Morphological and Molecular Diagnosis of Necator americanus and Ancylostoma ceylanicum Recovered from Villagers in Northern Cambodia

Human hookworm infections caused by adult Ancylostoma spp. and Necator americanus are one of the most important tropical diseases. We performed a survey of intestinal helminths using the Kato-Katz fecal examination technique targeting 1,156 villagers residing in 2 northern provinces (Preah Vihear and Stung Treng) of Cambodia in 2018. The results revealed a high overall egg positive rate of intestinal helminths (61.9%), and the egg positive rate of hookworms was 11.6%. Nine of the hookworm egg positive cases in Preah Vihear Province were treated with 5–10 mg/kg pyrantel pamoate followed by purging with magnesium salts, and a total of 65 adult hookworms were expelled in diarrheic stools. The adult hookworms were analyzed morphologically and molecularly to confirm the species. The morphologies of the buccal cavity and dorsal rays on the costa were observed with a light microscope, and the nucleotide sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene were analyzed. The majority of the hookworm adults (90.7%) were N. americanus, whereas the remaining 9.3% were Ancylostoma ceylanicum, a rare hookworm species infecting humans. The results revealed a high prevalence of hookworm infections among people in a northern part of Cambodia, suggesting the necessity of a sustained survey combined with control measures against hookworm infections.     

Ancylostoma ceylanicum,a re-emerging but neglected parasitic zoonosis

Although Ancylostoma ceylanicum is known to be an endemic and widely distributed hookworm of dogs and cats in Asia, its contribution to human morbidity as a potentially zoonotic hookworm remains largely unexplored. Since its discovery by Lane (1913) as a ‘new parasite’ of humans a century ago, the hookworm has been regarded as a ‘rare’ and ‘abnormal’ parasite and largely overlooked in surveys of human parasites. Recent molecular-based surveys in Asia, however, have demonstrated that A. ceylanicum is the second most common hookworm species infecting humans, comprising between 6% and 23% of total patent hookworm infections. In experimentally induced infections, A. ceylanicum mimics the clinical picture produced by the anthroponotic hookworms of ‘ground itch’ and moderate to severe abdominal pain in the acute phase. Natural infections with A. ceylanicum in humans have been reported in almost all geographical areas in which the hookworm is known to be endemic in dogs and cats, however for the majority of reports, no clinical data are available. Much like the anthroponotic hookworm species, patent A. ceylanicum adults can isolate within the jejunum to produce chronic infections that on occasion, may occur in high enough burdens to produce anaemia. In addition, the hookworm can act much like Ancylostoma caninum and be found lower in the gastrointestinal tract leading to abdominal distension and pain, diarrhoea and occult blood in the faeces accompanied by peripheral eosinophilia. Whether A. ceylanicum is capable of producing both classical hookworm disease and evoking morbidity through an uncontrolled allergic response in some individuals remains unascertained. Future investigations combining the use of molecular diagnostic tools with clinical and pathological data will shed further light on its role as a human pathogen. The control of this zoonosis necessitates an integrated and inter-sectorial “One Health” approach be adopted in communities where large numbers of dogs share a close relationship with humans.     

Ancylostoma ceylanicum infections in humans in Vietnam

Ancylostoma ceylanicum is recognized as the only zoonotic hookworm species that is able to mature into adult stage in the human intestine. While human infections caused by this hookworm species have been reported from neighboring countries and this hookworm is prevalent in dogs in Vietnam, human infection has never been reported in Vietnam. The present study, therefore, aimed to identify human infections with A. ceylanicum in Vietnam. A total of 526 fecal samples from the residents in Long An Province were collected and the presence of hookworm eggs was detected by the Kato-Katz method. The results indicated that the overall prevalence of human hookworm infection was 85/526 (16.2%). After filter paper culture, 3rd stage larvae were successfully obtained from 48 egg-positive samples. The larvae were identified for their species using semi-nested PCR-RLFP on the cox1 gene. As a result, two hookworm species were confirmed; single species infections with Necator americanus or A. ceylanicum, and mixed infections with both species were found in 47.9%, 31.3%, and 20.8% of the samples, respectively.     

