Perfiles de expresión de los genes ERG11, MDR1 y AFR1 en Cryptococcus neoformans var. grubii aislados de pacientes con HIV / Gene expression profiles of ERG11, MDR1 y AFR1 in Cryptococcus neoformans var. grubbi isolated from HIV patients

Introducción. El fluconazol es el antifúngico más utilizado para la prevención y el tratamiento de infecciones causadas por el género Cryptococcus, agente etiológico de la criptococosis. La resistencia al fluconazol en los aislamientos de Cryptoccocus neoformans puede hacer fracasar el tratamiento y generar recaídas de la infección. Objetivo. Evaluar los perfiles de expresión de los genes AFR1, MDR1 y ERG11 en aislamientos clínicos de C. neoformans var. grubii, durante la respuesta in vitro a la inducción con fluconazol. Materiales y métodos. Se estudiaron 14 aislamientos de C. neoformans var. grubii provenientes de pacientes con HIV, de los cuales 6 eran sensibles al fluconaol y 8 presentaban sensibilidad disminuida. Los niveles de expresión de los genes ERG11, AFR1 y MDR1 se determinaron mediante PCR en tiempo real. Resultados. Los aislamientos resistentes al fluconazol mostraron sobreexpresión de los genes AFR1 y MDR1, mientras que la expresión de los fenotipos de resistencia evaluados se mantuvo homogénea en ERG11, en todos los aislamientos de C. neoformans var. grubii. Conclusiones. La sobreexpresión de los genes AFR1 y MDR1 que codifican las bombas de eflujo, contribuye a la resistencia al fluconazol en los aislamientos estudiados. Sin embargo, los patrones de resistencia que se registran en este hongo, sumado a los casos de recaídas en pacientes con HIV, no pueden atribuirse únicamente a los casos de resistencia por exposición al fármaco. Otros mecanismos podrían también estar involucrados en este fenómeno, como la resistencia emergente (resistencia mediante otros genes ERG) y la heterorresistencia, los cuales deben ser estudiados en estos aislamientos.

Introduction: Fluconazole is the most used antifungal drug for prevention and treatment of Cryptococcus spp. infections, the etiological agent of cryptococcosis. Resistance to fluconazole among Cryptococcus neoformans isolates can lead to treatment failure and generate relapses. Objective: To evaluate the expression profles of the AFR1, MDR1 and ERG11 genes in C. neoformans var. grubii clinical isolates during the in vitro response to fluconazole induction. Materials and methods: Fourteen C. neoformans var. grubii isolates recovered from HIV patients were studied, in which 6 showed sensitivities to fluconazole and 8 decreased sensitivity. The expression levels of ERG11, AFR1 and MDR1 genes were determined by real-time PCR from extracted mRNA. Results: AFR1 and MDR1 genes from C. neoformans var. grubii were overexpressed in fluconazole resistant isolates, whereas ERG11 maintains homogeneous expression in all the evaluated resistance phenotypes of C. neoformans var. grubii isolates. Conclusions: The overexpression of AFR1 and MDR1 genes, which codify for efflux pumps, contributes to fluconazole resistance in the studied isolates. However, the resistance patterns in this fungus and the relapse cases in HIV patients cannot be attributed solely to the exposure to the drug. Heteroresistance and the emerging resistance (resistance through other ERG genes), might be other mechanisms involved in this phenomenon, which must be studied in these isolations.

Criptococosis diseminada por terapia biológica, se debe gestionar el riesgo / Disseminated cryptococcosis by biological therapy: We must manage the risk

Introducción. Se han descrito múltiples efectos adversos con el uso de la terapia biológica para enfermedades autoinmunitarias, muchos de ellos secundarios al estado de inmunosupresión, como las infecciones bacterianas, fúngicas o virales. Caso clínico. Se presenta el caso de una mujer de 64 años con diagnóstico comprobado de criptococosis diseminada secundaria al uso de tofacitinib. Se descartaron otras causas de inmunosupresión, como infección por el virus de la inmunodeficiencia humana (HIV). Tres años antes se le había diagnosticado artritis reumatoide y se encontraba en tratamiento farmacológico con un agente biológico que inhibe las enzimas JAK. Se han descrito muy pocos casos de criptococosis pulmonar y meníngea en este tipo de pacientes. Conclusión. Este reporte de caso es útil para que otros médicos tratantes tengan presente la posibilidad de este tipo de infección fúngica invasora asociada con la terapia biológica y el enfoque de gestión de riesgo.

Introduction: Multiple adverse effects have been described for the biological therapy in autoimmune diseases including many secondary to immunosuppression producing bacterial, fungal, or viral infections. Clinical case: We present the case of a 64-year-old female patient with proven disseminated cryptococcosis secondary to the use of tofacitinib. Other possible causes of immunosuppression such as the human immunodeficiency virus (HIV) were ruled out. The patient had been in treatment for rheumatoid arthritis diagnosed three years before. This drug is a biological agent that inhibits JAK enzymes. Very few cases of pulmonary and meningeal cryptococcosis in this type of patient have been described in the literature. Conclusion: This case report should be useful for other clinicians to bear in mind the possibility of this type of invasive fungal infection associated with biological therapy and to take a risk-management approach.

Aislamiento de Cryptococcus neoformans en excrementos de paloma de Castilla (Columba livia) provenientes de lugares públicos de El Salvador / Isolation of Cryptococcus neoformans from droppings of the Feral Pigeon (Columba livia) found in public places in El Salvador

Resumen Objetivo: El objetivo de este estudio fue aislar levaduras pertenecientes al complejo de especies Cryptococcus neoformans de los excrementos de paloma de Castilla (Columba livia) recolectados de plazas y parques públicos de El Salvador. Métodos: Las muestras se sembraron en medios de cultivos convencionales y a las colonias confirmadas se les efectuó una tipificación mediante la técnica de restricción enzimática del gen URA5. Resultados: De un total de 66 muestras analizadas, tres estaban positivas por levaduras pertenecientes al complejo de especies Cryptococcus neoformans. El estudio molecular agrupó los aislamientos en los tipos moleculares VNI y VNII; ambos corresponden a la especie Cryptococcus neoformans sensu stricto. Conclusión: En los sitios estudiados, la presencia de esta levadura es muy reducida, probablemente debido a factores ambientales. Se presenta el primer reporte de Cryptococcus neoformans sensu stricto, genotipos VNI y VNII en El Salvador, esta especie es de relevancia en salud pública por el ser el responsable de más del 90% de los casos de criptococosis a nivel mundial.

