Hookworm Anthelmintic Resistance: Novel Fecal Polymerase Chain Reaction Ancylostoma caninum Benzimidazole Resistance Marker Detection in a Dog.

A 4 yr old castrated male greyhound presented with a history of chronic (>3 wk) intermittent diarrhea. Initial fecal analysis identified infection with Ancylostoma caninum. Despite treatment with routine anthelmintics, the dog remained persistently A caninum positive for several months. A novel fecal gastrointestinal real-time polymerase chain reaction (qPCR) parasite panel detected A caninum and the genetic benzimidazole (BZ) F167Y resistance marker in multiple samplings over 48 hr. This finding, together with the dog's clinical signs (diarrhea) and lack of response to routine anthelmintics, prompted treatment with cyclooctadepsipeptide emodepside, a drug currently not registered for dogs in the United States. The dog's clinical signs resolved and post-treatment fecal qPCR testing was negative. However, 5 mo later, retesting with fecal qPCR detected A caninum and concurrent BZ resistance marker, as well as Giardia. A presumptive diagnosis of re-infection was made and the emodepside treatment was continued. The dog again reverted to undetected (A caninum and the 167 resistance marker) on reassessment fecal qPCR. This case report describes the use of a novel fecal qPCR panel for gastrointestinal parasites, persistent hookworm and BZ F167Y resistance marker detection in a dog, and highlights the importance of a stepwise approach to clinical management, treatment, and retesting.

Benzimidazole F167Y polymorphism in the canine hookworm, Ancylostoma caninum: Widespread geographic, seasonal, age, and breed distribution in United States and Canada dogs.

Surveillance data for Ancylostoma spp. and the A. caninum benzimidazole treatment resistance associated F167Y polymorphism using molecular diagnostics was obtained in a large population of dogs from the United States and Canada. Real-time PCR (qPCR) for Ancylostoma spp. and allele-specific qPCR detecting a single nucleotide polymorphism (SNP) F167Y was used in 262,872 canine stool samples collected between March and December of 2022. Ancylostoma spp. was found at an overall prevalence of 2.5% (6538/262,872), with the highest prevalence in the Southern US, 4.4% (4490/103,095), and the lowest prevalence in Canada 0.6% (101/15,829). The A. caninum F167Y polymorphism was found with the highest prevalence (13.4%, n = 46/343) in the Western US and the lowest in Canada at 4.1% (4/97). The F167Y polymorphism was detected every month over the 10-month collection period. Seasonal distribution showed a peak in June for both Ancylostoma spp. (3.08%, 547/17,775) and A. caninum F167Y (12.25%, 67/547). However, the A. caninum F167Y polymorphism prevalence was highest in September (13.9%, 119/856). Age analysis indicates a higher prevalence of both hookworm infections and occurrence of resistant isolates in puppies. The breeds with the highest F167Y polymorphism prevalence in Ancylostoma spp. detected samples were poodles (28.9%), followed by Bernese Mountain dogs (25%), Cocker spaniels (23.1%), and greyhounds (22.4%). Our data set describes widespread geographic distribution of the A. caninum benzimidazole resistance associated F167Y polymorphism in the United States and Canada, with no clear seasonality compared to the Ancylostoma spp. prevalence patterns. The F167 polymorphism was present in all geographic areas with detected hookworms, including Canada. Our study highlights that the F167Y polymorphism is represented in many dog breeds, including greyhounds.

Nationwide USA re-analysis of amplicon metabarcoding targeting ß-tubulin isoform-1 reveals absence of benzimidazole resistant SNPs in Ancylostoma braziliense, Ancylostoma tubaeforme and Uncinaria stenocephala.

