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.

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.

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.

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 percent to 15 percent for the former and from 0 percent to 70 percent for the latter species. Human infection increased from 35.5 percent to 61.9 percent in the locality occupied by B. straminea, and decreased from 40.3 percent to 20.8 percent 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

Factors involved in Schistosoma mansoni infection in rural areas of northeast Brazil

Two contiguous villages in Tracunhaem country (State of Pernambuco) endemic for schistosomiasis, were studied: Itapinassu (138 inhabitants) and Sao Joaquim (91 inhabitants). Agriculture predominates in the former region while ceramics is the main activitiy in the latter. Although no statistical difference was found regarding prevalence, severe infection (<400 epg) predominated in Itapinassu, probably related to the kind of occupation. No association was found between parasite burden and severity of disease, in spite of the high infection rates for Schistosoma mansoni in both communities (aprox. 60 per cent). Typical epidemiological features of schistosomiasis such as age-realted prevalences and intensities of infection (high in children, low in adults) were also mutual characteristics. Nutritional status determined through anthropometric evaluation was carried out by measuring specific anthropometric indicators. A deficit of energy intake, as well as vitamin A and riboflavin deficiencies were detected. The prevalence of moderate or severe undernutrition in patients under 18 years old was 21.9 per cent in Itapinassu and 24.1 per cent in Sao Joaquim. In this group as association was found between prevalence of schistosomiasis and chronic undernutrition. Similarly, for patients over 18 years old the prevalence of undernutrition was higher than 20 per cent. However, in this case no association between nutritional status and either prevalence of schistosomiasis or parasite burden could be detected. The two communities had not been treated for eight years.

The bioecology of snail vectors for schistosomiasis in Brazil

Aspectos ecológicos de duas espécies brasileiras de Biomphalaria, transmissoras da esquistossomose mansônica, säo discutidos no presente artigo B. glabrata e B. straminea tem sido intensamente estudadas no Brasil e em outros países desde a década de 50. Este artigo enfatiza três momentos que marcaram o desenvolvimento histórico de nosso conhecimento sobre a biologia destes caramujos: 1 o acervo antigo acumulado de estudos de laboratório e de campo; 2 o desenvolvimento de modelos ecológicos quantitativos; 3 o desenvolvimento de metodologia alternativa para o cultivo dos caramujos em condiçoes seminaturais.

Long-term controlled field experiment on the competition between two species of biomphalaria (mollusca, basommatophora), the snail vectors of schistosoma mansoni in northeastern Brazil / Estudo experimental de campos sobre a competiçao entre duas espécies de biomphalaria (mollusca, basommatophora), vetores do schistosoma mansoni no nordeste do Brasil

Um estudo experimental de campo sobre a interaçao populacional entre Biomphalaria glabrata (populaçao-alvo) e Biomphalaria straminea (competidor) foi realizado no município de Alhandra, PB, no período de 1980 a 1989. Os resultados deste experimento nos levam a considerar que é possível a induçao de um processo competitivo entre B. glabrata e B straminea, uma vez que em nove dos criadouros trabalhados até o final, seis deles tiveram as populaçoes da espécie endêmica totalmente substituídas pelo caramujo competidor. O processo competitivo ocorreu mais eficazmente naqueles criadouros sujeitos à dessecaçao, o que parece ter favorecido a espécie competidora, que resiste bem mais aos períodos de seca. Nao houve regressao do processo, ou seja, em todos os riachos onde B. straminea se estabeleceu, o caramujo alvo nao voltou a proliferar

Dynamics of snail populations of biompharia glabrata and B. straminea under semi-natural conditions

Curvas de crescimento e taxas de mortalidade para populaçoes de Biompharia glabrata e B. straminea, criadas em condiçoes semi-naturais, sao descritas no presente trabalho. As populaçoes de caramujos foram observadas durante 220 semanas em canaletas de cimento montadas no laboratório. Dados foram coletadas a cada intervalo de 20 semanas. As densidades brutas de crescimento foram significativamente diferentes para cada populaçao estudada. B. glabrata mostrou crescimento inferior às duas cepas de B. straminea. A cepa R3 desta última espécie mostrou o melhor desempenho, sendo capaz de viver em metade do volume de água necessária a B. glabrata quando ambas est*o submetidas a pressao populacional

Epidemiologia da esquistossomose mansônica no Engenho Bela Rosa, Município de Sao Lourenço da Mata, Pernambuco, Brasil / Epidemiology of mansonic schistosomiasis in Bela Rosa Sugar Mill, Municipality of Sao Lourenço da Mata, Pernambuco, Brasil

Bela Rosa, um pequeno engenho localizado no município de Sao Lourenço da Mata, estado de Pernambuco, representa uma típica unidade produtora de açúcar situada na grande regiao da mata úmida do Nordeste do Brasil. Os 370 habitantes do engenho vivem em níveis social e econômico muito baixos. A regiao é conhecida pelos seus elevados níveis de prevalência da esquistossomose e foi submetida nestes últimos anos a diferentes tipos de programas de controle de infecçao. Utilizando dados de 1967 a 1986 e mais aqueles obtidos por um estudo epidemiológico transversal feito em 1989, os autores descrevem a tendência evolutiva da endemicidade da esquistossomose na área