María Jesús Ruiz-Serrano

In Vitro Activities of Linezolid against Clinical Isolates of Mycobacterium tuberculosis That Are Susceptible or Resistant to First-Line Antituberculous Drugs

ABSTRACT We evaluated 117 isolates of Mycobacterium tuberculosis for susceptibility to linezolid by the proportion and E-test methods. Linezolid showed high in vitro activity, with all the strains inhibited by ≤1 μg of the drug per ml. E-test MICs were at least 4 dilutions lower than their equivalents by the standard proportion method.

Rapid Direct Detection of Multiple Rifampin and Isoniazid Resistance Mutations in Mycobacterium tuberculosis in Respiratory Samples by Real-Time PCR

ABSTRACT Rapid detection of resistance in Mycobacterium tuberculosis can optimize the efficacy of antituberculous therapy and control the transmission of resistant M. tuberculosis strains. Real-time PCR has minimized the time required to obtain the susceptibility pattern of M. tuberculosis strains, but little effort has been made to adapt this rapid technique to the direct detection of resistance from clinical samples. In this study, we adapted and evaluated a real-time PCR design for direct detection of resistance mutations in clinical respiratory samples. The real-time PCR was evaluated with (i) 11 clinical respiratory samples harboring bacilli resistant to isoniazid (INH) and/or rifampin (RIF), (ii) 10 culture-negative sputa spiked with a set of strains encoding 14 different resistance mutations in 10 independent codons, and (iii) 16 sputa harboring susceptible strains. The results obtained with this real-time PCR design completely agreed with DNA sequencing data. In all sputa harboring resistant M. tuberculosis strains, the mutation encoding resistance was successfully detected. No mutation was detected in any of the susceptible sputa. The test was applied only to smear-positive specimens and succeeded in detecting a bacterial load equivalent to 103 CFU/ml in sputum samples (10 acid-fast bacilli/line). The analytical specificity of this method was proved with a set of 14 different non-M. tuberculosis bacteria. This real-time PCR design is an adequate method for the specific and rapid detection of RIF and INH resistance in smear-positive clinical respiratory samples.

In Vitro Activities of Six Fluoroquinolones against 250 Clinical Isolates of Mycobacterium tuberculosis Susceptible or Resistant to First-Line Antituberculosis Drugs

ABSTRACT Two hundred fifty isolates of Mycobacterium tuberculosis were evaluated for susceptibility to ciprofloxacin, ofloxacin, levofloxacin, grepafloxacin, trovafloxacin, and gemifloxacin (SB-265805). Levofloxacin, ciprofloxacin, and grepafloxacin showed the greatest activity (MIC for 90% of strains tested [MIC90] 1 μg/ml), although ofloxacin also showed good activity, with an MIC90 of 2 μg/ml. Trovafloxacin and gemifloxacin showed lower in vitro activity, with MIC90s of 64 and 8 μg/ml, respectively.

Systematic Survey of Clonal Complexity in Tuberculosis at a Populational Level and Detailed Characterization of the Isolates Involved

ABSTRACT Clonally complex infections by Mycobacterium tuberculosis are progressively more accepted. Studies of their dimension in epidemiological scenarios where the infective pressure is not high are scarce. Our study systematically searched for clonally complex infections (mixed infections by more than one strain and simultaneous presence of clonal variants) by applying mycobacterial interspersed repetitive-unit (MIRU)–variable-number tandem-repeat (VNTR) analysis to M. tuberculosis isolates from two population-based samples of respiratory (703 cases) and respiratory-extrapulmonary (R+E) tuberculosis (TB) cases (71 cases) in a context of moderate TB incidence. Clonally complex infections were found in 11 (1.6%) of the respiratory TB cases and in 10 (14.1%) of those with R+E TB. Among the 21 cases with clonally complex TB, 9 were infected by 2 independent strains and the remaining 12 showed the simultaneous presence of 2 to 3 clonal variants. For the 10 R+E TB cases with clonally complex infections, compartmentalization (different compositions of strains/clonal variants in independent infected sites) was found in 9 of them. All the strains/clonal variants were also genotyped by IS6110-based restriction fragment length polymorphism analysis, which split two MIRU-defined clonal variants, although in general, it showed a lower discriminatory power to identify the clonal heterogeneity revealed by MIRU-VNTR analysis. The comparative analysis of IS6110 insertion sites between coinfecting clonal variants showed differences in the genes coding for a cutinase, a PPE family protein, and two conserved hypothetical proteins. Diagnostic delay, existence of previous TB, risk for overexposure, and clustered/orphan status of the involved strains were analyzed to propose possible explanations for the cases with clonally complex infections. Our study characterizes in detail all the clonally complex infections by M. tuberculosis found in a systematic survey and contributes to the characterization that these phenomena can be found to an extent higher than expected, even in an unselected population-based sample lacking high infective pressure.

