Doxycycline in the treatment of human onchocerciasis: Kinetics of Wolbachia endobacteria reduction and of inhibition of embryogenesis in female Onchocerca worms

Recently, experts have warned that mass treatment with ivermectin alone may not interrupt the transmission of Onchocerca. Hence, additional drugs are needed, such as antibiotics acting on symbiotic endobacteria of the filariae, the causative agents of onchocerciasis. Based on animal experiments, human onchocerciasis was treated with doxycycline, and preliminary observations published in 2001 in The Lancet showed sterility in female worms by depletion and marked reduction in symbiotic Wolbachia endobacteria from the filariae. Here, a detailed kinetic analysis of the features of the worms, following administration or not of doxycycline to the patients is reported. Sixty-three onchocerciasis patients in Ghana were treated with 100 mg doxycycline daily for 6 weeks and 2 or 6 months later with ivermectin. Onchocercomas were extirpated 2, 6, 11 and 18 months after the onset of treatment and the filariae were examined by immunohistology and PCR. The analysis showed: (i) progressive depletion of Wolbachia from adult worms and microfilariae by doxycycline over a period of 6 months; (ii) inhibition of embryogenesis by doxycycline after 6 months with respect to all embryo stages followed by decline in microfilariae after 11 months; (iii) reduction in spermatozoa in the female genital tract by doxycycline, whereas spermiogenesis was only partly reduced after 11 and 18 months; (iv) no relevant macro- or microfilaricidal activity; (v) depletion/marked reduction in endobacteria and inhibition of embryogenesis were sustained until 18 months after doxycycline and 12 months after co-administration of ivermectin; (vi) no severe adverse side effects were seen. Due to its long-lasting inhibition of embryogenesis, doxycycline presents an additional strategy for the treatment of onchocerciasis and control of Onchocerca microfilariae transmission. Extension of the existing registration will not require much time or high cost. Treatment of individual patients can be considered immediately.     

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     

Repeated high doses of avermectins cause prolonged sterilisation, but do not kill, Onchocerca ochengi adult worms in African cattle

Background

Ivermectin (Mectizan?, Merck and CO. Inc.) is being widely used in the control of human onchocerciasis (Onchoverca volvulus) because of its potent effect on microfilariae. Human studies have suggested that, at the standard dose of 150 μg/kg an annual treatment schedule of ivermectin reversibly interferes with female worm fertility but is not macrofilaricidal. Because of the importance of determining whether ivermectin could be macrofilaricidal, the efficacy of high and prolonged doses of ivermectin and a related avermectin, doramectin, were investigated in cattle infected with O. ochengi.

Methods

Drugs with potential macrofilaricidal activity, were screened for the treatment of human onchocerciasis, using natural infections of O. ochengi in African cattle. Three groups of 3 cows were either treated at monthly intervals (7 treatments) with ivermectin (Ivomec^®, Merck and Co. Inc.) at 500 μg/kg or doramectin (Dectamax^®, Pfizer) at 500 μg/kg or not treated as controls. Intradermal nodules were removed at 6 monthly intervals and adult worms were examined for signs of drug activity.

Results

There was no significant decline in nodule diameter, the motility of male and female worms, nor in male and female viability as determined by the ability to reduce tetrazolium, compared with controls, at any time up to 24 months from the start of treatments (mpt). Embryogenesis, however, was abrogated by treatment, which was seen as an accumulation of dead and dying intra-uterine microfilariae (mf) persisting for up to 18 mpt. Skin mf densities in treated animals had fallen to zero by <3 mpt, but by 18 mpt small numbers of mf were found in the skin of some treated animals and a few female worms were starting to produce multi-cellular embryonic stages. Follow-up of the doramectin treated group at 36 mpt showed that mf densities had still only regained a small proportion of their pre-treatment levels.

Conclusion

These results have important implications for onchocerciasis control in the field. They suggest that ivermectin given at repeated high does may sterilise O. volvulus female worms for prolonged periods but is unlikely to kill them. This supports the view that control programmes may need to continue treatments with ivermectin for a period of decades and highlights the need to urgently identify new marcofiliaricidal compounds.     

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.     

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.     

Status of Onchocerciasis Transmission after More Than a Decade of Mass Drug Administration for Onchocerciasis and Lymphatic Filariasis Elimination in Central Nigeria: Challenges in Coordinating the Stop MDA Decision

Background

This study was undertaken in five onchocerciasis/lymphatic filariasis (LF) co-endemic local government areas (LGAs) in Plateau and Nasarawa, Nigeria. Annual MDA with ivermectin had been given for 17 years, 8 of which were in combination with albendazole. In 2008, assessments indicated that LF transmission was interrupted, but that the MDA had to continue due to the uncertain status of onchocerciasis transmission. Accordingly, assessments to determine if ivermectin MDA for onchocerciasis could be stopped were conducted in 2009.

