Comparison of Brucella immunoglobulin M and G flow assays with serum agglutination and 2-mercaptoethanol tests in the diagnosis of brucellosis.

The diagnostic value of Brucella IgM/IgG flow assays was evaluated in comparison with serum agglutination and 2-mercaptoethanol tests by testing a selection of serum samples submitted to the laboratory because of clinical suspicion of brucellosis. All 39 admission and 11 follow-up samples that agglutinated in the serum agglutination test tested positive in the flow assay, whereas all 20 serum agglutination negative samples with clinical suspicion of brucellosis, 23 control samples from healthy individuals and 20 control samples from cases with chronic hepatitis tested negative in the flow assay. The Brucella IgM and IgG flow assays were slightly more sensitive than the agglutination tests in discriminating between specific IgM and IgG antibodies. The Brucella IgM and IgG flow assays are easy-to-perform and quick assays that can be used for the diagnosis of brucellosis. The flow assays are very useful, especially in rural settings where brucellosis is widespread and where well-equipped laboratories to perform the laboratory tests are not readily available.     

The value of ELISA vs. negative Coombs findings in the serodiagnosis of human brucellosis

This study was conducted to assess the value of ELISA findings in relation to negative findings in the Brucella-antihuman globulin (Coombs) test and in relation to the clinical condition of patients. One hundred and thirty three serum specimens, representing the same number of patients, submitted for serologic testing for brucellosis and showing negative Coombs, were tested by ELISA to determine their Brucella-IgG, -IgM and -IgA antibodies. Concordant negative results between Coombs and ELISA were found in 95 (71.4%) patients whose medical records also did not reveal suggestive clinical signs or symptoms of brucellosis. The elevated ELISA readings in the remaining 38 (28.6%) patients were distributed as follows: IgG + IgM + IgA in 1 patient, IgG + IgM in 8 patients, IgG alone in 24 patients and IgM alone in 5 patients. The clinical review of these patients indicated no current disease in 21 (15.8%), scanty evidence of brucellosis in 8 (6.0%) and suggestive or sufficient evidence in 9 (6.8%). Thus, ELISA is the test of choice to resort to in the case of clinical suspicion of brucellosis, even when the Coombs test shows negative findings.     

The evaluation of a user-friendly lateral flow assay for the serodiagnosis of human brucellosis in Kazakhstan

Serum samples from all patients with culture-confirmed brucellosis including those with chronic disease from Kazakhstan tested positive in the serum agglutination test for titers ≥1:25 and reacted in the Brucella immunoglobulin M/immunoglobulin G lateral flow assay (LFA) confirming the high sensitivity of these assays. The strong reactivity in the LFA observed for the majority (92.1%) of the samples from the patients with culture-confirmed brucellosis together with the user-friendliness of the assay procedure makes the LFA ideal for the confirmation of brucellosis in endemic areas in Kazakhstan. The Rose Bengal test lacked sensitivity in particular for patients with chronic brucellosis therefore limiting its value as a quick screening assay. The study emphasizes the importance of the LFA as a useful, rapid, and easy-to-perform tool in the diagnostic testing of brucellosis.     

Recombinant OMP28 antigen-based indirect ELISA for serodiagnosis of bovine brucellosis

Brucellosis is a zoonosis of both public health and economic importance in many developing countries including India. Early detection and segregation of the infected animals are important in order to control the disease. Serodiagnostic tests for brucellosis is mainly based on detection of antibodies developed against lipopolysaccharide (LPS) component of cell. In this study we evaluated a protein antigen, 28 kDa outer membrane protein (OMP28), of Brucella melitensis as an alternative to LPS. Recombinant OMP28 was produced in Escherichia coli system. The efficacy of purified OMP28 was studied in an indirect enzyme-linked immunosorbent assay (ELISA) for diagnosis of brucellosis in field sera collected from different regions of country. Using known negative and known positive serum samples it was found that OMP28 is immunoreactive to Brucella infected cattle, sheep, goat and dog sera. Three hundred and eighty two cattle sera were screened by OMP28 antigen-based ELISA and the results were compared to rose Bengal plate agglutination Test (RBPT). Recombinant OMP28 antigen-based ELISA has shown sensitivity of 88.7%, specificity of 93.8% and accuracy of 92.9%. It was concluded that recombinant B. melitensis OMP28 could be used as a protein antigen for diagnosis of brucellosis in domestic animals.     