Antifungal Susceptibility of 596 Aspergillus fumigatus Strains Isolated from Outdoor Air, Hospital Air, and Clinical Samples: Analysis by Site of Isolation

ABSTRACT We analyzed the activities of six antifungal drugs (amphotericin B, itraconazole, voriconazole, posaconazole, caspofungin, and micafungin) against 596 Aspergillus fumigatus strains isolated from outdoor air, hospital air, and clinical samples. We did not find differences among the susceptibilities by site of isolation.

Espondilodiscitis tuberculosa o enfermedad de Pott: experiencia en un hospital general

Fundamentos La enfermedad de Pott es una manifestacion infrecuente de tuberculosis extrapulmonar. Los datos sobre localizacion, diagnostico y tratamiento son muy dispares segun los centros y paises. Pacientes Y METodo Revisamos nuestra experiencia con la enfermedad de Pott durante un periodo de 7 anos (1993-1999), de forma retrospectiva y basandonos en datos clinicos, bacteriologicos y microbiologicos. Encontramos 14 casos de enfermedad de Pott. Revisamos las siguientes variables: edad, sexo, signos y sintomas de presentacion, reaccion de la tuberculina (PPD), datos microbiologicos, histopatologicos, de extension, tratamiento y evolucion clinica. Resultados El numero de casos de tuberculosis con diagnostico microbiologico durante este periodo fue de 1.400 (4 casos por cada 1.000 ingresos). De ellos, 1.047 casos (74,8%) fueron pulmonares y 353 (25,2%) extrapulmonares. Encontramos 14 casos de enfermedad de Pott, cinco de ellos con cultivo positivo (un 1,4% extrapulmonares). La edad media de nuestros pacientes fue de 58 anos. El tiempo de latencia hasta el diagnostico vario entre 2 y 720 dias. El dolor fue el sintoma de presentacion mas referido. La localizacion dorsal fue la mas frecuente (8/14). Encontramos tuberculosis en otra localizacion en 6 casos y PPD+ en 7 pacientes (50%). En cinco de los 8 casos en que se realizo, el cultivo de puncion-aspiracion con aguja fina (PAAF) o biopsia fue diagnostico, y la histologia fue congruente en ocho de los 11 casos en que pudo llevarse a cabo. Asimismo, encontramos discitis en 7 casos, absceso a partes blandas en 9 pacientes (2 abscesos del psoas) y en 9 casos fue necesario realizar una intervencion quirurgica (en tres de los cuales fue diagnostica). Conclusiones La enfermedad de Pott es una forma actualmente infrecuente de tuberculosis extrapulmonar en nuestro medio, incluso en pacientes infectados por el virus de la inmunodefiencia humana (VIH). La localizacion dorsal, la discitis y las formas evolucionadas con necesidad de planteamiento quirurgico siguen siendo habituales.

Evaluation of the upgraded amplified mycobacterium tuberculosis direct test (gen-probe) for direct detection of mycobacterium tuberculosis in respiratory and non-respiratory specimens

We evaluated the upgraded Amplified Mycobacterium Tuberculosis Direct Test kit (AMTD) (Gen-Probe Inc.) for the direct detection of Mycobacterium tuberculosis in respiratory and non-respiratory specimens, and compared the results between the traditional 30,000 RLUs cutoff criteria (C) and three equivocal ranges (30,000-100,000, R1; 30,000-500,000, R2; and 30,000-1,000,000, R3). We tested 663 respiratory and 238 non-respiratory samples from 464 patients. The gold standard was considered to be the combination of culture and clinical data. One hundred and nineteen samples were from 56 patients with pulmonary tuberculosis, and 36 samples were from 19 patients with extrapulmonary tuberculosis. When C criteria was applied, the sensitivity and specificity values were 90.8 and 93.0% for respiratory specimens, while they were 88.9 and 92.1% for non-respiratory specimens (p = NS). The sensitivity was significantly higher in smear-positive specimens (96.7%) than in smear-negative ones (81.0%) (p < 0.05). When compared with C criteria, the overall sensitivity was maintained at 90.3% for R1 criteria, and slightly decreased to 89.7% for R2 and R3 criteria (p = NS). Overall, specificity increased significantly from 92.9% (C) to 97.5% (R1), 99.1% (R2), and 99.2% (R3). Application of R2 or R3 criteria improved significantly the specificity of the test with little decrease in sensitivity.

