Comparison of immune responses to Loa loa stage-specific antigen extracts in Loa loa-exposed BALB/c mice upon clearance of infection

Background: Different immune mechanisms are capable of killing developmental stages of filarial nematodes and these mechanisms are also likely to vary between the primary and a challenge infection. However, the lack of a detailed analysis of cytokine, chemokine and immunoglobulin levels in human loiasis is still evident. Therefore, detailed analysis of immune responses induced by the different developmental stages of Loa loa in immune-competent BALB/c mice will aid in the characterization of distinct immune responses that are important for the immunity against loiasis. Methods: Different developmental stages of L. loa were obtained from human peripheral blood (microfilariae, MF), the transmitting vector, Chrysops (larval stage 3, L3) and infected immune-deficient BALB/cRAG2γc−/− mice (L4, L5, adult worms). Groups of wildtype BALB/c mice were then injected with the isolated stages and after 42 days postinfection (pi), systemic cytokine, chemokine and immunoglobulin levels were determined. These were then compared to L. loa-specific responses from in vitro re-stimulated splenocytes from individual mice. All parameters were determined using Luminex technology. Results: In a pilot study, BALB/c mice cleared the different life stages of L. loa within 42 days pi and systemic cytokine, chemokine and munoglobulin levels were equal between infected and naive mice. Nevertheless, L. loa-specific re-stimulation of splenocytes from mice infected with L5, MF or adult worms led to induction of Th2, Th17 and chemokine secretion patterns. Conclusions: This study shows that although host immunity remains comparable to naive mice, clearance of L. loa life-cycle development stages can induce immune cell memory leading to cytokine, chemokine and mmunoglobulins secretion patterns which might contribute to immunity and protection against reinfection

Differential susceptibility of Onchocerca volvulus microfilaria to ivermectin in two areas of contrasting history of mass drug administration in Cameroon: relevance of microscopy and molecular techniques for the monitoring of skin microfilarial repopulation within six months of direct observed treatment

Background Ivermectin is an excellent microfilaricide against Onchocerca volvulus. However, in some regions, long term use of ivermectin has resulted in sub-optimal responses to the treatment. More data to properly document the phenomenon in various contexts of ivermectin mass drug administration (IVM-MDA) is needed. Also, there is a need to accurately monitor a possible repopulation of skin by microfilariae following treatment. Skin snip microscopy is known to have a low sensitivity in individuals with light infections, which can be the case following treatment. This study was designed with two complementary objectives: (i) to assess the susceptibility of O. volvulus microfilariae to ivermectin in two areas undergoing IVM-MDA for different lengths of time, and (ii) to document the repopulation of skin by the O. volvulus microfilariae following treatment, using 3 independent diagnostic techniques. Method Identified microfilaridermic individuals were treated with ivermectin and re-examined after 1, 3, and 6 months using microscopy, actin real-time PCR (actin-qPCR) and O-150 LAMP assays. Susceptibility to ivermectin and trends in detecting reappearance of skin microfilariae were determined using three techniques. Microscopy was used as an imperfect gold standard to determine the performance of actin-qPCR and LAMP. Results In Bafia with over 20 years of IVM-MDA, 11/51 (21.6%) direct observe treated microfilaridemic participants were still positive for skin microfilariae after 1 month. In Melong, with 10 years of IVM-MDA, 2/29 (6.9%) treated participants were still positive. The microfilarial density reduction per skin biopsy within one month following treatment was significantly lower in participants from Bafia. In both study sites, the molecular techniques detected higher proportions of infected individuals than microscopy at all monitoring time points. LAMP demonstrated the highest levels of sensitivity and real-time PCR was found to have the highest specificity. Conclusion Patterns in skin mirofilariae clearance and repopulation were established. O. volvulus worms from Bafia with higher number of annual MDA displayed a lower clearance and higher repopulation rate after treatment with ivermectin. Molecular assays displayed higher sensitivity in monitoring O. volvulus microfilaridemia within six months following treatment

Impact of repeated mass ivermectin administration using a community directed approach on L. loa infection in Chrysops silacea of the rain forest and forest savanna of Cameroon

