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A real-time PCR method targeting the small subunit of the rRNA gene was developed for the detection of Strongyloides stercoralis DNA in faecal samples, including an internal control to detect inhibition of the amplification process. The assay was performed on a range of well-defined control samples (n=145), known positive faecal samples (n=38) and faecal samples from a region in northern Ghana where S. stercoralis infections are highly endemic (n=212), and achieved 100% specificity and high sensitivity. The use of this assay could facilitate monitoring the prevalence and intensity of S. stercoralis infections during helminth intervention programs. Moreover, the use of this assay in diagnostic laboratories could make the introduction of molecular diagnostics feasible in the routine diagnosis of S. stercoralis infections, with a two-fold increase in the detection rate as compared with the commonly used Baermann sedimentation method.

Transmission of Schistosoma mansoni depends on fecal eggs reaching water, but the way this happens is poorly understood. We studied the role of hygienic bathing after defecation in the contamination of water with S. mansoni eggs. Individuals in an endemic community in Northern Senegal (n=991) were examined for S. mansoni infection and a random sample (22%) was interviewed about stool disposal practices and hygienic behavior. We assessed the presence and viability of S. mansoni eggs adhering to the peri-anal region of 13 infected volunteers, by counting the miracidia in the water they had used for hygienic washing; for 10 of them (77%) miracidia were demonstrated. From the population infection distribution, average number of defecations per day, proportion of individuals bathing after defecation, and association between miracidial counts and infection intensity, we calculated a daily population miracidial output of approximately 30,000 through hygienic bathing. For comparison, one complete stool reaching the water was calculated to yield approximately 2500 miracidia. Thus, 12 individuals in this population should defecate into the water every day to produce the same number of miracidia as through hygienic bathing. Our results suggest a major role of hygienic bathing after defecation in the transmission of S. mansoni.

Objective: To examine the relationship of past and current intestinal helminth infections with asthma, allergic rhinoconjunctivitis, atopic dermatitis and atopy., Methods: Cross-sectional study of 1320 children aged 4-14 years from two Cuban municipalities. Helminth infections were determined by stool examination and parental questionnaire. Asthma, rhinoconjunctivitis and atopic dermatitis were diagnosed by International Study of Asthma and Allergies in Childhood questionnaire, asthma additionally by spirometry, atopy by skin prick testing., Results: Questionnaire-based frequencies were 21% for asthma, 14% for allergic rhinoconjunctivitis and 8% for atopic dermatitis. According to spirometry, 4% had asthma; 20% had a positive skin prick test. A history of infection for Enterobius vermicularis was associated with increased risk of atopic dermatitis (OR 1.88, P = 0.001) and allergic rhinoconjunctivitis (OR 1.34, P = 0.046), and hookworm with increased risk of allergic rhinoconjunctivitis (OR 2.77, P = 0.021). A positive stool examination for Ascaris lumbricoides infection was negatively associated with atopic dermatitis (OR 0.22, P = 0.007). Asthma and atopy were unrelated to helminth infections., Conclusion: Current A. lumbricoides infection protects against atopic dermatitis in Cuban children, while past infection with E. vermicularis and hookworm are risk factors for allergic rhinoconjunctivitis and/or atopic dermatitis. Apparently, interactions differ depending on the type of helminth and atopic disease and on the time of helminth infestation.

A multiplex real-time PCR assay for the detection and quantification of Schistosoma mansoni and S. haematobium DNA in faecal samples was developed and evaluated as an alternative diagnostic method to study the epidemiology of schistosomiasis. Primers and probes targeting the cytochrome c oxidase gene were designed for species-specific amplification and were combined with an internal control. Using positive control DNA extracted from adult Schistosoma worms and negative control samples (n=150) with DNA from a wide range of intestinal microorganisms, the method proved to be sensitive and 100% specific. For further evaluation, duplicate stool specimens with varying S. mansoni egg loads were collected in northern Senegal from pre-selected individuals (n=88). The PCR cycle threshold values, reflecting parasite-specific DNA loads in faeces, showed significant correlation with microscopic egg counts both for S. mansoni in stool and S. haematobium in urine. The Schistosoma detection rate of PCR (84.1%) was similar to that of microscopy performed on duplicate stool samples (79.5%). The simple faecal sample collection procedure and the high throughput potential of the multiplex real-time PCR provide a powerful diagnostic tool for epidemiological studies on schistosomiasis in remote areas, with possibilities for extension to other helminths or protozoa using additional molecular targets.

