schistosomiasis is a major human helminth infection endemic in developing countries where children harbor the greatest number of parasites . urogenital schistosomiasis , caused by infection with s. haematobium , is the most prevalent in sub - saharan africa and affected children suffer stunted growth , impaired memory and cognitive development , anemia , haematuria , and reduced physical fitness . current control programs target school children , typically aged 6 years old and above and younger pre - school age children ( i.e. , 5 years and below ) are not treated . this is partly because they have been regarded as being at very low risk of infection and infection - associated morbidity and partly because , until recently , there has been lack of safety data on praziquantel in this age group . yet , the reality is that exclusion of these children from control programs leaves them at high risk of developing chronic pathology as has been shown in high endemic foci . this health inequality is now being addressed with the realization that preschool age children do experience significant levels of infection [ 37 ] . the who has recently formally recognized that these children should be considered for treatment   and studies on the safety and efficacy of praziquantel , as well as methods of diagnosis and drug delivery in this age group [ 6 , 911 ] , have been carried out . the next stage is to assess the consequences of praziquantel treatment beyond the reduction of infection , particularly the effects on schistosome - specific responses as well as responses to bystander antigens . indeed , praziquantel treatment of school children and adults has been shown to enhance immune responses associated with resistance to reinfection with schistosomes in endemic areas [ 12 , 13 ] but immunological studies in pre - school children are scarce . moreover , antihelminthic treatment has been associated with an increase in atopy [ 14 , 15 ] , as a result of alterations in the immunological balance established by the parasites . these studies were conducted in schoolchildren and the effect of antihelminthic treatment on allergic sensitization in younger children remains to be determined . schistosome infections have also been reported to both exacerbate and ameliorate responses associated with resistance to plasmodium falciparum , the causative agent of malaria [ 16 , 17 ] . nonetheless , previous studies in schoolchildren aged 618 years old showed that antihelminthic treatment of s. haematobium infection did not alter immune responses to the malaria vaccine candidates merozoite surface protein ( msp ) 1 and 2 . thus , the first aim of this study was to determine the short - term antihelminthic effects on immunological markers of exposure to schistosome ( igm directed against schistosome cercariae , egg , and adult worms ) and malaria ( igm directed against plasmodium total schizont ) infections as well as the effects on antibody responses associated with resistance to infection / reinfection ( ige and igg4 directed against schistosome cercariae , egg , and adult worms , igg directed against plasmodium vaccine candidates msp-1 and msp-2 [ 16 , 2123 ] ) in children aged 35 years in zimbabwe . we also determined the effects of antihelminthic treatment on allergic sensitization to the house dust mite d. pteronyssinus , the most prevalent allergen in zimbabwe . the second aim of this study was to identify antigens serologically recognized on the adult worm by these children ( individually ) both before and 6 weeks after treatment , to assess whether antischistosome antibody changes following treatment were due to qualitative changes in antigens recognised by the host immune system as has been shown in adults . the study population was recruited from magaya , chitate , and chipinda villages in the mashonaland east province of zimbabwe where s. haematobium is endemic . populations from the 3 villages and selection criteria have been described elsewhere [ 9 , 26 , 27 ] . forty - one ( 41 ) children were included in this study ; 31 had received praziquantel treatment while 10 children , whose parents declined treatment for religious reasons , formed the untreated control group . all the treated children who were egg positive at baseline ( n = 4 ) had cleared infection at the six weeks follow up . there was no significant difference between treated / untreated children in terms of village of residence ( z = 1.037 , p = 0.463 ) and their parents were all subsistent farmers . all the children were also part of a larger study investigating the safety of praziquantel and they reported no adverse effects 24 hours following treatment . three children provided enough blood samples to enable proteomic analyses on schistosome antigens serologically recognised