Lymphatic filariasis: new insights and prospects for control Thomas B. Nutman

Lymphatic filariasis: new insights and prospects for control Thomas B Nutman Although lymphatic filariasis remains among the major causes of disability among the tropical infectious diseases, dramatic advances have been made in the approach to its diagnosis, epidemiology and treatment, in our understanding of the molecular composition of the parasites that cause these infections, and in the factors underlying the pathology seen Superimposing the tools of modern epidemiology, immunology, and molecular biology on field-based clinical trials has allowed the emergence of the concept of elimination of lymphatic filariasis Much of the important new research emphasizes parasite development in the context of the host response, the importance of both the adult worm and other factors in the pathogenesis of lymphatic filarial disease, the role the Wolbachia endosymbiont holds as both a target for drug treatment and in inducing post-treatment reactions, and the various principles underlying the implementation of control programs Curr Opin Infect Dis 14:539±546 # 2001 Lippincott Williams & Wilkins Helminth Immunology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA Correspondence to Thomas B Nutman MD, Head, Helminth Immunology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-0425, USA Tel: +1 301 496 5398; fax: +1 301 480 3757; e-mail: tnutman@niaidnihgov Current Opinion in Infectious Diseases 2001, 14:539±546 Abbreviation IFNg interferon-gamma # 2001 Lippincott Williams & Wilkins 0951-7375 Introduction Among the approximately 129 million patients infected with one of three lymph-dwelling lariae ± Wuchereria bancrofti, Brugia malayi, or B timori ± W bancrofti is responsible for the overwhelming majority (*115 million) [1 ] Although there are many differing clinical manifestations of longstanding infection with the lymphatic lariases (hydrocele, recurrent adenolymphangitis, lymphedema, elephantiasis or tropical pulmonary eosinophilia ± see [2 ] for a comprehensive review), the most intriguing (from an immunological perspective) is a subclinical condition associated with high levels of circulating micro lariae (or parasite antigen) Indeed, in areas where W bancrofti or B malayi are endemic, the overwhelming majority of infected individuals have few overt clinical manifestations of their larial infection despite the presence of circulating micro lariae (or parasite antigen) in the peripheral blood Although they may be clinically asymptomatic, virtually all persons with patent W bancrofti or B malayi infection have some degree of subclinical disease that includes microscopic hematuria or proteinuria [3], dilated (and tortuous) lymphatics when imaged [4,5,6 ] and, in men with W bancrofti infection, scrotal lymphangiectasia [7 ] This subclinical majority notwithstanding, current estimates suggest that lymphatic lariasis, among the reported tropical diseases, is responsible for the third most global burden of disease (behind malaria and tuberculosis) with a burden estimated at 49610 6 disability-adjusted life years lost [8] The parasite W bancrofti and the two Brugia species are transmitted by mosquitoes Each goes through a complex life cycle that includes an infective larval stage (L3) carried by the insects and an adult worm stage that can be found either in the lymph nodes or adjacent lymphatics The offspring of the adults, the micro lariae (220±280 mm long and 5±7 mm wide), circulate in the blood These micro lariae then can be ingested by a biting mosquito where they develop in the arthropod over a 1±2 week period into infective larvae, which are in turn capable of re-initiating the life cycle Parasite development Many aspects of the developmental biology of larial parasites await elucidation This lack of insight has stemmed, in part, from the lack of consistent in-vitro methods for culturing the early life cycle stages of the parasite [9 ], particularly using de ned conditions A serum-free system has been developed that supports the 539