Objectives: To determine the prevalence of intestinal parasite infections and their risk factors in children in urban and rural settings in two Cuban municipalities., Methods: A total of 1320 Cuban schoolchildren aged 4-14 were tested by stool examination for intestinal parasite infections and evaluated by parental questionnaire for a number of common environmental, sanitary, socioeconomic and behavioural risk factors. Multivariate regression was applied to examine the relationship between the respective parasite infections and the risk factors., Results: Prevalences of intestinal parasite infections were 58% in Fomento and 45% in San Juan y Martínez; for helminth infections, these were 18% and 24% and for protozoa infections, 50% and 29%, respectively. Helminth infections were associated with high parental education (maternal: OR 0.68, CI 0.50-0.93; paternal: OR 0.71, CI 0.52-0.96), absence of toilet (OR 1.57, CI 1.12-2.19), consumption of water from a well or river (OR 0.56, CI 0.41-0.77) and eating unpeeled/unwashed fruit (OR 1.37, CI 1.01-1.87); protozoa infections were only associated with high maternal education (OR 0.72, CI 0.57-0.91)., Conclusions: Paediatric intestinal parasite infections are still prevalent in certain areas in Cuba and associated with a number of common environmental, socioeconomic and sanitary risk factors.

Background: HIV-1-negative children with malaria have reversible lymphocyte and CD4 count decreases. We assessed the impact of malaria parasitemia on the absolute CD4 count in both HIV-1-infected and non-HIV-infected adults., Methods: In Ndola, Zambia, at the health-center level, we treated 327 nonpregnant adults for confirmed, uncomplicated, clinical malaria. We assessed HIV-1 status, CD4 count, and HIV-1 viral load (if HIV-1-infected) at enrollment and at 28 and 45 days after treatment., Results: After successful antimalarial treatment, the median CD4 count at day 28 of follow-up increased from 468 to 811 cells/microL in HIV-1-negative and from 297 to 447 cells/microL in HIV-1-positive patients (paired t test, P < 0.001 for both). CD4 count increment was inversely correlated with CD4 count at day 0 in both HIV-1-negative (P < 0.001) and HIV-1-positive patients (P = 0.03). After successful treatment, the proportion of patients with CD4 count <200/microL at day 45 decreased from 9.6% to 0% in HIV-1-negative and from 28.7% to 13.2% in HIV-1-positive malaria patients (P < 0.001 for both). In patients with detectable but mostly asymptomatic parasitemia, CD4 count and, if HIV-1-infected, viral load at day 45 of follow-up were similar to those observed at enrollment., Conclusion: Interpretation of absolute CD4 count might be biased during or just after a clinical malaria episode. Therefore, in malaria-endemic areas, before taking any decision on the management of HIV-1-positive individuals, their malaria status should be assessed.

The transmission of intestinal schistosomiasis presumes that faecal materials containing viable schistosome eggs reach natural water bodies infested with snail intermediate host. So far there is little knowledge about the contamination dynamics of streams with schistosome eggs. We conducted a pilot study on defecating behaviour and hygienic practices in an Schistosoma mansoni endemic focus in Northern Senegal. Questionnaires were used to obtain quantitative data on hygienic practices and the use of latrines in 59 children. Although the community was well endowed with pit latrines, most of the children declared that they usually defecated somewhere else, in particular near the streams where the vegetation offers hideouts. Observations based on mapping of defecation sites showed that a considerable number of stools were left just a few meters from the riverbank, thus bearing a high risk of being washed off into the water. All these practices can easily lead to contamination of water bodies with schistosomae eggs. In order to improve hygienic practices and reduce fecal pollution of the environment, a health education model respecting local beliefs and customs would be indispensable.

We evaluated the awareness of and knowledge about intestinal schistosomiasis in a highly infected rural community of northern Senegal where a variety of health information and education activities had taken place for 7 years as a component of different research and control programmes. As the infection had been introduced only recently, an initial 'zero' knowledge can be assumed. Most of the health education activities had been performed with adapted messages through local health and community workers. By a questionnaire, 566 individuals were asked simple questions on symptoms, mode of transmission, the sources of information and health-seeking behaviour. About 86% of the respondents stated that they knew what schistosomiasis was, and 92% that in case of illness they would seek treatment at the health centre. However, only half of the people accurately quoted symptoms associated with intestinal schistosomiasis: diarrhoea, abdominal pain and bloody stools. The majority of respondents realized that the disease was somehow linked with water and (lack of) hygiene, but only 44% of respondents reported water contact as the source of infection. Ultimately, only 30% of the respondents gave adequate answers about both symptoms and mode of transmission. We conclude that even intense and long-lasting education efforts for a specific and straightforward problem as schistosomiasis are not enough to have profound impact on the knowledge of rural traditional communities.

