Hookworm infection in central China: morphological and molecular diagnosis.

BACKGROUND: Necator americanus is one of the major etiological agents of human ancylostomiasis. Historically, the epidemiology of ancylostomiasis in Henan Province of central China and the molecular characteristics of N. americanus have been poorly understood. METHODS: In this study, we report a case of ancylostomiasis in Zhengzhou city of Henan Province. We also review the epidemiology of ancylostomiasis in Henan Province from 1949 to 2020. In addition, the complete mitochondrial (mt) genome of one clinical isolate is fully characterized using Illumina sequencing. All available mt genomes of hookworms in GenBank were included to reconstruct the phylogeny using both maximum likelihood (ML) and Bayesian inference (BI) methods. RESULTS: A total of three worms were collected from the patient. These worms were identified as N. americanus based on morphological characteristics as well as confirmed by genotyping with the barcoding gene cox1. Although ancylostomiasis cases have dropped substantially in recent years, hookworm infection is still a public health problem in underdeveloped areas and remote rural areas in Henan Province. The mt genome features of the N. americanus contained 12 protein-coding genes (PCGs), 22 transfer RNA genes, two ribosomal RNA genes, and a major non-coding region. The nad1 gene showed high sequence variability among isolates, which is worth considering for future genetic studies of N. americanus. Phylogenetic analyses support the monophyly of hookworm isolates from different hosts and distinct geographical locations. CONCLUSIONS: The mt genome of N. americanus presented here will serve as a useful data set for studying population genetics and phylogenetic relationships of hookworms. Positive measures for preventing and controlling ancylostomiasis are required by both health services and individuals in Henan Province.

Genotypic profile of benzimidazole resistance associated with SNP F167Y in the beta-tubulin gene of Necator americanus helminths obtained from Brazilian populations.

Necator americanus is a worm that parasites the small intestine of humans and is highly prevalent in regions with poor sanitary conditions. The main strategy to control this helminth is by mass benzimidazole administration, however, periodic use of these drugs can select strains of parasites resistant to treatment. Single nucleotide polymorphisms (SNPs) in the beta-tubulin isotype 1 gene located at codons 167, 198 and 200 have been associated with benzimidazole resistance in some nematodes. Previously, our group detected the presence of some of these SNPs in populations of soil-transmitted helminths collected in different locations in Brazil. Here, we evaluated the SNP at codon 167, which has recently been shown to be associated with failure of benzimidazoles to treat N. americanus. Our ARMS-PCR analyses were performed using 524 single N. americanus eggs from 48 patients' feces collected in six Brazilian states; however, we did not detect any mutated samples at codon 167. This study builds on previous work, helping us monitor the presence of resistance-related genotypes in Brazilian helminth populations. The data presented here can assist in the implementation of future control strategies.

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.

Old friends and friendly fire: Pregnancy, hookworm infection, and anemia among tropical horticulturalists.

OBJECTIVES: Despite public health concerns about hookworm infection in pregnancy, little is known about immune profiles associated with hookworm (Necator americanus and Ancylostoma duodenale) infection during pregnancy. Fetal tolerance requirements may constrain maternal immune response to hookworm, thereby increasing susceptibility to new infections or increasing hemoglobin loss. To explore this possibility, we study systemic immune response and hemoglobin levels in a natural fertility population with endemic helminthic infection. METHODS: We used Bayesian multilevel models to analyze mixed longitudinal data on hemoglobin, hookworm infection, reproductive state, eosinophils, and erythrocyte sedimentation rate (ESR) to examine the effects of pregnancy and hookworm infection on nonspecific inflammation, cellular parasite response, and hemoglobin among 612 Tsimane women aged 15-45 (1016 observations). RESULTS: Pregnancy is associated with lower eosinophil counts and lower eosinophil response to hookworm, particularly during the second and third trimesters. Both hookworm and pregnancy are associated with higher ESR, with evidence for an interaction between the two causing further increases in the first trimester. Pregnancy is moderately associated with higher odds of hookworm infection (OR: 1.23, 95% CI: 0.83 to 1.83). Pregnancy and hookworm both decrease hemoglobin and may interact to accentuate this effect in the first-trimester of pregnancy (Interaction: ß: -0.30 g/dL; CI: -0.870 to 0.24). CONCLUSIONS: Our findings are consistent with a possible trade-off between hookworm immunity and successful pregnancy, and with the suggestion that hookworm and pregnancy may have synergistic effects, particularly in the first trimester.

