Repurposing Auranofin as a Lead Candidate for Treatment of Lymphatic Filariasis and Onchocerciasis

Two major human diseases caused by filariid nematodes are onchocerciasis, or river blindness, and lymphatic filariasis, which can lead to elephantiasis. The drugs ivermectin, diethylcarbamazine (DEC), and albendazole are used in control programs for these diseases, but are mainly effective against the microfilarial stage and have minimal or no effect on adult worms. Adult Onchocerca volvulus and Brugia malayi worms (macrofilariae) can live for up to 15 years, reproducing and allowing the infection to persist in a population. Therefore, to support control or elimination of these two diseases, effective macrofilaricidal drugs are necessary, in addition to current drugs. In an effort to identify macrofilaricidal drugs, we screened an FDA-approved library with adult worms of Brugia spp. and Onchocerca ochengi, third-stage larvae (L3s) of Onchocerca volvulus, and the microfilariae of both O. ochengi and Loa loa. We found that auranofin, a gold-containing drug used for rheumatoid arthritis, was effective in vitro in killing both Brugia spp. and O. ochengi adult worms and in inhibiting the molting of L3s of O. volvulus with IC₅₀ values in the low micromolar to nanomolar range. Auranofin had an approximately 43-fold higher IC₅₀ against the microfilariae of L. loa compared with the IC₅₀ for adult female O. ochengi, which may be beneficial if used in areas where Onchocerca and Brugia are co-endemic with L. loa, to prevent severe adverse reactions to the drug-induced death of L. loa microfilariae. Further testing indicated that auranofin is also effective in reducing Brugia adult worm burden in infected gerbils and that auranofin may be targeting the thioredoxin reductase in this nematode.     

Reproductive Status of Onchocerca volvulus after Ivermectin Treatment in an Ivermectin-Na?ve and a Frequently Treated Population from Cameroon

Background

For two decades, onchocerciasis control has been based on mass treatment with ivermectin (IVM), repeated annually or six-monthly. This drug kills Onchocerca volvulus microfilariae (mf) present in the skin and the eyes (microfilaricidal effect) and prevents for 3–4 months the release of new mf by adult female worms (embryostatic effect). In some Ghanaian communities, the long-term use of IVM was associated with a more rapid than expected skin repopulation by mf after treatment. Here, we assessed whether the embryostatic effect of IVM on O. volvulus has been altered following frequent treatment in Cameroonian patients.

Methodology

Onchocercal nodules were surgically removed just before (D0) and 80 days (D80) after a standard dose of IVM in two cohorts with different treatment histories: a group who had received repeated doses of IVM over 13 years, and a control group with no history of large-scale treatments. Excised nodules were digested with collagenase to isolate adult worms. Embryograms were prepared with females for the evaluation of their reproductive capacities.

Principal Findings

Oocyte production was not affected by IVM. The mean number of intermediate embryos (morulae and coiled mf) decreased similarly in the two groups between D0 and D80. In contrast, an accumulation of stretched mf, either viable or degenerating, was observed at D80. However, it was observed that the increase in number of degenerating mf between D0 and D80 was much lower in the frequently treated group than in the control one (Incidence Rate Ratio: 0.25; 95% CI: 0.10–0.63; p = 0.003), which may indicate a reduced sequestration of mf in the worms from the frequently treated group.

Conclusion/Significance

IVM still had an embryostatic effect on O. volvulus, but the effect was reduced in the frequently treated cohort compared with the control population.     

Dynamics of Onchocerca volvulus Microfilarial Densities after Ivermectin Treatment in an Ivermectin-na?ve and a Multiply Treated Population from Cameroon

Background/Objective

Ivermectin has been the keystone of onchocerciasis control for the last 25 years. Sub-optimal responses to the drug have been reported in Ghanaian communities under long-term treatment. We assessed, in two Cameroonian foci, whether the microfilaricidal and/or embryostatic effects of ivermectin on Onchocerca volvulus have been altered after several years of drug pressure.

Methods

We compared the dynamics of O. volvulus skin microfilarial densities after ivermectin treatment in two cohorts with contrasting exposure to this drug: one received repeated treatment for 13 years whereas the other had no history of large-scale treatments (referred to as controls). Microfilarial densities were assessed 15, 80 and 180 days after ivermectin in 122 multiply treated and 127 ivermectin-naïve individuals. Comparisons were adjusted for individual factors related to microfilarial density: age and number of nodules.

