Comparison of plasma total homocysteine determinations in 9 French hospital laboratories by using 6 different methods

A lot of methods are now available for total plasma homocysteine (tHcy) determination. Commercial kits using immunoassay, easier to use, begin to supplant in-house laboratory methods. Our aim is to evaluate the interchangeability of tHcy measurements in 9 French hospital laboratories. Six different method types were used: 2 gas chromatography-mass spectrometry (GC-MS), 2 HPLC with fluorescence detection subdivided in one in-house method and one commercial kit (Bio-Rad ), 3 fluorescence polarization immunoassays (FPIA), 1 enzyme immunoassay, 1 amino acid analyser, 1 capillary electrophoresis coupled with laser-induced fluorescence detection (EC-LIF). Each laboratory analysed 41 patient's plasma samples in which 8 samples contained added homocystine. Results were analysed for imprecision, recovery, and methodological differences. The mean among-laboratory imprecision (CV) ranged from 12.5 to 18% in function of plasma sample type and was identical to the mean among-method variation. In terms of recovery, we obtained underestimated results with immunoassays. The bias relative to the GC-MS method was less than 12.5% except for two laboratories, one using FPIA assay and the other EC-LIF. In conclusion, the interchangeability of tHcy results between laboratories is not satisfactory and does not allow us to evaluate cardiovascular risk linked to moderate increases of tHcy.     

Chloroquine cardiotoxicity: clinicopathologic features in three patients and comparison with three patients with Fabry disease

BACKGROUND: Microscopic features of chloroquine cardiotoxicity are similar to those of Fabry disease. The purpose of the study was to compare clinicopathologic findings in both disorders. METHODS: Patients with a diagnosis of chloroquine cardiotoxicity or Fabry disease were identified who had undergone endomyocardial biopsy or autopsy at Mayo Clinic Rochester (1976-2000). Clinical information was collected from medical records and letters from referring physicians. Light and electron microscopy were performed in all cases. RESULTS: Three patients (two women, one man) with chloroquine cardiotoxicity ranged in age from 53 to 73 years. Chloroquine was given for rheumatoid arthritis in two and systemic lupus erythematosus in one. Three patients (two men, one woman) with Fabry disease ranged in age from 58 to 76 years. Two had angiokeratomas, but only one had a previous diagnosis of Fabry disease. All six patients presented with dyspnea. Light microscopy from all six revealed myocyte enlargement due to perinuclear vacuolization. By transmission electron microscopy, all six showed abundant myelinoid figures within involved myocytes. Curvilinear bodies were observed in two patients with chloroquine cardiotoxicity and in none with Fabry disease. CONCLUSIONS: Patients with cardiac dysfunction due to chloroquine cardiotoxicity or Fabry disease have similar ages, presenting clinical symptoms, cardiac light microscopy and sarcoplasmic myelinoid bodies ultrastructurally. Patients with Fabry disease may not have a personal or family history of the disease. Similarly, a history of chloroquine usage may not be known to the pathologist. In these settings, the presence of curvilinear bodies ultrastructurally is useful for the diagnosis of chloroquine cardiotoxicity.     

Detecting patients with Alzheimer''s disease suitable for drug treatment: comparison of three methods of assessment.