Abstract Aim: The objective of this study was to isolate yeast that belonged to the Cryptococcus neoformans species complex from the feces of the Feral Pigeon (Columba livia), from public places in El Salvador. Methods: Samples were seeded in conventional culture media and confirmed colonies were typed using the enzyme restriction technique of the URA5 gene. Results: Of a total of 66 samples analyzed, three were positive for yeasts that belonged to the Cryptococcus neoformans species complex. The molecular study grouped the isolates in the molecular types VNI and VNII; both belonging to the species Cryptococcus neoformans sensu stricto. Conclusions: In the studied sites the presence of this yeast is very low, probably due to environmental factors. We present the first report of Cryptococcus neoformans sensu stricto genotypes VNI and VNII in El Salvador. This specie is relevant in public health for being responsible for more than 90% of cases of cryptococcosis worldwide.

Hemolymph of triatomines presents fungistatic activity against Cryptococcus neoformans and improves macrophage function through MCP-I/TNF-α increase

Triatomines are blood-feeding arthropods belonging to the subfamily Triatominae (Hemiptera; Reduviidae), capable of producing immunomodulatory and water-soluble molecules in their hemolymph, such as antimicrobial peptides (AMPs). In this work, we evaluated the antifungal and immunomodulatory activity of the hemolymph of Meccus pallidipennis (MPH) and Rhodnius prolixus (RPH) against Cryptococcus neoformans. Methods: We assessed the activity of the hemolymph of both insects on fungal growth by a minimum inhibitory concentration (MIC) assay. Further, RAW 264.7 macrophages were cultivated with hemolymph and challenged with C. neoformans. Then, their phagocytic and killing activities were assessed. The cytokines MCP-1, IFN-γ, TNF-α, IL-10, IL-12, and IL-6 were measured in culture supernatants 4- and 48-hours post-infection. Results: Both hemolymph samples directly affected the growth rate of the fungus in a dose-dependent manner. Either MPH or RPH was capable of inhibiting fungal growth by at least 70%, using the lowest dilution (1:20). Treatment of RAW 264.7 macrophages with hemolymph of both insects was capable of increasing the production of MCP-I and TNF-α. In addition, when these cells were stimulated with hemolymph in the presence of C. neoformans, a 2- and a 4-fold increase in phagocytic rate was observed with MPH and RPH, respectively, when compared to untreated cells. For the macrophage killing activity, MPH decreased in approximately 30% the number of viable yeasts inside the cells compared to untreated control; however, treatment with RPH could not reduce the total number of viable yeasts. MPH was also capable of increasing MHC-II expression on macrophages. Regarding the cytokine production, MCP-I and TNF-α, were increased in the supernatant of macrophages treated with both hemolymphs, 4 and 48 hours after stimulation. Conclusion: These results suggested that hemolymph of triatomines may represent a source of molecules capable of presenting antifungal and immunomodulatory activity in macrophages during fungal infection.(AU)

Criptococosis diseminada en paciente inmunocompetente imitando un cáncer de pulmón metastásico / Disseminated cryptococcosis in an immunocompetent patient imitating metastatic lung cancer

El cáncer de pulmón es la principal causa de muerte en el mundo por cáncer, y en Colombia es la segunda. Su pronóstico es pobre cuando se ha documentado enfermedad metastásica en el sistema nervioso central. El diagnóstico se basa en el resultado definitivo de patología. Aunque los hallazgos imagenológicos pueden ser muy sugestivos de malignidad, hay reportes de otras enfermedades que pueden imitar cáncer, tales como infecciones o tumores benignos, los cuales pueden llevar a adoptar conductas terapéuticas inapropiadas. Las infecciones fúngicas como las producidas por Criptococcus neoformans, son capaces de generar lesiones que pueden imitar neoplasias. El objetivo de esta publicación es reportar el caso de un hombre a quien inicialmente se le sospechó un carcinoma pulmonar metastásico al sistema nervioso central, y finalmente se le diagnosticó una criptococosis diseminada posterior a su fallecimiento

Lung cancer is the leading cause of death from cancer in the world and the second in Colombia, its prognosis is bad when the diagnosis of metastatic disease in the central nervous system is documented. The diagnosis is based on the definitive pathologic result. Although the imaging findings can be highly suggestive of malignancy, there are reports of other conditions that can mimic lung cancer, such as infections or benign tumors, which can lead to inappropriate treatment. Fungal infections such as those caused by Criptococcus neoformans are capable of generating lesions that can mimic neoplasms. The objective of this article is to report the case of a man who was initially diagnosed with metastatic lung carcinoma to the central nervous system, and was finally diagnosed with disseminated cryptococcosis after his death

A case of AIDS complicated with Pneumocystis jirovecii pneumonia and disseminated infection of Talaromyces marneffei and Cryptococcus neoformans / 中国热带医学

