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Diagnostic algorithm for evaluating cases of potential macrocyclic lactone-resistant heartworm
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Background
The emergence of macrocyclic lactone resistance in canine heartworm poses a substantial threat to what is currently the only effective, FDA-approved available method of prevention. Further study of the biotypes is necessary to understand the mechanism of resistance and evaluate novel prevention options. Identifying cases of drug-resistan...
Citations
... Immediately after diagnosis, arthropod anti-feeding formulations were applied to the dog and guidelines for home restriction were given to the owners to minimize the possible transmission of the infection, although the diagnosis was made at the end of November, i.e. a month when the transmission risk is low in Italy (Genchi et al., 2011). The presence of a ML-resistant strain of D. immitis was investigated based on a well-established algorithm (Moorhead et al., 2017;Diakou and Prichard, 2021). As the dog was microfilaraemic, the first approach was the microfilariae suppression test (MFST) as previously described with recently proposed slight modifications (Diakou and Prichard, 2021). ...
... AR resistance in the gastrointestinal nematodes (GINs) of livestock is typically measured by the fecal egg count reduction test (FECRT) [9]. Similarly, concentrations of microfilariae in skin snips have been used to evaluate anthelmintic efficacy against Onchocerca volvulus in humans [10], and microfilarial counts in canine blood are used for diagnosing resistance in the heartworm, Dirofilaria immitis [5,11]. However, these approaches measure the production of parasite progeny by adult parasitic stages, and do not allow an accurate enumeration of the parasite burden [12]. ...
Long-term intensive use of anthelmintics for parasite control of livestock, companion animals, and humans has resulted in widespread anthelmintic resistance, a problem of great socioeconomic significance. But anthelmintic therapy may also select for other biological traits, which could have implications for anthelmintic performance. Here, we highlight recent examples of changing parasite dynamics following anthelmintic administration, which do not fit the definition of anthelmintic resistance. We also consider other possible examples in which anthelmintic resistance has clearly established, but where coselection for other biological traits may have also occurred. We offer suggestions for collecting more information and gaining a better understanding of these phenomena. Finally, we propose research questions that require further investigation and make suggestions to help address these knowledge gaps.
... The first indication for a veterinarian to consider that a case is worth investigating for resistance is when a dog under consistent preventives becomes heartworm-positive. In an integrated and practical description of how to deal with suspected cases, Moorhead et al. [61] proposed an "algorithm" that helps to navigate the veterinarian through the steps needed to solve such dilemmas. This algorithm describes the consecutive actions needed to obtain a clearer picture of the susceptibility nature of the parasites involved in a suspected LOE case. ...
Dirofilaria immitis infection is one of the most severe parasitic diseases in dogs. Prevention is achieved by the administration of drugs containing macrocyclic lactones (MLs). These products are very safe and highly effective, targeting the third and fourth larval stages (L3, L4) of the parasite. Until 2011, claims of the ineffectiveness of MLs, reported as “loss of efficacy” (LOE), were generally attributed to owners’ non-compliance, or other reasons associated with inadequate preventative coverage. There was solid argumentation that a resistance problem is not likely to occur because of (i) the great extent of refugia, (ii) the complexity of resistance development to MLs, and (iii) the possible large number of genes involved in resistance selection. Nevertheless, today, it is unequivocally proven that ML-resistant D. immitis strains exist, at least in the Lower Mississippi region, USA. Accordingly, tools have been developed to evaluate and confirm the susceptibility status of D. immitis strains. A simple, in-clinic, microfilariae suppression test, 14-28 days after ML administration, and a “decision tree” (algorithm), including compliance and preventatives’ purchase history, and testing gaps, may be applied for assessing any resistant nature of the parasite. On the molecular level, specific SNPs may be used as markers of ML resistance, offering a basis for the validation of clinically suspected resistant strains. In Europe, no LOE/resistance claims have been reported so far, and the existing conditions (stray dogs, rich wildlife, majority of owned dogs not on preventive ML treatment) do not favor selection pressure on the parasites. Considering the genetic basis of resistance and the epizootiological characteristics of D. immitis, ML resistance neither establishes easily nor spreads quickly, a fact confirmed by the current known dispersion of the problem, which is limited. Nevertheless, ML resistance may propagate from an initial geographical point, via animal and vector mobility, to other regions, while it can also emerge as an independent evolutionary process in a new area. For these reasons, and considering the current chemoprophylaxis recommendations and increasing use of ML endectoparasiticides as a potential selection pressure, it is important to remain vigilant for the timely detection of any ML LOE/resistance, in all continents where D. immitis is enzootic.
