Rapid pathogen identification and antimicrobial susceptibility testing in in vitro endophthalmitis with matrix assisted laser desorption-ionization Time-of-Flight Mass Spectrometry and VITEK 2 without prior culture

Abstract

PurposePrompt clinical diagnosis and initiation of treatment are critical in the management of infectious endophthalmitis. Current methods used to identify causative agents of infectious endophthalmitis are mostly inefficient, owing to suboptimal sensitivity, length, and cost. Matrix Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) can be used to rapidly identity pathogens without a need for culture. Similarly, automated antimicrobial susceptibility test systems (AST, VITEK 2) provide accurate antimicrobial susceptibility profiles. In this proof-of-concept study, we apply these technologies for the direct identification and characterization of pathogens in vitreous samples, without culture, as an in vitro model of infectious endophthalmitis.MethodsVitreous humor aspirated from freshly enucleated porcine eyes was inoculated with different inocula of Staphylococcus aureus (S. aureus) and incubated at 37°C. Vitreous endophthalmitis samples were centrifuged and pellets were directly analyzed with MALDI-TOF MS and VITEK 2 without prior culture. S. aureus colonies that were conventionally grown on culture medium were used as control samples. Time-to-identification, minimum concentration of bacteria required for identification, and accuracy of results compared to standard methods were determined.ResultsMALDI-TOF MS achieved accurate pathogen identification from direct analysis of intraocular samples with confidence values of up to 99.9%. Time from sample processing to pathogen identification was <30 minutes. The minimum number of bacteria needed for positive identification was 7.889x106 colony forming units (cfu/μl). Direct analysis of intraocular samples with VITEK 2 gave AST profiles that were up to 94.4% identical to the positive control S. aureus analyzed per standard protocol.ConclusionOur findings demonstrate that the direct analysis of vitreous samples with MALDI-TOF MS and VITEK 2 without prior culture could serve as new, improved methods for rapid, accurate pathogen identification and targeted treatment design in infectious endophthalmitis. In vivo models and standardized comparisons against other microbiological methods are needed to determine the value of direct analysis of intraocular samples from infectious endophthalmitis with MALDI-TOF MS and VITEK 2.

Classification of pathogens by Raman spectroscopy combined with generative adversarial networks

“…Staphylococcus hominis, Vibrio alginolyticus, and Bacillus licheniformis collected from marine environments were used in this study. Single colonies of each pure culture from LB agar plates were inoculated into 10 mL LB, followed by cultivation overnight at 37 °C in a shaker incubator (Coconnier et al, 1993). After 12 h of incubation, the cells were harvested by centrifuging at 12,000 rpm for 2 min and were then resuspended in sterile physiological saline twice.…”

“…However, studies on identification methods for marine pathogens are very rare (Cantrell et al, 2020). The identification of pathogens by traditional methods is mainly based on the morphological and physiological characteristics and biochemical reactions of colonies (Chun et al, 2019). Such methods are not only time consuming and laborious but also tedious and complex, and their effectiveness in isolating similar pathogens is not high (Zhao et al, 2018).…”

Update on Current Microbiological Techniques for Pathogen Identification in Infectious Endophthalmitis

“…The mammalian eye has the feature of acting as its own culture bottle with, albeit limited, nutritional media, and can serve as a reservoir for pathogenic growth. Stemming from this principle, the potential utility of MALDI-TOF MS for direct analysis of infectious endophthalmitis samples, was previously explored in a proof-of-concept study by the authors of this review [ 49 ]. In an in vitro model of bacterial endophthalmitis, vitreous humor aspirated from freshly enucleated porcine eyes was inoculated and incubated at 37 °C with different inocula of S. aureus , and minimally processed with centrifugation to form bacterial pellets for direct analysis with matrix solution α-cyano-4-hydroxycinnamic acid.…”

“…VITEK 2 AST has been shown to have a high degree of agreement with standard methods for determining the minimum inhibitory concentration (MIC) of antibiotics, with a gain-of-time of hours to days, and high reproducibility [ 62 , 63 ]. The authors of this review demonstrated that VITEK 2 can be used to directly analyze vitreous humor samples from in vitro models of S. aureus endophthalmitis without prior culture, with up to 94.4% concordance with conventional methods [ 49 ]. Time to result attainment was 8–9.25 h with direct analysis with VITEK 2, in contrast to the multiple days needed via conventional methods [ 37 , 38 ].…”

Clinical Characteristics and Outcomes in 314 Japanese Patients with Bacterial Endophthalmitis: A Multicenter Cohort Study from J-CREST

“…Despite recent advances in the science and technology for the identification of pathogens using polymerase chain reaction (PCR) [ 39 , 40 ] and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry [ 41 , 42 ], new antibiotics, and minimally invasive vitrectomy, bacterial endophthalmitis remains a severe intraocular infection that causes a high risk of vision loss. Recently, a large cohort study in Japanese patients with postoperative endophthalmitis after cataract surgery showed that the incidence of endophthalmitis was 0.025% [ 12 ].…”