Nationwide sampling by Venkatesan and colleagues (2023) described the resistance status of the canine hookworm, Ancylostoma caninum, to benzimidazoles across the USA via ß-tubulin isotype-1 amplicon metabarcoding. In this study, we aimed to use the existing public amplicon metabarcoding data and mine it for the presence of ß-tubulin isotype-1 sequences that belong to hookworm species other than A. caninum. Through bioinformatics analysis we assigned species to A. caninum, Ancylostoma braziliense, Ancylostoma tubaeforme and Uncinaria stenocephala. All non-A. caninum sequences contained only the benzimidazole susceptible residues of ß-tubulin isotype-1. Using two ß-tubulin isotype-1 metabarcoding sequence data (assay targeting 134 and 167 codons, and assay targeting 198 and 200 codons), 2.0% (6/307) and 2.9% (9/310) individual samples had hookworms other than A. caninum (A. braziliense n = 5, A. tubaeforme n = 4 and U. stenocephala n = 2), respectively. We identified one sample containing A. braziliense in each of the Northeastern region and Midwestern region, and in three samples from the Southern region. Presence of A. tubaeforme in dog faeces is considered as pseudoparasitism. There were no statistically significant regional differences for the distribution of each species, for either of the two assays independently or combined (χ2 tests, P > 0.05). Our work demonstrates the utility of the amplicon metabarcoding for the identification of species through antemortem assays, thus resolving the dilemma of assigning hookworm species based on either post-mortem or egg sizes for the identification of hookworms.

Molecular identification and genetic diversity of zoonotic hookworm infections in domestic dogs from northeastern, Thailand.

Hookworm infections remain a significant public health concern in tropical and subtropical regions, including Thailand. This study investigated the species and genetic diversity of hookworm infections in domestic dogs from northeastern Thailand. The molecular analysis focused on amplifying and sequencing specific regions of ribosomal RNA genes (ITS1-5.8S-ITS2 region) and the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene in hookworm larvae recovered from 21 domestic dog stool samples. Among 21 larvae (one larva per infected dog) analyzed, 14 had sequences identical to Ancylostoma caninum, and 7 showed sequences almost identical to Ancylostoma ceylanicum. Phylogenetic analysis of cox1 sequences placed A. caninum and A. ceylanicum in separate clades. The median-joining network of A. caninum cox1 sequences from Thailand showed high haplotype diversity and belonged to the same cluster as sequences from Australia while forming separate clusters from those of A. caninum samples from the USA. The available published A. ceylanicum cox1 sequences (n = 33), in combination with seven sequences in the present study, represented 15 haplotypes distributed among three clusters. Interestingly, A. ceylanicum sequences from dogs and humans shared the same haplotypes. These findings are crucial for recognizing the potential for zoonotic transmission, highlighting the necessity for targeted control measures, and increasing awareness among pet owners and healthcare professionals to mitigate the risk of hookworm transmission to humans.

New insight into genetic diversity of zoonotic-potential Ancylostoma ceylanicum in stray cats living in Bangkok, Thailand, based on deep amplicon sequencing.

AIMS: This study aimed to characterize feline hookworms from stray cats living in Bangkok. METHODS AND RESULTS: A total of 56 hookworm-positive faecal samples were identified for hookworm species by using PCR targeting the ITS1, 5.8S, and ITS2 fragment and qPCR targeting ITS2. Of 56 samples, 96.4% (54/56) were identified as Ancylostoma ceylanicum and 1.8% (1/56) as Ancylostoma caninum. With qPCR, 89.3% (50/56) were identified as single A. ceylanicum infection and 5.4% (3/56) as coinfection of A. ceylanicum and A. caninum. For genetic characterization of A. ceylanicum, 10 samples were pooled, and the partial COI gene was amplified, followed by deep amplicon sequencing. Five pooled samples were analysed, and 99.73% were identified with A. ceylanicum sequences, which were allocated into 19 haplotypes (AC01-AC19). Genetic diversity findings for A. ceylanicum in Asia revealed that three of eight haplotypes considered of zoonotic significance occurred in humans, dogs, and cats, including haplotypes H01, H20, and H21. The predominant haplotype in this study, AC01, was clustered with H01-a zoonotic haplotype. CONCLUSIONS: The diversity obtained by deep amplicon sequencing supported that the A. ceylanicum community had high genetic variation. Deep amplicon sequencing was a useful method to determine source, zoonotic potential, and host-parasite relationship.

