Detection and gram discrimination of bacterial pathogens from aqueous and vitreous humor using real-time PCR assays.
OBJECTIVE
To develop and apply real-time PCR protocols to the detection and classification of the Gram status of bacterial pathogens in aqueous and vitreous humor collected from clinically suspected intraocular infections.


METHODS
The analytical specificity of two PCR assays, SYBR Green 16S rDNA-Based Universal PCR (SGRU-PCR), and a Multiplex Gram-Specific TaqMan-Based PCR (MGST-PCR), was determined with 31 clinically important pathogens, including 20 Gram-positive and 11 Gram-negative. Analytical sensitivity was determined with a 10-fold dilution of Staphylococcus epidermidis and Escherichia coli DNA. Assays were further tested on aqueous (n = 10) and vitreous humor (n = 11) samples collected from patients with clinically diagnosed intraocular infections.


RESULTS
DNA was amplified from all control bacterial isolates when using SGRU-PCR. MGST-PCR correctly classified the Gram status of all these isolates. The SGRU-PCR limit of detection of S. epidermidis and E. coli DNA was 100 fg/μL (E = 0.82 and 0.86; r(2) = 0.99) and for MGST-PCR, 1 pg/μL (E = 0.66 and 0.70; r(2) = 0.99. For clinical intraocular samples, positivity of culture was 47.6% and for real-time PCR assays, 95.2%. Gram classification was achieved in 100% of MGST-PCR-positive samples. Among microbiologically negative samples, real-time PCR assays were positive in 90% of cases. The false-positive rate in control aqueous was 3.2%, and control samples of vitreous were negative.


CONCLUSIONS
The real-time PCR assays demonstrated good correlation, with culture-proven


RESULTS
With the use of these methods, bacterial detection was improved from 47.6% to 95.3%, demonstrating them to be sensitive, rapid tests for diagnosis of bacterial endophthalmitis.
Bispo
Paulo José Martins
PJ
Laboratório Especial de Microbiologia Clínica (LEMC), Infectious Diseases Division, Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazil.
de Melo
Gustavo Barreto
GB
Hofling-Lima
Ana Luisa
AL
Pignatari
Antonio Carlos Campos
AC
D001082:Aqueous Humor / Q000382:microbiology*; D001419:Bacteria / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000032:analysis; D009877:Endophthalmitis / Q000175:diagnosis / Q000382:microbiology*; D015818:Eye Infections, Bacterial / Q000175:diagnosis / Q000382:microbiology*; D005840:Gentian Violet; D006801:Humans; D010619:Phenazines; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D014822:Vitreous Body / Q000382:microbiology*
D004269:DNA, Bacterial; C035863:Gram's stain; D010619:Phenazines; D012336:RNA, Ribosomal, 16S; D005840:Gentian Violet