540 Tropical and travel-associated diseases development of the infective stage larvae (L3) into viable fourth-stage larvae [10], providing the capability, at least, to examine the minimum requirements for early parasite development in the mammalian host Parasite genetics and molecular biology The larial nuclear genome (based primarily on work done with B malayi) is estimated to be around 100 million base pairs, about the same size as that of the free living nematode Caenorhabditis elegans The genome is AT rich (71%) and is divided among ve pairs of chromosomes (four pairs of autosomes and one pair of sex chromosomes) Within the parasite two other genomes are found, the rst being a mitochondrial genome and the second being the genome of the bacterial endosymbiont (see below) The Filarial Genome Project, established in 1994, focused on gene discovery using an expressed sequence tag approach Thus far, more than 22 200 expressed sequence tags have been completed, and more than 7000 genes have been identi ed [11 ] There is a new initiative to sequence the entire genome as this approach is now more feasible given the advances in sequencing technologies The population biology of any of the lariae is poorly understood, and approaches relying on repeated sequences (used primarily for diagnostics) of DNA have failed to consistently identify signi cant diversity among related isolates [12,13] Recently, microsatellite loci from B malayi have been able to reliably distinguish among isolates from differing geographic locations [14 ] This methodologic `proof of principle' should help to address important population genetic questions in larial biology such as drug susceptibility, emergence of new isolates, genetic bases of periodicity, among others Additional attention has also been focused on individual molecules that may be targets for chemotherapy [15,16] and vaccine development [17,18,19 ]; moreover, there has been the molecular characterization of parasite molecules that may serve to modulate host immune functions such as T cell cytokine production or antigen presentation [20,21], induction of apoptosis [22] or those parasite transcription factors that may be regulated by host growth factors [23] Endosymbiont With the reemergence of interest in the intracellular Wolbachia endosymbiont of larial parasites [24,25] and the recent demonstration of their potential as targets for macro laricidal chemotherapy [26,27,28±32], there has been renewed attention paid to the biological makeup of the larial Wolbachiae [11,28,33,34,35] Indeed, there is not only a Wolbachia Genome Consortium in place to sequence the entire B malayi endosymbiont [11 ], but also, by using comparisons of the wsp, 16S rrna, and ftsz gene sequences, the phylogenetic relationships among various larial Wolbachiae and those of arthropods have been made [28,33,35] Moreover, there remains the possibility of transferring the endosymbionts from lariae to insect cell lines, already being utilized for maintaining insect endosymbionts, a process that will provide both material and methods to enable the study of the molecular organization of this critical intracellular bacterium [11 ] Clinical disease and its socioeconomic burdens Although many of the manifestations of lymphatic lariasis have been known for centuries, some much needed attention has recently been paid to several important clinical aspects These include an examination of the sexual dysfunction and reproductive health associated with genital disease in lymphatic lariasis [36,37,38] These studies have demonstrated quite clearly that the degree of sexual dysfunction, particularly among men with hydroceles and lymph scrotum [36,37], is great and relatively unexplored Moreover, qualitatively collected data on school age children in a larial endemic region of India suggested that hydoceles and adenolymphangitis played a major role in poor performance in school, absenteeism and dropout [39] As part of an ongoing effort to assess the economic burden of lymphatic lariasis, in India, for example, lymphatic lariasis has been estimated to be responsible for the loss of about 063% of the per capita gross national product [40 ] The economic impact was equally important in Ghana where it was found that in some communities more than 20% of all elective surgery was for hydrocele operations [41,42] Much of these and other data have been codi ed and reviewed by Haddix et al [43 ] Pathogenesis Recent evidence from detailed clinical, surgical and imaging (ultrasound or lymphoscintigraphy) observations and from immunohistologic analysis of tissue from lymphedematous limbs of affected patients has suggested that compromised lymphatic function and lymphangiectasia play a signi cant role in mediating some of the pathology associated with lymphatic larial infections [7,44] This type of pathology appears to be related to the presence of viable adult worms in the lymphatics or lymph nodes [44] Moreover, bacteriologic studies of tissue from lymphedematous limbs of affected patients [45] has implicated bacterial or fungal superinfections of limbs superimposed on already compromised lymphatic function as playing a signi cant role in triggering some episodes of adenolymphangitis which in turn may exacerbate the chronic obstructive changes in the lymphatics of affected patients These studies have

