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CASE REPORT  
Year : 2018  |  Volume : 11  |  Issue : 3  |  Page : 95-99
Subcutaneous dirofilariasis of the cheek with microfilaremia


Department of Microbiology, Kempegowda Medical College, Bengaluru, Karnataka, India

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Date of Web Publication10-Dec-2019
 

   Abstract 


Dirofilaria is a filarial nematode of domestic and wild animals. Human subcutaneous dirofilariasis is an accidental zoonotic infection. Infection with Dirofilaria repens has been reported from various regions of the world, mainly from Europe, Asia, and Africa. Culex, Aedes, and Anopheles mosquitoes serve as the vectors for transmission of this infection. In India, a probable focus of human infection with D. repens has been identified in Kerala, costal Karnataka, and Maharashtra. Herein, we report a rare case of subcutaneous dirofilariasis of the cheek with microfilaremia attributed to D. repens in a male patient aged 25 years hailing from Mandya.

Keywords: Dirofilaria repens, dirofilariasis, zoonosis

How to cite this article:
Murthy NS, Gopi A, Bele-Kenge P. Subcutaneous dirofilariasis of the cheek with microfilaremia. Ann Trop Med Public Health 2018;11:95-9

How to cite this URL:
Murthy NS, Gopi A, Bele-Kenge P. Subcutaneous dirofilariasis of the cheek with microfilaremia. Ann Trop Med Public Health [serial online] 2018 [cited 2020 Feb 20];11:95-9. Available from: http://www.atmph.org/text.asp?2018/11/3/95/272552



   Introduction Top


Dirofilaria (Railliet and Henry, 1911) are natural filarial parasites infesting dogs, cats, raccoons, and foxes.[1] Dirofilariasis is a group of parasitoses caused by the species Dirofilaria immitis, D. repens, D. tenuis, Dirofilaria striata, D. subdermata, and D. ursi belonging to the genus Dirofilaria. Human dirofilariasis has been reported worldwide due to D. immitis and D. repens.[1] It qualifies as one of the rapidly emerging zoonotic diseases in India.[2] Transmission takes place when a potential mosquito vector containing the infective third-stage larva (L3) bites susceptible hosts during a blood meal.[3] Local climatic change, which favors mosquito-genic condition, attributes to the possible human transmission of this parasite.[4] The L3 larva of D. repens wanders through the body of the human host and customarily lodges in the subcutaneous plane, while D. immitis frequently ends up in the subconjunctival region and rarely in the human lung.[5] Human Dirofilariasis is reported as an aberrant migration of the worm in humans worldwide, presenting with subcutaneous and ocular lesions.[1],[6] We report a case of subcutaneous Dirofilariasis of the cheek in a 25-year-old man caused by D. repens.


   Case Report Top


A 25-year-old male from Mandya working as a steward in a regional hotel presented to a tertiary care center in Bengaluru with complaints of a painless swelling in the right cheek region for the past 3 months. There was no history of fever/weight loss.

On examination, soft-tissue enlargement was seen in the right cheek region 1 cm behind the right commissure of the lip [Figure 1].
Figure 1: Swelling in the right cheek

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Intraoral examination revealed a solitary, painless, mobile, firm, subcutaneous nodule measuring about 2 cm × 1.5 cm located in the right buccal mucosa. There was no sinus or discharge from the swelling. The overlying mucosa was normal in appearance [Figure 2].
Figure 2: Intraoral photograph showing the oral swelling with intact mucosa

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A chest X-ray done showed no significant clinical findings. Ultrasound of the subcutaneous nodule showed a well-defined hypoechoic lesion measuring 2 cm × 1 cm × 1.8 cm in the right cheek subcutaneous plane with no internal vascularity. Linear mobile echogenic strands were noted within the lesion.

The lesion was excised under local anesthesia through intraoral approach in the department of oral surgery and a live worm was extracted en masse and sent to the microbiology laboratory for identification. 70% (v/v) ethanol solution was used for the transport of the worm.

Preliminary microbiological examination revealed a cylindrical, unsegmented nematode worm of length 10.8–11 cm [Figure 3].
Figure 3: Cylindrical, unsegmented worm

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Next, the worm was kept immersed in a clearing agent (lacto phenol) for about 2 h and mounted on a slide using Canada balsam oil for further identification of the worm morphology. The worm was examined for the presence of key morphological markers of identification as described by Levine.[7]

The total thickness of the worm at the level of its largest diameter was found to be 336 μm. Cuticle was found to be 20 μm thick. Anterior end of the worm was bluntly rounded and greater in diameter compared to the posterior end.

