Abstract
Background
Dengue virus (DENV) is the most prevalent mosquito-borne virus worldwide, with approximately half of the world’s population at risk of infection. Although it has been shown that parasitic infections can influence viral co-infections the role of schistosomiasis has not yet been explored. The objective of this exploratory study was to investigate the influence of schistosome infection on DENV infection in Madagascar.
Methods
Between March 2020 and October 2022 we recruited participants in the regions of Boeny and Atsinanana to assess the seroprevalence of DENV in the country using highly specific tests for the detection of IgG antibodies and investigated the influence of schistosome infections on DENV infections through a plaque reduction neutralisation test (PRNT). For this, additional participants were recruited in Haute Matsiatra between July 2022 and March 2023, Poisson regression models were used to assess the association of the PRNT results with schistosome infections.
Results
For the first time, we report a low seroprevalence of DENV (up to 3.3%) in areas with a high prevalence (> 50%) of schistosome infection. Additionally, we could demonstrate that sera derived from schistosome-infected individuals exert a significant antiviral activity against DENV infection (up to 27.5%). A Poisson regression analysis revealed that, among the possible factors assessed, the schistosome infection status was the only factor associated with the inhibitory effects against DENV infection in the PRNT. Finally, we could observe that highest IgE level were found in participants showing the greatest reduction in viral infection in the PRNT.
Conclusions
Our data suggest that schistosomiasis might play a protective role against DENV infections. These findings offer new perspectives regarding how chronic parasitic infections affect the dynamics of DENV infections in Africa.
Graphical Abstract
Supplementary Information
The online version contains supplementary material available at 10.1186/s40249-025-01370-0.
Keywords: Dengue virus, Schistosomiasis, Seroprevalence, Madagascar, Co-infection, Parasitic infections, Arboviruses
Background
Dengue virus (DENV) is the most prevalent mosquito-borne virus worldwide and has historically not been considered endemic in Africa, despite the presence of the main arthropod vectors on the continent [1]. Approximately half of the world’s population is at risk of infection, and more than 400 million infections are estimated to occur each year, accounting for approximately 1.1 million disability-adjusted life-years (DALYs) [2–5]. Overall, the true burden of dengue and other arboviral diseases in Africa is still unclear.
Parasitic infections modulate immune responses, and have been described to influence the outcome of secondary infections, including viral infections [6–9]. Among the hypothesised factors influencing the host immune system, the T helper cell 2 (Th2) response elicited by parasitic infections [including the increased production of interleukin-4 (IL-4), IL-13, and immunoglobulin E (IgE)] has been proposed to play an important role [9]. Interestingly, a review on the seroprevalence of DENV infections in Africa reported high heterogeneity across the continent and identified a trend of the highest prevalence in those countries where schistosomiasis levels are the lowest [10, 11].
Schistosomiasis is caused by helminths of the genus Schistosoma [11, 12]. Approximately 779 million people are at risk of infection worldwide, and over 250 million people are infected, with the majority living in sub-Saharan Africa [11]. This results in an estimated global disease burden of about 1.7 million DALYs [11, 13], with the highest burden of the disease associated mostly with the chronic course of infection [11, 14]. While the initial response to the parasite is mainly a type 1-mediated response [characterized by IFN-γ, tumor necrosis factor (TNF), and IL-12 secretion], high modulation of dendritic cells and macrophage activation contribute to a switch toward a type 2-response/regulatory phase [15–17]. Monocytes, dendritic cells, and macrophages are among the primary target cells of DENV [18, 19].
Madagascar is one of the countries with the highest prevalence of schistosomiasis globally, with over 50% of the population infected [11] and a high likelihood of chronic infections. Mosquito-borne diseases, such as malaria and dengue, have been described only in coastal areas. However, reports of DENV infections in Madagascar are scarce, despite conducive climate conditions and the presence of multiple competent arthropod species. To date, two major DENV outbreaks have been described in 2006 and 2020 [20, 21].
The aim of this exploratory study was to investigate the potential influence of schistosome infections in endemic areas on the occurrence of DENV infections. The specific objectives were (i) to assess the seroprevalence of DENV in Madagascar and (ii) to evaluate the influence of schistosome infections on DENV infections.
Methods
Study setting and data collection
Participants were recruited from the regions Boeny (16°06′50″S 46°45′24″E), Atsinanana (19°19′49"S 48°58′49"E), and Haute Matsiatra (21°26′38″S 47°05′16″E). Eligibility criteria for all studies were informed consent, willingness to comply with the protocol, and age ≥ 18 years for Boeny and Haute Matsiatra, or ≥ 5 years for Atsinanana. A general exclusion criterion was the history of epilepsy or convulsive episodes. For Boeny, the additional exclusion criteria of fever (temporary exclusion), a history of transfusion or congenital anaemia were applied; for Atsinanana and Haute Matsiatra, a history of bleeding/haemorrhage, current pregnancy as well as current suspected and/or confirmed coronavirus disease 2019 (COVID-19) infection and COVID-19 vaccination within the past two weeks before recruitment. For the regions Boeny [22] and Atsinanana [23], recruitment was previously described in detail. Participants were recruited from a primary healthcare centre in Ankazomborona in the Boeny region (March 2020–January 2021) and through home-based surveys in Vatomandry in the Atsinanana region (July–October 2022). From each participant 9 ml of venous blood was collected, and serum aliquots were stored at −80 ℃. In Haute Matsiatra (July 2022–March 2023), participants with different degrees of hepatosplenic schistosomiasis [schistosome-negative (Sneg), schistosome-positive (Spos) without liver fibrosis (Spos-NF), Spos with moderate liver fibrosis (Spos-MF), and Spos with severe liver fibrosis (Spos-SF)] were recruited at the main hospital [Centre Hospitalier Universitaire(CHU) Tambohobe Fianarantsoa], with pre-recruitment conducted at the primary level of healthcare and subsequent referral to CHU. A urine sample was taken and a rapid point-of-care circulating cathodic antigen test (POC-CCA) was performed to screen for schistosome infections. Disease severity in infected participants was assessed by staging the degree of liver fibrosis via ultrasound and segregating participants into the four corresponding groups. From each participant 5 ml of venous blood was collected to obtain an aliquot of serum, and 9 ml of venous blood was collected to obtain an aliquot of plasma, which were stored at −80 °C. All the samples were shipped on dry ice to the Bernhard Nocht Institute for Tropical Medicine (BNITM), Hamburg, Germany, where they were stored at −80 °C until further use. Background characteristics such as socio-demographic information, clinical history, and personal habits were provided in a pseudonymised format and later merged with the test results.
