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

Abstract

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.

Keywords: Enterobiasis vermicularis; PCR; pinworm infection; ribosomal DNA–rDNA; stool (DNA) test.

Figure 1

The figure shows the entire sample processing workflow, from the sample collection to the final diagnosis of Enterobius vermicularis infection.

Figure 2

The major and minor multigene families of the ribosomal DNA (rDNA) of Enterobius vermicularis. In (A) is reported the major family; it is arranged in tandemly repeated clusters (r-DNA-repeat), separated by non-transcribed spacer regions (NTS). In (B) is reported the minor gene family; it is also organized in tandem repeated clusters (r-DNA-repeat), separated by non-transcribed spacer regions (NTS). ETS, external transcribed spacers region; ITS, internal transcribed spacers region.

Figure 3

Analysis of Enterobius vermicularis SL1 of the 5S spacer region using the Nucleotide Blast tool. The records with the highest alignment score with the query sequence (bars in red) corresponded to different E. vermicularis isolates, while those with the lower alignment score (in green), correspond to other organisms. The two segments at both the ends are highly specific for E. vermicularis. The first tract corresponds to nucleotides 1–223 (223 bp) and the second one to nucleotides 341–839 (499 bp) of the 5S spacer region.

Figure 4

Analysis with Primer3plus software using the nucleotides 1–310 of Enterobius vermicularis spacer region. The software generates 10 different options of external primers. The couples first, second, fourth, sixth, and seventh do not include the SL1 sequence, while the others do. Among the latter, the tenth couple was chosen for the external primers because it picked the shorter tract (181 bp, nucleotides 82–263) including the target sequence. Assembled reads from Sanger sequencing of the positive control 1 showing perfect homology with the expected E. vermicularis amplicon with the selected pairs of primers.

Figure 5

Analysis of the sequences obtained using our primers in the NCBI database. Both external (A), and internal (B), primers select DNA sequences highly specific for Enterobius vermicularis, with no cross-reaction with other organisms.

Figure 6

Location of the external (Ext fw and Ext rv) and internal primers (Int fw and Int rv) in relation to the SL1 region and the target sequence.

Figure 7

Electrophoresis of the PCR products was obtained after the first PCR (left panel) and the nested PCR (right panel) carried out on one positive control. In the left panel, one band of 181 bp is observed, on the right panel three bands: 182 and 116 bp from the nested PCR, while the lower band from primer dimers.

Figure 8

Nested PCR in the study population. On the left are reported the results in the infested patients, while on the right there are those in healthy controls.