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-
1.
O’Connell EM, Nutman TB, 2016. Molecular diagnostics for soil-transmitted helminths. Am J Trop Med Hyg 95: 508.
-
2.
Pilotte N, Papaiakovou M, Grant JR, Bierwert LA, Llewellyn S, McCarthy JS, Williams SA, 2016. Improved PCR-based detection of soil transmitted helminth infections using a next-generation sequencing approach to assay design. PLoS Negl Trop Dis 10: e0004578.
-
3.
Stracke K, Clarke N, Awburn CV, Vaz Nery S, Khieu V, Traub RJ, Jex AR, 2019. Development and validation of a multiplexed-tandem qPCR tool for diagnostics of human soil-transmitted helminth infections. PLoS Negl Trop Dis 13: e0007363.
-
4.
Autier B, Gangneux JP, Robert-Gangneux F, 2021. Evaluation of the Allplex™ GI-Helminth (I) Assay, the first marketed multiplex PCR for helminth diagnosis. Parasite 28: 33.
-
5.
Kines KJ, Fox M, Ndubuisi M, Verocai GG, Cama V, Bradbury RS, 2021. Inactivating effects of common laboratory disinfectants, fixatives, and temperatures on the eggs of soil transmitted helminths. Microbiol Spectr 9: e0182821.
-
6.
McHardy IH, Wu M, Shimizu-Cohen R, Couturier MR, Humphries RM, 2014. Detection of intestinal protozoa in the clinical laboratory. J Clin Microbiol 52: 712–720.
-
7.
Ramos F, Zurabian R, Morán P, Ramiro M, Gómez A, Clark CG, Melendro EI, García G, Ximénez C, 1999. The effect of formalin fixation on the polymerase chain reaction characterization of Entamoeba histolytica. Trans R Soc Trop Med Hyg 93: 335–336.
-
8.
DeBurger BA, Hanna S, Mortensen JE, 2018. Evaluation of alternate parasite transport systems for the BD MAX™ enteric parasite panel. Diagn Microbiol Infect Dis 92: 204–205.
-
9.
Qvarnstrom Y, Benedict T, Marcet PL, Wiegand RE, Herwaldt BL, da Silva AJ, 2018. Molecular detection of Cyclospora cayetanensis in human stool specimens using UNEX-based DNA extraction and real-time PCR. Parasitology 145: 865–870.
-
10.
Kaisar MM, Brienen EA, Djuardi Y, Sartono E, Yazdanbakhsh M, Verweij JJ, Supali T, Van Lieshout L, 2017. Improved diagnosis of Trichuris trichiura by using a bead-beating procedure on ethanol preserved stool samples prior to DNA isolation and the performance of multiplex real-time PCR for intestinal parasites. Parasitology 144: 965–974.
-
11.
Papaiakovou M, Pilotte N, Baumer B, Grant J, Asbjornsdottir K, Schaer F, Hu Y, Aroian R, Walson J, Williams SA, 2018. A comparative analysis of preservation techniques for the optimal molecular detection of hookworm DNA in a human fecal specimen. PLoS Negl Trop Dis 12: e0006130.
-
12.
Ayana M, Cools P, Mekonnen Z, Biruksew A, Dana D, Rashwan N, Prichard R, Vlaminck J, Verweij JJ, Levecke B, 2019. Comparison of four DNA extraction and three preservation protocols for the molecular detection and quantification of soil-transmitted helminths in stool. PLoS Negl Trop Dis 13: e0007778.
-
13.
Jex AR, Waeschenbach A, Hu M, Van Wyk JA, Beveridge I, Littlewood DT, Gasser RB, 2009. The mitochondrial genomes of Ancylostoma caninum and Bunostomum phlebotomum – two hookworms of animal health and zoonotic importance. BMC Genomics 10: 1–0.
-
14.
Shi X et al., 2018. Comparative analysis of Ancylostoma ceylanicum mitochondrial genome with other Ancylostoma species. Infect Genet Evol 62: 40–45.
-
15.
Xie Y, Xu Z, Zheng Y, Li Y, Liu Y, Wang L, Zhou X, Zuo Z, Gu X, Yang G, 2019. The mitochondrial genome of the dog hookworm Ancylostoma caninum (Nematoda, Ancylostomatidae) from Southwest China. Mitochondrial DNA B Resour 4: 3002–3004.
-
16.