Recombinant VirB5 protein as a potential serological marker for the diagnosis of bovine brucellosis

The molecular tag vaccine against Brucella abortus and serological testing are the main methods of prevention of brucellosis used currently. They can discriminate vaccinated animals and humans from those naturally infected. In this study, we constructed a gene deletion mutant strain, B. abortus S19 virB5 with a molecular tag. Recombinant VirB5 was expressed and purified for evaluation as a diagnostic reagent for bovine brucellosis. In total, 400 sera samples were tested using a VirB5 antigen-based enzyme-linked immunosorbent assay (ELISA) and the results were compared with those of the standard tube agglutination test (SAT). This showed that the sensitivity was 88.2%, specificity was 97.8% and accuracy was 94.8%. Recombinant VirB5 could also be used to discriminate B. abortus-infected mice from mice infected with the B. abortus S19 virB5 mutant strain. It was concluded that recombinant VirB5 could be used as a potential antigen and serological marker for the diagnosis of bovine brucellosis.     

A repA-based ELISA for discriminating cattle vaccinated with Brucella suis 2 from those naturally infected with Brucella abortus and Brucella melitensis

The commonest ways of diagnosing brucellosis in animals include the Rose-Bengal plate agglutination test, the buffered plate agglutination test (BPA), the slide agglutination test, the complement fixation test, and the indirect enzyme linked immunosorbent assay (I-ELISA). However, these methods cannot discriminate the Brucella vaccine strain (Brucella suis strain 2; B. suis S2) from naturally acquired virulent strains. Of the six common Brucella species, Brucella melitensis, Brucella abortus, and B. suis are the commonest species occurring in China. To develop an ELISA assay that can differentiate between cows inoculated with B. suis S2 and naturally infected with B. abortus and B. melitensis, genomic sequences from six Brucella spp. (B. melitensis, B. abortus, B. suis, Brucella canis, Brucella neotomae and Brucella ovis) were compared using Basic Local Alignment Search Tool software. One particular gene, the repA-related gene, was found to be a marker that can differentiate B. suis from B. abortus and B. melitensis. The repA-related gene of B. suis was PCR amplified and subcloned into the pET-32a vector. Expressed repA-related protein was purified and used as an antigen. The repA-based ELISA was optimized and used as specific tests. In the present study, serum from animals inoculated with the B. suis S2 vaccine strain had positive repA-based ELISA results. In contrast, the test-positive reference sera against B. abortus and B. melitensis had negative repA-based ELISA results. The concordance rate between B. abortus antibody-negative (based on the repA-based ELISA) and the Brucella gene-positive (based on the ‘Bruce ladder’ multiplex PCR) was 100%. Therefore, the findings suggest that the repA-based ELISA is a useful tool for differentiating cows vaccinated with the B. suis S2 and naturally infected with B. abortus and B. melitensis.     

Indirect fluorescent antibody test versus enzyme-linked immunosorbent assay and agglutination tests in the serodiagnosis of patients with brucellosis

Anti-Brucella IgG, IgM and IgA in sera from patients with blood culture positive for B. melitensis and controls were measured by indirect fluorescent antibody (IFA) test and the findings compared with those of enzyme-linked immunosorbent assay (ELISA) and microagglutination test (MAT). Brucella melitensis and B. abortus antigens from three vendors (BioMerieux, Wellcome and Oxoid) and from reference strains (Ames, Iowa) were used in IFA and MAT while a whole cell heat-killed B. melitensis antigen was used in ELISA. Statistical analysis showed comparable results when using B. melitensis or B. abortus antigen, in IFA, from the same manufacturer but there were subtle differences among antigens from different manufacturers. Correlation between IFA and ELISA titers was poor, due to differences in the levels of these titers. However, the percentage of sensitivity, specificity, predictive positive, and predictive negative at different titers indicated the most reliable discriminative titers to be as follows: ELISA IgG 1 : 800 (100% for all), IgM 1 : 400 (100%, 93%, 100%, 100%, respectively) and IgA 1 : 200 (95%, 100%, 100%, 94%, respectively); IFA IgG 1 : 320 (95%, 93%, 95%, 93%, respectively) and IgM 1 : 80 (95%, 100%, 100%, 94%, respectively). IFA IgA showed either poor sensitivity or specificity at all titers. These findings and the subjective reading of IFA limit its value in Brucella diagnosis while the MAT showed high false negatives (5%–40%). Thus, ELISA proves to be the most reliable test for the diagnosis of patients with brucellosis.     

