**4. 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; 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 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).

Molecular Diagnosis and Monitoring of Benzimidazole Susceptibility of Human Filariids 411

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 (Roos et al, 1995; Elard et al, 1998; Elard et al, 1999; Silvestre et al, 2001; Silvestre and Humbert, 2002).

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

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.

The structural organization of homologous *tubb* genes of two filarial nematode parasites, *B. pahangi* (Guenette et al, 1991) and *D. immitis* (Bourguinat et al, 2011), has been shown for the establishment of complete coding sequences that span 9 discrete exons: exon 1 (Met1 to Lys19), exon 2 (Phe20 to Asp55), exon 3 (Gly56 to Gln131), exon 4 (Gly132 to Lys174), exon 5 (Val175 to Leu228), exon 6 (Val229 to Gln292), exon 7 (Met293 to Arg324), exon 8 (Glu325 to Thr386) and exon 9 (Ala387 to Glu448). The homology is 78% at DNA level due to bias of codon usage and insertion/deletion of intron sequences (Fig. 3). Among these, the exons 4 and 5 confer hypothetical point mutation at amino acid positions Phe167Tyr (or TTT/TAT) and Phe200Tyr (or TTC/TAC), based only on the second nucleotide base changed in the codons. In the homologous segment of its closely

(Gly132 to Lys174) and 5 (Val175 to Leu228), with flanking intron sequences (Fig. 3) shares the homology at DNA level with *B. malayi* and *B. pahangi* (93% similarity), compared to *O. volvulus* and *D. immitis* (76% similarity) (Bhumiratana et al, 2010; Pechgit et al, 2011). This target DNA has been proved useful for designing Wb*tubb* locus-specific primers to discriminate between Wb*tubb* and other homologs of human and animal filariids. Based on its usefulness in molecular diagnosis and monitoring of the infection carrying the benzimidazole-sensitive or resistant phenotypes, the PCR applications of this molecular marker for *W. bancrofti* have been well documented (Hoti et al, 2003; 2009;

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 (Table 3). As a result of the existence of genetically stable

*-tubulin* (Wb*tubb*) gene that possesses two distinct exons, 4

*-tubulin* gene as molecular marker and other

*-tubulin* gene can be obtained from the

*-tubulin* (*tubb*) gene that

The nematode parasites possess the single-copy homologous

nucleotide sequences of the homologous

Meanwhile, the homologous sequences of *B. malayi* 

Schwab et al, 2005; Bhumiratana et al, 2010; Pechgit et al, 2011).

**4.2 Polymerase chain reaction-based approaches** 

related taxa, *W. bancrofti*

TIGR genome database (http://www.tigr.org/tdb/e2k1/bma1).


Fig. 3. ClustalW alignment of the filarial *β-tubulin* gene. The partial nucleotide sequence representatives (accession no. and positions): *Brugia malayi* (BRQD553TR, 3-789), *Brugia pahangi* (M36380, 2267-3054), *Wuchereria bancrofti* (AY705383, 109-916), *Onchocerca volvulus* (AF019886, 1582-2400) and *Dirofilaria immitis* (HM596854, 1462-2244) are shown as coding (upper case) and non-coding (lower case) sequences. The deduced amino acid sequences for the conserved domains are shown for all taxa aligned; *D. immitis* and *O. volvulus* have one amino acid substituted at position Ala218Thr. The gap is performed on the maximum homology (insertion/deletion), which represents conserved (•) and degenerate nucleotide residues and the regions designed to amplify specifically the target sequences based on the Wb*tubb* primer sets (light-gray boxes), both forward () and reverse (). Hypothetically, two amino acid substitutions at positions Phe167Tyr and Phe200Tyr (dark-gray boxes) retained in DNA fragments (141 and 174 bp) could be identified using the PCR detection system described by Bhumiratana et al (2010) and Pechgit et al (2011).