Methods

We evaluated nodule, microfilarial (mf) skin snip, and antibody (IgG4 response to OV16) prevalence in adults and children in six sentinel sites where baseline data from the 1990s were available. We applied the 2001 WHO criteria for elimination of onchocerciasis that defined transmission interruption as an infection rate of <0.1% in children (using both skin snip and OV16 antibody) and a rate of infective (L3) blackflies of <0.05%.

Results

Among adult residents in sentinel sites, mean mf prevalence decreased by 99.37% from the 1991–1993 baseline of 42.95% (64/149) to 0.27% (2/739) in 2009 (p<0.001). The OV16 seropositivity of 3.52% (26/739) among this same group was over ten times the mf rate. No mf or nodules were detected in 4,451 children in sentinel sites and ‘spot check’ villages, allowing the exclusion of 0.1% infection rate with 95% confidence. Seven OV16 seropositives were detected, yielding a seroprevalence of 0.16% (0.32% upper 95%CI). No infections were detected in PCR testing of 1,568 Simulium damnosum s.l. flies obtained from capture sites around the six sentinel sites.

Conclusion

Interruption of transmission of onchocerciasis in these five LGAs is highly likely, although the number of flies caught was insufficient to exclude 0.05% with 95% confidence (upper CI 0.23%). We suggest that ivermectin MDA could be stopped in these LGAs if similar results are seen in neighboring districts.     

Effect of Two or Six Doses 800 mg of Albendazole Every Two Months on Loa loa Microfilaraemia: A Double Blind,Randomized, Placebo-Controlled Trial

BackgroundLoiasis is a parasitic infection endemic in the African rain forest caused by the filarial nematode Loa loa. Loiasis can be co-endemic with onchocerciasis and/or lymphatic filariasis. Ivermectin, the drug used in the control of these diseases, can induce serious adverse reactions in patients with high L loa microfilaraemia (LLM). A drug is needed which can lower LLM below the level that represents a risk so that ivermectin mass treatment to support onchocerciasis and lymphatic filariasis elimination can be implemented safely.MethodologySixty men and women from a loiasis endemic area in Cameroon were randomized after stratification by screening LLM (≤30000, 30001–50000, >50000) to three treatment arms: two doses albendazole followed by 4 doses matching placebo (n = 20), six doses albendazole (n = 20) albendazole or 6 doses matching placebo (n = 20) administered every two months. LLM was measured before each treatment and 14, 18, 21 and 24 months after the first treatment. Monitoring for adverse events occurred three and seven days as well as 2 months after each treatment.

Principal Findings

None of the adverse events recorded were considered treatment related. The percentages of participants with ≥ 50% decrease in LLM from pre-treatment for ≥ 4 months were 53%, 17% and 11% in the 6-dose, 2-dose and placebo treatment arms, respectively. The difference between the 6-dose and the placebo arm was significant (p = 0.01). The percentages of participants with LLM < 8100 mf/ml for ≥4 months were 21%, 11% and 0% in the 6-dose, 2-dose and placebo treatment arms, respectively.

Conclusions/ Significance

The 6-dose regimen reduced LLM significantly, but the reduction was insufficient to eliminate the risk of severe and/or serious adverse reactions during ivermectin mass drug administration in loiasis co-endemic areas.     

The impact of ivermectin on onchocerciasis in villages co-endemic for lymphatic filariasis in an area of onchocerciasis recrudescence in Burkina Faso

In Burkina Faso, onchocerciasis was no longer a public health problem when the WHO Onchocerciasis Control Programme in West Africa closed at the end in 2002. However, epidemiological surveillance carried out from November 2010 to February of 2011, showed a recrudescence of infection in the Cascades Region. This finding was made at a time when ivermectin, a drug recommended for the treatment of both onchocerciasis and lymphatic filariasis, had been distributed in this area since 2004 for the elimination of lymphatic filariasis. It was surprising that ivermectin distributed for treating lymphatic filariasis had not prevented the recrudescence of onchocerciasis. Faced with this situation, the aim of our study was to evaluate the effectiveness of ivermectin on the onchocerciasis parasite. The percentage reduction in microfilarial load after treatment with ivermectin was used as a proxy measure for assessing possible resistance. A cohort study was carried out with 130 individuals who had tested positive for microfilariae of Onchocerca volvulus in 2010 using microscopic examination of skin-snip biopsies from five endemic villages. Subjects were followed from July 2011 to June 2012. The microfilarial load of each individual was enumerated by skin-snip biopsy in 2010, prior to the first ivermectin treatment against onchocerciasis under community guidelines. All individuals received two ivermectin treatments six months apart. In 2012, the microfilarial loads were determined again, six months after the second round of ivermectin and the reductions in parasite loads were calculated to measure the impact of the drug. The percentage reduction of the microfilarial loads ranged from 87% to 98% in the villages. In all villages, there was a statistically significant difference between the average microfilarial loads in 2010 and 2012. The level of reduction of microfilarial loads suggests that ivermectin is effective against the recrudescent population of O. volvulus in Cascades Region of Burkina Faso. Further investigations would be necessary to determine the causes of the recrudescence of onchocerciasis. (For French language abstract, see S1 Alternative Language Abstract—Translation of the Abstract into French by the authors.)     

