Schistosome Infection Intensity Research Articles

Soluble CD23 Levels are Inversely Associated with Atopy and Parasite-Specific IgE Levels but Not with Polyclonal IgE Levels in People Exposed to Helminth Infection

Background: Protective acquired immunity against helminths and allergic sensitisation are both characterised by high IgE antibody levels. Levels of IgE antibodies are naturally tightly regulated by several mechanisms including binding of the CD23 receptor. Following observations that helminth infections and allergic sensitisation may co-present, the current study aims to investigate the relationship between the soluble CD23 (sCD23) receptor, parasite-specific IgE responses and allergic sensitisation in people exposed to the helminth parasite Schistosoma haematobium. Methods: A cohort of 434 participants was recruited in two villages with different levels of S. haematobium infection in Zimbabwe. Serum levels of the 25-kDa fragment of sCD23 were related to levels of schistosome infection intensity, allergen (house dust mite, HDM) and schistosome-specific IgE, total IgE and skin sensitisation to HDM. Results: sCD23 levels rose significantly with schistosome infection intensity but declined significantly with schistosome-specific IgE levels. Furthermore, sCD23 levels were negatively associated with skin sensitisation and IgE reactivity against HDM, but showed no relationship with total IgE. Conclusion: The results are consistent with the suppression of parasite and allergen-specific IgE levels by sCD23. Further mechanistic studies will determine the relevance of this potential regulatory mechanism in the development of helminth-specific immune responses in atopic individuals.

Atopy Is Inversely Related to Schistosome Infection Intensity: A Comparative Study in Zimbabwean Villages with Distinct Levels of Schistosoma haematobium Infection

Background: The hygiene hypothesis suggests that parasitic infections protect against allergic diseases by modulating the host’s immune responses. Experimental studies indicate that this protection depends on the intensity of parasitic infection, but this observation has not been tested in human populations. The aim of this study is to investigate whether the intensity of Schistosoma haematobium infection is related to atopic responses and whether this relationship differs between populations with distinct parasite transmission dynamics. Methods: The study was conducted in two villages with different Schistosoma haematobium transmission dynamics, i.e. high (n = 365) and low (n = 307) transmission. Allergic reactivity to the common house dust mite (Dermatophagoides pteronyssinus) was measured by skin prick tests and allergen-specific IgE and IgG4 quantified by enzyme-linked immunosorbent assay. Atopic responses were related to current infection intensity and schistosome transmission levels. Results: Schistosome infection intensity was negatively associated with the skin prick reactivity, mite-specific IgE and the ratio IgE/IgG4 in the high-transmission village. However, when only low levels of infection were analyzed in the 2 villages, there was no correlation between mite-specific responses and infection intensity. Conclusion: The relationship between schistosome infection and atopic responses is dependent on the intensity of current schistosome infection. Thus, consistent with results from animal models, with an increasing parasite burden, the immunoregulation of immune responses to allergens appears to become more pronounced.

Schistosome infection intensity is inversely related to auto-reactive antibody levels.

In animal experimental models, parasitic helminth infections can protect the host from auto-immune diseases. We conducted a population-scale human study investigating the relationship between helminth parasitism and auto-reactive antibodies and the subsequent effect of anti-helminthic treatment on this relationship. Levels of antinuclear antibodies (ANA) and plasma IL-10 were measured by enzyme linked immunosorbent assay in 613 Zimbabweans (aged 2–86 years) naturally exposed to the blood fluke Schistosoma haematobium. ANA levels were related to schistosome infection intensity and systemic IL-10 levels. All participants were offered treatment with the anti-helminthic drug praziquantel and 102 treated schoolchildren (5–16 years) were followed up 6 months post-antihelminthic treatment. ANA levels were inversely associated with current infection intensity but were independent of host age, sex and HIV status. Furthermore, after allowing for the confounding effects of schistosome infection intensity, ANA levels were inversely associated with systemic levels of IL-10. ANA levels increased significantly 6 months after anti-helminthic treatment. Our study shows that ANA levels are attenuated in helminth-infected humans and that anti-helminthic treatment of helminth-infected people can significantly increase ANA levels. The implications of these findings are relevant for understanding both the aetiology of immune disorders mediated by auto-reactive antibodies and in predicting the long-term consequences of large-scale schistosomiasis control programs.

