Response to therapy in patients with cryptococcosis and AIDS: association with in vitro susceptibility to fluconazole / Respuesta al tratamiento en pacientes con criptococosis y sida: relación con la sensibilidad in vitro al fluconazol

Background. The implications of the Cryptococcus neoformans resistance to fluconazole on patient therapy have not been fully elucidated due to the discordant results found in published studies. Aims. To establish the influence of C. neoformans resistance to fluconazole in the therapy of individuals with cryptococcosis and AIDS. Methods. This study retrospectively compared the clinical course of patients with cryptococcosis according to the level of fluconazole resistance of their C. neoformans isolates. Results. This study included 71 episodes of cryptococcosis, defined as those isolates of C. neoformans obtained from patients with mycosis, of which 36 isolates were sensitive to fluconazole, 20 susceptible dose-dependent (SDD), and 15 were resistant. There were 5 treatment failures in the consolidation phase; two occurred in patients who had a susceptible strain, 2 in patients who had SDD strains, and one in a patient who had a resistant strain. During the maintenance treatment, relapses occurred in 4 of 33 patients (12%), seen during the follow-up period, none of which occurred in the group with resistant isolates. There were no significant differences in survival time free of treatment failure (p = 0.65) or survival time free of failure or relapse (p = 0.38). These results were not affected when tested in a Cox model that included age, CD4T lymphocyte counts, and use of antiretroviral therapy. Conclusions. In HIV patients with cryptococcosis, the resistance of C. neoformans appeared not to increase the risk of failure or relapse during treatment (AU)

Antecedentes. Las implicaciones de la resistencia de Cryptococcus neoformans al fluconazol en el tratamiento de pacientes infectados con esta levadura no han sido completamente definidas debido a hallazgos discordantes obtenidos previamente. Objetivos. Dilucidar la influencia de la resistencia de C. neoformans al fluconazol en el tratamiento de los pacientes con criptococosis y sida. Métodos. En este estudio se compara retrospectivamente la evolución clínica de los pacientes con criptococosis según el grado de resistencia al fluconazol de los aislamientos de C. neoformans obtenidos de ellos. Resultados. Se incluyeron 71 episodios de criptococosis definidos por el aislamiento de C. neoformans de pacientes con la micosis, que se distribuyeron de la siguiente manera: 36 aislamientos fueron sensibles, 20 sensibles dosis-dependiente y 15 resistentes. En la fase de consolidación, cinco fallos en el proceso de tratamiento tuvieron lugar: dos en pacientes con aislamientos sensibles, dos en pacientes con aislamientos dosis-dependiente y uno en un paciente con un aislamiento resistente. Durante la fase de mantenimiento se presentaron 4 recurrencias en los 33 pacientes que tuvieron seguimiento (12%), ninguna de las cuales ocurrió en el grupo con aislamientos resistentes. No se encontraron diferencias estadísticamente significativas en el tiempo de supervivencia de los casos sin fallo terapéutico (p = 0.65) o en el tiempo de supervivencia de los casos sin fallo terapéutico o recaída (p = 0.38). Estos resultados no se modificaron cuando fueron evaluados en un modelo de regresión de Cox en el que se incluyeron la edad, el conteo de linfocitos T CD4 y el uso de terapia antirretroviral. Conclusiones. En pacientes con VIH y criptococosis la resistencia de C. neoformans a fluconazol parece no incrementar el riesgo de fallo terapéutico o recaída (AU)

Validation and clinical application of a nested PCR for paracoccidioidomycosis diagnosis in clinical samples from Colombian patients

Paracoccidioidomycosis is a systemic and endemic mycosis, restricted to tropical and subtropical areas of Latin America. The infection is caused by the thermal dimorphic fungus Paracoccidioides brasiliensisand Paracoccidioides lutzii. The diagnosis of paracoccidioidomycosis is usually performed by microscopic examination, culture and immunodiagnostic tests to respiratory specimens, body fluids and/or biopsies; however these methods require laboratory personnel with experience and several days to produce a result. In the present study, we have validated and evaluated a nested PCR assay targeting the gene encoding the Paracoccidioides gp43membrane protein in 191 clinical samples: 115 samples from patients with proven infections other than paracoccidioidomycosis, 51 samples as negative controls, and 25 samples from patients diagnosed with paracoccidioidomycosis. Additionally, the specificity of the nested PCR assay was also evaluated using purified DNA isolated from cultures of different microorganisms (n= 35) previously identified by culture and/or sequencing. The results showed that in our hands, this nested PCR assay for gp43 protein showed specificity and sensitivity rates of 100%. The optimized nested PCR conditions in our laboratory allowed detection down to 1 fg of P. brasiliensisDNA.