BACKGROUND. Therapy to enhance cholinergic function in the brain is under evaluation for the treatment of Alzheimer's disease. Tetrahydroaminoacridine (tacrine) has recently received a product licence in the United States of America for the treatment of Alzheimer's disease, and the licence application in the United Kingdom will shortly be reviewed. It is therefore possible that this drug will become available for use in the UK in due course. There will then be a need for screening procedures for a large number of elderly patients to decide whether or not they have dementia and, if so, whether it is the result of Alzheimer's disease and is suitable for treatment with the new drug. METHOD. A total of 246 patients aged 75 years or over in two general practices in Bristol were assessed to investigate the potential workload such screening would engender. Three different assessment schedules for the diagnosis of dementia were compared--the mini-mental state examination, the Kew test, and the abbreviated mental test score. RESULTS. None of the assessment schedules was found to be particularly onerous, with median times for administration of five, three and two minutes, respectively. A score of 23 or less on the mini-mental state examination was taken as the main cut-off point for further evaluation. Sixty six patients obtained this score--in 25 the low score reflected factors other than dementia, and 11 others declined further assessment. Of the remaining 30 patients only four had probable Alzheimer's disease at an appropriate level of severity for treatment, and lived with a carer who could ensure compliance and monitor side effects. Two of these patients were receiving conflicting medical treatment and a third declined therapy, leaving only one person for whom treatment could be prescribed. CONCLUSION. It seems likely that of those medically suitable for treatment, it may not be possible to prescribe tacrine for an appreciable proportion. Nevertheless, all potential patients should be screened as the procedures involved are not onerous and at least some of those found suitable for treatment are likely to benefit from this new approach.     

Evaluation and comparison of fluorescence polarization assay with three of the currently used serological tests in diagnosis of human brucellosis

Fluorescence polarization assay (FPA) is a method that has been used for the diagnosis of brucellosis in animals for many years. To test its possible usefulness for the diagnosis of human brucellosis, 230 sera from patients with clinical signs of brucellosis and positive serological tests (Rose Bengal, Standard Agglutination Test, iELISA), and 305 sera from a healthy population with no clinical/epidemiological/serological evidence were examined with FPA. By using ROC analysis, the cut-off value was estimated at 99 mP, with 93.5% sensitivity (95% CI 89.5–96.3) and 96.1% specificity (95% CI 93.2–97.9). The pairwise comparison of ROC curves between FPA and iELISA and between FPA and RBT revealed no significant statistic difference (P < 0.05). On the contrary it revealed a significant statistic difference between FPA and SAT (P > 0.05). SAT also had the lowest sensitivity (81.7%) among the three tests used in case definition while iELISA had a sensitivity of 90.8% and RBT a sensitivity of 88.7%. The Kappa analysis showed that FPA has a very good agreement (0.92) with the “status of the disease” and with iELISA (0.837). According to our results, FPA seems to be a valuable method for the diagnosis of brucellosis in humans. Taking into consideration the advantages of the method such as the speed of results obtaining, the objectivity of results interpretation, as well as the cost, FPA could be considered as a replacement for other established methods. However, further studies are needed to assess the reproducibility of FPA.     

Radioisotopic assay of total L-homocysteine in plasma and urine: application to serial determinations

The authors report here a simple radioenzymatic determination of total L-homocysteine in plasma and urine. L-homocysteine-containing disulfides were reduced with dithioerythritol. L-homocysteine was then condensed by S-adenosyl L-homocysteine hydrolase to 14C-adenosine to form S-14C adenosyl L-homocysteine that was separated from 14C-adenosine by paper descendant chromatography. The concentration of the total plasma L-homocysteine of 45 normal subjects was 8.04 +/- 0.26 (mean +/- SEM) mumol/l and total L-homocysteine concentration in urine was 0.59 +/- 0.06 mumol/mmol of creatinine (25 subjects). This method is as sensitive than the other methods described in the literature but more rapid and less expensive.     