Risk factors for infection with soil-transmitted helminths during an integrated community level water, sanitation, and hygiene and deworming intervention in Timor-Leste.

Water, sanitation and hygiene interventions have been advocated as important complements to deworming programs to improve soil-transmitted helminth control. Evidence for the impact of water, sanitation and hygiene on soil-transmitted helminth infections is mixed, and based mainly on cross-sectional studies. In this study, we assessed associations between individual- and household-level water, sanitation and hygiene variables and soil-transmitted helminth infections, using data collected during the 2 year follow-up study period of the WASH for WORMS randomised controlled trial in Timor-Leste. Data were collected across four surveys, conducted at 6 monthly intervals in 23 communities. We analysed water, sanitation and hygiene and sociodemographic variables as risk factors for infection with Necator americanus, Ascaris spp., and undifferentiated soil-transmitted helminth infection, using generalised linear mixed models to account for clustering at community, household and participant levels. Water, sanitation and hygiene risk factors were examined both concurrently and with a 6 month lag period that coincided with the most recent deworming. The analysis included 2333 participants. Factors associated with N. americanus infection included age group, male sex (adjusted odds ratio (aOR) 3.1, 95% confidence interval (CI) 2.4-4.2), working as a farmer (aOR 1.7, 95% CI 1.2-2.4), and completing secondary school or higher (aOR 0.29, 95% CI 0.16-0.53). Risk factors for Ascaris spp. infection included age group, living in a dwelling with more than six people (aOR 1.6, 95% CI 1.1-2.3), having a tube well or borehole as the household water source (aOR 3.7, 95% CI 1.3-10.8), and using a latrine shared between households 6 months previously (aOR 2.3, 95% CI 1.2-4.3). Handwashing before eating was protective against infection with any soil-transmitted helminth (aOR 0.79, 95% CI 0.65-0.95). In the context of regular deworming, few water, sanitation and hygiene-related factors were associated with soil-transmitted helminth infections. Future research examining the role of water, sanitation and hygiene in soil-transmitted helminth transmission is required, particularly in low transmission settings after cessation of deworming. Identifying improved indicators for measuring water, sanitation and hygiene behaviours is also a key priority.

Use of quantitative PCR to assess the efficacy of albendazole against Necator americanus and Ascaris spp. in Manufahi District, Timor-Leste.

BACKGROUND: Soil-transmitted helminths (STHs) including Ascaris lumbricoides, Necator americanus, Ancylostoma spp. and Trichuris trichiura are cause of significant global morbidity. To mitigate their disease burden, at-risk groups in endemic regions receive periodic mass drug administration using anthelmintics, most commonly albendazole and mebendazole. Assessing the efficacy of anthelmintic drugs is important for confirming that these regimens are working effectively and that drug resistance has not emerged. In this study we aimed to characterise the therapeutic efficacy of albendazole against Ascaris spp. and N. americanus in Timor-Leste, using a quantitative polymerase chain reaction (qPCR) method for parasite detection and quantification. RESULTS: A total of 314 participants from 8 communities in Timor-Leste provided stool samples before and 10-14 days after the administration of a single 400 mg dose of albendazole. Helminth infection status and infection intensity (measured in Ct-values and relative fluorescence units) were determined using qPCR. Efficacy was determined by examining the cure rates and infection intensity reduction rates. Albendazole was found to be highly efficacious against Ascaris spp., with a cure rate of 91.4% (95% CI: 85.9-95.2%) and infection intensity reduction rate of 95.6% (95% CI: 88.3-100%). The drug was less efficacious against N. americanus with a cure rate of 58.3% (95% CI: 51.4-64.9%) and infection intensity reduction rate of 88.9% (95% CI: 84.0-97.0%). CONCLUSIONS: The observed cure rates and infection intensity reduction rates obtained for Ascaris spp. and to a lower extent N. americanus, demonstrate the continued efficacy of albendazole against these species and its utility as a mass chemotherapy agent in Timor-Leste. Furthermore, this study demonstrates the usefulness of qPCR as a method to measure the efficacy of anthelminthic drugs. Additional research is necessary to translate Ct-values into eggs per gram in a systematic way. TRIAL REGISTRATION: Australian and New Zealand Clinical Trials Registry 12614000680662 (registered 27 June 2014).

