Cytokine production in allergic and Trichuris trichiura-infected children from an urban region of the Brazilian northeast.

The relationship between the cellular immune response during Trichuris trichiura infection and asthma has not yet been established. In this study, the cytokines interleukin (IL)-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ and IL-17A were evaluated in asthmatic children harboring T. trichiura. For this assessment, asthmatic and non-asthmatic children (ISAAC questionnaire) were submitted to parasitological tests and blood samples were cultured (mitogen stimulation) for cytokine measurements in the supernatant. Asthma frequencies were similar in infected and uninfected children, but IL-4, IL-6, TNF-α and IL-10 levels were high in the infected asthmatic children. Additionally, infected non-asthmatic children exhibited high levels of these cytokines in relation to uninfected non-asthmatic children; however, cytokine levels were lower when compared with infected and asthmatic children. Therefore, T. trichiura infection positively modulated the pro- and anti-inflammatory cytokines in asthmatic children, but a background of asthma seemed to narrow the production of cytokines induced by this helminth.

Quality control of the slides by Kato-Katz method for the parasitological diagnosis of schistosomiasis infection by Schistosoma mansoni / Controle de qualidade das lâminas pelo método Kato-Katz para o diagnóstico parasitológico da esquistossomose mansônica

ABSTRACT Introduction: Kato-Katz is a laboratory method recommended by the Brazilian Ministry of Health (BMH) and the World Health Organization (WHO) as the gold standard for the diagnosis of human infection by Schistosoma mansoni. The method has great clinical and epidemiological relevance because it allows the parasite load quantification of the infected patient by calculating the number of eggs per gram (EPG) of feces. This classification may also be used to estimate the intensity of infection in the communities, to measure the impact of disease control measures, as well as to establish quality parameters for reading the slides. Objective: To describe the correct laboratory procedures for the parasitological diagnosis of S. mansoni infection by the Kato-Katz method based on the quality control protocol established by the Laboratory and Reference Service in Schistosomiasis/Centro de Pesquisa Aggeu Magalhães (CPqAM)/Fundação Oswaldo Cruz (Fiocruz)/BMH. Methods: We describe: 1) the technical steps for fecal sample preparation and reading the slides; 2) the technical limitations; 3) the standard operating procedure (SOP) to be adopted by laboratories; 4) the methodology for the internal and external quality control of the reading slides results; and 5) the tolerance limits accepted for such control. Conclusion: This study provides the laboratory which performs the diagnosis of schistosomiasis using the Kato-Katz method with parameters to implement a diagnostic service that can be evaluated internally and externally. The establishment of a quality protocol enables the comparison of data and the identification of failures in the operational procedure, which can be corrected by training personnel and taking actions for the problems identified.

RESUMO Introdução: O Kato-Katz é o método laboratorial adotado pelo Ministério da Saúde (MS) e pela Organização Mundial da Saúde (OMS) como padrão-ouro para o diagnóstico da infecção humana pelo Schistosoma mansoni, sendo uma ferramenta de relevância clínica e epidemiológica, visto que permite classificar a carga parasitária do indivíduo infectado pelo cálculo de ovos por grama de fezes (OPG). Essa classificação pode também ser utilizada para estimar a intensidade da infecção nas comunidades, mensurar o impacto de medidas de controle da doença bem como estabelecer parâmetros de qualidade para a leitura das lâminas. Objetivo: Descrever os procedimentos laboratoriais corretos para o diagnóstico parasitológico da infecção pelo S. mansoni pelo método Kato-Katz a partir do protocolo de controle de qualidade estabelecido pelo Laboratório e pelo Serviço de Referência em Esquistossomose/Centro de Pesquisa Aggeu Magalhães (CPqAM)/Fundação Oswaldo Cruz (Fiocruz)/MS. Método: São descritas: 1) as etapas técnicas para o preparo das amostras de fezes e a leitura das lâminas; 2) as limitações da técnica; 3) o procedimento operacional padrão (POP) a ser adotado pelos laboratórios; 4) a metodologia para o controle de qualidade interno e externo da leitura das lâminas; e 5) os limites de tolerância aceitos para tal controle. Conclusão: Este trabalho instrumentaliza os laboratórios que realizam o diagnóstico da esquistossomose pelo método Kato-Katz com parâmetros para implantar um serviço diagnóstico passível de ser avaliado interna e externamente. O estabelecimento de um protocolo de qualidade viabiliza a comparação de dados e a identificação de falhas no procedimento operacional, que poderão ser corrigidas por meio de capacitação de pessoal e tomada de medidas para os problemas identificados.

The schisto track: a system for gathering and monitoring epidemiological surveys by connecting geographical information systems in real time.

