Integrated Histological and Molecular Analysis of Filarial Species and Associated Wolbachia Endosymbionts in Human Filariasis Cases Presenting Atypically in Thailand.

Atypical presentations of filariasis have posed diagnostic challenges due to the complexity of identifying the causative species and the difficulties in both diagnosis and treatment. In this study, we present the integrative histological and molecular analysis of seven atypical filariasis cases observed in regions of nonendemicity of Thailand. All filariasis cases were initially diagnosed based on histological findings. To confirm the causative species, molecular characterization based on both filarial mitochondrial (mt 12S rRNA and COI genes) and nuclear ITS1 markers was performed, together with the identification of associated Wolbachia bacterial endosymbionts. Among the cases studied, Brugia pahangi (N = 3), Brugia malayi (N = 1), Dirofilaria sp. "hongkongensis" (N = 2), and a suspected novel filarial species genetically related to Pelecitus copsychi (N = 1) were identified. By targeting the 16S rRNA gene, Wolbachia was also molecularly amplified in two cases of infection with Dirofilaria sp. "hongkongensis." Phylogenetic analysis further revealed that the detected Wolbachia could be classified into supergroups C and F, indicating the high genetic diversity of this endosymbiont in Dirofilaria sp. "hongkongensis." Furthermore, this study demonstrates the consistency between histological findings and species identification based on mitochondrial loci rather than on the nuclear ITS1. This suggests the utility of mitochondrial markers, particularly COI, as a highly sensitive and reliable diagnostic tool for the detection and differentiation of filarial species in clinical specimens. Precise identification of the causative species will facilitate accurate diagnosis and treatment and is also essential for the development of epidemiological and preventive strategies for filariasis.

Subconjunctival Dirofilaria masquerading as nodular scleritis.

A man in his late 50s presented with a gradually enlarging, painless, reddish mass on the white portion of his left eye for 2 weeks. His best-corrected visual acuity was 20/20 in both eyes. Slit-lamp examination showed a congested, nodular, elevated lesion on the temporal bulbar conjunctiva with two pustule-like elevations. Anterior segment optical coherence tomography showed a subconjunctival solid mass rather than an abscess or a cyst. Scleral deroofing was performed and a long thread-like object resembling a dead worm was identified. The worm was removed intact, and its histopathology confirmed the diagnosis of Dirofilaria Peripheral blood smear did not show any microfilariae. No recurrences or new lesions were observed during the follow-up examinations at 1 and 5 months post-surgery. This case highlights the importance of considering a parasitic aetiology in cases of nodular or infectious scleritis.

Ocular Dirofilariasis in Migrant from Sri Lanka, Australia.

We describe a case of imported ocular dirofilariasis in Australia, linked to the Hong Kong genotype of Dirofilaria sp., in a migrant from Sri Lanka. Surgical extraction and mitochondrial sequences analyses confirmed this filarioid nematode as the causative agent and a Dirofilaria sp. not previously reported in Australia.

Ecological niche modeling analysis (Cx. pipiens), potential risk and projection of Dirofilaria spp. infection in Greece.

Vector-borne diseases continue to increase worldwide. Dirofilariosis is one of the most common vector-borne zoonotic diseases, mainly caused by Dirofilaria spp. (D. immitis and D. repens) and spread by culicid mosquitoes of different species. Greece is one of the countries in southern Europe where it is traditionally endemic, and its distribution is not homogeneous. The aim of this study was to develop an environmental model for Greece that reflects the suitability of the ecological niche for Dirofilaria spp. infection risk and its projection until 2080. For this purpose, we used the potential distribution of suitable habitats for Culex pipiens calculated using an ecological niche model (ENM) and the potential number of generations of Dirofilaria spp. The ecological niche model of Cx. pipiens in Greece showed good predictive power (AUC=0.897) with the parasite at a resolution of 1 km2. The variables that contributed most to the model were mean annual temperature, rivers and human footprint. The highest risk of infection was found in coastal areas and in riverside areas of the main river basins, as well as in irrigated areas of the mainland and peninsular regions and in the whole territory of island areas, and the lowest risk was found in areas of higher altitude. A positive relationship was found between the risk of dirofilariosis and the location of infected dogs, with 86.65% located in very high and high risk areas. In 2080, the percentage of territory gained by Cx. pipiens will increase by 261.52%. This model provides a high predictive value, predicted presence, and risk of Dirofilaria spp. infection and can serve as a tool for the management and control of this disease.