At all the study sites, data were collected through case report forms (CRFs), which were administered via face-to-face interviews, either directly through eCRFs or by double data entry, according to data quality standards. The CRF data were managed using REDCap® electronic data capture tools version 15.0.33 (Vanderbilt University, Nashville, USA) hosted at the BNITM. The questionnaire was developed ad-hoc for the studies in collaboration with all the implementing partners. After finalisation, a pilot test was run during the training sessions, and additional adaptations were included if needed.
Sample size considerations
Sample size calculations were based on the premise that this study represents a secondary analysis of data originally collected for other objectives.
For the seroprevalence analysis, on the basis of previously available data from Madagascar, we assumed a DENV seroprevalence of 10%. To detect a 5% difference in the prevalence of DENV between subgroups with 80% power, we estimated the minimum required sample size to be 864 participants. Given the availability of overall 1478 participants, we included this total number.
To analyse the average reduction in plaque counts, the minimum required sample size was estimated using the non-parametric Guenther method for the Mann–Whitney test [24], as we anticipated a skewed distribution of plaque counts. To achieve 80% power for detecting small (Cohen's d = 0.2), medium (d = 0.5), and large (d = 0.8) effect sizes, a minimum of 412, 67, and 27 participants per group, respectively, were required. Sample size calculations were conducted using the "pwrss" package in R software version 4.4.2 (R Core Team, Vienna, Austria).
Laboratory testing
Different methodologies have been applied to detect the presence of schistosomes and previous DENV infections (diagnostic assays), whereas experimental in vitro assays have been established to assess the influence of schistosome infections on DENV infections (experimental assays).
Diagnostic assays
Schistosome PCR testing. DNA extraction and PCR were performed as previously described [22]. Briefly, DNA was extracted from 1 ml serum using the QIAamp MinElute ccfDNA Mini Kit, following the manufacturer’s instructions (Qiagen, Hilden, Germany). The extracted DNA was stored at −20 °C until further use. Semi-quantitative PCR (qPCR), which is based on a previously published protocol [25], was performed to simultaneously detect S. mansoni and S. haematobium infections. The readout resulted from the RotorGene 6000 Software v.7.87 (Qiagen, Hilden, Germany). Results with a clean sigmoid curve were considered positive.
POC-CCA testing. POC-CCA was performed on site according to the manufacturer’s instructions (Rapid Medical Diagnostics, Pretoria, South Africa) to detect schistosome infections. Two drops (100 µl) of urine were transferred to the POC-CCA test cassette and the results were read after 20 min.
Pan-DENV IgG ELISA. Sera were screened with the pan-DENV (NS1) IgG ELISA (Panadea Diagnostics, Hamburg, Germany) to identify samples with antibodies targeting DENV NS1. Afterwards, samples that tested positive in the screening were subjected to the pan-DENV (NS1) IgG SE ELISA (Panadea Diagnostics, Hamburg, Germany). The specificity enhancer in this kit suppresses signals from cross-reactive orthoflavivirus antibodies against DENV NS1, leaving only dengue-type-specific signals. Sera were diluted 1∶51 and incubated overnight at 4 °C in a sealed, moist environment with horseradish peroxidase (HRP)-labelled recombinant DENV NS1 antigen (serotypes 1–4), following the manufacturer’s instructions. The optical density (OD) was assessed at 450/620 nm, and the difference in the OD450–OD620 was calculated for each sample. The positivity threshold for samples from Boeny was manually defined as OD > 0.14 and as OD > 0.175 for samples from Atsinanana. All samples that tested positive with the pan-DENV (NS1) IgG ELISA were subjected to the pan-DENV (NS1) IgG SE ELISA. Samples that tested positive according to the pan-DENV (NS1) IgG SE ELISA were ultimately considered positive for dengue-type-specific antibodies. Samples that tested positive according to the pan-DENV (NS1) IgG ELISA but negative according to the pan-DENV (NS1) IgG SE ELISA were considered as being positive for DENV NS1 cross-reactive antibodies.
Experimental assays
Plaque reduction neutralization test (PRNT). Non-heat-inactivated serum was diluted 1∶8 with Dulbecco's Modified Eagle Medium, mixed with an equal volume of DENV2 (strain UVE/DENV-2/2018/RE/47099) and incubated for 1 h at 37 °C. The virus concentration used was adjusted to a range of 10–100 plaque forming units (PFU)/well, with 10 being the minimum plaque count when the DENV complex-reactive mouse monoclonal antibody D1-11(3) (IgG2a; GeneTex, Irvine, USA) was used as a positive neutralization control (final concentration: 1 µg/ml). The virus-serum mixture was transferred onto a Vero E6 monolayer (2.5 × 105 cells/24-well) to allow infection for 1 h at 37 °C. The mixture was aspirated, and the cells were overlaid with 1.5% carboxymethyl cellulose in Minimum Essential Medium supplemented with 2% fetal bovine serum and incubated for six days at 37 °C in 5% CO2. Afterwards, the cells were fixed with 10% formaldehyde and stained with 1% crystal violet solution as previously described [26]. The plaque count for each sample was assessed and used for further analysis.
