Unmodified gold nanoparticles for direct and rapid detection of Mycobacterium tuberculosis complex

ObjectivesThis work aims to develop rapid nano-gold assay prototypes for specific detection of Mycobacterium tuberculosis complex (MTBC).Design and methodsSpherical gold nanoparticles (AuNPs, 14 nm) were synthesized by citrate reduction method and characterized by spectrophotometry and SEM. MTB 16s rDNA regions were amplified by PCR and amplicons were detected using genus- and species-specific oligotargeters and AuNPs. In a second prototype, MTBC unamplified genomic DNA was directly detected using species-specific oligo-targeters and AuNPs.ResultsDetection limits were 1 ng for PCR product and 40 ng for genomic DNA. The nano-gold prototype detected 45 positive genomic DNA samples which were also positive with automated liquid culture system (BACTEC? MGIT?) and semi-nested PCR (100% concordance). Following DNA extraction, using standard procedures, the TB nano-gold prototype turnaround time is about 1 h.ConclusionsWe have developed nano-gold assay prototype for direct and inexpensive detection of MTBC. The developed prototypes are simple, sensitive, rapid and can substitute PCR-based detection. The developed assay may show potential in the clinical diagnosis of TB especially in developing countries.     

Native-valve endocarditis caused by Mycobacterium chelonae, misidentified as polymicrobial gram-positive bacillus infection

Mycobacterium chelonae, a species of rapidly growing mycobacteria, may grow in routine blood culture media and stain as gram-positive bacilli, which may cause diagnostic confusion. A patient with native-valve endocarditis caused by M. chelonae, which was misidentified as various gram-positive bacilli, is presented.     

Comparison of real-time polymerase chain reaction and conventional biochemical methods for identification of Mycobacterium chelonae–Mycobacterium abscessus group to the species level

The Mycobacterium chelonae–Mycobacterium abscessus group (MCAG) is the most common cause of infections because of rapidly growing mycobacteria. Rapid identification of MCAG to the species level is essential for choosing empiric antibiotic treatment and for public health measures. In this study, we compared the performance of a single-tube multiplex, real-time polymerase chain reaction (PCR) assay to 3 biochemical tests for species-level identification of 46 MCAG isolates. We show that real-time PCR provides the most accurate results for rapid species-level identification of MCAG.     

Evaluation of PyroMark Q24 pyrosequencing as a method for the identification of mycobacteria

We evaluated PyroMark Q24 (QIAGEN) pyrosequencing as a method for the identification of mycobacteria, with potential application in clinical practice. Sequence data from the hypervariable region A of the 16S rRNA gene (43 and 35 bp sequences) were obtained using PyroMark Q24, and a similarity search was performed automatically with PyroMark IdentiFire software. Of the 148 mycobacterial type strains tested, 138 (93.2%) were accurately identified to single or clade species level, including complex level. From the remaining 10 strains, 3 (Mycobacterium gilvum, Mycobacterium goodi, and Mycobacterium thermoresistible) showed poor sequencing quality of homopolymers. For 6 other strains (Mycobacterium cosmeticum, Mycobacterium flavescens, Mycobacterium pallens, Mycobacterium hodleri, Mycobacterium xenopi, and Mycobacterium crocinum), the sequences were unreadable from the middle, and Sanger sequencing indicated biallelic site. Finally, a 40 bp sequence for Mycobacterium gordonae could not be obtained despite repeated attempts. PyroMark Q24 provided accurate identification of multiple mycobacterial strains isolated from common clinical settings, but additional gene sequencing is required to distinguish species identified as a group or complex.     

