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Molecular Biology Applied to the Laboratory Diagnosis of Bacterial Endophthalmitis

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Molecular Biology Applied to the Laboratory Diagnosis of Bacterial Endophthalmitis ATUALIZAÇÃO CONTINUADA Molecular biology applied to the laboratory diagnosis of bacterial endophthalmitis Biologia molecular aplicada ao diagnóstico laboratorial de endoftalmite bacteriana Paulo José Martins Bispo1 ABSTRACT Ana Luisa Höfling-Lima2 Antonio Carlos Campos Pignatari3 Bacterial endophthalmitis is a serious but uncommon intraocular infection which frequently results in vision loss. Early diagnosis and appropriate therapy are associated with better visual outcome. Conventional micro- biological methods are currently used for microbiological characterization of eyes with suspected endophthalmitis. However, the sensitivity of bacterial detection from aqueous and vitreous humor using microbiology techniques is poor, and time-consuming to confirm the results. The appli- cation of molecular methods enhances significantly laboratory confir- mation of bacterial endophthalmitis, demanding a shorter time to draw a definitive result and thereby promoting the early initiation of a more specific therapy to limit the empirical or unnecessary use of broad- spectrum antibiotics. PCR-based techniques, including post-PCR me- thods such RFLP, DNA probe hybridization and DNA sequencing have been successfully used for the diagnostic elucidation of clinically sus- pected bacterial endophthalmitis cases, showing promising application in the routine practice of ocular microbiology laboratories. Keywords: Endophthalmitis/etiology; Eye infections, bacterial; Molecular diagnostic techni- ques; Polymerase chain reaction INTRODUCTION Endophthalmitis is an inflammation of intraocular tissues, which can result from the introduction of a bacterial agent in the posterior segment of the eye. It requires urgent medical attention because it is a potentially Trabalho realizado na Universidade Federal de São destructive condition for the eye, and even with therapeutic and surgical Paulo - UNIFESP - São Paulo (SP) - Brazil. intervention, it can lead to partial or complete vision loss after a few days 1 Biomédico, Pós-graduando (Mestrado) do Laborató- of inoculation(1-2). Regarding the acquisition mechanism, endophthalmitis is rio Especial de Microbiologia Clínica - LEMC e Labo- ratório ALERTA, Disciplina de Infectologia, Depar- classified as: (1) postoperative (acute or delayed-onset), (2) posttraumatic, tamento de Medicina, Universidade Federal de São Pau- (3) endogenous and (4) miscellaneous (e.g., secondary to keratitis)(3). Pos- lo - UNIFESP - São Paulo (SP) - Brazil. 2 Professora Titular do Departamento de Oftalmologia da toperative endophthalmitis is the most common presentation and it is fre- UNIFESP - São Paulo (SP) - Brazil. quently associated with cataract surgery. Causative pathogens generally 3 Professor Titular da Disciplina de Infectologia do De- partamento de Medicina e Diretor do Laboratório Espe- originate from the normal conjunctival and eyelid flora. In the Endophthal- cial de Microbiologia Clínica - LEMC - UNIFESP- São mitis Vitrectomy Study (EVS), the recovery of gram-positive bacteria Paulo (SP) - Brazil. (94.2%) was far greater than gram-negative (6.5%) in acute postoperative Correspondence address: Paulo José Martins Bispo. Rua Leandro Dupret, 188 - Vila Clementino - São Paulo (SP) endophthalmitis cases, where staphylococci, streptococci and enterococci CEP 04025-010 (4) E-mail: [email protected] were more frequently isolated . Posttraumatic endophthalmitis is mainly Recebido para publicação em 11.09.2008 caused by normal ocular flora and environmental isolates. Staphylococci Última versão recebida em 31.03.2009 and B. cereus are the most common isolates(5-6). Endogenous endophthal- Aprovação em 04.04.2009 mitis is a less common category, corresponding to 2 to 8% of all endoph- Arq Bras Oftalmol. 2009;72(5):734-40 72(5)10.pmd 734 16/11/2009, 13:36 Molecular biology applied to the laboratory diagnosis of bacterial endophthalmitis 735 thalmitis cases. As a consequence of hematogenous dissemi- The need for PCR in ophthalmology nation, specially in compromised hosts or intravenous drug The rapid identification of the causal agent and early insti- abusers, any pathogen causing bacteremia/sepsis could gain tution of a specific antibiotic treatment are associated with access to the posterior segment of the eye resulting in infection. better visual outcomes in endophthalmitis(2). Microbiological However, it has been shown that the most common bacterial endophthalmitis diagnosis is performed routinely by culture isolates are Staphylococcus aureus, group B streptococci, and microscopic examination of the vitreous (VH) and aqueous Streptococcus pneumoniae Listeria monocytogene Klebsiel- , , (AH) humors. However, conventional