... The first indication for a veterinarian to consider that a case is worth investigating for resistance is when a dog under consistent preventives becomes heartworm positive. In an integrated and practical description of how to deal with suspected cases, Moorhead et al. [57] proposed an "algorithm" that helps navigate the veterinarian through the steps needed to solve such dilemmas. This algorithm describes the consecutive actions needed to get a clearer picture of the susceptibility nature of the parasites involved in a LOE suspected case. ...
Dirofilaria immitis infection is one of the most severe parasitic diseases of dogs. Prevention is achieved by the administration of drugs containing macrocyclic lactones (MLs). These products are very safe and highly effective, targeting the third and fourth larval stages (L3, L4) of the parasite. Until 2011, claims of ineffectiveness of MLs, reported as "Lack of Efficacy" (LOE), were generally attributed to owners' non-compliance, or other reason for inadequate preventative coverage. There was solid argumentation that a resistance problem is not likely to occur because of i) the great extent of refugia, ii) the complexity of resistance development to MLs, and iii) the possible big number of genes involved in resistance selection. Nevertheless, today it is unequivocally proven that ML resistant D. immitis strains exist, at least in the Lower Mississippi region, USA. Accordingly, tools have been developed, to evaluate and confirm the susceptibility status of D. immitis strains. A simple, in-clinic, microfilariae suppression test, 14-28 days after ML administration, and a "decision tree" (al-gorithm), including compliance and preventatives' purchase history, and testing gaps, may be applied for assessing any resistant nature of the parasite. On the molecular level, specific SNPs may be used as markers of ML resistance, offering a basis for validation of clinically suspected resistant strains. In Europe, no LOE/resistance claims have been reported so far, and the existing conditions (stray dogs, rich wildlife, majority of owned dogs not on preventive MLs treatment) do not favor selection pressure on the parasites. Considering the genetic basis of resistance and the epizootiolog-ical characteristics of D. immitis, ML resistance neither establishes easily nor spreads quickly, a fact confirmed by the current known dispersion of the problem, which is limited. Nevertheless, ML resistance may propagate from an initial geographical point, via animal and vector mobility, to other regions, while it can also emerge as an independent evolutionary process in a new area. For these reasons and considering the current chemoprophylaxis recommendations and increasing use of ML endectoparasiticides as a potential selection pressure, it is important to remain vigilant for timely detection of any ML LOE/resistance, in all continents where D. immitis is enzootic.
... The detection of bona fide ML resistance in D. immitis infected animals is a major clinical challenge. The only patient-side test that can predict drug resistance is the microfilaria suppression test which involves measuring the number of microfilariae in blood before and after ML treatment [4,5]. Laboratory-based larval migration inhibition assays measuring the ability of L3 to migrate through a fine mesh in the presence of drugs have also been described [6,7]. ...
Prevention of canine heartworm disease caused by Dirofilaria immitis relies on chemoprophylaxis with macrocyclic lactone anthelmintics. Alarmingly, there are increased reports of D. immitis isolates with resistance to macrocyclic lactones and the ability to break through prophylaxis. Yet, there is not a well-established laboratory assay that can utilize biochemical phenotypes of microfilariae to predict drug resistance status. In this study we evaluated laboratory assays measuring cell permeability, metabolism, and P-glycoprotein-mediated efflux. Our assays revealed that trypan blue, propidium iodide staining, and resazurin metabolism could detect differences among D. immitis isolates but none of these approaches could accurately predict drug susceptibility status for all resistant isolates tested. P-glycoprotein assays suggested that the repertoire of P-gp expression is likely to vary among isolates, and investigation of pharmacological differences among different P-gp genes is warranted. Further research is needed to investigate and optimize laboratory assays for D. immitis microfilariae, and caution should be applied when adapting cell death assays to drug screening studies for nematode parasites.
... If the infections establish and become patent then resistance is confirmed (Bourguinat et al., 2015;Maclean et al., 2017;Pulaski et al., 2014). Alternatively, microfilarial suppression tests (MFST) can be used to confirm ML-resistance in field isolates (Geary et al., 2011;Moorhead et al., 2017). This test is based on the high-expected reductions in MF following the administration of a microfilaricidal dose of an ML drug (ivermectin or milbemycin oxime), with reduction in MF counts of < 75 % being considered indicative of resistance (Geary et al., 2011). ...