Findings

Two weeks post ivermectin, microfilarial density dropped equally (98% reduction) in the ivermectin-naïve and multiply treated groups. Between 15 and 180 days post ivermectin, the proportion of individuals with skin microfilariae doubled (from 30.8% to 67.8%) in controls and quadrupled (from 19.8% to 76.9%) in multiply treated individuals but the mean densities remained low in both sites. In fact, between 15 and 80 days, the repopulation rate was significantly higher in the multiply treated individuals than in the controls but no such difference was demonstrated when extending the follow-up to 180 days. The repopulation rate by microfilariae was associated with host factors: negatively with age and positively with the number of nodules.

Conclusion

These observations may indicate that the worms from the multi-treated area recover mf productivity earlier but would be less productive than the worms from the ivermectin-naïve area between 80 and 180 days after ivermectin. Moreover, they do not support the operation of a strong cumulative effect of repeated treatments on the fecundity of female worms as previously described.     

Advances and challenges in predicting the impact of lymphatic filariasis elimination programmes by mathematical modelling

Background

Infection with the filarial nematode Onchocerca volvulus can lead to severe dermatitis, visual impairment, and ultimately blindness. Since the currently used drug, ivermectin does not have macrofilaricidal or strong permanent sterilising effects on the adult worm, more effective drugs are needed to complement the use of ivermectin alone. Wolbachia endosymbiotic bacteria in filariae have emerged as a new target for treatment with antibiotics which can lead to long -term sterilization of the adult female filariae.

Methods

In the Central Region of Ghana, 60 patients were recruited, allocated into four groups and treated with 200 mg doxycycline per day for 2 weeks, 4 weeks, 6 weeks respectively. Untreated patients served as controls. Some of the treated patients and the untreated controls were given 150 μg/kg ivermectin 8 months after the start of doxycycline treatment.

Results

A follow up study 18 months post treatment showed that when using doxycycline alone there was a significant reduction of microfilarial (mf) loads in patients treated for either 4 or 6 weeks. However, there was no significant difference between the untreated controls and those given the 2 weeks regimen. Although no significant difference was demonstrated between the 4 and 6 weeks regimens, there was a trend observed, in that, microfilarial reduction appeared to have been greater following the 6 weeks regimen. Twelve months after ivermectin (i.e. 20 months after doxycycline) treatment, 8 out of 11 ivermectin-alone treated patients were mf-positive. In contrast, 1 out of the 7 patients treated for 4 weeks with doxycycline and none of the 4 patients treated for 6 weeks doxycycline (who were available for re-examination) were mf-positive after the combined treatment of doxycycline plus ivermectin treatment.

Conclusion

Treatment of onchocerciasis with doxycycline for 4 weeks is effective. Nonetheless, mf reduction appeared to be greater in the 6 weeks regimen. It is recommended that until further studies are carried out i.e. 4 weeks treatment with doxycycline is proven equivalent to the 6 weeks, selected groups of onchocerciasis patients should be treated for 6 weeks with doxycycline. As discussed earlier, this treatment should be accompanied by two doses of ivermectin.     

Genetic selection of low fertile Onchocerca volvulus by ivermectin treatment

Background

Onchocerca volvulus is the causative agent of onchocerciasis, or “river blindness”. Ivermectin has been used for mass treatment of onchocerciasis for up to 18 years, and recently there have been reports of poor parasitological responses to the drug. Should ivermectin resistance be developing, it would have a genetic basis. We monitored genetic changes in parasites obtained from the same patients before use of ivermectin and following different levels of ivermectin exposure.

Methods and Findings

O. volvulus adult worms were obtained from 73 patients before exposure to ivermectin and in the same patients following three years of annual or three-monthly treatment at 150 µg/kg or 800 µg/kg. Genotype frequencies were determined in β-tubulin, a gene previously found to be linked to ivermectin selection and resistance in parasitic nematodes. Such frequencies were also determined in two other genes, heat shock protein 60 and acidic ribosomal protein, not known to be linked to ivermectin effects. In addition, we investigated the relationship between β-tubulin genotype and female parasite fertility. We found a significant selection for β-tubulin heterozygotes in female worms. There was no significant selection for the two other genes. Quarterly ivermectin treatment over three years reduced the frequency of the β-tubulin “aa” homozygotes from 68.6% to 25.6%, while the “ab” heterozygotes increased from 20.9% to 69.2% in the female parasites. The female worms that were homozygous at the β-tubulin locus were more fertile than the heterozygous female worms before treatment (67% versus 37%; p = 0.003) and twelve months after the last dose of ivermectin in the groups treated annually (60% versus 17%; p<0.001). Differences in fertility between heterozygous and homozygous worms were less apparent three months after the last treatment in the groups treated three-monthly.

