Development and Validation of a Novel Diagnostic Test for Human Brucellosis Using a Glyco-engineered Antigen Coupled to Magnetic Beads

Brucellosis is a highly contagious zoonosis and still a major human health problem in endemic areas of the world. Although several diagnostic tools are available, most of them are difficult to implement especially in developing countries where complex health facilities are limited. Taking advantage of the identical structure and composition of the Brucella spp. and Yersinia enterocolitica O:9 O-polysaccharide, we explored the application of a recombinant Y. enterocolitica O:9-polysaccharide-protein conjugate (OAg-AcrA) as a novel antigen for diagnosis of human brucellosis. We have developed and validated an indirect immunoassay using OAg-AcrA coupled to magnetic beads. OAg-AcrA was produced and purified with high yields in Y. enterocolitica O:9 cells co-expressing the oligosaccharyltransferase PglB and the protein acceptor AcrA of Campylobacter jejuni without the need for culturing Brucella. Expression of PglB and AcrA in Y. enterocolitica resulted in the transfer of the host O-polysaccharide from its lipid carrier to AcrA. To validate the assay and determine the cutoff values, a receiver-operating characteristic analysis was performed using a panel of characterized serum samples obtained from healthy individuals and patients of different clinical groups. Our results indicate that, using this assay, it is possible to detect infection caused by the three main human brucellosis agents (B. abortus, B. melitensis and B. suis) and select different cutoff points to adjust sensitivity and specificity levels as needed. A cutoff value of 13.20% gave a sensitivity of 100% and a specificity of 98.57%, and a cutoff value of 16.15% resulted in a test sensitivity and specificity of 93.48% and 100%, respectively. The high diagnostic accuracy, low cost, reduced assay time and simplicity of this new glycoconjugate-magnetic beads assay makes it an attractive diagnostic tool for using not only in clinics and brucellosis reference laboratories but also in locations with limited laboratory infrastructure and/or minimally trained community health workers.布鲁氏菌病（Brucellosis）是一种高传染性人畜共患病，目前仍是全球流行地区的主要人类健康问题。尽管现有多种诊断工具，但多数难以开展，尤其是在医疗设施匮乏的发展中国家。研究团队利用布鲁氏菌属（Brucella spp.）与小肠结肠炎耶尔森菌O:9型（Yersinia enterocolitica O:9）的O多糖（O-polysaccharide）结构与组成一致的特点，探索了重组小肠结肠炎耶尔森菌O:9型多糖-蛋白结合物（OAg-AcrA）作为新型抗原用于人类布鲁氏菌病诊断的应用前景。本研究开发并验证了一种将OAg-AcrA偶联至磁珠的间接免疫测定法。研究人员在共表达空肠弯曲杆菌（Campylobacter jejuni）寡糖转移酶（oligosaccharyltransferase）PglB与蛋白受体AcrA的小肠结肠炎耶尔森菌O:9菌株中，高效生产并纯化得到了OAg-AcrA，无需培养布鲁氏菌。在小肠结肠炎耶尔森菌中表达PglB与AcrA后，可将宿主O多糖从其脂质载体转移至AcrA蛋白。为验证该检测方法并确定临界值，研究团队使用一组来自健康个体与不同临床组别患者的定型血清样本，开展了受试者工作特征（receiver-operating characteristic, ROC）曲线分析。结果显示，利用该检测方法可检出三类主要的人类布鲁氏菌病原体（流产布鲁氏菌、羊种布鲁氏菌以及猪种布鲁氏菌），并可根据需求调整临界值以优化灵敏度与特异度水平。当临界值设为13.20%时，检测灵敏度为100%，特异度为98.57%；临界值设为16.15%时，检测灵敏度与特异度分别为93.48%与100%。这种新型糖结合物-磁珠检测法诊断准确率高、成本低、耗时短且操作简便，不仅适用于临床机构与布鲁氏菌病参考实验室，也适合在实验室基础设施有限或社区卫生工作人员培训不足的地区推广使用。