Surprisingly low cure rates were repeatedly observed after treatment with a standard dosage of praziquantel in a recently established Schistosoma mansoni focus in northern Senegal. In 4 discrete cohorts from the same population, cure rates were 18-36% and egg count reduction rates were 77-88%. Data and material of 920 compliant subjects from all 4 cohorts were further analyzed to identify possible host-related factors associated with low cure rates. The lowest cure rates were found in the highest egg count groups. However, in low and moderate egg count groups, drug efficacy was also below normal values. Cure rates were similar in males and females, showed no seasonal variation, and were independent of previous praziquantel treatment. They were significantly higher in adults than in children, also after allowing for intensity of infection. Individual water contact behavior and specific humoral immune responses were examined in 2 extreme subgroups, either without significant egg count reduction or showing complete parasitologic cure. There was no significant difference in frequency and duration of water contact between those individuals with complete cure and those that showed little effect of praziquantel treatment. Levels of IgG, IgG1, IgG3, IgG4, IgM, and IgE against adult worm antigen were not different between the 2 subgroups. Thus, the abnormally frequent failure of treatment observed in this focus could not be associated with any host-related factor, other than age and pretreatment egg counts.

Day-to-day fluctuations of both circulating anodic antigen (CAA) and circulating cathodic antigen (CCA) in serum and urine were examined simultaneously in a group of Schistosoma mansoni-infected individuals from Burundi and compared with each other and with fecal egg count fluctuations. Significant correlations were found between fecal egg counts and circulating antigens (CAA and CCA) and between circulating antigen levels in serum and urine samples. The cumulative percentage of positive results after three samplings was highest for urine CCA detection, followed by fecal egg counts, serum CCA, serum CAA, and urine CAA detection, respectively. It was demonstrated that circulating antigen levels in both serum and urine showed less fluctuation than fecal egg counts, except for urine CAA levels. The serum CAA detection assay in particular, although less sensitive in this low endemic area in Burundi, gave very constant measurements over a period of one week. Our results indicate that detection of circulating antigens in a single serum or urine sample provides a quantitatively more stable diagnosis of S. mansoni infection than fecal egg counts based on a single stool examination.

A monoclonal antibody-based enzyme-linked immunosorbent assay detecting Schistosoma mansoni circulating soluble egg antigen (CSEA) was applied in epidemiological studies. The serum CSEA levels were determined for 2 populations with a high prevalence (> 95%) and high intensity of infection as determined by faecal egg counts. In one population (Maniema, Zaire) transmission had been occurring for several decades, while in the other population (Ndombo, Senegal) transmission had started only recently. CSEA could be detected in 88% and 70% of the serum samples from Maniema and Ndombo, respectively. The sensitivity of the CSEA assay increased with rising egg count. The age-related CSEA profiles of the Maniema population followed a pattern similar to that of egg counts and of the adult worm antigen CAA (circulating anodic antigen). However, the recently infected Ndombo population showed a clearly different profile: while the CSEA prevalence reached a peak in children and adolescents, the mean CSEA levels did not vary significantly in the different age groups. CSEA levels were significantly lower in Ndombo than in Maniema. As egg antigens in serum are thought to be in part, or even primarily, derived from eggs in the tissues, these findings indicate a relatively smaller tissue egg load in Ndombo than in Maniema.

Serum levels of the adult schistosome circulating anodic antigen (CAA) were compared in 2 populations, both living in an area with extremely high transmission levels of Schistosoma mansoni. In one focus (Maniema, eastern Zaire) transmission has been established for several decades, while in the other focus (Ndombo, northern Senegal) transmission started only recently. While parasite egg counts in the 2 populations were virtually similar, including analogous age-related distributions, serum levels of CAA were approximately 5 times higher in the chronically exposed community. This difference in antigen level was most pronounced in adolescents and adults. As the level of CAA is assumed to be a direct reflection of worm burden, these findings suggest higher parasite fecundity in the recently exposed community. It is not very likely that these observations could be explained solely by differences in clearance mechanisms caused by a variation in experience of infection. The relationship between circulating antigen levels and egg counts was consistent for all age groups in the Maniema population, while in the Ndombo population only children showed a pattern similar to that in the chronically exposed community.

