Kim Vereecken

Multiplex real-time PCR for the detection and quantification of Schistosoma mansoni and S. haematobium infection in stool samples collected in northern Senegal

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.

Epidemiology of mixed Schistosoma mansoni and Schistosoma haematobium infections in northern Senegal.

Due to the large overlap of Schistosoma mansoni- and Schistosoma haematobium-endemic regions in Africa, many people are at risk of co-infection, with potential adverse effects on schistosomiasis morbidity and control. Nonetheless, studies on the distribution and determinants of mixed Schistosoma infections have to date been rare. We conducted a cross-sectional survey in two communities in northern Senegal (n=857) to obtain further insight into the epidemiology of mixed infections and ectopic egg elimination. Overall prevalences of S. mansoni and S. haematobium infection were 61% and 50%, respectively, in these communities. Among infected subjects, 53% had mixed infections and 8% demonstrated ectopic egg elimination. Risk factors for mixed infection - i.e. gender, community of residence and age - were not different from what is generally seen in Schistosoma-endemic areas. Similar to overall S. mansoni and S. haematobium infections, age-related patterns of mixed infections showed the characteristic convex-shaped curve for schistosomiasis, with a rapid increase in children, a peak in adolescents and a decline in adults. Looking at the data in more detail however, the decline in overall S. haematobium infection prevalences and intensities appeared to be steeper than for S. mansoni, resulting in a decrease in mixed infections and a relative increase in single S. mansoni infections with age. Moreover, individuals with mixed infections had higher infection intensities of both S. mansoni and S. haematobium than those with single infections, especially those with ectopic egg elimination (P<0.05). High infection intensities in mixed infections, as well as age-related differences in infection patterns between S. mansoni and S. haematobium, may influence disease epidemiology and control considerably, and merit further studies into the underlying mechanisms of Schistosoma infections in co-endemic areas.

Associations between specific antibody responses and resistance to reinfection in a Senegalese population recently exposed to Schistosoma mansoni.

We examined associations between schistosome‐specific antibody responses and reinfection in Senegalese individuals recently exposed to Schistosoma mansoni. The effects of treatment, age, intensity of infection and duration of exposure on schistosome‐specific antibody responses were also investigated by comparing immune responses in individuals exposed for less than 3 years with responses in people exposed for more than 8 years. All individuals were bled before treatment as well as 6 and 12 weeks after. We used a statistical model that included interaction terms between time, age, infection intensity and duration of exposure. The overall patterns of most specific antibody responses by age were similar to those previously published for S. mansoni, Schistosoma japonicum and Schistosoma haematobium infections in different endemic areas. In general, a boost in specific antibody responses against adult worm antigen (SWA) was observed at 6 weeks after treatment whereas the majority of isotype responses against egg antigen (SEA) were not affected by treatment. Our analysis showed that the effect of treatment on schistosome‐specific antibody responses is influenced by age, infection intensity and duration of exposure. We found no evidence that treatment matures the specific antibody response of children recently infected with S. mansoni. Our results indicate that the build‐up of potentially protective immunoglobulin E (IgE) responses was associated with duration of exposure, or, in other words, experience of infection. Interestingly, in recently exposed individuals there was a significant association between IgA responses to SWA and resistance to reinfection. Resistance to reinfection and production of IgA‐SWA was associated with adulthood independently of exposure patterns, suggesting that susceptibility to S. mansoni and the development of protective immune responses is age‐dependent.

Bladder Morbidity and Hepatic Fibrosis in Mixed Schistosoma haematobium and S. mansoni Infections: A Population-Wide Study in Northern Senegal

Background The global distribution map of schistosomiasis shows a large overlap of Schistosoma haematobium- and S. mansoni-endemic areas in Africa. Yet, little is known about the consequences of mixed Schistosoma infections for the human host. A recent study in two neighboring co-endemic communities in Senegal indicated that infection intensities of both species were higher in mixed than in single infections. Here, we investigated the relationship between mixed Schistosoma infections and morbidity in the same population. So far, this has only been studied in children. Methods Schistosoma infection was assessed by microscopy. Schistosoma-specific morbidity was assessed by ultrasound according to WHO guidelines. Multivariable logistic regression models were used to identify independent risk factors for morbidity. Principal Findings Complete parasitological and morbidity data were obtained from 403 individuals. Schistosoma haematobium-specific bladder morbidity was observed in 83% and S. mansoni-specific hepatic fibrosis in 27% of the participants. Bladder morbidity was positively associated with S. haematobium infection intensity (OR = 1.9 (95% CI 1.3–2.9) for a 10-fold increase in intensity). Moreover, people with mixed infections tended to have less bladder morbidity than those with single S. haematobium infections (OR = 0.3 (95% CI 0.1–1.1)). This effect appeared to be related to ectopic S. mansoni egg elimination in urine. Hepatic fibrosis on the other hand was not related to S. mansoni infection intensity (OR = 0.9 (95% CI 0.6–1.3)), nor to mixed infections (OR = 1.0 (95% CI 0.7–1.7)). Conclusions/Significance This is the first population-wide study on the relationship between mixed Schistosoma infections and morbidity. Mixed infections did not increase the risk of S. mansoni-associated morbidity. They even tended to reduce the risk of S. haematobium-associated morbidity, suggesting a protective effect of S. mansoni infection on bladder morbidity. These unexpected results may have important consequences for schistosomiasis control in co-endemic areas and warrant further investigation.