同型半胱氨酸和MTHFR基因多态性与Alzheimer病的关系

目的探讨同型半胱氨酸(Hcy)和5,10 亚甲基四氢叶酸还原酶(MTHFR)基因多态性与Alzheimer病的关系.方法运用多聚酶链反应限制性内切酶片段长度多态性技术(PCR RFLP)和荧光偏振法(FPIA)检测66例阿尔茨海默病及143例正常人MTHFR基因多态性和血浆总Hcy水平.结果 (1)AD病人甲基四氢叶酸还原酶基因中,基因型C/T占56.06%,明显高于对照组的34.97%(P<0.01,RR=0.355),C/C占39.39%,明显低于对照组的62.94%(P<0.01),T/T占4.25%,与对照组2.09%无明显差异(P>0.05). AD病人中甲基四氢叶酸还原酶基因等位基因C的频率为67.43%、相对危险率(RR)为0.594,T的频率为32.57%、与对照组的C为80.42%、T为19.58%有显著性差异(P≤0.05).(2)AD病例组与对照组血浆Hcy分别为14.72±6.2μmol/L和10.9±2.4μmol/L,两者差异有显著差异(P<0.05).AD患者血浆总Hcy水平显著高于正常组.结论 MTHFR基因突变及高同型半胱氨酸血症与Alzheimer病发生有一定关系.     

Evaluation of in situ methods used to detect Mycobacterium avium subsp. paratuberculosis in samples from patients with Crohn's disease

In common with other diagnostic tests, detection of mycobacteria in tissue by microscopic examination is susceptible to spectrum bias. Since Crohn's disease is defined by the absence of detectable pathogenic organisms, the use of in situ techniques to search for Mycobacterium avium subsp. paratuberculosis in Crohn's disease samples requires validation of methods in a paucibacillary setting. To generate paucibacillary infection, C57BL/6 mice were artificially infected with Mycobacterium avium subsp. paratuberculosis strain K10 and M. tuberculosis H37Rv, yielding tissues harboring fewer than one bacillus per oil immersion field. Serial sections of organs were then studied by cell wall-based staining techniques (Ziehl-Neelsen and auramine rhodamine) and nucleic acid-based staining techniques (in situ hybridization [ISH] and indirect in situ PCR [IS PCR]). Microscopic examination and measurement of morphometric parameters of bacilli revealed that for all methods, Mycobacterium avium subsp. paratuberculosis bacilli were observed to be shorter, smaller, and less rod shaped than M. tuberculosis bacilli. Ziehl-Neelsen, auramine rhodamine stains, ISH targeting rRNA, and IS-PCR targeting the IS900 element afforded comparable sensitivities, but for all methods, visualization of individual bacterial forms required magnification x1,000. Auramine rhodamine staining and IS-PCR generated positive signals in negative controls, indicating the nonspecificity of these assays. Together, our results indicate that detection of Mycobacterium avium subsp. paratuberculosis bacilli in tissue requires oil immersion microscopy, that rRNA-ISH provides sensitivity and specificity comparable to those of Ziehl-Neelsen staining, and that the microscopic detection limit for Mycobacterium avium subsp. paratuberculosis in tissue is governed more by bacterial burden than by staining method.     

A comparison of two molecular methods for diagnosing leptospirosis from three different sample types in patients presenting with fever in Laos

ObjectivesTo compare two molecular assays (rrs quantitative PCR (qPCR) versus a combined 16SrRNA and LipL32 qPCR) on different sample types for diagnosing leptospirosis in febrile patients presenting to Mahosot Hospital, Vientiane, Laos.MethodsSerum, buffy coat and urine samples were collected on admission, and follow-up serum ∼10 days later. Leptospira spp. culture and microscopic agglutination tests (MAT) were performed as reference standards. Bayesian latent class modelling was performed to estimate sensitivity and specificity of each diagnostic test.ResultsIn all, 787 patients were included in the analysis: 4/787 (0.5%) were Leptospira culture positive, 30/787 (3.8%) were MAT positive, 76/787 (9.7%) were rrs qPCR positive and 20/787 (2.5%) were 16SrRNA/LipL32 qPCR positive for pathogenic Leptospira spp. in at least one sample. Estimated sensitivity and specificity (with 95% CI) of 16SrRNA/LipL32 qPCR on serum (53.9% (33.3%–81.8%); 99.6% (99.2%–100%)), buffy coat (58.8% (34.4%–90.9%); 99.9% (99.6%–100%)) and urine samples (45.0% (27.0%–66.7%); 99.6% (99.3%–100%)) were comparable with those of rrs qPCR, except specificity of 16SrRNA/LipL32 qPCR on urine samples was significantly higher (99.6% (99.3%–100%) vs. 92.5% (92.3%–92.8%), p <0.001). Sensitivities of MAT (16% (95% CI 6.3%–29.4%)) and culture (25% (95% CI 13.3%–44.4%)) were low. Mean positive Cq values showed that buffy coat samples were more frequently inhibitory to qPCR than either serum or urine (p <0.001).ConclusionsSerum and urine are better samples for qPCR than buffy coat, and 16SrRNA/LipL32 qPCR performs better than rrs qPCR on urine. Quantitative PCR on admission is a reliable rapid diagnostic tool, performing better than MAT or culture, with significant implications for clinical and epidemiological investigations of this global neglected disease.     

