Elisa Tagliani

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.

The new Xpert MTB/RIF ultra: Improving detection of Mycobacterium tuberculosis and resistance to Rifampin in an assay suitable for point-of-care testing

ABSTRACT The Xpert MTB/RIF assay (Xpert) is a rapid test for tuberculosis (TB) and rifampin resistance (RIF-R) suitable for point-of-care testing. However, it has decreased sensitivity in smear-negative sputum, and false identification of RIF-R occasionally occurs. We developed the Xpert MTB/RIF Ultra assay (Ultra) to improve performance. Ultra and Xpert limits of detection (LOD), dynamic ranges, and RIF-R rpoB mutation detection were tested on Mycobacterium tuberculosis DNA or sputum samples spiked with known numbers of M. tuberculosis H37Rv or Mycobacterium bovis BCG CFU. Frozen and prospectively collected clinical samples from patients suspected of having TB, with and without culture-confirmed TB, were also tested. For M. tuberculosis H37Rv, the LOD was 15.6 CFU/ml of sputum for Ultra versus 112.6 CFU/ml of sputum for Xpert, and for M. bovis BCG, it was 143.4 CFU/ml of sputum for Ultra versus 344 CFU/ml of sputum for Xpert. Ultra resulted in no false-positive RIF-R specimens, while Xpert resulted in two false-positive RIF-R specimens. All RIF-R-associated M. tuberculosis rpoB mutations tested were identified by Ultra. Testing on clinical sputum samples, Ultra versus Xpert, resulted in an overall sensitivity of 87.5% (95% confidence interval [CI], 82.1, 91.7) versus 81.0% (95% CI, 74.9, 86.2) and a sensitivity on sputum smear-negative samples of 78.9% (95% CI, 70.0, 86.1) versus 66.1% (95% CI, 56.4, 74.9). Both tests had a specificity of 98.7% (95% CI, 93.0, 100), and both had comparable accuracies for detection of RIF-R in these samples. Ultra should significantly improve TB detection, especially in patients with paucibacillary disease, and may provide more-reliable RIF-R detection. IMPORTANCE The Xpert MTB/RIF assay (Xpert), the first point-of-care assay for tuberculosis (TB), was endorsed by the World Health Organization in December 2010. Since then, 23 million Xpert tests have been procured in 130 countries. Although Xpert showed high overall sensitivity and specificity with pulmonary samples, its sensitivity has been lower with smear-negative pulmonary samples and extrapulmonary samples. In addition, the prediction of rifampin resistance (RIF-R) in paucibacillary samples and for a few rpoB mutations has resulted in both false-positive and false-negative results. The present study is the first demonstration of the design features and operational characteristics of an improved Xpert Ultra assay. This study also shows that the Ultra format overcomes many of the known shortcomings of Xpert. The new assay should significantly improve TB detection, especially in patients with paucibacillary disease, and provide more-reliable detection of RIF-R. IMPORTANCE The Xpert MTB/RIF assay (Xpert), the first point-of-care assay for tuberculosis (TB), was endorsed by the World Health Organization in December 2010. Since then, 23 million Xpert tests have been procured in 130 countries. Although Xpert showed high overall sensitivity and specificity with pulmonary samples, its sensitivity has been lower with smear-negative pulmonary samples and extrapulmonary samples. In addition, the prediction of rifampin resistance (RIF-R) in paucibacillary samples and for a few rpoB mutations has resulted in both false-positive and false-negative results. The present study is the first demonstration of the design features and operational characteristics of an improved Xpert Ultra assay. This study also shows that the Ultra format overcomes many of the known shortcomings of Xpert. The new assay should significantly improve TB detection, especially in patients with paucibacillary disease, and provide more-reliable detection of RIF-R.

A standardised method for interpreting the association between mutations and phenotypic drug resistance in Mycobacterium tuberculosis

A clear understanding of the genetic basis of antibiotic resistance in Mycobacterium tuberculosis is required to accelerate the development of rapid drug susceptibility testing methods based on genetic sequence. Raw genotype–phenotype correlation data were extracted as part of a comprehensive systematic review to develop a standardised analytical approach for interpreting resistance associated mutations for rifampicin, isoniazid, ofloxacin/levofloxacin, moxifloxacin, amikacin, kanamycin, capreomycin, streptomycin, ethionamide/prothionamide and pyrazinamide. Mutation frequencies in resistant and susceptible isolates were calculated, together with novel statistical measures to classify mutations as high, moderate, minimal or indeterminate confidence for predicting resistance. We identified 286 confidence-graded mutations associated with resistance. Compared to phenotypic methods, sensitivity (95% CI) for rifampicin was 90.3% (89.6–90.9%), while for isoniazid it was 78.2% (77.4–79.0%) and their specificities were 96.3% (95.7–96.8%) and 94.4% (93.1–95.5%), respectively. For second-line drugs, sensitivity varied from 67.4% (64.1–70.6%) for capreomycin to 88.2% (85.1–90.9%) for moxifloxacin, with specificity ranging from 90.0% (87.1–92.5%) for moxifloxacin to 99.5% (99.0–99.8%) for amikacin. This study provides a standardised and comprehensive approach for the interpretation of mutations as predictors of M. tuberculosis drug-resistant phenotypes. These data have implications for the clinical interpretation of molecular diagnostics and next-generation sequencing as well as efficient individualised therapy for patients with drug-resistant tuberculosis. A comprehensive basis for interpreting mutations to predict antibiotic resistance in tuberculosis http://ow.ly/hhwJ30g9jCY

