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Comparison of polymerase chain reaction and standard microbiological techniques in presumed bacterial corneal ulcers

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Abstract

To assess utility of PCR in the diagnosis of bacterial corneal ulcer and to compare sensitivity and specificity of this technique with conventional laboratory methods. A prospective nonrandomized investigative study conducted on 122 eyes of presumed bacterial keratitis. Samples were collected for bacterial and fungal culture and Gram stain smear. A separate sample was taken for PCR with 26 gauge needle and was dipped directly into eppendorff tube with lysis buffer in it. Diagnosis of culture proven bacterial keratitis was established in 53 (43.4 %) and most common bacterial isolate was staphylococcal sp. (83 %). Direct microscopic examination of Gram stained smear revealed presence of bacteria in 24 (23.9 %) specimens and PCR positivity was evident in 56 (45.5 %). In preantibiotic treated eyes culture was positive in 15 (30 %), Gram stain in 9 (18 %), and PCR in 18 (36 %). The same for untreated (fresh) eyes, positivity of culture as well as PCR was noted in 38 (52.7 %) and that of Gram stain was noted in 20 (27.7 %). Sensitivity of Gram stain and PCR was 45.28 and 88.68 % respectively; whereas specificity was 92.75 % for Gram stain and 86.96 % for PCR. The average time taken for PCR reaction was 4–8 h while culture reporting took at least 24–48 h. Our findings suggest that PCR is a good adjunct modality to the “Gold Standard” technique in the diagnosis of bacterial corneal ulcer.

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References

  1. Whitcher JP et al (1997) Corneal ulceration in developing world–a silent epidemic. Br J Ophthalmol 81:622–623

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  2. Brillant LB, Pokhrel RP et al (1985) Epidemiology of blindness in Nepal. Bull World Health Organ 63:375–386

    Google Scholar 

  3. Srinivasan M, Christine AG et al (1997) Epidemiology and etiological diagnosis corneal ulceration in Madurai, south India. Br J Ophthalmol 81:965–971

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Dandona R, Dandona L (2003) Corneal blindness in a south Indian population : need for health promotion strategies. Br J Ophthalmol 87:133–141

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. Dunlop AA, Wright ED et al (1994) Suppurative corneal ulceration in Bangladesh. A study of 142 cases examining the microbiological diagnosis, clinical and epidemiological features of bacterial and fungal keratitis. Aust N Z J Ophthalmol 22:105–110

    Article  CAS  PubMed  Google Scholar 

  6. Satpathy G, Vishalakshi P (1995) Ulcerative keratitis: microbial profile and sensitivity pattern-a five year study. Ann Ophthalmol 27:301–306

    Google Scholar 

  7. Khanal B, Deb M, Panda A et al (2005) Laboratory diagnosis in ulcerative keratitis. Ophthalmic Res 37:123–127

    Article  CAS  PubMed  Google Scholar 

  8. Sharma S, Kunimoto DY, Gopinathan U et al (2002) Evaluation of corneal scraping smear examination methods in the diagnosis of bacterial and fungal keratitis: a survey of eight years of laboratory experience. Cornea 21(7):643–647

    Article  PubMed  Google Scholar 

  9. Mullis K, Faloona F, Scharf S, Saiki R et al (1986) Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol 51(1):263–273

    Article  CAS  PubMed  Google Scholar 

  10. Rehman DA (1993) The identification of uncultured microbial pathogens. J Infect Dis 168:1–8

    Article  Google Scholar 

  11. Garcia-Ferrer FJ, Blatt AN, Laycock KA et al (1995) Molecular biologic techniques in ophthalmic pathology. Ophthalmol Clin North Am 8:25–36

    Google Scholar 

  12. Haykin PG, Tobal K, McIntyre G et al (1994) The diagnosis of delayed post-operative endophthalmitis by polymerase chain reaction of bacterial DNA in vitreous samples. J Med Microbiol 40:408–415

    Article  Google Scholar 

  13. McCabe KM, Knan G, Zhang YH et al (1995) Amplification of bacterial DNA using highly conserved sequrnces: automated analysis and potential for molecular triage of sepsis. Pediatrics 95:165–169

    CAS  PubMed  Google Scholar 

  14. Katcher HL, Schwartz I et al (1994) A distinctive property of Tth DNA polymerase: enzymatic amplification in the presence of phenol. Biotechniques 16:84–92

    CAS  PubMed  Google Scholar 

  15. Wiedbrank DL, Werner JC et al (1995) Inhibition of PCR by vitreous and aqueous fluids. J Clin Microbiol 33:2643–2646

    Google Scholar 

  16. Dworkic LL, Gibler TM et al (2002) Real time quantitative polymerase chain reaction. Diagnosis of infectious posterior uveitis. Arch Ophthalmol 120:1534–1539

