Ocular Dirofilariasis Case in Thailand Confirmed by Molecular Analysis to Be Caused by Dirofilaria immitis

ABSTRACT.

A rare ocular dirofilariasis case along with the clinical characteristics, treatment, and outcome is reported. A whitish roundworm (10.6 cm long and 0.5 mm width) emerged from the pterygium, a triangular tissue growth on the cornea of the eye, of a male patient. The worm had a rounded anterior part, mouth without lips, smooth cuticular surface, and short rounded posterior tail with spicules: these features suggested that it was a male Dirofilaria sp. Molecular identification confirmed that the worm belonged to Dirofilaria immitis. This is the first molecular confirmation that D. immitis is a causative agent of ocular dirofilariasis in Thailand: dirofilariasis is a newly emerging zoonotic disease. Physicians should be alert to zoonotic filarial worms and knowledgeable about treatment of this disease.

Human dirofilariasis, an emerging vector-borne zoonotic disease, is commonly caused by two species of helminths Dirofilaria repens and Dirofilaria immitis (Nematoda: Onchocercidae).^1,2 Mosquitoes are the vectors and humans are rare accidental hosts.^1,2 In the human body, the Dirofilaria life cycle is not completed and microfilariae have not been found.^1,3 Dirofilaria repens can cause subcutaneous and ocular dirofilariasis, whereas D. immitis causes pulmonary and heart lesions, mainly in carnivores and sporadically in humans.^1,4 In addition, rare ocular dirofilariasis cases caused by D. immitis have been reported from Australia,³ Iran,^5,6 Italy,⁷ and Ukraine.⁸ In Thailand, human cases of dirofilariasis have been reported. These consist of a case of pulmonary dirofilariasis,⁹ two ocular dirofilariasis cases caused by Dirofilaria sp.¹⁰ and D. repens¹¹ with morphological identification, and one case as a result of an unidentified Dirofilaria sp. with molecular similarity to a novel Dirofilaria species causing human and canine infections in Hong Kong.¹²

In this study, DNA sequences from portions of the mitochondrially encoded 12S ribosomal RNA (12S rRNA) and cytochrome c oxidase subunit I (cox1) genes were obtained from a worm surgically removed from one ocular dirofilariasis patient. The sequences were identified as being from D. immitis. The phylogenetic relationship between D. immitis and other Dirofilaria species is also discussed.

A 52-year-old male Thai patient, living in Tard Province, eastern Thailand, 315 km from Bangkok, had suffered from chronic irritation of both eyes for more than 10 years. The patient had a history of exposure to mosquito bites around his home, in an area where there were also domestic and stray dogs. The patient also had previously used topical drug treatment, provided by a general medical clinic, for pterygium, a triangular tissue growth on the cornea of both eyes.

In January of 2020, the patient consulted with an ophthalmologist at Chumphae General Hospital, Khon Kaen, Thailand, with sudden marked pain and irritation of the left eye. Additional symptoms included slightly blurred vision, photophobia, and massive production of tears. Examination revealed mild swelling of the pterygium of the left eye. Slit-lamp examination found severe inflammation of the pterygium with marked chemosis, papillary reaction of upper and lower palpebral conjunctiva, clear cornea, no inflammatory cells in anterior chamber, and normal fundus. Visual acuity was 20/20 in right eye and 20/70 in the left eye. The right eye showed a small head of simple pterygium without inflammation. Early diagnosis was recorded as severe inflammation of the pterygium of the left eye and the patient was treated with the anti-inflammatory drug tobramycin (0.3%) plus 0.1% dexamethasone one drop, four times a day.

