Enzyme-Linked Immunosorbent Assay for Detecting Syphilis Dry Blood Spot Samples

Objective:To explore the feasibility of enzyme-linked immunosorbent assay(ELISA)in detecting syphilis dry blood spots.Methods:Based on dry blood spot samples,laboratory linear dial,laboratory basic dial,laboratory interference dial,and laboratory precision dial were constructed.The linear range,sensitivity,specificity,precision and other performances of ELISA for detecting syphilis dry blood spot samples were comprehensively evaluated,and the stability of dry blood spot samples at 37℃was detected.In addition,112 suspected syphilis antibody-positive plasma samples were selected as the control,and dry blood spot samples were prepared accordingly.The clinical sensitivity,clinical specificity,positive predictive value and negative predictive value of dry blood spot samples and plasma samples in ELISA for syphilis detection were compared,and the consistency and correlation between the two samples were analyzed by the Kappa consistency test and Spearman rank correlation analysis.Results:The results of the linear analysis showed that the serial dilution of dry blood spot samples in ELISA for syphilis antibody ranged from 23to 27,and there was a good linear range[R2=0.9862(P<0.05)].Sensitivity and specificity analysis showed that the positive coincidence rate and negative coincidence rate of the two detection methods were 100%(15/15).The results of the interference dial test showed that ELISA based on dry blood spot samples could accurately detect 6 syphilis antibody samples from 18samples,and the detection accuracy rate was 100.00%(6/6).The results of the precision test showed that the RSD of syphilis antibody detection in dry blood spot samples with different dilution times(23,25and 27)was 0.24%to 3.87%between spots,0.06%to4.07%between batches and 0.49%to 3.88%between days.Within 7 days,the inter-day RSD of dry blood spots with different dilution times(23,25,27)were 0.27%,0.65%and 0.95%,respectively.The clinical sensitivity,clinical specificity,positive predictive value and negative predictive value of dry blood spot samples in ELISA detection of syphilis antibody were 96.00%(72/75),100.00%(37/37),100.00%(72/72)and 92.50%,respectively.The results of the Kappa consistency test and Spearman rank correlation analysis showed that the Kappa value of the two methods was 0.941(P<0.01),and the correlation coefficientρbetween the S/CO ratios of the two methods was 0.211(P<0.01).The comparison of S/CO ratio results showed that the distribution characteristics of the S/CO ratio between the two methods were similar,and the ratio distribution was relatively concentrated.Conclusion:using dry blood spot samples to perform ELISA for syphilis detection has good precision,strong anti-interference ability and excellent stability.Although false-positive results appear in weak positive samples,it still has a high application value in ELISA for syphilis antibody detection,which can provide an important reference for disease diagnosis.

肺结核患者ELISPOT试验结果的影响因素分析

目的 本试验旨在分析肺结核患者酶联免疫斑点试验（ELISPOT）结果的影响因素.方法 选取西安市结核病胸部肿瘤医院住院患者共252例,其中肺结核患者130例,非结核患者122例.入院24小时内抽取患者清晨空腹抗凝静脉血,应用结核杆菌感染T细胞酶联免疫斑点诊断试剂盒进行斑点计数,并对结果进行影响因素进行分析.结果 酶联免疫斑点试验（ELISPOT）无论在男性患者与女性患者,≤16岁或＞60岁患者与16岁～60岁患者,胸部CT形成空洞患者与无空洞患者,病变范围＜2个亚段患者与≥2个亚段患者,试验结果显示均无显著性差异.结论 肺结核患者ELISPOT试验结果不受性别、年龄、空洞、病变范围等因素的影响.

