INTRODUCTION AND LITERATURE REVIEW
Onchocerciasis is the disease resulting from infection with the filarial nematode Onchocerca volvulus, which is transmitted to man through the bite of the infected black flies belonging to the genus, Simulium (Family: Simuliidae) (WHO, 1991, Okuliez, 2008). The global distribution of onchocerciasis includes endemic areas in tropical Africa, where 99% of the total infected persons are found, Central and South America (WHO, 1987). It is estimated that 85.5 million people are at risk and that 17.7 million people are infected with O. Volvulus (WHO, 1995). Onchocerciasis is one of the leading causes of loss of sight in the world, responsible for about 270,000 blind and 500 000 partly sighted people (WHO, 1995).
The current global burden of the disease showed that an estimated 123 million persons were at risk of contacting the disease and 17 – 18 million were infected (WHO, 1999; Hoerauf et al., 2003; Boatin and Richrads, 2006). According to IJPD (2009), more than 30 million Nigerians in 32 states and the Federal Capital Territory are estimated to be at risk for onchocerciasis. About 360,000 people are projected to be blind (Abiose et al., 1993). The disease is present in all the states including the Federal Capital territory with the exception of Lagos, Rivers, and Akwa–Ibom states where the infections are sporadic.
The vectors breed along fast-flowing rivers where infection intensity and morbidity are highest. Onchocerciasis is also known colloquially as river blindness (WHO, 1985). The manifestations of onchocerciasis are predominantly dermal, lymphatic and ocular in character (Mackenzie et al., 1995), but several other features of uncertain association, etiology or pathogenesis have also been described, including low body weight, general debility, diffuse musculoskeletal pain and, in Africa, epilepsy and hyposexual dwarfism (WHO, 1987a; Nwoke, 1992; Kipp et al., 1994; Shu and Okonkwo, 1998). The consequences of onchocerciasis have repercussions beyond the individual and directly affect the family, community and country. In the usually remote, rural areas of the Savannah zones of Africa, the effects of river blindness have led to the decline and dessertation of villages, where it is without doubt the most important disease afflicting the communities. Blindness rates of 5-10% reduce the viability of communities; when the majority of men over 40 years of age are blind, villages rapidly cease to be economically viable. Populations move to healthier local environments, away from the rivers where the vectors breed but where the soil is usually not so fertile (WHO, 1987, 1995a; Anderson and Fuglesang,1978; Abiose et al., 1993; Murdoch et al., 2002). Furthermore, since blindness leads to a reduction of some 10 years in life expectancy, onchocerciasis is a disease that not only disables but is indirectly responsible for considerable premature mortality. In persons with prolonged intense infections, the skin lesions and itching are responsible for much chronic misery and disfigurement, and can lead to a degree of social isolation, with detrimental psychological effects (Nwoke, 1990). In Achi, an onchocerciasis-endemic community, in South Eastern Nigeria, the disease effects not only reduces the village economics but also has led to the disruption of most families because the able bodied men flee at the fear of getting blind (Okonkwo et al., 1991). In another community also in South Eastern Nigeria, Amazigo (1994) reported that the skin lesions of onchocerciasis causes not only great discomfort but reduces the chances of a girl getting married. Thus, the numerous problems posed by onchocerciasis have led to numerous efforts to control this parasitic disease and treat the millions of people already infected.
Previous efforts at halting the disease include the aerial spraying of biodegradable larvicides in West Africa- a co-operative effort of World Health Organisation -related groups, and the use of drug treatments, Diethylcarbamazine citrate (DEC) and Suramin, which can have severe adverse effects. These drugs were deemed unsuitable for mass use because they produce only short-term or unsatisfactory suppression of microfilariae and patients require close medical supervision during administration. DEC has been the standard theraphy for over three decades (WHO, 1987). It must be given orally daily for seven to ten days and is frequently accompanied by severe reactions, including deterioration of onchocercal eye lesions, and may itself even cause blindness and death. DEC is microfilaricidal only. Suramin kills adult worms and is therefore macrofilaricidal (WHO, 1995). It is/was the only macrofilaricidal drug and has to be given intravenously once a week for several weeks. Its administration may be accompanied by severe rash , diarrhoea, neurotoxicity, nephrotoxicity and sometimes death ( Stein et al., 1989; La Rocca et al., 1990; Voogt et al., 1993). It was apparent that the development of a new drug that may be administered orally as a single dose for mass chemotherphy was to be a major research goal. This search for a new drug for onchocerciasis led to the development of ivermectin (IVM) by Merck Sharp and Dohme (MSD). Other drugs which have shown limited promises include Amocarzine (CGP 6140) which is an antifilarial anthelmintic isolated from amoscanate active against adult worms of O. volvulus. Amocarzine is toxic to mitochondria and causes inhibition of respiration. The basis for its selective toxicity appears to be preferential drug uptake by the filarial worm (Kohler et al., 1992). Moxidectin – a milbemycin drug used in veterinary medicine has shown macrofilaricidal activity in animal screens and has been shown to be safe in preliminary (phase 1) human trials (WHO, 2000, Cotreau et al., 2003). It is being evaluated for Phase II clinical trial, in which 192 persons infected with onchocerciasis are enrolled. Following the period, moxidectin will hopefully be available to endemic countries by the year 2012 (Eeezzuduemhoi and Wilson, 2008)
Ivermectin, a broad range antiparasitic agent, has been developed for veterinary use and widely used in veterinary medicine. First treatment of onchocerciasis with ivermectin was performed in 1982. IVM has displaced DEC as a microfilaricidal drug (Goa et al., 1991). In a single oral dose of 150 ug/kg bodyweight, ivermectin is superior to DEC in elimininating high parasite loads and it has a more prolonged suppressive effect on skin and ocular microfilariae (Goa et al., 1991). Ivermectin causes less severe reactions and importantly, no ocular deficiency, and is therefore useful for mass distribution.
Ivermectin (derived from Streptomyces avermitilis) is the current drug of choice for the treatment of onchocerciasis. Ivermectin, a medicine capable of killing the parasite embryos (the microfilariae) circulating in the organism of patients and temporarily interrupting the nematode’s reproduction, is the only acceptable treatment used for onchocerciasis mass chemotherapy.
Already IVM is being distributed world-wide and several million individuals in several continents (WHO Exp. Committee, 1995a) have been dosed with encouraging results (Chabala et al., 1980; Soboslay et al., 1987, 1991; Taylor and Green 1989). Microfilarial loads are reduced to 20% of pretreatment levels for up to one year after single dose ivermectin therapy (White et al., 1987). In Ghana, skin microfilariae reduced to 96% after 2 months of a large-scale community treatment with ivermectin (Remme et al., 1989). In Liberia, skin microfilaria load reduced by 86% after a large-scale community treatment with ivermectin (Pacque et al., 1990a). Likewise ocular levels of microfilariae in the anterior chamber decreased to 20% of pretreatment values 4 months after ivermectin therapy, but rose 39% at 12 months (Dadzie et al., 1990).
In Achi community, ivermectin treatment started since 1990 on a yearly basis. After the first mass treatment, up to 82% reduction of skin microfilariae load was recorded, followed by the second treatment with 76% reduction observed (Okonkwo et al., 1991). However, subsequent treatment did not show much further reduction but rather a small percentage of the treated patients maintained their skin microfilarial load. Thus, it was not clear if this was due to malreabsorption of the drug by the host or reinfection of the parasite or resistance to the drug by the parasite. It is already evident that development of resistance by tissues or organisms to drugs is an evolutionary adaptation that puts at risk every tumouricidal, pesticidal and parasiticidal agent and resistance of parasites and infectious disease organisms to drugs and antibiotics is as old as chemotherapy itself.
Despite all efforts, there is still great frustration in cancer chemotherapy due to development of drug resistance. Malaria is now present in 102 countries, is responsible for 100 million clinical cases and 1 to 2 million deaths each year (Oaks, 1991) because, the lethal form of human malaria caused by Plasmodium falciparum has developed resistance to chloroquine in many areas worldwide. The P-glycoprotein (P- signifies permeability), which is a membrane-bound molecule has been implicated in multidrug resistant cancer cells and also in P. falciparum resistance to chloroquine. Thus, it was the keeping in mind the possibilty that parasite resistance to ivermectin may develop and pose a big threat both to current control activities and future plans that we thought it necessary to carry out this research work.
1.2 Justification of the Study
In Achi community, ivermectin treatment started since 1990/1991 on a yearly basis. After the first mass treatment, up to 82% reduction of skin microfilariae load was recorded in some villages followed by the second treatment with 76% reduction observed (Okonkwo et al., 1991). However, subsequent treatment did not show much further reduction but rather a small percentage of the treated patients maintained their skin microfilarial load. Thus, it was not clear if this was due to malreabsorption of the drug by the host or reinfection of the parasite or resistance to the drug by the parasite. It is already evident that development of resistance by tissues or organisms to drugs is an evolutionary adaptation that puts at risk every tumouricidal, pesticidal and parasiticidal agent and resistance of parasites and infectious disease organisms to drugs and antibiotics is as old as chemotherapy itself.
Despite all efforts, there is still great frustration in cancer chemotherapy due to development of drug resistance. Malaria is now present in 102 countries, is responsible for 100 million clinical cases and 1 to 2 million deaths each year (Oaks et al., 1991) because, the lethal form of human malaria caused by Plasmodium falciparum has developed resistance to chloroquine in many areas worldwide. The P-glycoprotein (P- signifies permeability), which is a membrane-bound molecule has been implicated in multidrug resistant cancer cells and also in P. falciparum resistance to chloroquine. Thus, it was the keeping in mind the possibilty that parasite resistance to ivermectin may develop and pose a big threat both to current control activities and future plans that we thought it necessary to carry out this research work
Due to the alarming rate in drug resistance, millions of people are concerned about the cause of the increase, the effects it has on humans and how these effects or problems can be controlled. In the treatment of malaria with chlororquine, drug resistance has been a major challenge which has kept malaria disease in the lime light up till today. Several causes has been attributed of which the most common is the misuse of drugs , malreabsorption of drugs, as well as development of enzymes that inactivate drugs as well as a host of others. Yet, each cause must be proved scientifically.
Since 1990 in Achi (an onchocerciasis-endemic community), South –Eastern Nigeria, annual Ivermectin treatment has been on-going and some treated patients still harbor a lot of microfilaria (Personal observation, WORLD BANK/UNDP/WHO ONCHO PROJECT, Achi, Enugu state, 1990 -1993). Also, the doubling of cases of infection in certain communities of Ghana between 2000 and 2005, in spite of annual treatments, created fear of the emergence of ivermectin-resistant strains (Flechet, 2008). This phenomenom is not new as it has been experienced in the treatment of malaria with chloroquine, thus the development of resistance to ivermectin is of high relevance especially in health development policies. There is therefore the need to start early and search for the resistant indices as observed in the treatment of other diseases like malaria and cancer.
Using highly molecular approaches to search for resistance indices, I intend to collect nodules and microfilaria from various ivermectin-treated levels of patients in Achi, South –Eastern Nigeria. Using the collagenase technique, isolate the adult worms. The female worms will be prepared as protein samples and used for SDS-PAGE (Sodium Dodecyle Sulphate Gel Electrophoresis) and DNA analysis. Furthermore, using immune- blotting and PCR (Polymerase Chain Reaction) techniques, the P-glycoprotein related proteins and mdr genes will be sought for and investigations into the possibility of resistance determined using relevant statistical packages.
1.3 Objectives of the Study
The objectives of the investigations described in this research work are threefold. Due to the wide margin of the disease manifestations observed with the patients and the few cases of the persistent microfilaridermiae, we tried to obtain a better understanding of what role epidemiological circumstances (occupational roles, age, sex and proximity to river) plays on the parasitic load of some onchocerciasis patients in Achi and Amansea, South Eastern Nigeria.
Secondly, an investigation was made to evaluate the effect of ivermectin on the uteri status of O. volvulus worms collected from treated and untreated onchocerciasis patients in Achi and Amansea.
Thirdly and chiefly, we searched for the presence of the P-glycoprotein (resistant factor) in the O. volvulus worms (adults and microfilariae) from these patients. If the P-glycoprotein is present, to determine its level or expression in O. volvulus worms collected from responsive and non-responsive onchocerciasis patients treated with ivermectin and investigate if there is any relationship between P-glycoprotein levels in worms and epidemiological circumstances and finally to check if there are already resistance signals. The mdr genes which code for the P-glycoprotein will also be investigated into.
In this write up, the field work was always presented before the laboratory work but a general conclusion which summarized all the discussions was made. The need for a follow up study based on the several questions arising from this research work is also discussed.
Thus, the objectives of this research work include to:
1) Identify the onchocerciasis- affected communities of Achi and Amansea towns and determine their endemicity levels.
2) Investigate the roles of epidemiological circumstances (occupational roles, age, sex, and proximity to river) on the parasitic load of some patients in Achi and Amansea.
3) Evaluate the effect of ivermectin treatment on the uteri status of O.volvulus.
