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PLoS Neglected Tropical Diseases Jun 2020The canine hookworms Ancylostoma braziliense, Ancylostoma ceylanicum, Ancylostoma caninum and Uncinaria stenocephala are not only capable of producing morbidity and...
The canine hookworms Ancylostoma braziliense, Ancylostoma ceylanicum, Ancylostoma caninum and Uncinaria stenocephala are not only capable of producing morbidity and mortality in dogs but are also neglected tropical zoonoses. Each hookworm species differs considerably in its geographical distribution, life cycle, biology, pathogenic impacts on both canine and human hosts, zoonotic potential, and response to treatment with anthelminthics. Here we describe the development and validation of two Taq-Man based multiplex PCR assays capable of detecting and differentiating all four canine hookworm species in faeces of naturally infected dogs. The analytical sensitivity of both assays was assessed using 10-fold serial dilutions of synthetic gene block fragments containing individual sequence targets of each hookworm species. The sensitivity of the assays and ability to detect mixed species infections were compared to a conventional PCR-Restriction Fragment Length Polymorphism based-approach when applied to laboratory and field samples from endemic areas. The qPCRs detected at least one species of hookworms in 82.4% of PCR-RFLP-negative but microscopy-positive samples. The qPCRs detected an additional 68% mixed infections with different species of canine hookworms, and additional single species infection with A. caninum (47%), U. stenocephala (33%) and A. ceylanicum (0.02%) that were missed by PCR-RFLP. These multiplex qPCR assays will assist field based epidemiological surveillance studies towards an accurate and sensitive monitoring of canine hookworm infections in dogs, to inform their species-specific zoonotic risks to populations living in endemic areas, globally.
Topics: Ancylostoma; Ancylostomatoidea; Ancylostomiasis; Animals; DNA, Helminth; Dog Diseases; Dogs; Feces; Hookworm Infections; Humans; Multiplex Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Sensitivity and Specificity; Zoonoses
PubMed: 32542036
DOI: 10.1371/journal.pntd.0008392 -
Parasitology Research Jul 2020Marsupials of the genus Didelphis, such as black-eared opossums (Didelphis aurita), are common synanthropic animals in urban areas of Brazil. These marsupials are...
Marsupials of the genus Didelphis, such as black-eared opossums (Didelphis aurita), are common synanthropic animals in urban areas of Brazil. These marsupials are frequently parasitized by numerous helminth species, including ancylostomatid nematodes. This study aimed to report the occurrence of Ancylostoma caninum in black-eared opossums captured in an urban environment of Southeastern Brazil and discuss the potential impact of these findings for public health. From January to June 2019, we collected fecal samples from 49 restrained opossums and evaluated by a simple flotation method; Helminth eggs were observed at different magnifications and identified according to morphological and morphometric features. Genomic DNA was extracted from Ancylostomatidae eggs and screened by duplex PCR for Ancylostoma spp. and Necator americanus using primers that amplify a region of internal transcribed spacer 2 and the 28S ribosomal RNA (ITS2-28S rRNA). Ancylostoma spp. eggs were detected in 65.3% (32/49) of the animals. Sequence analysis revealed 100% homology with A. caninum sequences from GenBank. Our results demonstrate a new host-parasite interaction for A. caninum, suggesting that black-eared opossums may participate in the zoonotic cycle of this parasite in urban areas of Brazil.
Topics: Ancylostoma; Ancylostomiasis; Animals; Brazil; Cities; Didelphis; Feces; Genome, Helminth; Parasite Egg Count; Prevalence
PubMed: 32435896
DOI: 10.1007/s00436-020-06708-1 -
International Journal For Parasitology.... Aug 2020Ancylostoma caninum is the most prevalent intestinal nematode of dogs, and has a zoonotic potential. Multiple-drug resistance (MDR) has been confirmed in a number of A....