One day after treatment, the patient returned to the eye clinic with marked pain of left eye, redness of the pterygium head, and he reported seeing a whitish worm extending from the left pterygium lesion. Severe pain periodically occurred when the worm appeared. However, slit-lamp examination at the clinic did not find a parasite. Then, an anesthetic, tetracaine hydrochloride (0.5%), was dropped in the left eye every 5 minutes. One hour later, a whitish roundworm emerged from the pterygium and was removed using a Jeweler Forceps (Figure 1B). Stool examination using the modified formalin ethyl acetate concentration technique¹³ revealed no parasites or their propagules. Complete blood count showed 29% eosinophilia. The patient was followed up 1 week after parasite removal, left eye pain had decreased, with no change of visual acuity, which may be due to pterygium. The patient had fully recovered 1 month later.

Figure 1.

Dirofilaria immitis worm found in ocular structures of the patient. (A) Swelling of pterygium of left eye. (B) Whitish color of roundworm that emerged from the pterygium site (arrowhead). (C) The worm removed from the left eye. (D) Round anterior part of the worm. (E) Coiled tail with spicules (arrowhead). This figure appears in color at www.ajtmh.org.

In normal saline, the worm was seen as a long, slender, whitish roundworm 10.6 cm in length and 0.5 mm in width. Microscopic examination showed it to be a male roundworm with a rounded anterior part, mouth lacking lips, smooth cuticular surface, and short, rounded tail with spicules (Figure 1). The recovered roundworm was kept in 70% ethanol and stored at −70°C until used for DNA extraction. The authors have complied with all the ethics guidelines applicable. This study was approved by the Khon Kaen University Ethics Committee for Human Research (HE631605). Informed consent was obtained from the patient.

Genomic DNA was extracted from the posterior part of worm using the NucleoSpin^® tissue kit (Macherey-Nagel GmbH & Co. KG, Duren, Germany) according to the manufacturer’s instructions. Genomic DNA was subjected to polymerase chain reaction (PCR) using newly developed primers to amplify a portion of the 12S rRNA gene (DiroFP_12S: 5′-GGTTATGGTTTTGATAAGGTTATG-3′ and DiroRP_12S: 5′-GAAACAAGTTTCCTTACCTCTACC-3′) and of the cox1 gene (Diro1FP_COI: 5′-GAATATTTTTTGTGGAATGAC-3′ and Diro2RP_COI: 5′-ATTACTTTCTATGATAACCATAACC-3′). Each PCR contained 0.5 µL of each primer (10 µM), 2.5 µL of 10 × FastStart High Fidelity Reaction Buffer with 18 mM MgCl₂ (Roche, Mannheim, Germany), 0.5 µL of dNTP mix (10 mM), 0.125 units of FastStart High Fidelity Enzyme Blend (Roche), 5 µL of Dirofilaria DNA template, and sterile deionized water to reach a final volume of 25 µL. The PCR cycling conditions were the same for both pairs of primers as follows: preincubation for 2 minutes at 94°C, followed by 10 cycles of 94°C for 1 minute (denaturation), 40°C for 1 minute (annealing), 68°C for 2 minutes (extension), then 30 cycles of 94°C for 1 minute (denaturation), 45°C for 1 minute (annealing), 68°C for 2 minutes (extension), and final extension at 68°C for 7 minutes.

The amplified products were shipped to Apical Scientific Sdn Bhd, Selangor, Malaysia, for DNA sequencing in both directions using the PCR primers as sequencing primers. The resulting sequences were aligned and compared with representative nucleotide sequences of Dirofilaria species from GenBank using the multiple sequence alignment program ClustalW within BioEdit Sequence Alignment Editor version 7.0.9.18.¹⁴ Similarity searches against known Dirofilaria species sequences were performed using the Basic Local Alignment Search Tool (BLAST) at the National Center for Biotechnology Information website (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Both sequences in this study have been deposited in the GenBank database under the accession numbers: MW512514 for the partial 12S rRNA gene and MW577348 for the partial cox1 gene. BLAST search results indicated that the sequences were from D. immitis. For the 12s rRNA gene, the partial sequence (665 bp [base pair]) was 100% identical (100% coverage) with D. immitis from Australia (GenBank accession no. AJ537512) and 91.78% similar (100% coverage) to those of D. repens from Vietnam, Poland, Italy, Hungary, Spain, France, and Romania (GenBank accession nos. KX265090, KX265088, KX265077, KX265069, KX265062, KX265059, and KX265051). The partial cox1 gene sequence (1,606 bp) obtained was 99.88% similar (100% coverage) to that of D. immitis from Australia (GenBank accession no. AJ537512) and 90.80% similar (100% coverage) to that of D. repens from Croatia (GenBank accession no. KX265049).

