Precision medicine and public health interventions: tuberculosis as a model?

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Pseudo-outbreak of Mycobacterium gordonae in a teaching hospital: importance of strictly following decontamination procedures and emerging issues concerning sterilization

Aim of this study was to investigate a pseudo-outbreak of Mycobacterium gordonae analyzing isolates detected from clinical and environmental samples. Mycobacterium gordonae was detected in 7 out of 497 broncho-alveolar lavage (BAL) samples after bronchoscopy procedure in patients admitted to a teaching hospital between January and April 2013. During this pseudo-outbreak clinical, epidemiological, environmental and molecular investigations were performed. None of the patients met the criteria for non-tuberculous mycobacterial (NTM) lung disease and were treated for M. gordonae lung disease. Environmental investigation revealed M. gordonae in 3 samples: in tap water and in the water supply channel of the washer disinfector. All the isolates were subjected to genotyping by pulsed-field gel electrophoresis (PFGE). The PFGE revealed that only patients' isolates presented the same band pattern but no correlation with the environmental strain was detected. Surveillance of the outbreak and the strict adherence to the reprocessing procedure and its supplies resulted afterwards in no detection of M. gordonae in clinical respiratory samples. Clinical surveillance of patients was crucial to establish the start of NTM treatment. Regular screening of tap water and endoscopic equipment should be adopted to compare the clinical strains with the environmental ones when an outbreak occurs

The Roles Of MicroRNAs On Tuberculosis Infection: Meaning Or Myth?

The central proteins for protection against tuberculosis are attributed to interferon-γ, tumor necrosis factor-α, interleukin (IL)-6 and IL-1β, while IL-10 primarily suppresses anti-mycobacterial responses. Several studies found alteration of expression profile of genes involved in anti-mycobacterial responses in macrophages and natural killer (NK) cells from active and latent tuberculosis and from tuberculosis and healthy controls. This alteration of cellular composition might be regulated by microRNAs (miRNAs). Albeit only 1% of the genomic transcripts in mammalian cells encode miRNA, they are predicted to control the activity of more than 60% of all protein-coding genes and they have a huge influence in pathogenesis theory, diagnosis and treatment approach to some diseases. Several miRNAs have been found to regulate T cell differentiation and function and have critical role in regulating the innate function of macrophages, dendritic cells and NK cells. Here, we have reviewed the role of miRNAs implicated in tuberculosis infection, especially related to their new roles in the molecular pathology of tuberculosis immunology and as new targets for future tuberculosis diagnostics

Whole-Genome Sequences of Two NDM-1-Producing Pseudomonas aeruginosa Strains Isolated in a Clinical Setting in Albania in 2018

Isolation of metallo-β-lactamase-producing, carbapenem-resistant, Pseudomonas aeruginosa strains is increasingly being documented worldwide; their presence constitutes a public health threat. Here, we report draft genome sequences of two New Delhi metallo-β-lactamase-1-producing, multidrug-resistant, P. aeruginosa strains of sequence type 235 that were isolated from the surgical wound of two patients hospitalized in the same ward

Analytical evaluation of QuantiFERON- Plus and QuantiFERON- Gold In-tube assays in subjects with or without tuberculosis

The QuantiFERON-TB Gold Plus (QFT-Plus) represents the new QuantiFERON-TB Gold In-tube (QFT-GIT) to identify latent tuberculosis infection (LTBI). The main differences is the addition of a new tube containing shorter peptides stimulating CD8 T-cells. Aim of this study is to evaluate the accuracy of QFT-Plus compared with QFT-GIT in a cross sectional study of individuals with or without tuberculosis (TB). We enrolled 179 participants: 19 healthy donors, 58 LTBI, 33 cured TB and 69 active TB. QFT-Plus and QFT-GIT were performed. The two tests showed a substantial agreement. Moreover we found a similar sensitivity in active TB and same specificity in healthy donors. A higher proportion of the LTBI subjects responded to both TB1 and TB2 compared to those with active TB (97% vs 81%). Moreover, a selective response to TB2 was associated with active TB (9%) and with a severe TB disease, suggesting that TB2 stimulation induces a CD8 T-cell response in absence of a CD4-response. In conclusion, QFT-Plus and QFT-GIT assays showed a substantial agreement and similar accuracy for active TB detection. Interestingly, a higher proportion of the LTBI subjects responded concomitantly to TB1 and TB2 compared to those with active TB, whereas a selective TB2 response associated with active TB

miRNA Signatures in Sera of Patients with Active Pulmonary Tuberculosis.

Several studies showed that assessing levels of specific circulating microRNAs (miRNAs) is a non-invasive, rapid, and accurate method for diagnosing diseases or detecting alterations in physiological conditions. We aimed to identify a serum miRNA signature to be used for the diagnosis of tuberculosis (TB). To account for variations due to the genetic makeup, we enrolled adults from two study settings in Europe and Africa. The following categories of subjects were considered: healthy (H), active pulmonary TB (PTB), active pulmonary TB, HIV co-infected (PTB/HIV), latent TB infection (LTBI), other pulmonary infections (OPI), and active extra-pulmonary TB (EPTB). Sera from 10 subjects of the same category were pooled and, after total RNA extraction, screened for miRNA levels by TaqMan low-density arrays. After identification of "relevant miRNAs", we refined the serum miRNA signature discriminating between H and PTB on individual subjects. Signatures were analyzed for their diagnostic performances using a multivariate logistic model and a Relevance Vector Machine (RVM) model. A leave-one-out-cross-validation (LOOCV) approach was adopted for assessing how both models could perform in practice. The analysis on pooled specimens identified selected miRNAs as discriminatory for the categories analyzed. On individual serum samples, we showed that 15 miRNAs serve as signature for H and PTB categories with a diagnostic accuracy of 82% (CI 70.2-90.0), and 77% (CI 64.2-85.9) in a RVM and a logistic classification model, respectively. Considering the different ethnicity, by selecting the specific signature for the European group (10 miRNAs) the diagnostic accuracy increased up to 83% (CI 68.1-92.1), and 81% (65.0-90.3), respectively. The African-specific signature (12 miRNAs) increased the diagnostic accuracy up to 95% (CI 76.4-99.1), and 100% (83.9-100.0), respectively. Serum miRNA signatures represent an interesting source of biomarkers for TB disease with the potential to discriminate between PTB and LTBI, but also among the other categories

