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Research Letters |

Novel Etiological Agent: Molecular Evidence for Trematode-Induced Anterior Uveitis in Children FREE

Sivakumar R. Rathinam, FAMS, PhD; Lalan Kumar Arya, MSc; Kim R. Usha, MD; Lalitha Prajna, MD; Veena Tandon, PhD
[+] Author Affiliations

Author Affiliations: Uveitis Service (Dr Rathinam) and Orbit and Ocular Prosthetics Services (Dr Usha), Aravind Eye Hospital and P. G. Institute of Ophthalmology, and Department of Microbiology, Aravind Medical Research Foundation (Mr Arya and Dr Prajna), Madurai, and Parasitology Laboratory, Department of Zoology, School of Life Sciences, North Eastern Hill University, Shillong (Dr Tandon), India.


Arch Ophthalmol. 2012;130(11):1481-1484. doi:10.1001/archophthalmol.2012.729.
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Published online

Histopathological analysis has provided support that trematode infections can cause a characteristic granulomatous anterior uveitis in children from South India.1 Southeast Asian populations are exposed to at least 70 species of foodborne and waterborne trematodes. The burden of disease and current distribution of the parasites within the population are largely unknown, however.2 Serologic testing is unreliable mainly because of cross-reactive antigens.2 Fecal egg identification likewise has limited utility because humans act as accidental hosts. Molecular diagnostic studies, in contrast, can identify individual species of trematodes involved in site-specific infections. In this study, we have applied polymerase chain reaction–based techniques to identify the trematode Procerovum varium as a cause of granulomatous anterior uveitis in children from South India.

A 9-year-old boy from the village of Jagathapatinum in Tamil Nadu, India, came to the hospital with a 2-week history of left eye redness, which he said started after bathing in the village pond. There was no history of trauma. Visual acuity was 20/20 OU and intraocular pressures were normal bilaterally. Slitlamp examination of the left eye revealed a normal conjunctiva and cornea. A whitish granuloma was seen in the anterior chamber at the 6-o’clock position (Figure 1A). Fundus examination findings were normal. Systemic examination revealed a normal complete blood cell count and no evidence of tuberculosis, syphilis, or sarcoidosis. Stool and urine examination findings were unremarkable. Following informed consent, both aqueous fluid and the granuloma were aspirated using a 25-gauge needle under general anesthesia. The aqueous fluid was sent for cellular analysis, which revealed a predominance of eosinophils, consistent with our previously published work1 (Figure 1B). Extraction of DNA from the granuloma was performed using a Qiagen polymerase chain reaction purification kit. Snails from the same village pond were collected for cercarial isolation in our laboratory (Figure 1C and D). The snails that released the cercarial larvae were sent to the Zoological Survey of India, Calcutta, India, where they were identified as Melanoides tuberculata. Real-time polymerase chain reaction was performed using the SYBR-Green assay on both the granuloma and cercarial DNA. A standard protocol3 was followed, using the universal forward primer AP101 (5′-AGAGCGCAGCCAACTGTGTGA-3′) and reverse primer AP101 (5′-TGCCACGTCCTAGCATCAGCC-3′). Nuclease-free water was used as a negative control and Fasciola gigantica DNA was the positive control4 (Figure 2). The amplified DNA was loaded on a 2% agarose gel and found to be a 369–base pair fragment of the ribosomal DNA spanning the internal transcribed spacer 2 region. Bidirectional sequencing and Basic Local Alignment Search Tool analysis were carried out using a standard protocol.3 Both the environmental cercaria and the granuloma revealed maximal similarity with P varium —a trematode of the family Heterophyidae (Figure 2D).

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Figure 1. Clinical and histopathological findings and snails with cercarial larvae. A, Anterior chamber granuloma in a 9-year-old boy. B, Aqueous fluid showing numerous eosinophils (E), neutrophil (N), lymphocyte (L), and a single macrophage (M) (hematoxylin-eosin, original magnification ×200). C, Melanoides tuberculata snails collected from the pond thought to be the focus of infection. D, Light microscopic image of the cercaria larval stage recovered from the snails (original magnification ×200).

Place holder to copy figure label and caption
Graphic Jump Location

Figure 2. Molecular confirmation of trematode etiology. A, Real-time polymerase chain reaction amplification plot of ribosomal DNA from the internal transcribed spacer 2 region, showing the positive control (Fasciola gigantica DNA, 100 pg) (a), granuloma DNA from the patient (b), cercarial DNA (c), and the negative control (nuclease-free water) (d). Rn indicates normalized reporter. B, Dissociation curve, showing the positive control (F gigantica DNA, 100 pg) (a), granuloma DNA from the patient (b), cercarial DNA (c), and the negative control (nuclease-free water) (d). C, Gel electrophoresis of the amplified DNA checked on 2% agarose gel. Lane 1 indicates a 100–base pair (bp) DNA ladder marker; lane 2, negative control; lane 3, positive control; lane 4, granuloma DNA; and lane 5, cercarial DNA. D, Representative electropherogram confirming the sequence of the trematode Procerovum varium. The numbers above the bases indicate the nucleotide sequence of the internal transcribed spacer 2 region of the trematode.

Granulomatous uveitis has traditionally raised concern for tuberculosis infection among patients seen in regional hospitals of South India. Many South Indian children with granulomatous anterior uveitis have received antituberculosis treatment, which has proven ineffective.1,5 Notably, these patients typically describe a strong temporal association between the onset of symptoms and having bathed in regional ponds or waterways. Recently, our histopathological study of affected ocular tissues suggested the presence of a trematode infection,1 which we have now identified as P varium. Snails act as the first intermediate host for this trematode in regional ponds, where they release cercarial larvae to infect the fish, which act as second intermediate hosts. Infected fish then transmit the trematode to birds.6 Fish-eating birds are the definitive hosts, while infected children become accidental hosts. Our data, together with the clustering of cases of granulomatous anterior uveitis in children from South Indian villages, suggest that trematode infection may be common in areas where fish and waterfowl infection is endemic.

