Giovanni Fadda

Elevated Levels of C-Reactive Protein at Discharge in Patients With Unstable Angina Predict Recurrent Instability

BACKGROUND In a group of patients admitted for unstable angina, we investigated whether C-reactive protein (CRP) plasma levels remain elevated at discharge and whether persistent elevation is associated with recurrence of instability. METHODS AND RESULTS We measured plasma levels of CRP, serum amyloid A protein (SAA), fibrinogen, total cholesterol, and Helicobacter pylori and Chlamydia pneumoniae antibody titers in 53 patients admitted to our coronary care unit for Braunwald class IIIB unstable angina. Blood samples were taken on admission, at discharge, and after 3 months. Patients were followed for 1 year. At discharge, CRP was elevated (>3 mg/L) in 49% of patients; of these, 42% had elevated levels on admission and at 3 months. Only 15% of patients with discharge levels of CRP <3 mg/L but 69% of those with elevated CRP (P<0.001) were readmitted because of recurrence of instability or new myocardial infarction. New phases of instability occurred in 13% of patients in the lower tertile of CRP (</=2.5 mg/L), in 42% of those in the intermediate tertile (2.6 to 8.6 mg/L), and in 67% of those in the upper tertile (>/=8.7 mg/L, P<0.001). The prognostic value of SAA was similar to that of CRP; that of fibrinogen was not significant. Chlamydia pneumoniae but not Helicobacter pylori antibody titers significantly correlated with CRP plasma levels. CONCLUSIONS In unstable angina, CRP may remain elevated for at >/=3 months after the waning of symptoms and is associated with recurrent instability. Elevation of acute-phase reactants in unstable angina could represent a hallmark of subclinical persistent instability or of susceptibility to recurrent instability and, at least in some patients, could be related to chronic Chlamydia pneumoniae infection.

Predictors of Mortality in Patients with Bloodstream Infections Caused by Extended-Spectrum-β-Lactamase-Producing Enterobacteriaceae: Importance of Inadequate Initial Antimicrobial Treatment

ABSTRACT Bloodstream infections (BSI) caused by extended-spectrum β-lactamase (ESBL)-producing organisms markedly increase the rates of treatment failure and death. We conducted a retrospective cohort analysis to identify risk factors for mortality in adult in-patients with BSI caused by ESBL-producing Enterobacteriaceae (ESBL-BSI). Particular attention was focused on defining the impact on the mortality of inadequate initial antimicrobial therapy (defined as the initiation of treatment with active antimicrobial agents >72 h after collection of the first positive blood culture). A total of 186 patients with ESBL-BSI caused by Escherichia coli (n = 104), Klebsiella pneumoniae (n = 58), or Proteus mirabilis (n = 24) were identified by our microbiology laboratory from 1 January 1999 through 31 December 2004. The overall 21-day mortality rate was 38.2% (71 of 186). In multivariate analysis, significant predictors of mortality were inadequate initial antimicrobial therapy (odds ratio [OR] = 6.28; 95% confidence interval [CI] = 3.18 to 12.42; P < 0.001) and unidentified primary infection site (OR = 2.69; 95% CI = 1.38 to 5.27; P = 0.004). The inadequately treated patients (89 of 186 [47.8%]) had a threefold increase in mortality compared to the adequately treated group (59.5% versus 18.5%; OR = 2.38; 95% CI = 1.76 to 3.22; P < 0.001). The regimens most commonly classified as inadequate were based on oxyimino cephalosporin or fluoroquinolone therapy. Prompt initiation of effective antimicrobial treatment is essential in patients with ESBL-BSI, and empirical decisions must be based on a sound knowledge of the local distribution of pathogens and their susceptibility patterns.

Mechanisms of Azole Resistance in Clinical Isolates of Candida glabrata Collected during a Hospital Survey of Antifungal Resistance