A cluster of multidrug-resistant Mycobacterium tuberculosis among patients arriving in Europe from the Horn of Africa: a molecular epidemiological study

Summary Background The risk of tuberculosis outbreaks among people fleeing hardship for refuge in Europe is heightened. We describe the cross-border European response to an outbreak of multidrug-resistant tuberculosis among patients from the Horn of Africa and Sudan. Methods On April 29 and May 30, 2016, the Swiss and German National Mycobacterial Reference Laboratories independently triggered an outbreak investigation after four patients were diagnosed with multidrug-resistant tuberculosis. In this molecular epidemiological study, we prospectively defined outbreak cases with 24-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) profiles; phenotypic resistance to isoniazid, rifampicin, ethambutol, pyrazinamide, and capreomycin; and corresponding drug resistance mutations. We whole-genome sequenced all Mycobacterium tuberculosis isolates and clustered them using a threshold of five single nucleotide polymorphisms (SNPs). We collated epidemiological data from host countries from the European Centre for Disease Prevention and Control. Findings Between Feb 12, 2016, and April 19, 2017, 29 patients were diagnosed with multidrug-resistant tuberculosis in seven European countries. All originated from the Horn of Africa or Sudan, with all isolates two SNPs or fewer apart. 22 (76%) patients reported their travel routes, with clear spatiotemporal overlap between routes. We identified a further 29 MIRU-VNTR-linked cases from the Horn of Africa that predated the outbreak, but all were more than five SNPs from the outbreak. However all 58 isolates shared a capreomycin resistance-associated tlyA mutation. Interpretation Our data suggest that source cases are linked to an M tuberculosis clone circulating in northern Somalia or Djibouti and that transmission probably occurred en route before arrival in Europe. We hypothesise that the shared mutation of tlyA is a drug resistance mutation and phylogenetic marker, the first of its kind in M tuberculosis sensu stricto. Funding The Swiss Federal Office of Public Health, the University of Zurich, the Wellcome Trust, National Institute for Health Research (NIHR) Oxford Biomedical Research Centre (BRC), the Medical Research Council, BELTA-TBnet, the European Union, the German Center for Infection Research, and Leibniz Science Campus Evolutionary Medicine of the Lung (EvoLUNG).

Half of rifampicin-resistant Mycobacterium tuberculosis complex isolated from tuberculosis patients in Sub-Saharan Africa have concomitant resistance to pyrazinamide

Background Besides inclusion in 1st line regimens against tuberculosis (TB), pyrazinamide (PZA) is used in 2nd line anti-TB regimens, including in the short regimen for multidrug-resistant TB (MDR-TB) patients. Guidelines and expert opinions are contradictory about inclusion of PZA in case of resistance. Moreover, drug susceptibility testing (DST) for PZA is not often applied in routine testing, and the prevalence of resistance is unknown in several regions, including in most African countries. Methods Six hundred and twenty-three culture isolates from rifampicin-resistant (RR) patients were collected in twelve Sub-Saharan African countries. Among those isolates, 71% were from patients included in the study on the Union short-course regimen for MDR-TB in Benin, Burkina Faso, Burundi, Cameroon, Central Africa Republic, the Democratic Republic of the Congo, Ivory Coast, Niger, and Rwanda PZA resistance, and the rest (29%) were consecutive isolates systematically stored from 2014–2015 in Mali, Rwanda, Senegal, and Togo. Besides national guidelines, the isolates were tested for PZA resistance through pncA gene sequencing. Results Over half of these RR-TB isolates (54%) showed a mutation in the pncA gene, with a significant heterogeneity between countries. Isolates with fluoroquinolone resistance (but not with injectable resistance or XDR) were more likely to have concurrent PZA resistance. The pattern of mutations in the pncA gene was quite diverse, although some isolates with an identical pattern of mutations in pncA and other drug-related genes were isolated from the same reference center, suggesting possible transmission of these strains. Conclusion Similar to findings in other regions, more than half of the patients having RR-TB in West and Central Africa present concomitant resistance to PZA. Further investigations are needed to understand the relation between resistance to PZA and resistance to fluoroquinolones, and whether continued use of PZA in the face of PZA resistance provides clinical benefit to the patients.