Fig. 3. ClustalW alignment of the filarial *β-tubulin* gene. The partial nucleotide sequence representatives (accession no. and positions): *Brugia malayi* (BRQD553TR, 3-789), *Brugia pahangi* (M36380, 2267-3054), *Wuchereria bancrofti* (AY705383, 109-916), *Onchocerca volvulus* (AF019886, 1582-2400) and *Dirofilaria immitis* (HM596854, 1462-2244) are shown as coding (upper case) and non-coding (lower case) sequences. The deduced amino acid sequences for the conserved domains are shown for all taxa aligned; *D. immitis* and *O. volvulus* have one amino acid substituted at position Ala218Thr. The gap is performed on the maximum homology (insertion/deletion), which represents conserved (•) and degenerate nucleotide residues and the regions designed to amplify specifically the target sequences based on the Wb*tubb* primer sets (light-gray boxes), both forward () and reverse (). Hypothetically, two amino acid substitutions at positions Phe167Tyr and Phe200Tyr (dark-gray boxes) retained in DNA fragments (141 and 174 bp) could be identified using the PCR detection

system described by Bhumiratana et al (2010) and Pechgit et al (2011).

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

1995; 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. Meanwhile, the homologous sequences of *B. malayi -tubulin* gene can be obtained from the TIGR genome database (http://www.tigr.org/tdb/e2k1/bma1).

the advantageous fitness of benzimidazole-resistant genotypes in the population (Roos et al,

The structural organization of homologous *tubb* genes of two filarial nematode parasites, *B. pahangi* (Guenette et al, 1991) and *D. immitis* (Bourguinat et al, 2011), has been shown for the establishment of complete coding sequences that span 9 discrete exons: exon 1 (Met1 to Lys19), exon 2 (Phe20 to Asp55), exon 3 (Gly56 to Gln131), exon 4 (Gly132 to Lys174), exon 5 (Val175 to Leu228), exon 6 (Val229 to Gln292), exon 7 (Met293 to Arg324), exon 8 (Glu325 to Thr386) and exon 9 (Ala387 to Glu448). The homology is 78% at DNA level due to bias of codon usage and insertion/deletion of intron sequences (Fig. 3). Among these, the exons 4 and 5 confer hypothetical point mutation at amino acid positions Phe167Tyr (or TTT/TAT) and Phe200Tyr (or TTC/TAC), based only on the second nucleotide base changed in the codons. In the homologous segment of its closely related taxa, *W. bancrofti -tubulin* (Wb*tubb*) gene that possesses two distinct exons, 4 (Gly132 to Lys174) and 5 (Val175 to Leu228), with flanking intron sequences (Fig. 3) shares the homology at DNA level with *B. malayi* and *B. pahangi* (93% similarity), compared to *O. volvulus* and *D. immitis* (76% similarity) (Bhumiratana et al, 2010; Pechgit et al, 2011). This target DNA has been proved useful for designing Wb*tubb* locus-specific primers to discriminate between Wb*tubb* and other homologs of human and animal filariids. Based on its usefulness in molecular diagnosis and monitoring of the infection carrying the benzimidazole-sensitive or resistant phenotypes, the PCR applications of this molecular marker for *W. bancrofti* have been well documented (Hoti et al, 2003; 2009; Schwab et al, 2005; Bhumiratana et al, 2010; Pechgit et al, 2011).

#### **4.2 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 (Table 3). As a result of the existence of genetically stable

Molecular Diagnosis and Monitoring of Benzimidazole Susceptibility of Human Filariids 413

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; 2009; 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 Wb*tubb* 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 advantages on how to circumvent some common 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.* 

*–tubulin* gene, the nested PCR amplification can work well with the

haploid genome. Therefore, the amplification is performed using the Wb*tubb* 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) and Hoti et al (2009). More specific, based on our experience, the Wb*tubb* 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 2drug 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.

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

*–tubulin* gene which is single copy in

*W. bancrofti*

**5. Future perspectives** 

*bancrofti* parasite population is amplified based on the

Fig. 4. A purposed scheme for PCR detection of *W. bancrofti* benzimidazole-susceptible isolates in human blood and mosquito.


5'modifications with additional recognition sequences: a*Bam*H I (GGATCC) and b*Eco*R I (GAATTC). c dRetrieved *Wuchereria bancrofti* genome accession nos.: AY705383 and GU19071824. eRetrieved *Brugia pahangi* genome accession nos.: M36380. f

Retrieved *Wuchereria bancrofti* genome accession nos.: EF190199-190209, EF492870-492878.

Table 4. The *β-tubulin* isotype 1 gene-specific primers used in the PCR amplification of *W. bancrofti* benzimidazole-susceptible isolates

*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; 2009; 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 Wb*tubb* 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 advantages on how to circumvent some common 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 Wb*tubb* 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) and Hoti et al (2009). More specific, based on our experience, the Wb*tubb* 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 2drug 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.
