Kohler Christian, Dunachie Susanna J, Müller Elke, Kohler Anne, Jenjaroen Kemajittra, Teparrukkul Prapit, Baier Vico, Ehricht Ralf, Steinmetz Ivo
Friedrich Loeffler Institut for Medical Microbiology, Greifswald, Germany.
Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.
PLoS Negl Trop Dis. 2016 Jul 18;10(7):e0004847. doi: 10.1371/journal.pntd.0004847. eCollection 2016 Jul.
The environmental bacterium Burkholderia pseudomallei causes the infectious disease melioidosis with a high case-fatality rate in tropical and subtropical regions. Direct pathogen detection can be difficult, and therefore an indirect serological test which might aid early diagnosis is desirable. However, current tests for antibodies against B. pseudomallei, including the reference indirect haemagglutination assay (IHA), lack sensitivity, specificity and standardization. Consequently, serological tests currently do not play a role in the diagnosis of melioidosis in endemic areas. Recently, a number of promising diagnostic antigens have been identified, but a standardized, easy-to-perform clinical laboratory test for sensitive multiplex detection of antibodies against B. pseudomallei is still lacking.
In this study, we developed and validated a protein microarray which can be used in a standard 96-well format. Our array contains 20 recombinant and purified B. pseudomallei proteins, previously identified as serodiagnostic candidates in melioidosis. In total, we analyzed 196 sera and plasmas from melioidosis patients from northeast Thailand and 210 negative controls from melioidosis-endemic and non-endemic regions. Our protein array clearly discriminated between sera from melioidosis patients and controls with a specificity of 97%. Importantly, the array showed a higher sensitivity than did the IHA in melioidosis patients upon admission (cut-off IHA titer ≥1:160: IHA 57.3%, protein array: 86.7%; p = 0.0001). Testing of sera from single patients at 0, 12 and 52 weeks post-admission revealed that protein antigens induce either a short- or long-term antibody response.
Our protein array provides a standardized, rapid, easy-to-perform test for the detection of B. pseudomallei-specific antibody patterns. Thus, this system has the potential to improve the serodiagnosis of melioidosis in clinical settings. Moreover, our high-throughput assay might be useful for the detection of anti-B. pseudomallei antibodies in epidemiological studies. Further studies are needed to elucidate the clinical and diagnostic significance of the different antibody kinetics observed during melioidosis.
环境细菌类鼻疽伯克霍尔德菌可引发传染病类鼻疽，在热带和亚热带地区病死率很高。直接检测病原体可能存在困难，因此需要一种有助于早期诊断的间接血清学检测方法。然而，目前针对类鼻疽伯克霍尔德菌抗体的检测方法，包括参考间接血凝试验（IHA），缺乏敏感性、特异性和标准化。因此，血清学检测目前在地方病流行地区的类鼻疽诊断中并未发挥作用。最近，已鉴定出一些有前景的诊断抗原，但仍缺乏一种用于敏感多重检测类鼻疽伯克霍尔德菌抗体的标准化、易于操作的临床实验室检测方法。
在本研究中，我们开发并验证了一种可用于标准96孔板形式的蛋白质微阵列。我们的阵列包含20种重组和纯化的类鼻疽伯克霍尔德菌蛋白，这些蛋白先前被鉴定为类鼻疽血清诊断候选物。我们总共分析了来自泰国东北部类鼻疽患者的196份血清和血浆，以及来自类鼻疽流行和非流行地区的210份阴性对照。我们的蛋白质阵列能够清晰地区分类鼻疽患者血清与对照，特异性为97%。重要的是，该阵列在类鼻疽患者入院时显示出比IHA更高的敏感性（IHA临界滴度≥1:160：IHA为57.3%，蛋白质阵列为86.7%；p = 0.0001）。对入院后0、12和52周的单例患者血清进行检测发现，蛋白质抗原可诱导短期或长期抗体反应。
我们的蛋白质阵列为检测类鼻疽伯克霍尔德菌特异性抗体模式提供了一种标准化、快速、易于操作的检测方法。因此，该系统有潜力改善临床环境中类鼻疽的血清诊断。此外，我们的高通量检测方法可能有助于在流行病学研究中检测抗类鼻疽伯克霍尔德菌抗体。需要进一步研究以阐明类鼻疽期间观察到的不同抗体动力学的临床和诊断意义。
