**3. Parasite infection and drug responsiveness**

### **3.1 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;

**applied or used in demonstrated areas**

products;

 Cannot differentiate larval stages (L1, L2 or L3) in infected mosquitoes

laboratory personnel and accessory equipments for DNA preparation and analysis of PCR

**References** 

2005

2010

Fischer et al, 2003 Kanjanavas et al,

Nuchprayoon et al, 2005; 2007 Rao et al, 2006 Mishra et al, 2007 WHO, 2009 Bhumiratana et al,

Pechgit et al, 2011 Takagi et al, 2011

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

mosquito infection rates, and to monitor and evaluate the infections or drug responses in individuals or in the target population under the

circumstances described above; Can differentiate the status between infection

(microfilaremia) and disease, occurrence (new infection) and recurrence, or sensitivity and

 Detection limit of as low as one Mf per blood volume tested (up to 1 ml), or one juvenile larva (L1, L2 or L3) per pooled

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

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

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;

mosquitoes (up to 100)

(Kyelem et al, 2008; Ottesen et al, 2008; WHO, 2008; 2010).

**3. Parasite infection and drug responsiveness 3.1 Microfilaremia and drug-responsive microfilaremia** 

resistance;

Lymphatic Filariasis

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 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: 2007; Churcher et al, 2008). Eventually, it may reduce the genetic variation in the population.

#### **3.2 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)

Molecular Diagnosis and Monitoring of Benzimidazole Susceptibility of Human Filariids 409

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; Kwa et al, 1995; 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 (2003, 2009) reported that the polymorphism in the codon of this residue in *W. bancrofti* populations representing geographically distant areas of India,

**4. Molecular diagnosis and monitoring of benzimidazole susceptibility** 

level by nucleotide substitution for the codon for amino acid position 200 of the

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,

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; 1994; 1995; 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

**4.1 Parasite beta-tubulin encoding gene as molecular marker** 

1988; Lacey and Gill, 1994; Robinson et al, 2004).

showed that *W. bancrofti* isolates from wide geographic areas of India had codon for Phe (TTC) at position 200, suggesting that the parasite might be genetically sensitive to benzimidazole. Similarly, Bhumiratana et al (2010) and Petchgit et al (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.

*-tubulin* isotype 1 gene. The nucleotide sequence data

*-tubulin*

through sequencing exon 5 region of

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; 2004; Oliveira-Menezes et al, 2007). The adult worm loads that are age-dependent (Lammie et al, 1994; Rajendran et al, 2002) 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; 2004; 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 (Rajendran et al, 2002). 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; 1999; Ismail et al, 1998; Horton et al, 2000; Ottesen, 2000; Mani et al, 2002; 2004; Molynuex et al, 2003; Yongyuth et al, 2006).

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; 2004; Oliveira-Menezes et al, 2007). The adult worm loads that are age-dependent (Lammie et al, 1994; Rajendran et al, 2002) 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.

Molynuex et al, 2003; Yongyuth et al, 2006).

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; 2004; 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 (Rajendran et al, 2002). 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; 1999; Ismail et al, 1998; Horton et al, 2000; Ottesen, 2000; Mani et al, 2002; 2004; 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; Kwa et al, 1995; 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 (2003, 2009) reported that the polymorphism in the codon of this residue in *W. bancrofti* populations representing geographically distant areas of India, through sequencing exon 5 region of *-tubulin* isotype 1 gene. The nucleotide sequence data showed that *W. bancrofti* isolates from wide geographic areas of India had codon for Phe (TTC) at position 200, suggesting that the parasite might be genetically sensitive to benzimidazole. Similarly, Bhumiratana et al (2010) and Petchgit et al (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.