2007年牡丹江市布鲁氏菌血清检测结果分析

目的　了解布鲁氏菌感染状况，进一步控制布鲁氏菌病疫情。方法　采用虎红平板凝集试验（RBPT）和试管凝集试验（SAT）依据GB 15988-1995《布鲁氏菌病诊断标准及处理原则》。虎红试验为阳性，同时SAT试验1∶100（?荻）及以上者，判定布鲁氏菌血清检测阳性。结果　2007年牡丹江市疾病预防控制中心布病检测门诊共检测126人，感染率为38.89%； 以职业性接触感染率最高，感染率为48.83%，其次是食源性接触感染，感染率为21.43%；不同病因的布鲁氏菌感染率差异有统计学意义（?字2=12.06，P0.01）；血检男性93人，阳性率44.09%；女性33人，阳性率24.24%，男女间阳性率差异有统计学意义（?字2=32.27，P0.005），男性的布鲁氏菌感染率高于女性。结论　2007年牡丹江市布鲁氏菌病门诊血检阳性率高达38.89%，明显高于近年来的调查结果。应及时建立严格的牲畜检疫制度，倡导需产品接触人群来取有效的个人布病防护措施。     

Pulmonary Brucellosis

SUMMARY Pulmonary abnormalities in brucellosis are rare. We report onnine cases (five adults and four children) with pulmonary brucellosis.All presented with fever, cough and mucopurulent sputum, andmost had abnormal signs in the chest. Radiography of the chestshowed pneumonic patches or consolidation in five patients,pleural effusion in three, granuloma of the lung in one anda picture of interstitial pneumonitis in one. All the patientshad a brucella agglutination titre of 1:320 or more, and anelevated titre in the brucella-specific enzyme linked immunosorbentassay of IgM, IgG and IgA. Blood cultures grew Brucella melitensisin six patients while the pleural fluid aspirate grew the sameorganism in two of three patients. Treatment with oral oxytetracycline,doxycycline, rifampicin, trimethoprim-sulfamethoxazole alone,in combination with each other or together with intramuscularstreptomycin was successful in all patients. All our patientsrecovered and none relapsed.     

布鲁氏菌病患者血液中布鲁氏菌与临床症状和抗体水平关联性研究

目的 应用巢式聚合酶链式反应（PCR）方法检测布鲁氏菌病（布病）患者血液中布鲁氏菌DNA,分析其与临床症状和抗体水平之间的关系。方法 对门诊就诊患者进行个案调查,包括临床症状、既往史、接触史、用药史等。布病可疑患者,采集血液,分离血清,进行血清试管凝集试验（SAT）,检测布鲁氏菌抗体,按照《布鲁氏菌病诊断》（WS 269—2019）进行布病临床诊断。同时对患者抗凝全血进行细菌DNA提取,应用巢式PCR方法检测血液中布鲁氏菌DNA。对不同症状、抗体水平血液中布鲁氏菌细菌数之间的关系进行统计学分析。结果 118例布病患者中,男性78例,女性40例。患者的临床症状主要有发热、乏力、头痛、胸背酸痛、腰痛、四肢酸痛、关节肌肉疼痛、出汗、失眠和睾丸痛等。118例布病患者血液标本中,布鲁氏菌DNA阳性率为63.56%(75/118),布鲁氏菌细菌数中位数(MED)为7拷贝/mL血液,四分位数(IQR)为2～17拷贝/mL血液。118例布病患者中,SAT检测阳性率为38.14%(45/118)。118例布病患者中,新发病例和复诊病例分别占55.08%(65/118)和44.92%(53/118),新发...     

