In vitro maintenance of Mansonella perstans microfilariae and its relevance for drug screening

doi:10.1016/j.exppara.2019.107769

Experimental Parasitology

Volume 206, November 2019, 107769

https://doi.org/10.1016/j.exppara.2019.107769 Get rights and content

Highlights

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DMEM or RPMI medium supplemented with 10% FBS cultured on feeder cells supported survival of 99.8% of mf for 20 days.
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Addition of 10% FBS to cell free culture improved worm motility.
•
Mefloquine and artesunate had the highest microfilaricidal activity at a concentration of 10 
•
For the first time, this study showed that mefloquine displayed complete microfilaricidal activity.
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This study provides an experimental platform for research on the neglected tropical pathogen Mansonella perstans.

Abstract

Background

Mansonellosis arises from infections with threadlike filarial nematodes in millions of individuals, especially in sub-Saharan Africa. Since infections present no overt clinical symptoms but attenuate immune responses that might lead to increased susceptibility and worsened disease course of concomitant infections, it is truly a neglected tropical disease. Nevertheless, only few studies focus on identifying suitable safe drugs for its control and little is known about the requirements for in vitro maintenance of the Mansonella perstans transmission stage. This study, therefore, evaluated the survival of M. perstans microfilariae (mf) using in vitro conditions that have been shown to promote survival of Loa loa, a closely related filarial nematode. Furthermore, the in vitro microfilaricidal effect of 15 agents was assessed on this helminth.

Methods

The ability of two basic culture media; Dulbecco's Modified Eagle's Medium (DMEM) and Roswell Park Memorial Institute (RPMI-1640) supplemented with 10% fetal bovine serum (FBS) and a monkey kidney epithelial cell line (LLC-MK₂) to support the survival of M. perstans microfilariae was investigated. Subsequently, 6 anti-helminthics, 5 anti-malarials, 1 anti-microbacterial, 2 trypanocidals and 1 anti-cancer agent were tested in vitro against mf. The suitability of the culture media as well as the effect of the anti-infective agents on mf survival was assessed by scoring their motility.

Results

FBS supplement and additional LLC-MK₂ cells significantly improved the survival of mf in DMEM and RPMI-1640 culture. In detail, RPMI-1640 supplemented with 10% FBS and LLC-MK₂ cells sustained the maintenance of mf for at least 20 days (100.00 ± 0.00% survival). In co-cultures with LLC-MK₂ cells without serum, M. perstans mf were maintained in DMEM and RPMI-1640 medium with a motility above 99% by day 5. Mefloquine displayed the highest microfilaricidal effect in vitro followed by artesunate.

Conclusion

Both RPMI and DMEM in the presence of LLC-MK₂ cells are suitable for the maintenance of M. perstans mf in vitro. In absence of the feeder cells, the addition of 10% FBS to RPMI-1640 medium improved the parasite survival rate and motility. The microfilaricidal activity of mefloquine and artesunate on M. perstans mf was documented for the first time in this study and can therefore be considered as reference for further screening of agents against this parasite stage.

Graphical abstract

Introduction

Mansonellosis is a clinical condition caused by the nematodes of the genus Mansonella. Its morbidity is mainly contributed by Mansonella perstans among populations in endemic areas despite the existence of the two other species: M. ozzardi and M. streptocerca. Adult Mansonella spp. worms live in tissues, body cavities or the dermis of their mammalian host (Simonsen et al., 2011). The females produce microfilariae (mf) which migrate to the blood or skin. Mansonellosis is generally considered asymptomatic because about 90% of infected subjects may exhibit little or no clinical manifestations (Asio et al., 2009; Hoerauf, 2009; Simonsen et al., 2011). When symptomatic, the major reported signs include pruritus, swellings (similar to Calabar swellings), fever and pain in bursae and/or joint synovia, in serous cavities, in the liver region and elephantoid scrotum as well as extreme exhaustion and psychological symptoms (Agbolade et al., 2005). Moreover, recently we showed that M. perstans modulates host immune responses (Ritter et al., 2018) which might be responsible for the increased susceptibility and worsened disease course of HIV, tuberculosis (TB) and malaria as well as lowered efficacy of Bacillus Calmette-Guérin vaccination against TB (Muhangi et al., 2007; Hillier et al., 2008; Elliott et al., 2010; Stensgaard et al., 2016; Mhimbira et al., 2017). Thus it is an urgent need to control these infections in endemic areas and discover novel treatment strategies against M. perstans.

