Paolo Miotto

Use of Genotype MTBDR Assay for Molecular Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Clinical Strains Isolated in Italy

ABSTRACT Mycobacterium tuberculosis is one of the leading causes of death worldwide, and multidrug-resistant tuberculosis (MDR-TB) is associated with a high case fatality rate. Rapid identification of resistant strains is crucial for the early administration of appropriate therapy, for prevention of development of further resistance, and to curtail the spread of MDR strains. The Genotype MTBDR (Hain Lifescience, Nehren, Germany) is a reverse hybridization line probe assay designed for the rapid detection of rpoB and katG gene mutations in clinical isolates. The ability of this technique to correctly identify resistant and MDR-TB strains was tested on 206 isolates from the Italian drug resistance surveillance system. This panel included the majority of MDR strains isolated in Italy in the past 3 years. The results of the test were compared to conventional drug susceptibility test performed on isolated strains and verified by sequencing the regions of interest of the bacterial genome. The rate of concordance between the results of the MTBDR and those obtained with “in vitro” sensitivity was 91.5% (130 of 142) for rifampin and 67.1% (116 of 173) for isoniazid. We also applied this test directly to a panel of 36 clinical specimens collected from patients with active TB. The MTBDR correctly identified the two cases of MDR-TB included in the panel. These results show that the MTBDR test is useful in the detection and management of tuberculosis when MDR disease is suspected.

PhyResSE: a Web Tool Delineating Mycobacterium tuberculosis Antibiotic Resistance and Lineage from Whole-Genome Sequencing Data

ABSTRACT Antibiotic-resistant tuberculosis poses a global threat, causing the deaths of hundreds of thousands of people annually. While whole-genome sequencing (WGS), with its unprecedented level of detail, promises to play an increasingly important role in diagnosis, data analysis is a daunting challenge. Here, we present a simple-to-use web service (free for academic use at http://phyresse.org). Delineating both lineage and resistance, it provides state-of-the-art methodology to life scientists and physicians untrained in bioinformatics. It combines elaborate data processing and quality control, as befits human diagnostics, with a treasure trove of validated resistance data collected from well-characterized samples in-house and worldwide.

Mycobacterium tuberculosis Pyrazinamide Resistance Determinants: a Multicenter Study

ABSTRACT Pyrazinamide (PZA) is a prodrug that is converted to pyrazinoic acid by the enzyme pyrazinamidase, encoded by the pncA gene in Mycobacterium tuberculosis. Molecular identification of mutations in pncA offers the potential for rapid detection of pyrazinamide resistance (PZAr). However, the genetic variants are highly variable and scattered over the full length of pncA, complicating the development of a molecular test. We performed a large multicenter study assessing pncA sequence variations in 1,950 clinical isolates, including 1,142 multidrug-resistant (MDR) strains and 483 fully susceptible strains. The results of pncA sequencing were correlated with phenotype, enzymatic activity, and structural and phylogenetic data. We identified 280 genetic variants which were divided into four classes: (i) very high confidence resistance mutations that were found only in PZAr strains (85%), (ii) high-confidence resistance mutations found in more than 70% of PZAr strains, (iii) mutations with an unclear role found in less than 70% of PZAr strains, and (iv) mutations not associated with phenotypic resistance (10%). Any future molecular diagnostic assay should be able to target and identify at least the very high and high-confidence genetic variant markers of PZAr; the diagnostic accuracy of such an assay would be in the range of 89.5 to 98.8%. IMPORTANCE Conventional phenotypic testing for pyrazinamide resistance in Mycobacterium tuberculosis is technically challenging and often unreliable. The development of a molecular assay for detecting pyrazinamide resistance would be a breakthrough, directly overcoming both the limitations of conventional testing and its related biosafety issues. Although the main mechanism of pyrazinamide resistance involves mutations inactivating the pncA enzyme, the highly diverse genetic variants scattered over the full length of the pncA gene and the lack of a reliable phenotypic gold standard hamper the development of molecular diagnostic assays. By analyzing a large number of strains collected worldwide, we have classified the different genetic variants based on their predictive value for resistance which should lead to more rapid diagnostic tests. This would assist clinicians in improving treatment regimens for patients. Conventional phenotypic testing for pyrazinamide resistance in Mycobacterium tuberculosis is technically challenging and often unreliable. The development of a molecular assay for detecting pyrazinamide resistance would be a breakthrough, directly overcoming both the limitations of conventional testing and its related biosafety issues. Although the main mechanism of pyrazinamide resistance involves mutations inactivating the pncA enzyme, the highly diverse genetic variants scattered over the full length of the pncA gene and the lack of a reliable phenotypic gold standard hamper the development of molecular diagnostic assays. By analyzing a large number of strains collected worldwide, we have classified the different genetic variants based on their predictive value for resistance which should lead to more rapid diagnostic tests. This would assist clinicians in improving treatment regimens for patients.