Evaluation of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Identification of Nontuberculous Mycobacteria from Clinical Isolates

ABSTRACT Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) for the identification of nontuberculous mycobacterial (NTM) isolates was evaluated in this study. Overall, 125 NTM isolates were analyzed by MALDI-TOF and GenoType CM/AS. Identification by 16S rRNA/hsp65 sequencing was considered the gold standard. Agreements between MALDI-TOF and GenoType CM/AS with the reference method were, respectively, 94.4% and 84.0%. In 17 cases (13.6%), results provided by GenoType and MALDI-TOF were discordant; however, the reference method agreed with MALDI-TOF in 16/17 cases (94.1%; P = 0.002).

Evaluation of GeneXpert MTB/RIF for the detection of Mycobacterium tuberculosis and resistance to rifampin in clinical specimens.

OBJECTIVESnGeneXpert (GX) is a novel real-time polymerase chain reaction assay for the simultaneous detection of Mycobacterium tuberculosis (MTB) and resistance to rifampin (RIF). We evaluated the performance of GX for direct detection of MTB in respiratory and non-respiratory specimens and assessed the ability of the assay to detect resistance to RIF in non-tuberculosis (TB) endemic country.nnnMETHODSnWe analyzed 595 clinical samples in a 1550-bed tertiary hospital in Madrid, Spain. Specimens were processed using GX, auramine smear, conventional culture, and drug phenotypic susceptibility testing (DST) with MGIT SIRE (BD).nnnRESULTSnOf the 595 clinical samples, 305 (51.3%) were non-respiratory and 290 (48.7%) were respiratory. In total, MTB was isolated in 81 specimens, 71 of which were positive with GX. The sensitivity, specificity, PPV, and NPV of the GX were: 97.1%, 98.6%, 95.7% and 99.1% for respiratory samples and 33.3%, 99.7%, 80.0% and 97.3% for non-respiratory. GX detected 8 RIF-resistant samples, but only 5 were confirmed by DST.nnnCONCLUSIONSnGX is an accurate, easy-to-apply and rapid test to detect MTB, especially in smear-positive respiratory samples. Although is a useful tool for the rapid identification of RIF-resistant MTB strains, the rifampin resistant results should be confirmed by DST.

LCx: a diagnostic alternative for the early detection of Mycobacterium tuberculosis complex.

This study aims to evaluate the performance of a new diagnostic method (LCx Tuberculosis Assay, Abbott Laboratories) based on Ligase Chain Reaction (LCR) technology, for the detection of Mycobacterium tuberculosis in respiratory and non-respiratory specimens and compare it with standard microbiological data and the clinical diagnosis of tuberculosis. Nine hundred specimens were collected from patients with a high suspicion of tuberculosis (740 respiratory samples and 160 non-respiratory specimens). The study was divided into two separate groups: samples washed and distilled water (207 samples) and unwashed samples that were directly resuspended in phosphate buffer (693 samples). The overall sensitivity, specificity, positive and negative predictive values of samples washed with distilled water after decontamination with SDS-NaOH were: 54%, 100%, 100%, and 94%, respectively. If these results were divided according to origin of specimens, the sensitivity, specificity, positive and negative predictive values in respiratory and non-respiratory samples were 54.5%, 100%, 100%, 94% and 50 100%, 100%, 93%, respectively. In contrast, for the non-washed samples, values were 85%, 95%, 80% and 98%, respectively. Respiratory and non-respiratory samples gave values of 84%, 96%, 77%, and 97.5% versus 89%, 99%, 94%, and 98%. The LCx M. tuberculosis assay is a novel, semi-automated assay and a rapid and highly specific technique for screening all forms of tuberculosis, including non-respiratory forms.