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Moxifloxacin is an attractive drug for the treatment of isoniazid-resistant rifampicin-susceptible tuberculosis (TB) or drug-susceptible TB complicated by isoniazid intolerance. However, co-administration with rifampicin decreases moxifloxacin exposure. It remains unclear whether this drug-drug interaction has clinical implications. This retrospective study in a Dutch TB centre investigated how rifampicin affected moxifloxacin exposure in patients with isoniazid-resistant or -intolerant TB. Moxifloxacin exposures were measured between 2015 and 2020 in 31 patients with isoniazid-resistant or -intolerant TB receiving rifampicin, and 20 TB patients receiving moxifloxacin without rifampicin. Moxifloxacin exposure, i.e. area under the concentration-time curve (AUC0-24h), and attainment of AUC0-24h/minimal inhibitory concentration (MIC) > 100 were investigated for 400 mg moxifloxacin and 600 mg rifampicin, and increased doses of moxifloxacin (600 mg) or rifampicin (900 mg). Moxifloxacin AUC0-24h and peak concentration with a 400 mg dose were decreased when rifampicin was co-administered compared to moxifloxacin alone (ratio of geometric means 0.61 (90% CI (0.53, 0.70) and 0.81 (90% CI (0.70, 0.94), respectively). Among patients receiving rifampicin, 65% attained an AUC0-24h/MIC > 100 for moxifloxacin compared to 78% of patients receiving moxifloxacin alone; this difference was not significant. Seven out of eight patients receiving an increased dose of 600 mg moxifloxacin reached the target AUC0-24h/MIC > 100. This study showed a clinically significant 39% decrease in moxifloxacin exposure when rifampicin was co-administered. Moxifloxacin dose adjustment may compensate for this drug-drug interaction. Further exploring the impact of higher doses of these drugs in patients with isoniazid resistance or intolerance is paramount.

Background Tuberculosis (TB) remains a serious public health threat worldwide. Theoretically ultimate resolution of whole genome sequencing (WGS) for Mycobacterium tuberculosis complex (MTBC) strain classification makes this technology very attractive for epidemiological investigations. Objectives To summarize the evidence available in peer-reviewed publications on the role and place of WGS in detection of TB transmission. Sources A total of 69 peer-reviewed publications identified in Pubmed database. Content Evidence from >30 publications suggests that a cut-off value of fewer than six single nucleotide polymorphisms between strains efficiently excludes cases that are not the result of recent transmission and could be used for the identification of drug-sensitive isolates involved in direct human-to-human TB transmission. Sensitivity of WGS to identify epidemiologically linked isolates is high, reaching 100% in eight studies with specificity (17%–95%) highly dependent on the settings. Drug resistance and specific phylogenetic lineages may be associated with accelerated mutation rates affecting genetic distances. WGS can be potentially used to distinguish between true relapses and re-infections but in high-incidence low-diversity settings this would require consideration of epidemiological links and minority alleles. Data from four studies looking into within-host diversity highlight a need for developing criteria for acceptance or rejection of WGS relatedness results depending on the proportion of minority alleles. Implications WGS will potentially allow for more targeted public health actions preventing unnecessary investigations of false clusters. Consensus on standardization of raw data quality control processing criteria, analytical pipelines and reporting language is yet to be reached.

Delamanid, a-first-in-class bicyclic nitroimidazole, was recently approved for multidrug-resistant tuberculosis treatment. Pitted against the hope for improving treatment outcomes is the threat of the rapid resistance emergence. This review provides information on the mechanisms of action, resistance emergence, and drug susceptibility testing (DST) for delamanid. Delamanid resistance has already been reported in both in vitro experiments and clinical settings. Although mutations conferring delamanid resistance have been identified in fbiA, fbiB, fbiC, ddn, and fgd1 genes of Mycobacterium tuberculosis, knowledge about the molecular resistance mechanisms is limited, and there remains no standardized DST method. The rapid acquisition of delamanid resistance emphasizes the need for optimal use of new drugs, the need for drug resistance surveillance, and a comprehensive understanding of drug resistance mechanisms. Further studies are necessary to investigate genetic and phenotypic changes that determine clinically relevant delamanid resistance to help develop a rapid delamanid DST.

The global control of Tuberculosis remains elusive, and Bacillus Calmette-Guerin (BCG) -the most widely used vaccine in history-has proven insufficient for reversing this epidemic. Several authors have suggested that the mass presence of vaccinated hosts might have affected the Mycobacterium tuberculosis (MTB) population structure, and this could in turn be reflected in a prevalence of strains with higher ability to circumvent BCG-induced immunity, such as the recent Beijing genotype. The effect of vaccination on vaccine-escape variants has been well-documented in several bacterial pathogens; however the effect of the interaction between MTB strains and vaccinated hosts has never been previously described. In this study we show for the first time the interaction between MTB Beijing-genotype strains and BCG-vaccinated hosts. Using a well-controlled murine model of progressive pulmonary tuberculosis, we vaccinated BALB/c mice with two different sub-strains of BCG (BCG-Phipps and BCG-Vietnam). Following vaccination, the mice were infected with either one of three selected MTB strains. Strains were selected based on lineage, and included two Beijing-family clinical isolates (strains 46 and 48) and a well-characterized laboratory strain (H37Rv). Two months after infection, mice were euthanized and the bacteria extracted from their lungs. We characterized the genomic composite of the bacteria before and after exposure to vaccinated hosts, and also characterized the local response to the bacteria by sequencing the lung transcriptome in animals during the infection. Results from this study show that the interaction within the lungs of the vaccinated hosts results in the selection of higher-virulence bacteria, specifically for the Beijing genotype strains 46 and 48. After exposure to the BCG-induced immune response, strains 46 and 48 acquire genomic mutations associated with several virulence factors. As a result, the bacteria collected from these vaccinated hosts have an increased ability for immune evasion, as shown in both the host transcriptome and the histopathology studies, and replicates far more efficiently compared to bacteria collected from unvaccinated hosts or to the original-stock strain. Further research is warranted to ascertain the pathways associated with the genomic alterations. However, our results highlight novel host-pathogen interactions induced by exposure of MTB to BCG vaccinated hosts.