before versus after treatment . permission to conduct the study in the region was obtained from the provincial medical director . institutional and ethical approval was received from the university of zimbabwe and the medical research council of zimbabwe respectively . in addition , the study received ethical approval from the world health organization 's research ethics review committee . at the beginning of the study , parents and guardians of participating children had the aims and procedures of the project explained fully in the local language , shona , and written consent was obtained from participants ' parents / guardians before enrolment into the study . after collection of all samples , all compliant participants were offered anti - helminthic treatment with the recommended dose of praziquantel ( 40  mg / kg of body weight ) . lyophilized soluble s. haematobium adult ( swap ) , cercariea ( cap ) , and egg ( sea ) antigens were obtained from the theodor bilharz institute ( giza , egypt ) and reconstituted in ultra pure water as recommended by the manufacturer . recombinant antigens ( from plasmodium merozoite surface protein , msp ) were msp-119 antigen , the 19  kda c - terminal domain of msp1 , and msp-2 which has 2 serotypes : ch150 that belongs to serotype a ( 3d7-like ) , and dd2 that belongs to serotype b ( fc27-like ) . a standard indirect enzyme - linked immunosorbent assay ( elisa ) was optimized as described previously [ 26 , 27 ] and used to quantify levels of antibodies directed against swap , sea , and cap ( igm , ige , and igg4 ) ; p. falciparum antigens ( igm and igg ) , and derp 1 allergen ( ige and igg4 ) both before and 6 weeks after treatment .   for schistosome - specific antibodies , microtitre plates were coated overnight at 4c with 100  l / well of antigen at 5  g / ml ( 10  g / ml for sea ) in carbonate bicarbonate buffer ( ph = 9.6 ) and then washed once with pbs/0.03% tween20 ( which was used for all subsequent washes ) . 100  l of the serum samples was then added diluted at 1  :  20 for ige ( anti - sea , swap and cap ) ; 1  :  100 for igm anti - swap and igg4 anti - cap and swap ; 1  :  400 for igm anti - cap and sea and igg4 anti - sea . plates were incubated for 2 hours at 37c . after washing 3 times , 100  l of antihuman horse - radish peroxide conjugated ige ( sigma ) , igm ( dako ) or igg4 ( the binding site ) was added diluted ( with blocking buffer ) at 1  :  1000 except for igm anti - swap ( 1  :  2000 ) , igg4 anti - cap and swap ( 1  :  500 ) , and ige anti - swap ( 1  :  250 ) . after 1 hour incubation at 37c followed by 6 washes , 100  l of the substrate ( abts ) was added . the reaction was stopped , using 25% hcl , after 15  min incubation at room temperature for igm and igg4 and 30  min incubation at 37c for ige . absorbance readings of all wells were performed at 405  nm .   for plasmodium - specific responses , the same protocol was followed but 50  l / well of 1  g / ml ( crude schizont ) or 50  ng / ml ( recombinant antigens ) was used to coat plates . all samples were diluted 1  :  100 for all assays and secondary antibodies ( sigma ) diluted 1  :  1000 ( igm and igg ) for crude antigens and 1  :  2000 ( igg ) for recombinant antigens . the colorimetric reaction time was 15 minutes at room temperature for all assays .   for derp1-specific responses , plates were coated with 50  l / well of 5  g / ml and samples diluted 1  :  10 . detection antibodies ( ige from sigma and igg4 from the binding site ) were diluted 1  :  1000 and the reaction time for the substrate was 30 minutes at 37c . all laboratory assays for the pre- and posttreatment time points were run at the same time and by the same individual . all samples were assayed in duplicate and 3 sera from european children ( negative controls ) who have never been exposed to schistosome or malaria infections were included on each plate and were used to set up cutoff points for igm status ( ods of 0.2 , 0.24 , and 0.55 , resp . a blank control containing no sera was included in duplicate on each plate and the background absorbance of reagents in the absence of serum was subtracted from all readings . a pool of responders ( positive controls ) was run on each plate and readings were comparable across plates ; that is , the % cvs were less than 20% for all comparisons and readings were below the critical values as determined by reed et al . ; one hundred g of antigen ( swap ) , diluted 1  :  1 in lithium dodecyl sulphate , was run on a 10% gel using sodium dodecyl sulphate ( sds ) buffer . the gel ( 7  cm ) was subsequently divided longitudinally into 2 parts , the first to be used for western blotting and the second for protein identification . the same gel for western blot and protein identification was used to exclude