microbiological tech- la spp., Escherichia coli, Pseudomonas aeruginosa, and Neis- niques are frequently insufficient to confirm clinical cases seria meningitidis(7). suspected of endophthalmitis, and the time necessary to obtain The correct and early identification of the etiological agent culture results can vary from 2 to 12 days. The consequences by analysis of vitreous and/or aqueous humor is important for for eyes not treated can be dreadful, the lack or delay of a the institution of an early and effective antibiotic therapy, and microbiological laboratory confirmation can lead to the wrong for the prevention of the inappropriate use of broad-spectrum use of some ophthalmic pharmacological therapy with poten- antibiotics, thereby minimizing the potential emergence of tial ocular toxicity, as well as the use of broad-spectrum anti- resistant bacterial strains. However, the institution of treatment biotics for several days, being the correct antibiotic therapy for patients with clinical signs of intraocular infection, presen- initiated only after the definitive identification of the causa- ting negative cultures is frequently experienced in the ophthal- tive microorganism(2,9-10). mologic routine. Sometimes, the clinical signs are secondary In clinical cases suspected of endophthalmitis, cultures to non-infectious causes, such as immune system reactions, show a positivity of about 25% to 56%(11-15). The low sensiti- chemical or physical aggression, vasculitis or neoplasia, which vity of microbiological culture is due to various factors such may be impossible to differentiate of an infection(2). Therefore, as small quantity of specimen, fixation of microorganisms in the therapeutic scheme instituted for each patient can be a solid surfaces (intraocular lens, lens fragments, capsule) and difficult choice, which can sometimes lead to a therapy that is consequent decrease of cells in the vitreous/aqueous humor, deleterious and potentially toxic to ocular tissues, producing use of antibiotics before the collection of clinical material undesirable effects or that is actually needed and advantageous and the presence of fastidious microorganisms such as agents in the case of an intraocular inflammatory infectious process. causing endophthalmitis(14). The application of molecular tech- Use of molecular techniques for the diagnosis of niques based on PCR increases significantly the cases of endo- infectious diseases phthalmitis laboratorial confirmed by and has prompted their growing utilization in this setting. Therefore, in the present The introduction of molecular biology techniques in diag- work, the results of studies that utilized molecular techniques nostic medicine, such as the polymerase chain reaction (PCR), for the laboratory microbiological characterization of bacte- and its application in clinical microbiology, established a rial endophthalmitis cases are compiled, serving as a reference new era in the detection and characterization of microorga- for those who aim to implement or utilize this type of methodo- nisms. PCR makes it possible to detect microorganisms that are difficult to detect using traditional microbiological me- logy in the routine practice of clinical ophthalmology services. thods and to reduce the time necessary for a confirmatory Molecular detection and identification in cases of laboratory report, which is an important improvement in the bacterial endophthalmitis characterization of microorganisms involved in serious and rapidly developing infections. Molecular methods have been Usually, the detection of bacteria of clinical interest by applied in the identification of antibiotic resistance mecha- molecular biology laboratories is completed by the amplifi- nisms, detection of fastidious microorganisms, viral genoty- cation of the 16S rRNA gene which codes for the small subunit ping and quantification, rapid diagnosis of bacterial and fun- of ribosomal RNA. Post-PCR analysis, utilizing hybridiza- gal infections in immunocompromised patients, epidemiolo- tion with DNA probes, restriction analysis of PCR products gical studies, and control of infection(8). Despite all the be- by RFLP (restriction fragment length polymorphism), or direct nefits of PCR use in the diagnosis of infectious diseases, bac- sequencing of these products allow the species identification terial culture has the advantages of allowing antibiotic sus- of the pathogen(9,11,14). Recently, a platform that utilizes mass ceptibility testing, important in establishing the therapeutic spectrometry of ionized PCR products (including products of course, as well as storing the cultures in reference microorga- the 16S rRNA gene) was developed which is capable of inte- nisms collection making it possible to carry out later studies grating in the same system, identification of the microorganism, related to mechanisms of drug
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