Beijing strains of Mycobacterium tuberculosis (lineage 2) have been associated with drug-resistance and transmission of tuberculosis worldwide. Most of the Beijing strains identified in the Colombian Pacific coast have exhibited a multidrug resistant (MDR) phenotype. We sought to evaluate the clonality and sublineage of Beijing strains circulating in Southwestern Colombia. Thirty-seven Beijing strains were identified through spoligotyping out of 311 clinical isolates collected in 9 years from 2002-2010. Further analysis by MIRU-VNTR 24 loci was conducted for the Beijing strains. For sublineage classification, deletions of RD105, RD207, and RD131 and point mutations at fbpB, mutT2, and acs were evaluated. Drug-resistance associated mutations to first-and second-line anti-TB drugs were also evaluated. Additionally, two Beijing strains were Illumina-whole genome sequenced (one MDR and one drug-susceptible). Among the 37 Beijing strains characterized, 36 belonged to the SIT190 type from which 28 were MDR, four pre-extensively drug resistant (XDR) TB, and four XDR-TB. The remaining strain was SIT1 and drug susceptible. MIRU-VNTR analysis allowed the identification of three Beijing clusters and two unique strains. Beijing strains were confirmed as "modern" sublineage. The mutations rpoB S531L and katG S315T were the most common among MDR strains. Moreover, the two strains evaluated by whole genome sequencing (WGS) shared most of the genetic features with the sublineage 2.2.1 "modern" Beijing previously characterized from Asian strains. WGS analysis of the MDR strain revealed the presence of eight SNPs previously reported in other MDR "Beijing-like" strains from Colombia. The presence of "modern" Beijing strains in Southwestern Colombia, most of them with MDR phenotype, suggests a different origin of this M. tuberculosis sublineage compared to other Beijing strains found in neighboring South American countries. This work may serve as a genetic baseline to study the evolution and spread of M. tuberculosis Beijing strains in Colombia, which play an important role in the propagation of MDR-TB.

International audience; Bedaquiline, a new antituberculosis drug, has already been used in >50 countries. The emergence of bedaquiline resistance is alarming, as it may result in the rapid loss of this new drug. This article aims to review currently identified mechanisms of resistance and the emergence of bedaquiline resistance, and discuss strategies to delay the resistance acquisition. In vitro and clinical studies as well as reports from compassionate use have identified the threat of bedaquiline resistance and cross-resistance with clofazimine, emphasizing the crucial need for the systematic surveillance of resistance. Currently known mechanisms of resistance include mutations within the atpE, Rv0678, and pepQ genes. The development of standardized drug susceptibility testing (DST) for bedaquiline is urgently needed. Understanding any target and non-target-based mechanisms is essential to minimize resistance development and treatment failure and help to develop appropriate DST for bedaquiline and genetic-based resistance screening.

A low pH was assumed to be required for the activity of pyrazinoic acid (the active form of pyrazinamide) against Mycobacterium tuberculosis, but recently activity has been demonstrated at neutral pH. Renewed interest in pyrazinamide has led to an increasing number of potential targets and the suspicion that pyrazinamide is a 'dirty drug'. However, it is our opinion that the recent demonstration that pyrazinoic acid is active against PanD provides an alternative explanation for the secret of pyrazinamide's unusual activity. In this article we propose that PanD is the primary target of pyrazinoic acid but expression of pyrazinoic acid susceptibility requires an intact stress response. As the mycobacterial stress response requires the interaction of a number of genes, disruption of any could result in an inability to enter the susceptible phenotype. We believe this model can explain most of the recent observations of the seemingly diverse spectrum of activity of pyrazinamide.

Background Whole 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. Aim To compare the impact of variability of several WGS analysis pipelines used internationally to detect epidemiologically linked TB cases. Methods From 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. Results The 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. Conclusion Concordance 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.

Beijing strains ofMycobacterium tuberculosis(lineage2) have been associated with drug-resistance and transmission of tuberculosis worldwide. Most of the Beijing strains identified in the Colombian pacific coast exhibited a multidrug resistant (MDR) phenotype, in contrast with the phenotype observed in Beijing isolates from other South-American countries. We wanted to evaluate the clonality and genetic background of the Beijing strains isolated in Colombia that belong mostly to the spoligo-international type (SIT) 190. Out of 37 Beijing stains characterized in an 8-years period, we identified five Beijing clones; 36 that belong to the SIT190 type and only one to SIT1. Two loci in VNTR typing: MIRU 39 and QUB11b exhibited the highest level of variation among these strains. Out of the 37 Beijing strains, only one was drug susceptible, 28 represented MDR-TB, four extensively drug resistant TB (XDR-TB) and four pre-XDR-TB. The mutationsrpoBS531L andkatGS315T1 were the most common among the MDR strains as reported elsewhere. Whole genome sequencing analysis allowed us to classify them as modern lineage Beijing strains, sharing up to 76 out of the 275 SNPs described in Beijing strains, as identified worldwide by Schürchet al;including 54 non-synonymous SNPs and 23 silent mutations. We were also able to confirm the presence of 8 specific SNPs that were so far only found in the Beijing strains from Colombia. The presence of modern Beijing strains, most of them representing MDR-TB, suggests a different origin of thisM. tuberculosislineage compared to other Beijing strains found in neighboring countries, such as Peru. The specific 8-SNP signature confirmed the identity of these Colombian Beijing MDR strains. This work may serve as a genetic baseline to study the evolution and spread ofM. tuberculosisBeijing strains in Colombia, which play an important role in the control of MDR-TB.