BACKGROUND: Using the Android platform as a notification instrument for diseases and disorders forms a new alternative for computerization of epidemiological studies. OBJECTIVE: The objective of our study was to construct a tool for gathering epidemiological data on schistosomiasis using the Android platform. METHODS: The developed application (app), named the Schisto Track, is a tool for data capture and analysis that was designed to meet the needs of a traditional epidemiological survey. An initial version of the app was finished and tested in both real situations and simulations for epidemiological surveys. RESULTS: The app proved to be a tool capable of automation of activities, with data organization and standardization, easy data recovery (to enable interfacing with other systems), and totally modular architecture. CONCLUSIONS: The proposed Schisto Track is in line with worldwide trends toward use of smartphones with the Android platform for modeling epidemiological scenarios.

Study of the snail intermediate hosts for Schistosoma mansoni on Itamaracá Island in northeast Brazil: spatial displacement of Biomphalaria glabrata by Biomphalaria straminea.

In 2012 a malacological survey of the breeding sites of Biomphalaria glabrata and B. straminea , the two intermediate host snails of Schistosoma mansoni , was carried out on Itamaraca Island in Pernambuco, Brazil. This study has now been extended by studying the competition between the two species. Snails were collected and dissected to identify the species and tests were performed to verify S. mansoni infection. Student's t test was used to compare the proportion between the two species and their breeding sites and a parasitological survey was conducted among local residents, using the Kato-Katz method. The spatial distribution of the two snail species was determined using TerraView, while a snail density map was constructed by Kernel estimate. The survey identified two breeding sites for B. glabrata with 17 specimens and 19 breeding sites for B. straminea with 459 snails, all of them negative for S. mansoni infection. The statistical analysis revealed that the proportion of the numbers of specimens and breeding sites of B. straminea (37.84 ± 9.01) were significantly greater than those of B. glabrata (8.50 ± 6.50). Parasitological examinations from 41 residents diagnosed two cases of schistosomiasis with parasite loads of 60 and 84 eggs per 1 g of stool, respectively. This indiction of a competitive process between the two snail species requires monitoring of schistosomiasis in the resident and travelling human populations occupying this environment, which could potentially result in social and economic changes on the island risking its attraction as a centre for eco-tourism.

Spatial distribution of Schistosoma mansoni infection before and after chemotherapy with two praziquantel doses in a community of Pernambuco, Brazil.

Praziquantel chemotherapy has been the focus of the Schistosomiasis Control Program in Brazil for the past two decades. Nevertheless, information on the impact of selective chemotherapy against Schistosoma mansoni infection under the conditions confronted by the health teams in endemic municipalities remains scarce. This paper compares the spatial pattern of infection before and after treatment with either a 40 mg/kg or 60 mg/kg dose of praziquantel by determining the intensity of spatial cluster among patients at 180 and 360 days after treatment. The spatial-temporal distribution of egg-positive patients was analysed in a Geographic Information System using the kernel smoothing technique. While all patients became egg-negative after 21 days, 17.9% and 30.9% reverted to an egg-positive condition after 180 and 360 days, respectively. Both the prevalence and intensity of infection after treatment were significantly lower in the 60 mg/kg than in the 40 mg/kg treatment group. The higher intensity of the kernel in the 40 mg/kg group compared to the 60 mg/kg group, at both 180 and 360 days, reflects the higher number of reverted cases in the lower dose group. Auxiliary, preventive measures to control transmission should be integrated with chemotherapy to achieve a more enduring impact.

Spatial distribution of Schistosoma mansoni infection before and after chemotherapy with two praziquantel doses in a community of Pernambuco, Brazil

Praziquantel chemotherapy has been the focus of the Schistosomiasis Control Program in Brazil for the past two decades. Nevertheless, information on the impact of selective chemotherapy against Schistosoma mansoni infection under the conditions confronted by the health teams in endemic municipalities remains scarce. This paper compares the spatial pattern of infection before and after treatment with either a 40 mg/kg or 60 mg/kg dose of praziquantel by determining the intensity of spatial cluster among patients at 180 and 360 days after treatment. The spatial-temporal distribution of egg-positive patients was analysed in a Geographic Information System using the kernel smoothing technique. While all patients became egg-negative after 21 days, 17.9 percent and 30.9 percent reverted to an egg-positive condition after 180 and 360 days, respectively. Both the prevalence and intensity of infection after treatment were significantly lower in the 60 mg/kg than in the 40 mg/kg treatment group. The higher intensity of the kernel in the 40 mg/kg group compared to the 60 mg/kg group, at both 180 and 360 days, reflects the higher number of reverted cases in the lower dose group. Auxiliary, preventive measures to control transmission should be integrated with chemotherapy to achieve a more enduring impact.