Cytokine profiling. Sera were subjected, in duplicate, to fluorescent bead measurement of cytokines and chemokines using the Human T Helper Cytokine Panel V02 (Cat # 741028), the Human Anti-Virus Response Panel (Cat # 740390), and the Human Anti-Virus Response Panel V02 (Cat # 741270), following the manufacturer’s instructions (BioLegend, San Diego, USA). The standard curves were optimized automatically by the software LEGENDplex Data Analysis Software Suite version 2025-05-01 (Qognit, San Carlos, USA) and verified manually.
IgE ELISA. The plasma samples were subjected to the Human IgE ELISA (Invitrogen, Waltham, USA), following the manufacturer’s instructions. Sample duplicates were bound to antibodies absorbed onto the microwells, before the addition of HRP-conjugated anti-human IgE antibodies. A colourful signal was measured at OD450. Sample IgE concentrations were determined via a standard curve for reference.
Statistical analysis
For numerical variables, medians and interquartile ranges (IQRs) are reported. For categorical variables, absolute and relative frequencies are reported. Seroprevalence rates with 95% confidence intervals (CIs) and crude prevalence odds ratios (cPORs) with 95% CIs were estimated using the R package epiR. Chi-square or Fisher’s exact tests were used to compare estimates between subgroups. The CIs of the median of cytokine concentrations were computed using the R package DescTools.
Comparisons of plaque counts, cytokine and IgE concentrations between groups were made via non-parametric Mann-Whitney-Wilcoxon or Kruskal-Wallis tests when applicable. Non-parametric tests were applied to provide more robust results accounting for small sample sizes and skewed distributions of outcome variables. Boxplots were used for graphical representation of plaque counts and IgE concentrations.
A multivariable Poisson regression model with correction for overdispersion was used to assess the effect of schistosome positivity on plaque counts and the influence of other covariables on this estimate. The percentage of reduction in the plaque count was calculated as (1–RR) × 100%. All analyses were performed using R software version 4.4.2 (R Core Team, Vienna, Austria).
Results
Description of the study population
Overall, this study included 1543 serum and 65 plasma samples from participants in the three regions of Madagascar (Fig. 1).
Fig. 1.
Inclusion flowchart of participants and samples for the performed experiments. DENV dengue virus, Sneg schistosome-negative, Spos schistosome-positive, PRNT plaque reduction neutralisation test, Spos-NF schistosome-positive without liver fibrosis, Spos-MF schistosome-positive with moderate liver fibrosis, Spos-SF schistosome-positive with severe liver fibrosis
The study sample characteristics for the three regions involved are reported in Table 1. The population of Boeny (n = 488) was characterised by 55% females (n = 266), a median age of 28 years (IQR: 21–40) and mostly farmers (77%, n = 372). The prevalence of schistosome infection was 65% (n = 315), as assessed by qPCR. The population of Atsinanana (n = 990) was characterised by 56% females (n = 557), a median age of 42 years (IQR: 32–54) and mostly farmers (59%, n = 563). Chronic diseases were self-reported by 5% (n = 51) of the participants. The prevalence of schistosome infection was 55% (n = 548), assessed by qPCR. Alcohol consumption and smoking were reported by 36% (n = 358) and 9% (n = 87) of the participants, respectively. The population of Haute Matsiatra (n = 65) was characterised by 68% females (n = 44) and a median age of 38 years (IQR: 27–49). Farming was reported by only 20% (n = 13) of the participants. Alcohol consumption was reported by 11% (n = 7). The prevalence of schistosome infection was 77% (n = 50) on the basis of the POC-CCA results. The participants were also assessed for other infections (HBV, HCV, or HIV), but only one participant was co-infected with HBV.
Table 1.