A therapeutic trial, involving 130 Schistosoma mansoni-infected children, with no previous history of antischistosomal treatment, was carried out to evaluate the efficacy of two different dose regimens of praziquantel. The study was carried out because low cure rates were described in this recently established (1990) S. mansoni focus in northern Senegal, following treatment with a standard dosage of 40 mg/kg. The subjects were randomly allocated into two groups: one group (1) received 40 mg/kg in one oral dose, the other group (2) was treated with two oral doses of 30 mg/kg at a 6-hr interval. Parasitologic examination and circulating anodic antigen (CAA) detection were performed before, 10 days, three, six, and 21 weeks after chemotherapy. No significant differences in cure rates were found between the two groups. Six weeks after treatment, 34% and 44% of the individuals were found to be stool negative in group 1 and group 2, respectively. However, only 10-15% became completely negative according to the serum CAA antigen assay. Mean egg counts were reduced by 99% in both groups. Antigen detection confirmed the parasitologic results. Fewer side effects were observed in the group treated with 2 x 30 mg/kg, which may be explained by split dosage administration. Our study shows that the low cure rates observed in this area could not be improved by using a higher dosage of praziquantel.

Quantitative enzyme-linked immunosorbent assays (ELISAs) for the detection of circulating anodic antigen (CAA) and circulating cathodic antigen (CCA) in serum and urine were applied as an epidemiologic tool in a recent, intense focus of Schistosoma mansoni in Senegal. Both CAA and CCA in serum and CCA in urine were found in 94%, 83%, and 95%, respectively, of the population, of which 91% were positive on stool examination. Circulating antigens were also detectable in sera and urines of most egg-negative individuals. The sensitivities of the urine CCA and serum CAA ELISA were substantially higher than that of a single egg count, and increased with egg output. The CAA and CCA levels correlated well with egg counts and with each other. The age-related evolution of antigen levels followed a similar pattern as egg counts, providing supplementary evidence for a genuine reduction of worm burdens in adults in spite of the supposed absence of acquired immunity in this recently exposed community. The antigen:egg ratios decreased in adults, suggesting lower worm fecundity in children. This would be compatible with a density-dependent reduction of fecundity, but not with anti-fecundity immunity in adults that perhaps has not yet developed in this new focus.

Schistosoma mansoni was first reported in the area of Richard Toll (Senegal) in 1988 and spread rapidly in the community, after a series of human-engineered ecologic changes. A random population sample (n = 422) from Ndombo, a village near Richard-Toll, was studied in 1991 by stool examination (four Kato slides from two stool samples) and antigen detection in urine and blood. Stool-positive individuals were treated with 40 mg/kg of praziquantel. A house-to-house interview regarding side effects was conducted 24 hr after treatment. Two hundred ninety-eight subjects were re-examined 10 days (antigen detection) and 12 weeks (egg counts, antigen detection) after treatment. Before treatment, positive egg counts were found in 91% of the subjects, with 41% excreting more than 1,000 eggs per gram (epg) of feces. Treatment of 352 individuals caused serious but transient side effects (colic, vomiting, urticaria, and edema), the frequency of which increased with increasing egg counts. The parasitologic cure rate 12 weeks after treatment was only 18%, the frequency of egg counts with more than 1,000 epg decreased to 5%, and the mean egg count of those remaining positive was reduced by 86%. Antigen detection in serum 10 days and 12 weeks after treatment remained positive in 90% of the subjects, although titers decreased sharply. The low cure rates may be due to intense transmission and/or undeveloped immune responses in this recently exposed population. However, reduced drug susceptibility of the parasite strain has now been confirmed in one local isolate.

Schistosoma mansoni was introduced in the Senegal basin around 1988, due to man-made ecological changes. Since 1991, we investigate a recent but very intense focus, Ndombo, a village near the city of Richard Toll where the outbreak was first described. Four cohorts, each a random sample (+/- 400 subjects each) from this community, were examined and followed up after treatment, starting at 8 month intervals over a 2-year period. Each cohort is examined parasitologically (Kato-Katz), clinically, serologically (circulating antigen and antibody profiles); treated with praziquantel 40 mg/kg; followed up 6-10 weeks, one and two years after treatment; and monitored for water contact patterns and local snail densities. In the first cohort, the prevalence was 91%, with a mean egg count of 663 epg. Prevalences are near 100% in all age groups, but egg counts decline strongly in adults. Antigen detection in serum and urine confirmed that the egg counts genuinely reflect variations of worm burdens, not e.g. of worm fecundity. This is surprising, as in this focus acquired immunity in adults should not have yet developed according to current hypothesis. The antigen detection assays (CAA/CCA) showed high sensitivity and quantitative power, and promising perspectives as a research tool and possibly as a method for non-invasive diagnosis and screening in urine. Epidemiological in subsequent cohorts were highly similar, although seasonal variations were observed possibly due to transmission fluctuations. Anti-AWA and anti-SEA IgE levels increased with age, while IgG4 peaked in the age-group 10 years and correlated well with egg counts.(ABSTRACT TRUNCATED AT 250 WORDS)

