Molecular Diagnosis and Monitoring of Benzimidazole Susceptibility of Human Filariids

Adisak Bhumiratana1,2,3, Apiradee Intarapuk3, Danai Sangthong3, Surachart Koyadun4, Prapassorn Pechgit1 and Jinrapa Pothikasikorn5 1Department of Parasitology and Entomology, Faculty of Public Health, Mahidol University, Bangkok 2Center for EcoHealth Disease Modeling and Intervention Development Research, Faculty of Public Health, Mahidol University, Bangkok 3Environmental Pathogen Molecular Biology and Epidemiology Research Unit, Faculty of Veterinary Medicine, Mahanakorn University of Technology, Bangkok 4Ministry of Public Health, Department of Disease Control, Office of Disease Prevention and Control 11 Nakhon Si Thammarat 5Department of Microbiology, Faculty of Science, Mahidol University, Bangkok Thailand


Introduction
Lymphatic filarial nematode parasites, mainly Wuchereria bancrofti and Brugia malayi, are causing agents of lymphatic filariasis in humans, which can be effectively treated with antifilarial drugs including diethylcarbamazine (DEC) and ivermectin. Albendazole, an effective benzimidazole compound, acts as a board-spectrum anthelminthic drug, and when combined with either one of antifilarial drugs, it exerts synergistic effects on reduction of peripheral microfilaremia in lymphatic filariasis cases. However, the varying parasite infection levels in those treated with DEC or ivermectin alone or in combination with albendazole are due to differences in drug responses. The additional clearance of infection with albendazole relative to what is observed with DEC or ivermectin alone suggests that albendazole has different parasite target(s). The homologous -tubulin gene of human and veterinary filariids that β-tubulin homologs have conserved domains structurally related to other orthologs among the nematodes, cestodes, trematodes and vertebrate hosts, is responsible for benzimidazole susceptibility. The genetic inheritance of resistance in nematode parasites can undergo under selection of benzimidazole compounds in a way that albendazole resistance mechanism involves one of two single amino acid substitutions from phenylalanine to tyrosine in parasite β-tubulin at position 167 or 200. This genetically-stable marker has shown promise for molecular diagnosis and monitoring of W. bancrofti infections that carry responsible genotypes associated with benzimidazole susceptibility or resistance. In particular, this approach can augment the surveillance and monitoring of mass treatment impacts on the parasite populations in target areas where long-running elimination programs for lymphatic filariasis are implemented at a large-scale by using a regionally-adopted combination therapy with antifilarial drugs, recommended by the World Health Organization.

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Current Topics in Tropical Medicine 398 2. Lymphatic filariasis towards elimination 2.1 Factors that favor elimination Lymphatic filariasis (LF) is a mosquito-borne parasitic disease caused by three main species of thread-like filarial nematode parasites belonging to the superfamily Filarioidea, which are Wuchereria bancrofti and Brugia malayi, and lesser extent by Brugia timori. These endoparasitic nematode lifestyles have a highly conservative life-cycle development sequence in which they develop their metamorphosis in both human and mosquito (Fig. 1). The adult worms that cause clinical manifestations of the disease are dioecious. Male and female worms have separate reproductive system in their pseudocoelomatic body cavity and they live in the After the induction of L3 infection in susceptible human host, the mature filarial female worm (A) that possesses fecundicity and fertilization can produce microfilariae (Mf). These offsprings develop from fertilized eggs (B) in the uterus of female worm. They are ingested during the bite of mosquito vector, and consequently, a 5-stage molting progression (B) initially starts after exsheatment of the Mf in mosquito's midgut (C). In this regard, the probable mechanisms involve: (a) proposed anterior of line of weakness, (b) internal digestion of the sheath, and (c) exsheatment of anterior end. Third stage larvae (L3) possess post-infective stage development in mosquito thoracic muscle and then migrate to the proboscis. They are transmitted by infective mosquito during a blood meal, and that they become the L4 (or L5) and mature adult worms in the lymphatic system in human.

