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A previously healthy man developed pulmonary symptoms 2 weeks after starting treatment with a tumour necrosis factor (TNF) inhibitor. A negative interferon-gamma release assay (IGRA) test was obtained prior to TNF inhibitor exposure, without consideration of the fact that the patient was already immunosuppressed and had a previous positive IGRA test 17 months earlier. The patient was treated for pneumonia twice but did not achieve remission. His physical health progressively deteriorated over the following months. Malignancy was suspected but not found. Eight months after the onset of symptoms, Mycobacterium tuberculosis was found in samples from mediastinal lymph nodes, and the patient was diagnosed with multidrug-resistant tuberculosis (MDR-TB).This case illustrates the diagnostic challenge of TB, the need to raise awareness of the increased risk of TB in patients treated with TNF inhibitors and the need to increase knowledge regarding the effect of immunosuppressive agents on IGRA tests.

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Nontuberculous mycobacteria (NTM) are emerging as notable causative agents of opportunistic infections. To examine clinical significance, species distribution, and temporal trends of NTM in Denmark, we performed a nationwide register-based study of all unique persons with NTM isolated in the country during 1991-2022. We categorized patients as having definite disease, possible disease, or isolation by using a previously validated method. The incidence of pulmonary NTM increased throughout the study period, in contrast to earlier findings. Mycobacterium malmoense, M. kansasii, M. szulgai, and M. avium complex were the most clinically significant species based on microbiologic findings; M. avium dominated in incidence. This study shows the need for surveillance for an emerging infection that is not notifiable in most countries, provides evidence to support clinical decision-making, and highlights the importance of not considering NTM as a single entity.

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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.

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In this case report, we present case reports for two nurses, both working in departments of respiratory medicine, who developed tuberculosis (TB). For each individual case, whole genome sequencing (WGS) revealed only one specific match within a genomic distance of <6 single-nucleotide polymorphisms. The subsequent epidemiological investigations confirmed that both nurses had relevant exposures to their corresponding match 1139 and 1704 days before presenting with TB symptoms, respectively. Twenty-two studies were identified that reported using genotyping to identify occupational transmission of Mycobacterium tuberculosis to healthcare workers. Only two studies applied WGS, both conducted in resource-rich countries, comparable to the present Danish investigation. When comparing the two WGS studies to the other studies that used older genotyping techniques, WGS provided a higher resolution and much more detailed information. Consequently, the epidemiological investigations were more straightforward. In conclusion, WGS is a powerful tool for determining whether M. tuberculosis transmission is occupational as demonstrated for the two cases in this study.

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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.

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OBJECTIVES: This study aimed to analyze mortality, risk factors, and causes of death among people with tuberculosis (TB). METHODS: This is a population-based cohort study with patients with TB ≥18 years notified from 1990 to 2018 in Denmark, compared with sex- and age-matched controls. Mortality was assessed in Kaplan-Meier models and risk factors for death were estimated in Cox proportional hazards models. RESULTS: Overall mortality was twofold higher among people with TB compared with controls up to 15 years after TB diagnosis (hazard ratio [HR]: 2.18, 95% confidence interval [CI]: 2.06-2.29, P <0.0001). Danes with TB were three times more likely to die than migrants (adjusted HR: 3.13, 95% CI: 2.84-3.45, P <0.0001). Risk factors for death included living alone, being unemployed, having low income, and comorbidities such as mental illness with substance abuse, lung diseases, hepatitis, and HIV. TB was the most common cause of death (21%), followed by chronic obstructive pulmonary disease (7%), lung cancer (6%), alcoholic liver disease (5%), and mental illness with substance abuse (4%). CONCLUSION: People with TB had substantially inferior survival up to 15 years after TB diagnosis, in particular, socially disadvantaged Danes with TB with specific comorbidities. This may reflect unmet needs for enhanced treatment of other medical/social conditions during TB treatment.

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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.

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BACKGROUND: The global vision is a world free of tuberculosis (TB). Even in resource-rich TB low-incidence settings, we need more focus on the role of social risk factors to end the TB epidemic. METHODS: Nationwide, retrospective register-based, case-control study from 1990 to 2018, including all TB patients in Denmark ≥18 years old (n = 9581) matched 1:3 on sex and age with population controls. TB risk factors were assessed in logistic regression models and estimated by odds ratio (OR). RESULTS: All TB patients had considerably lower socio-economic status compared with controls (P < 0.0001). Among ethnic Danes, TB was mostly found among males, persons between 35 and 65 years, those living alone, those with low educational level, persons on social welfare benefits and those with low income. Conversely, for migrants, being younger, sex and living alone were less important, whereas having children was protective. In an adjusted multivariable regression model among Danes, key risk factors for TB were being on disability pension (OR = 2.7) and cash benefits (OR = 4.7). For migrants, fewer social risk factors increased TB risk, although low income and cash benefits did (OR = 3.1). CONCLUSION: Even today in a resourceful setting, socio-economic status drives disparities in health. In our study, multifactorial social deprivation was highly associated with TB. Especially household structure, education, employment and income were important risk factors that should be addressed in the future to accelerate TB control and end the TB epidemic.