@#Abstract: AIDS combined with Pneumocystis jirovecii pneumonia (PJP) and disseminated infections of Talaromyces marneffei and Cryptococcus neoformans are rare. This paper summarizes and analyzes the diagnosis and treatment of an AIDS patient with multiple fungal infections for reference. A 79-year-old male patient was admitted to the hospital with "stool habit change for more than 20 days". The white blood cell count was 4.57×109/L, the percentage of neutrophils was 81.8%, the absolute count of CD4+ lymphocytes was 6/μL, and the CD4/CD8 ratio was 0.17. HIV antibody positive was confirmed by CDC. The cerebrospinal fluid and alveolar lavage fluid were positive for Cryptococcus neoformans capsular antigen, and Pneumocystis jirovecii was found by the bronchoalveolar lavage fluid stained with hexamine silver. The cerebrospinal fluid culture was positive for Cryptococcus neoformans, and the blood culture was positive for Cryptococcus neoformans and Talaromyces marneffei. CT showed that bronchovascular bundles in both lungs were more thick, patchy and cable-like high-density shadows were seen in both lungs, and the edges were blurred. Nodular and cable-like high-density shadows were seen in the posterior apical segment of the left upper lobe, with clear margins. Infection of both lungs was considered, and secondary pulmonary tuberculosis occurred in the left upper lobe. After admission, the patient was treated with various anti-bacterial and fungal drugs due to recurrent fever, but the effect was not effective. The fever symptoms of the patient could not be significantly improved, and his condition continued to worsen, and he eventually died. The patient with AIDS complicated with bacterial and fungal infection, especially PJP infection in serious condifiton and has a poor prognosis for rapid development, so clinical attention should be paid to. 

Disseminated cryptococcosis caused by Cryptococcus neoformans a case report and review / 中国热带医学

@#Abstract: To analyze the clinical, therapeutic and laboratory characteristics of disseminated cryptococcosis caused by Cryptococcus neoformans invading the blood stream in patient with liver cirrhosis and splenectomy. A 30-year-old male underwent splenectomy plus pericardial devascularization due to "splenomegaly and hypersplenism" in March in 2016. The patient had intermittent fever after operation for many times, and successively accompanied with back pain, left lower limb abscess and right hip pain. The highest body temperature was 39 ℃. CT and MRI revealed the lung lesion and multiple bone destruction. During that period, the effect of antibiotics was not good. On April 19th, 2017, Gram's stain, India ink stain, API 32C, Vitek 2 Compact, ribosomal ITS and IGS sequence analysis were performed to identify the strain isolated from the pus and blood stream. The serum of the patient was detected for cryptococcal antigen. Antifungal susceptibility test was used to determine drug sensitivity and minimum inhibitory concentration (MIC). The Cryptococcus neoformans isolated from fresh pus specimen showed a prominent, thick capsule after India ink stain. The colonies isolated from pus and blood stream were identified Cryptococcus neoformans using API 32C, Vitek 2 Compact, and sequence analysis of rDNA ITS and IGS. Cryptococcal capsule antigen was positive. The minimal inhibitory concentrations of 5-Flucytosine, amphotericin B, fluconazole, itriconazole, voriconazole against the isolate were <4 μg/mL, <0.5 μg/mL, 4 μg/mL, ≤0.25 μg/mL, 0.125 μg/mL respectively. The patient was initially treated with intravenous amphotericin B and flucytosine. After anti-Cryptococcus treatment for two months, the patient clinically improved, and the lesions were reduced on a follow-up CT scan. The patient made a full functional recovery after treatment for six months. Cryptococcosis has hidden onset, atypical clinical symptoms and lack of specificity. Blood stream is the main channel for Cryptococcus to spread and involve many organs of the whole body, including skin, bone and so on. Therefore, early use of blood culture to monitor blood flow dissemination, actively removing the primary focus and cutting off the infection route in time and carrying out effective anti-Cryptococcus treatment are conducive to the patient's early recovery.

Coinfección por Cryptococcus neoformans en paciente trasplantado renal con COVID-19 / Cryptococcus neoformans co-infection in a kidney transplant patient with COVID-19

Resumen La infección por el síndrome respiratorio agudo severo por coronavirus 2 (SARS-CoV-2), causante de la enfermedad COVID-19, representa una de las emergencias sanitarias más deletéreas de las últimas dos décadas; de la que aún existen innumerables incógnitas acerca del curso y manejo de presentaciones atípicas. Por lo anterior, resulta importante publicar casos clínicos de interés en población especial como lo son los pacientes receptores de trasplante renal, quienes pueden tener un mayor riesgo de contraer la enfermedad dado su estado de inmunosupresión, así como por el contacto frecuente con el sistema de atención médica. La coinfección con otros virus respiratorios no es infrecuente en población trasplantada con COVID-19, y la asociación con gérmenes oportunistas como Micobacterias, Citomegalovirus, Pneumocystis jirovecii y hongos, tal como el Cryptococcus neoformans es desconocida, pero es posible que dicha asociación pudiese empeorar la severidad del compromiso sistémico y pulmonar. Se presenta el caso de un hombre de 45 años, con antecedentes de trasplante renal de donante cadavérico, que ingresó al servicio de urgencias del Hospital de San José; el presentó síndrome de dificultad respiratoria aguda (SDRA) y falla ventilatoria por infección por SARS-CoV-2. Además, se confirmó infección sistémica por Cryptococcus neoformans sin compromiso del sistema nervioso central. Se inició manejo con ventilación mecánica invasiva, glucocorticoide, anfotericina B liposomal; fallece a los 12 días de su ingreso.

Abstract The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes the disease COVID-19, represents one of the most deleterious health emergencies of the last two decades; of which there are still innumerable unknowns about the course and handling of atypical presentations. Therefore, it is important to publish clinical cases of interest in a special population, such as kidney transplant recipients, who may have a greater risk of contracting the disease due to their immunosuppression and frequent contact with the medical care system. Co-infection with other respiratory viruses is not uncommon in the transplanted population with COVID-19, and the association with opportunistic germs such as Mycobacteria, Cytomegalovirus, Pneumocystis jirovecii and fungi, such as Cryptococcus neoformans, is unknown, yet it is possible that this association could worsen the severity of the systemic and pulmonary involvement. We present the case of a 45-year-old deceased-donor kidney transplant recipient man who was admitted to the emergency service; he presented acute respiratory difficulty syndrome (ARDS) and ventilatory failure due to infection by SARS-CoV-2. It was also confirmed that he had systemic infection by Cryptococcus neoformans without compromise of the central nervous system. It was necessary to use invasive mechanical ventilation, glucocorticoid, liposomal amphotericin B; he died 12 days after admission.

