A comparative analysis of preservation techniques for the optimal molecular detection of hookworm DNA in a human fecal specimen

Marina Papaiakovou*, Nils Pilotte, Ben Baumer, Jessica Grant, Kristjana Asbjornsdottir, Fabian Schaer, Yan Hu, Raffi Aroian, Judd Walson, Steven A. Williams

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

24 Citations (Scopus)


Background: Proper collection and storage of fecal samples is necessary to guarantee the subsequent reliability of DNA-based soil-transmitted helminth diagnostic procedures. Previous research has examined various methods to preserve fecal samples for subsequent microscopic analysis or for subsequent determination of overall DNA yields obtained following DNA extraction. However, only limited research has focused on the preservation of soil-transmitted helminth DNA in stool samples stored at ambient temperature or maintained in a cold chain for extended periods of time. Methodology: Quantitative real-time PCR was used in this study as a measure of the effectiveness of seven commercially available products to preserve hookworm DNA over time and at different temperatures. Results were compared against “no preservative” controls and the “gold standard” of rapidly freezing samples at -20°C. The preservation methods were compared at both 4°C and at simulated tropical ambient temperature (32°C) over a period of 60 days. Evaluation of the effectiveness of each preservative was based on quantitative real-time PCR detection of target hookworm DNA. Conclusions: At 4°C there were no significant differences in DNA amplification efficiency (as measured by Cqvalues) regardless of the preservation method utilized over the 60-day period. At 32°C, preservation with FTA cards, potassium dichromate, and a silica bead two-step desiccation process proved most advantageous for minimizing Cqvalue increases, while RNA later, 95% ethanol and Paxgene also demonstrate some protective effect. These results suggest that fecal samples spiked with known concentrations of hookworm-derived egg material can remain at 4°C for 60 days in the absence of preservative, without significant degradation of the DNA target. Likewise, a variety of preservation methods can provide a measure of protection in the absence of a cold chain. As a result, other factors, such as preservative toxicity, inhibitor resistance, preservative cost, shipping requirements, sample infectivity, and labor costs should be considered when deciding upon an appropriate method for the storage of fecal specimens for subsequent PCR analysis. Balancing logistical factors and the need to preserve the target DNA, we believe that under most circumstances 95% ethanol provides the most pragmatic choice for preserving stool samples in the field.

Original languageEnglish
Article numbere0006130
JournalPLoS Neglected Tropical Diseases
Issue number1
Publication statusPublished - Jan 2018

Bibliographical note

Funding Information:
This study was funded through a grant to the DeWorm3 Project, which is funded by a grant to the Natural History Museum from the Bill and Melinda Gates Foundation (OPP1129535, PI JW). The hookworm colony maintained at UMass Medical School, was funded by a NIAID (Health/National Institute of Allergy and Infectious Diseases) grant, R01 AI056189 to RA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors would like to thank Samara Loewenstein for technical support and Susan Haynes for logistical support.

Publisher Copyright:
© 2018 Papaiakovou et al.


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