Bacteriuria and phenotypic antimicrobial susceptibility testing in 45 min by point-of-care Sysmex PA-100 System: first clinical evaluation.

PURPOSE: This study compared the results of the new Sysmex PA-100 AST System, a point-of-care analyser, with routine microbiology for the detection of urinary tract infections (UTI) and performance of antimicrobial susceptibility tests (AST) directly from urine. METHODS: Native urine samples from 278 female patients with suspected uncomplicated UTI were tested in the Sysmex PA-100 and with reference methods of routine microbiology: urine culture for bacteriuria and disc diffusion for AST. RESULTS: The analyser delivered bacteriuria results in 15 min and AST results within 45 min. Sensitivity and specificity for detection of microbiologically confirmed bacteriuria were 84.0% (89/106; 95% CI: 75.6-90.4%) and 99.4% (155/156; 95% CI: 96.5-100%), respectively, for bacterial species within the analyser specifications. These are Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterococcus faecalis and Staphylococcus saprophyticus, which are common species causing uncomplicated UTI. Overall categorical agreement (OCA) for AST results for the five antimicrobials tested in the Sysmex PA-100 (amoxicillin/clavulanic acid, ciprofloxacin, fosfomycin, nitrofurantoin and trimethoprim) ranged from 85.4% (70/82; 95%CI: 75.9-92.2%) for ciprofloxacin to 96.4% (81/84; 95% CI: 89.9-99.3%) for trimethoprim. The Sysmex PA-100 provided an optimal treatment recommendation in 218/278 cases (78.4%), against 162/278 (58.3%) of clinical decisions. CONCLUSION: This first clinical evaluation of the Sysmex PA-100 in a near-patient setting demonstrated that the analyser delivers phenotypic AST results within 45 min, which could enable rapid initiation of the correct targeted treatment with no further adjustment needed. The Sysmex PA-100 has the potential to significantly reduce ineffective or unnecessary antibiotic prescription in patients with UTI symptoms.

In Vitro Effect of Three-Antibiotic Combinations plus Potential Antibiofilm Agents against Biofilm-Producing Mycobacterium avium and Mycobacterium intracellulare Clinical Isolates.

Patients with chronic pulmonary diseases infected by Mycobacterium avium complex (MAC) often develop complications and suffer from treatment failure due to biofilm formation. There is a lack of correlation between in vitro susceptibility tests and the treatment of clinical isolates producing biofilm. We performed susceptibility tests of 10 different three-drug combinations, including two recommended in the guidelines, in biofilm forms of eight MAC clinical isolates. Biofilm developed in the eight isolates following incubation of the inoculum for 3 weeks. Then, the biofilm was treated with three-drug combinations with and without the addition of potential antibiofilm agents (PAAs). Biofilm bactericidal concentrations (BBCs) were determined using the Vizion lector system. All selected drug combinations showed synergistic activity, reducing BBC values compared to those treated with single drugs, but BBC values remained high enough to treat patients. However, with the addition of PAAs, the BBCs steadily decreased, achieving similar values to the combinations in planktonic forms and showing synergistic activity in all the combinations and in both species. In conclusion, three-drug combinations with PAAs showed synergistic activity in biofilm forms of MAC isolates. Our results suggest the need for clinical studies introducing PAAs combined with antibiotics for the treatment of patients with pulmonary diseases infected by MAC.

Activity of Antibiotics and Potential Antibiofilm Agents against Biofilm-Producing Mycobacterium avium-intracellulare Complex Causing Chronic Pulmonary Infections.

Nontuberculous mycobacteria (NTM) cause lung infections in patients with underlying pulmonary diseases (PD). The Mycobacteriumavium-intracellulare complex (MAC) is the most frequently involved NTM. The MAC-PD treatment is based on the administration of several antibiotics for long periods of time. Nonetheless, treatment outcomes remain very poor. Among the factors involved is the ability of MAC isolates to form biofilm. The aim of the study was to assess the in vitro activity of different antibiotics and potential antibiofilm agents (PAAs) against MAC biofilm. Four antibiotics and six PAAs, alone and/or in combination, were tested against planktonic forms of 11 MAC clinical isolates. Biofilm was produced after 4 weeks of incubation and analyzed with the crystal violet assay. The antibiotics and PAAs were tested by measuring the absorbance (minimum biofilm inhibition concentrations, MBICs) and by performing subcultures (minimum biofilm eradication concentrations, MBECs). The clarithromycin/amikacin and clarithromycin/ethambutol combinations were synergistic, decreasing the MBECs values compared to the individual antibiotics. The amikacin/moxifloxacin combination showed indifference. The MBIC values decreased significantly when PAAs were added to the antibiotic combinations. These results suggest that antibiotic combinations should be further studied to establish their antibiofilm activity. Moreover, PAAs could act against the biofilm matrix, facilitating the activity of antibiotics.