Reaching consensus on drug resistance conferring mutations

OBJECTIVE/BACKGROUND Molecular-based, rapid drug-susceptibility tests are needed to guide the appropriate use of new drugs and new therapeutic regimens at the programmatic level, and to prevent a further increase in the incidence of drug-resistant tuberculosis (TB). Experts have recognized the need for a global, curated, and standardized analysis and data-sharing platform that provides a one-stop data source for clinically relevant genotypic and phenotypic information on Mycobacterium tuberculosis. METHODS To this purpose, the Relational Sequencing TB Data Platform (ReSeqTB) consortium has critically reviewed the most inclusive set of published data on mutations associated with drug resistance in M. tuberculosis to date, and has graded a comprehensive list of globally prevalent mutations based on the strength of their association with drug resistance. RESULTS AND CONCLUSIONS ReSeqTB serves as a single repository for the compilation, curation, and validation of existing and newly created sequences and metadata on M. tuberculosis strains and will use the currently reviewed data set, validated by international experts, as a starting point until sufficient new sequence data are accumulated. This initiative is supported by a global partnership of academic institutions, public health agencies, and nongovernmental organizations including the Critical Path Institute, FIND, the World Health Organization, the New Diagnostics Working Group, the U.S. Centers for Disease Control and Prevention, and the National Institute of Allergy and Infectious Diseases and it is financially supported by the Bill & Melinda Gates Foundation. Key strengths of the ReSeqTB Database include the following:Successful execution of such an extensive database platform requires substantial collaboration from scientists investigating the genetic basis for drug resistance worldwide, and from developers with expertise in database design and implementation.

Culture and Next-generation sequencing-based drug susceptibility testing unveil high levels of drug-resistant-TB in Djibouti: results from the first national survey

Djibouti is a small country in the Horn of Africa with a high TB incidence (378/100,000 in 2015). Multidrug-resistant TB (MDR-TB) and resistance to second-line agents have been previously identified in the country but the extent of the problem has yet to be quantified. A national survey was conducted to estimate the proportion of MDR-TB among a representative sample of TB patients. Sputum was tested using XpertMTB/RIF and samples positive for MTB and resistant to rifampicin underwent first line phenotypic susceptibility testing. The TB supranational reference laboratory in Milan, Italy, undertook external quality assurance, genotypic testing based on whole genome and targeted-deep sequencing and phylogenetic studies. 301 new and 66 previously treated TB cases were enrolled. MDR-TB was detected in 34 patients: 4.7% of new and 31% of previously treated cases. Resistance to pyrazinamide, aminoglycosides and capreomycin was detected in 68%, 18% and 29% of MDR-TB strains respectively, while resistance to fluoroquinolones was not detected. Cluster analysis identified transmission of MDR-TB as a critical factor fostering drug resistance in the country. Levels of MDR-TB in Djibouti are among the highest on the African continent. High prevalence of resistance to pyrazinamide and second-line injectable agents have important implications for treatment regimens.

Reaching consensus on drug resistance conferring mutations (Part 1)

Objective/background: Molecular-based, rapid drug-susceptibility tests are needed to guide the appropriate use of new drugs and new therapeutic regimens at the programmatic level, and to prevent a further increase in the incidence of drug-resistant tuberculosis (TB). Experts have recognized the need for a global, curated, and standardized analysis and data-sharing platform that provides a one-stop data source for clinically relevant genotypic and phenotypic information on Mycobacterium tuberculosis. Methods: To this purpose, the Relational Sequencing TB Data Platform (ReSeqTB) consortium has critically reviewed the most inclusive set of published data on mutations associated with drug resistance in M. tuberculosis to date, and has graded a comprehensive list of globally prevalent mutations based on the strength of their association with drug resistance. Results and Conclusions: ReSeqTB serves as a single repository for the compilation, curation, and validation of existing and newly created sequences and metadata on M. tuberculosis strains and will use the currently reviewed data set, validated by international experts, as a starting point until sufficient new sequence data are accumulated. This initiative is supported by a global partnership of academic institutions, public health agencies, and nongovernmental organizations including the Critical Path Institute, FIND, the World Health Organization, the New Diagnostics Working Group, the U.S. Centers for Disease Control and Prevention, and the National Institute of Allergy and Infectious Diseases and it is financially supported by the Bill & Melinda Gates Foundation. Key strengths of the ReSeqTB Database include the following: A user-friendly interface designed for nonexpert or expert operability. A standardized and validated analysis pipeline for variant analyses of M. tuberculosis next-generation sequencing (NGS) data. Access to data beyond the published literature with dynamic and iterative updates of new data generated by global surveillance and clinical trials. A well-developed legal structure to ensure intellectual property rights and data ownership remain with contributors. A structured data-sharing architecture to restrict access to sensitive or unpublished data sets. Metadata standardization using CDISC: supports global, platform-independent data standards that enable information system interoperability. An emphasis on data quality and rigorous, expert curation with multiple quality control checks for whole-genome sequencing and other metadata. Validation of NGS analysis output by an expert committee with grading of resistance conferring mutations based on rigorous statistical standards. Regulatory-compliant analysis pipeline and database architecture. Successful execution of such an extensive database platform requires substantial collaboration from scientists investigating the genetic basis for drug resistance worldwide, and from developers with expertise in database design and implementation.