- Introduction
- Material and Methods
- Acquisition of parasite material.
- Ethical approval.
- Wild-type (WT) and mutant-type (MT) plasmid constructs.
- Diagnosis of resistance-associated mutations in A. lumbricoides, T. trichiura, and N. americanus.
- Molecular assay applied to field hookworm samples.
- Allele frequency and sequence analysis.
- EHA for hookworms.
- Application of the EHA to human hookworms.
- Statistical analyses.
- Results
- Discussion
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
WHO, 1999. Informal Consultation on Monitoring Drug Efficacy in the Control of Schistosomiasis and Intestinal Nematodes. Geneva: World Health Organization, 1–59.
-
2.
Brooker S, Clements AC, Bundy DA, 2006. Global epidemiology, ecology and control of soil-transmitted helminth infections. Adv Parasitol 62: 221–261.
-
3.
Hotez PJ, Kamath A, 2009. Neglected tropical diseases in sub-Saharan Africa: review of their prevalence, distribution, and disease burden. PLoS Negl Trop Dis 3: e412.
-
4.
Hotez PJ, 2009. A constitutional amendment for deworming. PLoS Negl Trop Dis 3: e454.
-
5.
Chan MS, 1997. The global burden of intestinal nematode infections–fifty years on. Parasitol Today 13: 438–443.
-
6.
Molyneux DH, Hotez PJ, Fenwick A, Newman RD, Greenwood B, Sachs J, 2009. Neglected tropical diseases and the Global Fund. Lancet 373: 296–297.
-
7.
Albonico M, Stoltzfus RJ, Savioli L, Chwaya HM, d'Harcourt E, Tielsch JM, 1999. A controlled evaluation of two school-based anthelminthic chemotherapy regimens on intensity of intestinal helminth infections. Int J Epidemiol 28: 591–596.
-
8.
Albonico M, Montresor A, Crompton DW, Savioli L, 2006. Intervention for the control of soil-transmitted helminthiasis in the community. Adv Parasitol 61: 311–348.
-
9.
Bennett A, Guyatt H, 2000. Reducing intestinal nematode infection: efficacy of albendazole and mebendazole. Parasitol Today 16: 71–74.
-
10.
Taylor-Robinson D, Jones A, Garner P, 2009. Does deworming improve growth and school performance in children? PLoS Negl Trop Dis 3: e358.
-
11.
Kaplan RM, Vidyashankar AN, 2012. An inconvenient truth: global worming and anthelmintic resistance. Vet Parasitol 186: 70–78.
-
12.
Kwa MS, Veenstra JG, Roos MH, 1994. Benzimidazole resistance in Haemonchus contortus is correlated with a conserved mutation at amino acid 200 in beta-tubulin isotype 1. Mol Biochem Parasitol 63: 299–303.
-
13.
Silvestre A, Cabaret J, 2002. Mutation in position 167 of isotype 1 beta-tubulin gene of Trichostrongylid nematodes: role in benzimidazole resistance? Mol Biochem Parasitol 120: 297–300.
-
14.
Ghisi M, Kaminsky R, Maser P, 2007. Phenotyping and genotyping of Haemonchus contortus isolates reveals a new putative candidate mutation for benzimidazole resistance in nematodes. Vet Parasitol 144: 313–332.
-
15.
Mottier M, Prichard RK, 2008. Genetic analysis of a relationship between macrocyclic lactone and benzimidazole anthelmintic selection on Haemonchus contortus. Pharmacogenet Genomics 18: 129–140.
-
16.
Albonico M, Bickle Q, Ramsan M, Montresor A, Savioli L, Taylor M, 2003. Efficacy of mebendazole and levamisole alone or in combination against intestinal nematode infections after repeated targeted mebendazole treatment in Zanzibar. Bull World Health Organ 81: 343–352.
-
17.
Flohr C, Tuyen LN, Lewis S, Minh TT, Campbell J, Britton J, Williams H, Hien TT, Farrar J, Quinnell RJ, 2007. Low efficacy of mebendazole against hookworm in Vietnam: two randomized controlled trials. Am J Trop Med Hyg 76: 732–736.
-
18.
Keiser J, Utzinger J, 2008. Efficacy of current drugs against soil-transmitted helminth infections: systematic review and meta-analysis. JAMA 299: 1937–1948.
-
19.
Montresor A, 2011. Cure rate is not a valid indicator for assessing drug efficacy and impact of preventive chemotherapy interventions against schistosomiasis and soil-transmitted helminthiasis. Trans R Soc Trop Med Hyg 105: 361–363.
-
20.
