Detection of viable but non-culturable Pseudomonas aeruginosa in cystic fibrosis by qPCR: a validation study.

BACKGROUND: Routine culture-based diagnosis of Pseudomonas aeruginosa lung infection in Cystic Fibrosis (CF) patients can be hampered by the phenotypic variability of the microorganism, including its transition to a Viable But Non-Culturable (VBNC) state. The aim of this study was to validate an ecfX-targeting qPCR protocol developed to detect all viable P. aeruginosa bacteria and to identify VBNC forms in CF sputum samples. METHODS: The study involved 115 P. aeruginosa strains of different origins and 10 non-P. aeruginosa strains and 88 CF sputum samples, 41 Culture-Positive (CP) and 47 Culture-Negative (CN). Spiking assays were performed using scalar dilutions of a mixture of live and dead P. aeruginosa ATCC 9027 and a pooled P. aeruginosa-free sputum batch. Total DNA from sputum samples was extracted by a commercial kit, whereas a crude extract was obtained from the broth cultures. Extracellular DNA (eDNA) interference was evaluated by comparing the qPCR counts obtained from DNase-treated and untreated aliquots of the same samples. The statistical significance of the results was assessed by the Wilcoxon test and Student's t test. RESULTS: The newly-developed qPCR protocol identified 96.6% of the P. aeruginosa isolates; no amplification was obtained with strains belonging to different species. Spiking assays supported protocol reliability, since counts always matched the amount of live bacteria, thus excluding the interference of dead cells and eDNA. The protocol sensitivity threshold was 70 cells/ml of the original sample. Moreover, qPCR detected P. aeruginosa in 9/47 CN samples and showed higher bacterial counts compared with the culture method in 10/41 CP samples. CONCLUSIONS: Our findings demonstrate the reliability of the newly-developed qPCR protocol and further highlight the need for harnessing a non-culture approach to achieve an accurate microbiological diagnosis of P. aeruginosa CF lung infection and a greater understanding of its evolution.

Risk Factors and Impact on Clinical Outcome of Multidrug-Resistant Acinetobacter Baumannii Acquisition in Cardiac Surgery Patients.

OBJECTIVES: Acinetobacter baumannii recently has emerged as an important nosocomial pathogen. The aim of this study was to assess the impact on mortality of multidrug-resistant A. baumannii (MDR-AB) infection/colonization in patients undergoing cardiac surgery and to investigate microbiologic characteristics, epidemiologic spread of this pathogen, and the relative containment measures. DESIGN: Single-center, retrospective cohort study of prospectively collected data. SETTING: Cardiac surgery tertiary-care center. PARTICIPANTS: Patients with positive MDR-AB cultures from September 1, 2009 to December 31, 2011. INTERVENTIONS: Bivariate and multivariate analyses were performed to individualize the risk factors for MDR-AB-infections in cardiac surgery patients. To evaluate the MDR-AB attributable mortality, a retrospective matched cohort study was performed. Incidence density ratio (IDR) was calculated to compare the MDR-AB infection/colonization before and after the introduction of preventive measures adopted following the first cases. MEASUREMENTS AND MAIN RESULTS: MDR-AB acquisition occurred in 14 patients (0,6%) of 2385 patients. At the multivariate analyses, preoperative use of inotropic drugs (OR 18.2, 95% CI 4.6-71.9) and logistic EuroSCORE (OR 1.09, 95% CI 1.06-1.13) were found as independent risk factors. Patients with MDR-AB had 57% cumulative in-hospital mortality; no statistical differences in mortality were observed in the matched group. IDR revealed a significantly decreased incidence of infection/colonization (0.3 per 1,000 days of stay compared with 0.03/1,000 days of stay, p = 0.0001) after the containment measures became effective. CONCLUSIONS: Sicker patients are more susceptible to be infected by A. baumannii, but mortality is not significantly higher compared with other patients with similar characteristics. Adequate measures are fundamental to control the spread of the infection.

Epidemiology, clinical characteristics, and outcome of candidemia in a tertiary referral center in Italy from 2010 to 2014.