Prevention of infection with canine heartworm (Dirofilaria immitis) is based on the compliant administration of macrocyclic lactone (ML) drugs. Resistance to ML drugs is well documented in D. immitis; however, there remains a paucity of information on the spatial distribution and prevalence of resistant isolates. This project aims to improve understanding of ML-resistance by using a population genetic approach. We developed a large panel of microsatellite loci and identified 12 novel highly polymorphic markers. These 12, and five previously published markers were used to screen pools of microfilariae from 16 confirmed drug-susceptible, 25 confirmed drug-resistant, and from 10 suspected drug-resistant field isolates. In isolates where microfilarial suppression testing indicated resistance, Spatial Principal Component Analysis (sPCoA), Neighbor Joining Trees and Bayesian clustering all revealed high genetic similarity between pre- and post-treatment samples. Somewhat surprisingly, the Neighbor Joining tree and sPCoA generated using pairwise Nei’s distances did not reveal clustering for resistant isolates, nor did it reveal state-level geographic clustering from samples collected in Georgia, Louisiana or Mississippi. In contrast, Discriminant Analysis of Principle Components was able to discriminate between susceptible, suspected-resistant and resistant samples. However, no resistance-associated markers were detected, and this clustering was driven by the combined effects of multiple alleles across multiple loci. Additionally, we measured unexpectedly large genetic distances between different passages of laboratory strains that originated from the same source infection. This finding strongly suggests that the genetic makeup of laboratory isolates can change substantially with each passage, likely due to genetic bottlenecking. Taken together, these data suggest greater than expected genetic variability in the resistant isolates, and in D. immitis overall. Our results also suggest that microsatellite genotyping lacks the sensitivity to detect a specific genetic signature for resistance. Future investigations using genomic analyses will be required to elucidate the genetic relationships of ML-resistant isolates.
... In vitro assays measuring ML effects on larval migration or microfilarial motility have not proven informative for this phenotype Maclean et al., 2017), supporting the hypothesis that the effects of IVM (and presumably other MLs) are caused by alteration of the host-parasite interface (Geary and Moreno, 2012). However, a surrogate in vivo assay measuring the effect of ML treatment on microfilarial burden in the peripheral circulation has been proposed as an alternative diagnostic test for ML resistance Moorhead et al., 2017) based on the presumption that the mechanism of resistance is conserved in all larval stages. ...
Macrocyclic lactone (ML) anthelmintics are the most important class of anthelmintics because of our high dependence on them for the control of nematode parasites and some ectoparasites in livestock, companion animals and in humans. However, resistance to MLs is of increasing concern. Resistance is commonplace throughout the world in nematode parasites of small ruminants and is of increasing concern in horses, cattle, dogs and other animals. It is suspected in Onchocerca volvulus in humans. In most animals, resistance first arose to the avermectins, such as ivermectin (IVM), and subsequently to moxidectin (MOX). Usually when parasite populations are ML-resistant, MOX is more effective than avermectins. MOX may have higher intrinsic potency against some parasites, especially filarial nematodes, than the avermectins. However, it clearly has a significantly different pharmacokinetic profile. It is highly distributed to lipid tissues, less likely to be removed by ABC efflux transporters, is poorly metabolized and has a long half-life. This results in effective concentrations persisting for longer in target hosts. It also has a high safety index. Limited data suggest that anthelmintic resistance may be overcome, at least temporarily, if a high concentration can be maintained at the site of the parasites for a prolonged period of time. Because of the properties of MOX, there are reasonable prospects that strains of parasites that are resistant to avermectins at currently recommended doses will be controlled by MOX if it can be administered at sufficiently high doses and in formulations that enhance its persistence in the host. This review examines the properties of MOX that support this contention and compares them with the properties of other MLs. The case for using MOX to better control ML-resistant parasites is summarised and some outstanding research questions are presented.
