Javier Aznar

Influence of human immunodeficiency virus type 1 infection on the natural course of chronic parenterally acquired Hepatitis C

The aim of the present study was to investigate the possible role of human immunodeficiency virus (HIV) infection in the natural course of chronic hepatitis C. Seventy-six adult patients with chronic parenterally acquired hepatitis C virus (HCV) infection examined from 1989 to 1993 were enrolled; of these 32 (42.1 %) were HIV positive and 44 (57.9 %) were HIV negative. Serum HCV RNA quantitation was carried out by polymerase chain reaction in a well-characterized group (n=20; 11 HIV positive and 9 HIV negative). Distribution of histological findings in liver biopsies from both HIV-infected and noninfected patients was similar. However, within 15 years after initial HCV infection, 8 of 32 (25 %) HIV-positive patients developed cirrhosis, in comparison with only 2 of 31 (6.5 %) patients in the HIV-negative group (p<0.05); similar incidences of cirrhosis were found in both patient groups within 5 and 10 years after HCV infection. Most of the HIV-negative cirrhotic patients (9 of 11) developed cirrhosis in a time interval longer than 15 years. Finally, HCV load was almost ten times higher (1 10-fold dilution) in the HIV-positive group, but this difference did not reach statistical significance in this small study population. These results suggest that HIV infection can alter the natural course of chronic parenterally acquired hepatitis C, causing an unusually rapid progression to cirrhosis.

Reliability of the E-Test Method for Detection of Colistin Resistance in Clinical Isolates of Acinetobacter baumannii

ABSTRACT We compared the E-test to the broth microdilution method for testing the susceptibility of 115 clinical isolates of Acinetobacter baumannii to colistin. Twenty-two (19.1%) strains were resistant to colistin and 93 (80.8%) strains were susceptible according to the reference broth microdilution method. A categorical agreement of 98.2% was found, with only two (1.7%) very major errors. Agreement within 1 twofold dilution between the E-test and the broth microdilution was 16.5%. Complete agreement was found for the strains for which MICs fell within the range of 0.25 to 1 μg of colistin/ml. However, there was poor concordance, particularly in extreme dilutions with higher MICs by the E-test method.

Sequential measurements of procalcitonin levels in diagnosing ventilator-associated pneumonia

The utility of procalcitonin levels to improve the accuracy of clinical and microbiological parameters in diagnosing ventilator-associated pneumonia (VAP) was evaluated. Sequential measurement of procalcitonin and C-reactive protein levels and the calculation of the simplified Clinical Pulmonary Infection Scores (CPIS) were performed in 44 patients mechanically-ventilated for >48 h with neither active infection for the duration or suspicion of VAP. Patients who developed extrapulmonary infection were excluded. In total, 20 cases were suspected of having VAP and diagnosis was microbiologically confirmed in nine. In patients with confirmed VAP, procalcitonin levels were higher than in those without VAP. C-reactive protein levels and CPIS were lower in patients without suspected VAP, but could not discriminate confirmed and nonconfirmed suspicion of VAP. The best sensitivity and specificity (78 and 97%, respectively) corresponded to procalcitonin. The CPIS resulted in the same sensitivity, but had a lower specificity (80%). C-reactive protein had the worst sensitivity (56%), but a good specificity (91%). A CPIS ≥6 combined with serum levels of procalcitonin ≥2.99 ng·mL−1 did not improve the sensitivity (67%), but resulted in 100% specificity. Procalcitonin might be useful in the diagnosis of ventilator-associated pneumonia. Combined values of Clinical Pulmonary Infection Scores and procalcitonin below the cut-off points excluded false-positive diagnoses of ventilator-associated pneumonia.