Lymphatic filariasis Nutman 541 shown that the acute process usually starts in the skin and then spreads along the lymphatics to the lymph nodes Based on these observations, some have proposed that the syndrome be renamed dermatolymphangioadenitis [46 ] Although the importance of any one among the many factors involved in the pathogenesis of lymphedema, hydroceles, and elephantiasis remains still a matter of debate, the relative paucity of pathology among those with `active' or patent infection ± the presence of micro lariae and adult worms (as assessed by circulating antigen assays, polymerase chain reaction of blood, or ultrasonography) ± and its association with the inability of patient cells to proliferate or produce interferongamma (IFNg) in response to parasite antigen has been consistently found [47] A useful paradigm with which to understand the changes that the immune responses undergo over time is to divide the larial infection into `acute' (or early) and chronic phases [48,49] The `early' or prepatent infections appear to be associated with establishment of parasite-speci c lymphocyte proliferation, cytokine production with a mixed (interleukin-2, IFNg, interleukin-4, interleukin-5) phenotype, marked eosinophilia, and elevated levels of parasite-speci c IgE These ndings have been bolstered by the studies in permissive animal models (eg jirds, cats, dogs, monkeys and chimpanzees) in which most of these features have been seen early in infection With the onset of patency (development of adult worms and appearance of micro- lariae in the blood), certain immune responses are profoundly altered [48,49] Most notably, there is a diminution of parasite-speci c lymphocyte proliferation, interleukin-2 and IFNg production and an increase in anti larial IgG4 and the production of the regulatory cytokines interleukin-10 and perhaps transforming growth factor-b The development of this downregulated immune response is determined not only by the duration of infection but also by parasite burden (number of adult worms and micro lariae), both of which can be a function of the intensity of transmission [50 ] In areas of intense transmission, infections are acquired at younger ages and higher parasite burdens likely occur Immunology The most recent immunological studies on lymphatic dwelling lariae fall into two discrete groups First are the studies in animal models (particularly rodents) which have been increasingly used to examine the fundamental nature of the immune response to infection, particularly as it relates to tolerance induction [51,52,53,54±57], innate immunity and resistance to infection [58,59±63] Studies in humans have focused recently on the nature of protective immunity to lymphatic lariasis [49,64,65,66 ] and the evolution of the immune response underlying this protection [67 ] The immunological basis for protective immunity in humans has been notoriously dif cult to de ne in part because diagnostic tools were not suf ciently sensitive to determine that an individual was truly free of infection and in part because longitudinally collected information on exposure and infection status was generally unavailable for at risk populations With these as caveats, protective immunity to the micro lariae of W bancrofti appeared to be related directly to levels of antibody to the micro larial sheath [68] In two separate studies examining the humoral responses in so-called `endemic normal' individuals ± those with a clear history of exposure but lacking clinical disease and circulating antigenemia ± resistance to infection was associated with both anti-carbohydrate antibodies of the IgG2 and IgG3 isotypes [65] or with antibodies directed against L3 surface antigens [64] Using assessments of both cellular and antibody responses in a cohort of subjects studied longitudinally over a 17-year period in the Cook Islands, endemic normal individuals appeared to maintain their ability to respond to parasite antigen with cytokine, proliferative and antibody responses to a much greater degree than did patently infected individuals [67 ] Modulation of the immune response in larial infections has been examined in animal models in the context of (1) CD4+ T cell subset differentiation (Th1 versus Th2) [53,55±57], or (2) antigen presenting cell function [51,52,53,54] Clearly associated with the modulation of the immune response is diminished expression of the important costimulatory molecule CD80 [51 ] on antigen presenting cells, antigen presenting cell nitric oxide production [52,69] and the recruitment and presence of `alternatively activated' macrophages that inhibit parasite-speci c lymphocyte proliferation [53,54] Using a murine model in which micro lariae were used to drive the immune response, even in the presence of induced IFNg, the Th2 response predominated [56] Taken together, these and other [55,57] studies suggest several basic mechanisms by which the immune response to lymphaticdwelling lariae become downregulated In murine studies examining both innate and adaptive immune responses as mediators of protective immunity in lymphatic lariasis, attention has been focused on the role of the interleukin-4, interleukin-5 and IFNg in mediating resistance to infection While no consensus has emerged, both interleukin-4 [60±62] and interleukin- 5 [63] have been shown to play a major role In addition, the nding that B1 B lymphocytes, cells that appear to be induced in response to phosphorylcholine containing antigens from the larial parasites, can mediate protection provides a link between the adaptive and innate immune mechanisms [58,59]

542 Tropical and travel-associated diseases Diagnosis Although the detection of the micro lariae in the blood has been the mainstay of the diagnosis of lymphatic lariasis, the identi cation of the motile adult worms within the lymphatics (termed the ` laria dance sign') using high-frequency ultrasound in conjunction with Doppler has been found quite useful for diagnosis of bancroftian lariasis Although the transfer of ultrasound diagnosis from W bancrofti to B malayi infection was assumed likely, to date ultrasonography has failed to detect the adult B malayi adults [70 ] For W bancrofti infection, at least, both of these methods have been largely supplanted by detection of circulating larial antigen There are currently two commercially available tests, one in an enzyme-linked immunosorbent assay format [71] and the other a rapid-format immunochromatographic card test; each of these assays have sensitivities that range from 96 to 100% and speci cities that approach 100% [72] Because there are currently no tests for circulating antigens in brugian lariasis, there have been attempts to improve the diagnosis of brugian lariasis To this end, recombinantly-produced antigens with speci city for B malayi have been identi ed and shown, in preliminary experiments, to improve the diagnostic sensitivities for these infections [73,74 ] Polymerase chain reaction based assays for DNA of W bancrofti and B malayi in blood have also been developed, with the method being of equivalent or greater sensitivity than standard parasitologic methods The utility of these molecular diagnostics, however, has not been well assessed using blood obtained when micro lariae are not present (eg day blood of nocturnally periodic lariae) In a number of recent studies, methodologies for detection of DNA free of intact parasites have been developed for B malayi infections [75,76,77 ], although such techniques have been less successful in bancroftian lariasis [78] Occasionally, diagnostic aspiration for cytologic examination or as a consequence of excisional biopsy results in the identi cation of micro lariae in the specimens [79±83] Treatment In support largely of efforts to assess the impact of treatment on transmission with an eye toward control and possible elimination of lymphatic lariasis as a public health problem, a number of studies using single dose treatment with diethylcarbamazine, albendazole, or ivermectin alone or in various combinations have been performed in multiple geographic settings [42,84±88] Based on the cumulative data from 17 studies conducted around the world with a cumulative total of more than 90 000 individuals (with primarily W bancrofti infection), a review of the safety and ef cacy of the regimens to be used in the programs to eliminate lymphatic lariasis (albendazole + ivermectin or albendazole + diethylcarbamazine) suggests that the addition of albendazole to either diethylcarbamazine or ivermectin is no less safe than therapy with a single medication [89 ] and may provide additional anti larial ef cacy along with salutary effects on intestinal helminth infections [90,91 ] The macro laricidal effects of diethylcarbamazine or albendazole alone or in combination therapy ± assessed directly by ultrasound or indirectly by