Regulatory and activated T cells in human Schistosoma haematobium infections.

Acquired immunity against helminths is characterised by a complex interplay between the effector Th1 and Th2 immune responses and it slowly manifests with age as a result of cumulative exposure to parasite antigens. Data from experimental models suggest that immunity is also influenced by regulatory T cells (Treg), but as yet studies on Treg in human schistosome infections are limited.This study investigated the relationship between schistosome infection intensity and the two cell populations regulatory T cells (Treg: CD4+(dim)CD25+(high)FOXP3+CD127low), and activated (Tact: CD4+CD25+FOXP3−) T cells in Zimbabweans exposed to Schistosoma haematobium parasites. Participants were partitioned into two age groups, young children (8–13 years) in whom schistosome infection levels were rising to peak and older people (14+ years) with declining infection levels. The relationship between Tact proportions and schistosome infection intensity remained unchanged with age. However Treg proportions rose significantly with increasing infection in the younger age group. In contrast Treg were negatively correlated to infection intensity in the older age group.The relative proportions of regulatory T cells differ significantly between young individuals in whom high infection is associated with an enhanced regulatory phenotype and older infected patients in whom the regulatory response is attenuated. This may influence or reflect different stages of the development of protective schistosome acquired immunity and immunopathogenesis.

Human helminth co-infection: No evidence of common genetic control of hookworm and Schistosoma mansoni infection intensity in a Brazilian community

Strong statistical associations between soil-transmitted helminths and schistosomes are frequently observed in co-endemic human populations, although the underlying explanations remain poorly understood. This study investigates the contribution of host genetics and domestic environment to hookworm and Schistosoma mansoni infection intensity and evaluates the role of genetic and non-genetic factors in co-variation of infection intensity. Detailed genealogical information allowed assignment of 1303 individuals living in the Brazilian community of Americaninhas, Minas Gerais state, to 25 pedigrees (containing between two and 1159 members) residing in 303 households. The prevalence of co-infection with both hookworms and schistosomes was high (38.5%), with significant correlation between Necator americanus and S. mansoni faecal egg counts. Bivariate variance component analysis demonstrated a modest but significant species-specific heritability for intensity of N. americanus ( h 2 = 0.196) and S. mansoni infection ( h 2 = 0.230). However, after accounting for demographic, socio-economic and household risk factors, no evidence for common genetic control of intensity of hookworm and schistosome infection was observed. There was some evidence for residual clustering within households but the majority (63%) of the covariance between N. americanus and S. mansoni infection intensity remained specific to the individual and could not be explained by shared genes, shared environment or other shared demographic, socio-economic or environmental risk factors. Our results emphasize the importance of exposure to hookworm and schistosome infection in driving the association between levels of infection with these species in hosts resident in areas of high transmission and suggest that much of this common exposure occurs outside the home.

Present and future schistosomiasis control activities with support from the Schistosomiasis Control Initiative in West Africa

Since 2004 the West African countries of Burkina Faso, Mali and Niger have implemented national schistosomiasis and soil-transmitted helminthiasis control programmes with financial and technical support from the Schistosomiasis Control Initiative (SCI). In the first three years of the control programmes, nearly 13.5 million doses of praziquantel and albendazole have been administered against schistosomiasis and soil-transmitted helminthiasis with coverage rates varying between 67.0% and 93.9%. These treatments have resulted in a reduction of the prevalence and intensity of Schistosoma infection in the sentinel cohorts that were set up to monitor and evaluate the national control programmes. The challenges currently faced by these national control programmes are the ability to maintain the reduction in morbidity achieved thus far due to the mass treatment campaigns and ensuring sustainability. For reinforcement of surveillance, the establishment of a geographical information system is suggested in order to contribute towards enhanced sustainability of these programmes. Our new working hypothesis is that targeted control accompanied by periodic mass treatment campaigns (every two to three years) can contribute to maintaining the low levels of morbidity achieved thus far. The implementation of integrated neglected tropical disease control programmes in these countries will provide means to ensure the financial sustainability of control activities for the years to come.

