Angela Cardaccia

Diagnostic Performance of a Multiple Real-Time PCR Assay in Patients with Suspected Sepsis Hospitalized in an Internal Medicine Ward

ABSTRACT Early identification of causative pathogen in sepsis patients is pivotal to improve clinical outcome. SeptiFast (SF), a commercially available system for molecular diagnosis of sepsis based on PCR, has been mostly used in patients hospitalized in hematology and intensive care units. We evaluated the diagnostic accuracy and clinical usefulness of SF, compared to blood culture (BC), in 391 patients with suspected sepsis, hospitalized in a department of internal medicine. A causative pathogen was identified in 85 patients (22%). Sixty pathogens were detected by SF and 57 by BC. No significant differences were found between the two methods in the rates of pathogen detection (P = 0.74), even after excluding 9 pathogens which were isolated by BC and were not included in the SF master list (P = 0.096). The combination of SF and BC significantly improved the diagnostic yield in comparison to BC alone (P < 0.001). Compared to BC, SF showed a significantly lower contamination rate (0 versus 19 cases; P < 0.001) with a higher specificity for pathogen identification (1.00, 95% confidence interval [CI] of 0.99 to 1.00, versus 0.94, 95% CI of 0.90 to 0.96; P = 0.005) and a higher positive predictive value (1.00, 95% CI of 1.00 to 0.92%, versus 0.75, 95% CI of 0.63 to 0.83; P = 0.005). In the subgroup of patients (n = 191) who had been receiving antibiotic treatment for ≥24 h, SF identified more pathogens (16 versus 6; P = 0.049) compared to BC. These results suggest that, in patients with suspected sepsis, hospitalized in an internal medicine ward, SF could be a highly valuable adjunct to conventional BC, particularly in patients under antibiotic treatment.

Primary brain abscess with Nocardia farcinica in an immunocompetent patient

In this paper, we describe a case of an immunocompetent patient with cerebral nocardiosis. The onset was with loss of strength, paresthesia and focal epilepsy of the left arm. MRI showed on T2-weighted sequences a hyperintense central area of pus surrounded by a well-defined hypointense capsule and surrounding edema; on T1-weighted sequences a hypointense necrotic cavity with ring enhancement following administration of intravenous gadolinium. The patient underwent surgical excision of the abscess but culture from the specimen was negative. After 40 days of empirical antimicrobial therapy he developed neurological deterioration with focal epilepsy. A new MRI documented an enlargement of the hypointense lesion in the right frontal-parietal region. A second craniotomy with drainage of the abscess was performed; cultures yielded Nocardia farcinica. Therapy with trimethoprim/sulfamethoxazole, amikacin and meropenem was given for 35 days, and clinical and radiological improvement was observed. Home therapy was done with oral trimethoprim/sulfamethoxazole. Currently, 5 months from the second surgery, the patient can walk with support and no new episodes of epilepsy occurred. Side effects were absent from therapy. The MRI appearance of the brain lesion has improved, with a decrease in size, surrounding edema and ring enhancement.

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 better than C-reactive protein, erythrocyte sedimentation rate, and white blood cell count in predicting DNAemia in patients with sepsis

Abstract Background: Procalcitonin (PCT) levels can be used to predict bacteremia and DNAemia in patients with sepsis. In this study, the diagnostic accuracy of PCT in predicting blood culture (BC) results and DNAemia, as detected by real-time PCR (RT-PCR), was compared with that of other markers of inflammation commonly evaluated in patients with suspected sepsis, such as C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and white blood cell (WBC) count. Methods: A total of 571 patients for whom BC, blood RT-PCR, PCT, CRP, ESR, and WBC count were requested for laboratory diagnosis of sepsis were included in the study. Receiver operating characteristic curve analysis was performed to compare the ability of the above biomarkers to predict BC and blood RT-PCR results. Results: A total of 108 pathogens were identified by BC (79 pathogens, 14.5% positive rate) and/or RT-PCR (90 pathogens, 16.5% positive rate), after exclusion of 26 contaminated samples. The PCT areas under the curve (AUCs) in predicting BC (0.843; 95% CI 0.796–0.890; p < 0.0001) and RT-PCR (0.916; 95% CI 0.888-0.945; p < 0.0001) results were significantly greater than AUCs found for CRP, ESR, and WBC count. Conclusions: PCT showed a better diagnostic accuracy than CRP, ESR, and WBC count in predicting DNAemia and bacteremia in patients with suspected sepsis.

Diagnosis of infective endocarditis: comparison of the LightCycler SeptiFast real-time PCR with blood culture

Infective endocarditis (IE) is a lifethreatening condition, in which identification of the causative pathogen is pivotal for subsequent effective therapy. Blood culture (BC) is considered the gold standard for microbiological diagnosis of IE (Watkin et al., 2003) but in up to one third of patients with suspected IE, BC can be negative due to a number of possible factors, such as prior antimicrobial therapy, the presence of fastidious microorganisms, or a low bacterial load (Lamas & Eykyn, 2003; Naber & Erbel, 2007). Identification of pathogens from heart valve tissue by real-time PCR has been shown to be a reliable technique (Lang et al., 2004; Mencacci et al., 2011). Several studies have demonstrated the diagnostic value of PCR in IE patients (Kühn et al., 2011; Zaloudı́ková et al., 2011), but few studies (Casalta et al., 2009) have evaluated the diagnostic utility of the commercially available PCR-based system SeptiFast (SF, Roche Diagnostics) for testing blood specimens. The SF system is able to detect and differentiate between a wide range of Gram-negative and Gram-positive bacteria, as well as some fungi, commonly involved in systemic infections (Lehmann et al., 2008). The aim of this study was to assess the diagnostic performance of the SF system, compared with BC, using blood specimens from patients with IE.

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.

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.

A prediction model for real-time PCR results in blood samples from febrile patients with suspected sepsis.

Sepsis, a systemic, deleterious host response to infection that leads to organ dysfunction, is a potentially deadly condition needing prompt identification of the causative organisms and early appropriate antimicrobial therapy. Among non-culture-based diagnostic methods, SeptiFast (SF) can be employed to speed bacterial and fungal DNA detection, but it suffers from poor sensitivity and high cost. The aim of the present study, performed in 285 febrile patients, was to develop a prediction model to restrict the SF assay to clinical cases with a high probability of positive SF results. The prevalence of SF results positive for a pathogen was 17.2 %. Independent predictors of positive results were: blood sampling within 12 h after the onset of fever [odds ratio (OR) 20.03; 95 % confidence interval (CI) 6.87-58.38; P<0.0001]; ≥0.5 ng serum procalcitonin (PCT) ml(-1) (OR 18.52; 95 % CI 5.12-67.02; P<0.0001); body temperature ≥38 °C (OR 3.78; 95 % CI 1.39-10.25; P = 0.009); ≤3 g serum albumin dl(-1) (OR 3.40; 95 % CI 1.27-9.08; P = 0.014); and ≥13 000 white blood cells mm(-3) (OR 2.75; 95 % CI 1.09-7.69; P = 0.05). The model showed good calibration (Hosmer-Lemeshow chi-squared 1.61; P = 0.978). Area under the receiving operating characteristic curve was 0.944 (95 % CI 0.914-0.973; P<0.0001). These results suggest that a prediction model based on PCT and a few other routinely available laboratory and clinical variables could be of help in selecting patients with a high probability of SF-positive results.