In vitro susceptibility patterns for rapidly growing nontuberculous mycobacteria in the United States

Antimicrobial susceptibility testing for rapidly growing mycobacteria (RGM) is uncommon or only performed in large reference laboratories. Here we developed a cumulative antibiogram for 14 RGM using the largest sample size to date (N = 3860). All RGM showed 82% to 100% susceptibility to amikacin. Mycobacterium abscessus showed low percentages of susceptibility to most antimicrobials; of antimicrobials without interpretations, the minimum inhibitory concentration-90 for clofazimine was low (≤0.5mg/L). All three subspecies had ≤2.6% rrl resistance mutations, however intact erm(41) was detected in 70% to100% of M. abscessus abscessus and bolletii. Mycobacterium chelonae had a similar susceptibility pattern to M. abscessus subsp. massiliense and Mycobacterium immunogenum except that it was susceptible to tobramycin (87%). Mycobacterium fortuitum complex and similar organisms showed higher frequency of susceptibility to fluoroquinolones, beta-lactams, linezolid, and trimethoprim/sulfamethoxazole. Although relatively small published RGM antibiograms showed substantial variance, a comprehensive antibiogram can help influence treatment and monitoring patterns of resistance.     

Peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) assay for specific detection of Mycobacterium immunogenum and DNA-FISH assay for analysis of pseudomonads in metalworking fluids and sputum

Specific and rapid detection and quantification of mycobacteria in contaminated metalworking fluid (MWF) are problematic due to complexity of the matrix and heavy background co-occurring microflora. Furthermore, cross-reactivity among neighboring species of Mycobacterium makes species differentiation difficult for this genus. Here, we report for the first time a species-specific peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) method for Mycobacterium immunogenum, a non-tuberculous Mycobacterium species prevalent in MWF and implicated in occupational lung disease hypersensitivity pneumonitis and pseudo-outbreaks. A novel species-specific 14-bp PNA probe was designed for M. immunogenum based on its 16S rRNA gene sequence and was validated for specificity, by testing against a panel of other phylogenetically closely related rapidly growing mycobacteria and representative species of gram-positive, gram-negative, and acid fast organisms. In addition, a DNA-FISH protocol was optimized for co-detection of Pseudomonas, the most predominantly co-occurring genus in contaminated MWF. Reliable quantification for both the test organisms was achieved at or above a cell density of 10(3)cellsml(-1), a recognized minimum limit for microscopic quantification. The mycobacterial PNA-FISH assay was successfully adapted to human sputum demonstrating its potential for clinical diagnostic applications in addition to industrial MWF monitoring, to assess MWF-associated exposures and pseudo-outbreaks.     

Selection,characterization, and application of DNA aptamers for the capture and detection of Salmonella enterica serovars

Sensitive and specific pre-analytical sample processing methods are needed to enhance our ability to detect and quantify food borne pathogens from complex food and environmental samples. In this study, DNA aptamers were selected and evaluated for the capture and detection of Salmonella enterica serovar. Typhimurium. A total of 66 candidate sequences were enriched against S. Typhimurium outer membrane proteins (OMPs) with counter-selection against Escherichia coli OMPs and lipopolysaccharides (LPS). Specificity of the selected aptamers was evaluated by gel-shift analysis against S. Typhimurium OMP. Five Salmonella-specific aptamer candidates were selected for further characterization. A dilution-to-extinction capture protocol using pure cultures of S. Typhimurium further narrowed the field to two candidates (aptamers 33 and 45) which showed low-end detection limits of 10–40 CFU. DNase protection assays applied to these aptamers confirmed sequence-specific binding to S. Typhimurium OMP preparations, while South-Western blot analysis combined with mass spectrometry identified putative membrane proteins as targets for aptamer binding. Aptamer 33 was bound to magnetic beads and used for the capture of S. Typhimurium seeded into whole carcass chicken rinse samples, followed by detection using quantitative real-time RT-PCR. In a pull-down assay format, detection limits were 10¹–10² CFU S. Typhimurium/9 mL rinsate, while in a recirculation format, detection limits were 10²–10³ CFU/25 mL rinsate. Reproducible detection at <10¹ S. typhimurium CFU/g was also achieved in spike-and-recovery experiments using bovine feces. The pull-down analysis using aptamer 33 was validated on 3 naturally infected chicken litter samples confirming their applicability in the field. This study demonstrates the applicability of Salmonella specific aptamers for pre-analytical sample processing as applied to food and environmental sample matrices.     