    Article  Google Scholar 

  17. Alderberg JM, Wittwer C (1995) Use of Polymerase chain reaction in the diagnosis of ocular disease. Curr Opin Ophthalmol 6:80–85

    Article  Google Scholar 

  18. Van Galder RN (2001) Application of PCR to diagnosis of ophthalmic diseases. Surv Ophthalmol 46:248–258

    Article  Google Scholar 

  19. Osakabe Y, Yaguchi C, Miyai T et al (2006) Detection of streptococcus species by PCR in infectious crystalline keratopathy. Cornea 25:1227–1230

    Article  PubMed  Google Scholar 

  20. Vengayil S, Panda A, Satpathy G (2009) Polymerase chain reaction-guided diagnosis of mycotic keratitis: a prospective evaluation of its efficacy and limitations. Invest Ophthalmol Vis Sci 50(1):152–156

    Article  PubMed  Google Scholar 

  21. Wilson KH (1994) Detection of culture-resistant bacterial pathogens by amplification and sequencing of ribosomal DNA. Clin Infect Dis 18:958–962

    Article  CAS  PubMed  Google Scholar 

  22. Cockerill FR III (1999) Genetic methods for assessing antimicrobial resistance. Antimicrob Agents Chemother 43:199–212

    PubMed Central  CAS  PubMed  Google Scholar 

  23. Murakami K, Minamide W, Wada K et al (1991) Identification of methicillin-resistant strains of staphylococci by polymerase chain reaction. J Clin Microbiol 29:2240–2244

    PubMed Central  CAS  PubMed  Google Scholar 

  24. Khan MA, Ahmad S, Banu N (2010) Molecular characterisation of methicillin-resistant Staphylococcus aureus (MRSA) from keratitis patients: a microbiological analysis. Br J Ophthalmol 94(8):994–998

    Article  PubMed  Google Scholar 

  25. Satake S, Clark N, Rimland D et al (1997) Detection of vancomycin-resistant enterococci in fecal samples by PCR. J Clin Microbiol 35:2325–2330

    PubMed Central  CAS  PubMed  Google Scholar 

  26. Nachamkin I, Kang C, Weinstein MP et al (1997) Detection of resistance to isoniazid, rifampin, and streptomycin in clinical isolates of mycobacterium tuberculosis by molecular methods. Clin Infect Dis 24:894–900

    Article  CAS  PubMed  Google Scholar 

  27. Michele Knox C, Cevellos Vickey (1998) 16S ribosomal DNA typing for identification of pathogens in patients with bacterial keratitis. J Clin Microbiol 36:3492–3496

    Google Scholar 

  28. Inoue T, Ohashi Y (2013) Utility of real-time PCR analysis for appropriate diagnosis for keratitis. Cornea 32(1):S71–S76

    Article  PubMed  Google Scholar 

  29. Eleinen KG, Mohalhal AA, Elmekawy HE et al (2012) Polymerase chain reaction-guided diagnosis of infective keratitis - a hospital-based study. Curr Eye Res 37(11):1005–1011

    Article  PubMed  Google Scholar 

  30. Subrayan V, Peyman M, Lek YS et al (2010) Assessment of polymerase chain reaction in the detection of pseudomonas aeruginosa in contact lens-induced severe infectious keratitis. Eye Contact Lens 36(4):201–203

    Article  PubMed  Google Scholar 

  31. Prabhasawat P, Leelaporn A, Tesavibul N et al (2010) Molecular identification by 16S rDNA sequencing using excised corneal tissues: a useful diagnostic tool for refractory keratitis. Jpn J Ophthalmol 54(1):97–100

    Article  PubMed  Google Scholar 

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Authors and Affiliations

  1. Cornea, OSD and Refractive Surgery, Dr Rajendra Prasad Center for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India

    Anita Panda, Tajinder Pal Singh & Meenakshi Wadhwani

  2. Department of Ocular Microbiology, Dr Rajendra Prasad Center for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India

    Geeta Satpathy

  3. Department of Microbiology, North DMC Medical College, Malkaganj, Delhi, India

    Monika Matwani

  4. Hindu Rao Hospital, North DMC Medical College, Malkaganj, Delhi, 110007, India

    Anita Panda

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  1. Anita Panda
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  2. Tajinder Pal Singh
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  3. Geeta Satpathy
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  4. Meenakshi Wadhwani
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  5. Monika Matwani
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Correspondence to Anita Panda.

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Panda, A., Pal Singh, T., Satpathy, G. et al. Comparison of polymerase chain reaction and standard microbiological techniques in presumed bacterial corneal ulcers. Int Ophthalmol 35, 159–165 (2015). https://doi.org/10.1007/s10792-014-9925-9

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  • DOI: https://doi.org/10.1007/s10792-014-9925-9

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