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.

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.

Candidatus Dirofilaria Hongkongensis Infections in Humans During 2005 to 2020, in Kerala, India.

We tried to determine the epidemiology and species of human dirofilariasis observed at two tertiary care hospitals in Kerala. We searched the hospital database to identify cases of dirofilariosis from January 2005 to March 2020. Along with human isolates, one dog Dirofilaria isolate was also subjected to PCR and sequencing of pan filarial primers cytochrome oxidase subunits 1 and 12S rDNA. We documented 78 cases of human dirofilariosis. The orbit, eyelid, and conjunctiva were the most commonly affected sites. Molecular characterization identified one dog and five human isolates as Candidatus Dirofilaria Hongkongensis. A rare case of subconjunctival infestation by B. malayi was also documented. Human dirofilariosis is a public health problem in the state of Kerala in India, and it is mostly caused by Candidatus Dirofilaria Hongkongensis. We propose that all diroifilaria isolates are subjected to sequencing for identification.

Molecular detection of Dirofilaria spp. and host blood-meal identification in the Simulium turgaicum complex (Diptera: Simuliidae) in the Aras River Basin, northwestern Iran.

BACKGROUND: Blackflies (Diptera: Simuliidae) are known as effective vectors of human and animal pathogens, worldwide. We have already indicated that some individuals in the Simulium turgaicum complex are annoying pests of humans and livestock in the Aras River Basin, Iran. However, there is no evidence of host preference and their possible vectorial role in the region. This study was conducted to capture the S. turgaicum (s.l.), to identify their host blood-meals, and to examine their potential involvement in the circulation of zoonotic microfilariae in the study areas. METHODS: Adult blackflies of the S. turgaicum complex were bimonthly trapped with insect net in four ecotopes (humans/animals outdoors, irrigation canals, lands along the river, as well as rice and alfalfa farms) of ten villages (Gholibaiglou, Gungormaz, Hamrahlou, Hasanlou, Khetay, Khomarlou, Larijan, Mohammad Salehlou, Parvizkhanlou and Qarloujeh) of the Aras River Basin. A highly sensitive and specific nested PCR assay was used for detection of filarial nematodes in S. turgaicum (s.l.), using nuclear 18S rDNA-ITS1 markers. The sources of blood meals of engorged specimens were determined using multiplex and conventional cytb PCR assays. RESULTS: A total of 2754 females of S. turgaicum (s.l.) were collected. The DNA of filarial parasites was detected in 6 (0.62%) of 960 randomly examined individuals. Sequence analysis of 420 base pairs of 18S rDNA-ITS1 genes identified Dirofilaria spp. including 5 D. immitis and 1 D. repens. Importantly, all filarial positive specimens have been captured from humans and animals outdoors. Cytb-PCR assays showed that in all ecotypes studied, members of the S. turgaicum complex had preferably fed on humans, dogs, bovids, and birds, respectively. CONCLUSIONS: To the best of our knowledge, this is the first report of D. immitis/D. repens detection in blackflies. Results showed that S. turgaicum (s.l.) was the most abundant (97%) and anthropophilic (45%) blackfly in all studied ecotypes/villages and that DNA of Dirofilaria spp. was detected in the flies taken from six villages. Dirofilariasis is a common zoonosis between humans and carnivores, with mosquitoes (Culicidae) as the principal vectors. Further investigations are needed to demonstrate that blackflies are actual vectors of Dirofilaria in the studied region.

Human case of subcutaneous nodule because of a novel genetic variation of Dirofilaria sp.

A 75-year-old man presented with a 1-cm large elastic soft subcutaneous nodule on the left side of the umbilicus, which when excised showed presence of a helminthic form within the granulomatous lesions. Morphologically, the helminth was considered to be of the genus Dirofilaria, and the patient showed increased serum antibody titer against canine filaria. The partial DNA sequence of the mitochondrial 12S rRNA gene locus of this clinical isolate showed the highest nucleotide identity (89.6%) with Dirofilaria repens; however, the phylogenetic analysis addressed the haplotype and Dirofilaria ursi as outgroups of the clusters of D. repens and Dirofilaria immitis, which are the causal agents of most human dirofilariasis. As like bear filaria D. ursi, a wide variety of other carnivore-parasitizing filaria species have rarely been reported in humans. The newly detected genetic haplotype in this case may correspond to one of these species of Dirofilaria, though the genetic references are not available thus far.

A rare case of human pulmonary dirofilariasis with nodules mimicking malignancy: approach to diagnosis and treatment.

