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Heartworm disease caused by the nematode Dirofilaria immitis is one of the most important parasitoses of dogs. The treatment of the infection is long, complicated, risky and expensive. Conversely, prevention is easy, safe, and effective and it is achieved by the administration of macrocyclic lactones (MLs). In recent years, D. immitis strains resistant to MLs have been described in Southern USA, raising concerns for possible emergence, or spreading in other areas of the world. The present study describes the first case of ML-resistant D. immitis in a dog in Europe. The dog arrived in Rome, Italy, from USA in 2023. Less than 6 months after its arrival in Italy, the dog tested positive for D. immitis circulating antigen and microfilariae, despite it having received monthly the ML milbemycin oxime (plus an isoxazoline) after arrival. The microfilariae suppression test suggested a resistant strain. Microfilariae DNA was examined by droplet digital PCR-based duplex assays targeting four marker positions at single nucleotide polymorphisms (SNP1, SNP2, SNP3, SNP7) which differentiate resistant from susceptible isolates. The genetic analysis showed that microfilariae had a ML-resistant genotype at SNP1 and SNP7 positions, compatible with a resistant strain. It is unlikely that the dog acquired the infection after its arrival in Europe, while it is biologically and epidemiologically plausible that the dog was already infected when imported from USA to Europe. The present report highlights the realistic risk of ML-resistant D. immitis strains being imported and possibly transmitted in Europe and other areas of the world. Monitoring dogs travelling from one area to another, especially if they originate from regions where ML-resistance is well-documented, is imperative. Scientists, practitioners, and pet owners should be aware of the risk and remain vigilant against ML-resistance, in order to monitor and reduce the spreading of resistant D. immitis.

Abstract Background Dirofilaria immitis causes dirofilariosis, a potentially fatal condition in canids. Dirofilaria infections can be prevented with a macrocyclic lactone (ML) prophylactic regimen. However, some D. immitis isolates have become resistant to MLs. Genetic changes on the P-glycoprotein 11 gene, encoding an ABCB transporter, have been linked to the ML-resistant phenotypes and have been proposed as markers of drug resistance. However, nothing is known about the expression and the localization of this transporter in D. immitis, despite its strong link to ML-resistant phenotypes. Methods We examined the clinically validated D. immitis P-glycoprotein 11 (DimPgp-11) single nucleotide polymorphism (SNP) via MiSeq analysis in three ML-susceptible isolates (Missouri, MP3 and Yazoo) and two ML-resistant isolates (JYD-34 and Metairie), and correlated the data with previously published MiSeq results of USA laboratory-maintained D. immitis isolates. The level of the expression of the DimPgp-11 messenger RNA transcript was analyzed by droplet digital PCR (ddPCR) and compared in the USA laboratory-maintained isolates, namely the ML-susceptible Missouri and Berkeley isolates, the putative ML-susceptible Georgia III and Big Head isolates and the ML-resistant isolate JYD-34. The immunolocalization of DimPgp-11 was visualized in the microfilaria (mf) life stage of the Missouri isolate using confocal microscopy. Results The results confirmed that the SNP found on DimPgp-11 is differentially expressed in the USA laboratory-maintained isolates. The ML-susceptible isolates had an alternate allele frequency of between 0% and 15%, while it ranged between 17% and 56% in the ML-resistant isolates. The constitutive expression of DimPgp-11 was similar in the Berkeley, Georgia III and Big Head isolates, while it was significantly decreased in the ML-resistant JYD-34 isolate (P

Nematode parasites enter their definitive host at the developmentally arrested infectious larval stage (iL3), and the ligand-dependent nuclear receptor DAF-12 contributes to trigger their development to adulthood. Here, we characterized DAF-12 from the filarial nematodes Brugia malayi and Dirofilaria immitis and compared them with DAF-12 from the non-filarial nematodes Haemonchus contortus and Caenorhabditis elegans. Interestingly, Dim and BmaDAF-12 exhibit high sequence identity and share a striking higher sensitivity than Hco and CelDAF-12 to the natural ligands Δ4- and Δ7-dafachronic acids (DA). Moreover, sera from different mammalian species activated specifically Dim and BmaDAF-12 while the hormone-depleted sera failed to activate the filarial DAF-12. Accordingly, hormone-depleted serum delayed the commencement of development of D. immitis iL3 in vitro. Consistent with these observations, we show that spiking mouse charcoal stripped-serum with Δ4-DA at the concentration measured in normal mouse serum restores its capacity to activate DimDAF-12. This indicates that DA present in mammalian serum participate in filarial DAF-12 activation. Finally, analysis of publicly available RNA sequencing data from B. malayi showed that, at the time of infection, putative gene homologs of the DA synthesis pathways are coincidently downregulated. Altogether, our data suggest that filarial DAF-12 have evolved to specifically sense and survive in a host environment, which provides favorable conditions to quickly resume larval development. This work sheds new light on the regulation of filarial nematodes development while entering their definitive mammalian host and may open the route to novel therapies to treat filarial infections.