Direct Detection of Rifampin- and Isoniazid-Resistant Mycobacterium tuberculosis in Auramine-Rhodamine-Positive Sputum Specimens by Real-Time PCR

ABSTRACT Our objective was to evaluate the feasibility of a molecular assay based on a real-time PCR technique, carried out with a LightCycler instrument (Roche Biochemicals), to identify Mycobacterium tuberculosis bacilli and to detect rifampin and isoniazid resistance in DNA extracts from sputum samples. We studied three genes: rpoB, which is associated with rifampin resistance, and katG and inhA, which are associated with isoniazid resistance. A total of 205 sputum samples collected from 108 patients diagnosed with pulmonary tuberculosis with positive auramine-rhodamine-staining (AR) sputum samples, were tested. The sensitivities of the LightCycler PCR assay for the positive AR specimens was 97.5% (200 of 205) for rpoB and inhA genes and 96.5% (198 of 205) for the katG gene. For the total number of patients tested, the sensitivity was 100% (108 of 108 patients) for rifampin, whereas the sensitivity was 98.1% (106 of 108 patients) for isoniazid. Full agreement was found with the Bactec MGIT 960 method and the genotype inferred from the LightCycler data for rifampin. The phenotypic method for isoniazid reported 13 resistant strains (≥0.1 μg/ml). In seven (53.8%) strains there was a concordance between both methods, but we found that six (46.2%) strains reported as resistant by the phenotypic method were determined to be susceptible by real-time PCR. For the 75 strains reported as susceptible by the phenotypic method, the concordance with the LightCycler data was 100%. Our results demonstrate that rifampin-resistant M. tuberculosis could be detected in DNA extracted from auramine-rhodamine-positive sputum samples in a single-tube assay that took less than 3 h to perform for a collection of auramine-rhodamine-positive specimens obtained from patients with culture-documented pulmonary tuberculosis. Similarly, this occurs in half of the isoniazid-resistant M. tuberculosis DNA extracted from auramine-rhodamine-positive specimens.

In Vitro Activities of Tigecycline, Minocycline, and Colistin-Tigecycline Combination against Multi- and Pandrug-Resistant Clinical Isolates of Acinetobacter baumannii Group

Acinetobacter baumannii has emerged worldwide as an important nosocomial and opportunistic pathogen, particularly in intensive care units ([7][1]). Treatment is difficult due to the high rate of multidrug resistance (including carbapenems) ([4][2], [7][1]). As a result, polymyxins have been

Improved real-time PCR for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis clinical isolates

In this study we designed two pairs of probes for the detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis with real-time PCR procedures. One pair of probes spans the region between codon 510 and 528 of the rpoB gene, and the other one screens for mutation at the regulatory region of the inhA gene. We have evaluated these probes in combination with two other pairs of probes previously described to detect mutations in 20 susceptible and 53 unique resistant M. tuberculosis clinical isolates. We were able to detect nine different mutations affecting five codons of the rpoB gene, two different mutations at codon 315 of the katG gene and a nucleotide substitution (C209T) in the regulatory region of the inhA gene within two hours turnaround.

Rapid detection and identification of Staphylococcus aureus from blood culture specimens using real-time fluorescence PCR

Molecular surveillance of pathogens has shown the need for rapid and dependable methods for the detection and identification of organisms of clinical and epidemiologic importance. Staphylococcus aureus, one of the most frequent causes of human infections, was used as a model organism to develop and refine a real-time fluorescence PCR assay and enhanced DNA purification method. One hundred clinical isolates of S. aureus, verified by biochemical reactions and latex agglutination and 90 negative control clinical isolates were screened in the assay. Moreover, fifty blood broth samples from blood culture bottles showing Gram-positive cocci in clusters on direct Grams stain and 25 showing Gram-negative bacilli were screened. The probes, constructed from the nuc gene, correctly detected all S. aureus genomes present without cross-reaction to negative controls. The speed and ease of this approach will make it adaptable to identification of many bacterial pathogens and provide potential for adaptation to direct detection from other types of clinical specimens.

