Utilización de métodos moleculares para detectar ADN de Pnuemocystis jirovecii y genes de resistencia a trimetoprima sulfametoxazol en materiales respiratorios de pacientes de Argentina
Pneumonia caused by Pneumocystis jirovecii (PjP) is an infection caused by no-cultivable fungus. Consequently, the gold standard for diagnosing PjP involves direct observation using staining or immunofluorescence techniques. Additionally, assessing the fungus's resistance to trimethoprim-sulfam...
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| Formato: | Tesis doctoral acceptedVersion |
| Lenguaje: | Español |
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Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica
2024
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| Acceso en línea: | http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=posgraafa&cl=CL1&d=HWA_7892 https://repositoriouba.sisbi.uba.ar/gsdl/collect/posgraafa/index/assoc/HWA_7892.dir/7892.PDF |
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| Sumario: | Pneumonia caused by Pneumocystis jirovecii (PjP) is an infection caused by no-cultivable fungus. Consequently, the gold standard for diagnosing PjP involves direct observation using staining or immunofluorescence techniques. Additionally, assessing the fungus's resistance to trimethoprim-sulfamethoxazole (TMP/SMX), the primary treatment for PjP, requires methods no culture-based.\nThis study aimed to advance the epidemiology and rapid diagnosis of PjP and gene mutations in linked to TMP/SMX resistance in circulating P. jirovecii strains in Argentina. A real-time PCR (qPCR) assay was developed to amplify a fragment of the mitochondrial large subunit ribosomal RNA (mtLSU RNA) gene specific to P. jirovecii using the TaqMan system. Two additional PCR assays were used to amplify the DHPS and DHFR genes associated with drug resistance.\nThe qPCR?s limit of detection was determined to be 220 copies/25 ?l reaction (8.8 copies/?l), with an analytical specificity of 100% against other fungal species, mycobacteria, and respiratory bacteria. We analyzed 101 respiratory samples from patients treated in four hospitals from Buenos Aires (CABA) and 37 oropharyngeal washes from healthy individuals. The ROC curve analysis, comparing direct examination results with qPCR Ct values, showed an AUC of 0.96 (95% CI, 0.93 to 0.99), demonstrating excellent diagnostic accuracy. A Ct value of 36.07 (?2,200 copies of the gene/25 ?l) provided the highest likelihood of active PjP, with a sensitivity of 90.70% (95% CI, 78.40 to 96.32) and specificity of 95.79% (95% CI, 89.67 to 98.35). Ct values between > 36 and 40, referred to as the "gray zone," did not definitively distinguish between active infection and colonization.\nTo evaluate the utility of qPCR in diagnosing and epidemiologically studying PjP in Argentina, and detecting strains with DHPS and DHFR mutations, we analyzed 316 samples from 232 patients across 24 institutions in 13 jurisdictions. Direct examination identified 93 confirmed PjP cases, while qPCR detected an additional 16 probable cases, marking a percentage increase of 17.2% in diagnoses. Fifty-seven percent of cases were in CABA, with the rest distributed in other jurisdictions. The median age of patients was 42 years, with a male-to-female ratio of 1.4:1. The qPCR exhibited a sensitivity of 93.5%, specificity of 88.5%, a positive predictive value (PPV) of 84.5%, and a negative predictive value (NPV) of 95.3%.\nFour strains with non-synonymous DHPS gene mutations were found in four patients (three from CABA and one from Tucumán), three strains had two mutations (A165G and C171), while one had a single mutation (A165G). The synonymous DHFR gene mutations (T312C) were detected in 23 strains from 17 patients across six jurisdictions, with three patients harboring both: mutated and wild- type strains. The prevalence of strains with DHPS mutations was 3.7%, and those with DHFR mutations were 15.6% among 109 patients with confirmed or probable PjP.\nThe qPCR efficiency of molecular diagnosis according to the immunological status of the patients, showed improved sensitivity in HIV-positive patients (PLHIV), with a specificity, PPV, and NPV of\n4\n94.7%, 72.1%, 85.5%, and 88.6%, respectively. For patients with other immunodeficiencies (POIS) or without apparent immunocompromise (SICA), sensitivity was 88.9%, with a specificity of 97.0%, and PPV and NPV of 80.0% and 98.4%, respectively.\nThe qPCR-PjP technique with a Ct ? 36 enables rapid and specific detection of P. jirovecii DNA in respiratory samples from both immunocompromised and non-immunocompromised patients. Ct values >36-40 necessitate additional clinical interpretation, potentially involving further samples or supplementary tests such as the detection of ?-D-glucan (BDG) or lactate dehydrogenase (LDH). Most confirmed/probable PjP cases occurred in adults, with a slight male predominance, mainly in CABA and other densely populated areas with high HIV incidence. The prevalence of non-synonymous DHPS mutations associated with TMP/SMX resistance was low (3.2%) and not widely distributed. In contrast, DHFR mutations were more frequent and widespread but did not appear to affect TMP/SMX resistance. The qPCR method was more sensitive in PLHIV but less so in POIS and SICA, though the latter had higher specificity, making a negative result highly indicative of the absence of PjP. All identified mutated strains were in PLHIV, including most DHFR mutations. |
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