Applying whole genome sequencing to predict phenotypic drug resistance in Mycobacterium tuberculosis: leveraging 20 years of nationwide data from Denmark.

Infection with Mycobacterium tuberculosis remains one of the biggest causes of death from a single microorganism worldwide, and the continuous emergence of drug resistance aggravates our ability to cure the disease. New improved resistance detection methods are needed to provide adequate treatment, such as whole genome sequencing (WGS), which has been used increasingly to identify resistance-conferring mutations over the last decade. The steadily increasing knowledge of resistance-conferring mutations increases our ability to predict resistance based on genomic data alone. This study evaluates the performance of WGS to predict M. tuberculosis complex resistance. It compares WGS predictions with the phenotypic (culture-based) drug susceptibility results based on 20 years of nationwide Danish data. Analyzing 6,230 WGS-sequenced samples, the sensitivities for isoniazid, rifampicin, ethambutol, and pyrazinamide were 82.5% [78.0%-86.5%, 95% confidence interval (CI)], 97.3% (90.6%-99.7%, 95% CI), 58.0% (43.2%-71.8%, 95% CI), and 60.5% (49.0%-71.2%, 95% CI), respectively, and specificities were 99.8% (99.7%-99.9%, 95% CI), 99.8% (99.7%-99.9%, 95% CI), 99.4% (99.2%-99.6%, 95% CI), and 99.9% (99.7%-99.9%, 95% CI), respectively. A broader range of both sensitivities and specificities was observed for second-line drugs. The results conform with previously reported values and indicate that WGS is reliable for routine resistance detection in resource-rich tuberculosis low-incidence and low-resistance settings such as Denmark.

Genomic Epidemiology of a Major Mycobacterium tuberculosis Outbreak: Retrospective Cohort Study in a Low-Incidence Setting Using Sparse Time-Series Sampling.

Since 1992, Denmark has documented the largest outbreak of tuberculosis in Scandinavia ascribed to a single genotype, termed C2/1112-15. As of spring 2017, the International Reference Laboratory of Mycobacteriology in Copenhagen has collected and identified isolates from more than a thousand cases belonging to this outbreak via routine mycobacterial interspersed repetitive units-variable number of tandem repeats typing. Here, we present a retrospective analysis of the C2/1112-15 dataset, based on whole-genome data from a sparse time series consisting of 5 randomly selected isolates from 23 years of sampling. Even if these data are derived from only 12% of the collected isolates, we have been able to extract important key information, such as mutation rate and conserved single-nucleotide polymorphisms to identify discrete transmission chains, as well as the possible historical origins of the outbreak.

Mycobacterium chimaera in Heater-Cooler Units in Denmark Related to Isolates from the United States and United Kingdom.

Mycobacterium chimaera was present at high rates (>80%) in heater-cooler units (HCUs) from all 5 thoracic surgery departments in Denmark. Isolates were clonal to HCU-associated isolates from the United States (including some from patients) and United Kingdom. However, M. chimaera from 2 brands of HCU were genetically distinct.

Substantial molecular evolution and mutation rates in prolonged latent Mycobacterium tuberculosis infection in humans.

The genome of Mycobacterium tuberculosis (Mtb) of latently infected individuals may hold the key to understanding the processes that lead to reactivation and progression to clinical disease. We report here analysis of pairs of Mtb isolates from putative prolonged latent TB cases. We identified two confirmed cases, and used whole genome sequencing to investigate the mutational processes that occur over decades in latent Mtb. We found an estimated mutation rate between 0.2 and 0.3 over 33 years, suggesting that latent Mtb accumulates mutations at rates similar to observations from cases of active disease.

Dual-centre evaluation of the FluoroType MTBDR version 2 assay for detection of Mycobacterium tuberculosis complex and resistance-conferring mutations in pulmonary and extrapulmonary samples from Denmark, Germany and Sierra Leone.