Loiasis is an endemic filarial infection in the rainforest zone of West and Central Africa. Repeated annual community-directed treatment with ivermectin (CDTI) delivered for several years to control onchocerciasis has been shown to reduce the prevalence and intensity of Loiasis in some co-endemic areas. However, the impact of these multiple rounds of CDTI on entomological indicators of loiasis transmission is not known, and was therefore assessed in this study in areas with contrasting histories of CDTI. The study was conducted in the East, North-west and South-west 1 CDTI project sites of Cameroon. Two communities per CDTI project were selected for fly collection and dissection. Ivermectin treatment coverage was documented in these areas, and this was correlated to infection and infective rates. A total of 7029 female were collected from 6 communities of the 3 CDTI projects (East, North-west, and South-west 1) and from 2 communities in a non-CDTI district (East). biting densities and parous rates were significantly reduced in the North-west and South-west sites post-CDTI, while in the East, biting densities were similar in non-CDTI and CDTI sites, with higher parous rates observed in the non-CDTI site. Infection and infective rates in the East non-CDTI site were 4.4% and 1.8% respectively, as compared to 3.3% and 1.3% in the CDTI site after 10 ivermectin rounds (there were no baseline data for the latter). In the North-west site, significant reductions in infection and infective rates from 10.2% and 4.2% respectively, to 3.5% and 1.2 (after 9 rounds of ivermectin treatment), were recorded following CDTI. In the South-west, infection rate significantly increased from 1.74% to 2.8% and infective rate remained statistically unchanged after 14 rounds of CDTI (0.45% - 0.40%). Similar trends in Mean Head L3 were observed except in the East site where this indicator was similar in both CDTI and control sites. Only in the North-west site did monthly transmission potentials decrease significantly. This study demonstrated that the impact of repeated annual treatment with ivermectin for the control of onchocerciasis using community directed delivery approach on the entomological indicators of loiasis varies with bioecological zones. Community directed treatment with ivermectin induced a significant reduction in the entomological indicators of loiasis in the North-West project site which lies in forest savanna area. A non-significant decrease was observed in the East project site and in contrast, a significant increase was observed in the South-West 1 project site which both lies in the rainforest zones. [Abstract copyright: © 2024 Published by Elsevier Ltd on behalf of World Federation of Parasitologists.

Correction to: Differential susceptibility of Onchocerca volvulus microfilaria to ivermectin in two areas of contrasting history of mass drug administration in Cameroon: relevance of microscopy and molecular techniques for the monitoring of skin microfilarial repopulation within six months of direct observed treatment

After publication of the original article [1], the authors identified an error in the author name of Manuel Ritter. The given name and family name were erroneously transposed. The original article has been corrected. (BMC Infectious Diseases, (2020), 20, 1, (726), 10.1186/s12879-020-05444-2

Mapping lymphatic filariasis in Loa loa endemic health districts naïve for ivermectin mass administration and situated in the forested zone of Cameroon

Background The control of lymphatic filariasis (LF) caused by Wuchereria bancrofti in the Central African Region has been hampered by the presence of Loa loa due to severe adverse events that arise in the treatment with ivermectin. The immunochromatographic test (ICT) cards used for mapping LF demonstrated cross-reactivity with L. loa and posed the problem of delineating the LF map. To verify LF endemicity in forest areas of Cameroon where mass drug administration (MDA) has not been ongoing, we used the recently developed strategy that combined serology, microscopy and molecular techniques. Methods This study was carried out in 124 communities in 31 health districts (HDs) where L. loa is present. At least 125 persons per site were screened. Diurnal blood samples were investigated for circulating filarial antigen (CFA) by FTS and for L. loa microfilariae (mf) using TBF. FTS positive individuals were further subjected to night blood collection for detecting W. bancrofti. qPCR was used to detect DNA of the parasites. Results Overall, 14,446 individuals took part in this study, 233 participants tested positive with FTS in 29 HDs, with positivity rates ranging from 0.0% to 8.2%. No W. bancrofti mf was found in the night blood of any individuals but L. loa mf were found in both day and night blood of participants who were FTS positive. Also, qPCR revealed that no W. bancrofti but L.loa DNA was found with dry bloodspot. Positive FTS results were strongly associated with high L. loa mf load. Similarly, a strong positive association was observed between FTS positivity and L loa prevalence. Conclusions Using a combination of parasitological and molecular tools, we were unable to find evidence of W. bancrofti presence in the 31 HDs, but L. loa instead. Therefore, LF is not endemic and LF MDA is not required in these districts

