Background

Loiasis, caused by the filarial nematode Loa loa and transmitted from human to human by tabanids, is exclusively endemic in central Africa. In infected individuals, adult female worms release embryos called microfilariae (mfs) which circulate in the bloodstream according to a diurnal cycle, with densities occasionally surpassing 100,000 mfs per mL of blood (mf/mL).

Although loiasis is historically considered as a benign disease, recent findings suggest that microfilaremic individuals have a significantly shortened life expectancy [1]. In addition, studies in the Republic of Congo showed that high L. loa microfilarial density (MFD) was significantly associated with proteinuria [2], cognitive impairment [3], and increased vascular stiffness [4].

Eosinophilia exceeding 5,000 /μL is common among subjects with loiasis. Eosinophils, known to express tissue factor, may trigger blood coagulation [5], leading to thrombosis. Reports indicate that arterial and venous thrombotic events can occur in patients with parasite-related hypereosinophilia [6], with documented instances of thrombosis [7] and micro-emboli formation in capillary vessels [8], occurring both spontaneously and as part of post-ivermectin (IVM) Loa-related serious adverse events (SAEs). Indeed, IVM mass drug administration in onchocerciasis endemic regions may cause SAEs such as life-threatening encephalopathies in patients with high L. loa MFD above 30,000 mf/mL, due to its potent microfilaricidal effect [9]. Evidence from primate models suggests an association between post-IVM encephalopathy and hemostatic dysregulation, characterized by degenerating mfs in small vessels associated with fibrin deposition, endothelial damages, and hemorrhages [10]. Similarly, humans experiencing post-ivermectin encephalopathy often exhibit conjunctival and retinal hemorrhages [11].

A potential dysregulation of hemostasis may contribute to the excess morbidity and mortality associated with loiasis and its treatment with IVM. However, data on this phenomenon remains limited and unexplored in humans. The objective of the present study was to assess for the first time the impact of both L. loa MFD and IVM on human hemostasis and inflammation blood parameters.

Methods

Results

Twenty males and 18 females aged between 28 and 67 years (median age: 43 years old) participated in the study.

Table 1 presents the blood counts and the inflammation and hemostasis parameters according to the participants’ L. loa MFD categories. There was a trend towards increased leucocyte, granulocyte and eosinophil counts with increasing MFD (p = 0.011, p = 0.013 and p<0.001, respectively, Cuzick’s tests for trend). Median eosinophil counts were significantly higher in patients with a MFD exceeding 20,000 mf/mL compared to those with no mf (1,016/μL vs 111/μL, p<0.001). Similarly, median platelet counts were also significantly higher in the same group (254,500/μL vs 180,000/μL, p = 0.044). Inflammation and hemostasis parameters did not significantly differ between groups. Linear regression analysis of D-dimers by MFD, adjusted on age and sex, did not show any significant associations. Median ESR were consistently increased across all groups (normal values: < 20 mm/h). Gamma-globulins were also elevated in all groups, with medians from 19 to 23 g/L (normal values: < 11 g/L). Clonal gamma-globulin expression was suspected in the serum protein electrophoresis in 3 patients, each in separate groups.

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Table 1. Blood count, inflammation, and hemostasis parameters according to L. loa microfilaremia.

https://doi.org/10.1371/journal.pntd.0012491.t001

Eighteen individuals with an MFD between 20 and 19,999 mf/mL received a single dose of IVM and were re-examined at D4 (Table 2). Post-treatment, the median MFD significantly decreased from 3,605 mf/mL to 1,810 mf/mL. Median eosinophil counts rose from 171/μL to 1,095/μL (p<0.001). Various inflammation markers changed significantly: CRP, ESR, fibrinogen and alpha-1-globulin levels increased (p = 0.038, p = 0.010, p = 0.002 and p = 0.044, respectively), while albumin level decreased p = 0.044). Among hemostatic parameters, median D-dimers significantly increased, from 582 ng/mL to 745 ng/mL after IVM (p = 0.024) with a median relative variation of +34.7% (interquartile range: 1.44–72.5%). Prothrombin fragment 1+2 did not significantly vary (252 versus 469 pmol/L, p = 0.313). No new clonal gamma-globulin expression on protein electrophoresis was suspected, while previously identified clonal expression in treated patients did not change after IVM.