OBJECTIVES: We evaluated the ability of FluoroType MTBDR version 2 (FTv2; Hain Lifescience), a second-step real-time PCR assay, to simultaneously detect Mycobacterium tuberculosis complex (MTBC) DNA and mutations conferring resistance to rifampicin (RIF) and isoniazid (INH), in pulmonary and extrapulmonary samples from patients and compared them with corresponding cultures. METHODS: FTv2 MTBC was evaluated on 1815 and 432 samples from Denmark (DK) and Germany (DE), respectively. RIF and INH resistance mutations were assessed in the German samples and 110 samples from Sierra Leone and subsequently compared to phenotypic antimicrobial susceptibility testing and a composite reference DNA (CRD) based on the GenoType MTBDR line-probe assay and Sanger sequencing or whole-genome sequencing. RESULTS: Of the 584 (557 smear-negative) Danish and 277 (85 smear-negative) German sputum samples, 42 (16) and 246 (54) were culture positive, and 44 (18) and 222 (35) were FTv2 positive, providing an FTv2 sensitivity and specificity of 0.86 (0.63) and 0.98 (DK), 0.90 (0.65) and 1.00 (DE), respectively. The count, sensitivities, and specificities for all pulmonary samples were 1434, 0.79, and 0.99 (DK) and 347, 0.86, and 1.00 (DE), respectively; for extrapulmonary samples, 381, 0.33, 0.99 (DK) and 83, 0.50, and 1.00 (DE). The valid count, sensitivity, and specificity compared with CRD for detecting resistance mutations were RIF 355, 0.99, 0.96, and INH 340, 1.00, and 0.98, respectively. DISCUSSION: FTv2 reliably detects MTBC DNA in pulmonary and extrapulmonary samples and detects resistance mutations for INH and RIF resistance in inhA promoter, katG, and rpoB genes.

The geographic diversity of nontuberculous mycobacteria isolated from pulmonary samples: an NTM-NET collaborative study.

A significant knowledge gap exists concerning the geographical distribution of nontuberculous mycobacteria (NTM) isolation worldwide. To provide a snapshot of NTM species distribution, global partners in the NTM-Network European Trials Group (NET) framework (www.ntm-net.org), a branch of the Tuberculosis Network European Trials Group (TB-NET), provided identification results of the total number of patients in 2008 in whom NTM were isolated from pulmonary samples. From these data, we visualised the relative distribution of the different NTM found per continent and per country. We received species identification data for 20 182 patients, from 62 laboratories in 30 countries across six continents. 91 different NTM species were isolated. Mycobacterium avium complex (MAC) bacteria predominated in most countries, followed by M. gordonae and M. xenopi. Important differences in geographical distribution of MAC species as well as M. xenopi, M. kansasii and rapid-growing mycobacteria were observed. This snapshot demonstrates that the species distribution among NTM isolates from pulmonary specimens in the year 2008 differed by continent and differed by country within these continents. These differences in species distribution may partly determine the frequency and manifestations of pulmonary NTM disease in each geographical location.

Can molecular methods detect 1% isoniazid resistance in Mycobacterium tuberculosis?

Patients may harbor both drug-susceptible and -resistant bacteria, representing heteroresistance. We studied mixtures of isoniazid-resistant and -susceptible Mycobacterium tuberculosis strains. Conventional drug susceptibility testing was the most sensitive method of detection, whereas the line probe assay and sequencing were not able to detect the clinically relevant 1% proportion of resistant bacteria.

Rifampin heteroresistance in Mycobacterium tuberculosis cultures as detected by phenotypic and genotypic drug susceptibility test methods.

Tuberculosis patients may harbor both drug-susceptible and -resistant bacteria, i.e., heteroresistance. We used mixtures of rifampin-resistant and -susceptible Mycobacterium tuberculosis strains to simulate heteroresistance in patient samples. Molecular tests can be used for earlier discovery of multidrug resistance (MDR), but the sensitivity to detect heteroresistance is unknown. Conventional phenotypic drug susceptibility testing was the most sensitive, whereas two line probe assays and sequencing were unable to detect the clinically important 1% resistant bacteria.

Successful Direct Whole Genome Sequencing and Revivification of Freeze-Dried Nontuberculous Mycobacteria after More than Half a Century of Storage.