Correspondence: Dr Rathinam, Uveitis Service, Aravind Eye Hospital and P. G. Institute of Ophthalmology, 1, Anna Nagar, Madurai, Tamil Nadu 625 020, India (rathinam@aravind.org).

Author Contributions: All the authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Conflict of Interest Disclosures: None reported.

Funding/Support: This work was supported by the Aravind Medical Research Foundation.

Additional Contributions: Shanthi Rathakrishnan, MD, read the hematoxylin-eosin–stained slide of aqueous fluid.

Rathinam SR, Usha KR, Rao NA. Presumed trematode-induced granulomatous anterior uveitis: a newly recognized cause of intraocular inflammation in children from south India.  Am J Ophthalmol. 2002;133(6):773-779
PubMed   |  Link to Article
Johansen MV, Sithithaworn P, Bergquist R, Utzinger J. Towards improved diagnosis of zoonotic trematode infections in Southeast Asia.  Adv Parasitol. 2010;73:171-195
PubMed
Gomes AL, Melo FL, Werkhauser RP, Abath FG. Development of a real time polymerase chain reaction for quantitation of Schistosoma mansoni DNA.  Mem Inst Oswaldo Cruz. 2006;101:(suppl 1)  133-136
PubMed   |  Link to Article
Prasad PK, Goswami LM, Tandon V, Chatterjee A. PCR-based molecular characterization and insilico analysis of food-borne trematode parasites Paragonimus westermani, Fasciolopsis buski, and Fasciola gigantica from Northeast India using ITS2 rDNA.  Bioinformation. 2011;6(2):64-68
PubMed   |  Link to Article
Rajamohan M, Srikanth K, Raghuraman V, Srinivasan R, Nelson Jesudasan CA. Conglomerate tubercle: myth or reality?  Tamil Nadu Ophthalmic Assoc J. 1998;38:23-24
Umadevi K, Madhavi R. Observations on the morphology and life-cycle of Procerovum varium (Onji & Nishio, 1916) (Trematoda: Heterophyidae).  Syst Parasitol. 2000;46(3):215-225
PubMed   |  Link to Article

Figures

Place holder to copy figure label and caption
Graphic Jump Location

Figure 1. Clinical and histopathological findings and snails with cercarial larvae. A, Anterior chamber granuloma in a 9-year-old boy. B, Aqueous fluid showing numerous eosinophils (E), neutrophil (N), lymphocyte (L), and a single macrophage (M) (hematoxylin-eosin, original magnification ×200). C, Melanoides tuberculata snails collected from the pond thought to be the focus of infection. D, Light microscopic image of the cercaria larval stage recovered from the snails (original magnification ×200).

Place holder to copy figure label and caption
Graphic Jump Location

Figure 2. Molecular confirmation of trematode etiology. A, Real-time polymerase chain reaction amplification plot of ribosomal DNA from the internal transcribed spacer 2 region, showing the positive control (Fasciola gigantica DNA, 100 pg) (a), granuloma DNA from the patient (b), cercarial DNA (c), and the negative control (nuclease-free water) (d). Rn indicates normalized reporter. B, Dissociation curve, showing the positive control (F gigantica DNA, 100 pg) (a), granuloma DNA from the patient (b), cercarial DNA (c), and the negative control (nuclease-free water) (d). C, Gel electrophoresis of the amplified DNA checked on 2% agarose gel. Lane 1 indicates a 100–base pair (bp) DNA ladder marker; lane 2, negative control; lane 3, positive control; lane 4, granuloma DNA; and lane 5, cercarial DNA. D, Representative electropherogram confirming the sequence of the trematode Procerovum varium. The numbers above the bases indicate the nucleotide sequence of the internal transcribed spacer 2 region of the trematode.

Tables

References

Rathinam SR, Usha KR, Rao NA. Presumed trematode-induced granulomatous anterior uveitis: a newly recognized cause of intraocular inflammation in children from south India.  Am J Ophthalmol. 2002;133(6):773-779
PubMed   |  Link to Article
Johansen MV, Sithithaworn P, Bergquist R, Utzinger J. Towards improved diagnosis of zoonotic trematode infections in Southeast Asia.  Adv Parasitol. 2010;73:171-195
PubMed
Gomes AL, Melo FL, Werkhauser RP, Abath FG. Development of a real time polymerase chain reaction for quantitation of Schistosoma mansoni DNA.  Mem Inst Oswaldo Cruz. 2006;101:(suppl 1)  133-136
PubMed   |  Link to Article
Prasad PK, Goswami LM, Tandon V, Chatterjee A. PCR-based molecular characterization and insilico analysis of food-borne trematode parasites Paragonimus westermani, Fasciolopsis buski, and Fasciola gigantica from Northeast India using ITS2 rDNA.  Bioinformation. 2011;6(2):64-68
PubMed   |  Link to Article
Rajamohan M, Srikanth K, Raghuraman V, Srinivasan R, Nelson Jesudasan CA. Conglomerate tubercle: myth or reality?  Tamil Nadu Ophthalmic Assoc J. 1998;38:23-24
Umadevi K, Madhavi R. Observations on the morphology and life-cycle of Procerovum varium (Onji & Nishio, 1916) (Trematoda: Heterophyidae).  Syst Parasitol. 2000;46(3):215-225
PubMed   |  Link to Article

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