Characterization of Candida parapsilosis complex isolates

Candida parapsilosis former groups II and III have recently been established as independent species, named Candida orthopsilosis and Candida metapsilosis, respectively. We investigated the distribution of C. parapsilosis complex species in 122 isolates from blood and other sources in a southern Spain tertiary-care hospital, and we examined the relationship between species, site of isolation and biofilm positivity. We also evaluated the planktonic MICs and sessile MICs (SMICs) of voriconazole, amphotericin B and anidulafungin. One hundred and eleven isolates (91%) were categorized as C. parapsilosis sensu stricto, whereas ten isolates (8.2%) were categorized as C. orthopsilosis and one (0.8%) as C. metapsilosis. Biofilm positivity was observed in 58.5% (65 of 111) of C. parapsilosis sensu stricto isolates vs. 0% (0 of 11) of C. orthopsilosis and C. metapsilosis isolates (p <0.01). There was no difference in biofilm production among C. parapsilosis sensu stricto isolates from blood and other sources. MIC values showed that all isolates were susceptible to voriconazole and amphotericin B, whereas two isolates (1.8%) of C. parapsilosis sensu stricto were non-susceptible to anidulafungin. However, the MIC₉₀ value of voriconazole was higher (0.125 mg/L) for C. orthopsilosis than for C. parapsilosis sensu stricto (0.03 mg/L). In contrast to planktonic cells, the SMICs show that amphotericin B and anidulafungin are moderately effective against the biofilm of C. parapsilosis sensu stricto, whereas voriconazole is ineffective.

Fine-tuning the space, time, and host distribution of mycobacteria in wildlife

BackgroundWe describe the diversity of two kinds of mycobacteria isolates, environmental mycobacteria and Mycobacterium bovis collected from wild boar, fallow deer, red deer and cattle in Doñana National Park (DNP, Spain), analyzing their association with temporal, spatial and environmental factors.ResultsHigh diversity of environmental mycobacteria species and M. bovis typing patterns (TPs) were found. When assessing the factors underlying the presence of the most common types of both environmental mycobacteria and M. bovis TPs in DNP, we evidenced (i) host species differences in the occurrence, (ii) spatial structuration and (iii) differences in the degree of spatial association of specific types between host species. Co-infection of a single host by two M. bovis TPs occurred in all three wild ungulate species. In wild boar and red deer, isolation of one group of mycobacteria occurred more frequently in individuals not infected by the other group. While only three TPs were detected in wildlife between 1998 and 2003, up to 8 different ones were found during 2006-2007. The opposite was observed in cattle. Belonging to an M. bovis-infected social group was a significant risk factor for mycobacterial infection in red deer and wild boar, but not for fallow deer. M. bovis TPs were usually found closer to water marshland than MOTT.ConclusionsThe diversity of mycobacteria described herein is indicative of multiple introduction events and a complex multi-host and multi-pathogen epidemiology in DNP. Significant changes in the mycobacterial isolate community may have taken place, even in a short time period (1998 to 2007). Aspects of host social organization should be taken into account in wildlife epidemiology. Wildlife in DNP is frequently exposed to different species of non-tuberculous, environmental mycobacteria, which could interact with the immune response to pathogenic mycobacteria, although the effects are unknown. This research highlights the suitability of molecular typing for surveys at small spatial and temporal scales.

West Nile virus past infections in the general population of Southern Spain

OBJECTIVE To analyze the prevalence of past and recent infections by West Nile virus (WNV) and the risk factors associated with WNV exposure in a representative population from southern Spain. METHODS Sample size was established for an estimated prevalence of past WNV infections of 5 +/- 2.5% in 504 subjects. A pre-stratification was performed according to age distribution and place of residence. After random telephone solicitation and acquisition of informed consent, a serum sample was collected and an epidemiologic survey performed on all participating subjects. Samples were tested with ELISA-IgG and MAC-ELISA to detect specific IgG and IgM antibodies; results were confirmed by the plaque reduction neutralization test (PRNT). Multivariate analysis using a forward stepwise logistic regression model was performed to assess potential risk factors associated with WNV exposure. RESULTS Prevalence of past WNV infections confirmed by PRNT in the 504 participants was 0.6%, affecting mainly older persons (mean age 65 +/- 23 vs. 34 +/- 22 years; P = 0.018), those living in rural areas (5.4% vs. 0% in urban areas; P = 0.01), and individuals with risk professions (prevalence 2.8% vs. 0%; P = 0.048). None of the five recent infections detected by MAC-ELISA was confirmed by PRNT. CONCLUSIONS These results strongly suggest past circulation and exposure of the human population to WNV in southern Spain.