Performance characteristics of the Euroimmun enzyme-linked immunosorbent assay kits for Brucella IgG and IgM

Brucella IgG and IgM ELISA kits manufactured by Euroimmun (Lubeck, Germany) were evaluated in a reference laboratory setting. Intraassay coefficient of variation (CV) values were ≤10% for positive sera and ≤12% for negative sera; interassay CVs were ≤12% for positive sera and ≤20% for negative sera. The tube agglutination test (TAT) was performed on 51 sera exhibiting various ELISA reactivity profiles. All 18 sera negative for both IgG and IgM by ELISA were TAT negative (titer <1:80), whereas 31 (94%) of 33 sera positive for IgG and/or IgM were TAT positive; the 2 discordant sera were IgG positive IgM negative by ELISA. None of 41 sera from healthy laboratory employees were ELISA IgG positive, whereas 1 (2%) of 41 was ELISA IgM positive. Similarly, 0 of 149 potentially cross-reactive sera (containing rheumatoid factor or antibodies to selected Gram-negative bacteria) was ELISA IgG positive, whereas 4 (3%) of 149 were ELISA IgM positive. These findings demonstrate the acceptable performance of the Euroimmun ELISAs for Brucella antibodies.     

A modified indirect fluorescent antibody test for the diagnosis of brucellosis

We adapted the conventional indirect fluorescent antibody (IFA) test to assay IgM and IgG Brucella-specific antibodies to differentiate acute from chronic infections rather than measure total antihuman globulin specific antibodies. The results were compared with the slide agglutination test (SAT) used for screening and the quantitative microagglutination test (MAT). Of a total of 118 randomly selected samples sent for anti-Brucella antibodies received at a general hospital laboratory, 58 (47.9%) were found to be positive for IFA-IgG test but not necessarily by other tests. Eleven of these cases were positive for Brucella melitensis by culture. Sixty serum samples found negative for Brucella antibodies by IFA and other tests were of patients with medical conditions other than brucellosis. Fifty serum samples from healthy blood donors were negative for Brucella spp. antibodies by all the three tests. The IFA test was found to be a more sensitive test than MAT and distinguished an acute infection from chronic disease.     

Pulmonary brucellosis

Pulmonary abnormalities in brucellosis are rare. We report on nine cases (five adults and four children) with pulmonary brucellosis. All presented with fever, cough and mucopurulent sputum, and most had abnormal signs in the chest. Radiography of the chest showed pneumonic patches or consolidation in five patients, pleural effusion in three, granuloma of the lung in one and a picture of interstitial pneumonitis in one. All the patients had a brucella agglutination titre of 1:320 or more, and an elevated titre in the brucella-specific enzyme linked immunosorbent assay of IgM, IgG and IgA. Blood cultures grew Brucella melitensis in six patients while the pleural fluid aspirate grew the same organism in two of three patients. Treatment with oral oxytetracycline, doxycycline, rifampicin, trimethoprim-sulfamethoxazole alone, in combination with each other or together with intramuscular streptomycin was successful in all patients. All our patients recovered and none relapsed.     

布鲁菌在Bact／Alert血培养仪上生长曲线特点

目的观察布鲁菌在Bact／Alert血培养仪上生长曲线特点,帮助预测该菌株生长,协助临床快速诊断布鲁菌病。方法对血培养阳性,经血清学检测布鲁菌凝集试验阳性确诊的15例布鲁菌病患者的流行病学、临床资料、实验室资料进行回顾分析。结果15例患者均有不规则发热、动物接触史。15例患者的血培养细菌生长曲线具有相同的特点：阳性报警时间72h左右,迟缓期较长,生长期较短,生长期对应的纵轴较短,稳定期平缓。结论布鲁菌病临床表现多样,临床医师对该病的认识不足容易误诊,因此对发热患者应重视血液病原学检验。实验室工作人员通过观察血培养细菌生长曲线有助于加快对该菌的预测。工作人员应增强自身防护意识,防止实验室感染。     

Evaluation of the Virclia® automated chemiluminescent immunoassay system for diagnosing pneumonia caused by Mycoplasma pneumoniae

Background

Mycoplasma pneumoniae is considered an important etiologic agent of community‐acquired pneumonia (CAP) in outpatients. We aimed to evaluate the diagnostic accuracy of a quick automated chemiluminescent immunoassay (CLIA) for M. pneumoniae in a population‐based prospective study of CAP.

