Selection of new diagnostic markers for Dirofilaria repens infections with the use of phage display technology.

Dirofilaria repens is a parasitic nematode causing vector-borne disease (dirofilariasis), considered an emerging problem in veterinary and human medicine. Although main hosts are carnivores, particularly dogs, D. repens shows high zoonotic potential. The disease spreads uncontrollably, affecting new areas. Since there is no vaccine against dirofilariasis, the only way to limit disease transmission is an early diagnosis. Currently, diagnosis depends on the detection of microfilariae in the host bloodstream using modified Knott's test or multiplex PCR. However, the efficacy of tests relying on microfilariae detection is limited by microfilariae periodic occurrence. Therefore, a new reliable diagnostic test is required. Our study aimed to select new diagnostic markers for dirofilariasis with potential application in diagnostics. We focused on single epitopes to ensure high specificity of diagnosis and avoid cross-reactivity with the other parasite infections common in dogs. Using phage display technology and 12-mer peptides library, we selected epitopes highly reactive with IgG from sera of infected dogs. Additionally, our study presents the possibility of detecting D. repens specific cell-free DNA in dogs with no microfilaria but high IgG and IgM antibody levels against parasite somatic antigen.

Concurrent subcutaneous and ocular infections with Dirofilaria repens in a Polish patient: a case report in the light of epidemiological data.

Dirofilariasis is an emerging zoonosis caused by nematodes of the genus Dirofilaria, most often D. repens and D. immitis. The main final hosts and reservoirs of pathogens are dogs. The intermediate hosts and vectors of infection are female mosquitoes (Culicidae). Human is an accidental host in which the parasite does not usually mature. Over the past 20 years, the range of Dirofilaria spp. in Europe has expanded. We present an unusual case of multifocal dirofilariasis of mixed subcutaneous-ocular course caused by D. repens in a 52-year-old Polish patient who was probably infected in Spain or Croatia, where she stayed one year before the onset of symptoms. Surgical removal of the nematodes followed by treatment with Ivermectin in a single dose of 1200 µg and Doxycycline 200 mg daily for 7 days resulted in complete recovery. We believe that all cases of human dirofilariasis, especially in countries where the disease is not frequent at present, should be registered for epidemiological purposes. Moreover, due to the widening of the range of D. repens and D. immitis occurrence and the possibility of atypical courses of infection with both nematodes, diagnostics should include the species identification of the parasite.

Dirofilaria immitis infection in the Pelagie archipelago: The southernmost hyperendemic focus in Europe.

Dirofilaria immitis and Dirofilaria repens are mosquito-borne filarioids of zoonotic concern, which primarily cause canine heartworm disease (HWD) and subcutaneous dirofilariosis, respectively. The geographical distribution of these nematodes is constantly changing, due to many factors such as the colonization by new invasive mosquito species, the increased movement of animals and the absence of chemoprophylactic strategies in non-endemic regions. Although HWD has always been considered endemic in northern Italy, an increase in the prevalence of this disease has been recorded in the last decades in central and southern regions. We describe the southernmost hyperendemic European focus of heartworm disease in the Pelagie archipelagos. From June to November 2020, 157 dogs and 46 cats were sampled in Linosa and Lampedusa islands for the detection of Dirofilaria immitis and Dirofilaria repens by modified Knott's, rapid antigen and molecular tests. Mosquitoes were collected using CDC light and BG sentinel-2 traps and aspirators, and tested for host blood meal and Dirofilaria spp. DNA. Out of 56 dogs in Linosa and 101 in Lampedusa, 33 (i.e. 58.9%) and 8 (i.e. 7.9%) were positive to D. immitis and D. repens, respectively. Three cats scored positive to D. immitis (i.e. 17.6%) in Linosa. Six mosquito species were identified, and the abdomen of a non-engorged Aedes albopictus was positive for D. immitis and human DNA. The results suggest that D. immitis infection could spread to new previously non-endemic territories in southern Europe, representing a real threat to animal and human health.

Dirofilaria immitis and Dirofilaria repens in mosquitoes from Corsica Island, France.