Epidemiological and molecular updates on hookworm species in dogs from southern Italy.

BACKGROUND: The zoonotic hookworms Ancylostoma caninum and Uncinaria stenocephala are widespread soil-transmitted helminths in dogs in Europe. Given the veterinary and public health importance of hookworms in dogs and the recent changes in the molecular epidemiology of some species, there is a need to continuously monitor the epidemiological and molecular prevalence of these parasites also at the "local" level. The present study aimed to update the epidemiological scenario of hookworm infections in both owned and stray dogs in southern Italy and to discriminate between different hookworm species (A. caninum and U. stenocephala) through molecular analyses. For this purpose, a retrospective analysis was performed over 10 years (2011-2021), including a total of 7008 owned dogs and 5642 stray dogs referred to our laboratory for copromicroscopic examinations. Moreover, 72 faecal samples, from dogs naturally infected by hookworms, were used to discriminate between A. caninum and U. stenocephala using two PCR protocols. Prior to molecular analyses, a subsample of 40/72 positive faecal samples was used for morphometric investigations on hookworm eggs. RESULTS: The results of the ten-year retrospective analysis (2011-2021) showed an overall prevalence of hookworm infection of 9.16%, specifically 5.1% in owned dogs and 14.2% in stray dogs. Logistic regression showed a significant association between positivity to hookworms and the variable "puppies" both in stray (13.84%; OR = 2.4) and owned (7.07%; OR = 2.2) dogs. The results of molecular analyses showed that positivity was confirmed only in 21/72 samples, specifically, 6 samples using protocol A and 19 with protocol B. Sequencing revealed 15 samples positive to U. stenocephala and 6 to A. caninum. CONCLUSIONS: The findings of this study showed a high prevalence of hookworm infections in dogs in southern Italy, updating the epidemiological scenario of the last decade. Moreover, the results of the study revealed the first identification of hookworm species in dogs in Italy by molecular studies, highlighting that U. stenocephala is more prevalent than A. caninum.

Unambiguous identification of Ancylostoma caninum and Uncinaria stenocephala in Australian and New Zealand dogs from faecal samples.

Hookworms (Ancylostomatidae) are well-known parasites in dogs due to their health impacts and zoonotic potential. While faecal analysis is the traditional method for detection, improvements in husbandry and deworming have decreased their prevalence in urban owned dogs. Drug resistance in Ancylostoma caninum is becoming a discussion point in small animal practices across the region. This study aimed to identify hookworm species present in Australian and New Zealand dogs using molecular techniques. The ITS-2 and isotype-1 ß-tubulin assays were used to identify and quantify hookworm species. Results showed absence of coinfection in Australian samples from Greater Sydney region belonging either to A. caninum or Uncinaria stenocephala, while New Zealand samples were a mixture of A. caninum and U. stenocephala. The amplified isotype-1 ß-tubulin sequences exhibited susceptibility to benzimidazole drugs. Rare mutations were identified in A. caninum and U. stenocephala sequences, representing a small percentage of reads. This study highlights the importance of molecular techniques in accurately identifying and quantifying hookworm species in dog populations.

Molecular variability of the Ancylostoma secreted Protein-2 (Aca-asp-2) gene from Ancylostoma caninum contributes to expand information on population genetic studies of hookworms.