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human lymphatic system, i.e., lymphatic vessels, with 5-15 years of life expectancy (Table 1). Only when its fecundic lifespan is capable of mating does the lymphatic-dwelling female worm produce advanced stage of sheathed larvae called "microfilariae". These short-living offsprings then penetrate the blood circulation. For a complete life cycle, the microfilariae are ingested by susceptible female mosquito during a blood meal in which they can develop further into larval stages L1-L2 to infective L3 stage. Transmission occurs when the infected mosquito transmits this L3 stage to susceptible persons during other blood meals. The naturally acquired transmission is associated with both intrinsic and extrinsic factors that can regulate the parasitic worm burdens in an endemic population (Fig. 2). Fig. 2. An explanatory model of human-parasite-vector interactions and favorable factors that influence its adaptation in hosts. The lymphatic filarial infections in humans depend on extrinsic factors of incoming L3 inocula and host recognition and location, and a proportion of adult worms with fecundicity and mating probability. Transmission is influenced by host-vector combination (host location and recognition of mosquito feeding habits), microfilarial loads in blood with timing of microfilariae release, and vectoral capacity (longevity and low refractoriness). Long lifespan of the mosquito and timing of L3 release are favorable factors of transmission.
As such, understanding of how these filarial nematode parasites can be removed from the human hemisphere is to understand basically the biology of their life cycle. How do the parasite taxa succeed their complete life cycle in the certain conditions under which they evoke host exploitation strategies? The biology of the parasitic symbionts can disclosure the parasite diversity and fitness shaped by hosts and environmental constraints that the www.intechopen.com Please use Adobe Acrobat Reader to read this book chapter for free.
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parasites can evolve the adaptation, i.e., the ability to control the physiology and behavior of their host for their own benefit. It has been known so far their distribution among hosts and strategies of host exploitation are restricted in number of host species. The W. bancrofti, which is highly host-specific parasite, is sessile to human while other zoonotic Brugia taxa exploit other non-human reservoir hosts. This host-specificity variation does not account for the origin of the parasitism as they succeed their parasitism and share common transmission pattern and complex life cycle in vertebrate hosts and arthropods. The geographical variation, on the other hand, can be restrained by selective pressures from the hosts, or the physio-chemical environments such as therapeutic agents and insecticides, or due to phylogenetic constraints. Such this selection can be explained by the experimental infections, mainly using Brugia species in rodents (e.g., jirds, hamsters, rats and mice), dogs, ferrets, cats and monkeys. All of which were proposed not just for understanding vertebrate immunology, pathology and chemotherapy, but also for exploring the interactions of the parasite and host. Among these host-parasite systems, B. pahangi that infects naturally cats in the Southeast Asia is experimentally appropriate for studies of infection and disease dynamics (Table 1). Because of their conservation of complex life-cycle development between feline and human lymphatic filarial parasites, it induces the infection and clinical sequelae in susceptible cats, and hence, resembles that of Brugia and Wuchereria in humans. However, the parasite does not always succeed their population diversity by increasing its fitness in the hosts (Fig. 2). Its population dynamic is primarily influenced by naturally acquired immunity (i.e., a type of concomitant immunity), which plays a significant role in host selection pressure to restrict a parasitic worm burden (Bundy et al, 1991;Grenfell et al, 1991;Mitchell, 1991;Grenfell and Micheal, 1992). This immunity against incoming L3 cannot remove them from the infection in humans but restrict a number of L3 by a production of concomitant immunity. Variability of adult worm burden results in a proportion of microfilaremic and amicrofilaremic persons in the population. The circulating microfilariae have short life cycle in human blood circulation; a proportion of the microfilariae can be removed by anti-microfilarial antibody www.intechopen.com Please use Adobe Acrobat Reader to read this book chapter for free. Just open this same document with Adobe Reader. If you do not have it, you can download it here. You can freely access the chapter at the Web Viewer here. (Simonsen et al, 1996;Ravindran et al, 2003). They develop two molts (L1 and L2) and induce the infection in susceptible mosquito vectors while the infected mosquitoes can regulate melanization involved in innate immune defense and wound healing to the penetration of filarial nematodes (Zou et al, 2010;Castillo et al, 2011). In blood-engorged mosquitoes that harbor high microfilarial density, they can induce death due to the vector intolerance against development of juvenile forms, and consequently, this leads to loss of L3. Furthermore, release of the L3 does not always permit a passage at an equal number during other blood meal taken by the infective mosquito due to the vector tolerance. Thus, they are naturally killed by host immune, vector barrier, and physical environment; these contributing factors can favor the LF elimination in humans. As was, LF becomes one of six potentially eradicable diseases of which the criteria for assessing their eradicability (CDC, 1993) are based not only on the scientific feasibility of understanding the biological information mentioned earlier and practical use of the public health interventions and other methods to be applied or used in existing national control program, but also on the political will or popular support of executing the implementation of LF control strategies. First, the scientific feasibility depends basically on the disease vulnerability. Unlike the W. bancrofti that exists only in humans, the B. malayi spends an enzoonotic life-cycle in which the parasite is transmitted by the potent mosquito and thrive in domestic cats as non-human reservoir, and a per se epizoonotic cycle in which the parasite is transmitted and thrive through which the mosquito vector takes blood meal from infected cats, and subsequently, transmit the infective stage to humans during other blood meal. Controversially, the humans carrying B. malayi infection can serve as a source for the infection to permit a passage to the animal reservoir through bites of the mosquito vector. Nonetheless, both diseases do not always ease their spread into the population as a result of the naturally-induced immunity and duration of microfilarial production in susceptible individuals or communities (Maizels and Lawrence, 1991;Ottesen, 1992) (Fig. 2). More important, the infections can be both easily diagnosed using advanced tools and effectively treated with the antifilarial drugs that are safe, inexpensive and easily deployed. The feasibility of elimination has been shown that the operation of the pragmatic diagnostic methods or interventions is demonstrable at a large scale in the target populations. Last, the political will/popular support is pivotal for the program manager to capture information required to analyze situations of the perceived burden of LF that is figured out by a large number of suffering and disabled persons worldwide (WHO, 2002;: the details are available at the websites of the Global Program to Eliminate Lymphatic Filariasis (GPELF) (http://www.who.int/lymphatic_filariasis/disease/en/) and Global Alliance to Eliminate Lymphatic Filariasis (http://www.filariasis.org). Also, the National Program to Eliminate Lymphatic Filariasis (PELF) by which the resource mobilization and funding structure are administered must be allied, by adopting the GPELF's strategies, to practical considerations of interventions, methods, logistic supplies (medical and field-work), delivery processes, expenditures (unit costs and costeffectiveness), integration of control activities into existing health systems services (or other health development programs), socio-economic impacts on gaining health benefits, community awareness and acceptance, and ecological disturbances to target disease, nontarget disease and the environment. For the resource-limited countries as allied nations, a national budget plan that notifies outsources and fund raising needs the subsidy of the internationally collaborative program.

