Erik Svensson

Can Molecular Methods Detect 1% Isoniazid Resistance in Mycobacterium tuberculosis?

ABSTRACT 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

ABSTRACT 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.

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.

Nontuberculous mycobacteria in Denmark, incidence and clinical importance during the last quarter-century

Disease caused by nontuberculous mycobacteria (NTM) is reported to increase due to an ageing population and a rise in the proportion of immunosuppressed patients. We did a retrospective cohort study of NTM-disease in the Danish population through a quarter-century to determine the disease burden and trends in annual incidence rates. 524,119 clinical specimens were cultured for mycobacteria from 1991 through 2015 at the International Reference Laboratory of Mycobacteriology in Denmark. Among these, 8,227 NTM strains were identified from 3,462 patients and distributed according to microbiological disease criteria. We observed no increase in NTM disease incidence or proportion of patients with positive NTM cultures during the study period (Quasi-Poisson regression, pu2009=u20090.275 and 0.352 respectively). Annual incidence rates were 1.20/105 for definite NTM disease, 0.49/105 for possible NTM disease and 0.88/105 for NTM colonization. The incidence rate of NTM disease was highest in children aged 0-4 years (5.36/105/year), predominantly with cervical Mycobacterium avium complex (MAC) adenitis. Surprisingly, based on more than half a million clinical specimens cultured for mycobacteria in Denmark through 25 years, the NTM disease burden and trend in incidence in the Danish population has not increased opposed to numerous internationals reports.

Occupational Tuberculosis in Denmark through 21 Years Analysed by Nationwide Genotyping.

Tuberculosis (TB) is a well-known occupational hazard. Based on more than two decades (1992–2012) of centralized nationwide genotyping of all Mycobacterium tuberculosis culture-positive TB patients in Denmark, we compared M. tuberculosis genotypes from all cases notified as presumed occupational (N = 130) with M. tuberculosis genotypes from all TB cases present in the country (N = 7,127). From 1992 through 2006, the IS6110 Restriction Fragment Length Polymorphism (RFLP) method was used for genotyping, whereas from 2005 to present, the 24-locus-based Mycobacterial Interspersed Repetitive Unit-Variable Number of Tandem Repeat (MIRU-VNTR) was used. An occupational TB case was classified as clustered if the genotype was 100% identical to at least one other genotype. Subsequently, based on genotype, time period, smear positivity, geography, susceptibility pattern, and any reported epidemiological links between the occupational cases and any potential source cases, the occupational case was categorized as confirmed, likely, possible or unlikely occupationally infected. Among the 130 notified presumed occupational cases, 12 (9.2%) could be classified as confirmed and 46 (35.4%) as unlikely, accounting for nearly half of all cases (44.6%). The remaining 72 cases (55.4%) were categorized as possible. Within this group, 15 cases (11.5%) were assessed to be likely occupational. Our study shows that genotyping can serve as an important tool for disentangle occupational TB in high-income low incidence settings, but still needs to be combined with good epidemiological linkage information.

Optimisation of a murine splenocyte mycobacterial growth inhibition assay using virulent Mycobacterium tuberculosis

In the absence of a validated correlate of protection or robust animal models for human tuberculosis, Mycobacterial growth inhibition assays (MGIAs) aim to assess vaccines ability to inhibit mycobacterial growth in-vitro. We optimised a reproducible murine splenocyte MGIA based on in-vitro infection with ufeffvirulent Mycobacterium tuberculosis (M.tb) Erdman. We identified splenocyte viability as a problem in state-of-art MGIA protocols, which can be improved by simple changes in culture conditions (viability increase from 21% to 46% at last day of culture). The growth inhibitory potential in mice immunised with either BCG, H56:CAF01 or H56:CAF01 administered side-by-side with BCG was significantly better compared to placebo in all groups (0.3 log10 CFU [±0.2, pu2009=u20090.049], 0.5 [±0.2, pu2009=u20090.016] and 0.6 [±0.1, pu2009=u20090.0007], respectively) corresponding to the levels of in-vivo protection. Unexpectedly the CAF01 adjuvant control group also induced significant growth inhibition of 0.3 log10 CFU (±0.2, pu2009=u20090.047). Finally, we explored vaccine-associated T cell effector functions. Despite presence of high levels of vaccine-specific T cells, we found no increase in CD4+ T cell number or cytokine expression profile, nor a difference in cytokine levels in the supernatant after four days culture with or without M.tb. Spontaneous IFN-γ release correlated with growth inhibition levels (pu2009=u20090.02), however the cellular source was not found.

