Data_Sheet_1_Development of a Gold Nanoparticle-Based Lateral-Flow Immunoassay for Pneumocystis Pneumonia Serological Diagnosis at Point-of-Care.pdf

Pneumocystis jirovecii pneumonia (PcP) is a major human immunodeficiency virus (HIV)-related illness, rising among immunocompromised non-HIV patients and in developing countries. Presently, the diagnosis requires respiratory specimens obtained through invasive and costly techniques that are difficult to perform in all patients or implement in all economic settings. Therefore, the development of a faster, cost-effective, non-invasive and field-friendly test to diagnose PcP would be a significant advance. In this study, recombinant synthetic antigens (RSA) of P. jirovecii’s major surface glycoprotein (Msg) and kexin-like serine protease (Kex1) were produced and purified. These RSA were applied as antigenic tools in immunoenzymatic assays for detection of specific anti-P. jirovecii antibodies (IgG and IgM) in sera of patients with (n = 48) and without (n = 28) PcP. Results showed that only IgM anti-P. jirovecii levels were significantly increased in patients with PcP compared with patients without P. jirovecii infection (p ≤ 0.001 with both RSA). Thus, two strip lateral flow immunoassays (LFIA), based on the detection of specific IgM anti-P. jirovecii antibodies in human sera samples, were developed using the innovative association of P. jirovecii’s RSA with spherical gold nanoparticles (AuNPs). For that, alkanethiol-functionalized spherical AuNPs with ca. ~40 nm in diameter were synthetized and conjugated with the two RSA (Msg or Kex1) produced. These AuNP-RSA conjugates were characterized by agarose gel electrophoresis (AGE) and optimized to improve their ability to interact specifically with serum IgM anti-P. jirovecii antibodies. Finally, two LFIA prototypes were developed and tested with pools of sera from patients with (positive sample) and without (negative sample) PcP. Both LFIA had the expected performance, namely, the presence of a test and control red colored lines with the positive sample, and only a control red colored line with the negative sample. These results provide valuable insights into the possibility of PcP serodiagnosis at point-of-care. The optimization, validation and implementation of this strip-based approach may help to reduce the high cost of medical diagnosis and subsequent treatment of PcP both in industrialized and low-income regions, helping to manage the disease all around the world.

肺孢子虫肺炎（Pneumocystis jirovecii pneumonia，简称PcP）是一种重要的与人类免疫缺陷病毒（HIV）相关的疾病，其发病率在非HIV免疫受损患者以及发展中国家呈上升趋势。目前，该疾病的诊断需要通过侵入性且昂贵的呼吸样本获取技术，这些技术在所有患者中实施或在所有经济环境中应用都存在困难。因此，开发一种快速、经济、非侵入性且适合现场使用的PcP诊断测试将是一项重大进步。在本研究中，我们制备并纯化了肺孢子虫主要表面糖蛋白（Msg）和kexin样丝氨酸蛋白酶（Kex1）的重组合成抗原（RSA）。这些RSA被用作免疫酶联测定中的抗原工具，用于检测PcP患者（n = 48）及非PcP患者（n = 28）血清中的特异性抗-P. jirovecii抗体（IgG和IgM）。结果显示，与未感染P. jirovecii的患者相比，PcP患者的IgM抗-P. jirovecii水平显著升高（p ≤ 0.001，均以RSA为依据）。因此，基于检测人类血清样本中特异性IgM抗-P. jirovecii抗体的原理，开发了两条基于RSA与球形金纳米颗粒（AuNPs）创新结合的条带式横向流动免疫测定（LFIA）。为此，合成了直径约为40 nm的烷硫醇功能化球形AuNPs，并将其与两个RSA（Msg或Kex1）交联。这些AuNP-RSA交联物通过琼脂糖凝胶电泳（AGE）进行表征，并优化以提高其与血清IgM抗-P. jirovecii抗体的特异性相互作用能力。最终，开发了两个LFIA原型，并使用来自PcP患者（阳性样本）和非PcP患者（阴性样本）的血清混合物进行了测试。两种LFIA均表现出预期的性能，即在阳性样本中存在测试线和对照线，而在阴性样本中仅存在对照线。这些结果为PcP现场血清诊断的可能性提供了宝贵的见解。对该条带式方法的优化、验证和实施可能有助于降低PcP在工业化和低收入地区医疗诊断及其后续治疗的成本，从而有助于全球范围内管理该疾病。