CHAPTER 1 INTRODUCTION

Transcription

1 1 CHAPTER 1 INTRODUCTION 1.1 BACKGROUND Lymphatic filariasis is a mosquito- borne parasitic disease caused by thread like, filaroid nematode worms that include Wucheraria bancrofti, Brugia malayi, and Brugia timori. They lodge in the lymphatic system, a network of nodes and vessels that maintain the body s fluid balance and an essential component of body s immune system. It is an endemic disease prevalent mostly in the tropical and subtropical regions of the world. Lymphatic filariasis also known as, Elephantiasis puts at risk more than a billion people across the globe. A recent survey estimates about 120 million infected people of which over 40 million of them are seriously incapacitated and disfigured by this dreadful blight (Molyneux et al 2003). Though mapping of its prevalence is ongoing, statistics for many countries are old, very sketchy or non existent. One third of the people infected with the disease live in India, one third are in Africa, and most of the remainder are in Pacific, South Asia and the Americas. While Bancroftian infections are common in Africa and India, Brugian filaraiasis is restricted to south east, eastern Asia and Timor Islands. Having both Bancroftian and Brugian filariasis, India alone contributes to 45% of the global burden with the latter localized to Tamil Nadu, Kerala, Andhra Pradesh, Madhya Pradesh, Assam and some parts of Orissa.

2 2 In tropical and subtropical areas where lymphatic filariasis is well established the prevalence of infection continues to increase. A primary cause for this endemicity is uncontrolled urbanization leading to accumulation of sewage and waste water that provides an ideal breeding ground for mosquitoes that transmit the disease. In most obvious manifestations, lymphatic filariasis causes enlargement of the entire leg or arm and the genitals. In addition, even more common than the overt abnormalities is hidden internal damage to the kidneys and lymphatic system (Ottesen et al 1997). In endemic communities, around 10-50% of men and upto 10% of women could be affected. Hence WHO identified lymphatic filariasis as the second leading cause of permanent disability. The psychological and social stigma associated with the disease is immense and varies widely from place to place. The degree of suffering is associated with the severity and visibility of the disease (Evan et al 1993; Mujinja et al 1997). Thus Filariasis is considered to be a terrible disease, as sufferers are shunned by their family to an extent that they will be considered unsuitable as marriage partners. As the social stigma associated with filariasis is burdensome, the latter has been identified along with Schistosomiasis, Leishmaniasis, Leprosy and Onchocerciasis, as diseases that should receive special attention under a "gender and tropical diseases" research program (WHO 1995). Besides this, Global Programme for Elimination of Lymphatic Filariasis (GPELF) was also launched in the year 2002, with an aim to eliminate this parasite curse by Thus the ultimate objective of GPELF is to eliminate this public health problem to make the world free of one of the most stigmatizing and disabling disease (Molyneux 2009).

3 3 1.2 REVIEW OF LITERATURE Filarial Parasites Filariasis is a chronic helminth disease caused by arthropod transmitted nematode parasites. The filarial nematodes that infect humans belong to the phylum Nematoda under family Filarioidea of the order Spirurida (Azevedo 1964). There are seven principal species of filariae that infect humans, and these are typically grouped into three categories. The worms like Wuchereria bancrofti, Brugia malayi and Brugia timori are the causative agents of lymphatic filariasis. The adult worm that causes lymphatic obstruction resides in the lymphatics and produces larval form called microfilaria that can be seen in the blood. Onchocerciasis or river blindness is another similar disease caused by nematode parasite Onchocerca volvulus. These adult worm live in the subcutaneous nodules or in the deeper skeletal tissues associated with major bones. The microfilariae are present in the skin and eyes that lead to severe lesions or visual impairment. The remaining filariae that infect humans including Loa loa, Mansonella perstans, Mansonella ozzardi, and Mansonella streptocerca are comparatively less important in causing the disease, although Loa loa can provoke temporary inflammatory swelling (calabar swelling), hypereosinophillia and other allergic reactions. Loa loa has the habit of migrating widely throughout the body and it also crosses the eye and surrounding tissues (WHO 1992). The adult worms of B. malayi and W. bancrofti have minute, thread like structure with a smooth cuticle that forms the exoskeleton of the parasites and helps in the maintenance of morphology, motility, nutrition and protection from environmental factors. Adult worms exhibit a marked sexual dimorphism with female worms are larger and longer than the male worms. Both adult male and female worms have periodic annulations and numerous small spherical protuberances on its surface (Araujo et al 1995). The adult

4 4 male measures 40 mm in length and 0.1 mm in diameter, whereas the female worms are mm in length and mm in diameter (Nanduri and Kazura 1989). Microfilariae are µm in the case of W. bancrofti and µm in the case of Brugian species Global Distribution of Lymphatic Filariasis Lymphatic Filariasis (LF) has a wide geographic distribution with long history. The disease is found in more than 80 countries in the tropics through regions of South and Central America, Central Africa, Eastern Mediterranean, Southeast Asia and Western Pacific. LF is the world s second leading cause of permanent long-term disability and its prevalence continues to increase. Asia and the Western Pacific regions bear the burden most, since two out of three cases globally occur in these regions (WHO 1995). It affects approximately 120 million people of which, 107 million are infected with W. bancrofti, 13 million with B. malayi and at least 1.2 billion people are at the risk of infection (Michael and Bundy 1997). It has been estimated that 43 million individuals show physical disabilities due to either lymphoedema and hydrocele or the newly recognized sub-clinical abnormalities of lymphatic and renal function, with Bancroftian filariasis accounting for almost 40 million of these cases (Ottesen et al 1997; Molyneux et al 2003; Molyneux and Zagaria 2002). B. malayi infection is widely distributed almost throughout the Asian countries like China, Korea, India, Indonesia, Malaysia, Philippines, Sri Lanka. On contrary, B. timori infection occurs in Indonesia (Islands of Alor, Flores, and Timor) and W. bancrofti has a larger distribution like Asia (China, India, Indonesia, Japan, Malaysia, Philippines, South-East Asia, Sri Lanka), Tropical Africa, Central and South America, Pacific Islands (Figure 1.1).