antigen detection assays ± have demonstrated a consistent but partial effect [44,89,92,93 ] Regimens of higher dosage, longer duration or newer combinations have been suggested, as has the addition of antibiotics to standard single dose therapy (as has been used recently for onchocerciasis [27 ]) New approaches to anti larial chemotherapy, which include targeting the endosymbiont of the lymphatic larial parasites with antibiotics, particularly the tetracyclines [29,32,94,95], appear to be the most promising way forward in macro laricidal therapy of the lymphdwelling lariae Although clinical studies in lymphatic lariasis have not been completed, data from Onchocerca volvulus (in humans) and larial infections in animal models suggest that this approach should be feasible [27,29±32] Further, it is possible that the release of the intracellular endosymbionts and their lipopolysaccharide is responsible for the early in ammatory reactions seen following treatment of lymphatic lariasis [26,96] Agents such as UMF-078 [97] and an extract from a medicinal plant [98] have been shown to be active against the adult worms of B pahangi; in other drug screens, however, neither phosphoramidate inhibitors of b-tubulin [99] nor a set of anticancer agents [100] have been shown to possess activity against the lymph dwelling lariae Control The development of a global program to eliminate lariasis came following a resolution by the World Health Assembly in 1997 [26,90,91,101,102] The principal goals of the program are to interrupt transmission of infection and alleviate and prevent the disability caused by the disease [91 ] Primarily using community wide distribution [103] of yearly single dose combination chemotherapy ± diethylcarbamazine + albendazole or ivermectin + albendazole (made possible by a donation of albendazole by SmithKline Beecham/GlaxoSmith- Kline (Greenford, Middlesex, UK) with an additional commitment of Merck & Company (Whitehouse Station, New Jersey, USA) to expand the donation of ivermectin to African countries in which onchocerciasis and lymphatic lariasis are co-endemic) ± for transmission

Lymphatic filariasis Nutman 543 interruption [103] and strategies focusing on hygiene and decreasing secondary bacterial and fungal infections for morbidity control, the Global Lymphatic Filariasis Programme was launched in the year 2000 [104±106] As part of this control program, detailed distribution maps of prevalence of lymphatic lariasis were deemed a priority Using a variety of approaches including Geographic Information Systems [107], climate generated risk pro les [108 ], detection of circulating antigen [109,110], rapid assessments using questionnaires involving hydrocele or lymphedema presence [111], rates of hydrocelectomy as a proxy for hydrocele prevalence or the more traditional methods of micro larial surveys and clinical examinations [112±115], better mapping of lymphatic lariasis distribution is underway Other tools developed for the control effort include mathematical models for describing transmission dynamics [116,117] and its control; social and economic projections for quantifying the impact of lymphatic lariasis on society and the cost and bene ts of implementing control [43,118,119]; training in clinical management with an emphasis on morbidity control [44,120,121]; and consideration of methods to certify elimination [122] An ancillary bene t from these control programs will be the treatment of hookworm and other intestinal parasite infections by albendazole and ivermectin [89,123,124], and the treatment of ectoparasites (lice/ scabies) by ivermectin [125 ] and the hope of improved nutrition and cognitive function [91,126] Conclusion The elimination of lymphatic lariasis as a public health problem as a concept has emerged on the shoulders of extremely sound basic and clinical research The programs based on its implementation will require redoubled research efforts to provide, among others, new targets for intervention, improved methods for assessing infection status at both the individual and community level, testing for emergence of drug resistance, and operational answers to the problem of drug distribution, cost and socioeconomic impact of lymphatic lariasis References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1 Michael E The population dynamics and epidemiology of lymphatic filariasis In: Lymphatic filariasis Tropical medicine: science and practice, Vol 1 Nutman TB (editor) London: Imperial College Press; 2000 pp 41±82 A comprehensive examination of the population dynamics based on