Molecular Identification of Hookworm Isolates in Humans,Dogs and Soil in a Tribal Area in Tamil Nadu,India

BackgroundHookworms (Necator americanus and Ancylostoma duodenale) remain a major public health problem worldwide. Infections with hookworms (e.g., A. caninum, A. ceylanicum and A. braziliense) are also prevalent in dogs, but the role of dogs as a reservoir for zoonotic hookworm infections in humans needs to be further explored.Conclusions/SignificanceIn our study we regularly detected the presence of A. caninum DNA in the stool of humans. Whether this is the result of infection is currently unknown but it does warrant a closer look at dogs as a potential reservoir.     

Necator americanus and Ancylostoma duodenale: life history parameters and epidemiological implications of two sympatric hookworms of humans

The life history data available for two species of hookworm that commonly infect humans, Necator americanus and Ancylostoma duodenale, were compiled. The data were then analyzed in the light of two ecological theories: r and K selection and the constraints of body size on reproduction. Field and laboratory experiments have shown that N. americanus is smaller than A. duodenale and produces fewer but larger eggs. The former is less virulent but the latter is hardier during its free-living stages and can infect orally as well as via the skin. Such species differences reflect the greater opportunism of A. duodenale and the greater degree of accommodation to the human host of N. americanus. A. duodenale optimizes the probability of finding a host and invading it, while N. americanus optimizes the chance of survival once within the host. On the basis of population density, longevity, and fecundity, N. americanus is the more K selected of the two species. However, the theory of r and K selection alone is insufficient to explain the different adaptations of closely related and sympatric parasites with complex life histories. N. americanus does not show greater competitive ability in its freeliving stage, and its mortality is not demonstrably more dependent on density than A. duodenale, as would be predicted by r and K selection theory as presently applied. Epidemiological differences between the two hookworm species are related to such life history parameters as lifespan, fecundity, age-specific survivorship, and density relationships.     

Real-time PCR demonstrates Ancylostoma duodenale is a key factor in the etiology of severe anemia and iron deficiency in Malawian pre-school children

Background

Hookworm infections are an important cause of (severe) anemia and iron deficiency in children in the tropics. Type of hookworm species (Ancylostoma duodenale or Necator americanus) and infection load are considered associated with disease burden, although these parameters are rarely assessed due to limitations of currently used diagnostic methods. Using multiplex real-time PCR, we evaluated hookworm species-specific prevalence, infection load and their contribution towards severe anemia and iron deficiency in pre-school children in Malawi.

Methodology and Findings

A. duodenale and N. americanus DNA loads were determined in 830 fecal samples of pre-school children participating in a case control study investigating severe anemia. Using multiplex real-time PCR, hookworm infections were found in 34.1% of the severely anemic cases and in 27.0% of the non-severely anemic controls (p<0.05) whereas a 5.6% hookworm prevalence was detected by microscopy. Prevalence of A. duodenale and N. americanus was 26.1% and 4.9% respectively. Moderate and high load A. duodenale infections were positively associated with severe anemia (adjusted odds ratio: 2.49 (95%CI 1.16–5.33) and 9.04 (95%CI 2.52–32.47) respectively). Iron deficiency (assessed through bone marrow examination) was positively associated with intensity of A. duodenale infection (adjusted odds ratio: 3.63 (95%CI 1.18–11.20); 16.98 (95%CI 3.88–74.35) and 44.91 (95%CI 5.23–385.77) for low, moderate and high load respectively).

Conclusions/Significance

This is the first report assessing the association of hookworm load and species differentiation with severe anemia and bone marrow iron deficiency. By revealing a much higher than expected prevalence of A. duodenale and its significant and load-dependent association with severe anemia and iron deficiency in pre-school children in Malawi, we demonstrated the need for quantitative and species-specific screening of hookworm infections. Multiplex real-time PCR is a powerful diagnostic tool for public health research to combat (severe) anemia and iron deficiency in children living in resource poor settings.     