5 5 Figure 1.1 Global Distribution of Bancroftian and Brugian Filariasis A map of the world showing the areas where Bancroftian filariasis is endemic and, in the inset, where Brugian filariasis is endemic. Large areas of endemicity are shaded but small foci are indicated with asterisks (*). Adapted from Zagaria and Savioli (2002) In India an estimated 374 million people live in endemic area and about 45 million are infected. Bancroftian filariasis is prevalent in Andhra Pradesh, Tamilnadu, Pondichery, Goa, Karnataka, Orissa, Madhya Pradesh, Gujarat, Uttar Pradesh, Bihar, Assam and West Bengal. Brugian filariasis is mostly found in Kerala, Assam, Madhya Pradesh, Andhra Pradesh, Tamilnadu and scattered pockets of Orissa (Rao 1982) Vectors There exists over seventy species of mosquitoes in the genera Culex, Anopheles, Aedes and Mansonia that infect humans with the disease.

6 6 The most important vectors of W. bancrofti are Culex quinquefasciatus, Anopheles gambiae, Anopheles funestus, Aedes polynesiensis, Aedes scapularis, Aedes pseudoscutellaris etc. For B. malayi, Anopheles barbirostris, Anopheles sinensis, Anopheles donaldi and several species of Aedes and Mansonia serve as vectors, while B. timori infection is transmitted through Anopheles barbiosrtis Periodicity Microfilariae are the larval forms that are vital for the disease transmittance. These parasite stages are absent in the peripheral blood during the day and appear only at night, especially at between 10 p.m. to 2 a.m, and get ingested by mosquito during the blood meal. This phenomenon is referred to as nocturnal periodicity (Gupta et al 1990). When not in circulation, they generally reside in the capillaries and blood vessels of the lung. This may be due to differences in oxygen tension between the arterial and venous blood in the lungs (Burren 1972; Nanduri and Kazura 1989). Thus if the human host reverses the sleep and wake up cycle, the periodicity of microfilariae also gets changed (MacKenzie 1882). In case of B. malayi, there are two strains-one nocturnally periodic and the other nocturnally sub-periodic Life Cycle of the Filarial Worm Filarial nematodes are complex digenetic parasites that are highly host specific, with the requirement of a primary human vertebrate host and a secondary invertebrate mosquito host. Within the lymphatics, the adult male and female worms mate and begin to produce millions of first stage-larvae called microfilariae (mf) (L1) 60 days post infection. The mf pass out of the lymphatic system into the circulatory blood system, from which a minority are taken up in the blood meal of feeding mosquitoes. Further, maturation in the mosquito occurs when mf leave the mid-gut and migrate into the flight

7 7 muscles where they moult twice to form infective stage larva L3. Depending on the species and environmental conditions, 7-14 days are required for the L1 to develop into L-3. The mature L3 then move from the flight muscles to the proboscis of the mosquito to be ready to invade a host when the mosquito bites subsequently. Unlike malaria, the infective stage is not directly injected into the skin of the new host. It is deposited onto the skin whilst the mosquito is feeding and finds its way through the skin, usually via the puncture made by the mosquito. The infective stage larvae (L3) enter the human host following a mosquito bite and migrate through the subcutaneous tissue to the nearest lymphatic vessel. They lie in the afferent vessel of the lymph node and undergo maturation by molting first to fourth stage larvae at around 7 days post-infection and then molting to adulthood around 30 days post infection. The prepatent period, which commences with the entry of L-3 into definitive host and ends with the detection of mf in peripheral blood, is around days (Eberhard and Lammie 1991). The extraordinary life-span of adult filarial nematodes within the mammalian host (estimated to be 8-16 years), combined with an estimated microfilarial life-span of more than 100 days, ensures the spread of disease (Leeuwin 1962). The life cycle of the human filarial parasite B. malayi is shown in Figure 1.2. The complex life cycle of the filarial parasite thus makes the endemic inhabitants a compulsory host for any of the parasite developmental stage even if most of them are asymptomatic. These individuals are the ones who are at an alarming risk of developing pathogenesis. Hence the availability of an amicable diagnosis is the need of the hour as this can rescue the masses in the endemic regions from crippling disability and suffering.

8 8 Figure 1.2 Life cycle of human lymphatic filarial parasite Brugia malayi (Adapted from FilGenNet web page: fgn/filgen.html) Diagnosis of Human Lymphatic Filariasis During the past few years many diagnostic tools have been developed for both the initial diagnosis and post treatment follow-ups. A variety of diagnostic tools for adult and microfilaria detection by molecular diagnosis based on the detection of parasitic DNA by Polymerase Chain Reaction or DNA probes, immunological diagnosis for the detection of filarial antigen or antibody have been done (Wamae 1994).