BACKGROUND: Dirofilaria immitis and Dirofilaria repens are the main causative agents of heartworm disease and subcutaneous dirofilariasis in domestic and wild canids, respectively. Both pathogens have zoonotic potential and are transmitted by mosquitoes. The present study aimed to determine the transmission period, prevalence and diversity of Dirofilaria spp. vectors from endemic areas of Corsica (France). METHODS: A monthly point data model based on average temperature recorded by four meteorological stations during 2017 was used to calculate the Dirofilaria transmission period. From June to September 2017, female mosquitoes (n = 1802) were captured using Biogents® Sentinel 2 traps lured with carbon dioxide and BG-Lure™ or octanol. Mosquitoes were identified to species level, pooled accordingly, and screened using multiplex real-time qPCR to detect D. immitis and D. repens. RESULTS: The monthly point data model showed the possible transmission of Dirofilaria spp. from the third week in May to the last week in October in the studied area. Mosquitoes were identified as Ochlerotatus caspius (n = 1432), Aedes albopictus (n = 199), Culex pipiens sensu lato (n = 165) and Aedes vexans (n = 6) and were grouped into 109 pools (from 1 to 27 specimens, mean 11.4 ± 0.7), of which 16 scored positive for Dirofilaria spp. (i.e., n = 13; estimated infection rate [EIR] = 1.1% for D. immitis and n = 3; EIR = 0.2% for D. repens). Specifically, 6 (i.e., EIR = 3.8%) of 15 pools of Ae. albopictus were positive for D. immitis, 2 of 14 of Cx. pipiens s.l. were positive for D. immitis and D. repens, respectively, and 8 of 77 pools of Oc. caspius were positive for D. immitis (i.e., n = 6; EIR = 0.4%) and D. repens (i.e., 2; EIR = 0.1%). The highest mosquito infection rate was recorded in July (EIR = 2.5%), then in June (EIR = 1.3%) and September (EIR = 0.6%). CONCLUSIONS: The data suggest that both Dirofilaria species are endemic and occur possibly in sympatry in the studied area in Corsica, highlighting the need to implement preventive chemoprophylaxis and vector control strategies to reduce the risk of these filarioids in dog and human populations.

A rare clinical presentation of human Dirofilaria repens infection as a pseudo-tumour of the epididymis - Case Report.

Introduction. Epididymal dirofilariasis is one of the unusual manifestations of this zoonosis. In Slovakia, this is the third case out of 20 Dirofilaria repens infected patients in whom the worm (the parasite) was identified in the epididymis. The patient felt a painless tumour about 2 cm in size on the left testicle. During ultrasound examination, the radiologist observed a cyst containing a live worm within the epididymis. After mechanical stimulation, the movement characteristic for filarial worms (´filarial dance sign´) appeared. An orchiectomy was performed at the Department of Urology in the University Hospital in Kosice. Histopathology confirmed a parasitic cyst with a worm, and based on characteristic morphological features, the parasite was identified as Dirofilaria repens. Objective. The aim of case report is to alert physicians to the possibility dirofilarial infection of the epididymis, where tumors and cystic structures often occur and to present clinical signs of the disease.

Case Report: Molecular Detection of Dirofilaria repens in an Italian Patient after a Stay in Tanzania.

A 35-year-old man was admitted to a hospital in the south of Italy because of a periocular nodule and subpalpebral edema. The patient reported having been stayed in Tanzania five months before. Hematologic parameters were within the normality range, the Acanthocheilonema viteae ELISA did not detect significant levels of antifilarial IgG, and no further symptoms were described. The surgical inspection of the nodule led to the isolation of two filarioid parasites, identified as Dirofilaria repens by scanning electron microscope (SEM), and then by molecular assays. Knott's test did not reveal microfilaremia, whereas loop-mediated isothermal amplification and PCR detected D. repens DNA. The patient was treated with doxycycline, and he was found no more positive at the follow-up.

Human dirofilariosis in Austria: the past, the present, the future.