Onchocerca ochengi male worms implanted in SCID mice and gerbil : relationship between microfilaridermia status of cows, nodular worm viability and fertility and worm survival in the rodents

Background Current treatment options for onchocerciasis are sub-optimal, prompting research and development of a safe cure (macrofilaricide). Onchocerca ochengi, a parasite of cattle, is used as a close surrogate for the human parasite O. volvulus in a murine model for pre-clinical screening of macrofilaricides. Skin from naturally infected cattle have been used in previous studies as a reliable source of parasite material. However, there is limited knowledge on how source-related factors such as the microfilaridermia status of the cattle, the nodule load and nodular worm viability may affect survival of male O. ochengi worms implanted in the rodent hosts. Such relationships were investigated in this study. Methods Dermal tissue and nodules were obtained from Gudali cattle, dissected and cultured to obtain migrating microfilariae (mf) and male worms. Emerged male worms were implanted into SCID mice and Gerbils (Meriones unguiculatus) and recovery rates were determined upon 42 days post implantation. Finally, nodules were processed for histology and embryogram analyses to assess the nodular worm viability and fertility, respectively. Results Of the 69 cattle sampled, 24 (34.8%) were mf+ and 45 (65.2%) were mf–. The mean nodule loads were 180.5 ± 117.7 (mf+) and 110.6 ± 102.7 (mf-) (p = 0.0186). The mean male worm harvest from nodules were 76.8 ± 120.3 and 47.2 ± 33.4 (p = 0.2488) for mf+ and mf– cattle, respectively. The number of male worms per 100 nodules were 57/100 and 46/100 nodules for mf+ and mf– cows, respectively. Female worms from nodules of mf– cows had higher counts of both normal and abnormal embryos with higher proportions of dead nodular worms evinced by histology compared to those from mf+ cows. A total of 651 worms were implanted into mice and gerbils, out of which 129 (19.81%) were recovered. Logistic regression analysis indicated that the microfilaridermia status of the cattle (presence of mf) (OR = 4.3319; P = 0.001) is the single most important predictor of the success of male worm recovery after implantation into rodents. Conclusion Microfilaridermic cattle provide a promising source of adult O. ochengi. Male worms from this group of cattle have a better success rate of survival in a murine implant model. Nevertheless, in the programmatic point of view, amicrofilaridermic Gudali cattle would still constitute an important source of O. ochengi male worms with relatively good viability after implantation into rodents

Multiple lineages of nematode-Wolbachia symbiosis in supergroup F and convergent loss of bacterioferritin in filarial Wolbachia

© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.The intracellular endosymbiotic proteobacteria Wolbachia have evolved across the phyla nematoda and arthropoda. In Wolbachia phylogeny, supergroup F is the only clade known so far with members from both arthropod and filarial nematode hosts and therefore can provide unique insights into their evolution and biology. In this study, 4 new supergroup F Wolbachia genomes have been assembled using a metagenomic assembly and binning approach, wMoz and wMpe from the human filarial parasites Mansonella ozzardi and Mansonella perstans, and wOcae and wMoviF from the blue mason bee Osmia caerulescens and the sheep ked Melophagus ovinus respectively. A comprehensive phylogenomic analysis revealed two distinct lineages of filarial Wolbachia in supergroup F, indicating multiple horizontal transfer events between arthropod and nematode hosts. The analysis also reveals that the evolution of Wolbachia-filaria symbioses is accompanied by a convergent pseudogenization and loss of the bacterioferritin gene, a phenomenon found to be shared by all filarial Wolbachia, even those outside supergroup F. These observations indicate that differences in heme metabolism might be a key feature distinguishing filarial and arthropod Wolbachia. The new genomes provide a valuable resource for further studies on symbiosis, evolution, and the discovery of new antibiotics to treat mansonellosis.publishersversionepub_ahead_of_prin

Heterogeneity in the in vitro susceptibility of Loa loa microfilariae to drugs commonly used in parasitological infections.