In this study, 28 "historical" clinical freeze-dried nontuberculous mycobacterial isolates collected from 1948 to 1957, were analyzed by investigating their viability and performing whole genome sequencing (WGS) on DNA extracted (i) directly from freeze-dried cells versus (ii) after culturing, to determine cell properties and DNA quality after centuries of freeze-dried storage. The isolated DNA was sequenced on the Illumina MiSeq platform and data quality evaluated analyzing the per-base quality scores of paired-end sequencing reads as well as the overall contiguity of resulting de novo assemblies. After 72 years in storage, all freeze-dried isolates were viable, and showed no signs of cell damage and limited signs of contamination when reculturing. They were recultured without problems and identified through WGS with only four of 13 parameters showing statistical significance based on sequence data obtained directly from the freeze-dried cells versus after reculturing, indicating no DNA degradation. Thus, mycobacteria can be whole genome sequenced successfully directly from freeze-dried material without prior recultivation, saving laboratory time and resources, and emphasizing the value of freeze-drying for long-term storage. Our study lays the groundwork for further genomic investigations of freeze-dried bacterial isolates, and the approximately 4,000 historical isolates in our collection will provide a unique opportunity to investigate mycobacterial DNA from a variety of NTM species unexposed to antimicrobials, some maybe still undescribed species. IMPORTANCE The genus Mycobacterium was described more than a century ago and new species are continuously identified and described. There is an ongoing discussion about an increase in the incidence of disease caused by nontuberculous mycobacteria (NTM). How the different bacteria looked before exposure to antibiotics can only be investigated by looking at strains from before the antibiotic era. Strains from that era will be stored in different ways, for example by freeze-drying. The question is how to investigate these strains, and if they are still viable, whether they need to be cultured, and if that changes the DNA. Here, we test all these parameters on freeze-dried strains and show that NGS can be applied directly without culturing.

A cohort study of the long-term outcome of latent tuberculosis infection among socially marginalized people in a low-incidence country.

BACKGROUND: Tuberculosis (TB) prevalence is high among socially marginalized citizens in Denmark, and management of latent TB infection (LTBI) may be part of preventing new cases. Patients with LTBI are offered either preventive treatment (TPT) or follow-up chest x-rays, but knowledge about the long-term outcome in terms of active TB is sparse. METHODS: We performed a retrospective cohort study investigating the long-term outcomes for socially marginalized citizens who were diagnosed with LTBI or who had a positive interferon-gamma release assay (IGRA) but were lost to follow-up. Information on TB examinations, diagnostics, and treatment along with data on death were gathered from medical records from the date of positive IGRA to February 1, 2021. RESULTS: We identified 119 patients with LTBI, 18 of which (15.1%) were diagnosed with TB during the follow-up period (mean, 4.5 years). TPT was completed by 36.1% and the TB incidence rate ratio of those completing TPT to those who did not was 0.78 (confidence interval, 0.25-2.17; P =.6). Of the patients with TB, 16 of 18 achieved treatment success. CONCLUSION: High rates of TB development are found among socially marginalized citizens with LTBI. Overall incidence of TB was not significantly reduced by administration of TPT, although TB did not develop in the first 2 years following TPT.

First outbreak of multidrug-resistant tuberculosis (MDR-TB) in Denmark involving six Danish-born cases.

BACKGROUND: Denmark is a low-incidence country for tuberculosis (TB) and multidrug-resistant (MDR) TB at 5 and 0.05 cases per 100,000 population, respectively. Until 2018, the transmission of MDR-TB was nonexistent except for a few pairwise related family cases. In this study, we describe the first MDR-TB outbreak in Denmark. METHODS: On the basis of genotyping of all Mycobacterium tuberculosis (Mtb) culture-positive cases in Denmark spanning 3 decades, 6 molecular- and epidemiologically linked Danish-born cases were identified as the first cluster of an MDR-TB in Denmark. The primary case was diagnosed posthumously in 2010 followed by 5 epidemiologically linked cases from 2018 to 2019. RESULTS AND CONCLUSION: Through a combination of routine Mtb genotyping and clinical epidemiological surveillance data, we identified the first Danish MDR-TB outbreak spanning 10 years and were able to disclose the specific transmission pathways in detail, which helped guide the outbreak investigations. The occurrence of an MDR-TB outbreak in a resource-rich low TB incidence setting such as Denmark highlights the importance of a collaborative control system combining classic contact tracing; timely identification of drug-resistant TB through rapid diagnostics; and a close collaboration between clinicians and classical- and molecular epidemiologists for the benefit of TB control.

Detection of Mycobacterium tuberculosis complex in pulmonary and extrapulmonary samples with the FluoroType MTBDR assay.

