Development and validation of a long-read metabarcoding platform for the detection of filarial worm pathogens of animals and humans.

BACKGROUND: Filarial worms are important vector-borne pathogens of a large range of animal hosts, including humans, and are responsible for numerous debilitating neglected tropical diseases such as, lymphatic filariasis caused by Wuchereria bancrofti and Brugia spp., as well as loiasis caused by Loa loa. Moreover, some emerging or difficult-to-eliminate filarioid pathogens are zoonotic using animals like canines as reservoir hosts, for example Dirofilaria sp. 'hongkongensis'. Diagnosis of filariasis through commonly available methods, like microscopy, can be challenging as microfilaremia may wane below the limit of detection. In contrast, conventional PCR methods are more sensitive and specific but may show limited ability to detect coinfections as well as emerging and/or novel pathogens. Use of deep-sequencing technologies obviate these challenges, providing sensitive detection of entire parasite communities, whilst also being better suited for the characterisation of rare or novel pathogens. Therefore, we developed a novel long-read metabarcoding assay for deep-sequencing the filarial nematode cytochrome c oxidase subunit I gene on Oxford Nanopore Technologies' (ONT) MinION™ sequencer. We assessed the overall performance of our assay using kappa statistics to compare it to commonly used diagnostic methods for filarial worm detection, such as conventional PCR (cPCR) with Sanger sequencing and the microscopy-based modified Knott's test (MKT). RESULTS: We confirmed our metabarcoding assay can characterise filarial parasites from a diverse range of genera, including, Breinlia, Brugia, Cercopithifilaria, Dipetalonema, Dirofilaria, Onchocerca, Setaria, Stephanofilaria and Wuchereria. We demonstrated proof-of-concept for this assay by using blood samples from Sri Lankan dogs, whereby we identified infections with the filarioids Acanthocheilonema reconditum, Brugia sp. Sri Lanka genotype and zoonotic Dirofilaria sp. 'hongkongensis'. When compared to traditionally used diagnostics, such as the MKT and cPCR with Sanger sequencing, we identified an additional filarioid species and over 15% more mono- and coinfections. CONCLUSIONS: Our developed metabarcoding assay may show broad applicability for the metabarcoding and diagnosis of the full spectrum of filarioids from a wide range of animal hosts, including mammals and vectors, whilst the utilisation of ONT' small and portable MinION™ means that such methods could be deployed for field use.

Retrospective review of patients with myasthenia gravis switched from plasma exchange therapy to efgartigimod treatment.

INTRODUCTION/AIMS: Therapeutic plasma exchange (TPE) is sometimes used as maintenance therapy for the treatment of myasthenia gravis (MG). Efgartigimod is a newly approved monoclonal antibody targeting the neonatal Fc receptor, effectively reducing immunoglobulin G levels in the treatment of MG. The aim of this study was to describe the clinical experience of switching patients from maintenance TPE treatment to efgartigimod infusions. METHODS: A retrospective review of medical records was performed on patients previously treated with maintenance TPE for the diagnosis of MG and subsequently switched to efgartigimod infusions. Clinical characteristics and response to treatment switch were described. RESULTS: Five of seven patients demonstrated improvement on Myasthenia Gravis Foundation of America-post intervention status, one was unchanged and one was in pharmacological remission. This was reflected in pre- and postswitch MG activities of daily living and MG manual muscle testing scores. All patients have continued on efgartigimod therapy. The duration of treatment with efgartigimod at the time of this review ranged from 1 to 13 months. Recurrent uncomplicated infections were noted in two patients on efgartigimod therapy. Maintenance dosing regimens of efgartigimod varied based on clinical response to treatment and side effects. DISCUSSION: In this series, efgartigimod appeared effective and well tolerated in patients switched from TPE.

Paraproteinemic neuropathies.