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Mass chemotherapy as elimination strategy
Ideally, the success of controlling the disease depends definitely on the objectives and ultimate goals of disease control spectrum (CDC, 1993); with this regard, the rational management of LF elimination differs with control ( Table 2). As recommended by the World Health Organization (WHO), two pillars of global elimination strategies emphasize interruption of transmission and elimination of the infection in humans, and the other largescale morbidity control to prevent disease and disability (WHO, 1999a;. Principle outcomes of the GPELF reduce numbers of microfilaremic persons and disease cases as preventing new infection introduced among the population at risk of, or affected with, the infection. To meet this criteria, the GPELF proposes a mainstay of elimination strategy effectively available for mass drug administration (MDA) in target population; a combined treatment with diethylcarbamzine citrate (DEC) 6 mg/kg plus 400 mg albendazole (in W. bancrofti transmssion areas where Onchocerca volvulus is not coendemic), or with 200 g ivermectin plus 400 mg albendazole (in W. bancrofti transmssion areas where O. volvulus is coendemic) (Ottesen et al, 1997;Ottesen, 2000;WHO, 1999a;2000;. An annual MDA with coverage of 60-80% for 4-5 years is considered to be effective enough to interrupt transmission in control areas in the absence of vector control (Gyapong et al, 2005). Also, new options for mass treatment of at risk population are effectively available: DEC-fortified salt for 1 year; and a combination of single annual dose of albendazole plus DEC, followed by DEC-fortified salt (Weaver et al, 2011). The ample supply and distribution of DEC-fortified salt can be administered in some countries.