ABSTRACT The increasing use of azole antifungals for the treatment of mucosal and systemic Candida glabrata infections has resulted in the selection and/or emergence of resistant strains. The main mechanisms of azole resistance include alterations in the C. glabrata ERG11 gene (CgERG11), which encodes the azole target enzyme, and upregulation of the CgCDR1 and CgCDR2 genes, which encode efflux pumps. In the present study, we evaluated these molecular mechanisms in 29 unmatched clinical isolates of C. glabrata, of which 20 isolates were resistant and 9 were susceptible dose dependent (S-DD) to fluconazole. These isolates were recovered from separate patients during a 3-year hospital survey for antifungal resistance. Four of the 20 fluconazole-resistant isolates were analyzed together with matched susceptible isolates previously taken from the same patients. Twenty other azole-susceptible clinical C. glabrata isolates were included as controls. MIC data for all the fluconazole-resistant isolates revealed extensive cross-resistance to the other azoles tested, i.e., itraconazole, ketoconazole, and voriconazole. Quantitative real-time PCR analyses showed that CgCDR1 and CgCDR2, alone or in combination, were upregulated at high levels in all but two fluconazole-resistant isolates and, to a lesser extent, in the fluconazole-S-DD isolates. In addition, slight increases in the relative level of expression of CgSNQ2 (which encodes an ATP-binding cassette [ABC] transporter and which has not yet been shown to be associated with azole resistance) were seen in some of the 29 isolates studied. Interestingly, the two fluconazole-resistant isolates expressing normal levels of CgCDR1 and CgCDR2 exhibited increased levels of expression of CgSNQ2. Conversely, sequencing of CgERG11 and analysis of its expression showed no mutation or upregulation in any C. glabrata isolate, suggesting that CgERG11 is not involved in azole resistance. When the isolates were grown in the presence of fluconazole, the profiles of expression of all genes, including CgERG11, were not changed or were only minimally changed in the resistant isolates, whereas marked increases in the levels of gene expression, particularly for CgCDR1 and CgCDR2, were observed in either the fluconazole-susceptible or the fluconazole-S-DD isolates. Finally, known ABC transporter inhibitors, such as FK506, were able to reverse the azole resistance of all the isolates. Together, these results provide evidence that the upregulation of the CgCDR1-, CgCDR2-, and CgSNQ2-encoded efflux pumps might explain the azole resistance in our set of isolates.

Biofilm Production by Candida Species and Inadequate Antifungal Therapy as Predictors of Mortality for Patients with Candidemia

ABSTRACT Nosocomial Candida bloodstream infections rank among infections with highest mortality rates. A retrospective cohort analysis was conducted at Catholic University Hospital to estimate the risk factors for mortality of patients with candidemia. We reviewed records for patients with a Candida bloodstream infection over a 5-year period (January 2000 through December 2004). Two hundred ninety-four patients (42.1% male; mean age ± standard deviation, 65 ± 12 years) were studied. Patients most commonly were admitted with a surgical diagnosis (162 patients [55.1%]), had a central venous catheter (213 [72.4%]), cancer (118 [40.1%]), or diabetes (58 [19.7%]). One hundred fifty-four (52.3%) patients died within 30 days. Of 294 patients, 168 (57.1%) were infected by Candida albicans, 64 (21.7%) by Candida parapsilosis, 28 (9.5%) by Candida tropicalis, and 26 (8.8%) by Candida glabrata. When fungal isolates were tested for biofilm formation capacity, biofilm production was most commonly observed for isolates of C. tropicalis (20 of 28 patients [71.4%]), followed by C. glabrata (6 of 26 [23.1%]), C. albicans (38 of 168 [22.6%]), and C. parapsilosis (14 of 64 [21.8%]). Multivariable analysis identified inadequate antifungal therapy (odds ratio [OR], 2.35; 95% confidence interval [95% CI], 1.09 to 5.10; P = 0.03), infection with overall biofilm-forming Candida species (OR, 2.33; 95% CI, 1.26 to 4.30; P = 0.007), and Acute Physiology and Chronic Health Evaluation III scores (OR, 1.03; 95% CI, 1.01 to 1.15; P < 0.001) as independent predictors of mortality. Notably, if mortality was analyzed according to the different biofilm-forming Candida species studied, only infections caused by C. albicans (P < 0.001) and C. parapsilosis (P = 0.003) correlated with increased mortality. Together with well-established factors, Candida biofilm production was therefore shown to be associated with greater mortality of patients with candidemia, probably by preventing complete organism eradication from the blood.