Development of an enzymatic assay for sphingomyelin with rapid and automatable performances: Analysis in healthy subjects and coronary heart disease patients

BackgroundSphingomyelin (SM) is an important choline group-containing phospholipid and is considered to be an independent risk factor for coronary heart disease.MethodsWe have developed a specific enzymatic assay for SM measurement with rapid and automatable performances by using two-reagent system involving sphingomyelinase. We performed within-run and between-run precision, linearity test, detection limit, recovery test and interference to validate this assay. Then, we measured the serum SM concentration in 194 healthy subjects and 141 consecutive patients undergoing coronary angiography.ResultsThe within-run and between-run coefficients of variation for SM concentrations were 1.1–1.3% and 1.0–1.2%, respectively. Quantitative measurements to a lower limit of 30 μmol/L were shown to be possible. The recoveries of the exogenously added SM to the control samples were 98.7%–101.5%. No effect was observed after the addition of some interference materials. The mean ± SD of the serum SM concentration in the 194 healthy subjects was 553.3 ± 100.1 μmol/L. We found that the SM concentration was significantly higher among an acute coronary syndrome subjects than among the healthy subjects (P < 0.01) and that the serum SM concentrations were significantly correlated with the serum magnesium concentration.ConclusionsWe have developed a rapid and automatable enzymatic assay for SM that enables the automatic measurement of choline-containing phospholipids. This assay may be useful for various types of biochemical and clinical research.     

A real-time multiplex PCR for the identification and typing of Vibrio cholerae

We report the development and validation of a duo-triplex real-time polymerase chain reaction (PCR) for the rapid identification and typing of Vibrio cholerae. The PCR assay targets a species-specific toxR gene present in all strains of V. cholerae and used as a marker for the species wbeO1 and wbfO139, encoding the O1 and O139 somatic antigens, and ctxA, encoding cholera toxin (CT). The two tcpA variants associated with the classical and El-Tor biotypes are used to infer biotype. The assay was evaluated using 178 isolates comprising eight different Vibrio species, including 122 isolates of V. cholerae. The PCR results of 171/178 (96.1%) isolates were concordant with the serotyping, biotyping, and expected CT results. Variants of toxR (n = 3), nonfunctional wbeO1 (n = 1), and CT-negative isolates of V. cholerae O1 (n = 3) were likely explanations for the mismatched results. This duo-triplex real-time PCR is a reproducible and robust assay for the rapid identification and typing of V. cholerae belonging to the highly pathogenic, pandemic lineages.     

Visual Format for Detection of Mycobacterium tuberculosis and M. bovis in Clinical Samples Using Molecular Beacons