Surveillance and monitoring systems
As for presenting dynamics of the infection and disease in nature (Grenfell et al, 1991;Srividya et al, 1991;Ottesen, 1992;Meyrowitsch et al, 1995), the parasites cause a wide spectrum of clinical manifestations in the affected population that are characterized by asymptomatic microfilaremia, acute lymphatic inflammation and chronic lymphatic pathology (WHO, 1992a;1992b;. Susceptible persons develop clinically LF as a result of prolonged exposure to multiple infective bites of potent mosquitoes from several months to years. On the other hand, the people living for at least 6 months are at the greatest risk for the infection. The chronic filariasis cases represent a tip of the iceberg, as microfilaremic carriers are the reservoir of the infection to others. The prevalence and intensity of the infection in humans depends mainly upon a number of microfilaremic persons and a geometric mean of microfilarial loads in the affected population. These proximate measures are indicative of the degrees of endemicity. The prevalence and geographical distribution of the disease are important not just for determination of its potential transmission in mosquito, but also for diagnosis and surveillance in different endemic settings. Therefore, the recognition of what is the filarial origin of the disease and where the affected individuals or communities are is fundamental for public health importance to identify the solution and control rationale. Diagnostic approaches that emphasize the detection and specific identification of microfilaremic infection in the individual by using most standard microscopic methods are important component of LF control program, which is aimed to reduce number of microfilaremic persons. The suitable blood collection and microscopic diagnosis in health settings can be effectively available to identify anyone infected, and subsequently, treated with antifilarial drug regimens. However, this specific objective of control is less important as the elimination is desired not just to interrupt transmission and clear human infection on a large-scale, but also to reduce the morbidity attributed to the www.intechopen.com Please use Adobe Acrobat Reader to read this book chapter for free. Just open this same document with Adobe Reader. If you do not have it, you can download it here. You can freely access the chapter at the Web Viewer here. disease and hence improve both personal-focused hygiene and health care to all beneficiaries in target population at risk (Table 2).

Objective
-To assess whether the control program achieves its goals or desired outcomes (e.g., reduction in microfilaremia and/or disease prevalence) -To assess whether the elimination program achieves its goals or desired outcomes † (e.g., reduction in microfilaremia prevalence, antigenemia prevalence)

Process indicators as options for monitoring and evaluation tools
-Coverage of selective treatment and follow-up, as mosquito net utilization, insecticide spraying and reservoir control can be useful indicators -Mass treatment coverage, drug compliance coverage (e.g., adverse drug reactions prevalence and severe adverse event reports), KAP survey 3.1 Objective -To assess how well the various components of the control program are functioning (e.g., the number of newly and follow-up microfilaremic persons treated with antifilarial drugs) -To assess how well the various components of the elimination program are functioning ‡ (e.g., the number of new infections, the number of drug tablets distributed) Adapted from the GPELF (http://www.who.int/lymphatic_filariasis/disease/en/). †, ‡ Outcome indicators can be optionally used for monitoring systems; longitudinal surveillance of populations in sentinel sites, cross-sectional "spot checks" in other sites, and auxiliary "background" surveillance. KAP -Knowledge, attitudes and practices.  Weil & Liftis, 19871997Freedman et al, 1997Ramzy et al, 1999Bhumiratana et al, 1999Nguyen et al, 1999Phantana et al, 1999Simonsen et al, 1999Omar et al, 2000Pani et al, 2000Sunish et al, 2002 More & Copeman, 1990Turner et al, 1993Chanteau et al, 1994a1994bLammie et al, 1994McCarthy et al, 1995Rocha et al, 1996Nicolas, 1997Ismail et al, 1998Eberhard, 1997Simonsen and Dunyo, 1999Nuchprayoon et al, 2003Bhumiratana et al, 2004  To meet this objective, a large-scale transmission control requires a current magnitude and geographical distribution of the disease in the at-risk population. To understand the extent to which the target population needs to be designed for MDA and monitored whether the MDA implementation is effective, such surveillance and monitoring systems are required. To identify the communities with, or at risk of, the infection, for instance, the direct assessment techniques (Table 3) are required for practical use both in initial surveillance for filarial infection and in monitoring and evaluating the effectiveness of mass treatment, as part of the GPELF (WHO, 1999a;1999b;Ottesen, 2000). In this regard, the mass treatment with more effective antifilarial drug regimens as well as the availability of other existing and alleviating control measures has been deliberately implemented to meet such these highly achievable objectives of the elimination. Nonetheless, in addition to what is recommended by WHO, the GPELF requires for ground-breaking development of systems, protocols and tools that will be able to be convincingly applied to or routinely used in the PELF to fix undesirable events of mass treatment impacts in different complex epidemiological settings (Kyelem et al, 2008;Ottesen et al, 2008;WHO, 2008;.