Effect of losartan with folic acid on plasma homocysteine and vascular ultrastructural changes in spontaneously hypertensive rats

Elevated homocysteine (Hcy) is a high risk factor of hypertension due to its function in endothelial dysfunction. Its level in the blood is strongly influenced by folic acid. In order to investigate the effects of losartan with folic acid on plasma level of Hcy and vascular ultrastructural changes, thirty spontaneously hypertensive rats (SHR) involved and randomly divided into three groups (n=10): SHR-C group (control), SHR-L group (losartan 25 mg·kg^-1·d^-1), SHR-L+Y group (losartan 25 mg·kg^-1·d^-1 + folic acid 0.4 mg·kg^-1·d^-1). Another 10 Wistar Rats involved as WKY-C group for normal control. The level of plasma Hcy was measured dynamically by LS-MS, the vascular ultrastructural changes were analyzed by light and electron microscopy. Moreover, the thickness and area of aorta was measured. The results showed the Hcy levels in four groups were WKY-C 7.49 ± 1.95 μmol/L; SHR-C 8.45 ± 1.90 μmol/L; SHR-L 8.28 ± 2.11 μmol/L; SHR-L+Y 7.53 ± 2.02 μmol/L at 80 days. There was no significant change for plasma Hcy (P>0.05). The morphological change showed the subendothelial space didn’t increased significantly, the endothelial cells have a more smooth and intact cellular membrane in SHR-L+Y group. In conclusion, Losartan combined with folic acid could improve arterial endothelial structure in SHR which has no significant correlation with plasma Hcy.     

Increased concentrations of homocysteine and asymmetric dimethylarginine and decreased concentrations of nitric oxide in the plasma of patients with Alzheimer's disease

Vascular risk factors increase the risk of developing Alzheimer's disease. Increased concentrations of circulating homocysteine are associated with an increased risk of both vascular disease and Alzheimer's disease. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase. There is an increase in the concentration of ADMA in the circulation in vascular disease. We measured the concentrations of homocysteine, ADMA and nitric oxide (as nitrate and nitrite) in the plasma of 25 patients with Alzheimer's disease and 25 control subjects. There was a highly significant increase in the plasma concentration of homocysteine (P<0.001) and ADMA (P<0.0001) and a highly significant decrease in the plasma concentration of nitric oxide (P<0.0001) among the Alzheimer's patients. In the combined patient and control groups a highly significant positive correlation was found between the plasma concentrations of homocysteine and ADMA (r=0.782, P<0.0001). In addition, significant negative correlations were detected between the plasma concentration of nitric oxide and the plasma concentration of homocysteine (r=-0.592, P<0.0001) and ADMA (r=-0.789, P<0.0001). These significant correlations were found to persist, even when they were restricted to the Alzheimer's patients. The inhibition of endothelial nitric oxide synthesis by ADMA impairs cerebral blood flow, which may contribute to the development of Alzheimer's disease. Endothelial dysfunction is also associated with atherosclerosis and stroke, which are important risk factors for Alzheimer's disease. Inflammation plays an important role in Alzheimer's disease and the inhibition of endothelial nitric oxide by ADMA may increase the concentration of inflammatory mediators in the brain. The inhibition of neuronal nitric oxide synthesis by ADMA may cause cognitive dysfunction in Alzheimer's disease.     