Anti-malaria humoral responses in children exposed to Plasmodium falciparum and Schistosoma haematobium

Antibody responses directed against the Plasmodium falciparum antigens, total extract, anti-merozoite surface protein-3 (MSP3b) and glutamate-rich protein (Glurp-R0) were studied in 42 children exposed to both Schistosoma haematobium and P. falciparum infections. The association between levels of the anti-malaria IgG subclasses and IgM with host age, sex, schistosome infection intensity and schistosome specific antibodies was studied before chemotherapeutic treatment of schistosome infections. This showed a significant negative association between schistosome infection intensity and levels of IgG1, IgG3, and IgG4 directed against malaria total extract antigen, and a positive association between levels of anti-schistosome soluble egg antigen IgG2, IgG3, and IgG4 and levels of the same subclasses directed against malaria total extract antigens. The effect of treating schistosome infections with praziquantel on malaria specific responses was also studied. This treatment resulted in increases in significant IgG4 levels against MSP3b and IgM against Glurp R0. Treatment also resulted in a significant decrease in IgG4 levels against Glurp R0. Host age, sex or pre-treatment infection intensity was not associated with the magnitude of change in the two IgG4 responses while males showed a significantly higher increase in levels of IgM. The results suggest cross reactivity between schistosome and malaria antigens in this population.

Bayesian spatial analysis and disease mapping: tools to enhance planning and implementation of a schistosomiasis control programme in Tanzania

To predict the spatial distributions of Schistosoma haematobium and S. mansoni infections to assist planning the implementation of mass distribution of praziquantel as part of an on-going national control programme in Tanzania. Bayesian geostatistical models were developed using parasitological data from 143 schools. In the S. haematobium models, although land surface temperature and rainfall were significant predictors of prevalence, they became non-significant when spatial correlation was taken into account. In the S. mansoni models, distance to water bodies and annual minimum temperature were significant predictors, even when adjusting for spatial correlation. Spatial correlation occurred over greater distances for S. haematobium than for S. mansoni. Uncertainties in predictions were examined to identify areas requiring further data collection before programme implementation. Bayesian geostatistical analysis is a powerful and statistically robust tool for identifying high prevalence areas in a heterogeneous and imperfectly known environment.

Cytokine production in whole blood cultures from a fishing community in an area of high endemicity for Schistosoma mansoni in Uganda: the differential effect of parasite worm and egg antigens.

The human host is continuously exposed to the egg and the adult worm developmental stages of Schistosoma mansoni during chronic infections with the parasite. To assess the cytokine responses induced by these different costimulating stages and how they are influenced by host age and infection intensity, whole blood samples from a cross-sectional cohort of 226 members of a Ugandan fishing community who had been resident in an area with high transmission of S. mansoni for the previous 10 years or from birth were stimulated with S. mansoni egg antigen (SEA) or worm antigen (SWA). SWA-specific gamma interferon (IFN-gamma) production increased with age, and the levels of SWA- and SEA-specific interleukin 3 (IL-3) were weakly correlated with schistosome infection intensity. The production of most cytokines was little affected by age or infection intensity but was either SEA or SWA specific. One hundred thirty-two members of the cohort coproduced IL-5 and IL-13 specifically in response to SWA, whereas only 15 produced these cytokines, and at much lower levels, in response to SEA. IL-10, IL-4, and IFN-gamma were also produced in response to SWA, whereas the response to SEA consisted almost exclusively of IL-10. Our results suggest that, in contrast to what has been described for the murine model of S. mansoni and during acute human infections, chronic intense exposure to and infection with S. mansoni in this cohort resulted in very low levels of response to SEA in vitro in the presence of a vigorous and mixed Th1-Th2 response to SWA.

On the calculation of intestinal schistosome infection intensity

The effects of using different methods to calculate individual infection intensities on the age-infection distribution of Schistosoma mansoni field data are demonstrated. Methods are tested on a maximum of three stool samples per person collected on three consecutive days; the methods considered for the calculation of individual infection intensities are the geometric mean (GM), arithmetic mean (AM) and pseudo geometric mean (GM of stool samples instead of replicates). In addition, the effects of calculating the infection intensity for each age group using either AMs or GMs are compared. Differences occur in the shape of the age-infection profiles obtained by using either the arithmetic or geometric group mean. When using the AM, peak infection intensity occurs in a younger age group compared to using the GM, and all three methods of calculating individual infection intensity give the first peak of infection in the same age group. However, differences occur in the position of the second peak which occurs earlier with the two GMs than with the AM. Bootstrapping procedures show that the individual AM, gives a different age group for the first peak of infection at least 25% of the time when compared to either of the GMs, and 31% of the time for the second peak, while the two GMs give the same peak age groups around 90–92% of the time for both peaks. When using the GM, to calculate infection intensity for each age group, there are no differences between the three methods used to calculate individual infection intensity. This is confirmed by bootstrapping procedures. The results are discussed in relation to the distribution of parasites and levels of parasite aggregation. The implications of the results for field studies are also discussed.

