**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

Fig. 1. A development sequence of filarial nematode parasite in human and mosquito (A-D). 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.

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

Fig. 1. A development sequence of filarial nematode parasite in human and mosquito (A-D). 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.

**2. Lymphatic filariasis towards elimination** 

**2.1 Factors that favor elimination** 

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

Molecular Diagnosis and Monitoring of Benzimidazole Susceptibility of Human Filariids 401

(Simonsen et al, 1996; 2008; 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

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; 2008; 2010): 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

contributing factors can favor the LF elimination in humans.

internationally collaborative program.

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).


aFor both host-parasite systems, infection patterns encapsulate a spectrum ranging from asymptomatic persistent microfilaremia to symptomatic amicrofilaremia or complete refractoriness to infection. Adapted from Grenfell et al (1991).

Table 1. Correlation between feline and human lymphatic filariasis

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

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

**Parameter Cat Human** 

Longevity (years) 7-8 5-15 Mature female worm length (cm) 3-7 5-10

Table 1. Correlation between feline and human lymphatic filariasis

Typical infection/disease sequlaea Infection –

Adapted from Grenfell et al (1991).

Parasite *Brugia pahangi Wuchereria bancrofti* 

Lymphatics Blood Mosquito

Pathology

aFor both host-parasite systems, infection patterns encapsulate a spectrum ranging from asymptomatic persistent microfilaremia to symptomatic amicrofilaremia or complete refractoriness to infection.

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

Loss of infection –

*B. malayi, B. timori* 

Lymphatics Blood Mosquito

Infection – Loss of infection –

Pathology

dynamics (Table 1).

Location of : Adult Microfilariae

Vector

(Simonsen et al, 1996; 2008; 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; 2008; 2010): 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.

Molecular Diagnosis and Monitoring of Benzimidazole Susceptibility of Human Filariids 403

disease and hence improve both personal-focused hygiene and health care to all

**Control Elimination** 



health problem

indicator

and antigenemia rate - Disease rate is not useful


(e.g., reduction in

distributed)

**elimination program achieves its goals or desired outcomes†**

microfilaremia prevalence, antigenemia prevalence)


prevalence and severe adverse event reports), KAP survey


beneficiaries in target population at risk (Table 2).

**2. Outcome indicators as options for monitoring tools** 

**3. Process indicators as options for monitoring and evaluation tools** 

surveillance.

**1. Definition** - Reduction of microfilaremia

prevalence

population)

useful indicator

useful indicators

**3.1 Objective** - **To assess how well the various** 

drugs)

KAP - Knowledge, attitudes and practices.

**2.1 Objective** - **To assess whether the control** 



infective bite per annum which acquire the infection) as mosquito infection rate is not

**program achieves its goals or desired outcomes** (e.g., reduction in microfilaremia and/or disease prevalence)


**components of the control program are functioning** (e.g., the number of newly and follow-up microfilaremic persons treated with antifilarial

Adapted from the GPELF (http://www.who.int/lymphatic\_filariasis/disease/en/).

Table 2. Rational approaches to lymphatic filariasis control and elimination

**†,‡**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"

#### **2.2 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; 2002; 2008; 2010). 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; 1999; Ottesen, 2000; WHO, 1999a; 2000; 2001; 2002). 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.

#### **2.3 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; 1994). 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

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; 2002; 2008; 2010). 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; 1999; Ottesen, 2000; WHO, 1999a; 2000; 2001; 2002). 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.

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; 1994). 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

**2.2 Mass chemotherapy as elimination strategy** 

**2.3 Surveillance and monitoring systems** 

disease and hence improve both personal-focused hygiene and health care to all beneficiaries in target population at risk (Table 2).


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.

Table 2. Rational approaches to lymphatic filariasis control and elimination

Molecular Diagnosis and Monitoring of Benzimidazole Susceptibility of Human Filariids 405

**applied or used in demonstrated areas**

 Cannot differentiate the status between infection and disease, occurrence and recurrence, or sensitivity and resistance Primarily requires standardization and quality control of lots of venous blood

 Costly, laborintensive and intrusive

> samples (subsequently prepared for sera or plasma) that are collected,

transported, stored and analyzed; Also requires welltrained field workers and

laboratory personnel

 Too costly, laborintensive, intrusive, in-house developed

 Primarily requires standardization and quality control of lots of samples (finger-prick or venous blood, or pooled wild-caught mosquitoes) that are

collected,

transported, stored and analyzed; Also requires welltrained field workers or

tool

**References** 

1990

1995

al, 2003

2004; 2005

More & Copeman,

Turner et al, 1993 Chanteau et al, 1994a; 1994b Lammie et al, 1994 McCarthy et al,