variations due to experimental settings . the proteins on the piece of gel used for identification were stained with silver nitrate while the rest were transferred onto a 0.45  m nitrocellulose membrane . proteins were transferred onto a nitrocellulose membrane using a wet system , in transfer buffer made of 20% methanol , 25  mm tris , and 192  mm glycine . the membrane was then blocked with 2% bsa in tbs/0.05% tween20 for 3 hours , washed 2 times in tbs/0.05% tween/0.2% triton x-100 , and cut into strips . on each strip , 1.5  ml of individual serum diluted 1  :  50 in blocking solution was added and all strips were incubated overnight at 4c . after 3 washes , 1.5  ml of hrp - conjugated ige antibody ( sigma ) was added and the strips incubated for 2 hours at room temperature . after 4 washes , the strips were incubated in ecl plus reagent system for 5 minutes , before being exposed on hyperfilm . the film was developed and bands were matched to those on the silver - stained piece of gel . bands on the silver stained piece of gel that matched those on the western blots were excised , reduced with farmer 's reagent , and subjected to in - gel trypsin digestion using standard protocols . the resulting tryptic peptides were solubilized in 0.5% formic acid and fractionated on a nanoflow uhplc system ( thermo rslcnano ) . peptide separation was performed on a pepmap c18 reversed phase column ( lc packings ) , using a 585%  v / v acetonitrile gradient ( in 0.5%  v / v formic acid ) run over 45  min at a flow rate of 0.2  ul / min . eluate was analysed by online electrospray ionisation ( esi ) mass spectrometry using an amazon ion trap ms / ms ( bruker daltonics ) operating a continuous duty cycle of survey ms scan followed by up to five ms / ms analyses of the most abundant peptides , choosing the most intense multiply charged ions with dynamic exclusion for 120  s. ms data was processed using data analysis software ( bruker ) and the automated matrix science mascot daemon server ( v2.1.06 ) . protein identifications were assigned using the mascot search engine to interrogate schistosome protein sequences in the ncbinr database , allowing a mass tolerance of 0.3  da for both ms and ms / ms analyses . since the data did not fulfill the assumptions for parametric tests and data transformations could not overcome this , nonparametric tests were used for all statistical analyses . the nonparametric wilcoxon signed - rank tests for paired samples were performed to compare pre- and posttreatment antibody raw data ( optical densities ) , ratios ( ige / igg4 ) , and infection intensities . for comparisons between groups of different individuals ( i.e. , infected versus uninfected , igm negative versus igm positive , etc . ) , mann - whitney u tests were performed . the cutoffs for igm anti - schistosome or antischizont antigens were calculated as the mean absorbance plus 2 standard deviations of readings from european negative controls . all statistical analyses were conducted in pasw statistics ( formerly spss ) 17 and p values of < 0.05 were considered significant . a sequential bonferroni correction was used to identify results that were significant in the context of the multiple statistical comparisons . at baseline , 38 children provided urine samples and 13.2% ( ci95 4.428 ) were schistosome egg positive ( mean egg count of 10 eggs/10  ml ) . of the 41 children included in this study , 17.5% ( ci95 7.332.8 ) showed immunological evidence of recent exposure to egg antigens ( positive anti - sea igm ) , 39% ( ci95 24.255.5 ) to cercariae antigens ( positive anti - cap igm ) , and 22% ( ci95 10.637.6 ) to adult schistosome antigens ( positive anti - swap igm ) . none of the children were p. falciparum positive as determined by thin and thick smears or by the paracheck serological test but 21% ( ci95 7.142.1 ) showed immunological evidence of recent exposure to parasite antigens ( positive anti - schizont igm ) . no significant difference in plasmodium - specific antibody levels was observed between schistosome egg positive versus egg negative children ( z = 0.132 , p = 0.935 ; z = 0.742 , p = 0.505 ; z = 0.579 , p = 0.62 ; z = 1.354 , p = 0.202 ; z = 1.180 , p = 0.271 , resp . , for msp119 , dd2 , ch150 , schizont - igm , and schizont igg ) . similarly , there was no significant difference in levels of derp1-specific responses between schistosome egg positive versus egg negative ( z = 0.173 , p = 0.4315 ; z = 1.152 , p = 0.1245 ; z = 1.36 , p = 0.087 , resp . , for ige , igg4 , and ige / igg4 ratio ) .   