Conclusions

The results indicate that ivermectin is causing genetic selection on O. volvulus. This genetic selection is associated with a lower reproductive rate in the female parasites. We hypothesize that this genetic selection indicates that a population of O. volvulus, which is more tolerant to ivermectin, is being selected. This selection could have implications for the development of ivermectin resistance in O. volvulus and for the ongoing onchocerciasis control programmes.     

Macrofilaricidal Activity after Doxycycline Only Treatment of Onchocerca volvulus in an Area of Loa loa Co-Endemicity: A Randomized Controlled Trial

Background

The risk of severe adverse events following treatment of onchocerciasis with ivermectin in areas co-endemic with loiasis currently compromises the development of control programmes and the treatment of co-infected individuals. We therefore assessed whether doxycycline treatment could be used without subsequent ivermectin administration to effectively deliver sustained effects on Onchocerca volvulus microfilaridermia and adult viability. Furthermore we assessed the safety of doxycycline treatment prior to ivermectin administration in a subset of onchocerciasis individuals co-infected with low to moderate intensities of Loa loa microfilaraemia.

Methods

A double-blind, randomized, field trial was conducted of 6 weeks of doxycycline (200 mg/day) alone, doxycycline in combination with ivermectin (150 µg/kg) at +4 months or placebo matching doxycycline + ivermectin at +4 months in 150 individuals infected with Onchocerca volvulus. A further 22 individuals infected with O. volvulus and low to moderate intensities of Loa loa infection were administered with a course of 6 weeks doxycycline with ivermectin at +4 months. Treatment efficacy was determined at 4, 12 and 21 months after the start of doxycycline treatment together with the frequency and severity of adverse events.

Results

One hundred and four (60.5%) participants completed all treatment allocations and follow up assessments over the 21-month trial period. At 12 months, doxycycline/ivermectin treated individuals had lower levels of microfilaridermia and higher frequency of amicrofilaridermia compared with ivermectin or doxycycline only groups. At 21 months, microfilaridermia in doxycycline/ivermectin and doxycycline only groups was significantly reduced compared to the ivermectin only group. 89% of the doxycycline/ivermectin group and 67% of the doxycycline only group were amicrofilaridermic, compared with 21% in the ivermectin only group. O. volvulus from doxycycline groups were depleted of Wolbachia and all embryonic stages in utero. Notably, the viability of female adult worms was significantly reduced in doxycycline treated groups and the macrofilaricidal and sterilising activity was unaffected by the addition of ivermectin. Treatment with doxycycline was well tolerated and the incidence of adverse event to doxycycline or ivermectin did not significantly deviate between treatment groups.

Conclusions

A six-week course of doxycycline delivers macrofilaricidal and sterilizing activities, which is not dependent upon co-administration of ivermectin. Doxycycline is well tolerated in patients co-infected with moderate intensities of L. loa microfilariae. Therefore, further trials are warranted to assess the safety and efficacy of doxycycline-based interventions to treat onchocerciasis in individuals at risk of serious adverse reactions to standard treatments due to the co-occurrence of high intensities of L. loa parasitaemias. The development of an anti-wolbachial treatment regime compatible with MDA control programmes could offer an alternative to the control of onchocerciasis in areas of co-endemicity with loiasis and at risk of severe adverse reactions to ivermectin.

Trial Registration

Controlled-Trials.com ISRCTN48118452     

Emodepside targets SLO-1 channels of Onchocerca ochengi and induces broad anthelmintic effects in a bovine model of onchocerciasis

Onchocerciasis (river blindness), caused by the filarial worm Onchocerca volvulus, is a neglected tropical disease mostly affecting sub-Saharan Africa and is responsible for >1.3 million years lived with disability. Current control relies almost entirely on ivermectin, which suppresses symptoms caused by the first-stage larvae (microfilariae) but does not kill the long-lived adults. Here, we evaluated emodepside, a semi-synthetic cyclooctadepsipeptide registered for deworming applications in companion animals, for activity against adult filariae (i.e., as a macrofilaricide). We demonstrate the equivalence of emodepside activity on SLO-1 potassium channels in Onchocerca volvulus and Onchocerca ochengi, its sister species from cattle. Evaluation of emodepside in cattle as single or 7-day treatments at two doses (0.15 and 0.75 mg/kg) revealed rapid activity against microfilariae, prolonged suppression of female worm fecundity, and macrofilaricidal effects by 18 months post treatment. The drug was well tolerated, causing only transiently increased blood glucose. Female adult worms were mostly paralyzed; however, some retained metabolic activity even in the multiple high-dose group. These data support ongoing clinical development of emodepside to treat river blindness.     

