A PCR-based method for the diagnosis of Enterobius vermicularis in stool samples, specifically designed for clinical application

• Published in: Frontiers in microbiology Vol. 13; p. 1028988
• Main Authors: Ummarino, Aldo, Caputo, Michele, Tucci, Francesco Antonio, Pezzicoli, Gaetano, Piepoli, Ada, Gentile, Annamaria, Latiano, Tiziana, Panza, Anna, Calà, Nicholas, Ceglia, Antonio Pio, Pistoio, Giovanni, Troiano, Vincenzo, Pucatti, Michela, Latiano, Anna, Andriulli, Angelo, Tucci, Antonio, Palmieri, Orazio
• Format: Journal Article
• Language: English
• Published: Frontiers Media S.A 17.11.2022
• Subjects: Enterobiasis vermicularis; Microbiology; PCR; pinworm infection; ribosomal DNA–rDNA; stool (DNA) test
• ISSN: 1664-302X; 1664-302X
• DOI: 10.3389/fmicb.2022.1028988

Background Enterobius vermicularis ( E. vermicularis ) is a nematode that infects up to 200 million people worldwide, despite effective medications being available. Conventional diagnostic tests are hindered by low sensitivity and poor patient compliance. Furthermore, no biomolecular techniques are available for clinical application. The aim of this study was to develop a procedure specifically designed for clinical application to detect E. vermicularis by means of PCR. Materials and methods Two subject groups were taken into account: a group of 27 infected patients and a control group of 27 healthy subjects. A nested-PCR was performed on fecal samples to detect E. vermicularis . Due to the intrinsic difficulties of the fecal matrix, several countermeasures were adopted to ensure the efficient performance of the method: (a) a large amount of feces for the extraction process (20 g instead of 200 mg); (b) a combination of chemical and physical treatments to grind the fecal matrix; (c) an additional purification process for the negative samples after the first nested-PCR; and (d) the selection of a very specific target region for the PCR. Results Due to the lack of overlap with other organisms, a sequence of the 5S ribosomal DNA (rDNA) spacer region including the tract SL1 was chosen to design appropriate external and internal primers. The first nested-PCR detected E.vermicularis in 19/27 samples from infected patients. After further purification, 5/8 of the negative samples resulted positive at the second PCR. Conversely, all the samples from healthy controls resulted negative to both PCRs. Sensitivity and specificity of the method were, respectively, 88.9% and 100%. Conclusion The results prove the high diagnostic accuracy of the proposed method, addressing and overcoming the challenges posed by both conventional tests and PCR-based approaches. Therefore, the method can be proposed for clinical application.