Dirofilaria immitis is a parasitic nematode and causes dirofilariosis, a potentially fatal pulmonary infection which primarily infects canids. Dirofilariosis infections are controlled via prophylactic macrocyclic lactone (ML) regimens. Recent evidence has confirmed the development of ML-resistant isolates in the USA, which are genetically distinct from wildtype populations. Single nucleotide polymorphisms (SNP) associated with ML-resistant phenotypes were clinically validated in USA populations. In this study, 3 USA laboratory-maintained isolates (Berkeley, Georgia II, and WildCat) and 11 randomly selected European clinical samples from 7 hosts were analyzed. The samples tested were fresh microfilaria (mf) in blood or adult worms preserved in ethanol. The samples underwent MiSeq sequencing of the top 9 SNP associated with ML resistance. The results provide the first genotypic analysis of the three USA laboratory-maintained isolates and any European samples. The European clinical samples show no genomic evidence of ML resistance based on the 9 SNP. The early adoption of genotyping of clinical D. immitis samples could provide an early indication of the potential development of ML resistance and aid to distinguish clinical cases of heartworm infection due to ML resistance from those due to a lack compliance with the recommended treatments, as has been seen in North America.

The egg reduction rate (ERR) is the current standard mean to assess the efficacy of drugs against human soil-transmitted helminths (STHs; Ascaris lumbricoides, Trichuris trichiura and hookworm). Although the timing of post-treatment sampling is pivotal for a readily interpretation of drug efficacy, there is lack empirical data that allows recommending the optimal time point for a follow-up egg counting. In the present study, we re-analyzed both the kinetics of worm expulsion and egg output for Ascaris lumbricoides following a single oral dose of albendazole in a series of studies previously conducted in Kenyan communities. The results indicate that it takes up to 10 days post-treatment before the expulsion of both adult male and female Ascaris worms is completed, approximately 20% of the worms being expelled between day 7 and 10 post-treatment. The sequential analysis of the egg out put, indicated a poor ERR (89.4%) at day 7 post-treatment, but a 100% ERR at day 14 and 21 post-treatment. Based on our findings we recommend to wait at least 14 days after an albendazole treatment before conducting the follow-up egg count. Any sampling before this time point may result in biased ERR estimates, due the release of residual eggs from moribund or degenerating worms. Keywords: Ascaris lumbricoides, Worm expulsion, Egg reduction rate, Anthelmintic resistance, Albendazole

BACKGROUND:A DNA extraction and preservation protocol that yields sufficient and qualitative DNA is pivotal for the success of any nucleic acid amplification test (NAAT), but it still poses a challenge for soil-transmitted helminths (STHs), including Ascaris lumbricoides, Trichuris trichiura and the two hookworms (Necator americanus and Ancylostoma duodenale). In the present study, we assessed the impact of different DNA extraction and preservativation protocols on STH-specific DNA amplification from stool. METHODOLOGY AND PRINCIPAL FINDINGS:In a first experiment, DNA was extracted from 37 stool samples with variable egg counts for T. trichiura and N. americanus applying two commercial kits, both with and without a prior bead beating step. The DNA concentration of T. trichiura and N. americanus was estimated by means of qPCR. The results showed clear differences in DNA concentration across both DNA extraction kits, which varied across both STHs. They also indicated that adding a bead beating step substantially improved DNA recovery, particularly when the FECs were high. In a second experiment, 20 stool samples with variable egg counts for A. lumbricoides, T. trichiura and N. americanus were preserved in either 96% ethanol, 5% potassium dichromate or RNAlater and were stored at 4°C for 65, 245 and 425 days. DNA was extracted using the DNeasy Blood & Tissue kit with a bead beating step. Stool samples preserved in ethanol proved to yield higher DNA concentrations as FEC increased, although stool samples appeared to be stable over time in all preservatives. CONCLUSIONS:The choice of DNA extraction kit significantly affects the outcome of NAATs. Given the clear benefit of bead beating and our validation of ethanol for (long-term) preservation, we recommend that these aspects of the protocol should be adopted by any stool sampling and DNA extraction protocol for downstream NAAT-based detection and quantification of STHs.