一种新型结核分枝杆菌感染快速检测方法的应用

目的探讨酶联免疫斑点试验(ELISPOT)快速诊断结核病的价值。方法选取15例确诊为活动性肺结核的患者和100例入境人员的外周抗凝全血标本,采用ELISPOT检测试剂盒(T SPOT-TB)测定结核杆菌特异抗原刺激反应的效应T淋巴细胞数量。结果TSPOT-TB检测结核病的敏感性为93.3%,特异性为100.0%,阳性预测值为100.0%,阴性预测值为81.8%。采用该方法检测的结核潜伏感染率与病例来源呈密切相关,而与年龄和性别无相关性。结论TSPOT-TB的敏感性和特异性均较高,可用于结核感染的快速检测,但无法判断结核的活动性或潜伏感染。

滤纸干血片样品用于自愿咨询检测HIV抗体的研究

目的研究在自愿咨询检测HIV抗体工作中,用采集末梢血制成滤纸干血片替代采集静脉血的可行性。方法采集200例自愿咨询受检者的手指末梢血及静脉血(抗凝全血和非抗凝血)。将手指末梢血和静脉抗凝全血分别制成滤纸干血片样品,非抗凝血则分离出血清,并在不同条件下保存。用ELISA法和免疫印迹法检测HIV抗体,将检测结果进行比较。结果各类滤纸干血片与静脉血清经ELISA法检测,定性结果一致。与血清的测量结果相比,直径6 mm的滤纸干血片OD值有显著性差异(P<0.05)。直径10 mm的滤纸干血片OD值无显著性差异(P>0.05),滤纸干血片样品用生理盐水和pH7.4的PBS洗脱均可,300μL的生理盐水洗脱效果更好。滤纸干血片保存1周的结果稳定。用免疫印迹法检测,滤纸干血片与静脉血清判读结论一致。结论采集末梢血制成的滤纸干血片,以ELISA法检测HIV抗体,结果可靠、稳定,且操作简便,运输和保存无生物危害,可替代采集静脉血用于自愿咨询HIV抗体检测工作。

结核感染T细胞斑点试验在骨与关节结核诊断中的价值

目的探讨结核感染T细胞斑点试验(T-SPOT TB)在临床诊断骨与关节结核的应用价值。方法收集154例疑似骨与关节结核患者外周抗凝血标本,依据细菌学检验、病理学结果及临床表现判定有无结核分枝杆菌感染;其中确诊骨与关节结核73例,非结核感染81例,分别进行T-SPOT TB和结核抗体(TB-Ab)检测,并分析T-SPOT TB在骨与关节结核诊断中的敏感度和特异度。结果 73例确诊骨与关节结核中,64例T-SPOT TB阳性;81例非结核中,72例T-SPOT TB阴性;T-SPOT TB检测敏感度为87.67%(64/73),TB-Ab为41.10%(30/73),T-SPOT TB诊断骨与关节结核的敏感度显著高于TB-Ab(P<0.001);T-SPOT TB检测特异度为88.89%(72/81),TB-Ab为71.60%(58/81),T-SPOT TB特异度也高于TB-Ab(P=0.006)。在结核组中,涂阳与涂阴两组T-SPOT TB阳性检出率分别为95.45%(21/22)及86.49%(32/37),差异无统计学意义(P>0.05)。在结核组中,病理诊断阳性率92.16%(47/51)与T-SPOT TB总阳性率87.67%(64/73)比较差异无统计学意义(P>0.05)。结论 T-SPOT TB在诊断骨与关节结核中具有良好的敏感度和特异度,具有重要临床应用价值。

Levels of v5 and v6 CD44 splice variants in serum of patients with colorectal cancer are not correlated with pT stage,histopathological grade of malignancy and clinical features

AIM:This study was designed to compare the levels of v5 and v6 splice variants of CD44 evaluated using EITSA test in the serum of patients with colorectal cancer in different stages of progression of the disease estimated in pT stage according to WHO score,histopathological grade of malignancy and some clinicopathological features. METHODS:The serum obtained from 114 persons with colorectal adenocarcinomas was examined using ELISA method,pT stage and grade of malignancy of the tumour were examined in formalin fixed and paraffin embedded materials obtained during operation. RESULTS:Only the level of CD44 v5 in the serum of patients before operation with G2 pT4 tumour was lower than that in other probes and the difference was statistically significant. We did not find any other correlations between the level of v5 and v6 CD44 variants and other evaluated parameters. CONCLUSION:The level of CD44 v5 and v6 estimated by ELISA test in the serum can not be used as a prognostic factor in colorectal cancer.

