Application of synthetic recombinant multi-epitope antigens and gold nanoparticles for a Pneumocystis pneumonia rapid diagnostic test

Abstract

Introduction: Pneumocystis jirovecii pneumonia (PcP) is an important morbidity/mortality factor in immunocompromised patients, whose diagnosis requires respiratory specimens obtained by invasive and costly techniques. Thus, the development of a rapid, cost-effective, minimally invasive and field-friendly test to diagnose PcP would be a significant clinical advance.

Materials and methods: Surface proteins as the major surface glycoprotein (Msg) and kexin- like serine protease (Kex1) are specific of P. jirovecii with reactive antigenic properties. Newly recombinant synthetic antigens (RSA), involving multi antigenic regions of these proteins, were designed based on the study of the immunogenicity of the carboxyl-terminal domain of Msg [1] and the entire sequence of Kex1. RSAs were purified by affinity chromatography with immobilized nickel ions and applied as antigenic tools in indirect ELISA assays that were optimized for detection of circulating antibodies anti-P. jirovecii in sera of patients (N = 122), with and without the disease. These RSAs were conjugated with 40 nm diameter functionalized spherical gold nanoparticles (AuNPs) and these bionanoconjugates were used to detect specific anti-P. jirovecii antibodies in patients’ sera by agarose gel electrophoresis. A device for PcP rapid diagnostic test, based on a strip immunochromatographic format, is being optimized and validated in view of clinical implementation. This study was approved by the ethical committee from Instituto de Higiene e Medicina Tropical that, because this was a retrospective observational study, waived informed consent.

Results: Indirect ELISA assays showed that the IgM anti-P. jirovecii levels were significantly increased in patients with PcP, compared with patients without PcP (p < 0.001 with both RSA). Assays with the bionanoconjugates formed by AuNPs and the Msg RSA confirmed recognition of these bionanoconjugates by patients’ sera containing anti-P. jirovecii antibodies in agarose gel electrophoresis. Results obtained until now with the strip immunochromatographic test, support the viability of the platform to be developed.

Discussion and conclusions: The two RSAs function as biomarkers of infection by detection of IgM anti-P. jirovecii levels in patients serum specimens and AuNP-Msg bionanoconjugates showed applicability in a serological platform for PcP diagnosis. The improvement and validation of this strip-based test could enable a faster and inexpensive screening/diagnosis of this opportunistic infectious disease, helping better-tailored therapeutic interventions, the disease control and retrenchment to healthcare systems.

Acknowledgements

The authors acknowledge funding from Fundação para a Ciência e a Tecnologia (FCT- UID/Multi/04413/2013 & SFRH/BD/108433/2015) and Gilead GÉNESE (PGG/001/2014) to this study. This work was also supported by the Applied Molecular Biosciences Unit-UCIBIO financed by national funds from FCT/MCTES (UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER - 007265).