Hookworm infection is a major public health problem in many regions of the world. Given the high levels of host morbidity and even mortality of the host caused by these infections, it is crucial to understand the genetic structure of hookworm populations. This understanding can provide insights into the ecology, transmission patterns, mechanisms of drug resistance, and the development of vaccines and immunotherapeutic strategies. Previously, we examined presumably neutral molecular markers, such as microsatellites and COI (Cytochrome C oxidase subunit 1) in Brazilian populations of Ancylostoma caninum. Here we analyze the molecular variability of a genomic fragment of the Aca-asp-2 (Ancylostoma secreted protein-2) gene from Ancylostoma caninum. This gene is a highly expressed and activated following the infection of the L3 larvae in the host. We obtained individuals of A. caninum from five different geographic locations in Brazil, sequenced and analyzed parts of the gene. The results revealed extensive polymorphism at this fragment, especially in the intronic region, indicating low selective pressure acting on these sequences. However, we also observed irregular distributions of nucleotides and polymorphisms in the coding region of this gene, resulting in the identification of 27 alleles. The data presented here contribute to expanding the understanding of population genetic studies of hookworms.

Epidemiology of soil-transmitted helminths using quantitative PCR and risk factors for hookworm and Necator americanus infection in school children in Dak Lak province, Vietnam.

BACKGROUND: Soil-transmitted helminth (STH) infection is driven by a complex interaction of demographic, socioeconomic and behavioural factors, including those related to water, sanitation and hygiene (WASH). Epidemiological studies that measure both infection and potential risk factors associated with infection help to understand the drivers of transmission in a population and therefore can provide information to optimise STH control programmes. METHODS: During October and November 2019, we conducted a cross-sectional survey of the prevalence and intensity of STH infection and associated risk factors among 7710 primary-school-age children from 64 primary schools across 13 districts in Dak Lak province, Vietnam. Quantitative PCR (qPCR) was used to detect and quantify STH infections. RESULTS: The predominant STH species was the hookworm Necator americanus (overall cluster-adjusted prevalence of 13.7%), and its prevalence was heterogeneously distributed across surveyed schools (0% to 56.3%). All other STH species had a prevalence of less than 1%. Using mixed-effects logistic regression, we found that the adjusted odds ratio (aOR) was significantly higher for both infection and moderate-to-heavy-intensity infection with N. americanus among children from multiple ethnic minority groups, compared to children from the majority group (Kinh). Adjusted odds of infection with N. americanus were also higher in children who reported practising open defecation at school (aOR 1.42, 95% CI 1.05, 1.93, P = 0.02) and in those who had an unimproved household water supply (aOR 1.28, 95% CI 1.04, 1.57, P = 0.02). Conversely, children with a flushing household toilet had a reduced risk of infection (aOR 0.58, 95% CI 0.47, 0.70, P < 0.01), as did those whose primary female carer attended secondary (aOR 0.65, 95% CI 0.51, 0.84, P < 0.01) or tertiary education (aOR 0.39, 95% CI 0.24, 0.63, P < 0.01). CONCLUSIONS: This study is the largest reported prevalence survey of STH infections conducted using qPCR as a diagnostic technique. The findings of higher adjusted odds of infection amongst ethnic minority children highlight that STH control programmes may not be reaching certain population groups and that additional culturally appropriate approaches may be required. Additionally, the associations between specific WASH factors and infection indicate potential programmatic targets to complement preventive chemotherapy programmes.

Emergence of Ancylostoma caninum parasites with the benzimidazole resistance F167Y polymorphism in the US dog population.