9 Detection of microfilariae (mf) by direct and concentration methods One of the gold standards in the diagnosis of lymphatic filariasis is the detection of microfilariae in peripheral blood samples. A simple method for the identification of mf is the Giemsa staining of the capillary blood collected by finger prick. For epidemiologic screening, l of capillary ( fingerstick ) blood can be dried on a slide, stained with Giemsa, and examined under a microscope. Sensitivity of mf detection has been further improved by concentration methods in which 1 ml of venous blood is filtered through a 3-5 m Nuclepore filter (Nuclepore Corporation, Pleasanton, CA) (Turner et al 1993). Other commonly used techniques include counting chamber, Knott s concentration and DEC provocative day test (Denham 1995). The nocturnal periodicity of the mf in many endemic areas demands blood sample collection late at night a situation not acceptable by community members and health workers. In this scenario, diagnostic tools were further upgraded by techniques like detection of filarial antigen and antibodies in the peripheral blood of infected individuals Immunodiagnostic assay Immunodiagnostic methods are currently under evaluation for the diagnosis of filarial infections in addition to the conventional night blood smear. The diagnosis of either circulating antigen or antibody is based on heterologous/ homologous, crude or fractionated antigens, recombinant antigens and monoclonal antibodies (Mabs) Detection of filarial antigen Based on the filarial antigenaemia that is associated with filarial infection, many diagnostic tools have been developed using both monoclonal

10 10 and polyclonal antibodies raised against various antigens. A number of monoclonal antibodies have been developed against parasite antigens and were successfully used to identify circulating parasite antigens in the patients. Mab E34 raised against W. bancrofti mf ES antigen was able to detect filarial antigen associated with active infection (Reddy et al 1984). Monoclonal antibodies developed against phosphorylcholine, antigens from other related parasites and a recombinant antigen have been shown to detect circulating parasite antigens in Bancroftian infections (Dissanayake et al 1984; Forsyth et al 1985; Lal et al 1987; Theodore and Kaliraj 1996). Although these tests are useful to diagnose filarial infections, due to lack of high specificity for microfilaremic individuals, a suitable antigen detection system had to be developed. The first commercial assay Trop Bio Og4C3 antigen test, produced by TropBio Pvt Ltd, Townsville, Australia. (More and Copeman 1990) was based on the monoclonal antibody raised against the Onchocerca gibsoni antigen, that showed a high specificity for the W. bancrofti antigen. Og4C3 assay is a marker for active filarial infections with adult worms since it detects antigen from both adult worm and microfilariae (Chanteau et al 1994a, b). In our lab Og4C3 detection was evaluated and for the epidemiological screening, a method was standardized by collecting blood samples into filter paper strips for this assay (Lalitha et al 1998). The reported diagnostic sensitivity of the Og4C3 assay for W. bancrofti varies from 73% to 100% (Lammie et al 1994; Rocha et al 1996), suggesting that the sensitivity of the Og4C3 may be reduced when microfilaria count is very low. Other commercially available kit is the ICT card test, which uses monoclonal antibody to AD12 of Dirofilaria immitis (Weil and Liftis 1987) (now marketed as BINAX Filariasis NOW). Sensitivity of this test appears to be lower in infected persons who are mf negative or those with ultra-low mf densities. The greatest advantage of these techniques over the mf detection is

11 11 that they can be used in the diurnal blood samples making the sample collection more easy (Faris et al 1998). A major development at our laboratory, was the development of Wb-SXP-1 antigen capture ELISA that can diagnose both Brugian and Bancroftian filariasis with a senstivity of around 83 % and 88.3% respectively (Lalitha et al 2002). This has been further enhanced by the development of a rapid flow through immuno filtration test based on the Wb SXP-1 antigen for the diagnosis of Brugian (90.8%) and Bancroftian (91.4%) filariasis (Vijayabasker et al 2004). These circulating antigen detection assays were particularly useful in the assessment of efficiency treatment with DEC or Ivermectin in the infected patients (Eberhand et al 1997; Weil et al 1998) Filarial antibody detection Detection of antifilarial antibodies against homologous and heterologous antigens in the sera is another tool for diagnosis. However, detection of total antifilarial IgG antibody is nonspecific and totally ineffective to diagnose current infection. This class of antibodies has been further substituted by more specific antibodies of the IgG4 isotype in the diagnosis of W. bancrofti infection (Lal and Ottesen 1988; Kwan-Lim et al 1990; Estambale et al 1994). An anti-wb-sxp-1 IgG4 ELISA was developed at our centre and this assay was found to be 100% sensitive for patients with patent W. bancrofti infections (Rao et al 2000). Until recently, there was no comparable rapid test for Brugian filariasis and diagnosis relied upon the traditional method of nocturnal mf detection with its poor sensitivity. A recently released dipstick test called Brugia Rapid, developed in Malaysia, showed 97% sensitivity, 99% specificity, 97% positive predictive value and 99% negative predictive value. It detects IgG4 antibody, not filarial antigen, and is not specific to Brugia

12 12 species. Besides this, it reacts with most cases (but interestingly not all) of W. bancrofti, but not with other common helminth and protozoal parasites (Rahmah et al 2001) Homologous antigens These antigens can be obtained either by fractionation of the crude worm extracts or by recombinant DNA technology. Several fractionated homologous antigens from W. bancrofti and B. malayi are preferred over the heterologous antigens, since they inhibit even the diminished cross reactivity among the different nematode species. Kaliraj et al (1981a), fractionated human filarial serum (FSI) and the antibody was used to detect circulating antigen by counter immuno electrophoresis (CIEP), and it was found that all the mf carrier patients had circulating filarial antigens, but none of the other sera from those with helminthes showed the presence of CFA. Purified surface antigens of the bovine filarial parasite S. digitata were sensitive and specific in the detection of antibodies in filarial sera and showed least cross reactivity with other parasitic infections compared with crude antigens (Theodore and Kaliraj 1990). Though several homologous antigens have been characterized and proved to be of diagnostic value, they remain unsuitable for field applications, due to scarcity of parasitic material, lack of animal models for W. bancrofti and tedious fractionation procedures Heterologous antigen based detection The non-availability of the homologous antigens in filariasis has led to the use of closely related helminth heterologous antigens for diagnosis due to their identical nature and specificity (Maizels et al 1985). Heterologous antigens include soluble whole worm antigens or mf antigen extracts, excretory-secretary (ES) antigens and circulating immune complex antigens (CIC). Parasite excretory-secretary products and circulating immune