В статті представлено результати наукового пошуку щодо епізоотологічних особливостей, діагностики та ефективністі лікування м'ясоїдних, хворих на дирофіляріоз. Дирофіляріоз – інфекційне паразитарне захворювання, що характеризується проблемами серця, печінки та нирок. Хвороба вражає собак, котів, диких тварин, а іноді і людей. Збудником цього захворювання є нематоди Dirofilaria immitis, Dirofilaria repens та інші. Вони мають прозоре тіло, вкрите прозорою мембраною. Великі гельмінти довжиною 25-30 см, живі, личинки (мікрофілярії) довжиною 0,22-0,29 мм, діаметром дорівнює розміру еритроцита. Dirofilaria immitis вражає правий шлуночок серця, легеневу артерію, викликаючи важкі напади легеневої вени, правого шлуночка та правого передсердя. Дірофілярії зазвичай знаходяться в незвичних для паразитів місцях: в очах, мозку, шлунку, підшкірному просторі, хребті. Личинки потрапляють у кров господаря через хоботок комахи та розвиваються в організмі тварини. Тривалість життя личинки триває близько 7-8 місяців. Один дирофілярій виробляє 30 000 личинок на день. Мікрофілярії циркулюють у крові вагітних протягом трьох років. Максимальна кількість мікродирофілярів у периферичній крові тварин вранці та ввечері, що відповідає двом пікам у центральній нервовій системі. Пряма мікроскопія краплі свіжої відібраної крові від м’ясоїдних тварин при малому збільшенні є найпростішим, зручним і швидким методом діагностики дирофіляріозу. Також рекомендуємо застосовувати метод дослідження за Кноттом, за Куликовим. Також необхідно проводити розтин грудної порожнини і оглядати перикард та інші тканини на наявність гельмінтів. Профілактика зараження тварин дирофіляріями ґрунтується в першу чергу на перериванні трансмісивної передачі інвазії та складається з декількох напрямків: винищення комарів, виявлення та дегельмінтизація інвазованих домашніх собак, запобігання контакту комарів з домашніми тваринами. Необхідна щомісячна обробка тварин проти комах, доброякісна годівля, своєчасна вакцинація та дегельмінтизація тварин Препарати «Івермектин», «Мільбеміцин» та «Діронет 500» мають пролонговану дію. Застосування даних препаратів з інтервалом 30-35 днів згубно діє на мікрофілярії, що дозволяє не допускати зараження інших м’ясоїдних і запобігає поширенню дирофіляріозу.
Filarial worms are a unique group of parasites with importance in both human and veterinary medicine. These parasites are typically long‐lived and difficult to detect, often causing chronic disease states over a period of years and, for these reasons, effective diagnostic testing is crucial for their control. Adult filarial worms tend to occupy inaccessible anatomical sites within the host, but microfilariae disperse widely in the blood or skin to allow uptake and transmission by the hematophagous insects necessary to complete the life cycle, and the detection of this microscopic stage represents a fundamental form of diagnostic testing. Immunodiagnostic and DNA‐based tests have since been developed for several filarial species, as well as methods for visualizing adult parasites in situ . All these techniques carry their own distinct strengths and weaknesses, so reliable diagnosis often requires a strategic combination of tests. Accurate diagnosis is important for potentially fatal infections like canine heartworm and is also essential for identifying emergent zoonoses, like Onchocerca lupi , and potential animal reservoirs, as with Brugia malayi . Accurate parasite detection and identification is useful not only in clinical settings but also greatly assists research efforts. This chapter will review the diagnostic methods available for some of the most common species of filarial nematodes in small animal veterinary medicine.
Anthelmintic resistance is a significant threat to livestock production systems worldwide and is emerging as an important issue in companion animal parasite management. It is also an emerging concern for the control of human soil-transmitted helminths and filaria. An important aspect of managing anthelmintic resistance is the ability to utilise diagnostic tests to detect its emergence at an early stage. In host-parasite systems where resistance is already widespread, diagnostics have a potentially important role in determining those drugs that remain the most effective. The development of molecular diagnostics for anthelmintic resistance is one focus of the Consortium for Anthelmintic Resistance and Susceptibility (CARS) group. The present paper reflects discussions of this issue that occurred at the most recent meeting of the group in Wisconsin, USA, in July 2019. We compare molecular resistance diagnostics with in vivo and in vitro phenotypic methods, and highlight the advantages and disadvantages of each. We assess whether our knowledge on the identity of molecular markers for resistance towards the different drug classes is sufficient to provide some expectation that molecular tests for field use may be available in the short-to-medium term. We describe some practical aspects of such tests and how our current capabilities compare to the requirements of an ‘ideal’ test. Finally, we describe examples of drug class/parasite species interactions that provide the best opportunity for commercial use of molecular tests in the near future. We argue that while such prototype tests may not satisfy the requirements of an ‘ideal’ test, their potential to provide significant advances over currently-used phenotypic methods warrants their development as field diagnostics.