A real-time polymerase chain reaction (PCR) assay for the direct identification of Mycobacterium tuberculosis and M. bovis using molecular beacons was developed. The assay was modified for use in regular thermal cyclers. Molecular beacons that were specific for M. tuberculosis (Tb-B) and M. bovis (Bo-B) were designed. The fluorescence of the target PCR product-molecular beacon probe complex was detected visually using a transilluminator. The results were then compared with those of conventional multiplex PCR (CM-PCR) assays and biochemical identification. The detection limit of Tb-B and Bo-B beacons was 500 fg and 50 fg by the visual format and real-time PCR assay, respectively, compared with 5 pg by CM-PCR assay. Pulmonary and extrapulmonary samples were examined. The agreement between culture and the two assays was very good in sputum samples and fair in extrapulmonary samples. The agreement between clinical diagnoses with the two assays was moderate in extrapulmonary samples. There was very good agreement between CM-PCR and visual format assays for all samples used in the study. Concordance in the identification of isolates by the visual, CM-PCR assay, and biochemical identification was seen. Hence, the use of molecular beacon detection of M. tuberculosis and M. bovis in clinical samples is feasible by setting up two asymmetric PCRs concurrently. The assay is sensitive, specific, simple to interpret, and takes less than 3 hours to complete.For effective treatment of tuberculosis (TB), rapid and accurate diagnosis is essential. Conventional polymerase chain reaction (PCR)-based assays designed to detect Mycobacterium tuberculosis that involve electrophoresis based analysis of amplicons are relatively fast but are laborious and the potential risk of inadvertent dispersal of amplicons leading to contamination of untested samples exists. To overcome these difficulties a number of probes, including TaqMan probes, molecular beacons, and side-by-side probes, that can report the amplification of the correct amplicon in sealed tubes have been developed.^1,2,3 Several different instruments that measure increase in fluorescence of these probes while simultaneously performing amplification have also become available. Although such real-time PCR assays are more quantitative than conventional ones, the instruments used are not commonly available in resource-poor locations. Since such localities are often where tuberculosis is more prevalent, it is important to develop sealed tube assays that can be implemented on conventional PCR machines but have the simplicity and accuracy of probe-based detection.In the previously developed tests different coding and intergenic regions from the M. tuberculosis genome, such as devR, IS6110, IS986, RNA polymerase gene, and ribosomal RNA gene have been used as targets for amplification using molecular beacons or fluorescent probes.^4,5,6,7 One of the limitations of these tests is that they have exclusively focused on M. tuberculosis. However, other closely related bacteria such as M. bovis have been often associated clinically with human and bovine samples in practice.^8,9,10 Therefore an ideal test should distinguish between M. tuberculosis and M. bovis. We used the mce3 operon that has a differential organization in the M. tuberculosis and M. bovis genome as a suitable target for the assay.¹¹To develop a simple sealed tube test that can reliably detect M. tuberculosis in clinical samples using conventional PCR instrument for amplification and a common lamp for transillumination, we used molecular beacons as probes. However, the signal intensity in PCRs performed with molecular beacons is often limited because the two product strands of the amplicon bind to each other excluding the probe from the target strand. We improved the fluorescence intensity of these reactions by performing asymmetric PCR that produced more of the molecular beacon target strand than of the opposite strand. The optimizations were performed using a real-time instrument. We show that our assay is robust, specific, sensitive, and can be completed in less than 3 hours. The presence of mycobacteria in the sample can be conclusively established by visualizing the fluorescent amplicon in a blue light transilluminator by the naked eye. We also compared and correlated our endpoint visual format assay with acid-fast bacilli (AFB) smear microscopy; a gel-based conventional multiplex PCR (CM-PCR) assay, culture, and clinical diagnosis. The simplified visual format assay was found to be an accurate predictor of the presence of M. tuberculosis and M. bovis in clinical samples.     

Novel real-time PCR assay for rapid prenatal diagnosis of Down syndrome: A prospective study of 563 amniocytes

ObjectiveTo explore the possibilities of a novel real-time PCR assay for rapid prenatal diagnosis of Down syndrome in clinical settings.Design and methodsThis duplex real-time PCR assay is based on relative quantification of DSCR4 gene on chromosome 21 by using RABIF gene on chromosome 1 as a reference. For each sample, the differences in threshold cycles between DSCR4 and RABIF genes (Delta Ct, ΔCt) were detected, and a calibrated ΔCt value (ΔΔCt, ΔCt _sample – ΔCt _{internal control}) was analyzed. Overall, 563 amniotic fluid samples from patients were blindly tested for fetal chromosome analysis and their ΔΔCt values were evaluated according to the karyotyping results.ResultsChromosome analysis revealed that 12 fetuses had trisomy 21 and 551 others were normal in chromosome 21. The ΔΔCt values of trisomy 21 fetuses were significantly lower than those of normal ones (p-value < 0.001) and no overlapping was shown: lower than ? 0.49 for trisomy 21 and above ? 0.30 for a normal one.ConclusionsΔΔCt value could be used as a direct diagnostic index in real-time PCR assay; this novel assay is applicable for rapid prenatal diagnosis of Down syndrome in clinical settings.     