Genotype MTBDRplus: a Further Step toward Rapid Identification of Drug-Resistant Mycobacterium tuberculosis

In a previous publication ([1][1]), we evaluated the Genotype MTBDR (GT-MTBDR; Hain Lifescience, Nehren, Germany) assay as a rapid diagnostic tool for multidrug resistance (MDR) detection in Mycobacterium tuberculosis isolates and clinical specimens. The main limitation of the test was the low

Diagnostic Performance of the New Version (v2.0) of GenoType MTBDRsl Assay for Detection of Resistance to Fluoroquinolones and Second-Line Injectable Drugs: a Multicenter Study

ABSTRACT Resistance to fluoroquinolones (FLQ) and second-line injectable drugs (SLID) is steadily increasing, especially in eastern European countries, posing a serious threat to effective tuberculosis (TB) infection control and adequate patient management. The availability of rapid molecular tests for the detection of extensively drug-resistant TB (XDR-TB) is critical in areas with high rates of multidrug-resistant TB (MDR-TB) and XDR-TB and limited conventional drug susceptibility testing (DST) capacity. We conducted a multicenter study to evaluate the performance of the new version (v2.0) of the Genotype MTBDRsl assay compared to phenotypic DST and sequencing on a panel of 228 Mycobacterium tuberculosis isolates and 231 smear-positive clinical specimens. The inclusion of probes for the detection of mutations in the eis promoter region in the MTBDRsl v2.0 test resulted in a higher sensitivity for detection of kanamycin resistance for both direct and indirect testing (96% and 95.4%, respectively) than that seen with the original version of the assay, whereas the test sensitivities for detection of FLQ resistance remained unchanged (93% and 83.6% for direct and indirect testing, respectively). Moreover, MTBDRsl v2.0 showed better performance characteristics than v1.0 for the detection of XDR-TB, with high specificity and sensitivities of 81.8% and 80.4% for direct and indirect testing, respectively. MTBDRsl v2.0 thus represents a reliable test for the rapid detection of resistance to second-line drugs and a useful screening tool to guide the initiation of appropriate MDR-TB treatment.

Genome-Wide Discovery of Small RNAs in Mycobacterium tuberculosis

Only few small RNAs (sRNAs) have been characterized in Mycobacterium tuberculosis and their role in regulatory networks is still poorly understood. Here we report a genome-wide characterization of sRNAs in M. tuberculosis integrating experimental and computational analyses. Global RNA-seq analysis of exponentially growing cultures of M. tuberculosis H37Rv had previously identified 1373 sRNA species. In the present report we show that 258 (19%) of these were also identified by microarray expression. This set included 22 intergenic sRNAs, 84 sRNAs mapping within 5′/3′ UTRs, and 152 antisense sRNAs. Analysis of promoter and terminator consensus sequences identified sigma A promoter consensus sequences for 121 sRNAs (47%), terminator consensus motifs for 22 sRNAs (8.5%), and both motifs for 35 sRNAs (14%). Additionally, 20/23 candidates were visualized by Northern blot analysis and 5′ end mapping by primer extension confirmed the RNA-seq data. We also used a computational approach utilizing functional enrichment to identify the pathways targeted by sRNA regulation. We found that antisense sRNAs preferentially regulated transcription of membrane-bound proteins. Genes putatively regulated by novel cis-encoded sRNAs were enriched for two-component systems and for functional pathways involved in hydrogen transport on the membrane.