BACKGROUND: Anthelmintic resistance to benzimidazole has been detected in the canine hookworm, Ancylostoma caninum. Benzimidazole resistance is believed to have developed originally in greyhounds, but has also been detected in non-greyhound pet dogs. The aim of this study was to validate a probe-based allele-specific real-time PCR tests for the F167Y polymorphism on the ß-tubulin isotype-1 gene and to determine the geographic distribution. METHODS: Allele-specific real-time PCR tests were established and validated to detect the codon 167 polymorphism in the Ancylostoma caninum ß-tubulin isotype-1gene. Additionally, real-time PCR tests were validated for Ancylostoma spp. and Uncinaria stenocephala. Two nucleic acid extraction protocols were validated including mechanical disruption of parasite structures in stool. The frequency of the F167Y single nucleotide polymorphism (SNP) was determined in hookworm confirmed stool samples. Samples with the resistant 167Y genotype were confirmed by ß-tubulin gene sequencing and allele frequencies were determined. RESULTS: The Ancylostoma spp. and A. caninum F167Y allele-specific real-time PCR tests were highly sensitive and specific when tested against synthetic DNA, spiked samples, and characterized parasites. Using an optimized total nucleic acid extraction protocol, 54 of 511 (10.6%) were found to contain the benzimidazole resistance allele. All 55 samples containing hookworms with the resistance mutation were confirmed by ß-tubulin gene sequencing. The majority of resistant hookworms (44 resistant, 183 tested; 24.4%) originated from Florida, five from California (103 tested, 4.9%), three from Idaho (40 tested, 7.5%), two from Nevada (22 tested, 9.1%), and one sample from Hawaii (13 tested, 7.7%). Resistant genotypes were found in 14 different dog breeds including eight in Greyhounds. Allele-frequency determination revealed resistance allele frequencies between 1 and 100% with 58% above 50%. CONCLUSIONS: This data strongly supports recent findings of benzimidazole resistant canine hookworms present throughout the general US pet dog population.

Molecular evidence of hookworms in public environment of Bangladesh.

Accurate diagnosis by precise identification of causative agents is essential for the effectiveness of any control interventions. Despite high zoonotic potential, available literature on hookworms in Bangladesh is still scarce and nonspecific. The objective of this study was to determine the occurrence of hookworms in public locations across northeastern Bangladesh (Sylhet metropolitan area) using integrated parasitological and molecular assays. A total of 130 samples (80 soil and 50 environmental canine feces) were collected and examined using modified flotation technique and formalin-ether sedimentation methods. Modified plate culture was used to isolate larvae. The identification was made based on morphometric features and confirmed by amplifying the ITS region of the nuclear rDNA. Overall, 66.2% (86/130) of examined samples were positive for hookworms infection. Characteristic eggs (61-68 × 29-37 µm) and/or larvae of hookworms were observed in 73.8% (59/80) soils and 54.0% (27/50) environmental fecal samples. Rhabditiform larvae (0.48-0.54 × 0.04-0.07 mm) were observed in cultured samples. Genetic analysis of rDNA sequences revealed the presence of Ancylostoma caninum and Ancylostoma ceylanicum. In this study, hookworms' contamination of the public environment was substantial. To the best of our knowledge, this is the first molecular proof of A. caninum and A. ceylanicum observed in urban public environment in Bangladesh.

A Pilot Comparison of Fixatives for Hookworm Real-time Polymerase Chain Reaction.

Polymerase chain reaction (PCR) is increasingly used in the diagnosis of soil-transmitted helminth infections. Despite this, few studies have evaluated the impact of different fecal fixatives on the outcome of fecal helminth qPCR analysis, and none have evaluated the effect of commercial parasitology fixatives commonly used in diagnostic laboratories. We fixed dog feces containing Ancylostoma spp. hookworm eggs in zinc polyvinyl alcohol (Zn-PVA) and Total-Fix, and with 70% ethanol (EtOH) as a control. DNA was extracted at timepoints 11, 33, 64, and 94 days and subjected to Ancylostoma spp. quantitative PCR (qPCR). A linear regression model was created to assess the effect of preservative types on the temporal change of qPCR quantification cycle number (Cq) values, accounting for variances among individual animals. Fixation in 70% EtOH least affected Cq values over 94 days. Total-Fix preservation yielded a higher Cq overall, but there was no significant difference compared with 70% EtOH fixation. Fixation in Zn-PVA resulted in significantly (P < 0.001) higher Cq values than 70% EtOH after only 33 days and loss of amplification at 64 days. Consistent with other helminth fixation studies, 70% EtOH performed well in preserving hookworm DNA over 94 days. Total-Fix provided a comparable alternative for qPCR analysis for hookworm. Fixation in Zn-PVA resulted in loss of detectable hookworm DNA at 64 days, as determined by qPCR.

Assessing the efficacy of albendazole against hookworm in Vietnam using quantitative PCR and sodium nitrate flotation.

