Broad-Range Bacterial and Fungal DNA Amplification on Vitreous Humor from Suspected Endophthalmitis Patients
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ORIGINAL RESEARCH ARTICLE
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Broad-Range Bacterial and Fungal DNA Amplification on Vitreous Humor from Suspected Endophthalmitis Patients Betsy Varghese,1 Camilla Rodrigues,1 Minal Deshmukh,1 S. Natarajan,2 Paritosh Kamdar3 and Ajita Mehta1 1 2 3
Department of Microbiology and Research, P.D. Hinduja National Hospital and Medical Research Center, Mumbai, India Aditya Jyot Eye Hospital, Mumbai, India Jashem Eye Institute, Mumbai, India
Abstract
Background: Prompt and accurate diagnosis of infectious endophthalmitis is crucial for rapid and effective treatment. By identifying whether the causative pathogen is bacterial or fungal, a rational approach for the use of antibacterials or corticosteroids, respectively, can be followed. Aim: To assess the clinical utility of broad-range bacterial and fungal DNA amplification in the detection of endophthalmitis (postoperative, posttraumatic, and endogenous). Methods: In a prospective study, vitreous humor samples from 70 patients with the clinical diagnosis of presumed endophthalmitis, and from 30 patients undergoing surgery for non-infectious causes, were subjected to routine microbiologic and molecular investigation. DNA extracted from a 50μL sample was amplified by primers targeting the conserved 16S and 18S ribosomal RNA gene sequences of bacteria and fungi, respectively. Reagents for bacterial DNA amplification were decontaminated of endogenous DNA using 8-methoxypsoralen and long wave UV treatment. Results and Discussion: A total of 35 specimens were positive for bacteria or fungi by culture. Of these, Gram-positive organisms were isolated in 19 specimens, Gram-negative organisms in 13 specimens and fungi in 3 specimens. Pseudomonas species, coagulase-negative Staphylococcus, and Streptococcus species were the main etiological agents isolated. Bacterial DNA amplification resulted in 49 positive specimens, compared with 32 positive specimens by culture; and fungal DNA amplification resulted in 11 positive specimens, compared with 3 positive specimens by culture. All control specimens were negative for both culture and DNA amplification. Conclusion: DNA extracted using a single-extraction protocol from 50μL of vitreous humor and amplified with broad-range bacterial and fungal primers will enable the rapid differentiation (within 14 hours) between bacterial and fungal endophthalmitis and allow tailoring of therapy to individual patients.
Background In endophthalmitis, it is important for the ophthalmologist to determine whether the origin of inflammation is bacterial, fungal, or sterile. Early distinction of the pathogenic agent, with commencement of the appropriate therapy, may result in better visual outcome. Although culture investigations of aqueous and vitreous humor (VH) are of immense value in endophthalmitis diagnosis, they are often negative as a result of various factors.[1,2] Therefore,
a more rapid and sensitive technique is required for the early detection of endophthalmitis. In infections where the spec
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