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

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 ±â€¯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.

[Contribution of the PCR assay to the diagnosis of Mansonella ozzardi in endemic areas of Argentina]. / Aporte de la técnica de PCR en el diagnóstico de Mansonella ozzardi en zonas endémicas de la Argentina.

Mansonella ozzardi is a tissue-dwelling parasitic nematode, the causative agent of mansonelliasis in almost all Latin American countries. It has been described along the Argentine Yungas region. The microscopic diagnosis can yield false-negative test results at low microfilaremia levels. The aim of this study was to optimize the molecular diagnostic technique and compare it with the Knott's method and standard blood smear procedures (thin blood films and thick smears) in 92 blood samples of individuals from an endemic area. The PCR technique followed by the sequencing of the amplified product yielded 100 % sensitivity compared to the Knott's test, which is considered a reference method. Seven more cases of this parasitosis could only be identified with the molecular technique.

Phase III Clinical Trial to Evaluate Ivermectin in the Reduction of Mansonella ozzardi infection in the Brazilian Amazon.

The treatment of mansonelliasis is still a challenge because there are few clinical trials for the treatment of the disease. This double-blind, randomized, placebo-controlled study (phase III clinical trial) was conducted to evaluate the effectiveness of a single oral dose of ivermectin (0.15 mg/kg) in the reduction of the Mansonella ozzardi microfilaraemia and the occurrence of adverse effects in infected people compared with the control group treated with placebo. A total of 49 microfilaraemic patients were randomly selected from the municipality of Lábrea, State of Amazonas, in the Brazilian Amazon. Among them, 40 patients have concluded the study, 19 treated with ivermectin and 21 treated with placebo. In the first and third days after the treatment, all the patients were clinically evaluated, and the diagnostic and quantification of blood microfilariae through blood filtration in polycarbonate membranes was performed. A significant reduction of the microfilaraemia (99.9%) was observed in the patients who received ivermectin. Slight changes in laboratory test results, without clinical importance, were seen in treated and control groups. Our results suggest that ivermectin is effective and safe for the treatment of infections caused by M. ozzardi.

Successful long-term maintenance of Mansonella perstans in an in vitro culture system.

BACKGROUND: Approximately 114 million people are infected with Mansonella perstans in large proportions of Africa. In contrast to other filariae that infect humans, M. perstans-infected individuals show no distinct pathology or specific clinical picture, indicating a well-tuned adaptation to the host. In addition, since M. perstans adult worms reside in serous cavities which are difficult to access, research has been hindered and there is a paucity of knowledge about the biology of M. perstans, especially the development of the different life stages as well as M. perstans-driven immune responses. Thus in this study, an in vitro culture system was developed which allows an in-depth analysis of M. perstans. RESULTS: Culicoides species were caught in Ediki (Kumba), Southwest Region within Cameroon following a blood meal on a microfilaremic donor that had 1500 microfilariae/ml of peripheral blood and kept in captivity for 12 days at 23 °C. In a pilot experiment, 15 infective larvae were obtained from the midges and co-cultured with a confluent monolayer of monkey kidney epithelial cells (LLC-MK2) in DMEM medium supplemented with 10% FBS for up to 77 days. The resulting survival rates of 33% revealed that the cell-conditioned medium was suitable for long-term maintenance of M. perstans worms. To confirm these preliminary observations, 249 infective larvae were cultured for 50 days and their development was monitored daily and microscopically graded for motility. In total, 170 (68.3%) filariae survived and 124 (49.8%) larvae moulted between days 21-30 to become L5 stage larvae which were motile and showed continuous vigorous movement. CONCLUSION: We have established an in vitro culture system for the generation and long-term maintenance of viable M. perstans worms. This technique will be an important tool to study parasite biology and development, the role in host immunity, and might be helpful to discover novel treatment strategies against this filariae.

Update on the distribution of Mansonella perstans in the southern part of Cameroon: influence of ecological factors and mass drug administration with ivermectin.

