New test for the diagnosis of bacterial endophthalmitis.
BACKGROUND
Diagnosis of bacterial endophthalmitis (BE) often fails due to: (1) insufficient volumes of vitreous fluid (VF) and aqueous humour (AH); (2) lack of sensitivity of culture; (3) antibiotic treatments; (4) polymerase chain reaction (PCR) cross-contamination; and (5) limitations on the interpretation of the real-time PCR melting curve. We developed a fast real-time (f-real-t) PCR to improve the performance of the laboratory diagnosis of BE.


METHODS
The following samples were processed after adding an internal control: phosphate buffered saline (PBS); VF, AH and cell suspensions spiked with Bacteria (Bac); VF and AH from patients with endophthalmitis; and VF and AH from non-infective patients. DNA was extracted (MagNA Pure) and added to four tubes containing selected primers and probes for the identification and quantification of all Bac and eight genera by f-real-t PCR. Diagnostic performances based on direct microscopic examination, culture and f-real-t PCR were compared.


RESULTS
The f-real-t PCR detected at least 0.01 colony-forming units (CFU) of Bac/microl with no cross-reactivity with fungi. Correlation with culture-positive results was 100%. Sixty per cent of BE samples tested culture-positive, but f-real-t PCR tested positive for 90%. Samples from non-infective cases were negative.


CONCLUSIONS
The f-real-t PCR detected and quantified Bac, Staphylococci, Streptococci, Haemophilus, Pseudomonas, Enterobacteria, Acinetobacter, Propionibacteriacae and Corynebacteria in one run. Cultures required several hours to days (with a non-negligible number of false-negative results) and the f-real-t PCR was completed in 90 min. The f-real-t PCR is presented as a new tool for the diagnosis of BE: its usefulness requires validation with larger series of samples.
Goldschmidt
P
P
Laboratoire du Centre National d'Ophtalmologie des Quinze-Vingts, 28 rue de Charenton, 75012 Paris France. pablogol@aol.com
Degorge
S
S
Benallaoua
D
D
Basli
E
E
Batellier
L
L
Boutboul
S
S
Allouch
C
C
Borderie
V
V
Laroche
L
L
Chaumeil
C
C
D001082:Aqueous Humor / Q000382:microbiology; D015373:Bacterial Typing Techniques / Q000379:methods; D004269:DNA, Bacterial / Q000032:analysis; D009877:Endophthalmitis / Q000175:diagnosis* / Q000382:microbiology; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D014822:Vitreous Body / Q000382:microbiology
D004269:DNA, Bacterial