The effect of patient choice of intervention on health outcomes

Background:Patient preference may influence intervention effects, but has not been extensively studied. Randomized controlled design (N = 1075) assessed outcomes when women (60 years+) were given a choice of two formats of a program to enhance heart disease management.Methods:Randomization to “no choice” or “choice” study arms. Further randomization of “no choice” to: 1) Group intervention program format, 2) Self-Directed program format, 3) Control Group. “Choice” arm selected their preferred program format. Baseline, four, twelve, and eighteen month follow-up data were collected. Two analyses: health outcomes for choice compared to being randomized; and preference effect on treatment efficacy.Results:Women who chose a format compared to being assigned a format had better psychosocial functioning at four months (p = 0.02) and tended toward better physical functioning at twelve months (p = 0.07). At eighteen months women who chose versus being assigned a format had more symptoms measured as: number (p = 0.004), frequency (p = 0.006) and bother (p = 0.004). At four months women who preferred the Group format had better psychosocial functioning when assigned the Group format than when they were assigned the Self-Directed format (p = 0.03). At eighteen months women preferring a Group format had more symptoms: number (p = 0.001), frequency (p = 0.001), bother (p = 0.001) when assigned the Group format than when assigned the Self-Directed format.Conclusions:Choice and preference for the Group format each enhanced psychosocial and physical functioning up to one year. Despite the preference for Group format, over the longer term (eighteen months) cardiac symptoms were fewer when assigned the Self-Directed format.     

A 96-well plate assay for high-throughput analysis of holocarboxylase synthetase activity

BackgroundHolocarboxylase synthetase (HCS) catalyzes the covalent binding of biotin to both carboxylases and histones. Biotinylated carboxylases and biotinylated histones play crucial roles in the metabolism of fatty acids, amino acids, and glucose, and in gene regulation and genome stability, respectively. HCS null mammals are not viable whereas HCS deficiency is linked to developmental delays in humans and phenotypes such as short life span and low stress resistance in Drosophila.MethodsHCS-dependent biotinylation of the polypeptide p67 was detected and quantified in a 96-well plate format using IRDye-streptavidin and infrared spectroscopy.ResultsBiotinylation of p67 by recombinant HCS (rHCS) and HCS from human cell extracts depended on time, temperature, and substrate concentration, all consistent with enzyme catalysis rather than non-enzymatic biotinylation. The Michaelis–Menten constant of rHCS for p67 was 4.1 ± 1.5 μmol/l. The minimal concentration of rHCS that can be detected by this assay is less than 1.08 nmol/l. Jurkat cells contained 0.14 ± 0.02 U of HCS activity [μmol of biotinylated p67 formed/(nmol/l HCS h)] in 400 μg of total protein.ConclusionsWe developed a 96-well plate assay for high-throughput analysis of HCS activity in biological samples and studies of synthetic and naturally occurring HCS inhibitors.     

Identification of a novel IRGM promoter single nucleotide polymorphism associated with tuberculosis

BackgroundHuman immunity-related GTPase M (IRGM) is found to play an important role in defense against intracellular pathogen Mycobacterium tuberculosis in vitro by regulating autophagy. To verify whether single nucleotide polymorphisms (SNPs) in the promoter region of IRGM gene are associated with tuberculosis (TB) 1.7 kb IRGM promoter region was sequenced and SNP analysis was conducted in TB patients and healthy controls.MethodsA simple and rapid procedure for extracting DNA from clotted-blood was developed in this study. A 1.7 kb IRGM promoter region was amplified and sequenced for nucleotide polymorphism search. Then, 3 SNPs were selected and analyzed in 216 TB patients and 275 healthy subjects by ligase detection reaction technique.ResultsDNA extracted by our method was of high quality and suitable for PCR, sequencing, and genotyping. We identified 29 polymorphisms in the 1.7 kb IRGM promoter region, including 11 novel polymorphisms not yet reported. Large population analysis showed that frequencies of ? 1208A allele (P = 0.031), ? 1208AA genotype (P = 0.042), and ? 1208A/?1161C/?947C (P = 0.035) and ? 1208G/?1161C/?947C (P = 0.030) haplotypes in cases were significantly different from those in controls.ConclusionsIn 1.7 kb IRGM promoter region, only ? 1208A/G polymorphism is associated with susceptibility to TB.     