BACKGROUND: Dirofilariosis is a vector-borne parasitosis caused by filarial nematodes of the genus Dirofilaria. In humans, who represent accidental hosts, dirofilariosis is mostly caused by Dirofilaria repens and Dirofilaria immitis. In Austria, the first reported case occurred in 1978. Since then, several (case) reports have been published. METHODS: A systematic and retrospective review of collected published cases and new, unpublished confirmed cases of human dirofilariosis occurring in Austria was performed. A nematode was extracted from the eyelid of a previously unreported case and subsequently characterized histologically and using molecular biology techniques. RESULTS: Data on a total of 39 cases of human dirofilariosis in Austria occurring between 1978 and 2020 are summarized. Over the past four decades the incidence has markedly increased, in particular after 1998. Of the 39 patients, men and women were equally affected, and the mean age was 47.1 years. The area most frequently affected was the head (38.5% of cases). Confined ocular involvement was observed in 23.1% of cases, and nematodes were isolated from the neck/trunk, extremities and the genito-inguinal area in 25.6, 15.4 and 15.4% of patients, respectively. Microfilariae were detected in two cases. Of the 39 patients, only 73.9% tested positive for anti-filarial antibodies and 56.3% for eosinophilia, despite successful isolation of a nematode; consequently, these measures did not represent reliable markers for dirofilariosis. Most patients had a travel history to countries endemic for Dirofilaria species. One patient who had not traveled abroad represented the only autochthonous case recorded to date. Dirofilaria repens was the predominant species, identified in 89.7% of cases. In the newly reported case of subcutaneous dirofilariosis, a live non-gravid Dirofilaria repens adult female of 12 cm length was isolated from the eyelid of the patient, and a video of the extraction is provided. CONCLUSIONS: The incidence of human dirofilariosis cases has increased strikingly over the last four decades in Austria. More cases can be expected in the foreseeable future due to changes in human behavior and (travel) activities as well as climate changes and the associated alterations in the availability of the natural reservoir, the vectors and the intrinsic characteristics of the parasite.

A case of inguinal hernia associated with atypical Dirofilaria repens infection in a dog.

BACKGROUND: Dirofilaria repens is a filarioid nematode transmitted by mosquitoes. Adult D. repens are typically localized in the subcutaneous tissue of the host, but other, atypical localizations have also been reported. There have been several reports of clinical cases involving an association of parasites and hernias in both animals and humans. However, it is unclear if parasitic infection can act as a triggering factor in the development of hernias. METHODS: A 12-year-old dog was referred to a private veterinarian clinic in Satu Mare, northwestern Romania due to the presence of a swelling in the lateral side of the penis (inguinal area). The dog underwent hernia repair surgery during which four long nematodes were detected in the peritoneal serosa of the inguinal hernial sac. One female specimen was subjected to genomic DNA extraction to confirm species identification, based on amplification and sequencing of a 670-bp fragment of the cytochrome c oxidase subunit 1 (cox1) gene. Treatment with a single dose of imidacloprid 10% + moxidectin 2.5% (Advocate, Bayer AG) was administered. RESULTS: The nematodes were morphologically identified as adult D. repens, and the BLAST analyses revealed a 100% nucleotide similarity to a D. repens sequence isolated from a human case in Czech Republic. CONCLUSIONS: We report a case of an atypical localization of D. repens in the peritoneal cavity of a naturally infected pet dog with inguinal hernia and discuss the associations between hernia and parasitic infections.

The first case of periorbital human dirofilariasis in the Czech Republic.

Dirofilaria repens and Dirofilaria immitis are the most common filarial species affecting humans in Europe. Dirofilaria repens causes subcutaneous or ocular infection, whereas D. immitis is responsible mainly for the pulmonary form. In this report, we present the first human case of periorbital dirofilariasis in the Czech Republic. A 58-year-old woman suffered from an eyelid oedema, redness and pain in the left eye. After excising the parasite from her eyelid, all clinical symptoms disappeared. Based on the morphology and cytochrome oxidase I sequencing, the parasite was identified as D. repens. Histology revealed that the excised worm was female with absent microfilariae in uteri. With respect to the length of the incubation period and the sequence identity with a known Czech isolate, we concluded that D. repens was most likely of autochthonous origin.