13 13 complexes (CICs) help in antibody detection, as they are more speciesspecific than crude somatic extracts (Kaushal et al 1984) ES antigen based antibody assays Release of macromolecules by parasites into their environment both in vitro and in vivo has been reported in lymphatic filariasis (Kaushal et al 1982). ES products have been studied with respect to function, vaccination potential, pathogenicity and ability to serve as antigen targets for diagnostic tests. ES antigens are released by the living adult worm and thus may induce higher antibody titers than the somatic extracts of the worms. Due to nonavailability of sufficient parasite material from W. bancrofti, a heterologous ES antigen from B. malayi has been used to diagnose Bancroftian filariasis. The WbE34 monoclonal antibody raised against W. bancrofti microfilarial ES antigen detected the filarial antigen in W. bancrofti and B. malayi infected sera (Reddy et al 1989). Filarial antigen detection system Seva-Filachek developed based on B. malayi mf ES antigens identified occult filarial infections (Harinath et al 1996). Secretory acetyl cholinesterase (75 kda and 45 kda) from S. digitata microfilariae, purified by affinity chromatography or DEAE sepharose column is a potential antigen to diagnose human filariasis (Sharma and Rathur 1999). Circulating immune complexes (CIC): CICs occurring in most of the parasitic diseases are capable of modulating the immune responses (Barnett 1986) and are formed in circulation or tissues, as a result of interaction between exogenous and endogenous antigens and their corresponding antibodies. CICs in the sera of filaraemics were demonstrated using polyclonal antibodies raised against adult S. digitata worms (Dissanayake et al 1982). The polyclonal antibodies were reactive with the antigens derived from CIC and bound to adult worms, but not the microfilariae.

14 DNA detection method Nucleic acid probes and gene amplification are used to detect the parasite genetic material to diagnose the parasitic infections (Weiss 1995). Repeat sequences like HhaI (McReynolds et al 1986), Bm15 (Sim et al 1986) 320bp repeat sequences of Brugia malayi (Williams et al 1988) and 969bp in W. bancrofti (Siridewa et al 1996) present in the parasite genome are identified as potential targets for genus and species specific identification. Blood, plasma and paraffin-embedded tissue samples of the W. bancrofti infected individuals are used for DNA analysis by PCR with high sensitivity (McCarthy et al 1996; Zhong et al 1996). PCR diagnosis on blood could be useful in areas where multifilarial parasitism occurs such as Central Africa or India, as specific probes now exist for W. bancrofti, B. malayi and L. loa (Nicolas 1996). Though the sensitivity is high, these assays appear to be positive only when circulating mf are detectable (Williams et al 1996). Detection of W. bancrofti larvae in the mosquitoes to monitor infection rates was developed previously (Dissanayake et al 1991). This method was based on non radioactive probe that uses chemiluminescent substrate. Identification of parasites in the mosquitoes will be useful in the vector control strategy employed in the endemic areas. Field application of PCR based assays for monitoring Wuchereria bancrofti infection in Africa has been reviewed by Ramzy (2002). Quantification of PCR amplified product of Brugia Hha I repeat DNA using a semiautomated Q PCR was reported by Rao et al (2002). Although these methods are highly sensitive, the costs of the reagents, requirement of sophisticated instruments make it unsuitable for large-scale field evaluation. Besides this, precautions should be taken to avoid the risk of contamination of reagents with target DNA to prevent false positive results.

15 Ultrasound Ultrasonography has been used to locate the adult W. bancrofti worm in the scrotal lymphatics of the infected patients using a 50 MHz sectorial transducer (Amaral et al 1994). This is possible because the live adult worms have a distinctive pattern of movement called the filaria dance sign where the parasite is seemingly attached to the endothelium (Suresh et al 1997). Ultrasound examination of the scrotal area of infected men provides the only noninvasive method with which to directly monitor the macrofilaricidal efficacy of antifilarial drugs (Dreyer et al 1995). However, this test is not suitable for routine field studies in view of the technical expertise involved Lymphatic imaging Lymphangiography Alterations in lymphatic anatomy of filarial patients were observed initially using lymphangiography (Sen and Ellappan 1968). However, the method is technically demanding, time consuming, invasive and uses oil based contrast material for imaging that can induce local morbidity aggravating the pathology (Kobayashi and Miller 1987). Thus this technique is not feasible for field studies Lymphoscintigraphy In lymphoscintigraphy, radiolabeled albumin or dextran injected intradermally or subcutaneously is traced by gamma camera (Witte et al 1993). Studies using this technique demonstrated the presence of lymphatic abnormalities in Microfilaraemics (Freedman et al 1994). Thus this technique provide clues on the lymphatic system in individuals at risk. The advantages of the method is the ease to perform and the noninvasiveness of the technique.