Validation and clinical application of a nested PCR for paracoccidioidomycosis diagnosis in clinical samples from Colombian patients.

Paracoccidioidomycosis is a systemic and endemic mycosis, restricted to tropical and subtropical areas of Latin America. The infection is caused by the thermal dimorphic fungus Paracoccidioides brasiliensis and Paracoccidioides lutzii. The diagnosis of paracoccidioidomycosis is usually performed by microscopic examination, culture and immunodiagnostic tests to respiratory specimens, body fluids and/or biopsies; however these methods require laboratory personnel with experience and several days to produce a result. In the present study, we have validated and evaluated a nested PCR assay targeting the gene encoding the Paracoccidioides gp43 membrane protein in 191 clinical samples: 115 samples from patients with proven infections other than paracoccidioidomycosis, 51 samples as negative controls, and 25 samples from patients diagnosed with paracoccidioidomycosis. Additionally, the specificity of the nested PCR assay was also evaluated using purified DNA isolated from cultures of different microorganisms (n=35) previously identified by culture and/or sequencing. The results showed that in our hands, this nested PCR assay for gp43 protein showed specificity and sensitivity rates of 100%. The optimized nested PCR conditions in our laboratory allowed detection down to 1fg of P. brasiliensis DNA.

Response to therapy in patients with cryptococcosis and AIDS: Association with in vitro susceptibility to fluconazole.

BACKGROUND: The implications of the Cryptococcus neoformans resistance to fluconazole on patient therapy have not been fully elucidated due to the discordant results found in published studies. AIMS: To establish the influence of C. neoformans resistance to fluconazole in the therapy of individuals with cryptococcosis and AIDS. METHODS: This study retrospectively compared the clinical course of patients with cryptococcosis according to the level of fluconazole resistance of their C. neoformans isolates. RESULTS: This study included 71 episodes of cryptococcosis, defined as those isolates of C. neoformans obtained from patients with mycosis, of which 36 isolates were sensitive to fluconazole, 20 susceptible dose-dependent (SDD), and 15 were resistant. There were 5 treatment failures in the consolidation phase; two occurred in patients who had a susceptible strain, 2 in patients who had SDD strains, and one in a patient who had a resistant strain. During the maintenance treatment, relapses occurred in 4 of 33 patients (12%), seen during the follow-up period, none of which occurred in the group with resistant isolates. There were no significant differences in survival time free of treatment failure (p=0.65) or survival time free of failure or relapse (p=0.38). These results were not affected when tested in a Cox model that included age, CD4T lymphocyte counts, and use of antiretroviral therapy. CONCLUSIONS: In HIV patients with cryptococcosis, the resistance of C. neoformans appeared not to increase the risk of failure or relapse during treatment.

Validation and clinical application of a molecular method for identification of Histoplasma capsulatum in human specimens in Colombia, South America.

The conventional means of diagnosis of histoplasmosis presents difficulties because of the delay to the time that the diagnosis is made, indicating the need for the implementation of molecular assays. We evaluated 146 clinical samples from 135 patients suspected of having histoplasmosis using a previously reported nested PCR assay for the Histoplasma capsulatum-specific 100-kDa protein (the Hc100 PCR). In order to determine the specificity of this molecular test, we also used samples from healthy individuals (n = 20), patients suspected of having respiratory disease with negative fungal cultures (n = 29), and patients with other proven infections (n = 60). Additionally, a sizable collection of DNA from cultures of H. capsulatum and other medically relevant pathogens was studied. A panfungal PCR assay that amplified the internal transcribed spacer 2 region was also used to identify all fungal DNAs. All PCR-amplified products were sequenced. Of the 146 clinical samples, 67 (45.9%) were positive by culture and PCR, while 9 samples negative by culture were positive by PCR. All the sequences corresponding to the 76 amplified products presented > or =98% identity with H. capsulatum. The Hc100 PCR exhibited a sensitivity of 100% and specificities of 92.4% and 95.2% when the results were compared to those for the negative controls and samples from other proven clinical entities, respectively; the positive predictive value was 83% and the negative predictive value was 100%; the positive and negative likelihood rates were 25 and 0, respectively. These results suggest that the Hc100 nested PCR assay for the detection of H. capsulatum DNA is a useful test in areas where mycosis caused by this organism is endemic.