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.

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.

Chronic respiratory disease, inhaled corticosteroids and risk of non-tuberculous mycobacteriosis.

BACKGROUND: Chronic respiratory disease and inhaled corticosteroid (ICS) therapy for chronic obstructive pulmonary disease (COPD) increase the risk of pneumonia. Few data are available on the association of these risk factors with non-tuberculous mycobacterial (NTM) pulmonary disease. METHODS: This study examined chronic respiratory diseases and ICS use as risk factors in a population-based case-control study encompassing all adults in Denmark with microbiologically confirmed NTM pulmonary disease between 1997 and 2008. The study included 10 matched population controls per case. Conditional logistic regression was used to compute adjusted ORs for NTM pulmonary disease with regard to chronic respiratory disease history. RESULTS: Overall, chronic respiratory disease was associated with a 16.5-fold (95% CI 12.2 to 22.2) increased risk of NTM pulmonary disease. The adjusted OR for NTM disease was 15.7 (95% CI 11.4 to 21.5) for COPD, 7.8 (95% CI 5.2 to 11.6) for asthma, 9.8 (95% CI 2.03 to 52.8) for pneumoconiosis, 187.5 (95% CI 24.8 to 1417.4) for bronchiectasis, and 178.3 (95% CI 55.4 to 574.3) for tuberculosis history. ORs were 29.1 (95% CI 13.3 to 63.8) for patients with COPD on current ICS therapy and 7.6 (95% CI 3.4 to 16.8) for patients with COPD who had never received ICS therapy. Among patients with COPD, ORs increased according to ICS dose, from 28.1 for low-dose intake to 47.5 for high-dose intake (more than 800 µg/day). The OR was higher for fluticasone than for budesonide. CONCLUSION: Chronic respiratory disease, particularly COPD treated with ICS therapy, is a strong risk factor for NTM pulmonary disease.

Diabetes, glycemic control, and risk of tuberculosis: a population-based case-control study.

OBJECTIVE: To examine the association between diabetes, glycemic control, and risk of tuberculosis (TB). RESEARCH DESIGN AND METHODS: We conducted a population-based case-control study in Northern Denmark. Cases of active TB were all individuals with a first-time principal hospital diagnosis of TB between 1980 and 2008. Each case subject was matched with up to five population control subjects with similar age, sex, place and length of residence in Denmark, and country of emigration. We computed odds ratios (ORs) for a first-time TB diagnosis among people with and without diabetes using regression to control for other comorbidities, alcoholism, immunosuppressive medications, and socioeconomic markers. RESULTS: We identified 2,950 patients, including 156 diabetic individuals (5.3%), with active TB, and 14,274 population control subjects, of which 539 had diabetes (3.8%). The adjusted OR for active TB among subjects with diabetes was 1.18 (95% CI 0.96-1.45) compared with nondiabetic individuals. We found a similar risk increase from diabetes in the 843 (29%) TB case subjects who were immigrants; adjusted OR = 1.23 (95% CI 0.78-1.93). In a subset with laboratory data, diabetic individuals with an HbA(1c) <7.0, 7-7.9, and ≥8.0% had ORs of 0.91 (0.51-1.63), 1.05 (0.41-2.66), and 1.19 (CI 0.61-2.30), respectively, compared with individuals without diabetes. CONCLUSIONS: In the low TB-burden country of Denmark, the TB risk increase associated with diabetes is substantially lower than previously suggested. We found no evidence for any association between TB and dysglycemia.

Tuberculous meningitis in Denmark: a review of 50 cases.

BACKGROUND: Tuberculous meningitis is the most severe manifestation of extrapulmonary tuberculosis with a high mortality rate and a high rate of sequelae among survivors. The aim of this study is to assess the current epidemiology, clinical features, diagnostic procedures, treatment and outcome in patients with tuberculous meningitis in Denmark, a country with a low tuberculosis incidence. METHODS: A nationwide retrospective study was conducted, comprising all patients notified with tuberculous meningitis (TBM) in Denmark from 2000-2008. Medical records were reviewed using a standardised protocol. RESULTS: Fifty patients, including 12 paediatric patients, were identified. 78% of the patients were immigrants from countries of high tuberculosis endemicity. 64% of all patients had a pre-existing immunosuppressive condition; 10% were HIV positive, 48% were HIV seronegative and 42% had an unknown HIV status. Median symptom duration before admission was 14 days in the Danish patient population and 20 days in the immigrant group. Biochemical analysis of cerebrospinal fluid (CSF) samples revealed pleocytosis in 90% with lymphocyte predominance in 66%. Protein levels were elevated in 86%. The most common findings on neuro-radiological imaging were basal meningeal enhancement, tuberculomas and hydrocephalus. Lumbar puncture was performed on 42 patients; 31 of these specimens (74%) had a positive CSF culture for mycobacteria and 9.5% were smear positive for acid-fast bacilli. The overall mortality rate was 19% and 48% of the remaining patients had neurological sequelae of varying degree. CONCLUSION: TBM is a rare but severe manifestation of extrapulmonary TB in Denmark. The clinician must be prepared to treat empirically if the suspicion of TBM has arisen to improve treatment outcome.