An environmental assessment and risk map of Ascaris lumbricoides and Necator americanus distributions in Manufahi District, Timor-Leste.

BACKGROUND: In Timor-Leste there have been intermittent and ineffective soil-transmitted helminth (STH) deworming programs since 2004. In a resource-constrained setting, having information on the geographic distribution of STH can aid in prioritising high risk communities for intervention. This study aimed to quantify the environmental risk factors for STH infection and to produce a risk map of STH in Manufahi district, Timor-Leste. METHODOLOGY/PRINCIPAL FINDINGS: Georeferenced cross-sectional data and stool samples were obtained from 2,194 participants in 606 households in 24 villages in the Manufahi District as part of cross sectional surveys done in the context of the "WASH for Worms" randomised controlled trial. Infection status was determined for Ascaris lumbricoides and Necator americanus using real-time quantitative polymerase chain reaction. Baseline infection data were linked to environmental data obtained for each household. Univariable and multivariable multilevel mixed-effects logistic regression analysis with random effects at the village and household level were conducted, with all models adjusted for age and sex. For A. lumbricoides, being a school-aged child increased the odds of infection, whilst higher temperatures in the coolest quarter of the year, alkaline soils, clay loam/loam soils and woody savannas around households were associated with decreased infection odds. For N. americanus, greater precipitation in the driest month, higher average enhanced vegetation index, age and sandy loam soils increased infection odds, whereas being female and living at higher elevations decreased the odds of infection. Predictive risk maps generated for Manufahi based upon these final models highlight the high predicted risk of N. americanus infection across the district and the more focal nature of A. lumbricoides infection. The predicted risk of any STH infection is high across the entire district. CONCLUSIONS/SIGNIFICANCE: The widespread predicted risk of any STH infection in 6 to 18 year olds provides strong evidence to support strategies for control across the entire geographical area. As few studies include soil texture and pH in their analysis, this study adds to a growing body of evidence suggesting these factors influence STH infection distribution. This study also further supports that A. lumbricoides prefers acidic soils, highlighting a potential relatively unexplored avenue for control. TRIAL REGISTRATION: ClinicalTrials.gov ACTRN12614000680662.

First Molecular Identifications of Necator americanus and Ancylostoma ceylanicum Infecting Rural Communities in Lower Myanmar.

Hookworms are enteric parasitic roundworms infecting an estimated 400 million persons worldwide. Herein, we provide the first molecular identifications of human hookworms from certain parts of rural Lower Myanmar. DNA was extracted from hookworm-positive stool samples, as determined by microscopy. DNA sequences of the partial internal transcribed spacer 1, full length 5.8S gene, and partial internal transcribed spacer 2 were determined and compared with available hookworm sequences from public databases. Of the 11 polymerase chain reaction-positive samples, eight (Bago Region, N = 4; Mon State, N = 4) yielded sequences with high similarity to those of Necator americanus A further three sequences (Mon State, N = 2; Bago Region, N = 1) showed high similarity with those of Ancylostoma ceylanicum The latter is primarily a parasite of dogs and represents a zoonosis. Given that different species of hookworms exhibit different epidemiological and biological characteristics, accurate identification is essential for the planning and execution of effective control programs for hookworm infections.

Restoration of hookworm egg development after prolonged storage in stool suspension.

Hookworm infection is still prevalent in southern Thailand despite control measures. Hookworm eggs submerged for an extended period under water from rainfall or in latrines may not survive, but they may recover their ability to develop into infective larvae when exposed to atmospheric air. This study examined the survival of the hookworm eggs in stool suspension and the restoration of development capability after prolonged storage. In stool mass, eggs developed normally and yielded infective filariform larvae (FL) in 7 days. On the contrary, in 1:10 stool suspension, hookworm eggs were found to remain at the 4-8 cell stage; degenerated eggs were observed after 15 days of storage, and the number of degenerated eggs reached 80 % on day 30. Aeration of the suspension, or transferring to a Petri dish or agar plate, restored the capacity of eggs stored for up to 15 days to develop into FL; thereafter, the capacity declined sharply. Retardation of egg development under water or in stool suspension may be due to a lack of atmospheric air. Use of "night soil" from latrines as fertilizer may be one factor in maintaining hookworm transmission, as worm eggs can undergo normal development upon exposure to atmospheric air.

