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QuantiFERON-TB Gold Plus (QFT-Plus) is the most widely used interferon gamma release assay (IGRA) for the diagnosis of latent tuberculosis infection (LTBI). The aim of this study was to compare QFT-Plus results by enzyme-linked immunosorbent assay (ELISA) on the SkyLab system with those obtained with chemiluminescence immunoassay (CLIA) on the Liaison XL analyzer. Agreement between the two assays was evaluated on 419 QFT-Plus blood samples and was found to be substantial (75.4%); higher agreement was found for positive (95.4%) and negative (80.4%) results, while most discordances were due to ELISA-indeterminate/CLIA-determinate results. According to Italian Clinical Microbiologist Association recommendations, in samples (n = 79) with a borderline result in ELISA (0.20 to 0.70 IU/ml), CLIA median values statistically increased (from 0.29 to 0.59 IU/ml for TB1 and from 0.32 to 0.60 IU/ml for TB2) but remained in the borderline range. Linear regression analysis indicated a substantial correlation between ELISA and CLIA for antigen tubes TB1 (Pearson's r = 0.8666) and TB2 (Pearson's r = 0.8728), but CLIA produced higher values than ELISA. Receiver operating characteristic (ROC) analysis showed that the optimal cutoff value in CLIA was 0.45 IU/ml for TB1 and 0.46 IU/ml for TB2. In conclusion, automated QFT-Plus with CLIA is comparable to QFT-Plus performed by ELISA. Within the linearity range of the test, CLIA detects higher quantitative values than ELISA, resulting in a higher number of determinate results and the conversion of samples that were close to the cutoff into positive borderline results. A higher cutoff for QFT-CLIA needs to be defined based on clinical diagnostic criteria.
Assuntos
Tuberculose Latente , Mycobacterium tuberculosis , Ensaio de Imunoadsorção Enzimática , Humanos , Testes de Liberação de Interferon-gama , Tuberculose Latente/diagnóstico , Luminescência , Medições Luminescentes , Teste TuberculínicoRESUMO
The long incubation time required for Mycobacteria detection may allow cultures to become overgrown by contaminating organisms. Therefore, samples need to be decontaminated before solid and liquid culture. MYCO-TB is a ready-to-use digestion and decontamination kit with single-sample formulation developed by Copan. Sample processing time (3 minutes) is shorter than that of other commercial kits. The aim of this study was to compare the performance of MYCO-TB with MycoPrep, both based on N-acetyl-Lcysteine and sodium hydroxide solution, in terms of culture contamination and Mycobacterial detection by culture. We tested 162 respiratory samples: the overall proportions of contamination of both liquid and solid media were 1.8% for MYCO-TB and 1.8% for MycoPrep. Mycobacterial growth was detected without significant differences in times to positivity (TTP) in liquid culture: 10.5 days for MYCO-TB and 11.1 days for MycoPrep. Samples decontaminated with MYCO-TB were suitable for molecular assays such as Xpert MTB/RIF Ultra and GenoType CMdirect. Extending decontamination times (up to 10 minutes) with MYCO-TB of 20 Mycobacteria-positive specimens did not produce any difference in TTP in liquid culture or in Ultra IS1081/IS6110 probe Ct values. In conclusion, the MYCO-TB kit proved to be effective for the rapid digestion and decontamination of respiratory materials for the detection of Mycobacteria, making it possible to reduce the manual skills required and lower the risk of contamination. Longer decontamination time could be used for samples with a high level of contamination, such as those from cystic fibrosis patients.
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Técnicas Bacteriológicas , Mycobacterium tuberculosis , Kit de Reagentes para Diagnóstico , Tuberculose , Técnicas Bacteriológicas/métodos , Técnicas Bacteriológicas/normas , Descontaminação , Humanos , Mycobacterium tuberculosis/isolamento & purificação , Kit de Reagentes para Diagnóstico/normas , Sensibilidade e Especificidade , Tuberculose/diagnóstico , Tuberculose/microbiologiaRESUMO
Sputum acid-fast bacilli smear conversion is a fundamental index of treatment response and reduced infectivity in patients with pulmonary tuberculosis (P-TB). To date, there are no models to predict the time to sputum conversion based on patient characteristics. This study aims to ascertain the time to sputum conversion in patients with smear-positive P-TB under treatment, and the variables associated with time to smear conversion. We retrospectively evaluated the time to sputum smear conversion of 89 patients with smear-positive P-TB undergoing treatment at the S. Orsola-Malpighi University Hospital, Bologna (Italy), a referral centre for the diagnosis of TB. Multivariate Cox regression analysis was performed to document variables independently associated with time to conversion. Median time to sputum smear conversion was 24 days (IQR 12-54); the sputum smear converted within the first 2 months of treatment in 78.7% patients. Multivariate Cox regression analysis showed that older age, high baseline mycobacterial load detected by Xpert MTB/RIF, and severity of lung involvement are predictors of persistent smear positivity. The identification of risk factors delaying smear conversion allowed us to develop predictive models that may greatly facilitate the management of smear-positive patients in terms of the duration of respiratory isolation and treatment.