16 16 Because of the high cost and this method is unsuitable for routine field studies. Even though each diagnostic tool has its own merits and demerits in terms of specificity and sensitivity, the Og4C3 antigen test seems to be a good compromise for the diagnosis of Bancroftian filariasis. The other commercially available antigen detection test, ICT card test is now widely accepted as the tool of choice for survey work because of its simplicity and on the spot detection methodology. Diagnosing an individual for the presence of filarial parasite or its components at an early stage may prevent the development to symptomatic pathology. Hence a sound knowledge of control and elimination strategies are a must to combat symptomatic pathogenesis in the endemic areas Control and Elimination Strategy for Lymphatic Filariasis Global programme to eliminate lymphatic filariasis Due to the lack of control tools and cost-effective strategies, filariasis has been persistent in the endemic countries. An initiative of WHO (1997) to combat this nematode blight led to the development of the global programme for the elimination of lymphatic filariasis (focusing on disease caused by W. bancrofti) as an important strategy to rescue the people who are facing severe embarrassment owing to the disease. This program was recommended earlier by the International Task Force for Disease Eradication in 1993 based on the idea that Lymphatic Filariasis was one of six diseases that could, in theory, be eradicated. Therefore, the Global Programme to Eliminate Lymphatic Filariasis (GPELF) coordinated by the World Health Organization (WHO) was launched in The two principal goals of the GPELF are: a) to interrupt transmission of infection; and (b) to alleviate and prevent the suffering and disability caused by the disease.

17 Interruption of transmission (Treatment regimes) Mass Drug Administration (MDA) has been adopted in endemic countries with an aim to interrupt the transmission of lymphatic filariasis by treating all members of the endemic community at the same time that reduces the blood microfilaria levels significantly. This was done with the idea of treating pre as well as post patent infection. The strategy prescribes: (i) Annual treatment with single dose of two drugs given together (albendazole plus either Ivermectin or diethylcarbamazine (DEC) for 4-6 years, and (ii) Exclusive use of DEC-fortified table or cooking salt for 1-2 years Prevention of disability The purpose of GPELF in preventing disability caused by lymphatic filariasis - lymphedema and hydrocoele lies in: (i) Community home-based self care for lymphedema through support services and (ii) Access to surgery for lymphatic filarial patients with hydrocoele Treatment and disease management Chemotherapy Chemotherapy for filariasis relies heavily on the filaricidal drugs, DEC (diethyl carbamazine) and Ivermectin. Though the exact mechanism of action of DEC is not known, DEC has a broad spectrum of activity against intestinal helminthes and is also effective as a macro- and microfilaricide. The chemical name of DEC is 1-diethylcarbamyl-4-methyl piperazine, formulated and commercially marketed under the trade names Hetrazan, Banocide and Notezine. Since its introduction in 1947, a single dose of DEC is essentially the only drug used for treating lymphatic filariasis in giving long-term reductions in microfilaria levels (~90% reduction of pre-treatment levels even one year after treatment). An annual single dose of 6 mg per kg of body

18 18 weight was effective in reducing mf prevalence (Kimura and Mataika 1996). The target of DEC was shown to be cholinergic receptors in the muscles of the parasite causing paralysis and dislocation of the parasite in the host (Langhman and Kramer 1980). Further DEC causes alterations in the microfilarial surface membranes and makes the parasite susceptible for destruction by host defence mechanisms (Hawking 1979; Mackanzie and Kron 1985). Due to adverse reactions of DEC, this drug is not recommended for treatment in areas of filarial infections where coexistence of Onchocerciasis or Loasis is found Ivermectin when given as a single dose effectively reduces microfilaremia by ~90% just as safely as DEC (Cao et al 1997). This drug is commercially available under the trade name Stromectol by Merck and Co., Inc. It disrupts the neuromuscular activity of the parasites by binding to glutamate gated chloride channels and has been extensively used in the clinical trials of Onchocerciasis (Chodakewitz 1995). A single dose administration of 400 g per kg of body weight was shown to be effective (Moulia-Pelat et al 1994). This is the most important break through in the development of new treatment regimens in lymphatic filariasis. Thus, onceyearly, single-dose treatment with either DEC or Ivermectin has become a feasible control strategy to reduce microfilaremia and thus prevent the onset of filarial infections. A major advancement in chemotherapy came from the discovery that, single doses of DEC and Ivermectin administered together were more effective than either drug alone, and were equally safe (Cao et al 1997). Subsequent studies have shown that this combination-effect is not just limited to DEC-plus Ivermectin but also found for Ivermectin-plus-Albendazole (Addiss et al 1997; Ismail et al 1997) and DEC-plus-Albendazole (Ismail et al 1997).

19 19 Albendazole has already been widely recognized as a powerful antiparasitic agent for many other types of parasitic infections such as Ascaris, Trichuris, Hookworms, Strongyloides, Onchocerca, Cysticercosis, Echinicoccosis, Trichomonads and Microsporidia (Ottesen et al 1999). It disrupts microtubule formation by affecting the normal function of helminth s gut cells and its ability to obtain nutrients. The repeated high dose of Albendazole was shown to have macrofilaricidal effect against W. bancrofti (Jayakody et al 1993). No clinical adverse reactions in the course of treatment suggest that it has a primary effect on the adult parasite, resulting in slow decrease in microfilaremia. The simultaneous administration of Albendazole and Ivermectin was shown to be effective in the reduction of microfilariae in Bancroftian filariasis than does Ivermectin alone (Ismail et al 2001). An analogue of ubiquinone (2, 3-Dimethoxy-5-methyl1-1, 4-benzoquinone) was shown to produce irreversible paralysis in the adult parasites and microfilariae of the bovine filarial parasite Setaria digitata and W. bancrofti (Sivan and Kaleysa Raj 1999). In addition to these Benzimidazole compounds have also been used in the treatment of cattle infected with O. gibsoni. Apart from these new annual treatment tools, there is an alternative public health strategy for reducing microfilaremia that utilizes DEC-fortified salt as a replacement for normal table/cooking salt (Gelband 1994). Studies especially, in China and India, indicates that utilization of such salt for 4 months to 4 years leads to dramatic reduction in microfilaremia and subsequent interruption of transmission (Subramanyam and Venkateswaralu 1996). The use of Filaricidal drugs, their mode of action and the associated toxic effects are summarized in Table 1.1.