The diagnostic evaluation of a peripheral neuropathy includes testing for the presence of monoclonal gammopathy, which can be found in about 10% of patients with peripheral neuropathy. Our role, as physicians, is to determine whether the neuropathy is directly related to the gammopathy or whether the co-occurrence of these two disorders is purely coincidental. The evaluating physician needs to be familiar with the different types of neuropathies associated with monoclonal gammopathies, their clinical and electrodiagnostic characteristics, and their appropriate diagnostic evaluation and management. Testing for monoclonal protein disorders includes serum protein electrophoresis (SPEP) and immunofixation of blood, and in some cases of urine, as well as measurement of free light chains and quantitative immunoglobulins. Specific antibody testing is directed by paraprotein type and neuropathy phenotype. Patients with abnormal free light chains in association with sensory and autonomic neuropathy should be evaluated for AL amyloidosis. When a lambda monoclonal protein is identified together with a clinical phenotype of chronic inflammatory demyelinating neuropathy (CIDP), a diagnosis of polyneuropathy, organomegaly, endocrinopathy, monoclonal plasma cell disorder, skin changes (POEMS) syndrome should be considered. Patients with IgM paraprotein associated neuropathy should be assessed for distal acquired demyelinating sensorimotor (DADS) neuropathy, with or without anti myelin associated glycoprotein (MAG) antibody or CANOMAD syndrome. In many cases, a monoclonal gammopathy of uncertain significance (MGUS) is incidental and unrelated to the neuropathy. Collaboration with oncology is critical in evaluating patients with monoclonal proteins to assess for underlying plasma cell neoplasms or B cell lymphomas.

Neuroprognostication: Guillain-Barré Syndrome.

Guillain-Barré syndrome is an immune-mediated disease of the peripheral nerves characterized by rapidly progressing symmetric weakness, areflexia, and albuminocytological dissociation. Most patients reach their nadir within 2 weeks. Disease severity can be mild to severe, with 20% of patients requiring mechanical ventilation. Intravenous immunoglobulin and plasma exchange are equally effective treatments. Monitoring strength, respiratory function, blood pressure, and heart rate, as well as pain management and rehabilitative therapy are important aspects of management. About 20% of patients require assistance to walk at 6 months. Older age, preceding diarrhea, and lower Medical Research Council (MRC) sum scores predict poor outcome. Death from cardiovascular and respiratory complications can occur in the acute or recovery phases of the illness in 3 to 7% of the patients. Risk factors for mortality include advanced age and disease severity at onset. Neuropathic pain, weakness, and fatigue can be residual symptoms; risk factors for these include axonal loss, sensory involvement, and severity of illness.

Zoonotic helminths of dogs and risk factors associated with polyparasitism in Grenada, West Indies.

Canine soil-transmitted helminths (STHs) cause important zoonoses in the tropics, with varying degrees of intensity of infection in humans and dogs. This study aimed to investigate the prevalence and associated risk factors for STHs in community dogs residing in Grenada, West Indies. In May 2021, 232 canine fecal samples were examined for zoonotic helminths by microscopy (following flotation), and genomic DNA from a subset of 211 of these samples were subjected to multiplex qPCR for the detection and specific identification of hookworms, Toxocara spp. and Strongyloides. Microscopic examination revealed that 46.5% (108/232, 95% CI 40­52.9), 9% (21/232, 95% CI 5.35­12.7) and 5.2% (12/232, 95% CI 2.3­8) of the samples contained eggs of Ancylostoma spp., Toxocara spp. and Trichuris vulpis, respectively. Multiplex qPCR revealed that, 42.2% (89/211, 95% CI 35.5­48.8) were positive for at least 1 zoonotic parasite. Of these, 40.8% (86/211, 95% CI 34.1­47.3) of samples tested positive for Ancylostoma spp., 36% (76/211, 95% CI 29.5­42.9) were positive for A. caninum, 13.3% (28/211, 95% CI 9­18.6) for A. ceylanicum, 5.7% for T. canis (12/211, 95% CI 2.97­8.81) and 1% (2/211, 95% CI 0­2.26) for Strongyloides spp. (identified as S. stercoralis and S. papillosus by conventional PCR-based Sanger sequencing). Using a multiple logistic regression model, a low body score and free-roaming behaviour were significant predictors of test-positivity for these parasitic nematodes in dogs (P < 0.05). Further studies of zoonotic STHs in humans should help elucidate the public health relevance of these parasites in Grenada.

Ancylostoma ceylanicum Hookworms in Dogs, Grenada, West Indies.