BACKGROUND: Mansonellosis remains one of the most neglected of tropical diseases and its current distribution in the entire forest block of southern Cameroon is unknown. In order to address this issue, we have surveyed the distribution of Mansonella perstans in different bioecological zones and in addition, elucidated the influence of multiple rounds of ivermectin (IVM) based mass drug administration (MDA). METHODS: A mixed design was used. Between 2000 and 2014, both cross-sectional and longitudinal surveys were carried out in 137 communities selected from 12 health districts belonging to five main bioecological zones of the southern part of Cameroon. The zones comprised of grassland savanna (GS), mosaic forest savanna (MFS), forested savanna (FS), deciduous equatorial rainforest (DERF) and the dense humid equatorial rainforest (DHERF). The survey was carried out in some areas with no treatment history as well as those currently under IVM MDA. Individuals within the participatory communities were screened for the presence of M. perstans microfilariae (mf) in peripheral blood by the calibrated thick film method to determine both prevalence and geometric mean intensities at the community level. RESULTS: Apart from sporadic cases in savanna areas, distribution of M. perstans was strongly linked to the equatorial rainforest zones. Before CDTI, the highest mean prevalence (70.0 %) and intensity (17,382.2 mf/ml) were obtained in communities in Mamfes' DHERF areas followed by communities in the DHERF zone of Lolodorf (53.8 % and 7,814.8 mf/ml, respectively). A longitudinal survey in Mamfe further showed that M. perstans infections had reduced by 34.5 % in DERF (P < 0.001) but not DHERF zones after ten years of IVM MDA. Further data from the cross-sectional study revealed that there was a decrease in prevalence in DHERF zones only after ten years of MDA. In DERF zones however, the infection was relatively lower after four years of MDA. CONCLUSIONS: The distribution of M. perstans in the southern part of Cameroon varies with bioecological zones and IVM MDA history. The zones with high prevalence and intensities lie in forested areas while those with low endemicity are in the savanna areas. MDA with ivermectin induced significant reduction in the endemicity of mansonellosis in the decidious equatorial rainforest. In contrast, the prevalence and intensity remained relatively high and stable in the dense humid equatorial rainforest zones even after a decade of mass drug administration with ivermectin. Since it is known that M. perstans down-regulates host's immune system, the findings from this work would be useful in designing studies to understand the impact of M. perstans on host immune response to vaccination and co-infection with other pathogens such as Mycobacterium spp. and Plasmodium spp. in areas of contrasting endemicities.

Mansonella ozzardi: a neglected New World filarial nematode.

Mansonella ozzardi (Nematoda: Onchocercidae) is an understudied filarial nematode, originally described by Patrick Manson in 1897, that can be transmitted by two families of dipteran vectors, biting midges (most of them members of the genus Culicoides) and black flies (genus Simulium). With a patchy geographic distribution from southern Mexico to northwestern Argentina, human infection with M. ozzardi is highly prevalent in some of the Caribbean islands, along riverine communities in the Amazon Basin, and on both sides of the border between Bolivia and Argentina. There is no clinical entity unequivocally associated with M. ozzardi infection, although fever, arthralgia, headache, cold lower extremities, and itchy cutaneous rashes are occasionally mentioned in case report series. More recently, ocular manifestations (especially keratitis) have been associated with mansonelliasis, opening an important area of investigation. Here, we briefly review the biology, epidemiology, pathogenesis, and clinical aspects of M. ozzardi infection and point to some existing knowledge gaps, aiming to stimulate a research agenda to help filling them.

Review of the genus Mansonella Faust, 1929 sensu lato (Nematoda: Onchocercidae), with descriptions of a new subgenus and a new subspecies.

Based on material deposited in museum collections, twelve species within Mansonella sensu lato were examined and their descriptions amended. Based on additional morphological details, the erection of the new monotypic subgenus Filyamagutia Bain & Uni for M. (F.) akitensis (Uni, 1983), and the new combination M. (Pseudolitomosa) musasabi (Yamaguti, 1941) Bain & Uni are proposed. A new subspecies, M. (Tetrapetalonema) atelensis amazonae Bain & Guerrero is described and a key to the seven subgenera of Mansonella is provided. Furthermore, the elevation of Sandnema to full genus rank comprising the two species S. digitatum (Chandler, 1929) n. comb. and S. sunci (Sandground, 1933) n. comb., is proposed. Host and geographic records for the species of Mansonella and Sandnema are included.

Development of Mansonella ozzardi in a black fly species of the Simulium sanguineum group from Eastern Vaupés, Colombia.

Development of the microfilaria of Mansonella ozzardi to the infective stage in a species of the Simulium sanguineum group from the Mitú area, Comisaría del Vaupés, Colombia is described. Development was synchronous, and by day 6 third-stage larvae were observed in the head of flies which had fed on two naturally infected volunteers. Simulium biting activity was high from January-March, during the dry season. Only 261 blood-fed Culicoides were collected from an infected volunteer; 40% of 43 dissected on day 0 had ingested microfilariae. However, only one of 129 C. caprilesi specimens dissected after day 2 was positive, containing a second-stage larva thought to be M. ozzardi. It is concluded that Simulium, rather than Culicoides are the principal vectors of mansonelliasis in the Mitú area.

Mansonella ozzardi in Haiti II. Arthropod vector studies.