Onchocerciasis-associated epilepsy: an update and future perspectives

Onchocerciasis-associated epilepsy (OAE) is an important neglected public health problem in areas with high ongoing onchocerciasis transmission. The risk that children in such areas develop epilepsy is related to their Onchocerca volvulus microfilarial (mf) load. Before the implementation of mass treatment with ivermectin, microfilariae were detected in cerebrospinal fluid (CSF). More recently, neither O. volvulus microfilariae nor DNA were detected in CSF or brain tissue; however, these samples were obtained years after seizure onset. It is possible that during fever-induced increased blood–brain barrier permeability, microfilariae enter the brain and, upon dying, cause an inflammatory reaction inducing seizures. Including OAE in the onchocerciasis disease burden estimation may mobilise extra resources for onchocerciasis disease elimination and treatment/care of OAE-affected persons/families.     

Contribution of migrant coffee labourers infected with Onchocerca volvulus to the maintenance of the microfilarial reservoir in an ivermectin-treated area of Mexico

Background

Since 1991, in Mexico, ivermectin has been administered twice a year to all residents in the onchocerciasis endemic foci which are mainly located in the coffee growing areas. However, the presence of a potentially infected itinerant seasonal labour force which is not treated regularly could jeopardise the attainment of the 85% coverage which is the present target for elimination of the disease.

Methods

The prevalence and intensity of Onchocerca volvulus microfilariae (mf), as well as their transmission from humans to vectors, were assessed during the coffee planting-clearing and harvesting seasons of 1997–1998, and 1998–1999 in two localities (I and II) of Southern Chiapas, Mexico, which regularly receive an influx of untreated migrant coffee labourers.

Results

Localities I and II had, respectively, an average of 391 (± 32) and 358 (± 14) resident inhabitants, and 70 (± 52) and 498 (± 289) temporary labourers. The ratio of migrants to residents ranged from 0.1:1 in locality I to 2.4:1 in locality II. The proportion of infected Simulium ochraceum s.l. parous flies was significantly lower in locality I than in locality II, and significantly higher during the stay of the migrants than before their arrival or after their departure. Parity and infection were higher in May-July than in November-February (in contrast with the latter being typically considered as the peak onchocerciasis transmission season by S. ochraceum s.l.).

Conclusion

The presence of significant numbers of untreated and potentially infected migrants may contribute to ongoing transmission, and their incorporation into ivermectin programmes should be beneficial for the attainment of the elimination goals of the regional initiative. However, the possibility that the results also reflect transmission patterns for the area cannot be excluded and these should be analyzed further.     

Onchocerca volvulus-specific antibody and cellular responses in onchocerciasis patients treated annually with ivermectin for 30 years and exposed to parasite transmission in central Togo

BackgroundAnnual mass drug administrations (MDA) of ivermectin will strongly reduce Onchocerca volvulus microfilariae (mf) in the skin and in the onchocerciasis patients’ eyes. Ivermectin treatment will also affect the expression of immunity in patients, such that activated immune defenses may help control and contribute to clearance of mf of O. volvulus. Longitudinal surveys are a prerequisite to determining the impact of ivermectin on the status of anti-parasite immunity, notably in risk zones where parasite transmission and active O. volvulus infections persist.Methodology/Principal findingsOnchocerciasis patients were treated annually with ivermectin and their Onchocerca volvulus antigen (OvAg) specific IgG and cellular responses were investigated before and at 30 years post initial ivermectin treatment (30yPT).Repeated annual ivermectin treatments eliminated persisting O. volvulus microfilariae (mf) from the skin of patients and abrogated patent infections. The OvAg-specific IgG1 and IgG4 responses were diminished at 30yPT to the levels observed in endemic controls. Prior to starting ivermectin treatment, OvAg-induced cellular productions of IL-10, IFN-γ, CCL13, CCL17 and CCL18 were low in patients, and at 30yPT, cellular cytokine and chemokine responses increased to the levels observed in endemic controls. In contrast, mitogen(PHA)- induced IL-10, IFN-γ, CCL17 and CCL18 cellular production was diminished. This divergent response profile thus revealed increased parasite antigen-specific but reduced polyclonal cellular responsiveness in patients. The transmission of O. volvulus continued at the patients’ location in the Mô river basin in central Togo 2018 and 2019 when 0.58% and 0.45%, respectively, of Simulium damnosum s.l. vector blackflies carried O. volvulus infections.Conclusions/SignificanceRepeated annual ivermectin treatment of onchocerciasis patients durably inhibited their patent O. volvulus infections despite ongoing low-level parasite transmission in the study area. Repeated MDA with ivermectin affects the expression of immunity in patients. O. volvulus parasite-specific antibody levels diminished to levels seen in infection-free endemic controls. With low antibody levels, antibody-dependent cellular cytotoxic responses against tissue-dwelling O. volvulus larvae will weaken. O. volvulus antigen inducible cytokine and chemokine production increased in treated mf-negative patients, while their innate responsiveness to mitogen declined. Such lower innate responsiveness in elderly patients could contribute to reduced adaptive immune responses to parasite infections and vaccines. On the other hand, increased specific cellular chemokine responses in mf-negative onchocerciasis patients could reflect effector cell activation against tissue invasive larval stages of O. volvulus. The annual Simulium damnosum s.l. biting rate observed in the Mô river basin was similar to levels prior to initiation of MDA with ivermectin, and the positive rtPCR results reported here confirm ongoing O. volvulus transmission.     