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Table 2. Blood count, inflammation, and hemostasis parameters at baseline and 4 days after IVM treatment in patients with L. loa microfilaremia.

https://doi.org/10.1371/journal.pntd.0012491.t002

Discussion

In this pilot biological study, we describe for the first time hemostasis and inflammation parameters associated with loiasis. Our results show a remarkable trend towards increased leukocytes and granulocytes, mainly driven by elevated eosinophils, with increasing L. loa MFD. In a 2022 biological study in Gabon [13], eosinophil counts were also positively correlated with L. loa MFD, with an estimated increase in eosinophil counts every 10-fold increase in parasitemia (p-adj. = 0.012, ß-estimate: 0.17[0.04–0.31]). Chronic eosinophilia, a characteristic feature of loiasis, is known for its ability to trigger severe health conditions, such as endomyocardial fibrosis, tropical pulmonary eosinophilia, and thromboembolic events, consequently increasing the risk of mortality associated with loiasis [1].

The notable increase in platelets with increasing MFDs suggests their probable role in filarial infections. Prior research has established that platelets mediate the immunological killing of L. loa mfs [14,15], while data in lymphatic filariasis also demonstrated mfs’ ability to modulate platelet functions [16].

Furthermore, our study highlighted a polyclonal elevation of gamma-globulins across all MFD groups, suggesting a systemic immune response. This phenomenon is consistent with a previous study in Ghana, were 75% of participants had gamma-globulins > 16 g/L [17]. This hypergammaglobulinemia might be explained by the influence of genetic as well as environmental factors. ESR were increased in all groups, possibly driven by hypergammaglobulinemia, which is known to accelerate erythrocyte sedimentation.

Interestingly, while inflammation and hemostasis parameters did not significantly vary with L. loa MFD at baseline, treatment with IVM resulted in significant alterations in several inflammatory markers (such as CRP, ESR, fibrinogen, albumin and alpha-1-globulins). While post-IVM CRP increase has been reported previously [18], we document for the first time significant changes in a range of inflammatory parameters. We observed a marked increase in eosinophils, consistent with previous reports of interleukin-5 (IL-5)-driven eosinophilia following microfilarial killing by IVM [19].

The increase in D-dimers observed on D4 post-IVM suggests both enhanced fibrinolysis and coagulation activation which happen simultaneously. This could be attributed to the close relationship between hemostasis and inflammation, where inflammation induces fibrin formation in the extravascular space, facilitating immune cells functions [20]. Consequently, secondary fibrinolysis generates D-dimers, which correlate with inflammation severity. However, this correlation is modest, as previous studies found a Spearman’s correlation coefficient of 0.18 between CRP and D-dimers, indicating a limited response of D-dimers to CRP elevation [21]. Therefore, in addition to inflammation, the potential involvement of thrombotic processes in the increase of D-dimers after IVM cannot be ruled out, potentially both driven by intravascular dying mfs and inflammation-induced hypercoagulable condition.

In addition to previous reports of post-IVM retinal emboli [11], this hypothesis is supported by autopsies of post-IVM encephalopathies in baboon models, which show abnormalities ranging from intravascular degenerating mfs and vessel wall infiltration by immune cells such as eosinophils, to fibrin deposition and endothelial disruption with extravascular hemosiderin deposits [10,22]. These findings provide a plausible pathological basis for the observed post-IVM treatment biological changes, potentially contributing to the pathogenesis of post-IVM SAEs.

Despite the valuable insights gained, our study has limitations. The small sample size and absence of preexisting data require cautious interpretation of our findings, highlighting the need for larger dedicated studies to validate our observations and understand their clinical implications. However, our results, particularly in baseline comparisons, may guide further research with adequate sample sizes, especially to explore the impact of loiasis on the hemostatic system. Additionally, potential confounding by other health conditions among participants, particularly in a low-resource healthcare setting, warrants consideration. Despite this, hematological changes showed a significant increase trend with L. loa MFD, aligning with previous findings [13]. However, as only L. loa MFD was assessed, potential confounding by other parasitic infections sensitive to IVM cannot be ruled out. Finally, our reliance on a single post-IVM evaluation timepoint may have limited our ability to detect biological abnormalities occurring outside this timeframe.

In conclusion, this pilot study identified several biological changes potentially associated with loiasis, occurring both spontaneously and after treatment with IVM. Further dedicated studies are needed to confirm these results, which might participate in loiasis excess mortality as well as post-IVM SAEs pathogenesis.

Acknowledgments

We thank all the participants who agreed to take part in this study as well as the village chiefs and health workers involved.