Amedeo Moretti

Rapid identification of bacterial and fungal pathogens from positive blood cultures by MALDI-TOF MS.

Sepsis is a syndrome characterized by a systemic inflammatory response due to severe infection. Early detection of causal agents and appropriate antimicrobial treatment reduce mortality. Conventional microbiological methods often do not provide time critical results for an optimal early management. We used an in-house protocol based on Tween 80 to process 109 positive blood cultures for bacteria and yeast identification by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS), and results were compared to standard reference or automated methods. MALDI-TOF MS correctly identified 91.7% of the isolates. Correct identification was obtained for 57/62 (91.9%) aerobic/facultative anaerobic Gram-positive isolates, 53 (85.5%) at species level, and 4 (6.4%) at the genus level; 32/32 (100%) aerobic/facultative anaerobic Gram-negative isolates, 31 (96.9%) at species level, and 1 (3.1%) at the genus level; 7/7 (100%) obligate anaerobes, all at the genus level; 3/7 (42.8%) fungi, all at genus level. Overall, the median identification time of MALDI-TOF MS vs reference standard methods was significantly shorter: median (interquartile range) 7.1h (4.7-10.2) vs 48.1h (32.5-50.0), p<0.0001. MALDI-TOF MS is a valuable tool for rapid identification of pathogens in septic patients. An in-house protocol based on Tween 80 can be used to process positive blood cultures.

Procalcitonin Predicts Real-Time PCR Results in Blood Samples from Patients with Suspected Sepsis

Background Early diagnosis and rapid bacterial identification are of primary importance for outcome of septic patients. SeptiFast® (SF) real-time PCR assay is of potential utility in the etiological diagnosis of sepsis, but it cannot replace blood culture (BC) for routine use in clinical laboratory. Procalcitonin (PCT) is a marker of sepsis and can predict bacteremia in septic patients. The aim of the present study was to investigate whether PCT serum levels could predict SF results, and could help screening febrile patients in which a SF assay can improve the etiological diagnosis of sepsis. Methods From 1009 febrile patients with suspected sepsis, 1009 samples for BC, SF real-time PCR, and PCT determination were obtained simultaneously, and results were compared and statistically analysed. Receiver operating characteristic (ROC) curves were generated to determine the area under the curve and to identify which cut-off of PCT value produced the best sensitivity to detect SF results. Results Mean PCT values of sera drawn simultaneously with samples SF positive (35.42±61.03 ng/ml) or BC positive (23.14±51.56 ng/ml) for a pathogen were statistically higher than those drawn simultaneously with SF negative (0.84±1.67 ng/ml) or BC negative (2.79±16.64 ng/ml) samples (p<0.0001). For SF, ROC analysis showed an area under the curve of 0.927 (95% confidence interval: 0.899–0.955, p<0.0001). The PCT cut-off value of 0.37 ng/ml showed a negative predictive value of 99%, reducing the number of SF assays of 53.9%, still identifying the 96.4% of the pathogens. Conclusion PCT can be used in febrile patients with suspected sepsis to predict SF positive or negative results. A cut-off value of 0.37 ng/ml can be considered for optimal sensitivity, so that, in the routine laboratory activity, SF assay should not be used for diagnosis of sepsis in an unselected patient population with a PCT value <0.37 ng/ml.

Comparison of conventional culture with SeptiFast real-time PCR for microbial pathogen detection in clinical specimens other than blood.

Early detection of aetiological agents is pivotal for adequate therapy for bacterial infections. Although culture is still considered the mainstay for laboratory diagnosis, it often lacks sensitivity, especially in patients already treated with antibiotics. The present study investigated the potential clinical utility of the commercial real-time-PCR-based system SeptiFast (SF), originally intended for diagnosis of sepsis from blood specimens, in the aetiological diagnosis of other bacterial infections, in patients undergoing antibiotic therapy. A total of 53 non-blood specimens were analysed for microbial pathogen detection by conventional culture and with SF real-time PCR: 19 (35.8%) synovial fluids, 9 (17.0%) cardiac valve tissues and 25 (47.2%) purulent exudates from various body sites. Overall, the number of specimens positive for a pathogen by SF (26/53; 49.1%) was significantly greater (P=0.001) than that of specimens positive by culture (10/53; 18.9%). In particular, SF was superior to culture for pathogen detection in cardiac valve tissues and synovial fluids. The analysis of concordance showed a fair agreement between the two methods (kappa value=0.314; 95% confidence interval=0.531-0.097). Even with the limitation of the low number of specimens, this study confirmed the great potential of diagnosing bacterial infections by a molecular approach, and indicates that the real-time PCR SF system can be used for specimens other than blood, from patients undergoing antibiotic treatment.