at baseline , there was no significant difference in mean antibody levels ( all isotypes ) between children who were subsequently treated and those who were not . furthermore , infection levels were comparable between the two groups ( z = 0.146 , p = 0.884 for mean infection intensity and  = 0.043 , p = 0.662 for prevalence ) . six weeks after treatment , all of the treated children were schistosome egg negative ( all the egg positive children had cleared infection ) , resulting in a significant fall in the mean egg count ( z = 1.626 , p = 0.034 ) . on the other hand , infection intensity increased for the one child who was egg positive at baseline in the untreated group ( 73 to 166 eggs per 10  ml ) . there were significant changes in levels of the majority of antibodies in the treated children between the two time points as shown in table 2 and detailed below . overall , the percentage of children with positive cap- and swap - igm ( marker of exposure ) increased after treatment ( 39% to 63.4% and 22% to 43.9% , resp . ) . adult schistosome worm and cercariae - specific igm titers , but not egg - specific igm , rose significantly in treated children ( figure 1(a ) ) . similarly , adult worm and egg - specific ige titers also increased significantly following treatment ( figure 1(b ) ) . the figures ( figures 1(a ) and 1(b ) ) further illustrate that there was no clear distinction between children who were egg positive and negative at baseline . overall , adult worm specific igg4 titers ( figure 1(c ) ) decreased following treatment , but the decline only occurred in children with mild infection at baseline ( 0.3 to 10.7 mean eggs/10  ml ) and not in egg negative children . the decline was however not statistically significant after bonferroni correction ( table 2 ) . in contrast , levels of all anti - schistosome antibodies ( except egg - specific igg4 which declined ) did not change significantly in the 6 weeks period for untreated children ( figures 1(a)1(c ) and table 1 ) . no significant changes occurred in the ratio ige / igg4 against all the 3 parasite life cycle stages ( z = 1.118 , p = 0.453 ; z = 1.139 , p = 0.128 ; and z = 0.02 , p = 0.492 , resp . , for adult worm , cercariae and egg antigens ) in either group ( treated or untreated ) . a significant increase in ige and igg4 against derp1 was observed in treated children ( figure 2 ) . no significant change was observed in the ratio ige / igg4 in both groups ( z = 0.431 , p = 0.333 and z = 0.357 , p = 0.361 , resp . , for treated and untreated groups ) . a decrease occurred in titres of igm and igg antibodies against the total schizont antigen following praziquantel ( pzq ) treatment ( figure 3(a ) ) . similarly there was a significant decline in igg levels against all the malaria vaccine candidates msp-1 ( msp119 ) and msp-2 ( dd2 and ch150 ) after treatment ( figure 3(b ) ) . untreated children also exhibited a decrease in igg against dd2 and msp119 , and igm against the total schizont over the same time frame . the before treatment and after treatment western blot assays were conducted on the same membrane to exclude any variation that might arise from the use of different antigen preparations . only 1 band ( ~80  kda ) was recognized by all serum samples collected before treatment ( figure 4 ) . six weeks following treatment , sera from treated children recognized an additional band ( ~100  kda ) which was absent on the blot from the untreated child . furthermore , the image master analysis showed that the 80  kda band was enhanced after treatment ( the bands were quantified by their pixel intensities ) . the potential identities of the proteins recognized , and the corresponding hit scores , are listed in table 3 . with increasing calls for the inclusion of pre - school children and infants in mass chemotherapy programmes for schistosome control [ 1 , 4 , 6 , 9 , 11 ] , and studies in school children and adults suggesting that antihelminthic treatment not only affects immune responses directed against schistosomes but may also affect immune responses directed against unrelated antigens [ 13 , 14 , 32 ] , the current study aimed to investigate immunological consequences of praziquantel treatment in young pre - school age children . the study shows significant changes in the levels of anti - schistosome adult worm igm and ige antibody titres 6 weeks following praziquantel treatment in children aged 35 years old living in an s. haematobium endemic area . there was a decline in anti - schistosome worm igg4 levels following treatment , essentially driven by children presenting with s. haematobium eggs in urine at baseline . however , this decline was not statistically significant following bonferroni correction , possibly due to the small sample size . the change in anti - schistosome antibody levels in children who were egg negative at baseline suggests that these children may have had prepatent infection , single sex infection , and/or low levels of infection undetected by the egg