Is sarcoidosis a rickettsiosis? An archival study.

BACKGROUND: Based on earlier research, Rickettsia helvetica could possibly be involved in the pathogenesis of sarcoidosis. Rickettsiae are transmitted to humans by a tick vector, Ixodes ricinus; this tick is highly prevalent in Northern Europe. We aimed to investigate the association between evidence of rickettsiae and sarcoidosis in histological samples. METHODS: We included formalin-fixed, paraffin-embedded mediastinal lymph node biopsies from 52 ethnic Danish patients with sarcoidosis and compared these with 50 biopsies from ethnic Danish patients with mediastinal lymphadenopathy of other causes. Samples were analysed for: (1) rickettsial DNA by real-time polymerase chain reaction (PCR) and (2) rickettsial rDNA (ribosomal DNA) by a specific fluorescence in situ hybridization technique (FISH). RESULTS: Rickettsia was not detected in biopsies by real-time PCR and/or FISH analyses. CONCLUSION: Our results do not support the hypothesis that Rickettsia is involved in the pathogenesis of sarcoidosis.

First-in-man open clinical trial of a combined rdESAT-6 and rCFP-10 tuberculosis specific skin test reagent.

BACKGROUND: Tuberculin is still the only available skin test reagent for the diagnosis of mycobacterial infection. The product has a remarkable sensitivity, but poor specificity. Previous studies, including two human phase I clinical trials, have indicated that rdESAT-6 has a potential as an improved skin test reagent. Animal studies have shown that the sensitivity may be increased by inclusion of the genetically related CFP-10 antigen in the preparation without loosing specificity. METHODOLOGY: In this study a Lactococcus fermented, recombinant skin test reagent consisting of a 1ratio1 wt/wt of rdESAT-6 and CFP-10 was manufactured according to GMP standards and tested for the first time in 42 healthy adult volunteers. The two doses of 0.01 microg or 0.1 microg were injected intradermally by the Mantoux technique with 6 or 12 weeks interval. No serious adverse events and only mild adverse reactions were reported. The reagent elicited a positive skin test reaction after the first injection in one participant, who most likely was latently infected with M. tuberculosis as indicated by an appreciable IFN gamma response just below the Quantiferon(R) cut-off level at the screening visit. None of the remaining participants in the four groups had any skin test reactions and sensitisation by the reagent could therefore be excluded. CONCLUSION: The investigational skin test reagent rdESAT-6 and CFP-10 appeared safe and non-sensitising in this first-in-man clinical trial in human volunteers and can now be tested in larger clinical trials involving individuals with latent M. tuberculosis infection or active TB disease. TRIAL REGISTRATION: ClinicalTrials.gov NCT00793702.

Nontuberculous pulmonary mycobacteriosis in Denmark: incidence and prognostic factors.

RATIONALE: Few population-based data are available regarding nontuberculous mycobacteria (NTM) pulmonary disease epidemiology and prognosis. OBJECTIVES: To examine NTM pulmonary colonization incidence, disease incidence, and prognostic factors. METHODS: All adults in Denmark with at least one NTM-positive pulmonary specimen during 1997 to 2008 were identified using national medical databases and were categorized as having possible or definite NTM disease or colonization. MEASUREMENTS AND MAIN RESULTS: We calculated annual age-standardized NTM incidence rates and adjusted hazard ratios (HR) of death associated with patient age, sex, comorbidity, NTM species, and NTM disease status. Of 1,282 adults with 2,666 NTM-positive pulmonary specimens, 335 (26%) had definite NTM disease, 238 (19%) possible disease, and 709 (55%) colonization only. NTM incidence rates decreased until 2002, followed by an increase from 2003 to 2008 (mean annual rate per 100,000 person-years: NTM colonization, 1.36; NTM disease, 1.08). Five-year mortality after definite NTM disease was 40.1%. After controlling for potential confounders, 5-year mortality for definite NTM disease was slightly higher than for NTM colonization (adjusted hazard ratio [HR], 1.15; 95% confidence interval [CI], 0.90-1.51). Mycobacterium xenopi was associated with worse prognosis (adjusted HR, 1.51; 95% CI, 0.99-2.33) than the reference Mycobacterium avium complex. High comorbidity level (HR, 2.97), age greater than or equal to 65 years (HR, 9.17), and male sex (female sex HR, 0.73) were predictors of death. CONCLUSIONS: NTM disease incidence has remained unchanged in Denmark over the past 12 years. Patients with NTM colonization and disease have similarly poor prognosis. Negative prognostic factors include high levels of comorbidity, advanced age, male sex, and M. xenopi.