Ancylostoma ceylanicum hookworms are recognized agents of human infection in the Asia-Pacific region. We investigated prevalence of zoonotic hookworm infections in dogs in Grenada in 2021; 40.8% were infected by hookworms, including Ancylostoma ceylanicum. Surveillance of this parasite in dogs and humans is needed in tropical/subtropical countries in the Americas.

Zoonotic Soil-Transmitted Helminths in Free-Roaming Dogs, Kiribati.

Soil-transmitted helminths are highly prevalent in the Asia-Pacific region. We report a 96.5% prevalence of zoonotic soil-transmitted helminths in dogs in Kiribati. We advocate for urgent implementation of treatment and prevention programs for these zoonotic pathogens, in line with the Kiribati-World Health Organization Cooperation Strategy 2018-2022.

Canine Leishmaniasis Control in the Context of One Health.

Dogs are the main reservoir of Leishmania infantum and in some countries have been regularly culled as part of government policy to control visceral leishmaniasis. At the 13th Symposium of the Companion Vector-Borne Diseases World Forum in Windsor, UK, March 19-22, 2018, we consolidated a consensus statement regarding the usefulness of dog culling as a means of controlling visceral leishmaniasis. The statement highlighted the futility of culling infected dogs, whether healthy or sick, as a measure to control the domestic reservoir of L. infantum and reduce the risk for visceral leishmaniasis.

A cluster-randomised controlled trial comparing school and community-based deworming for soil transmitted helminth control in school-age children: the CoDe-STH trial protocol.

BACKGROUND: Current guidelines and targets for soil-transmitted helminth (STH) control focus on school-based deworming for school-age children, given the high risk of associated morbidity in this age group. However, expanding deworming to all age groups may achieve improved STH control among both the community in general and school-age children, by reducing their risk of reinfection. This trial aims to compare school-based targeted deworming with community-wide mass deworming in terms of impact on STH infections among school-age children. METHODS: The CoDe-STH (Community Deworming against STH) trial is a cluster-randomised controlled trial (RCT) in 64 primary schools in Dak Lak province, Vietnam. The control arm will receive one round of school-based targeted deworming with albendazole, while in the intervention arm, community-wide mass deworming with albendazole will be implemented alongside school-based deworming. Prevalence of STH infections will be measured in school-age children at baseline and 12 months following deworming. The primary outcome is hookworm prevalence in school-age children at 12 months, by quantitative PCR. Analysis will be intention-to-treat, with outcomes compared between study arms using generalised linear and non-linear mixed models. Additionally, cost-effectiveness of mass and targeted deworming will be calculated and compared, and focus group discussions and interviews will be used to assess acceptability and feasibility of deworming approaches. Individual based stochastic models will be used to predict the impact of mass and targeted deworming strategies beyond the RCT timeframe to assess the likelihood of parasite population 'bounce-back' if deworming is ceased due to low STH prevalence. DISCUSSION: The first large-scale trial comparing mass and targeted deworming for STH control in South East Asia will provide key information for policy makers regarding the optimal design of STH control programs. TRIAL REGISTRATION: ACTRN12619000309189 .

Spirorchiidiasis in marine turtles: the current state of knowledge.

Blood flukes of the family Spirorchiidae are important disease agents in marine turtles. The family is near cosmopolitan in distribution. Twenty-nine marine species across 10 genera are currently recognized, but taxonomic problems remain and it is likely that more species will be discovered. Spirorchiids infect the circulatory system, where they and their eggs cause a range of inflammatory lesions. Infection is sometimes implicated in the death of the turtle. In some regions, prevalence in stranded turtles is close to 100%. Knowledge of life cycles, important for control and epidemiological studies, has proven elusive until recently, when the first intermediate host identifications were made. Recent molecular studies of eggs and adult worms indicate that a considerable level of intrageneric and intraspecific diversity exists. The characterization of this diversity is likely to be of importance in exploring parasite taxonomy and ecology, unravelling life cycles, identifying the differential pathogenicity of genotypes and species, and developing antemortem diagnostic tools, all of which are major priorities for future spirorchiid research. Diagnosis to date has been reliant on copromicroscopy or necropsy, which both have significant limitations. The current lack of reliable antemortem diagnostic options is a roadblock to determining the true prevalence and epidemiology of spirorchiidiasis and the development of effective treatment regimes.

