Use of polymerase chain reaction (PCR) and DNA probe hybridization to determine the Gram reaction of the infecting bacterium in the intraocular fluids of patients with endophthalmitis.
OBJECTIVE
To evaluate polymerase chain reaction (PCR) combined with DNA probe hybridization to determine the Gram reaction of the bacterium in intraocular specimens from patients with infectious endophthalmitis.


METHODS
Fifty-seven intraocular specimens - 17 aqueous humor (AH) and 40 vitreous fluid (VF) - from 55 patients with clinically diagnosed infectious endophthalmitis and 25 control intraocular specimens from non-infectious ocular disorders (10 AH and 15 VF) were evaluated by microscopy, culture and PCR-DNA probe hybridization to detect the Gram reaction of the bacterium.


RESULTS
PCR-DNA probe hybridization was specific and sensitive to detect 30 fg of both gram-positive and gram-negative bacterial DNA. None of the controls showed bacteria by microscopy, culture or PCR. Of the 57 intraocular specimens, conventional microbiological methods could detect a bacterial aetiology in 32 (56.1%), while PCR-DNA probe hybridization could detect 52 (91.2%) specimens. This difference was statistically significant (P= 0.003). In bacteriologically positive specimens, there was absolute correlation of the Gram reaction between the results of smear and culture methods and PCR-DNA probe hybridization. Of the 25 bacteriologically negative specimens, 20 (80%) were positive by PCR-DNA probe hybridization, of which seven (35%) were gram-positive, 12 (60%) gram-negative and one (5%) positive by both. Results of PCR on AH and VF were not significantly different.


CONCLUSIONS
PCR and DNA probe hybridization to determine the Gram reaction of the bacterium in intraocular fluids is a specific and sensitive method in the diagnosis of bacterial endophthalmitis. AH is an ideal specimen for PCR, since its collection is a simple and safe office procedure.
Anand
A R
AR
Microbiology Research Centre, Sankara Nethralaya, 18 College Road, Chennai, 600006, India.
Madhavan
H N
HN
Therese
K L
KL
D001082:Aqueous Humor / Q000382:microbiology*; D015342:DNA Probes; D004269:DNA, Bacterial / Q000032:analysis; D009877:Endophthalmitis / Q000175:diagnosis* / Q000382:microbiology; D006090:Gram-Negative Bacteria / Q000235:genetics / Q000302:isolation & purification*; D016905:Gram-Negative Bacterial Infections / Q000175:diagnosis / Q000382:microbiology; D006094:Gram-Positive Bacteria / Q000235:genetics / Q000302:isolation & purification*; D016908:Gram-Positive Bacterial Infections / Q000175:diagnosis / Q000382:microbiology; D006801:Humans; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014822:Vitreous Body / Q000382:microbiology
D015342:DNA Probes; D004269:DNA, Bacterial