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 Table of Contents  
CASE REPORT
Year : 2021  |  Volume : 1  |  Issue : 1  |  Page : 154-155

Usefulness of mitochondrial deoxyribonucleic acid barcoding in identification of Chrysomya bezziana ophthalmomyasis: A case report


1 Department of Orbit and Oculoplasty, Aravind Eye Hospital and PG Institute of Ophthalmology, Madurai, Tamil Nadu, India
2 Department of Paediatric Ophthalmology, Aravind Eye Hospital and PG Institute of Ophthalmology, Madurai, Tamil Nadu, India
3 Scientist ‘F’ and Officer in Charge ICMR-Vector Control Research Centre, Indian Council of Medical Research, Department of Health Research, Ministry of Health and Family Welfare, Madurai, Tamil Nadu, India

Date of Submission31-May-2020
Date of Acceptance05-Aug-2020
Date of Web Publication31-Dec-2020

Correspondence Address:
Dr. Meghana Tanwar
Aravind Eye Hospital and PG Institute of Ophthalmology, 1, Anna Nagar Main Road, Sathamangalam, Madurai - 625 020, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijo.IJO_1758_20

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  Abstract 


We report a case of ophthalmomyiasis profunda with a complete destruction of ocular tissues in an old diabetic lady who presented with sudden onset of bleeding from a progressively increasing, painless swelling in her left eye since two weeks. She was primarily treated with Ivermectin, paraffin dressing, manual removal of maggots, and then total exenteration. Deoxyribonucleic acid (DNA) barcoding analysis supported by DNA sequencing accurately identified the species as Chrysomya bezziana based on the mitochondrial cytochrome c oxidase 1 gene. This case report demonstrates the usefulness of DNA barcoding, complementing the conventional identification methods, in accurate identification of maggots of ophthalmic significance.

Keywords: Chrysomya bezziana, cytochrome oxidase gene, DNA barcoding, DNA sequencing, orbital myiasis


How to cite this article:
Tanwar M, Chakrabarty S, Chowdhury G, Kim U, Rajaiah P. Usefulness of mitochondrial deoxyribonucleic acid barcoding in identification of Chrysomya bezziana ophthalmomyasis: A case report. Indian J Ophthalmol Case Rep 2021;1:154-5

How to cite this URL:
Tanwar M, Chakrabarty S, Chowdhury G, Kim U, Rajaiah P. Usefulness of mitochondrial deoxyribonucleic acid barcoding in identification of Chrysomya bezziana ophthalmomyasis: A case report. Indian J Ophthalmol Case Rep [serial online] 2021 [cited 2021 Mar 3];1:154-5. Available from: https://www.ijoreports.in/text.asp?2021/1/1/154/305505



Identification of larval instars is of paramount importance in forensic, veterinary, and medical sciences. A morphological taxonomy is often challenging due to the convergent evolution of these ectoparasites.[1] Polymerase chain reaction (PCR) based assays with DNA barcoding, targeting mitochondrial genomes, namely, cytochrome b (CB) and cytochrome c oxidase 1 (CO-1), are newer and more specific modalities for taxonomy.[2],[3] They are widely used for complementing conventional morphology-based identification methods. We report here a case of ophthalmomyiasis profunda with Chrysomya bezziana, where the organism was identified by DNA barcoding followed by genomic sequencing of the third-instar larvae, in addition to morphological identification.


  Case Report Top


An 80-year-old diabetic lady, presented to us on 30th of November 2018, with bleeding from a progressively enlarging, painless swelling in her left eye for two weeks. She admitted to have lost vision in that eye a year ago but had not sought medical help. She belonged to a low socio-economic background, had two goats at home and lived alone.

Her left orbit was severely inflamed with the complete destruction of the lids and the globe [Figure 1]a. Live larvae were attached to the necrotic tissue. There was active serosanguineous oozing from the necrotic mass. The right eye showed mild non-proliferative diabetic retinopathy and her ENT examination was unremarkable. She had a poor glycaemic control with a glycosylated hemoglobin level of 7.1%.
Figure 1: (a) Clinical picture of the presenting mass in the left orbit with a maggot being extracted. (b) CT Scan (3D reconstruction) showing distorted facial and ocular architecture. (c) CT scan (axial view) showing destruction of ocular integrity with involvement of deeper orbital tissues; (d) Gross morphology of the extracted maggots

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Orbital CT scan [Figure 1]b and [Figure 1]c revealed disorganized orbital tissues, which were filled with larvae. The orbital walls, periorbital sinuses, and the cranium were normal. A few larvae were collected [Figure 1]d and sent for species identification. In addition to the classical morphological identification techniques, DNA-based barcoding attempts were carried out targeting the CO-1 gene. Subsequently, the PCR amplified product was sequenced for further identification using BLAST analysis, which confirmed the parasite as C. bezziana, Diptera: Calliphoridae (Query cover: 93% Identical: 78.32%; GenBank Accession Number - MT787554).

She received oral Ivermectin (12 mg, single dose) with intravenous antibiotics for 5 days. Insulin injections were added to her anti-diabetic regimen. Paraffin dressing and manual removal of maggots were performed daily for a week. She underwent exenteration of the left orbit on the 8th day because of the persistence of larval instars. The post-operative period was uneventful with the left socket showed healthy granulating tissue and no evidence of residual infestation.