Background characteristics of the study sample, segregated by recruitment region
| Boeny (n = 488) | Atsinanana (n = 990) | Haute Matsiatra (n = 65) | |
|---|---|---|---|
| Age, years | |||
| Median (IQR) | 28 (21–40) | 42 (32–54) | 38 (27–49) |
| Age groups, years | |||
| 0–17 | NA | 23 (2%) | NA |
| 18–29 | 255 (52%) | 174 (18%) | 19 (29%) |
| 30–44 | 137 (28%) | 346 (35%) | 21 (32%) |
| 45–59 | 61 (13%) | 289 (29%) | 19 (29%) |
| 60 + | 35 (7%) | 158 (16%) | 6 (9%) |
| Sex | n = 988* | ||
| Female | 266 (55%) | 557 (56%) | 44 (68%) |
| Male | 222 (45%) | 431 (44%) | 21 (32%) |
| Occupation | n = 484* | n = 961* | |
| Non-farmer | 112 (23%) | 398 (41%) | 52 (80%) |
| Farmer | 372 (77%) | 563 (59%) | 13 (20%) |
| Alcohol consumption | |||
| No | NA | 632 (64%) | 58 (89%) |
| Yes | NA | 358 (36%) | 7 (11%) |
| Smoking | |||
| No | NA | 903 (91%) | NA |
| Yes | NA | 87 (9%) | NA |
| Chronic disease (self-reported)† | n = 987* | ||
| No | NA | 936 (95%) | NA |
| Yes | NA | 51 (5%) | NA |
| Schistosome infection status§ | |||
| Negative | 173 (35%) | 442 (45%) | 15 (23%) |
| Positive | 315 (65%) | 548 (55%) | 50 (77%) |
| Degree of liver fibrosis | |||
| Sneg | NA | NA | 15 (23%) |
| Spos-NF | NA | NA | 22 (34%) |
| Spos-MF | NA | NA | 18 (28%) |
| Spos-SF | NA | NA | 9 (14%) |
| No assessment | NA | NA | 1 (1%) |
| Test performed | |||
| Seroprevalence | 488 (100%) | 990 (100%) | NA |
| PRNT | NA | 821 (83%)¶ | 65 (100%) |
IQR interquartile range, Sneg schistosome-negative, Spos schistosome-positive, Spos-NF schistosome-positive without liver fibrosis, Spos-MF schistosome fibrosis with moderate fibrosis, Spos-SF schistosome-positive with severe fibrosis, PRNT plaque reduction neutralisation test, qPCR semi-quantitative polymerase chain reaction, POC-CCA point-of-care circulating cathodic antigen
*Participants with missing information were not included
†Self-reported chronic diseases included asthma, allergy, cough, dermatosis, diabetes, elephantiasis, high blood pressure, joint pain, kidney disease, memory loss, palpitations, rheumatism, stomach ache, tuberculosis, and vaginal discharge
§The schistosome infection status was assessed using qPCR for samples collected in Boeny and Atsinanana. POC-CCA was used to assess samples collected in Haute Matsiatra
¶Due to limited availability of samples, PRNT was only performed on a smaller subset
Assessment of DENV IgG seroprevalence in the regions of Boeny and Atsinanana
Using the pan-DENV (NS1) IgG ELISA, a prevalence of DENV NS1-reactive IgG antibodies of 10.0% (95% CI: 7.5–13.1) and 16.9% (95% CI: 14.6–19.4) was detected in Boeny and Atsinanana, respectively. When the results were adjusted with the pan-DENV (NS1) IgG SE ELISA, a DENV-specific seroprevalence of 3.3% (95% CI: 1.9–5.3) and 3.2% (95% CI: 2.2–4.5) was detected in both regions, respectively (Fig. 2). Therefore, cross-reactive antibodies against DENV NS1 were detected in 6.8% (n = 33) and 13.6% (n = 135) of the participants, respectively.
Fig. 2.
Study sites in Madagascar for DENV seroprevalence and PRNT. The prevalence of DENV NS1 IgG antibodies was assessed in two coastal regions of Madagascar (Boeny, n = 488; Atsinanana, n = 990); seroprevalence was not assessed for the third region (Haute Matsiatra, n = 65), as no relevant DENV transmission has been reported in the mountain areas of Madagascar. DENV dengue virus, PRNT plaque reduction neutralisation test
The DENV seroprevalence estimates stratified by various variables with cPOR are reported in Table 2. The DENV seroprevalence did not significantly differ by sex, age group, or region of origin, based on chi-square and Fisher’s exact tests. However, the seroprevalence of DENV was significantly lower among farmers with a cPOR of 0.5 (95% CI: 0.3–0.9). The cPOR of DENV seroprevalence among schistosome-infected participants was 0.9 (95% CI: 0.5–1.6), which was lower than that among non-infected participants.
Table 2.
DENV seroprevalence and crude prevalence odds ratios
| DENV positive, n | Total, n | Prevalence (%) | cPOR (95% CI) | P-value | |
|---|---|---|---|---|---|
| Age groups* | |||||
| 0–17 | 2 | 23 | 8.7 (1.1–28.0) | 3.4 (0.7–16.3) | 0.145 |
| 18–29 | 15 | 429 | 3.5 (2.0–5.7) | 1.3 (0.6–2.8) | 0.565 |
| 30–44 | 13 | 483 | 2.7 (1.4–4.6) | Ref | |
| 45–59 | 14 | 350 | 4.0 (2.2–6.6) | 1.5 (0.7–3.3) | 0.325 |
| 60 + | 4 | 193 | 2.1 (0.6–5.2) | 0.8 (0.3–2.4) | 0.790 |
| Sex | |||||
| Female | 26 | 823 | 3.2 (2.1–4.6) | Ref | |
| Male | 22 | 653 | 3.4 (2.1–5.1) | 1.1 (0.6–1.9) | 0.821 |
| Occupation | |||||
| Non-farmer | 24 | 510 | 4.7 (3.0–6.9) | Ref | |
| Farmer | 22 | 935 | 2.4 (1.5–3.5) | 0.5 (0.3–0.9) | 0.015 |
| Schistosome infection status | |||||
| Negative | 21 | 615 | 3.4 (2.1–5.2) | Ref | |
| Positive | 27 | 863 | 3.1 (2.1–4.5) | 0.9 (0.5–1.6) | 0.760 |
| Region | |||||
| Boeny | 16 | 488 | 3.3 (1.9–5.3) | Ref | |
| Atsinanana | 32 | 990 | 3.2 (2.2–4.5) | 1.0 (0.5–1.8) | 0.962 |
DENV dengue virus, cPOR crude prevalence odds ratio, CI confidence interval, Ref reference
*P-value: Fisher’s exact test
The seroprevalence in Haute Matsiatra was not assessed as dengue, and other mosquito-transmitted infections, are neither regularly reported nor expected to occur in this area because of the altitude of the region.
Effects of soluble serum factors from schistosome-infected participants on DENV infection
A PRNT was performed on a total of 886 participants divided into two groups. The first group included 821 participants from Atsinanana, on which the seroprevalence was assessed. As expected, sera from individuals with pre-existing DENV antibodies (n = 26) or cross-reactive antibodies to DENV NS1 (n = 108) presented a significant reduction in the PRNT (Fig. 3). To rule out confounding effects arising from pre-existing immunity, these sera were removed from further analysis.