Antigen detection assay with parasite specific monoclonal antibodies for diagnosis of lymphatic filariasis

BackgroundLymphatic filariasis is a painful and profoundly disfiguring disease. Infection is usually acquired in childhood but its visible manifestations occur later in life, causing temporary or permanent disability. The importance of developing effective assays to diagnose, monitor and evaluate human lymphatic filariasis has been emphasized by the WHO.MethodsHigh-affinity monoclonal antibodies (mAbs) specific for recombinant filarial antigen WbSXP-1 were developed. An ELISA based capture assay using monoclonal and polyclonal antibodies for WbSXP-1 was used for detection of circulating filarial antigen.ResultsHigh-affinity monoclonal antibodies (mAbs) were developed that specifically binds both W. bancrofti and B. malayi mf antigens. Two mAbs (1F6H3 and 2E12E3) of subclass IgG2a and IgM showed high affinity, avidity and reactivity to recombinant and mf native antigen. Both the mAbs were used in combination as capture antibodies and polyclonal as detection antibody to develop the assay. The assay showed very high sensitivity towards W. bancrofti mf positive samples compared to endemic normal samples (P < 0.0001).ConclusionA capture assay using high-affinity monoclonal antibodies for WbSXP-1 was developed for the detection of filarial circulating antigen in clinical samples from bancroftian infection. Besides, this would also help in epidemiological studies in endemic areas of filarial infections.     

Cutaneous Mycobacterium chelonae infection following autologous peripheral blood stem cell transplantation for POEMS syndrome

Although hematopoietic stem cell transplantation (HSCT) may increase the curability of refractory hematologic diseases, it requires complication management due to a long-term immunocompromised state. We experienced a case who received an autologous peripheral blood stem cell transplantation (Auto-PBSCT) for POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes) and developed cutaneous Mycobacterium chelonae infection. It is clear that attention needs to be paid to prevent bacterial, fungal and viral infection after HSCT. It is also important to keep in mind that tuberculous and nontuberculous mycobacteria (NTM), in rare cases, lead to lethal complications.     

Comparison of 1,3-β-d-glucan with galactomannan in serum and bronchoalveolar fluid for the detection of Aspergillus species in immunosuppressed mechanical ventilated critically ill patients

PurposeInvasive pulmonary aspergillosis (IPA) is an important cause of morbidity/mortality in immunocompromised critically ill patients. New diagnostic strategies for early detection of IPA include the noninvasive biomarkers 1,3-β-d-glucan (BDG), serum, and bronchoalveolar (BAL) fluid galactomannan (GM). The aim of this study was to compare these markers for early detection of IPA in immunosuppressed critically ill patients.MethodsBetween December 2014 and December 2015, 49 immunosuppressed patients with respiratory failure were treated at our intensive care unit (ICU). We compared the BDG Fungitell assay with GM Platelia assay in serum and BAL for early detection of IPA. All tests were performed initially after admission at the ICU.ResultsIn our study with 49 patients, 13 (26%) had probable IPA. These patients had a higher Acute Physiology And Chronic Health Evaluation II score (28 vs 23, P < .001), Sequential Organ Failure Assessment score (16 vs 14, P < .001), more neutropenia (77% vs 30%, P < .001), worse Horowitz Index (99 vs 73 P < .020), a longer ICU stay (26 vs 17 days, P < .044), and a higher mortality rate (77% vs 58%, P < .001) as compared with patients without probable IPA.The used biomarker BDG presented in patients with probable IPA showed significantly higher levels as compared with patients without probable IPA (375 [103-1000 pg/mL; P < .001] vs 64 [30-105 pg/mL; P < .001]).Comparison of BDG with GM showed that positive serum GM could be detected in only 4 (30%), whereas positive BAL GM could be detected in 12 (92%; mean optical density index, 3.7) of 13 probable IPA cases.These results can be expressed as an overall sensitivity of 88% and a specificity of 82% for probable IPA using the BDG Fungitell assay, a sensitivity of 35% and a specificity of 70% using the serum GM Platelia assay, and a sensitivity of 70% and a specificity of 94% using the BAL GM Platelia assay. The negative predictive values of the used tests were 94% for the BDG Fungitell assay, 94% for the serum GM Platelia assay, and 90% for the BAL GM Platelia assay.Conclusion1,3-β-d-Glucan may be a useful marker for patients under surveillance at risk for IPA. In critically ill patients with immunosuppression, early diagnosis of IPA may be improved by BDG as compared with serum GM. However, diagnostic performance and accuracy increase when BDG is run in parallel with GM from BAL; moreover, the association of the 2 parameters has also the advantage of detecting early and reliable IPA.     

