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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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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I28-R145-HWA_7892 |
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dspace |
| institution |
Universidad de Buenos Aires |
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I-28 |
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R-145 |
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Repositorio Digital de la Universidad de Buenos Aires (UBA) |
| language |
Español |
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spa |
| topic |
Pneumocystis jirovecii Detección molecular Resistencia Ciencias de la vida |
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Pneumocystis jirovecii Detección molecular Resistencia Ciencias de la vida Toranzo, Adriana Inés Utilización de métodos moleculares para detectar ADN de Pnuemocystis jirovecii y genes de resistencia a trimetoprima sulfametoxazol en materiales respiratorios de pacientes de Argentina |
| topic_facet |
Pneumocystis jirovecii Detección molecular Resistencia Ciencias de la vida |
| description |
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. |
| author2 |
Di Conza, José |
| author_facet |
Di Conza, José Toranzo, Adriana Inés |
| format |
Tesis doctoral Tesis doctoral acceptedVersion |
| author |
Toranzo, Adriana Inés |
| author_sort |
Toranzo, Adriana Inés |
| title |
Utilización de métodos moleculares para detectar ADN de Pnuemocystis jirovecii y genes de resistencia a trimetoprima sulfametoxazol en materiales respiratorios de pacientes de Argentina |
| title_short |
Utilización de métodos moleculares para detectar ADN de Pnuemocystis jirovecii y genes de resistencia a trimetoprima sulfametoxazol en materiales respiratorios de pacientes de Argentina |
| title_full |
Utilización de métodos moleculares para detectar ADN de Pnuemocystis jirovecii y genes de resistencia a trimetoprima sulfametoxazol en materiales respiratorios de pacientes de Argentina |
| title_fullStr |
Utilización de métodos moleculares para detectar ADN de Pnuemocystis jirovecii y genes de resistencia a trimetoprima sulfametoxazol en materiales respiratorios de pacientes de Argentina |
| title_full_unstemmed |
Utilización de métodos moleculares para detectar ADN de Pnuemocystis jirovecii y genes de resistencia a trimetoprima sulfametoxazol en materiales respiratorios de pacientes de Argentina |
| title_sort |
utilización de métodos moleculares para detectar adn de pnuemocystis jirovecii y genes de resistencia a trimetoprima sulfametoxazol en materiales respiratorios de pacientes de argentina |
| publisher |
Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica |
| publishDate |
2024 |
| url |
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 |
| work_keys_str_mv |
AT toranzoadrianaines utilizaciondemetodosmolecularesparadetectaradndepnuemocystisjiroveciiygenesderesistenciaatrimetoprimasulfametoxazolenmaterialesrespiratoriosdepacientesdeargentina |
| _version_ |
1851374248869756928 |
| spelling |
I28-R145-HWA_78922025-11-06 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. Fil: Toranzo, Adriana Inés. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Buenos Aires, Argentina Di Conza, José Canteros, Cristina Elena Toranzo, Adriana Inés 2024-11-05 La neumonía causada por Pneumocystis jirovecii (PjP) es una infección fúngica cuyo agente etiológico es un hongo que no puede ser cultivado in vitro. Debido a esto, el estándar de oro para el diagnóstico de esta micosis consiste en la observación directa mediante coloraciones o técnicas de inmunofluorescencia. Además, la determinación de la resistencia del hongo a la trimetoprima-sulfametoxazol (TMP/SMX), tratamiento de primera línea para la PjP, también requiere métodos que no se basen en cultivos.\nEl objetivo de este trabajo fue contribuir a la epidemiología y al diagnóstico rápido de la PjP, así como identificar mutaciones en los genes asociados a la resistencia a TMP/SMX en las cepas de P. jirovecii circulantes en Argentina. Para ello, se estandarizó una PCR en tiempo real (qPCR) que amplifica un fragmento del gen de la subunidad mayor del ribosoma mitocondrial (mtLSU RNA) específico de P. jirovecii en un sistema Taqman, junto con dos PCR que amplifican los genes DHPS y DHFR, relacionados con la resistencia.