Molecular detection of Ancylostoma duodenale, Ancylostoma ceylanicum, and Necator americanus in humans in northeastern and southern Thailand.

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.

Human hookworm infections commonly cause socioeconomic and public health problems, with approximately 1 billion persons infected worldwide [1]. Hookworm infections cause iron deficiency anemia, resulting in mental retardation and growth insufficiency in children [2]. Ancylostoma duodenale and Necator americanus are the 2 common species that cause human infections [3]. The former is common in the Middle East, Northern Africa, India, Australia, and Europe, while N. americanus is widespread in the western hemisphere, sub-Saharan Africa, eastern Asia, and southeast Asia [4]. In addition, zoonotic hookworms such as Ancylostoma ceylanicum, Ancylostoma braziliense, and Ancylostoma caninum have been reported as potentially significant public health threats in many areas [5]. Hookworm infections are still highly prevalent in Thailand [6,7] and are generally diagnosed by finding larvae/eggs in fecal preparations. Morphological identification of hookworm larvae to species is difficult and molecular identification is a useful tool in this regard. Zoonotic hookworm disease caused by A. ceylanicum was detected by copro-molecular methods in central Thailand [6,8] and Lao PDR [9]. A molecular approach to identify the causative species in other parts of Thailand is still lacking. Here, we report molecular identification of hookworm species that infect humans in the northeastern (NE) and southern Thailand. This genetic data is important as a part of continuing investigations into epidemiology of hookworms in Thailand.

The study was conducted between 2011 and 2013. Hookworm larvae were collected from 30 fecal samples using the agar plate culture technique [10]. Ten fecal samples were collected from patients in Srinagarind Hospital, Faculty of Medicine, Khon Kaen University, NE Thailand and 20 samples from rural villagers, Nakhon Si Thammarat, southern Thailand. A single larval hookworm from each fecal sample was kept in 95% ethanol until used for DNA extraction. Oral or written informed consent was obtained from patients and legal guardians. This study was approved by the Khon Kaen University Ethics Committee for Human Research (HE551247), Khon Kaen, Thailand.

DNA was extracted separately from each larva using a Genomic DNA Mini Kit (Macherey-Nagel GmbH & Co., Düren, Germany) according to the manufacturer's instructions. The partial ITS1, full length 5.8S gene, and partial ITS2 ribosomal DNA regions were amplified from each larva using PCR and the primers RTHW1F (forward): 5'GAT GAG CAT TGC WTG AAT GCC G-3'and RTHW1R (reverse): 5'GCA AGT RCC GTT CGA CAA ACA G-3'[8]. PCR conditions were as follows; initial denaturation at 94℃ for 5 min, followed by 35 cycles of 95℃ for 30 sec (denaturation), 65℃ for 30 sec (annealing), and 72℃ for 30 sec (extension), and a final extension at 72℃ for 10 min. The reaction was carried out in a 25 µl volume containing PCR 1x FastStart High Fidelity Reaction Buffer (Roche Applied Science, Mannheim, Germany), 1.8 mM MgCl2, 0.2 mM of each deoxyribonucleotide triphosphate, 0.2 µM of each primer, and 0.625 units of FastStart High Fidelity Enzyme Blend (Roche Applied Science), respectively. The PCR product was run on a 1% agarose gel, cut out, and purified for DNA sequencing, which was performed using the MegaBACE™ 1000 DNA Analysis System (GE Healthcare, Piscataway, New Jersey, USA). The specific primers above were used as sequencing primers. The nucleotide sequences were analyzed by BLAST-N search via NCBI and the DNA alignment using Clustal-W [11].

Amplicon sizes were approximately 485 bp (typical of N. americanus) or 380 bp (typical of Ancylostoma spp.). Sequences showed extremely high similarities (99-100%) with hookworm sequences in the GenBank database. Of the 10 samples from NE Thailand, 6 were N. americanus (Khon Kaen, n=3; Mukdaharn, n=1; Roi ET, n=1; Loei, n=1) (different from AF217891 at a single base), 3 were A. ceylanicum, (Khon Kaen, n=2; Mahasarakham, n=1) (identical with AB501355), and 1 was A. duodenale (Loei) different from AB501348 at 2 bases). All 20 samples from Nakhon Si Thammarat, southern Thailand, were N. americanus (different from AF217891 at a single base) (n=20). All sequences obtained in this study were deposited in the GenBank data base under the accession no. KC896796-KC896825 (Table 1).

Table 1

Hookworm sequences deposited in Genbank database

In the present study, molecular analysis was used to confirm human infections with 2 species of human hookworms, namely, N. americanus and A. duodenale found in NE and southern Thailand. In addition, 1 species of animal hookworm, namely, A. ceylanicum, was found in NE Thailand. In NE Thailand, N. americanus was the main hookworm identified, but A. duodenale and A. ceylanicum were also found. In southern Thailand, only N. americanus was detected. Our results complement previous reports from the central part of Thailand [6,7]. A survey of gastrointestinal parasites of dogs and humans in communities in Bangkok revealed A. ceylanicum and A. caninum in dogs and N. americanus and A. ceylanicum in humans [6]. Recently, a cohort study to identify the incidence and risk factors of hookworm infections was conducted in a rural community, central Thailand. N. americanus was the most common hookworm identified there. A. duodenale and A. ceylanicum were also detected [7]. We confirmed that the principal hookworm species infecting humans in Thailand is N. americanus. However, A. duodenale and A. ceylanicum infections were also detected in NE Thailand. The finding of the predominance of N. americanus in southern Thailand is consistent with the study in Peninsular Malaysia [12], where N. americanus was more common than A. ceylanicum, whereas A. duodenale infection was not found. In contrast, a study in Lao PDR found that A. duodenale, and the animal hookworms, A. caninum and A. ceylanicum were slightly more prevalent than N. americanus [8]. Thailand, Lao PDR, and Malaysia are neighboring countries. The geographical differences in the species of hookworms causing human infections might possibly be associated with parasite behavior, ethnicity, climate, temperature, and environmental factors [13,14].

In conclusion, our study revealed evidence of both human and animal hookworms among people in various areas of Thailand.