Recent Developments in SARS-CoV-2 Neutralizing Antibody Detection Methods

The ongoing Coronavirus disease 19 pandemic has likely changed the world in ways not seen in the past.Neutralizing antibody(NAb)assays play an important role in the management of the severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)outbreak.Using these tools,we can assess the presence and duration of antibody-mediated protection in naturally infected individuals,screen convalescent plasma preparations for donation,test the efficacy of immunotherapy,and analyze NAb titers and persistence after vaccination to predict vaccine-induced protective effects.This review briefly summarizes the various methods used for the detection of SARS-CoV-2 NAbs and compares their advantages and disadvantages to facilitate their development and clinical application.

Detection of anti-Helicobacter pyloriantibodies in serum and duodenal fluid in peptic gastroduodenal disease

AIM:To study the diagnosis of Helicobacter pylori(H pylori) infection through the determination of serum levels of anti- H pylori IgG and IgA antibodies,and the levels of anti-H pylori IgA antibodies in duodenal fluid. METHODS:Data were collected from 93 patients submitted to upper digestive endoscopy due to dyspeptic symptoms. The patients were either negative(group A)or positive (group B)to H pylori by means of both histological detection and urease tests.Before endoscopy,peripheral blood was collected for the investigation of anti-H pylori IgG and IgA antibodies.To perform the urease test,biopsies were obtained from the gastric antrum.For the histological evaluation,biopsies were collected from the gastric antrum (greater and lesser curvatures)and the gastric body. Following this,duodenal fluid was collected from the first and second portions of the duodenum.For the serological assaying of anti-Hpylori IgG and IgA,and anti-Hpylori IgA in duodenal fluids,the ELISA method was utilized. RESULTS:The concentration of serum IgG showed sensitivity of 64.0%,specificity of 83.7%,positive predictive value of 82.0%,negative predictive value of 66.6% and accuracy of 73.1% for the diagnosis of H pylori infection.For the same purpose,serum IgA showed sensitivity of 72.0%, specificity of 65.9%,positive predictive value of 72.0%, negative predictive value of 67.4% and accuracy of 69.8%. If the serological tests were considered together,i.e.when both were positive or negative,the accuracy was 80.0%, sensitivity was 86.6%,specificity was 74.2%,positive predictive value was 74.2% and negative predictive value was 86.6%.When values obtained in the test for detecting IgA in the duodenal fluid were analyzed,no significant difference(P=0.43)was observed between the values obtained from patients with or without H pylori infection. CONCLUSION:The results of serum IgG and IgA tests for H pylori detection when used simultaneously,are more efficient in accuracy,sensitivity and negative predictive value, than those when used alone.The concentration of IgA antibodies in duodenal fluid is not useful in identifying patients with or without H pylori.

结核分枝杆菌γ-干扰素酶联免疫斑点试验在菌阴肺结核中的诊断价值

目的通过检测结核分枝杆菌γ-干扰素酶联免疫斑点试验的结果,探讨其在菌阴肺结核诊断及鉴别诊断中的意义。方法将研究对象分为菌阳肺结核、菌阴肺结核、肺部肿瘤、肺炎4组。所有患者分别进行结核分枝杆菌γ-干扰素酶联免疫斑点试验、结核抗体三项测定及结核菌素试验。结果菌阳肺结核、菌阴肺结核、肺部肿瘤、肺炎4组患者PPD试验阳性率为84.8%、83.3%、28.6%、33.3%;结核抗体试验的阳性率为84.8%、66.6%、28.6%、33.3%;ELISPOT试验阳性率为93.9%、93.3%、7.1%、3.3%。结论结核杆菌感染T细胞酶联免疫斑点试验在肺结核诊断,可能优于结核菌素试验及结核抗体测定,对于菌阴肺结核的诊断与鉴别诊断具有临床意义。

Do Occupational Factors Render Army Service a High-Leptospirosis-Risk Occupation?