Sequence differences in the internal transcribed spacer ribosomal DNA among four species of hookworm (Ancylostomatoidea: Ancylostoma)

The two ribosomal DNA internal transcribed spacers (1 and 2) of the hookworms Ancylostoma caninum, A. tubaeforme, A. ceylanicum and A. duodenale were sequenced. The sequence lengths were similar among the four species, except that A. ceylanicum had slightly longer (by 5–7 bp) internal transcribed spacer 1 and 2 sequences. The predicted secondary structure of the internal transcribed spacer 2 precursor rRNA was similar for all species, despite interspecific differences in primary sequence ranging from 0.9% to 13.2%. Interspecific differences in internal transcribed spacer 1 sequence ranged from 0.9% to 7.5%. A cladistic analysis of the sequence data, using the human hookworm Necator americanus as the outgroup, provided little resolution of the phylogenetic relationships, except that A. ceylanicum occurred on a branch external to the other three species. Nonetheless, internal transcribed spacers 1 and 2 may provide useful phylogenetic information at higher taxonomic levels within the superfamily Ancylostomatoidea.     

Epidemiological and genetic data supporting the transmission of Ancylostoma ceylanicum among human and domestic animals

Background

Currently, information on species-specific hookworm infection is unavailable in Malaysia and is restricted worldwide due to limited application of molecular diagnostic tools. Given the importance of accurate identification of hookworms, this study was conducted as part of an ongoing molecular epidemiological investigation aimed at providing the first documented data on species-specific hookworm infection, associated risk factors and the role of domestic animals as reservoirs for hookworm infections in endemic communities of Malaysia.

Methods/Findings

A total of 634 human and 105 domestic canine and feline fecal samples were randomly collected. The overall prevalence of hookworm in humans and animals determined via microscopy was 9.1% (95% CI = 7.0–11.7%) and 61.9% (95% CI = 51.2–71.2%), respectively. Multivariate analysis indicated that participants without the provision of proper latrine systems (OR = 3.5; 95% CI = 1.53–8.00; p = 0.003), walking barefooted (OR = 5.6; 95% CI = 2.91–10.73; p<0.001) and in close contact with pets or livestock (OR = 2.9; 95% CI = 1.19–7.15; p = 0.009) were more likely to be infected with hookworms. Molecular analysis revealed that while most hookworm-positive individuals were infected with Necator americanus, Ancylostoma ceylanicum constituted 12.8% of single infections and 10.6% mixed infections with N. americanus. As for cats and dogs, 52.0% were positive for A. ceylanicum, 46.0% for Ancylostoma caninum and 2.0% for Ancylostoma braziliense and all were single infections.

Conclusion

This present study provided evidence based on the combination of epidemiological, conventional diagnostic and molecular tools that A. ceylanicum infection is common and that its transmission dynamic in endemic areas in Malaysia is heightened by the close contact of human and domestic animal (i.e., dogs and cats) populations.     

Spatial heterogeneity of parasite co-infection: Determinants and geostatistical prediction at regional scales

Multiple parasite infections are widespread in the developing world and understanding their geographical distribution is important for spatial targeting of differing intervention packages. We investigated the spatial epidemiology of mono- and co-infection with helminth parasites in East Africa and developed a geostatistical model to predict infection risk. The data used for the analysis were taken from standardised school surveys of Schistosoma mansoni and hookworm (Ancylostoma duodenale/Necator americanus) carried out between 1999 and 2005 in East Africa. Prevalence of mono- and co-infection was modelled using satellite-derived environmental and demographic variables as potential predictors. A Bayesian multi-nominal geostatistical model was developed for each infection category for producing maps of predicted co-infection risk. We show that heterogeneities in co-infection with S. mansoni and hookworm are influenced primarily by the distribution of S. mansoni, rather than the distribution of hookworm, and that temperature, elevation and distance to large water bodies are reliable predictors of the spatial large-scale distribution of co-infection. On the basis of these results, we developed a validated geostatistical model of the distribution of co-infection at a scale that is relevant for planning regional disease control efforts that simultaneously target multiple parasite species.     

