Loiasis, caused by the filarial worm Loa loa, is transmitted by tabanid flies ( Chrysops sp.) in Central Africa and West Africa. Demographic data suggest that tens of millions of people are exposed and 15 million are actually infected [ 1 ]. A notable characteristic of loiasis is that female worms can produce millions of microfilariae (mf) circulating in the blood, with infected individuals potentially harboring very high microfilarial densities (MFD) exceeding 100,000 mf/ml of blood. Additionally, loiasis is a chronic infection as adult worms live up to 20 years, and the MFD of untreated individuals remains fairly stable over time [ 2 , 3 , 4 ]. Significant eosinophilia is commonly associated, which may contribute to coagulation and cardiac disorders [ 5 , 6 ].

Although historically loiasis has been described as causing benign manifestations, such as pruritus, subconjunctival migration of the adult worms ( "eye worm" ), and transient episodes of angioedema ( "Calabar swellings" ), retrospective population-based cohort studies in Cameroon and the Republic of Congo demonstrated that individuals with high L. loa MFD had a significantly reduced life-expectancy and that the risk of premature death may be proportional to the individual’s MFD [ 7 , 8 ]. Recent results show that loiasis has a significant burden of disease [ 9 ], and can induce complications affecting different organs such as the cardiovascular system, central nervous system, and kidneys [ 10 , 11 , 12 , 13 ], which may partially explain this excess mortality.

Although the possible presence of splenic nodules in patients with loiasis has been described for a long time [ 14 ], data on the impact of filariasis on the spleen are scarce [ 15 ]. It has been reported that the spleens of monkeys infected with L. loa present nodules that gradually disappear after an antifilarial treatment with diethylcarbamazine [ 16 ]. More recently, splenic lesions have been found in 8% of European cases and 1.9% of Gabonese cases of loiasis, respectively [ 17 , 18 ]. The pathogenic processes underlying the development of these nodules remain unclear. However, several cases of hypoechoic splenic nodular lesions associated with granulomas containing mf have been reported in loiasis patients [ 14 , 19 ]. In one case, treatment with ivermectin led to regression of splenic lesions [ 20 ]. In another patient, who died after receiving diethylcarbamazine treatment, foci of necrosis containing degenerated mf were found in the spleen [ 21 ].

In the context of polyparasitism among individuals living in Central Africa, particularly with the presence of malaria and hyperimmune tropical splenomegaly (also referred to as chronic malarial splenomegaly), which coexists with a significant prevalence of sickle cell disease, we aimed to consider loiasis as another potential factor that may have a deleterious effect on the spleen. Furthermore, sickle cell disease poses the dual challenge of splenomegaly during crises and secondary involution due to multiple infarctions; the ultimate consequence being the decreased functionality of the spleen against infections.

A meticulous examination of spleen volume (SV) was undertaken to test the hypothesis that L. loa MFD might be associated with smaller SV, owing to alteration of the reticuloendothelial system. Our objective was to characterize the association between L. loa microfilaremia and lower SV. We also aimed to describe the splenic ultrasound characteristics in a general population residing in rural areas of the Republic of Congo, and to characterize factors associated with anomalies in volume, size [understood here as a bi-dimensional measurement, namely height, length, width, and cranio-caudal distance (CCD)], and the presence of lesions, including loiasis and malaria.

This study provides a comprehensive description of the bi-dimensional sizes and volume of the spleen and the factors influencing its dimensions variations in a rural population in Central Africa. Notably, we demonstrated for the first time the impact of L. loa MFD on splenic volume, and provide a global overview of the respective roles of loiasis and malaria on spleen dimensions with estimates suggesting that L. loa mf may have a great influence on splenic dimensions.

A strength of this study lies in the consideration of various comorbidities known to be associated with splenic involvement, including parasitic infections such as malaria, sickle cell disease, and hematological comorbidities. Our findings are consistent with the existing literature regarding the physiological influence of BMI and sex on SV and the splenic involution with age. Additionally, we tried to account for numerous common exposure factors in this region, such as STH and other parasites ( O. volvulus, M. perstans, S. haematobium), but given the absence of very low number of cases, their impact should still be investigated in further studies. The fact that no case of schistosomiasis was found is probably due to the low prevalence of this disease in the study area, as shown by surveys conducted on school-age children in 2015 (prevalence of 3% in the Sibiti Health District) and to the fact that praziquantel is available for individual treatment in the health structures. Regarding hookworm infections, our results might be due to technical issues, even if the microscopists were experienced.

