RESUMEN
The ability to detect SARS-CoV-2 in the upper respiratory tract ceases after 2 to 3 weeks post-symptom-onset in most patients. In contrast, SARS-CoV-2 can be detected in the stool of some patients for greater than 4 weeks, suggesting that stool may hold utility as an additional source for diagnosis. We validated the Cepheid Xpert Xpress SARS-CoV-2 and Hologic Panther Fusion real-time RT-PCR assays for detection of viral RNA in stool specimens and compared performance. We utilized remnant stool specimens (n = 79) from 77 patients with gastrointestinal symptoms. Forty-eight patients had PCR-confirmed COVID-19, and 29 either were nasopharyngeal/oropharyngeal PCR negative or presented for reasons unrelated to COVID-19 and were not tested. Positive percent agreement between the Cepheid and Hologic assays was 93% (95% confidence interval [CI]: 81.1% to 98.2%), and negative percent agreement was 96% (95% CI: 89% to 0.99%). Four discrepant specimens (Cepheid positive only, n = 2; Hologic positive only, n = 2) exhibited average cycle threshold (CT ) values of >37 for the targets detected. Of the 48 patients with PCR-confirmed COVID-19, 23 were positive by both assays (47.9%). For the negative patient group, 2/29 were positive by both assays (6.9%). The two stool PCR-positive, nasopharyngeal/oropharyngeal PCR-negative patients were SARS-CoV-2 IgG positive. Our results demonstrate acceptable agreement between two commercially available molecular assays and support the use of stool PCR to confirm diagnosis when SARS-CoV-2 is undetectable in the upper respiratory tract.
Asunto(s)
Betacoronavirus/genética , Técnicas de Laboratorio Clínico , Infecciones por Coronavirus/diagnóstico , Heces/virología , Neumonía Viral/diagnóstico , Reacción en Cadena de la Polimerasa , COVID-19 , Prueba de COVID-19 , Vacunas contra la COVID-19 , Técnicas de Laboratorio Clínico/métodos , Técnicas de Laboratorio Clínico/normas , Técnicas de Laboratorio Clínico/estadística & datos numéricos , Humanos , Límite de Detección , Pandemias , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa/normas , Reacción en Cadena de la Polimerasa/estadística & datos numéricos , ARN Viral/análisis , ARN Viral/genética , Reproducibilidad de los Resultados , SARS-CoV-2RESUMEN
The fungal pathogen Histoplasma capsulatum causes a spectrum of disease, ranging from local pulmonary infection to disseminated disease. The organism seeks residence in macrophages, which are permissive for its survival. Hypoxia-inducible factor 1α (HIF-1α), a principal regulator of innate immunity to pathogens, is necessary for macrophage-mediated immunity to H. capsulatum in mice. In the present study, we analyzed the effect of HIF-1α in human macrophages infected with this fungus. HIF-1α stabilization was detected in peripheral blood monocyte-derived macrophages at 2 to 24 h after infection with viable yeast cells. Further, host mitochondrial respiration and glycolysis were enhanced. In contrast, heat-killed yeasts induced early, but not later, stabilization of HIF-1α. Since the absence of HIF-1α is detrimental to host control of infection, we asked if large amounts of HIF-1α protein, exceeding those induced by H. capsulatum, altered macrophage responses to this pathogen. Exposure of infected macrophages to an HIF-1α stabilizer significantly reduced recovery of H. capsulatum from macrophages and produced a decrement in mitochondrial respiration and glycolysis compared to those of controls. We observed recruitment of the autophagy-related protein LC3-II to the phagosome, whereas enhancing HIF-1α reduced phagosomal decoration. This finding suggested that H. capsulatum exploited an autophagic process to survive. In support of this assertion, inhibition of autophagy activated macrophages to limit intracellular growth of H. capsulatum Thus, enhancement of HIF-1α creates a hostile environment for yeast cells in human macrophages by interrupting the ability of the pathogen to provoke host cell autophagy.
Asunto(s)
Histoplasma/inmunología , Histoplasmosis/inmunología , Subunidad alfa del Factor 1 Inducible por Hipoxia/inmunología , Macrófagos/inmunología , Proteínas Asociadas a Microtúbulos/inmunología , Animales , Autofagia , Histoplasmosis/genética , Histoplasmosis/microbiología , Histoplasmosis/fisiopatología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Macrófagos/microbiología , Masculino , Ratones , Proteínas Asociadas a Microtúbulos/genética , Fagosomas/inmunología , Fagosomas/microbiologíaRESUMEN
Hypoxia-inducible factor (HIF)-1α is a transcription factor that regulates metabolic and immune response genes in the setting of low oxygen tension and inflammation. We investigated the function of HIF-1α in the host response to Histoplasma capsulatum because granulomas induced by this pathogenic fungus develop hypoxic microenvironments during the early adaptive immune response. In this study, we demonstrated that myeloid HIF-1α-deficient mice exhibited elevated fungal burden during the innate immune response (prior to 7 d postinfection) as well as decreased survival in response to a sublethal inoculum of H. capsulatum The absence of myeloid HIF-1α did not alter immune cell recruitment to the lungs of infected animals but was associated with an elevation of the anti-inflammatory cytokine IL-10. Treatment with mAb to IL-10 restored protective immunity to the mutant mice. Macrophages (MÏs) constituted most IL-10-producing cells. Deletion of HIF-1α in neutrophils or dendritic cells did not alter fungal burden, thus implicating MÏs as the pivotal cell in host resistance. HIF-1α was stabilized in MÏs following infection. Increased activity of the transcription factor CREB in HIF-1α-deficient MÏs drove IL-10 production in response to H. capsulatum IL-10 inhibited MÏ control of fungal growth in response to the activating cytokine IFN-γ. Thus, we identified a critical function for MÏ HIF-1α in tempering IL-10 production following infection. We established that transcriptional regulation of IL-10 by HIF-1α and CREB is critical for activation of MÏs by IFN-γ and effective handling of H. capsulatum.