Risk of sensitization in healthy adults following repeated administration of rdESAT-6 skin test reagent by the Mantoux injection technique.

Limited specificity of the tuberculin skin test incited the development of the intradermal Mycobacterium tuberculosis-specific rdESAT-6 skin test. Animal studies have shown, however, that there is a possible risk of sensitization when repeated injections of rdESAT-6 are given. The aim of this phase 1 open clinical trial was to assess the sensitization risk and safety of repeated administration of rdESAT-6 reagent in 31 healthy adult volunteers. Three groups of volunteers received two fixed doses of 0.1 microg rdESAT-6 28, 56 or 112 days apart, respectively. After the second injection, the diameter of induration and/or redness at the injection site was measured and taken as a possible sensitization reaction if >5mm. In vitro interferon gamma (IFN-gamma) responses were measured as supportive evidence. Local adverse reactions at the injection site and adverse events were recorded. One out of 31 (3%) volunteers showed a positive skin reaction (sensitization) upon a second injection of rdESAT-6 after 28days and an increased IFN-gamma response to ESAT-6. For 7 (23%) of the volunteers, local adverse reactions related to the product were registered, but all reactions were mild and predictable. In conclusion, repeated injections of the rdESAT-6 skin test reagent are safe, and sensitization occurs at a low rate, especially if the time span between succeeding doses is wide.

Stability of DNA patterns and evidence of Mycobacterium tuberculosis reactivation occurring decades after the initial infection.

Two hundred three freeze-dried strains of Mycobacterium tuberculosis collected during the 1960s were compared with 4102 strains collected during the 1990s, and 14 DNA patterns identified among the "historical strains" were 100% identical to patterns identified among the "recent strains." They were isolated from 41 and 40 patients who had tuberculosis during the 1960s and 1990s, respectively. The patients' mean age differed by >30 years, a finding strongly suggesting that the patients from the 1990s experienced reactivation of M. tuberculosis infection acquired during the 1960s. The half-life of IS6110 DNA patterns during latency was estimated to be 36 years (95% confidence interval, 25-54 years). Thus, this comparison of historical and recent strains yields molecular epidemiologic evidence of M. tuberculosis reactivation spanning decades and suggests that the rate of change of DNA patterns during latency is much longer than that during active disease. This has important implications for the interpretation of clustering, especially for the extent of recent transmission.

Incidence and clinical significance of non-tuberculous mycobacteria isolated from clinical specimens during a 2-y nationwide survey.

A 2-y nationwide survey of patients in Denmark with non-tuberculous mycobacteria (NTM) cultures was undertaken. Patients were identified by means of records held at the International Reference Laboratory of Mycobacteriology, Statens Serum Institut, Denmark. The objectives were to identify isolated NTM to species level, to describe the incidence of the various species and to evaluate the clinical significance of pulmonary NTM isolates other than M. avium complex (MAC) and M. gordonae. Identification was performed by means of hybridization or sequencing of 16S rDNA. The clinical significance of pulmonary NTM isolates was evaluated by means of questionnaires concerning patients (was sent to the clinicians!) patients who had NTM isolated for the first time using bacteriologic, radiographic and clinical criteria. A total of 1110 specimens (2.1%) from 525 patients grew NTM. After MAC (n = 198) and M. gordonae (n = 168), most patients had M. abscessus (n = 21), M. malmoense (n = 20) and M. xenopi (n = 17) isolated. Of the pulmonary patients, 50.6% met bacteriologic criteria, 75.3% radiographic criteria and 53.4% clinical criteria for significant infection. Almost half of the pulmonary patients met all the criteria for significant NTM infection that could be evaluated. Clinically significant infection was associated with underlying disease in most patients.

Molecular evidence of endogenous reactivation of Mycobacterium tuberculosis after 33 years of latent infection.

Since Robert Koch described the cause of tuberculosis in 1882, the natural history of the disease after primary infection has been subject to debate. Only approximately 10% of infected individuals develop active disease, which may appear years to decades after infection. Late onset has been attributed to the endogenous reactivation of dormant bacteria. However, this has not been documented by molecular means for latencies of more than a few years. In Denmark, we have recently recultured 205 freeze-dried Mycobacterium tuberculosis strains obtained from 1961 through 1967. These "historical" strains are analyzed by DNA restriction fragment-length polymorphism testing, and their DNA patterns are compared with those of 4008 recently obtained clinical specimens. This has, surprisingly, yielded molecular evidence of M. tuberculosis reactivation after 33 years of latent infection. A father and son who developed tuberculosis in 1961 and in 1994, respectively, were the only patients infected with strains that share an identical DNA pattern.