1887

Abstract

Microsporidia are obligate intracellular parasites that infect eukaryotic cells and have emerged as major opportunistic human pathogens. Due to the difficulties in definitive laboratory diagnosis and insufficient knowledge, ocular microsporidiosis is infrequently reported in India. To improve diagnostic facilities, we have developed a novel duplex PCR (dPCR) for the simultaneous identification of both genera and species of isolates with microsporidian aetiology that cause keratitis. The material scraped from the corneas of 12 clinically diagnosed microsporidial keratitis patients was subjected to routine microbiological examinations and molecular diagnosis using a novel dPCR that targeted the small-subunit rRNA gene (SSU-rRNA) of microsporidia and using genus- and species-specific primers. Of the 12 corneal scrapes, 6 showed positive results in smears, while dPCR provided positive amplification with both pan-microsporidial and species-specific primers for 9 corneal scrapes. The results were validated by sequencing and analysis. The sensitivity of this novel dPCR method was higher than that of conventional microscopy in the diagnosis of corneal microsporidial infection. dPCR with specific primers is potentially more sensitive, specific and depends less on more complicated methods for exact identification of the aetiology of microsporidial keratitis.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.051722-0
2013-04-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/jmm/62/4/553.html?itemId=/content/journal/jmm/10.1099/jmm.0.051722-0&mimeType=html&fmt=ahah

References

  1. Abreu-Acosta N., Lorenzo-Morales J., Leal-Guio Y., Coronado-Alvarez N., Foronda P., Alcoba-Florez J., Izquierdo F., Batista-Díaz N., Del Aguila C., Valladares B. 2005; Enterocytozoon bieneusi (microsporidia) in clinical samples from immunocompetent individuals in Tenerife, Canary Islands, Spain. Trans R Soc Trop Med Hyg 99:848–855 [View Article][PubMed]
    [Google Scholar]
  2. Baker M. D., Vossbrinck C. R., Didier E. S., Maddox J. V., Shadduck J. A. 1995; Small subunit ribosomal DNA phylogeny of various microsporidia with emphasis on AIDS related forms. J Eukaryot Microbiol 42:564–570 [View Article][PubMed]
    [Google Scholar]
  3. Bharathi M. J., Ramakrishnan R., Meenakshi R., Padmavathy S., Shivakumar C., Srinivasan M. 2007; Microbial keratitis in South India: influence of risk factors, climate, and geographical variation. Ophthalmic Epidemiol 14:61–69 [View Article][PubMed]
    [Google Scholar]
  4. Cali A., Meisler D. M., Lowder C. Y., Lembach R., Ayers L., Takvorian P. M., Rutherford I., Longworth D. L., McMahon J. T., Bryan R. T. 1991; Corneal microsporidioses: characterization and identification. J Protozool 38:215S–217S[PubMed] [CrossRef]
    [Google Scholar]
  5. Das S., Sharma S., Sahu S. K., Nayak S. S., Kar S. 2012; Diagnosis, clinical features and treatment outcome of microsporidial keratoconjunctivitis. Br J Ophthalmol 96:793–795 [View Article][PubMed]
    [Google Scholar]
  6. Didier E. S. 2005; Microsporidiosis: an emerging and opportunistic infection in humans and animals. Acta Trop 94:61–76 [View Article][PubMed]
    [Google Scholar]
  7. Fedorko D. P., Hijazi Y. M. 1996; Application of molecular techniques to the diagnosis of microsporidial infection. Emerg Infect Dis 2:183–191 [View Article][PubMed]
    [Google Scholar]
  8. Franzen C., Müller A. 1999; Molecular techniques for detection, species differentiation and phylogenetic analysis of microsporidia. Clin Microbiol Rev 12:245–285