4) Evaluate the effect of ivermectin treatment on the uteri status of O. volvulus worms collected from treated and untreated onchocerciasis patients in Achi.
5) Search for the presence of the P-glycoprotein (resistant factor) in the O. volvulus worms from these patients. If the P-glycoprotein is present, to determine its level or expression in O. volvulus worms collected from responsive and non-responsive onchocercerciasis patients treated with ivermectin.
6) Determine the level of homology between the mdr genes found in O. volvulus with other mdr genes.
1.4 Literature Review
1.4.1 THE BIOLOGY OF THE PARASITE ONCHOCERCA VOLVULUS
Onchocerca volvulus is a thin nematode worm found as a parasite in human beings. It is transmitted by black flies of the genus Simulium. The adult worms (females 30-80 cm, males 3-5 cm) live in fibrous nodules, some of which are subcutaneous and palpable while others lie deep in the connective and muscular tissues. They have a life span of some 9-14 years. The females produce abundant microfilariae (250-300 µm in length), which migrate from the nodules to invade the skin, eyes and some other organs. They cause most of the disease manifestations of onchocerciasis and have a life span of about 6-24 months. The microfilariae ingested from the skin by blood-feeding Simulium vectors develop over 6-12 days, without multiplication, to form infective larvae (L3) which can be inoculated into a new host when the fly feeds subsequently. In the human host they moult twice, again without multiplying, to reach the adult stage; the first microfilariae produced by adult females may appear in the skin some 10-15 months after infection (Manson Bahr and Bell, 1991). Microfilaria can survive for 30 months in the skin. There is evidence of transplacental transmission of microfilariae so that the foetus is infected in utero (Brinkman et al, 1976).
The infective larvae of O. volvulus moult to the L4 stage within 3-7 days of arriving in the human host and the moult from L4 to the juvenile adult stage probably occurs 4-6 weeks later. The route followed by immature worms is unknown, they appear to be attracted to existing nodules and may settle on their surface to form satelite or composite nodules. The proportion of infective larvae inoculated that develop into adult worms is unknown. Young, old and calcified dead worms are often associated in the same nodule. Onchocercomata or nodules are found in distinct sites of predilection in the body. On average, 80% of the nodules contain one or two male and two or three female worms. Accumulation of more than 50 worms can occur, but this is the exception. In contrast to the sessile female worms, male O. volvulus regularly leave the nodules. In excised onchocercomata, a striking predominance of female worms is often observed as a result of this migration in the host of a proportion of the male worms. It is assumed that the migratory instincts and possibly the reproductive activity of male worms decrease with age in areas where transmission has been interrupted over a long period since a sex ratio of about 1:1 is found in the nodules of persons harbouring an aging worm population. Inactive old male worms are often clearly separated from non-gravid female worms, although they remain in the same nodule (Schulz – Key et al., 1987; Nelson, 1991; Duke, 1990).
With regards to mating, shedding of oocytes into the uteri may be a prerequisite for the stimulation of males to mate since sperm is scanty in female worms with empty uteri. So far, nothing is known about the pheromones or other stimuli that attract males to the females in the nodules. On the other hand, in many females, oocytes are released from the narrow ovaries into the wide lumen of the uteri independently of the presence of male worms. Large numbers of degenerating and shrinking oocytes may accumulate and subsequently be reabsorbed when the gravid females do not mate. Nematode sperm are short-lived, insemination normally continues during the early phase of embryogenesis. Only 10-15% of worms are found typically entangled in the mating position and, gravid females are regularly found deserted after insemination (Schulz-Key, 1988). Spermatozoa transferred to the female worms show amoeboid movements which enable them to force their way through a stream of embryos or oocytes moving in the opposite direction until they reach the posterior parts of the uteri. Schulz-Key (1988) and Duke et al. (1991) variously estimated the development of oocyte into a mature microfilaria within the female to take 3-12 weeks.
The reproduction of O. volvulus occurs in asynchronous cycles lasting 2-4 months each (Schulz-Key, 1988). Such cyclic reproduction has been observed for Onchocerca.ochengi, Onchocerca.gibsoni, and Onchocerca species in red deer and roan antelope, and may be typical for species with skin-dwelling microfilariae. However, Duke, (1990) observed that female worms shed oocytes continually while awaiting insemination.
Female O. volvulus shows a heterogeneous distribution of uterine/developmental stages. These stages are classified into six groups: Oocytes and five developmental stages of the embryos. Primary oocytes are elongate cells, clusters of which are attached in situ to the rachis of the posterior parts of the ovaries, in all mature female worms but, on average, fewer than two-thirds of the females actually contain embryonic stages and microfilariae. Secondary oocytes are more rounded cells lying separately in the oviduct. Embryonic stages: Two-cell and four-cell stages are co-ordinated to the group of small morulae. Small morulae has an average size of 15:8 µm. Big morulae in the subsequent group are more rounded and measure about 20:13 µm. Normally developed morulae consist of coherent cells filling out the whole space within the egg-shell. Then, is the stage of the advanced embryo which shows a lateral incubation indicating the beginning organization of the embryo. The larvae gradually become longer and slimmer. Embryos exceeding a circle in the egg-shell are co-ordinated to the subsequent stage of the coiled microfilaria, also called “brezel-stage’’. The final stage was represented by the stretched microfilaria which had cast off the egg-shell. However, in all groups, pathological alterations and deformities could be observed occurring naturally or due to drug effects. In the oocytes for example, while normal oocytes showed well defined nuclear membranes, in the deformed oocytes due to shrinkage of the cytoplasm, the shape of the nucleus becomes indistinct and the space within the egg shell will only be partially filled out and sometimes the egg-shell itself will be deformed. The size of the cells in abnormal two- and four-cell stages are often in-equal, the cell membranes are partially dissolved and the cells are sometimes separated from each other, the cytoplasm granular, turbid or caseous. Pathologically altered embryos of the further developed morulae often consist of cells of inequal size some of which do have stunted growth and others might show abnormal increase. Also, single cells are often separated from the embryos which become irregular in outline and disorganised with necrotic areas. The space within the egg shell are not completely filled out, occasionally egg-shells of normal size could be found containing only single cells or remnants of them. In the deformed advanced embryos, a similar segregation of cells can be observed and sometimes longitudinal clefts of the embryos indicates abnormal development and gradual dissolution of cell membranes. Abnormal coiled microfilariae are deformed when they show excrescences, are underdeveloped or have enlarged nuclei. In the pathologically altered microfilariae, the nuclear column seems to be interrupted at several sections by the dissolution of the cytoplasma and sometimes motile microfilariae show abnormal big nuclei and vacuoles (Schulz-Key et al., 1990).
1.4.2 Reproductive potential of O.volvulus
Embryogram techniques can be used to assess the reproductive capacity of a female worm because it quantifies the number of intrauterine stages actually present in a female worm, but it cannot indicate how many microfilariae are actually produced or released per day. Schulz-Key (1990) observed worms maintained in-vitro and suggests that 700-1500 microfilariae per female are released into the host on average per day, i.e. only a small proportion of the microfilariae developed in utero actually leave the female worms. In contrast to other filarial species, microfilariae of O. volvulus are not expelled by the female worm but leave it actively one by one. It takes at least 5-10 seconds for a microfilaria to leave the female worm when it has arrived at the vulva. Microfilaria that stay in the uteri gradually degenerate and are then reabsorbed. Thus an embryogram can provide precise information on the dynamics of reproduction by assessing the number of intrauterine stages present, the prevalence of abnormal forms, whether a cycle has just started or whether it is expiring, Whether a female worm has recently been inseminated and how many sperms are actually present the effects of drugs.
The reproductive life span of O. volvulus has been estimated by longitudinal skin -snip surveys undertaken in villages under vector control and by analysis of trends in community microfilarial load (the geometric mean number of microfilariae per skin snip among persons aged 20 years and over; including those with zero count) using the mathematical model ONCHOSIM. ONCHOSIM uses the technique of “stochastic microsimulation’’, which involves the explicit simulation of the individual life histories of both human hosts and adult parasites. Models based on microsimulation are flexible in design, which makes it easy to specify and simulate alternative assumptions. Furthermore, they can provide detailed output in the same format as field observations; this is useful in the validation of the model, while it makes the model output more understandable to decision-makers.
In ONCHOSIM, the most important variables are (i) human factors, namely population dynamics (birth, death, immigration), and heterogeneity in exposure to the vector; (ii) vector factors; such as vector density; biting rates and seasonal variation; (iii) the life history of the parasite in the host (life span, pre-patent period, age-specific microfilarial output, mating) ; (iv) larval uptake by flies as a function of human microfilarial load; (v) the development of blindness and associated excess mortality; (vi) the timing and coverage of epidemiological surveys and ivermectin treatment ;(vii) the timing and effectiveness of larviciding and (viii) the microfilaricidal and possible macrofilaricidal effect of ivermectin (Habbema et.al., 1992).
The mean duration of reproductive life has been estimated at 9-11 years, and 95% of adults do not reproduce for longer than 13-14 years (Plaisier et.al. 1991). Furthermore, Schulz – Key (1990) and Duke (1993) from their various observations showed that considerable numbers of female worms are hidden in deep-lying, impalpable nodules, and imply that a high proportion of infective larvae fail to develop into adult worms, and that in many infected persons, tens or hundreds of thousands of microfilariae must die and be disposed in the body each day.
1.4.3 Vector biology
Onchocerca volvulus is transmitted to man through the bite of infected black-flies belonging to the genus Simulium (WHO, 1987; Nelson, 1991) which breed along fast flowing water bodies. The female blackfly ingest O. volvulus microfilariae (embryonic form) from humans and acts as an intermediary for the development of infective larvae. Transmission occurs when infected flies take a blood meal. Reactions to the bites of Simulium sp themselves can be severe and allergic reactions occur.Very little is known about the development of O. volvulus in man. This is in part due to onchocerciasis being an end of the road disease, with the most infected communities living in remote under doctored areas where there are no facilities for detailed autopsy studies. But of most importance is the lack of a small animal model. Much more information on the development of Onchocerca has been obtained from studies of related species in Simulium and cattle, especially by Bianco and his colleagues (Bianco et al., 1980; Bianco et al., 1990). However, Following an approximately 2-week period these larvae can be transmitted back to the host during subsequent bites. The main vectors, Simulium damnosum, S.neavei, S.ochraceum, S.metallicum, and S.exiguum, are complexes of sibling species which do not otherwise form a taxonomically close group of species.
In Africa and the Southern Arabian peninsula, onchocerciasis is associated mainly with members of the S.damnosum complex, and to a lesser extent with the S.neavei group. These two groups have been known to be broadly distributed (WHO, 1987; Crosskey, 1987 and 1990), but S.albivirgulatum, the vector in the “Cuvette centrale’’ focus of Zaire, is the only vector species outside these two taxonomic groups. In the Americas, Simulium ochraceum, a species complex of at least three cytospecies, is considered to be the primary vector in all five foci in Guatemala and Mexico, while S.metallicum s.I. and S.callidum play secondary roles. In South America, the known vectors are S.exiguum s.I., S.guianense, S. incrustatum, S.metallicum s.I., S.oyapockense s.I., and S.quadrivittatum, S.limbatum is strongly suspected of acting as a vector. The vectors breed in fast flowing and well oxygenated rivers and streams with nutrients in the savannah and rain forest area. They have the ability to travel hundreds of kilometres in flight and wind currents. Their life span is about 4 weeks (Eezzuduemhoi and Wilson, 2008).
The aquatic stages of black flies inhabit a wide variety of streams in different bio-climatic zones that reflect the ecological requirements of each species. These range from tiny rivulets no more than a few inches wide for S. ochraceum to very large rivers for some members of the S. damnosum complex. In general, breeding occurs in swift running, well oxygenated, unpolluted water. The larvae attach to various submerged supports (vegetation, rocks, debris, etc.) by means of a posterior sucker armed with hooks. The duration of the aquatic stages depends on water temperature and requires 10-12 days for the West African species and up to 30 days for S. ochraceum at the higher elevation in Guatemala. Larval simulidae have plumose fans on their mouthparts that filter out particulate food from the flowing water. Ingestion is indiscriminate and larvae will take in insecticide particles as well as other matter. Female black flies bite from dawn to dusk usually outside of houses. Except for S. ochraceum, which feeds mostly above the waist, vector black flies prefer to feed on the lower parts of the body. Biting is not constant throughout the day and unimodal or bimodal biting curves have been described for several species.
The abundance of flies may show marked seasonal variations related to the productivity of the breeding sites. Fly production usually fluctuates according to the water level and flow rate. For example, the high water levels during wet season in Guatemala make the streams unsuitable for attachment of larvae and peak populations occur at the end of the rainy season when streams subside. In Africa, adult fly populations may vary inversely or directly with water level and flow rate depending on the species and the stream type.