Infectivity assay of bovine rotavirus: evaluation of plaque and end-point methods in comparison with immunofluorescent cell assay

Three different methods, namely plaque assay, immunofluorescent cell (IFC) count and end-point dilution (TCID50) were evaluated for quantitative infectivity assay of the cell culture adapted UK strain of bovine rotavirus in secondary calf kidney (CK) cells and BGM cell line. Plaque and IFC count techniques were found equally efficient for infectivity titration of bovine rotavirus. Addition of trypsin into maintenance medium enhanced the sensitivity of the TCID50 method. Both CK and BGM cells served as efficient assay cells for infectivity assay of bovine rotavirus by IFC count and TCID50 methods, whereas, for plaque assay, only CK cells were found suitable.     

血浆同型半胱氨酸水平与糖耐量减低患者周围神经病变的相关性研究

目的 探讨血浆同型半胱氨酸水平与糖耐量减低(IGT)周围神经病变的相关性.方法 根据常规神经传导检测结果,选取IGT患者80例,其中40例伴有周围神经病变(IGT-PN),40例无周围神经病变(IGT-NPN),同时选取40名健康者作为对照.采用酶速率法分别对3组受试者进行血浆同型半胱氨酸水平检测,通过多伦多临床评分系统(TCSS)对神经病变的严重程度进行评分与分级.结果 研究对象中,IGT组血浆同型半胱氨酸水平均高于健康对照组.IGT-PN组血浆同型半胱氨酸水平为(14.2±2.7) μmol/L,显著高于IGT-NPN组的(12.3±2.6) μmol/L,其差异具有统计学意义(P＜0.05).回归分析表明血浆同型半胱氨酸水平对IGT合并周围神经病变具有独立作用.血浆同型半胱氨酸水平与TCSS评分呈显著正相关.结论 血浆同型半胱氨酸可能具有促进IGT患者伴发周围神经病变的作用,其水平与IGT患者周围神经病变的严重程度相关.     

Detection of toxoplasmosis in patients with end-stage renal disease by enzyme-linked immunosorbent assay and polymerase chain reaction methods

Toxoplasmosis caused by Toxoplasma gondii is an opportunistic infection. In healthy individuals, the infection is largely asymptomatic, but in immunocompromised people the parasite can become widely disseminated, causing severe toxoplasmosis. In patients undergoing haemodialysis, the phagocytic process shows a highly significant impairment. Therefore, this study aimed to investigate toxoplasmosis in patients with end-stage renal disease (ESRD) undergoing haemodialysis in Ahvaz hospitals, southwest of Iran. A total of 280 patients and 100 healthy subjects participated in this study. The presence of serum IgM and IgG antibodies against T. gondii was detected by ELISA and the presence of Toxoplasma parasites in whole blood was evaluated by GRA6 PCR. Anti-T. gondii IgG antibodies were detected in 82 (29.3 %) haemodialysis patients and 26 (26 %) controls. In addition, anti-T. gondii IgM antibodies were detected in 7.9 % of patients and in 4 % of controls. For both the antibodies, the differences were statistically significant (P?<?0.05). PCR was performed with DNA extracted from blood samples of all patients and controls. PCR gave positive results with four of the 280 blood samples from patients but none for the control blood samples. The results revealed a high percentage of positivity for Toxoplasma antibodies in patients with ESRD undergoing haemodialysis and also confirmed the parasite in whole blood, indicating disseminated infection in these patients. Patients undergoing dialysis have a higher rate of active infection with Toxoplasma likely due to reactivation of a chronic infection. Thus, parasitological examinations of ESRD patients should be periodically carried out for monitoring and evaluating the possible dissemination of toxoplasmosis during haemodialysis.     

