Abstract INTRODUCTION: In most Strongyloides stercoralis infected individuals, nematoidosis occurs asymptomatically, but in immunocompromised patients, it can cause hyperinfection. Serological techniques seem to be a good alternative for detecting this parasite. METHODS The frequency of seropositivity for strongyloidiasis in Alfenas, MG, was estimated using the enzyme linked immunosorbent assay on blood samples, between May and August of 2015. RESULTS: Out of 258 samples tested, 53.9% were positive, and the frequency of seropositive individuals was higher in the peripheral districts of the municipality. CONCLUSIONS: The results indicate high seropositivity rates for strongyloidiasis among the residents of Alfenas city.

SUMMARY Visceral leishmaniasis is a systemic and chronic disease and dogs are the main reservoir of the etiologic agent, Leishmania infantum (syn L. chagasi). A serological and molecular investigation of canine visceral leishmaniasis (CVL) was performed in the municipality of Alfenas, located in the southern region of Minas Gerais, where the disease is not endemic. Samples from 87 dogs were submitted to serological tests including the Dual Path Platform (DPP (r) ) CVL Bio-Manguinhos rapid test, an in-house enzyme-linked immunosorbent assay (ELISA) and an immunofluorescence antibody test (IFAT), as well as molecular techniques such as a conventional polymerase chain reaction (PCR) with the RV1/RV2 primers and a quantitative PCR (qPCR) with the LinJ31, Ldon and DNApol primers. Of the 87 serum samples, eight (9.2%) were positive for Leishmania using the DPP rapid test, but only four (4.6%) were confirmed by ELISA and two (2.3%) by IFAT. In these two serologically confirmed cases, spleen and liver samples were positive by all the employed molecular and parasitological procedures performed on spleen samples. When whole blood samples were used in the molecular assays, two samples (2.3%) were positive only by qPCR. DNA extracted and amplified from the spleens of seropositive dogs was sequenced, showing 100% of similarity with the Leishmania infantum (syn L. chagasi) sequence. Thus, the first cases of CVL have been confirmed in the Alfenas region, suggesting the importance of canine surveys in non-endemic municipalities for CVL to monitor disease progression and to prevent outbreaks.

Isolados de Cryptosporidium identificados em quatorze amostras de fezes, coletadas de cinco pacientes com infecção por HIV e de nove crianças imunocompetentes, residentes no estado de São Paulo, Brasil, foram submetidos a análise molecular por Nested-PCR, seguido da caracterização genética por polimorfismo do tamanho do fragmento de restrição (RFLP). A análise foi baseada na digestão, com a enzima de restrição RsaI, de um fragmento de DNA amplificado do gene que codifica a proteína de parede do oocisto de Cryptosporidium (COWP). Baseado nesta análise, quando comparadas aos padrões utilizados, quatro amostras foram identificadas como Cryptosporidium parvum, oito como Cryptosporidium hominis e duas apresentaram um perfil correspondente ao de Cryptosporidium meleagridis. O uso de métodos moleculares pode ser útil para identificar a fonte das infecções e os fatores de risco relacionados à infecção por Cryptosporidium em nossas comunidades.

Cryptosporidium isolates identified in fourteen stool samples, collected from five HIV-infected patients and nine immunocompetent children, living in the Sate of São Paulo, Brazil, were submitted to a molecular analysis using a nested PCR followed of restriction fragment length polymorphism (RFLP), for genetic characterization. The analysis was based on digestion with RsaI restriction enzyme of a DNA fragment amplified from the Cryptosporidium oocyst wall protein (COWP) gene. Based on this analysis, four samples were identified as Cryptosporidium parvum, eight as Cryptosporidium hominis and two presented a profile that correspondedto Cryptosporidium meleagridis when compared to the standards used in the analysis. The use of molecular methods can be helpful to identify source of infections and risk factors related to Cryptosporidium infection in our communities.