Fig. 3.
Verification of PRNT establishment. PRNT results for 713 sera (exclusion of n = 108 with DENV NS1 cross-reactive IgG antibodies), depicted by plaque count of samples and stratified by presence of DENV NS1 IgG antibodies (n positive = 26, n negative = 687, P = 0.013, estimated with Mann-Whitney-Wilcoxon test; median positive = 25, median negative = 37) (panel A). PRNT results for 795 sera (exclusion of n = 26 with DENV-specific NS1 IgG antibodies), depicted by plaque count and stratified by presence of DENV NS1 cross-reactive IgG antibodies (n positive = 108, n negative = 687, P < 0.001, estimated with Mann-Whitney-Wilcoxon test; median positive = 18, median negative = 37) (panel B). PRNT plaque reduction neutralisation test, DENV dengue virus
We then observed a statistically significant plaque count reduction of 14.3% (RR: 0.857; 95% CI: 0.800–0.919) among schistosome-infected participants compared with Sneg (Fig. 4a). A Poisson regression analysis revealed that, among the possible factors assessed, the schistosome infection status was the only factor associated with the inhibitory effects against DENV infection in the PRNT (Fig. 4b).
Fig. 4.
The effect of Spos sera from Atsinanana and Haute Matsiatra on DENV infections. PRNT results for 687 sera, depicted by plaque count and stratified by presence of schistosome infection (n positive = 365, n negative = 322, P < 0.001, estimated with Mann-Whitney-Wilcoxon test; median positive = 34, median negative = 41.5) (panel A). PRNT results for 65 sera, depicted by plaque count and stratified by presence of schistosome infection (n positive = 50, n negative = 15, P = 0.007, estimated with Mann-Whitney-Wilcoxon test; median positive = 36, median negative = 43) (panel C). PRNT results for 65 sera, depicted by plaque count and stratified by presence of schistosome infection and degree of liver fibrosis (n Sneg = 15, n Spos-NF = 22, n Spos-MF = 18, n Spos-SF = 9, P = 0.022, estimated with Kruskal-Wallis test; median Sneg = 43, median Spos-NF = 33, median Spos-MF = 40.5, median Spos-SF = 32) (panel E). Multivariable Poisson regression models with correction for overdispersion for the Schistosoma positivity estimate (panel B, panel D) and Schistosoma positivity with degrees of liver fibrosis (panel F) (crude and adjusted for relevant covariables) depicted with 95% CI; Sneg = reference category. Spos schistosome-positive, DENV dengue virus, PRNT plaque reduction neutralisation test, Spos-NF schistosome-positive without liver fibrosis, Spos-MF schistosome-positive with moderate fibrosis, Spos-SF schistosome-positive with severe fibrosis, CI confidence interval, CD chronic disease
Effects of soluble serum factors from participants with different degrees of liver fibrosis on DENV infection
In the second group, which included 65 participants from Haute Matsiatra, we assessed whether sera from participants, affected by varying degrees of fibrosis, would have a differential influence on the assay. A plaque count reduction of 20.0% (RR: 0.800; 95% CI: 0.682–0.944) was observed among infected participants in the PRNT (Fig. 4c). In this group, the Poisson regression analysis confirmed that schistosome infection was the only factor significantly associated with the plaque count reduction (Fig. 4d). When the samples were stratified by the degree of liver fibrosis, a non-linear association with disease severity was observed (P = 0.022, Fig. 4e). The greatest antiviral effects in the PRNT were observed between Sneg and Spos-NF at 21.8% (RR: 0.782; 95% CI: 0.645–0.948), and Sneg and Spos-SF at 27.5% (RR: 0.725; 95% CI: 0.559–0.932). Collectively, these data indicated that the degree of liver fibrosis was the only factor significantly associated with a reduction in DENV infectivity in vitro (Fig. 4f).
Contribution of secreted cytokines and IgE to the antiviral activity of schistosome-positive sera
Next, we aimed to characterise the soluble factors potentially associated with the observed protective effects of schistosomiasis sera against DENV infection. Given the quantity and type of samples needed, we quantitatively assessed the levels of a selected panel of cytokines related to the chronic immune response to schistosomes (e.g., IL-4, IL-13, and IL-5) and DENV infections (e.g., IFN-α2 and TNF-α). Furthermore, we targeted IgE due to its specific role in parasitic infections.
First, a subset (n = 40) of sera from Atsinanana with comparable socio-demographic characteristics was selected on the basis of the PRNT results: (i) Sneg samples with plaque counts similar to the median (n = 10), (ii) Spos samples with the highest plaque counts (n = 15), and (iii) Spos samples with the lowest plaque counts (n = 15). Few differences among the cytokine profiles of the three groups were observed (Supplementary Table 1). Upon combining the two Spos groups, we observed a mild decrease in the levels of pro-inflammatory cytokines (IL-1β, IFN-α2, IL-6, and TNF-α) among the Spos sera. However, statistically significant differences were identified only for IFN-α2 (P = 0.014) and TNF-α (P = 0.029) (Supplementary Table 2). Second, a subset of sera from Haute Matsiatra (n = 40) was selected for further testing: (i) Sneg samples (n = 8), (ii) Spos-NF samples (n = 11), (iii) Spos-MF samples (n = 12), and (iv) Spos-SF samples (n = 9). No significant differences were observed in the cytokine concentrations when the four groups were analysed separately or when the three Spos groups were combined, based on Mann-Whitney-Wilcoxon and Kruskal-Wallis tests. However, a decrease in TNF-α with increasing disease severity was observed (Supplementary Table 3).