Immunisation with a Multivalent,Subunit Vaccine Reduces Patent Infection in a Natural Bovine Model of Onchocerciasis during Intense Field Exposure

Human onchocerciasis, caused by the filarial nematode Onchocerca volvulus, is controlled almost exclusively by the drug ivermectin, which prevents pathology by targeting the microfilariae. However, this reliance on a single control tool has led to interest in vaccination as a potentially complementary strategy. Here, we describe the results of a trial in West Africa to evaluate a multivalent, subunit vaccine for onchocerciasis in the naturally evolved host-parasite relationship of Onchocerca ochengi in cattle. Naïve calves, reared in fly-proof accommodation, were immunised with eight recombinant antigens of O. ochengi, administered separately with either Freund''s adjuvant or alum. The selected antigens were orthologues of O. volvulus recombinant proteins that had previously been shown to confer protection against filarial larvae in rodent models and, in some cases, were recognised by serum antibodies from putatively immune humans. The vaccine was highly immunogenic, eliciting a mixed IgG isotype response. Four weeks after the final immunisation, vaccinated and adjuvant-treated control calves were exposed to natural parasite transmission by the blackfly vectors in an area of Cameroon hyperendemic for O. ochengi. After 22 months, all the control animals had patent infections (i.e., microfilaridermia), compared with only 58% of vaccinated cattle (P = 0.015). This study indicates that vaccination to prevent patent infection may be an achievable goal in onchocerciasis, reducing both the pathology and transmissibility of the infection. The cattle model has also demonstrated its utility for preclinical vaccine discovery, although much research will be required to achieve the requisite target product profile of a clinical candidate.     

Phenotypic evidence of emerging ivermectin resistance in Onchocerca volvulus

Background

Ivermectin (IVM) has been used in Ghana for over two decades for onchocerciasis control. In recent years there have been reports of persistent microfilaridermias despite multiple treatments. This has necessitated a reexamination of its microfilaricidal and suppressive effects on reproduction in the adult female Onchocerca volvulus. In an initial study, we demonstrated the continued potent microfilaricidal effect of IVM. However, we also found communities in which the skin microfilarial repopulation rates at days 90 and 180 were much higher than expected. In this follow up study we have investigated the reproductive response of female worms to multiple treatments with IVM.

Methods and Findings

The parasitological responses to IVM in two hundred and sixty-eight microfilaridermic subjects from nine communities that had received 10 to 19 annual doses of IVM treatment and one pre-study IVM-naïve community were followed. Skin snips were taken 364 days after the initial IVM treatment during the study to determine the microfilaria (mf) recovery rate. Nodules were excised and skin snips taken 90 days following a second study IVM treatment. Nodule and worm density and the reproductive status of female worms were determined. On the basis of skin mf repopulation and skin mf recovery rates we defined three categories of response—good, intermediate and poor—and also determined that approximately 25% of subjects in the study carried adult female worms that responded suboptimally to IVM. Stratification of the female worms by morphological age and microfilarial content showed that almost 90% of the worms were older or middle aged and that most of the mf were produced by the middle aged and older worms previously exposed to multiple treatments with little contribution from young worms derived from ongoing transmission.

Conclusions

The results confirm that in some communities adult female worms were non-responsive or resistant to the anti-fecundity effects of multiple treatments with IVM. A scheme of the varied responses of the adult female worm to multiple treatments is proposed.     