BACKGROUND:To work towards reaching the WHO goal of eliminating soil-transmitted helminth (STH) infections as a public health problem, the total number of children receiving anthelmintic drugs has strongly increased over the past few years. However, as drug pressure levels rise, the development of anthelmintic drug resistance (AR) is more and more likely to appear. Currently, any global surveillance system to monitor drug efficacy and the emergence of possible AR is lacking. Consequently, it remains unclear to what extent the efficacy of drugs may have dropped and whether AR is already present. The overall aim of this study is to recommend the best diagnostic methods to monitor drug efficacy and molecular markers to assess the emergence of AR in STH control programs. METHODS:A series of drug efficacy trials will be performed in four STH endemic countries with varying drug pressure (Ethiopia and Brazil: low drug pressure, Lao PDR: moderate drug pressure and Tanzania: high drug pressure). These trials are designed to assess the efficacy of a single oral dose of 400 mg albendazole (ALB) against STH infections in school-aged children (SAC) by microscopic (duplicate Kato-Katz thick smear, Mini-FLOTAC and FECPAKG2) and molecular stool-based diagnostic methods (quantitative PCR (qPCR)). Data will be collected on the cost of the materials used, as well as the time required to prepare and examine stool samples for the different diagnostic methods. Following qPCR, DNA samples will also be submitted for pyrosequencing to assess the presence and prevalence of single nucleotide polymorphisms (SNPs) in the β-tubulin gene. These SNPs are known to be linked to AR in animal STHs. DISCUSSION:The results obtained by these trials will provide robust evidence regarding the cost-efficiency and diagnostic performance of the different stool-based diagnostic methods for the assessment of drug efficacy in control programs. The assessment of associations between the frequency of SNPs in the β-tubulin gene and the history of drug pressure and drug efficacy will allow the validation of these SNPs as a marker for AR in human STHs. TRIAL REGISTRATION:The trial was retrospectively registered the 7th of March 2018 on Clinicaltrials.gov (ID: NCT03465488).

Abstract Background Strains of Dirofilaria immitis suspected of lack of efficacy (LOE) to macrocyclic lactone (ML) preventive drugs have been increasingly reported in dogs by practicing veterinarians since 2005 in the Lower Mississippi Delta region. If proven, and not controlled in the early stages, the emergence of ML drug resistance threatens to become a widespread problem in the US that may limit the effectiveness of current preventive drug treatment methods. Methods To validate practice reports, a statewide survey of Louisiana veterinarians was done to define the extent of the problem and identify focal `hotspots’ of reported ML LOEs using Geographic Information Systems (GIS) methods. The present study then utilized microfilariae (Mf) from two canine field cases from different state locations that fit criteria for a high index of suspicion of LOE against heartworms by ML drugs. Blood containing Mf from the canine field cases was used to infect and produce L3 in Aedes aegypti for experimental infection of two groups of dogs, each of which contained two laboratory dogs, one treated with prophylactic ivermectin (12 μg/kg) monthly for 6 months at twice the label dose (6 μg/kg), and one untreated control. Results Both treated and untreated dogs from Group I and Group II developed patent D. immitis infections by 218 DPI and 189 DPI, respectively, as evidenced by a positive occult heartworm antigen test and microfilaremia by the Knott’s test. Mf counts gradually increased post-patency in test and control dogs. Infective larvae raised from microfilariae from the treated Group I dog were used to successfully establish a second generation isolate, confirming heritability of resistance in the face of a monthly ivermectin challenge dose of 24 μg/kg, given monthly for 3 months. Conclusions These experimental infection studies provide in vivo evidence of the existence of ML drug resistance in dogs infected by D. immitis L3 from suspect field LOE cases in the Lower Mississippi Delta. Results encourage further work on mechanisms underlying the emergence of ML resistance in D. immitis and development of evidence-based resistance management strategies for heartworm preventives in order to extend the useful life of current drugs.

