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Metronidazole Resistance in Clostridium difficile Is Heterogeneous

ABSTRACT At our institution, the prevalence of clinical isolates of Clostridium difficile with resistance to metronidazole is 6.3%. We observed that initial metronidazole MICs of 16 to 64 mg/liter against toxigenic, primary fresh C. difficile isolates, as determined by agar dilution, decreased to 0.125 mg/liter after the isolates were thawed. In this study, we examined the possibility of heterogeneous or inducible resistance. Totals of 14 metronidazole-resistant and 10 metronidazole-susceptible clinical isolates of toxigenic C. difficile were studied. The isolates were investigated for the presence of nim genes by PCR. After the isolates were thawed, susceptibility testing was done by agar dilution, by disc diffusion using a 5-μg metronidazole disc, and by the Etest method. An experiment for determining the effect of prolonged exposure to metronidazole was applied to all resistant isolates and to susceptible control strains. None of the isolates presented the nim genes. All initially metronidazole-resistant C. difficile isolates became susceptible after thawing; however, they presented slow-growing subpopulations within the inhibition zones of both the disk and the Etest strip. All metronidazole-susceptible isolates remained homogeneously susceptible by both methods. After prolonged exposure in vitro to metronidazole, no zone of inhibition was found around the 5-μg disk in any of the metronidazole-resistant isolates, and the MICs as determined by the Etest method ranged from 0.125 to >256 mg/liter, with colonies growing inside the inhibition zone. Our results indicate that (i) resistance to metronidazole was not due to the presence of nim genes, (ii) resistance to metronidazole in toxigenic C. difficile isolates is heterogeneous, and (iii) prolonged exposure to metronidazole can select for in vitro resistance. We recommend routine performance of the disk diffusion method (5-μg metronidazole disk) with primary fresh C. difficile isolates in order to ensure that metronidazole-heteroresistant populations do not go undetected.

The undiagnosed cases of Clostridium difficile infection in a whole nation: where is the problem?

Underdiagnosis of Clostridium difficile infection (CDI) because of lack of clinical suspicion or the use of non-sensitive diagnostic techniques is a known problem whose real magnitude has not yet been quantified. In order to estimate the extent of this underdiagnosis, we performed C. difficile cultures on all unformed stool specimens sent-irrespective of the type of request-to a series of laboratories in Spain on a single day. The specimens were cultured, and isolates were characterized at a central reference laboratory. A total of 807 specimens from 730 patients aged ≥ 2 years were selected from 118 laboratories covering 75.4% of the Spanish population. The estimated rate of hospital-acquired CDI was 2.4 episodes per 1000 admissions or 3.8 episodes per 10,000 patient-days. Only half of the episodes occurred in patients hospitalized for >2 days. Two of every three episodes went undiagnosed or were misdiagnosed, owing to non-sensitive diagnostic tests (19.0%) or lack of clinical suspicion and request (47.6%; mostly young people or non-hospitalized patients). The main ribotypes were 014/020 (20.5%), 001 (18.2%), and 126/078 (18.2%). No ribotype 027 strains were detected. Strains were fully susceptible to metronidazole and vancomycin. CDI was underdiagnosed in diarrhoeic stools in a high proportion of episodes, owing to the use of non-sensitive techniques or lack of clinical suspicion, particularly in people aged <65 years or patients with community-acquired diarrhoea. C. difficile toxins should be routinely sought in unformed stools of any origin sent for microbiological diagnosis. The ribotype 027 clone has not yet disseminated in Spain.

Group A streptococcal bacteremia : a 10-year prospective study

&NA; Abbreviations used in this article: GAS, group A Streptococcus; IVDU, intravenous drug users; non‐IVDU, non‐intravenous drug users; STSS, Streptococcal toxic shock syndrome.

Prolonged viral shedding in pandemic influenza A(H1N1): clinical significance and viral load analysis in hospitalized patients

The clinical significance of prolonged viral shedding (PVS) and viral load (VL) dynamics has not been sufficiently assessed in hospitalized patients with pandemic 2009 influenza A(H1N1). We performed a prospective study of adults with confirmed influenza A(H1N1) virus infection admitted to our hospital from 20 September 2009 to 31 December 2009. Consecutive nasopharyngeal swabs were collected every 2 days during the first week after diagnosis, and then every week or until viral detection was negative. Relative VL was measured on the basis of haemagglutinin and RNaseP gene analysis. PVS was defined as positive detection of influenza A(H1N1) virus by real-time RT-PCR at day 7 after diagnosis. We studied 64 patients: 16 (25%) presented PVS. The factors associated with PVS were admission to the intensive-care unit (69% vs. 33%, p 0.02), purulent expectoration (75% vs. 44%, p 0.04), higher dosage of oseltamivir (62.5% vs. 27%, p 0.016), corticosteroid treatment (50% vs. 21%, p 0.05), mechanical ventilation (MV) (50% vs. 12.5%, p 0.004), and longer stay (34 vs. 7 median days, p 0.003). Multivariate analysis revealed the factors independently associated with PVS to be immunosuppression (OR 5.15; 95% CI 1.2-22.2; p 0.03) and the need for MV (OR 11.7; 95% CI 2.5-54.4; p 0.002). VL at diagnosis correlated negatively with age and septic shock. VL dynamics of patients with acute respiratory distress syndrome and/or mortality were very different from those of other patients. PVS was detected in 25% of hospitalized patients with pandemic 2009 influenza A(H1N1) and was strongly associated with immunosuppression and the need for MV. Diagnostic VL and viral clearance varied with the clinical course.