Adult females can disperse considerable distances from the breeding sites by flight on a prevailing wind, or by both methods. In Onchocerciasis Control Programme of the Upper Volta river basin, West Africa, flies have travelled distances of 150 km or more to re-invade areas where breeding was apparently controlled. The Central American vectors have maximum flight ranges of 10-15 km. The longevity of adult females is not known precisely but probably does not exceed a month for both S. damnosum and S. ochraceum. A higher proportion of older females are found within 1-10 km of breeding sites: this makes transmission more intense at the stream banks where people congregate to fish, bathe, wash cloths, and obtain drinking water.
There is a marked difference between the situations in Africa and the Americas with regards to the distribution of Simulium spp. Vectors and potential vectors, and that of the disease. In Africa, wherever anthropophilic members of the two vector complexes occur, the human population suffers from some degree of onchocerciasis. In contrast, in the Americas, potential vectors occur widely outside the areas in which onchocerciasis is endemic. In Central America, the primary and secondary vectors are much more widespread than endemic onchocerciasis. In South America, human-biting blackflies occur over vast areas in the absence of the disease; however, the missing element here is not only the parasite but also a significant human population. It is probable that current changes in settlement pattern together with long-distance migrations of large numbers of people, such as gold-miners, will eventually lead to the establishment of new foci.
1.4.4 Vector ecology
In Africa, the spread of onchocerciasis has resulted from human activity and consequent environmental changes such as deforestation, resulting in the conversion of forest habitats into savanah and the creation of artificial breeding sites. In West Africa, such changes have resulted in a shift of the distribution area of the savannah species into forest zones.
Simulium damnosum was originally considered to be a fairly uniform species, differing biologically in different bioclimatic zones. However, since the mid-1960s it has become apparent that it is a complex of morphologically similar (sibling) species which can be distinguished by the banding patterns of the larval chromosomes. At present, over 40 different cytological forms have been described, half of which have been named without adequate morphological and cytological study.
In West Africa, west of Nigeria, the S. damnosum complex has received detailed study, all of these species are either known or suspected (S. dieguernse) vectors. In addition to the formally named species, numerous additional cytospecies have been described in West Africa and given vernacular names (Vajime and Gregory, 1990). The situation in Central and East Africa is much more complex. In contrast to West Africa, the cytospecies of the S. damnosum complex have not been fully studied and described. Many have very restricted distributions, they are often zoophilic, and many are not known to be vectors of O. volvulus. However, it is clear that species of the S. damnosum complex are responsible for most of the transmission of onchocerciasis that occurs in Ethiopia, Malawi and the United Republic of Tanzania.
With regards to vector capacity and transmission in West Africa, under natural conditions, there are significant variations in transmission both between and within species, the differences observed being the result mainly of factors such as longevity, trophic preferences and the relative abundance of the various hosts. In the forest zone of southern Guinea and Sierra Leone, the mean number of infective larvae morphologically indistinguishable from O. volvulus and associated with species of the S. sanctipauli subcomplex is six. Cytological identification confirms that these areas are populated mainly by S. leonese. In most of the basins of the western zone of the OCP, the equivalent values are four L3 larva for S. yahense and five for the other forest species. In the Savannah region, O. volvulus is associated with S. sirbanum and S. damnosum s.s., and the average number of L3 larvae per infective fly is just over two.
Extensive experimental studies of vector competence show that all species of the S.damnosum complex in West Africa (with the exception of S.dierguerense,which has not been studied) are capable of transmitting O.volvulus. However, the compatibility of vectors and parasites may depend on their respective origins. Thus the vector competence of the main species of S.damnosum s.I. may differ for the main strains of O. volvulus (Savannah and forest). The highest parasite yields under normal conditions of transmission occur with species of the S.sanctipauli subcomplex and with S.yahense, which is consistently a more efficient vector than S.squamosum. The lowest parasite yields are found among Savannah vectors (S.sirbanum and S.damnosum s.s.). Considerable differences exist when vectors and parasites strains are of different geographical origins (cross-transmission). Under experimental conditions, forest parasite strains develop poorly or not at all in Savannah vectors (S.sirbanum and S.damnosum s.s.).The parasite yields obtained from different species of the S.sanctipauli subcomplex are high, in general, irrespective of the geographical origin of the parasite strains concerned. In order to quantify the relationship between the potential infectivity of S.damnosum s.s. and the intensity of O. volvulus infection of the human host, the OCP conducted an experiment in the Savannah focus of Asubende, Ghana, in which blackflies were engorged on 40 volunteers with a wide range of microfilarial loads. A clear relationship was found between vector infectivity and skin microfilarial load: persons with a low intensity of infection contributed only little to transmission but the potential infectivity of the vectors increased rapidly with the skin microfilarial load of the host. However, even with the most heavily infected patients, fewer than 50% of the flies became infected. These results, while valid for S.damnosum s.s., cannot be extrapolated to other vector species because of differences in the relationship between vector infectivity and skin microfilarial loads. Such differences are of great importance for onchocerciasis control since they should determine the relative effectiveness of large-scale ivermectin treatment programmes in preventing O. volvulus transmission (De Leon and Duke, 1966; Dalmat, 1955; Dadzie et al., 1989, 1990; Davies and Crosskey, 1991; Dozie and Nwoke, 2002).
Figure 1: The life cycle of Onchocerca volvulus.
1.4.5 ONCHOCERCIASIS: DISEASE MANIFESTATIONS
The itchy disease, known as craw-craw in the then Gold Coast, was in 1875 associated with onchocercal microfilariae found in the skin of patients by O’Neil, and in 1893, skin nodules from patients were found by Leuckart to contain adult worms. Sir Albert Cook reported the occurrence of onchocerciasis in Uganda in 1899. Robbles became interested in its manifestation as a serious eye disease in Guatemala in 1915 while Pacheco Luna established its association with blindness in 1918.
188.8.131.52 Basic symptoms of onchocerciasis
The major presenting symptoms of onchocerciasis are dermal, ocular, lymphatic and systemic in nature, arising from the infiltration of microfilariae throughout body tissues. Several other features of uncertain association, etiology or pathogenesis have also been described, including low bodyweight, general debility, diffuse musculoskeletal pain and in Africa, epilepsy and hyposexual dwarfism. A number of individuals, the so-called ‘endemic normals’, do not show clinical symptoms nor is there any parasitological evidence of onchocerciasis despite ongoing exposure to the parasite (Ward et al., 1988) and (Nutman et al.,1991). Host-parasite interaction at an immunological level is responsible for the clinical outcome of the infection (Williams et al., 1986 and Mackenzie et al., 1987). Marked geographical variations in the clinical picture exist, which may be related to the different pathogenicity of O. volvulus parasite strains (WHO, 1987; Zimmerman et al., 1992), vector biting habits (WHO, 1987) and host ethnic (Molea et al.,1984), genetic (Brattig et al.,1986) and immunological factors. Human onchocerciasis differs as regards clinical manifestations in the savannah and the rain forest of West Africa (Anderson et al., 1974). The skin lesions are more pronounced in the forest zones, whereas the blindness rate and the ocular manifestations are severe in the savannah. Microfilariae of the putative savannah strain are more pathogenic than those from the forest strain, if injected into the eyes of rabbits (Duke and Anderson, 1972). The pathogenesis of onchocerciasis arises from two factors namely the effect due to adult worms and those due to the microfilariae. The adult effects are minimal in early stages of light infection as the adult worms lie freely in the subcutaneous tissues (Duke, 1990). The main significant pathology arises from the reaction to dead microfilariae in the skin and the eyes. In the skin the condition is called onchodermatitis, the basic lesion is the cellular reaction to the dead microfilariae (Manson-Bahr and Bell, 1991).
The extent and distribution of skin and lymphatic lesions permit classification of the disease into generalized and local forms. Generalized onchocerciasis is the usual presentation, characterized by fairly symmetrical lesions which may be more marked in the lower, or less commonly, the upper part of the body. The local form is asymmetric and may be confined to one limb and the adjacent area or to a circumscribed part of the body. Acute manifestations of localised onchodermatitis occur in new residents and in people from outside the endemic areas; the chronic form of localized onchodermatitis is synonymous with hyperactive onchodermatitis or sowda and is characterised by frequent acute exacerbation. The traditional teaching is that microfilariae can be found at all levels in the dermis but tend to be most numerous at the dermal-epidermal junction. Intact microfilariae excite little inflammatory reaction. Vuong et al., (1988) recently studied the skin lesions in people living in the West African forest and savannah and found that the microfilariae of O. volvulus were found predominantly in the lymphatic channels of the dermis surrounded by minor areas of inflammatory reaction. Reactions thought to represent successive stages of an inflammatory process were observed around extralymphatic microfilariae ( WHO, 1987, 1995b; Chijioke, 2009). The pathogenesis of onchocerciasis arises from two factors namely the effect due to adult worms and those due to the microfilariae. The adult effects are minimal in early stages of light infection as the adult worms lie freely in the subcutaneous tissues (Duke, 1990). The main significant pathology arises from the reaction to dead microfilariae in the skin and the eyes.
184.108.40.206 Skin manifestations
Although some individuals with onchocerciasis may have clinically normal skin, others have intense pruritus which is the most common early manifestation of onchocerciasis, developing after the prepatent period. The itching may lead to excoriation and secondary infection and may involve any part of the body. The first visible changes in the skin other than evidence of scratching, is an alteration in the pigmentation with areas of hyper- and hypo-pigmentation (WHO, 1987, and Duke, 1990). After years of chronic infection, atrophy of the skin develops. In severe cases a condition called lizard skin, characterized by a thin epidermis with a shiny fragile appearance, may develop. The normal dermal structure is replaced by a scar tissue and elasticity is lost. Another characteristic aspect of onchocercal skin disease is leopard skin, a spotty depigmentation occurring on the anterior part of the lower extremities.(Manson-Bahr and Bell, 1991; WHO, 1995)
In Africa, onchodermatitis is typically ‘generalized’- diffuse, and characteristically maximal on the lower trunk, pelvic girdle and thighs. Skin lesions in Guatemala and Mexico are in general milder than in Africa. An unusual feature of chronic hyperreactive localized onchodermatitis, often called sowda, is found in low prevalence also in Africa and America. Sowda in patients is characterized by an intense itching, usually asymetrical, well-circumscribed onchodermatitis with pustules and crust, oedema, pachydermia, darkening of the skin and considerable enlargement of the local lymph nodes, and no or few microfilariae in the skin (Gasparini 1962).
In most endemic areas of Africa and in Ecuador (Guderian et al., 1984), the majority of nodules are found on the pelvic girdle. In Central America, nodules are more common on the head, which may be due to the vectors biting more frequently on the upper parts of the body (WHO, 1987). Nodules measure 0.5 – 2 cm in diameter, but some may be larger with diameters exceeding 6cm. New nodules tend to develop around older nodules. Usually nodules are painless and cause little trouble. Onchocerca nodules may be confused with lymph nodes, lipomas, foreign body granulomas, sebaceous and dermoid cysts, ganglia and histoplasmosis or cysticercosis nodules. Recently a managable clinical classification and grading system of the cutaneous changes has been developed, enabling objective comparism between different geographical areas (Murdoch et al., 1993). Under this classification, the main categories of onchocerca skin disease are defined as follows; acute papular onchodermatitis, chronic papular onchodermatitis, lichenified onchodermatitis, atrophy and depigmentation. But two important points regarding this classification are that, first, it is based on clinical findings consistent with cutaneous onchocerciasis but not necessarily specific or diagnostic of the disease. Secondly, the categories are not mutually exclusive and one pattern may coexist with, or evolve into another. This scheme also includes a method of grading for use in recording the clinical severity of lesions, the clinical activity in terms of pruritus and excoriation, and the extent of distribution over the body. Its practical usefulness is currently being evaluated.
Subdermal nodules called “onchocercomata”, which are most easily seen over bony prominences are another commonly reported manifestation of onchocerciasis (Okuliez et al., 2004). In Africa, onchocercomata are often found over the bony prominences of the torso, and hips, where as in South America, where it is sometimes called “Robles disease”, (WHO, 2001), the predominant strain typically produce nodules in the head and shoulders (Wolf et al., 2003). Cases of onchocercomata presenting as a breast mass or as deep nodules in the pelvis have been described (Okuliez et al., 2004; Zarich et al., 2004). An angiogenic protein produced by the adult female is thought to contribute to the formation of the nodules. The presence of onchocercomata does not correlate with microfilariae load (Okuliez et al., 2004).