Ongoing Transmission of Onchocerca volvulus after 25 Years of Annual Ivermectin Mass Treatments in the Vina du Nord River Valley,in North Cameroon

Background

Recent reports of transmission interruption of Onchocerca volvulus, the causing agent of river blindness, in former endemic foci in the Americas, and more recently in West and East Africa, raise the question whether elimination of this debilitating disease is underway after long-term treatment of the population at risk with ivermectin. The situation in Central Africa has not yet been clearly assessed.

Methods and findings

Entomologic data from two former endemic river basins in North Cameroon were generated over a period of 43 and 48 months to follow-up transmission levels in areas under prolonged ivermectin control. Moreover, epidemiologic parameters of animal-borne Onchocerca spp. transmitted by the same local black fly vectors of the Simulium damnosum complex were recorded and their impact on O. volvulus transmission success evaluated. With mitochondrial DNA markers we unambiguously confirmed the presence of infective O. volvulus larvae in vectors from the Sudan savannah region (mean Annual Transmission Potential 2009–2012: 98, range 47–221), but not from the Adamawa highland region. Transmission rates of O. ochengi, a parasite of Zebu cattle, were high in both foci.

Conclusions/significance

The high cattle livestock density in conjunction with the high transmission rates of the bovine filaria O. ochengi prevents the transmission of O. volvulus on the Adamawa plateau, whereas transmission in a former hyperendemic focus was markedly reduced, but not completely interrupted after 25 years of ivermectin control. This study may be helpful to gauge the impact of the presence of animal-filariae for O. volvulus transmission in terms of the growing human and livestock populations in sub-Saharan countries.     

Uncertainty Surrounding Projections of the Long-Term Impact of Ivermectin Treatment on Human Onchocerciasis

Background

Recent studies in Mali, Nigeria, and Senegal have indicated that annual (or biannual) ivermectin distribution may lead to local elimination of human onchocerciasis in certain African foci. Modelling-based projections have been used to estimate the required duration of ivermectin distribution to reach elimination. A crucial assumption has been that microfilarial production by Onchocerca volvulus is reduced irreversibly by 30–35% with each (annual) ivermectin round. However, other modelling-based analyses suggest that ivermectin may not have such a cumulative effect. Uncertainty in this (biological) and other (programmatic) assumptions would affect projected outcomes of long-term ivermectin treatment.

Methodology/Principal Findings

We modify a deterministic age- and sex-structured onchocerciasis transmission model, parameterised for savannah O. volvulus–Simulium damnosum, to explore the impact of assumptions regarding the effect of ivermectin on worm fertility and the patterns of treatment coverage compliance, and frequency on projections of parasitological outcomes due to long-term, mass ivermectin administration in hyperendemic areas. The projected impact of ivermectin distribution on onchocerciasis and the benefits of switching from annual to biannual distribution are strongly dependent on assumptions regarding the drug''s effect on worm fertility and on treatment compliance. If ivermectin does not have a cumulative impact on microfilarial production, elimination of onchocerciasis in hyperendemic areas may not be feasible with annual ivermectin distribution.

Conclusions/Significance

There is substantial (biological and programmatic) uncertainty surrounding modelling projections of onchocerciasis elimination. These uncertainties need to be acknowledged for mathematical models to inform control policy reliably. Further research is needed to elucidate the effect of ivermectin on O. volvulus reproductive biology and quantify the patterns of coverage and compliance in treated communities.     

A Simple Isothermal DNA Amplification Method to Screen Black Flies for Onchocerca volvulus Infection

Onchocerciasis is a debilitating neglected tropical disease caused by infection with the filarial parasite Onchocerca volvulus. Adult worms live in subcutaneous tissues and produce large numbers of microfilariae that migrate to the skin and eyes. The disease is spread by black flies of the genus Simulium following ingestion of microfilariae that develop into infective stage larvae in the insect. Currently, transmission is monitored by capture and dissection of black flies and microscopic examination of parasites, or using the polymerase chain reaction to determine the presence of parasite DNA in pools of black flies. In this study we identified a new DNA biomarker, encoding O. volvulus glutathione S-transferase 1a (OvGST1a), to detect O. volvulus infection in vector black flies. We developed an OvGST1a-based loop-mediated isothermal amplification (LAMP) assay where amplification of specific target DNA is detectable using turbidity or by a hydroxy naphthol blue color change. The results indicated that the assay is sensitive and rapid, capable of detecting DNA equivalent to less than one microfilaria within 60 minutes. The test is highly specific for the human parasite, as no cross-reaction was detected using DNA from the closely related and sympatric cattle parasite Onchocerca ochengi. The test has the potential to be developed further as a field tool for use in the surveillance of transmission before and after implementation of mass drug administration programs for onchocerciasis.     