Multicentre study on the reproducibility of MALDI-TOF MS for nontuberculous mycobacteria identification.

The ability of MALDI-TOF for the identification of nontuberculous mycobacteria (NTM) has improved recently thanks to updated databases and optimized protein extraction procedures. Few multicentre studies on the reproducibility of MALDI-TOF have been performed so far, none on mycobacteria. The aim of this study was to evaluate the reproducibility of MALDI-TOF for the identification of NTM in 15 laboratories in 9 European countries. A total of 98 NTM clinical isolates were grown on Löwenstein-Jensen. Biomass was collected in tubes with water and ethanol, anonymized and sent out to the 15 participating laboratories. Isolates were identified using MALDI Biotyper (Bruker Daltonics). Up to 1330 MALDI-TOF identifications were collected in the study. A score ≥ 1.6 was obtained for 100% of isolates in 5 laboratories (68.2-98.6% in the other). Species-level identification provided by MALDI-TOF was 100% correct in 8 centres and 100% correct to complex-level in 12 laboratories. In most cases, the misidentifications obtained were associated with closely related species. The variability observed for a few isolates could be due to variations in the protein extraction procedure or to MALDI-TOF system status in each centre. In conclusion, MALDI-TOF showed to be a highly reproducible method and suitable for its implementation for NTM identification.

Inducible and Acquired Clarithromycin Resistance in the Mycobacterium abscessus Complex.

PURPOSE: Clarithromycin was considered the cornerstone for the treatment of Mycobacterium abscessus complex infections. Genetic resistance mechanisms have been described and many experts propose amikacin as an alternative. Nevertheless, clarithromycin has several advantages; therefore, it is necessary to identify the non-functional erm(41) allele to determine the most suitable treatment. The aims of this study were to characterize the molecular mechanisms of clarithromycin resistance in a collection of Mycobacterium abscessus complex isolates and to verify the relationship between these mechanisms and the antibiogram. MATERIALS AND METHODS: Clinical isolates of M. abscessus complex (n = 22) from 16 patients were identified using four housekeeping genes (rpoB, secA1, sodA and hsp65), and their genetic resistance was characterized by studying erm(41) and rrl genes. Nine strains were recovered from the clinical isolates and subjected to E-test and microdilution clarithromycin susceptibility tests, with readings at 3, 7 and 14 days. RESULTS: We classified 11/16 (68.8%) M. abscessus subsp. abscessus, 4/16 (25.0%) M. abscessus subsp. bolletii, and 1/16 (6.3%) M. abscessus subsp. massiliense. T28 erm(41) allele was observed in 8 Mycobacterium abscessus subps. abscessus and 3 Mycobacterium abscessus subsp. bolletii. One strain of M. abscessus subsp. bolletii had an erm(41) gene truncated and was susceptible to clarithromycin. No mutations were observed in rrl gene first isolates. In three patients, follow-up of initial rrl wild-type strains showed acquired resistance. CONCLUSIONS: Most clinical isolates of M. abscessus complex had inducible resistance to clarithromycin and total absence of constitutive resistance. Our findings showed that the acquisition of resistance mutations in rrl gene was associated with functional and non-functional erm(41) gene. Caution is needed when using erm(41) sequencing alone to identify M. abscessus subspecies. This study reports an acquired mutation at position 2057 of rrl gene, conferring medium-low clarithromycin constitutive resistance.

Utilización de diferentes técnicas de biología molecular integradas en un algoritmo de identificación de micobacterias no tuberculosas / Use of different PCR-based techniques integrated into a non-tuberculous identification algorithm

Introducción El objetivo del presente trabajo fue demostrar la utilidad de un algoritmo de identificación de micobacterias no tuberculosas (MNT) que integra diferentes técnicas de biología molecular y características fenotípicas básicas. Además se ha realizado una actualización del algoritmo de interpretación del análisis del patrón de restrición de hsp65 (PRA hsp65).Métodos La manera elegida de trabajar consistió en la identificación mediante hibridación con sondas de ADN seguido de PRA hsp65 en aquellos aislados que no pudieron ser identificados mediante hibridación con sondas de ADN. En caso necesario se realizó secuenciación del 16S rDNA y hsp65.ResultadosSe aislaron 236 MNT. De ellos, 102 (43,2%) aislados fueron identificados mediante hibridación con sondas de ADN y 76 (32,2%) mediante PRA hsp65. En los 58 (24,5%) aislados restantes se secuenció 16S rDNA, lo cual permitió la identificación de 53 (22,4%). Para 5 (2,1%) aislados se secuenció hsp65 y permitió la identificación de un aislado más. Cuatro (1,7%) aislados no pudieron ser identificados. Tres nuevos patrones de PRA hsp65 fueron encontrados. Siete aislamientos hibridaron con la sonda AccuProbe Mycobacterium avium complex Identification pero no lo hicieron con las sondas específicas de especie incluidas en el MAC. Cinco y 2 aislados fueron identificados como M. intracellulare y Mycobacterium colombiense, respectivamente. Conclusión Este esquema de trabajo nos permitió la identificación de casi todas las MNT encontradas en este estudio, incluyendo especies recientemente descritas(AU)

