Abstract
Diagnosis of Pneumocystis pneumonia (PcP) relies on the detection of P. jirovecii in respiratory specimens obtained by invasive techniques. Thus, the development of a serological test is urgently needed as it will allow the diagnosis of PcP using blood, an inexpensive and non-invasive specimen. This study aims to combine the production of a multi-epitope synthetic recombinant antigen (RSA) and an ELISA test for detection of anti-P. jirovecii antibodies, in order to develop a new approach for PcP diagnosis. The RSA was selected and designed based on the study of the immunogenicity of the carboxyl-terminal domain of the major surface glycoprotein. This antigen was purified and used as an antigenic tool in an ELISA technique for detection of Ig, IgG and IgM antibodies anti-P. jirovecii (patent-pending no. PT109078). Serum specimens from 88 patients previously categorized in distinct clinical subgroups and 17 blood donors, were analysed. The IgM anti-P. jirovecii levels were statistically increased in patients with PcP (p = 0.001) and the ELISA IgM anti-P. jirovecii test presented a sensitivity of 100% and a specificity of 80.8%, when associated with the clinical diagnosis criteria. This innovative approach, provides good insights about what can be done in the future serum testing for PcP diagnosis.
Highlights
Diagnosis of Pneumocystis pneumonia (PcP) relies on the detection of P. jirovecii in respiratory specimens obtained by invasive techniques
This study analysed serum specimens from a universe of 105 persons who were previously categorized into five groups. 50 serum specimens from patients with PcP (47.62%), 11 serum specimens from patients colonized with P. jirovecii (10.48%), 19 serum specimens from patients without P. jirovecii or other fungal infections (18.09%), eight serum specimens from patients without P. jirovecii infection but with other fungal diseases (7.62%) and 17 serum samples from blood donors (16.19%), were analysed (Table 1)
The three potential immunogenic regions selected, Msg1696–1851 (126–176 aa) which is located at the terminal fraction of the MsgB portion, Msg2596–2712 (426–464 aa) and Msg2896–3033 (526–571 aa), both integrated at the C-termini of the MsgC portion, were identified according to the sequence with GenBank accession no
Summary
Diagnosis of Pneumocystis pneumonia (PcP) relies on the detection of P. jirovecii in respiratory specimens obtained by invasive techniques. The RSA was selected and designed based on the study of the immunogenicity of the carboxyl-terminal domain of the major surface glycoprotein This antigen was purified and used as an antigenic tool in an ELISA technique for detection of Ig, IgG and IgM antibodies anti-P. jirovecii The IgM anti-P. jirovecii levels were statistically increased in patients with PcP (p = 0.001) and the ELISA IgM anti-P. jirovecii test presented a sensitivity of 100% and a specificity of 80.8%, when associated with the clinical diagnosis criteria. The standard laboratory diagnosis of PcP relies on microscopic visualization of stained P. jirovecii organisms and/or DNA detection by PCR in respiratory specimens, such as bronchoalveolar lavage (BAL) These specimens are obtained by invasive techniques that carry an associated risk of complications and are not easy to perform in patients with respiratory failure or in children[6]. The Pneumocystis antigen that has received the most attention is the major surface glycoprotein (Msg), which contains shared and species-specific epitopes, elicits humoral and www.nature.com/scientificreports/
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