Detection and Gram Discrimination of Bacterial Pathogens from Aqueous and Vitreous Humor Using Real-Time PCR Assays

Laboratório Especial de Microbiologia Clínica (LEMC), Infectious Diseases Division, Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazil.
Investigative ophthalmology & visual science (Impact Factor: 3.66). 02/2011; 52(2):873-81. DOI: 10.1167/iovs.10-5712
Source: PubMed

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

  • [Show abstract] [Hide abstract]
    ABSTRACT: The subband gap optical absorption spectra of amorphous hydrogenated silicon p-i-n solar cells were investigated by photocurrent spectroscopy in the CPM (constant photocurrent method) mode. A comparative study was made of thin (standard) and thick p-i-n junctions as well as junctions with slightly n- and p-doped active layers. Characteristic dependencies on the applied bias voltages (forward and reverse bias and short-circuit case) were observed. A critical discussion of these results in comparison to standard measurements (I-V curves under illumination, spectral response) is given. It is found that Urbach energies (slope of the exponential tail) are not voltage bias dependent and show, in the cases observed, the expected enhancement in p- nu -n and p- pi -n diodes with respect to p-i-n diodes.
    Photovoltaic Specialists Conference, 1988., Conference Record of the Twentieth IEEE; 02/1988
  • [Show abstract] [Hide abstract]
    ABSTRACT: To determine the usefulness of real-time polymerase chain reaction (PCR) assays in the diagnosis of postoperative bacterial endophthalmitis in clinically diagnosed infectious cases and to test for bacterial DNA in control samples collected from noninfected eyes. Federal University of São Paulo, Brazil. Evaluation of diagnostic test or technology. This study comprised patients with clinically diagnosed infectious endophthalmitis after cataract surgery and vitreous samples (from noninflamed eyes obtained through vitrectomy) and aqueous samples (at end of phacoemulsification) from control patients at a single university setting. Universal and gram-specific real-time PCR, Gram staining, and culture were performed. Sensitivity and cycle thresholds were determined. Clinical and microbiologic data were also assessed. The study evaluated 11 patients with infectious endophthalmitis (9 vitreous and 7 aqueous samples), 12 control vitreous samples, and 50 control aqueous samples. Gram and culture identified 80% and 75%, respectively, of patients with infectious endophthalmitis. Real-time PCR assays were positive in 91% of patients with a clinical diagnosis of endophthalmitis using aqueous samples, vitreous samples, or both. None of the 12 vitreous controls were positive by PCR. Two aqueous control samples were positive by real-time PCR. The cycle threshold cutoff value was 36 for universal PCR (sensitivity 93.8%; specificity 100%) and 38 for gram-specific PCR (sensitivity 93.8%; specificity 100%). Gram-positive microorganisms prevailed, and visual acuity varied according to the causative bacteria. Real-time PCR provided fast and accurate diagnosis of bacterial endophthalmitis. As a quantitative technique, it may be useful in distinguishing between contamination and infection based on the cycle thresholds value.
    Journal of Cataract and Refractive Surgery 07/2011; 37(7):1244-50. DOI:10.1016/j.jcrs.2011.01.025 · 2.55 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Visual loss in infectious posterior uveitis or panuveitis can occur if proper therapy is delayed because of diagnostic uncertainty. Some disorders, such as acute retinal necrosis and bacterial endophthalmitis, can be rapidly progressive, and therefore require prompt and accurate diagnosis to guide initial therapy. Other more slowly evolving infections, such as toxoplasmic chorioretinitis or fungal endophthalmitis, can be worsened by empiric use of corticosteroids without specific antimicrobial coverage. Key ocular diagnostic features are helpful but highly variable with overlap with both non-infectious uveitis and neoplastic masquerades, even for key signs such as hypopyon. Close examination of the fundus with attention to color, location, size, border, and opacity of lesions and associated arteriolitis or frosted branch angiitis is helpful in the diagnosis of chorioretinitis. Ultrasonography is an important tool in the evaluation of eyes with suspected endophthalmitis, especially those with intracapsular infection or focal infected deposits. Testing of intraocular fluid can be extremely useful but suffers from inaccessibility, poor sensitivity, and test selections dependent on a presumptive diagnosis, which may be wrong. The dilemma for clinician is to make the correct diagnosis of a rare, blinding, variegated disease quickly enough to intercede with specific therapy or to apply empiric therapy in a sufficiently skilled manner to avert disaster and confirm the diagnosis by response to treatment. When non-infectious uveitis is in the differential, empiric corticosteroids must sometimes be used, at great risk, if clinical examination, ancillary testing, and any available intraocular diagnostic tests have failed to confirm a diagnosis.
    Eye (London, England) 11/2011; 26(2):194-201. DOI:10.1038/eye.2011.299 · 1.90 Impact Factor
Show more