220.127.116.11 Ocular manifestations
Ocular involvement is common and blindness (resulting from complications such as sclerosing keratitis, iridocyclitis, chorioretinitis and optic atrophy) is the most severe sequela, occurring particularly in patients carrying a high microfilarial load (living or dead) for prolonged periods. These can be seen with a slit lamp and can involve any part of the eye from the conjunctiva and cornea to the uvea and the posterior segment, including the optic nerve and retina. The earliest sign of occular involvement in onchocerciasis is the invasion of the eye by microfilariae. Conjuctival reactions with hyperaemia, chemosis, and epiphora are often present in severely infected patients. A typical feature of punctate keratitis are snowflake opacities of about 0.5 mm in diameter representing dead microfilariae surrounded by an inflammatory infiltrate. These infiltrates usually resolve in a few weeks without sequelae (O’Day and Mackenzie, 1985). In later stages, sclerosing keratitis may develop. A fibrovascular pannus and an inflamatory infiltrate develop initially in the interpalpebral fissure and inferiorly. After several years, the entire cornea may become opaque, vascularized and pigmented leading to blindness. Anterior uveitis can also be found, which in severe cases often leads to inferior, anterior and posterior synchiae. A characteristic pear-shaped deformity of the pupil, caused by inferior-posterior synechiae, is also called oncho-pupil (Anderson et al., 1974b; Dadzie et al., 1990; Murdoch et al., 2002). Other complications of severe uveitis include secondary cataract and secondary glaucoma. A higher prevalence of skin snip positive individuals among younger glaucoma patients (10-39 years) compared with older glaucoma patients has been reported (Berghout, 1973). Since these patients lack signs of active uveitis or synechia, a primary role for onchocerciasis in glaucoma has been suggested. However, the relevance of onchocerciasis as a primary cause of glaucoma remains to be established. Onchocercal ocular pathology is usually bilateral but not necessaryl symmetrical and results mainly from local death of microfilariae and the host inflammatory response to Wolbachia antigens (Higazi et al., 2005)
It was commonly believed at one time that active optic neuritis was rare in patients suffering from onchocerciasis, but this lesion has been observed in a number of subjects in a community-based ivermectin study carried out in Nigeria (Abiose et al., 1993). Clinical experience has shown that the active optic neuritis associated with onchocerciasis lasts from several weeks to one year or more. It appears as a congested suffused disc with or without swelling, while postneuritic optic atrophy is often associated with scarring and pigment disturbance at the disc margin. Associated dense vascular sheathing may extend along retinal vessels for a considerable distance beyond the optic nerve head. Primary optic atrophy may also occur, and may be partial or complete. The reported prevalence of optic atrophy has varied between 1% and 4% in the hyperendemic rain forest and savannah communities of Cameroon to between 6% and 9% in the Guinea Savannah of Northern Nigeria.
Blindness is the most serious consequence of onchocerciasis, and can result from lesions that affect different parts of the eye. In the savannah areas of West-Africa, blindness is prevalent in 2-15%, and was previously attributed mainly to sclerosing keratitis (WHO, 1987), but a recent study has shown that optic nerve disease plays a significant role in some localities (Abiose, 1993). In the rain forest, blindness is less prevalent (up to 2.4%) and is said to be due mainly to posterior segment disease (WHO, 1987, Meyers et al., 1977). Recently, using DNA techniques, an association between ocular pathology and parasite strains has been observed (Zimmerman et al., 1992). Relatively few serious eye lesions occur in South-America (WHO, 1987; Molea et al., 1984). Blindness has an enormous social and economical impact. It strikes mainly at economically active adults in the prime of life, and eventually it may even result in villages and fertile lands being abandoned (WHO, 1987).
18.104.22.168 Other manifestations
In lymphatic onchocerciasis, with heavy microfilarial infection, some of the parasites pass to the lymph nodes draining the area, leading to lymphadenitis with accompanying fibrosis. Enlarged lymph nodes develop especially in the inguino-femoral region, which in combination with wrinkled skin give rise to ‘hanging groin’ and predispose to herniae. Sometimes elephantiasis of the scrotum may occur (WHO, 1987; Duke, 1990).
Onchocerciasis can be considered as a systemic disease. Small numbers of microfilariae can be found not only in skin, eyes and lymph nodes, but also in many deeper organs, including the liver, kidney, spleen, pancreas, lung, peripheral nerves and arteries. Microfilariae have also been found in tears, blood, urine, cerebrospinal fluid, sputum, vaginal secretions, and peritoneal fluid (Meyers et al., 1977). In hyperendemic areas, patients with severe onchocerciasis may lose weight (Burnham, 1991; Kirkwood et al., 1983). The significance of systemic manifestations should not be underestimated. Systemic effects of onchocerciasis may lessen productivity of an endemic region by a process distinct from effects of visual impairment (Burnham, 1991) and may even lead to excess mortality (Kirkwood et al., 1983).
1.4.6 Geographical distribution and epidemiology
22.214.171.124 GEOGRAPHICAL DISTRIBUTION
Onchocerciasis is still endemic in 34 countries, 26 endemic areas in tropical Africa, where 99% of the total infected persons are found, 6 in the Region of the Americas, and 2 in the Eastern Mediterranean Region. However, in Africa, in the original core area of the OCP (Cote d’Ivoire, Burkina Faso, Mali, Ghana, Togo, and Benin), the disease has declined dramatically in both prevalence and public health importance. The main public health problem remains in the countries of sub – Saharan Africa outside the OCP area, where the disease is both widely prevalent and severe in terms of blindness and skin lesions, and where there is additionally the risk that Savannah blackflies will become established in degraded forest habitats. New foci have been found in the Americas, and the disease may spread still further as infected workers continue to exploit areas of virgin forest.
Current estimates suggest that about 17.7 million are infected, out of which 270 000 are blind; in addition, a further 500 000 are severely visually disabled (WHO, 1995). Onchocerca volvulus adult worms can live for over a decade in skin nodules of affected humans, releasing millions of microfilariae that cause debilitating itching and blindness (Richards et al., 2001). An estimated 37 million people are infected and there are 46,000 new cases of blindness annually (http://www.apoc.bf/, 2005).
In Nigeria, currently it is estimated that a few more than three million people are infected of whom about 100 000 are blind as a result of onchocerciasis. Nigeria has the largest population of any sub-Saharan African country, also has the largest number of infected persons, but it is a measure of the lack of accuracy of country-wide endemicity figures that the estimate of nearly 7 million infected persons made by the WHO Expert Committee on Onchocerciasis in its third report (WHO, 1987) was reduced to about 3 million in the national sample survey conducted in Nigeria in 1988-1989. The disease varies in endemicity in Nigeria but it is present in all states and the Federal Capital Territory, with the exception of Lagos, Rivers and Akwa-Ibom states, where infections are sporadic. However, Abanobi and Anosike (2000) observed that there is lack of detailed and accurate statistics on the prevalence of onchocerciasis in Nigeria and can be mostly attributed to the problems encountered in the epidemiological surveys. Some of these problems include language barriers between the investigators and the population at risk, lack of
Figure 2. Geographical Distribution of Onchocerciasis
(Bjorn, Onchocerciasis; Elimination on the Horizon. MD, Mectizan Donation Programme, Georgia, USA. GLOBAL REVIEW JOURNAL: ONCHOCERCIASIS, LEPROSY and HIV/AIDS, pp 114 – 116).
trained man-power, and absence of more sensitive diagnostic techniques to detect sub-clinical infections (Enyenihi, 1982).
In central Africa, the serious blinding form of the disease extends from the eastern Nigerian states of Taraba, Adamawa and Borno across northern Cameroon into the six south-western prefectures of Chad, the three north-western prefectures of the Central African Republic, and south-eastern Sudan. In Uganda, deforestation following subsistence agricultural development and timber exploitation has reduced the cover available to the S.neavi vector, thereby reducing the transmission of O.volvulus, but if S.damnosum colonizes these foci, transmission will return. In Ethiopia , there is substantial disfiguring skin disease, especially in coffee and tea plantations but there appears to be no blindness due to onchocerciasis. In Malawi and Sudan, endemic zones have been identified while in Angola, Liberia, and Zaire, the current situation is uncertain (WHO, 1995).
With regards to the Americas, Studies have revealed the existence of Onchocerciasis in Yemen, and although there are reports of onchocerciasis in Saudi Arabia, transmission in that country has not been confirmed. It is probable that cases in Saudi Arabia have been imported. Furthermore, no vector of onchocerciasis have been identified hence it is unlikely that there is any significant focus of the disease in the country.
About 261 660 people are at risk in Mexico and over 25 000 cases have been reported. In Guatemala, active foci are concentrated on the western slopes of the volcanic range and in the regions of Chimaltenango, Solola and Suchitepequez, 30% of the communities are hyperendemic. In Venezuela, onchocerciasis was first recognised in 1948, In Columbia in 1965, in Brazil, 1967, and in Ecuador in 1982. Since 1985, there has been no convincing evidence of any expansion of the existing foci. In Amazonas state in Venezuela, studies indicate that the geographical distribution of competent vectors is considerably larger than that of the disease, so that, if infected individuals migrate, new foci could be created (WHO, 1995). In Brazil, onchocercal foci are located in the northern part of Amazonas state and in the western part of Roraima State, which borders Venezuela. In Colombia, the main known focus is the Lopez de Mucay area on the Pacific coast, where 16 300 individuals are at risk of infection in 155 communities. In Ecuador, the onchocerciasis focus is located in the north-western coastal province of Esmeraldas. The major focus involves blacks and Chachi Amerindians living in the Santiago River basin. There were 192 known infected communities in 1993, and 20 089 individuals at risk of infection (WHO, 1995).
126.96.36.199 Epidemiology of onchocerciasis
The epidemiology, of onchocerciasis is that of a vector-borne disease of which human beings are the only vertebrate host. Infection with O.volvulus, like other filarial infections, is also characterized by coincidence between the degree of human infection and the intensity of exposure to infected vectors.
However, the epidemiology of onchcocerciasis is not uniform throughout its distribution because different disease patterns are associated with different variants or strains of the parasite, with differences in the vector competence and feeding characteristics of local blackfly populations, with the abundance of the vector and with differences in the human host responses to the parasite. These factors, together with those related to environmental, geographical, social and demographic influences, increase the complexity of the epidemiology of the disease in the different areas of its distribution.
Factors influencing the epidemiology of onchocerciasis can be divided into those relating to the host, the parasite and the vector, but behavioural and community factors also need to be considered.
With respect to host factors, there are no known sex differences in acquisition of infection, and age merely determines cumulative exposure to infection. In individuals with sowda lesions, there are apparent variations in the immune response to infection. Parasite factors such as genotype may explain the pattern of disease in certain foci, for example, two different types of O. volvulus (forest and Savannah) exist in Africa; this is of importance in setting priorities for control measures.
Vector factors are important inasmuch as they affect the transmission of the parasite. Transmission rates may vary both seasonally and by geographical location. Vector abundance depends on hydrological conditions, which determine the number and productivity of blackfly larval habitats. Vector density, is also determined by dispersal habits. Ecological factors such as prevailing winds and humidity, also contribute to passive dispersal and migration, some species of African savannah flies travel up to 400km from their breeding sites. There are also major differences between vector species in their feeding habits, for example, in the degree of preference for human as opposed to animal hosts. Furthermore, the intensity of microfilarial infection in the skin may play a critical role in determining the infection of the vector, since each species has an infection threshold.
Individuals who frequently visit the breeding sites or whose work requires them to spend long periods on the river bank (e.g fishermen, farmers) tend to have very severe manifestations of onchocerciasis. Prevalence of onchocerciasis is lowest in the first decade of life, after which it rises steeply to reach a peak usually, in the third decade of life (WHO, 1976; Onwuliri et al., 1987). Male / female differences in prevalence, intensity of infection and clinical manifestation of disease have all been observed (Brabin, 1990). Behavioural and community factors are most important in the planning, implementation, and evaluation of control measures. In the savannah areas, the intensity of exposure to transmission is determined by the distance between a community and a fly breeding site and by the presence or absence of other human settlements in the intervening area; these considerations have led to the characterization of villages as first-, second-, and third line. Furthermore. Males are generally more affected than females, though gender-related differences may not appear till a certain age.
Another important determinant of intensity of infection is the density of the human population in relation to the vector population emerging from local breeding sites, as is the presence of cattle near rivers, since it reduces the contact of the human population with zoophilic vectors of O.volvulus. Additionally, the regular inoculation of the human population with bovine /animal Onchocerca L3 larvae may provide an immunological stimulus to the host and thus help prevent infection with O.volvulus.
Wherever onchocerciasis exists at a high intensity and endemicity, it is a serious threat to the health of the populations concerned and an impediment to socio-economic development. The two factors responsible for the major public health impact of the disease are the serious eye lesions, which occur when the intensity of infection is high and where the strain of the parasite is pathogenic for the eye, and the prominent skin lesions. The socio-economic consequences of onchocerciasis are most marked in the hyperendemic belt that extends across sub-Saharan Africa, excluding the West African countries in the original OCP area, where the burden of onchocercal blindness has been greatly reduced as a result of control.
Within Africa, blindness rates in hyperendemic communities not under control may rise to 15%, and up to 40% of adults may show severe ocular impairment. When there are high rates of visual impairment, communities become unstable, their agricultural capacity declines, and eventually the villages are abandoned. Populations move to healthier local environments, away from the rivers where the vectors breed but where the soil is usually not so fertile. Furthermore, since blindness leads to a reduction of some 10 years in life expectancy, onchocerciasis is a disease that not only disables but is indirectly responsible for considerable premature mortality. In Guinea for example, the impact of onchocerciasis-related blindness on the household is severe, most often resulting in the family’s inability to support itself. The disruption of family life is directly related to stress within the household, contributing to its destitution. The extra burdens placed on other members of the family, once the main breadwinner is blind and can no longer continue with normal activities, have adverse effects on their physical, psychological and emotional health. The older children often choose to migrate, fearful of becoming blind themselves.
