Roberto Alonso

Reassessment of Clostridium difficile Susceptibility to Metronidazole and Vancomycin

ABSTRACT Clostridium difficile is the most frequently identified enteric pathogen in patients with nosocomially acquired, antibiotic-associated diarrhea. The drugs most commonly used to treat diseases associated with C. difficile are metronidazole and vancomycin. Most clinical laboratories assume that all C. difficile isolates are susceptible to metronidazole and vancomycin. We report on the antimicrobial susceptibilities of 415 C. difficile isolates to metronidazole and vancomycin over an 8-year period (1993 to 2000). The overall rate of resistance to metronidazole at the critical breakpoint (16 μg/ml) was 6.3%. Although full resistance to vancomycin was not observed, the overall rate of intermediate resistance was 3.1%. One isolate had a combination of resistance to metronidazole and intermediate resistance to vancomycin. Rates of resistance to metronidazole and vancomycin were higher among isolates from human immunodeficiency virus-infected patients. Molecular typing methods proved the absence of clonality among the isolates with decreased susceptibilities to the antimicrobials tested.

In Vitro Activity of Ramoplanin against Clostridium difficile, Including Strains with Reduced Susceptibility to Vancomycin or with Resistance to Metronidazole

ABSTRACT We evaluated the in vitro activity of ramoplanin, an antimicrobial compound that inhibits cell wall synthesis by acting at the level of lipid intermediate formation, against Clostridium difficile. We included strains with reduced susceptibilities to vancomycin (vancomycin-intermediate [Vani] strains) or with resistance to metronidazole (Mtzr), in order to assess the potential utility of ramoplanin for the treatment of C. difficile-associated diarrhea. We tested the activity of ramoplanin against a total of 105 nonduplicate clinical isolates of toxigenic C. difficile, including 8 Vani isolates and 6 Mtzr isolates, obtained from our laboratory. Ramoplanin was active against all strains tested at concentrations ranging from 0.03 to 0.5 μg/ml (MICs at which 50 and 90% of isolates were inhibited, 0.25 μg/ml; geometric mean MIC, 0.22 μg/ml). All isolates, independently of their levels of susceptibility to vancomycin or metronidazole, were considered susceptible to ramoplanin (MICs, ≤0.5 μg/ml).

Rapid detection of toxigenic Clostridium difficile from stool samples by a nested PCR of toxin B gene

Toxigenic Clostridium difficile is the aetiologic agent of most cases of antibiotic-associated diarrhoea and pseudomembranous colitis. The present standard method for C. difficile diagnosis is a cytotoxicity assay, performed on human fibroblast cultures. It is time consuming and requires special facilities. A nested-PCR assay detecting toxin B gene within a few hours was designed. One hundred and two stool samples were collected during four months. All samples were processed for toxin B-PCR, cultured for C. difficile and tested for cytotoxicity. This approach achieved 99% concordance with the cytotoxic assay. The sensitivity and specificity for the new PCR assay were 96.3% and 100% respectively. The procedure described is easy to perform, does not require special equipment and has produced excellent results. It deserves serious consideration for routine clinical microbiology laboratory use.

Evidence of Nosocomial Stenotrophomonas Maltophilia Cross-Infection in a Neonatology Unit Analyzed by Three Molecular Typing Methods

OBJECTIVE To characterize the epidemiological relationships among Stenotrophomonas maltophilia isolates in the neonatology unit of our institution over a 4-month period in which an increased number of isolates was observed. SETTING The neonatology ward in a 2,000-bed university hospital in Madrid, Spain. DESIGN A retrospective molecular epidemiological analysis using three different typing methods, arbitrarily primed polymerase chain reaction (PCR), pulsed-field gel electrophoresis, and enterobacterial repetitive intergenic consensus-PCR, was performed with 11 isolates obtained from seven neonates over a 4-month period. Presumed unrelated isolates also were included as controls. A similarity dendrogram was obtained, to analyze the genetic relatedness among the isolates. RESULTS All isolates from the neonates, except one, showed a remarkably high homology among their typing patterns for the three methods assayed and clustered in the relatedness dendrogram at 96% similarity. The unrelated strains selected as controls were unclustered. The index case was considered to be a newborn who had an S. maltophilia isolate from a culture drawn on the day of admission to the neonatology unit and which was included in the clustered similarity group. CONCLUSIONS Such a high genetic similarity among the isolates, together with the presence of an index case who had been colonized or infected by S. maltophilia before arrival at our institution, constitutes the first evidence of nosocomial cross-transmission of this microorganism.