Geerts S, Gryseels B, 2000. Drug resistance in human helminths: current situation and lessons from livestock. Clin Microbiol Rev 13: 207–222.
-
21.
Diawara A, Drake LJ, Suswillo RR, Kihara J, Bundy DA, Scott ME, Halpenny C, Stothard JR, Prichard RK, 2009. Assays to detect beta-tubulin codon 200 polymorphism in Trichuris trichiura and Ascaris lumbricoides. PLoS Negl Trop Dis 3: e397.
-
22.
Albonico M, Wright V, Bickle Q, 2004. Molecular analysis of the beta-tubulin gene of human hookworms as a basis for possible benzimidazole resistance on Pemba Island. Mol Biochem Parasitol 134: 281–284.
-
23.
Champetier de Ribes GFM, Desormeaux AM, Eyma E, Montagut P, Champagne C, Pierre J, Pape JW, Raccurt CP, 2005. Intestinal helminths in school children in Haiti in 2002. Bull Soc Pathol Exot 98: 127–132.
-
24.
Kotze AC, Coleman GT, Mai A, McCarthy JS, 2005. Field evaluation of anthelmintic drug sensitivity using in vitro egg hatch and larval motility assays with Necator americanus recovered from human clinical isolates. Int J Parasitol 35: 445–453.
-
25.
Dobson RJ, Griffiths DA, Donald AD, Waller PJ, 1987. A genetic model describing the evolution of levamisole resistance in Trichostrongylus colubriformis, a nematode parasite of sheep. IMA J Math Appl Med Biol 4: 279–293.
-
26.
Waller PJ, Dobson RJ, Donald AD, Griffiths DA, Smith EF, 1985. Selection studies on anthelmintic resistant and susceptible populations of Trichostrongylus colubriformis of sheep. Int J Parasitol 15: 669–676.
-
27.
Schwab AE, Boakye DA, Kyelem D, Prichard RK, 2005. Detection of benzimidazole resistance-associated mutations in the filarial nematode Wuchereria bancrofti and evidence for selection by albendazole and ivermectin combination treatment. Am J Trop Med Hyg 73: 234–238.
-
28.
Barrère V, Alvarez L, Suarez G, Ceballos L, Moreno L, Lanusse C, Prichard RK, 2012. Relationship between increased albendazole systemic exposure and changes in single nucleotide polymorphisms on the β-tubulin isotype 1 encoding gene in Haemonchus contortus. Vet Parasitol 186: 344–349.
-
29.
Eng JK, Blackhall WJ, Osei-Atweneboana MY, Bourguinat C, Galazzo D, Beech RN, Unnasch TR, Awadzi K, Lubega GW, Prichard RK, 2006. Ivermectin selection on beta-tubulin: evidence in Onchocerca volvulus and Haemonchus contortus. Mol Biochem Parasitol 150: 229–235.
-
30.
Schwenkenbecher JM, Albonico M, Bickle Q, Kaplan RM, 2007. Characterization of beta-tubulin genes in hookworms and investigation of resistance-associated mutations using real-time PCR. Mol Biochem Parasitol 156: 167–174.
-
31.
Elard L, Humbert JF, 1999. Importance of the mutation of amino acid 200 of the isotype 1 beta-tubulin gene in the benzimidazole resistance of the small-ruminant parasite Teladorsagia circumcincta. Parasitol Res 85: 452–456.
-
32.
Schwab AE, Churcher TS, Schwab AJ, Basanez MG, Prichard RK, 2006. Population genetics of concurrent selection with albendazole and ivermectin or diethylcarbamazine on the possible spread of albendazole resistance in Wuchereria bancrofti. Parasitol 133: 589–601.
-
33.
Blaxter ML, De Ley P, Garey JR, Liu LX, Scheldeman P, Vierstraete A, Vanfleteren JR, Mackey LY, Dorris M, Frisse LM, Vida JT, Thomas WK, 1998. A molecular evolutionary framework for the phylum nematoda. Nature 392: 71–75.
-
34.
Kotze AC, Clifford S, O'Grady J, Behnke JM, McCarthy JS, 2004. An in vitro larval motility assay to determine anthelmintic sensitivity for human hookworm and Strongyloides species. Am J Trop Med Hyg 71: 608–616.
-
35.
von Samson-Himmelstjerna G, Coles GC, Jackson F, Bauer C, Borgsteede F, Cirak VY, Demeler J, Donnan A, Dorny P, Epe C, Harder A, Hoglund J, Kaminsky R, Kerboeuf D, Kuttler U, Papadopoulos E, Posedi J, Small J, Varady M, Vercruysse J, Wirtherle N, 2009. Standardization of the egg hatch test for the detection of benzimidazole resistance in parasitic nematodes. Parasitol Res 105: 825–834.
