Material and methods
Serum samples. Serum samples were collected using vacuum tube systems after a garroting period of 1 minute or less. Five ml of total blood was harvested with a dry tube without additives and without gel seed. After clot retraction was observed, the samples were centrifuged at 3000 rpm for 10 minutes at room temperature. Samples were stored at -80 °C.[11] All samples were classified and catalogued, including clinical and demographic information from each patient.
Molecular diagnosis. The CHIVK infection status previously confirmed by qRT-PCR by our group.[12]
Biochemical analysis. The biochemical analysis was performed on a fully automatic analyzer, Cobas® 6000 module (Cobas C501, Roche Diagnostics International Ltd., Rotkreuz, SWZ), using kits provided by Roche Diagnostics International Ltd. The dosage of the biochemical profile was made by kinect enzymatic and colorimetric methods as dictated by Roche Diagnostics International Ltd.[13,14,15]
Statistical analysis. To evaluate clinical association among renal markers and hepatic alterations, we performed the Student’s t-test, on data from patients that presented symptoms and markers alterations under a statistic significance of p <0.05. Some comparisons were not done because of a lack of proper patient records or a lack of sample material. Because gene expression and viral genome replication show logarithmic variation, we used a Kolmogorov-Smirnov test (K-S test) to verify correlations of clinical test results with Cts obtained from the same patients. The K-S, is a non-parametric test on the equality of continuous and one-dimensional probability distributions that can be used to compare a sample with a reference probability distribution (uniamostral K-S test) or two samples with each other (bi-lateral K-S test).[16]
A critical p >0.05 level was assumed as indication of difference in distributions between changes in clinical markers and Cts of patients.