A rationale for schistosomiasis control in elementary schools of the rainforest zone of pernambuco, Brazil.

BACKGROUND: Since its beginning in 1999, the Schistosomiasis Control Program within the Unified Health System (PCE-SUS) has registered a cumulative coverage of just 20% of the population from the Rainforest Zone of Pernambuco (ZMP), northeast Brazil. This jeopardizes the accomplishment of the minimum goal of the Fifty-Fourth World Health Assembly, resolution WHA54.19, of providing treatment for schistosomiasis and soil-transmitted helminthiases (STH) to 75% of school-aged children at risk, which requires attending at least 166,000 residents in the 7-14 age range by year 2010 in that important endemic area. In the present study, secondary demographic and parasitological data from a representative municipality of the ZMP are analyzed to provide evidence that the current, community-based approach to control schistosomiasis and STH is unlikely to attain the WHA-54.19 minimum goal and to suggest that school-based control actions are also needed. METHODOLOGY/PRINCIPAL FINDINGS: Data available on the PCE-SUS activities related to diagnosis and treatment of the population from the study municipality were obtained from the State Secretary of Health of Pernambuco (SES/PE) for 2002-2006, complemented by the Municipal Secretary of Health (SMS) for 2003-2004. Data from a school-based stool survey carried out by the Schistosomiasis Reference Service of the Oswaldo Cruz Foundation (SRE/Fiocruz) in 2004 were used to provide information on infection status variation among school-aged children (7-14 years). According to the SES, from 2004 to 2006, only 2,977 (19.5%) of the estimated 15,288 residents of all ages were examined, of which 396 (13.3%) were positive for Schistosoma mansoni. Among these, only 180 (45.5%) were treated. According to the SMS, of the 1,766 examined in the 2003-2004 population stool survey 570 (32.3%) were children aged 7-14 years. One year later, the SRE/Fiocruz school survey revealed that the infection status among those children remained unchanged at 14%-15% prevalence. By 2006, the school-aged population was estimated at 2,981, of which 2,007 (67.3%) were enrolled as pupils. CONCLUSIONS: It is suggested that in the most troubled municipalities individual diagnosis and treatment should be concentrated in school-aged children rather than the whole population. School-based actions involving teachers and children's families may help the health teams to scale up control actions in order to attain the WHA-54.19 minimum goal. This strategy should involve health and education organs and include both enrolled and non-enrolled children.

Treatment of human acute schistosomiasis with oxamniquine induces an increase in interferon-gamma response to Schistosoma mansoni antigens.

Patients with acute schistosomiasis were studied before and after oxamniquine treatment. They had been exposed to cercariae 5 to 9 weeks before, and presented compatible clinical manifestations, eosinophilia, and high levels of total IgE. Interferon-gamma (IFN-gamma) and interleukin-4 were measured by ELISA in whole blood samples under soluble egg antigen or soluble adult worm preparation stimulation. After treatment, the reduction of leukocytosis and eosinophilia were not significant, but total IgE levels decreased significantly, in contrast to IFN-gamma levels that were significantly increased. The oxamniquine treatment of acute schistosomiasis patients is followed by an improvement of a Th1 response in vitro. If this response has a protective aspect is unknown, and some investigations need to be realized.

Treatment of human acute schistosomiasis with oxamniquine induces an increase in interferon-gamma response to Schistosoma mansoni antigens

Patients with acute schistosomiasis were studied before and after oxamniquine treatment. They had been exposed to cercariae 5 to 9 weeks before, and presented compatible clinical manifestations, eosinophilia, and high levels of total IgE. Interferon-gamma (IFN-gamma) and interleukin-4 were measured by ELISA in whole blood samples under soluble egg antigen or soluble adult worm preparation stimulation. After treatment, the reduction of leukocytosis and eosinophilia were not significant, but total IgE levels decreased significantly, in contrast to IFN-gamma levels that were significantly increased. The oxamniquine treatment of acute schistosomiasis patients is followed by an improvement of a Th1 response in vitro. If this response has a protective aspect is unknown, and some investigations need to be realized.