-
36.
Coles GC, Bauer C, Borgsteede FH, Geerts S, Klei TR, Taylor MA, Waller PJ, 1992. World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) methods for the detection of anthelmintic resistance in nematodes of veterinary importance. Vet Parasitol 44: 35–44.
-
37.
Albonico M, Wright V, Ramsan M, Haji HJ, Taylor M, Savioli L, Bickle Q, 2005. Development of the egg hatch assay for detection of anthelminthic resistance in human hookworms. Int J Parasitol 35: 803–811.
-
38.
Kotze AC, Lowe A, O'Grady J, Kopp SR, Behnke JM, 2009. Dose-response assay templates for in vitro assessment of resistance to benzimidazole and nicotinic acetylcholine receptor agonist drugs in human hookworms. Am J Trop Med Hyg 81: 163–170.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 1255 | 867 | 42 |
| Full Text Views | 741 | 15 | 0 |
| PDF Downloads | 335 | 20 | 0 |
Molecular and Biological Diagnostic Tests for Monitoring Benzimidazole Resistance in Human Soil-Transmitted Helminths
Aïssatou Diawara
Aïssatou Diawara
Institute of Parasitology, McGill University, Ste. Anne-de-Bellevue, Canada; Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, Georgia
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Jan M. Schwenkenbecher
Jan M. Schwenkenbecher
Institute of Parasitology, McGill University, Ste. Anne-de-Bellevue, Canada; Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, Georgia
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Ray M. Kaplan
Ray M. Kaplan
Institute of Parasitology, McGill University, Ste. Anne-de-Bellevue, Canada; Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, Georgia
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Roger K. Prichard
Roger K. Prichard
Institute of Parasitology, McGill University, Ste. Anne-de-Bellevue, Canada; Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, Georgia
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In endemic countries with soil-transmitted helminths mass drug administration with albendazole or mebendazole are being implemented as a control strategy. However, it is well known in veterinary helminths that the use of the same benzimidazole drugs can place selection on the β-tubulin gene, leading to resistance. Given the concern that resistance could arise in human soil-transmitted helminths, there is an urgent need to develop accurate diagnostic tools for monitoring resistance. In this study, we developed molecular assays to detect putative resistance genetic changes in Ascaris lumbricoides, Trichuris trichiura, and hookworms, and we optimized an egg hatch assay for the canine hookworm Ancylostoma caninum and applied it to Necator americanus. Both assays were tested on field samples. The molecular assays demonstrated their reproducibility and capacity to detect the presence of worms carrying putative resistance-associated genetic changes. However, further investigations are needed to validate our molecular and biological tests on additional field isolates.
Author Notes
Financial support: This work was supported by a grant from the Atlanta Research and Education Foundation, Inc. in collaboration with the Centers for Diseases Control and Prevention (CDC), and the research at the Institute of Parasitology was supported by the Québec Centre for Host Parasite Interactions/FQRNT.
Authors' addresses: Aïssatou Diawara and Roger K. Prichard, Institute of Parasitology, McGill University, Ste-Anne-de-Bellevue, Quebec, Canada, E-mails: aissatou.diawara@mail.mcgill.ca and roger.prichard@mcgill.ca. Jan M. Schwenkenbecher and Ray M. Kaplan, Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA, E-mails: janschwenkenbecher@yahoo.de and rkaplan@uga.edu.
- Introduction
- Material and Methods
- Acquisition of parasite material.
- Ethical approval.
- Wild-type (WT) and mutant-type (MT) plasmid constructs.
- Diagnosis of resistance-associated mutations in A. lumbricoides, T. trichiura, and N. americanus.
- Molecular assay applied to field hookworm samples.
- Allele frequency and sequence analysis.
- EHA for hookworms.
- Application of the EHA to human hookworms.
- Statistical analyses.
- Results
- Discussion
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
WHO, 1999. Informal Consultation on Monitoring Drug Efficacy in the Control of Schistosomiasis and Intestinal Nematodes. Geneva: World Health Organization, 1–59.
-
2.
Brooker S, Clements AC, Bundy DA, 2006. Global epidemiology, ecology and control of soil-transmitted helminth infections. Adv Parasitol 62: 221–261.
-
3.
Hotez PJ, Kamath A, 2009. Neglected tropical diseases in sub-Saharan Africa: review of their prevalence, distribution, and disease burden. PLoS Negl Trop Dis 3: e412.
-
4.