GenoType MTBDRsl performance on clinical samples with diverse genetic background

We evaluate the performance of the GenoType® MTBDRsl (Hain Lifescience Nehren, Germany) for the detection of second-line resistant tuberculosis and we correlate the frequency of mutations to different Mycobacterium tuberculosis genotypes. We tested 175 strains and 59 clinical specimens interpreting the results according to the Standards for Reporting of Diagnostic Accuracy recommendations. All the strains were also investigated by spoligotyping and Mycobacterial Interspersed Repetitive Units–Variable Number of Tandem Repeats typing. The performances of the MTBDRsl in detecting resistance to fluoroquinolones (FQ), second-line injectable drugs (SLID), and ethambutol (EMB) on clinical isolates were similar (specificity ∼99%, sensitivity ∼70%, and positive predictive value (PPV) ∼99%). Of the 59 respiratory specimens, three samples were classified as “indeterminate”. The specificity in detecting resistances was similar for FQs and EMB 100% (95% CI 92.7–100%) and 100% (95% CI 83.9–100%), respectively with a PPV of 100% (95% CI 64.6–100%) and 100% (95% CI 87.9–100%), respectively. Detection of SLID showed a specificity of 89.1% (95% CI 77.0–95.3%) and a PPV of 58.3% (95% CI 32.0–80.7%). Sensitivity for FQ-resistance detection was 100% (95% CI 64.6–100%), whereas for SLID and EMB it was 89.1% (95% CI 77.0-95.3%) and 86.1% (95% CI 71.3-93.9%), respectively. We detected a significant association between mutations in the rrs gene and Beijing lineage. The MTBDRsl can be used to “rule in” extensively drug-resistant strains of tuberculosis in a high risk group; the low sensitivity and negative predicted value (NPV) make confirmation by conventional drug susceptibility testing mandatory when mutations are not identified. NPV for SLID is higher in Beijing strains, showing that the predictive values of the molecular tests are related to the genetic background.

A Genome-Wide Identification Analysis of Small Regulatory RNAs in Mycobacterium tuberculosis by RNA-Seq and Conservation Analysis

We propose a new method for smallRNAs (sRNAs) identification. First we build an effective target genome (ETG) by means of a strand-specific procedure. Then we propose a new bioinformatic pipeline based mainly on the combination of two types of information: the first provides an expression map based on RNA-seq data (Reads Map) and the second applies principles of comparative genomics leading to a Conservation Map. By superimposing these two maps, a robust method for the search of sRNAs is obtained. We apply this methodology to investigate sRNAs in Mycobacterium tuberculosis H37Rv. This bioinformatic procedure leads to a total list of 1948 candidate sRNAs. The size of the candidate list is strictly related to the aim of the study and to the technology used during the verification process. We provide performance measures of the algorithm in identifying annotated sRNAs reported in three recent published studies.

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.

Early tuberculosis treatment monitoring by Xpert® MTB/RIF

To the Editors: Progress made to improve laboratory capacity for tuberculosis (TB) diagnosis led to the development of molecular assays that are now replacing conventional microscopy and culture-based methods on a large scale [1, 2]. Unfortunately, current molecular techniques detect both live and dead bacteria, and a positive result does not imply the viability of the pathogen. Indeed, DNA can persist for a long period after bacterial death and nucleic acid from dead bacteria is equally amplifiable. Therefore, molecular assays are unsuitable for treatment monitoring and/or for infection control purposes. We report an innovative approach to selectively amplify DNA derived from viable Mycobacterium tuberculosis in clinical specimens, which is useful for monitoring mycobacterial load in pulmonary TB patients during anti-TB treatment. The protocol is based on pre-treatment of samples with propidium monoazide (PMA; Biotium Inc., Hayward, CA, USA), a chemical compound that can intercalate the DNA of non-viable (or membrane-damaged) organisms but is excluded from viable bacteria. After light activation, PMA binds covalently to the DNA, preventing its amplification by PCR [3]. After light exposure, unbound PMA is not able to interact further with DNA molecules. The assay was first optimised using acid-fast bacilli (AFB)-negative sputum samples spiked with dead or live mycobacteria at different concentrations. In brief, live Mycobacterium fortuitum cells were added to N -acetyl-cysteine decontaminated sputum specimens negative for AFB by smear microscopy at a final concentration of 106 bacteria·mL−1. An aliquot of this laboratory-made sample was treated to heat kill the M. fortuitum cells. PMA stock solution was prepared and stored at -20°C and protected from light exposure, until use, as recommended by the manufacturers. PMA was added as a pre-treatment at a final concentration of 500 μM and incubated for 30 min at 4°C in the …