Ancylostoma caninum is the most prevalent intestinal nematode of dogs, and has a zoonotic potential. Multiple-drug resistance (MDR) has been confirmed in a number of A. caninum isolates, including isolate Worthy 4.1F3P, against all anthelmintic drug classes approved for hookworm treatment in dogs in the United States (US). The cyclooctadepsipeptide emodepside is not registered to use in dogs in the US, but in a number of other countries/regions. The objective of this study was to evaluate the efficacy of emodepside + praziquantel, as well as three commercial products that are commonly used in the US for treatment of hookworms, against a suspected (subsequently confirmed) MDR A. caninum isolate Worthy 4.1F3P. 40 dogs infected on study day (SD) 0 with 300 third-stage larvae, were randomly allocated to one of five treatment groups with eight dogs each: pyrantel pamoate (Nemex®-2), fenbendazole (Panacur® C), milbemycin oxime (Interceptor®), emodepside + praziquantel tablets and non-treated control. Fecal egg counts (FEC) were performed on SDs 19, 20, 22, 27, 31 and 34. All treatments were administered as per label requirements on SD 24 to dogs in Groups 1 through 4. Two additional treatments were administered on SDs 25 and 26 to dogs in Group 2 as per label requirements. Dogs were necropsied on SD 34 and the digestive tract was removed/processed for worm recovery and enumeration. The geometric mean (GM) worm counts for the control group was 97.4, and for the pyrantel pamoate, fenbendazole, milbemycin oxime, and emodepside + praziquantel groups were 74.8, 72.0, 88.9, and 0.4, respectively. These yielded efficacies of 23.2%, 26.1%, and 8.8%, and 99.6%, respectively. These data support previous findings of the MDR status of Worthy 4.1F3P as treatments with pyrantel pamoate, fenbendazole and milbemycin oxime lacked efficacy. In sharp contrast, Worthy 4.1F3P was highly susceptible to treatment with emodepside + praziquantel.
Topics: Ancylostomatoidea; Ancylostomiasis; Animals; Anthelmintics; Depsipeptides; Dog Diseases; Dogs; Drug Combinations; Drug Resistance, Multiple; Hookworm Infections; Intestines; Macrolides; Praziquantel; Pyrantel; Treatment Outcome
PubMed: 32403053
DOI: 10.1016/j.ijpddr.2020.04.003 -
The American Journal of Tropical... Jul 2020Hookworm infections are classified as the most impactful of the human soil-transmitted helminth (STH) infections, causing a disease burden of ∼4 million...
Hookworm infections are classified as the most impactful of the human soil-transmitted helminth (STH) infections, causing a disease burden of ∼4 million disability-adjusted life years, with a global prevalence of 406-480 million infections. Until a decade ago, epidemiological surveys largely assumed and as the relevant human hookworm species implicated as contributing to iron-deficiency anemia. This assumption was based on the indistinguishable morphology of the spp. eggs in stool and the absence of awareness of a third zoonotic hookworm species, . The expanded use of molecular diagnostic assays for differentiating hookworm species infections during STH surveys has now implicated , a predominant hookworm of dogs in Asia, as the second most common hookworm species infecting humans in Southeast Asia and the Pacific. Despite this, with the exception of sporadic case reports, there is a paucity of data available on the impact of this emerging zoonosis on human health at a population level. This situation also challenges the current paradigm, necessitating a One Health approach to hookworm control in populations in which this zoonosis is endemic. Here, we have summarized the available research studies and case reports on human infections in Southeast Asia and the Pacific after 2013 using a systematic review approach. We summarized eight research articles and five clinical case studies, highlighting the importance of future in-depth investigation of zoonotic infections using sensitive and cost-effective diagnostic tools.
Topics: Ancylostoma; Ancylostomiasis; Animals; Asia, Southeastern; Dog Diseases; Dogs; Feces; Humans; Neglected Diseases; One Health; Pacific Islands; Parasite Egg Count; Zoonoses
PubMed: 32342850
DOI: 10.4269/ajtmh.20-0060 -
Gastroenterology Dec 2020
Topics: Albendazole; Ancylostoma; Ancylostomiasis; Animals; Antinematodal Agents; Duodenum; Endoscopy, Digestive System; Erythrocyte Transfusion; Hemoglobins; Humans; Infant; Male; Mebendazole; Melena
PubMed: 32311357
DOI: 10.1053/j.gastro.2020.04.021 -
Scientific Reports Feb 2020Hookworms are intestinal parasites that cause major public health problems, especially in developing countries. To differentiate eggs from different hookworm species, it...
