Direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry and real-time PCR in a combined protocol for diagnosis of bloodstream infections: a turnaround time approach

ABSTRACT Bloodstream infections (BSIs) are serious infections associated with high rates of morbidity and mortality. Every hour delay in initiation of an effective antibiotic increases mortality due to sepsis by 7%. Turnaround time (TAT) for conventional blood cultures takes 48 h, forcing physicians to streamline therapy by exposing patients to broad-spectrum antimicrobials. Our objective was (1) to evaluate the accuracy and TAT of an optimized workflow combining direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and in-house real-time polymerase chain reaction (PCR) for bacterial identification and antimicrobial resistance profiling directly from positive blood bottles for diagnosing bloodstream infections and (2) to verify the effect of reporting results to medical staff. A total of 103 BSI episodes from 91 patients admitted to three hospitals in São Paulo, Brazil were included. TAT from molecular versus conventional methods was measured and compared. Our protocol showed an overall agreement of 93.5% for genus and 78.5% for species identification; 74.2% for methicillin resistance detection, 89.2% for extended-spectrum β-lactamase profiling, 77.8% for metallo-β-lactamase profiling, and 100% for carbapenemase profile and vancomycin-resistance detection when compared with conventional testing. TAT of molecular sample processing according to our protocol was 38 h shorter than conventional methods. Antimicrobial interventions were possible in 27 BSI episodes. Antimicrobial discontinuation was achieved in 12 BSI episodes while escalation of therapy occurred in 15 episodes. Antimicrobial therapy was inadequate in three (12%) BSI episodes diagnosed using results of molecular testing. Our in-house rapid protocol for identifying both bacteria and antimicrobial resistance provided rapid and accurate results, having good agreement with conventional testing results. These results could contribute to faster antimicrobial therapy interventions in BSI episodes.

Nocardial scleritis: A case report and a suggested algorithm for disease management based on a literature review.

PURPOSE: To report a case of nocardial scleritis and to propose a logical treatment algorithm based on a literature review. OBSERVATIONS: It is important to suspect a nocardial infection when evaluating anterior unilateral scleritis accompanied by multiple purulent or necrotic abscesses, especially in male patients with a history of chronic ocular pain and redness, trauma inflicted by organic materials, or recent ophthalmic surgery. A microbiological investigation is essential. In positive cases, a direct smear reveals weakly acid-fast organisms or Gram-positive, thin, beading and branching filaments. Also, the organism (usually) grows on blood agar and Lowenstein-Jensen plates. An infection can generally be fully resolved by debridement of necrotic areas and application of topical amikacin drops accompanied by systemic sulfamethoxazole-trimethoprim. CONCLUSIONS AND SIGNIFICANCE: Together with the case report described, we review data on a total of 43 eyes with nocardial scleritis. Our proposed algorithm may afford a useful understanding of this sight-threatening disease, facilitating easier and faster diagnosis and management.

Risk factors for KPC-producing Klebsiella pneumoniae: watch out for surgery.

This study describes the molecular characteristics and risk factors associated with carbapenem-resistant Klebsiella pneumoniae strains. Risk factors associated with KPC-producing K. pneumoniae strains were investigated in this case-control study from May 2011 to May 2013. Bacterial identification was performed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Antimicrobial susceptibility was determined by broth microdilution. Carbapenemase production was assessed by both modified Hodge test (MHT) and ertapenem hydrolysis using MALDI-TOF MS. The presence of ß-lactamase-encoding genes was evaluated by PCR and DNA sequencing. Alterations in genes encoding K. pneumoniae outer membrane proteins were analysed by PCR and DNA sequencing as well as SDS-PAGE. Genetic relatedness among strains was determined by pulsed-field gel electrophoresis. This study included 94 patients. Longer hospitalisation, mechanical ventilation, catheters, and previous surgery were associated with KPC-producing K. pneumoniae. Sixty-eight strains showed resistance to carbapenems. Carbapenemase production was detected by MHT in 67 K. pneumoniae strains and by MALDI-TOF MS in 57. The presence of the blaKPC-2 gene was identified in 57 strains. The blaKPC-2 gene was not found in 11 carbapenem-resistant K. pneumoniae; instead, the blaCTX-M-1-like, blaCTX-M-2-like, blaCTX-M-8 like, blaCTX-M-14-like and blaSHV- like genes associated with OmpK35 and OmpK36 alterations were observed. Thirty-three KPC-producing K. pneumoniae strains were clonally related, and patients infected with these strains had a higher mortality rate (78.78 %). Our results show that KPC-producing K. pneumoniae was associated with several healthcare-related risk factors, including recent surgery.