    [Google Scholar]
  9. Franzen C., Müller A. 2001; Microsporidiosis: human diseases and diagnosis. Microbes Infect 3:389–400 [View Article][PubMed]
    [Google Scholar]
  10. Friedberg D. N., Ritterband D. C. 1999; Ocular microsporidiosis. In The Microsporidia and Microsporidiosis pp. 213–314 Edited by Wittner M., Weiss L. M. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  11. Ghosh K., Weiss L. M. 2009; Molecular diagnostic tests for microsporidia. Interdiscip Perspect Infect Dis 2009:926521[PubMed]
    [Google Scholar]
  12. Hartskeerl R. A., Schuitema A. R., DeWachter R. 1993; Secondary structure of the small subunit ribosomal RNA sequence of the microsporidium Encephalitozoon cuniculi . Nucleic Acids Res 21:1489 [View Article][PubMed]
    [Google Scholar]
  13. Hartskeerl R. A., Van Gool T., Schuitema A. R., Didier E. S., Terpstra W. J. 1995; Genetic and immunological characterization of the microsporidian Septata intestinalis Cali, Kotler and Orenstein, 1993: reclassification to Encephalitozoon intestinalis . Parasitology 110:277–285 [View Article][PubMed]
    [Google Scholar]
  14. Joseph J., Murthy S., Garg P., Sharma S. 2006a; Use of different stains for microscopic evaluation of corneal scrapings for diagnosis of microsporidial keratitis. J Clin Microbiol 44:583–585 [View Article][PubMed]
    [Google Scholar]
  15. Joseph J., Vemuganti G. K., Garg P., Sharma S. 2006b; Histopathological evaluation of ocular microsporidiosis by different stains. BMC Clin Pathol 6:6 [View Article][PubMed]
    [Google Scholar]
  16. Joseph J., Sharma S., Murthy S. I., Krishna P. V., Garg P., Nutheti R., Kenneth J., Balasubramanian D. 2006c; Microsporidial keratitis in India: 16S rRNA gene-based PCR assay for diagnosis and species identification of microsporidia in clinical samples. Invest Ophthalmol Vis Sci 47:4468–4473 [View Article][PubMed]
    [Google Scholar]
  17. Katinka M. D., Duprat S., Cornillot E., Méténier G., Thomarat F., Prensier G., Barbe V., Peyretaillade E., Brottier P. other authors 2001; Genome sequence and gene compaction of the eukaryote parasite Encephalitozoon cuniculi . Nature 414:450–453 [View Article][PubMed]
    [Google Scholar]
  18. Menotti J., Cassinat B., Porcher R., Sarfati C., Derouin F., Molina J. M. 2003; Development of a real-time polymerase-chain-reaction assay for quantitative detection of Enterocytozoon bieneusi DNA in stool specimens from immunocompromised patients with intestinal microsporidiosis. J Infect Dis 187:1469–1474 [View Article][PubMed]
    [Google Scholar]
  19. Peyretaillade E., Biderre C., Peyret P., Duffieux F., Méténier G., Gouy M., Michot B., Vivarès C. P. 1998; Microsporidian Encephalitozoon cuniculi, a unicellular eukaryote with an unusual chromosomal dispersion of ribosomal genes and a LSU rRNA reduced to the universal core. Nucleic Acids Res 26:3513–3520 [View Article][PubMed]
    [Google Scholar]
  20. Pieniazek N. J., Da Silva A. J., Slemenda S. B., Visvesvara G. S., Kurtti T. J., Yasunaga C. 1996; Nosema trichoplusiae is a synonym of Nosema bombycis based on the sequence of the small subunit ribosomal RNA coding region. J Invertebr Pathol 67:316–317 [View Article][PubMed]
    [Google Scholar]
  21. Polley S. D., Boadi S., Watson J., Curry A., Chiodini P. L. 2011; Detection and species identification of microsporidial infections using SYBR Green real-time PCR. J Med Microbiol 60:459–466 [View Article][PubMed]
    [Google Scholar]