Human dirofilariasis - Unforeseen lesion in subcutaneous nodules: Case series from a tertiary care hospital, Wayanad.

Human dirofilariasis is a type of zoonotic infection caused by the Dirofilarial species. It is a type of roundworm which is commonly seen in dogs, cats and is transmitted to humans by mosquito bite. Microfilaria is rarely produced in humans. Although clinically they can have varied presentations, it usually presents as ocular or subcutaneous nodules. Definitive diagnosis requires histopathological findings of this worm. We hereby report three cases of Dirofilariasis from a tertiary care hospital in Wayanad.

Genetic diversity and characterization of Wolbachia endosymbiont in canine filariasis.

Canine filariasis is caused by nematodes from the family Onchocercidae, which is transmitted by arthropod vectors. The disease is commonly found in Southeast Asia and exists worldwide. Some filarial nematodes are associated with intracellular bacteria of the genus Wolbachia, which plays an important role in embryogenesis, molting, and the long-term survival of adult worms. This study aims to characterize Wolbachia sp. and determine the association between Wolbachia and canine filarial nematode species in Thailand. A total of 46 dog blood samples that were naturally infected with filarial nematodes were obtained to identify filarial nematode species by Giemsa stained under a light microscope and confirmed using the molecular technique. In order to characterize Wolbachia sp., the nested PCR assay targeting the 16S rRNA gene showed that all samples of Dirofilaria immitis and fifteen samples of Candidatus Dirofilaria hongkongensis were grouped into Wolbachia supergroup C. In addition, all samples of Brugia spp. and five samples of Candidatus Dirofilaria hongkongensis were classified into Wolbachia supergroup D. The genetic diversity analysis conducted using the 16S rRNA gene revealed a similar result when analyzed through phylogenetic tree analysis. This is the first genetic diversity study of Wolbachia of Candidatus Dirofilaria hongkongensis in infected dogs in Thailand.

A rare subcutaneous periorbital zoonotic infection in a 42-year-old female patient.

Human dirofilariasis represents a zoonotic infectious disorder caused by parasites belonging to the Dirofilaria genus, which includes numerous species with a large variability regarding the host specificity, life cycle, and clinical manifestations. This disease appears to be a vector-borne parasitosis that is spread through insects - intermediate hosts (usually mosquitos) - and affects several carnivores as definitive hosts. Humans represent an unsuitable host for the parasite to complete its life cycle, being unable to release microfilariae in the blood as the inoculated larvae fail to reach sexual maturity. Therefore, humans are unable to transmit the infection to other humans, neither directly nor through an intermediate host. The current case report indicates a rare encounter of a Dirofilaria repens infection in a 42-year-old female patient living in an urban area (Craiova, Romania), who developed intermittent right periorbital edema after a previous trip to Greece. Over the course of one month, the right periorbital edema gradually remitted, and a firm, round lump developed in the external inferior right periorbital region. There were no similar clinical manifestations among other family members. Her medical history showed no other complaints or any serious general health problems. Also, the patient denied having any pets at home. The blood tests did not reveal any abnormalities. The exact source of infection could not be identified, but the chances for this infection to be related to the history of traveling to Greece are high, given that this region is one of the most important endemic areas in Europe.

Genetic diversity of vector-borne pathogens in ixodid ticks infesting dogs from Pakistan with notes on Ehrlichia canis, Rickettsia raoultii and Dirofilaria immitis detection.