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The emergence and spread of resistant tuberculosis (TB) pose a threat to public health, so it is necessary to diagnose the drug-resistant forms in a clinically short time frame and closely monitor their transmission. In this study, we carried out a first whole genome sequencing (WGS)-based analysis of multidrug resistant (MDR) M. tuberculosis strains to explore the phylogenetic lineages diversity, drug resistance mechanisms, and ongoing transmission chains within the country. In total, 65 isolates phenotypically resistant to at least rifampicin and isoniazid collected in the Czech Republic in 2005-2020 were enrolled for further analysis. The agreement of the results obtained by WGS with phenotypic drug susceptibility testing (pDST) in the determination of resistance to isoniazid, rifampicin, pyrazinamide, streptomycin, second-line injectables and fluoroquinolones was more than 80%. Phylogenetic analysis of WGS data revealed that the majority of MDR M. tuberculosis isolates were the Beijing lineage 2.2.1 (n = 46/65; 70.8%), while the remaining strains belonged to Euro-American lineage. Cluster analysis with a predefined cut-off distance of less than 12 single nucleotide polymorphisms between isolates showed 19 isolates in 6 clusters (clustering rate 29.2%), located mainly in the region of the capital city of Prague. This study highlights the utility of WGS as a high-resolution approach in the diagnosis, characterization of resistance patterns, and molecular-epidemiological analysis of resistant TB in the country.

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BackgroundPregnancy increases the risk of tuberculosis (TB), however, data on TB epidemiology in pregnant women are limited.AimTo guide possible interventions, we analysed risk factors for TB in pregnant and post-partum women.MethodsWe conducted a nationwide retrospective register-based case-control study from January 1990 to December 2018 in Denmark. Cases were women diagnosed with TB during their pregnancy or in the post-partum period. We selected two control groups: pregnant or post-partum women without TB, and non-pregnant women with TB. Differences were assessed by chi-squared or Fisher's exact test. Risk factors for TB were identified through logistic regression and estimated by odds ratio (OR).ResultsWe identified 392 cases, including 286 pregnant and 106 post-partum women. Most were migrants (n = 366; 93%) with a shorter median time spent in Denmark (2.74 years; interquartile range (IQR): 1.52-4.64) than non-pregnant TB controls (3.98 years; IQR: 1.43-8.51). Cases less likely had a Charlson comorbidity index ≥ 2compared with non-pregnant TB controls (p < 0.0001), and had no increased risk of severe disease (p = 0.847). Migrants from other World Health Organization regions than Europe, especially Africa (OR: 187; 95%CI: 125-281) had persistently higher odds of TB.ConclusionsIn Denmark, the risk of TB in pregnant and post-partum women is increased in migrant women who have stayed in the country a median time of approximately 3 years. We recommend increased focus on TB risk during pregnancy and suggest evaluating targeted TB screening of selected at-risk pregnant women to promote early case finding and prevent TB among mothers and their newborn children.

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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.

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Beijing genotype Mycobacterium tuberculosis strains associate with increased virulence, resistance and/or higher transmission rates. This study describes a specific Beijing strain predominantly identified in the Panamanian province of Colon with one of the highest incidences of tuberculosis in the country. Retrospective mycobacterial interspersed repetitive unit/variable number of tandem repeats analysis of 42 isolates collected between January and August 2018 allowed to identify a cluster (Beijing A) with 17 (40.5%) Beijing isolates. Subsequent prospective strain-specific PCR-based surveillance from September 2019 to March 2020 confirmed the predominance of the Beijing A strain (44.1%) in this province. Whole-genome sequencing revealed higher-than-expected diversity within the cluster, suggesting long-term prevalence of this strain and low number of cases caused by recent transmission. The Beijing A strain belongs to the Asian African 3 (Bmyc13, L2.2.5) branch of the modern Beijing sublineage, with their closest isolates corresponding to cases from Vietnam, probably introduced in Panama between 2000 and 2012.

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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.

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

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BACKGROUND: Whole genome sequencing (WGS) has enabled the development of new approaches to track Mycobacterium tuberculosis (Mtb) transmission between tuberculosis (TB) cases but its utility may be challenged by the discovery that Mtb diversifies within hosts. Nevertheless, there is limited data on the presence and degree of within-host evolution. METHODS: We profiled a well-documented Mtb transmission cluster with three pulmonary TB cases to investigate within-host evolution and describe its impact on recent transmission estimates. We used deep sequencing to track minority allele frequencies (<50·0% abundance) during transmission and standard treatment. FINDINGS: Pre-treatment (n = 3) and serial samples collected over 2 months of antibiotic treatment (n = 16) from all three cases were analysed. Consistent with the epidemiological data, zero fixed SNP separated all genomes. However, we identified six subclones between the three cases with an allele frequency ranging from 35·0% to 100·0% across sampling intervals. Five subclones were identified within the index case pre-treatment and shared with one secondary case, while only the dominant clone was observed in the other secondary case. By tracking the frequency of these heterogeneous alleles over the two-month therapy, we observed distinct signatures of drift and negative selection, but limited evidence for de novo mutations, even under drug pressure. INTERPRETATION: We document within-host Mtb diversity in an index case, which led to transmission of minority alleles to a secondary case. Incorporating data on heterogeneous alleles may refine our understanding of Mtb transmission dynamics. However, more evidence is needed on the role of transmission bottleneck on observed heterogeneity between cases.

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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.

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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.

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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.

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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.

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