Prevalencia y susceptibilidad de Cryptococcus neoformans aislados del suelo y excretas de palomas en la ciudad de Maceió ­ AL, Brasil / Prevalência e suscetibilidade de Cryptococcus neoformans isolados do solo e de excretas de pombos na cidade de Maceió ­ AL, Brasil

Introducción: Cryptococcus neoformans es una levadura encapsulada, que se encuentra en fuentes ambientales, incluyendo excrementos de palomas. Tiene una gran relevancia clínica porque es el agente etiológico de la criptococosis, considerada una causa importante de mortalidad en personas inmunocomprometidas en todo el mundo. Objetivo: determinar la prevalencia y susceptibilidad de C. neoformans aislados del suelo y excremento de palomas en la ciudad de Maceió ­ Alagoas, Brasil. Metodología: Se recolectaron 150 muestras (50 de excretas secas, 50 de excretas frescas y 50 del suelo) y cultivaron en Agar Dextrosa Sabouraud. Las colonias aisladas sugestivas de C. neoformans se sometieron a identificación y caracterización mediante análisis morfológicos, actividad de la enzima fenol-oxidasa, sensibilidad a la cicloheximida, desarrollo a 37°C, hidrólisis de urea, asimilación de carbono y nitrógeno y fenotipificación en medio canavanina-glicina-azul de bromotimol. La prueba de susceptibilidad antifúngica se realizó utilizando la técnica de difusión en agar. Resultados: se obtuvieron 36 (24%) muestras positivas para C. neoformans, de las cuales 33 fueron de excretas secas y 3 del suelo cercano al excremento. El perfil de susceptibilidad fue de 100.0% para anfotericina B y 87.4% para ketoconazol, no obstante, hubo un alto porcentaje de resistencia a fluconazol (91.5%) e itraconazol (80.0%). Conclusión: estos hallazgos confirman que las excretas de palomas secas son reservorios de C. neoformans en el medio ambiente, caracterizando un problema de salud única. Además, la anfotericina B exhibió una alta actividad in vitro, representando buena alternativa en el tratamiento de la criptococosis.

Introdução: Cryptococcus neoformans é um fungo leveduriforme encapsulado, encontrado em fontes ambientais, incluindo excretas de pombos. Apresenta grande relevância clínica por ser agente etiológico da criptococose, considerada importante causa de mortalidade em indivíduos imunocomprometidos em todo o mundo. Objetivo: determinar a prevalência e suscetibilidade de C. neoformans isolados do solo e de excretas de pombos na cidade de Maceió, Alagoas. Metodologia: foram coletadas 150 amostras (50 de excretas secas, 50 de excretas frescas e 50 do solo) e cultivadas em Ágar Sabouraud Dextrose. As colônias isoladas sugestivas de C. neoformans foram submetidas à identificação e caracterização por meio de análises morfológicas, atividade da enzima fenoloxidase, sensibilidade à cicloheximida, termotolerância à 37ºC, hidrólise da ureia, assimilação de carbono e nitrogênio e quimiotipagem em ágar L-canavanina-glicina-azul de bromotimol. O teste de suscetibilidade antifúngica foi realizado por meio da técnica de difusão em ágar. Resultados: foram obtidas 36 (24%) amostras positivas para C. neoformans, das quais 33 (91,6%) foram provenientes de excretas secas e 3 (8,3%) do solo próximo a excrementos. O perfil de suscetibilidade foi de 100,0% para a anfotericina B e 87,4% para o cetoconazol, não obstante, verificou-se alto percentual de resistência ao fluconazol (91,8%) e itraconazol (80%). Conclusão: estes achados confirmam que excretas de pombos secas são reservatórios de C. neoformans no ambiente, caracterizando um problema de saúde única. Além disso, a anfotericina B apresentou elevada atividade, in vitro, representando boa alternativa no tratamento da criptococose.

Características clínicas de la criptococosis cerebral en pacientes infectados por el VIH: Hospital Nacional de Paraguay años 2012 a 2020 / Clinical characteristics of cerebral cryptococcosis in HIV-infected patients: Hospital Nacional de Paraguay years 2012 to 2020

RESUMEN Introducción: el cuadro clínico de la infección por el VIH refleja la inmunodeficiencia progresiva desde fases iniciales a veces asintomáticas hasta las finales cuando clínicamente corresponde a estadios más avanzados de inmunosupresión, con linfocitos T CD4 < 100cel /µL, el síndrome de inmunodeficiencia adquirida, en el que ya aparecen las enfermedades definitorias, una de ellas la criptococosis cerebral. Objetivos: describir las características clínicas de la criptococosis cerebral en pacientes infectados por el VIH internados en el servicio de Clínica Médica del Hospital Nacional ­ Itauguá, Paraguay de 2012 a 2020. Metodología: se realizó un estudio observacional descriptivo, retrospectivo de corte transversal, en pacientes de ambos sexos mayores de 18 años. Resultados: se estudiaron 35 pacientes, 69 % fueron varones, con mediana de edad 43 años. El 100 % se hallaba sin tratamiento antirretroviral al ingreso, 74 % no se conocían infectados por el VIH y el 26 % no tenía adherencia al tratamiento. El cuadro evolutivo en promedio fue de 20 días (2 a 90 días), el 91 % de los síntomas al ingreso fueron neurológicos, siendo el predominante la cefalea, que se presentó en forma aislada en el 28 % y el resto con otro síntoma neurológico. La carga viral promedio fue 867.464 copias/mL, la mediana del nivel CD4 34 cél/mm3 y el 100 % < 150 cél/mm3. El promedio de los hallazgos del líquido cefalorraquídeo fueron glucorraquia 37 mg/dl, proteinorraquia 174 mg/dl, la celularidad 46 % ≤ 6 cél/µl, 98 % con predominio mononuclear. Todos fueron tratados con anfotericina B y de mantenimiento con fluconazol. La mediana de los días de internación fue 30 días y la mortalidad de 26 %, siendo los factores de mal pronóstico: consulta tardía, no conocidos infectados por VIH y mayor carga viral. Conclusiones: se pudo evidenciar mayoría de los casos en pacientes varones, adultos jóvenes, en estadio sida, 100 % sin tratamiento en antirretrovirales y como factores de mal pronóstico la consulta tardía y la alta carga viral. Es válida la sospecha de concomitancia criptococosis meníngea-VIH, en pacientes con cefalea crónica aun en pacientes no conocidos infectados con VIH.