BACKGROUND: Vector-/tick-borne pathogens (V/TBPs) pose a potential threat to human and animal health globally. Information regarding canine V/TBPs is scarce and no specific study has been conducted so far to explore the microbial diversity within ticks infesting dogs from Pakistan. Herein, this knowledge gap is addressed by assessing the genetic diversity and prevalence pattern of V/TBPs in ixodid ticks with special implications for public and canine health. METHODS: A total of 1150 hard ticks were collected from 300 dogs across central Khyber Pakhtunkhwa (KP), Pakistan. After morpho-molecular identification, 120 tick samples were screened for the presence of V/TBPs by amplifying 16S rRNA/gltA (Rickettsia/Ehrlichia and Wolbachia sp.), 18S rRNA (Theileria sp.) and cox1 (Dirofilaria sp.) genes through PCR followed by sequencing and phylogenetic study. RESULTS: In toto, 50 ixodid ticks (50/120, 41.7%) were found positive for V/TBPs DNA. The detected V/TBPs were categorized into five genera and eight species, viz. Ehrlichia (E. canis and Ehrlichia sp.), Rickettsia (R. massiliae, R. raoultii and Rickettsia sp.), Theileria (T. annulata), Dirofilaria (D. immitis) and Wolbachia (Wolbachia sp.). The pathogen prevalence patterns showed that R. massiliae was the most prevalent zoonotic V/TBP (19.5%), followed by E. canis (10.8%), Rickettsia sp. (7.5%), R. raoultii (6.7%), T. annulata (5.8%), D. immitis (5.8%), Wolbachia sp. (4.2%) and Ehrlichia sp. (3.3%), respectively. Among the screened tick species, most Rhipicephalus sanguineus sensu lato samples were found positive for V/TBP DNA (20/20,100%) followed by Rh. turanicus sensu stricto (13/20, 65%), Hyalomma dromedarii (8/20, 40%), Rh. haemaphysaloides (6/20, 30%), Hy. excavatum (2/20, 10%) and Rh. microplus (1/20, 5%). Co-occurrence of V/TBP was also detected in tick specimens (single V/TBP infection: 32 ticks; double and triple: 13 and 5 tick samples). The detected pathogens shared a phylogenetic relationship with similar isolates published in NCBI GenBank from Old and New World countries. CONCLUSION: Ixodid ticks infesting dogs harbor a diverse array of V/TBPs including zoonotic agents from Pakistan. Furthermore, the presence of D. immitis in ticks that infest dogs raises the possibility that this parasite has either attained its dead-end host (i.e. the tick) while feeding on dogs or has expanded its range of intermediate/paratenic hosts. Further research work is needed to investigate the epidemiology and confirm the vector competence of screened tick species for these pathogens from Pakistan.

An updated list of mosquito species in Armenia and Transcaucasian region responsible for Dirofilaria transmission: A review.

Canine dirofilariasis is a world-wide distributed mosquito-borne helminthiasis that has a potential zoonotic impact. This disease is a serious problem in Southern Caucasus region, including Armenia. Average extensity of Dirofilaria infection in canids in Armenia is 26.9%. At present, 77 mosquito species (Diptera: Culicidae) are presumed to have a role in the transmission of dirofilariasis. 80 species of Culicidae family mosquitoes are registered in Southern Caucasus Region, and 31 of them are described in Armenia. The following species of Culicidae can serve as vectors of Dirofilaria spp. in Armenia: Aedes vexans, Ae. caspius, Ae. albopictus, Ae. geniculatus, Culex pipiens, Cx. theileri, Cx. modestus, Anopheles maculipennis s.s., An. claviger, An. hyrcanus, An. superpictus, Coquillettidia richardi, Culiseta annulata and Uranotaenia unguiculta. We see a risk of transmission in active foci of dirofilariasis, as well as for human infection in Armenia. Comprehensive research should be conducted on detection of microfilariae in the target mosquito species and in target localities.

POPULATIONS AND SITE SELECTION OF DIROFILARIA URSI (NEMATODA: ONCHOCERCIDAE) IN AMERICAN BLACK BEARS (URSUS AMERICANUS).

