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.