Background Kazakhstan remains a high-burden TB prevalence country with a concomitent high-burden of multi-drug resistant tuberculosis. For this reason, we performed an in depth genetic diversity and population structure characterization of Mycobacterium tuberculosis complex (MTC) genetic diversity in Kazakhstan with both patient and community benefit. Methods A convenience sample of 700 MTC DNA cultures extracts from 630 tuberculosis patients recruited from 12 out of 14 regions in Kazakhstan, between 2010 and 2015, was independently studied by high-throughput hybridization-based methods, TB-SPRINT (59-Plex, n = 700), TB-SNPID (50-Plex, n = 543). DNA from 391 clinical isolates was successfully typed by two methods. To resolve the population structure of drug-resistant clades in more detail two complementary assays were run on the L2 isolates: an IS6110-NTF insertion site typing assay and a SigE SNP polymorphism assay. Results Strains belonged to L2/Beijing and L4/Euro-American sublineages; L2/Beijing prevalence totaled almost 80%. 50% of all samples were resistant to RIF and to INH., Subtyping showed that: (1) all L2/Beijing were “modern” Beijing and (2) most of these belonged to the previously described 94–32 sublineage (Central Asian/Russian), (3) at least two populations of the Central Asian/Russian sublineages are circulating in Kazakhstan, with different evolutionary dynamics. Conclusions For the first time, the global genetic diversity and population structure of M. tuberculosis genotypes circulating in Kazakhstan was obtained and compared to previous local studies. Results suggest a region-specific spread of a very limited number of L2/Beijing clonal complexes in Kazakhstan many strongly associated with an MDR phenotype. Electronic supplementary material The online version of this article (10.1186/s12879-019-4201-2) contains supplementary material, which is available to authorized users.

BackgroundDrug-susceptibility testing (DST) of Mycobacterium tuberculosis complex (MTBC) isolates by the Mycobacteria Growth Indicator Tube (MGIT) approach is the most widely applied reference standard. However, the use of WGS is increasing in many developed countries to detect resistance and predict susceptibility. We investigated the reliability of WGS in predicting drug susceptibility, and analysed the discrepancies between WGS and MGIT against the first-line drugs rifampicin, isoniazid, ethambutol and pyrazinamide.MethodsDST by MGIT and WGS was performed on MTBC isolates received in 2016/2017. Nine genes and/or their promotor regions were investigated for resistance-associated mutations: rpoB, katG, fabG1, ahpC, inhA, embA, embB, pncA and rpsA. Isolates that were discrepant in their MGIT/WGS results and a control group with concordant results were retested in the MGIT, at the critical concentration and a lower concentration, and incubated for up to 45 days after the control tube became positive in the MGIT.ResultsIn total, 1136 isolates were included, of which 1121 were routine MTBC isolates from the Netherlands. The negative predictive value of WGS was ≥99.3% for all four first-line antibiotics. The majority of discrepancies for isoniazid and ethambutol were explained by growth at the lower concentrations, and for rifampicin by prolonged incubation in the MGIT, both indicating low-level resistance.ConclusionsApplying WGS in a country like the Netherlands, with a low TB incidence and low prevalence of resistance, can reduce the need for phenotypic DST for ∼90% of isolates and accurately detect mutations associated with low-level resistance, often missed in conventional DST.

Multi-and extensively drug-resistant tuberculosis (M/XDR-TB) has become an increasing threat not only in countries where the TB burden is high but also in affluent regions, due to increased international travel and globalization. Carbapenems are earmarked as potentially active drugs for the treatment of Mycobacterium tuberculosis. To better understand the potential of carbapenems for the treatment of M/XDR-TB, the aim of this review was to evaluate the literature on currently available in vitro, in vivo, and clinical data on carbapenems in the treatment of M. tuberculosis and to detect knowledge gaps, in order to target future research. In February 2018, a systematic literature search of PubMed and Web of Science was performed. Overall, the results of the studies identified in this review, which used a variety of carbapenem susceptibility tests on clinical and laboratory strains of M. tuberculosis, are consistent. In vitro, the activity of carbapenems against M. tuberculosis is increased when used in combination with clavulanate, a BLaC inhibitor. However, clavulanate is not commercially available alone, and therefore, it is impossible in practice to prescribe carbapenems in combination with clavulanate at this time. Few in vivo studies have been performed, including one prospective, two observational, and seven retrospective clinical studies to assess the effectiveness, safety, and tolerability of three different carbapenems (imipenem, meropenem, and ertapenem). We found no clear evidence at the present time to select one particular carbapenem among the different candidate compounds to design an effective M/XDR-TB regimen. Therefore, more clinical evidence and dose optimization substantiated by hollow-fiber infection studies are needed to support repurposing carbapenems for the treatment of M/XDR-TB.

Contains fulltext : 170267.pdf (Publisher’s version ) (Open Access) INTRODUCTION: Success rates for treatment of extensively drug resistant tuberculosis (XDR-TB) are low due to limited treatment options, delayed diagnosis and inadequate health care infrastructure. Areas covered: This review analyses existing programmes of prevention, diagnosis and treatment of XDR-TB. Improved diagnostic procedures and rapid molecular tests help to select appropriate drugs and dosages. Drugs dosages can be further tailored to the specific conditions of the patient based on quantitative susceptibility testing of the M. tuberculosis isolate and use of therapeutic drug monitoring. Pharmacovigilance is important for preserving activity of the novel drugs bedaquiline and delamanid. Furthermore, biomarkers of treatment response must be developed and validated to guide therapeutic decisions. Expert commentary: Given the currently poor treatment outcomes and the association of XDR-TB with HIV in endemic regions, a more patient oriented approach regarding diagnostics, drug selection and tailoring and treatment evaluation will improve treatment outcome. The different areas of expertise should be covered by a multidisciplinary team and may involve the transition of patients from hospitalized to home or community-based treatment.

For the past decades, an acidic pH has been used to render Mycobacterium tuberculosis susceptible to pyrazinamide for in vitro testing. Here, we show that at the standard breakpoint concentration and reduced culture temperatures, pyrazinamide (PZA) is active against tuberculosis (TB) at neutral pH. This finding should help unravel the mechanism of action of PZA and allow drug susceptibility testing (DST) methods to be optimized.