Automated determinations of thyroid and gastric complement-fixing antibody; comparison with the fluorescent antibody and manual complement-fixation methods

An automated technique has been established for the screening and titration of sera for complement-fixing antibody to gastric (parietal) cell and thyroid microsomal antigens. The automated method is of equal sensitivity to the manual complement-fixation technique but slightly less sensitive compared to the indirect fluorescent antibody method. The automated method has the advantage over the manual complement-fixation test of greater accuracy and reproducibility and the element of subjective interpretation is removed. It is less demanding on technician skill. The automated method requires only small amounts of sera but it consumes a considerable amount of antigen. Human antigen has been used in the present study.

The automated method for complement-fixation reactions has some advantages over the manual method for both routine clinical purposes and for the research laboratory.

     

Mucoid nitrate-negative Moraxella nonliquefaciens from three patients with chronic lung disease

Mucoid strains of Moraxella nonliquefaciens were recovered from the sputa of three indigenous Australians with chronic lung disease. These atypical strains failed to reduce nitrate, and one strain produced beta-lactamase. While the mucoid phenotype of M. nonliquefaciens has rarely been reported, the mucoid nitrate-negative biovar has never been previously reported.     

Role of plasma homocysteine and lipoprotein (a) in coronary artery disease

This study looks at the possible role of some non-traditional risk factors for premature coronary artery disease (CAD) and assesses the presence of relationship between these factors and the traditional cardiovascular risk factors. The study subjects (n=45) are divided into three groups comprising 15 premature CAD patients without traditional cardiovascular risk factors (group I); 15 premature CAD patients with one or more traditional cardiovascular risk factors (group II); and 15 healthy normal control subjects matched for age and sex (group III). Estimation of plasma homocysteine (Hcy) and plasminogen activator inhibitor-1 (PAI-1) is performed by enzyme-linked immunosorbent assay (ELISA); plasma folic acid by radioimmunoassay; plasma lipoprotein a (Lpa) by turbidimetry; and plasma lipids by colorimetry. Results showed a significant association between elevated Hcy and low folate levels and premature CAD in both patient groups. Also, a significant association was seen between elevated PAI-1 and CAD in the two patient groups, and between CAD and high levels of Hcy and triglycerides, as well as a low level of high-density lipoprotein cholesterol. Lpa showed significant association with premature CAD only in group II. Thus, Hcy, folic acid and PAI-1 might serve as independent risk factors for premature CAD in patients both with and without traditional coronary risk factors. However, Lpa might confer an additional coronary risk factor only in the presence of traditional risk factors.     

Multicenter comparison of three commercial methods for quantification of human immunodeficiency virus type 1 RNA in plasma.

Three procedures for the quantification of human immunodeficiency virus type 1 (HIV-1) RNA from plasma were compared at three laboratories. The comparison involved the Quantiplex branched DNA assay (version 1.0) by Chiron Diagnostics, the NASBA-QT assay by Organon Teknika, and the Amplicor Monitor assay by Roche Molecular Systems. The laboratories performed each of the three assays with the same sets of reconstructed HIV-1-infected human plasma samples, cross-sectionally collected clinical plasma samples and longitudinally collected plasma samples from patients starting zidovudine therapy. Analysis of the reconstruction panel results for interlaboratory variation demonstrated that no laboratory differences in results were detected for any of the assays. A comparison of the reproducibilities of duplicate samples analyzed by batch and in separate assay runs demonstrated that the reproducibilities of the test results were similar within one assay and appeared to be independent of the HIV-1 concentration. The best reproducibility was obtained with the Quantiplex assay, but all three assays demonstrated equal reliability, which was independent of batched or unbatched analysis of replicate samples. Differences in the absolute concentrations calculated were observed for the assays, in particular in the analysis of reconstructed samples. In all assays, similar changes in plasma HIV-1 RNA concentrations were determined for longitudinally collected clinical samples.