IgE levels were assessed in the same subset of plasma from Haute Matsiatra (n = 40). Overall, the median IgE level steadily increased with increasing disease severity in schistosomiasis participants (Supplementary Fig. 1). The highest median concentration was found in the Spos-SF group. In the same group, the greatest reduction in viral infection was observed in the PRNT.
Discussion
This study reports for the first time a low seroprevalence of DENV (up to 3.3%) in areas with a high prevalence of schistosome infection (> 50%) in multiple regions of Madagascar and a significant antiviral activity against DENV infection in vitro in sera derived from schistosome-infected individuals (up to 27.5%).
Compared with previous studies in Madagascar, the lower seroprevalence of DENV detected in this study [27, 28] can be explained by the different methodologies used. The seroprevalence assessment in our study is based on a recently marketed Fc-receptor-based assay, allowing for improved pan-DENV serology. This assay enables differential detection of IgG antibodies elicited by contact with different orthoflaviviruses. Using this approach, we demonstrate that 6–14% of all DENV seropositive tests are due to cross-reactive antibodies against DENV NS1. This finding indicates the presence of other orthoflaviviruses circulating in the area. Hence, we speculate that the differences observed in our study might be due to the broad cross-reactivity of widely used tests for the detection of IgG antibodies against multiple orthoflaviviruses. The specific assays used here, in contrast, provide a more accurate and realistic assessment of the DENV seroprevalence in the area. We observed a tendency toward a lower DENV prevalence in schistosome-infected individuals. Additionally, we observed a decreased prevalence of DENV in farmers. As a population in direct and continuous contact with stagnating waters, farmers are expected to be at high risk of DENV infection [29], which contrasts our findings. Nevertheless, farmers were described in previous studies to be among the groups with the highest prevalence of schistosomiasis [23, 30].
Importantly, our results strongly support a protective effect of sera from schistosome-infected individuals (reduction of 14.3%), and those presenting with no fibrosis or high disease severity (reduction of up to 27.5%), against DENV infections in vitro. Although the PRNT is conventionally considered the gold standard of serological tests for characterising and quantifying circulating levels of DENV-specific neutralising antibodies [31, 32], its experimental design allows the visualisation of the influence of other soluble factors on viral infectivity [33]. This feature supports its validity for use in our study. Collectively, these data suggest that the degree of liver fibrosis, specifically either no fibrosis or severe fibrosis, which represent the two extremes of the immune response spectrum during Schistosoma infections, is more likely associated with the secretion of soluble mediators that confer protection against DENV infection in vitro. In contrast, moderate fibrosis does not show this association.
Analysis of IgE levels across different participant groups revealed increased concentrations in the Spos-SF group, which also presented the greatest antiviral activity in the PRNT group, suggesting a possible connection between increased IgE levels and anti-DENV activity.
To our knowledge, this is one of the first studies leveraging a well-controlled and stratified epidemiological survey in an endemic country, with a targeted phenotypic approach to characterise the effect of pre-existing schistosome infections on the disease outcome of acute viral infections. Importantly, leveraging a large cohort of human samples from endemic areas for both schistosome and arboviral infections, this study supports for the first time a possible association between schistosome and DENV infections. Despite the value of this proof-of-concept, the limitations of our study must be considered. Due to the unexpectedly low DENV seroprevalence, the sample size, although nearly doubled compared with our initial assumptions, was still insufficient to perform a precise risk factor analysis. Furthermore, sampling occurred in rural settings that are ideal for schistosomiasis but might be less favourable for DENV infections, which are more frequently described in urban settings [2, 34]. The use of samples from other studies limited the ad hoc sampling design, preventing the investigation of specific indicators that might have strengthened some of the results. In addition, the PRNT was conducted on a non-human cell line, so conclusions may have limited applicability to humans. Moreover, the limited availability of samples did not allow further investigations into soluble factors responsible for the observed reduction in DENV infectivity.
Conclusions
In conclusion, our study suggests that schistosomiasis may exert a protective effect against DENV infections, as sera from infected participants significantly inhibited viral infection in vitro. By combining a well-controlled epidemiological survey with experimental laboratory-based methodologies, we shed light upon potential interactions between co-circulating infections that could influence infectious disease dynamics on the African continent. These findings open up new avenues for studies aimed at understanding the underlying mechanisms of protection and the interplay of chronic schistosomiasis and DENV.
Supplementary Information
Acknowledgements
We are grateful to all the participants, as well as the field teams including drivers, data clerks, healthcare workers, technicians, and community workers who contributed to the success of the study. Special thanks to all the country authorities who allowed the implementation of this study and were supportive of its activities.
Abbreviations
- BNITM
Bernhard Nocht Institute for Tropical Medicine
- CHU
Centre Hospitalier Universitaire
- CI
Confidence interval
- COVID-19
Coronavirus disease 2019
- cPOR
Crude prevalence odds ratio
- CRF
Case report form
- DALYs
Disability-adjusted life-years
- DENV
Dengue virus
- DNA
Deoxyribonucleic acid
- eCRF
Electronic case report form
- ELISA
Enzyme-linked immunosorbent assay
- HBV
Hepatitis B virus
- HCV
Hepatitis C virus
- HIV
Human immunodeficiency virus
- HRP
Horseradish peroxidase
- IFN
Interferon
- Ig
Immunoglobulin
- IL
Interleukin
- IQR
Interquartile range
- NS
Non-structural
- OD
Optical density
- PCR
Polymerase chain reaction
- PFU
Plaque forming units
- POC-CCA
Point-of-care circulating cathodic antigen
- PRNT
Plaque reduction neutralisation test
- qPCR
Semi-quantitative polymerase chain reaction
- Ref
Reference
- RR
Risk ratio
- Sneg
Schistosome-negative
- Spos
Schistosome-positive
- Spos-MF
Schistosome-positive with moderate liver fibrosis
- Spos-NF
Schistosome-positive without liver fibrosis
- Spos-SF
Schistosome-positive with severe liver fibrosis
- Th
T helper cell
- TNF
Tumor necrosis factor
Author contributions
DF and RAR supervised the studies in Madagascar. DF, TaR, RR, NGS, JM, and RAR contributed to the conceptualisation of the field-based studies. PS, DF, and LB conceptualised the experimental part of the study. The main writers of the manuscript were JCH, DF, and PS. DF, JCH, TaR, ARR, NMN, ZTR, TiR, TRR, JH, NR, AR, JMK, and VM contributed to the field implementation, data and sample collection. DF, PS, LB, and CD supervised the laboratory analysis. Mainly JCH, and partly ARR, NMN, ZTR, SL, SR, LW, and CE performed the processing of the samples and laboratory testing. AR, JH, PR, AJ, and IK contributed to data management and cleaning. JCH and IK performed the data analysis. DF and partly PS and JM contributed to the acquisition of the required funding for the study. All authors revised and approved the manuscript before submission.