Procalcitonin Levels in Gram-Positive, Gram-Negative, and Fungal Bloodstream Infections

Procalcitonin (PCT) can discriminate bacterial from viral systemic infections and true bacteremia from contaminated blood cultures. The aim of this study was to evaluate PCT diagnostic accuracy in discriminating Gram-positive, Gram-negative, and fungal bloodstream infections. A total of 1,949 samples from patients with suspected bloodstream infections were included in the study. Median PCT value in Gram-negative (13.8 ng/mL, interquartile range (IQR) 3.4–44.1) bacteremias was significantly higher than in Gram-positive (2.1 ng/mL, IQR 0.6–7.6) or fungal (0.5 ng/mL, IQR 0.4–1) infections (P < 0.0001). Receiver operating characteristic analysis showed an area under the curve (AUC) for PCT of 0.765 (95% CI 0.725–0.805, P < 0.0001) in discriminating Gram-negatives from Gram-positives at the best cut-off value of 10.8 ng/mL and an AUC of 0.944 (95% CI 0.919–0.969, P < 0.0001) in discriminating Gram-negatives from fungi at the best cut-off of 1.6 ng/mL. Additional results showed a significant difference in median PCT values between Enterobacteriaceae and nonfermentative Gram-negative bacteria (17.1 ng/mL, IQR 5.9–48.5 versus 3.5 ng/mL, IQR 0.8–21.5; P < 0.0001). This study suggests that PCT may be of value to distinguish Gram-negative from Gram-positive and fungal bloodstream infections. Nevertheless, its utility to predict different microorganisms needs to be assessed in further studies.

Bactericidal activity of oxacillin and glycopeptides against Staphylococcus aureus in patients with endocarditis: Looking for a relationship between tolerance and outcome

BackgroundThere is no clear relationship between in vitro bactericidal activity tests and clinical outcome. We studied bactericidal activity of oxacillin, vancomycin and teicoplanin against Staphylococcus aureus isolates in patients with endocarditis and then we sought to determine if there was a relationship between in vitro bactericidal activity and clinical outcome.MethodsMinimal bacteriostatic and minimal bactericidal concentrations were determined for Staphylococcus aureus strains isolated from patients with endocarditis following standardized methods. Medical records were reviewed retrospectively to collect data on antimicrobial susceptibility at admission, antimicrobial therapy, need for surgery, embolic events and outcome.Results and DiscussionSixty-two Staphylococcus aureus strains were studied in 62 patients with endocarditis. Overall, 91.9% definite, 21% methicillin resistant and 72.6% cured. Surgery was performed in 32.3% and embolic events were documented in 64.5%. Tolerance to oxacillin and teicoplanin was more common than vancomycin tolerance among methicillin susceptible Staphylococcus aureus. Among methicillin resistant Staphylococcus aureus teicoplanin was shown to have a higher rate of tolerance than vancomycin. No statistically significant differences on clinical outcome between oxacillin tolerant and oxacillin non tolerant Staphylococcus aureus infections were observed. Tolerance to oxacillin did not adversely affect clinical outcomes of patients with methicillin susceptible Staphylococcus aureus endocarditis treated with a combination of antimicrobials including oxacillin. The cure rate was significantly lower among patients with methicillin resistant Staphylococcus aureus endocarditis.ConclusionsIn vitro bactericidal test results were not valid predictors of clinical outcome. Physicians need to use additional parameters when treating patients with staphylococcal endocarditis.

In vitro Antimicrobial Activity of Ampicillin-Ceftriaxone and Ampicillin-Ertapenem Combinations Against Clinical Isolates of Enterococcus faecalis with High Levels of Aminoglycoside Resistance

This paper reports on the in vitro antimicrobial activity of ampicillin-ceftriaxone and ampicillin-ertapenem combinations against five strains of E. faecalis with high-level aminoglycoside resistance recovered from blood of septicemic patients. Double disk diffusion test and time killing curves were used. A bacteriostatic synergistic effect between ampicillin and ceftriaxone was detected using the disk diffusion assay for three of the five enterococcal strains studied. With the same three isolates enhanced bactericidal activity was also observed using time killing experiments. Overall, for these three strains, after 24 hr of contact, a decrease ≥ 2 log10 from the initial bacterial inoculum was registered with most ampicillin-ceftriaxone combinations, reaching with some of them a colony reduction ≥ 3 log10. This bactericidal interaction was negatively influenced increasing the bacterial inoculum. In all five isolates neither a bacteriostatic nor a bactericidal cooperation was observed for ampicillin combined with 2 mg/l of ertapenem. This investigation broadened the evidence of antimicrobial synergism in vitro between ampicillin and ceftriaxone in selected strains of Enterococcus faecalis with high-level aminoglycoside resistance.

