Direct identification of mycobacteria from clinical specimens by multiplex real-time PCR.

AIMS: To directly identify clinically relevant mycobacteria from clinical specimens, we have developed a multiplex real-time PCR assay with hydrolysis probes that can identify 20 mycobacterial species. METHODS AND RESULTS: The assay was initially evaluated using 248 strains, including both reference strains and clinical isolates. Then, the assay was implemented according to a scheme in our laboratory. The scheme based on the clinical differences between the Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) consisted of three stepwise PCRs. MTC and NTM were differentially detected in the step 1 PCR, and the NTM species were identified in the step 2 and step 3 PCRs. During a 2·5-year period, 1877 isolates of MTC (1142 directly recovered from clinical specimens) and 596 isolates of NTM (143 directly recovered from clinical specimens) were detected, and the species of 590 (99·0%) of the 596 NTM isolates were identified. CONCLUSIONS: Our experience shows that this is a new paradigm for rapidly and accurately identifying clinically relevant mycobacteria, in which a multiplex real-time PCR assay is directly applied to clinical specimens in a stepwise fashion. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first report of a multiplex real-time PCR assay for identifying clinically important mycobacterial species directly from clinical specimens and its application in a clinical microbiology laboratory.

MICB polymorphisms and haplotypes with MICA and HLA alleles in Koreans.

Major histocompatibility complex (MHC) class I chain-related gene B (MICB) is located within the human MHC class I region. The location of MICB in the MHC region may imply the presence of linkage disequilibrium with polymorphic MICA and human leukocyte antigen (HLA) loci. MICB is also polymorphic; however, MICB polymorphisms have not been investigated in Koreans. Using sequence-based typing (SBT), we estimated the allelic frequencies of MICB and haplotypes with MICA, HLA-B, and HLA-DRB1 at high resolution in a population of 139 unrelated Korean individuals. Eight MICB alleles were identified. The most frequent allele was MICB*005:02/*010 (57.2%), followed by *002 (11.5%), *004 (8.3%), *005:03 (8.3%), and *008 (6.8%). The most common two-locus haplotypes were MICB*005:02/*010-MICA*010 (19.4%), MICB*005:02/*010-DRB1*15:01 (6.5%), and MICB*005:02/*010-B*15:01 (10.4%); the most common three-locus haplotypes were B*15:01-MICA*010-MICB*005:02/*010 (5.8%) and MICA*010-MICB*005:02/*010-DRB1*04:06 (10.4%); and the most common four-locus haplotype was B*15:01-MICA*010-MICB*005:02/*010-DRB1*04:06 (5.8%). This is the first study to provide information about MICB allele frequencies and haplotypes with HLA in Koreans. These study results should help understand mechanisms of disease association between the MICB locus and neighboring loci in Koreans.

HLA-C*03:93, a novel HLA-C*03 allele identified by sequence-based typing.

The new allele C*03:93 showed one nucleotide difference with C*03:04:01 at codon 140 (GCT/ACT).

MICA polymorphisms and haplotypes with HLA-B and HLA-DRB1 in Koreans.

Major histocompatibility complex (MHC) class I chain-related gene A (MICA) is located within the human MHC, centromeric to HLA-B and telomeric to HLA-DRB1. The location of MICA in the MHC indicates the presence of linkage disequilibrium with human leukocyte antigen (HLA). Like HLA, MICA is highly polymorphic; however, the information available for MICA polymorphisms is not as comprehensive as that for HLA polymorphisms. We estimated the allelic frequencies of MICA and haplotypes with HLA-B and HLA-DRB1 at high-resolution in a population of 139 unrelated Korean individuals by applying the newly developed method of sequence-based typing (SBT). A total of 17 MICA alleles were identified. The most frequent allele was MICA*010 (19.4%), followed by alleles *00201 (17.6%), *00801 (14.7%), *01201 (9.4%), *004 (8.3%) and *049 (7.9%). The most common two- and three-locus haplotypes were HLA-B*1501-MICA*010 (10.4%), MICA*010-HLA-DRB1*0406 (5.8%) and HLA-B*1501-MICA*010-HLA-DRB1*0406 (5.8%). This is the first study to provide such high-resolution information on the distribution of haplotypes comprising MICA, HLA-B and HLA-DRB1 in Korean individuals, a level of resolution made possible by use of the SBT method. The results of this study should help determine the mechanisms underlying diseases associated with MICA polymorphisms in Korean individuals.