20 20 Table 1.1 Anti filarial drugs used in chemotherapy Drugs Mode of action Toxic effect Macrofilaricidal Suramin Macro and Microfilaricidal Diethylcarbamazine (DEC) Microfilaricidal Ivermectin Metrifonate Antihelminthes Albendazole Levamisole Unknown Sensitizes mf to phagocytosis Activates GABA pathways and chloride channel permeability Inhibits cholinesterases and paralyses the worm Inhibitory effects on tubulin polymerization which results in the loss of cytoplasmic microtubules Interferes with the carbohydrate metabolism and inhibits the production of succinate dehydrogenase, causing paralysis of the worms Nausea Fever, nausea, headache Rarely nausea and abdominal pain Rarely nausea and vomiting Unusual Unusual Thus, multiple tools are now available to bring down microfilaremia levels in affected communities to values where it is unlikely that transmission can occur. Although the drug combinations that are used in the filariasis elimination programs might prevent the development of resistance to any individual drug, clearly this is an area of research that merits further investigation. Along with the chemotherapy strategies for the elimination of microfilaria, the other important aspect of treatment lies in the

21 21 maintenance of careful hygiene in infected persons to reduce the incidence and severity of secondary (e.g. bacterial) infections Disease management and control Through regular exercise and elevation of the limbs, the subclinical lymphatic damage of the microfilaremics can be prevented from the risk of developing clinically apparent lymphatic disease. Severe elephantiasis of male external genitalia is managed effectively by surgical excision of affected tissue and split skin graft (Ollapallil and Walters 1995). Hygiene and skin care are essential to prevent secondary bacterial infections in lymphoedema and patients with clinical elephantiasis. Periodical cleaning of the affected limb and topical application of antibiotics results in suppression of adenolymphangitis (Shenoy et al 1995). Use of pneumatic pumps with rhythmic cycles of pressure (available from AMLA Mediequip, New Delhi, India) was effective in the disease management of limb elephantiasis. Complex physical therapy, heat treatment and nodo-venous anasthemosis were the other methods of lymphoedema management (Manokaran and Jamal 1996) Vector control Apart from the treatment and personal care, vector control is another important strategy for the elimination of lymphatic filariasis. Reduction in vector density has a long term effect in transmission interruption. Vector control strategies include the use of insecticide-treated clothing, wearing of protective clothing, and a correct use of DEET-based topical insect repellents. It has been suggested that practical insect avoidance measures, combined with pyrethroid-treated nets and clothing, and DEETbased topical repellents, can achieve almost 100% protection against biting arthropods (Croft et al 2001). Vector control by highly active and persistent

22 22 insect growth regulator can also be a very effective strategy (Yapabandara et al 2001). Since mosquitoes have gained resistance to chemicals like DTT, biological weapons like biocides from Bacillus sphaericus and Bacillus thuringiensis might play a major role in vector control (Porter et al 1993). Since disease management by controlling the mosquito vectors and mass drug administration has its own limitations, recent studies have suggested that vector control integrated with the mass drug administration will be potential initiative in the success of filariasis elimination programme (Burket et al 2006) Genome Based Approach for the Control of Lymphatic Filariasis Helminth genomics Since all the filarial nematodes are closely related, intensive genome analysis of one species would serve as the model in the study of other filarial species. Therefore B.malayi was chosen as a model in the genome analysis as it can complete its life cycle in the laboratory animal models. The types of sequence data that are available fall into three basic categories: a) Complete or nearly complete genome sequences, which are normally released as contigs of overlapping sequence reads; b) Genome-survey sequence (GSS) tags, which are generated by skimming genomic sequences (either random clones or bacterial artificial chromosome [BAC] ends); and c) Expressed sequence tags (ESTs), which are generated from mrnas expressed during one or more stages of the parasite life cycle.

23 23 The purpose of genome analysis was to identify genes and their products to shed light on the important parasitic processes of development, metabolism, invasion, host evasion and so on (Tarleton and Kissinger 2001) Filarial Genome Project (FGP) FGP was initiated in 1994 by the World Health Organization, with four main goals: (1) to identify a large number of novel genes from new cdna libraries from all life cycle stages of B. malayi; (2) to construct genomic libraries and map the Brugia genome; (3) to devise and implement globally accessible database for display; and (4) to establish and foster partnerships between laboratories in non-endemic and endemic countries to aid in the training of scientists from endemic nations in DNA sequencing, mapping and all aspects of genomics. FGP was initiated by the WHO with a core funding by WHO Tropical Diseases Research (TDR), and has attracted support from other agencies such as the Edna McConnell Clark Foundation, Medical Research Council (UK), New England Biolabs, and institutional sources (Blaxter 1995) to target five disease organisms such as Filaria, Schistosoma, Leishmania, Trypanosoma brucei and T. cruzi. The WHO sponsored an international collaboration of seven endemic and non-endemic laboratories (Table 1.2) to implement a program of gene discovery, genome mapping, and post-genomic analysis of B. malayi (Blaxter et al 1999). The availability of all the life cycle stages was the determining factor in selecting Brugia malayi over other filarial nematodes for the genome sequencing. B.malayi nuclear genome is organized as five chromosomes pairs, four autosomal and one XY sex determination pair. Male worms possess 8 small, one large and one medium elements and female worms possess 8 small and 2 large elements (Sim et al 1987). As an outcome of the