Reliability of current estimates of schistosomiasis prevalence in the Rainforest Zone of the state of Pernambuco, Northeastern Brazil

The Program for Schistosomiasis Control within the Unified Health System (PCE-SUS) was implemented by 1999 in the Rainforest Zone or "Zona da Mata" of Pernambuco (ZMP) aiming to carry out biennial stool surveys of whole populations through municipal health organs followed by treatment of the positives through the local units of the Family Health Program (PSF). Yearly reports from the Health Department of Pernambuco State (SES/PE) from 2002 to 2004 on the PCE-SUS surveys were assessed to evaluate whether the current estimates of prevalence in the municipalities of the ZMP are based on reliable samples so as to allow considerations on the real situation of schistosomiasis in that area. The surveys carried out in that period did not follow the major principles underlying sampling design, thus posing problems in both precision and validity of the estimates. Only 12 out of 43 municipalities had minimally reliable estimates: five with moderate prevalence (10-50 percent) and seven with low prevalence (< 10 percent). Surveys with appropriate sampling procedures aimed either at representative target groups (school-aged children) or communities are recommended for the ZMP and other endemic areas not only to provide reliable information on the current situation of schistosomiasis but also to plan adequate control strategies.

Attaining the minimum target of resolution WHA 54.19 for schistosomiasis control in the Rainforest Zone of the state of Pernambuco, Northeastern Brazil

Resolution 19 of the 54th World Health Assembly (WHA-54.19) urged member nations to promote preventive measures, ensure treatment and mobilize resources for control of schistosomiasis and soil-transmitted helminthiases (STH). The minimum target is to attend 75 percent of all school-age children at risk by year 2010. The Brazilian Ministry of Health (MoH) recommends biennial surveys of whole communities and treatment of the positives through the Schistosomiasis Control Program within the Unified Health System (PCE-SUS). However, by 2004 the PCE-SUS had covered only 8.4 percent of the 1.2 million residents in the Rainforest Zone of Pernambuco (ZMP). Six of the 43 municipalities still remained unattended. Only three of the municipalities already surveyed reached coverage of 25 percent or more. At least 154 thousand children in the 7-14 years old range have to be examined (and treated if positive) within the next five years to attend the minimum target of the WHA 54.19 for the ZMP. To make this target feasible, it is suggested that from 2006 to 2010 the PCE-SUS actions should be complemented with school-based diagnosis and treatment, involving health and educational organs as well as community associations to include both children in schools and non-enrolled school-age children.

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.

Development of molecular approaches for the identification of transmission sites of schistosomiasis.

Primers targeting the gene encoding the small subunit rRNA were designed to amplify DNA from Schistosoma mansoni with high specificity. Three PCR systems were developed: conventional PCR, two-step nested PCR (NPCR) and single-tube nested PCR (STNPCR). The limits of detection of parasite DNA for the conventional PCR, NPCR and STNPCR were 10 pg, 0.1 fg and 1 fg, respectively. The assays were highly specific for S. mansoni and did not recognise DNA from closely related non-schistosome trematodes. Using pools of Biomphalaria molluscs, PCR, NPCR and STNPCR were positive in 6/16 (37.5%), 15/16 (93.8%) and 13/16 (81.3%) of the tested samples, respectively, whereas the observation of cercariae shedding after exposure to light was able to detect S. mansoni infection in 6/16 (37.5%) of the pools. Thus, the molecular detection systems had a higher level of sensitivity than standard screening of intermediate hosts by cercarial shedding when DNA was purified from pools of snails collected from endemic areas. These PCR protocols have potential to be used as tools for monitoring of schistosome transmission.

Reliability of current estimates of schistosomiasis prevalence in the Rainforest Zone of the state of Pernambuco, Northeastern Brazil.

The Program for Schistosomiasis Control within the Unified Health System (PCE-SUS) was implemented by 1999 in the Rainforest Zone or "Zona da Mata" of Pernambuco (ZMP) aiming to carry out biennial stool surveys of whole populations through municipal health organs followed by treatment of the positives through the local units of the Family Health Program (PSF). Yearly reports from the Health Department of Pernambuco State (SES/PE) from 2002 to 2004 on the PCE-SUS surveys were assessed to evaluate whether the current estimates of prevalence in the municipalities of the ZMP are based on reliable samples so as to allow considerations on the real situation of schistosomiasis in that area. The surveys carried out in that period did not follow the major principles underlying sampling design, thus posing problems in both precision and validity of the estimates. Only 12 out of 43 municipalities had minimally reliable estimates: five with moderate prevalence (10-50%) and seven with low prevalence (< 10%). Surveys with appropriate sampling procedures aimed either at representative target groups (school-aged children) or communities are recommended for the ZMP and other endemic areas not only to provide reliable information on the current situation of schistosomiasis but also to plan adequate control strategies.

Attaining the minimum target of resolution WHA 54.19 for schistosomiasis control in the Rainforest Zone of the state of Pernambuco, Northeastern Brazil.