Rocha et al, 1996 Nicolas, 1997 Ismail et al, 1998 Eberhard, 1997 Simonsen and Dunyo, 1999 Nuchprayoon et

Bhumiratana et al,

Chanteau et al,

Siridewa et al,

Williams et al,

Zhong et al, 1996 Ramzy et al, 1997 Nicolas et al, 1999 Cox-singh et al,

Thanomsub et al,

Pradeep Kumar et

Lizotte et al, 1994 McCarthy et al,

1994c

1996

1996

1996

2000

2000

al, 2002

**Tools Advantages for the applications Chief obstacles when** 

 Commercially available diagnostic test kit, and

Filariasis;

principally the same when use in qualitative and quantitative detection of active *W. bancrofti* infection, but more sensitive and specific than the ICT

 Highly reproducible when lots of large-scale serum/plasma samples (100 l each of freshly prepared or frozen samples) are analyzed in the public health reference laboratory or research institutes in order to assess the human infection rates, and to monitor and evaluate the infection or drug responses in individuals or in the target population under the circumstances described above;

 Very highly sensitive and specific for the microfilaremic infections with *W. bancrofti* and *B. malayi* in humans and mosquitoes distinguishable from other filarial nematode parasites such as *O. volvulus*, *D.* 

*immitis*, *D. repens* and *B.* 

Highly reproducible and

assess the human and

practical when lots of samples as low as 20 l human blood, individual mosquito (dissected or whole body), or mosquito pool, are analyzed in the public health reference laboratory or research institutes in order to

*pahangi*;

**Og4C3 ELISA** 

**Polymerase chain reaction (PCR)** 


**applied or used in demonstrated areas**

 Indicate, but not quantify, the level of the circulating antigens; somehow, provides falsenegative

identifications with the infections harboring very low antigen titers; Cannot differentiate the status between infection and disease, occurrence and recurrence, or sensitivity and resistance Primarily requires standardization and quality control of lots of samples (finger-prick blood) that are collected and analyzed

Costly;

**References** 

1997

2005

1999

1999

2004

2003

2003

2003

2009

al, 2003

Weil & Liftis, 1987 Weil et al, 1987; 1996; 1997 Freedman et al,

Ramzy et al, 1999 Bhumiratana et al, 1999; 2002; 2004;

Nguyen et al, 1999 Phantana et al,

Simonsen et al,

Omar et al, 2000 Pani et al, 2000;

Sunish et al, 2002;

Braga et al, 2003 Engelbrecht et al,

Koyadun et al,

Nuchprayoon et

Siriaut et al, 2005 Ruberanziza et al,

Foo et al, 2011

**Tools Advantages for the applications Chief obstacles when** 

 Simple-to-use rapid diagnostic test kit commercially available for use in qualitative detection of *W. bancrofti* adult worm circulating antigens present in whole blood/serum/plasma samples; stronger positive test line equivalent to higher

antigen levels;

of active infection (antigenemic) whether microfilaremia is present, or the treatment is given; Highly reproducible and practical when lots of largescale blood samples (100 l each) are analyzed either under the field conditions by not-well-trained field workers or in laboratory settings (with 100 l each of serum/plasma samples freshly prepared or frozen) by laboratory personnel in order to assess the human infection rates in areas known as endemic for *W. bancrofti* or

respective areas of emergence/reemergence; Suitable for rapid assessment survey to detect the early infection in endemic carriers including migrants, refugees, any persons who work in endemic areas (mine in rainforest and border) for years, or visitors to the areas; Suitable to monitor and evaluate the infection in humans inhabiting in risk areas in initial surveillance before MDA; drug responses during MDA; and the new infection in post-MDA areas whether they are certified as eliminated areas

 Sensitive and specific for anytime-of-day determination

**ICT Filariasis** 


Molecular Diagnosis and Monitoring of Benzimidazole Susceptibility of Human Filariids 407

Simonsen et al, 1996; 2008; 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

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: 2007; Churcher et al, 2008).

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)

plus DEC or albendazole plus ivermectin.

Eventually, it may reduce the genetic variation in the population.

**3.2 Benzimidazole-susceptibility of the parasite** 


Table 3. Direct determination tools for use as part of the Global Program to Eliminate Lymphatic Filariasis

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; 2010).
