Abstract
Pneumocystis jirovecii is a fungus responsible for human Pneumocystis pneumonia, one of the most severe infections encountered in immunodepressed individuals. The diagnosis of Pneumocystis pneumonia continues to be challenging due to the absence of specific symptoms in infected patients. Moreover, the standard diagnostic method employed for its diagnosis involves mainly PCR-based techniques, which besides being highly specific and sensitive, require specialized personnel and equipment and are time-consuming. Our aim is to demonstrate an optical biosensor methodology based on surface plasmon resonance to perform such diagnostics in an efficient and decentralized scheme. The biosensor methodology employs poly-purine reverse-Hoogsteen hairpin probes for the detection of the mitochondrial large subunit ribosomal RNA (mtLSU rRNA) gene, related to P. jirovecii detection. The biosensor device performs a real-time and label-free identification of the mtLSU rRNA gene with excellent selectivity and reproducibility, achieving limits of detection of around 2.11 nM. A preliminary evaluation of clinical samples showed rapid, label-free and specific identification of P. jirovecii in human lung fluids such as bronchoalveolar lavages or nasopharyngeal aspirates. These results offer a door for the future deployment of a sensitive diagnostic tool for fast, direct and selective detection of Pneumocystis pneumonia disease.
Highlights
Pneumocystis jirovecii is an atypical fungus exhibiting pulmonary tropism responsible for human Pneumocystis pneumonia (PcP)
We have demonstrated the efficiency of a surface plasmon resonance (SPR) biosensor for the direct and rapid detection of Pneumocystis pneumonia in human fluid samples without amplification or labelling steps in a reduced volume (50 μL)
The triplex approach ensured better and stronger capture of the mtLSU rRNA gene as compared to the traditional approach based on duplex hybrids through linear DNA probes
Summary
Pneumocystis jirovecii is an atypical fungus exhibiting pulmonary tropism responsible for human Pneumocystis pneumonia (PcP). PcP is one of the most serious and potentially fatal infections encountered in AIDS patients. With the currently rising number of patients receiving immunosuppressive therapies for malignancies such as cancer, allogeneic organ transplantations and autoimmune diseases, PcP is becoming more and more common in non-HIV immunosuppressed individuals [1]. PcP remains an important cause of morbidity and mortality worldwide with a mortality rate for HIV-infected patients ranging between 11 and 53% [2,3]. The clinical presentation of PcP may differ from HIV-infected patients to other immunocompromised patients, and there are no specific symptoms or signs. There is no universally agreed approach to the initial management of patients with suspected PcP, and many institutions treat patients empirically, while others pursue a definitive microbiological diagnosis [5]
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