Genomic analysis of globally diverse Mycobacterium tuberculosis strains provides insights into the emergence and spread of multidrug resistance.

Multidrug-resistant tuberculosis (MDR-TB), caused by drug-resistant strains of Mycobacterium tuberculosis, is an increasingly serious problem worldwide. Here we examined a data set of whole-genome sequences from 5,310 M. tuberculosis isolates from five continents. Despite the great diversity of these isolates with respect to geographical point of isolation, genetic background and drug resistance, the patterns for the emergence of drug resistance were conserved globally. We have identified harbinger mutations that often precede multidrug resistance. In particular, the katG mutation encoding p.Ser315Thr, which confers resistance to isoniazid, overwhelmingly arose before mutations that conferred rifampicin resistance across all of the lineages, geographical regions and time periods. Therefore, molecular diagnostics that include markers for rifampicin resistance alone will be insufficient to identify pre-MDR strains. Incorporating knowledge of polymorphisms that occur before the emergence of multidrug resistance, particularly katG p.Ser315Thr, into molecular diagnostics should enable targeted treatment of patients with pre-MDR-TB to prevent further development of MDR-TB.

Molecular analysis of genetic mutations among cross-resistant second-line injectable drugs reveals a new resistant mutation in Mycobacterium tuberculosis.

Mutations causing mono and cross-resistance among amikacin, kanamycin and capreomycin of second-line injectable drugs (SLIDs) namely are not well understood. We investigated 124 isolates of Mycobacterium tuberculosis for mutations within rrs, eis, tlyA and efflux pump (Rv1258c and Rv0194) genes involved in resistance towards SLIDs. The distribution of mutations across these genes were significantly different in strains with mono-resistance or cross-resistance. A new mutation G878A was found in rrs gene, among strains with capreomycin mono-resistant, or in strains with cross-resistance of capreomycin, kanamycin and amikacin. This mutation was associated with the Euro-American X3 lineage (P < 0.0001). Mutations in the two efflux genes Rv1258c and Rv0194 were confined to strains with only capreomycin/amikacin/kanamycin cross-resistance. We further investigated the minimum inhibitory concentration of capreomycin on isolates with new G878A mutation ranging from 8 µg/mL to 64 µg/mL. Inclusion of G878A on new molecular assays could increase the sensitivity of capreomycin resistance detection.

Multidrug-Resistant Tuberculosis Treatment Outcomes in Relation to Treatment and Initial Versus Acquired Second-Line Drug Resistance.

BACKGROUND: Resistance to second-line drugs develops during treatment of multidrug-resistant (MDR) tuberculosis, but the impact on treatment outcome has not been determined. METHODS: Patients with MDR tuberculosis starting second-line drug treatment were enrolled in a prospective cohort study. Sputum cultures were analyzed at a central reference laboratory. We compared subjects with successful and poor treatment outcomes in terms of (1) initial and acquired resistance to fluoroquinolones and second-line injectable drugs (SLIs) and (2) treatment regimens. RESULTS: Of 1244 patients with MDR tuberculosis, 973 (78.2%) had known outcomes and 232 (18.6%) were lost to follow-up. Among those with known outcomes, treatment succeeded in 85.8% with plain MDR tuberculosis, 69.7% with initial resistance to either a fluoroquinolone or an SLI, 37.5% with acquired resistance to a fluoroquinolone or SLI, 29.3% with initial and 13.0% with acquired extensively drug-resistant tuberculosis (P < .001 for trend). In contrast, among those with known outcomes, treatment success increased stepwise from 41.6% to 92.3% as the number of drugs proven effective increased from ≤1 to ≥5 (P < .001 for trend), while acquired drug resistance decreased from 12% to 16% range, depending on the drug, down to 0%-2% (P < .001 for trend). In multivariable analysis, the adjusted odds of treatment success decreased 0.62-fold (95% confidence interval, .56-.69) for each increment in drug resistance and increased 2.1-fold (1.40-3.18) for each additional effective drug, controlling for differences between programs and patients. Specific treatment, patient, and program variables were also associated with treatment outcome. CONCLUSIONS: Increasing drug resistance was associated in a logical stepwise manner with poor treatment outcomes. Acquired resistance was worse than initial resistance to the same drugs. Increasing numbers of effective drugs, specific drugs, and specific program characteristics were associated with better outcomes and less acquired resistance.

Prevalence of and risk factors for resistance to second-line drugs in people with multidrug-resistant tuberculosis in eight countries: a prospective cohort study.