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.
Topics: Ancylostoma; Ancylostomiasis; Animals; Brazil; DNA, Helminth; Feces; Genotype; Humans; Intestines; Necator americanus; Necatoriasis
PubMed: 32080267
DOI: 10.1038/s41598-020-59874-8 -
Journal of Helminthology Jan 2020Glutathione S-transferases (GSTs) are a detoxifying enzyme family that is essential for parasite blood-feeding and survival, and represent potential targets for hookworm...
Glutathione S-transferases (GSTs) are a detoxifying enzyme family that is essential for parasite blood-feeding and survival, and represent potential targets for hookworm vaccine development. Multiple GST-encoding complementary DNAs (cDNAs) have been cloned from Ancylostoma caninum and Necator americanus, but there are no reports about the cloning of this enzyme from Ancylostoma ceylanicum, the animal-derived zoonotic hookworm. To study the molecular nature and tissue localization of GST of A. ceylanicum (Ace-GST), we designed primers based on the GST gene sequence of A. ceylanicum in GenBank, amplified the Ace-GST cDNA by reverse transcription polymerase chain reaction, and analysed its homology and genetic evolution relationship. The amplified product was cloned into the pET-32a vector and transformed into Escherichia coli BL21 (DE3) for expression. To prepare anti-GST polyclonal antibodies, the recombinant protein was purified and used to immunize Kunming mice. The level of immunoglobulin G (IgG) antibody in the serum of immunized mice was detected by indirect enzyme-linked immunosorbent assay, and the Ace-GST localization in adult worm was determined using the immunofluorescence method. The results showed that the full-length cDNA encoding Ace-GST was 468 bp, which had the highest homology with Ac-GST-1 (60.1%) and clustered into one branch (v-class) with Ac-GST-1 and Na-GST-1 in a phylogenetic tree. Mice immunized with recombinant Ace-GST showed specific IgG antibody response. Immunolocalization revealed that natural Ace-GST is mainly located in the epidermis, muscle and intestine of the adult. These results may lay a foundation for further studies on the biological function of Ace-GST.
Topics: Ancylostoma; Ancylostomiasis; Animals; Antibodies, Helminth; Antigens, Helminth; Cloning, Molecular; DNA, Helminth; Escherichia coli; Glutathione Transferase; Immunohistochemistry; Phylogeny; Recombinant Proteins; Transformation, Bacterial
PubMed: 31959266
DOI: 10.1017/S0022149X20000012 -
Parasites & Vectors Jan 2020Japan is one of the few countries believed to have eliminated soil-transmitted helminths (STHs). In 1949, the national prevalence of Ascaris lumbricoides was 62.9%,...
BACKGROUND
Japan is one of the few countries believed to have eliminated soil-transmitted helminths (STHs). In 1949, the national prevalence of Ascaris lumbricoides was 62.9%, which decreased to 0.6% in 1973 due to improvements in infrastructure, socioeconomic status, and the implementation of national STH control measures. The Parasitosis Prevention Law ended in 1994 and population-level screening ceased in Japan; therefore, current transmission status of STH in Japan is not well characterized. Sporadic cases of STH infections continue to be reported, raising the possibility of a larger-scale recrudescence of STH infections. Given that traditional microscopic detection methods are not sensitive to low-intensity STH infections, we conducted targeted prevalence surveys using sensitive PCR-based assays to evaluate the current STH-transmission status and to describe epidemiological characteristics of areas of Japan believed to have achieved historical elimination of STHs.
METHODS
Stool samples were collected from 682 preschool- and school-aged children from six localities of Japan with previously high prevalence of STH. Caregivers of participants completed a questionnaire to ascertain access to water, sanitation and hygiene (WASH), and potential exposures to environmental contamination. For fecal testing, multi-parallel real-time PCR assays were used to detect infections of Ascaris lumbricoides, Necator americanus, Ancylostoma duodenale and Trichuris trichiura.
RESULTS
Among the 682 children, no positive samples were identified, and participants reported high standards of WASH.