Preventive chemotherapy (PC), consisting of the regular distribution of anthelmintics to populations or groups of populations at risk, is the primary tool used to control soil-transmitted helminth (STH) infections. This strategy, whilst cost-effective, raises the concern of potential emergence of drug resistance. The efficacy of anthelmintics against STH infections is measured using cure rate (CR) and egg reduction rate (ERR), using microscopy-based techniques such as the Kato-Katz thick smear. However, Kato-Katz has low sensitivity, especially for low-intensity infections, and requires fresh samples that need to be processed quickly. Realtime quantitative PCR (qPCR), which is more sensitive, is emerging as a "gold standard" for STH diagnostics given its higher sensitivity (important in low prevalence settings) and ability to differentiate hookworm species, while sodium nitrate flotation (SNF) may provide a low-cost more sensitive and practical alternative to Kato-Katz in the field. In this study, we examined the efficacy of a locally manufactured brand of albendazole 400 mg ("Alzental") against hookworm in Dak Lak province, Vietnam, using both qPCR and SNF. For qPCR, formulae to convert qPCR cycle threshold (Ct) values into eggs per gram of faeces (EPG) were utilised to determine efficacy calculations, and these values directly compared with efficacy values generated using SNF. Factors associated with CR and ERR were examined, and Alzental tablet quality was assessed by comparing with an Australian TGA-approved equivalent "Eskazole" tablet. We observed a CR and ERR of 64.9% and 87.5% respectively using qPCR, and 68.4% and 67.6% respectively using SNF. The tablet composition of Alzental was comparable to Eskazole in terms of active albendazole drug concentration with no evidence of impurities. This study demonstrates that the efficacy of Alzental against hookworm is within the range of previously reported studies for albendazole 400 mg. The study also demonstrates the value of qPCR and SNF as alternatives to standard Kato-Katz methodology for assessment of anthelmintic efficacy.

A case of severe hookworm infection. / 临床极重症钩虫感染1例.

With the improvement of sanitation, the infection rate of hookworm is greatly reduced and the severe infected case is rarely reported. Combined morphological and molecular biological examinations, a severe hookworm infection patient was diagnosed in Department of Laboratorial Examination, Quanzhou First Affiliated Hospital of Fujian Medical University. The morphological methods such as direct fecal smear microscopy, saturated brine flotation and hookworm larvae culture methods were used to identify the eggs and larvae from stool samples of the patient. There were a large number of hookworm eggs in patient's stool samples, and the average count was 60 840 per gram by modified Kato method, which belonged to severe hookworm infection. Meanwhile, to distinguish the hookworm species, the semi-nested RT-PCR assay was employed to detect hookworm internal transcribed spacer series from eggs in patient's stool samples, and the result showed that the hookworm species was confirmed to be Necator americanus.

Arthrostoma leucurus sp. n. (Nematoda: Ancylostomatidae), A New Hookworm Species Isolated from Asian Badger in China.

PURPOSE: To date, ten validated Arthrostoma species were reported. Here, a new hookworm species was found from Asian badger (Meles leucurus). METHODS: Nineteen hookworms (9 males and 10 females) were collected from the small intestine of two Asian badgers in Xinjiang Uygur Autonomous Region, northwestern China. The hookworms were morphologically examined according to key taxonomic characters, such as anterior extremity direction, structures of oral opening (cutting plates or teeth), vulva location, buccal capsule anatomy (integrated or formed by articulating plates), the length of spicule and gubernaculum, number of plates of buccal capsule, and presence or absence of vulvar papillae. RESULTS: The hookworm species from Asian badger, here named as Arthrostoma leucurus sp. n., was different from the previously described ten Arthrostoma species. The phylogenetic tree based on the cox1 gene showed that Arthrostoma leucurus sp. n. formed a separate clade, as a sister group to Ancylostoma and Uncinaria species. CONCLUSION: Arthrostoma leucurus sp. n., the eleven validated Arthrostoma species, was identified from Asian badger in China.