Value of a single galactomannan determination (Platelia) for the diagnosis of invasive aspergillosis in non-hematological patients with clinical isolation of Aspergillus spp.

We studied 75 patients with non-hematological conditions from whom Aspergillus spp. were recovered from clinical specimens during the period March 2003 to August 2006. The patients were classified according to EORTC criteria and the presence of galactomannan (Platelia Aspergillus) in their sera was evaluated. Ten of these patients (13.3%) had proven or probable invasive aspergillosis, i.e., chronic obstructive pulmonary disease in five (50%), HIV infection in one (10%), lymphoma in one (10%), liver transplant in one (10%), solid malignancies in one (10%), and corticosteroid treatment in one (10%). The sensitivity, specificity, and positive and negative predictive values for the detection of galactomannan, using cut-offs of > or =0.5 ng/ml and > or =1 ng/ml were 60%/50%, 89.23%/100%, 46.15%/100%, and 93.55%/92.86% (p=0.001 and p<0.001), respectively. The determination of galactomannan in the sera of non-neutropenic patients could prove to be a useful microbiological finding when diagnosing invasive aspergillosis.

Candida biomarkers in patients with candidaemia and bacteraemia

OBJECTIVES Microbiological strategies are necessary to help clinicians discontinue empirical antifungal therapy in patients with suspected invasive candidiasis. Culture methods and biomarkers each show low sensitivity. We analysed the value of combining different biomarkers as a decision-making tool for discontinuing empirical antifungal treatment. METHODS We studied stored serum samples from 31 patients with candidaemia (Candida albicans 40%, Candida tropicalis 20%, Candida parapsilosis 18%, Candida glabrata 12% and other 10%) and 50 patients with bacteraemia at Gregorio Marañón Hospital, Madrid, Spain. C. albicans germ tube antibody (CAGTA), mannan antigens (MN), antimannan antibodies (AMN) and (1→3)-β-d-glucan (BDG) were assayed using the manufacturers and alternative cut-offs to improve the accuracy of the tests. RESULTS The sensitivity of the biomarkers when used alone was low (58%-84%), but specificity was high (65.8%-92.0%). The best combinations were CAGTA and BDG using cut-offs of 1/80 and 80 pg/mL, respectively (sensitivity 96.8% and specificity 84%), and CAGTA and MN using cut-offs of 1/80 and 75 pg/mL, respectively (sensitivity 93.5% and specificity 86.0%). The sensitivity of both combinations was 100% for C. albicans, C. tropicalis and C. parapsilosis, but only combinations including BDG detected Candida krusei. The negative predictive values (NPVs) of both combinations were, respectively, 97.7% and 95.6% (prevalence of candidaemia, 23.6%). For a prevalence of candidaemia of 5% and 10%, the NPV reached 99.8% and 99.6%. CONCLUSIONS The combinations of CAGTA and BDG or CAGTA and MN had a very high NPV at the alternative cut-offs and could be used in antifungal stewardship programmes as a decision-making tool for discontinuing unnecessary empirical therapy in patients with suspected candidaemia.

In Vitro Activity of Linezolid against Clostridium difficile

ABSTRACT We examined the in vitro activity of linezolid against Clostridium difficile, including isolates with reduced susceptibility to metronidazole or vancomycin. The MIC at which 50% of the isolates were inhibited (MIC50) and MIC90 were 0.5 and 2 μg/ml, respectively (range, 0.03 to 4 μg/ml). MICs were always ≤ 4 μg/ml, and thus, all isolates were considered susceptible.

Immune monitoring of anti cytomegalovirus antibodies and risk of cytomegalovirus disease in heart transplantation.