Resolution 19 of the 54th World Health Assembly (WHA-54.19) urged member nations to promote preventive measures, ensure treatment and mobilize resources for control of schistosomiasis and soil-transmitted helminthiases (STH). The minimum target is to attend 75% of all school-age children at risk by year 2010. The Brazilian Ministry of Health (MoH) recommends biennial surveys of whole communities and treatment of the positives through the Schistosomiasis Control Program within the Unified Health System (PCE-SUS). However, by 2004 the PCE-SUS had covered only 8.4% of the 1.2 million residents in the Rainforest Zone of Pernambuco (ZMP). Six of the 43 municipalities still remained unattended. Only three of the municipalities already surveyed reached coverage of 25% or more. At least 154 thousand children in the 7-14 years old range have to be examined (and treated if positive) within the next five years to attend the minimum target of the WHA 54.19 for the ZMP. To make this target feasible, it is suggested that from 2006 to 2010 the PCE-SUS actions should be complemented with school-based diagnosis and treatment, involving health and educational organs as well as community associations to include both children in schools and non-enrolled school-age children.

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.

Origin and diversification of the human parasite Schistosoma mansoni.

Schistosoma mansoni is the most widespread of the human-infecting schistosomes, present in 54 countries, predominantly in Africa, but also in Madagascar, the Arabian Peninsula, and the Neotropics. Adult-stage parasites that infect humans are also occasionally recovered from baboons, rodents, and other mammals. Larval stages of the parasite are dependent upon certain species of freshwater snails in the genus Biomphalaria, which largely determine the parasite's geographical range. How S. mansoni genetic diversity is distributed geographically and among isolates using different hosts has never been examined with DNA sequence data. Here we describe the global phylogeography of S. mansoni using more than 2500 bp of mitochondrial DNA (mtDNA) from 143 parasites collected in 53 geographically widespread localities. Considerable within-species mtDNA diversity was found, with 85 unique haplotypes grouping into five distinct lineages. Geographical separation, and not host use, appears to be the most important factor in the diversification of the parasite. East African specimens showed a remarkable amount of variation, comprising three clades and basal members of a fourth, strongly suggesting an East African origin for the parasite 0.30-0.43 million years ago, a time frame that follows the arrival of its snail host. Less but still substantial variation was found in the rest of Africa. A recent colonization of the New World is supported by finding only seven closely related New World haplotypes which have West African affinities. All Brazilian isolates have nearly identical mtDNA haplotypes, suggesting a founder effect from the establishment and spread of the parasite in this large country.

Evaluation of tests based on the antibody response to keyhole limpet haemocyanin and soluble egg antigen to differentiate acute and chronic human schistosomiasis mansoni.

Specific IgG and IgM responses to soluble egg antigen (SEA) and keyhole limpet haemocyanin (KLH) were measured by ELISA in patients with acute and chronic schistosomiasis. The tests based upon IgM and IgG antibodies responses to KLH presented the best diagnostic discrimination, and can be used in conjunction with clinical and epidemiological data to the differential diagnosis of acute schistosomiasis.

Evaluation of tests based on the antibody response to keyhole limpet haemocyanin and soluble egg antigen to differentiate acute and chronic human schistosomiasis mansoni

Specific IgG and IgM responses to soluble egg antigen (SEA) and keyhole limpet haemocyanin (KLH) were measured by ELISA in patients with acute and chronic schistosomiasis. The tests based upon IgM and IgG antibodies responses to KLH presented the best diagnostic discrimination, and can be used in conjunction with clinical and epidemiological data to the differential diagnosis of acute schistosomiasis.

Longitudinal Study on the Natural Infection of Biomphalaria straminea and B. glabrata by Schistosoma mansoni in an Endemic Area of Schistosomiasis in Pernambuco, Brazil.

The abundance of snail hosts and the rates of infection with Schistosoma mansoni were monitored monthly for four years in two representative localities subjected to repeated chemotherapy of infected persons. Snail abundance varied from 1.0 to 4.4 collected per person/minute/station for Biomphalaria straminea and from 0.1 to 7.0 for B. glabrata. Infection rates of snails in nature varied from 0% to 15% for the former and from 0% to 70% for the latter species. Human infection increased from 35.5% to 61.9% in the locality occupied by B. straminea, and decreased from 40.3% to 20.8% in that occupied by B. glabrata. No relationship could be detected between human infection and the snail variables. Despite seasonal variations, natural infection persisted throughout the monitoring period in both snail species. It reached remarkably high levels in B. straminea when compared to those obtained by other authors probably because of differences in methodology. It is recommended that longitudinal studies should be carried out focally and periodically to avoid underestimating the prevalence of schistosome infection in snails.