The cuticle shows the presence of longitudinal striations and transverse ridges [Figure 4] and [Figure 5]. The posterior end lacks preanal papillae and spicules [Figure 6]. The anterior end had a simple mouth and showed the presence of an anterior vulva [Figure 5] and [Figure 7].
Figure 4: Longitudinal striations in the cuticle

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Figure 5: Transverse ridges and the anterior vulval opening

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Figure 6: Posterior end of the worm

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Figure 7: Anterior end of the worm

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One microliter of blood from the patient was subjected to concentration by Knott's technique[8],[9] for detection of microfilaria in the peripheral blood circulation. One milliliter of ethylenediaminetetraacetic acid (EDTA) blood sample was mixed with 9 ml of 2% formalin in a 15-ml tube and centrifuged for 5 min at 500 ×g. The supernatant was poured off and 2 × 10 μl of the sediment was transferred to two slides. Leishman and Giemsa differential staining techniques were used, respectively, to stain the two slides. The stained slides showed the presence of nonsheathed microfilaria as depicted in [Figure 8] and [Figure 9].
Figure 8: Microfilaria of Dirofilaria repens (Giemsa staining, ×1000)

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Figure 9: Microfilaria of Dirofilaria repens (Leishman's staining, ×1000)

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A fragment from the worm and the peripheral blood containing microfilaria were subjected to molecular identification by conventional polymerase chain reaction (PCR). After careful demounting, the nematode worm was subjected to serial washings in xylene (three times) and absolute ethanol (six times). After drying, the worm sample was taken in 200 μl of TE buffer (10 mM Tris-Cl, pH 7.5, 1 mM EDTA) and sonicated for 2 min. The sonicated worm sample was directly used as positive control for PCR. Meanwhile, the DNA was extracted from blood samples using Geneasy Blood DNA isolation kit (Bhat Biotech, Karnataka, Bengaluru, India) according to the manufacturer's instructions, and 2 μl of the sonicated sample/extracted DNA was used as template. Blood sample of a healthy person was used as negative control or blank for PCR.

Conventional PCR was performed using pan-filarial primers described by Rishniw et al.[10] with forward primer DIDR-F1 (5′-AGT GCG AAT TGC AGA CGC ATT GAG-3′) and reverse primer DIDR-R1 (5′-AGC GGG TAA TCA CGA CTG AGT TGA-3′), which amplify different fragment lengths of the internal transcribed spacer region 2 (ITS2) of the ribosomal DNA from D. immitis, Acanthocheilonema reconditum, D. repens, Amphiachyris dracunculoides, Brugia pahangi, B. malayi, B. timori, Mansonella ozzardi, and Onchocerca volvulus.[10]

Each PCR reaction consisted of 25 μL of 2× PCR master mix (Bhat Biotech), 0.4 μM of each primer, and 2 μL of DNA template at a total volume of 50 μL. The PCR procedure consisted of denaturation step at 95°C for 5 min and 35 cycles of denaturation (45 s at 94°C), annealing (45 s at 58°C), and extension (45 s at 72°C), together with final extension (10 min at 72°C) in DNAmp thermocycler (Bhat Biotech).

Electrophoresis: The PCR products were resolved on a 1% agarose gel containing 0.5 μg/ml ethidium bromide solution at 100 V and 250 mA for 45 min and were photographed on an ultraviolet transilluminator. The PCR-amplified products yielded a band at 484 base pairs specific to D. repens[10] [Figure 10].
Figure 10: Polymerase chain reaction analysis of worm and blood samples. Lane 1: DNA isolated from blood sample of a healthy person (negative control), Lane 2: Sonicated worm sample (positive control), Lane 5: DNA size marker (Bhat Biotech), and Lanes 3, 4, 6, and 7: two peripheral blood samples drawn from the same patient run in duplicate

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   Discussion Top


Human dirofilariasis is a rare helminthic infection. The first case of human dirofilariasis in India was reported from Kerala in 1976.[11] Subcutaneous dirofilariasis predominantly presents as a single migratory nodule in India.[2],[3],[4]

The identity of D. repens can be confirmed by its morphological characteristics. Mitochondrial 12S rRNA gene sequencing can be done for molecular confirmation since no information on 18S rRNA gene sequences of D. repens is registered in the GenBank.[12] Misidentification of Dirofilaria species is likely among cases diagnosed only by morphology owing to the difficulties in the identification in the immature stage of Dirofilaria parasites and because of poor sampling conditions. Therefore, the use of pan-filarial primers for conventional PCR would be cost-effective as well as specific for the diagnosis of filarial nematodes.[10]

