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.


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. D irofilariosis, caused by Dirofilaria repens or D. immitis nematodes, is a zoonotic filarial infection transmitted through the bite of various mosquitoes. The most frequent manifestations in humans are subcutaneously migrating worms and formation of nodules in various body parts (1). Increasing numbers of human D. repens infections have been reported from Europe, Africa, and Asia (2,3). Austria was considered nonendemic, until the first autochthonous case in a human was reported in 2006 (4) from the most eastern province, the Burgenland, where D. repens nematodes were recently also found for the first time in 2 Anopheles mosquito species (5). We describe a case of imported ocular dirofilariosis caused by the recently newly proposed species Candidatus Dirofilaria hongkongensis (6).
The patient, a 38-year-old woman, had recurrent eyelid swelling in both eyes and conjunctival inflammation with Appendix Figure, panel A, https://wwwnc.cdc.gov/EID/ article/23/8/17-0423-Techapp1.pdf). She visited numerous physicians and, upon various putative diagnoses (ranging from sicca syndrome to burnout syndrome), she received corresponding therapies, including antibiotics, steroids, and acupuncture. From January 2012 on, the eyelid swellings were accompanied by a creeping sensation and occurred more often. In early August 2012, she sought care at the emergency department of a university eye clinic in Vienna, Austria. She had a moving object in her left eye. Slit lamp examination revealed a white slender worm moving subconjunctivally in the temporal part of the left eye (online Technical Appendix Figure,  Results of serologic testing for filariae were negative before and after extraction of the worm, as were results for testing of EDTA blood for microfilariae. Blood test results, including differential blood counts, were within reference ranges throughout the case history. The patient had returned from a 7-week stay in India, including the areas of Goa, Maharashtra, Delhi, and Uttar Pradesh, 3 months before initial onset of symptoms. Her travel history of the preceding 3 years included 4 more trips to India of several weeks each; a 2-week stay in Israel (October 2010); and a 2-week stay in Dubai, United Arab Emirates (July 2009). For confirmation of the morphologic identification, we isolated DNA from a 1-cm piece of the worm after homogenization by using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany). We amplified fragments of the cytochrome C oxidase subunit I (COI) with panfilarial primers COXfw 5′-GCKTTTCCTCGTGTTATGC-3′/ COXrev 5′-CCAGCCAAAACAGGAACAG-3′ and 12S rRNA with panfilarial primers Panfil-12S-F 5′-GTTC-CAGAATAATCGGCTA-3′/Panfil-12S-R 5′-ATTGAC-GGATGRTTTGTACC-3′ (7). We sequenced amplicons and subjected them to phylogenetic analyses (online Technical Appendix). All sequence data were submitted to Gen-Bank (accession nos. KY750548-KY750550).
The 329 bp COI fragment (accession no. KY750548) showed 99%-100% identity to 2 sequences from Candidatus Dirofilaria hongkongensis (accession nos. KX265050 and JX187591). Identity to D. repens sequences was 95%-96%, to D. immitis 89%, and to Onchocerca spp. up to 92%. The 466 bp mitochondrial 12S rDNA fragment (accession no. KY750549) showed 99% identity to Candidatus Dirofilaria hongkongensis sequences from case-patients in India (accession no. KX265050) and Hong Kong (accession no. KY750550), the latter derived from original material of the first description of Candidatus Dirofilaria hongkongensis (6). Identity to a Dirofilaria sp. from a patient returning from India and Sri Lanka and to Dirofilaria sp. Thailand II, recently reported among dogs in Thailand (accession nos. KX265092 and KX265093) (8), was also 99%. Phylogenetic analysis using the COI sequence clearly placed the sequence into the Candidatus Dirofilaria hongkongensis cluster, the sister taxon to D. repens (Figure). Although D. immitis shows virtually identical COI sequences from 4 continents, genetic variability in D. repens-like parasites is obviously much higher, possibly associated with varying zoonotic potentials, reservoirs, and vectors; however, molecular data on Dirofilaria are still scarce.
In this case, Candidatus Dirofilaria hongkongensis was most likely acquired in India. An infection in Austria seems unlikely because, until now, only 1 singular autochthonous Dirofilaria infection has been reported, and that case was classic D. repens infection (4). Dubai is considered nonendemic for Dirofilaria spp. parasites, whereas Israel is known to be endemic for D. repens nematodes (1,3), but the patient's trips to these countries were much longer ago than her latest trip to India. Moreover, all cases from India or Sri Lanka analyzed by us so far represented Candidatus Dirofilaria hongkongensis (8,9; S. Poppert, unpub. data), suggesting that this species is widely distributed on the Indian subcontinent. In fact, whether classical D. repens infection occurs in India at all is unclear. Infections with Candidatus Dirofilaria hongkongensis nematodes might take a similar course as infections with classical D. repens; however, a case of meningoencephalitis caused by nematodes of this candidate species also has been described (9). Dirofilaria spp. parasites isolated from human case-patients should be investigated by molecular methods to establish an exact species diagnosis, especially if infections were acquired outside Europe.
T he foodborne zoonotic pathogen Shiga toxin (Stx)producing Escherichia coli (STEC) is responsible for human diseases ranging from uncomplicated diarrhea to the life-threatening hemolytic-uremic syndrome (HUS) (1). Stx production is the most determining virulence factor implicated in HUS, and the intimin, encoded by eae, is the most common adherence factor in HUS-associated STEC (1). Stx2d is a Stx2 variant in which cytotoxicity is increased (from 35-to 350-fold) by the action of elastase in intestinal mucus (2). This mucus-enhanced toxicity is termed "activation," and activatable Stx2d proteins are designated Stx2dact. Stx2dact production and Stx2dact genotype (stx2d) have been associated primarily with eaenegative STEC and considered a predictor for severe clinical outcome in such infections (3).
All STEC strains received or isolated in the Reference and Research Laboratory of Food and Waterborne Bacterial Infections (Majadahonda, Spain) are routinely tested for stx1 and stx2 subtypes by a PCR subtyping method (4). For serotyping, O antigen is identified with both commercial antiserum and PCR (5), and H antigen is identified by PCR amplification of the fliC gene (6) and further sequencing of the PCR product. During 2012-2016, stx2d was identified in 7 (3%) of 236 STEC strains isolated from patients with HUS and/or diarrhea in Spain (193 eae-positive and 43 eaenegative strains). Six were eae-negative non-O157 STEC belonging to serotypes O73:H18 (2 strains), O91:H21, O148:H8, O181:H49, and ONT:H21. Strikingly, the other stx2d-positive strain identified (CNM-2140/12) belonged to serotype O157:H7 and contained stx2d in combination with stx2c, apart from eae. The strain had been isolated from a 2-year-old boy with bloody diarrhea July 2012, and it fermented sorbitol after overnight incubation on sorbitol Mac-Conkey agar (Becton Dickinson, Sparks, MD, USA). We confirmed Stx2 production using the enzyme immunoassay kit SHIGA TOXIN QUIK CHEK (TechLab, Blacksburg, VA, USA). The activatable property of the toxin was confirmed by partial sequencing of the stx2 gene (4), analysis of the resulting nucleotide sequence by comparison with the published stx2 reference sequences, and comparison of the resulting amino acid sequences. The nucleotide sequence of stx2d (GenBank accession no. MF094370) was 100% identical to that of strain 06-5231 (O55:H7, GenBank accession no. EF584538). The sequence was translated to amino acids (online Technical Appendix Figure, https:// wwwnc.cdc.gov/EID/article/23/8/17-0570-Techapp1.pdf),