EpHLA software: a timesaving and accurate tool for improving identification of acceptable mismatches for clinical purposes

The HLAMatchmaker algorithm, which allows the identification of “safe” acceptable mismatches (AMMs) for recipients of solid organ and cell allografts, is rarely used in part due to the difficulty in using it in the current Excel format. The automation of this algorithm may universalize its use to benefit the allocation of allografts. Recently, we have developed a new software called EpHLA, which is the first computer program automating the use of the HLAMatchmaker algorithm. Herein, we present the experimental validation of the EpHLA program by showing the time efficiency and the quality of operation. The same results, obtained by a single antigen bead assay with sera from 10 sensitized patients waiting for kidney transplants, were analyzed either by conventional HLAMatchmaker or by automated EpHLA method. Users testing these two methods were asked to record: (i) time required for completion of the analysis (in minutes); (ii) number of eplets obtained for class I and class II HLA molecules; (iii) categorization of eplets as reactive or non-reactive based on the MFI cutoff value; and (iv) determination of AMMs based on eplets' reactivities. We showed that although both methods had similar accuracy, the automated EpHLA method was over 8 times faster in comparison to the conventional HLAMatchmaker method. In particular the EpHLA software was faster and more reliable but equally accurate as the conventional method to define AMMs for allografts. CONCLUSION: The EpHLA software is an accurate and quick method for the identification of AMMs and thus it may be a very useful tool in the decision-making process of organ allocation for highly sensitized patients as well as in many other applications.     

Pharmacokinetic analysis of two new sustained-release products of diltiazem designed for twice-and once-daily treatment

The pharmacokinetics of two new sustained-release (SR) products of diltiazem, Dilapress 120 mg tablets and Dilapress 240 mg tablets, was analysed and characterized in three different studies, in comparison to the following diltiazem SR formulations: Cardizem Retard, Cardizem SR, and Cardizem CD. Dilapress 120, designated for twice-daily dosing, was found to be bioequivalent to Cardizem SR and to Cardizem Retard with mean (±SD) relative bioavailability values of 99 ± 27% and 113 ± 38%, respectively. Dilapress 240, designed for once-a-day treatment, was found to have a slower absorption rate than Cardizem SR and its extent of absorption was 56 ± 19% relative to that of Cardizem SR. However, the bioavailability of Dilapress 240 relative to that of Cardizem CD was 118±46%, indicating that the bioavailability of Cardizem CD relative to that of Cardizem SR was only 54±29%. Diltiazem is partially available due to a saturable liver first-pass effect. A high dose of Cardizem SR may partially escape this first-pass effect and, thus, achieve a higher extent of absorption than a slower SR product. Consequently, SR products of diltiazem designed for once-daily treatment may not reach the saturation stage in the liver first-pass effect process that diltiazem is susceptible to. Consequently, a twice-daily SR product of diltiazem cannot serve as a reference for extent of absorption assessments of a once-daily SR product.     

Evaluation of Low-Colony-Number Counts of Mycobacterium tuberculosis on Solid Media as a Microbiological Marker of Cross-Contamination