A commercially available multiplex real-time PCR for detection of pathogens in cardiac valves from patients with infective endocarditis

Infective endocarditis (IE) is a life-threatening condition, burdened by high mortality. Current guidelines recommend that, in case of negative culture result, tissues from excised heart valves or vegetations from patients with suspected IE should be referred for broad-range bacterial PCR and sequencing. In this proof-of-concept study, the diagnostic utility of the commercially available multiplex real-time PCR system SeptiFast (SF), performed on cardiac valves, was evaluated in a selected population of 20 patients with definite IE of known origin, in comparison with culture. A significant difference was found between SF and culture in the rate of pathogen detection (19 versus 3 respectively; chi-square 14.06; P=0.0002). SF sensitivity was 95%; specificity, 100%; positive predictive value (PPV), 100%; and negative predictive value (NPV), 83.3%. Culture sensitivity was 15%; specificity, 100%; PPV, 100%; and NPV, 22.7%. SF assay, performed on culture-negative excised heart valves, can be useful for the etiological diagnosis of IE.

Comparison of the BD Phoenix System with the Cefoxitin Disk Diffusion Test for Detection of Methicillin Resistance in Staphylococcus aureus and Coagulase-Negative Staphylococci

ABSTRACT The BD Phoenix system was compared to the cefoxitin disk diffusion test for detection of methicillin (meticillin) resistance in 1,066 Staphylococcus aureus and 1,121 coagulase-negative staphylococcus (CoNS) clinical isolates. The sensitivity for Phoenix was 100%. The specificities were 99.86% for S. aureus and 88.4% for CoNS.

Fatal Nocardia farcinica Bacteremia Diagnosed by Matrix-Assisted Laser Desorption-Ionization Time of Flight Mass Spectrometry in a Patient with Myelodysplastic Syndrome Treated with Corticosteroids

Nocardia farcinica is a Gram-positive weakly acid-fast filamentous saprophytic bacterium, an uncommon cause of human infections, acquired usually through the respiratory tract, often life-threatening, and associated with different clinical presentations. Predisposing conditions for N. farcinica infections include hematologic malignancies, treatment with corticosteroids, and any other condition of immunosuppression. Clinical and microbiological diagnoses of N. farcinica infections are troublesome, and the isolation and identification of the etiologic agent are difficult and time-consuming processes. We describe a case of fatal disseminated infection in a patient with myelodysplastic syndrome, treated with corticosteroids, in which N. farcinica has been isolated from blood culture and identified by Matrix-Assisted Laser Desorption-Ionization Time of Flight Mass Spectrometry. The patient died after 18 days of hospitalization in spite of triple antimicrobial therapy. Nocardia farcinica infection should be suspected in patients with history of malignancy, under corticosteroid therapy, suffering from subacute pulmonary infection,and who do not respond to conventional antimicrobial therapy. Matrix-Assisted Laser Desorption-Ionization Time of Flight Mass Spectrometry can be a valuable tool for rapid diagnosis of nocardiosis.

Aqueous Humor Antimicrobial Activity: In Vitro Analysis after Topical 0.5% Chloramphenicol Application.

ABSTRACT Purpose: To assess aqueous humor antimicrobial activity in vitro after topical 0.5% chloramphenicol application. Materials and methods: This investigation included 63 eyes from 65 cataract surgery patients. The study group of 48 eyes received preoperatively four topical applications of 0.5% chloramphenicol. The control group of 15 eyes was given no topical applications. Aqueous humor samples were collected for in vitro antimicrobial analysis using Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Pasteurella multocida organisms by means of disk diffusion test. Results: No inhibition halo was observed around all aqueous humor samples from all chloramphenicol-treated patients, irrespective of the sample quantity added to the paper disks, with no significant difference from aqueous humor from untreated control patients. Conclusions: Aqueous humor displayed no bactericidal effect against any of the microorganisms evaluated after topical 0.5% chloramphenicol application.