Hotez PJ, 2009. A constitutional amendment for deworming. PLoS Negl Trop Dis 3: e454.
-
5.
Chan MS, 1997. The global burden of intestinal nematode infections–fifty years on. Parasitol Today 13: 438–443.
-
6.
Molyneux DH, Hotez PJ, Fenwick A, Newman RD, Greenwood B, Sachs J, 2009. Neglected tropical diseases and the Global Fund. Lancet 373: 296–297.
-
7.
Albonico M, Stoltzfus RJ, Savioli L, Chwaya HM, d'Harcourt E, Tielsch JM, 1999. A controlled evaluation of two school-based anthelminthic chemotherapy regimens on intensity of intestinal helminth infections. Int J Epidemiol 28: 591–596.
-
8.
Albonico M, Montresor A, Crompton DW, Savioli L, 2006. Intervention for the control of soil-transmitted helminthiasis in the community. Adv Parasitol 61: 311–348.
-
9.
Bennett A, Guyatt H, 2000. Reducing intestinal nematode infection: efficacy of albendazole and mebendazole. Parasitol Today 16: 71–74.
-
10.
Taylor-Robinson D, Jones A, Garner P, 2009. Does deworming improve growth and school performance in children? PLoS Negl Trop Dis 3: e358.
-
11.
Kaplan RM, Vidyashankar AN, 2012. An inconvenient truth: global worming and anthelmintic resistance. Vet Parasitol 186: 70–78.
-
12.
Kwa MS, Veenstra JG, Roos MH, 1994. Benzimidazole resistance in Haemonchus contortus is correlated with a conserved mutation at amino acid 200 in beta-tubulin isotype 1. Mol Biochem Parasitol 63: 299–303.
-
13.
Silvestre A, Cabaret J, 2002. Mutation in position 167 of isotype 1 beta-tubulin gene of Trichostrongylid nematodes: role in benzimidazole resistance? Mol Biochem Parasitol 120: 297–300.
-
14.
Ghisi M, Kaminsky R, Maser P, 2007. Phenotyping and genotyping of Haemonchus contortus isolates reveals a new putative candidate mutation for benzimidazole resistance in nematodes. Vet Parasitol 144: 313–332.
-
15.
Mottier M, Prichard RK, 2008. Genetic analysis of a relationship between macrocyclic lactone and benzimidazole anthelmintic selection on Haemonchus contortus. Pharmacogenet Genomics 18: 129–140.
-
16.
Albonico M, Bickle Q, Ramsan M, Montresor A, Savioli L, Taylor M, 2003. Efficacy of mebendazole and levamisole alone or in combination against intestinal nematode infections after repeated targeted mebendazole treatment in Zanzibar. Bull World Health Organ 81: 343–352.
-
17.
Flohr C, Tuyen LN, Lewis S, Minh TT, Campbell J, Britton J, Williams H, Hien TT, Farrar J, Quinnell RJ, 2007. Low efficacy of mebendazole against hookworm in Vietnam: two randomized controlled trials. Am J Trop Med Hyg 76: 732–736.
-
18.
Keiser J, Utzinger J, 2008. Efficacy of current drugs against soil-transmitted helminth infections: systematic review and meta-analysis. JAMA 299: 1937–1948.
-
19.
Montresor A, 2011. Cure rate is not a valid indicator for assessing drug efficacy and impact of preventive chemotherapy interventions against schistosomiasis and soil-transmitted helminthiasis. Trans R Soc Trop Med Hyg 105: 361–363.
-
20.
Geerts S, Gryseels B, 2000. Drug resistance in human helminths: current situation and lessons from livestock. Clin Microbiol Rev 13: 207–222.
-
21.
Diawara A, Drake LJ, Suswillo RR, Kihara J, Bundy DA, Scott ME, Halpenny C, Stothard JR, Prichard RK, 2009. Assays to detect beta-tubulin codon 200 polymorphism in Trichuris trichiura and Ascaris lumbricoides. PLoS Negl Trop Dis 3: e397.
-
22.
Albonico M, Wright V, Bickle Q, 2004. Molecular analysis of the beta-tubulin gene of human hookworms as a basis for possible benzimidazole resistance on Pemba Island. Mol Biochem Parasitol 134: 281–284.
-
23.
Champetier de Ribes GFM, Desormeaux AM, Eyma E, Montagut P, Champagne C, Pierre J, Pape JW, Raccurt CP, 2005. Intestinal helminths in school children in Haiti in 2002. Bull Soc Pathol Exot 98: 127–132.
-
24.