ABSTRACT Introduction: the clinical picture of HIV infection reflects the progressive immunodeficiency from the initial phases, sometimes asymptomatic to the final, when clinically it corresponds to more advanced stages of immunosuppression, with CD4 T lymphocytes <100cells / µL, the acquired immunodeficiency syndrome, in the one that already appears the defining diseases, one of them the cerebral cryptococcosis. Objectives: to describe the clinical characteristics of cerebral cryptococcosis in HIV-infected patients admitted to the Medical Clinic Service of the Hospital Nacional - Itauguá, Paraguay from 2012 to 2020. Methodology: a descriptive, retrospective, cross-sectional observational study was carried out in patients of both sexes older than 18 years. Results: 35 patients were studied, 69 % were male, with a median age of 43 years. 100 % were without antiretroviral treatment upon admission, 74 % were not known to be infected with HIV, and 26 % had no adherence to treatment. The evolutionary picture on average was 20 days (2 to 90 days), 91 % of the symptoms on admission were neurological, the predominant being headache, which occurred in isolation in 28% and the rest with another neurological symptom . The mean viral load was 867,464 copies / mL, the median CD4 level was 34 cells / mm3 and 100 % <150 cells / mm3. The mean cerebrospinal fluid findings were glucorrhachia 37 mg / dl, protein sprain 174 mg / dl, cellularity 46 % ≤ 6 cells / µl, 98% predominantly mononuclear. All were treated with amphotericin B and maintenance with fluconazole. The median number of days of hospitalization was 30 days and mortality was 26 %, with poor prognosis factors being: late consultation, unknown HIV-infected patients, and higher viral load. Conclusions: most of the cases could be evidenced in male patients, young adults, in the AIDS stage, 100 % without antiretroviral treatment, and late consultation and high viral load as poor prognostic factors. The suspicion of meningeal cryptococcosis-HIV concomitance is valid in patients with chronic headache even in unknown patients infected with HIV.

Criptococosis y linfocitopenia T CD4 idiopática: Reporte de un caso / Cryptococcosis and idiopathic CD4 T lymphocytopenia: A case report

Resumen La linfocitopenia T CD4 idiopática (LCI) es un síndrome clínico inusual que se caracteriza por un déficit de células T CD4+ circulantes en ausencia de infección por VIH u otra condición de inmunosupresión. Los pacientes con dicha enfermedad pueden presentarse asintomáticos o con infecciones oportunistas, las más frecuentes son por criptococo, micobacterias o virales como herpes zoster. Presentamos el caso de un hombre de 32 años, sin antecedentes, en quien se descartó infección por retrovirus, con recuento de linfocitos T CD4+ menor a 300 células/m3; se diagnosticó LCI posterior al diagnóstico de criptococomas cerebrales mediante hallazgos imagenológicos los cuales fueron congruentes con estudios microbiológicos.

Summary Idiopathic CD4 T lymphocytopenia (ICL) is an unusual clinical syndrome characterized by a deficit of circulating CD4 + T cells in the absence of HIV infection or another immunosuppression condition. Patients with this disease may present asymptomatic or with opportunistic infections, the most frequent are cryptococcus, mycobacteria or viral such as herpes zoster. We present a case of a 32-year-old man with no prior disease, in whom retrovirus infection was discarded, with CD4 + T lymphocyte count less than 300 cells/m3; ICL was diagnosed after the diagnosis of brain cryptococomas by imaging findings which were consistent with microbiological studies.

Cryptococcosis by Cryptococcus neoformans/Cryptococcus gattii Species Complexes in non-HIV-Infected Patients in Southeastern Brazil

Abstract INTRODUCTION: The clinical manifestations of cryptococcosis are usually associated with the infecting agents Cryptococcus neoformans (CN) and C. gattii (CG) species complexes and the host. In this study, non-HIV-infected patients, at a university hospital in southeastern Brazil, had epidemiological and clinical data associated with cryptococcal disease and isolated Cryptococcus species: CN - 24 patients and CG - 12 patients. METHODS: The comparison was comprised of demographic data, predisposing factors, clinical and laboratory manifestations, and outcomes of cryptococcosis patients treated between 2000 and 2016. Immunocompetent and immunosuppressed patients were also compared, irrespective of the infecting species. Cryptococcus spp. were genotyped by PCR-RFLP analysis of the URA5 gene. RESULTS: Infections by the CN species complex (100% VNI genotype) were associated with drug immunosuppression and fungemia, and patients infected with the CG species complex (83% VG II and 17% VGI genotypes) had more evident environmental exposure and higher humoral response. CN and CG affected patients with or without comorbidities. CONCLUSIONS: Diabetes mellitus, other chronic non-infectious diseases, and alcoholism were likely predisposing factors for infection by both CN and CG species. Immunocompetent patients, independent of the infecting Cryptococcus species complexes, showed a higher occurrence of meningitis and a trend toward less fungal dissemination and longer survival than immunosuppressed hosts.