BACKGROUND:The benzimidazole (BZ) anthelmintics, albendazole (ABZ) and mebendazole (MBZ) are the most common drugs used for treatment of soil-transmitted helminths (STHs). Their intensive use increases the possibility that BZ resistance may develop. In veterinary nematodes, BZ resistance is caused by a single nucleotide polymorphism (SNP) in the β-tubulin isotype 1 gene at codon position 200, 167 or 198, and these SNPs have also been correlated with poor response of human Trichuris trichiura to BZ treatment. It is important to be able to investigate the presence of resistance-associated SNPs in STHs before resistance becomes clinically established. METHODS:The objective of this study was to develop new genotyping assays to screen for the presence of β-tubulin SNPs in T. trichiura and Ascaris lumbricoides. Rapid, simple and accurate genotyping assays were developed based on the SmartAmp2 method. Primer sets were optimized and selected to distinguish the SNP-variant genotypes. After initial optimization on control plasmids, the feasibility of the assay was assessed in field samples from Haiti and Panama. Finally, spiked fecal samples were assessed to determine the tolerance of Aac polymerase to fecal inhibitors. FINDINGS:Rapid SNP genotyping assays were developed to target β-tubulin polymorphisms in T. trichiura and A. lumbricoides. The assays showed high sensitivity and specificity in field samples and also demonstrated high tolerance to PCR inhibitors in fecal samples. CONCLUSION:These assays proved to be robust and efficient with the potential to be used as field tools for monitoring SNPs that could be associated with BZ resistance. However, further work is needed to validate the assays on large numbers of field samples before and after treatment.

BACKGROUND:Soil-transmitted helminths (STHs) are the most prevalent intestinal helminths of humans, and a major cause of morbidity in tropical and subtropical countries. The benzimidazole (BZ) drugs albendazole (ABZ) and mebendazole (MBZ) are used for treatment of human STH infections and this use is increasing dramatically with massive drug donations. Frequent and prolonged use of these drugs could lead to the emergence of anthelmintic resistance as has occurred in nematodes of livestock. Previous molecular assays for putative resistance mutations have been based mainly on PCR amplification and sequencing. However, these techniques are complicated and time consuming and not suitable for resource-constrained situations. A simple, rapid and sensitive genotyping method is required to monitor for possible developing resistance to BZ drugs. METHODS:To address this problem, single nucleotide polymorphism (SNP) detection assays were developed based on the Smart amplification method (SmartAmp2) to target codons 167, 198, and 200 in the β-tubulin isotype 1 gene for the hookworm Necator americanus. FINDINGS:Diagnostic assays were developed and applied to analyze hookworm samples by both SmartAmp2 and conventional sequencing methods and the results showed high concordance. Additionally, fecal samples spiked with N. americanus larvae were assessed and the results showed that the Aac polymerase used has high tolerance to inhibitors in fecal samples. CONCLUSION:The N. americanus SmartAmp2 SNP detection assay is a new genotyping tool that is rapid, sensitive, highly specific and efficient with the potential to be used as a field tool for monitoring SNPs associated with BZ resistance. However, further validation on large numbers of field samples is required.

The anthelmintics ivermectin (IVM) and moxidectin (MOX) display differences in toxicity in several host species. Entrance into the brain is restricted by the P-glycoprotein (P-gp) efflux transporter, while toxicity is mediated through the brain GABA(A) receptors. This study compared the toxicity of IVM and MOX in vivo and their interaction with GABA(A) receptors in vitro. Drug toxicity was assessed in Mdr1ab(-/-) mice P-gp-deficient after subcutaneous administration of increasing doses (0.11-2.0 and 0.23-12.9 µmol/kg for IVM and MOX in P-gp-deficient mice and half lethal doses (LD(50)) in wild-type mice). Survival was evaluated over 14-days. In Mdr1ab(-/-) mice, LD(50) was 0.46 and 2.3 µmol/kg for IVM and MOX, respectively, demonstrating that MOX was less toxic than IVM. In P-gp-deficient mice, MOX had a lower brain-to-plasma concentration ratio and entered into the brain more slowly than IVM. The brain sublethal drug concentrations determined after administration of doses close to LD(50) were, in Mdr1ab(-/-) and wild-type mice, respectively, 270 and 210 pmol/g for IVM and 830 and 740-1380 pmol/g for MOX, indicating that higher brain concentrations are required for MOX toxicity than IVM. In rat α1β2γ2 GABA channels expressed in Xenopus oocytes, IVM and MOX were both allosteric activators of the GABA-induced response. The Hill coefficient was 1.52±0.45 for IVM and 0.34±0.56 for MOX (p