  Discussion Top


Ophthalmomyiasis is an uncommon parasitic infestation in humans. Orbital myiasis (ophthalmomyiasis profunda) is a severe form of ophthalmomyiasis where there is damage to ocular and periocular structures which can proceed to bone destruction.[4] Early diagnosis of C. bezziana ophthalmomyiasis is especially important as the second and third larval instars can eat through intact bone and can easily invade vital structures unless early intervention is planned.[5]

There could be multiple reasons for the late presentation of this case of ophthalmomyiasis. A combination of a low socio-economic status and family estrangement are perhaps the most important causes. Another contributory factor is her poor control of diabetes mellitus, which not only predisposes one to larval infestations but also to sensory neuropathies. This neuropathy may explain the apparent analgesia and the advanced stage at presentation.

The aim of treatment is maggot removal. Maggots resist removal by digging into deeper tissue and holding on with their hook-like structures. Attempted forceful removal may result in incomplete extraction, leading to granulomatous inflammation and calcification.[6] They are therefore asphyxiated with petroleum jelly, paraffin, or dehydrated using turpentine oil or hypertonic saline prior to manual extraction. Oral Ivermectin (200 μg/kg) may also be useful.[6]

There have been reports which have tried to detail the light microscopic and scanning electron microscopic findings of the larval instars but it is technically difficult and requires an experienced entomologist.[7] Genetic sequencing using taxon barcodes as proposed by Herbert et al. is an extremely sensitive and specific technique for species identification and diagnosis.[3]

For performing barcoding in our case, whole body DNA extraction was carried out using QIAGEN kit (QIAGEN, Hilden, Germany) as described by the manufacturer. Thereafter, the CO-1 gene was amplified using universal primers as described by Simon et al.[8] The amplified gene product was DNA sequenced using custom services. The sequence-based percent identity was found to be 78.32% and the complementing evidence of morphological identification helped us to identify the sample as C. bezziana.

A single case of external ophthalmomyiasis by Sarcophaga argyrostoma has been reported where sequencing of CO-1 gene has been used to identify the organism.[9] Sequencing of the mitochondrial CB genome has been used by contemporary investigators to identify Chrysomya bezziana ophthalmomyiasis but our literature review revealed that this is the first reported case where taxon barcodes based on the CO-1 gene have been used for this organism.[10]

The main advantage of targeting the CO-1 gene for taxonomy is that the universal primers for this gene are extremely robust which enables the recovery of its 5' end from most animals.[3] However, the CB gene has been shown to offer greater informative value in a smaller fragment than CO-1.[2]


  Conclusion Top


Orbital myiasis is a very rare entity but still remains a public health issue and is encountered by general ophthalmologists. Screw worms produce an extremely invasive infestation that can be life-threatening. Opthalmologists find it extremely difficult to identify ectoparasites from morphological features alone. The study emphasizes the usefulness of DNA barcoding in complementing the classical morphology-based identification of parasites for early intervention, if one has access to such facilities.

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.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Szpila K, Hall MJR, Wardhana AH, Pape T. Morphology of the first instar larva of obligatory traumatic myiasis agents (Diptera: Calliphoridae, Sarcophagidae). Parasitol Res 2014;113:1629-40.  Back to cited text no. 1
    
2.
Tobe SS, Kitchener A, Linacre A. Cytochrome b or cytochrome c oxidase subunit I for mammalian species identification—An answer to the debate. Forensic Sci Int Genet Suppl Ser 2009;2:306-7.  Back to cited text no. 2
    
3.
Hebert PD, Cywinska A, Ball SL, deWaard JR. Biological identifications through DNA barcodes. Proc Biol Sci 2003;270:313-21.  Back to cited text no. 3
    
4.
Fabio F. Omar L. Myiasis. Clin Microbiol Rev 2012;25:79-105.  Back to cited text no. 4
    
5.
Sutherst RW, Spradbery JP, Maywald GF. The potential geographical distribution of the Old World screw-worm fly, Chrysomya bezziana. Med Vet Entomol 1989;3:273-80.  Back to cited text no. 5
    
6.
Zhou X, Kambalame DM, Zhou S, Guo X, Xia D, Yang Y, et al. Human Chrysomya bezziana myiasis: A systematic review. PLoS Negl Trop Dis 2019;13:e0007391.  Back to cited text no. 6
    
7.
Sukontason KL, Piangjai S, Boonsriwong W, Bunchu N, Ngern-klun R, Vogtsberger RC, et al. Observations of the third instar larva and puparium of Chrysomya bezziana (Diptera: Calliphoridae). Parasitol Res 2006;99:669-74.  Back to cited text no. 7
    
8.
Simon C, Frati F, Beckenbach A, Crespi B, Liu H, Flook P. Evolution, weighting and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction “primers”. Ann Entomol Soc Am 1994;87:651-701.  Back to cited text no. 8
    
9.
Graffi S, Peretz A, Wilamowski A, Schnur H, Akad F, Naftali M. External ophthalmomyiasis caused by a rare infesting larva, Sarcophaga argyrostoma. Case Rep Ophthalmol Med 2013;2013:850865.  Back to cited text no. 9
    
10.
Nabie R, Spotin A, Poormohammad B. Ophthalmomyiasis caused by Chrysomya bezziana after periocular carcinoma. Emerg Infect Dis 2019;25:2123-4.  Back to cited text no. 10
    


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