Assuntos
Mycobacterium tuberculosis , Escarro , Tuberculose Pulmonar , Fatores Etários , Humanos , Itália/epidemiologia , Estudos Retrospectivos , Fatores de Risco , Sensibilidade e Especificidade , Escarro/microbiologia , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/epidemiologiaRESUMO
Mycobacterium saskatchewanense is a species of pigmented slow-growing Non-Tuberculous Mycobacteria (NTM), positive for Mycobacterium avium complex (MAC) by AccuProbe system. MAC organisms have frequently been isolated from different medical devices. This is the first study reporting isolation of M. saskatchewanense from medical devices and highlights the importance of correctly identifying the NTMs that often colonize sanitary water. GenoType Mycobacterium CM CE-IVD kit (CM) was used as the first step of NTM strain identification, and all positive cultures were found to be components of MAC. Then, GenoType NTM-DR CE-IVD kit (NTM-DR) was used to differentiate the different species. Sub-culture on solid media were used for: (i) phenotypical confirmation by colony morphology and Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) mass spectrometry; (ii) molecular confirmation by Next Generation Sequencing. All positive cultures were identified as M. intracellulare by CM and NTM-DR assays, whereas colony morphology showed bright yellow scotochromogenic growth. MALDI-TOF analyses identified the strains as M. saskatchewanense with a high score, and identification was confirmed by NGS analysis based on the hsp-65 region. This paper suggests that it is important to actively monitor NTM contamination in medical devices that use sanitary water, to prevent the possibility of patients becoming infected.
Assuntos
Infecções por Mycobacterium não Tuberculosas , Mycobacterium , Humanos , Micobactérias não Tuberculosas/genética , Complexo Mycobacterium avium/genética , Infecções por Mycobacterium não Tuberculosas/microbiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , ÁguaRESUMO
BACKGROUND: Clinical interpretation of trace results by Xpert MTB/RIF Ultra assay (Ultra) used as an initial diagnostic test for tuberculosis (TB) may be challenging. The aim of the study was to evaluate the frequency and epidemiology of trace readouts in routine clinical practice in a low TB prevalence setting and to propose guidance on how to manage patients with trace calls considering the data available (clinical, radiological, bacteriological etc.). MATERIALS AND METHODS: A retrospective, observational, monocentric study was conducted at IRCCS Azienda Ospedaliero-Universitaria of Bologna, Italy between November 2017-December 2020. Presumptive TB patients with at least one Ultra trace result during diagnostic workup before treatment were included in the study. Patients with ongoing anti-TB treatment at the time of the trace call result or with no clinical data available were excluded from the study. RESULTS: Fifty-nine presumptive TB patients with Ultra trace readouts were included in the study (mean age 37.0 years, 61% males). Four patients had a history of TB in the last 2 years. Twenty-five (42.4%) of the 59 samples with trace results were respiratory material. 57/59 (96.6%) patients started anti-TB treatment soon after obtaining trace results, based on clinical, radiological or other information available, while for two patients with a recent history of TB the trace result did not lead to anti-TB treatment. Culture was positive for M. tuberculosis for 31/59 (52.5%) samples with trace calls: 13/25 (52.0%) were respiratory samples and 18/33 (54.5%) non-respiratory samples. The clinical and/or radiological findings of 47/57 (82.4%) patients given anti-TB therapy improved during treatment. CONCLUSION: In low TB incidence settings, Ultra trace calls in presumptive TB patients should be considered as true-positive and treatment should be started promptly, except in cases of recent history of TB, where careful evaluation of other diagnostic criteria is necessary before starting anti-TB treatment. A decisional algorithm for clinical management is proposed.