Studies on acquired resistance of the cotton rat against microfilariae of Litomosoides carinii. 2. Injection of microfilariae during prepatency

Cotton rats infected by infective third-stage larvae of Litomosoides carinii were treated at increasing time intervals by a threefold injection of living homologous microfilariae (mf) during the prepatent period. Starting with the first treatment 3, 4 or 5 weeks p.i. seven animals remained completely and two almost mf-negative (1 or 2 mf/mm3 each only once) until 16 weeks p.i. Starting 6, 7 or 8 weeks p.i. six animals developed a normal level of parasitaemia between 42 and 436 mf/mm3, two animals developed a continuous level of 1-2 mf/mm3. The number of fertile adult worms shedding great numbers of microfilariae in the pleural cavity was equal in all animals. However, in mf-negative animals the lung capillary blood showed, in the geometric mean, only 0.6% of the mf-concentration seen in mf-positive animals. The hypothesis is proposed that microfilariae accumulating primarily in the lung capillaries absorb all aggressive components specifically reacting with microfilarial antigens, i.e. neutralize the immune response against them to enable the development of the parasitaemia in the peripheral blood.     

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.     

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.     

Corallopyronin A for short-course anti-wolbachial,macrofilaricidal treatment of filarial infections

Current efforts to eliminate the neglected tropical diseases onchocerciasis and lymphatic filariasis, caused by the filarial nematodes Onchocerca volvulus and Wuchereria bancrofti or Brugia spp., respectively, are hampered by lack of a short-course macrofilaricidal–adult-worm killing–treatment. Anti-wolbachial antibiotics, e.g. doxycycline, target the essential Wolbachia endosymbionts of filariae and are a safe prototype adult-worm-sterilizing and macrofilaricidal regimen, in contrast to standard treatments with ivermectin or diethylcarbamazine, which mainly target the microfilariae. However, treatment regimens of 4–5 weeks necessary for doxycycline and contraindications limit its use. Therefore, we tested the preclinical anti-Wolbachia drug candidate Corallopyronin A (CorA) for in vivo efficacy during initial and chronic filarial infections in the Litomosoides sigmodontis rodent model. CorA treatment for 14 days beginning immediately after infection cleared >90% of Wolbachia endosymbionts from filariae and prevented development into adult worms. CorA treatment of patently infected microfilaremic gerbils for 14 days with 30 mg/kg twice a day (BID) achieved a sustained reduction of >99% of Wolbachia endosymbionts from adult filariae and microfilariae, followed by complete inhibition of filarial embryogenesis resulting in clearance of microfilariae. Combined treatment of CorA and albendazole, a drug currently co-administered during mass drug administrations and previously shown to enhance efficacy of anti-Wolbachia drugs, achieved microfilarial clearance after 7 days of treatment at a lower BID dose of 10 mg/kg CorA, a Human Equivalent Dose of 1.4 mg/kg. Importantly, this combination led to a significant reduction in the adult worm burden, which has not yet been published with other anti-Wolbachia candidates tested in this model. In summary, CorA is a preclinical candidate for filariasis, which significantly reduces treatment times required to achieve sustained Wolbachia depletion, clearance of microfilariae, and inhibition of embryogenesis. In combination with albendazole, CorA is robustly macrofilaricidal after 7 days of treatment and fulfills the Target Product Profile for a macrofilaricidal drug.     

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.     

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.     

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.     

Rapid molecular assays for specific detection and quantitation of Loa loa microfilaremia

Background

Accurate diagnosis of Loa loa infection is essential to the success of mass drug administration efforts to eliminate onchocerciasis and lymphatic filariasis, due to the risk of fatal encephalopathic reactions to ivermectin occurring among highly microfilaremic Loa-infected individuals living in areas co-endemic for multiple filarial species.