  22. Raynaud L., Delbac F., Broussolle V., Rabodonirina M., Girault V., Wallon M., Cozon G., Vivares C. P., Peyron F. 1998; Identification of Encephalitozoon intestinalis in travelers with chronic diarrhea by specific PCR amplification. J Clin Microbiol 36:37–40[PubMed]
    [Google Scholar]
  23. Reddy A. K., Balne P. K., Gaje K., Garg P. 2011; PCR for the diagnosis and species identification of microsporidia in patients with keratitis. Clin Microbiol Infect 17:476–478 [View Article][PubMed]
    [Google Scholar]
  24. Schwartz D. A., Visvesvara G. S., Diesenhouse M. C., Weber R., Font R. L., Wilson L. A., Corrent G., Serdarevic O. N., Rosberger D. F. other authors 1993; Pathologic features and immunofluorescent antibody demonstration of ocular microsporidiosis (Encephalitozoon hellem) in seven patients with acquired immunodeficiency syndrome. Am J Ophthalmol 115:285–292[PubMed] [CrossRef]
    [Google Scholar]
  25. Sharma S., Das S., Joseph J., Vemuganti G. K., Murthy S. 2011; Microsporidial keratitis: need for increased awareness. Surv Ophthalmol 56:1–22 [View Article][PubMed]
    [Google Scholar]
  26. Silveira H., Canning E. U. 1995; Vittaforma corneae n. comb. for the human microsporidium Nosema corneum Shadduck, Meccoli, Davis & Font, 1990, based on its ultrastructure in the liver of experimentally infected athymic mice. J Eukaryot Microbiol 42:158–165 [View Article][PubMed]
    [Google Scholar]
  27. Slamovits C. H., Fast N. M., Law J. S., Keeling P. J. 2004; Genome compaction and stability in microsporidian intracellular parasites. Curr Biol 14:891–896 [View Article][PubMed]
    [Google Scholar]
  28. Van de Peer Y., Ben Ali A., Meyer A. 2000; Microsporidia: accumulating molecular evidence that a group of amitochondriate and suspectedly primitive eukaryotes are just curious fungi. Gene 246:1–8 [View Article][PubMed]
    [Google Scholar]
  29. Vavra J., Larsson J. I. R. 1999; Structure of the Microsporidia. In The Microsporidia and Microsporidiosis pp. 7–84 Edited by Wittner M., Weiss L. M. Washington, DC: American Society for Microbiology; [CrossRef]
    [Google Scholar]
  30. Vivarès C. P., Méténier G. 2000; Towards the minimal eukaryotic parasitic genome. Curr Opin Microbiol 3:463–467 [View Article][PubMed]
    [Google Scholar]
  31. Vossbrinck C. R., Maddox J. V., Friedman S., Debrunner-Vossbrinck B. A., Woese C. R. 1987; Ribosomal RNA sequence suggests microsporidia are extremely ancient eukaryotes. Nature 326:411–414 [View Article][PubMed]
    [Google Scholar]
  32. Vossbrinck C. R., Baker M. D., Didier E. S., Debrunner-Vossbrinck B. A., Shadduck J. A. 1993; Ribosomal DNA sequences of Encephalitozoon hellem and Encephalitozoon cuniculi: species identification and phylogenetic construction. J Eukaryot Microbiol 40:354–362 [View Article][PubMed]
    [Google Scholar]
  33. Witner M., Weiss L. M. 1999 The Microsporidia and Microsporidiosis Washington, DC: American Society for Microbiology;
    [Google Scholar]
  34. Woese C. R. 1987; Bacterial evolution. Microbiol Rev 51:221–271[PubMed]
    [Google Scholar]
  35. Zhu X., Wittner M., Tanowitz H. B., Cali A., Weiss L. M. 1994; Ribosomal RNA sequences of Enterocytozoon bieneusi, Septata intestinalis and Ameson michaelis: phylogenetic construction and structural correspondence. J Eukaryot Microbiol 41:204–209 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.051722-0
Loading
/content/journal/jmm/10.1099/jmm.0.051722-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error