Considerando a escassez de antígenos quimicamente definidos, realmente úteis e confiáveis para aplicação na soroepidemiologia da esquistossomose em larga escala, foi proposto, neste trabalho, um método alternativo para a seleção de clones de cDNA que expressam proteínas com putativo potencial diagnóstico na esquistossomose. Empregando anticorpos específicos contra uma fração proteica de 31/32 kDa (Sm31/32), purificados através da dissociação de imunocomplexos, foram selecionados cinco clones de cDNA a partir de genoteca de verme adulto de Schistosoma mansoni. O seqüenciamento parcial destes clones demonstrou que todos eram relacionados ao S. mansoni: dois apresentaram homologia com a proteína de choque térmico de 70 kDa e os demais com glutationa S-transferase, "homeodomain protein" e uma etiqueta de seqüência expressa (EST). Este último foi o clone que melhor reagiu, durante o processo de seleção, com os anticorpos anti-Sm31/32 dissociados de imunocomplexos. Baseado na seqüência de aminoácidos deste clone, dois peptídeos foram quimicamente sintetizados e analisados separadamente frente a misturas de soros de indivíduos normais e de pacientes com esquistossomose mansoni. Ambos os peptídeos demonstraram uma intensa reatividade somente contra a mistura de soros positivos, sugerindo que estes peptídeos podem ser úteis como antígenos para o diagnóstico da esquistossomose mansoni.

Considering the scarcity of defined antigens, actually useful and reliable for use in the field studies, we propose an alternative method for selection of cDNA clones with potential use in the diagnosis of schistosomiasis. Human antibodies specific to a protein fraction of 31/32 kDa (Sm31/32), dissociated from immune complexes, are used for screening of clones from an adult worm cDNA library. Partial sequencing of five clones, selected through this strategy, showed to be related to Schistosoma mansoni: two were identified as homologous to heat shock protein 70, one to glutathione S-transferase, one to homeodomain protein, and one to a previously described EST (expressed sequence tag) of S. mansoni. This last clone was the most consistently reactive during the screening process with the anti-Sm31/32 antibodies dissociated from the immune complexes. The complete sequence of this clone was obtained and the translation data yielded only one ORF (open reading frame) that code for a protein with 57 amino acids. Based on this amino acid sequence two peptides were chemically synthesized and evaluated separately against a pool of serum samples from schistosomiasis patients and non-schistosomiasis individuals. Both peptides showed strong reactivity only against the positive pool, suggesting that these peptides may be useful as antigens for the diagnosis of schistosomiasis mansoni.

A study was undertaken to search for DNA recombinant Schistosoma mansoni proteins responsible for eliciting an antibody response from the host at a very early phase after infection. A S. mansoni adult worm cDNA expression library was screened using pooled sera from baboons with four weeks of infection. Based on their specific reactivity with the S. mansoni infected sera and no reactivity when tested against the pre-infection sera from the same baboons, four clones were selected for further studies. Sequence analysis revealed that they were homologous to the S. mansoni heat shock protein 70 (hsp70). The insert sizes of the four selected clones varied from 1150 to 2006 bp. The preliminary characterization for antibody reactivity against a panel of baboon sera showed that the longest clone was the most reactive, eight out of eight acute and three out of four chronic sera reacting positively to this clone. The shortest clone was the least reactive. Our results suggest that the S. mansoni hsp70 elicits an early and strong antibody response in baboons and that antibodies to this protein can be detected in chronically infected animals. Therefore S. mansoni hsp70 may be a valid target for immunodiagnosis. However further studies are needed to identify the portion of the hsp70 that best fits the requirements for a valuable diagnostic antigen.

The high sensitivity and the ability to diagnose schistosomiasis in a very early phase after infection have indicated the detection of IgM antibodies to Schistosoma mansoni gut antigens by the immunofluorescence test (IgM-IFT) as a useful serological test for epidemiological studies in low endemic areas. When applied in a follow-up study for two years, higher rates of seroconversion from IFT negative to positive were observed during the summer months, suggesting seasonal transmission of schistosomiasis in the rural area of the municipality of Itariri (São Paulo, Brazil). In each survey, blood samples from about 600 schoolchildren were collected on filter paper and submitted to IgM-IFT. When the blood samples were classified for the IgM antibody levels, according to the intensity of fluorescent reaction observed at fluorescence microscopy, and correlated to the egg counts in the Kato-Katz positive patients, no association was observed. This observation might suggest that the intensity of fluorescence observed in the IgM-IFT, as an indicator of IgM antibody levels, could not be an useful seroepidemiological marker for classifying areas of low endemicity according to degrees of infection.