The epidemiology of Taenia spp. infection and Taenia solium cysticerci exposure in humans in the Central Highlands of Vietnam.

BACKGROUND: Vietnam is endemic for taeniasis and T. solium cysticercosis. Despite this, information on the epidemiological characteristics of the diseases in the Central Highlands of Vietnam are poorly described. The aims of this study were to determine the epidemiological characteristics of taeniasis (Taenia spp.) and T. solium cysticerci exposure in humans in Dak Lak province in the Central Highlands, Vietnam. METHODS: This cross-sectional study was carried out in six villages in three districts of Dak Lak. A total of 190 households were visited. From each household, between one and five individuals were asked to donate a single faecal and blood sample and respond to a questionnaire. Serum samples were subjected to lentil lectin purified glycoprotein enzyme-linked immunoelectrotransfer blot assay to detect antibodies against T. solium cysticerci. Multiplex real-time PCR was used to detect Taenia spp. infection in faecal samples. A fixed-effects logistic regression model was developed to identify factors associated with the probability of Taenia spp. infection or T. solium cysticerci exposure risk. The contribution of each of identified factor was quantified using population attributable fractions. RESULTS: The prevalence of seroexposure to T. solium in Dak Lak was 5% (95% CI 3% to 8%). Consumption of raw vegetables, sourcing drinking water from lakes, streams or ponds and the practice of outdoor defaecation were identified as primary risk factors for the prevalence of T. solium cysticerci exposure, while consuming undercooked pork and beef, pork tongue and observing Taenia proglottids in stool were associated with Taenia spp. infection. Consumption of raw vegetables attributed to 74% of T. solium cysticerci exposure-positive cases and consumption of undercooked beef attributed to 77% of taeniasis cases in these communities. CONCLUSIONS: The prevalence of T. solium seroexposure in Dak Lak is consistent with those reported in other regions of Vietnam. The identified risk factors associated with the prevalence of T. solium seroexposure and taeniasis infection in Dak Lak are modifiable and thus advocate for targeted community intervention programs to mitigating these risks.

Molecular characterization of Hepatozoon canis from farm dogs in Pakistan.

Hepatozoon canis is a tick-borne pathogen of canids, which is distributed worldwide. However, very little is known about this protozoan parasite in Pakistan. This study provides the first molecular evidence of H. canis from farm dogs from three agro-ecological zones of Punjab, Pakistan. A conventional PCR targeting the 18S rRNA gene was used to characterize H. canis from farm dogs from three districts, namely Kasur, Rawalpindi, and Muzaffargarh, in Punjab. Of 341 blood samples tested, 155 (45.5%) were positive for H. canis, 73 (61.3%) from Kasur, 46 (42.5%) from Rawalpindi, and 36 (31.5%) from Muzaffargarh. Phylogenetic analyses revealed that 18S rRNA sequences of H. canis from this study clustered in three clades with those of H. canis from previously published studies to the exclusion of all other Hepatozoon spp. included in the analysis. This study provides the first insight into H. canis from farm dogs in Pakistan. Furthermore, it lays a foundation for future studies of the parasite to assess the impact of canine hepatozoonosis in dogs from various agro-ecological zones in Pakistan where pet ownership of dogs is increasing.

Ancylostoma ceylanicum Hookworm in the Solomon Islands.

Although hookworm is highly prevalent in the Solomon Islands, the species involved are unknown. We initiated this study in response to finding Ancylostoma ceylanicum hookworm in a peacekeeper in Australia who had returned from the Solomon Islands. Kato-Katz fecal surveys performed in 2013 and 2014 in 2 village groups in East Malaita, Solomon Islands, identified hookworm-positive samples. These specimens were tested by cytochrome oxidase 1 (cox-1) gene multiplex PCR and sequenced. Of 66 positive specimens, 54 (81.8%) contained only Necator americanus, 11 (16.7%) contained only A. ceylanicum, and 1 (1.5%) contained both species. A. duodenale was not found. Haplotype analysis of cox-1 sequences placed all human isolates (99% bootstrap support) of A. ceylanicum within the zoonotic clade rather than the human-specific clade. This study confirms that A. ceylanicum is endemic in the East Malaita region of this Pacific Island nation. The strain of the A. ceylanicum in this region can be shared among humans, dogs, and cats.