Co-infection with loiasis remains a potential problem in control programs targeting filarial infections. The effects of many anti-parasitic drugs often administered to Loa loa infected people are not well documented. This study compared the in vitro activity of several of these drugs on the viability of L. loa microfilariae (mf). Human strain L. loa mf were isolated from baboon blood using iso-osmotic Percoll gradient, and cultured in RPMI 1640/10% FBS with antimalarial drugs (mefloquine, amodiaquine, artesunate, chloroquine and quinine), anthelmintics (ivermectin, praziquantel, flubendazole and its reduced and hydrolyzed metabolites), two potential trypanocidal agents (fexinidazole and Scynexis-7158) and the anticancer drug imatinib. The drug concentrations used varied between 0.156 μg/ml and 10 μg/ml. Mf motility (CR = 50% immotility) and a metabolic viability assay (MTT) were used to assess the effects of these drugs on the parasites. Mf in control cultures showed only a slight reduction in motility after 5 days of culture. Active inhibition of Loa loa motility was seen with mefloquine and amodiaquine (CR values of 3.87 and 4.05 μg/ml, respectively), immobilizing > 90% mf within the first 24 hours: mefloquine killed the mf after 24 hours of culture at concentrations ≥ 5 μg/ml. SCYX-7158 also induced a concentration-dependent reduction in mf motility, with > 50% reduction in mf motility seen after 5 days at 10 μg/ml. The anticancer drug imatinib reduced mf motility at 10 μg/ml from the first day of incubation to 55% by day 5, and the reduction in motility was concentration-dependent. Praziquantel and fexinidazole were inactive, and FLBZ and its metabolites, as well as ivermectin at concentrations > 5 μg/ml, had very minimal effects on mf motility over the first 4 days of culture. The considerable action of the anti-malarial drugs mefloquine and amodiaquine on Loa mf in vitro highlights the possibility of repurposing the existing anti-infectious agents for the development of drugs against loiasis. The heterogeneity in the activity of anti-parasitic agents on Loa loa mf supports the need for further investigation using animal models of loiasis

Urine metabolites for the identification of Onchocerca volvulus infections in patients from Cameroon

Background: The tropical disease onchocerciasis (river blindness), caused by Onchocerca volvulus filarial nematodes, is targeted for elimination by mass treatment with nematocidal and antimicrobial drugs. Diagnosis of O. volvulus infections is based on counts of skin-borne microfilariae, but additional diagnostic tools, e.g. worm- or host-derived small RNAs, proteins or metabolites, are required for high-throughput screening. N-acetyltyramine-O,beta-glucuronide (NATOG) was suggested as a biomarker for onchocerciasis but its viability as diagnostic tool has been challenged. Methods: We performed a screening program of urine samples from individuals from Cameroon infected with O. volvulus, Loa loa, Mansonella perstans or a combination thereof. Urine metabolites were measured by liquid chromatography-mass spectrometry (LC-MS). Principle component analysis (PCA) revealed that onchocerciasis causes complex changes of the urine metabolome. Results: The mean NATOG content was elevated in urine of O. volvulus-infected compared with non-infected individuals, but NATOG levels showed considerable variation. However, 13.8% of all O. volvulus-infected individuals had high NATOG levels never reached by individuals without filarial infections or only infected with L. loa or M. perstans. Therefore, the identification of individuals with high NATOG levels might be used to screen for the elimination of onchocerciasis after mass drug application. Additional metabolites, including a compound identified as cinnamoylglycine, had high PC1/PC2 loadings in the data set. Mean levels of cinnamoylglycine were increased in O. volvulus-infected individuals, and 17.2% of all O. volvulus individuals had elevated cinnamoylglycine levels not reached by the controls. Conclusions: On an individual level, NATOG alone had poor discriminative power distinguishing infected from non-infected individuals. However, 13.8% of all O. volvulus-infected individuals had NATOG levels never reached by individuals without filarial infections or infected with only L. loa or M. perstans. Discrimination of O. volvulus infections from controls or individuals suffering from multiple infections was improved by the measurement of additional metabolites, e.g. cinnamoylglycine. Thus, measuring a combination of urine metabolites may provide a way to assess onchocerciasis on the population level. This provides the possibility to design a strategy for large-scale onchocerciasis epidemiological screening programs based on urine rather than invasive techniques