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Mycobacterium tuberculosis , Tuberculose Pulmonar , Tuberculose , Adulto , Feminino , Humanos , Masculino , Mycobacterium tuberculosis/genética , Estudos Retrospectivos , Sensibilidade e Especificidade , Escarro , Tuberculose/diagnóstico , Tuberculose/tratamento farmacológico , Tuberculose/epidemiologia , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/epidemiologiaRESUMO
Legionella spp. are Gram-negative bacteria that inhabit freshwater environments representing a serious risk for human health. Legionella pneumophila (Lp) is the species most frequently responsible for a severe pneumonia known as Legionnaires' disease. Lp consists of 15 serogroups (Sgs), usually identified by monoclonal or polyclonal antibodies. With regard to Lp serogrouping, it is well known that phenotyping methods do not have a sufficiently high discriminating power, while genotypic methods although very effective, are expensive and laborious. Recently, mass spectrometry and infrared spectroscopy have proved to be rapid and successful approaches for the microbial identification and typing. Different biomolecules (e.g., lipopolysaccharides) adsorb infrared radiation originating from a specific microbial fingerprint. The development of a classification system based on the intra-species identification features allows a rapid and reliable typing of strains for diagnostic and epidemiological purposes. The aim of the study was the evaluation of Fourier Transform Infrared Spectroscopy using the IR Biotyper® system (Bruker Daltonik, Germany) for the identification of Lp at the serogroup (Sg) level for diagnostic purposes as well as in outbreak events. A large dataset of Lp isolates (n = 133) and ATCC reference strains representing the 15 Lp serogroups were included. The discriminatory power of the instrument's classifier, was tested by Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA). All isolates were classified as follows: 12/133 (9.0%) as Lp Sg1 and 115/133 (86.5%) as Lp Sg 2-15 (including both ATCC and environmental Lp serogroup). Moreover, a mis-classification for 2/133 (1.5%) isolates of Lp Sg 2-15 that returned as Lp Sg1 was observed, and 4/133 (3.0%) isolates were not classified. An accuracy of 95.49% and an error rate of 4.51% were calculated. IR Biotyper® is able provide a quick and cost-effective reliable Lp classification with advantages compared with agglutination tests that show ambiguous and unspecific results. Further studies including a larger number of isolates could be useful to implement the classifier obtaining a robust and reliable tool for the routine Lp serogrouping. IR Biotyper® could be a powerful and easy-to-use tool to identify Lp Sgs, especially during cluster/outbreak investigations, to trace the source of the infection and promptly adopt preventive and control strategies.
RESUMO
Extra-pulmonary mycobacterial infections are characterized by a paucibacillary nature and extra-pulmonary samples consist of different matrices; the processing of these samples requires a high level of manual skills and non-standardized procedures. The aim of this study was to compare the performance of MYCO-TB with MycoPrep on extra-pulmonary samples in terms of Mycobacteria detection, culture contamination and suitability for molecular assay. This prospective study was conducted on 201 extra-pulmonary samples from suspected cases of mycobacterial infection. Specimens were divided into two equal aliquots; one was decontaminated with MYCO-TB the other with MycoPrep. The contamination rate of liquid cultures was significantly different: 2.5% (5/201) for MYCO-TB and 7.5% (15/201) for MycoPrep (p = 0.036). At least 1 Mycobacterium tuberculosis complex (MTBc) positive culture was detected in 6 specimens treated with MYCO-TB and 8with MycoPrep, without significant differences in times to positivity (TTP) in liquid culture. No Xpert MTB/RIF Ultra invalid results were obtained with samples decontaminated with MYCO-TB. The MYCO-TB kit had greater activity than MycoPrep in the digestion and decontamination of extra-pulmonary specimens for the detection of Mycobacteria, supporting the use of MYCO-TB in this type of sample. Ready-to use reagents, rapid protocol and single-sample formulation of MYCO-TB reduced the level of manual skills required as well as the risk of sample contamination.