Dados de prevalência e incidência da esquistossomose foram estimados, acompanhando-se um grupo de escolares residentes em área rural do município de Itariri (São Paulo, Brasil), por um período de 2 anos, com cinco inquéritos, um a cada semestre, realizados no primeiro semestre de cada ano entre março e abril e no segundo, entre setembro e outubro. O hospedeiro intermediário do Schistosoma mansoni na área é a Biomphalaria tenagophila. A infecção pelo S. mansoni foi determinada pelo método parasitológico de Kato-Katz, através do exame de três lâminas, e os resultados analisados comparativamente aos da reação de imunofluorescência para detecção de anticorpos IgM (RIF-IgM). Foram encontrados nos cinco inquéritos índices de prevalência de 8,6%, 6,8%, 9,9%, 5,8% e 17,2% pelo método parasitológico, e de 56,5%, 52,6%, 60,8%, 53,5% e 70,1%, pelo sorológico. As médias geométricas do número de ovos por grama de fezes (opg) foram baixas: 57,8, 33,0, 35,6, 47,3 e 40,9 opg, respectivamente do 1º ao 5º inquérito. Dos 299 escolares que se submeteram à RIF-IgM nos cinco inquéritos, 40% apresentaram-se positivos e 22% negativos no decorrer do acompanhamento. Soroconversão de negativo para positivo pela RIF-IgM, indicando provável infecção recente, foi observado com maior frequência nos inquéritos realizados no primeiro semestre (pós-verão), que no segundo semestre. A pesquisa de ovos nas fezes não mostrou sensibilidade para detectar essa sasonalidade. Os resultados indicam a superioridade da RIF-IgM para determinação de incidência de S. mansoni em áreas de baixa endemicidade.

For a period of 2 years, five follow-up measures of prevalence and incidence rates were estimated in a prospective study of S. mansoni infection in a group of schoolchildren who were living in a rural area of the Municipality of Itariri (São Paulo, Brazil), where schistosomiasis is transmitted by Biomphalaria tenagophila. Infection was determined by the examination of three Kato-Katz stool slides, and the parasitological findings were analyzed in comparison to serological data. In the five surveys, carried out at 6-month intervals (March-April and September-October), the prevalences were, respectively, 8.6, 6.8, 9.9, 5.8 and 17.2% by the Kato-Katz, and 56.5, 52.6, 60.8, 53.5 and 70.1% by the immunofluorescence test (IFT). Geometric mean egg counts were low: 57.8, 33.0, 35.6, 47.3 and 40.9 eggs per gram of feces, respectively. Of the total of 299 schoolchildren, who submitted five blood samples at 6-month intervals, one for each survey, 40% were IFT-positive throughout the study, and 22% were IFT-negative in all five surveys. Seroconversion from IFT negative to positive, indicating newly acquired S. mansoni infection, was observed more frequently in surveys carried out during March-April (after Summer holidays), than during September-October. Seasonal trends were not statistically significant for detection of S. mansoni eggs in stool. The results indicate that the use of IgM-IFT is superior to parasitological methods for detection of incidence of S. mansoni infection in areas with low worm burden.

O potencial diagnóstico dos anticorpos IgM e IgA contra antígenos do tubo digestivo do Schistosoma mansoni, detectados através da reação de imunofluorescência indireta utilizando-se cortes parafinados de vermes adultos, foi avaliado, comparativamente aos resultados do exame parasitológico de fezes, para fins epidemiológicos em áreas de baixa endemicidade para a esquistossomose. Amostras de sangue em papel de filtro foram coletadas de escolares, residindo em duas localidades diferentes dentro do município de Itariri (São Paulo, Brasil), com características epidemio-lógicas distintas em relação à esquistossomose. Os dados parasitológicos e sorológicos foram comparados aos obtidos com um outro grupo de escolares, residentes em uma área não endêmica para esquistossomose. Os resultados demonstraram a falta de sensibilidade do método parasitológico para detecção de indivíduos com baixa carga parasitária e indicam a potencialidade do método sorológico como importante instrumento a ser incorporado aos programas de controle e de vigilância da esquistossomose, para verificação da real situação da esquistossomose em áreas de baixa endemicidade.