Whole genome sequencing (WGS) can be used for molecular typing and characterisation ofMycobacterium tuberculosiscomplex (MTBC) strains. We evaluated the systematic use of a WGS-based approach for MTBC surveillance involving all European Union/European Economic Area (EU/EEA) countries and highlight the challenges and lessons learned to be considered for the future development of a WGS-based surveillance system.WGS and epidemiological data of patients with rifampicin (RR) and multi-drug resistant (MDR)-tuberculosis (TB) were collected from EU/EEA countries between January 2017 and December 2019. WGS-based genetic relatedness analysis was performed using a standardised approach including both core genome multilocus sequence typing (cgMLST), and single nucleotide polymorphism (SNP)-based calculation of distances on all WGS data that fulfilled minimum quality criteria to ensure data comparability.From 25 countries, 2218 RR/MDR-MTBC isolates were collected. Fifty-six cross-border clusters with increased likelihood of recent transmission (≤5 SNPs distance) comprising a total of 316 RR/MDR-MTBC isolates were identified. The cross-border clusters included between two and thirty resistant isolates from two to six countries unravelling different RR/MDR-TB transmission patterns in Western and Eastern EU.This pilot study shows that a WGS-based surveillance system is not only feasible but can efficiently elucidate the dynamics of in-country and cross-border RR/MDR-TB transmission across EU/EEA countries. Lessons learned from this study highlight how the establishment of an EU/EEA centralised WGS-based surveillance system for TB will require strengthening of national integrated systems performing prospective WGS surveillance and the development of clear procedures to facilitate international collaboration for the investigation of cross-border clusters.

DNA methylation is an epigenetic modification of the genome involved in regulating crucial cellular processes, including transcription and chromosome stability. Advances in PacBio sequencing technologies can be used to robustly reveal methylation sites. The methylome of the Mycobacterium tuberculosis complex is poorly understood but may be involved in virulence, hypoxic survival and the emergence of drug resistance. In the most extensive study to date, we characterise the methylome across the 4 major lineages of M. tuberculosis and 2 lineages of M. africanum, the leading causes of tuberculosis disease in humans. We reveal lineage-specific methylated motifs and strain-specific mutations that are abundant globally and likely to explain loss of function in the respective methyltransferases. Our work provides a set of sixteen new complete reference genomes for the Mycobacterium tuberculosis complex, including complete lineage 5 genomes. Insights into lineage-specific methylomes will further elucidate underlying biological mechanisms and other important phenotypes of the epi-genome.

Antibiotic treatment of tuberculosis takes ≥6 months, putting a major burden on patients and health systems in large parts of the world. Treatment beyond 2 months is needed to prevent tuberculosis relapse by clearing remaining, drug-tolerantMycobacterium tuberculosisbacilli. However, the majority of patients treated for only 2–3 months will cure without relapse and do not need prolonged treatment. Assays that can identify these patients at an early stage of treatment may significantly help reduce the treatment burden, while a test to identify those patients who will fail treatment may help target host-directed therapies.In this review we summarise the state of the art with regard to discovery of biomarkers that predict relapse-free cure for pulmonary tuberculosis. Positron emission tomography/computed tomography scanning to measure pulmonary inflammation enhances our understanding of “cure”. Several microbiological and immunological markers seem promising; however, they still need a formal validation. In parallel, new research strategies are needed to generate reliable tests.

Background Patients with Mycobacterium tuberculosis isolates sharing identical DNA fingerprint patterns can be epidemiologically linked. However, municipal health services in the Netherlands are able to confirm an epidemiological link in only around 23% of the patients with isolates clustered by the conventional variable number of tandem repeat (VNTR) genotyping. This research aims to investigate whether whole genome sequencing (WGS) is a more reliable predictor of epidemiological links between tuberculosis patients than VNTR genotyping. Methods VNTR genotyping and WGS were performed in parallel on all Mycobacterium tuberculosis complex isolates received at the Netherlands National Institute for Public Health and the Environment in 2016. Isolates were clustered by VNTR when they shared identical 24-loci VNTR patterns; isolates were assigned to a WGS cluster when the pair-wise genetic distance was ≤ 12 single nucleotide polymorphisms (SNPs). Cluster investigation was performed by municipal health services on all isolates clustered by VNTR in 2016. The proportion of epidemiological links identified among patients clustered by either method was calculated. Results In total, 535 isolates were genotyped, of which 25% (134/535) were clustered by VNTR and 14% (76/535) by WGS; the concordance between both typing methods was 86%. The proportion of epidemiological links among WGS clustered cases (57%) was twice as common than among VNTR clustered cases (31%). Conclusion When WGS was applied, the number of clustered isolates was halved, while all epidemiologically linked cases remained clustered. WGS is therefore a more reliable tool to predict epidemiological links between tuberculosis cases than VNTR genotyping and will allow more efficient transmission tracing, as epidemiological investigations based on false clustering can be avoided.

Real time PCR has become a dominant method for the highly sensitive detection of pathogens in clinical material. Real time PCR can generate a fluorescence signal by using fluorescence labelled probes, allowing us to detect and semi quantify the amount of amplified DNA. Here we test the variability of the detection system and cost implications of three different versions of the LightCycler® 480 (LC480), focusing on the intensity of fluorescence and Cq in monoplex and multiplex rtPCRs. For gastro-intestinal pathogens there was no correlation between the intensity of fluorescence and the Cq value in the different LC480 types. For probes with the dyes FAMTM, HEXTM, Cy5 and Red610 a higher fluorescence intensity was seen in LC480 type II and III compared to LC480 type I. After lowering the probe concentration for the Cy5 dye three-fold (from 0.3μM to 0.1μM) the Cq value remains the same and the intensity of fluorescence decreases. For the LC480 type II and III the difference in fluorescence intensity was much more extreme. The concentration of the different labelled probes can be lowered at least six-fold in LC480 type II and III cyclers while maintaining a fluorescence intensity as high as achieved in the LC480 type I with undiluted probe. In conclusion, the strength of the fluorescence signal of the LightCycler® 480 type III is superior to that of LightCycler® 480 types I and II, allowing the use of lower probe concentrations for all dyes, particularly for the dyes Red610 and Cy5. This results in a two thirds reduction in PCR probe costs. Switching to these newer machines for real-time PCR can reduce dye labelled probe consumption and thus reduce costs significantly.

SUMMARY We are far away from the days when tuberculosis (TB) accounted for 1 in 4 deaths during the 19th century. However, Mycobacterium tuberculosis complex (MTBC) strains are still the leading cause of morbidity and mortality by a single infectious disease, with 9.6 million cases and 1.5 million deaths reported. One-third of the world's population is estimated by the WHO to be infected with latent TB. During the last decade, several studies have aimed to define the characteristics of dormant bacteria in these latent infections. General features of the shift to a dormant state encompass several phenotypic changes that reduce metabolic activity. This low metabolic state is thought to increase the resistance of MTBC strains to host/environmental stresses, including antibiotic action. Once the stress ceases (e.g., interruption of treatment), dormant cells can reactivate and cause symptomatic disease again. Therefore, a proper understanding of dormancy could guide the rational development of new treatment regimens that target dormant cells, reducing later relapse. Here, we briefly summarize the latest data on the genetics involved in the regulation of dormancy and discuss new approaches to TB treatment.