Assuntos
Mycobacterium tuberculosis/isolamento & purificação , Tuberculose/diagnóstico , Técnicas Bacteriológicas/métodos , Técnicas de Cultura de Células , Descontaminação , Testes Diagnósticos de Rotina , Humanos , Estudos ProspectivosRESUMO
Effective treatment of tuberculosis is frequently hindered by the emerging antimicrobial resistance of Mycobacterium tuberculosis. The present study evaluates monocyclic ß-lactam compounds targeting the mycobacterial cell wall remodeling. Novel N-thio-ß-lactams were designed, synthesized, and characterized on the L,D-transpeptidase-2, a validated target in M. tuberculosis. The candidates were evaluated in biochemical assays identifying five compounds presenting target-specific kinetic constants equal or superior to meropenem, a carbapenem currently considered for tuberculosis therapy. Mass spectrometry in line with the crystal structures of five target-ligand complexes revealed that the N-thio-ß-lactams act via an unconventional mode of adduct formation, transferring the thio-residues from the lactam ring to the active-site cysteine of LdtMt2. The resulting stable adducts lead to a long-term inactivation of the target protein. Finally, the candidates were evaluated in vitro against a drug-susceptible and multidrug-resistant clinical isolates of M. tuberculosis, confirming the antimycobacterial effect of these novel compounds.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Peptidil Transferases/antagonistas & inibidores , beta-Lactamas/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium tuberculosis/enzimologia , Peptidil Transferases/metabolismo , beta-Lactamas/síntese química , beta-Lactamas/químicaRESUMO
The MDR/MTB ELITe MGB® Kit on the ELITe InGenius® platform (ELITechGroup SpA, Italy) is the first system for simultaneous detection of the Mycobacterium tuberculosis complex (MTBc) genome and the main mutations responsible for resistance to Isoniazid (inhA, katG) and Rifampicin (rpoB), from decontaminated and heat inactivated samples. In this study we compared the performance of the MDR/MTB ELITe MGB® Kit (ELITe) with culture in 100 pulmonary and 160 extra-pulmonary samples. The sensitivity and specificity of ELITe compared to culture for pulmonary samples were 98.0% and 98.0% respectively; for extra-pulmonary samples the overall sensitivity was 86.3% (80% for urine, 85% for biopsy and gastric aspirate and 95% for cavitary fluid) and specificity was 100%. Genotypic Isoniazid and Rifampicin susceptibility typing was feasible in 96% of sputum MTBc-positive samples and 43% of extra-pulmonary samples; all samples were found to be drug susceptible by phenotypic and ELITe (100% agreement). Detection of mutations in the rpoB, kat G or inhA genes was evaluated on 300 spiked samples (60 per biological matrix) and all resistance profiles were correctly identified by ELITe. Molecular agreement between ELITe and Xpert was 98.0% and 93.3% for pulmonary and extra-pulmonary samples, respectively. In conclusion, our results provide evidence to support the use of MDR/MTB ELITe MGB® Kit in combination with ELITe InGenius® for the diagnosis of MTBc and the detection of Rifampicin and Isoniazid resistance-related mutations in both pulmonary and extra-pulmonary samples. This system simplifies the laboratory workflow, shortens report time and is an aid in choosing appropriate therapeutic treatment and patient management.
Assuntos
Farmacorresistência Bacteriana , Isoniazida/farmacologia , Kit de Reagentes para Diagnóstico , Rifampina/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Pulmonar/diagnóstico , Antibióticos Antituberculose/uso terapêutico , Biópsia , Genótipo , Humanos , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Curva ROC , Reprodutibilidade dos Testes , Estudos Retrospectivos , Sensibilidade e Especificidade , Temperatura , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Pulmonar/tratamento farmacológicoRESUMO
Legionella spp. are widespread bacteria in aquatic environments with a growing impact on human health. Between the 61 species, Legionella pneumophila is the most prevalent in human diseases; on the contrary, Legionella non-pneumophila species are less detected in clinical diagnosis or during environmental surveillance due to their slow growth in culture and the absence of specific and rapid diagnostic/analytical tools. Reliable and rapid isolate identification is essential to estimate the source of infection, to undertake containment measures, and to determine clinical treatment. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS), since its introduction into the routine diagnostics of laboratories, represents a widely accepted method for the identification of different bacteria species, described in a few studies on the Legionella clinical and environmental surveillance. The focus of this study was the improvement of MALDI-TOF MS on Legionella non-pneumophila species collected during Legionella nosocomial and community surveillance. Comparative analysis with cultural and mip-gene sequencing results was performed. Moreover, a phylogenetic analysis was carried out to estimate the correlations amongst isolates. MALDI-TOF MS achieved correct species-level identification for 45.0% of the isolates belonging to the Legionella anisa, Legionella rubrilucens, Legionella feeleii, and Legionella jordanis species, displaying a high concordance with the mip-gene sequencing results. In contrast, less reliable identification was found for the remaining 55.0% of the isolates, corresponding to the samples belonging to species not yet included in the database. The phylogenetic analysis showed relevant differences inside the species, regruped in three main clades; among the Legionella anisa clade, a subclade with a divergence of 3.3% from the main clade was observed. Moreover, one isolate, identified as Legionella quinlivanii, displayed a divergence of 3.8% from the corresponding reference strain. However, these findings require supplementary investigation. The results encourage the implementation of MALDI-TOF MS in routine diagnostics and environmental Legionella surveillance, as it displays a reliable and faster identification at the species level, as well as the potential to identify species that are not yet included in the database. Moreover, phylogenetic analysis is a relevant approach to correlate the isolates and to track their spread, especially in unconventional reservoirs, where Legionella prevention is still underestimated.
