PCR-based approach for qualitative molecular analysis of six neurotropic pathogens.

In the last few years, polymerase chain reaction analysis is frequently required to improve the detection of pathogen infections in central nervous system as a potential cause of neurological disorders and neuropsychiatric symptoms. The goal of this paper is to set up a fast, cheap and reliable molecular approach for qualitative detection of six neurotropic pathogens. A method based on PCR has been designed and implemented to guarantee the qualitative DNA detection of herpes simplex virus types 1 and 2 (HSVI/II), Epstein-Barr virus (EBV), cytomegalovirus (CMV), varicella-zoster virus (VZV), rubella virus (RUBV) and Toxoplasma gondii in the cerebrospinal fluid, where otherwise they are barely detectable. Each PCR assay was tested using dilutions of positive controls, which demonstrated a sensitivity allowing to detect up to 102 copies/ml in PCR and 10 copies/ml in real-time PCR for each pathogen. Once been set up, the protocol was applied to evaluate the cerebrospinal fluid from 100 patients with suspected infectious diseases of the central nervous system and 50 patients without any infection. The method allowed to identify 17 positive cerebrospinal fluid with polymerase chain reaction and 22 with real-time PCR (RT-PCR), respectively. Therefore, application of RT PCR allows a fast and sensitive evaluation of neurotropic DNA pathogens in the course of diagnostic routine within neurological units.

In vitro evaluation of different antimicrobial combinations against carbapenemase-producing Klebsiella pneumoniae: the activity of the double-carbapenem regimen is related to meropenem MIC value.

Objectives and methods: We evaluated the in vitro activity of different antimicrobial combinations with and without colistin against 39 carbapenem-resistant Klebsiella pneumoniae (CR-Kp) strains (colistin + meropenem/doripenem, colistin + tigecycline, colistin + rifampicin, gentamicin + meropenem, gentamicin + tigecycline and the double-carbapenem regimen meropenem + ertapenem) using the chequerboard method. The triple combination colistin + meropenem + tigecycline was also tested. In addition, killing studies were performed for meropenem + ertapenem. Results: Gentamicin-based combinations showed a high level of synergy. Meropenem + ertapenem was synergic in 12/39 (30.7%) of the strains, whereas based on killing studies 1 × MIC meropenem + 1 × MIC ertapenem and 2 × MIC meropenem + 1 × MIC ertapenem combinations were bactericidal and synergic at 24 h [mean area under the bactericidal curve (AUBC) 54.9 ±âŸ26.1 and 44.2 ±âŸ15.3 compared with 1 × MIC meropenem (134.5 ±âŸ40.1) and 2 × MIC meropenem (126.4 ±âŸ5.4), respectively, P < 0.0001]. When the results were stratified according to meropenem MIC, we found that the degree of synergy significantly increased for isolates with lower meropenem (and not ertapenem) MICs, up to an MIC of 128 mg/L. Among colistin-containing combinations, synergy was observed in 18/39 (46.1%), 33/34 (97%), 24/39 (61.5%) and 17/39 (43.5%) of the strains for colistin + meropenem, colistin + rifampicin, colistin + tigecycline and colistin + doripenem, respectively, including colistin-resistant strains. Colistin + meropenem + tigecycline at subinhibitory concentrations resulted in the absence of growth of 37/39 strains (94.8%). Conclusions: Our in vitro data suggest that colistin might be a valid therapeutic option against CR-Kp, even in the presence of colistin resistance, whereas the double-carbapenem regimen represents a viable option when colistin is not recommended, especially if the meropenem MIC is ≤ 128 mg/L. Since traditional antimicrobial susceptibility reports are not sufficiently informative for clinicians, synergy testing as well as actual meropenem MIC evaluation should always be performed in the case of CR-Kp infections.

A seven-year prospective study on spondylodiscitis: epidemiological and microbiological features.