24 24 FGP, B. malayi was the third nematode and the first parasitic nematode to be fully sequenced, following the model organisms C. elegans and C. briggsae (Ghedin et al 2004). The ~90-95 megabase (Mb) genome of the human filarial parasite B. malayi was sequenced and predicted to contain ~11,500 protein coding genes (Ghedin et al 2007). Table 1.2 Collaborators of FGP 1 Department of Biological Sciences, Clark Science Center, Smith College, Northampton, MA USA 2 Institute of Cell, Animal and Population Biology, University of Edinburgh, Edinburgh, UK EH93JT 3 Centre for Biotechnology, Anna University, Chennai, , India 4 Research and Training Centre on Vectors of Diseases, Faculty of Science Building, Ain Shams University, Abbassia Square, Cairo, Egypt 5 Department of Molecular Microbiology and Immunology, School of Hygiene and Public Health, Johns Hopkins, University, Baltimore, MD 21205, USA 6 New England Biolabs, 32 Tozer Road, Beverly, MA , USA 7 Department of Parasitology, Faculty of Medicine, University of Indonesia, Salewba 6, Jakarta 10430, Indonesia An important out put of FGP with tremendous implications has been the availability of parasite stage specific cdna libraries. This along with the advancements in recombinant DNA technology made the novel gene identification and recombinant protein characterization, an easier task. Thus in-house strategies were initiated globally, to evaluate the diagnostic and prophylactic capabilities of filarial antigens.

25 Recombinant proteins Recombinant DNA technology facilitated the possibility of expressing the target genes in different host cells, providing the scientists with copious amounts of proteins. The choice of host system depends on many factors, such as the size, structure and stability of the gene product, and the requirements of post-translational modifications for biological activity. Among the many systems available for heterologous protein production, the Gram-negative bacterium Escherichia coli happens to be an ideal host, due to its well-characterized genetics and the availability of an increasingly large number of cloning vectors and mutant host strains. Apart from this, the other hosts used for recombinant protein production range from simple prokaryotic organisms (bacteria) to unicellular eukaryotic organisms such as yeast and the more complex eukaryotic insect and mammalian cells. The T-7 expression system especially prset vectors have been used at our centre to express most of the genes that are identified through FGP T-7 expression system Apart from the gene of interest, E. coli expression vector should contain, an origin of replication, selectable marker (like a gene that confers antibiotic resistance), a promoter and a transcription terminator. Bacteriophage T7 has been used in the study of DNA replication due to its linear chromosome and its ability to encode its own DNA polymerase. T7 promoters also require a special RNA polymerase; as a result, they were incorporated into a number of cloning / expression vectors. The prset vectors are gene expression systems based on T7 RNA polymerase (Studier and Moffatt 1986). Transcription by T7 polymerase is selective and active that, almost all of the cell s resources are converted to target gene expression and the desired product can comprise more than the rest of the total cell protein after few hours of induction, thus leading to higher expression of

26 26 genes cloned under T7 promoter. prset A vector must be used in a strain carrying a chromosomal T7 RNA polymerase gene that is under the control of a lac promoter. Typically, production hosts contain a prophage ( DE3) encoding the enzyme under control of the IPTG or lactose -inducible lacuv5 promoter. The constitutive expression of high levels of almost any protein is toxic to the bacteria. The protein itself may be toxic, or the simple competition for cellular resources can lead to poor growth of the bacterial host, and in some cases, cell death. Therefore, it is necessary to regulate the production of high levels of recombinant protein. This vector also contains a nucleotide sequence that encodes a metal binding domain, a series of six consecutive histidine amino acids expressed as N-terminal fusion to the protein of interest. This metal binding domain (six-tagged histidine moieties) on the fusion peptide has high affinity for the divalent ions (like nickel, copper and cobalt) and facilitates one-step purification of the protein using (IMAC) immobilized metal affinity columns (Crowe et al 1995). Due to its small size, histidine tag is non-immunogenic and histidine tagged recombinant proteins can be used in immunological studies without employing tedious proteolytic cleavage procedures generally required in other recombinant fusion protein systems. However, if necessary enterokinase cleavage site facilitates the removal of histidine tag. The other advantages of the vector include its small size, presence of ribosomal binding site, multiple cloning site and ampicillin resistance marker (Appendix 3). Genes identified and characterized at our centre with their functions are given in Table 1.3.

27 27 Table 1.3 Genes identified and their functional significance Genes Functional importance BmSXP-1 Diagnosis WbSXP-1 Diagnosis BmR1 Diagnosis Abundant larval Transcript-2 (WbALT) Prophylaxis Cuticular collagen (BmCol-2) Prophylaxis Venom (Vespid) Allergen Homolog (Wb VAH) Prophylaxis Bm Transglutaminase Prophylaxis Nucleotide diphosphate kinase (NDK) Drug target N-acetyltransferase Drug target Cyclophilin Drug target Wb Thioredoxin Antioxidant Bm-TPx (Thioredoxin peroxidase) Antioxidant Catalase Antioxidant Bm-GST (Glutathione S- Transferase) Antioxidant Bm-SOD (Superoxide dismutase) Antioxidant Bm-MIF Immune evasion Bm-tgh-2 Immune evasion Bm-Serpin Immune evasion Cystatin Immune evasion Bm- 33 Immune evasion A major set back to filariasis research has been the lack of an animal model. The experimental usage of Jirds or Gerbils (Meriones unguiculatus), that are the permissive hosts in case of Brugia still suffer, due to the lack of appropriate reagents. Humans are the only definitive host for lymphatic filariasis caused by W. bancrofti (accounting for 90% of the cases), and no animal models are available to study this parasite development. Inspite