Different epidemiological pattern of canine dirofilariosis in two neighboring countries in Central Europe-the Czech Republic and Slovakia.

The known data resulting from individual surveys of canine dirofilariosis point to the great differences in the epidemiological situation among countries where Dirofilaria parasites emerged approximately at the same time. In this regard, the Czech Republic and Slovakia, neighboring countries situated in Central Europe, could serve as an illustrative example of such a situation. The present study aimed to assess the prevalence of canine dirofilariosis in both countries and to discuss the reasons for potential differences shown. Between October and December 2019, 429 dogs from the Czech Republic and 644 from Slovakia were examined for canine dirofilariosis using the Knott test for microfilariae detection and conventional PCR for the species determination. The results' analyses showed notable differences. While in the Czech Republic autochthonous Dirofilaria repens cases are reported sporadically and Dirofilaria immitis infections have been confirmed only as imported so far, in Slovakia, both Dirofilaria species seem to have become endemic. Concretely, in the Czech Republic, microfilariae were detected in the peripheral blood of 8 dogs (1.86%): in seven, D. repens was confirmed, and in one dog, mixed infection with D. repens and D. immitis was diagnosed. Seven infected animals came from the eastern part of the country neighboring Slovakia. In Slovakia, microfilariae were detected in 68 (10.56%) dogs examined. DNA analysis confirmed D. repens mono-infection in 38 (5.90%) dogs, single D. immitis infection in 21 (3.26%) animals, and both Dirofilaria species were detected in 9 (1.40%) samples. Although we are unable to determine the cause of the differences, our study confirmed that the long-registered low number of canine dirofilariosis cases in the Czech Republic is not due to insufficient investigation (monitoring), but due to a low prevalence of the parasite in this area.

Extensive Chorio-retinal Damage Due to Dirofilaria Repens- Report of a Case.

Purpose: To report a case of ocular dirofilariasis causing extensive chorio-retinal damage.Method: Surgical removal of the worm and identification of the same by microscopic examinationand polymerase chain reaction.Results: The worm was identified as Dirofilaria repens.Conclusion: Ocular dirofilariasis is an arthropod borne parasitic infestation affecting the subconjunctival tissue and anterior chamber. Vitreous involvement in Dirofilaria is very rare. Dirofilaria repens can cause extensive chorio-retinal damage resembling diffuse unilateralsubacute neuroretinitis.

The first case of autochthonous subcutaneous dirofilariasis (Dirofilaria repens) in a dog from Bialowieza (NE Poland) and possible threat posed to inhabitants of Bialowieza Primeval Forest area.

We present the first case of dirofilariasis in a dog from Bialowieza village located in the primeval European forest-Bialowieza Primeval Forest (NE Poland). Molecular analysis of adult nematode specimens isolated from subcutaneous tissue confirmed the infection with Dirofilaria repens. An adult male dog has not travelled out of the Bialowieza village for at least five years; therefore, we assume this is the autochthonous case of the disease. We discuss possible inter- and intra-species transmission routes of dirofilariasis on this territory, which is inhabited by diverse community of wild carnivores, domestic animals, and humans. We also discuss the likely sources of the disease in this, highly biodiverse unique European forest complex. We underline the lack of attention to this problem and its importance for veterinary, wildlife, and human health safety.

Detection and cross-reaction of Dirofilaria repens using a commercial heartworm antigen test kit.