Direct international normalized ratio determination using multicalibrators is more responsive than the conventional method for measuring prothrombin time.

Direct international normalized ratio (INR) determination using certified INR plasmas was shown to improve precision and accuracy. We evaluated the utility of a multicalibrator in determining INR. INR values were measured in 493 blood samples from patients subjected to anticoagulation therapy (320) and control subjects (173). Study was performed using CA-7000 coagulation analyzer (Sysmex, Japan) with Thromborel S (Dade Behring, Germany). Direct INR values were obtained using PT-Multi Calibrator (Dade Behring) composed of five lyophilized calibrant plasmas. Conventional INR values were calculated from mean normal prothrombin time and instrument/reagent-specific international sensitivity index (ISI). We compared the difference between the INR results obtained with the two methods. The mean INR value of direct INR method was significantly higher than that of conventional method. The differences in values (direct INR - conventional INR) generated using the two methods increased in proportion to the INR values. Elevation of INR was observed in data obtained with the direct INR method, compared with conventional INR values. Accordingly, we conclude that direct INR method is more responsive than conventional method in determining INR.

Novel HLA-Cw*06 allele, Cw*0612, identified by sequence-based typing.

In this report, we describe the identification of an human leucocyte antigen-Cw*06 (HLA-Cw*06) nucleotide sequence variant, a new HLA-Cw*0612. The new allele was detected during routine HLA typing by high-resolution sequence-based typing. Allele Cw*0612 showed one nucleotide difference with Cw*0602 at codon 153 (GCG-->ACG) resulting in an amino acid change from alanine to threonine.

CT versus sonography for monitoring radiofrequency ablation in a porcine liver.

OBJECTIVE: The objective of this study was to compare CT and sonography for monitoring radiofrequency (RF) lesions in porcine livers. SUBJECTS AND METHODS: RF lesions (n = 12) were created in three pig livers by applying 13 min of current to a multielectrode RF probe with a target temperature of 95 degrees C. Helical unenhanced CT and corresponding axial sonography were performed before ablation, at 2 min, 8 min, and immediately after ablation. Contrast-enhanced CT was performed after ablation. CT scans and sonograms were evaluated by blinded observers for conspicuity of the RF lesion, edge detection (visibility of liver-lesion interface), and artifacts. Hounsfield units were recorded at every time interval, and radiologic-pathologic correlation for lesion size and configuration was performed. RESULTS: Mean size of RF lesions was 3. 03 +/- 0.9 cm. On CT, RF lesions had consistently lower attenuation values than surrounding liver (22.2 H lower than liver at 8 min, p < 0.0001). Echogenicity was variable with sonography (hypoechoic = 59%, hyperechoic = 25%, isoechoic = 16%). Unenhanced CT significantly improved conspicuity, edge detection of RF lesions, and decreased artifacts compared with sonography (p < 0.05). Contrast-enhanced CT improved RF lesion detection, border discrimination, and artifacts compared with sonography (p < 0.05). Unenhanced CT had the best correlation to pathologic size (r = 0.74), followed by contrast-enhanced CT (r = 0.72) and sonography (r = 0.56). Contrast-enhanced CT best correlated with lesion shape, but slightly overestimated size because of areas of ischemia peripheral to the RF lesion. CONCLUSION: In this animal model, unenhanced CT was an effective way to monitor RF ablation compared with sonography because of increased lesion discrimination, reproducible decreased attenuation during ablation, and improved correlation to pathologic size.

Metastatic breast cancer.

The unfortunate reality of metastatic breast cancer is that all treatment is palliative in nature. This is a disease that currently has no cure and for which therapy is directed towards accentuating survival and relieving symptoms. Current technology allows the prediction and detection of metastases earlier and with greater accuracy. These achievements need to be consolidated by the discovery of innovative therapies that can alter the inevitable outcome of this disease.

Chronic cryptogenic sensory polyneuropathy: clinical and laboratory characteristics.