Introduction The aim of the present work was to demonstrate the utility of a non-tuberculous mycobacteria (NTM) identification algorithm, which integrates different PCR-based techniques and basic phenotypic features. Moreover, the algorithm for pattern restriction analysis of hsp65 (hsp65 PRA) interpretation has been updated. Methods The workflow chosen consisted of the identification by a DNA hybridization probe method, followed by PCR-restriction enzyme analysis of hsp65 (hsp65 PRA) in those isolates that cannot be identified by hybridization probes. If necessary, 16S rRNA gene and hsp65 gene sequencing were used for speciation. Results A total of 236 NTM were collected, in which 102 (43.2%) isolates were identified by DNA specific probes and 76 (32.2%) isolates were identified with hsp65 PRA. Partial sequencing of the 16S rRNA gene was used for species identification of the remaining 58 (24.5%) isolates. Fifty-three (22.4%) were identified using this method. Five isolates (2.1%) were submitted for partial sequencing of hsp65 gene and one isolate was identified with this method. Four strains (1.7%) could not be identified at species level. Three new PRA patterns were found. Seven isolates tested positive with the AccuProbe Mycobacterium avium complex identification test but did not test positive with the M. avium or Mycobacterium intracellulare specific probes. Five and two of these isolates were identified as M. intracellulare and Mycobacterium colombiense, respectively. Conclusion This approach allowed us to identify almost all NTM isolates found in this study, including some recently described species(AU)

[Use of different PCR-based techniques integrated into a non-tuberculous identification algorithm]. / Utilización de diferentes técnicas de biología molecular integradas en un algoritmo de identificación de micobacterias no tuberculosas.

INTRODUCTION: The aim of the present work was to demonstrate the utility of a non-tuberculous mycobacteria (NTM) identification algorithm, which integrates different PCR-based techniques and basic phenotypic features. Moreover, the algorithm for pattern restriction analysis of hsp65 (hsp65 PRA) interpretation has been updated. METHODS: The workflow chosen consisted of the identification by a DNA hybridization probe method, followed by PCR-restriction enzyme analysis of hsp65 (hsp65 PRA) in those isolates that cannot be identified by hybridization probes. If necessary, 16S rRNA gene and hsp65 gene sequencing were used for speciation. RESULTS: A total of 236 NTM were collected, in which 102 (43.2%) isolates were identified by DNA specific probes and 76 (32.2%) isolates were identified with hsp65 PRA. Partial sequencing of the 16S rRNA gene was used for species identification of the remaining 58 (24.5%) isolates. Fifty-three (22.4%) were identified using this method. Five isolates (2.1%) were submitted for partial sequencing of hsp65 gene and one isolate was identified with this method. Four strains (1.7%) could not be identified at species level. Three new PRA patterns were found. Seven isolates tested positive with the AccuProbe Mycobacterium avium complex identification test but did not test positive with the M. avium or Mycobacterium intracellulare specific probes. Five and two of these isolates were identified as M. intracellulare and Mycobacterium colombiense, respectively. CONCLUSION: This approach allowed us to identify almost all NTM isolates found in this study, including some recently described species.

Evaluation of the BacT/ALERT PZA kit in comparison with the BACTEC 460TB PZA for testing Mycobacterium tuberculosis susceptibility to pyrazinamide.

OBJECTIVES: To compare the performance of the BacT/ALERT PZA kit (BioMerieux, Marcy l'Etoile, France) with the radiometric BACTEC 460TB PZA test (Becton-Dickinson method) for testing Mycobacterium tuberculosis susceptibility to pyrazinamide. METHODS: A total of 50 M. tuberculosis strains were tested. Thirty of these strains had been previously considered pyrazinamide-susceptible and 20 pyrazinamide-resistant by BACTEC 460TB. RESULTS: Final overall agreement was 100%. Time needed for the susceptibility test was 6.69 days for the BacT/ALERT PZA kit versus 8.07 days for the BACTEC 460TB PZA test. CONCLUSIONS: BacT/ALERT PZA test is an excellent alternative to BACTEC 460TB for pyrazinamide susceptibility testing.