Microfilaremia and drug-responsive microfilaremia
In an endemic population that represents the infection and disease dynamics in nature, the asymptomatic microfilaremia is a stage of the active infection with filarial adult worms (Srividya et al, 1991;Meyrowitsch et al, 1995), which regulate host immune responses (hypoimmune responsiveness) in infected individuals (Bundy et al, 1991;Grenfell et al, 1991;Maizels and Lawrence, 1991;Mitchell, 1991;Grenfell and Micheal, 1992;Ottesen, 1992; www.intechopen.com Please use Adobe Acrobat Reader to read this book chapter for free. Just open this same document with Adobe Reader. If you do not have it, you can download it here. You can freely access the chapter at the Web Viewer here. Simonsen et al, 1996;Ravindran et al, 2003). This phenomenon results in immunotolerance, i.e., a prolonged induction of the balance of immune defense to the parasites stimulated by the adult worms, in most asymptomatic microfilaremic persons. The female adult worm involves in regulation of the host microfilaremia. The fecundicity (a period of pregnancy) allows its fertilization to produce a diverse number of the offspring microfilariae. Although the proportion of microfilariae can be removed from the blood circulation in patients, there are the plenty of microfilariae, which circulate in the peripheral blood and show the appearance both nocturnally and diurnally. This microfilarial periodicity or circadian cycle of the parasite in humans is clinically unimportant for treatment but very important for its epidemiologic implication, which plays a significant role in diagnosis, surveillance and epidemiology. The parasite infection is the foundation for the processes that not only determine the infection prevalence but also monitor and evaluate the effectiveness of the treatment with the antifilarial drugs in infected individuals. In this regard, the amount of microfilaremia seems to be a function of naturally-acquired infection loads, which refers to as the most viable microfilariae, and drug-responsive microfilaremia refers to as the affected parasite population that harbors a diverse range of viable and nonviable microfilariae (Pechgit et al, 2011). These outcome indicators are useful for monitoring and evaluating the benzimidazole susceptibility of the filarial nematode parasites in the population in areas of the PELF implementing the MDA 2-drug regimen either albendazole plus DEC or albendazole plus ivermectin. However, the MDA 2-drug regimen is not the only factor that shapes the parasite population under complex epidemiological settings. Of note, the W. bancrofti populations have ability to provoke the genetic variability that shows the important implications in the endemic populations targeted by the MDA (Pradeep Kumar, 2000). The existence of genetic diversity of W. bancrofti populations that has greater heterogeneity under DEC therapy and vector control gives rise to questioning about the development of drug resistance in LF, which possibly occurs in the target populations. The selection pressure is an intensity of selection affecting the frequency of genes in a parasite population. The selection that increases or decreases the susceptibility of the parasite population depends on the frequency of the alleles involved. The genetic polymorphism occurring in the W. bancrofti population under the selection pressure(s) may evoke gradually under specified conditions to yield the fitness, which can be determined by a genotype in the parasite population. The increase in the parasite fitness can be estimated by the equilibrium frequencies of the alleles (genotypes) at heterozygote advantage in a hypothethical population. That is, rapid establishment of advantageous alleles in the W. bancrofti population, called "selection sweep", may evoke with advantageous drug-resistant genotypes epidemiologically linked to other factors shaped by the host and environment (Schwab et al, 2006(Schwab et al, : 2007Churcher et al, 2008). Eventually, it may reduce the genetic variation in the population.