Zoonotic Echinostome Infections in Free-Grazing Ducks in Thailand

Free-grazing ducks play a major role in the rural economy of Eastern Asia in the form of egg and meat production. In Thailand, the geographical location, tropical climate conditions and wetland areas of the country are suitable for their husbandry. These environmental factors also favor growth, multiplication, development, survival, and spread of duck parasites. In this study, a total of 90 free-grazing ducks from northern, central, and northeastern regions of Thailand were examined for intestinal helminth parasites, with special emphasis on zoonotic echinostomes. Of these, 51 (56.7%) were infected by one or more species of zoonotic echinostomes, Echinostoma revolutum, Echinoparyphium recurvatum, and Hypoderaeum conoideum. Echinostomes found were identified using morphological criteria when possible. ITS2 sequences were used to identify juvenile and incomplete worms. The prevalence of infection was relatively high in each region, namely, north, central, and northeast region was 63.2%, 54.5%, and 55.3%, respectively. The intensity of infection ranged up to 49 worms/infected duck. Free-grazing ducks clearly play an important role in the life cycle maintenance, spread, and transmission of these medically important echinostomes in Thailand.     

Copro-molecular identification of intestinal nematode infections in a rural community in East Malaysia

Soil-transmitted helminth (STH) infections are the most common intestinal parasitic infections of medical importance in humans. The infections are widely distributed throughout tropical and subtropical countries, particularly among disadvantaged and underprivileged communities. In Malaysia, STH infections is highly prevalent, especially among the indigenous groups. However, species identification through molecular studies is still lacking. Using molecular techniques, this study was conducted to identify STH species that infect humans. Faecal samples were collected from three Iban tribal longhouses in the rural area of Sarikei Division, Sarawak. Positive faecal samples by microscopy were subjected to PCR and sequenced for species identification. Based on the microscopy examination, the most prevalent STH infection was Ascaris, followed by hookworm and Trichuris. All microscopy-positive samples were then PCR-amplified. The amplicons were sequenced, aligned, and compared with other sequences in the GenBank database. The results showed that Ascaris lumbricoides was the predominant STH species, followed by Trichuris trichiura, Ancylostoma duodenale, and Necator americanus. Our findings were contradictory to past studies, as we showed that A. duodenale was more abundant than N. americanus. Socioeconomic improvement and health education programs should be included in the management and prevention of public health strategies against STH.     

Rapid Detection and Identification of Human Hookworm Infections through High Resolution Melting (HRM) Analysis

Background

Hookworm infections are still endemic in low and middle income tropical countries with greater impact on the socioeconomic and public health of the bottom billion of the world''s poorest people. In this study, a real-time polymerase chain reaction (PCR) coupled with high resolution melting-curve (HRM) analysis was evaluated for an accurate, rapid and sensitive tool for species identification focusing on the five human hookworm species.

Methods

Real-time PCR coupled with HRM analysis targeting the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA as the genetic marker was used to identify and distinguish hookworm species in human samples. Unique and distinct characteristics of HRM patterns were produced for each of the five hookworm species. The melting curves were characterized by peaks of 79.24±0.05°C and 83.00±0.04°C for Necator americanus, 79.12±0.10°C for Ancylostoma duodenale, 79.40±0.10°C for Ancylostoma ceylanicum, 79.63±0.05°C for Ancylostoma caninum and 79.70±0.14°C for Ancylostoma braziliense. An evaluation of the method''s sensitivity and specificity revealed that this assay was able to detect as low as 0.01 ng/µl hookworm DNA and amplification was only recorded for hookworm positive samples.

Conclusion

The HRM assay developed in this study is a rapid and straightforward method for the diagnosis, identification and discrimination of five human hookworms. This assay is simple compared to other probe-based genotyping methods as it does not require multiplexing, DNA sequencing or post-PCR processing. Therefore, this method offers a new alternative for rapid detection of human hookworm species.     

