Figure 1.
Standardization of qRT-PCR using the SYBR Green system.
A) Standard amplification curve generated by10 fold serial dilutions of DNA from blood spiked with T. cruzi epimastigotes DNA (from 5.104 to 5.10−3 parasites/mL) and negative controls, threshold = 0.12, efficiency = 1.05. C) Linear regression curve and regression coefficient, R2 = 0.986. B). Melting curve analysis of amplicons of samples represented in A: Tm = 89.66±0.25°C.
Table 1.
Sensitivities of the qualitative PCRa, xenodiagnosisb and blood culturec in Chagas disease with/without HIV infection.
Table 2.
Results of xenodiagnosisa, C-PCRb and qRT-PCRc in the three groups of patientsd (CO, CR, RE).
Figure 2.
C-PCR and qRT-PCR in the blood of patients' groups (CR, CO and RE).
A) Number of T. cruzi/mL (log10) in blood by C-PCR. CR (n = 17); CO (n = 16) and RE (n = 5). The line represents the median. Comparative analysis among the groups showed a statistically significant difference (P<0.001) by Kruskal–Wallis test. Comparing the groups, a statistically significant difference was observed between CR×CO, P<0.001; CR×RE, P<0.001; and CO×RE, P>0.05) (Dunn s Multiple Comparison test). B) Number of T. cruzi/mL (log10) in blood by qRT-PCR. CR (n = 57); CO (n = 29) and RE (n = 5). Comparison of the qRT-PCR results observed in the three groups of patients with Chagas disease. The results revealed a statistically significant difference (P<0.001) by Kruskal–Wallis test, and comparison between the groups showed statistically significant differences between CR×CO (P<0.001), CR×RE (P<0.001), and CO×RE, (P<0.05) (Dunn's Multiple Comparison test).
Table 3.
Spearman's rank correlation coefficients observed between tests: blood culture, xenodiagnosis, competitive PCR, quantitative real-time PCR.
Figure 3.
Amplification of T. cruzi DNA extracted from blood of four patients with HIV/T. cruzi coinfection (CO).
Colored continuous lines represent CO patients (4.83–142.4 parasites/mL), and black line represents the positive control, 5×10−14 parasites/mL. A) Dashed lines represent the controls without Chagas disease, and the negative controls for the reagents and room of DNA application. B) Dashed lines represent the negative control individual without Chagas disease for each patient. Dotted lines represent the negative controls for the reagents and DNA application.
Figure 4.
Correlation between number of parasites/mL of blood by competitive PCR (C-PCR) and real-time PCR (qRT-PCR).
Correlation between number of T. cruzi/mL in 38 paired samples from patients infected with HIV/T. cruzi with or without Chagas disease reactivation. Spearman's correlation index (rs) = −0.725, P<0.001.
Figure 5.
Correlation between the number of parasites/mL of blood (qRT-PCR) and CD4+/CD8+ T cell ratio.
Spearman correlation index (rs) = −0.484, P = 0.007 by analysis of 30 samples from patients infected with HIV/T. cruzi with and without Chagas disease reactivation.
Figure 6.
Correlation between number of T. cruzi/mL of blood (qRT-PCR) and HIV RNA copies/mL of plasma).
Spearman's correlation coefficient (rs) = 0.584, P = 0.007 (Analysis of 20 samples from patients infected with HIV/T. cruzi with and without Chagas disease reactivation). In this Figure, 8 samples were superimposed in the lower limit of viral load and number of parasites/mL.