Funding
Open Access funding enabled and organized by Projekt DEAL. Work from DF was supported by the German Centre for Infection Research (DZIF) under Grant TI 03.907 and Grant 8008803819; and the German Federal Ministry of Education and Research (BMBF) as part of the German Alliance for Global Health Research (GLOHRA) under Grant O1KA21O9A. Work in the PS laboratory was supported by the Free and Hanseatic City of Hamburg and the German Research Foundation (DFG Deutsche Forschungsgemeinschaft) under Grant 499961789. PS and LW are associated with the DFG Collaborative Research Center (CRC) 1648 (SFB 1648/1 2024–5 12741711).
Data availability
Research data supporting the findings of this study are available upon reasonable request from the corresponding author.
Declarations
Ethics approval and consent to participate
Ethical clearance was obtained from the National Ethics Committee of Madagascar (N°23 MSANP/CERBM, 05/03/2018; N°137 MSANP/SG/AMM/CERBM, 13/06/2023; N°136 MSANP/SG/AMM/CERBM, 13/06/2023) and the Ethics Committee of the Hamburg State Medical Chamber in Germany (PV7019-4419-BO-ff, 30/05/2023; 2022-100793-BO-ff, 17/07/2023; 2021-100743-BO-ff, 08/06/2023). All participants were informed about the aims of the study and its procedures in the local language (Malagasy). Study participation was voluntary, and no monetary incentives were offered. Written informed consent for participation was obtained from all participants. The participants had the right to withdraw from the study at any time without reason. Those positive for schistosome infection at recruitment were offered a treatment of 40 mg/kg praziquantel.
Consent for publication
All co-authors agreed with the text and gave consent for publication.
Competing interests
CD is the founder and shareholder of Panadea Diagnostics GmbH. All the other authors report that there are no competing interests to declare.
Contributor Information
Pietro Scaturro, Email: pietro.scaturro@leibniz-liv.de.
Daniela Fusco, Email: fusco@bnitm.de.
References
- 1.Amarasinghe A, Kuritsky JN, Letson GW, Margolis HS. Dengue virus infection in Africa. Emerg Infect Dis. 2011;17(8):1349–54. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.World Health Organization. Dengue and severe dengue. https://www.who.int/news-room/fact-sheets/detail/dengue-and-severe-dengue. Accessed 18 Feb 2025.
- 3.Vicco A, McCormack C, Pedrique B, Ribeiro I, Malavige GN, Dorigatti I. A scoping literature review of global dengue age-stratified seroprevalence data: estimating dengue force of infection in endemic countries. EBioMedicine. 2024;104:105134. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL, et al. The global distribution and burden of dengue. Nature. 2013;496(7446):504–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Deng J, Zhang H, Wang Y, Liu Q, Du M, Yan W, et al. Global, regional, and national burden of dengue infection in children and adolescents: an analysis of the Global Burden of Disease Study 2021. eClinicalMedicine. 2024. 10.1016/j.eclinm.2024.102943. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Bullington BW, Klemperer K, Mages K, Chalem A, Mazigo HD, Changalucha J, et al. Effects of schistosomes on host anti-viral immune response and the acquisition, virulence, and prevention of viral infections: a systematic review. PLoS Pathog. 2021;17(5):e1009555. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Nkurunungi G, Nassuuna J, Natukunda A, Zirimenya L, Walusimbi B, Zziwa C, et al. The effect of intensive praziquantel administration on vaccine-specific responses among schoolchildren in Ugandan schistosomiasis-endemic islands (POPVAC A): an open-label, randomised controlled trial. Lancet Glob Health. 2024;12(11):e1826–37. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Wolday D, Gebrecherkos T, Arefaine ZG, Kiros YK, Gebreegzabher A, Tasew G, et al. Effect of co-infection with intestinal parasites on COVID-19 severity: A prospective observational cohort study. EClinicalMedicine. 2021;39:101054. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Yazdanbakhsh M, Matricardi PM. Parasites and the hygiene hypothesis. Clin Rev Allergy Immunol. 2004;26(1):15–23. [DOI] [PubMed] [Google Scholar]
- 10.Simo FBN, Bigna JJ, Kenmoe S, Ndangang MS, Temfack E, Moundipa PF, et al. Dengue virus infection in people residing in Africa: a systematic review and meta-analysis of prevalence studies. Sci Rep. 2019;9:13626. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.McManus DP, Dunne DW, Sacko M, Utzinger J, Vennervald BJ, Zhou XN. Schistosomiasis. Nat Rev Dis Primers. 2018;4(1):13. [DOI] [PubMed] [Google Scholar]
- 12.World Health Organization. Schistosomiasis. https://www.who.int/news-room/fact-sheets/detail/schistosomiasis. Accessed 17 Feb 2025.