Causes of neuropathy in patients referred as "idiopathic neuropathy".

INTRODUCTION: The etiology of neuropathy was idiopathic in 20%-30% of patients despite thorough investigation, based on results from the 1980s and 1990s. Since then, new etiologies have been recognized, and skin biopsy has been used to confirm small-fiber neuropathy. METHODS: The authors reviewed the charts of 373 patients with idiopathic neuropathy who were referred to a neuropathy center between 2002 and 2012. RESULTS: Among the 284 eligible patients, 93 (32.7%) remained idiopathic. The most common cause was impaired glucose metabolism (72 patients, 25.3%), including diabetes in 26 and prediabetes in 46. Other etiologies were chronic inflammatory demyelinating polyneuropathy (CIDP) in 57 (20%) and monoclonal gammopathy in 20 (7%), as well as toxic, Sjögren disease, celiac disease, other immune-mediated diseases, vitamin B12 deficiency, amyloidosis, vitamin B1 and B6 deficiency, vasculitis, hypothyroidism, hereditary, Lyme disease, and anti-sulfatide antibody. CONCLUSIONS: The major causes of undiagnosed neuropathies were impaired glucose metabolism, CIDP, and monoclonal gammopathies. Despite thorough evaluation 32.7% remained idiopathic. Muscle Nerve 53: 856-861, 2016.

Comparative pathogenesis of eosinophilic meningitis caused by Angiostrongylus mackerrasae and Angiostrongylus cantonensis in murine and guinea pig models of human infection.

This study investigated comparatively the pathogenicity of experimental infection of mice and guinea pigs, with Angiostrongylus mackerrasae and the closely related species A. cantonensis. Time course analyses showed that A. mackerrasae causes eosinophilic meningitis in these hosts, which suggests that the species has the potential to cause meningitis in humans and domestic animals. Both A. mackerrasae and the genetically similar A. cantonensis caused eosinophilic meningitis in mice at two time points of 14 and 21 days post infection (dpi). The brain lesions in mice infected with A. mackerrasae were more granulomatous in nature and the parasites were more likely to appear degenerate compared with lesions caused by A. cantonensis. This may indicate that the mouse immune system eliminates A. mackerrasae infection more effectively. The immunologic responses of mice infected with the two Angiostrongylus species was compared by assessing ex vivo stimulated spleen derived T cells and cytokines including interferon-gamma, interleukin 4 and interleukin 17 on 14 and 21 dpi. The results were similar for mice infected with A. cantonensis and A. mackerrasae. Serum from the infected animals with either A. cantonensis or A. mackerrasae recognized total soluble antigen of A. cantonensis female worms on Western blot.

Features of Blastocystis spp. in xenic culture revealed by deconvolutional microscopy.

Blastocystis spp. are common human enteric parasites with complex morphology and have been reported to cause irritable bowel syndrome (IBS). Deconvolutional microscopy with time-lapse imaging and fluorescent spectroscopy of xenic cultures of Blastocystis spp. from stool samples of IBS patients and from asymptomatic, healthy pigs allowed observations of living organisms in their natural microbial environment. Blastocystis organisms of the vacuolated, granular, amoebic and cystic forms were observed to autofluorescence in the 557/576 emission spectra. Autofluorescence could be distinguished from fluorescein-conjugated Blastocystis-specific antibody labelling in vacuolated and granular forms. This antibody labelled Blastocystis subtypes 1, 3 and 4 but not 5. Surface pores of 1 µm in diameter were observed cyclically opening and closing over 24 h. Vacuolated forms extruded a viscous material from a single surface point with coincident deflation that may demonstrate osmoregulation. Tear-shaped granules were observed exiting from the surface of an amoebic form, but their origin and identity remain unknown.

Molecular analysis of the genera Hapalotrema Looss, 1899 and Learedius Price, 1934 (Digenea: Spirorchiidae) reveals potential cryptic species, with comments on the validity of the genus Learedius.