Asunto(s)
Histoplasmosis/inmunología , Subunidad alfa del Factor 1 Inducible por Hipoxia/inmunología , Interleucina-10/inmunología , Macrófagos/inmunología , Animales , Western Blotting , Proteína de Unión a CREB/inmunología , Modelos Animales de Enfermedad , Citometría de Flujo , Histoplasma/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
BACKGROUND Extrapulmonary small cell carcinoma (SmCC) is a relatively rare clinical entity constituting only 2.5-5% of SmCCs. Recently, evidence has emerged that high-risk types of human papillomavirus (HPV) might play an etiologic role in oropharyngeal SmCC, similar to squamous cell carcinoma. CASE REPORT Here, we present a case of tonsillar SmCC that presented as combined SmCC-squamous cell carcinoma in a cervical lymph node, raising the possibility that the SmCC-component represents disease progression. CONCLUSIONS This case lends further support to the importance of HPV in the development of oropharyngeal SmCC and suggests a mechanism of disease progression.
Asunto(s)
Carcinoma de Células Pequeñas/patología , Carcinoma de Células Escamosas/patología , Tonsila Palatina/virología , Infecciones por Papillomavirus/diagnóstico , Neoplasias Tonsilares/patología , Carcinoma de Células Pequeñas/diagnóstico por imagen , Carcinoma de Células Escamosas/diagnóstico por imagen , Femenino , Humanos , Ganglios Linfáticos/patología , Metástasis Linfática , Persona de Mediana Edad , Faringitis/etiología , Neoplasias Tonsilares/diagnóstico por imagenRESUMEN
Human pathogenic fungi are highly adaptable to a changing environment. The ability to adjust to low oxygen conditions is crucial for colonization and infection of the host. Recently, the impact of mammalian hypoxia-inducible factor-1α (HIF-1α) on fungal immunity has emerged. In this review, the role of hypoxia and HIF-1α in fungal infections is discussed regarding the innate immune response.
Asunto(s)
Hongos/inmunología , Hongos/patogenicidad , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/patología , Inmunidad Innata , Micosis/inmunología , Micosis/patología , Animales , Modelos Animales de Enfermedad , Hongos/crecimiento & desarrollo , HumanosRESUMEN
Histoplasma capsulatum, an environmental fungus, is the most common endemic pulmonary mycosis in the USA. Disease is most frequently observed in immunocompromised patients living in endemic areas. We present the mechanisms of fungal recognition, innate immune response and adaptive immune response that lead to protection or exacerbation of disease. Current understanding of these mechanisms is the result of a continuing dialogue between clinical observations and murine studies. Mice are a powerful model to study the immune response to H. capsulatum alone or in the presence of immunomodulatory drugs. Vigilance for histoplasmosis should be exercised with novel immunosuppressive agents that target the important immune pathways identified here.
Asunto(s)
Histoplasma/inmunología , Histoplasmosis/epidemiología , Histoplasmosis/inmunología , Enfermedades Pulmonares Fúngicas/epidemiología , Enfermedades Pulmonares Fúngicas/inmunología , Inmunidad Adaptativa , Animales , Modelos Animales de Enfermedad , Enfermedades Endémicas , Histoplasma/aislamiento & purificación , Histoplasmosis/microbiología , Histoplasmosis/patología , Humanos , Inmunidad Innata , Huésped Inmunocomprometido , Enfermedades Pulmonares Fúngicas/microbiología , Enfermedades Pulmonares Fúngicas/patología , Ratones , Infecciones Oportunistas/epidemiología , Infecciones Oportunistas/inmunología , Infecciones Oportunistas/microbiología , Infecciones Oportunistas/patología , Estados Unidos/epidemiologíaRESUMEN
Luciferase reporter plasmids (pGL3 backbone, Promega) have been utilized to characterize the transcriptional effects of the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other aryl hydrocarbon receptor (AhR) ligands. Following ligand activation, the AhR and its dimerization partner AhR nuclear translocator (ARNT) regulate transcription by binding dioxin response elements (DREs) in regulatory regions of dioxin-sensitive genes. Upon sequencing of our luciferase reporters, we unexpectedly identified a DRE core motif within the multiple cloning site (mcsDRE) of the pGL3 luciferase plasmid backbone in a subset of our reporters. Therefore, the objective of this study was to determine if the mcsDRE inadvertently influences reporter activity. Utilizing deletional analysis we determined that the mcsDRE did significantly alter the transcriptional effect induced by TCDD. Since many chemicals have been shown to interact with the AhR and influence transcription through the DRE, the presence of the mcsDRE in the pGL3 luciferase plasmid may inappropriately influence promoter and enhancer analysis. As such, insertion of regulatory elements into pGL3 reporters should be designed to avoid retaining the mcsDRE core motif (GCGTG) and currently utilized pGL3 reporters should be evaluated for the presence of the mcsDRE.