Epidemiological studies found that hepatitis E virus genotype 3 (HEV-3) infection was associated with chronic hepatitis and cirrhosis in immunocompromised patients. Our study aimed to investigate the relationship between the host immunosuppressive status and the occurrence of HEV-related chronic hepatitis. Here we describe a successful experimental study, using cynomolgus monkeys previously treated with tacrolimus, a potent calcineurin inhibitor immunosuppressant, and infected with a Brazilian HEV-3 strain isolated from naturally infected pigs. HEV infected monkeys were followed up during 160 days post infection (dpi) by clinical signs; virological, biochemical and haematological parameters; and liver histopathology. The tacrolimus blood levels were monitored throughout the experiment. Immunosuppression was confirmed by clinical and laboratorial findings, such as: moderate weight loss, alopecia, and herpes virus opportunistic infection. In this study, chronic HEV infection was characterized by the mild increase of liver enzymes serum levels; persistent RNA viremia and viral faecal shedding; and liver histopathology. Three out of four immunosuppressed monkeys showed recurrent HEV RNA detection in liver samples, evident hepatocellular ballooning degeneration, mild to severe macro and microvesicular steatosis (zone 1), scattered hepatocellular apoptosis, and lobular focal inflammation. At 69 dpi, liver biopsies of all infected monkeys revealed evident ballooning degeneration (zone 3), discrete hepatocellular apoptosis, and at most mild portal and intra-acinar focal inflammation. At 160 dpi, the three chronically HEV infected monkeys showed microscopic features (piecemeal necrosis) corresponding to chronic hepatitis in absence of fibrosis and cirrhosis in liver parenchyma. Within 4-months follow up, the tacrolimus-immunosuppressed cynomolgus monkeys infected with a Brazilian swine HEV-3 strain exhibited more severe hepatic lesions progressing to chronic hepatitis without liver fibrosis, similarly as shown in tacrolimus-immunosuppressed solid organ transplant (SOT) recipients. The cause-effect relationship between HEV infection and tacrolimus treatment was confirmed in this experiment.

Determination of urinary catecholamines and metanephrines is important for diagnosis of pheochromocytoma. Liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed for the determination of catecholamines and metanephrines in human urine using deuterated internal standards (IS). In this method, 1000 µL of urine samples were treated by liquid-liquid extraction using ethyl acetate and subjected to LC-MS/MS analysis using positive electrospray ionization (ESI+). A BDS HYPERSILTM C18 column (125 mm × 3 mm, 3 µm) was used and mobile phase was water:methanol (98:2, v/v) with 0.25% of formic acid at 200 µ L min −1 . The method has a chromatographic running time of approximately 10 min. The average of recovery was 92.9-106.1% for epinephrine, 94.6-107.7% for norepinephrine, 98.0-108.8% the dopamine, 94.2-105.1% the metanephrine, 97.0-106.0% for normetanephrine. The precision was lower than 7.0% for all analytes. Furthermore, this method has been implemented successfully in routine laboratory due to its easy execution and excellent precision. ]]>

No abstract

Zika still poses a threat to global health owing to its association with serious neurological conditions and the absence of a vaccine and treatment. Sofosbuvir, an anti-hepatitis C drug, has shown anti-Zika effects in animal and cell models. Thus, this study aimed to develop and validate novel LC-MS/MS methods for the quantification of sofosbuvir and its major metabolite (GS-331007) in human plasma and cerebrospinal (CSF) and seminal fluid (SF), and apply the methods to a pilot clinical trial. The samples were prepared by liquid-liquid extraction and separated using isocratic mode on Gemini C 18 columns. Analytical detection was performed using a