Regarding the abnormalities observed in our adult population, chronic malaria exposure, as indicated by Plasmodium IgG levels, was positively and gradually associated with splenomegaly, and age. Subsequently, we sought to categorize individuals with the HMS entity. HMS are mainly based on an ancient definition by Fakunle et al. [ 31 ]although it has been questioned [ 32 ]. Apart from splenomegaly and reduction of splenic volume following antimalarial treatment, it includes several biologic abnormalities, for which different threshold can be found across the literature. When used, the cut-off for total plasmatic IgM varies between 2.5 and 3.5 g/L, or+ 2 SD above normal value when the latter is known [ 31 , 35 ]. For anti- Plasmodium-IgG levels, one can apply the threshold of > 1/800 when outdated indirect immunofluorescence techniques are applied, otherwise it is most frequently considered relevant when elevated without specification. For this reason, we proposed two sets of criteria—one stringent and one more permissive—to provide a range for this potential issue in this region, and estimated that they represented between 1.4 and 5.3% of our study population. The figures appear close to those found in other African countries [ 36 , 37 , 38 , 39 , 40 , 41 ].

Regarding the association with small spleens, particularly those ≤ 80 cm ³, which have been associated in one study with excess mortality from pneumococcal infections [ 30 ], we demonstrated a gradual association with L. loa MFD, with high odds ratio (5.8 to 17.9) for individuals with MFD > 30,000 mf/ml, depending on the SV cutoff. Although not all subgroup analyses revealed significant gradient effects, the fact that aORs were consistently increased in individuals with microfilaremia, regardless of their MFD, and that the association was highly significant in those belonging to the highest MFD category, supports the hypothesis of a deleterious effect on the spleen. The primary mechanism might be purely mechanical, directly correlated with circulating L. loa mf causing chronic splenic microembolization (akin to sickle cell disease) and leading to secondary atrophy of the reticuloendothelial system (RES), and this could be influenced or not by hemostatic disorders consecutive to hypereosinophilia. The impairment of the RES, by circulating immune complexes present in loiasis and a chronic generation of opsonins and other mediators of phagocytosis may also contribute to an RES atrophy.

Nonetheless, it is crucial to interpret our results within the context of an absence of direct translation between AH and functional hyposplenia. Furthermore, while some studies suggest an association between very small spleens and functional impairment, there is also evidence to the contrary, such as the preserved functionality of small spleen implants post-splenectomy, which underscores the complexity of this relationship and the need for cautious interpretation [ 42 , 43 , 44 ]. While the presence of HJB strongly indicates splenic impairment, the absence of HJB is not indicative of functional splenic tissue [ 45 ]; nevertheless it remains the reference method widely used to assess splenic function. In our study, the clear association between HJB and AH (and its possible gradient effect) reinforces the validity of our results and suggests that those individuals are at higher risk of functional hyposplenia. Given this context, functionality assessment using cytometric measurement of circulating memory B lymphocytes is required in future studies.

Two other results are worth highlighting. First, no association was found between SV and STH or history of eyeworm, which we considered as a proxy for L. loa adult worm burden during life. This supports the hypothesis that the impact of L. loa on the spleen is due to the presence of blood mf and to the MFD, and not to the number of adult worms present. Second, AH was associated with low loiasis pan-IgG-RDT reactivity. This result should be interpreted cautiously given that the test we have used has been recently investigated by Veletzky et al., and was found to be often falsely negative in individuals with very high L. loa MFDs [ 46 ]. Therefore, the association between AH and low RDT reactivity might be explained by high MFDs.

We emphasize that our findings highlight an association rather than establish causality. The possibility of reverse causation was considered, with the main hypothesis that functional hyposplenism might arise from a cause unrelated to L. loa, and that this condition can induce the presence or an increase in MFD, as seen in primate models of L. loa where high MFDs are allowed by splenectomy. However, parasitological surveys conducted over decades show consistent stability in microfilarial distribution in populations, suggesting that such a condition would have a stable prevalence over time and an evenly distribution in central Africa. At the individual level, the MFD is also plateauing after 15 years in individuals and remains stable after. The underlying condition would therefore occur in children or young adults, and would not worsen after adulthood. We cannot identify a condition meeting those criteria (high prevalence stable over decades, absence of worsening after adulthood) to the best of our knowledge. Nevertheless, we plan future studies with measurements of memory B lymphocytes, monitoring of spleen size over time, including HIV status, and assessing bacterial infection trends in relation with spleen dimension evolution over time to further elucidate these possibilities. While splenectomy is associated with increased MFD due to complete loss of splenic filtration, functional hyposplenia represents a partial reduction in splenic activity rather than its complete absence. This may explain why an increase in mf/ml is not observed, as compensatory or adaptative mechanisms could mitigate the effects of reduced splenic function.