In Nigeria, Nwoke, (1990) reported that in persons with prolonged intense infections, the skin lesions and itching are responsible for much chronic misery and disfigurement, and can lead to a degree of social isolation, with detrimental psychological effects. Also reported in Nigeria is the social stigmatization and rejection of those with skin disease because of the belief that it is caused by dirtiness (Amazigo and Obikeze, 1991). As a result of onchocerciasis, quality of life is reduced and the problems could be compounded by conditions like multiple infections, malnutrition and starvation (Ukoli, 1984).
In the Americas, the socioeconomic consequences of onchocerciasis have not been fully studied and thus requires further study particularly with regard to the impact of blindness and skin disease on productivity.
In the southern Venezuelan foci, cultural interpretation of the origin of some of the disease signs and symptoms, as well as the mechanisms of onchocerciasis transmission, are sometimes a source of conflict between communities. These cultural factors also determine the social acceptance of the changes in physical appearance associated with the disease, although individuals with severe skin manifestations (e.g. hanging groin) are socially isolated (Petralanda et al.,, 1993). In the Amazon Basin, a major consequence of the exploitation of forested areas through mining activities will be infection of migrant workers with O.volvulus, that over the next decade, the movement of these workers into previously non-endemic areas of South America, where potential vectors exist may have severe long-term social and economic consequences for several countries in the region. The socio-economic impact of this disease could be summarized as loss of labour (Prost and Prescott, 1984; Prost, 1986) mortality and decreased life expectancy of infected persons (Prost and Vangelade, 1981; Duke, 1990) migration and depopulation of endemic areas which are usually fertile lands (WHO 1976), social stigmatization of persons affected with dermal lesion (Nwoke 1990, Amazigo and Obikeze 1991) as well as loss of productivity due to morbidity (WHO 1997; Benton 1998). Until recently, the blindness and skin pathology caused by heavy infections constituted a major public health problem in many parts of tropical Africa, Yemen and Latin America. This consideration led to the establishment of the Onchocerciasis Control Program (OCP) (Molyneux, 1995) in West Africa, the Onchocerciasis Elimination Program in the America (Blanks et al., 1998) and the African Program for Onchocerciasis Control (APOC) (Remme 1995 and WHO 1999). All the three programmes have to rely on the regular (OEPA semi-annually, OCP, both annually and semiannually, and APOC annually) distribution of ivermectin to lower the microfilarial load in affected communities and thereby reduce transmission and mitigate the clinical manifestations of the infections (Goa et al., 1991).
Figure 3: Onchocercal blindness: soon to be consigned to the history books?
(Adapted from TDR THIRTEENTH PROGRAMME REPORT, 2001)
Figure 4: Disfiguring skin lesion due to onchocerciasis.
1.4.8 CONTROL OF ONCHOCERCIASIS
Previous efforts at halting onchocerciasis include the aerial spraying of biodegradable larvicides in West Africa-( a co-operative effort of World Health Organization-related groups) and the use of drug treatments-(only two anti-onchocerciasis compounds, Diethylcarbamazine citrate (DEC) and Suramin).
Vector control is based on the insecticide treatment of rivers in which the larvae of vector species develop. It rarely takes more than 1-2 weeks for the pre-adult stages to develop from eggs to pupa, and this means that insecticide must usually be applied weekly. In Africa, because of the large areas and number of breeding sites to be treated, insecticides are sprayed mainly from the air. Ground treatment may, however, be undertaken in accessible and isolated areas. Following the eradication of S. neavei from the Kodera Valley in Kenya by the application of DDT, French entomologists undertook large-scale trials in West Africa on larviciding of S.damnosum. The conclusion of these field studies and pilot projects was that ground control was quite successful in reducing the biting blackfly population as long as control activities were maintained, but the impact on transmission was limited by the regular influx of flies from surrounding untreated areas. Much larger areas would need to be treated to obtain a lasting effect.
With this objective in mind, experiments were carried out by the ORSTOM (Institut Francais de Recherche Scientifique pour le Development en Cooperation) entomologists assigned to OCCGE (Organisation de Cordination et de Cooperation pour la Lutte contre les Grandes Endemies), and a control programme was started in 1962 in an area covering parts of Cote d’Ivore, Burkina Faso (then Upper Volta) and Mali, with financial support from the European Development Fund (EDF) and the French Aid and Co-operation Fund. With the emerging indications that control was possible, health authorities in the countries most affected invited the scientific community to study the operational aspects of a large-scale control programme. This led to a technical meeting in Tunis in July 1968, of experts in Public health, parasitology, epidemiology, entomology, ophthalmology, economics, sociology and medical geography under the auspices of a joint USAID/OCCGE/WHO.The meeting emphasised that a mass campaign against onchocerciasis should aim at the eradication of this endemic disease. After a series of other joint meetings involving Government of Ghana, OCCGE, EDF, USAID, FAO, the World Bank, UNDP, and WHO, an initial programme known as the PAG Mission (Preparatory Assistance Mission) was initiated with regards to the involved countries- Governments of Dahomey (now Benin), Ghana, Cote d’Ivoire, Mali, Niger, Togo and Upper Volta (now Burkina Faso).
The objectives of the PAG Mission centered on aerial larviciding over an area then believed to be sufficiently large to avoid re-invasion of infective blackflies. The larviciding was to be conducted continuously for 20 years, the period considered necessary for the adult worm reservoir in the human population to die out in the absence of renewed infection. The control area defined by the PAG Mission was confined to the Savannah zones where the blinding form of onchocerciasis was prevalent, and excluded the forest zones characterized by the milder form of the disease. This area delineated for control corresponded largely also to that identified at that 1968 Tunis meeting (WHO, 1995). Following a planning meeting in Accra in October 1972 which discussed the reports of the PAG mission, the representatives of the governments of the seven participating countries signed on November 1, 1973, ‘Agreement governing the operations of the Onchocerciasis Control Programme in the Volta River Basin area’ with the World Health Organisation as the executing agency of the programme. This thus led the existence of the OCP (Onchocerciasis Control Programme).
So far, the benefits of the OCP are enormous. Currently vector control (OCP operations) has achieved the virtual interruption of transmission in 90% of the original OCP area and the incidence of infection in children has been reduced by 99% . 30 million people have been protected from onchocercal disease – 10 million in the original Programme area where the threat has been virtually eliminated with the disappearance of the parasite reservoir in humans after 14 years of vector control, and 20 million in the extension areas where transmission has been halted by aerial larviciding and where the reservoir will disappear by the turn of the century. It is estimated that 9 million children have been born within the OCP area since operations began and none of them has ever run the risk of contracting onchocercal blindness, and will never do so within the OCP area. Thus, by the year 2000 or before, the numbers of children protected have grown to 15 million. When the programme started, there were those seriously infected with onchocerciasis and therefore in grave danger of developing ocular manifestations, but among them, 1.25 million are no longer infected; this figure is expected to increase to 2 million before or by the year 2000. With regards to blindness, more than 100 000 cases of blindness have so far been averted by OCP control operations, a figure that will rise to 150 000 before the end of the century. Therefore, application of larvicide to the breeding sites of the blackfly is currently the only means of interrupting transmission sufficiently to allow the human reservoir of O. volvulus eventually to die out, and will remain so until elimination of the adult worm can be achieved by community-wide application of drugs.
International programs supported by the World Health Organization and many other groups have worked to control the impact of onchocerciasis using vector control with insecticides beginning in 1974 and mass drug administration (MDA) with ivermectin (IVM, brand name Mectizan) beginning in 1987 (Peters et al., 2004). IVM is a highly effective microfilaricide and inhibits female worm microfilarial production for several months. Annual IVM MDA reduces morbidity (Ejere et al., 2001; Tielsch and Beeche, 2004) and lowers transmission (Boussinesq et al., 1997; Collins et al., 1992). From 1974 to 2002, the Onchocerciasis Control Programme (OCP) in West Africa greatly decreased O. volvulus transmission in the 11 OCP countries and prevented 600,000 cases of blindness (Molyneux et al., 1995, Boatin and Richard, 2006 and Basanez et al., 2006). IVM without vector control has been the principal tool for the Onchocerciasis Elimination Program of the Americas (1992–present) (Boatin and Richards, 2006) and the African Programme for Onchocerciasis Control (1995–present). In the Americas, where O. volvulus is less common, the Onchocerciasis Elimination Program has substantially reduced transmission to eliminate the disease.
Diethylcarbamazine developed in 1947, is a microfilaricide (it kills microfilariae) and must therefore be given repeatedly as long as the patient harbours fertile female worms. It must be given orally daily for seven to ten days. It provokes severe and sometimes dangerous systemic (Mazzoti) reactions and aggravate existing ocular lesions, or precipitate new ones, as a result of the sudden, massive death of the microfilariae. DEC may itself even cause blindness and death ( Boatin and Richard, 2006).
Suramin, available as from 1920 for the treatment of sleeping sickness, was shown in the late 1940s also to be effective as a macrofilaricide (it kills the adult onchocercal worm). It is the only macrofilaricidal drug and has to be given intravenously once a week for several weeks up to two months. Its administration may be accompanied by severe rash, anaphylactic shock, kidney, liver and gastrointestinal complications and sometimes death (Manson-Bahr and Bell, 1991).
These drugs were deemed unsuitable for mass use because they produce only short-term or unsatisfactory suppression of microfilariae and patients require close medical supervision during administration. It was therefore apparent that the development of a new drug that is safe and effective, preferably one that may be administered orally as a single dose for mass chemotherapy must continue to be a major research goal. This search for a new drug for onchocerciasis therapy has led to the development of ivermectin(IVM).
Towards the end of last decade, some other drugs for the treatment of onchocerciasis evolved. Amocarzine (CGP 6140) is an antifilarial anthelmintic isolated from amoscanate. It is active against adult worms of O.volvulus. Amorcazine is toxic to mitochondria and causes inhibition of respiration. The basis for its selective toxicity appears to be preferential drug uptake by the filarial worm (Kohler et al., 1992).
Moxidectin is a milbemycin drug used in veterinary medicine for the prevention of heartworms and intestinal worms in cats, horses, cattle and sheep. Moxidectin is a fermentation product from Streptomyces cyaneogriseus spp. Noncyanogenic. It is chemically related to avermectins. However, moxidectin has a longer lasting microfilaricidal effect than ivermectin by virtue of its much longer half-life, 20 days as compared to 2 days (Tagboto and Townsend, 1996; WHO, 2000). Moxidectin also shows promising macrofilaricidal activity in animal screens and has been shown to be safe in preliminary (Phase 1) human trials (WHO, 2000; Cotreau et al., 2003). Moxidectin is being evaluated in phase II clinical trial, in which 192 persons infected with onchocerciasis are enrolled. Following the period, moxidectin will hopefully be available to endemic countries by the end of this year 2012 (Eezzuduemhoi and Wilson, 2008).
188.8.131.52 Antibiotic therapy
The break-through of using antibiotic to target the bacterial symbiont of the parasite has identified a novel treatment and target that offers a superior therapeutic altermative to current anthelminthic drugs (Johnston and Taylpr, 2007; Hoerauf et al., 2009). The rationale for this novel treatment is the antibiotic targeting of Wolbachia a bacterial endosymbiont of filarial parasites. Treatment with doxycycline for six (6) weeks in a controlled trial eliminated Wolbachia from adult worms resulting in suppression of embryogenesis, and most importantly death of adult worms (Hoerauf et al., 2001). In contrast ivermectin only works against late- stage developing microfilariae still in the uterus, and it has little or no effect on early-stage embryos. These suggest that infected patients who permanently leave in areas of enedemicity should be offered, in addition to ivermectin, a 4 – 6 week course of doxycycline (100-200 mg per day) to achieve long term amicrofilaridermia (Udall, 2007). However, caution should accompany the concurrent use of ivermectin and doxycycline, because these agents have not been formally studied for interactions (Udall 2007). The outcome of doxycycline therapy has several advantages including the elimination of the inflammatory inducing bacteria (Leiser et al., 2002), and avoidance of potential adverse reactions to nematode products associated with a rapid- kill as observed in loasis co-infection (Hoerauf et al., 2009; Taylor and Hoerauf, 1999; Hoerauf et al., 2001, 2003). However, a major obstacle to the use of antibiotic therapy in control of onchocerciasis is the length of the treatment regimen, which is considered to be logistically imcompatible with the community directed treatment strategies used in filariasis control and contraindications for children > 9 yrs and pregnancy (Taylor et al., 2009). In consideration of all these, Ivermectin thus remains a more acceptable therapy for onchocerciasis.