Rapid and quantitative detection of CpG-methylation status using TaqMan PCR combined with methyl-binding-domain polypeptide

ObjectivesTo assess the medical applicability of CpG methylation as molecular markers for cancer diagnosis, we established a new system to determine DNA methylation based on TaqMan PCR combined with a methyl-binding-domain polypeptide 2.Design and methodsWe evaluated the diagnostic applicability of this approach by examining the methylation status of two tumor suppressor genes, RASSF1A and APC, in 10 paired hepatocellular carcinoma (HCC) and the corresponding non-tumor liver tissues.ResultsMethylation levels of total 20 clinical samples measured by the TaqMan PCR assay showed a significantly positive correlation (R = 0.814, P < 0.0005 for RASSF1A, R = 0.736, P < 0.00001 for APC) with those calculated by bisulfite sequencing. The methylated DNA amount measured by our TaqMan PCR system precisely replicated the methylation status estimated by direct sequencing.ConclusionsThis suggests our method may serve as a reliable and easy-to-use tool for cancer diagnosis using methylated genes as biomarkers.     

Comparative study between anion-exchange HPLC and homogeneous assay methods in regard to the accuracy of high- and low-density lipoprotein cholesterol measurement

Objectives:A convenient method based on anion-exchange HPLC was recently developed to determine cholesterol levels of lipoproteins (HDL, LDL, IDL, VLDL, and chylomicron). The present study was performed to compare this HPLC method to homogenous assay in regard to measurement accuracy of HDL and LDL cholesterol.Design and methods:Serum samples (n = 105), including three samples from cholestasis patients, were measured by homogenous assay with Cholestest-LDL and CholestestN-HDL (Daiichi Chemicals, Tokyo) and by HPLC as reported previously (J Lipid Res 2003; 44: 1404–12).Results:The homogenous assay for HDL cholesterol correlated strongly with the HPLC method for HDL cholesterol (r = 0.976). Two samples from cholestasis patients could not be measured by homogenous assay but were measured by HPLC. The homogenous assay for LDL cholesterol correlated modestly with the HPLC method for LDL cholesterol (r = 0.823). Three outlier samples, from cholestasis patients with serum cholesterol levels > 17 mmol/L, were observed in this correlation analysis. Homogenous assay data showed that these LDL cholesterol levels were 15.2–34.7 mmol/L. However, HPLC data showed that these LDL cholesterol levels were 3.6–8.2 mmol/L, and that the major lipoprotein fractions were VLDL and IDL. The difference in LDL cholesterol levels (homogenous assay data minus HPLC data) was positively correlated with VLDL cholesterol levels.Conclusions:When measuring samples from cholestasis patients, homogenous assay may give inaccurate results. In contrast, the HPLC method is likely to be capable of accurately measuring HDL and LDL cholesterol levels without the involving VLDL.