Antigen enzyme-linked immunosorbent assay tests are widely used for the diagnosis of heartworm infection in dogs. While commercially-available heartworm antigen tests have high sensitivity and specificity, false-negative test results can occur in dogs with low worm burdens, female-only infections, or prior to patency. The use of immune complex dissociation (ICD) methods have demonstrated increased sensitivity in the detection of Dirofilaria immitis antigens and the reversal of false-negative antigen results. However, there are concerns pertaining to false-positive antigen results due to infections of other nematode parasites, especially post-ICD. Therefore, this study evaluated the effect of heat-treatment on serum samples of dogs experimentally-infected with Dirofilaria repens during the course of infection, to assess for potential cross-reactivity on heartworm antigen tests. Archived serum samples from three dogs experimentally-infected with D. repens were utilized. All samples were tested for cross-reactivity pre- and post-heat-treatment using the DiroCHEK® Heartworm Antigen test kit throughout infection (day -9 through 404 days post-infection; dpi). All heat-treated samples tested false-positive starting at 164 dpi and continuing through 404 dpi, thereby testing positive prior to patency. No cross-reactivity was observed for any dog at any time point prior to heat-treatment. Our results suggest that the ICD method decreased the specificity of heartworm antigen tests and caused cross-reactivity of serum from dogs experimentally infected with D. repens. In conclusion, heat-treatment of serum in areas co-endemic for D. repens and D. immitis has limited clinical value, and should be used with caution.

Potential Mosquito Vectors of Dirofilaria immitis and Dirofilaira repens (Spirurida: Onchocercidae) in Aras Valley, Turkey.

Dirofilaria immitis (Leidy, 1856) and Dirofilaria repens (Railliet & Henry, 1911) are mosquito-borne filarial nematodes that primarily affect dogs, causing heartworm disease and subcutaneous dirofilariosis. The canine heartworm is reported in different provinces in Turkey. However, studies about the transmitting mosquito species are limited. Hence, this study aimed to investigate potential vectors of D. immitis and D. repens in Aras Valley, Turkey. In total, 17,995 female mosquitoes were collected from eight villages during three mosquito seasons (2012-2014) in Aras Valley, located in north-eastern Turkey. A total of 1,054 DNA pools (527 abdomen and 527 head-thorax) were tested with Dirofilaria primers by multiplex-polymerase chain reaction (PCR). Aedes caspius was the most abundant species in collection sites with 90%; this was followed by Culex theileri Theobald, 1903 (Diptera: Culicidae) (7.31%), Anopheles maculipennis Meigen 1818 (Diptera: Culicidae) (1.28%), Culex pipiens Linnaeus, 1758 (Diptera: Culicidae) (0.43%), (Anopheles) hyrcanus (Pallas, 1771) (Diptera: Culicidae) (0.37%), Aedes vexans (Meigen, 1830) (Diptera: Culicidae) (0.25%), and Culiseta annulata Schrank, 1776 (Diptera:Culicidae) (0.02%). Dirofilaria immitis and D. repens were detected in mosquito pools from five villages. The total Dirofilaria spp. estimated infection rate was 1.33%. The highest estimated infection rate was found in Ae. vexans (6.66%) and the lowest was in Ae. caspius (1.26%). The results show that An. maculipennis sl, Ae. caspius, Ae. vexans, Cx. theileri and Cx. pipiens are potential vectors of D. immitis and D. repens with DNA in head-thorax pools; An. hyrcanus is also a likely vector, but Dirofilaria DNA was found only in abdomen pools for the study area. This study revealed new potential vector species for D. immitis. Mosquitoes with natural infections of D. repens were reported for the first time in Turkey.

Usual or unusual presentations of Dirofilaria repens in two sibling dogs: a case report.

This study describes two different manifestations of Dirofilaria repens infection in sibling dogs with microfilaremia. Dog 1, asymptomatic, harbored a gravid female of D. repens on the parietal layer of tunica vaginalis of one testicle and showed a marked circulating eosinophilia (3.3·103/µL). Both testicles were normal in shape and size without any gross lesions. Dog 2 had a pyotraumatic dermatitis. The cases were confirmed by PCR and sequencing. The sequences obtained showed 100% identity with those of D. repens isolated from human scrotum in Croatia. The treatment with moxidectin 2.5% and imidacloprid 10%/kg was effective in eliminating microfilariae after just one application, as demonstrated by negative modified Knott's tests and PCR analyses of blood samples. This status was maintained during the post-treatment observation period. The classical localization of D. repens in dogs is in subcutaneous tissues, within nodules or free; however, it can also occur with some frequency in testicles, as described in humans. The infection can be associated with circulating eosinophilia or pyotraumatic dermatitis, as reported in this study. Thus, in endemic areas, it is advisable to carefully inspect the removed testicles at neutering since parasite localization can take place without any macroscopic changes. Moreover, in the case of circulating eosinophilia or pyotraumatic dermatitis, investigations should include modified Knott's test and PCR to ensure that D. repens is not the cause of these alterations. Rapid and sensitive tests for the early detection of infected animals would help to prevent or limit the spread of this zoonosis.