We sought to determine whether quantitative assessment of anti-cytomegalovirus (CMV) antibodies could be useful to identify patients at risk of cytomegalovirus (CMV) disease after heart transplantation (HT). 75 patients who underwent HT at a single health care center were prospectively studied. Induction therapy included 2 doses of daclizumab and maintenance tacrolimus (n=42) or cyclosporine (n=29), mycophenolate mofetil and prednisone. All patients received prophylaxis with gancyclovir or valganciclovir. Anti-CMV intravenous immunoglobulin (CMV-IG) was added in high risk patients (CMV D+/R- serostatus). Serial determinations of anti-CMV antibodies, immunoglobulins (IgG, IgA, IgM) and IgG-subclasses were analysed. CMV infection was based on detection of the virus by antigenemia. CMV disease consisted of detection of signs or symptoms attributable to this microorganism. Ten patients (13.3%) developed CMV disease. Mean time of development of CMV disease was 3.4+/-1.6 months. In Cox regression analysis, patients with low baseline anti-CMV titers (<4.26 natural logarithm of titer, RH: 8.1, 95%CI: 1.93-34.1, p=0.004) and recipients with 1-month post-HT IgG hypogammaglobulinemia (IgG<500 mg/dl, RH: 4.49, 95%CI: 1.26-15.94, p=0.02) were at higher risk of having CMV disease. Despite use of prophylactic CMV-IG, D+/R- patients showed significantly lower titers of anti-CMV antibodies at 7 d, 30 d and 90 d post HT as compared with HT recipients without infections. Four out of 6 of these patients developed late CMV disease. Monitoring of specific anti-CMV antibodies on the bedside warrants further evaluation as a potential tool to identify heart transplant recipients at higher risk of CMV disease.

New Automated Chemiluminescence Immunoassay for Simultaneous but Separate Detection of Human Immunodeficiency Virus Antigens and Antibodies

ABSTRACT The recently launched Liaison XL Murex HIV Ab/Ag assay (DiaSorin S.p.A) uses chemiluminescence immunoassay technology for the combined qualitative determination of p24 antigen of HIV-1 and specific antibodies to both HIV-1 and HIV-2. We studied 571 serum samples from those submitted to our laboratory for HIV screening. The samples were divided into 3 subsets: subset A, 365 samples collected prospectively during 1 week; subset B, 158 samples from confirmed HIV-positive patients; and subset C, 48 samples with a positive screening result but a negative or indeterminate confirmatory test result. Our standard screening/confirmatory algorithm was used as a reference. In subset A (prospective), 5 samples were positive and 360 negative by the standard procedure. Liaison XL Murex HIV Ab/Ag correctly identified all 5 positive samples (100%) and 357 negative samples (99.2%). In subset B (confirmed positive), all 158 positive samples were in total agreement in both procedures. In subset C (screen positive only), Liaison XL Murex HIV Ab/Ag yielded accurate results in 42 out of 48 samples (87.5%). Global sensitivity and specificity for Liaison XL Murex HIV Ab/Ag (all subsets included) were 98.3% and 98.5%, respectively. Considering only nonselected prospective samples and confirmed positive samples (subsets A and B), the corresponding sensitivity and specificity values were 100% and 99.2%, respectively. The new fully automated HIV screening test showed high sensitivity and specificity compared to our standard algorithm. Its added advantage of being able to detect HIV-1 and HIV-2 antibodies and p24 antigen separately could prove useful in the diagnosis of early infections.

A Prospective Monitoring Study of Cytomegalovirus Infection in Non-Immunosuppressed Critical Heart Surgery Patients.

Background Reactivation of cytomegalovirus (CMV) has been reported occasionally in immnunocompetent patients in the intensive care unit (ICU). The epidemiology and association of CMV infection with adverse outcome is not well defined in this population. Patients undergoing major heart surgery (MHS) are at a particularly high risk of infection. CMV infection has not been systematically monitored in MSH-ICU patients. Methods We assessed CMV plasma viremia weekly using a quantitative polymerase chain reaction assay in a prospective cohort of immunocompetent adults admitted to the MHS-ICU for at least 72 hours between October 2012 and May 2013. Risk factors for CMV infection and its potential association with continued hospitalization or death by day 30 (composited endpoint) were assessed using univariate and multivariate logistic regression analyses. Results CMV viremia at any level was recorded in 16.5% of patients at a median of 17 days (range, 3-54 days) after admission to the MHS-ICU. Diabetes (adjusted OR, 5.6; 95% CI, 1.8-17.4; p=0.003) and transfusion requirement (>10 units) (adjusted OR, 13.7; 95% CI, 3.9-47.8; p<0.001) were independent risk factors associated with CMV reactivation. Reactivation of CMV at any level was independently associated with the composite endpoint (adjusted OR, 12.1; 95% CI, 2.3-64; p=0.003). Conclusion Reactivation of CMV is relatively frequent in immunocompetent patients undergoing MHS and is associated with prolonged hospitalization or death.