Trematode Fluke Procerovum varium as Cause of Ocular Inflammation in Children, South India

Abstract

Trematodes are recognized as a group of emerging parasites in tropical countries. We identified a trematode as a cause of ocular granulomas that developed in children who bathed in ponds or rivers in South India. DNA was isolated from patients' surgically excised granulomas and from the trematode cercariae (larvae) released by the snail Melanoides tuberculata in water in which the children bathed. Real-time and conventional PCRs were performed that targeted ribosomal DNA regions spanning the internal transcribed spacer 2 and 28S sequences of this trematode. The PCR-amplified products were subjected to bidirectional sequencing. Analysis of sequences for the granuloma samples and the trematode cercariae showed maximum sequence similarity with Procerovum varium (family Heterophyidae). Our results confirmed the etiology of the ocular infection, implicating snail vectors as environmental risk factors for ocular parasitosis.

Keywords: India; cercaria; child; eye disease; fresh water; granuloma; lakes; parasites; ponds; real-time PCR; ribosomal DNA; rivers; sequencing; trematode; uveitis.

Figure 1

Eleven district sites (gray shading) where snails were collected in the state of Tamil Nadu, India, for testing as part of a study of ocular granulomas in children. Inset shows location of Tamil Nadu in India.

Figure 2

Clinical photographs of patients’ eyes in study of ocular granulomas in children, South India. A) Left eye of a 14-year-old boy with a distinct subconjunctival granuloma; B) left eye of a 7-year-old boy with distinct grayish-white granuloma in the eye’s anterior chamber.

Figure 3

Real-time PCR amplification of ocular granuloma DNA obtained from patients infected with trematodes, South India. Gel electrophoresis was performed on 2% agarose gel by using Power SYBR Green Real-Time PCR (Applied Biosystems, Warrington, UK). A) Lanes 1–4 show subconjunctival granuloma DNA; lane 5, negative control; lane 6, 100-bp DNA marker. Arrow indicates 369-bp amplified DNA product. B) Lanes 1–5 show anterior chamber granuloma DNA; lane 6, negative control; lane 7, 100-bp DNA marker. Arrow indicates 369-bp amplified DNA product. C) BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) analysis output of patient granuloma DNA sequence showing maximum identity with internal transcribed spacer 2 region gene sequence of the Procerovum species resembling GenBank reported sequence EU826639.1 from Vietnam.

Figure 4

Snail and trematode cercaria from study of ocular inflammation in children, South India. A) Melanoides tuberculata snails collected from a pond that was the focus of the infection. B) Staining and light microscopy image of the cercaria larval stage recovered from the snails (original magnification ×200).

Figure 5

PCR amplification of trematode cercaria DNA obtained from Melanoides tuberculata snails in study of ocular inflammation in children, South India. Gel electrophoresis was performed on 1.5% agarose gel. A) Internal transcribed spacer 2 region; arrow indicates 369-bp amplified DNA product. Lanes 1–3, trematode cercariae DNA; lane 4, negative control; lane 5, 100-bp DNA marker. B) Internal transcribed spacer 2 region; arrow indicates 539-bp–amplified DNA product. Lanes 1–4, trematode cercariae DNA; lane 5, negative control; lane 6, 100-bp DNA marker. C) 28S rDNA region; arrow indicates 715-bp amplified DNA product. Lanes 1–4, trematode cercariae DNA; lane 5, negative control; lane 6, 100-bp DNA marker.

Figure 6

BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) analysis output of environmental trematode cercaria DNA sequences from South India. A) Internal transcribed spacer 2 DNA sequence shows maximum identity with Procerovum species and resembles GenBank reported sequence EU826639.1 from Vietnam. B) 28S rDNA sequence shows maximum identity with Procerovum varium and resembles GenBank reported sequence HM004184.1 from Thailand.