A total of 4,846 (84.5±87.15, 3-429: mean±SD, range) subadult or adult Dirofilaria ursi were recovered from 56 American black bears (Ursus americanus) in Ontario, 1975-77. Yearling bears had fewer worms than older bears; age classes greater than 2.5 yr had similar numbers of worms. Dead worms comprised 3.7% of all worms. There was one dead worm in total in 10 yearling bears and consistently more dead worms in older bears. The occurrence of worms within local sites in bears differed over time. Increasing numbers of worms appeared first in peritracheal sites (Site 1) following 1-3 seasons of transmission, in abundance in perirenal sites (Site 2) following the second season of transmission, and in lateral and ventral trunk and abdomen plus medio-proximal legs (Site 3) following three and four seasons of transmission. Few worms occupied other sites (Site 4). The proportion of worms recovered from the four sites was 46, 30, 19.8, and 3.9%, respectively. Dead worms comprised only 3.2% of worms in the first three definitive sites but 14% of worms in other sites. These data are consistent with primary, secondary, and tertiary site preferences for mature D. ursi in this species. Circulating microfilaremia in peripheral blood was periodic in 7/10 experiments and revealed a pattern of variable numbers during the day, highest in the evening and lowest during the night. The time of high density of microfilariae in peripheral blood was synchronous with the feeding cycle of Simulium venustum, the vector of D. ursi. Immediately postmortem, microfilariae were at highest densities in blood of lungs regardless of their relative density in peripheral blood. These data can direct diagnostic efforts to maximize detection of adult D. ursi in tissues and larvae in blood samples of naturally infected bears.

Subconjunctival Dirofilariasis presenting as orbital cellulitis.

Background: Dirofilariasis is an emerging zoonosis in India. Most of the cases from India have been reported from the states of Kerala, coastal Karnataka and Maharashtra, and a few from the North India, Orissa and Assam. Dogs, cats, foxes and other wild animals are definitive hosts for dirofilaria. Human ocular parasitosis is prevalent in geographical areas where environmental factors and poor sanitary conditions favor parasitism between humans and animals. However, in recent years, migrating population have facilitated the spread of certain parasitic diseases from endemic to non-endemic areas. We report a case of subconjunctival dirofilariasis in a 91-year-old man, who presented with features of orbital cellulitis. An intact live worm, measuring 13.5 cm, was extracted from the subconjunctival space, following which there was prompt resolution of symptoms. Purpose: Given the increase in the frequency of dirofilariasis in humans in recent years, medical practitioners should bear in mind the possibility of ocular dirofilariasis when a patient presents with an ocular or orbital inflammatory lesion. Synopsis: This video illustrates various presentations of ocular dirofilaria and surgical extraction of a live worm from the subconjunctival space. Highlights: Lifecycle and human ocular manifestations of dirofilaria ared elucidated. Rare presentation of subconjunctival dirofilariasis as orbital cellulitis and its resolution following removal of the live worm has been demonstrated. Video link: https://youtu.be/Nj8unz0S85M.

Human dirofilariosis in the 21st century: A scoping review of clinical cases reported in the literature.

Human dirofilariosis is a clinical entity caused by infection with nematode species of the genus Dirofilaria. The traditional picture depicts the disease as a sporadic event associated with the presence of a single immature worm causing a nodular lesion. With the aim to reassess this paradigm, establishing a more accurate picture of the disease and homogenize criteria, a scoping review was conducted by searching, screening and analysing published clinical cases of human dirofilariosis worldwide during the 21st century. After extracting data from 305 publications containing 576 case reports, results showed that human dirofilariosis is currently caused by five Dirofilaria species (mainly D. repens). Maturation was not uncommon, since 42.95% of the parasites recovered were described as mature worms, most of them females, 26.42% of which contained micofilariae in the uterus. Moreover, six microfilaremic cases have been described. The predominant clinical manifestation was the presence of a worm encapsulated within a nodule, but there is a considerable variety of accompanying symptoms depending on anatomical location and type of dirofilariosis. Parasites/nodules were found in 71 different anatomical locations, being the traditional nomenclature of human dirofilariosis unable to properly cover this complex situation. Delay in seeking medical assistance (patient perception) and the frequency of wrong clinical suspicions (doctor knowledge), strongly influenced clinical management. The initial suspicion in cases of subcutaneous and pulmonary dirofilariosis is predominantly a tumour, while in the ocular dirofilariosis a parasite (but not directly Dirofilaria) is mostly suspected. Surgery is usually applied, regardless of the use of non-invasive techniques during preoperative management and although its use is still limited, molecular approach is the most accurate technique to establish a species-level diagnosis. Accurate epidemiological, parasitological and clinical information while handling and reporting human clinical cases is a need for physicians and researchers to improve and standardize the clinical management of human dirofilariosis.