count technique . indeed , recent studies in pre - school children using a rapid diagnostic test based on cercariae antigens indicate that infection levels are about 3 times higher than prevalence obtained by the egg count . consistent with these findings , the current study shows a prevalence of 39% based on cap - igm , which is 3 times higher than the 13% prevalence obtained based on egg count . the possibility of prepatent and/or single sex infections was further supported by the increase in the percentage of children with positive cap- and swap - igm after treatment . the increase in anti - worm and anti - cercariae igm responses is consistent with reported changes in adult worm ( and cross - reactive ) antigens exposed to the immune system following the killing of adult worms by praziquantel . antiegg responses have been proposed as a diagnostic tool for schistosome infections in young children where egg counts are less reliable due to low levels of patent infection or to single sex infections . in this study , antiegg igm responses did not change following treatment , suggesting that while antiegg responses may be good markers of the presence of a patent schistosome infection before treatment , they may not be a reliable tool for use in evaluating the efficacy of treatment . this study showed that , consistent with findings in older individuals , praziquantel treatment boosts the anti - worm ige immune responses associated with protection , suggesting that this young age group may also benefit immunologically from anti - schistosome chemotherapy , which reduces reinfection in older children and adults by stimulating ige responses [ 13 , 32 ] . the increase in ige titres was accompanied by an increase in the number of parasite antigens recognised , suggesting that treatment results in the enhancement of antigen recognition as already reported for older children and adults . hidden  antigens or enhanced affinity maturation of ige responses . the proteins identified after treatment were essentially constituents of the parasite surface and musculature , consistent with the praziquantel disruptive action on them [ 3638 ] . among these was the myosin heavy chain , which has been previously recognized by pooled sera from older individuals with a longer history of infection . two distinct fragments of this protein have previously been associated with protection against reinfection in mice vaccinated with radiation - attenuated cercariae [ 40 , 41 ] . s. mansoni heat shock protein 70 ( hsp70 ) homologue was also identified , consistent with previous results from pooled samples of older individuals . hsps are proteins that are expressed by all eukaryotic cells to maintain cell homeostasis under stress conditions . furthermore , because they are suspected of being inducers of multiple pathways of immunity , these proteins may be explored as potential adjuvants in vaccine development against cancer and infections ( reviewed by segal et al . ) . this approach presents the limitation that all the parasite antigens described were recognised by ige . although parasite - specific ige antibodies have been associated with protection [ 21 , 32 , 43 ] , vaccine candidates eliciting ige responses in helminth endemic areas have recently been shown to induce urticaria . although  desensitization  of the ige - inducing vaccine is a possibility to overcome this problem , identification of antigens recognized by other isotypes   may be more realistic . nevertheless , characterising parasite antigens eliciting ige responses gives the information on what to  avoid  or  take with precaution  in vaccine design . furthermore , given the similarities between anti - helminth and anti - allergen responses , comparative studies ( schistosome antigens versus allergens ) would provide the spectrum of antigen recognition and inform on possible cross - reactivity . this would narrow the choice of antigen vaccine candidates and minimise the risk of sensitization . in this study , anti - plasmodium responses declined in both treated and untreated children , suggesting that this change was not due to praziquantel treatment . since the decline was both in igm responses directed against the schizont ( marker of exposure to plasmodium infection ) and igg responses directed against the vaccine candidates msp-1 and msp-2 , it is likely that the decline was reflecting normal dynamics of plasmodium antibodies in the absence of infection . levels of antibodies to several plasmodium antigens vary with the seasonality of parasite transmission , often being higher during periods of high transmission than at the end of a low transmission season . furthermore , levels of anti - plasmodium antibodies directed against both crude and recombinant antigens tend to be higher in