To date, no safe and effective drug is registered for the treatment of mansonellosis cases (Bregani et al., 2006). Due to the contradictory findings on the effect of diethylcarbamazine (DEC) in clearing Acanthocheilonema perstans mf from the bloodstream (Strohschneider, 1956; Adolph et al., 1962), not much interest has been shown for the use of this agent to clear the filaria. This is compounded by the consequence of the Mazzotti reaction that DEC induces in patients with onchocerciasis; another filarial infection frequently co-endemic with Mansonellosis (Shenoy et al., 2000). Furthermore, although decreases of M. perstans mf have been drastically reduced upon prolonged administration of high doses of mebendazole in combination with levamizol in O. volvulus and M. perstans co-infected Zairian individuals (Maertens and Wéry, 1975), these regimens are not suitable for mass treatment purposes (Simonsen et al., 2011). As reviewed by Simonsen et al. (2011), numerous trials evaluated traditional antifilarial drugs such as ivermectin or other benzimidazoles like albendazole and thiabendazole but the findings from those studies were inconclusive, highlighting the need for safe and effective drugs. Identification and development of such drugs require in vitro culture systems in which parasite stages can be maintained to assess the efficacy of potential antifilarial agents. We previously reported an in vitro culture system which allows the long-term maintenance of M. perstans larvae and the development of M. perstans L3 into L5 stages (Njouendou et al., 2017). Although no attempt to culture and maintain M. perstans mf in vitro has been previously performed, different stages of Loa loa, a filarial nematode with blood dwelling mf like M. perstans, were successfully maintained in vitro (Zofou et al., 2018). In addition, the applied in vitro culture conditions have been used to assess the microfilaricidal properties of antiparasitic agents against L. loa mf (Njouendou et al., 2018) and infective larvae (Fombad et al., 2019). In this study, we designed eight culturing scenarios to optimise M. perstans mf viability and survival in vitro and selected one of the suitable culture conditions to test the efficacy of different agents against M. perstans. Thus, the findings here describe a platform that could be subsequently used to elucidate novel microfiraicidal drugs against M. perstans.

Section snippets

Ethical considerations

Ethical clearance was obtained from the National Institutional Review board, Yaoundé (REF: N° 2015/09/639/CE/CNERSH/SP) and administrative clearance from the Delegation of Public Health, South West Region (Re: R11/MINSANTE/SWR/RDPH/PS/259/382) after approval of the protocol. Special consideration was taken to minimize any health risks of the participant and involvement was strictly voluntary. The objectives of the study were explained in detail to each individual willing to participate after

Optimization of the in vitro culture condition for the maintenance of M. perstans microfilaria

The effect on the motility of M. perstans mf under different culture conditions was assessed either in RPMI (Fig. 1a) or DMEM medium (Fig. 1b) in the presence or absence of monkey kidney epithelial feeder cells (LLC-MK₂) and 10% fetal bovine serum (FBS). Statistical analysis is documented in Table 1.

In the presence of LLC-MK₂ feeder cells, with or without FBS, all microfilariae had 95%–100% motility for the entire observational period (20 days), independent of the type of the medium used (Fig. 1

Discussion

This study was conducted to investigate the suitability of culture medium (DMEM or RPMI), supplemented with 10% FBS and monkey kidney epithelial feeder cells in the in vitro maintenance and survival of M. perstans mf. Furthermore, we aim to validate the appropriate conditions for drug screening by assessing the microfilaricidal properties of 15 antiparasitic agents. Globally, the survival of M. perstans mf either in DMEM or RPMI medium was found to be improved by the presence of monkey kidney

Conclusions

The basic medium DMEM or RPMI supplemented with 10% FBS cultured on monkey kidney epithelial feeder cells supported survival of 99.8% of M. perstans mf for 20 days. The addition of 10% FBS to cell free culture improve the worm motility. From the primary screening, mefloquine and artesunate had the highest microfilaricidal activity at a concentration of 10 μg/ml. Mefloquine displayed complete microfilaricidal activity at a concentration ≥5 μg/ml and its activity on M. perstans mf documented for

Author's contributions

AJN, PE and SW developed the protocol. CAK, AJN, MEE, DBT, PCN and NVTG carried out the parasite production and in vitro studies. AJN, MR and SW performed data analysis. MR, AJN, FJ, PE, KP, LEL, SW and JT drafted and edited the manuscript. All authors read and approved the final manuscript.