Ac-SAA-1, an immunodominant 16 kDa surface-associated antigen of infective larvae and adults of Ancylostoma caninum

A cDNA encoding a surface-associated antigen was cloned from an Ancylostoma caninum infective larvae (L(3)) cDNA library by immunoscreening with pooled human immune sera. The sera were obtained from individuals living in an Ancylostoma duodenale hookworm-endemic region of China, who had light intensity infections and high antibody titers against A. caninum L(3). Ancylostoma caninum surface-associated antigen-1 is encoded by an 843 bp mRNA with a predicted open reading frame of 162 amino acids. Recombinant Ancylostoma caninum surface-associated antigen-1 was expressed in Escherichia coli and used to prepare a specific antiserum. A Western blot with anti-Ancylostoma caninum surface-associated antigen-1 specific antiserum showed that native Ancylostoma caninum surface-associated antigen-1 protein is expressed by both L(3) and adult hookworms; RT-PCR confirmed that the mRNA is transcribed in both stages. In adult hookworms, the protein localised to the basal layer of the cuticle and hypodermis of adult worms. Serological analysis determined that recombinant Ancylostoma caninum surface-associated antigen-1 protein is recognised by 61% of human sera from a Necator americanus hookworm endemic area in China, indicating the antigen is immunodominant. Anti-Ancylostoma caninum surface-associated antigen-1 antiserum partially inhibited (46.7%) invasion of hookworm L(3) into dog skin in vitro. Together these results suggest that Ancylostoma caninum surface-associated antigen-1 offers promise as a protective vaccine antigen.     

Molecular Variation in the Paragonimus heterotremus Complex in Thailand and Myanmar

Paragonimiasis is an important food-borne parasitic zoonosis caused by infection with lung flukes of the genus Paragonimus. Of the 7 members of the genus known in Thailand until recently, only P. heterotremus has been confirmed as causing human disease. An 8th species, P. pseudoheterotremus, has recently been proposed from Thailand, and has been found in humans. Molecular data place this species as a sister species to P. heterotremus, and it is likely that P. pseudoheterotremus is not specifically distinct from P. heterotremus. In this study, we collected metacercariae of both nominal species (identification based on metacercarial morphology) from freshwater crabs from Phetchabun Province in northern Thailand, Saraburi Province in central Thailand, and Surat Thani Province in southern Thailand. In addition, we purchased freshwater crabs imported from Myanmar at Myawaddy Province, western Thailand, close to the Myanmar-Thailand border. The DNAs extracted from excysted metacercariae were PCR-amplified and sequenced for ITS2 and cox1 genes. The ITS2 sequences were nearly identical among all samples (99-100%). Phylogenies inferred from all available partial cox1 sequences contained several clusters. Sequences from Indian P. heterotremus formed a sister group to sequences from P. pseudoheterotremus-type metacercariae. Sequences of P. heterotremus from Thailand, Vietnam, and China formed a separate distinct clade. One metacercaria from Phitsanulok Province was distinct from all others. There is clearly considerable genetic variation in the P. heterotremus complex in Thailand and the form referred to as P. pseudoheterotremus is widely distributed in Thailand and the Thai-Myanmar border region.     

An anticoagulant peptide from the human hookworm, Ancylostoma duodenale that inhibits coagulation factors Xa and XIa

A full-length cDNA encoding an anticoagulant peptide, named AduNAP4, was cloned and identified from the human hookworm Ancylostoma duodenale. AduNAP4 has 104 amino acids including a predicted 23-residue signal peptide and shows ?50% similarity with other known nematode anticoagulant protein/peptide (NAP). AduNAP4 is extremely efficient at prolonging the activated partial thromboplastin time, and is an inhibitor of both fXa (K_i = 7.34 ± 1.74 nM) and fXIa (K_i = 42.45 ± 3.25 nM). No fXIa inhibitor has previously been described from other blood-feeding animals. Our results suggest that hookworms have evolved a potent mechanism that interferes with coagulation by inhibition of fXIa to facilitate its blood-feeding lifestyle.     