CONCLUSIONS
To our knowledge, this is the first STH-surveillance study in Japan to use sensitive molecular techniques for STH detection. The results suggest that recrudescence of STH infections has not occurred, and that declines in prevalence have been sustained in the sampled areas. These findings suggest that reductions in prevalence below the elimination thresholds, suggestive of transmission interruption, are possible. Additionally, this study provides circumstantial evidence that multi-parallel real-time PCR methods are applicable for evaluating elimination status in areas where STH prevalence is extremely low.
Topics: Adolescent; Ancylostoma; Ancylostomiasis; Animals; Ascariasis; Ascaris lumbricoides; Child; Child, Preschool; Feces; Female; Helminths; Humans; Hygiene; Japan; Male; Necator americanus; Necatoriasis; Soil; Surveys and Questionnaires; Trichuriasis; Trichuris
PubMed: 31915050
DOI: 10.1186/s13071-019-3875-z -
Emerging Infectious Diseases Jan 2020A 60-year-old man from South Korea underwent a colonoscopy. A juvenile female worm showing 3 pairs of teeth in the buccal cavity was recovered from the descending colon....
A 60-year-old man from South Korea underwent a colonoscopy. A juvenile female worm showing 3 pairs of teeth in the buccal cavity was recovered from the descending colon. Partial sequencing of the internal transcribed spacer region showed 100% identity with Ancylostoma caninum, the dog hookworm.
Topics: Ancylostoma; Ancylostomiasis; Animals; Humans; Male; Middle Aged; Phylogeny; Polymerase Chain Reaction; Republic of Korea; Sequence Analysis, DNA
PubMed: 31855538
DOI: 10.3201/eid2601.191335 -
Parasites & Vectors Dec 2019The canine hookworm, Ancylostoma caninum is the most prevalent and important intestinal nematode parasite of dogs in the USA. Hookworms are typically well controlled by...
BACKGROUND
The canine hookworm, Ancylostoma caninum is the most prevalent and important intestinal nematode parasite of dogs in the USA. Hookworms are typically well controlled by treatment with all commonly used anthelmintics that are approved for this use in dogs. However, in the past few years, cases of recurrent/persistent canine hookworm infections appear to have dramatically increased, suggesting that anthelmintic resistance (AR) may have evolved in this parasite. These cases are highly overrepresented by greyhounds, but multiple other breeds are also represented. The aim of this study was to characterize several of these suspected resistant isolates using in vitro, genetic and clinical testing to determine if these cases represent true anthelmintic resistance in A. caninum.
METHODS
Fecal samples containing hookworm eggs from three cases of persistent hookworm infections; one from a greyhound, one from a miniature schnauzer and one from a hound-mix, were received by our laboratory. These were then used to establish infections in laboratory dogs and to perform egg hatch assays (EHA) and larval development assays (LDA) for detecting resistance to benzimidazoles and macrocyclic lactones, respectively. Additional EHA and LDA were performed on eggs recovered from the laboratory-induced infections. Fecal egg count reduction tests were performed to detect resistance to pyrantel. Deep amplicon sequencing assays were developed to measure the frequency of non-synonymous single nucleotide polymorphisms (SNP) at codons 167, 198 and 200 of the A. caninum isotype-1 β-tubulin gene.
RESULTS
Resistance ratios for the three A. caninum isolates tested ranged from 6.0 to > 100 and 5.5 to 69.8 for the EHA and LDA, respectively. Following treatment with pyrantel, reduction in faecal egg counts was negative or 0%. Deep amplicon sequencing of the isotype-1 β-tubulin gene identified a high frequency of resistance-associated SNPs at codon 167 in all three resistant isolates and in two additional clinical cases.
CONCLUSIONS
These data conclusively demonstrate multiple anthelmintic resistance in multiple independent isolates of A. caninum, strongly suggesting that this is an emerging problem in the USA. Furthermore, evidence suggest that these resistant hookworms originate from racing greyhound farms and kennels, though additional research is needed to confirm this.
Topics: Ancylostoma; Ancylostomiasis; Animals; Anthelmintics; Breeding; Dog Diseases; Dogs; Drug Resistance, Multiple; Feces; Parasite Egg Count; Polymorphism, Single Nucleotide; Pyrantel; United States
PubMed: 31818311
DOI: 10.1186/s13071-019-3828-6