OBJECTIVES: Rifampicin (RIF) and isoniazid (INH) are the two most effective first-line antibiotic drugs for the treatment of tuberculosis (TB). The new FluoroType MTBDR (FT-MTBDR) real-time PCR is intended to detect INH and RIF resistance mutations as a second step following a primary Mycobacterium tuberculosis complex (MTBC) PCR. Here we evaluate the feasibility of the FT-MTBDR assay to detect simultaneously MTBC-specific DNA as well as to detect potential INH and RIF resistance through analysing inhA promotor, katG and rpoB sequences in one PCR reaction. METHODS: We analysed 3885 consecutive primary samples with FT-MTBDR and compared the results with microscopy and culture: 978 were from sputum, 2007 from other respiratory tract locations plus gastric lavages, and 875 from extrapulmonary locations, respectively. RESULTS: Overall, 176 samples were MTBC culture positive and 139 FT-MTBDR positive, providing a FT-MTBDR sensitivity of 0.714 (95% confidence interval 0.640-0.779) and specificity of 0.996 (0.994-0.998), respectively. For the 978 sputum, 96 were MTBC culture positive and 89 FT-MTBDR positive, sensitivity 0.854 (0.764-0.915) and specificity 0.992 (0.983-0.997). Of the 139 MTBC positive, 99 (71%) had interpretable genotypic resistance results for at least one drug, 92 (66%) for both drugs. DISCUSSION: The ability of FT-MTBDR to detect MTBC is adequate with the significant added feature of simultaneous genotypic resistance detection of both INH and RIF in a single PCR reaction.

Tuberculosis Drug Susceptibility, Treatment, and Outcomes for Belarusian HIV-Positive Patients with Tuberculosis: Results from a National and International Laboratory.

BACKGROUND: To cure drug-resistant (DR) tuberculosis (TB), the antituberculous treatment should be guided by Mycobacterium tuberculosis drug-susceptibility testing (DST). In this study, we compared conventional DST performed in Minsk, Belarus, a TB DR high-burden country, with extensive geno- and phenotypic analyses performed at the WHO TB Supranational Reference Laboratory in Copenhagen, Denmark, for TB/HIV coinfected patients. Subsequently, DST results were related to treatment regimen and outcome. METHODS: Thirty TB/HIV coinfected patients from Minsk were included and descriptive statistics applied. RESULTS: Based on results from Minsk, 10 (33%) TB/HIV patients had drug-sensitive TB. Two (7%) had isoniazid monoresistant TB, 8 (27%) had multidrug-resistant (MDR) TB, 5 (17%) preextensive drug-resistant (preXDR) TB, and 5 (17%) had extensive drug-resistant (XDR) TB. For the first-line drugs rifampicin and isoniazid, there was DST agreement between Minsk and Copenhagen for 90% patients. For the second-line anti-TB drugs, discrepancies were more pronounced. For 14 (47%) patients, there were disagreements for at least one drug, and 4 (13%) patients were classified as having MDR-TB in Minsk but were classified as having preXDR-TB based on DST results in Copenhagen. Initially, all patients received standard anti-TB treatment with rifampicin, isoniazid, pyrazinamide, and ethambutol. However, this was only suitable for 40% of the patients based on DST. On average, DR-TB patients were changed to 4 (IQR 3-5) active drugs after 1.5 months (IQR 1-2). After treatment adjustment, the treatment duration was 8 months (IQR 2-11). Four (22%) patients with DR-TB received treatment for >18 months. In total, sixteen (53%) patients died during 24 months of follow-up. CONCLUSIONS: We found high concordance for rifampicin and isoniazid DST between the Minsk and Copenhagen laboratories, whereas discrepancies for second-line drugs were more pronounced. For patients with DR-TB, treatment was often insufficient and relevant adjustments delayed. This example from Minsk, Belarus, underlines two crucial points in the management of DR-TB: the urgent need for implementation of rapid molecular DSTs and availability of second-line drugs in all DR-TB high-burden settings. Carefully designed individualized treatment regimens in accordance with DST patterns will likely improve patients' outcome and reduce transmission with drug-resistant Mycobacterium tuberculosis strains.

Recurrent tuberculosis in patients infected with the predominant Mycobacterium tuberculosis outbreak strain in Denmark. New insights gained through whole genome sequencing.