Real time PCR has become a dominant method for the highly sensitive detection of pathogens in clinical material. Real time PCR can generate a fluorescence signal by using fluorescence labelled probes, allowing us to detect and semi quantify the amount of amplified DNA. Here we test the variability of the detection system and cost implications of three different versions of the LightCycler® 480 (LC480), focusing on the intensity of fluorescence and Cq in monoplex and multiplex rtPCRs. For gastro-intestinal pathogens there was no correlation between the intensity of fluorescence and the Cq value in the different LC480 types. For probes with the dyes FAMTM, HEXTM, Cy5 and Red610 a higher fluorescence intensity was seen in LC480 type II and III compared to LC480 type I. After lowering the probe concentration for the Cy5 dye three-fold (from 0.3μM to 0.1μM) the Cq value remains the same and the intensity of fluorescence decreases. For the LC480 type II and III the difference in fluorescence intensity was much more extreme. The concentration of the different labelled probes can be lowered at least six-fold in LC480 type II and III cyclers while maintaining a fluorescence intensity as high as achieved in the LC480 type I with undiluted probe. In conclusion, the strength of the fluorescence signal of the LightCycler® 480 type III is superior to that of LightCycler® 480 types I and II, allowing the use of lower probe concentrations for all dyes, particularly for the dyes Red610 and Cy5. This results in a two thirds reduction in PCR probe costs. Switching to these newer machines for real-time PCR can reduce dye labelled probe consumption and thus reduce costs significantly.

BACKGROUND: Little is known about drug-resistant tuberculosis (TB) and its transmission in Papua, which has one of the highest rates of TB in Indonesia. DESIGN: We examined genotypic drug resistance patterns using multiplex ligation-dependent probe amplification and the degree of molecular clustering using 24-locus mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) among 199 consecutive pulmonary TB patients in Jayapura, Papua. Results : Drug resistance mutations were present in 30/198 (15.2%) patients: 16/144 (11.1%) primary cases and 14/51 (27.5%) retreatment cases. Genotypic resistance to rifampicin was found in 15 (7.6%) patients, to isoniazid in 19 (9.6%), to ethambutol in 7 (3.5%), and to streptomycin and second-line injectable drugs in 5 (2.5%) patients. Eight (4.0%) patients had multidrug-resistant TB, while no mutations were found for fluoroquinolones. The most common lineage found among all isolates was East-African Indian (n = 66, 33.7%), followed by Euro-American (n = 38, 19.4%). Drug resistance mutations were more common among Beijing strains than other lineages. Of the 30 drug-resistant isolates, 12 (40.0%) fell into four clusters that were separate from drug-susceptible clusters as determined using MIRU-VNTR. CONCLUSIONS: These are the first genotypic drug resistance data from Jayapura, Papua, showing moderate rates of resistance to first-line drugs and likely transmission of drug-resistant TB.

Simple effective tools to monitor the long treatment of tuberculosis (TB) are lacking. Easily measured host derived biomarkers have been identified but need to be validated in larger studies and different population groups. Here we investigate the early response in IP-10 levels (between day 0 and day 7 of TB therapy) to identify bacteriological status at diagnosis among 127 HIV-infected patients starting TB treatment. All participants were then classified as responding or not responding to treatment blindly using a previously described IP-10 kinetic algorithm. There were 77 bacteriologically confirmed cases and 41 Xpert MTB/RIF(R) and culture negative cases. Most participants had a measurable decline in IP-10 during the first 7 days of therapy. Bacteriologically confirmed cases were more likely to have high IP-10 levels at D0 and had a steeper decline than clinically diagnosed cases (mean decline difference 2231 pg/dl, 95% CI: 897-3566, p = 0.0013). Bacteriologically confirmed cases were more likely to have a measurable decline in IP-10 at day 7 than clinically diagnosed cases (48/77 (62.3%) vs 13/41 (31.7%), p < 0.001). This study confirms the association between a decrease in IP-10 levels during the first week of treatment and a bacteriological confirmation at diagnosis in a large cohort of HIV positive patients.

In many countries, Mycobacterium tuberculosis isolates are routinely subjected to variable-number tandem-repeat (VNTR) typing to investigate M. tuberculosis transmission. Unexpectedly, cross-border clusters were identified among African refugees in the Netherlands and Denmark, although transmission in those countries was unlikely. Whole-genome sequencing (WGS) was applied to analyze transmission in depth and to assess the precision of VNTR typing. WGS was applied to 40 M. tuberculosis isolates from refugees in the Netherlands and Denmark (most of whom were from the Horn of Africa) that shared the exact same VNTR profile. Cluster investigations were undertaken to identify in-country epidemiological links. Combining WGS results for the isolates (all members of the central Asian strain [CAS]/Delhi genotype), from both European countries, an average genetic distance of 80 single-nucleotide polymorphisms (SNPs) (maximum, 153 SNPs) was observed. The few pairs of isolates with confirmed epidemiological links, except for one pair, had a maximum distance of 12 SNPs. WGS divided this refugee cluster into several subclusters of patients from the same country of origin. Although the M. tuberculosis cases, mainly originating from African countries, shared the exact same VNTR profile, most were clearly distinguished by WGS. The average genetic distance in this specific VNTR cluster was 2 times greater than that in other VNTR clusters. Thus, identical VNTR profiles did not represent recent direct M. tuberculosis transmission for this group of patients. It appears that either these strains from Africa are extremely conserved genetically or there is ongoing transmission of this genotype among refugees on their long migration routes from Africa to Europe.