The diagnostic potential of circulating IgM and IgA antibodies against Schistosoma mansoni gut-associated antigens detected by the immunofluorescence test (IFT) on adult worm paraffin sections was evaluated comparatively to the fecal parasitological method, for epidemiological purposes in low endemic areas for schistosomiasis. Blood samples were collected on filter paper from two groups of schoolchildren living in two different localities of the municipality of Itariri (São Paulo, Brazil) with different histories and prevalences of schistosomiasis. The parasitological and serological data were compared to those obtained for another group of schoolchildren from a non-endemic area for schistosomiasis. The results showed poor sensitivity of the parasitological method in detecting individuals with low worm burden and indicate the potential of the serological method as an important tool to be incorporated into schistosomiasis control and vigilance programs for determining the real situation of schistosomiasis in low endemic areas.

The potential of an immunofluorescence test for detection of IgM antibodies against Schistosoma mansoni gut-associated antigens (IgM-IFT) was evaluated as a tool for studying aspects related to the schistosomiasis transmission in Ribeirão Pires, in the metropolitan area of the capital of the State of São Paulo, Brazil. Children from a school with about 400 students, 6 to 18 years, were followed-up for two years. In the five surveys, carried out at 6-month intervals, from October 92 to October 94, serological (IgM-IFT) prevalence indices of 5.3%, 5.8%, 6.2%, 2.9% and 3.3% were obtained. These indices were 7 to 10 times higher than the parasitological prevalence indices of 0.5%, 0.5%, 0.7%, 0.4% and 0% determined by the Kato-Katz method. Seroconversion from IFT negative to positive was indicating possible newly acquired S. mansoni infection in three children. But confirmation of infection by fecal examination was possible in only one child. The IgM-IFT can constitute a valuable tool for the improvement of the vigilance program in low endemic areas for schistosomiasis, better characterizing the S. mansoni transmission in such areas.

The high sensitivity and the possibility of automation of the enzyme-linked-immunosorbent-assay (ELISA) has indicated this technique as one of the most useful serological test for epidemiological studies. In the present study, an ELISA for detection of IgG antibodies against adult worm antigens (IgG-ELISA) was investigated for epidemiological purposes, in a rural area of the municipality of Itariri (São Paulo, Brazil). Blood on filter paper (1,180 samples) from about 650 schoolchildren were submitted to ELISA and the data compared to the results of the parasitological method of Kato-Katz and also to the IgM-IFT (immunofluorescence test for IgM antibodies to gut associated antigens). The prevalence rates respectively of 8.5%, 43.0%, and 56.2% by the Kato-Katz, IgG-ELISA, and IgM-IFT methods suggest the poor sensitivity of the parasitological method for detection of Schistosoma mansoni eggs in individuals with low worm burden, situation commonly observed in low endemic areas. These results can partially explain the poor degree of agreement between the IgG-ELISA and the Kato-Katz, as suggested by the Kappa index of 0.170. Otherwise, the Kappa index of 0.675 showed substantial agreement between the two serological tests. Some discrepancy of results between the two serological techniques must be better investigated.

Uma biblioteca de expressão de cDNA de verme adulto de S. mansoni foi selecionada utilizando-se soros de macacos babuínos em fase inicial da infecção. Os clones que reagiram positivamente com os soros de infecção recente foram examinados quanto à reatividade contra soros normais e soros de infecção heteróloga. Com a finalidade de se conseguir melhor discriminação entre reatividade positiva com o anticorpo específico e aquela devido aos anticorpos anti-E. coli residuais, um clone de cDNA não relacionado ao S. mansoni foi plaqueado em mistura com o clone positivo. O plano de fundo negativo proporcionado pelo clone não relacionado forneceu o contraste necessário para discriminar a reação positiva específica. Esta metodologia de caracterização dos clones de expressão permite alcançar rapidamente os clones que apresentam o perfil de reatividade desejada, para posterior sequenciamento, subclonagem e expressão

A S. mansoni adult worm cDNA expression library was screened with sera from baboons in a early phase after infection. The clones that were positive with the early infection sera were examined for reactivity with pre-infection sera and heterologous infection sera. In order to discriminate a positive antibody reaction from the reactivity due to residual anti-E. coli antibodies, an unrelated cDNA clone was plated with the positive clone. The unrelated clone provided the negative background and the contrast necessary to discern a positive antibody reaction. In this way, we were able to eliminate selected clones that were positive with the pre-infection sera or heterologous infection sera. This characterization of the expression library clones enabled us to quickly target only clones with the desired pattern of antibody reactivity for sequencing, subcloning, and expressing