Bloodstream Infections Caused by Extended-Spectrum-β-Lactamase-Producing Klebsiella pneumoniae: Risk Factors, Molecular Epidemiology, and Clinical Outcome

ABSTRACT Bloodstream infections caused by extended-spectrum-β-lactamase (ESBL)-producing Klebsiella pneumoniae isolates are a major concern for clinicians, since they markedly increase the rates of treatment failure and death. One hundred forty-seven patients with K. pneumoniae bloodstream infections were identified over a 5-year period (January 1999 to December 2003). The production of ESBLs in bloodstream isolates was evaluated by molecular methods. A retrospective case-case-control study was conducted to identify risk factors for the isolation of ESBL-producing K. pneumoniae or non-ESBL-producing K. pneumoniae isolates in blood cultures. Forty-eight cases infected with ESBL-producing K. pneumoniae isolates and 99 cases infected with non-ESBL-producing K. pneumoniae isolates were compared to controls. Risk factors for isolation of ESBL-producing K. pneumoniae isolates were exposure to antibiotic therapy (odds ratio [OR], 11.81; 95% confidence interval [CI], 2.72 to 51.08), age (OR, 1.14; 95% CI, 1.08 to 1.21), and length of hospitalization (OR, 1.10; 95% CI, 1.04 to 1.16). Independent determinants for isolation of non-ESBL-producing K. pneumoniae were previous urinary tract infection (OR, 8.50; 95% CI, 3.69 to 19.54) and length of hospitalization (OR, 1.07; 95% CI, 1.04 to 1.10). When the initial response was assessed at 72 h after antimicrobial therapy, the treatment failure rate for the ESBL-producing K. pneumoniae-infected group was almost twice as high as that of the non-ESBL-producing K. pneumoniae-infected group (31% versus 17%; OR, 2.19; 95% CI, 0.98 to 4.89). The 21-day mortality rate for all patients was 37% (54 of 147); it was 52% (25 of 48) for patients with ESBL-producing K. pneumoniae bloodstream infections and 29% (29 of 99) for patients with non-ESBL-producing K. pneumoniae bloodstream infections (OR, 2.62; 95% CI, 1.28 to 5.35). In summary, this investigation identifies epidemiological characteristics that distinguish ESBL-producing K. pneumoniae infections from non-ESBL-producing K. pneumoniae ESBL bloodstream infections.

Detection and Isolation of Mycobacterium avium Subspecies paratuberculosis from Intestinal Mucosal Biopsies of Patients with and without Crohn's Disease in Sardinia

OBJECTIVES:Sardinia is an island community of 1.6 million people. There are also about 3.5 million sheep and one hundred thousand cattle in which Johnes disease and Mycobacterium avium subspecies paratuberculosis infection are endemic. The present study was designed to determine what proportion of people in Sardinia attending for ileocolonoscopy with or without Crohns disease were infected with this pathogen.METHODS:Mycobacterium avium subspecies paratuberculosis was detected by IS900 PCR on DNA extracts of fresh intestinal mucosal biopsies as well as by isolation in culture using supplemented MGIT media followed by PCR with amplicon sequencing.RESULTS:Twenty five patients (83.3%) with Crohns disease and 3 control patients (10.3%) were IS900 PCR positive (p = 0.000001; Odds ratio 43.3). Mycobacterium avium subspecies paratuberculosis grew in cultures from 19 Crohns patients (63.3%) and from 3 control patients (10.3%) (p = 0.00001; Odds ratio 14.9). All patients positive by culture had previously been positive by PCR. Mycobacterium avium subspecies paratuberculosis first appeared in the liquid cultures in a Ziehl Neelsen (ZN) staining negative form and partially reverted through a rhodamine-auramine positive staining form to the classical ZN positive form. This resulted in a stable mixed culture of all 3 forms illustrating the phenotypic versatility of these complex chronic enteric pathogens.CONCLUSIONS:Mycobacterium avium subspecies paratuberculosis was detected in the majority of Sardinian Crohns disease patients. The finding of the organism colonizing a proportion of people without Crohns disease is consistent with what occurs in other conditions caused by a primary bacterial pathogen in susceptible hosts.

Species identification of Aspergillus, Fusarium and Mucorales with direct surface analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Accurate species discrimination of filamentous fungi is essential, because some species have specific antifungal susceptibility patterns, and misidentification may result in inappropriate therapy. We evaluated matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for species identification through direct surface analysis of the fungal culture. By use of culture collection strains representing 55 species of Aspergillus, Fusarium and Mucorales, a reference database was established for MALDI-TOF MS-based species identification according to the manufacturers recommendations for microflex measurements and MALDI BioTyper 2.0 software. The profiles of young and mature colonies were analysed for each of the reference strains, and species-specific spectral fingerprints were obtained. To evaluate the database, 103 blind-coded fungal isolates collected in the routine clinical microbiology laboratory were tested. As a reference method for species designation, multilocus sequencing was used. Eighty-five isolates were unequivocally identified to the species level (≥99% sequence similarity); 18 isolates producing ambiguous results at this threshold were initially rated as identified to the genus level only. Further molecular analysis definitively assigned these isolates to the species Aspergillus oryzae (17 isolates) and Aspergillus flavus (one isolate), concordant with the MALDI-TOF MS results. Excluding nine isolates that belong to the fungal species not included in our reference database, 91 (96.8%) of 94 isolates were identified by MALDI-TOF MS to the species level, in agreement with the results of the reference method; three isolates were identified to the genus level. In conclusion, MALDI-TOF MS is suitable for the routine identification of filamentous fungi in a medical microbiology laboratory.