Source of funding

This work is funded through a grant awarded to SW, MR, KP, LEL and AH from the German Research Foundation (DFG) (Grant DFG HO2009/10-1; HO, 2009/14-1) within the “African-German Cooperation Projects in Infectiology”. Furthermore, SW and AH are financially supported by the German Federal Ministry of Education and Research (BMBF initiative Research Networks for Health Innovations in Sub-Sahara Africa: TAKeOFF). AH and LEL are members of the Excellence Cluster Immunosensation (DFG, EXC 1023) and

Declaration of competing interest

The authors of this work have no financial, personal or professional interests that could have been construed to influence this manuscript.

Acknowledgement

We sincerely thank the chiefs and entire populations in all health districts surveyed for accepting and participating in this study. The authors are grateful to the participants for their voluntary participation in this study. We also thank the Cameroon Health System for use of all the facilities during the surveys.

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The findings presented here are based on the previous publication entilted “In vitro maintenance of Mansonella perstans microfilariae and its relevance for drug screening” [1].
Endemic communities of Mansonella perstans infections have been neglected since associated pathology remains undefined. Consequently, improvements in drug therapy have also been ignored despite a large number of infected individuals in areas of Cameroon. Thus, we established an in vitro system to culture M. perstans microfilariae (Mf); the transmission stage of infection. In short, we compared the ability of two renowned culture media (Dulbecco's Modified Eagle's Medium (DMEM) and Roswell Park Memorial Institute (RPMI-1640)) to sustain Mf in culture. Media were supplemented with 10% fetal bovine serum (FBS) and monkey kidney epithelial cells (LLC-MK₂) were used as feeder cells. As readout we assessed Mf survival and motility using a standardised microscopy assessment strategy. Moreover, this in vitro culture system was used to test susceptibility levels of microfilariae to different chemotherapeutic agents. Parasite motility was scored daily using a graded system and analysed using the average motility and area under the motility curve of M. perstans Mf. These datasets were analysed and discussed in detail in the related article entitled: “In vitro maintenance of Mansonella perstans microfilariae and its relevance for drug screening” [1].
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¹: Shared authorship.

View full text

In vitro maintenance of Mansonella perstans microfilariae and its relevance for drug screening

Highlights

Abstract

Background

Methods

Results

Conclusion

Graphical abstract

Introduction

Section snippets

Ethical considerations

Optimization of the in vitro culture condition for the maintenance of M. perstans microfilaria

Discussion

Conclusions

Author's contributions

Source of funding

Declaration of competing interest

Acknowledgement

Trans. R. Soc. Trop. Med. Hyg.

Trans. R. Soc. Trop. Med. Hyg.

Vaccine

Trans. R. Soc. Trop. Med. Hyg.

Biochem. Biophys. Res. Commun.

Trans. R. Soc. Trop. Med. Hyg.

Trans. R. Soc. Trop. Med. Hyg.

Biochem. Pharmacol.

Acta Trop.

Diagnosis and treatment of Acanthocheilonema perstans filariasis

Am. J. Trop. Med. Hyg.

Loa loa and Mansonella perstans: neglected human infections that need control in Nigeria

Afr. J. Biotechnol.

Cultivation of parasites

Tropenmed. Parasitol.

Emergence of mefloquine-resistant malaria in Africa without drug pressure

Lancet

Density gradient separation of Loa loa and Dipetalonema perstans microfilariae from infected patients

Ann. Soc. Belg. Med. Trop.

Perspectives on the in vitro culture of filariae

In Vitro Cell. Dev. Biol. Anim.

The microfilaricidal activity of ivermectin in vitro and in vivo

Effect of flubendazole on developing stages of Loa loa in vitro and in vivo: a new approach for screening filaricidal agents

Parasites Vectors

Adding “filaricide” to the Gleevec portfolio

Mansonella Perstans—the Importance of an Endosymbiont

Filariasis testing in a jird model: new drug leads from some old standbys

Am. J. Trop. Med. Hyg.

In vivo and in vitro efficacies of mebendazole, mefloquine and nitazoxanide against cyst echinococcosis

Parasitol. Res.