BACKGROUND: Human pulmonary dirofilariasis is a rare zoonosis caused by the dog worm Dirofilaria spp., a parasite transmitted by mosquitos and resulting in peripheral lung nodules. The filarial nematode enters the subcutaneous tissue, travels to the right ventricle and dies causing a small pulmonary infarction that may embolize through the pulmonary vessels and may appear as a solitary nodule. These nodules are usually incidentally identified in asymptomatic patients undergoing chest imaging studies, and are generally interpreted to be malignant. CASE PRESENTATION: We present the case report of a human dirofilariasis in a patient with multiple pulmonary nodules resected using video-assisted thoracic surgery (VATS). According to our literature review, this is the first case with double synchronous lung nodules reported in Italy. CONCLUSIONS: Minimally invasive resection with histologic examination may be the best approach for the diagnosis and treatment of pulmonary dirofilariasis. Polymerase Chain Reaction testing may provide a more accurate etiological diagnosis in case of an inconclusive pathology result.

Prevalence and genetic characterization of Dirofilaria lutrae Orihle, 1965 in North American river otters (Lontra canadensis).

Dirofilaria lutrae Orihle, 1965 is a subcutaneous filarial worm found in North American river otters (Lontra canadensis). Previous studies reported the geographical range of this worm to include Louisiana, Florida, and North Carolina, USA. Although D. lutrae does not cause disease in otters, it must be distinguished from Dirofilaria immitis (Leidy, 1856), which causes heartworm disease in otters. The goal of this study was to determine the prevalence of D. lutrae in several states and to investigate the phylogenetic relationship of D. lutrae and other Dirofilaria species. Adult D. lutrae were collected from 32 of 40 (80%) otters from North Carolina (n = 38), South Carolina (n = 1), and Kentucky (n = 1), whereas all otters from Georgia (n = 1), Pennsylvania (n = 2), Arkansas (n = 184) and California (n = 1) were negative for D. lutrae. A portion of the cytochrome c oxidase I (COI) was amplified from nine North Carolina specimens and one South Carolina specimen. Sequence analysis indicated that all but one were identical to each other and D. lutrae was phylogenetically most closely related to D. immitis; however, few Dirofilaria sequences are available for comparison. Because other subcutaneous parasites may be detected in otters, parasites should be carefully examined to confirm their identity. Finally, given the high prevalence of D. lutrae in otters in some southeastern states, microfilaria found in blood samples of otters must be correctly identified because treatment for D. lutrae is generally not warranted and some pharmaceutical treatments can cause mortality in otters.

Candidatus Dirofilaria hongkongensis as Causative Agent of Human Ocular Filariosis after Travel to India.

We report a human case of ocular Dirofilaria infection in a traveler returning to Austria from India. Analysis of mitochondrial sequences identified the worm as Candidatus Dirofilaria hongkongensis, a close relative of Dirofilaria repens, which was only recently described in Hong Kong and proposed as a new species.

Zoonotic intestinal parasites and vector-borne pathogens in Italian shelter and kennel dogs.

This study investigated the presence of zoonotic parasites and vector-borne pathogens in dogs housed in kennels and shelters from four sites of Italy. A total of 150 adoptable dogs was examined with different microscopic, serological and molecular methods. Overall 129 dogs (86%) were positive for one or more parasites and/or pathogens transmitted by ectoparasites. Forty-eight (32%) were positive for one infection, while 81 (54%) for more than one pathogen. The most common zoonotic helminths recorded were hookworms, roundworms and Capillaria aerophila, followed by mosquito-borne Dirofilaria spp. and Dipylidium caninum. One hundred and thirteen (77.9%), 6 (4.1%) and 2 (1.4%) dogs were positive for Rickettsia spp., Leishmania infantum and Anaplasma spp., respectively. The results show that dogs living in rescue facilities from the studied areas may be infected by many zoonotic internal parasites and vector-borne pathogens, and that control measures should be implemented.

Molecular characterization of human Dirofilaria isolates from Kerala.