Epidemiology and psycho-social aspects of onchocercal skin diseases in northeastern Nigeria

Background

Observations were made on the prevalence of onchocerciasis and Onchocercal Skin Diseases (OSD); frequency of occurrence and anatomical distribution of OSD in the Hawal River Valley, an established onchocerciasis endemic focus in north-eastern Nigeria.

Methods

Symptoms of OSD were diagnosed in 5 844 subjects using Rapid Assessment Method (RAM) while 1 479 of the subjects chosen from alternate households had their skin biopsies examined for active microfilariae of Onchocerca volvulus. Also, Focal Group Discussions (FGD) were conducted at the Health District levels.

Results

O. volvulus was recorded in (19.0%) and OSD in (43.8%) of the subjects. The Mantel-Haenszel test for linear association showed a close agreement between onchocerciasis prevalence and the rate of OSD (χ² = 3.93; p < 0.05). The various forms of OSD occurred in the order: CPOD (17.7%), APOD (9.9%), DPM (9.0%), LOD (7.0%) and ATR (3.1%). The overall frequency of occurrence of various symptoms of OSD on different anatomical locations showed the locations in descending order of occurrence as lower limbs (24.6%), upper limbs (21.3%), buttocks (19.9%), shoulder & neck (19.1%), abdomen and trunk (11.3%), backside (10.6), and 'other' sites (7.5%). The Focal Group Discussion (FGD) revealed the most worrisome consequences of OSD as social isolation of victims (31.3%), shame and low self esteem (22.7%) and high cost of medication (15.6%).

Conclusion

It is recommended that Onchocerciasis control programmes in the Hawal River Valley and any other focus with high incidence of OSD should incorporate an aspect that would address the anxiety and depression caused by various OSD lesions since they carry lots of psycho-social implications. This would increase acceptance and compliance of the target population. The classification criteria of onchocerciasis endemicity should be based on either or both of the O. volvulus and onchocercal skin disease burden of any community and no longer on O. volvulus parasitic infection rate alone.     

Onchocerciasis: The Pre-control Association between Prevalence of Palpable Nodules and Skin Microfilariae

Background

The prospect of eliminating onchocerciasis from Africa by mass treatment with ivermectin has been rejuvenated following recent successes in foci in Mali, Nigeria and Senegal. Elimination prospects depend strongly on local transmission conditions and therefore on pre-control infection levels. Pre-control infection levels in Africa have been mapped largely by means of nodule palpation of adult males, a relatively crude method for detecting infection. We investigated how informative pre-control nodule prevalence data are for estimating the pre-control prevalence of microfilariae (mf) in the skin and discuss implications for assessing elimination prospects.

Methods and Findings

We analyzed published data on pre-control nodule prevalence in males aged ≥20 years and mf prevalence in the population aged ≥5 years from 148 African villages. A meta-analysis was performed by means of Bayesian hierarchical multivariate logistic regression, accounting for measurement error in mf and nodule prevalence, bioclimatic zones, and other geographical variation. There was a strong positive correlation between nodule prevalence in adult males and mf prevalence in the general population. In the forest-savanna mosaic area, the pattern in nodule and mf prevalence differed significantly from that in the savanna or forest areas.

Significance

We provide a tool to convert pre-control nodule prevalence in adult males to mf prevalence in the general population, allowing historical data to be interpreted in terms of elimination prospects and disease burden of onchocerciasis. Furthermore, we identified significant geographical variation in mf prevalence and nodule prevalence patterns warranting further investigation of geographical differences in transmission patterns of onchocerciasis.     

Human Onchocerciasis: Modelling the Potential Long-term Consequences of a Vaccination Programme

Background

Currently, the predominant onchocerciasis control strategy in Africa is annual mass drug administration (MDA) with ivermectin. However, there is a consensus among the global health community, supported by mathematical modelling, that onchocerciasis in Africa will not be eliminated within proposed time frameworks in all endemic foci with only annual MDA, and novel and alternative strategies are urgently needed. Furthermore, use of MDA with ivermectin is already compromised in large areas of central Africa co-endemic with Loa loa, and there are areas where suboptimal or atypical responses to ivermectin have been documented. An onchocerciasis vaccine would be highly advantageous in these areas.

