Egg genotyping reveals the possibility of patent Ancylostoma caninum infection in human intestine.

Hookworms are intestinal parasites that cause major public health problems, especially in developing countries. To differentiate eggs from different hookworm species, it is necessary to use molecular methodologies, since the eggs are morphologically similar. Here, we performed the molecular identification of single hookworm eggs from six Brazilian states. Of the 634 eggs individually analyzed, 98.1% (622/634) represented Necator americanus, and surprisingly, 1.9% (12/634 eggs from the same patient) represented Ancylostoma caninum. DNA analysis of the A. caninum-positive stool sample revealed no contamination with animal feces. This is the first report of the presence of A. caninum eggs in human feces, which may have a direct implication for the epidemiology of hookworm infection caused by this species. This suggests the need for special attention regarding prophylaxis, as different reservoirs, previously not described, may have great relevance for the spread of A. caninum.

Quantitative Polymerase Chain Reaction for Diagnosis of Soil-Transmitted Helminth Infections: A Comparison with a Flotation-Based Technique and an Investigation of Variability in DNA Detection.

Appropriate diagnostic techniques are crucial to global soil-transmitted helminth (STH) control efforts. The recommended Kato-Katz method has low sensitivity in low-transmission settings. Quantitative polymerase chain reaction (qPCR) is a highly sensitive alternative diagnostic option. However, little is known about the variability in qPCR results, and there are few published comparisons between qPCR and other microscopy-based techniques such as sodium nitrate flotation (SNF). Using 865 stool samples collected from 571 individuals, we compared SNF and qPCR in terms of diagnostic sensitivity and infection intensity measurements. In addition, we conducted repeated examinations on a single Necator americanus-positive stool sample over a 6-month period. Results showed good diagnostic agreement between SNF and qPCR for Ascaris spp. (κ = 0.69, P < 0.001), and moderate agreement for hookworm (κ = 0.55, P < 0.001) and Trichuris spp. (κ = 0.50, P < 0.001). Quantitative polymerase chain reaction demonstrated higher sensitivity than SNF for Ascaris spp. (94.1% versus 68.1%) and hookworm (75.7% versus 66.9%) but not for Trichuris spp. (53.1% versus 81.3%), which had very low prevalence. Sodium nitrate flotation and qPCR infection intensity measurements were strongly correlated for Ascaris spp. (ρ = 0.82, P < 0.001) and moderately correlated for hookworm (ρ = 0.58, P < 0.001). Repeated examinations using qPCR showed that N. americanus cycle threshold values decreased significantly at 1 month and remained stable thereafter. Results confirm the high diagnostic sensitivity of qPCR for Ascaris spp. and hookworm, particularly for light-intensity infections, which is ideal for settings approaching transmission elimination. Results support the potential for qPCR to be used as a quantitative assay for STH. Further research is needed in settings where Trichuris trichiura is endemic.

[Differentiating the morphology of infective larvae between two human hookworms].

The morphological differentiation of the infective larvae between human Ancylostoma duodenale and Necator americanus is of great significance for the epidemiological survey of hookworm diseases and human parasitology teaching. Understanding of features of the oral spear and transverse lines on the tunica vaginalis is able to accurately differentiate the infective larvae between these two human hookworms.

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.

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.

Development and characterization of microsatellite markers for the canine hookworm, Ancylostoma caninum.

Microsatellites are repetitive genomic elements that show high levels of variation and therefore provide excellent tools to study the genetics of eukaryotic organisms. Hookworms are extremely common and important nematode parasites of humans and animals, causing potentially serious disease morbidity. Control of hookworms in dogs is achieved by frequent treatment with anthelmintics, and in humans, anthelmintics are frequently administered in a mass-treatment community-wide approach. Understanding the population genetics of hookworms has important implications for studies on the development and spread of drug resistance. We investigated the genome of Ancylostoma caninum for microsatellites by developing and then screening an enriched genomic library as well as by data mining published sequences of a whole genome shotgun library. Investigations revealed a high abundance of trinucleotide repeats. Dinucleotide repeats were characterized by a high number of AT, GA, and GT repeats. After testing and optimization of 68 markers, a panel of 34 polymorphic microsatellite markers were selected. Microsatellite analysis of hookworm isolates revealed a high degree of polymorphism, which was not influenced by the length of the repeats. This panel of microsatellite markers makes it possible to pursue investigations on the population genetics of A. caninum. Furthermore, a number of the markers demonstrated suitability for analysis of the human hookworm species Necator americanus and A. duodenale.

Influence of parasitic life style on the patterns of codon usage and base frequencies of Ancylostoma and Necator species.