Em pacientes com esquistossomose, são encontrados anticorpos contra grande número de antígenos parasitários, e aqueles contra formas evolutivas jovens do parasita demonstraram que eram eficientes marcadores imunológicos para o diagnóstico da esquistossomose. Padrões de queda de anticorpos IgM e IgG contra cercária e esquistossômulo foram aqui estudados, comparativamente aos dos anticorpos contra verme e ovo, em pacientes esquistossomóticos após quimioterapia, abordando aspectos soroepidemiológicos. Dados obtidos no estudo de 359 amostras de soros, pertencentes a pacientes infectados por Schistosoma mansoni, indivíduos não infectados e pacientes acompanhados pós-tratamento por um período de 12 a 15 meses, forneceram base para postular padrão geral da cinética de queda de anticorpos. Antes do tratamento, o padrão de anticorpos é representado por curva unimodal, que se modifica para curva bimodal após tratamento, e finaliza com curva unimodal semelhante à do grupo de indivíduos não infectados. Diferentes tipos de anticorpos foram classificados em quatro categorias, conforme características apresentadas na queda, e o anticorpo IgM anti-esquistossômulo foi considerado como pertencente à categoria de anticorpos com queda moderada, enquanto que os demais anticorpos contra formas evolutivas jovens do parasita, à categoria daqueles com queda lenta. A metodologia aqui empregada permite identificar anticorpos potencialmente mais adequados a serem detectados em programas para controle da esquistossomose ou de outras parasitoses no campo

Antibodies to a number of parasite antigens are found in schistosomiasis patients, and antibodies to early developmental stages were demonstrated to be efficient immunologic markers for the diagnosis of schistosomiasis. In the present study, decay patterns of IgM and IgG antibodies against cercariae and schistosomula were investigated, in comparison to antibodies against worms and eggs in schistosomiasis patients after chemotherapy, for an investigation of seroepidemiologic aspects. Data obtained in the study of 359 serum samples from patients with Schistosoma mansoni infection, noninfected individuals, and patients followed-up for a period of 12 to 15 months after treatment provided the basis to postulate a general pattern for the kinetics of antibody decay. Before treatment, the antibody pattern was represented by a unimodal curve, which shifted to a bimodal curve after treatment, and ended with a unimodal curve similar to that for the noninfected group. Different types of antibodies were classified into four categories according to their decay features, and anti-schistosomulum IgM was classified into the moderate-decay caterogy, whereas other antibodies to early parasite stages were classified into the slow-decay category. The present methodology permits the identification of the most suitable antibodies to be detected in field control programs for schistosomiasis or other parasitoses

Estudou-se a cinética de anticorpos anti-Toxocara em camundongos BALB/c infectados experimentalmente segundo três esquemas: Grupo I (GI): 25 camundongos infectados com dose única de 200 ovos embrionados de T. canis; grupo II (GII): 25 camundongos infectados com 150 ovos embrionados de T. canis, divididos em três doses de 50 ovos, administrados no 1º, 5º e 8º dias; Grupo III (GIII): 25 camundongos infectados com 150 ovos embrionados de T. canis, administrados em três doses de 50 ovos no 1º, 14º e 28º dias. Um grupo de 15 camundongos foi mantido nas mesmas condições, porém sem infecção, constituindo o grupo controle (GIV). No 30º, 50º, 60º, 75º, 105º e 180º dias pós-infecção três camundongos dos grupos GI, GII e GIII e dois do grupo controle foram sacrificados sob anestesia e sangrados para obtenção de soro. No 360º dia os animais restantes dos quatro grupos foram igualmente sacrificados. Pesquisou-se a presença de anticorpos anti-Toxocara nos soros obtidos utilizando-se teste imunoenzimático (ELISA) e empregando-se antígeno de excreção-secreção produzido por larvas de T. canis mantidas em cultura. Nos grupos GI e GII revelou-se a presença de anticorpos anti-Toxocara, em níveis elevados, pelo menos até o 180º dia após infecção. Nos camundongos do grupo GIII apenas no 30º dia pós-infecção foi possível detectar anticorpos anti-Toxocara.