Objective: Leptospirosis is a known zoonotic disease which is closely related to certain high-risk occupational group. This study aimed to identify the risk factors associated with seropositive leptospirosis among army personnel. Methodology: A cross sectional study was conducted involving 616 army personnel using a self-administered questionnaire. The questionnaire consisted of sociodemographic, occupational and environmental data as well as recreational activities history. Serum sample for leptospirosis was taken for screening with IgM Enzyme-linked Immunosorbent Assay (IgM ELISA) test. Positive tests were confirmed by Microscopic Agglutination Test (MAT). Results: All respondents were male, Malays and married with the mean age of 29.28 (7.15) years old. The mean duration of working was 11.50 (7.75). Majority of the respondents were non-officer and riflemen, smokers (455 (73.9%), stayed inside the camp and engaged in recreational activities. The mean duration of employment was 11.50 (7.75) years. The overall seroprevalence of leptospirosis was 16.2% (95% CI: 13.32, 19.15) and the predominant pathogenic serovar identified was Terengganu (38.2%). Those who had lower educational levels were more protective for positive leptospirosis compared to those who have only primary education (OR: 0.32;95% CI: 0.14, 0.76;p = 0.009). Conclusion: Despite being named as one of the high-risk occupations for leptospirosis, this study found no significant occupational factors related to leptospirosis seropositivity among army personnel.

The clinical significance evaluation of serum β_2-microglobulin for thyroid cancer patients

Objective： The aim of this study was to investigate the clinical value and relevance on the serum β2-microglobulin （β2-MG） of patients with thyroid cancer. Methods： One thousand and two normal cases, 95 thyroid cancer patients and 243 nodular goiter patients were selected to measure serum β2-MG levels using double-antibody sandwich enzyme-linked immunosorbent assay （ELISA）. Results： The positive rate of 7.78% in normal population （78/1002） and 31.57% in thyroid cancer patients （30/95）. There were significant differences between the normal population and thyroid cancer patients （X2 = 55.352; P = 0.000）. The positive rate of 7.81% in nodular goiter patients （19/243） and there were no significant differences between the normal population and nodular goiter patients （X2 = 0.0004; P = 0.986）, but significant differences between nodular goiter patients and thyroid cancer patients （x2 = 31.106; P = 0.000）. Meanwhile, the significant difference of the positive rate existed in between the various pathological types of thyroid cancer （X2 = 10.015; P = 0.007）, anaplastic thyroid cancer patients with the highest positive rate and The significant difference was found between the positive lymph node metastasis groups and negative lymph node metastasis groups （x2 = 4.441; P = 0.035）, the presence of distant metastasis group and absence of distant metastasis group （X2 = 9.795; P = 0.002）. Conclusion： Serum β2-MG levels and prognosis of thyroid cancer patients was negatively correlated. It showed important clinical value to detect the level of β2-MG in the early diagnosis, prognosis and the clinical observation for thyroid cancer patients.

Canine Herpesvirus Seroprevalence and Associated Factors in Dogs of Mexico

Canine herpesvirus (CHV-1) causes disease associated with high mortality in infect-ed puppies, which represents large financial losses for dog breeders. Since CHV-1 at the time of the study he had not been reported in Mexico, the main objective of this study was to determine the prevalence of antibodies against CHV-1 in canine kennels in the metropolitan area of Mexico City. A commercial enzyme-linked immuno-sorbent assay (ELISA) was used, and the results were compared to those of a viral neutralization test. The ELISA kit uses the complete viral particle as the antigen. The plaque reduction neutralization test was combined with the immunoperoxidase technique because of the low cytopathic effect of CHV-1. Neutralizing antibodies were also detected in 20 randomly selected samples. The prevalence of CHV-1 with ELISA was 87%. The concordance between ELISA and serum neutralization (SN) was 0.1129, the sensitivity of the ELISA against SN was 1.0 (100%), the positive predic-tive value was 0.39 (39%), and the negative predictive value was 1 (100%). These results show that ELISA is useful for monitoring the dog population for CHV-1;a positive test result requires confirmation with an SN test, and a negative ELISA result indicates a high probability of being SN-negative. The only variables that were sta-tistically associated with CHV-1 prevalence were breed and kennel. A statistically significant relationship between the degree of ELISA and SN titer was obtained, with a confidence level of 95%. None of the clinical presentation factors was statistically significant. These results suggest that most of the canine population studied in Mex-ico is in a herpesvirus latency state.

