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

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

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

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

Towards standardisation:comparison of five whole genome sequencing (WGS) analysis pipelines for detection of epidemiologically linked tuberculosis cases

BackgroundWhole genome sequencing (WGS) is a reliable tool for studying tuberculosis (TB) transmission. WGS data are usually processed by custom-built analysis pipelines with little standardisation between them.AimTo compare the impact of variability of several WGS analysis pipelines used internationally to detect epidemiologically linked TB cases.MethodsFrom the Netherlands, 535 Mycobacterium tuberculosis complex (MTBC) strains from 2016 were included. Epidemiological information obtained from municipal health services was available for all mycobacterial interspersed repeat unit-variable number of tandem repeat (MIRU-VNTR) clustered cases. WGS data was analysed using five different pipelines: one core genome multilocus sequence typing (cgMLST) approach and four single nucleotide polymorphism (SNP)-based pipelines developed in Oxford, United Kingdom; Borstel, Germany; Bilthoven, the Netherlands and Copenhagen, Denmark. WGS clusters were defined using a maximum pairwise distance of 12 SNPs/alleles.ResultsThe cgMLST approach and Oxford pipeline clustered all epidemiologically linked cases, however, in the other three SNP-based pipelines one epidemiological link was missed due to insufficient coverage. In general, the genetic distances varied between pipelines, reflecting different clustering rates: the cgMLST approach clustered 92 cases, followed by 84, 83, 83 and 82 cases in the SNP-based pipelines from Copenhagen, Oxford, Borstel and Bilthoven respectively.ConclusionConcordance in ruling out epidemiological links was high between pipelines, which is an important step in the international validation of WGS data analysis. To increase accuracy in identifying TB transmission clusters, standardisation of crucial WGS criteria and creation of a reference database of representative MTBC sequences would be advisable

Integrating pharmacokinetics and pharmacodynamics in operational research to end tuberculosis

Tuberculosis (TB) elimination requires innovative approaches. The new Global Tuberculosis Network (GTN) aims to conduct research on key unmet therapeutic and diagnostic needs in the field of TB elimination using multidisciplinary, multisectorial approaches. The TB Pharmacology section within the new GTN aims to detect and study the current knowledge gaps, test potential solutions using human pharmacokinetics informed through preclinical infection systems, and return those findings to the bedside. Moreover, this approach would allow prospective identification and validation of optimal shorter therapeutic durations with new regimens. Optimized treatment using available and repurposed drugs may have an increased impact when prioritizing a personcentered approach and acknowledge the importance of age, gender, comorbidities, and both social and programmatic environments. In this viewpoint article, we present an in-depth discussion on how TB pharmacology and the related strategies will contribute to TB elimination