infection rates were 69.2 percent (LE strain) and 96.7 percent (SJ strain) for B. glabrata and 0 percent for B. occidentalis. LS-PCR enabled early resistance diagnosis