Simultaneous quantification of isoniazid, rifampicin, ethambutol and pyrazinamide by liquid chromatography/tandem mass spectrometry.

A remediable cause of poor treatment response in drug-susceptible tuberculosis (TB) patients may be low plasma levels of one or more of the first-line anti-TB drugs. The aim of this work was to develop an accurate and precise LC-MS/MS method for simultaneous quantification of all four first-line anti-TB drugs in plasma suitable for therapeutic drug monitoring (TDM). To adjust for degradation and losses during sample preparation, isotopically labeled compounds were used as internal standards. Plasma samples spiked with internal standards were extracted using protein precipitation with methanol and acetonitrile. Simultaneous separation of all four drugs was accomplished with a Chromolith Reversed-Phase column and mobile phases consisting of water, methanol, ammonium acetate and formic acid with subsequent mass spectrometric quantification. The linear range of the calibration curve for isoniazid was 0.5-10 mg/L, for rifampicin 0.75-30 mg/L, for ethambutol 0.25-10 mg/L and for pyrazinamide 4-80 mg/L. The lower limit of quantification was 0.5 mg/L, 0.75 mg/L, 0.25 mg/L and 4.0 mg/L, respectively. Precision estimated by the coefficient of variation was <15% for all four drugs. The LC-MS/MS method can readily be used for simultaneous quantification of first-line anti-TB drugs in plasma and is well suited for TDM.

Clinical significance of 2 h plasma concentrations of first-line anti-tuberculosis drugs: a prospective observational study.

OBJECTIVES: To study 2 h plasma concentrations of the first-line tuberculosis drugs isoniazid, rifampicin, ethambutol and pyrazinamide in a cohort of patients with tuberculosis in Denmark and to determine the relationship between the concentrations and the clinical outcome. METHODS: After 6-207 days of treatment (median 34 days) 2 h blood samples were collected from 32 patients with active tuberculosis and from three patients receiving prophylactic treatment. Plasma concentrations were determined using LC-MS/MS. Normal ranges were obtained from the literature. Clinical charts were reviewed for baseline characteristics and clinical status at 2, 4 and 6 months after the initiation of treatment. At a 1 year follow-up, therapy failure was defined as death or a relapse of tuberculosis. RESULTS: Plasma concentrations below the normal ranges were frequently observed: isoniazid in 71%, rifampicin in 58%, ethambutol in 46%, pyrazinamide in 10% and both isoniazid and rifampicin in 45% of the patients. The plasma concentrations of isoniazid correlated inversely with the C-reactive protein level at the time of sampling (P = 0.001). During 1 year of follow-up, therapy failure occurred in five patients. Therapy failure occurred more frequently when the concentrations of isoniazid and rifampicin were both below the normal ranges (P = 0.013) and even more frequently when they were below the median 2 h drug concentrations obtained in the study (P = 0.005). CONCLUSIONS: At 2 h, plasma concentrations of isoniazid and rifampicin below the normal ranges were frequently observed. The inverse correlation between the plasma concentrations of isoniazid and C-reactive protein indicate a suboptimal treatment effect at standard dosing regimens. Dichotomization based on median 2 h drug concentrations was more predictive of outcome than dichotomization based on normal ranges.

A review of paediatric tuberculosis in Denmark: 10-year trend, 2000-2009.

Paediatric tuberculosis (TB) is a key indicator for recent transmission and presents a reservoir for the disease. We describe trends in epidemiology, microbiological characteristics and treatment outcome in Denmark between 2000 and 2009. Data were retrieved from the national TB surveillance system and the International Reference Laboratory of Mycobacteriology. In total, 323 TB cases were reported in children aged <15 years, accounting for 7.6% of all notified cases in Denmark. The overall incidence rate of childhood TB declined from 4.1 per 100,000 to 1.9 per 100,000 in the study period. Immigrant children comprised 79.6% of all cases, with the highest incidence rate of 94.1 per 100,000 children in 2001. In contrast to immigrant children, the majority of Danish children were aged <5 years and had a known exposure to TB. Pulmonary TB was the commonest presentation. Only half of the cases were culture confirmed. We observed an overall decreasing trend in the child to adult notification ratio, but a slight increase in the ratio when calculated specifically for ethnic Danes. Childhood TB needs continuous attention with a special focus on risk groups. Emphasis on improving early TB case detection, contact tracing and further implementation of preventive treatment is necessary.

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.

Diabetic ketoacidosis in Denmark Incidence and mortality estimated from public health registries.

The aims of this study were to estimate incidence of diabetic ketoacidosis and mortality from diabetic ketoacidosis using data from public health registries. Four thousand eight hundred and seven admissions in the period 1996-2002 and 137 deaths in the period 1996-2000 with a diagnosis of diabetic ketoacidosis were identified from the Danish National Patient Registry and Danish Cause of Death Registry, respectively. Annual incidence of diabetic ketoacidosis in the general population was estimated to 12.9 per 100,000, being higher in males than in females (14.4 versus 11.4 per 100,000, p<0.0001). Twelve percent of all patients were classified as Type 2 diabetes, predominantly in patients >50 years. Overall mortality was 4%, being higher in patients >70 years than in patients < or =70 years (15% versus 2%, p<0.0001). One or more additional somatic diagnoses were stated on 77% of the death certificates, most often a diagnosis of cardiovascular (47%) or infectious (30%) diseases. Compared to previous studies, the incidence in the general population seems to have remained unaltered the past 25 years, but may have decreased in younger patients. Older patients with diabetic ketoacidosis differed from younger patients in having a higher mortality and a larger proportion of patients classified as Type 2 diabetes.

Treatment of diabetic ketoacidosis in adults in Denmark: a national survey.

The aims of this study were to investigate management routines of diabetic ketoacidosis (DKA) in adult patients in departments of internal medicine in Denmark and to relate current routines of treatment to available evidence. A questionnaire requesting information on management routines of DKA was sent to all departments of internal medicine in Denmark responsible of managing DKA. Fifty-nine departments (88%) returned the questionnaire and/or a copy of their management protocol. At 19 departments (32%), all patients with DKA were managed in an intensive care unit (ICU). Twenty-four different insulin regimens and 21 fluid protocols were identified. Routines of insulin therapy varied in terms of doses and routes of administration. Fifty-eight departments (97%) used isotonic saline for hydration. Potassium supplements were administered as a separate infusion of either isotonic potassium-sodium-chloride (83%) or isotonic potassium-chloride (10%). Recommended volumes to be administered during the first 8h of treatment varied significantly (median 4800ml, range 3750-7700ml). Use of bicarbonate was endorsed by 80%. This study shows significant variations in management routines of DKA in Denmark. In many cases, the treatment routines employed are not supported by evidence from clinical trials. We recommend implementation of national and/or European guidelines for management of DKA in adult patients.