Production of a latex agglutination reagent for the rapid diagnosis of cryptococcal meningitis / Producción de un reactivo de látex para el diagnóstico rápido de criptococosismeníngea

Abstract Cryptococcosis is a fungal disease affecting more than one million people per yearworldwide. Its main etiological agents are Cryptococcus neoformans species complex and Cryp-tococcus gattii species complex. Cryptococcal meningitis (CM) is considered an AIDS-definingcondition. Rapid diagnosis by cryptococcal antigen assays, either the latex agglutination test(LA) or the lateral flow assay, is key to decreasing mortality due to cryptococcal disease. Theaim of the study was to develop a latex agglutination reagent (LA-ANLIS) for the rapid and reliable diagnosis of cryptococcosis in Argentina. This reagent will be produced in order to supplythe NMLN (National Mycology Laboratory Network). The evaluation of LA-ANLIS performanceand its comparison with the Cryptococcus Antigen Latex Agglutination Test System (LA-IMMY)(Immuno-Mycologics, Inc., USA) were conducted in 94 samples of cerebrospinal fluid. LA-ANLISand LA-IMMY compared exhibited 100% positive agreement and 97% negative agreement. LA-ANLIS showed 94% sensitivity and 97% specificity with the positive and negative predictivevalues of 94% and 97%, respectively. The LA-ANLIS is a reliable, reproducible and cost-effectivereagent, especially useful in countries where the commercial kit is not generally available andmust be obtained at a high cost. National production of reagents is the best choice for a reliableaccess to the rapid diagnosis of CM in Argentina.

Resumen La criptococosis es una enfermedad fúngica que afecta a más de un millón de personas por año en todo el mundo. Los principales agentes etiológicos pertenecen a los complejos de especies Cryptococcus neoformans y Cryptococcus gattii. La criptococosis meníngea (CM) se considera una enfermedad marcadora de sida. El diagnóstico rápido de esta enfermedad a través de la detección del antígeno de Cryptococcus, ya sea por aglutinación en partículas de látex o por inmunocromatografía, es clave para disminuir la mortalidad. El objetivo del presente estudio fue desarrollar un reactivo de aglutinación en partículas de látex para el diagnóstico rápido y certero de la CM en Argentina. Este reactivo (denominado en adelante LA-ANLIS) será producido para abastecer a la Red Nacional de Laboratorios de Micología. Se evaluó el desempeno del reactivo LA-ANLIS, y se realizó una comparación con el reactivo comercial Immuno-Mycologics, Inc. (en adelante, LA-IMMY) utilizando 94 muestras de líquido cefalorraquídeo. Hubo un 100% de acuerdo positivo y un 97% de acuerdo negativo entre los resultados obtenidos con los reactivos LA-ANLIS y LA-IMMY. El reactivo LA-ANLIS mostró una sensibilidad del 94% y una especificidad del 97%; los valores predictivos positivo y negativo fueron del 94 y del 97%, respectivamente. Se concluye que el LA-ANLIS es un reactivo confiable y rentable, que arroja resultados reproducibles, por lo que es especialmente útil en países donde los reactivos comerciales generalmente no están disponibles o sus costos son elevados. La producción nacional de reactivos es la mejor opción para asegurar el acceso de todos los hospitales al diagnóstico rápido de la CM en Argentina.

Frequency and geographical distribution of genotypes and mating types of Cryptococcus neoformans and Cryptococcus gattii species complexes in Argentina / Frecuencia y distribución geográfica de genotipos y tipos sexuales de los complejos de especies Cryptococcus neoformans y Cryptococcus gattii en Argentina

Abstract The aim of this work was to know the frequency and geographical distribution of genotypes and mating types of Cryptococcus neoformans and Cryptococcus gattii species complexes isolated from human infections in Argentina during the period from April 2009 to April 2011. A multicenter study was conducted, in which 372 isolates were obtained from 61 laboratories throughout the country. Of those, 98.8% of the isolates belonged to the C. neoformans species complex and 1.1% to the C. gattii species complex. Genotype VNI (MATa) was the most frequently isolated (n = 326, 87.6%), followed by VNII (MATa) (n = 22, 5.9%), the recently described VNII-VNIV (aADa) hybrid (n = 14, 3.8%), VNIV (MATa) (n=4, 1.1%), VNIII (aADa) hybrid (n = 1, 0.3%), and VNIII (aADa) hybrid (n = 1, 0.3%). The Argentine Central region showed the greatest number of cases and genotype diversity. Interestingly, a relative high frequency was observed in genotype VNII (MATa) in the Cuyo, Northeast and Northwest regions and, also in VNII-VNIV (aADa) hybrids in the Northwest region. C. gattii species complex was isolated at a low rate; 3 VGI (MATa) and 1 VGII (MATa) isolates were obtained from the Northwest and Central regions. In conclusion, this study shows that genotype frequencies seem to vary among regions in Argentina and reveals a relatively high frequency of rare hybrids in the Northwest region. Further regional clinical and environmental studies may help to elucidate if those varia-tions in frequencies are associated with the existence of regional ecological niches or any other regional factors.

Resumen El objetivo de! trabajo fue conocer la frecuencia y la distribución geográfica de genotipos y tipos sexuales de aislados pertenecientes a los complejos de especies Cryptococcus neoformansy Cryptococcus gattii obtenidos de infecciones humanas en Argentina. Entre abril de 2009 y abril de 2011 se realizó un estudio multicéntrico del que se obtuvieron 372 aislados de 61 laboratorios de diferentes zonas del país. El 98,8% de los aislados pertenecieron al complejo C. neoformansy el 1,1% al complejo C. gattii. El genotipo VNI (MATa) fue el más frecuente (n = 326; 87,6%), le siguieron VNII (MATa) (n = 22; 5,9%), el híbrido VNII-VNIV (aADa) (n = 14; 3,8%), VNIV (MATa) (n =4; 1,1%) y los híbridos VNIII (aADa) (n = 1; 0,3%) y VNIII (aADa) (n = 1; 0,3%). La región Centro mostró el mayor número de casos y la mayor diversidad de genotipos. Cabe destacar que el genotipo VNII (MATa) tuvo una frecuencia relativamente alta en las regiones de Cuyo, Noreste y Noroeste. En esta última región, también fue alta la frecuencia del híbrido VNII-VNIV (aADa). La frecuencia de aislamiento de miembros del complejo C. gattii fue baja: se obtuvieron 3 aislados VGI (MATa) y 1 VGII (MATa) de las regiones Centro y Noroeste. En conclusión, este estudio muestra que las frecuencias de genotipos varían entre las distintas regiones de Argentina y señala la presencia de híbridos poco comunes en una frecuencia relativamente alta dentro de la región Noroeste. Contar con mayor número de estudios clínicos y ambientales regionales podría ayudar a elucidar si tales variaciones están asociadas a la existencia de nichos ecológicos particulares o a algún otro factor regional.