The parasitism of Schistosoma mansoni (Digenea-Trematoda) in a naturally infected population of water rats, Nectomys squamipes (Rodentia-Sigmodontinae) in Brazil.

Schistosomiasis is a health problem in Brazil and the role of rodents in maintaining the schistosome life-cycle requires further clarification. The influence of Schistosoma mansoni on a population of Nectomys squamipes was studied by capture-recapture (1st phase, from June 1991 to November 1995) and removal (2nd phase, from April 1997 to March 1999) studies at Sumidouro, Rio de Janeiro, Brazil. During both phases coproscopic examinations were performed. At the 2nd phase the rodents were perfused and worms were counted. The population dynamics of parasites was studied. During the 1st phase, female reproductive parameters, longevity, recruitment and survivorship rates and migration patterns were studied in relation to schistosome prevalence. Water contamination (source of miracidia), abundance intermediate host and rodent migration were related to prevalence. The N. squamipes population was not obviously influenced by the infection, as shown by the high number of reproductive infected females, high longevity of infected individuals and the absence of a relationship between recruitment or survivorship rates and the intensity of schistosome infection. The data indicate that N. squamipes can increase transmission of S. mansoni in endemic areas and carry it to non-infected areas. Furthermore, this rodent can be used as an indicator of a transmission focus.

A statistical approach to schistosome population dynamics and estimation of the life-span of Schistosoma mansoni in man

Dynamic models which predict changes in the intensity of schistosome infection with host age are fitted to pre-intervention Schistosoma mansoni data from Kenya. Age-specific post-treatment-reinfection data are used to estimate the force of infection, thus enabling investigation of the rate of worm death. An empirical and statistical approach is taken to the model fitting: where possible, distributional properties and function relationships are obtained from the data rather than assumed from theory. Attempts are made to remove known sources of bias. Maximum likelihood techniques, employed to allow for error in both the pre-intervention and reinfection data, yield confidence intervals for the worm life-span (CI95% = 5.7-10.5 years) and demonstrate that the worm death rate is unlikely to vary with host age. The possibilities and limitations of fitting dynamic models to data are discussed. We conclude that a detailed, quantitative approach will be necessary if progress is to be made with the interpretation of epidemiological data and the models intended to describe them.

Immunodiagnosis of schistosomiasis mansoni in a low endemic area in Surinam by determination of the circulating antigens CAA and CCA

We evaluated the applicability of circulating antigen detection in serum and urine for the diagnosis of Schistosoma infections in a low endemic area. In total 389 individuals from Saramacca (Surinam) participated in the survey. Stool samples were examined using the Kato method, while circulating anodic antigen (CAA) and circulating cathodic antigen (CCA) were determined by highly specific monoclonal antibody-based ELISA's. Also schistosome specific IgM antibodies were measured by the indirect immunofluorescence assay, but the diagnostic performance of this test was found to be poor in this population. S. mansoni eggs were found in 29% of the examined cases, while CAA and CCA could be demonstrated in 23% and 17% of the serum samples and in 3% and 28% of the urine samples, respectively. Forty three percent of the study population was positive in at least one of these diagnostic assays, indicating that each individual test misses a substantial part of the subjects with an active infection. In most positive cases, intensities of infection were very low. As 204 individuals participated in all screening assays, diagnostic performance of each test was evaluated in this sub-population. The highest sensitivities were achieved with the urine-CCA assay and the parasitological examination, detecting 59 and 58 out of the 107 cases with an active infection, respectively. The serum-CAA assay detected 47 positive cases. Our results demonstrate that determination of circulating antigens, especially CCA in urine and CAA in serum, provides information additional to the parasitological examination, for the assessment of prevalence and intensity of Schistosoma infection in low endemic areas.