Shepherd C, Wangchuk P, Loukas A, 2018. Of dogs and hookworms: man’s best friend and his parasites as a model for translational biomedical research. Parasit Vectors 11: 1–6.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 3115 | 2433 | 62 |
| Full Text Views | 313 | 62 | 0 |
| PDF Downloads | 145 | 27 | 0 |
A Pilot Comparison of Fixatives for Hookworm Real-time Polymerase Chain Reaction
Richard Bradbury
Richard Bradbury
Federation University, Melbourne, Australia;
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Kengo Inagaki
Kengo Inagaki
Department of Pediatrics, University of Michigan, Ann Arbor, Michigan;
Department of Pediatrics, Division of Infectious Disease, Children’s of Mississippi, University of Mississippi Medical Center, Jackson, Mississippi;
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Department of Pediatrics, Division of Infectious Disease, Children’s of Mississippi, University of Mississippi Medical Center, Jackson, Mississippi;
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Gurbaksh Singh
Gurbaksh Singh
Department of Pediatrics, Division of Infectious Disease, Children’s of Mississippi, University of Mississippi Medical Center, Jackson, Mississippi;
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Urita Agana
Urita Agana
Department of Pediatrics, Division of Infectious Disease, Children’s of Mississippi, University of Mississippi Medical Center, Jackson, Mississippi;
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Kayla Patterson
Kayla Patterson
Department of Pediatrics, Division of Infectious Disease, Children’s of Mississippi, University of Mississippi Medical Center, Jackson, Mississippi;
Oak Ridge Institute for Science and Education, Oak Ridge, Tennessee;
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Oak Ridge Institute for Science and Education, Oak Ridge, Tennessee;
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Lacy Malloch
Lacy Malloch
Department of Pediatrics, Division of Infectious Disease, Children’s of Mississippi, University of Mississippi Medical Center, Jackson, Mississippi;
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Eduardo Rodriguez
Eduardo Rodriguez
Oak Ridge Institute for Science and Education, Oak Ridge, Tennessee;
Division of Parasitic Diseases and Malaria, Centers for Disease Control and Prevention, Atlanta, Georgia
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Division of Parasitic Diseases and Malaria, Centers for Disease Control and Prevention, Atlanta, Georgia
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Yvonne Qvarnstrom
Yvonne Qvarnstrom
Division of Parasitic Diseases and Malaria, Centers for Disease Control and Prevention, Atlanta, Georgia
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Charlotte V. Hobbs
Charlotte V. Hobbs
Department of Pediatrics, Division of Infectious Disease, Children’s of Mississippi, University of Mississippi Medical Center, Jackson, Mississippi;
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ABSTRACT.
Polymerase chain reaction (PCR) is increasingly used in the diagnosis of soil-transmitted helminth infections. Despite this, few studies have evaluated the impact of different fecal fixatives on the outcome of fecal helminth qPCR analysis, and none have evaluated the effect of commercial parasitology fixatives commonly used in diagnostic laboratories. We fixed dog feces containing Ancylostoma spp. hookworm eggs in zinc polyvinyl alcohol (Zn-PVA) and Total-Fix, and with 70% ethanol (EtOH) as a control. DNA was extracted at timepoints 11, 33, 64, and 94 days and subjected to Ancylostoma spp. quantitative PCR (qPCR). A linear regression model was created to assess the effect of preservative types on the temporal change of qPCR quantification cycle number (Cq) values, accounting for variances among individual animals. Fixation in 70% EtOH least affected Cq values over 94 days. Total-Fix preservation yielded a higher Cq overall, but there was no significant difference compared with 70% EtOH fixation. Fixation in Zn-PVA resulted in significantly (P < 0.001) higher Cq values than 70% EtOH after only 33 days and loss of amplification at 64 days. Consistent with other helminth fixation studies, 70% EtOH performed well in preserving hookworm DNA over 94 days. Total-Fix provided a comparable alternative for qPCR analysis for hookworm. Fixation in Zn-PVA resulted in loss of detectable hookworm DNA at 64 days, as determined by qPCR.
- Supplemental Materials (PDF 361 KB)
Author Notes
Financial support: This work was funded by the University of Mississippi Vice Chancellor’s Office for Research (CVH). The CDC provided laboratory support for PCR.
Disclaimer: The findings and conclusions presented in this manuscript are those of the authors and do not necessarily represent the views of the U.S. Centers for Disease Control and Prevention.
Authors’ addresses: Richard Bradbury, Federation University, Melbourne, Australia, E-mail: r.bradbury@federation.edu.au. Kengo Inagaki, Department of Pediatrics, University of Michigan, Ann Arbor, MI, E-mail: kinagaki@umc.edu. Charlotte V. Hobbs, Gurbaksh Singh, Urita Agana, Kayla Patterson, and Lacy Malloch, Department of Pediatrics, Division of Infectious Disease, Children’s of Mississippi, University of Mississippi Medical Center, Jackson, MS. E-mails: chobbs@umc.edu, gsingh2@umc.edu, uritaagana@gmail.com, knpatterson@umc.edu, and lmalloch@umc.edu. Eduardo Rodriguez, Oak Ridge Institute for Science and Education, Oak Ridge, TN, and Division of Parasitic Diseases and Malaria, Centers for Disease Control and Prevention, Atlanta, Georgia, E-mail: pvj5@cdc.gov. Yvonne Qvarnstrom, Division of Parasitic Diseases and Malaria, Centers for Disease Control and Prevention, Atlanta, Georgia, E-mail: bvp2@cdc.gov.
