Comparative sequence analysis of Mycobacterium leprae and the new leprosy-causing Mycobacterium lepromatosis.

Mycobacterium lepromatosis is a newly discovered leprosy-causing organism. Preliminary phylogenetic analysis of its 16S rRNA gene and a few other gene segments revealed significant divergence from Mycobacterium leprae, a well-known cause of leprosy, that justifies the status of M. lepromatosis as a new species. In this study we analyzed the sequences of 20 genes and pseudogenes (22,814 nucleotides). Overall, the level of matching of these sequences with M. leprae sequences was 90.9%, which substantiated the species-level difference; the levels of matching for the 16S rRNA genes and 14 protein-encoding genes were 98.0% and 93.1%, respectively, but the level of matching for five pseudogenes was only 79.1%. Five conserved protein-encoding genes were selected to construct phylogenetic trees and to calculate the numbers of synonymous substitutions (dS values) and nonsynonymous substitutions (dN values) in the two species. Robust phylogenetic trees constructed using concatenated alignment of these genes placed M. lepromatosis and M. leprae in a tight cluster with long terminal branches, implying that the divergence occurred long ago. The dS and dN values were also much higher than those for other closest pairs of mycobacteria. The dS values were 14 to 28% of the dS values for M. leprae and Mycobacterium tuberculosis, a more divergent pair of species. These results thus indicate that M. lepromatosis and M. leprae diverged approximately 10 million years ago. The M. lepromatosis pseudogenes analyzed that were also pseudogenes in M. leprae showed nearly neutral evolution, and their relative ages were similar to those of M. leprae pseudogenes, suggesting that they were pseudogenes before divergence. Taken together, the results described above indicate that M. lepromatosis and M. leprae diverged from a common ancestor after the massive gene inactivation event described previously for M. leprae.

A new Mycobacterium species causing diffuse lepromatous leprosy.

Mycobacterium leprae causes leprosy. M leprae strains collected worldwide have been genetically clonal, which poorly explains the varying severity and clinical features of the disease. We discovered a new Mycobacterium species from 2 patients who died of diffuse lepromatous leprosy (DLL). The Mycobacterium was purified from heavily infected, freshly frozen autopsy liver tissue followed by DNA extraction in 1 case. Paraffin-embedded skin tissue was used for DNA extraction in another case. Six genes of the organism were amplified by polymerase chain reaction, sequenced on cloning or from amplicons, and analyzed. Significant genetic differences with M leprae were found, including a 2.1% divergence of the 16S ribosomal RNA (rRNA) gene, a highly conserved marker of bacterial evolution, and 6% to 14% mismatches among 5 less conserved genes. Phylogenetic analyses of the genes of 16S rRNA, rpoB, and hsp65 indicated that the 2 most related organisms evolved from a common ancestor that had branched from other mycobacteria. These results and the unique clinicopathologic features of DLL led us to propose Mycobacterium lepromatosis sp nov. This species may account for some of the clinical and geographic variability of leprosy. This finding may have implications for the research and diagnosis of leprosy.

Critically ill cancer patients are not consistently hypercoagulable after craniotomy.

INTRODUCTION: Recent reports using thrombelastography have suggested that neurosurgical patients develop a hypercoagulable state in the postoperative period. Since venous thromboembolism is a potentially life threatening complication in these patients, we studied a similar population in our institution. METHODS: We conducted a prospective pilot study to evaluate postoperative coagulation changes in critically ill cancer patients after craniotomy. Data collected included demographics, diagnoses, severity of illness, all hematological information (coagulation tests included conventional and TEG), therapies, and complications. Analysis included descriptive statistics, and multivariate regression analysis. RESULTS: Eleven patients were included in the study. Mean age was 52 +/- 17 years, BMI 28 +/- 6.5, APACHE II and SOFA scores were 11.18 +/- 5.0 and 3.82 +/- 1.6 respectively. The Coagulation Index (CI), which is derived from the measured values of R, K, MA, and alpha angle was 1.22 +/- 3.5, R 4.2 +/- 1.6, K 2.0 +/- 2.1, MA 60.78 +/- 5.97, and alpha angle 66.88 +/- 14.9; while the Thrombodynamic Potential Index (TPI), which is derived from the measured values of K and MA only was 32.48 +/- 21. The CI correlated significantly with R, K, alpha angle, MA, TMA, TPI, PMA, E, A30 and A60 but not with the PTT, INR, or SOFA and APACHE II scores. One patient was hypocoagulable by CI and TPI values; in contrast, nine patients were hypercoagulable by TPI but only one by CI. There were no cases of VTE. CONCLUSIONS: Hypercoagulability as defined by the CI was not a common finding in this study. Although the TPI indicated hypercoagulability in a large number of patients, we do not believe it is a good tool to assess the patient's clotting status or predictor of thrombosis because in contrast to the CI, it does not take into account the enzymatic portions of the clotting cascade. A larger TEG study is warranted to determine the clinical significance of these changes in this and other populations.

