Multivariable regression analysis in Schistosoma mansoni-infected individuals in the Sudan reveals unique immunoepidemiological profiles in uninfected, egg+ and non-egg+ infected individuals

Background: In the Sudan, Schistosoma mansoni infections are a major cause of morbidity in schoolaged children and infection rates are associated with available clean water sources. During infection, immune responses pass through a Th1 followed by Th2 and Treg phases and patterns can relate to different stages of infection or immunity. Methodology: This retrospective study evaluated immunoepidemiological aspects in 234 individuals(range 4–85 years old) from Kassala and Khartoum states in 2011. Systemic immune profiles(cytokines and immunoglobulins) and epidemiological parameters were surveyed in n = 110 persons presenting patent S. mansoni infections (egg+), n = 63 individuals positive for S. mansoni via PCR in sera but egg negative (SmPCR+) and n = 61 people who were infection-free (Sm uninf). Immunoepidemiological findings were further investigated using two binary multivariable regression analysis. Principal Findings: Nearly all egg+ individuals had no access to latrines and over 90% obtained water via the canal stemming from the Atbara River. With regards to age, infection and an egg+ status was linked to young and adolescent groups. In terms of immunology, S. mansoni infection per se was strongly associated with increased SEA-specific IgG4 but not IgE levels. IL-6, IL-13 and IL-10 were significantly elevated in patently-infected individuals and positively correlated with egg load. In contrast, IL-2 and IL-1β were significantly lower in SmPCR+ individuals when compared to Sm uninf and egg+ groups which was further confirmed during multivariate regression analysis. Conclusions/Significance: Schistosomiasis remains an important public health problem in the Sudan with a high number of patent individuals. In addition, SmPCR diagnostics revealed another cohort of infected individuals with a unique immunological profile and provides an avenue for future studies on non-patent infection states. Future studies should investigate the downstream signalling pathways/mechanisms of IL-2 and IL-1β as potential diagnostic markers in order to distinguish patent from non-patent individuals

Distinct Schistosoma mansoni-Specific Immunoglobulin Subclasses Are Induced by Different Schistosoma mansoni Stages—A Tool to Decipher Schistosoma mansoni Infection Stages

Despite the existence of an effective medication against schistosomiasis, the disease remains a major health problem in affected areas, especially for those lacking appropriate sanitary facilities. Moreover, treatment cannot prevent re-infection since it is only effective on adult schistosome worms. Previous retrospective studies in the Sudan have discovered unique immuno-epidemiological profiles in uninfected individuals and those positive for Schistosoma mansoni via polymerase chain reaction (PCR) but egg-negative and those with eggs in their stool. Expanding on these data, serum samples from these individuals were further investigated for the presence of cercarial (SmCTF)-specific antibodies, which would indicate immune responses at the early stages of infection. Indeed, SmCTF IgG1, 2, 3 and 4 levels were significantly elevated in SmPCR+ individuals when compared to egg+ patients. Following multivariable regression analysis, including SmCTF-specific Igs, Schistosoma egg antigen (SEA)-specific and Schistosoma worm antigen (SWA)-specific immunoglobulins revealed a specific immunoglobulin (Ig) profile of individuals presenting different states of infection, which may be a useful future tool in order to identify egg− individuals and thereby prevent unnecessary treatments

Gender and age distribution of study participants.

<p>Study participants were grouped according to absence (<i>Sm</i> uninf = 61) or presence of <i>S</i>. <i>mansoni</i> DNA in blood (<i>Sm</i>PCR⁺ = 63) or <i>S</i>. <i>mansoni</i> eggs in stool samples (egg⁺ = 110). (A) Distribution of age within all three groups. Data shows box whiskers with median, interquartile ranges and outliers. Statistical significances between the indicated groups were obtained after Kruskal-Wallis and Mann-Whitney-U tests. (B) Correlation of age with egg number, statistical significances were tested using the Spearman correlation test.</p

Summary of binary multivariable regression analysis between the different patient groups: Relation of the covariates age, exposure and individual cytokines or Igs.

<p>Summary of binary multivariable regression analysis between the different patient groups: Relation of the covariates age, exposure and individual cytokines or Igs.</p

Higher levels of SEA-specific IgG4 but not IgE production in infected individuals.

<p>Serum from all three groups was analyzed for the concentration of total IgE (A) and the optical density of SEA-specific IgG4 (C) and IgE (E) using ELISA. (F) depicts the ratio of antigen-specific IgG4/IgE. Graphs show box whiskers with median, interquartile ranges and outliers. Statistical significances between the indicated groups were obtained after Kruskal-Wallis and Mann-Whitney-U tests. Levels of total IgE and SEA-specific IgG4 immunoglobulins were further correlated with egg load (B and D respectively) and tested for statistical significance using the Spearman correlation test.</p

Systemic IL-13 is elevated in patently-infected <i>S. mansoni</i> individuals.

<p>Serum from participants (n = 234) was analyzed for the production of IFN-γ (A), IL-2 (B), IL-4 (C), IL-5 (D) and IL-13 (E) using FlowCytomix technology. Graphs show box whiskers with median, interquartile ranges and outliers. Statistical significances between the indicated groups were obtained after Kruskal-Wallis and Mann-Whitney-U tests. IL-13 levels were correlated with the number of eggs (F) and tested for statistical significance using the Spearman correlation test.</p

Lower levels of systemic IL-1β in SmPCR⁺ individuals.

<p>Individual serum samples from all three groups were analyzed for the production of IL-12p70 (A), TNF-α (B), IL-8 (C) and IL-1β (D) using FlowCytomix technology or ELISA (IL-8). Graphs show box whiskers with median, interquartile ranges and outliers. Statistical significances between the indicated groups were obtained after Kruskal-Wallis and Mann-Whitney-U tests. IL-1β levels were correlated with the number of eggs (E) and tested for statistical significance using the Spearman correlation test.</p

Map of study area.

<p>Schematic representation of the regions of Kassala and Khartoum states in the Sudan. Red circles denote villages in which study participants resided.</p

Systemic levels of IL-10 or IL-9 showed opposing profiles in patently infected individuals.

<p>Serum from all three groups was analyzed for the production of IL-9 (A) and IL-10 (C) using FlowCytomix technology. Graphs show box whiskers with median, interquartile ranges and outliers. Statistical significances between the indicated groups were obtained after Kruskal-Wallis and Mann-Whitney-U tests. Levels of IL-9 (B) and IL-10 (D) were correlated with the number of eggs and tested for statistical significance using the Spearman correlation test.</p

Characteristics of study population.

<p>Characteristics of study population.</p