Anti-Toxocara antibody production and persistence were studied in experimental infections of BALB/c mice, according to three different schedules: Group I (GI) - 25 mice infected with 200 T. canis eggs in a single dose; Group II (GII) 25 mice infected with 150 T. canis eggs given in three occasions, 50 in the 1st, 50 in the 5th and 50 in the 8th days; Group III (GIII) - 25 mice also infected with 150 T. canis eggs, in three 50 eggs portions given in the 1st, 14th and 28th days. A 15 mice control group (GIV) was maintained without infection. In the 30th, 50th, 60th, 75th, 105th and 180th post-infection days three mice of the GI, GII and GIII groups and two mice of the control group had been sacrificed and exsanguinated for sera obtention. In the 360th day the remainder mice of the four groups were, in the same way, killed and processed. The obtained sera were searched for the presence of anti-Toxocara antibodies by an ELISA technique, using T. canis larvae excretion-secretion antigen. In the GI and GII, but not in the GIII, anti-Toxocara antibodies had been found, at least, up to the 180th post-infection day. The GIII only showed anti-Toxocara antibodies, at significant level, in the 30th post-infection day.

A esquistosomose humana, causada pelo Schistosoma mansoni, é altamente prevalente no Brasil e é usualmente diagnosticada através de exames de fezes, que são demorados. Os testes sorológicos nesta doença limitam-se aos estudos epidemiológicos, mostrando apenas o contato prévio com o parasita e não a infecção realmente ativa. Neste trabalho, nós padronizamos e comparamos um novo ensaio de Dot-ELISA, para detecção de IgM e IgG específicas contra antígenos de ovo e vermes de S. mansoni, comparando seus resultados com a técnica clássica de imunofluorescência (IFI), tanto para IgG ou IgM, utilizando antígenos similares, no estudo de uma população de 27 pacientes com quantificação de ovos nas fezes. A positividade do teste de Dot-ELISA foi de 96,3% para IgG e 88,9% para IgM, com antígeno de verme, e 92,6% para IgG e 90,9% para IgM com antígeno de ovo, que foi comparado com a IFI que apresentou resultados similares com antígeno de verme, 92,6% (IgG) e 96,3% (IgM) e menores com antígeno de ovo, 77,8% (IgG e IgM). Nosso grupo de pacientes claramente apresentava dois subgrupos, quanto à excreção de ovos nas fezes, com maior positividade dos testes sorológicos para o grupo composto por pacientes com maior excreção fecal de ovos. Quando comparamos a excreção quantitativa de ovos nas fezes com os títulos sorológicos, detectamos apenas correlação entre estes valores e o Dot-ELISA IgM (r=0,552 p=0.0127). Estes dados mostram que o teste de Dot-ELISA pode ser usado para a detecção de anticorpos específicos contra o S. mansoni em soros de pacientes suspeitos, para estudos epidemiológicos de prevalência; e, com maior purificação dos antígenos de ovos e amostragem ampliada, poderá tornar-se um teste útil para uma estimativa grosseira da carga parasitária em estudos epidemiológicos.

Human schistosomiasis, caused by Schistosoma mansoni, is highly prevalent in Brazil and usually diagnosed by time consuming stool analysis. Serological tests are of limited use in this disease, mainly for epidemiological studies, showing no discrimination between previous contact with the parasite and active infections. In the present study, we standardized and compared a Dot-ELISA for IgM and IgG antibodies against S. mansoni antigens from eggs and worms with a routine IgG and IgM immunofluorescence assay using similar antigens, in the study of sera from 27 patients who had quantified egg stool excretion. The positivity obtained for IgG Dot-ELISA was 96.3% and 88.9% for IgM Dot-ELISA with worm antigen and 92.6% and 90.9% with egg antigen. The IFI presented similar positivities using worm antigen, 92.6% (IgG) and 96.3% (IgM),and lower results with egg antigen, 77.8% (IgG and IgM). The patients studied were divided into two groups according to their egg excretion, with greater positivity of serological tests in higher egg excreters. When comparing the quantitative egg excretion and the serological titers of the patients, we detected a correlation only with IgM Dot-ELISA, with r=0.552 (p=0.0127). These data show that Dot-ELISA can be used for the detection of specific antibodies against S. mansoni in sera from suspected patients or in epidemiological studies and, with further purification of egg antigen and larger samples, IgM Dot-ELISA could be a possible tool for rough estimates of parasite burden in epidemiological studies.