OBJECTIVES: Recurrent tuberculosis (TB) is defined by more than one TB episode per patient and is caused by reinfection with a new M. tuberculosis (Mtb) strain or relapse with the previous strain. In Denmark, a major TB outbreak caused by one specific Mtb genotype "DKC2" is ongoing. Of the 892 patients infected with DKC2 between 1992 and 2014, 32 had recurrent TB with 67 TB episodes in total. METHODS: The 32 cases were evaluated in terms of number of single-nucleotide polymorphism (SNP) differences and time between episodes derived from whole-genome sequencing data. RESULTS: For four TB cases, the subsequent episodes could be confirmed as relapse and for one case as reinfection. Eight cases with SNP differences <6, theoretically indicating relapse, could be classified as likely reinfections based on phylogenetic analysis in combination with geographical data. Subsequent TB episodes for the remaining 19 cases could not be classified as relapse or reinfection even though they all had a SNP difference of <6 SNPs. CONCLUSIONS: In newer studies, investigating recurrent TB with the use of WGS, the number of SNPs has been used to distinguish between relapse and reinfection. The algorithm proposed for this is not valid in the Danish TB outbreak setting as our findings challenge the interpretation of few SNP differences as representing relapse. However, when including phylogenetic analysis and geographical data in the analysis, classification of 13 of the 32 cases were possible.

Complete Genome Sequence of Mycobacterium tuberculosis DKC2, the Predominant Danish Outbreak Strain.

The largest clonal outbreak of Mycobacterium tuberculosis infection in Scandinavia has been monitored by the International Reference Laboratory of Mycobacteriology (IRLM) since 1992. Here, we present the complete genome sequence of M. tuberculosis strain DKC2 substrain PP1, a representative isolate collected in 1993 from a Danish patient with pulmonary tuberculosis.

Set-up and validation of mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) analysis of Mycobacterium tuberculosis using BioNumerics software.

The objective was to describe and validate a new and alternative software procedure for 24-locus mycobacterial interspersed repetitive unit-variable number-tandem repeat (MIRU-VNTR) typing of Mycobacterium tuberculosis (Mtb) based on the multipurpose BioNumerics software. DNA from randomly selected isolates of Mtb from two European laboratories, including external control samples for MIRU-VNTR typing, were analysed. Samples were genotyped using the commercial 24-locus VNTR typing kit from GenoScreen. The PCR amplified fragments were separated by capillary electrophoresis. For the subsequent analyses, the currently used software GeneMapper was compared with BioNumerics. The endpoint was the level of concordance when comparing genotyping results obtained from BioNumerics with results obtained from GeneMapper and the ECDC proficiency study reference results. Also, the number of necessary manual standard size corrections and allele assignments in the two different software methods were compared. In total, 272 DNA samples, including the ECDC proficiency panel, were analysed. For all samples, there were 100% concordance of results. For a randomly selected set of 96 samples the numbers of manual corrections needed for size standards were 199 with GeneMapper versus zero for BioNumerics. The numbers of manual corrections for allele assignments were 122 with GeneMapper versus 16 with BioNumerics. In conclusion, we have validated the multipurpose software BioNumerics for standard 24-locus MIRU-VNTR typing and the software shows promising benefits in terms of simplification and minimization of hand-on time.

A Major Mycobacterium tuberculosis outbreak caused by one specific genotype in a low-incidence country: Exploring gene profile virulence explanations.

Denmark, a tuberculosis low burden country, still experiences significant active Mycobacterium tuberculosis (Mtb) transmission, especially with one specific genotype named Cluster 2/1112-15 (C2), the most prevalent lineage in Scandinavia. In addition to environmental factors, antibiotic resistance, and human genetics, there is increasing evidence that Mtb strain variation plays a role for the outcome of infection and disease. In this study, we explore the reasons for the success of the C2 genotype by analysing strain specific polymorphisms identified through whole genome sequencing of all C2 isolates identified in Denmark between 1992 and 2014 (n = 952), and the demographic distribution of C2. Of 234 non-synonymous (NS) monomorphic SNPs found in C2 in comparison with Mtb reference strain H37Rv, 23 were in genes previously reported to be involved in Mtb virulence. Of these 23 SNPs, three were specific for C2 including a NS mutation in a gene associated with hyper-virulence. We show that the genotype is readily transmitted to different ethnicities and is also found outside Denmark. Our data suggest that strain specific virulence factor variations are important for the success of the C2 genotype. These factors, likely in combination with poor TB control, seem to be the main drivers of C2 success.

[Strengthening of laboratories is a new component in the global strategy for tuberculosis control]. / Styrkelse af laboratorier er et nyt element i den globale tuberkulosekontrolstrategi.

Although old techniques remain important, new techniques offer new possibilities. Mutations conferring resistance to rifampin and isoniazid can be detected in primary specimens from infectious pulmonary cases. Infections can be detected with interferon-gamma release assays, and chains of transmission can be detected by mycobacteria interspersed repetitive units. Centralized diagnostics makes it possible to apply results of routine analyses in national and international surveillance.