BACKGROUND: The prevalence of extensively drug-resistant (XDR) tuberculosis is increasing due to the expanded use of second-line drugs in people with multidrug-resistant (MDR) disease. We prospectively assessed resistance to second-line antituberculosis drugs in eight countries. METHODS: From Jan 1, 2005, to Dec 31, 2008, we enrolled consecutive adults with locally confirmed pulmonary MDR tuberculosis at the start of second-line treatment in Estonia, Latvia, Peru, Philippines, Russia, South Africa, South Korea, and Thailand. Drug-susceptibility testing for study purposes was done centrally at the Centers for Disease Control and Prevention for 11 first-line and second-line drugs. We compared the results with clinical and epidemiological data to identify risk factors for resistance to second-line drugs and XDR tuberculosis. FINDINGS: Among 1278 patients, 43·7% showed resistance to at least one second-line drug, 20·0% to at least one second-line injectable drug, and 12·9% to at least one fluoroquinolone. 6·7% of patients had XDR tuberculosis (range across study sites 0·8-15·2%). Previous treatment with second-line drugs was consistently the strongest risk factor for resistance to these drugs, which increased the risk of XDR tuberculosis by more than four times. Fluoroquinolone resistance and XDR tuberculosis were more frequent in women than in men. Unemployment, alcohol abuse, and smoking were associated with resistance to second-line injectable drugs across countries. Other risk factors differed between drugs and countries. INTERPRETATION: Previous treatment with second-line drugs is a strong, consistent risk factor for resistance to these drugs, including XDR tuberculosis. Representative drug-susceptibility results could guide in-country policies for laboratory capacity and diagnostic strategies. FUNDING: US Agency for International Development, Centers for Disease Control and Prevention, National Institutes of Health/National Institute of Allergy and Infectious Diseases, and Korean Ministry of Health and Welfare.

Baseline insulin/glucose ratio as a marker for the clinical course of hyperglycemic critically ill children treated with insulin.

OBJECTIVE: The objective of this study was to investigate the relations of baseline insulin/glucose ratio to the clinical course of critically ill children. Such information will provide insight into the pathophysiologic mechanisms leading to hyperglycemia and will optimize preventive and therapeutic measures for hyperglycemia in critically ill children. METHODS: Sixty-four consecutively admitted critically ill children with hyperglycemia, defined as a blood glucose level higher than 8 mmol/L (>145 mg/dL) and treated with insulin according to a glucose-control protocol, were included. Demographic data and clinical and laboratory parameters were collected. Insulin sensitivity was investigated by calculating the ratio of insulin to the blood glucose level just before the start of insulin administration. Results are expressed as median (range). RESULTS: Sixty-four children (24 girls) 7.0 y of age (0.3-16.9 y) with various diagnoses were included. A hyperinsulinemic response, indicated by an increased insulin/glucose ratio (>18 pmol/mmol), was seen in 55% of children. The durations of insulin therapy, mechanical ventilation, and pediatric intensive care unit length of stay in children with a hyperinsulinemic response were longer than in children with a hypoinsulinemic response. CONCLUSION: Hyper- and hypoinsulinemic responses play a role in the occurrence of hyperglycemia in critically ill children. Each is associated with a particular clinical course after the initiation of insulin therapy. It would be worthwhile to further investigate if the insulinemic response to hyperglycemia, determined by the insulin/glucose ratio in combination with the type of organ dysfunction, could be used in clinical practice to determine the need for insulin therapy.

Management of hyperglycemia in the pediatric intensive care unit; implementation of a glucose control protocol.

OBJECTIVE: To evaluate a stepwise nurse-driven glucose control protocol for the treatment of hyperglycemia in critically ill pediatric patients. SETTING: Academic pediatric intensive care unit. DESIGN: Prospective observational study. PATIENTS: A total of 50 consecutively admitted critically ill children with hyperglycemia >8 mmol/L (>145 mg/dL) were included and treated according to the glucose control protocol. MEASUREMENTS AND MAIN RESULTS: Demographic data and clinical parameters were collected and different steps in the protocol were evaluated. Data were expressed as medians with interquartile ranges. Fifty children (28 boys), aged 3.5 yrs (range, 1.2 -9.3 yrs) were treated in 18 mos. Forty-two children had multiple organ failure. Eight children died. Insulin treatment was initiated 4 hrs after the first episode of hyperglycemia was documented (median blood glucose, 11.4 mmol/L, [207 mg/dL] [9.7-14.5 mmol/L, 176-264 mg/dL]). Blood glucose was <8 mmol/L (<145 mg/dL) within 12 hrs of initiating insulin therapy in 47 (94%) of 50 children (median, 5 hrs). Duration of treatment was 34 hrs (17-72 hrs) and the maximum insulin dose ranged between 20 and 200 mIU/kg/hr (median, 70 mIU/kg/hr). Episodes of severe hypoglycemia <2.2 mmol/L (<47 mg/dL) did not occur. CONCLUSION: The use of a stepwise nurse-driven glucose control protocol resulted in normoglycemia within 12 hrs for 94% of the children involved. Episodes of severe hypoglycemia did not occur. We conclude that the glucose control protocol is effective in treating hyperglycemia in critically ill children. Further studies are necessary to assess safety before the protocol could also be implemented in other pediatric intensive care units.