Methods

A total of 137 outpatients diagnosed with CAP were included in the study. Acute‐ and convalescent phase sera were analyzed for IgG and IgM to M. pneumoniae with both CLIA (VirClia^®) and ELISA immunoassays. Conventional serological criteria by quantitative ELISA were considered as reference standard. Sensitivity and specificity of the assay were assessed with the construction of receiver operating characteristic (ROC) curves, and the kappa index was used to evaluate the accuracy of the IgG and IgM determinations in the acute phase.

Results

Thirty‐eight patients were diagnosed with pneumonia by M. pneumoniae. ROC curves for IgG and IgM of convalescent and acute phase (C/A) quotients by the CLIA and ELISA assays were comparable. Specifically, for the CLIA, the best C/A quotient for IgG was 2.617 (sensitivity, 94.9%; specificity, 99.9%), and for IgM 1.400 (sensitivity, 65.8%; specificity, 100%). Regarding the acute phase, the best diagnostic accuracy for the CLIA was obtained with an IgG index of 1.120 (sensitivity, 89.5%; specificity, 73.7%). The CLIA was very simple to execute and required a minimum sample handling.

Conclusion

The accuracy of the Virclia^® assay for the diagnosis of M. pneumoniae infection in outpatients with CAP was equivalent to the quantitative ELISA. The CLIA was quicker to perform and displayed better analytic workability than conventional ELISA.

     

Profiles of brucella-specific immunoglobulin G subclasses in sera of patients with acute and chronic brucellosis

Serum specimens from patients with acute brucellosis (164), chronic brucellosis (22) and controls (75) were tested by ELISA for brucella-specific IgG, IgM, IgA and subclasses of IgG1 to 4 antibodies, Rose Bengal antigen slide agglutination (RB) and microagglutination (MA) tests. The RB and MA showed similar results and were positive in 100% and 64% of specimens from patients with acute and chronic brucellosis respectively. ELISA IgG and IgA were positive in sera from all patients with brucellosis while IgM was positive in 100% and 32% of specimens from patients with acute and chronic brucellosis respectively. Elevated IgG subclasses to brucella antigen were found in different proportions in the sera of patients with acute and chronic brucellosis. In patients with acute brucellosis, IgG1 was the predominant response (79%) followed by IgG3 (58%), IgG2 (36%) and IgG4 (14%). In contrast, IgG4 was the predominating subclass response (73%) in patients with chronic brucellosis followed by IgG1 (41%), IgG2 and IgG3 (27% each). When considering the most common elevated IgG subclasses, in each serum, either alone or in combination with each other, patients with acute brucellosis showed IgG1+IgG3 (24%), IgG1 (19%), IgG1+IgG2+IgG3 (16%) while patients with chronic brucellosis showed IgG4 (27%) and IgG1+IgG2+IgG3+IgG4 (18%). This study reveals that in addition to the difference in brucella-specific Ig class response in patients with acute (IgG, IgM, IgA) and chronic (IgG, IgA) brucellosis, the profiles of IgG subclasses are different where IgG1 predominates in the acute and IgG4 in the chronic stages of the disease.     