Avaliação das metodologias M.I.C.E.®, Etest® e microdiluição em caldo para determinação da CIM em isolados clínicos / Evaluation of M.I.C.E.TM, Etest® and CLSI broth microdilution methods for antimicrobial susceptibility testing of nosocomial bacterial isolates

INTRODUÇÃO: As fitas Oxoid® M.I.C.Evaluator® (M.I.C.E., Thermo Fisher Scientific, Basingstoke, UK), recém-lançadas no mercado brasileiro, representam uma alternativa rápida para a realização de testes de sensibilidade a antimicrobianos (TSA). OBJETIVO: Avaliar o desempenho da metodologia M.I.C.E. em relação à microdiluição em caldo (teste de referência) e ao Etest® (BioMérieux, Marcy l'Étoile, France). Material e métodos: Foram selecionados 160 isolados bacterianos, sendo P. aeruginosa (20), Acinetobacter spp. (20), K. pneumoniae (20), E. coli (20), S. aureus (20), Staphylococcus coagulase-negativa (20), E. faecalis (20) e E. faecium (20). Os TSAs foram realizados por microdiluição em caldo, Etest e M.I.C.E., seguindo-se as recomendações do Clinical Laboratory Standards Institute (CLSI, 2009) e dos respectivos fabricantes. Os resultados foram interpretados segundo os critérios estabelecidos pelo CLSI e comparados por análise de regressão. RESULTADOS: Avaliando-se todas as combinações de antimicrobianos vs. a espécie bacteriana, o desempenho da metodologia M.I.C.E. foi muito bom, apresentando uma concordância geral (variação na concentração inibitória mínima [CIM] ± 1-log2) > 90 por cento, exceto para cefotaxima (85 por cento) e vancomicina (76,3 por cento), quando em comparação com os resultados da metodologia de referência. Quando comparado com o Etest, a metodologia M.I.C.E. apresentou concordância geral > 96 por cento, com exceção para a combinação amoxicilina/ácido clavulânico (67,5 por cento). CONCLUSÃO: Os resultados do TSA obtidos pela metodologia M.I.C.E. apresentaram boa correlação com aqueles obtidos pela microdiluição em caldo e pelo Etest, indicando que essa metodologia é uma alternativa rápida para a determinação da CIM pelos laboratórios de microbiologia clínica. Atenção especial deve ser dada á determinação da CIM para a combinação amoxicilina/ácido clavulânico.

INTRODUCTION: The Oxoid® M.I.C.EvaluatorTM methodology (M.I.C.E., Thermo Fisher Scientific, Basingstoke, UK), recently released into the market, represents a rapid alternative to antimicrobial susceptibility testing. OBJECTIVE: The objective of this study was to evaluate the performance of M.I.C.E. methodology in relation to broth microdilution (reference test) and Etest® (BioMérieux, Marcy l'Étoile, France). Material and method: A total of 160 bacterial isolates were collected comprising the following species: P. aeruginosa (20), Acinetobacter spp. (20), K. pneumoniae (20), E. coli (20), S. aureus (20), coagulase-negative Staphylococcus (20), E. faecalis (20) and E. faecium (20). Following Clinical Laboratory Standands Institute (CLSI) standards (2009) and the manufacturers' recommendations, antimicrobial susceptibility testing was performed using broth microdilution method, Etest and M.I.C.E. The results were interpreted according to the criteria established by CLSI and compared through regression analysis. RESULTS: All antimicrobial combinations vs. bacterial species were evaluated and M.I.C.E. methodology yielded good results with general correlation (MIC variation ± 1-log2) > 90 percent, except for cefotaxime (85 percent) and vancomycin (76.3 percent) when compared with the reference method. The M.I.C.E. results compared to Etest showed general correlation (> 96 percent), except for amoxicillin/clavulanic acid (67.5 percent) combination. CONCLUSION: AST results obtained from M.I.C.E. methodology showed a good correlation with those from broth microdilution and Etest, which corroborates its time effectiveness in the determination of MIC. However, the combination of amoxicillin/clavulanic acid requires further attention.