Although five classes of chemotherapeutic agents are potentially available for the treatment of onchocerciasis, only one drug, ivermectin, is actually being used regularly for this purpose. Diethylcarbamazine (DEC) and suramin are considered unsuitable for large-scale therapy, while the two groups of potentially macrofilaricidal compounds, thioureas and benzimidazoles, are still at the stage of experimental drugs. Amocarzin (CGP 6140), the piperazonyl derivative of amoscanate, has been shown to have good macrofilaricidal actvity in animal infections, and has been developed to the stage of Phase II-III clinical trials (Poltera et al., 1991). Most of the trials have been carried out in Ecuador and Guatemala. Benzimidazoles have always shown good macrofilaricidal activity in screening models for filariasis. However, to date, this activity has not been observed in onchocerciasis patients. Benzimidazoles designed to act as anthelminthic agents against nematodes are poorly absorbed, they bind to the tubulin making up the microtubules of the mitotic spindles of the submembrane network, and so interfere with cell division and substrate transport. Multiple doses of mebendazole and albendazole given orally and flubendazole given by intramuscular injection show little or no macrofilaricidal activity in human onchocerciasis. Their primary action is a toxic effect on the embryonic stages of the parasite. Flubendazole when studied, induced severe inflammation and sterile abscesses at the injection site. A flubendazole prodrug (UMF, 078) has been developed that is macrofilaricidal by both oral and intramuscular routes in animals. When the prodrug is given intramuscularly as a suspension in oil, the inflammatory response seen with flubendazole itself is absent. Two or three doses of UMF 078 are required for full macrofilaricidal activity in animal models; this compound is currently entering the preclinical toxicology phase of development.
IVM is a chemical modification of one of a series of naturally occurring substances designated as avermectins. Avermectins are a family of macrocyclic lactones discovered and developed as anthelmintic agents at the Merck Research Laboratories in the middle to late 1970s (Burg et al., 1979; Egerton et al., 1979). They are naturally fermentation products synthesized by the soil bacterium, Streptomyces avermitilis. The avermectin-producing organism was initially isolated from a soil sample collected in Japan as part of a collaborative agreement between the Merck Research Laboratories and the Kitasato Institute in Tokyo (Burg et al., 1979). The anthelmintic activity of the test sample was detected by incorporating the fermentation broth into the diet of mice that had been experimentally infected with the intestinal nematode Heligmosomoides polygyrus (Nematospiroides dubius) and then monitoring for fecal egg output and presence of worms in the gut as an indication of efficacy. This screen, like many in vivo screens, allowed for simultaneous assessment of the compound’s efficacy, oral activity and bioavailability, and absence of toxic side effects. Subsequent investigations revealed that this class of compounds also possessed potent insecticidal activity (Ostlind et.al., 1979) but lacked antibacterial or antifungal properties.
Avermectin nomenclature is based on the biosynthetic variations that occur at the C-5 and the C-22,23 positions and the C-25 side chain. The C-5 hydroxy-substituted avermectin analogs (B series) are generally more potent anthelmintic agents than the C-methoxy derivatives (A series). Ivermectin (22,23-dihydroavermectin B1a)*, a semisynthetic avermectin analogs (Chabala et al., 1980) was introduced commercially in 1981 and rapidly became the drug of choice for treating a broad spectrum of conditions caused by nematode and arthropod parasites. Ivermectin is a highly lipophilic 16-membered macrocyclic lactone, selectively binds to postsynaptic glutamate-gated chloride ion channels in muscles and nerve cells of the invertebrate. Increased permeability of cell membrane to chloride ions leads to hyperpolarization of the nerve or muscle cell, paralysis, and death of the parasite (Eezzuduemhoi and Wilson, 2008).
Ivermectin (IVM) has activity against a wide range of nematode and arthropod ecto- and endo- parasites in domestic animals (e.g Poultry, dogs, goats, sheep, swine, cattle, horses): Example of these parasites include Strongylus Spp., Haemonchus Spp., Ancylostoma Spp. and Dirofilariae Spp. It has also been found to be active against human gastro-intestinal nematodes (Strongyloides Spp., Trichuris trichuria, Enterobius vermicularis) and filarial parasites especially O. volvulus. Ivermectin has also shown activity against various other nematodes including Wuchereria bancrofti (Diallo et al., 1987; Ottesen et al., 1990; Rout et al., 1989), Loa loa, Ascaris lumbricoides and Strongyloides stercoralis (Datry et al., 1991; Freedman et al., 1989; Naquira et al., 1989; Anosike, 2000)
Since its release in 1987, for the treatment of onchocerciasis, ivermectin has gained wide acceptance as a potent anthelmintic for the control of onchocerciasis. Ivermectin is safe, effective and relatively well tolerated drug for the treatment of onchocerciasis. Ivermectin is given as a single oral dose of 150 µg/kg of body weight once or twice a year. It should not be given to children under the age of 5 years or weighing less than 15 kg, during pregnancy, to mothers nursing infants during the first week of life, and in severe illness, as specified in the manufacturer’s exclusion criteria. Different regimens may be required to achieve different objectives, such as the improvement of specific severe ocular or skin manifestations, the control of transmission, and more pronounced effects (macrofilaricidal or sterilizing) on the adult worms. Ivermectin has no pharmacological activity in humans or intrinsic toxicity in single doses of up to 600 µg/kg of body weight or in multiple doses of 100-150 µg/kg of body weight given every 2 weeks, monthly or every 3 months up to a total dose of 1.8 mg/kg of body weight. The reactions seen from patients who took ivermectin were qualitatively similar to those observed following the use of diethylcarbamazine (i.e. the mazzotti reaction) but were much less frequent and less severe. The common side-effects includes itching and rash, musculoskeletal pain, relatively painless swelling (oedema) of the limbs and face, fever, and gland pain and swelling. There have been no life-threatening side effcets or mortality attributable to ivermectin. Severe reaactions are uncommon and those requiring the use of corticosteroids are rare. Most reactions can be managed with simple analgesics and antihistamines. If those receiving ivermectin are advised to rest in bed when feeling weak or dizzy, the incidence of severe symptomatic postural hypotension can be reduced from approximately 1 per 1000 to nearly zero. Reactions commence on the first day after treatment, most of the severe reactions occurring by the second day. There is a direct correlation between infection intensity and the severity of musculoskeletal pain, fever and lymphadenitis, but there is no such relationship for cutaneous reactions. Reactions are prominent in localised onchodermatitis and may be more frequent in non-resident visitors to endemic areas despite low skin microfilarial counts. Thus previous constraints on the use of diethylcarbamazine do not apply to ivermectin. Even on repeated dosings of individuals or populations , reactions are greatly diminished. However, reactions such as acute laryngeal oedema, attacks of asthma in known asthmatics, bullae, and the late development of abscesses have also been reported, but it might possibly represent pre-existing or coincidental illnesses that appeared at the same time as ivermectin administration. There have been no reports so far on the outcome of pregnancy or on the course of epilepsy.
Although the treatment of patients with concomitant nematode infections has not been fully studied, but investigations to date in onchocerciasis patients who also have loasis have not shown any increased morbidity or adverse experiences and there have been reports of Ascaris spp. and Taenia Spp. excretion from children with multiple worm infections (Okonkwo et al., 1991) field observation (Freedman et al., 1989; Anosike et al., 2007).
Ivermectin is microfilaricidal and thus has a potent, rapid action against skin microfilariae. Although it is still uncertain about the mechanism of action on O.volvulus, recent studies of the ivermectin receptor in the free-living nematode Caenorhabditis elegans show that in this nematode, ivermectin binds at extremely low concentrations to a membrane chloride channel normally controlled by glutamate.
Massive reductions in microfilarial counts occur during the first few days, and maximum reduction may however, not be achieved for 2 or more weeks. Skin microfilariae migrate into the dermis about 24 hours after ivermectin is given, and elicit little inflammatory reaction (Duke et al., 1991), unlike in the administration of diethylcarbamazine, when microfilariae migrate towards the epidermis as early as 1-6 hours after treatment and become the foci of microabscesses. These differences might account for the much less severe cutaneous manifestations seen after treatment with ivermectin as compared with diethylcarbamazine. Most microfilariae are probably killed in the lymph nodes after treatment with ivermectin, as their density there increases by a factor of about 1000 (Darge et al., 1991). Suggestively, ivermectin causes them to migrate from the subepidermal layer into the deeper layers of the dermis, then into fatty and connective tissue and finally into the regional lymph nodes. On the other hand, Vuong et al. (1988) found that if the microfilariae are predominantly resident in the lymphatics, they could either move actively into the lymph nodes when “mobilized’’ by ivermectin or be swept passively along the nodes. Almost all microfilariae in the nodes are dead or dying, surrounded by eosiniphils in the early stages, and later by histiocytes and giant cells. With ragards to the adult worms, ivermectin appears neither to be macrofilaricidal for O. volvulus nor to affect embryogenesis or spermatogenesis. Its most marked effect in the adult worms is a block in the release of stretched microfilariae from the uterus followed by microfilarial degeneration. These effects last for more than six months and are mainly responsible for the prolonged suppression of skin and ocular microfilariae counts. Other effects include a reduction in the percentage of nodules in which intact microfilariae are present in the nodular tissue. Multiple doses of the standard regimen have been given at varying intervals up to a total of 12 doses and 1.8mg/kg of body weight. Repeated yearly doses appear to have some macrofilaricidal activity, but the greatest effects against adult worms followed 11 doses given at 3-monthly intervals. This resulted in an excess mortality of the female worms (32.6% greater than in the control group who received no drug treatment), a reduction in the number of live male worms and in the proportion of inseminated females, and a cessation of microfilarial production (Duke, 1992). Ukaga et al. (2000) and Anosike et al. (2007) reported that though Ivermectin is not technically a macrofilaricde but they observed in the course of routine evaluation and monitoring of on-going Community Directed Distribution of Ivermectin (CDTI) in endemic communities in the rain forest of south-eastern Nigeria, that some villagers reported the disappearance or dissolution of onchocercal nodules after repeated treatment with ivermectin.
With regards to ocular changes, during the first 3-4 days after a single dose of ivermectin, the number of microfilariae in the anterior chamber of the eye either remains unchanged or increases temporarily. Corneal microfilarial are not increased in the first few days and a reduction in the number of ocular microfilariae does not occur for at least 2 weeks, and microfilariae may not be eliminated for 3 or more months.
After repeated doses of ivermectin (150µg/kg of body weight given annually or semi-annually): there is a significant reduction in ocular microfilarial loads and a 90% decrease in prevalence after 2-4 years. There is at least a 50% reduction in the prevalence of early iridocyclitis and a reduction of up to one-third in the prevalence of sclerosing keratitis after 2-4 years. There is less marked impact on posterior segment lesions than on anterior segment lesions . There is a significant reduction of about one-third in the overall incidence of optic atrophy, which is suggestive of a substantial reduction in O.volvulus-specific lesions. There is no impact on the prevalence of chorioretinitis. No significant benefit in terms of visual acuity is observed but there is a significant reduction in the occurrence of marked loss in paracentral visual fields, especially in those with pre-existing optic nerve disease. There is no evidence of a reduction in the prevalence of blindness and it may take a long time for the effect of ivermectin on the incidence of blindness to become apparent; These were some of the conclusions reached following four community-based studies in Cameroun, Ghana, Nigeria and Sierra Leone on the effect of repeated doses of ivermectin (WHO, 1993, unpublished document). However, so far for the control of onchocerciasis Ivermectin has proved to be both highly effective and well tolerated in clinical and community trials. Ivermectin treatment combined with vector control significantly reduced onchocercal dermatitis, microfilariae carrier rates and nodule prevalence
( Brieger et al., 1998; Seidenfaden et al., 1998; Emukah et al., 2004; Ndymugyenyi et al., 2004). This efficacy has made ivermectin to be acclaimed by all and it is being distributed world-wide. Three main bodies play a significant role in the stimulation and support of ivermectin distribution;
(1)The Mectizan Donation Program which was established in mid-1993 by the Carter Center to expand the activities of the Mectizan Expert Committee in support of non-governmental agencies involved in ivermectin distribution, create regional coalitions of individuals and organizations distributing ivermectin, to generate resources to support distribution, and to review methods and strategies designed to sustain distribution.
NGO Coordination Group for Ivermectin Distribution which is an open-ended group whose membership currently consists of Africare, Christoffel-Blindenmission, Helen Keller International Inc., the International Eye Foundation, Organisation pour la Prevention de la Cecite, the River Blindness Foundation and Sight Savers(formerly the Royal Commonwealth Society for the Blind),the Mectizan Donation Program and WHO. The Group which was established in December 1992 aims to promote world-wide interest in, and support for, the use of ivermectin in the treatment of onchocerciasis in endemic countries, and to assist interested countries or groups of countries in planning, implementing and evaluating ivermectin distribution programmes.
(2) Onchocerciasis Elimination Program in the Americas (OEPA) which is a multinational, multi agency and multi donor initiative, created in September 1991 by the Pan American Health Organization and aims at eliminating the severe pathological manifestations of the disease and to reduce morbidity in the Americas through the mass distribution of ivermectin.