Parametric analyses were used to investigate the nucleotide, codon, and amino acid composition of coding sequences corresponding to hook-worms. Ancylostoma caninum and Necator americanus. Although genomic research has become prevalent within the scientific community, few studies have dealt directly with parasitic species. Parasites have existed throughout the history of mankind due to their wide range of distribution in nature and their ability to evade immune detection. An AT nucleotide bias was identified in both A. caninum and N. americanus sequences. A similar AT bias was also identified in both datasets when considering relative synonymous codon usage. However, the codon bias was much more pronounced in N. americanus as compared to A. caninum. Bias was also present at the amino acid level, and appeared to be partially independent of the nucleotide-based biases. Analysis of parasite genomes will facilitate the development of vaccines against larval forms of parasites. Moreover, the examination of the parasite genes in general, will allow for a more in-depth understanding of the evolution of the parasites and parasitism.

Species-specific identification of human hookworms by PCR of the mitochondrial cytochrome oxidase I gene.

Significant differences in the life histories of the human hookworms Ancylostoma duodenale and Necator americanus necessitate their differentiation for epidemiological studies and the design of control programs. Current methods of identification require time-consuming, labor-intensive techniques. A polymerase chain reaction (PCR)-based method that enables rapid species identification is described. The mitochondrial cytochrome oxidase I genes of both species were sequenced, and species-specific primer sets were designed. The primers were used in PCR to amplify 585-bp fragments of the cytochrome oxidase gene from individual hookworm eggs, larvae, and adults. The technique was also able to identify mixed infections containing equal amounts of eggs from each species. The technique is rapid, technically simple, and sensitive and will permit the accurate identification of human hookworms in epidemiological field studies.

[Sequencing of cytochrome C oxidase 1 gene of Ancylostoma duodenale and Necator americanus].

AIM: To identify the genetic diversity between Ancylostoma duodenale and Necator americanus. METHODS: Mitochondrial cytochrome C oxidase subunit 1 (CO1) gene was amplified from genomic DNA of human hookworms collected from infected patients in Hejiang County, Sichuan Province, and the purified PCR products were directly sequenced by using Licor auto-sequencer. RESULTS: The PCR products were about 700 bp. Alignment of CO1 gene fragment sequences showed 89.7% similarity between Ancylostoma duodenale and Necator americanus, but still certain nucleotide variations (10.3%) existed. CONCLUSION: CO1 gene sequence can be used as a marker to identify the two species of human hookworms.

Hallazgo de ancylostoma duodenale en la localidad de Pororó, Paraguay

a) Se realizó un trabajo de prevalencia de parásitosintestinales, en la localidad de Pororó. Se encotro que el 93 porciento de la población estudiada estabaparasitada, 61 prciento poseían dos o más parásitos.Entre los principales parásitos intestinales encontrados están: blastocystis hominis 71,3 porciento, uncinaria 52,8 poriento y giardialamblia 19,7 porcientoTambién se realizó el cultivo de las heces por método de Harada-Mori, se encontro: necator americanusen el 68porciento de las muestras, ancylostomaduodenaleen el 36 porciento y strongyloides stercoralis en el4 porciento las cifras de ancylostoma duodenale son superior

Necator americanus (Nematoda: Ancylostomatidae) from Africa and Malaysia have different ITS-2 rDNA sequences.

The nucleotide sequences of the second internal transcribed spacer of rDNA were determined for adult worms of Necator americanus originating from Togo (Africa) and Sarawak (Malaysia). The length of the sequences of specimens from Togo (325 bp) were shorter than those from Sarawak (327 bp). There were six fixed genetic differences in the aligned sequences of N. americanus from Sarawak and Togo, excluding one or two polymorphic sites within the sequence of N. americanus from each geographical region. These findings suggest that there is either population variation in the sequence of N. americanus, or that N. americanus from the two countries may represent genetically distinct but morphologically similar (i.e. cryptic) species, however, comparison of the sequence differences among other hookworm species supports the latter conclusion.

Differentiation between the human hookworms Ancylostoma duodenale and Necator americanus using PCR-RFLP.

There are 2 major species of hookworms that infect humans. Ancylostoma duodenale and Necator americanus. Although traditionally considered to be identical for treatment purposes, there are significant life history differences between the species that must be considered for the rational design of chemotherapeutic and immunoprophylactic control strategies. However, identification of the species infecting a particular population has been problematic, as the eggs of the 2 species cannot be reliably differentiated by classical parasitological methods. A PCR-based technique for the differentiation of hookworm species that infect humans is reported. A fragment of the 3' untranslated region of the cAMP-dependent protein kinase catalytic subunit gene was amplified from A. duodenale and N. americanus genomic DNA using primers derived from the corresponding A. caninum cDNA. Digestion of the amplified DNA with the restriction enzymes HpaII, MboI, TaqI, and ThaI generated specific restriction fragment patterns unique to each species. The technique can distinguish between pure and mixtures of hookworm DNA and can amplify DNA from a single egg. The primers also amplify the fragment from the DNA of several other species of hookworms that infect humans and other animals. The technique is fast, simple, and hookworm specific and represents a considerable savings in time over current methods used for distinguishing between human hookworm infections.