Extraction of the worm en masse or complete surgical excision of the dirofilarial lesion is the treatment of choice for human dirofilariasis. Geldelman et al. suggest the use of a cryoprobe to curtail the excessive movement of the worm encountered during surgical removal.[13] Chemotherapy is usually not indicated in solitary nodules, as the presence of dirofilarial microfilaraemia is extremely rare in humans.[14] The present case showed peripheral microfilaraemia and responded to treatment with surgical removal of the swelling along with medical management using diethyl carbamazepine. Cheek peripheral smears performed 3 months after completion of medical therapy showed clearance of microfilaria. Combined ivermectin and doxycycline therapy has also been suggested to have antimicrofilarial and adulticidal effects, respectively, reserved for use in dirofilarial microfilaraemia and pulmonary dirofilariasis.[15]

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Acknowledgment

The authors thank Dr. G. C. Puttalakshmamma, Dr. G. S Mamatha, and Dr. Jaya Lakkundi from the Department of Parasitology at Karnataka Veterinary, Animal and Fisheries Sciences University, Hebbal, Bengaluru, for their dexterous confirmation of the identity of the nematode worm.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Simón F, Siles-Lucas M, Morchón R, González-Miguel J, Mellado I, Carretón E, et al. Human and animal dirofilariasis: The emergence of a zoonotic mosaic. Clin Microbiol Rev 2012;25:507-44.  Back to cited text no. 1
    
2.
Ghosh SK. Human dirofilariasis: A fast emerging zoonosis in India. Indian J Med Microbiol 2015;33:595-6.  Back to cited text no. 2
[PUBMED]  [Full text]  
3.
Sabu L, Devada K, Subramanian H. Dirofilariasis in dogs and humans in Kerala. Indian J Med Res 2005;121:691-3.  Back to cited text no. 3
    
4.
Reddy MV. Human dirofilariasis: An emerging zoonosis. Trop Parasitol 2013;3:2-3.  Back to cited text no. 4
[PUBMED]  [Full text]  
5.
Popescu I, Tudose I, Racz P, Muntau B, Giurcaneanu C, Poppert S, et al. Human Dirofilaria repens infection in Romania: A Case report. Case Rep Infect Dis 2012;2012:472976.  Back to cited text no. 5
    
6.
Genchi C, Kramer LH, Rivasi F. Dirofilarial infections in Europe. Vector Borne Zoonotic Dis 2011;11:1307-17.  Back to cited text no. 6
    
7.
Levine ND. Nematode Parasites of Domestic Animals and of Man. 2nd ed. Minneapolis: Burgess Publishing Co.; 1980.  Back to cited text no. 7
    
8.
Rojas A, Rojas D, Montenegro VM, Baneth G. Detection of Dirofilaria immitis and other arthropod-borne filarioids by an HRM real-time qPCR, blood-concentrating techniques and a serological assay in dogs from Costa Rica. Parasit Vectors 2015;8:170.  Back to cited text no. 8
    
9.
Magnis J, Lorentz S, Guardone L, Grimm F, Magi M, Naucke TJ, et al. Morphometric analyses of canine blood microfilariae isolated by the Knott's test enables Dirofilaria immitis and D. Repens species-specific and Acanthocheilonema (syn. Dipetalonema) genus-specific diagnosis. Parasit Vectors 2013;6:48.  Back to cited text no. 9
    
10.
Rishniw M, Barr SC, Simpson KW, Frongillo MF, Franz M, Dominguez Alpizar JL, et al. Discrimination between six species of canine microfilariae by a single polymerase chain reaction. Vet Parasitol 2006;135:303-14.  Back to cited text no. 10
    
11.
Joseph A, Thomas PG, Subramaniam KS. Conjunctivitis by Dirofilaria conjunctivae. Indian J Ophthalmol 1977;24:20-2.  Back to cited text no. 11
    
12.
Suzuki J, Kobayashi S, Okata U, Matsuzaki H, Mori M, Chen KR, et al. Molecular analysis of Dirofilaria repens removed from a subcutaneous nodule in a Japanese woman after a tour to Europe. Parasite 2015;22:2.  Back to cited text no. 12
    
13.
Geldelman D, Blumberg R, Sadun A. Ocular Loa loa with cryoprobe extraction of subconjunctival worm. Ophthalmology 1984;91:300-3.  Back to cited text no. 13
    
14.
Khurana S, Singh G, Bhatti HS, Malla N. Human subcutaneous dirofilariasis in India: A report of three cases with brief review of literature. Indian J Med Microbiol 2010;28:394-6.  Back to cited text no. 14
[PUBMED]  [Full text]  
15.
McCall JW, Genchi C, Kramer LH, Guerrero J, Venco L. Heartworm disease in animals and humans. In: Rollinson D, Hay SI, editors. Advances in Parasitology. Vol. 66. USA: Academic Press; 2008. p. 193-285.  Back to cited text no. 15
    

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Correspondence Address:
Dr. Neetha S Murthy
#3, Block-5, Health Department Layout, Dattagalli 2nd Stage, Ramakrishna Nagar, Mysore - 570 022, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ATMPH.ATMPH_692_16

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]



 

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