BackgroundDiagnosis 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.MethodsPerformance of Xpert was compared to acid-fast microscopic examination using Ziehl-Neelsen (ZN) stain in patients with culture-confirmed TB.Results386 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%).ConclusionsXpert 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.

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BackgroundThe 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.MethodsThe 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.ResultsDuring 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.ConclusionsOur 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%.

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.

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.