2014-2018年北京市海淀区布鲁氏菌病重点职业人群监测血清学结果分析

目的对北京市海淀区布鲁氏菌病（布病）重点职业人群监测的血清学结果进行分析,了解重点职业人群的感染状况,为疾病的防控提供参考信息。方法2014 — 2018年以海淀区选定区域牛羊散养户和动物疫病所的工作人员为布病重点人群,采集全血,按照《布鲁氏菌病诊断标准》（WS 269 — 2007）中标准试管凝集方法（SAT）对采集的血清中抗体进行检测,使用SPSS 22.0软件进行统计分析,分析方法采用χ2检验。结果共检测血清样本243例,布病抗体阳性 7例,阳性率为2.88%,7例均未发现布病症状,不同年份阳性率差异无统计学意义（使用Fisher精确检验法,P＞0.05）。 不同职业人群中,饲养员阳性率为3.03%;牛、羊均接触的人群血清阳性率为4.84%,只接触牛的人群阳性率为3.66%,只接触羊的人群阳性率为1.01%。结论北京市海淀区2014 — 2018年布病重点职业人群为兽医及饲养员,接触牲畜主要为牛、羊,应加强防控知识的宣传教育,做好动物的免疫,及早发现感染病例,掌握疫情趋势,做好布病的防治工作。     

2015年浙江省人间布鲁氏菌病监测结果分析

目的 了解浙江省人间布鲁氏菌病（布病）发病状况及流行规律,为制定防治策略提供依据。方法 疫情资料来自国家疾病监测信息报告管理系统的疫情报告,采用描述流行病学和统计学方法分析数据;布病重点职业人群血清学检测采用虎红平板凝集试验和试管凝集试验。结果 2015年浙江省共报告布鲁氏菌病病例98例,发病率为0.18/10万,发病年龄主要集中在30～65岁年龄组,男女性发病数比例为2.63:1;2015年全省检测重点人群6 815人,阳性160例,阳性者中新发患者84例,感染者35例,41例既往患者和感染者。发病到确诊平均间隔时间30 d。结论 浙江省布病疫情呈稳定趋势,应加强医务人员培训和健康教育等工作以有效控制疫情,防止发生慢性化患者。     

Analysis of laboratory and serological test results in patients with acute brucellosis during follow‐up

BackgroundThe laboratory test results and serum‐specific antibodies of patients with acute brucellosis initial infection were followed up and analyzed.Methods70 patients in Hohhot City, Inner Mongolia Autonomous Region, with acute brucellosis were followed up for 360 days. Serum samples were collected at 0, 15, 30, 60, 90, 180, and 360 days after diagnosis and analyzed by Rose Bengal plate test (RBPT), colloidal gold test paper (GICA), and test tube agglutination test (SAT). The serum‐specific antibodies IgG and IgM were detected.ResultsRBPT results: False negative (‐) gradually increased with the extension of the course of disease, with the largest change in 30–60 days after diagnosis, and the constituent ratio increased by 12.9%. GICA results: The false negative increased with the course of disease, and the constituent ratio of false negative was 20.0% after 180 days of diagnosis. SAT results: 1:100 positive showed a ladder like decrease with the increase in the course of disease, and the largest decrease was 90–180 days, with a decrease of 34.3% in the constituent ratio. 360 days after diagnosis, the constituent ratio of positive was only 14.3%. During the follow‐up period, the IgG average value fluctuated and the average IgM value decreased.ConclusionThe false‐negative results of RBPT, GICA, and SAT increased with the course of disease, and the false‐negative rates were higher than 20% after half a year. IgM level is beneficial to the early diagnosis of brucellosis, while IgG level is helpful to the judgment of brucellosis stage.     

2010年新疆维吾尔自治区阜康市布鲁氏菌血清检测结果分析

目的了解新疆阜康地区人群布鲁氏菌病(布病)的感染情况,调查人群布病的流行现状及特征,进一步控制布病疫情。方法对当地村民分群抽样进行布病流行病学调查,采血做虎红平板凝集和试管凝集试验检测布鲁氏菌血清抗体,按照WS 269-2007,血清凝集滴度1∶100(++)及以上者为阳性。结果 2010年阜康市检测665人,血清阳性率为1.65%(11/665)。不同性别和年龄布病检测阳性率差异无统计学意义(P>0.05);不同民族检测阳性率差异有统计学意义(P<0.05)。结论当地人间布病感染风险较大,畜群患布病是人感染布病的危险因素。