Phase I and pharmacologic study of PKI166, an epidermal growth factor receptor tyrosine kinase inhibitor, in patients with advanced solid malignancies.

PURPOSE: This phase I study was conducted to assess the tolerability, pharmacokinetics, and antitumor activity of the oral, selective epidermal growth factor receptor tyrosine kinase inhibitor PKI166 in patients with advanced solid malignancies. EXPERIMENTAL DESIGN: PKI166 was first given once daily continuously and in the second part of the study once daily for 2 weeks every 4 weeks to establish the maximum tolerated dose (MTD). Ten additional patients were studied at MTD to acquire additional safety information and characterize the effect of food intake on PKI166 pharmacokinetics. Pharmacokinetics of PKI166 were characterized after single and multiple doses at all dose levels. RESULTS: Fifty-four patients received a total of one hundred sixteen 28-day cycles of PKI166. Dose-limiting transaminase elevations were observed in two of seven and two of eight patients using 50 and 100 mg PKI166 continuously. In the second part with PKI166 once daily for 2 weeks every 4 weeks, MTD was set at 750 mg. Dose-limiting toxicity consisted of diarrhea, skin rash, and transaminase elevations. Pharmacokinetic analysis revealed fast absorption, a linear dose-response relationship without drug accumulation after multiple doses. At MTD, no significant influence of food intake on PKI166 pharmacokinetics was observed. Stable disease for more than two cycles was observed in 11 patients. CONCLUSIONS: PKI166 given once daily for 2 weeks every 4 weeks is well tolerated with linear pharmacokinetics, compatible with once daily dosing, and without significant effect of food intake on absorption. The recommended dose for further studies is 750 mg once daily for 2 weeks every 4 weeks.

Effectiveness and safety of ICL670 in iron-loaded patients with thalassaemia: a randomised, double-blind, placebo-controlled, dose-escalation trial.

BACKGROUND: Transfusional iron overload is a potentially fatal complication of the treatment of thalassaemia. We aimed to investigate short-term efficacy, pharmacokinetic/pharma- codynamic (PK/PD) relations, and safety of ICL670, a novel, tridentate, orally active iron chelator. METHODS: We enrolled 24 patients and divided them into three cohorts consisting of a minimum of seven individuals. Patients were admitted to a metabolic unit and consumed a diet with a defined content of iron. Two patients in each cohort were randomly allocated placebo. Five or more patients received one daily dose of ICL670 at 10, 20, or 40 mg x kg(-1) x day(-1), from day 1 to 12. Net iron excretion (NIE) was measured between days 1 and 12. Primary objectives included assessment of safety and tolerability (measured by adverse events and clinical laboratory monitoring), pharmacokinetics (measured as drug and drug-iron complex), and cumulative net iron excretion (measured by faecal and urine output minus food input). Analysis was for efficacy. FINDINGS: ICL670 was absorbed promptly and was detectable in the blood for 24 h. Exposure (area under the curve of plasma concentration) to ICL670 at pharmacokinetic steady state was proportional to dose. All three doses resulted in positive NIE. The NIE achieved at 20mg x kg(-1) day(-1) would prevent net iron accumulation in most patients transfused with 12-15 mL packed red-blood-cells kg(-1) month(-1), equivalent to 0.3-0.5 mg iron kg(-1) x day(-1). A linear relation (PK/PD) was recorded between exposure to ICL670 and total iron excretion, by contrast with placebo (r2=0.54, p<0.0001). Skin rashes were noted in four patients treated at 20 and 40 mg x kg(-1) x day(-1), and one patient also developed grade 2 transaminitis. INTERPRETATION: ICL670 given once daily at 20 mg/kg seems to be an effective orally active iron chelator and is reasonably well tolerated. Long-term studies are now necessary to establish the practical contribution of this drug.