BACKGROUND: The aim of this paper was to enlarge the available knowledge on clinical and etiological aspects of patients affected by spondylodiscitis. PATIENTS AND METHODS: All patients with spondylodiscitis admitted between January 2001 and December 2007 at the 1,300-bed University Hospital "Policlinico Umberto I" of Rome, Italy, were followed. Demographic characteristics, underlying diseases, invasive procedures, imaging studies, isolated microorganisms, treatment, complications, and outcome were recorded. RESULTS: Eighty-one patients of mean age 57.7 +/- 14.7 years with lumbosacral (72.8%), thoracic (14.8%), and cervical tract (12.3%) site of infection were included, of which 38 developed community-acquired (CA) spondylodiscitis and 43 developed hospital-acquired (HA) spondylodiscitis. Underlying disease was present in 49.4% of patients. HA spondylodiscitis was diagnosed earlier (46.8 +/- 49.7 days) than CA spondylodiscitis (65.0 +/- 55.4 days) (P < 0.05). The most frequently isolated microorganisms were Staphylococcus aureus (28 strains, 43.1%), coagulase-negative staphylococci (CNS) (eight strains, 12.3%), Pseudomonas aeruginosa (eight strains, 12.3%), and three methicillin-resistant S. aureus (MRSA) strains were isolated in CA spondylodiscitis. Fungi and yeasts, isolated in six patients, represented 9.2% of all strains but 17.6% when considering only HA spondylodiscitis. Over 85% of patients were managed by conservative treatment alone, and the treatment time depended on clinical and laboratory evidence. Poor outcome was recorded in 12 (14.8%) patients, and was associated with neurological deficit symptoms (relative risk [RR] 2.87; 95% confidence interval [CI] 1.02-8.07; P < 0.05) and the time between diagnosis and the onset of symptoms > or = 60 days (RR 2.65; 95% CI 0.92-7.59; P < 0.05). CONCLUSIONS: Infectious spondylodiscitis affects most frequently the elderly population, who are more exposed to healthcare contacts. Consequently, the infection etiology includes a growing proportion of multi-resistant bacteria and fungi.

Hospital-acquired infection surveillance in a neurosurgical intensive care unit.

A prospective study on hospital-acquired infection (HAI) was undertaken in the eight-bed neurosurgical intensive care unit (NSICU) of a teaching hospital in Rome, Italy. All patients admitted for >48 h between January 2002 and December 2004 were included. The infection control team collected the following data from all patients: demographic characteristics, patient origin, diagnosis, severity score, underlying diseases, invasive procedures, HAI, isolated micro-organisms and antibiotic susceptibilities. Overall, 323 patients were included in the study. Mean age was 55.5 years (range 17-91), and mean American Society of Anesthesiologists' score was 2.88. Seventy (21.7%) patients developed 132 NSICU HAIs: 43 pneumonias, 40 bloodstream infections (BSIs), 30 urinary tract infections (UTIs), 10 cases of meningitis associated with an external ventricular drain (EVD) and nine surgical site infections (SSIs). The SSI rate was high (5.6%), but a reduction was achieved during the three-year period. There were 7.2 bloodstream infection episodes per 1000 days of device exposure; 11.00 pneumonias per 1000 days of mechanical ventilation and 4.5 UTIs per 1,000 days of urinary catheterisation. Among patients with an EVD, the SSI relative risk was 11.3 [95% confidence intervals (CI) 4.2-30.6; P<0.01]. Sixty-one (18.9%) patients died. Logistic regression analysis showed that mortality was significantly associated with infection [odds ratio (OR)=2.28; 95%CI 1.11-4.71; P=0.02] and age (OR=1.04; 95%CI 1.01-1.06; P=0.002). Candida spp. were the leading cause of UTIs (40.0%) and the third most common cause of BSIs (12.7%). Antibiotic-resistant pathogens included meticillin-resistant staphylococci (77.5%), carbapenem-resistant Pseudomonas aeruginosa (36.4%), and extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (75.0%). Although the overall incidence of infection (21.7%) was within the range of published data, the associated mortality, the increasing severity of illness of patients, and the emergence of multi-drug-resistant organisms shows the need to improve infection control measures.

Conservative medical therapy of prosthetic joint infections: retrospective analysis of an 8-year experience.

Successful treatment of prosthetic joint infections often requires multiple surgical interventions and prolonged antimicrobial therapy. However, in certain situations, a surgical approach may not be in the best interest of the patient. A conservative approach was used to treat 34 patients with prosthetic joint infection between 1995 and 2003. Diagnosis of infection was based on clinical-microbiological evidence, confirmed by (99)Tc-labelled leukocyte scintigraphy, and involved 12 Staphylococcus aureus infections, nine Staphylococcus epidermidis infections, two Enterococcus faecalis infections, two mixed infections (S. aureus plus Pseudomonas aeruginosa; S. epidermidis plus E. faecalis), with the infecting pathogen being unidentified for nine patients. Most infections were treated initially with intravenous or intramuscular teicoplanin +/- ciprofloxacin or rifampicin, followed by oral ciprofloxacin or minocycline plus rifampicin. The mean duration of antimicrobial therapy was 41.2 weeks. Overall, only three patients did not respond to therapy, and infection was controlled in the remaining 31 patients. Among these, no relapse was observed in 17 patients during follow-up for 9-57 months; improvement with early (within 6 months of antibiotic discontinuation) or late relapse was observed in seven and three patients, respectively; two patients improved clinically, but continued to receive antibiotic therapy; and two patients whose condition improved initially were lost after a 6-month follow-up following discontinuation of antibiotics. No patient complained of side effects requiring discontinuation of antibiotic therapy. The study confirmed that suppression of infection, with salvage of the infected device in an acceptably functional state, can be achieved in selected cases.