Adult hookworms (Necator spp.) collected from researchers working with wild western lowland gorillas.

BACKGROUND: In general, studies on the diversity of strongylid nematodes in endangered host species are complicated as material obtained by non-invasive sampling methods has limited value for generic and species identification. While egg morphology barely allows assignment to family, the morphology of cultivated infective third stage larvae provides a better resolution at the generic level but cannot be used for exact species identification. Morphology-based taxonomic approaches greatly depend on the examination of adult worms that are usually not available. METHODS: Hookworm parasites in two European researchers, who participated in gorilla research in the Central African Republic, were expelled after anthelmintic treatment to the faeces, collected and morphologically examined. A male worm discharged naturally from a wild bonobo (Pan paniscus) in Congo was also examined for comparison. RESULTS: Two species of Necator were identified in researchers' faecal material: Necator americanus (Stiles, 1902) and N. gorillae Noda & Yamada, 1964; the latter species differed in having a smaller body, smaller buccal cavity and shorter spicules with spade-shaped membranes situated distally. Males of N. gorillae also possessed unusual cuticular thickenings on the dorsal side of the prebursal region of the body. These characters, shared with the male worm from the bonobo, correspond well to the description of N. gorillae described from gorillas in Congo. CONCLUSIONS: Based on the morphology of the hookworms recovered in this study and previous molecular analyses of larvae developed from both humans and western lowland gorillas (Gorilla gorilla gorilla) from this locality, we conclude that the researchers became infected with gorilla hookworms during their stay in the field. This is the first report of infection with a Necator species other than N. americanus in humans.

Humans and great apes cohabiting the forest ecosystem in central african republic harbour the same hookworms.

BACKGROUND: Hookworms are important pathogens of humans. To date, Necator americanus is the sole, known species of the genus Necator infecting humans. In contrast, several Necator species have been described in African great apes and other primates. It has not yet been determined whether primate-originating Necator species are also parasitic in humans. METHODOLOGY/PRINCIPAL FINDINGS: The infective larvae of Necator spp. were developed using modified Harada-Mori filter-paper cultures from faeces of humans and great apes inhabiting Dzanga-Sangha Protected Areas, Central African Republic. The first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA and partial cytochrome c oxidase subunit 1 (cox1) gene of mtDNA obtained from the hookworm larvae were sequenced and compared. Three sequence types (I-III) were recognized in the ITS region, and 34 cox1 haplotypes represented three phylogenetic groups (A-C). The combinations determined were I-A, II-B, II-C, III-B and III-C. Combination I-A, corresponding to N. americanus, was demonstrated in humans and western lowland gorillas; II-B and II-C were observed in humans, western lowland gorillas and chimpanzees; III-B and III-C were found only in humans. Pairwise nucleotide difference in the cox1 haplotypes between the groups was more than 8%, while the difference within each group was less than 2.1%. CONCLUSIONS/SIGNIFICANCE: The distinctness of ITS sequence variants and high number of pairwise nucleotide differences among cox1 variants indicate the possible presence of several species of Necator in both humans and great apes. We conclude that Necator hookworms are shared by humans and great apes co-habiting the same tropical forest ecosystems.

Integrated school-based surveillance for soil-transmitted helminth infections and lymphatic filariasis in Gampaha district, Sri Lanka.

We explored the practicality of integrating surveillance for soil-transmitted helminthiasis (STH, assessed by Kato-Katz) with transmission assessment surveys for lymphatic filariasis (LF) in two evaluation units (EUs) in Gampaha district, Sri Lanka (population 2.3 million). The surveys were performed 6 years after five annual rounds of mass drug administration with diethylcarbamazine and albendazole. Each transmission assessment survey tested children (N = 1,462 inland EU; 1,642 coastal EU) sampled from 30 primary schools. Low filarial antigenemia rates (0% and 0.1% for the inland and coastal EUs) suggest that LF transmission is very low in this district. The STH rates and stool sample participation rates were 0.8% and 61% (inland) and 2.8% and 58% (coastal). Most STH detected were low or moderate intensity Trichuris trichiura infections. The added cost of including STH testing was ∼$5,000 per EU. These results suggest that it is feasible to integrate school-based surveillance for STH and LF.