Background & objectives: Human dirofilariosis is a well-recognized zoonosis caused by several species of the genus Dirofilaria. The disease is prevalent among canines and human beings in Kerala. The objective of the present study was to confirm the human Dirofilaria isolates by molecular characterization. Methods: The worms or segments obtained from human sources were subjected to diagnostic polymerase chain reaction (PCR) targeting the cytochrome oxidase subunit 1 (COI) and 5S ribosomal RNA (rRNA) genes of Dirofilaria repens. The amplicons were sequenced and analyzed. Results: The filariid nematodes recovered from ocular as well as subcutaneous tissue of human origin were identified as D. repens based on PCR targeting COI as well as 5S rRNA genes. The phylogenetic analysis of the COI gene nucleotide sequence obtained in the present study showed that D. repens shared the closest evolutionary relationship with D. honkongensis. Interpretation & conclusions: Molecular identification of D. repens isolated from human source assumes significance from the point of zoonotic threat of this mosquito-borne nematode. Phylogenetic analysis revealed a close evolutionary relationship with Asian isolate of D. honkongensis. Timely detection and treatment of infection in dogs, together with mosquito control, should be an integral part of the control strategy of this disease.

The Mitochondrial Genomes of the Zoonotic Canine Filarial Parasites Dirofilaria (Nochtiella) repens and Candidatus Dirofilaria (Nochtiella) hongkongensis Provide Evidence for Presence of Cryptic Species.

BACKGROUND: Cutaneous dirofilariosis is a canine mosquito-borne zoonosis that can cause larva migrans disease in humans. Dirofilaria repens is considered an emerging pathogen occurring with high prevalence in Mediterranean areas and many parts of tropical Asia. In Hong Kong, a second species, Candidatus Dirofilaria hongkongensis, has been reported. The present study aimed to compare mitochondrial genomes from these parasites and to obtain population genetic information. METHODS AND FINDINGS: Complete mitochondrial genomes were obtained by PCR and Sanger sequencing or ILLUMINA sequencing for four worms. Cytochrome oxidase subunit 1 sequences identified three as D. repens (all from Europe) and one as C. D. hongkongensis (from India). Mitochondrial genomes have the same organization as in other spirurid nematodes but a higher preference for thymine in the coding strand. Phylogenetic analysis was in contradiction to current taxonomy of the Onchocercidae but in agreement with a recent multi-locus phylogenetic analysis using both mitochondrial and nuclear markers. D. repens and C. D. hongkongensis sequences clustered together and were the common sister group to Dirofilaria immitis. Analysis of a 2.5 kb mitochondrial genome fragment from macrofilaria or canine blood samples from Europe (42), Thailand (2), India (1) and Vietnam (1) revealed only small genetic differences in the D. repens samples including all European and the Vietnam sample. The Indian C. D. hongkongensis and the two Thai samples formed separate clusters and differences were comparatively large. CONCLUSION: Genetic differences between Dirofilaria spp. causing cutaneous disease can be considerable whereas D. repens itself was genetically quite homogenous. C. D. hongkongensis was identified for the first time from the Indian subcontinent. The full mitochondrial genome sequence strengthens the hypothesis that it represents an independent species and the Thai samples might represent another cryptic species, Candidatus Dirofilaria sp. 'Thailand II', or a quite divergent population of C. D. hongkongensis.

Genetic diversity of Dirofilaria spp. isolated from subcutaneous and ocular lesions of human patients in Ukraine.

This short communication describes the phylogenetic analysis of 48 Dirofilaria worms isolated from human patients in Ukraine. 102 cases were both of subcutaneous (47; 46.1%) and ocular (54; 52.9%) locations. Worms from 44 patients (15 subcutaneous and 29 ocular) were subjected to DNA extraction and amplification of a specific fragment of the 12S rRNA subunit, and sequences were used for phylogenetic analysis. Results showed that 13.8% of the ocular cases analyzed at molecular level were caused by Dirofilaria immitis. Very few cases of ocular human dirofilariosis due to D. immitis have been described in the literature to date, majority of them attributed to Dirofilaria repens. Our results show that ocular dirofilariosis cannot be excluded in areas of low endemicity for D. repens were D. immitis is also present.

Evidences of increasing risk of dirofilarioses in southern Italy.