PURPOSE: We evaluated the epidemiology, clinical characteristics and outcome of candidemia in a single institution from 2010 to 2014. METHODS: A retrospective observational study of all cases of candidemia was carried out at a University Hospital in Central Italy including five intensive care units (ICUs), 11 medical and 11 surgical wards. Data regarding demographic characteristics and clinical risk factors were collected from the patient's medical records. Antifungal susceptibility testing was performed and MIC results were interpreted according to species-specific clinical breakpoints. RESULTS: A total of 270 episodes of candidemia were identified. Overall incidence rate was 1.5 episodes/1000 hospital admissions. Although Candida albicans represented the most commonly isolated species, its percentage significantly decreased from 68 to 48 % (p = 0.040). The overall 30-day mortality was 35 %. The variables independently associated with a significant higher risk of mortality were: older age; being hospitalized in ICU or in medical wards vs surgical wards; being infected with C. albicans vs other species; the occurrence of septic shock, pneumonia and acute renal failure; the presence of a solid organ tumor or a chronic pulmonary disease. Conversely, an appropriate treatment was confirmed to be significantly associated with a lower risk of mortality. The overall resistance was low and it was noted only among triazoles. CONCLUSIONS: Our study shows that candidemia is a significant source of morbidity and mortality. The identification of risk factors associated with mortality along with the knowledge of local susceptibility may lead to a better management in terms of preventive and therapeutic measures.

Diagnostic value of C-reactive protein in discriminating fungal from nonfungal pulmonary infiltrates in patients with hematologic malignancies.

The objective of this study was to assess the utility of C-reactive protein (CRP) on differential diagnosis of pulmonary infiltrates occurring in 143 febrile patients with hematologic malignancies. Serum CRP level, measured on the first day of pneumonia, was significantly higher in patients with fungal lung infiltrates than in those with nonfungal pneumonia (22.3 mg/dl vs 7.3 mg/dl; p<0.0001). Predictive factors for fungal pneumonia were CRP level higher than 10 mg/dl, neutropenia longer than 10 days, and active underlying disease.

Risk assessment of patients with hematologic malignancies who develop fever accompanied by pulmonary infiltrates: a historical cohort study.

BACKGROUND: The mortality rate associated with fever accompanied by pulmonary infiltrates after chemotherapy for hematologic malignancies remains higher than the corresponding rate associated with febrile neutropenia without pulmonary infiltrates. Nonetheless, few studies have focused on the factors that predict outcome for patients with lung infiltrates. The purpose of the current study was to construct a risk model for clinical use by assessing the factors that affect outcome for patients with fever and pulmonary infiltrates. METHODS: A historical cohort of 110 patients with hematologic malignancies who developed fever and pulmonary infiltrates was examined. Using parameters for which data were available at the onset of lung infiltrates, univariate and multivariate analyses were performed to assess factors affecting outcome. After a value of one point was assigned to each significant variable, a prediction score was calculated for each patient; scores were used to generate a system for identifying patients with a low risk of death due to fever accompanied by pulmonary infiltrates. RESULTS: The crude mortality rate associated with pulmonary infiltrates was 23%; factors associated with cure included a favorable change in white blood cell counts (odds ratio [OR], 5.6; 95% confidence interval [CI], 1.7-18.9; P = 0.001), C-reactive protein levels < 10 mg/dL (OR, 4.6; 95% CI, 1.6-13.8; P = 0.001), and serum albumin levels > or = 3 g/dL (OR, 3.2; 95% CI, 1.4-7.3; P = 0.004). Low-risk patients (risk score, 2-3) and high-risk patients (risk score, 0-1) had survival rates of 95% and 46%, respectively (P < 0.0001). The risk model had a specificity of 88% and a positive predictive value of 95%. CONCLUSIONS: The risk model tested in the current study accurately predicted the survival of patients with hematologic malignancies who developed fever with pulmonary infiltrates. Once prospectively validated, the model could be used to select patients for trials involving novel diagnostic and therapeutic strategies.