BACKGROUND: Chronic sensory-predominant polyneuropathy (PN) is a common clinical problem confronting neurologists. Even with modern diagnostic approaches, many of these PNs remain unclassified. OBJECTIVE: To better define the clinical and laboratory characteristics of a large group of patients with cryptogenic sensory polyneuropathy (CSPN) evaluated in 2 university-based neuromuscular clinics. DESIGN: Medical record review of patients evaluated for PN during a 2-year period. We defined CSPN on the basis of pain, numbness, and tingling in the distal extremities without symptoms of weakness. Sensory symptoms and signs had to evolve for at least 3 months in a roughly symmetrical pattern. Identifiable causes of PN were excluded by history, physical examination findings, and results of laboratory studies. We analyzed clinical and laboratory data from patients with CSPN and compared findings in patients with and without pain. RESULTS: Of 402 patients with PN, 93 (23.1%) had CSPN and stable to slowly progressive PN syndrome. These patients presented with a mean age of 63.2 years and a mean duration of symptoms of 62.9 months. Symptoms almost always started in the feet and included distal numbness or tingling in 86% of patients and pain in 72% of patients. Despite the absence of motor symptoms at presentation, results of motor nerve conduction studies were abnormal in 60% of patients, and electromyographic evidence of denervation was observed in 70% of patients. Results of laboratory studies were consistent with axonal degeneration. Patients with and without pain were similar regarding physical findings and laboratory test abnormalities. Only a few patients (<5%) had no evidence of large-fiber dysfunction on physical examination or electrophysiologic studies. All 66 patients who had follow-up examinations (mean, 12.5 months) remained ambulatory. CONCLUSIONS: Cryptogenic sensory polyneuropathy is a common, slowly progressive neuropathy that begins in late adulthood and causes limited motor impairment. Isolated small-fiber involvement is uncommon in this group of patients. Management should focus on rational pharmacotherapy of neuropathic pain combined with reassurance of CSPN's benign clinical course.

Amyotrophic lateral sclerosis: abnormalities of the tongue on magnetic resonance imaging.

We compared the magnetic resonance images of the tongues of 16 amyotrophic lateral sclerosis (ALS) patients with those of 20 control patients and found the tongue in ALS patients is more frequently and more severely involved than suspected clinically, with major abnormalities of size, shape, position, and internal structure. The tongue size in ALS, as measured in the sagittal plane, can be reduced by as much as two-thirds of normal. The shape of the tongue in ALS tends to be rectangular or square rather than curved as is normal. As severity of the disease increases, the position of the tongue changes so that the bulk of the muscle falls away from the incisors and no longer is in contact with the hard or soft palate. The normal radial bands from the anterior floor of the mouth to the mucosal surface are often missing in ALS as are the two curvilinear bands that run parallel to the mucosal surface and intersect the radial bands. Also, there is a mottled disorganization of the internal structure of the tongue with areas of increased and decreased signal intensity.

Charge asymmetry of the purple membrane measured by uranyl quenching of dansyl fluorescence.

Purple membrane was covalently labeled with 5-(dimethylamino) naphthalene-1-sulfonyl hydrazine (dansyl hydrazine) by carbodiimide coupling to the cytoplasmic surface (carboxyl-terminal tail: 0.7 mol/mol bacteriorhodopsin) or by periodate oxidation and dimethylaminoborane reduction at the extracellular surface (glycolipids: 1 mol/mol). In 2 mM acetate buffer, pH 5.6, micromolar concentrations of UO2 +(2) were found to quench the dansyl groups on the cytoplasmic surface (maximum = 26%), while little quenching was observed at the extracellular surface (maximum = 4%). Uranyl ion quenched dansyl hydrazine in free solution at much higher concentrations. Uranyl also bound tightly to unmodified purple membrane, (apparent dissociation constant = 0.8 microM) as measured by a centrifugation assay. The maximum stoichiometry was 10 mol/mol of bacteriorhodopsin, which is close to the amount of phospholipid phosphorus in purple membrane. The results were analyzed on the assumptions that UO2 +(2) binds in a 1:1 complex with phospholipid phosphate and that the dansyl distribution and quenching mechanisms are the same at both surfaces. This indicates a 9:1 ratio of phosphate between the cytoplasmic and extracellular surfaces. Thus, the surface change density of the cytoplasmic side of the membrane is more negative than -0.010 charges/A2.