Low-colony-number counts on solid media are considered characteristic of cross-contamination, although they are normally observed in true-positive cultures from some groups of patients. The aim of this study was to evaluate low-yield growth cultures as a microbiological marker for cross-contamination. We evaluated 106 cultures with <15 colonies from 94 patients, and the proportions of false-positive cultures were 0.9% per sample and 1.1% per patient, which indicates that low-yield growth is not a reliable marker of cross-contamination.Isolation and identification of Mycobacterium tuberculosis from clinical specimens remain the definitive methods for diagnosing tuberculosis (TB). Occasionally, false-positive cultures occur due to contaminated clinical equipment, clerical errors, or cross-contamination of specimens (1, 3, 11, 14, 16). Indications of cross-contamination include culture results inconsistent with the patient''s clinical course, unexpected drug resistance, single culture-positive specimens, and solid-medium cultures with low-colony-number counts (4). A single positive culture is the most commonly reported indicator of false positivity. When molecular-strain-typing methods were used to investigate the source of single positive cultures, outbreaks of laboratory cross-contamination that had been unrecognized by clinicians or laboratory personnel were identified (7, 11). Drug treatment trials usually consider cultures yielding ≤10 colonies negative to avoid false-positive culture results. However, in cultures of specimens from some patients having active TB but minimal radiographic disease, having human immunodeficiency virus coinfection, or already receiving anti-TB treatment, growth of few colonies is normally observed (5, 8, 12). No studies have systematically evaluated the predictive value of low-colony-number counts of M. tuberculosis on solid media as a microbiological indicator (4).This study was conducted in the mycobacteriology laboratory of a large urban teaching hospital in Brazil, where about 30 specimens are processed daily and the M. tuberculosis positivity rate is 8%. From January 2003 to January 2005, 12,984 respiratory samples from 8,656 patients suspected of having TB were received. M. tuberculosis was isolated from 2,399 (18.5%) specimens obtained from 2,141 patients. Isolates from smear-negative, low-colony-number (<15 colonies) cultures with corresponding clinical information were included in the study; 106 isolates from 94 patients met these criteria. Quantification of growth and identification of isolates were done according to standard procedures (6, 9). To investigate cross-contamination episodes, the DNA fingerprint patterns of the 106 isolates were compared to those of positive specimens processed on the same day, resulting in the analysis of 279 isolates processed on 92 days.The rapid PCR-based epidemiological typing (RAPET) method was used to screen 279 isolates. The PCR mixture contained 20 mM Tris-HCl (pH 8,4), 50 mM KCl, 2.5 mM MgCl₂, 0.2 mM of each deoxynucleoside triphosphate, 40 pmol of the primer, and 1 U of Taq polymerase; the PCR primers and cycling conditions were as described by Yates et al. (17). PCR mixtures showing similar patterns were digested with HaeIII. Isolates that had similar RAPET patterns were subjected to restriction fragment length polymorphism (RFLP) analysis with IS6110, using a standardized method (15). Isolates were considered identical when they had matching RAPET fingerprint patterns which were confirmed by RFLP analysis. When an isolate from a low-growth-rate culture had the same fingerprint pattern as an isolate from a culture processed on the same day, the low-growth-rate culture was considered to be cross-contaminated, especially when the patient had no clinical indication of active TB and no epidemiological link with the presumed source of M. tuberculosis. When there were no isolates with matching fingerprint patterns and the patient had a clinical course consistent with active TB, the culture was considered to be a true positive.To investigate cross-contamination episodes, the RAPET PCR patterns from each of the isolates included in the study were compared to the RAPET PCR patterns of all culture-positive specimens processed concurrently. After RAPET, 16 isolates showed a molecular pattern similar to that of a true-positive specimen processed in the same batch. These isolates were subjected to the RAPET restriction enzyme analysis and RFLP analysis. After RAPET restriction analysis, only two of the isolates had patterns identical to that of a true-positive specimen (Fig. ​(Fig.1).1). Subsequently, RFLP analysis showed that the fingerprint patterns of each of the pairs were indistinguishable (Fig. ​(Fig.2).2). Thus, molecular results suggested the occurrence of two cross-contamination episodes.Open in a separate windowFIG. 1.Comparison of RAPET patterns of M. tuberculosis isolates from suspected false-positive cultures and isolates from cultures processed on the same day. Patterns generated by HaeIII digestion of PCR products are shown.Open in a separate windowFIG. 2.IS6110 RFLP patterns from M. tuberculosis isolates that showed similar patterns of PCR products after the first step of RAPET analysis. The rectangles represent the two possible cross-contaminated cultures.Next, demographic and clinical data for the paired patients were reviewed. The patients were not epidemiologically linked. Medical records showed that patients 1 and 2 had clinical courses consistent with active TB (Table ​(Table1).1). The second pair was assessed to involve a cross-contamination episode between patients 3 and 4, as patient 3 had been treated for TB for 2 years at the time the specimen was collected, whereas patient 4 had a clinical course inconsistent with active TB, had no other positive smears or cultures, and was not reported as a TB case by the responsible clinician.

TABLE 1.

Clinical and laboratory characteristics of patients whose M. tuberculosis cultures were suspected to be false positive and patients with isolates matching those of another patient whose specimen was processed on the same day

MR angiography for abdominal and thoracic aortic aneurysms: assessment before endovascular repair in patients with impaired renal function

OBJECTIVE: The aim of the study was to establish the feasibility of using MR angiography as the sole imaging technique before endovascular repair of abdominal or thoracic aortic aneurysms and to compare preprocedural measurements by MR angiography and digital subtraction angiography in patients with impaired renal function. CONCLUSION: MR angiography appears to be effective and reliable for use as the sole imaging method before endovascular repair of aortic aneurysms in patients with renal impairment.     

左氧氟沙星、加替沙星及莫西沙星对肺结核患者的早期及延长早期杀菌活性

目的:评估左氧氟沙星、加替沙星及莫西沙星等新氟喹诺酮类药物对肺结核患者的早期杀菌活性及延长早期杀菌活性。设计:随机开放临床试验。40例成年新诊断的涂阳肺结核患者(每组10例)分别每日单一接受异烟肼300mg,或左氧氟沙星1000mg,或加替沙星400mg,或莫西沙星400mg的7天治疗。治疗前2天及单药治疗7天期间,每日收集痰液进行定量培养。计量单药治疗开始的头2天(0-2天)及治疗后5天(2-7天)的菌量下降情况,以评估各药的早期及延长早期杀菌活性。对实验室工作人员关于病人治疗分组信息采用单盲法。结果:异烟肼的早期杀菌活性为0.67log10cfu/毫升/天,高于莫西沙星(0.33log10cfu/毫升/天)及加替沙星(0.35log10cfu/毫升/天)。与每日1000mg左氧氟沙星的早期杀菌活性(0.45log10cfu/毫升/天)相比,无统计学差异(P=0.14)。3种氟喹诺酮类药物的早期杀菌活性(0-2天)与延长早期杀菌活性(2-7天)相似。总体说来,氟喹诺酮类药物的延长早期杀菌活性高于异烟肼。结论:莫西沙星、加替沙星及高剂量左氧氟沙星,虽然略低于异烟肼,但均有良好的早期杀菌活性,并具有较高的延长早期杀菌活性。有理由对这些药物敏感的结核病人进行进一步的治疗研究。