28 28 of these shortcomings many parasite proteins have been evaluated for their diagnostic and prophylactic capabilities. Chitinase, cloned from the microfilarial stages of B.malayi has been shown to be a promising transmission blocking candidate (Fuhrman et al 1992). Reduction in the adult worm burden and blood microfilaraemia by Paramyosin (an immunodominant antigen derived from the L3 stage of the parasite) has been demonstrated in Jirds (Li et al 1993). Another promising candidate vaccine for filariasis is the Abundant Larval Transcript (ALT-1) cloned from B. malayi (Gregory et al 2000). Besides this, SXP-1, a multiple stage antigen cloned from B. malayi has been shown to provide partial protection in Jirds (Wang et al 1997). Recent studies on Wb VAH, from our centre (Anand et al 2007) suggested a predominance of IgG1, G2 and G3 isotype antibody reactivity in putatively immune individuals and G3 reactivity in chronically infected population. Wb VAH specific cellular responses in EN demonstrated a profound increase in PBMC proliferation supported by elevated IFN-γ in EN compared to other groups. Studies in experimental models are currently in progress to evaluate the protective efficacy of this antigen. Several other recombinant antigens have been evaluated for their diagnostic potential and reviewed in the immunodiagnosis section. Characterization of the recombinant proteins, structurally and functionally has provided insights into the host-parasite interactions (Schierack et al 2003; Stanley and Stein 2003; Winter et al 2003). Further, several studies including those from our centre, have used various recombinant filarial proteins in understanding the immune responses to recombinant antigens (Raman et al 1999; Gnanasekar et al 2002, Sasisekhar et al 2005a; Krushna et al 2009). However the applicability of the candidate Antigen in the disease spectrum of

29 29 a complicated disease like lymphatic filariasis depends to a great extent on the immune responses that operate among the endemic population Endemic Population- Spectrum of Responses A spectrum of responses is seen in the people living in an area endemic for lymphatic filariasis. These individuals can be broadly divided into 3 groups based on their clinical and immunological characteristics (Dasgupta 1984; Partono 1987; Ottesen 1992, 1993, 1994; Evans et al 1993; Roberts and Janovy 1996): (1) people who are exposed, but with no evidence of disease so called Endemic Normals (EN), (2) asymptomatic people with microfilaraemia termed as Microfilaremics (MF); (3) chronically infected people with disease manifestations such as chronic lymphoedema, hydrocele and elephantiasis Chronic Pathology (CP) Asymptomatic Amicrofilaraemic Endemic Normals (EN) Endemic normals are a group of individuals living in an area endemic for filariasis and remain free of symptoms or microfilaria inspite of being repeatedly exposed to the mosquito bite. These individuals have a high serum IgG levels compared to infected people (Ottesen et al 1984). The rationale that EN people being infection free might be due to the immunity they developed against invading L3 form of filarial worm (Maizels and Lawrence 1991). Filaria specific IgG4 antibody, an active marker for filarial infection is low in these group of individuals (Ottesen et al 1985). The total IgE, parasite specific IgE and eosinophil levels did not differ from those seen in carrier group of individuals and chronic patients whereas the levels of filarial specific IgG1, IgG2 and IgG3 isotypes are diminished (Yazdanbakhsh et al 1993).

30 Asymptomatic Microfilaraemics (MF) These groups of individuals remain as asymptomatic, inspite of the presence of micofilaria in the peripheral blood. Though asymptomatic, these individuals show some degree of subclinical disease such as haematuria and/or proteinuria with low-grade renal damage (WHO 1994), gross damage of lymphatics of the limbs (Freedmam et al 1994) and occasional filarial fever. Ultrasound examinations have shown that approximately half of the men with asymptomatic microfilaraemia have nests of motile adult worms in their scrotal lymphatics, the "filarial dance sign" (Amaral et al 1994). When compared to putatively immune normal individuals and chronically infected people microfilaraemics were found to have very low levels of filarial specific antibodies (Piessens et al 1980a; Ottesen et al 1982). One of the hallmark of filarial infections is that MF individuals with active microfilaremic infection show elevated levels of filarial-specific immunoglobulin of the IgG4 isotype (Kurniawan et al 1993). In humans, this is normally the least abundant IgG isotype, comprising only 5% of antibody in the bloodstream, however, in filarial patients IgG4 can be as much as 95% of the blood immunoglobulin. IgG4 is upregulated by the type 2 cytokine, IL-4. Levels of IgG4 fall rapidly following chemotherapy for filariasis, thus high levels of IgG4 are often diagnostic of active infection (Kurniawan et al 1995). IgG4 has been shown to act as either a blocking antibody (Hussain et al 1992) by competing with IgE for antigen binding (Lundgren et al 1989) or the high expression of IgG4 might induce a regulatory constraint to minimize IgE production (Maizels and Lawrence 1991). Filarial antigen specific hyporesponsiveness is the characteristic of these MF individuals (Mahanty et al 1997; Ravichandran et al 1997). Clonal anergy as a mechanism responsible for hyporesponsive state is supported by the observation of low numbers of parasite-specific T and B-lymphocytes in