Given the spread of Aedes albopictus from northern to southern Italy, and the lack of updated data on Dirofilaria infections, this study was carried out to assess the infection risk for dogs and cats in Apulia region. During a 2-year study, 175 A. albopictus female specimens and samples of blood from 427 dogs (309 privately owned dogs and 118 shelter dogs) and 12 cats were collected. All blood samples were subjected to a modified Knott method, to a test for the detection of circulating Dirofilaria immitis antigen, and to a Dirofilaria species-specific real-time PCR for the simultaneous detection of D. immitis and Dirofilaria repens, targeting on partial cytochrome oxidase subunit 1 and internal transcribed spacer-2, respectively. Two abdomen and one thorax pools from A. albopictus were positive for D. immitis, with minimum infection rates of 1.14 and 0.51, respectively, and a probability of a single positive specimen to be infected of P = 0.6 % (95 % confidence interval (CI) = 0.12-1.73). Out of 439 examined subjects, 22 (5.0 %) tested positive for Dirofilaria spp. in at least one diagnostic test. A specific D. immitis infestation rate of 3.5 % was found among the privately owned dogs, while shelter dogs tested positive only for D. repens with a prevalence of 3.4 %; one cat tested molecularly positive for D. immitis. There was a significantly higher rate of positivity among guard dogs for D. immitis (odds ratio, 6.24, 95 % CI, 1.26-25.28; P < 0.05). The increasing risk of D. immitis infection in southern Italy is supported by the noteworthy positivity of A. albopictus populations and the cat. Our data highlight the usefulness to include filarioid infestation in routine diagnosis.

A novel Dirofilaria species causing human and canine infections in Hong Kong.

Dirofilariasis is globally the commonest manifestation of zoonotic filariasis. We report the detection of a novel canine species causing human and canine dirofilariasis in Hong Kong. Three human cases occurring over 10 months were identified, one presenting with cervical lymphadenopathy, one with an abdominal subcutaneous mass, and one with a subconjunctival nodule. Transected worms recovered from the resected abdominal subcutaneous mass were morphologically compatible with Dirofilaria. The cox1 gene sequences of the three human isolates were identical; however, they were only 96.2% and 89.3% identical to the cox1 gene of Dirofilaria repens and Dirofilaria immitis, respectively. Sequencing of the 18S-ITS1-5.8S gene cluster was successful in the intact worm, and the nucleotide sequences were 94.0% and 94.9% identical to those of D. repens and D. immitis, respectively. Screening of the blood samples from 200 dogs and 100 cats showed the presence of the novel Dirofilaria species in 3% (6/200) of the dogs' but none of the cats' blood samples. Nucleotide sequences of the cox1 gene and 18S-ITS1-5.8S gene clusters of the dogs' samples were identical to those in the human samples. The sera of canines infected by this novel Dirofilaria species were negative when tested with the SNAP 4Dx D. immitis detection kit, except in the case of dogs with a mixed infection with D. immitis as detected by PCR. The results from this study suggest that this novel Dirofilaria species is a cause of filarial infection in humans and dogs in Hong Kong. We propose to name this Dirofilaria species "Candidatus Dirofilaria hongkongensis."

Development of loop-mediated isothermal amplification method for detecting Wuchereria bancrofti DNA in human blood and vector mosquitoes.

We have developed loop-mediated isothermal amplification (LAMP) method to detect Wuchereria bancrofti DNA. The sensitivity and specificity of LAMP method were equivalent to those of PCR method which detects SspI repeat sequence in W. bancrofti genomic DNA: both methods detected one thousandth of W. bancrofti DNA from one microfilaria (Mf), and did not cross-react with DNAs of Brugia malayi, B. pahangi, Dirofilaria immitis, human and Culex quinquefasciatus. We also examined the sensitivity of LAMP using the mimic samples of patient's blood or blood-fed mosquitoes containing one W. bancrofti Mf per sample. The LAMP method was able to detect W. bancrofti DNA in 1000 µl of blood or in a pool of 60 mosquitoes, indicating its usefulness in detecting/monitoring W. bancrofti infection in humans and vector mosquitoes in endemic areas.

Subcutaneous dirofilariosis in South-East Serbia--case report.

Human dirofilariosis is a relatively rare infection caused by filarial worms of the genus Dirofilaria. We herein report the first case of human subcutaneous dirofilariosis in the southeastern part of Serbia. A complete alive nematode was removed from a nodule in the periorbital region of a 50-year-old woman. The nematode was morphologically identified as a D. repens-like immature female. The diagnosis was confirmed with molecular methods. The patient was probably infected in the South-East Serbia as she had not travelled abroad, nor in other parts of Serbia such as Vojvodina, recently identified as a hyperendemic area for D. repens infection of dogs.

Human intraocular filariasis caused by Dirofilaria sp. nematode, Brazil.

A case of human intraocular dirofilariasis is reported from northern Brazil. The nematode was morphologically and phylogenetically related to Dirofilaria immitis but distinct from reference sequences, including those of D. immitis infesting dogs in the same area. A zoonotic Dirofilaria species infesting wild mammals in Brazil and its implications are discussed.