OBJECTIVETo develop a quantitative PCR method for detecting hookworm infection and quantification.

METHODSA real-time PCR method was designed based on the intergenic region II of ribosomal DNA of the hookworm Necator americanus. The detection limit of this method was compared with the microscopy-based Kato-Katz method. The real-time PCR method was used to conduct an epidemiological survey of hookworm infection in southern Fujian Province of China.

RESULTSThe real-time PCR method was specific for detecting Necator americanus infection, and was more sensitive than conventional PCR or microscopy-based method. A preliminary survey for hookworm infection in villages of Fujian Province confirmed the high prevalence of hookworm infections in the resident populations. In addition, the infection rate in women was significantly higher than that of in men.

CONCLUSIONSA real-time PCR method is designed, which has increased detection sensitivity for more accurate epidemiological studies of hookworm infections, especially when intensity of the infection needs to be considered.

Animals ; China ; epidemiology ; DNA, Helminth ; genetics ; Female ; Humans ; Male ; Microscopy ; Necator americanus ; genetics ; isolation & purification ; Necatoriasis ; diagnosis ; epidemiology ; genetics ; Nucleic Acid Amplification Techniques ; Real-Time Polymerase Chain Reaction ; Sensitivity and Specificity ; Sentinel Surveillance ; Sequence Analysis, DNA ; Sex Distribution

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.
Ancylostoma/*genetics/isolation & purification ; Ancylostomiasis/*diagnosis/parasitology ; Animals ; Base Sequence ; DNA, Intergenic/genetics ; DNA, Protozoan/genetics ; DNA, Ribosomal Spacer/genetics ; Diagnosis, Differential ; Humans ; Molecular Sequence Data ; Necator americanus/*genetics/isolation & purification ; Phylogeny ; Polymerase Chain Reaction/*methods ; Sequence Alignment ; Trichostrongylosis/*diagnosis/parasitology ; Trichostrongylus/*genetics/isolation & purification

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.
Ancylostoma/*genetics/isolation & purification ; Ancylostomiasis/*diagnosis/parasitology ; Animals ; Base Sequence ; DNA, Intergenic/genetics ; DNA, Protozoan/genetics ; DNA, Ribosomal Spacer/genetics ; Diagnosis, Differential ; Humans ; Molecular Sequence Data ; Necator americanus/*genetics/isolation & purification ; Phylogeny ; Polymerase Chain Reaction/*methods ; Sequence Alignment ; Trichostrongylosis/*diagnosis/parasitology ; Trichostrongylus/*genetics/isolation & purification

The 2 principal species of hookworms infecting humans are Necator americanus and Ancylostoma duodenale. Case studies on zoonotic hookworm infections with Ancylostoma ceylanicum and/or Ancylostoma caninum are known mainly from Asian countries. Of these 2 zoonotic species, only A. ceylanicum can develop to adulthood in humans. In the present study, we report a molecular-based survey of human hookworm infections present in southern and northeastern Thailand. Thirty larval hookworm samples were obtained from fecal agar plate cultures of 10 patients in northeastren Thailand and 20 in southern Thailand. Partial ITS1, 5.8S, and ITS2 regions of the ribosomal DNA genes were amplified using PCR. The amplicons were sequenced, aligned, and compared with other hookworm sequences in GenBank database. The results showed that, in Thailand, N. americanus is more prevalent than Ancylostoma spp. and is found in both study areas. Sporadic cases of A. ceylanicum and A. duodenale infection were seen in northeastern Thailand.
Ancylostoma/classification/genetics/*isolation & purification ; Ancylostomiasis/*epidemiology ; Animals ; Cluster Analysis ; DNA, Ribosomal Spacer/chemistry/genetics ; Feces/parasitology ; Humans ; Molecular Sequence Data ; Necator americanus/classification/genetics/*isolation & purification ; Necatoriasis/*epidemiology ; Phylogeny ; Polymerase Chain Reaction ; Prevalence ; RNA, Ribosomal, 5.8S/genetics ; Sequence Analysis, DNA ; Sequence Homology, Nucleic Acid ; Thailand/epidemiology