individuals carrying parasites than in those without parasites . the region of zimbabwe where the present study was conducted is mesoendemic for malaria with an incidence of 0.2 to 10 cases/1000/year . the absence of plasmodium parasites on blood smears of participants in this study and the decline in the levels of anti - plasmodium antibody responses may therefore reflect the end of a malaria transmission season in this area ( at the time of sample collection ) . in the present study , there was no significant difference for derp1 specific antibodies between schistosome egg positive and negative children at baseline . following treatment , derp1 specific ige and igg4 were significantly increased , resulting in the ratio ige / igg4 ( which is associated with allergic reactivity ) being not significantly changed . hagel and colleagues indicated that a certain  threshold  is required for parasite infection to suppress allergic reactivity , consistent with findings from animal models . the low levels of infections in the present study population would therefore explain the absence of significant changes in the ratio ige / igg4 against derp 1 allergen . the increase in allergen - specific ige responses in treated children may be due to cross - reactivity since levels of these antibodies directed against schistosome antigens also increased following treatment . however , cross - reactivity can not explain the increase in igg4 against derp1 in treated and , to a lesser extent , in untreated children , since the anti - schistosome igg4 declined . changes in antimite igg4 antibodies following seasonal changes in natural exposure to house dust mites have previously been reported . investigations on natural temporal variations in levels of exposure to mite were beyond the scope of this study but this finding requires further investigation . the current study presents the limitation that the sample size was small , which could have affected the statistical power . this was due to the limited accessibility of these young children in terms of blood collection , and also because treatment has only recently started in this age group . nevertheless , this study is one of the first to demonstrate a boost , due to praziquantel treatment , in anti - schistosome immune responses in pre - school age children . taken together , findings from this study suggest that praziquantel treatment of preschool children has comparable effects to those reported in studies on older children and adults , boosting antibody responses associated with putative resistance to schistosome infection / reinfection six weeks following chemotherapy . furthermore , serological recognition of schistosome antigens was enhanced following praziquantel treatment in these young children , and recognized antigens are associated with the development of acquired protective immunity . praziquantel treatment did not have a significant effect on plasmodium - specific responses , which showed temporal fluctuation in both untreated and treated individuals . furthermore , pzq treatment did not significantly alter allergen - specific responses which would mediate pathology in allergic disease . thus , the study suggests that pre - school age children may also benefit immunologically from praziquantel treatment .
urogenital schistosomiasis , due to schistosoma haematobium , is endemic in sub - saharan africa . 
 control is by targeted treatment with praziquantel but preschool age children are excluded from control programs . 
 immunological studies on the effect of treatment at this young age are scarce . in light of studies in older individuals showing that praziquantel alters antischistosome immune responses and responses to bystander antigens , this study aims to investigate how these responses would be affected by treatment at this young age . 
 antibody responses directed against schistosome antigens , plasmodium falciparum crude and recombinant antigens , and the allergen house dust mite were measured in children aged 3 to 5 years before and 6 weeks after treatment . 
 the change in serological recognition of schistosome proteins was also investigated . 
 treatment augmented antischistosome igm and ige responses . 
 the increase in ige responses directed against adult worm antigens was accompanied by enhanced antigen recognition by sera from the children . 
 antibody responses directed against plasmodium antigens were not significantly affected by praziquantel treatment nor were levels of allergen specific responses . 
 overall , praziquantel treatment enhanced , quantitatively and qualitatively , the antiworm responses associated with protective immunity but did not alter plasmodium - specific responses or allergen - specific responses which mediate pathology in allergic disease .