Benzimidazole-susceptibility of the parasite
This chapter emphasizes microfilaremia responsiveness in the population under the suppression of the PELF implementing MDA 2-drug regimen, 6 mg/kg DEC plus 400 mg albendazole. The microfilaremia responses against the DEC are the foundations of understanding how the albendazole exerts the effects on the parasite population in addition to what is observed by DEC alone. The DEC is known as the oldest of the antifilarial drugs used in the LF control. The single-dose drug acts as microfilaricide as does the effective ivermectin (de Silva et al, 1997;Ottesen, 2000;Molynuex et al, 2003;Ottesen et al, 2008) www.intechopen.com Please use Adobe Acrobat Reader to read this book chapter for free.
Just open this same document with Adobe Reader. If you do not have it, you can download it here. You can freely access the chapter at the Web Viewer here. while its macrofilaricidal activity is not definitely effective against adult worms (Eberhard et al, 1991;Norões et al, 1997;Dreyer et al, 1998;Rajendran et al, 2002;Oliveira-Menezes et al, 2007). The adult worm loads that are age-dependent (Lammie et al, 1994; are susceptible to treatment with the DEC alone or even combination with albendazole (Norões et al, 1997;Rajendran et al, 2002). It was seen that DEC alone disturbs the microfilarial sheath of some filarial species while it has effects on the oogenesis and fertilization of female adult worms. Nonetheless, little is known about the filarial nematode parasites whether they evoke resistance mechanism against DEC due to the lack of deepening its mechanism of action, particularly the availability in parasite tissues and the selectivity on parasite targets. Oliveira-Menezes et al (2007) demonstrated that DEC has minor effects on alterations of the cuticle or surface of both male and female adult worms; these responsible parasites were collected from the W. bancrofti-infected patients treated with DEC, as compared to those isolated from the untreated patients. Additionally, such alterations are seen in adult worms recovered from the patients treated with DEC plus albendazole. The possible explanation is that the potential adulticidal effect of albendazole relative to what is observed by the DEC alone. The subtle alterations imply the distinct morphologic characteristics of the parasite itself or the complex host-parasite interactions, and if implemented and continuously prolonged, the annual mass treatment with DEC plus albendazole has not yet become an apparent issue, particularly the impacts on the parasite population adaptation. Of note, the O. volvulus parasite develops the mechanism involved in resistance to ivermectin (Awadzi et al, 2004). Do the filarial nematode parasites have mimicry in the resistance mechanisms to ivermectin and albendazole? However, detailed study of the parasite resistance to albendazole has not been established. Focus is on the MDA 2-drug that acts as effective microfilaricide while its aberrant activity that influences microfilaremia response to its efficacy in microfilaricidal activity. Provided this phenomenon occurs, the expected outcomes of such drug failure will impact on solving the solutions and paving the implications of how they will adapt under the certain circumstances and how we will also mitigate their adaptation. Most studies enlightened the understanding of this effective deworming MDA 2-drug, which plays the significance of reduction of the infection prevalence. A single-dose combined treatment with DEC plus albendazole has short-and long-term effects on W. bancrofti microfilaremics in the endemic populations (Ismail et al, 1998;Ottesen et al, 1999;El Setouhy et al, 2004;Rajendran et al, 2004). Compared to those receiving DEC alone, an additional benefit of the combined drugs results in decline in annual cyclic infection prevalence due to progressive reduction in density of W. bancrofti microfilaremia. Although its macrofilaricidal effect on clearance of W. bancrofti antigenemia has been reported (McCarthy et al, 1995;Eberhard, 1997;Rajendran et al, 2002;Koyadun et al, 2003;Bhumiratana et al, 2004;Siriaut et al, 2005;Bhumiratana et al, 2005;Yongyuth et al, 2006), the DEC alone or co-administered with the albendazole does not clear rapidly the antigenemia. The MDA with the DEC alone will recover an increase in the antigenemia prevalence of W. bancrofti unless there is yearly-round MDA in the population . A 400 mg single oral-dose albendazole regimen is broad-spectrum effective against helminthiases (Albonico, 1994;de Silva et al, 1997;Beach et al, 1999;Ottesen et al, 1999;Horton, 2000) and, as coadministered orally with DEC, a synergistic and long-term effect on geohelminths has been proven useful for 'beyond-lymphatic filariasis' elimination program (Ottesen et al, 1997;Ismail et al, 1998;Horton et al, 2000;Ottesen, 2000;Mani et al, 2002;Molynuex et al, 2003;Yongyuth et al, 2006).

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Few reports established the evidence that the human filarial nematode parasites provoke molecular mechanism involved in benzimidazole sensitivity/resistance until recently findings of the genetically-induced resistance against benzimidazole compounds have been well documented in veterinary nematode parasites (Beech et al, 1994;Humbert et al, 2001;Bennett et al, 2002;Drogemuller et al, 2004;Robinson et al, 2004;Cole et al, 2006;Ghisi et al, 2007). Resistance to albendazole in veterinary nematodes is known to be caused by either one of two single amino acid substitutions from phenylalanine to tyrosine in parasite -tubulin at position 167 or 200. The genetically stable W. bancrofti -tubulin gene responsible for a molecular mechanism of drug resistance has been proposed as that of the veterinary helminth parasites is performed under selection of albendazole and ivermectin. The W. bancrofti population isolated from the patients treated with a combination of albendazole and ivermectin had significantly higher genotypic frequencies associated with resistance at position 200 (Schwab et al, 2005). A resistance mutation was not detected at position 167. Hoti et al (2003Hoti et al ( , 2009 (2011) demonstrated that the W. bancrofti population recovered from the dynamic cross-border migrant population from areas that have been targeted by the MDA 2-drug regimen (300 mg DEC plus 400 mg albendazole) elicits the genetic background of benzimidazole susceptibility; a resistance mutation has not been observed at position 167 or 200. However, the albendazole, anthelmintic benzimidazole, is being co-administered with an antifilarial drug such as DEC, part of the PELF implementing in many endemic countries. But this drug is known to result in the faster development of drug resistance in the veterinary nematode parasites and hence it is necessary to monitor drug sensitivity among the responsible W. bancrofti populations.