Crecimiento del complejo Cryptococcus neoformans/ Cryptococcus gattiien extractos de excretas de palomas / Growth of Cryptococcus neoformans/Cryptococcus gattii species complex in pigeon excreta / Crescimento do complexo Cryptococcus neoformans/ Cryptococcus gattii em extratos de excrementos de pombos

RESUMEN Cryptococcus neoformans/Cryptococcus gattii son agentes etiológicos de la criptococosis, el primero tiene distribución mundial y su hábitat principal son las excretas de palomas (Columba livia), mientras que el último se ha aislado de árboles y en pocas ocasiones de heces de aves. En este estudio se comparó en condiciones de laboratorio, el crecimiento de C. neoformans y C. gattii en extractos de excretas de paloma. Se utilizaron dos extractos de heces de aves cautivas: uno de una muestra en la que se aisló C. neoformans (extracto positivo) y otro de una muestra negativa para este hongo (extracto negativo), los cuales fueron inoculados con C. neoformans y C. gattii. Los extractos permitieron el desarrollo de las dos especies y, mostraron un patrón de crecimiento uniforme en el extracto negativo, mientras que, en el positivo, el crecimiento de C. neoformans fue mayor comparado con el de C. gattii (P = 0.0001). El crecimiento de C. gattii fue menor a un pH superior a 7,5, mientras que el crecimiento de C. neoformans no mostró diferencias significativas en los dos extractos independiente de las variaciones del pH. Se necesitan más estudios para elucidar la interacción de estas dos especies en las excretas de palomas.

ABSTRACT Cryptococcus neoformans/Cryptococcus gattii are etiologic agents of cryptococcosis, the former has a worldwide distribution, and its habitat is associated with pigeon excreta (Columba livia), whereas the latter it has been mainly associated with trees and rarely from the feces of birds. In this study was to compare, under laboratory conditions, the growth of C. neoformans and C. gattii in extracts of pigeon excreta. Two extracts of captive bird feces were used: one prepared from a sample in which C. neoformans was isolated (positive extract) and one from which sample was negative for this fungus (negative extract). Under laboratory conditions, the extracts allowed the development of both species. Both showed a uniform pattern of growth in the negative extract, whereas in the positive, C. neoformans growth was significantly higher than that of C. gattii (P = 0.0001). The growth of the C. gattii was lower in positive extracts and it seems to decrease at pH greater than 7.5, whereas the growth of C. neoformans showed no significant differences regardless of pH variations. Further studies are need to elucidate the interaction between these two species in the excreta of pigeon.

RESUMO Cryptococcus neoformans / Cryptococcus gattii são agentes etiológicos da criptococose, o primeiro tem distribuição mundial e seu habitat principal é a excreção de pombos (Columba livia), enquanto o último foi isolado de árvores e raramente de fezes de aves. Neste estudo, o crescimento de C. neoformans e C. gattii nos extratos de excrementos de pombo foi comparado em condições de laboratório. Foram utilizados dois extratos de fezes de aves em cativeiro: um de uma amostra em que C. neoformans (extrato positivo) foi isolado e outro de uma amostra negativa para esse fungo (extrato negativo), que foi inoculado com C. neoformans e C gattii Os extratos permitiram o desenvolvimento das duas espécies e mostraram um padrão de crescimento uniforme no extrato negativo, enquanto no positivo o crescimento de C. neoformans foi maior em comparação ao de C. gattii (P = 0,0001). O crescimento de C. gattii foi menor a pH maior que 7,5, enquanto o crescimento de C. neoformans não mostrou diferenças significativas nos dois extratos, independentemente das variações de pH. Mais estudos são necessários para elucidar a interação dessas duas espécies nos excrementos de pombos.

Sensibilidad in vitro de cepas chilenas de Cryptococcus neoformans de origen clínico / In vitro susceptibility of Chilean Cryptococcus neoformans strains of clinical source

Resumen Introducción: Las infecciones por levaduras del género Cryptococcus afectan principalmente a pacientes con déficit de la inmunidad mediada por células. Han sido escasos los estudios de sensibilidad realizados para este género en Chile. Objetivos: Determinar la sensibilidad in vitro de Cryptococcus sp a antifúngicos de uso habitual y evaluar la concordancia esencial entre sensibilidad determinada por microdilución en caldo y por difusión en agar con tiras comerciales. Materiales y Método: Estudio descriptivo de 21 cepas aisladas desde liquido céfalo-raquídeo y sangre. Las CIM50 y CIM90 para fluconazol, voriconazol y anfotericina B se determinaron por microdilución en caldo (Sensititre Yeast One®) y por difusión en agar con tiras comerciales (MIC Test Strips). Resultados: Todas las cepas correspondieron a C. neoformans. Los rangos de CIM50 y CIM90 para cada antifúngico estudiado fueron amplios por ambos métodos. La concordancia esencial entre microdilución y difusión en agar con tiras comerciales fue de 24, 62 y 29% para fluconazol, voriconazol y anfotericina B, respectivamente. Conclusiones: La prueba de Sensititre Yeast One® y la de difusión en agar con tiras comerciales, MIC Test Strips, tienen una pobre concordancia esencial para fluconazol y anfotericina B.