We read with great interest the mathematical model presented by Fofana et al. ([1][1]) on the role of pyrazinamide (PZA) in the emergence of multidrug-resistant tuberculosis (MDR TB), particularly as the results of their model mirror our concerns regarding the amplification of PZA resistance during

Since 2004, variable-number tandem-repeat (VNTR) typing of Mycobacterium tuberculosis complex isolates has been applied on a structural basis in The Netherlands to study the epidemiology of tuberculosis (TB). Although this technique is faster and technically less demanding than the previously used restriction fragment length polymorphism (RFLP) typing, reproducibility remains a concern. In the period from 2004 to 2015, 8,532 isolates were subjected to VNTR typing in The Netherlands, with 186 (2.2%) of these exhibiting double alleles at one locus. Double alleles were most common in loci 4052 and 2163b. The variables significantly associated with double alleles were urban living (odds ratio [OR], 1.503; 95% confidence interval [CI], 1.084 to 2.084; P = 0.014) and pulmonary TB (OR, 1.703; 95% CI, 1.216 to 2.386; P = 0.002). Single-colony cultures of double-allele strains were produced and revealed single-allele profiles; a maximum of five single nucleotide polymorphisms (SNPs) was observed between the single- and double-allele isolates from the same patient when whole-genome sequencing (WGS) was applied. This indicates the presence of two bacterial populations with slightly different VNTR profiles in the parental population, related to genetic drift. This observation is confirmed by the fact that secondary cases from TB source cases with double-allele isolates sometimes display only one of the two alleles present in the source case. Double alleles occur at a frequency of 2.2% in VNTR patterns in The Netherlands. They are caused by biological variation rather than by technical aberrations and can be transmitted either as single- or double-allele variants.

We describe the population structure of a representative collection of 3,133 Mycobacterium tuberculosis isolates, collected within the framework of a national resistance survey from 2007 in China. Genotyping data indicate that the epidemic strains in China can be divided into seven major complexes, of which 92% belonged to the East Asian (mainly Beijing strains) or the Euro-American lineage. The epidemic Beijing strains in China are closely related to the Beijing B0/W148 strain earlier described in Russia and a large cluster of these strains has spread national wide. The density of Beijing strains is high in the whole of China (average 70%), but the highest prevalence was found North of the Yellow river. The Euro-American lineage consists of three sublineages (sublineage_1, 2, and 3) and is more prevalent in the South. Beijing lineage showed the highest cluster rate of 48% and a significantly higher level of resistance to rifampicin (14%, p

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Background The ongoing epidemic of multidrug-resistant tuberculosis (MDR-TB) in Georgia highlights the need for more effective control strategies. A new regimen to treat MDR-TB that includes pyrazinamide (PZA) is currently being evaluated and PZA resistance status will largely influence the success of current and future treatment strategies. PZA susceptibility testing was not routinely performed at the National Reference Laboratory (NRL) in Tbilisi between 2010 and September 2015. We here provide a first insight into the prevalence of PZA resistant TB in this region. Methods Phenotypic susceptibility to PZA was determined in a convenience collection of well-characterised TB patient isolates collected at the NRL in Tbilisi between 2012 and 2013. In addition, the pncA gene was sequenced and whole genome sequencing was performed on two isolates. Results Out of 57 isolates tested 33 (57.9%) showed phenotypic drug resistance to PZA and had a single pncA mutation. All of these 33 isolates were MDR-TB strains. pncA mutations were absent in all but one of the 24 PZA susceptible isolate. In total we found 18 polymorphisms in the pncA gene. From the two major MDR-TB clusters represented (94–32 and 100–32), 10 of 15, 67.0% and 13 of 14, 93.0% strains, respectively were PZA resistant. We also identified a member of the potentially highly transmissive clade A strain carrying the characteristic I6L substitution in PncA. Another strain with the same MLVA type as the clade A strain acquired a different mutation in pncA and was genetically more distantly related suggesting that different branches of this particular lineage have been introduced into this region. Conclusion In this high MDR-TB setting more than half of the tested MDR-TB isolates were resistant to PZA. As PZA is part of current and planned MDR-TB treatment regimens this is alarming and deserves the attention of health authorities. Based on our typing and sequence analysis results we conclude that PZA resistance is the result of primary transmission as well as acquisition within the patient and recommend prospective genotyping and PZA resistance testing in high MDR-TB settings. This is of utmost importance in order to preserve bacterial susceptibility to PZA to help protect (new) second line drugs in PZA containing regimens.

Mycobacterium tuberculosis (Mtb) lineage identification and typing of clinical isolates in general is performed only retrospectively. The results are rarely linked to drug susceptibility testing (DST) or patient data. Consequently, the association between Mtb lineage, (multi)drug resistance and treatment history is not fully explored at the local level. Here we evaluated a new SNP based typing assay. We furthermore assessed the added value of genotyping of Mtb isolates for epidemiological purposes and guidance of tuberculosis (TB) control. Mtb lineage, DST profile and treatment history were determined for 399 samples at the National TB Reference Laboratory (NRL) in Tbilisi, Georgia by local staff. Data was shared electronically and analysis was performed remotely. Out of 399 isolates, 74 (74/399, 18.5%) were at least multidrug resistant (MDR)-TB, of which 63 (63/74, 85.1%) were members of three different Mtb Beijing lineages. Previous treatment was reported in 38/74 (51.4%) MDR(+) patients. The availability of this data allows associations with lineages. Notably, multidrug resistant TB was more strongly associated with the Beijing lineage than treatment history. Of all MDR-TB Beijing strains 56.7% (42/74) were members of a genetic cluster. This is most easily explained by (ongoing) MDR-TB transmission rather than drug resistance amplification. This knowledge is useful when designing intervention strategies for MDR-TB. Our study provides an example that on-site integrated Mtb genotyping is realistic and could support TB control activities.

Background Approximately 10 % of the Mycobacterium tuberculosis genome is made up of two families of genes that are poorly characterized due to their high GC content and highly repetitive nature. The PE and PPE families are typified by their highly conserved N-terminal domains that incorporate proline-glutamate (PE) and proline-proline-glutamate (PPE) signature motifs. They are hypothesised to be important virulence factors involved with host-pathogen interactions, but their high genetic variability and complexity of analysis means they are typically disregarded in genome studies. Results To elucidate the structure of these genes, 518 genomes from a diverse international collection of clinical isolates were de novo assembled. A further 21 reference M. tuberculosis complex genomes and long read sequence data were used to validate the approach. SNP analysis revealed that variation in the majority of the 168 pe/ppe genes studied was consistent with lineage. Several recombination hotspots were identified, notably pe_pgrs3 and pe_pgrs17. Evidence of positive selection was revealed in 65 pe/ppe genes, including epitopes potentially binding to major histocompatibility complex molecules. Conclusions This, the first comprehensive study of the pe and ppe genes, provides important insight into M. tuberculosis diversity and has significant implications for vaccine development. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2467-y) contains supplementary material, which is available to authorized users.