Molecular diagnosis and monitoring of benzimidazole susceptibility 4.1 Parasite beta-tubulin encoding gene as molecular marker
Molecular mechanisms of benzimidazole resistance in nematode parasites are hypothesized. However, detailed study of benzimidazole resistance in trichostrongylids found that the tubulin encoding gene involved in benzimidazole susceptibility is responsible for the genetic inheritance of resistance in the veterinary nematode parasites under selection with benzimidazole that involves one of two single amino acid substitutions from phenylalanine (Phe) to tyrosine (Tyr) in parasite -tubulin at position 167 or 200 (Beech et al, 1994;Kwa et al, 1993;Roos et al, 1990;Elard et al, 1996;Elard and Humbert, 1999;Humbert et al, 2001;von Samson-Himmelstjerna et al, 2002;Winterrowd et al, 2003;Drogemuller et al, 2004;Cole et al, 2006;Ghisi et al, 2007). The potential point mutation occurs at the DNA level by nucleotide substitution for the codon for amino acid position 200 of the -tubulin gene, a substitution of TTC (Phe) with TAC (Tyr). This irreversible change brings about distinguishment of the responsible parasite population between benzimidazole-sensitive and -resistant nematodes. This principal mechanism for benzimidazole resistance is postulate to involve changes in the selectivity of the benzimidazoles on the primary structure of β-tubulin molecules, a building block of the microtubule in the parasites (Lacey, 1988;Lacey and Gill, 1994;Robinson et al, 2004).

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Intriguingly, such mimicry in molecular mechanism for benzimidazole resistance in the filarial nematode parasites has been increasingly investigated, based basically on the molecular characterization of the homologous -tubulin gene retained in their genome and the advantageous fitness of benzimidazole-resistant genotypes in the population Elard et al, 1998;Elard et al, 1999;Silvestre et al, 2001;Silvestre and Humbert, 2002).
The nematode parasites possess the single-copy homologous -tubulin (tubb) gene that encodes a -tubulin polypeptide, 448 amino acids (Met1 to Glu448). Hypothetically similar to that of trichostrongylids, the binding of benzimidazoles to conserved domains (of the exons 4 to 6) leads to blocking an assembly of tubulin (TUBB), and thus disrupting structural formation of microtubule (cytoskeleton protein) in the nematode parasites. The nucleotide sequences of the homologous -tubulin gene as molecular marker and other related TUBB gene family of the nematode parasites can be retrieved from the genome databases: the GenBank at the National Center for Biotechnology Information (NCBI), http://www.ncbi.nlm.nih.gov/genbank/, the European Molecular Biology Laboratory (EMBL) http://www.ebi.ac.uk/, and the DNA DataBank of Japan (DDBJ) http://www.ddbj.nig.ac.jp/. The website of nematode and neglected genomics (http://www.nematodes.org/fgn/index.html) establishes genome database, especially for the filarial genome project (FGP), which includes published complete B. malayi genome.