Ancylostoma ceylanicum Infection in a Miniature Schnauzer Dog Breed.

PURPOSE: Canine hookworm disease is a global zoonotic parasitic disease caused by a variety of nematodes in families Ancylostomatidae, including Ancylostoma spp., Necator spp., and Uncinaria spp., in the small intestine (mainly the duodenum) of dogs. The disease is widely distributed in China. The purpose of this study is to systematically diagnose and treat canine hookworm disease through the case of miniaturization Schnauzer dog feed infected with A. ceylanicum, so as to provide experimental basis for subsequent prevention and control of canine hookworm disease. METHODS: In the current study, we isolated hookworm eggs from a diseased miniature schnauzer, then the polymerase chain reaction (PCR) was used to amplify the ITS1-5.8S-ITS2 gene sequence from genomic DNA extracted from hookworms. Phylogenetic analysis based on ITS1-5.8S-ITS2 gene sequence sequences was inferred using MEGA-X. After phylogenetic analysis, etiologic and symptomatic therapies were used to treat the canine hookworm disease. RESULTS: The sequencing results showed that the length of the ITS1-5.8S-ITS2 gene sequence was approximately 960 bp, and ITS1 and ITS2 were extracted to analyze similarity with other hookworms to build a phylogenetic tree. After phylogenetic analysis, the results showed that the diseased miniature schnauzer was infected by A. ceylanicum. Using etiologic and symptomatic therapies, the sick dog with an A. ceylanicum infection was also treated for 5 days. CONCLUSIONS: To our knowledge, this is the first report of diagnosis and treatment for canine hookworm disease in Guangzhou city. In addition, with the improvement of economic level, the scale of pet dog breeding is also increasing. The diagnostic methods and treatment schemes adopted in this report will help to standardize the prevention and control of canine hookworm disease.

Occurrence of Hookworm and the First Molecular and Morphometric Identification of Uncinaria stenocephala in Dogs in Central Europe.

PURPOSE: Hookworms are hematophagous parasitic nematodes that occur in the intestinal tract of various mammals, including humans. The objective of this work was to develop a two-step morphology-molecular analysis-based strategy to identify the genus and the species of eggs and larvae of the Ancylostomatidae family in dogs, which were kept in various living conditions in Slovakia. METHODS: Faecal samples were collected from 270 dogs kept in two different shelters (160 samples) and in a marginalised Roma community (110 samples). Faecal samples were processed using the flotation method. Microscopically positive faecal samples with hookworm eggs were subjected to a coproculture and the hatched larvae were identified morphometrically, prior to molecular testing. The faecal samples with hookworm´s eggs and individual larvae were identified by a molecular assay based on the amplification of the 18S ribosomal RNA gene fragment. Further, species-specific primer sets were designed for the internal transcribed spacer (ITS 1 region) and the mitochondrial cytochrome c oxidase subunit I (COX1) gene section. RESULTS: Hookworm eggs were microscopically detected in 9.6% (26/270) of the total number of faecal samples. The prevalence in the Roma settlement was higher, 14.5% (16/110), than in shelters, 6.3% (10/160). Using PCR and subsequent Sanger sequencing, we identified the canine hookworm species Uncinaria stenocephala in all positive samples. CONCLUSION: Our results have provided new data on the molecular identification of the neglected species U. stenocephala affecting dogs in Slovakia and supplemented the missing information on the prevalence and incidence of hookworms in dogs in Europe.

A case of severe hookworm infection / 中南大学学报(医学版)

With the improvement of sanitation, the infection rate of hookworm is greatly reduced and the severe infected case is rarely reported. Combined morphological and molecular biological examinations, a severe hookworm infection patient was diagnosed in Department of Laboratorial Examination, Quanzhou First Affiliated Hospital of Fujian Medical University. The morphological methods such as direct fecal smear microscopy, saturated brine flotation and hookworm larvae culture methods were used to identify the eggs and larvae from stool samples of the patient. There were a large number of hookworm eggs in patient's stool samples, and the average count was 60 840 per gram by modified Kato method, which belonged to severe hookworm infection. Meanwhile, to distinguish the hookworm species, the semi-nested RT-PCR assay was employed to detect hookworm internal transcribed spacer series from eggs in patient's stool samples, and the result showed that the hookworm species was confirmed to be Necator americanus.

