Differential diagnosis of Trichostrongylus and hookworm eggs via PCR using ITS-1 sequence

Abstract

Trichostrongylus eggs observed in cellophane-thick smears are difficult, in practice, to distinguish from hookworm eggs. In order to overcome these limitations, a molecular approach was conducted. A Trichostrongylus colubriformis adult worm was obtained from a human in Laos, which was identified morphologically. ITS-1 sequence of this worm was determined, and found to be most similar with that of T. colubriformis among the Trichostrongylus spp. reported so far. Then, this sequence was compared with those of human hookworm species, Ancylostoma duodenale and Necator americanus, and species-specific oligonucleotide primers were designed. Polymerase chain reaction (PCR) using these primers evidenced specifically amplified PCR products of Trichostrongylus sp., A. duodenale and N. americanus from the eggs of each (520 bp, 690 bp, and 870 bp, respectively). A species-specific PCR technique can be developed in order to study the epidemiology of Trichostrongylus spp. and hookworms in endemic areas.

Fig. 1

Trichostrongylus colubriformis adults collected from humans in Khamouane Province, Laos in 2003. (A) A female worm showing uterine eggs in one row. (B) Spicules of a male worm.

Fig. 2

Phylogenetic relationship of the ITS regions from Trichostrongylus spp by using the PAUP^* (version 4.0) program. The data were retrieved from GenBank except^* T. colubriformis, of which was obtained in this study.

Fig. 3

Nucleotide sequence alignment of the ITS-1 gene from Trichostrongylus colubriformis used in this study, Ancylostoma duodenale, and Necator americanus. The locations of the forward primers jmAD (5'-TGCGAAGTTCGCGTTTCGCTGAGCT-3'), jmNA (5'-CGTTAACATTGTATACCTGTACATAC-3'), and jhTsp (5'-TTATGTGCCACAAATGAAGA-3') are underlined in each line. Asterisks indicate identical nucleotides.

Fig. 4

Schematic representation of part of the rDNA transcriptional unit and relative locations of primers (NC5, NC2, jmAD, jmNA and jhTsp) used in this study. The region harboring ITS 1, ITS 2, and 5.8 S rDNA was amplified with NC5 and NC2 primers. The species-specific primers, jmAD, jmNA, and jhTsp were targeted to the region within the ITS-1 sequence.

Fig. 5

Specificity of ITS-PCR in the recognition of the genomic DNA of other bacteria, protozoa, Lane M, 100 bp ladder, 600 bp and 100 bp bands were shown. Lane 1; N. americanus, Lane 2; A. duodenale, Lane 3; Trichostrongylus colubriformis, Lane 4; Escherichia coli, Lane 5; Entamoeba histolytica, Lane 6; Giardia lamblia. Only specifically amplified PCR products were detected on lanes 1-3.