Identification of liver fibrosis in HIV/HCV-coinfected patients using a simple predictive model based on routine laboratory data

Summary.  We constructed noninvasive models to predict significant fibrosis (F ≥ 2) and advanced fibrosis (F ≥ 3) among human immunodeficiency virus (HIV)/hepatitis C virus (HCV)‐coinfected patients, naïve for anti‐HCV treatment. A total of 296 patients with liver biopsy were randomly assigned to an estimation group (EG = 226; 70%) and a validation group (VG = 70; 30%). We developed the Hospital Gregorio Marañón (HGM)‐1 index, based on platelet count, aspartate aminotransferase (AST) and glucose, to predict F ≥ 2 and the HGM‐2 index, based on platelet count, international normalized ratio, alkaline phosphatase and AST to predict F ≥ 3. The area under the receiver operating characteristic curves (AUROCs) of the HGM‐1 index for the EG and the VG were 0.807 and 0.712 respectively. The AUROCs of the HGM‐2 index for the EG and the VG were 0.844 and 0.815 respectively. With the HGM‐1 index applied to the VG, using best cutoff scores, the negative predictive value (NPV) to exclude F ≥ 2 was 54.5% and the positive predictive value (PPV) to confirm F ≥ 2 was 93.3%. With the HGM‐2 index applied to the VG, using best cutoff scores, the NPV to exclude F ≥ 3 was 92.3, and the PPV to confirm F ≥ 3 was 64.3%. Thus, HGM‐2 accurately predicted F ≥ 3 among HIV/HCV‐coinfected patients. HGM‐1 was less accurate at predicting F ≥ 2.

Hepatitis C virus infection is associated with endothelial dysfunction in HIV/hepatitis C virus coinfected patients.

Objective:To quantify serum levels of intercellular adhesion molecule-1 (sICAM-1) and vascular cell adhesion molecule-1 (sVCAM-1) in HIV/HCV coinfected patients to examine their association with several clinical and epidemiological characteristics and the therapeutic responsiveness to interferon (IFN)-α and ribavirin therapy (IFN-α + RBV). Design:Retrospective study. Methods:We carried out a cross-sectional study with 183 IFN-α-naive patients on HAART, and 24 healthy controls. We also analyzed 30 out of 183 patients on IFN-α + RBV for the duration of 48 weeks. Results:HIV/HCV coinfected patients had higher levels of sICAM-1 and sVCAM-1 than the healthy control group (P < 0.05). Patients with HCV-genotype 1, advanced fibrosis (F≥3) or moderate to severe activity grade (A≥2) had the highest values of sICAM-1 and sVCAM-1. When we carried out a multivariate analysis, we found a significant positive relationship between both HCV-genotype 1 and advanced fibrosis (F≥3) with sICAM-1 (R = 0.549; P < 0.001); and a significant positive relationship between HCV-genotype 1 and advanced fibrosis (F≥3) with sVCAM-1 (R = 0.624; P < 0.001). We also found a positive relationship of sICAM-1 or sVCAM-1 levels with transaminases and alkaline phosphatase circulation levels (P < 0.05). Nonresponder patients had higher sICAM-1 and sVCAM-1 serum levels, and patients with sustained virologic response had significantly lower levels of sICAM-1 (P = 0.001) and sVCAM-1 (P = 0.019). Conclusion:HIV and HCV coinfection induces alterations in sICAM-1 and sVCAM-1 serum levels, which were higher in patients with HCV-genotype 1 and advanced stage of HCV infection. However, response to IFN-α + RBV may reduce these cardiovascular risk markers.

Insulin resistance is associated with advanced liver fibrosis and high body mass index in HIV/HCV-coinfected patients.

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Resistance and virulence mutations in patients with persistent infection by pandemic 2009 A/H1N1 influenza