- 13.Li Q, Li YL, Guo SY, Li SZ, Wang Q, Lin WN, et al. Global trends of schistosomiasis burden from 1990 to 2021 across 204 countries and territories: findings from GBD 2021 study. Acta Trop. 2025;261:107504. [DOI] [PubMed] [Google Scholar]
- 14.Colley DG, Bustinduy AL, Secor WE, King CH. Human schistosomiasis. Lancet. 2014;383(9936):2253–64. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Hams E, Aviello G, Fallon PG. The schistosoma granuloma: friend or foe? Front Immunol. 2013;4:89. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Schramm G, Haas H. Th2 immune response against Schistosoma mansoni infection. Microbes Infect. 2010;12(12):881–8. [DOI] [PubMed] [Google Scholar]
- 17.Barron L, Wynn TA. Macrophage activation governs schistosomiasis-induced inflammation and fibrosis. Eur J Immunol. 2011;41(9):2509–14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.St. John AL, Abraham SN, Gubler DJ. Barriers to preclinical investigations of anti-dengue immunity and dengue pathogenesis. Nat Rev Microbiol. 2013;11(6):420–6. [DOI] [PubMed] [Google Scholar]
- 19.Kou Z, Quinn M, Chen H, Rodrigo WWSI, Rose RC, Schlesinger JJ, et al. Monocytes, but not T or B cells, are the principal target cells for dengue virus (DV) infection among human peripheral blood mononuclear cells. J Med Virol. 2008;80(1):134–46. [DOI] [PubMed] [Google Scholar]
- 20.Ratsitorahina M, Harisoa J, Ratovonjato J, Biacabe S, Reynes JM, Zeller H, et al. Outbreak of dengue and chikungunya fevers, Toamasina, Madagascar, 2006. Emerg Infect Dis. 2008;14(7):1135–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Institut Pasteur de Madagascar A. Investigation d’épidémie de dengue à Mahajanga du 4 au 9 mai 2020. https://www.pasteur.mg/investigation-depidemie-de-dengue-a-mahajanga-du-4-au-9-mai-2020/. Accessed 12 Mar 2025.
- 22.Gruninger SK, Rasamoelina T, Rakotoarivelo RA, Razafindrakoto AR, Rasolojaona ZT, Rakotozafy RM, et al. Prevalence and risk distribution of schistosomiasis among adults in Madagascar: a cross-sectional study. Infect Dis Poverty. 2023;12(1):44. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Krumkamp R, Remkes A, Hainasoa J, Rasamoelina T, Razafindrakoto AR, Razafindralava NM, et al. Prevalence of schistosome infection in a region of Madagascar regularly covered by Mass Drug Administration: a cross-sectional study. Manuscript submitted for publication. 2025.
- 24.Chow SC, Shao J, Wang H, Lokhnygina Y. Sample Size Calculations in Clinical Research. 3rd ed. New York: Chapman and Hall/CRC; 2017. p. 510. [Google Scholar]
- 25.Wichmann D, Panning M, Quack T, Kramme S, Burchard GD, Grevelding C, et al. Diagnosing schistosomiasis by detection of cell-free parasite DNA in human plasma. PLoS Negl Trop Dis. 2009;3(4):e422. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Scaturro P, Cortese M, Chatel-Chaix L, Fischl W, Bartenschlager R. Dengue virus non-structural protein 1 modulates infectious particle production via interaction with the structural proteins. PLoS Pathog. 2015;11(11):e1005277. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Schwarz NG, Girmann M, Randriamampionona N, Bialonski A, Maus D, Krefis AC, et al. Seroprevalence of antibodies against chikungunya, dengue, and Rift Valley fever viruses after febrile illness outbreak, Madagascar. Emerg Infect Dis. 2012;18(11):1780–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Broban A, Olive MM, Tantely ML, Dorsemans AC, Rakotomanana F, Ravalohery JP, et al. Seroprevalence of IgG antibodies directed against dengue, chikungunya and West Nile viruses and associated risk factors in Madagascar, 2011 to 2013. Viruses. 2023;15(8):1707. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Sievers BL, Hyder S, Claes F, Karlsson EA. Ingrained: rice farming and the risk of zoonotic spillover, examples from Cambodia. One Health. 2024;18:100696. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Bukenya GB, Nsungwa JL, Makanga B, Salvator A. Schistosomiasis mansoni and paddy-rice growing in Uganda: an emerging new problem. Ann Trop Med Parasitol. 1994. 10.1080/00034983.1994.11812880. [DOI] [PubMed] [Google Scholar]
- 31.World Health Organization. Informing vaccination programs: a guide to the design and conduct of dengue serosurveys. 2017; 50. https://iris.who.int/handle/10665/255650. Accessed 30 Jan 2025.
- 32.Maeda A, Maeda J. Review of diagnostic plaque reduction neutralization tests for flavivirus infection. Vet J. 2013;195(1):33–40. [DOI] [PubMed] [Google Scholar]
- 33.Zainal N, Tan KK, Johari J, Hussein H, Wan Musa WR, Hassan J, et al. Sera of patients with systemic lupus erythematosus cross-neutralizes dengue viruses. Microbiol Immunol. 2018;62(10):659–72. [DOI] [PubMed] [Google Scholar]
- 34.Kolimenakis A, Heinz S, Wilson ML, Winkler V, Yakob L, Michaelakis A, et al. The role of urbanisation in the spread of Aedes mosquitoes and the diseases they transmit—a systematic review. PLoS Negl Trop Dis. 2021;15(9):e0009631. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
Research data supporting the findings of this study are available upon reasonable request from the corresponding author.