Adult blood flukes of the genera Hapalotrema Looss, 1899 and Learedius Price, 1934 were collected from turtles off Queensland and the Hawaiian Islands. Specimens were identified as Hapalotrema pambanensis Mehrotra, 1973, H. synorchis Luhman, 1935, H. postorchis Rao, 1976 and Learedius learedi Price, 1934 on the basis of morphology. No major morphological differences were found between specimens from this study and previously published descriptions. DNA was also extracted and sequences obtained using custom spirorchiid-specific primers for the ITS2 and 28S rDNA regions, in order to confirm species identification and investigate phylogenetic relationships. Intraspecific genetic variation was generally low. However the ITS2 region of H. postorchis and to a lesser extent that of L. learedi showed considerable variation between specimens from the Pacific and Atlantic oceans. Further studies will be required to determine whether this variation should be considered inter- or intra-specific. Maximum likelihood phylogenetic analyses were completed for both sequenced genes. Learedius learedi was unequivocally nested among species of Hapalotrema, suggesting that the status of the genus Learedius may need to be reassessed.

High prevalence of Ancylostoma ceylanicum hookworm infections in humans, Cambodia, 2012.

Ancylostoma ceylanicum, a hookworm of canids and felids in Asia, is becoming the second most common hookworm infecting humans. In 2012, we investigated the prevalence and infection dynamics of and risk factors for hookworm infections in humans and dogs in a rural Cambodian village. Over 57% of the population was infected with hookworms; of those, 52% harbored A. ceylanicum hookworms. The greatest intensities of A. ceylanicum eggs were in persons 21-30 years of age. Over 90% of dogs also harbored A. ceylanicum hookworms. Characterization of the cytochrome oxidase-1 gene divided isolates of A. ceylanicum hookworms into 2 groups, 1 containing isolates from humans only and the other a mix of isolates from humans and animals. We hypothesize that preventative chemotherapy in the absence of concurrent hygiene and animal health programs may be a factor leading to emergence of A. ceylanicum infections; thus, we advocate for a One Health approach to control this zoonosis.

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.

A comparative field efficacy trial of three treatment programs against endo- and ectoparasites in naturally infected dogs.

Introduction: Tropical climates in remote Aboriginal and Torres Strait Islander communities in northern Australia are conducive to the transmission of canine helminths such as hookworms, as well as ectoparasites such as fleas and ticks. In addition to their veterinary importance, these parasites may present a zoonotic risk either directly, or as potential vectors for bacterial pathogens. These factors necessitate efficacious and effective antiparasitic treatment programs for community dogs. Methods: A cluster-randomised trial was performed on three islands in the Torres Strait to examine the short-term efficacy and medium-term effectiveness of three treatment programs. Treatments administered included oral oxibendazole/praziquantel (Paragard®) and oral afoxolaner (Nexgard®); topical moxidectin/imidacloprid (Advocate®) and imidacloprid/flumethrin collars (Seresto®); and off-label oral ivermectin (Bomectin®). Canine faecal samples were collected and examined for endoparasites by faecal flotation and real-time PCR at baseline, 7-11 days after treatment and 6 months later. Results: The proportion of dogs positive for Ancylostoma caninum at baseline and negative at day 7-11 was 9% (95% CI 4.4-17.4) for dogs treated with oxibendazole, 56.4% (95% CI 41-70.7) for moxidectin, and 89.7% (95% CI 73.6-96.4) for ivermectin. Faecal flotation results showed a greater than 90% egg reduction in 29.2% (95% CI 19.9-40.5) of dogs treated with oxibendazole, 79.4% (95% CI 63.2-89.7) for moxidectin, and 95% (95% CI 76.4-99.1) for off-label ivermectin. Elimination of ectoparasite infestation was observed at day 7-11 in 69.9% (95% CI 56.7-80.1) of dogs treated with afoxolaner, 80% (95% CI 60.9-91.1) with imidacloprid/flumethrin collars, and 0% (95% CI 0-11.7) for off-label ivermectin. Mixed effects modelling revealed only treatment group to be significantly associated with outcome measures. Discussion: Based on these study results, the poor efficacy of oxibendazole against A. caninum renders it inept for treatment, while ivermectin and moxidectin were suitable. Ivermectin was unsuitable for ectoparasite treatment due to its poor efficacy, while afoxolaner and imidacloprid/flumethrin collars appear suitable.