International programs supported by the World Health Organization and many other groups have worked to control the impact of onchocerciasis using vector control with insecticides beginning in 1974 and Mass Drug Administration (MDA) with ivermectin (IVM, brand name Mectizan) beginning in 1987 (Peters et al., 2004). Annual IVM MDA reduces morbidity (Ejere et al., 2001; Tielsch and Beeche, 2004) and lowers transmission (Boussinesq et al., 1997; Collins et al., 1992). From 1974 to 2002, the Onchocerciasis Control Programme (OCP) in West Africa greatly decreased O. volvulus transmission in the 11 OCP countries and prevented 600,000 cases of blindness (Molyneux, 1995; Boatin and Richard, 2006; Basanez et al., 2006). IVM without vector control has been the principal tool for the Onchocerciasis Elimination Program of the Americas (1992–present) (Boatin and Richards, 2006) and the African Programme for Onchocerciasis Control (1995–present). In the Americas, where O. volvulus is less common, the Onchocerciasis Elimination Program has substantially reduced transmission to eliminate the disease.
For more than a decade, ivermectin has been distributed world-wide and the number of treatments provided annually continues to grow at a dramatic rate. In 1990, 1.4 million people received ivermectin, in 1991, 2.8 million, in 1992, 5.3 million and in 1993, 9.2 million people (impressive but represents a relatively small proportion of the 100 million people at risk from the disease) (WHO, 1995). Various methods of distribution are being employed, each with its own advantages and disadvantages;
(3) Community-based distribution- where the village leader appoints someone to be trained as a community-based distributor to carry out the yearly treatment within the village. Active treatment campaigns – where organized teams travel to the endemic communities to treat people, a typical example of what obtains with the Oncho. project in Achi, south-eastern Nigeria.
Through passive treatment campaigns – where drugs are left at fixed health posts within the community, and patients are encouraged to visit such posts.
Although ivermectin does not, have such a decisive impact on transmission, as had originally been hoped, encouraging results have been obtained from some large scale community trials (Chabala et al., 1980; Taylor and Greene, 1989; Soboslay et al., 1987, 1991) with regards to skin microfilarial reductions. The efficacy of antifilarial drugs is measured by the reduction of microfilariae densities in the skin and by the observations of dead or degenerate parasites in the nodules and uteri contents of the female worms (Schulz – Key, 1987). This has been demonstrated in large scale community trials involving tens of thousands of onchocerciasis patients in Ghana comprising a study population of 24 575 people, a total of 14 991 (61.5%) received ivermectin and there was a 92% reductions in skin microfilarial load after 2 months (Remme et al., 1989). In Liberia, in 1987, a total of 7699/7956 were treated and 86% reductions in skin microfilariae was recorded after 6 months (Paque et al., 1990a), and in 1988, out of a total of 13977 people, 8062/8438 (eligible at 96%), 78% reductions was obtained after 12 months(Paque et al., 1990a).
In Nigeria, Ivermectin distribution in Nigeria started as early as in 1989 in different parts of the country under different auspices. The establishment of Onchocerciasis Operation Research (OOR) by TDR in 1990 was a major reorganization that brought success in the disease control in Nigeria (Nwoke and Dozie, 2001).
In 1992, TDR, OOR and Federal Ministry of Health funded a 5-group multicentre, Nigeria Scientists on Rapid Assessment method (RAM) to identify communities/zones eligible for large-scale treatment with ivermectin. Later, Rapid Epidemiological Mapping of Onchocerciasis (REMO) was adapted as a standard protocol for producing acceptable baseline results for the control of disease in Africa and Nigeria using only nodules as a basis for estimating endemicity. Thus an area with the community prevalence rates of nodules > 20% is a priority area where ivermectin mass treatment is indicated (Gemade et al., 1998). The achievements of the Nigerian CDTI programme with the bold back up provided for the National Onchocerciasis Control Programme (NOCP) by the River Blindness Foundation, LION club, The Global 2000 River Blindness Programme, and other NGOs; so far include:
- Treatment of over 17 million people since 1999 (in 1988 only 6270 people treated).
- Training of over 20,848 health staff at all levels.
- Adequate and free flow of ivermectin
- High level of awareness of the programme
- 75% to 80% of all CTDI communities were actively involved in community directed drug distributors’ selection, drug distribution, choice in distribution method, month and CDD training.
- 63% offered suitable incentives to their CDDS.
- Appreciation of the benefits of ivermectin
- Transmission has dramatically declined.
- Incidence of new infection is nearly zero in most foci.
- Today 33 states under treatment (in 1988 only 2 states were covered).
- Oncho control activities gradually intergrated into primary health care.
Treatment coverage rate increased per year by 8 to 10% (Nwoke and Dozie, 2001). Despite these successes recorded there are still other challenges and problems that must be addressed, such as more research needed and development efforts as well as the provision of substancial research grants and other necessary incentives for promoting investigation into various facets of the onchocerciaisis problems. Other crucial considerations such as the looming problem of drug and pesticides resistance as have been variously suggested. This work targets to plunge into the issue of resistance.
In Achi, an onchocerciasis-endemic zone in south-eastern Nigeria, a project supported by the UNDP/WORLD BANK/WHO with the aim of investigating the epidemiology and controlling onchocerciasis, started ivermectin distribution in October to December 1990 on a yearly basis. Achi, an area described as savannah mosaic revealed that microfilaridermia was found in 76% of the population and nodules in 62%. Skin and ocular lesions of onchocerciasis including sowda were common and S. damnosum complex were found breeding along the entire length of the Oji River and its tributaries. Biting occurred all year round but peaked in the months of March, April, and May (Okonkwo et al., 1991, 1992 unpublished).
Despite the wide spread use of ivermectin, there is considerable uncertainty surrounding the mechanism of action. In Achi, ivermectin administration has helped to reduce the microfilarial density of the population but some individuals who have been dosed still show high microfilarial levels. It is also evident that after frequent administration, most organisms (viruses and parasites in particular) seem to have the capacity to become resistant to drug originally used for their control. The reason for this is not known, since ivermectin has been used in Nigeria for quite a while, this might be innate. A further question is whether this is resistance will it be consistent and spread in the course of repeated mass treatments? as in the case of P.falciparum malaria and chloroquine.
The trials in Achi showed that in some of the onchocerciasis patients, the drug is not effective. While reductions in the skin microfilarial loads were observed after the first dose, by the second and third doses, further reductions were obtained but there were still some few patients without any reduction in their skin microfilarial loads observed)**Okoli, M.N. personal observations.
One reason for this persistent microfilariae may be reinvasion by infected Simulium flies or malreabsorption of the drug by the patients, but there is also the possibility that it may be the early signs of resistance development. Since 1995, the African Programme for Onchocerciasis Control (APOC) has been covering 19 of the continent’s 28 countries hit by the disease. Access to this treatment is possible for 70 million people and has significantly diminished the onchocerciasis-induced morbidity. However, the doubling of cases of infection in certain communities of Ghana between 2000 and 2005, in spite of annual treatments, created fear of the emergence of ivermectin-resistant strains. Such apprehension appears particularly justified in that a high degree of therapeutic cover is achieved during mass distribution campaigns and hence only a tiny part of the parasite population targeted remains unexposed to drug treatment pressure (Fletchet, 2008)
Parasites developing resistance to drugs directed against them have been one of the few ways that parasites are thriving to defeat man. Drugs which once could be counted on for protection against many parasites and infectious diseases are becoming less and less useful as resistance to them spreads. Drug resistance have been a major cause of setback in the treatment and control of many diseases.
The resistance of parasites and infectious disease organisms to drugs and antibiotics is as old as chemotherapy itself. In malaria chemotherapy, chloroquine was initially acclaimed as the answer to malaria control but currently chloroquine can no longer be used for non-immune patients in most endemic regions because of development of resistant strains of P.falciparum (Panisco and Keystone, 1990). Each year 400 million cases of malaria are reported and about two million children die from Plasmodium falciparum infection (WHO, 1994). The World Health Organization estimates that 300-500 million new cases of malaria are reported annually causing the death of about 2.5 million people ( Van der Westhyzen and Parkinson, 2005). The increasing prevalence and distribution of malaria has been attributed to a number of factors, one of them being the emergence of spread of drug resistant parasites. The chemotherapy of cancer has proved insurmountable because of the development of drug resistance more especially multi-drug resistance. It is already evident that development of resistance by tissues or organisms to drugs is an evolutionary adaptation that puts at risk every tumouricidal, pesticidal and parasiticidal agent. Drug resistance is defined as a change in the gene frequency of a population that is produced by drug selection whereby more drug is required to exact same effect than was required prior to selection. It is to be expected that when drug pressure is applied repeatedly and intensively, successive populations would move directionally towards less and less susceptibility. If the lack of susceptibility by a population is not the result of drug selection, then it is not resistance but only tolerance. Tolerance is defined as the innate lack of susceptibility that did not result from drug selection. Observations suggest a mechanism for drug resistance, whereby, there appears to be some barrier that kept the drug from reaching the interior of the cell, where it would have its lethal effect. Two possible theories were put forward to account for the evidence. One theory proposed that a permeability barrier prevented drug entry into the cells. The other suggested that an afflux pump, a mechanism that actively pumped drug out of the cell once it had got inside was at work in the resistant cells (Kartner and Ling, 1987). Whatever the actual mechanism, two points seemed clear, one was that the process of keeping drugs out of the cell would need to be rather nonspecific, that is able to cope with drugs of diverse molecular structure. The other was that because the cells surface membrane (the plasma membrane) is the first line of defence against the entry of drugs, the difference between drug sensitive and drug resistant cells would probably be found there. Multi-drug resistance was first noticed in cancer chemotherapy whereby tumour cells were simultaneously resistant – that is , cross resistant to completely unrelated drugs. Greater drug efflux is one of several features resistant malaria parasites share with drug-resistant cancer cells, in which the efflux is mediated by transport protein, the P-glycoprotein ( Juliano and Ling, 1976; Di Pietro et al., 1999; Hung et al.,1998; Rosenberg et al., 1997)
184.108.40.206 Molecular nature of resistance
Glycoproteins are compact molecules made up of protein and carbohydrates that are usually associated with the plasma membrane. In P-glycoprotein, P- signifies permeability. P-glycoproteins are evolutionarily well conserved membrane bound proteins which belong to the family of ATP binding cassette (ABC) transporters (Higgins and Gottesman, 1992) and widely represented in the animal kingdom. The P-glycoprotein located in the plasma membrane, consists of two similar halves, each with six transmembrane domains and an intracellular ATP binding sites (Endicott and Ling, 1989; Gottesman and Pastan, 1988). The P-glycoproteins are coded for by the mdr genes. Reported molecular weights of the P-glycoproteins ranges from 140-170 kDa and 180-210 kDa (Juliano and Ling, 1976; Endicott and Ling, 1989). However, recently, Thevenod et al., 1994, 1996) reported some MDR1 related proteins with molecular weights of about 65kDa and 80kDa. The P-glycoprotein is normally found expressed in the kidneys, adrenal glands, liver, and parts of the gastrointestinal tract of the normal adult (Kartner and Ling, 1989; Schinkel et al., 1994). The most consistent feature of cells with high level of multi-drug resistance (MDR) is the overproduction of the P-glycoproteins, thus increasing the outward transport of the drugs. The roles of the P-glycoproteins remain a mystery or more or less under controversy but they do transport peptides and in yeast, a lipopeptide (McGRath and Varsharsky, 1989). It is also thought that they provide a protective mechanism against exogenous toxins present in the diet and environment (Ames et al., 1990), by actively extruding structurally and functionally unrelated agents, they can rescue the cell, and the organism from toxic drug effects. This mechanism of drug resistance has been found in the resistance of tumour cells to Vinca alkaloids, epipodophyllo-toxins, and anthracyclines ( DeVita, 1989). A number of MDR cell lines have been isolated that show a good correlation between the levels of DNA amplification, increased mRNA expression and MDR. Working with MDR cell lines derived from human ovarian SKOV3 cells. At low levels of drug resistance (up to 64-fold relative resistance to vinblastine, and 16-fold relative resistance to vincristine in the cell line SKVLBO.03 selected with vinblastine), increase in P-glycoprotein mRNA and protein occurs without concomitant DNA amplification. P-glycoprotein gene amplification is only observed in
Figure 5: Schematic Structural Organization of P- glycoprotein
Di Pietro et al. (1999)
subsequent steps. Thus it appears that MDR cell lines can be selected that show no P-glycoprotein DNA or RNA amplification, but only with low levels of drug resistance. Subsequent selection for high levels of resistance results in DNA amplification, and over expression of P-glycoprotein mRNA. In general the levels of DNA, mRNA, and P-glycoprotein expression correlates reasonably well, but a number of exceptions have been reported. In addition to most commonly observed phenomenon of increased P-glycoprotein mRNA and protein levels as a result of DNA amplification, increase in mRNA and protein expression can also occur without P-glycoprotein gene amplification. This suggests that P-glycoprotein may be transcriptionally and/or translationally regulated and thus not an evolutionary process ( Akiyama et al., 1985, Shen et al., 1986; Van de Bliek et al., 1988 and Greenberger et al., 1988). Amplified DNA can often be observed by light microscopy, either as an extended chromosomal region (ECR) or as extra-chromosomal elements, called double minutes (DMs) and the same gene can be amplified either chromosomally or extrachromosomally in a single population of cells, but the two forms do not usually coexist within the same cell (Cherif et al., 1989) although a few examples to the contrary have been noted. A few different molecular mechanisms have been suggested by different workers (Carroll et al., 1988; Passananti et al., 1987). In one of the mechanisms, they proposed that the actual mechanism of DNA amplification on the molecular level occurs in a single cell whereby bidirectional replication at an origin generates a bubble that can undergo further rounds of unscheduled DNA replication, resulting in a nested set of partially replicated duplexes. It is then possible for linear duplex DNA to become detached from the structure if two replication forks can approach one another very loosely. Recombination within the same duplex could generate extrachromosomal circles, while multiple recombination among different duplexes could resolve the structure into an intrachromosomal linear array. Thus, an onion skin structure might give rise either to an ECR or to episome. The episome might then reintergrate giving HSR’s or remain extrachromosomal, perhaps evolving to form DMs (Carroll et al., 1988). The structural key features of a typical P-glycoprotein which are consistent with biochemical and immunohistochemical data depicts that it consists of about 1280 amino acids arranged in two similar halves ( Luurtsema et al., 2002).