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.

Modelling the ecological niche of hookworm in Brazil based on climate.

The distribution of hookworm in schistosomiasis-endemic areas in Brazil was mapped based on climate suitability. Known biological requirements of hookworm were fitted to data in a monthly long-term normal climate grid (18 x 18 km) using geographical information systems. Hookworm risk models were produced using the growing degree day (GDD) water budget (WB) concept. A moisture-adjusted model (MA-GDD) was developed based on accumulation of monthly temperatures above a base temperature of 15 °C (below which there is no lifecycle progression of Necator americanus) conditional on concurrent monthly values (rain/potential, evapotranspiration) of over 0.4. A second model, designated the gradient index, was calculated based on the monthly accumulation of the product of GDD and monthly WB values (GDD x WB). Both parameters had a significant positive correlation to hookworm prevalence. In the northeastern part of Brazil (the Caatinga), low hookworm prevalence was due to low soil moisture content, while the low prevalence in southern Brazil was related to low mean monthly temperatures. Both environmental temperature and soil moisture content were found to be important parameters for predicting the prevalence of N. americanus.

Generalized urticaria induced by the Na-ASP-2 hookworm vaccine: implications for the development of vaccines against helminths.

BACKGROUND: Necator americanus Ancylostoma-secreted protein 2 (Na-ASP-2) is secreted by infective hookworm larvae on entry into human hosts. Vaccination of laboratory animals with recombinant Na-ASP-2 provides significant protection against challenge infections. In endemic areas antibodies to Na-ASP-2 are associated with reduced risk of heavy N americanus infections. OBJECTIVE: To assess the safety and immunogenicity of recombinant Na-ASP-2 adjuvanted with Alhydrogel in healthy Brazilian adults previously infected with N americanus. METHODS: Participants were randomized to receive Na-ASP-2 or hepatitis B vaccine. Major IgG and IgE epitopes of the Na-ASP-2 molecule were mapped by using sera from these same subjects. Seroepidemiologic studies in adults and children residing in hookworm-endemic areas were conducted to assess the prevalence of IgE responses to Na-ASP-2. RESULTS: Vaccination with a single dose of Na-ASP-2 resulted in generalized urticarial reactions in several volunteers. These reactions were associated with pre-existing Na-ASP-2-specific IgE likely induced by previous hookworm infection. Surveys revealed that a significant proportion of the population in hookworm-endemic areas had increased levels of IgE to Na-ASP-2. Epitope mapping demonstrated sites on the Na-ASP-2 molecule that are uniquely or jointly recognized by IgG and IgE antibodies. CONCLUSION: Infection with N americanus induces increased levels of total and specific IgE to Na-ASP-2 that result in generalized urticaria on vaccination with recombinant Na-ASP-2. These data advance knowledge of vaccine development for helminths given their propensity to induce strong T(H)2 responses. Study data highlight the important differences between the immune responses to natural helminth infection and to vaccination with a recombinant helminth antigen.

Application of a real-time PCR method for detecting and monitoring hookworm Necator americanus infections in Southern China.

OBJECTIVE: To develop a quantitative PCR method for detecting hookworm infection and quantification. METHODS: A 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. RESULTS: The 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. CONCLUSIONS: A 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.

A study on the prevalence of hookworm infection in four districts of West Bengal and its linkage with anaemia.

Stool samples collected from the districts of Darjeeling, Jalpaiguri, Hooghly and Calcutta in West Bengal were examined for hookworm and associated parasites. It was found that maximum prevalence of hookworm was in Hooghly district (24.8%). N. americanus appeared to be more prevalent species in all the districts studied. The load ofhookworm infection appeared to be mostly in the mild range. Ascaris lumbricoides appeared to the most common accompanying infection. In the affected population, the degree of anaemia was mostly mild.

Eosinofilia en inmigrante africano asintomático / Asymptomatic eosinophilia in an African immigrant

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