\nSe evaluó el límite de detección de la qPCR, que fue de 220 copias/25 ?l de reacción (8,8 copias/?l) para un Ct promedio de 40, con una especificidad analítica del 100% frente a otras especies de hongos, micobacterias y bacterias que causan infecciones respiratorias. Para determinar el valor de corte (Ct) de la qPCR, se analizaron 101 muestras respiratorias de igual número de pacientes de 4 hospitales de la Ciudad Autónoma de Buenos Aires (CABA) y 37 lavados orofaríngeos (LOF) de 37 individuos sanos. Se compararon los resultados del examen directo con los valores de Ct, generando una curva ROC que mostró un AUC de 0,96 (IC del 95%, 0,93 a 0,99), lo que indica una excelente capacidad de la qPCR para discriminar entre pacientes con PjP activa y no enfermos. El Ct=36,07 (?2.200 copias del gen/25 ?l de reacción) mostró la mayor verosimilitud, con una sensibilidad del 90,70% (IC 95%, 78,40 a 96,32) y una especificidad del 95,79% (IC 95%, 89,67 a 98,35). En las muestras donde la qPCR tuvo un Ct entre >36 y 40, no pudimos confirmar si el paciente estaba cursando una PjP con baja carga fúngica o si estaba colonizado, lo que se denomina ?zona gris?.\nPara evaluar la utilidad de la qPCR en el diagnóstico y epidemiología de PjP en Argentina y detectar la circulación de cepas con mutaciones en los genes DHPS y DHFR, se analizaron un total de 316 muestras de 232 pacientes de 24 instituciones en 13 jurisdicciones. El examen directo detectó 93 casos de PjP probada, mientras que la qPCR identificó 16 casos adicionales (PjP probable), lo que representa un incremento del 17,2% en los casos diagnosticados. El 57% de los casos se diagnosticaron en CABA y el resto en otras jurisdicciones. La mediana de edad de los pacientes con PjP probada/probable fue de 42 años, y la relación de género (M/F) fue de 1,4/1. La qPCR presentó una sensibilidad del 93,5%, una especificidad del 88,5%, y un valor predictivo positivo (VPP) y negativo (VPN) del 84,5% y 95,3%, respectivamente.\nSe identificaron 4 cepas en 4 pacientes (3 de CABA y una de Tucumán) con mutaciones no sinónimas en el gen DHPS; de estas, 3 presentaban dos mutaciones (A165G y C171), y una de\n2\nCABA contaba con una mutación (A165G). Además, se detectó en 23 cepas de 17 pacientes (de seis jurisdicciones) una mutación sinónima (T312C) en el gen DHFR. En dos de estos pacientes coexistían la cepa mutada y la cepa wild-type. La prevalencia de cepas con mutaciones asociadas a resistencia en los 109 pacientes con PjP probada/probable fue del 3,7% (mutaciones no sinónimas) para DHPS y del 15,6% (mutaciones sinónimas) para DHFR.\nAl evaluar la eficiencia del diagnóstico molecular según el estatus inmunológico de los pacientes, en aquellos con HIV positivo (PLHIV), la qPCR incrementó la sensibilidad diagnóstica, con especificidad, VPP y VPN de 94,7%, 72,1%, 85,5% y 88,6%, respectivamente. En pacientes con otras causas de inmunodeficiencia (POIS) o sin inmunocompromiso aparente (SICA), la sensibilidad diagnóstica fue del 88,9%, con una especificidad del 97,0%, y VPP y VPN de 80,0% y 98,4%, respectivamente.\nLa técnica de qPCR-PjP con Ct ? 36 permite detectar de forma rápida y específica el ADN de P. jirovecii en muestras respiratorias de pacientes con y sin inmunocompromiso. Los valores de Ct >36-40 requieren interpretación clínica adicional, que puede incluir muestras adicionales del mismo paciente o pruebas complementarias como detección de ?-D-glucano (BDG) o lactato deshidrogenasa (LDH).\nLa mayoría de los casos de PjP probada/probable fueron en pacientes adultos, con un ligero predominio masculino, y se diagnosticaron en CABA y otras áreas con alta densidad poblacional y alta incidencia de HIV. La prevalencia de cepas con mutaciones no sinónimas en el gen DHPS asociadas a resistencia a TMP/SMX fue muy baja (3,2%) y, aparentemente, no están ampliamente distribuidas en el país. Por otro lado, las cepas con mutaciones "silentes" en el gen DHFR fueron más frecuentes y se distribuyeron más ampliamente, aunque estas mutaciones no influyen en la resistencia a TMP/SMX, nos permite reconocer los genotipos circulantes de P. jirovecii. La sensibilidad de la qPCR fue mayor en PLHIV, mientras que en POIS y SICA fue menor; sin embargo, en estos últimos, la especificidad es superior, y un resultado negativo prácticamente descarta la PjP. Las 4 cepas mutadas en el gen DHPS fueron identificadas en pacientes PLHIV, al igual que la mayoría de las mutaciones del gen DHFR. application/pdf Almuzara, Marisa Quiroga, Paula Afeltra, Javier Pneumocystis jirovecii Detección molecular Resistencia spa Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by-nc-nd/2.5/ar/ Ciencias de la vida Doctora de la Universidad de Buenos Aires en Ciencias de la salud Utilización de métodos moleculares para detectar ADN de Pnuemocystis jirovecii y genes de resistencia a trimetoprima sulfametoxazol en materiales respiratorios de pacientes de Argentina info:eu-repo/semantics/doctoralThesis info:ar-repo/semantics/tesis doctoral info:eu-repo/semantics/acceptedVersion 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 |