Kotze AC, Coleman GT, Mai A, McCarthy JS, 2005. Field evaluation of anthelmintic drug sensitivity using in vitro egg hatch and larval motility assays with Necator americanus recovered from human clinical isolates. Int J Parasitol 35: 445–453.
-
25.
Dobson RJ, Griffiths DA, Donald AD, Waller PJ, 1987. A genetic model describing the evolution of levamisole resistance in Trichostrongylus colubriformis, a nematode parasite of sheep. IMA J Math Appl Med Biol 4: 279–293.
-
26.
Waller PJ, Dobson RJ, Donald AD, Griffiths DA, Smith EF, 1985. Selection studies on anthelmintic resistant and susceptible populations of Trichostrongylus colubriformis of sheep. Int J Parasitol 15: 669–676.
-
27.
Schwab AE, Boakye DA, Kyelem D, Prichard RK, 2005. Detection of benzimidazole resistance-associated mutations in the filarial nematode Wuchereria bancrofti and evidence for selection by albendazole and ivermectin combination treatment. Am J Trop Med Hyg 73: 234–238.
-
28.
Barrère V, Alvarez L, Suarez G, Ceballos L, Moreno L, Lanusse C, Prichard RK, 2012. Relationship between increased albendazole systemic exposure and changes in single nucleotide polymorphisms on the β-tubulin isotype 1 encoding gene in Haemonchus contortus. Vet Parasitol 186: 344–349.
-
29.
Eng JK, Blackhall WJ, Osei-Atweneboana MY, Bourguinat C, Galazzo D, Beech RN, Unnasch TR, Awadzi K, Lubega GW, Prichard RK, 2006. Ivermectin selection on beta-tubulin: evidence in Onchocerca volvulus and Haemonchus contortus. Mol Biochem Parasitol 150: 229–235.
-
30.
Schwenkenbecher JM, Albonico M, Bickle Q, Kaplan RM, 2007. Characterization of beta-tubulin genes in hookworms and investigation of resistance-associated mutations using real-time PCR. Mol Biochem Parasitol 156: 167–174.
-
31.
Elard L, Humbert JF, 1999. Importance of the mutation of amino acid 200 of the isotype 1 beta-tubulin gene in the benzimidazole resistance of the small-ruminant parasite Teladorsagia circumcincta. Parasitol Res 85: 452–456.
-
32.
Schwab AE, Churcher TS, Schwab AJ, Basanez MG, Prichard RK, 2006. Population genetics of concurrent selection with albendazole and ivermectin or diethylcarbamazine on the possible spread of albendazole resistance in Wuchereria bancrofti. Parasitol 133: 589–601.
-
33.
Blaxter ML, De Ley P, Garey JR, Liu LX, Scheldeman P, Vierstraete A, Vanfleteren JR, Mackey LY, Dorris M, Frisse LM, Vida JT, Thomas WK, 1998. A molecular evolutionary framework for the phylum nematoda. Nature 392: 71–75.
-
34.
Kotze AC, Clifford S, O'Grady J, Behnke JM, McCarthy JS, 2004. An in vitro larval motility assay to determine anthelmintic sensitivity for human hookworm and Strongyloides species. Am J Trop Med Hyg 71: 608–616.
-
35.
von Samson-Himmelstjerna G, Coles GC, Jackson F, Bauer C, Borgsteede F, Cirak VY, Demeler J, Donnan A, Dorny P, Epe C, Harder A, Hoglund J, Kaminsky R, Kerboeuf D, Kuttler U, Papadopoulos E, Posedi J, Small J, Varady M, Vercruysse J, Wirtherle N, 2009. Standardization of the egg hatch test for the detection of benzimidazole resistance in parasitic nematodes. Parasitol Res 105: 825–834.
-
36.
Coles GC, Bauer C, Borgsteede FH, Geerts S, Klei TR, Taylor MA, Waller PJ, 1992. World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) methods for the detection of anthelmintic resistance in nematodes of veterinary importance. Vet Parasitol 44: 35–44.
-
37.
Albonico M, Wright V, Ramsan M, Haji HJ, Taylor M, Savioli L, Bickle Q, 2005. Development of the egg hatch assay for detection of anthelminthic resistance in human hookworms. Int J Parasitol 35: 803–811.
-
38.
Kotze AC, Lowe A, O'Grady J, Kopp SR, Behnke JM, 2009. Dose-response assay templates for in vitro assessment of resistance to benzimidazole and nicotinic acetylcholine receptor agonist drugs in human hookworms. Am J Trop Med Hyg 81: 163–170.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 1255 | 867 | 42 |
| Full Text Views | 741 | 15 | 0 |
| PDF Downloads | 335 | 20 | 0 |