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New test for the diagnosis of bacterial endophthalmitis
  1. Pablo Goldschmidt (pablogol{at}aol.com),
  2. Sandrine Degorge (s.degorge{at}quinze-vingts.fr),
  3. Djida Benallaoua,
  4. Elena Basli,
  5. Laurence Batellier,
  6. Sandrine Boutboul,
  7. Cécile Allouch,
  8. Vincent Borderie,
  9. Laurent Laroche,
  10. Christine Chaumeil (c.chaumeil{at}quinze-vingts.fr)
  1. Laboratoire du Centre National d'Ophtalmologie des Quinze-Vingts, France
  2. Laboratoire du Centre National d'Ophtalmologie des Quinze-Vingts, France
  3. Laboratoire du Centre National d'Ophtalmologie des Quinze-Vingts, France
  4. Service 5- Centre National d'Ophtalmologie des Quinze-Vingts, France
  5. Laboratoire du Centre National d'Ophtalmologie des Quinze-Vingts, France
  6. Service 5- Centre National d'Ophtalmologie des Quinze-Vingts, France
  7. Service 5- Centre National d'Ophtalmologie des Quinze-Vingts, France
  8. Service 5- Centre National d'Ophtalmologie des Quinze-Vingts, France
  9. Service 5- Centre National d'Ophtalmologie des Quinze-Vingts, France
  10. Laboratoire du Centre National d'Ophtalmologie des Quinze-Vingts, France

    Abstract

    Background: Diagnosis of bacterial endophthalmitis (BE) often fails due to: 1-reduced volumes of vitreous fluid (VF) and aqueous humor (AH); 2-lack of sensitivity of culture; 3-antibiotic treatments; 4-PCR cross-contamination and 5-limitations for real-time PCR melting curve interpretation. We developed an fast real-time-PCR to improve laboratory diagnosis performances.

    Methods: a-PBS, VF, AH and cell suspensions spiked with Bacteria; b-VF and AH from endophthalmitis, and c-VF and AH from non-infective disorders were processed after adding an internal control (IC). DNA was extracted (MagnaPure®) and introduced into 4 tubes containing selected primers and probes for the identification and quantification of all Bacteria (Bac) and of 8 Genera by fast real-t PCR (f-real-t PCR). Performances of diagnosis based on direct microscopic examination, culture and f-real-t PCR were compared.

    Results: The f-real-t PCR detected at least 0.01 CFU Bac/µl with no cross reactivity with fungi. Correlation with culture positive results was 100%. Sixty % of BE samples tested culture positive but f-real-t PCR tested positive for 90%. Samples from non-infective cases were negative.

    Conclusion: The f-real-t PCR detects and quantifies Bac, Staphylococci, Streptococci, Haemophilus, Pseudomonas, Enterobacteria, Acinetobacter, Propionibacteriacae and Corynebacteria in one run. Cultures required several hours to days (with non-negligible number of false negative results) and the f-real-t PCR was completed in 90 minutes. The f-real-t PCR appears as a new tool for the diagnosis of BE and its usefulness requires validation with larger series of samples.

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