Schistosomiasis japonica on Jishan Island, Jiangxi Province, People's Republic of China: persistence of hepatic fibrosis after reduction of the prevalence of infection with age

Hepatic fibrosis due to schistosomiasis japonica was examined by ultrasonography in a cross-sectional community study of 825 individuals on Jishan Island, Jiangxi Province, China. The prevalence of active infection was 39·4% with peak infection in the 10–19·9 years age group followed by a significant decline. A similar pattern was observed for intensity of infection. The prevalence of hepatomegaly in the midsternal line ⩾6 cm peaked at 60% in the fourth decade and remained elevated. A progressive increase in the severity of hepatic periportal fibrosis was observed with age, with advanced fibrosis peaking in the fifth decade. The proportion of individuals with advanced fibrosis was significantly greater in males than in females despite equivalent prevalence and intensity of schistosome infection. In addition, a positive association ( P < 0·01) was found between periportal fibrosis and both hepatomegaly ⩾6 cm and splenomegaly. This study suggests that the natural history of schistosomiasis japonica in this hyperendemic community in China is marked by persistence of hepatomegaly and schistosome-induced periportal fibrosis in adults despite a decrease in the prevalence of infection.

Seroepidemiology and serodiagnosis of schistosomiasis in Kenya using crude and purified egg antigens of Schistosoma mansoni in ELISA

The performance of antibody detection for the diagnosis of schistosomiasis has been evaluated in Kenya. Approximately 1500 blood samples from 3 areas with endemic schistosomiasis ( Schistosoma mansoni only, S. haematobium only, and a mixed infection area), and from a non-endemic control area, were tested for their antibody reactivity in an enzyme-linked immunosorbent assay (ELISA). The results were compared with infection status determined by parasitological examination. Two test antigens were used: unfractionated S. mansoni egg homogenate (SEA), and CEF6, a previously described, partially purified fraction of SEA containing 2 cationic antigens. The antigens prepared from eggs of Kenya and Puerto Rico S. mansoni isolates gave very similar results. Bloods from patients with S. haematobium infection cross-reacted significantly with the two S. mansoni antigen preparations, but reactivity against CEF6 appeared more specifically indicative of S. mansoni infection. Of 254 blood samples from schoolchildren in the non-endemic area, 100% gave ELISA optical density readings at 492 nm (OD 492) <0·20 against SEA, and 98% were <0·20 against CEF6. With 887 blood samples from subjects of all ages in the area endemic for S. mansoni alone, using an ELISA OD 492 cut-off point of 0·20, SEA and CEF6 had sensitivities of 94% and 97% respectively, and specificities of 64% and 59% respectively. Increasing the OD 492 cut-off value reduced the sensitivity and increased the specificity of both test antigens. Specificity of both antigens was poor with samples from 234 children in an area endemic for both S. mansoni and S. haematobium (<20% for both antigens at an OD 492 cut-off value of 0·20). It is suggested, with supporting evidence, that one reason for the apparently poor specificity of the serological testing in the endemic area is the inherently poor sensitivity of parasitological examinations of small volumes of stool. There was a significant positive correlation between blood elisa results and the number of eggs excreted by infected subjects in the area endemic for S. mansoni only. Highest correlation coefficients were obtained in children aged under 10 years, and CEF6 gave marginally higher correlation coefficients than SEA. The graphs of prevalence and intensity of schistosome infection drawn from serological results were similar in shape to the graphs of these 2 quantities based on parasitological results, and the results indicate that serology merits wider use as an epidemiological tool for determining infection status in schistosomiasis.

Nutritional status of children with schistosomiasis mansoni in two different areas of Machakos District, Kenya

A possible association between nutritional status and Schistosoma mansoni infection or morbidity was investigated by comparing anthropometric indices among 362 children from 3 primary schools in Machakos District, Kenya. Matithini was a prosperous school in an area (Kangundo) of moderate intensity of schistosome infection but low associated morbidity. A second area (Kambu) showed more severe schistosome-associated morbidity: in this area, Kitengei school was prosperous and with high intensities of schistosome infection, while Misuuni school was less prosperous and with low intensities of infection. Nutritional status was assessed by measurement and appropriate standardization of height, weight and skinfold thickness and by questionnaires concerning diet. Children in Kangundo were better nourished than those in Kambu. Within Kambu, children from Misuuni showed low mean skinfold thickness and low mean weight-for-height (‘wasting’): this was associated with a lack of dietary variety and of intake of animal products. In contrast, those from Kitengei showed low mean height-for-age (‘stunting’). The relationship between intensity of schistosome infection and nutritional indices, although significant, was complex and not readily interpretable. However, intensity of infection was also correlated with hepatomegaly, which was more clearly related to nutritional status. Depending on the school, children with hepatomegaly were significantly more stunted and/or wasted than those without, and had less variety in their diet. Possible reasons for the observed associations are discussed and, of various possibilities, the hypothesis is suggested that schistosome-associated morbidity leads to a subsequent nutritional defect. This hypothesis can now be tested by appropriate intervention studies.