Lastly, we estimated that nearly a quarter of the small spleen burden in our study population could be attributable to detectable microfilaremia. Although we mostly found hyperechoic macronodular rather than micronodular lesions—in relation with the use of a convex probe as detailed in the limits thereafter—we observed an association between MFD and the presence of US anomalies, providing indirect evidence of a potentially chronic deleterious effect of loiasis on the spleen. The only comparable cross-sectional study conducted in an endemic area was performed in Gabon [ 18 ]. This study did not find an effect of L. loa MFD on spleen dimensions, likely due to the limited sample size (216 participants) and, more critically, the small number of individuals with over 8000 mf/ml (10 participants). In contrast, our study included 97 individuals with more than 8000 mf/ml, and the observed significant differences were predominantly found in this subgroup with intermediate and high MFD levels. Further studies are needed, but we believe the lack of association in the Gabon study is primarily due to insufficient sample size. Regarding lesion characterization, the Gabon study reported hypoechoic macronodules in 3 participants (1.4%, 95% CI: 0.3, 4.0), all of whom were microfilaremic (3.6%, 95% CI: 0.7, 10.1). Hypoechoic micronodules, visible only with the linear probe, were observed in 3 participants (1.4%, 95% CI: 0.3, 4.0), all amicrofilaremic. This corresponds to a prevalence of 2.3% for micronodules among amicrofilaremic individuals. In comparison with our findings, it is noteworthy that the prevalence levels across different lesion types appear relatively consistent, as the confidence intervals overlap or are very similar. Importantly, as in the Gabon study, our only significant difference pertains to participants with hypoechoic macronodules, observed exclusively in microfilaremic individuals. In our study, the prevalence of hypoechoic macronodules was significantly higher among microfilaremic individuals compared to amicrofilaremic ones (1.7% vs. 0.3%, P= 0.026). However, given the small number of lesions observed, interpretation of this finding remains limited. As suggested in the Gabon study, hypoechoic lesions may be transient. A limitation of our study is that millimetric lesions or areas potentially corresponding to reticuloendothelial system (RES) atrophy could not be detected with our ultrasound probe. Since our main hypothesis revolves around mechanisms affecting the RES, incorporating CT scans would provide valuable insights. Unfortunately, implementing such imaging techniques in these regions is highly challenging. Future studies should focus specifically on hypoechoic lesions to determine whether they represent true spleen pathology or are merely indirect indicators of microfilarial presence.

Our study has some limitations. Although TBS is a reference technique and was prepared and read by highly experienced technicians in our study, we acknowledge this method has a lower sensitivity compared to concentration techniques, which could result in the underestimation of microfilaremia prevalence of M. perstans and L. loa. However, despite potential misclassification, our study was able to demonstrate a robust association between MFD and AH, underscoring the validity of our findings. We used a convex probe for ultrasonographic examination. Those low-frequency transducers are generally used for abdominal examination as they offer a better coverage of deep structures. However, we acknowledge that the use of a linear high-frequency probe could have allowed the identification of some additional micronodular lesions given its higher resolution in superficial structures, and that we may have missed some millimetric lesions. Therefore, the use of a linear probe could have enhanced the detection or identification of features indicative of RES atrophy, but the use of both probes was not feasible due to limitations in the availability of human resources time. Although very high microfilaremia (> 30,000 mf/ml) were significantly associated with AH (< 80, 110, 150 cm ³) in logistic analysis, the association could not be evidenced with the volume as a continuous variable using the linear regression model, likely due to a lack of power.

This study provides a comprehensive description of factors associated with splenic variation in dimension in a general population living in rural Central Africa. The first one was chronic exposure to malaria, leading to increased spleen volume and in some cases to HMS. The second one was L. loa microfilaremia, which was strongly associated with AH, accounting for up to 25% of them. If an association with functional hyposplenia remains to be proven, our results point towards this direction, suggesting that, given the size of this attributable fraction, loiasis may be responsible for a significant proportion of excess mortality following malarial and bacterial infections in Central Africa, particularly for encapsulated germs.

Despite these promising results, further studies are necessary to refine our findings and confirm an association with spleen functionality impairment, particularly through cohort studies, that should incorporate B memory cell typing and evaluate long term outcomes, in particular the risk of developing bacterial and malarial infections.