Phylogenetic trees were inferred in MEGA version 10.0.5.¹⁵ For the alignment of 12S rRNA gene sequences, the phylogeny was inferred using the maximum likelihood method and the best-fit model was the Hasegawa-Kishino-Yano model (HKY) using a discrete Gamma distribution (+G) with five rate categories; the 12S rRNA tree with the highest log likelihood (−1097.20) is shown in Figure 2A. For the partial cox1 gene alignment, the maximum likelihood method was used with the best-fit model, which was the Tamura-Nei model (T93) using a discrete Gamma distribution (+G) with five rate categories; the cox1 tree with the highest log likelihood (−1690.23) is shown in Figure 2B. Bootstrap percentages were estimated using 1,000 replications. The phylogenetic trees show that the sequences from this study have high similarity with those of D. immitis in GenBank. The 12S rRNA sequences of D. immitis cluster in a group with high bootstrap support (98%). The genus Dirofilaria appears as monophyletic, with each represented species forming a distinct lineage (Figure 2A). Dirofilaria repens forms the basal group whereas D. striata is a sister to D. immitis. Analysis of cox1 sequences similarly placed our sequence in a well-supported clade of D. immitis (100%). Again, the genus Dirofilaria appears as monophyletic, with each species forming a distinct lineage and D. ursi a sister to D. immitis, as shown in Figure 2B.

Figure 2.

Phylogenetic trees of roundworms in the genus Dirofilaria reconstructed using the maximum likelihood method based on partial 12S ribosomal RNA gene sequences (A) and partial cytochrome c oxidase subunit I gene sequences (B). Dirofilaria species obtained from GenBank are indicated with accession numbers and country codes (ISO 3166-1 alpha-3). Dirofilaria immitis sequences obtained from this study are presented in bold. Bootstrap scores, expressed as a percentage based on 1,000 replications, are indicated at each node. Scale bars indicate substitutions per nucleotide position.

Occasional orbital infection of humans by filarial worms is caused by several genera, that is, Loa, Dirofilaria, and Onchocerca.¹⁶ Dirofilaria repens is a typical agent of ocular dirofilariasis, but ocular disease is rarely caused by D. immitis.⁶ Orbital, subconjunctival, and intraocular infections are clinical forms of ocular dirofilariasis. The primary diagnosis of ocular dirofilariasis is by clinical signs, that is, a fibroma, atheroma, cyst, and/or a nodule/tumor that can change into an abscess containing the worm. Surgical excision is required to remove the worm.² Gross examination of the worm may not reveal the actual Dirofilaria species involved. Here, we report a case of ocular dirofilariasis caused by D. immitis with molecular confirmation of the causative species. PCR and sequencing should always be considered for identification of the species causing ocular dirofilariasis. The eye may become infected as a result of the typical worm migration during development from the venous circulation to the subcutaneous tissues,³ but the route of entering the eye needs to be clarified.

To our knowledge, the present study is the first to confirm by molecular analysis that D. immitis is a cause of ocular dirofilariasis in Thailand. This species must therefore be considered as causing a newly emerging zoonotic disease. Identification was done by sequencing DNA from an adult male worm recovered from a patient. DNA sequencing analysis can provide unambiguous identification of Dirofilaria species that cause human diseases.⁸ It is also important for epidemiological studies. Therefore, physicians should be alert to such zoonotic filarial worms and the additional danger posed by mosquito bites. They should also be aware of treatment and control of this ocular disease.