Multicenter laboratory evaluation of the MB/BacT Mycobacterium detection system and the BACTEC MGIT 960 system in comparison with the BACTEC 460TB system for susceptibility testing of Mycobacterium tuberculosis.

In this multicenter study, the reliability of two nonradiometric, fully automated systems, the MB/BacT and BACTEC MGIT 960 systems, for testing the susceptibilities of 82 Mycobacterium tuberculosis strains to isoniazid, rifampin, ethambutol, and streptomycin was evaluated in comparison with the radiometric BACTEC 460TB system. The arbitration of discrepant results was done by the reanalysis of the strain, the determination of the MIC, and the molecular characterization of some resistance determinants. The overall level of agreement with BACTEC 460TB results was 96% with the MB/BacT test and 97.2% with the BACTEC MGIT 960 system. With both methods, the level of agreement with BACTEC 460TB results was 96.3% for isoniazid, 98.8% for rifampin, and 98.8% for ethambutol. The level of agreement for streptomycin was 90.2% with MB/BacT and 97.5% with BACTEC MGIT 960. Overall, there were 11 very major errors and 2 major errors with the MB/BacT method and 5 very major errors and 2 major errors with the BACTEC MGIT 960 system. In general, the MB/BacT and BACTEC MGIT 960 systems showed good performance for susceptibility testing with first-line antituberculosis drugs.

Rapid detection of specific gene mutations associated with isoniazid or rifampicin resistance in Mycobacterium tuberculosis clinical isolates using non-fluorescent low-density DNA microarrays.

BACKGROUND: A new, fast 'low cost and density' DNA microarray (LCD array), designed for the detection of mutations that confer isoniazid or rifampicin resistance in Mycobacterium tuberculosis isolates, has been developed and was evaluated using 46 resistant clinical isolates from Barcelona. METHODS: LCD chips are pre-structured polymer supports using a non-fluorescent detection principle based on the precipitation of a clearly visible dark substrate. One LCD chip consists of eight identical microarrays, designed to detect mutations within the 90 bp rpoB region, codon 315 in the katG gene and the mabA-inhA regulatory region. A total of 22 strains with a katG 315 mutation, 19 strains with alterations in the mabA-inhA regulatory region and 16 strains with mutations in the rpoB region, characterized previously, were studied. RESULTS: The identification of S315T and S315N mutations using the LCD was 100% concordant with the sequencing data. A strain with the S315R mutation, which is not tiled on the LCD array, was detected by the absence of hybridization using the wild-type probe. Of 19 strains with low-level isoniazid resistance related to the mabA-inhA regulatory region, 18 were identified correctly. The detection of mutations in the rpoB region was 93.8% concordant with the sequencing data. One mabA-inhA and rpoB mutated strain showed a cross-hybridization. CONCLUSIONS: The LCD array protocol takes 45 min (15 min 'hands-on' time) after prior PCR amplification. Only minimal laboratory equipment is required. LCD arrays provide a rapid and economical method to characterize mutations in codon 315 of the katG gene, in the mabA-inhA regulatory region and in the rpoB gene.

Molecular analysis of isoniazid and rifampin resistance in Mycobacterium tuberculosis isolates recovered from Barcelona.

We studied the presence of mutations in the whole katG gene and specific regions of the oxyR-ahpC and mabA-inhA regulatory region in 61 Mycobacterium tuberculosis isoniazid-resistant isolates. An 81-bp region of the rpoB gene was also sequenced in 17 rifampin-resistant strains. Alterations in the katG gene were detected in 55% of the isolates. Mutation in codon 315 was the most prevalent (32%). Strains showed a high level of resistance, and most maintained a substantial catalase-peroxidase activity. Three strains with an isoniazid MIC of >or=32 microg/ml lacked catalase-peroxidase activity. Two of them had deletions in the catalytic domain of the KatG protein. One strain with deletion and three strains with mutations in the C-terminal domain showed low-level resistance and conserved the catalase-peroxidase activity. Mutations in the mabA-inhA regulatory region were identified in 32% of the isolates. All had low-level resistance, and the vast majority conserved catalase-peroxidase activity. Seventeen percent of the isoniazid-resistant isolates had no detectable alterations at the studied loci. Resistance to rifampin was associated with mutations in the 81-bp of the rpoB gene in all cases. IS6110 analysis indicated that recent transmission contributed substantially to the emergence of isoniazid- resistant tuberculosis in Barcelona through short transmission chains. A rapid genotypic assay, including the 315-katG codon and the -15 nucleotide of the mabA-inhA regulatory region, may cover 62% of isoniazid- resistant strains in Barcelona. In contrast, the targeting of the 81-bp region of rpoB would detect all our rifampin-resistant isolates.