The present vector studies show that the biting midge, Culicoides furens, serves as an intermediate host for Mansonella ozzardi in Haiti. A total of 3,430 C. furens were collected as they engorged on infected individuals. Under the maintenance conditions described herein, the microfilaria required 9 days to reach the infective stage. Approximately 43% of the midges survived, and from these 1,128 infective stage larvae were collected. This represents an overall infection rate of 0.76 larva per midge. Typically, there was a directly proportional relationship between the infection rate in midges and the level of microfilaremia in the individuals upon whom the C. furens fed. Also, the vector efficiency of C. furens collected from saltwater or freshwater breeding habitats was comparable.

Culicoides variipennis, a laboratory vector for the amazon form of Mansonella ozzardi.

The colonized midge, Culicoides variipennis, is shown to be a competent laboratory host for the Amazon form of Mansonella ozzardi. It raises the possibility that certain Culicoides spp. in the Amazon region may support development of the parasite and possibly play a role in the natural transmission cycle.

Assessment of Leptoconops bequaerti as a potential vector of Mansonella ozzardi in Haiti.

Experimental studies in Bayeux, Haiti showed that the biting midge, Leptoconops bequaerti, is capable of supporting the complete development of Mansonella ozzardi but only on a very limited scale. This suggests that the species may not be involved in the natural transmission cycle despite its abundance and pestiferous nature in certain areas of Haiti. A midge-holding container is described which markedly enhanced the survival of engorged L. bequaerti in the laboratory.

Assessment of Culicoides barbosai as a vector of Mansonella ozzardi in Haiti.

Studies in the southern peninsula of Haiti showed that the biting midge, Culicoides barbosai, was capable of supporting the development of Mansonella ozzardi to the infective stage. The known vector, Culicoides furens, also was encountered. Both species showed distinct biting site preferences, i.e., 98% of the midges that engorged on the arms and head were C. barbosai whereas C. furens was collected mostly from the lower legs. Nine days after engorgement, 19 infective larvae were recovered from 13 C. barbosai versus six larvae from four C. furens. It was judged that C. barbosai may be as importantly involved in the natural transmission cycle as C. furens in this community.

Population dynamics of Loa loa and Mansonella perstans infections in individuals living in an endemic area of the Congo.

A follow-up of Loa loa and Mansonella perstans microfilaremia was carried out in an adult population living in a highly endemic area of the Congo. Infection rates and parasite loads were found to be stable in the general population, both in the short-term (two months) and long-term (3-4 years) followup. The microfilarial status of most of the subjects examined did not change between tests. At the individual level, the microfilarial densities of L. loa and M. perstans also remained remarkably constant over time. This results in a qualitative and quantitative stability of the parasitic material available for the vectors.

Ivermectin in loiasis and concomitant O. volvulus and M. perstans infections.

Two clinical studies were carried out in Gabon, Africa to evaluate the efficacy, safety, and tolerability of ivermectin in the treatment of patients with Loa loa infection. In the first study, 35 patients received single oral doses of ivermectin, 5-200 mcg/kg body weight. Blood microfilariae levels did not decrease after a single oral 5, 10, 30, or 50 mcg/kg dose of ivermectin, but levels did decrease after doses of 100, 150, and 200 mcg/kg. The most efficacious dose was 200 mcg/kg; mean blood microfilariae levels decreased to 12% of mean pretreatment values by day 15 and remained decreased for 28 days. A second study evaluated the safety and efficacy of ivermectin in patients with multifilarial infections. All 17 patients had concomitant Loa loa and O. volvulus infection. M. perstans affected 5 of the patients. Sixteen patients also had infections due to intestinal nematodes. The patients each received single oral doses of 200 mcg/kg ivermectin. Ten days later, the mean Loa loa blood microfilariae level had decreased to 20% of the mean pretreatment level. O. volvulus dermal microfilariae densities were reduced to 2% of the pretreatment levels. A minimal increase in blood microfilaria levels was observed on day 28. In contrast, dermal microfilariae levels remained near zero for the duration of the study. Intestinal infection due to Ascaris was eradicated in all of the affected patients by day 23; efficacy against Trichuris and hookworm infections, however, was poor. All patients tolerated ivermectin well including those with multiple infections.

Simulium species of the amazonicum group as vectors of Mansonella ozzardi in the Brazilian Amazon.

Experimental infection with Mansonella ozzardi of common haematophagous Diptera collected at a Ticuna Indian village on the upper reaches of the Solimões river in the Brazilian Amazon, showed that Simulium amazonicum and Simulium n.sp. are capable of supporting full development of the parasite. Natural infections with this filaria were found in both species including infective larvae in Simulium n.sp. No development of M. ozzardi occurred in Mansonia amazonensis, Culicoides insinuatus or Lepiselaga crassipes (Tabanidae). The dimensions of developing larvae of M. ozzardi in both species of black-fly were recorded. Infective larvae of this species may easily be distinguished from those of Onchocerca volvulus, also transmitted in the Amazon by a species closely resembling S. amazonicum, by the presence of a bifid tail and higher anal ratio in M. ozzardi.