Gain of function mutations in CgPDR1 of Candida glabrata not only mediate antifungal resistance but also enhance virulence.

CgPdr1p is a Candida glabrata Zn(2)-Cys(6) transcription factor involved in the regulation of the ABC-transporter genes CgCDR1, CgCDR2, and CgSNQ2, which are mediators of azole resistance. Single-point mutations in CgPDR1 are known to increase the expression of at least CgCDR1 and CgCDR2 and thus to contribute to azole resistance of clinical isolates. In this study, we investigated the incidence of CgPDR1 mutations in a large collection of clinical isolates and tested their relevance, not only to azole resistance in vitro and in vivo, but also to virulence. The comparison of CgPDR1 alleles from azole-susceptible and azole-resistant matched isolates enabled the identification of 57 amino acid substitutions, each positioned in distinct CgPDR1 alleles. These substitutions, which could be grouped into three different “hot spots,” were gain of function (GOF) mutations since they conferred hyperactivity to CgPdr1p revealed by constitutive high expression of ABC-transporter genes. Interestingly, the major transporters involved in azole resistance (CgCDR1, CgCDR2, and CgSNQ2) were not always coordinately expressed in presence of specific CgPDR1 GOF mutations, thus suggesting that these are rather trans-acting elements (GOF in CgPDR1) than cis-acting elements (promoters) that lead to azole resistance by upregulating specific combinations of ABC-transporter genes. Moreover, C. glabrata isolates complemented with CgPDR1 hyperactive alleles were not only more virulent in mice than those with wild type alleles, but they also gained fitness in the same animal model. The presence of CgPDR1 hyperactive alleles also contributed to fluconazole treatment failure in the mouse model. In conclusion, this study shows for the first time that CgPDR1 mutations are not only responsible for in vitro/in vivo azole resistance but that they can also confer a selective advantage under host conditions.

Fatal Pulmonary Infection Due to Multidrug-Resistant Mycobacterium abscessus in a Patient with Cystic Fibrosis

ABSTRACT We report a case of fatal pulmonary infection caused byMycobacterium abscessus in a young patient with cystic fibrosis, who underwent bipulmonary transplantation after a 1-year history of severe lung disease. Fifteen days after surgery he developed septic fever with progressive deterioration in lung function. M. abscessus, initially isolated from a pleural fluid specimen, was then recovered from repeated blood samples, suggesting a disseminated nature of the mycobacterial disease. Drug susceptibility testing assay, performed on two sequential isolates of the microorganism, showed a pattern of multidrug resistance. Despite aggressive therapy with several antimycobacterial drugs, including clarithromycin, the infection persisted, and the patient died.

Rv1818c‐encoded PE_PGRS protein of Mycobacterium tuberculosis is surface exposed and influences bacterial cell structure

Identification of the novel PE multigene family was an unexpected finding of the genomic sequencing of Mycobacterium tuberculosis. Presently, the biological role of the PE and PE_PGRS proteins encoded by this unique family of mycobacterial genes remains unknown. In this report, a representative PE_PGRS gene (Rv1818c/PE_PGRS33) was selected to investigate the role of these proteins. Cell fractionation studies and fluorescence analysis of recombinant strains of Mycobacterium smegmatis and M. tuberculosis expressing green fluorescent protein (GFP)‐tagged proteins indicated that the Rv1818c gene product localized in the mycobacterial cell wall, mostly at the bacterial cell poles, where it is exposed to the extracellular milieu. Further analysis of this PE_PGRS protein showed that the PE domain is necessary for subcellular localization. In addition, the PGRS domain, but not PE, affects bacterial shape and colony morphology when Rv1818c is overexpressed in M. smegmatis and M. tuberculosis. Taken together, the results indicate that PE_PGRS and PE proteins can be associated with the mycobacterial cell wall and influence cellular structure as well as the formation of mycobacterial colonies. Regulated expression of PE genes could have implications for the survival and pathogenesis of mycobacteria within the human host and in other environmental niches.