Post-exposure prophylaxis (PEP) for leprosy is administered as one single dose of rifampicin (SDR) to the contacts of newly diagnosed leprosy patients. SDR reduces the risk of developing leprosy among contacts by around 60 % in the first 2–3 years after receiving SDR. In countries where SDR is currently being implemented under routine programme conditions in defined areas, questions were raised by health authorities and professional bodies about the possible risk of inducing rifampicin resistance among the M. tuberculosis strains circulating in these areas. This issue has not been addressed in scientific literature to date. To produce an authoritative consensus statement about the risk that SDR would induce rifampicin-resistant tuberculosis, a meeting was convened with tuberculosis (TB) and leprosy experts. The experts carefully reviewed and discussed the available evidence regarding the mechanisms and risk factors for the development of (multi) drug-resistance in M. tuberculosis with a view to the special situation of the use of SDR as PEP for leprosy. They concluded that SDR given to contacts of leprosy patients, in the absence of symptoms of active TB, poses a negligible risk of generating resistance in M. tuberculosis in individuals and at the population level. Thus, the benefits of SDR prophylaxis in reducing the risk of developing leprosy in contacts of new leprosy patients far outweigh the risks of generating drug resistance in M. tuberculosis. Electronic supplementary material The online version of this article (doi:10.1186/s40249-016-0140-y) contains supplementary material, which is available to authorized users.

Contains fulltext : 171243.pdf (Publisher’s version ) (Closed access) Mycobacterium tuberculosis genotype distribution is different between West and Central Indonesia, but there are no data on the most Eastern part, Papua. We aimed to identify the predominant genotypes of M. tuberculosis responsible for tuberculosis in coastal Papua, their transmission, and the association with patient characteristics. A total of 199 M. tuberculosis isolates were collected. Spoligotyping was applied to describe the population structure of M. tuberculosis, lineage identification was performed using a combination of lineage-specific markers, and genotypic clusters were identified using a combination of 24-locus-MIRU-VNTR and spoligotyping. A high degree of genetic diversity was observed among isolates based on their spoligopatterns. Strains from modern lineage 4 made up almost half of strains (46.9%), being more abundant than the ancient lineage 1 (33.7%), and modern lineage 2 (19.4%). Thirty-five percent of strains belonged to genotypic clusters, especially strains in the Beijing genotype. Previous TB treatment and mutations associated with drug resistance were more common in patients infected with strains of the Beijing genotype. Papua shows a different distribution of M. tuberculosis genotypes compared to other parts of Indonesia. Clustering and drug resistance of modern strains recently introduced to Papua may contribute to the high tuberculosis burden in this region.

Introduction: Tuberculosis remains a global health problem and pharmacokinetic variability has been postulated as one of the causes of treatment failure and acquired drug resistance. New developments enable implementation of therapeutic drug monitoring, a strategy to evaluate drug exposure in order to tailor the dose to the individual patient, in tuberculosis treatment.Areas covered: Literature on pharmacokinetics and pharmacodynamics of anti-tuberculosis drugs was explored to evaluate the effect of drug exposure in relation to drug susceptibility, toxicity and efficacy. New, down-sized strategies, like dried blood spot analysis and limited sampling strategies are reviewed. In addition, molecular resistance testing of Mycobacteria tuberculosis, combining a short turn-around time with relevant information on drug susceptibility of the causative pathogen was explored. Newly emerging host biomarkers provide information on the response to treatment.Expert opinion: Therapeutic drug monitoring can minimize toxicity and increase efficacy of tuberculosis treatment and prevent the development of resistance. Dried blood spot analysis and limited sampling strategies, can be combined to provide us with a more patient friendly approach. Furthermore, rapid information on drug susceptibility by molecular testing, and information from host biomarkers on the bacteriological response, can be used to further optimize tuberculosis treatment.

In Bulgaria during 2007-2011, a total of 188 MDR/ XDR M. tuberculosis isolates were characterized by drug- susceptibility testing and spoligotyping (31 in 2007, 31 in 2008, 39 in 2009, 47 in 2010, and 40 in 2011) and represent 72% of the 261 MDR/XDR M. tuberculosis isolates identi- fied during that period. The MDR/XDR strains were iso- lated from sputum of 181 (96.2%) patients, gastric lavage fluid from 3 (1.5%), bronchoalveolar lavage fluid from 2 (1%), pleural fluid from 1 (0.5%), and fistula swab sample from 1 (0.5%). The first MDR/XDR M. tuberculosis strain isolated per patient was analyzed for this study. Drug susceptibility of these strains was confirmed by the National Reference TB Laboratory, which used liq- uid culture at concentrations of 0.1 µg/mL for isoniazid, 1 µg/mL for rifampin, 5 µg/mL for ethambutol, 1 µg/mL for streptomycin, 2.5 µg/mL for capreomycin, 1 µg/mL for amikacin, 5 µg/mL for kanamycin, and 2 µg/mL for ofloxacin. Of the MDR/XDR strains, 77 (41%) were resis- tant to all first-line anti-TB drugs, 51 (27%) were resistant to isoniazid and rifampin; 38 (20%) to isoniazid, rifampin, and streptomycin; and 22 (12%) to isoniazid, rifampin, and ethambutol. Second-line drug-susceptibility testing was performed for 174 (81%) of the MDR strains. Of these, 140 (80%) were sensitive to all second-line anti-TB drugs and 20 (12%) were resistant to ofloxacin. Five percent (n = 9) of XDR strains had combined resistance to ofloxacin, ami - kacin, kanamycin, and capreomycin. To detect and genotype the Beijing strains, we used a spoligotyping kit (Isogen Bioscience BV, Maarssen, the Netherlands), and we performed single-nucleotide polymorphism typing by bead-based multiplex ligation- dependent probe amplification (MLPA) (8,9). We also screened for the presence of the Beijing genotype in 117 drug-sensitive strains collected from across the country in 2011, representing a convenience sample of ≈10%. Both methods identified 1 drug-sensitive M. tuberculosis strain