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
O’Connell EM, Nutman TB, 2016. Molecular diagnostics for soil-transmitted helminths. Am J Trop Med Hyg 95: 508.
-
2.
Pilotte N, Papaiakovou M, Grant JR, Bierwert LA, Llewellyn S, McCarthy JS, Williams SA, 2016. Improved PCR-based detection of soil transmitted helminth infections using a next-generation sequencing approach to assay design. PLoS Negl Trop Dis 10: e0004578.
-
3.
Stracke K, Clarke N, Awburn CV, Vaz Nery S, Khieu V, Traub RJ, Jex AR, 2019. Development and validation of a multiplexed-tandem qPCR tool for diagnostics of human soil-transmitted helminth infections. PLoS Negl Trop Dis 13: e0007363.
-
4.
Autier B, Gangneux JP, Robert-Gangneux F, 2021. Evaluation of the Allplex™ GI-Helminth (I) Assay, the first marketed multiplex PCR for helminth diagnosis. Parasite 28: 33.
-
5.
Kines KJ, Fox M, Ndubuisi M, Verocai GG, Cama V, Bradbury RS, 2021. Inactivating effects of common laboratory disinfectants, fixatives, and temperatures on the eggs of soil transmitted helminths. Microbiol Spectr 9: e0182821.
-
6.
McHardy IH, Wu M, Shimizu-Cohen R, Couturier MR, Humphries RM, 2014. Detection of intestinal protozoa in the clinical laboratory. J Clin Microbiol 52: 712–720.
-
7.
Ramos F, Zurabian R, Morán P, Ramiro M, Gómez A, Clark CG, Melendro EI, García G, Ximénez C, 1999. The effect of formalin fixation on the polymerase chain reaction characterization of Entamoeba histolytica. Trans R Soc Trop Med Hyg 93: 335–336.
-
8.
DeBurger BA, Hanna S, Mortensen JE, 2018. Evaluation of alternate parasite transport systems for the BD MAX™ enteric parasite panel. Diagn Microbiol Infect Dis 92: 204–205.
-
9.
Qvarnstrom Y, Benedict T, Marcet PL, Wiegand RE, Herwaldt BL, da Silva AJ, 2018. Molecular detection of Cyclospora cayetanensis in human stool specimens using UNEX-based DNA extraction and real-time PCR. Parasitology 145: 865–870.
-
10.
Kaisar MM, Brienen EA, Djuardi Y, Sartono E, Yazdanbakhsh M, Verweij JJ, Supali T, Van Lieshout L, 2017. Improved diagnosis of Trichuris trichiura by using a bead-beating procedure on ethanol preserved stool samples prior to DNA isolation and the performance of multiplex real-time PCR for intestinal parasites. Parasitology 144: 965–974.
-
11.
Papaiakovou M, Pilotte N, Baumer B, Grant J, Asbjornsdottir K, Schaer F, Hu Y, Aroian R, Walson J, Williams SA, 2018. A comparative analysis of preservation techniques for the optimal molecular detection of hookworm DNA in a human fecal specimen. PLoS Negl Trop Dis 12: e0006130.
-
12.
Ayana M, Cools P, Mekonnen Z, Biruksew A, Dana D, Rashwan N, Prichard R, Vlaminck J, Verweij JJ, Levecke B, 2019. Comparison of four DNA extraction and three preservation protocols for the molecular detection and quantification of soil-transmitted helminths in stool. PLoS Negl Trop Dis 13: e0007778.
-
13.
Jex AR, Waeschenbach A, Hu M, Van Wyk JA, Beveridge I, Littlewood DT, Gasser RB, 2009. The mitochondrial genomes of Ancylostoma caninum and Bunostomum phlebotomum – two hookworms of animal health and zoonotic importance. BMC Genomics 10: 1–0.
-
14.
Shi X et al., 2018. Comparative analysis of Ancylostoma ceylanicum mitochondrial genome with other Ancylostoma species. Infect Genet Evol 62: 40–45.
-
15.
Xie Y, Xu Z, Zheng Y, Li Y, Liu Y, Wang L, Zhou X, Zuo Z, Gu X, Yang G, 2019. The mitochondrial genome of the dog hookworm Ancylostoma caninum (Nematoda, Ancylostomatidae) from Southwest China. Mitochondrial DNA B Resour 4: 3002–3004.
-
16.
Shepherd C, Wangchuk P, Loukas A, 2018. Of dogs and hookworms: man’s best friend and his parasites as a model for translational biomedical research. Parasit Vectors 11: 1–6.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 3115 | 2433 | 62 |
| Full Text Views | 313 | 62 | 0 |
| PDF Downloads | 145 | 27 | 0 |