An epidemiological survey of Dirofilaria spp. and Acanthocheilonema spp. in dogs from the Republic of Moldova.

BACKGROUND: During the last decades, filarial infections caused by Dirofilaria spp. have spread rapidly within dog populations of several European countries. Increasing scientific interest in filariasis, and the availability of new diagnostic tools, has led to improved knowledge of the biology, morphology, and epidemiology of different species of filarial worms. However, data are still scarce for a number of countries, including the Republic of Moldova. Thus, we assessed the epidemiological status of canine filariasis in the Republic of Moldova to address part of this knowledge gap. METHODS: A total of 120 blood samples were collected between June 2018 and July 2019 from dogs originating from the cities of Cahul and Chisinau. The samples were examined microscopically, and multiplex polymerase chain reaction was performed to evaluate filarioid species diversity. RESULTS: Microscopic examination revealed that 12 dogs (10.0%) were positive for circulating microfilariae. The molecular test showed that one dog was positive for Acanthocheilonema reconditum (0.8%), one for Dirofilaria immitis (0.8%), six for Dirofilaria repens (5.0%), and four (3.3%) harboured a co-infection with D. immitis and D. repens. Prevalence was significantly higher in dogs aged ≥ 2 years. CONCLUSIONS: The epidemiological survey presented here for the Republic of Moldova confirmed the presence D. immitis, D. repens and A. reconditum in dogs that had not received any heartworm preventive.

Multiple diagnostic tests demonstrate an increased risk of canine heartworm disease in northern Queensland, Australia.

BACKGROUND: Canine heartworm (Dirofilaria immitis) is a life-threatening infection of dogs with a global distribution. Information on the prevalence of D. immitis and associated risk factors for canine heartworm antigen positivity-and thus disease-in Australia is scarce or outdated. The current reference method for D. immitis diagnosis in dogs is via the detection of heartworm antigen in blood using commercially available microwell-based enzyme-linked immunosorbent assays (ELISAs). Heat treatment of canine plasma prior to testing has been suggested to increase test sensitivity. The aim of the current study was to estimate the prevalence of D. immitis in dogs confined to shelters in Queensland, Australia. The impact of heat treatment on antigen test results was also assessed. METHODS: Blood samples (n = 166) were collected directly from dogs in seven shelters across Queensland (latitudinal span of approx. 1700 km) into EDTA blood collection tubes. A commercially available ELISA (DiroCHEK®) was used to detect canine heartworm antigen in untreated and heat-treated plasma. Whole blood was concurrently tested for the presence of microfilariae and D. immitis DNA using a modified Knott's test and real-time PCR, respectively. Risk factors (age, gender, source, location) associated with the odds of positivity for canine heartworm were assessed using binary logistic regression models. RESULTS: A total of 16 dogs (9.6%; 95% confidence interval [CI]: 5.9-15.2%) were positive for canine heartworm based on combined test results. Heat treatment did not impact on the positivity of D. immitis antigen within samples (Cohen's kappa = 0.98), but the optical density was significantly increased in paired plasma samples for D. immitis antigen-positive samples (Wilcoxon matched-pairs signed rank test, two-tailed P < 0.01). Location of the dog in a shelter in northern Queensland was the only risk factor significantly associated with the odds of a dog being more likely to be D. immitis antigen positive (odds ratio: 4.39; 95% CI: 1.26-13.51). All samples positive for the modified Knott's test were also positive for D. immitis DNA by PCR. CONCLUSIONS: This study demonstrated the presence of heartworm-positive dogs in shelters in Queensland, with positive animals significantly more likely to occur in northern Queensland than southern Queensland. Sustained testing for the presence of D. immitis microfilariae and antigen remain important diagnostic tools in areas with known and re-emerging canine heartworm activity.