Schistosoma mansoni was introduced in the Richard Toll area (Senegal) around 1988, probably due to man-made ecological changes in the Senegal river basin. Since 1991, we investigate the community of Ndombo, close to Richard Toll. Four random population samples of approximately 400 subjects are surveyed, starting at 8 months intervals. Each cohort is examined parasitologically (Kato-Katz), clinically, serologically (circulating antigen and antibody profiles); treated with praziquantel 40 mg/kg; and followed up 6-12 weeks, 1 and 2 years after treatment. Water contact patterns and snail densities are longitudinally surveyed. In the first cohort, prevalence of infection was 91%, with 41% excreting over 1000 eggs per gram (epg); the mean egg count was 646 epg, individual counts up to 24,000 epg. Prevalences remained almost 100%, but egg counts declined strongly in adults, in spite of continued exposure and the supposed lack of acquired immunity. Antigen detection in serum and urine confirmed that the egg counts genuinely reflect variations of worm burdens. Serum circulating anodic antigen (CAA) provided intriguing epidemiological information on worm burdens, while circulating cathodic antigen (CCA) showed promise for non-invasive diagnosis and screening. So far, similar epidemiological results were found in subsequent cohorts, although some variations were observed, possibly due to seasonal transmission fluctuations. IgE levels increased with age, while IgG4 peaked in the age-group 10-19 years. IgE and IgG4-levels against adult worm antigen (AWA) and soluble egg antigen (SEA) increased between cohort 1 and cohort 3 in almost all age-groups. In all 3 cohorts examined so far a strong correlation between IgG4 and pre-treatment egg-load was observed. Further follow-up and analysis, and comparison with chronically infected populations will provide insight in the development of acquired immunity. Abdominal discomfort was reported by 61% and diarrhoea by 33% of the subjects in the first cohort; mild hepatomegaly was found in 16%, splenomegaly in 0.5%. There was no correlation between frequency of symptoms and egg counts. This low morbidity, in spite of intense infections, was confirmed by ultrasound, and may be due to the recent nature of the focus. In the first cohort, 82% of treated subjects still excreted eggs 12 weeks after treatment, though egg counts declined strongly. Antigen detection confirmed these results. Parasitological negativation rates in subsequent cohorts, followed up sooner after treatment, improved but remained remarkably low. The low drug efficacy may be due to very rapid reinfection (though further reinfection after one year was limited), and/or to the lack of immunity in the population. Reduced susceptibility of the local schistosome strain can not be excluded, however. Praziquantel treatment provoked impressive but transient side effects (colics, vomiting, urticaria, oedema), the frequency of which correlated with intensity of infection.

The epidemiology of Schistosoma mansoni infection was investigated in Ndombo, a village in the epicenter of a very recent outbreak of schistosomiasis in northern Senegal. Repeated fecal egg counts and antigen detection in urine and serum were carried out in a random population sample (n = 422). Eggs were found in 91% of the subjects, with 41% excreting > 1,000 eggs per gram of feces (epg) (mean egg load of 646 epg). The prevalence was almost 100% in groups greater than five years of age. In spite of the supposed absence of acquired immunity, intensities of infection decreased strongly in adults. Antigen detection confirmed the high prevalence and intensity of infection and the age-related distribution of worm loads. The emergence of this new focus is probably due to the ecologic impact of newly built dams and the extension of irrigation projects in the Senegal basin.

A simple enzyme-linked immunosorbent assay (ELISA) for the identification of cultured mycobacteria belonging to the Mycobacterium tuberculosis complex, the Mycobacterium avium complex, and Mycobacterium kansasii has been developed (R. Schöningh, C. P. H. J. Verstijnen, S. Kuijper, and A. H. J. Kolk. J. Clin. Microbiol. 28:708-713, 1990). The test for the routine identification of cultured mycobacteria was introduced in five clinical laboratories located in Tanzania, Thailand, Vietnam, and The Netherlands. The ELISA can be conducted without an ELISA reader since the test can be read visually. The results of identification of 255 strains of the M. tuberculosis complex by microbiological means and by ELISA were compared; the specificity and the sensitivity were 100%. For the M. avium complex, the specificity was 100% and the sensitivity was 64%. All 26 M. kansasii strains tested could be identified as M. kansasii. The ELISA described here proved to be useful in both well- and modestly equipped laboratories and may replace the microbiological method of identification of M. tuberculosis and M. kansasii.