Methodology/Principal Findings

From a pool of over 1,800 L. loa microfilaria (mf) expressed sequence tags, 18 candidate L. loa mf-specific PCR targets were identified. Real-time PCR (qPCR) assays were developed for two targets (LLMF72 and LLMF269). The qPCR assays were highly specific for L. loa compared with related filariae and also highly sensitive, with detection limits of 0.1 pg genomic DNA, or 1% of DNA extracted from normal blood spiked with a single L. loa microfilaria. Using various DNA extraction methods with dried blood spots obtained from Cameroonian subjects with parasitologically proven loiasis, the LLMF72 qPCR assay successfully estimated mf burden in 65 of 68 samples (50–96,000 mf/mL by microscopy), including all 12 samples subjected to a simple 10-minute boiling extraction. Additionally, the assay detected low-level microfilaremia among 5 of 16 samples from patients thought to be amicrofilaremic by microscopy.

Conclusions/Significance

This novel, rapid, highly sensitive and specific qPCR assay is an important step forward in the laboratory diagnosis of L. loa infection.     

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.     

<Emphasis Type="Italic">Loa loa</Emphasis> encephalopathy temporally related to ivermectin administration reported from onchocerciasis mass treatment programs from 1989 to 2001: implications for the future

Of the 207 Serious Adverse Events (SAEs) following treatment with Mectizan^® (ivermectin, Merck, Sharpe &; Dohme) that were reported from 1989 to 2001 through the passive SAE surveillance system required of all onchocerciasis mass treatment programs, 65 were cases of 'Probable' or 'Possible' Loa loa Encephalopathy temporally Related to treatment with Mectizan^® (PLERM).A retrospective analysis of these 65 PLERM cases revealed that 97% were from southern Cameroon, 85% were male and 93% were being treated with ivermectin for the first time. The mean time to onset of symptoms was 1.7 days (95% CI: 1.3, 2.2) but the mean time to receiving medical attention after the onset of symptoms was 2.0 days (95% CI: 1.5, 2.6). Hospitalization was reported in 53 cases with a mean duration of 27.5 days (95% CI: 13.3, 41.6, n = 35). Clinical outcome was reported in 34 cases: 64.7% recovered fully, 11.8% had partial neurologic deficit and 23.5% died. For the 32 cases where quantitative L. loa data were reported, the arithmetic means with 95% confidence intervals were for 1) peripheral blood: pre-treatment – 164,250 mf/ml (79,537, 248,963; n = 4); post-treatment within 1 month – 3926 mf/ml (2,128, 5,725; n = 21) and within 5 to 6 months – 7800 mf/ml (3417, 12,183; n = 7); and for 2) cerebrospinal fluid: 32 mf/ml (7, 37; n = 10) within 1 month post-treatment.Pending further research on practical methods to exclude individuals with high intensity L. loa infection from onchocerciasis mass treatment programs, more emphasis should be placed on surveillance and monitoring to ensure early recognition, referral and management of SAEs, during the first 2 years when majority of the population is presumably naïve to ivermectin.     

Impact of two rounds of mass drug administration using diethylcarbamazine combined with albendazole on the prevalence of Brugia timori and of intestinal helminths on Alor Island, Indonesia

Background

Annual mass drug administration (MDA) using diethylcarbamizine (DEC, 6 mg/kg) combined with albendazole (alb, 400 mg) is recommended by the Global Programme to Eliminate Lymphatic Filariasis (GPELF). This strategy has been shown to be efficient in the of control bancroftian filariasis, but data on brugian filariasis as well as on the positive side effects on intestinal helminths are lacking.

Methods

The effect of one selective treatment and two rounds of MDA using DEC and alb on the prevalence and intensity of Brugia timori infection were studied on Alor island using a cross-sectional and a cohort approach. Before the campaign and ten months after each treatment cycle microfilariae (mf) were assessed by filtration of night blood. Before and ten months after MDA, stool samples were collected and the prevalence of intestinal helminths were determined.

Results

In all, the mf-rate dropped from 26.8% before any treatment to 3.8% following the second MDA. Almost all mf-positive, treated individuals showed very low mf densities. The crude prevalence of hookworm dropped from 25.3% to 5.9%. The reduction of prevalence of Ascaris lumbricoides (32.3% to 27.6%) and Trichuris trichiura (9.4% to 8.9%) was less pronounced. Within a cohort of 226 individuals, which was examined annually, the prevalence of A. lumbricoides dropped from 43.8% to 26.5% and of T. trichiura from 12.8% to 6.6%. The results indicate that this MDA approach reduces not only the mf prevalence of B. timori but also the prevalence of hookworm and to a lesser extent also of A. lumbricoides and T. trichiura.

Conclusion

The MDA using DEC and alb as recommended by GPELF is extremely effective for areas with brugian filariasis. The beneficial effect of MDA on intestinal helminths may strengthen the national programme to eliminate lymphatic filariasis in Indonesia and may set resources free which are otherwise used for deworming campaigns of schoolchildren.