Use of WGS in M. tuberculosis routine diagnosis

AbstractWhole Genome Sequencing (WGS) is becoming affordable with overall costs comparable to other tests currently in use to perform the diagnosis of drug resistant tuberculosis and cluster analysis. The WGS approach allows an “all-in one” approach providing results on expected sensitivity of the strains, genetic background, epidemiological data and indication of risk of laboratory cross-contamination.Although ideal, WGS from the direct diagnostic specimen is not yet standardized and up today the two most promising approaches are WGS from early positive liquid culture and targeted sequencing from diagnostic specimens using Next Generation Technology. Both have advantages and disadvantages. Sequencing from early MGIT requires positive cultures while targeted sequencing can be performed from a specimen positive for M. tuberculosis with a consistent gain in time to information. Aim of this study is to evaluate the feasibility and cost to use WGS with a centralized approach to speed up diagnosis of tuberculosis in a low incidence country.From March to September 2016 we collected and processed by WGS 89 early positive routine MGIT960 tubes. Time to diagnosis and accuracy of this technique were compared with the standard testing performed in the routine laboratory.An aliquot of 2ml of early positive MGIT was processed, starting with heat inactivation. DNA was then isolated by using the Maxwell 16 Cell DNA Purification Kit and Maxwell 16 MDx for automated extraction. Paired-end libraries of read-length 75–151bp were prepared using the Nextera XT DNA Sample Preparation kit, and sequenced on Illumina Miseq/Miniseq platform (based on the first available run). Total variant calling was performed according to the pipeline of the Phyresse web-tool.The DNA isolation step required 30′ for inactivation plus 30′ for extraction. The concentration obtained ranged from 0.1 to 1ng/μL, suitable for library preparation. Samples were sequenced with a turn around time of 24–48h. The percentage of reads mapped to H37Rv reference genome was 83% on average. Mean read coverage was 65×. Main challenge was the presence of non–mycobacterial DNA contamination in a variable amount. Lineage detection was possible for all cases, and mutations associated to drug resistance to antitubercular drugs were examined. We observed high diagnostic accuracy for species identification and detection of full drug resistance profile compared to standard DST testing performed in MGIT.Two events of recent transmissions including respectively three and two patients were identified and two laboratory cross-contamination were investigated and confirmed based on the analysis. Time to availability of report was around 72h from MGIT positivity compared to up to 6–9weeks for XDR-TB diagnosis with standard testing.In addition to speed, main advantages were the availability of a full prediction of resistance determinants for rifampicin resistant cases, the fast detection of potential cross-contaminations and clusters to guide epidemiological investigation and cross border tracing.Cost analysis showed that the cost per strain was approximately 150 Euro inclusive of staff cost, reagents and machine cost.WGS is a rapid, cost-effective technique that promises to integrate and replace the other tests in routine laboratories for an accurate diagnosis of DR-TB, although suitable nowadays for cultured samples only

First characterization of the CD4 and CD8 T-cell responses to QuantiFERON-TB Plus

Summary Introduction QuantiFERON ® -TB Gold Plus (QFT-Plus) is the new generation of QuantiFERON-TB Gold In-Tube test to identify latent tuberculosis infection (LTBI). QFT-Plus includes TB1 and TB2 tubes which contain selected Mycobacterium tuberculosis (Mtb) peptides designed to stimulate both CD4 and CD8 T-cells. Aim of this study is the flow cytometric characterization of the specific CD4 and CD8 T-cell responses to Mtb antigens contained within QFT-Plus. Methods We enrolled 27 active tuberculosis (TB) patients and 30 LTBI individuals. Following stimulation with TB1 and TB2, antigen-specific T-cells were characterized by flow cytometry. Data were also correlated with the grade of TB severity. Results TB1 mainly elicited a CD4 T-cell response while TB2 induced both CD4 and CD8 responses. Moreover, the TB2-specific CD4 response was detected for both active TB and LTBI patients, whereas the TB2-specific CD8 response was primarily associated with active TB (p = 0.01). Conclusions To our knowledge, we report the first characterization of the CD4 and CD8 T-cell response to QFT-Plus. CD8 T-cell response is mainly due to TB2 stimulation which is largely associated to active TB. These results provide a better knowledge on the use of this assay

Extensively Drug-Resistant Tuberculosis, Burkina Faso

Because data from countries in Africa are limited, we measured the proportion of extensively drug-resistant (XDR) tuberculosis (TB) cases among TB patients in Burkina Faso for whom retreatment was failing. Of 34 patients with multidrug-resistant TB, 2 had an XDR TB strain. Second-line TB drugs should be strictly controlled to prevent further XDR TB increase