BACKGROUND Pandemic 2009 influenza A/H1N1 (H1N1v) is resistant to adamantanes, leaving neuraminidase inhibitors as the only therapeutic option. Other mutations are considered to be associated with virulence and clinical severity. However, out of the surveillance programs, few studies analyze the presence of resistance/virulent H1N1v variants in certain clinical circumstances. OBJECTIVES To define the frequency and role of resistance and virulence mutations in a specific clinical circumstance-in patients with persistent infection by H1N1v. STUDY DESIGN Observational study of patients with persistent H1N1v infection admitted to our hospital. RESULTS NAI-resistance mutations were detected in 14.3% of cases with persistent infection (2/14), and in none of the non-persistent controls (0/15). These cases were initially infected with susceptible variants that acquired resistance at different time-points after therapy with oseltamivir (OTV). The first case (case 2) was an HIV-positive patient who rapidly acquired resistance 9 days after diagnosis (6 days on OTV) and whose infection resolved after standard OTV therapy. The second case (case 3) was a patient with chronic lymphocytic leukemia [corrected] and the longest viral persistence (59 days). The resistance mutation was detected in the specimen taken on day 37 after diagnosis (30 days on OTV). Once the resistance mutation was identified, OTV was substituted by zanamivir and the infection resolved. In addition to mutations encoding resistance, variants associated with virulence were also sought. The D225G mutation was not found in any case, whereas the D225E variant was identified in three persistent cases but also in two non-persistent ones. In one patient, the D225E substitution coincided with the H275 resistant mutation. CONCLUSIONS NAI-resistance mutations were detected, at rather different paces, in non-severe immunosuppressed cases with persistent infection by influenza A/H1N1v.

Can serum hyaluronic acid replace simple non-invasive indexes to predict liver fibrosis in HIV/Hepatitis C coinfected patients?

BackgroundHyaluronic acid (HA) serum levels correlate with the histological stages of liver fibrosis in hepatitis C virus (HCV) monoinfected patients, and HA alone has shown very good diagnostic accuracy as a non-invasive assessment of fibrosis and cirrhosis. The aim of this study was to evaluate serum HA levels as a simple non-invasive diagnostic test to predict hepatic fibrosis in HIV/HCV-coinfected patients and to compare its diagnostic performance with other previously published simple non-invasive indexes consisting of routine parameters (HGM-1, HGM-2, Forns, APRI, and FIB-4).MethodsWe carried out a cross-sectional study on 201 patients who all underwent liver biopsies and had not previously received interferon therapy. Liver fibrosis was determined via METAVIR score. The diagnostic accuracy of HA was assessed by area under the receiver operating characteristic curves (AUROCs).ResultsThe distribution of liver fibrosis in our cohort was 58.2% with significant fibrosis (F≥2), 31.8% with advanced fibrosis (F≥3), and 11.4% with cirrhosis (F4). Values for the AUROC of HA levels corresponding to significant fibrosis (F≥2), advanced fibrosis (F≥3) and cirrhosis (F4) were 0.676, 0.772, and 0.863, respectively. The AUROC values for HA were similar to those for HGM-1, HGM-2, FIB-4, APRI, and Forns indexes. The best diagnostic accuracy of HA was found for the diagnosis of cirrhosis (F4): the value of HA at the low cut-off (1182 ng/mL) excluded cirrhosis (F4) with a negative predictive value of 99% and at the high cut-off (2400 ng/mL) confirmed cirrhosis (F4) with a positive predictive value of 55%. By utilizing these low and high cut-off points for cirrhosis, biopsies could have theoretically been avoided in 52.2% (111/201) of the patients.ConclusionsThe diagnostic accuracy of serum HA levels increases gradually with the hepatic fibrosis stage. However, HA is better than other simple non-invasive indexes using parameters easily available in routine clinical practice only for the diagnosing of cirrhosis.

High plasma CXCL10 levels are associated with HCV-genotype 1, and higher insulin resistance, fibrosis, and HIV viral load in HIV/HCV coinfected patients

BACKGROUND CXCL10 may contribute to the host immune response against the hepatitis C virus (HCV), liver disease progression, and response to HCV antiviral therapy. The aim of our study was to analyze the relationship among virological, immunological, and clinical characteristics with plasma CXCL10 levels in human immunodeficiency virus (HIV)/HCV-coinfected patients. METHODS We carried out a cross-sectional study on 144 patients. CXCL10 and insulin were measured using an immunoassay kit. The degree of insulin resistance was estimated for each patient using the homeostatic model assessment (HOMA) method. Insulin resistance was defined as a HOMA index higher than or equal to 3.8. Aspartate aminotransferase (AST) to platelet ratio (APRI), FIB-4, Forns index, HGM1, and HGM2 were calculated. RESULTS The variables associated with log(10) CXCL10 levels by univariate analysis were age (b=0.013; p=0.023), prior AIDS-defining condition (b=0.127; p=0.045), detectable plasma HIV viral load (b=0.092; p=0.006), log(10) HOMA (b=0.216; p=0.002), HCV-genotype 1 (b=0.114; p=0.071), and liver fibrosis assessed by all non-invasive indexes (log(10) APRI (b=0.296; p=0.001), log(10) FIB-4 (b=0.436; p<0.001), log(10) Forns index (b=0.591; p<0.001), log(10) HGM1 (b=0.351; p=0.021), and log(10) HGM2 (b=0.215; p=0.018)). However, in multivariate analysis, CXCL10 levels were only associated with HOMA, detectable plasma HIV viral load, HCV-genotype 1 and FIB-4 (R-square=0.235; p<0.001). CONCLUSION Plasma CXCL10 levels were influenced by several characteristics of patients related to HIV and HCV infections, insulin resistance, and liver fibrosis, indicating that CXCL10 may play an important role in the pathogenesis of both HCV and HIV infections.