Experimental and natural infection of Simulium sanchezi by Mansonella ozzardi in the Middle Orinoco region of Venezuela.

Experimental and natural infections of Simulium sanchezi by Mansonella ozzardi were studied in the area of Síquita, Territorio Federal Amazonas, Venezuela. The microfilariae developed synchronously in the blackflies, reaching stage L3 in seven to eight days at temperatures between 23 degrees and 27 degrees C. Larvae in different stages of development, including infective forms, were found in 0.6% of 662 unfed wild-caught females. These results confirm that simuliids are the main vectors of M. ozzardi in the American continent.

Transfusional Mansonella perstans microfilariasis.

Mansonella perstans filariasis is widely distributed across the center of Africa and equatorial America. We describe a case of post-transfusional M. perstans microfilariasis in a young child, affected with severe Plasmodium falciparum malaria, admitted in Goundi Hospital in South of Chad. A decrease of M. perstans microfilariasis in the patient's blood was observed, with no subsequent development of either clinical symptoms or eosinophilia. We suggest that, in endemic areas, transfused M. perstans microfilariae may be cleared from the blood over relatively short periods of time. It is likely that only adult worms are responsible for symptoms and eosinophilia, whereas microfilariae in the bloodstream are unable to give clinical manifestations.

Effects of thiabendazole in Mansonella perstans filariasis.

Mansonella perstans is a human filarial parasite distributed across the center of Africa and equatorial America. Although M. perstans infection is asymptomatic in most individuals, a variety of symptoms have been described, including angioedema, pruritus, fever, ocular involvement, and serous cavities pain. Eosinophilia is found in many cases. Treatment with diethyl-carbamazine or mebendazole is often ineffective. We present a study on the effects of thiabendazole in the treatment of symptomatic M. perstans filariasis. Twenty-five patients were treated with thiabendazole at a single dose of 50 mg/kg for children and 3 g for adults. Sixteen out of 25 subjects repeated a second dose a week later. Parasite density, eosinophilia, and symptoms were significantly reduced after both one and two-step therapy in most patients. This study shows that thiabendazole may be effective in M. perstans infection. More studies are needed to determine a more effective dosage, or a putative combination treatment.

CD4+CD25hiFOXP3+ cells in cord blood of neonates born from filaria infected mother are negatively associated with CD4+Tbet+ and CD4+RORγt+ T cells.

BACKGROUND: Children who have been exposed in utero to maternal filarial infection are immunologically less responsive to filarial antigens, have less pathology, and are more susceptible to acquire infection than offspring of uninfected mothers. Moreover children from filaria infected mothers have been shown to be less responsive to vaccination as a consequence of an impairment of their immune response. However, it is not well known how in utero exposure to parasite antigens affects cellular immune responses. METHODOLOGY: Here, 30 pregnant women were examined for the presence of microfilaria of Loa loa and Mansonella perstans in peripheral blood. At delivery, cord blood mononuclear cells (CBMC) were obtained and the CD4+T cells were phenotyped by expression of the transcription factors Tbet, RORγt, and FOXP3. RESULTS: No significant difference was observed between newborns from infected versus uninfected mothers in the frequencies of total CD4+T cells and CD4+T cells subsets including CD4+Tbet+, CD4+RORγt+ T and CD4+CD25hiFOXP3+ T cells. However, there was a negative association between CD4+CD25hiFOXP3+T cells and CD4+Tbet+ as well as CD4+RORγt+ T cells in the infected group only (B = -0.242, P = 0.002; B = -0.178, P = 0.013 respectively). CONCLUSION: Our results suggest that filarial infection during pregnancy leads to an expansion of functionally active regulatory T cells that keep TH1 and TH17 in check.

Mansonella perstans filariasis in Africa.

Mansonella perstans is a vector-borne human filarial nematode, transmitted by tiny blood-sucking flies (biting midges). It is widespread in many parts of Sub-Saharan Africa and also occurs in parts of Central and South America. Despite the commonness of this parasite very few studies have been carried out on its epidemiology and on the morbidity resulting from it, and only few thorough drug trials have been conducted to look for effective and suitable drugs and drug regimens for treatment and control. Here, we review currently available knowledge on M. perstans infections in Africa, including documented aspects of biology, vectors, transmission, diagnosis, epidemiology, morbidity and treatment. It is concluded that there is an urgent need for more research on this widespread but greatly neglected infection in order to properly assess its public health significance and as a background for identifying and recommending optimal means and strategies for treatment and control.