Methodology/Principal Findings

We used a previously developed onchocerciasis transmission model (EPIONCHO) to investigate the impact of vaccination in areas where loiasis and onchocerciasis are co-endemic and ivermectin is contraindicated. We also explore the potential influence of a vaccination programme on infection resurgence in areas where local elimination has been successfully achieved. Based on the age range included in the Expanded Programme on Immunization (EPI), the vaccine was assumed to target 1 to 5 year olds. Our modelling results indicate that the deployment of an onchocerciasis vaccine would have a beneficial impact in onchocerciasis–loiasis co-endemic areas, markedly reducing microfilarial load in the young (under 20 yr) age groups.

Conclusions/Significance

An onchocerciasis prophylactic vaccine would reduce the onchocerciasis disease burden in populations where ivermectin cannot be administered safely. Moreover, a vaccine could substantially decrease the chance of re-emergence of Onchocerca volvulus infection in areas where it is deemed that MDA with ivermectin can be stopped. Therefore, a vaccine would protect the substantial investments made by present and past onchocerciasis control programmes, decreasing the chance of disease recrudescence and offering an important additional tool to mitigate the potentially devastating impact of emerging ivermectin resistance.     

Lymphatic Vascularisation and Involvement of Lyve-1+ Macrophages in the Human Onchocerca Nodule

Onchocerciasis, caused by the filarial nematode Onchocerca volvulus, is a parasitic disease leading to debilitating skin disease and blindness, with major economic and social consequences. The pathology of onchocerciasis is principally considered to be a consequence of long-standing host inflammatory responses. In onchocerciasis a subcutaneous nodule is formed around the female worms, the core of which is a dense infiltrate of inflammatory cells in which microfilariae are released. It has been established that the formation of nodules is associated with angiogenesis. In this study, we show using specific markers of endothelium (CD31) and lymphatic endothelial cells (Lyve-1, Podoplanin) that not only angiogenesis but also lymphangiogenesis occurs within the nodule. 7% of the microfilariae could be found within the lymphatics, but none within blood vessels in these nodules, suggesting a possible route of migration for the larvae. The neovascularisation was associated with a particular pattern of angio/lymphangiogenic factors in nodules of onchocerciasis patients, characterized by the expression of CXCL12, CXCR4, VEGF-C, Angiopoietin-1 and Angiopoietin-2. Interestingly, a proportion of macrophages were found to be positive for Lyve-1 and some were integrated into the endothelium of the lymphatic vessels, revealing their plasticity in the nodular micro-environment. These results indicate that lymphatic as well as blood vascularization is induced around O. volvulus worms, either by the parasite itself, e.g. by the release of angiogenic and lymphangiogenic factors, or by consecutive host immune responses.     

The Mbam drainage system and onchocerciasis transmission post ivermectin mass drug administration (MDA) campaign,Cameroon

BackgroundThe impact of large scale Mass Drug Adminstration (MDA) of ivermectin on active onchocerciasis transmission by Simulium damnosum, which transmits the parasite O. volvulus is of great importance for onchocerciasis control programmes. We investigated in the Mbam river system area, the impact of MDA of ivermectin on entomological indices and also verify if there are river system factors that could have favoured the transmission of onchocerciasis in this area and contribute to the persistence of disease. We compared three independent techniques to detect Onchocerca larvae in blackflies and also analyzed the river system within 9 months post-MDA of ivermectin.MethodSimulium flies were captured before and after 1, 3, 6 and 9months of ivermectin-MDA. The biting rate was determined and 41% of the flies dissected while the rest were grouped into pools of 100 flies for DNA extraction. The extracted DNA was then subjected to O-150 LAMP and real-time PCR for the detection of infection by Onchocerca species using pool screening. The river system was analysed and the water discharge compared between rainy and dry seasons.Principal findingsWe used human landing collection method (previously called human bait) to collect 22,274 adult female Simulium flies from Mbam River System. Of this number, 9,134 were dissected while 129 pools constituted for molecular screening. Overall biting and parous rates of 1113 flies/man/day and 24.7%, respectively, were observed. All diagnostic techniques detected similar rates of O. volvulus infection (P = 0.9252) and infectivity (P = 0.4825) at all monitoring time points. Onchocerca ochengi larvae were only detected in 2 of the 129 pools. Analysis of the river drainage revealed two hydroelectric dams constructed on the tributaries of the Mbam river were the key contributing factor to the high-water discharge during both rainy and dry seasons.ConclusionResults from fly dissection (Microscopy), real-time PCR and LAMP revealed the same trends pre- and post-MDA. The infection rate with animal Onchocerca sp was exceptionally low. The dense river system generate important breeding sites that govern the abundance of Simulium during both dry and rainy seasons.