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: 3.4). 02/2011; 52(2):873-81. DOI: 10.1167/iovs.10-5712
Source: PubMed


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.

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    • "The starting point for the design of the described nested-multiplex qPCR method was the work describing the application of the qPCR method to detect bacteria and fungi in biological materials separately – Bispo et al. described the PCR methodology in the detection of bacteria with Gram differentiation in the vitreous humor, and Sugita et al. described the PCR method for the detection of yeast and filamentous fungi in the eyeball when it is inflamed [10,11]. During the work carried out by our team, it was possible to combine the sequences of primers and probes described by the authors into a multiplex reaction for simultaneous detection of bacteria and fungi with their differentiation into Gram-negative bacteria, Gram-positive bacteria, yeast fungi, and filamentous fungi. "
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    ABSTRACT: Background The study describes the application of the PCR method for the simultaneous detection of DNA of Gram-negative bacteria, Gram-positive bacteria, yeast fungi and filamentous fungi in blood and, thus, a whole range of microbial etiological agents that may cause sepsis. Material for the study was sterile blood inoculated with four species of microorganisms (Escherichia coli, Staphylococcus aureus, Candida albicans and Aspergillus fumigatus) and blood collected from patients with clinical symptoms of sepsis. The developed method is based on nested-multiplex real-time PCR . Results Analysis of the obtained data shows that sensitivity of nested-multiplex real-time PCR remained at the level of 101 CFU/ml for each of the four studied species of microorganisms and the percentage of positive results of the examined blood samples from the patients was 70% and 19% for the microbiological culture method. The designed primers correctly typed the studied species as belonging to the groups of Gram-positive bacteria, Gram-negative bacteria, yeast fungi, or filamentous fungi. Conclusions Results obtained by us indicated that the designed PCR methods: (1) allow to detect bacteria in whole blood samples, (2) are much more sensitive than culture method, (3) allow differentiation of the main groups of microorganisms within a few hours.
    Full-text · Article · Jun 2014 · BMC Microbiology
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    • "and Fusarium spp. [7]. All the primers and probes were selected from the National Center for Biotechnology Information website (NCBI; and synthesized by Applied Biosystems (Foster City, CA, USA). "
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    ABSTRACT: Background Early identification of pathogens and antimicrobial resistance in bloodstream infections (BSIs) decreases morbidity and mortality, particularly in immunocompromised patients. The aim of the present study was to compare real-time polymerase chain reaction (PCR) with commercial kits for detection of 17 pathogens from blood culture (BC) and 10 antimicrobial resistance genes. Methods A total of 160 BCs were taken from bone marrow transplant patients and screened with Gram-specific probes by multiplex real-time PCR and 17 genus-specific sequences using TaqMan probes and blaSHV, blaTEM, blaCTX, blaKPC, blaIMP, blaSPM, blaVIM, vanA, vanB, and mecA genes by SYBR Green. Results Twenty-three of 33 samples identified by phenotypic testing were concordantly positive by BC and real-time PCR. Pathogen identification was discordant in 13 cases. In 12 of 15 coagulase-negative staphylococci, the mecA gene was detected and four Enterococcus spp. were positive for vanA. Two blaCTX and three blaSHV genes were found by quantitative PCR. The blaKPC and metallo-β-lactamase genes were not detected. Five fungal species were identified only by real-time PCR. Conclusions Real-time PCR could be a valuable complementary tool in the management of BSI in bone marrow transplants patients, allowing identification of pathogens and antimicrobial resistance genes.
    Full-text · Article · Apr 2013 · BMC Infectious Diseases
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    • "The use of PCR enabled a positive result to be obtained in 95% of patients. In this study the rate of false positive PCR results was low, occurring in 3.4% of patients [20]. "
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    ABSTRACT: A correct diagnosis of uveitis is often challenging, given the wide range of possible underlying conditions and the lack of typical phenotypes. Management decisions may be difficult in view of the risk of visual loss with either inappropriate or delayed therapy. Analysis of the vitreous may therefore be used to provide the clinician with valuable information. In this paper, we describe the main clinical situations in which vitreous sampling is indicated and provide some guidance to clinicians for tailoring their requests. These situations include suspected intraocular infection and suspected intraocular malignancy. We describe the principal tests carried out on vitreous samples, including cultures, polymerase chain reaction-based testing, and cytokine analysis. Limitations of the tests used are likely to become less as more advanced testing methods are introduced. The importance of selecting the appropriate investigations to support a clinical suspicion is emphasised, as is the interpretation of test results within a clinical context.
    Full-text · Article · Oct 2012 · Mediators of Inflammation
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