The Zoonotic Parasite Dirofilaria repens Emerged in the Baltic Countries Estonia, Latvia, and Lithuania in 2008-2012 and Became Established and Endemic in a Decade.

The zoonotic parasite Dirofilaria repens has spread toward north in Europe, and cases of autochthonous dirofilariosis caused by D. repens have emerged in the Baltic countries Estonia, Latvia, and Lithuania. We conducted a review on the emergence of dirofilariosis in humans and domestic dogs in these three countries in northeastern Europe. Based on the available literature and reports, the first finding in the Baltic countries was made in Latvia in 2008, followed by the first in Lithuania in 2010, and the first in Estonia in 2012. In all three countries, further findings were reported soon after the first reports. By the end of 2019, autochthonous human D. repens infections had been described from Latvia and Lithuania, and autochthonous canine D. repens infections had been described from all three Baltic countries. While no epidemiological studies estimating prevalence or incidence of the human infections have been published from the three countries, a substantial proportion of investigated dogs have tested positive for microfilariae in studies performed in Latvia and Lithuania. Dirofilariosis is an emerging zoonosis in northern Europe, and the summarized data confirm that D. repens has become established and endemic in the Baltic countries. The available data do not provide a good overview of the situation, and further epidemiological studies are needed. Awareness about the recently emerged zoonotic parasite should be increased among medical doctors, veterinarians, and the general public. Managing this zoonotic infection is a public health challenge that needs to be addressed using a One Health approach. Investigating the spread of D. repens in the Baltic countries could be useful for better preparedness for the anticipated further spread to the Nordic countries.

First report of Dirofilaria repens infection in a microfilaraemic cat from Romania.

The present study describes the first report of Dirofilaria repens infection with the presence of both microfilariae and adult nematodes in a cat from Northeastern Romania. Briefly, a 5-year-old male mixed breed cat was presented to a veterinary clinic in Iasi (Romania), for neutering, in early February 2020. During the surgery, two whitish worms were removed from the internal part of the scrotum. Two adult nematodes, one female and one male, were identified, on the basis of morphological features, as D. repens with whitish, cylindrical bodies, measuring 12.5 cm and 6.5 cm in length, respectively. At histology, the female nematode showed two cavitated structures containing myriads of variably arranged microfilariae. The male had a transversal diameter of 350 µm, a 10 µm thick cuticle and a ridge-period of 10 Μm. multiplex PCR confirmed the diagnosis of D. repens from both nematodes. The Knott's test revealed the presence of microfilariae of D. repens. Routine biochemistry panel was within range with one exception, urea serum level slightly increased. The haematology results revealed an increased number of neutrophils, lymphocytes and eosinophils. The cat had an infection with Otodectes cynotis as well. The cat was discharged with the following therapy recommended: oral doxycycline (10 mg/kg) for 30 days and topical moxidectin, monthly doses. After six months, the Knott's test gave negative results. Further studies should include new insights of D. repens infection in cats concerning its epidemiology, diagnosis and control.

A case of human Dirofilaria repens infection, causing an asymptomatic subcutaneous nodule.

We present a case of subcutaneous dirofilariasis, a vector-borne zoonotic disease, in a young woman from Austria. The diagnosis was confirmed by ultrasound and histology of the excised subcutaneous nodule. The parasite species was identified as Dirofilaria repens by polymerase chain reaction. We expect to see more cases of human dirofilariasis also due to climate change and associated increase of the spectrum of suitable mosquito vectors.