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BACKGROUND: The new Xpert MTB/RIF Ultra assay (Ultra, Cepheid, Sunnyvale, USA) is a cartridge-based automated diagnostic test that can simultaneously identify Mycobacterium tuberculosis complex (MTB) and resistance to Rifampicin (RIF). With respect to the previous version Xpert MTB/RIF assay (Xpert), IS6110/IS1081 repetitive elements probes have been added allowing the detection of lower MTB load, defined by the new semi-quantitative category "trace" with indeterminate RIF resistance. The aim of this study was to evaluate performance of the new version Ultra on Xpert-negative, but TB culture-positive clinical samples. METHODS: The de-identified frozen samples (-20 °C) collected over a 4-year period (February 2014-October 2017), which had previously resulted smear-negative, Xpert-negative but MTB culture-positive, were analyzed with Ultra. The de-frosted samples were loaded into the cartridge using the same process as the previous version, according to manufacturer's instruction. RESULTS: During the study period 382 MTB culture-positive samples were archived: 314 resulted Xpert-positive and 68 Xpert-negative. Thirty-one of the 68 Xpert-negative samples resulted positive with Ultra, with an overall improvement in MTB detection of 45.6%. Out of 36 Xpert-negative respiratory samples, 18 resulted Ultra-positive with the following semi-quantitative loads: "low"(n = 1), "very low"(n = 11), "trace"(n = 6), with an improvement in MTB detection of 50%. The best performance was achieved on bronchoalveolar lavage specimens (53.8%). Out of 32 Xpert-negative non-respiratory samples, 13 resulted Ultra-positive with the following semi-quantitative loads: "very low"(n = 7), "trace"(n = 6), with an improvement in MTB detection of 40.6%. The best performance was achieved on biopsies (55.6%) and lymph nodes (50%). The new category "trace" detected 12 out of the 31 Ultra-positive MTB samples; in the remaining 19 samples RIF susceptibility was determined with 100% concordance with the phenotypic susceptibility test. The mean time to positivity of samples found negative by Ultra was significantly longer in comparison to positive samples in liquid culture. CONCLUSIONS: Our results are consistent with the few studies published so far and confirm the better performance of Ultra compared to the previous version in both respiratory and non-respiratory smear-negative samples, with an overall improvement of 45.6%.
Assuntos
Antibióticos Antituberculose/farmacologia , Farmacorresistência Bacteriana , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Rifampina/farmacologia , Tuberculose/diagnóstico , Tuberculose/microbiologia , Testes Diagnósticos de Rotina , Humanos , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos Testes , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Diagnosis of pulmonary (PTB) and extra-pulmonary tuberculosis (EPTB) in smear-negative patients can be difficult. We assessed retrospectively the performance of Xpert MTB/RIF system (Xpert, Cepheid) in diagnosing smear-negative tuberculosis (TB), which represents the most common form of TB in a low incidence setting. METHODS: Performance of Xpert was compared to acid-fast microscopic examination using Ziehl-Neelsen (ZN) stain in patients with culture-confirmed TB. RESULTS: 386 Mycobacterium tuberculosis (MTB) culture-positive samples were detected out of 5170 specimens tested with smear microscopy, Xpert and culture: 323 were both culture- and Xpert-positive, and 63 culture-positive only. Of these, 234 (60.6%) were smear-negative. In addition Xpert detected 40 probable TB cases, based on clinical findings, which were culture-negative. Compared to culture, Xpert showed an overall sensitivity of 83.7% and a specificity of 99.1%; sensitivity was higher for respiratory samples (86.5%) than for non-respiratory samples (76.8%). Xpert sensitivity for smear-negative culture-confirmed TB was 73.1% and was not influenced by TB localization. As sensitivity of microscopy alone was poor (39.4%), Xpert improved both diagnosis of pulmonary TB (Δ = 36.5%) and extra-pulmonary TB (Δ = 63.4%). CONCLUSIONS: Xpert MTB/RIF is a sensitive method for rapid diagnosis of TB compared to the conventional ZN staining. Xpert can serve as a sensitive and time-saving diagnostic method for microbiological diagnosis of smear-negative TB in countries with a low TB prevalence.