The intensity of experimental schistosome infections modulates hepatic pathology.

Groups of mice infected with Schistosoma japonicum were killed 7, 10 or 15 weeks after infection. The size of hepatic granulomas and degree of hepatic fibrosis in individual mice were examined in relation to the number of worm pairs present using regression analysis. Mice with heavy S. japonicum infections had smaller circumoval granulomas in their livers than did mice with lighter infections. Hepatic fibrosis per unit infection, i.e., per worm pair or per egg, also decreased as the intensity of infection increased. These trends were similar in 8 strains of mice examined. No clear trends were found in rabbits or monkeys infected with S. japonicum. Hepatic fibrosis in S. haematobium-infected mice decreased with increasing intensity of infection, although this trend was significant only in infections of 1 year duration. C57BL/6 mice infected with S. mansoni often showed trends similar to those in S. japonicum-infected mice; however the findings in C3H and ICR mice infected with S. mansoni were more variable. Most of the data for the present report were from animals also described in previous publications. The relevance of these findings to schistosome infections in humans is unknown but the results are clearly pertinent to the analysis of hepatic pathology in experimental infections. Overall egg production by the worms was not affected and the number of eggs per worm pair recovered from the tissues was not generally influenced by the number of worm pairs present.

Hepatic fibrosis in rabbits infected with Japanese and Philippine strains of Schistosoma japonicum.

Symmers' clay pipestem fibrosis of the liver was produced by Schistosoma japonicum infection in the rabbit. Gross and microscopic portal fibrosis and portal vascular lesions resembled those in hepatosplenic schistosomiasis in man. Portal obstruction caused dilation of portal-systemic collateral veins but only minimal portal hypertension. The rapidity of development and prevalence of Symmers' fibrosis were related to the intensity of the schistosome infection. A mixed macronudular and micronodular cirrhosis of the liver was also present in most animals with Symmers' fibrosis, and other differences from the disease in man were present as well. Hepatic fibrosis regressed after the 30th week of infection, as judged from gross and microscopic observations and from measurement of hepatic collagen. The prevalence of Symmers' fibrosis and cirrhosis also decreased after the 30th week in the less heavily infected rabbits. The S. japonicum-infected rabbit is an imperfect model of human Symmers' fibrosis, but the rabbit is the only experimental model available other than the schistosome infected chimpanzee. The rapid production of severe fibrosis without the use of hepatotoxic agents, and the reversibility of the lesions, make this model of general interest for the study of hepatic fibrosis.

Schistosoma mansoni and S. haematobium infections in Egypt. IV. Hepatic lesions.

We performed 400 consecutive autopsies in Cairo, Egypt. The intensity of schistosome infection in these cases was measured by counting adult worms recovered by perfusion and dissection and by counting eggs in the tissues of infected cases. Symmers' clay pipestem fibrosis of the liver was clearly related to the presence and intensity of Schistosoma mansoni, but not S. haematobium, infection. Morphologic findings in cases with Symmers' fibrosis were comparable to those in Brazilian cases, and the intensity of S. mansoni infection associated with Symmers' fibrosis was similar in Brazil and Egypt. The fine bilharzial periportal fibrosis described by Hashem was not identified in our material, and Symmers' fibrosis was present in all cases of portal hypertension caused by schistosomiasis. Schistosome eggs were found concentrated in areas of portal fibrosis of cases with Symmers' fibrosis. In the absence of Symmers' fibrosis, eggs did not concentrate in large portal areas regardless of the intensity of infection or the presence of lesser degrees of portal fibrosis. We thus feel it unlikely that Symmers' fibrosis is formed by the fusion of fibrotic granulomas around the schistosome eggs.