observations and mathematical models 2 Kumaraswami V The clinical manifestations of lymphatic filariasis In: Lymphatic filariasis Tropical medicine: science and practice, Vol 1 Nutman TB (editor) London: Imperial College Press; 2000 pp 103±126 A detailed look at the clinical manifestations seen with infection with W bancrofti and B malayi 3 Dreyer G, Ottesen EA, Galdino E, et al Renal abnormalities in microfilaremic patients with bancroftian filariasis Am J Trop Med Hyg 1992; 46:745±751 4 Freedman DO, de Almeida Filho PJ, Besh S, et al Lymphoscintigraphic analysis of lymphatic abnormalities in symptomatic and asymptomatic human filariasis J Infect Dis 1994; 170:927±933 5 Freedman DO, Bui T, De Almeida Filho PJ, et al Lymphoscintigraphic assessment of the effect of diethylcarbamazine treatment on lymphatic damage in human bancroftian filariasis Am J Trop Med Hyg 1995; 52:258± 261 6 Witte CL, Witte MH, Unger EC, et al Advances in imaging of lymph flow disorders Radiographics 2000; 20:1697±1719 A comprehensive review of the modalities currently in place to examine lymphatics and lymph flow 7 Dreyer G, Noroes J, Figueredo-Silva J, Piessens WF Pathogenesis of lymphatic disease in bancroftian filariasis: a clinical perspective Parasitol Today 2000; 16:544±548 An important exposition of the clinical data suggesting that the adult worm induces lymphatic dysfunction and lymphangiectasia and that several important cofactors are required to induce the severe and chronic disease associated with lymphatic filariasis Beautiful illustrations of clinical pathology are also shown 8 Morel CM Reaching maturity: 25 years of the TDR Parasitol Today 2000; 16:522±528 9 Smith HL, Paciorkowski N, Babu S, Rajan TV Development of a serum-free system for the in vitro cultivation of Brugia malayi infective-stage larvae Exp Parasitol 2000; 95:253±264 An important methodological advance that may allow for a more detailed understanding of early parasite development 10 Smith HL Investigating development of infective stage larvae of filarial nematodes Front Biosci 2000; 5:E95±E102 11 Williams SA, Lizotte-Waniewski MR, Foster J, et al The filarial genome project: analysis of the nuclear, mitochondrial and endosymbiont genomes of Brugia malayi Int J Parasitol 2000; 30:411±419 An extremely important update on the filarial genome project as well as the status of the mitochondrial and Wolbachia genomes of B malayi This provides a framework for studies of sequencing the entire Brugia and Wolbachia genomes 12 Fadiel A, Lithwick S, Wanas MQ, Cuticchia AJ Influence of intercodon and base frequencies on codon usage in filarial parasites Genomics 2001; 74:197±210 13 Degrave WM, Melville S, Ivens A, Aslett M Parasite genome initiatives Int J Parasitol 2001; 31:531±535 14 Underwood AP, Supali T, Wu Y, Bianco AE Two microsatellite loci from Brugia malayi show polymorphisms among isolates from Indonesia and Malaysia Mol Biochem Parasitol 2000; 106:299±302 Microsatellite loci have been identified and used to demonstrate polymorphisms among related parasite isolates 15 Harris MT, Lai K, Arnold K, et al Chitin synthase in the filarial parasite, Brugia malayi Mol Biochem Parasitol 2000; 111:351±362 16 Rao UR, Salinas G, Mehta K, Klei TR Identification and localization of glutathione S-transferase as a potential target enzyme in Brugia species Parasitol Res 2000; 86:908±915 17 Zang X, Atmadja AK, Gray P, et al The serpin secreted by Brugia malayi microfilariae, Bm-SPN-2, elicits strong, but short-lived, immune responses in mice and humans J Immunol 2000; 165:5161±5169 18 Vasu C, Reddy MV, Harinath BC A 43-kDa circulating filarial antigen fraction of Wuchereria bancrofti in immunoprophylaxis against Brugia malayi in jirds Parasitol Int 2000; 48:281±288 19 Gregory WF, Atmadja AK, Allen JE, Maizels RM The abundant larval transcript- 1 and -2 genes of Brugia malayi encode stage-specific candidate vaccine antigens for filariasis Infect Immun 2000; 68:4174±4179 The identification and molecular characterization of two important larval transcripts and their ability to induce protective immunity in a permissive animal model 20 Manoury B, Gregory WF, Maizels RM, Watts C Bm-CPI-2, a cystatin homolog secreted by the filarial parasite Brugia malayi, inhibits class II MHC-restricted antigen processing Curr Biol 2001; 11:447±451 Identification of a parasite-encoded and secreted cysteine protease inhibitor that inhibits antigen processing Implications for evasion of the host immune response or of its modulation is discussed

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