Each half is joined by a highly divergent cytoplasmic region. Each half has a short hydrophilic amino-terminal segment, six hydrophobic membrane spanning domains that form three transmembrane loops and a hydrophilic carboxy-terminal region containing consensus sequences for a nucleotide-binding site, presumably responsible for adenosine triphosphate (ATP) binding and hydrolysis. There are several potential glycosylation sites on the first external loop near the amino terminal of the molecule. That each half of the molecule does not act independently to transport the drugs is suggested by the finding that inactivation of either one of the two nucleotide binding sites results in loss of functional activity (Azzaria et al., 1989). The P-glycoprotein molecule presumably forms a channel-like structure through which substrates, including the drugs involved in MDR, are transported actively from the cytoplasm and or the plasma membrane to the outside of the cell. All members of the human and rodent mdr gene families have high sequence homology, indicating a common structural and functional pattern. Mammalian P-glycoproteins are encoded by small gene families containing two members in humans, MDR1 and MDR3 or MDR2, three members in mouse, mdr1a or mdr3, mdr1b or mdr1, and mdr2, and three members in Hamster, Pgp1, Pgp2 and Pgp3 (Chen et al., 1986; Van de Bliek et al., 1986, 1988; Gros.et al., 1986b; 1988; Hsu et al., 1989; Devault and Gros, 1990; Schinkel et al., 1991). The P-glycoproteins has also been found in many other organisms e.g. invertebrates and parasitic protozoa ( Wilson et al., 1989). However, not all P-glycoproteins are involved in drug resistance, human MDR1 and mouse mdr1a and mdr1b can confer resistance, but MDR3 and mdr2 cannot (Gros et al., 1986, 1988; Schinkel et al., 1991; Devault and Gros, 1990). Hamster, pgp1 and pgp2 can confer resistance while pgp3 cannot. The mouse mdr2 gene (and its human MDR3 homolog) is involved in secretion of phosphatidylcholine into bile and is probably a phospholipid translocator. In Drosophila melanogaster three P-glycoprotein genes have been identified and at least one of them (Mdr49) has been suggested to be implicated in drug resistance. The parasite Leishmania has a P-glycoprotein gene family of at least six members, some of which are involved in drug resistance, e.g ltpgpA is involved in low level oxyanion resistance, which includes resistance to antimony, the drug of choice in treatment of leishmaniasis (Callahan and Beverly, 1991), and ldmdr is involved in resistance to several compounds included in the mammalian MDR spectrum. In some resistant malaria parasites, P.falciparum, amplification of the P-glycoprotein gene (pfmdr1) was observed and it was concluded that chloroquine resistance was caused by a P-glycoprotein-mediated mechanism (Foote et al., 1989; Wilson et al., 1989) but Wellems et al., 1990, and Wilson, (1993) disproved that when their results from the HB3xDd2 P.falciparum cross (Wellems et al., 1990) indicate that rapid efflux, chloroquine resistance phenotype is independent of the pfmdr1 and pfmdr2 genes, stating that the history of spread of chloroquine resistance suggests that acquisition of resistance involved very rare molecular events in P.falciparum, although the copy number of the pfmdr1 gene does correlate with the level of resistance in mefloquine- and halofantrine-resistant Plasmodium (Wilson, 1993). The malaria parasite’s P-glycoprotein homologue, Pgh1, is known to influence the sensitivity of malaria parasites to a diverse range of antimalarial drugs, but the mechanism by which it does so has remained obscure (Saliba et al., 2008).
However, it is still unknown whether this P-glycoprotein can be associated with drug resistance in nematodes and in ivermectin and onchocerciasis. Recently, a family of Pgp homologues have been described in nematodes. In the free-living Caenorrhabditis elegans and in the sheep parasite Haemonchus contortus, there is evidence of Pgp genes. In C.elegans four P-glycoprotein homologs have been identified (pgp-1, pgp-2, pgp-3 and pgp-4) and three have been analysed in detail. They share similarities with mammalian P-glycoprotein in their predicted protein structure (Lincke , 1992, personal communication; Broeks, A. ,unpublished results). The presence of an ivermectin resistant organism was confirmed in South Africa in November 1985, 33 months after the drug was introduced there. Since then, ivermectin resistance has been reported both in the field and in laboratory across the globe. In South Africa, Van Wyk and Malan, (1988) reported that four isolates of Haemonchus contortus from sheep to be resistant to ivermectin. In Brazil, resistance to ivermectin was detected in an H.contortus isolate from sheep. In New Zealand ivermectin resistance has been reported in Ostertagia spp. from goats ( Badger and Mckenna, 1990).
Recent studies on P-glycoprotein and ivermectin using mice, further confirms that P-glycoprotein have a wide variety of functions useful to the well being of the cells. The mouse mdr2 P-glycoprotein is indispensable for the secretion of phospholipids into bile (Smit et al., 1994). Based on the tissue distribution of the human MDR1 P-glycoprotein ( Cordon-cardo et al., 1989) it has been proposed that MDR P-glycoprotein plays a role in the protection of organisms against toxic xenobiotics, by active excretion of these compounds into bile, urine or intestinal lumen and by preventing accumulation in critical organs such as the brain. This postulation was proved when Schinkel and his co-workers (1994) generated an mdr1a P-glycoprotein-knock-out mice and administered it with ivermectin. The mice died of ivermectin toxicity at a concentration very low to affect the heterozygous mouse whose P-glycoproetin was intact (Schinkel et al., 1994). Furthermore, mice without mdr1a P-glycoprotein and orally injected with ivermectin (0.2mg/kg) retained the drug in especially brain tissue more than the heterozygous mice with their mdr1a intact. Thus, suggesting that the P-glycoprotein coded for by the mdr1a might confer tolerance to ivermectin in other organisms with a blood-brain barrier. Meanwhile, O. volvulus have no blood-brain barrier.
The possibillity that Pgp is involved in anthelmintic resistance is real. At least two common anthelmintics. Ivermectin and closantel, are potential substrates and resistance to both occurs independently in strains of trichostrongylid nematodes from grazing sheep. In H. contortus, no evidence of mdr gene amplification has been found in benzimidazole-resistant isolates. Levamisole-resistant isolates nor ivermectin-resistant isolates, rather Benzimidazole-resistance was found to entail a minimum of two genetic steps at separate ß-tubulin loci – first, through a series of steps at which different isotype 1 alleles are lost, followed by the loss of different isotype 2 alleles and other possible mechanisms (Roos et al., 1995). Drugs that cause gene amplification in tumour cells do not cause amplification of P-glycoprotein genes in C.elegans even at much higher concentrations (Lincke, personal communication). On the other hand, of the four P-glycoproteins genes (pgp-1, pgp-2, pgp-3, pgp-4) identified in C.elegans, three have been analysed in detail. Pgp-1 and pgp-3 are expressed through out the life cycle, and exclusively expressed in the intestinal cells and pgp-3 was found to be involved in colchicine and chloroquine resistance in C.elegans (Broeks et al., 1995).
Onchocerca volvulus is a parasitic nematode. Ivermectin is the current drug of choice for the treatment of onchocerciasis and ivermectin is a potential substrate. The possibility of resistance manifestations in the treatment of onchocerciasis patients with ivermectin is very real and it would be worth genuine practical consideration to ensure early surveillance so as to prevent another ‘chemotherapy shock’ or resistance outbreak like in the treatment of malaria.
However, the life cycle of O. volvulus (review in 1.1, and Schulz-Key et.al., 1987) is not as simple as that of C.elegans or H.contortus where you can maintain easily in-vitro or in-vivo in animal models. Up to date, only controlled efficacy tests requiring necrospy or FECR (Fecal egg count reduction) tests have been used to detect ivermectin resistance and because of practical considerations and expense, manufacturers recommended use-level has been employed as the threshold of resistance. Reliance upon necropsy following treatment cannot be routine procedure for resistance monitoring and it is unknown how sensitive FECR tests are at detecting ivermectin resistance at low frequencies. Five in-vitro tests to detect ivermectin resistance have been reported but non is used routinely ( Gill et al., 1991). All were tested against isolates that are resistant to the use-level of ivermectin and whether they are able to detect resistance when gene frequencies are low is unknown, moreover these methods cannot be easily applied in humans.
The use of the PCR pool screening technique will make it possible to detect now the level of transmission in a given area with a limited investment of human and material resources. This technique may thus be a valuable tool for the surveillance activities to be conducted in the OCP area following the cessation of active vector control in 2002. In addition, the ability to accurately measure infection rates in the vector population may prove useful in monitoring the effect of ivermectin-based onchocerciasis control programs (such as the Onchocerciasis Elimination Program in the Americas and the African Program for Onchocerciasis Control) on transmission. Furthermore, the pool screen PCR is an efficient means to screen large numbers of flies for the presence of O. volvulus larvae. It may thus prove useful in establishing that a given area is freed of O. volvulus transmission following successful control.
Drug resistance has been defined as a loss of the normal response to treatment and is heritable (Prichard et al., 1980). Ivermectin affects both the microfilariae, removing them from the skin, and the adult worms, inhibiting their reproduction for many weeks. Because of these various and prolonged effects, and because ivermectin activity involves host immunity, it is not reliable to assess efficacy in vitro. Phenotypic assessment of resistance needs to consider both skin microfilarial loads (repopulation of the skin by microfilariae), and worm fertility (by embryogram). A meta-analysis was conducted of both these outcomes after single-dose ivermectin following a systematic review of early clinical and field trials and fitted a mathematical model to the data . Results were compared with those obtained in a study of 10 repeatedly treated communities in Ghana (with >10 annual treatments) (Osei -Atweneboana et al., 2007). This study indicated continued high microfilaricidal activity of ivermectin but suggested that inhibition of reproduction by adult worms was impaired in some repeatedly treated communities in contrast to an ivermectin-naïve community. Subsequent repopulation of skin by microfilariae was faster than expected even after considering the inter-study variability of the (also ivermectin-naïve) meta-analysis. A model for repopulation rates was fitted to microfilarial temporal profiles after treatment for each person examined in one of the communities (treated for 10 years) to quantify the level of inter-individual variability in parasitological response. Ivermectin resistance is common in veterinary parasites and has a genetic basis associated with selection on ATP-binding cassette (ABC) transporters (e.g. P-glycoproteins) and β-tubulin. Onchocerca volvulus samples from communities in Ghana and Cameroon that have received many treatments have been found to have significant changes in similar genes (β-tubulin, P-glycoproteins and other ABC transporters) compared with worms isolated from treatment-naïve subjects or the same subjects prior to treatment (Bourginat et al., 2006). These genetic changes should be useful markers for ivermectin resistance.
Isoenzyme analysis has also been used to characterize ivermectin-resistant and susceptible strains. Using starch-gel electrophoresis it was found that one ivermectin-resistant isolate of H.contortus did not show activity for propionyl esterase when the susceptible isolate did (Echevarria et al., 1992), in the same test, another isolate multiply resistant to ivermectin and benzimidazoles did have propionyl esterasse activity. Whether the loss of propionyl esterase activity in the ivermectin-resistant isolate had anything to do with its resistance or whether it was only coincidental is not clear. Thus a major obstacle facing investigators searching for an effective monitoring against ivermectin resistance in O. volvulus is the lack of an elaborate, complete and controlled maintenance system for O. volvulus. But, since some of the factors responsible for most mdr resistance manifestations (like the P-glycoprotein coded for by the mdr genes or the loss of isotype 1 and 2 alleles respectively) have been discovered in some other organisms, it will be of primary importance to investigate also in O. volvulus for such factors. To date, resistance of O. volvulus to ivermectin has not been clearly described but constant surveillance must be maintained to ensure that any such development is detected after the cessation of control activities. The Onchocerca genome has to be studied ( Jolodar and Brattig, 2009; Jolodar, 2010).
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