We have developed a multiplex assay, based on multiplex ligation-dependent probe amplification (MLPA), that allows simultaneous detection of multiple drug resistance mutations and genotype-specific mutations at any location in the Mycobacterium tuberculosis genome. The assay was validated on a reference panel of well-characterized strains, and the results show that M. tuberculosis can be accurately characterized by our assay. Eighteen discriminatory markers identifying drug resistance ( rpoB , katG , inhA , embB ), members of the M. tuberculosis complex (16S rRNA, IS 6110 , TbD1), the principal genotypic group ( katG , gyrA ), and Haarlem and Beijing strains ( ogt , mutT2 , mutT4 ) were targeted. A sequence specificity of 100% was reached for 16 of the 18 selected genetic targets. In addition, a panel of 47 clinical M. tuberculosis isolates was tested by MLPA in order to determine the correlation between phenotypic drug resistance and MLPA and between spoligotyping and MLPA. Again, all mutations present in these isolates that were targeted by the 16 functional probes were identified. Resistance-associated mutations were detected by MLPA in 71% of the identified rifampin-resistant strains and in 80% of the phenotypically isoniazid-resistant strains. Furthermore, there was a perfect correlation between MLPA results and spoligotypes. When MLPA is used on confirmed M. tuberculosis clinical specimens, it can be a useful and informative instrument to aid in the detection of drug resistance, especially in laboratories where drug susceptibility testing is not common practice and where the rates of multidrug-resistant and extensively drug resistant tuberculosis are high. The flexibility and specificity of MLPA, along with the ability to simultaneously genotype and detect drug resistance mutations, make MLPA a promising tool for pathogen characterization.

Background In conjunction with the spread of HIV infection, tuberculosis (TB) remains a major cause of illness and death worldwide. The Ethiopian national report reveals that extra pulmonary tuberculosis is on the rise and that case detection rate is exceeding that of smear positive or negative cases in many parts of the country. Different studies indicated that host and/or pathogen related factors are associated with the rise of extra pulmonary cases. However, the reason for this is not clearly known in our setting. Methods Specimens were taken from clinically suspected extra pulmonary patients and confirmed by cytology, histopathology and culture. Deletion typing and Spoligotyping was utilized to identify the strains. The isolates were then assigned to lineage using conformal Bayesian network (rules model) algorithm and dendrograms were drawn using UPGMA methods. In addition, drug sensitivity test was done using the indirect proportion and 24 well plate methods. Results Out of the 200 clinically suspected extra pulmonary tuberculosis patients, 106 (53 %) were between 15 and 35 years of age and 167 (83.5 %) were new while 33 (16.5 %) were retreatment cases. The culture yield was 29.5 % (59). Of these only one was M. bovis and 58 were M. tuberculosis strains with 31 different spoligotype patterns grouped into seven clusters. The largest cluster (ST53) comprised 12 (20.3 %) isolates. There was higher clustering of CAS isolates in TBLN than in any other form of extra pulmonary tuberculosis cases. Resistance to rifampicin was higher (22 %) than that for INH, STM and EMB (8.1 %, 5 % and 3 % respectively). Out of the 37 isolates tested for resistance, only 2 isolates were resistant for both STM and INH and no MDR strain was found. Conclusions There is an ongoing active recent transmission among extra pulmonary tuberculosis in the study areas as shown by the presence of clusters. Although no MDR case was observed, there is a risk of emergence of MDR as noted from the high proportion of resistance to rifampicin. Detailed study at population level is recommended to monitor its trend.

Background Molecular resistance detection (MRD) of resistance to second-line anti-tuberculous drugs provides faster results than phenotypic tests, may shorten treatment and allow earlier separation among patients with and without second-line drug resistance. Methods In a decision-analytical model we simulated a cohort of patients diagnosed with TB in a setting where drug resistant TB is highly prevalent and requires initial hospitalization, to explore the potential benefits of a high-throughput MRD-assay for reducing potential nosocomial transmission of highly resistant strains, and total costs for diagnosis of drug resistance, treatment and hospitalization. In the base case scenario first-line drug resistance was diagnosed with WHO-endorsed molecular tests, and second-line drug resistance with culture and phenotypic methods. Three alternative scenarios were explored, each deploying high-throughput MRD allowing either detection of second-line mutations in cultured isolates, directly on sputum, or MRD with optimized markers. Results Compared to a base case scenario, deployment of high-throughput MRD reduced total costs by 17-21 %. The period during which nosocomial transmission may take place increased by 15 % compared to the base case if MRD had currently reported suboptimal sensitivity and required cultured isolates; increased by 7 % if direct sputum analysis were possible including in patients with smear-negative TB, and reduced by 24 % if the assay had improved markers, but was still performed on cultured isolates. Improved clinical sensitivity of the assay (additional markers) by more than 35 % would be needed to avoid compromising infection control. Conclusions Further development of rapid second-line resistance testing should prioritize investment in optimizing markers above investments in a platform for direct analysis of sputum. Electronic supplementary material The online version of this article (doi:10.1186/s12879-015-1205-4) contains supplementary material, which is available to authorized users.

In contrast to most other antimycobacterial drugs where--particularly in multidrug-resistant (MDR) strains--a limited number of resistance mutations dominate, pyrazinamide (PZA) resistance associated mutations remain highly diverse with limited clustering. This apparent lack of evolutionary selection for successful PZA resistance mechanisms deserves attention. A clear understanding of the epidemiology of PZA resistance acquisition and spread would be expected to result in important insights into how PZA might be better exploited in treatment regimens to minimize the amplification of Mycobacterium tuberculosis (MTB) drug resistance. We propose that PZA resistance typically induces a fitness cost that impairs MTB transmission. This would explain the lack of extensive clustering for PZA-resistant mutants. Our hypothesis also leads to a series of testable predictions which we outline that could confirm or refute our ideas.