Micro-Geographical Heterogeneity in Schistosoma mansoni and S. haematobium Infection and Morbidity in a Co-Endemic Community in Northern Senegal

Background Schistosoma mansoni and S. haematobium are co-endemic in many areas in Africa. Yet, little is known about the micro-geographical distribution of these two infections or associated disease within such foci. Such knowledge could give important insights into the drivers of infection and disease and as such better tailor schistosomiasis control and elimination efforts. Methodology In a co-endemic farming community in northern Senegal (346 children (0–19 y) and 253 adults (20–85 y); n = 599 in total), we studied the spatial distribution of S. mansoni and S. haematobium single and mixed infections (by microscopy), S. mansoni-specific hepatic fibrosis, S. haematobium-specific urinary tract morbidity (by ultrasound) and water contact behavior (by questionnaire). The Kulldorffs scan statistic was used to detect spatial clusters of infection and morbidity, adjusted for the spatial distribution of gender and age. Principal Findings Schistosoma mansoni and S. haematobium infection densities clustered in different sections of the community (p = 0.002 and p = 0.023, respectively), possibly related to heterogeneities in the use of different water contact sites. While the distribution of urinary tract morbidity was homogeneous, a strong geospatial cluster was found for severe hepatic fibrosis (p = 0.001). Particularly those people living adjacent to the most frequently used water contact site were more at risk for more advanced morbidity (RR = 6.3; p = 0.043). Conclusions/Significance Schistosoma infection and associated disease showed important micro-geographical heterogeneities with divergent patterns for S. mansoni and S. haematobium in this Senegalese community. Further in depth investigations are needed to confirm and explain our observations. The present study indicates that local geospatial patterns should be taken into account in both research and control of schistosomiasis. The observed extreme focality of schistosomiasis even at community level, suggests that current strategies may not suffice to move from morbidity control to elimination of schistosomiasis, and calls for less uniform measures at a finer scale.

Toxocara seropositivity, atopy and asthma: a study in Cuban schoolchildren

Evidence suggests that human toxocariasis (HT) could stimulate the onset of allergic diseases such as asthma. More specifically, in subjects having a hypothetical ‘atopic genotype’, HT could boost preexistent allergy symptoms. We tested the latter hypothesis in Cuba, a country where both asthma and HT are prevalent.

The role of hygienic bathing after defecation in the transmission of Schistosoma mansoni

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.

Associations between atopic markers in asthma and intestinal helminth infections in Cuban schoolchildren.

To cite this article: Vereecken K, Kanobana K, Wördemann M, Junco Diaz R, Menocal Heredia L, Ruiz Espinosa A, Nuñez FA, Rojas Rivero L, Bonet Gorbea M, Polman K. Associations between atopic markers in asthma and intestinal helminth infections in Cuban schoolchildren. Pediatric Allergy Immunology 2012: 23: 332–338.

The contribution of water contact behavior to the high Schistosoma mansoni Infection rates observed in the Senegal River Basin

BackgroundSchistosomiasis is one of the major parasitic diseases in the world in terms of people infected and those at risk. Infection occurs through contact with water contaminated with larval forms of the parasite, which are released by freshwater snails and then penetrate the skin of people. Schistosomiasis infection and human water contact are thus essentially linked, and more knowledge about their relationship will help us to develop appropriate control measures. So far, only few studies have related water contact patterns to infection levels.MethodsWe have conducted detailed direct water contact observations in a village in Northern Senegal during the first years of a massive Schistosoma mansoni outbreak to determine the role of human water contact in the extent of the epidemic.We quantified water contact activities in terms of frequency and duration, and described how these vary with age and sex. Moreover, we assessed the relationship between water contact- and infection intensity patterns to further elucidate the contribution of exposure to the transmission of schistosomiasis.ResultsThis resulted in over 120,000 recorded water contacts for 1651 subjects over 175 observation days. Bathing was the main activity, followed by household activities. Frequency and duration of water contact depended on age and sex rather than season. Water contacts peaked in adolescents, women spent almost twice as much time in the water as men, and water contacts were more intense in the afternoon than in the morning, with sex-specific intensity peaks. The average number of water contacts per person per day in this population was 0.42; the average time spent in the water per person per day was 4.3 minutes.ConclusionsThe observed patterns of water contact behavior are not unusual and have been described before in various other settings in sub-Saharan Africa. Moreover, water contact levels were not exceptionally high and thus cannot explain the extremely high S. mansoni infection intensities as observed in Northern Senegal. Comparison with fecal egg counts in the respective age and sex groups further revealed that water contact levels did not unambiguously correspond with infection levels, indicating that factors other than exposure also play a role in determining intensity of infection.

Individual and Combined Effects of pH and Lactic Acid Concentration on Listeria innocua Inactivation: Development of a Predictive Model and Assessment of Experimental Variability

ABSTRACT In food technology, organic acids (e.g., lactic acid, acetic acid, and citric acid) are popular preservatives. The purpose of this study was to separate the individual effects of the influencing factors pH and undissociated lactic acid on Listeria innocua inactivation. Therefore, the inactivation process was investigated under controlled, initial conditions of pH (pH0) and undissociated lactic acid ([LaH]0). The resulting inactivation curves consisted of a (sometimes negligible) shoulder period followed by a descent phase. In a few cases, a tailing phase was observed. Depending on the conditions, the descent phase contained one or two log-linear parts or had a convex or concave shape. In addition, the inactivation process was characterized by a certain variability, dependent on the severity of the conditions. Furthermore, in the neighborhood of the growth/no growth interface sometimes contradictory observations occurred. Overall, the individual effects of the influencing factors pH and undissociated lactic acid could clearly be distinguished and were also apparent based on fluorescence microscopy. Appropriate model types were developed and enabled prediction of which conditions of pH0 and [LaH]0 are necessary to obtain a predetermined inactivation (number of decimal reductions) within a predetermined time range.