A Cross-Sectional Study on Intestinal Parasitic Infections in Rural Communities,Northeast Thailand

Despite the existence of effective anthelmintics, parasitic infections remain a major public health problem in Southeast Asia, including Thailand. In rural communities, continuing infection is often reinforced by dietary habits that have a strong cultural basis and by poor personal hygiene and sanitation. This study presents a survey of the prevalence of intestinal parasitic infections among the people in rural Thailand. The community-based cross-sectional study was conducted in villages in Khon Kaen Province, northeastern Thailand, from March to August 2013. A total of 253 stool samples from 102 males and 140 females, aged 2-80 years, were prepared using formalin-ethyl acetate concentration methods and examined using light microscopy. Ninety-four individuals (37.2%) were infected with 1 or more parasite species. Presence of parasitic infection was significantly correlated with gender (P=0.001); nearly half of males in this survey (49.0%) were infected. Older people had a higher prevalence than younger members of the population. The most common parasite found was Opisthorchis viverrini (26.9%), followed by Strongyloides stercoralis (9.5%), Taenia spp. (1.6%), echinostomes (0.4%), and hookworms (0.4%). The prevalence of intestinal protozoa was Blastocystis hominis 1.6%, Entamoeba histolytica 0.8%, Entamoeba coli 0.8%, Balantidium coli 0.4%, Iodamoeba bütschlii 0.4%, and Sarcocystis hominis 0.4%. Co-infections of various helminths and protozoa were present in 15.9% of the people. The present results show that the prevalence of parasitic infections in this region is still high. Proactive education about dietary habits, personal hygiene, and sanitation should be provided to the people in this community to reduce the prevalence of intestinal parasite infections. Moreover, development of policies and programs to control parasites is needed.     

Intestinal Parasites among Wild Rodents in Northern Gangwon-do,Korea

To determine geographical patterns of natural parasite infections among wild rodents, a total of 46 wild rodents from 3 different localities in northern Gangwon-do (Province), Korea were examined for intestinal parasite infections. Along with nematodes such as hookworms and Syphacia spp., Plagiorchis muris (2 specimens) (Trematoda) were collected from striped field mice, Apodemus agrarius. In a Korean wood mouse, Apodemus peninsulae, the overall nematode infections were similar to A. agrarius, but an adult worm of Echinostoma hortense (Trematoda) was collected. In addition, 2 species of cestodes, i.e., Hymenolepis nana and Hymenolepis diminuta, were collected from A. agrarius. Through this survey, A. agrarius and A. peninsule were confirmed as the natural definite hosts for zoonotic intestinal helminths, i.e., P. muris, E. hortense, H. nana, and H. diminuta, in northern Gangwon-do, Korea. Considering increased leisure activities around these areas, seasonal and further comprehensive surveys on wild rodents seem to be needed to prevent zoonotic parasite infections.     

Necator americanus and helminth co-infections: further down-modulation of hookworm-specific type 1 immune responses

Background

Helminth co-infection in humans is common in tropical regions of the world where transmission of soil-transmitted helminths such as Ascaris lumbricoides, Trichuris trichiura, and the hookworms Necator americanus and Ancylostoma duodenale as well as other helminths such as Schistosoma mansoni often occur simultaneously.

Methodology

We investigated whether co-infection with another helminth(s) altered the human immune response to crude antigen extracts from either different stages of N. americanus infection (infective third stage or adult) or different crude antigen extract preparations (adult somatic and adult excretory/secretory). Using these antigens, we compared the cellular and humoral immune responses of individuals mono-infected with hookworm (N. americanus) and individuals co-infected with hookworm and other helminth infections, namely co-infection with either A. lumbricoides, Schistosoma mansoni, or both. Immunological variables were compared between hookworm infection group (mono- versus co-infected) by bootstrap, and principal component analysis (PCA) was used as a data reduction method.

Conclusions

Contrary to several animal studies of helminth co-infection, we found that co-infected individuals had a further downmodulated Th1 cytokine response (e.g., reduced INF-γ), accompanied by a significant increase in the hookworm-specific humoral immune response (e.g. higher levels of IgE or IgG4 to crude antigen extracts) compared with mono- infected individuals. Neither of these changes was associated with a reduction of hookworm infection intensity in helminth co-infected individuals. From the standpoint of hookworm vaccine development, these results are relevant; i.e., the specific immune response to hookworm vaccine antigens might be altered by infection with another helminth.