Scope and limitations of a multiplex conventional PCR for the diagnosis of S. stercoralis and hookworms.

OBJECTIVES: Describe the diagnostic characteristics of a conventional multiplex PCR for the diagnosis of S. stercoralis, N. americanus and Ancylostomas spp. METHODS: Fecal samples were collected from a cross-sectional study in Orán department, Salta province, Argentina. The stool samples were analyzed using concentration-sedimentation, Harada Mori, McMaster, and Baermann techniques. DNA was extracted from 50 mg fecal sample using the FastPrep® Spin Kit for Soil. Three pairs of primers were used for the amplification of three products of 101, 330, and 577 base pairs (bp) for S. stercoralis, N. americanus and Ancylostoma spp, respectively. The sensitivity and analytical specificity of multiplex PCR were evaluated, as well as the sensitivity and diagnostic specificity, using a composite standard and Bayesian approach. RESULTS AND CONCLUSIONS: Multiplex PCR did not present cross-reaction with other intestinal parasites, and the detection limit for multiplex PCR was between 2 and 20 pg of genomic DNA. In addition it presented a diagnostic sensitivity of 97.4% for S. stercoralis and 90.3% for hookworms with a specificity of 100% and 87.6%, respectively. PCR identified a higher proportion (p <0.01) of coinfections (15.3%) than microscopic techniques (3.5%). Also, multiplex PCR showed that there was a positive association between S. stercoralis and hookworms (odds ratio = 2.12). However, this association was due to N. americanus (odds ratio= 3.22), since no association was observed between S. stercoralis and Ancylostoma spp. Neither was an association observed between the two species of hookworms.

Characterization of T cell responses to co-administered hookworm vaccine candidates Na-GST-1 and Na-APR-1 in healthy adults in Gabon.

Two hookworm vaccine candidates, Na-GST-1 and Na-APR-1, formulated with Glucopyranosyl Lipid A (GLA-AF) adjuvant, have been shown to be safe, well tolerated, and to induce antibody responses in a Phase 1 clinical trial (Clinicaltrials.gov NCT02126462) conducted in Gabon. Here, we characterized T cell responses in 24 Gabonese volunteers randomized to get vaccinated three times with Na-GST-1 and Na-APR-1 at doses of 30µg (n = 8) or 100µg (n = 10) and as control Hepatitis B (n = 6). Blood was collected pre- and post-vaccination on days 0, 28, and 180 as well as 2-weeks after each vaccine dose on days 14, 42, and 194 for PBMCs isolation. PBMCs were stimulated with recombinant Na-GST-1 or Na-APR-1, before (days 0, 28 and 180) and two weeks after (days 14, 42 and 194) each vaccination and used to characterize T cell responses by flow and mass cytometry. A significant increase in Na-GST-1 -specific CD4+ T cells producing IL-2 and TNF, correlated with specific IgG antibody levels, after the third vaccination (day 194) was observed. In contrast, no increase in Na-APR-1 specific T cell responses were induced by the vaccine. Mass cytometry revealed that, Na-GST-1 cytokine producing CD4+ T cells were CD161+ memory cells expressing CTLA-4 and CD40-L. Blocking CTLA-4 enhanced the cytokine response to Na-GST-1. In Gabonese volunteers, hookworm vaccine candidate, Na-GST-1, induces detectable CD4+ T cell responses that correlate with specific antibody levels. As these CD4+ T cells express CTLA-4, and blocking this inhibitory molecules resulted in enhanced cytokine production, the question arises whether this pathway can be targeted to enhance vaccine immunogenicity.