Abstract
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.
Highlights
The fungus Pneumocystis jirovecii is a pathogen able to cause a fatal pneumonia (PcP) in immunocompromised patients worldwide (Barry and Johnson, 2001; Huang et al, 2011; Esteves et al, 2014; Matos et al, 2017)
The kexin-like serine protease 1 (Kex1) Recombinant Synthetic Antigens (RSA) consists of 110 amino acids composed by three regions with high-predicted antigenicity and reactivity from P. jirovecii’s Kex1 entire sequence, that were chosen and synthesized interconnected by two “linkers” of five glycine residues and expressed with a tail of six residues of histidine
The SDS-PAGE analysis showed that both RSA were expressed with their predicted molecular sizes (16.7 kDa for major surface glycoproteins (Msg) RSA and 22 kDa for Kex1 RSA) after IPTG induction (Figures 2A,B) and that they were successfully purified by immobilized metal-ion affinity chromatography (IMAC) (Figure 2C)
Summary
The fungus Pneumocystis jirovecii is a pathogen able to cause a fatal pneumonia (PcP) in immunocompromised patients worldwide (Barry and Johnson, 2001; Huang et al, 2011; Esteves et al, 2014; Matos et al, 2017). Despite all the advances in understanding P. jirovecii infection over the last years, in the twenty-first century the standard diagnosis of this disease still depends on the detection of P. jirovecii organisms through expensive and laborious technologies (cytochemical or immunofluorescent staining and/or PCR) applied to respiratory specimens obtained by invasive techniques, such as bronchoscopy (Alanio et al, 2016; Matos and Esteves, 2016; Matos et al, 2017; Tomás and Matos, 2018) These standard diagnosis methods, besides being difficult to implement in all economic settings, are not always possible to perform in patients with respiratory failure or in children (Alanio et al, 2016; Matos and Esteves, 2016; Matos et al, 2017; Tomás and Matos, 2018). To improve disease management worldwide, there is a need to develop and implement an alternative approach for the diagnosis of PcP that can reduce associated costs, the need for invasive procedures, and improves response time and specificity
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