Polymerase chain reaction-based approaches
In contrary to the antigen detection methods such as ICT Filariasis and Og4C3 ELISA that provide the proof of W. bancrofti antigenemic infection in human blood, the microfilarial DNA detection by PCR provides the evidence of W. bancrofti microfilaremic infection in human blood and mosquito (  W. bancrofti -tubulin gene, the nested PCR amplification can work well with the microfilaremic infection that responds to treatment with MDA 2-drug regimen (DEC plus albendaozle) (Pechgit et al, 2011). This newly developed PCR assay in addition to promising advanced tool (Hoti et al, 2003;Bhumiratana et al, 2010) has the potential benefits in the molecular diagnosis and monitoring of the infection, as compared to the other PCR amplification methods previously described elsewhere (Table 3). The concepts for PCR assays based on the Wbtubb locus-specific primers (Table 4) have been proposed in two applicable formats: the locus-specific nested PCR and allele-specific nested PCR. These applications have established the advantage s o n h o w t o c i r c u m v e n t s o m e c o m m o n counterintuitive problems of conventional PCR with regards to both parasite genome analysis and low-copy gene detection; such detailed study has been well established by Pechgit et al (2011). The W. bancrofti microfilarial DNA detection methods depends much on the purity and quantity of the microfilariae recovered from different blood sample preparations. The purified aggregate parasite number in the absence of human host white blood cells, for example, are ideal for the quality of DNA extract, which serves as target sequences in the PCR reactions. In general, most PCR methods for the detection of W. bancrofti distinguishable from other filarial nematode parasites in human and mosquito is based on the repetitive Ssp I sequences, which are highly copy number per haploid genome. However, PCR amplification based on this Ssp I locus provides the positive identifications of the parasite infection existed in specimens of choice. The assay does not determine the infection that responds to benzimidazole sensitivity/resistance; such responsible W.
bancrofti parasite population is amplified based on the -tubulin gene which is single copy in haploid genome. Therefore, the amplification is performed using the Wbtubb locus-specific nested PCR and allele-specific nested PCR that provides the proof of the W. bancrofti infection carrying benzimidazole-sensitive/resistant phenotypes; methodologically, the technical requirements for their applications have been described by Pechgit et al (2011) andHoti et al (2009). More specific, based on our experience, the Wbtubb locus-specific nested PCR with thermocycling modifications using touchdown and touchup cycles has been applied or used in detection and characterization of W. bancrofti infection both in human blood from patients untreated or treated with DEC plus albendazole and in wild-caught mosquito, provided such infections carrying benzimidazole-sensitive/resistant strains are the same source of the parasite population (Fig. 4). Hypothesis is that whether the parasite infection is genetically predisposed to the MDA 2drug regimen (DEC plus albendazole) in areas under suppression of PELF, it will have frequencies of benzimidazole-susceptible homozygous allele (SS) greater than benzimidazole-susceptible heterozygous allele (Sr) and homozygous resistant allele (rr), which are associated with albendazole resistance, unless the parasite fitness is increased. This also permit the monitoring and evaluation of the parasite fitness to better understand theoretically and hypothetically evolutionary biology and ecology of the parasite, by which the human hosts play a key as a major source of selective pressures on the adaptation of parasite population constrained by environmental conditions.

Future perspectives
The GPELF has been deployed into the endemic countries implementing MDA 2-drug regimes (i.e., single annual doses of albendazole in combination with DEC or ivermectin) to reduce microfilaremia prevalence to levels low enough (principally lower than transmission threshold) to interrupt transmission of the disease in the absence of vector control. Based on scientific information on drug resistance to anthelmintics, the issue of albendazole resistance www.intechopen.com Please use Adobe Acrobat Reader to read this book chapter for free.
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in the W. bancrofti parasite has assumed increasing importance since the GPELF is implemented on a large-scale in at-risk populations in different complex epidemiological settings, and predictably, the implementation phrase of the program will increase. Many studies have shown some vulnerability in how the parasite has the ability to evoke molecular mechanisms for resistance to anthelminthics as the nematodes of veterinary importance have developed resistance against both albendazole and ivermectin. Furthermore, there have been lines of evidence that the vulnerability of helminthiasis control programs that employ the MDA with these drugs is associated with several factors that facilitate the promotion of drugresistant strains. At the same time, the factors are considered concerning the new options of drug combinations with different parasite targets or modes of actions to keep active shelf-life of DEC because the filarial nematode parasites have long life-span and complex life-cycle development. Likewise, to better understand wha t i s r e la t i v e t o a c h iev e M D A ' s g o al t o confront growing trend of drug resistance, it is essential for the development of molecularly diagnosing and monitoring the benzimidazole sensitivity/resistance in areas of long-running PELF program implementation using albendazole plus DEC or albendazole plus ivermectin. More applicable tools which will be validated in effective manner are also required to explore the genetic basis for resistance to anthelminthics. [http://www.cdc.gov/mmwr/preview/mmwrhtml/00025967.htm] Chanteau, S., Moulia-Pelat, J.P., Glaziou, P., Nguyen, N.L., Luquiaud, P., Plichart, C., et al. (1994a). Og4C3 circulating antigen: a marker of infection and adult worm burden in Wuchereria bancrofti filariasis. J Infect Dis 170, 247250. Chanteau, S., Glaziou, P., Luquiaud, P., Plichart, C., Moulia-Pelat, J.P., Cartel, J.L. (1994b).