Abstract Background: Cryptococcus yeast infections primarily affect immunocompromised patients. There have been few susceptibility studies conducted for this genus in Chile. Aims: To determine the in vitro susceptibility to commonly used antifungals and evaluate the concordance between susceptibility determined by microdilution in broth and commercially available strips. Methods: Descriptive study of 21 Cryptococcus strains, isolated from cerebrospinal fluid and blood. The MIC50 and MIC90 for fluconazole, voriconazole and amphotericin B was determined by broth microdilution (Sensititre Yeast One®) and by commercial drug sensitivity strips (MIC Test Strips). Results: All strains corresponded to C. neoformans. The ranges of MIC50 and MIC90 for each antifungal studied were wide by both methods. The essential agreement between Sensititre Yeast One test and strips was 24, 62 and 29% for fluconazole, voriconazole and amphotericin B, respectively. Conclusions: The Sensititre Yeast One test and MIC Test Strips exhibited poor essential concordance, especially for fluconazole and amphotericin B.

Clinical and microbiological characteristics of cryptococcosis at an university hospital in China from 2013 to 2017

ABSTRACT Background: This study aims to explore the epidemiology, clinical profile and strain characteristics of cryptococcosis from 2013 to 2017 in a major teaching hospital in China. Methods: Trends in antifungal drug susceptibility of 217 consecutive non-repetitive cryptococcal isolates collected from patients of an university hospital in China were analyzed between 2013 and 2017. Of those, 98 isolates were conserved for identification by internal transcribed spacer (ITS) sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system. Multilocus sequence typing (MLST) was used to designate molecular types. Clinical characteristics of the 98 patients with cryptococcosis during the period of 2013-2017 were retrospectively evaluated. Results: There was a trend for gradual increase in the MIC range of fluconazole was from 2013 to 2017. The conserved 98 clinical cryptococcal isolates included 97 C. neoformans and one C. gattii, and 90 (91.8%) isolates belonged to ST5 genotype VNI. Out of the 98 patients with cryptococcosis, 28 (28.6%) were HIV-infected and 32 (32.7%) had no underlying diseases. HIV-infected patients had higher mortality than HIV-uninfected patients (28.6% vs 14.3%, p = 0.147). Conclusions: Most of the patients with cryptococcosis were not HIV-infected in this study, while patients with HIV had a higher mortality. Reduced susceptibility to fluconazole was observed among C. neoformans isolates, most of them belonged to ST5 genotype VNI having an impact on the effective dose of fluconazole.

Synergic effect of simvastatin in combination with amphotericin B against environmental strains of Cryptococcus neoformans from northeastern Brazil: a prospective experimental study

BACKGROUND: Statins are used as cholesterol-lowering drugs and may also have direct antimicrobial effects. OBJECTIVE: To evaluate synergic interactions between simvastatin and both amphotericin B and fluconazole, against environmental strains of Cryptococcus neoformans isolated from captive birds' droppings. DESIGNAND SETTING: Experimental study conducted at Federal University of Piauí, Parnaíba, in collaboration with Federal University of Triângulo Mineiro, Uberaba, Brazil. METHODS: Statin susceptibility tests of Cryptococcus neoformans samples were performed as prescribed in standards. Interactions of simvastatin with amphotericin and fluconazole were evaluated using the checkerboard microdilution method. Presence of these interactions was quantitatively detected through determining the fractional inhibitory concentration index (FICI). RESULTS: Isolates of Cryptococcus neoformans were obtained from 30 of the 206 samples of dry bird excreta (14.5%) that were collected from pet shops and houses. Ten isolates were selected for susceptibility tests. All of them were susceptible to amphotericin and fluconazole. All presented minimum inhibitory concentration (MIC) > 128 µg/ml and, thus, were resistant in vitro to simvastatin. An in vitro synergic effect was shown through combined testing of amphotericin B and simvastatin, such that six isolates (60%) presented FICI < 0.500. Two isolates showed considerable reductions in MIC, from 1 µg/ml to 0.250 µg/ml. No synergic effect was observed through combining fluconazole and simvastatin. CONCLUSION: These results demonstrate that simvastatin should be considered to be a therapeutic alternative, capable of potentiating the action of amphotericin B. However, further studies are necessary to clarify the real effect of simvastatin as an antifungal agent.

Multilocus sequence typing (MLST) of clinical and environmental isolates of Cryptococcus neoformans and Cryptococcus gattii in six departments of Colombia reveals high genetic diversity

Abstract INTRODUCTION: The average annual incidence of cryptococcosis in Colombia is 0.23 cases per 100,000 inhabitants in the general population, and 1.1 cases per 1000 in inhabitants with Acquired Immune Deficiency Syndrome (AIDS). In addition, the causal fungus has been isolated from the environment, with serotypes A-B and C in different regions. This study aims to determine the genetic association between clinical and environmental isolates of C. neoformans/C. gattii in Colombia. METHODS: Multilocus sequence typing (MLST) was used to identify possible clones, providing information about the epidemiology, ecology, and etiology of this pathogen in Colombia. RESULTS: A total of 110 strains, both clinical (n=61) and environmental (n=49), with 21 MLST sequence types (ST) of C. neoformans (n=14STs) and C. gattii (n=7STs) were identified. The STs which shared clinical and environmental isolate sources were grouped in different geographical categories; for C. neoformans, ST93 was identified in six departments, ST77 in five departments; and for C. gattii, ST25 was identified in three departments and ST79 in two. CONCLUSIONS: High genetic diversity was found in isolates of C. neoformans/gattii by MLST, suggesting the presence of environmental sources harboring strains which may be sources of infection for humans, especially in immunocompromised patients; these data contribute to the information available in the country on the distribution and molecular variability of C. neoformans and C. gattii isolates recovered in Colombia.

Synergistic effect of ibuprofen with itraconazole and fluconazole against Cryptococcus neoformans

The present study investigated the association of the non-steroidal anti-inflammatory drug ibuprofen with itraconazole, fluconazole and amphotericin B against Cryptococcus neoformans isolates. The minimal inhibitory concentration (MIC) was found according to M27-A3 protocol and in vitro interactions were evaluated using checkerboard microdilution method. Synergism was demonstrated between azoles and ibuprofen for most isolates. However, no synergistic effects were seen when amphotericin B was combined with ibuprofen. Therefore, our results suggest that ibuprofen presents clinical potential when combined with azole drugs in the treatment of cryptococcosis.