Accuracy of a new rapid diagnostic test for urinary antigen detection and assessment of drug treatment in opisthorchiasis

Background Screening for opisthorchiasis,a parasitic worm infection affecting many millions of people in Southeast Asia,has traditionally relied on faecal egg examination such as the formalin-ethyl acetate concentration technique(FECT)and Kato-Katz method.Although the urinary enzyme-linked immunosorbent assay(ELISA)has been used more recently,we developed a urinary antigen-based rapid diagnostic test(RDT)to simplify diagnosis and as a point-of-care testing(POCT)and field applications for surveillance and control of opisthorchiasis.Methods A urinaryOpisthorchis viverrini(OV)-RDT was developed using immunochromatographic methodology with a specific monoclonal antibody against OV.The diagnostic performance of the urinary OV-RDT was compared to that of quantitative faecal FECT and urinary antigen ELISA(n=493).Cross-reactivities of urinary OV-RDT with other helminthiases coexisted withO.viverrini were determined(n=96).A field trial in the application of urinary OV-RDT was compared with urinary antigen ELISA at baseline screening and assessment of drug treatment outcomes in opisthorchiasis(n=1629).The McNemar chi-square,Kruskal-Wallis and Cohen’s kappa coefficient(κ-value)tests were used for statistical analyses.Results Urinary OV-RDT had sensitivity of 94.2%and specificity of 93.2%,compared to faecal FECT.Urinary OV-RDT had high diagnostic agreement(Kappa=0.842-0.874,P<0.001)and quantitative correlation with urinary antigen ELISA(Kruskal-Wallis tests=316.2,P<0.0001)and faecal FECT(Kruskal-Wallis tests=362.3,P<0.0001).The positive rates by OV-RDT,ELISA and FECT were 48.9%,52.5%and 49.3%,respectively.Cross-reactions of urinary OV-RDT with other helminthiases were few(2%).Field trials of urinary OV-RDT yielded comparable prevalence ofO.viverrini between urinary OV-RDT(53.2%)and urinary antigen ELISA(54.0%).OV screening showed high diagnostic agreement(kappa>0.8,P<0.0001)between urinary OV-RDT and urinary antigen ELISA.The cure rates of opisthorchiasis at 1 month post-praziquantel treatment determined by urinary OV-RDT(86.6%)and urinary antigen ELISA(80.5%)were similar(P>0.05).Conclusions The urinary OV-RDT test has high potential as a new tool for screening and evaluating treatment outcomes in opisthorchiasis.The ease of sample collection and simplicity of urinary OV-RDT may facilitate mass screening,control and elimination of opisthorchiasis,thereby contributing to a reduction in the disease burden in Southeast Asia.

一种新型结核杆菌感染快速检测方法的应用

目的:探讨酶联免疫斑点试验(ELISPOT)快速诊断结核病的价值。方法:选取70例确诊为结核病的患者和60例对照组的外周抗凝全血标本,采用ELISPOT检测试剂盒(TSPOT-TB)测定结核杆菌特异抗原刺激反应的效应T淋巴细胞数量。结果:TSPOT-TB检测结核病的敏感性为95.7%,特异性为96.7%。结论:TSPOT-TB的敏感性和特异性均较高,可用于结核病感染的快速检测,是一种诊断肺结核的有价值的检测方法。

Serologic study on the outbreak of acute upper respiratory tract infections caused by adenovirus 3

From April to June,2004,an outbreak of acute upper respiratory tract infections(AURTI)occurred in the north area of Jiangsu Province,China.Twenty throat swabs were collected with 13 of them presenting an adenovirus(Ad)-like cytopathogenic effect on HEp-2.These were verified as Ad by the electron microscope,direct immunofluorescence assay and Ad primer-mediated PCR.Moreover,they were identified as adenovirus type 3(Ad3)by type-specific PCR and sequencing of the amplification products.Subsequent serologic studies were carried out to finally diagnose and document the outbreak.The neutralization test of paired serum of six in nine cases show obviously increased antibodies titers.The positive rate of IgM,IgG and recovery phase neutralization antibodies of the cases were 3.7%,44.4%and 59.5%respectively while those of the controls were 0%,8.3%and 33.3%respectively.The P values of Chi-Square were 0.510,0.018 and 0.226 respectively.The concordance between IgG detected by ELISA and neutralization anti bodies detected by the neutralization test was 61.4%and the P value of Kappa was 0.070.By the serologic study,we can definitively diagnose that this outbreak of acute respiratory infections was caused by Adenovirus 3.