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A Pilot Comparison of Fixatives for Hookworm Real-time Polymerase Chain Reaction

Richard BradburyFederation University, Melbourne, Australia;

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Kengo InagakiDepartment 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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Gurbaksh SinghDepartment of Pediatrics, Division of Infectious Disease, Children’s of Mississippi, University of Mississippi Medical Center, Jackson, Mississippi;

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Urita AganaDepartment of Pediatrics, Division of Infectious Disease, Children’s of Mississippi, University of Mississippi Medical Center, Jackson, Mississippi;

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Kayla PattersonDepartment 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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Lacy MallochDepartment of Pediatrics, Division of Infectious Disease, Children’s of Mississippi, University of Mississippi Medical Center, Jackson, Mississippi;

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Eduardo RodriguezOak 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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Yvonne QvarnstromDivision of Parasitic Diseases and Malaria, Centers for Disease Control and Prevention, Atlanta, Georgia

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Charlotte V. HobbsDepartment 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.

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Author Notes

Address correspondence to Richard Bradbury, Federation University (Berwick Campus), 100 Clyde Rd., Berwick, VIC, 3806, Australia. E-mail: r.bradbury@federation.edu.au or Charlotte V. Hobbs, Department of Pediatrics, Division of Infectious Disease, Children’s of Mississippi, University of Mississippi Medical Center, 2500 North State St., Jackson, MS 39216. E-mail: chobbs@umc.edu

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.

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