- ABSTRACT.
- INTRODUCTION
- MATERIALS AND METHODS
- Ethical statement and sample collection.
- Agar plate culture technique and worm collection.
- Formalin-ethyl acetate concentration technique.
- Spiking of human stool with S. stercoralis.
- Sources of DNA and DNA extraction.
- Primer and probe design.
- Real-time PCR.
- Droplet digital PCR assay for S. stercoralis detection.
- Analytical sensitivity and specificity of ddPCR.
- Statistical analysis.
- RESULTS
- DISCUSSION
- ACKNOWLEDGMENTS
- REFERENCES
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Development and Efficacy of Droplet Digital PCR for Detection of Strongyloides stercoralis in Stool
ABSTRACT.
Human strongyloidiasis is one of the neglected tropical diseases caused by infection with soil-transmitted helminth Strongyloides stercoralis. Conventional stool examination, a method commonly used for diagnosis of S. stercoralis, has low sensitivity, especially in the case of light infections. Herein, we developed the droplet digital polymerase chain reaction (ddPCR) assay to detect S. stercoralis larvae in stool and compared its performance with real-time PCR and stool examination techniques (formalin ethyl-acetate concentration technique [FECT] and agar plate culture [APC]). The ddPCR results showed 98% sensitivity and 90% specificity, and real-time PCR showed 82% sensitivity and 76.7% specificity when compared with the microscopic methods. Moreover, ddPCR could detect a single S. stercoralis larva in feces, and cross-reactions with other parasites were not observed. In conclusion, a novel ddPCR method exhibited high sensitivity and specificity for detection of S. stercoralis in stool samples. This technique may help to improve diagnosis, particularly in cases with light infection. In addition, ddPCR technique might be useful for screening patients before starting immunosuppressive drug therapy, and follow-up after treatment of strongyloidiasis.
Author Notes
Financial support: This work was financially supported by the young researcher development project of Khon Kaen University (2019) and Invitation Research fund (Grant number IN63250) Faculty of Medicine, Khon Kaen University, Thailand.
Authors’ addresses: Kantapong Iamrod, Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, E-mail: kantapongia@kkumail.com. Apisit Chaidee, Kulthida Y. Kopolrat, Chanika Worasith, Somchai Pinlaor, Paiboon Sithithaworn, and Nuttanan Hongsrichan, Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, and Cholangiocarcinoma Research Institute, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, E-mails: apisch@kku.ac.th, kulthida_kop@yahoo.com, chanika.w@kkumail.com, psomec@kku.ac.th, paibsit@gmail.com, and nuttho@kku.ac.th. Rucksak Rucksaken, Department of Veterinary Technology, Faculty of Veterinary Technology, Kasetsart University, Bangkok, Thailand, E-mail: lucidlucky@gmail.com. Phattharaphon Wongphutorn, Cholangiocarcinoma Research Institute, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, and Biomedical Science Program, Graduate School, Khon kaen University, Khon Kaen, Thailand, E-mail: phat_phutorn@hotmail.com. Kitti Intuyod, Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, E-mail: kittin@kku.ac.th. Jiraporn Sithithaworn, Faculty of Medicine, Mahasarakham University, Mahasarakham, Thailand, E-mail: jirapornsith@gmail.com.