Disseminated Mycobacterium celatum disease with prolonged pulmonary involvement

Mycobacterium celatum is a rare cause of human infection, causing disseminated disease in immunosuppressed individuals. Infections localized to the lungs and the lymph nodes have also been reported in immunocompetent individuals. The existing literature on the subject is limited as are experiences with treatment regimens and durations. In the case presented herein, two different treatment regimens were applied to an immunocompromised HIV-negative patient with primary skin involvement and extensive pulmonary involvement due to suspected relapse on isoniazid, ethambutol, and clarithromycin treatment. The treatment regimen was changed to azithromycin, ciprofloxacin, and pyrazinamide and the treatment duration was prolonged to a total of 24 months, with good effect.

Mycobacterium marinum infections in Denmark from 2004 to 2017: A retrospective study of incidence, patient characteristics, treatment regimens and outcome

Mycobacterium marinum (M. marinum) is a slowly growing nontuberculous mycobacterium. The incidence of M. marinum infections in Denmark is unknown. We conducted a retrospective nationwide study including all culture confirmed cases of M. marinum from 2004 to 2017 in Denmark. All available medical records were reviewed. Demographics, clinical characteristics, and treatment regiments were analyzed. Fifty-five patients were identified, 40 (72.7%) were men with a median age of 50 years. Aquatic exposure was reported by 48 (90.6%) of the patients. Site of infection was upper extremities in 49 (92.5%) patients and 49 (92.5%) had superficial infection. The median time from symptom presentation to diagnosis was 194 days. All patients received antibiotics. Median time of treatment duration among all patients was 112 days. Treatment outcome was classified as improved in 40 (75%), improved with sequela in 4 (7.6%) patients and only 3 patients (3.8%) were classified as failed. Infection with M. marinum is rare and there is a long delay from symptom manifestation to diagnosis. The infection is predominantly related to aquatic exposure. M. marinum should be a differential diagnose in patients with slow-developing cutaneous elements and relevant exposure. Treatment outcomes are overall good and severe sequela are rare.

Tuberculosis Transmission in Danish Children; A Nationwide Register-Based Study

Background: Tuberculosis (TB) remains a major public health issue among children worldwide. Data on TB transmission in children living in low-incidence countries is limited. Methods: We studied TB transmission in ethnic Danish children younger than 15 years of age between 2000 and 2013. Identification of children with TB disease and information on demographics and TB contacts were retrieved from the national TB surveillance register and the International Reference Laboratory of Mycobacteriology. Results: In total, 88 children with TB disease were identified in the study period, corresponding to a mean annual incidence of 6.9 per 1,000,000 children younger than 15 years of age. The male to female ratio was 1.3. Median age was 5 years (interquartile range, 3–8.5). Seventy-three (83%) children had a known TB contact of which 60% was among household contacts with recent TB, predominantly parents. Sixty-six (75%) children were classified as part of epidemiologic clusters. Thirty-five (40%) children had culture verified TB of which information on genotypes was available for 34 (97%). Of these, 35% belonged to cluster C2/1112–15, the most prevalent cluster among adult Danes. Conclusions: We found on-going TB transmission in Danish children within the households of a low TB incidence population. These findings emphasize the need for early diagnosis of TB in children, thorough contact tracing and increased focus on risk groups.BACKGROUNDnTuberculosis (TB) remains a major public health issue among children worldwide. Data on TB transmission in children living in low-incidence countries is limited.nnnMETHODSnWe studied TB transmission in ethnic Danish children younger than 15 years of age between 2000 and 2013. Identification of children with TB disease and information on demographics and TB contacts were retrieved from the national TB surveillance register and the International Reference Laboratory of Mycobacteriology.nnnRESULTSnIn total, 88 children with TB disease were identified in the study period, corresponding to a mean annual incidence of 6.9 per 1,000,000 children younger than 15 years of age. The male to female ratio was 1.3. Median age was 5 years (interquartile range, 3-8.5). Seventy-three (83%) children had a known TB contact of which 60% was among household contacts with recent TB, predominantly parents. Sixty-six (75%) children were classified as part of epidemiologic clusters. Thirty-five (40%) children had culture verified TB of which information on genotypes was available for 34 (97%). Of these, 35% belonged to cluster C2/1112-15, the most prevalent cluster among adult Danes.nnnCONCLUSIONSnWe found on-going TB transmission in Danish children within the households of a low TB incidence population. These findings emphasize the need for early diagnosis of TB in children, thorough contact tracing and increased focus on risk groups.