Real-time PCR in HIV/Trypanosoma cruzi coinfection with and without Chagas disease reactivation: association with HIV viral load and CD4 level

Abstract

Background: Reactivation of chronic Chagas disease, which occurs in approximately 20% of patients coinfected with HIV/Trypanosoma cruzi (T. cruzi), is commonly characterized by severe meningoencephalitis and myocarditis. The use of quantitative molecular tests to monitor Chagas disease reactivation was analyzed.

Methodology: Polymerase chain reaction (PCR) of kDNA sequences, competitive (C-) PCR and real-time quantitative (q) PCR were compared with blood cultures and xenodiagnosis in samples from 91 patients (57 patients with chronic Chagas disease and 34 with HIV/T. cruzi coinfection), of whom 5 had reactivation of Chagas disease and 29 did not.

Principal findings: qRT-PCR showed significant differences between groups; the highest parasitemia was observed in patients infected with HIV/T. cruzi with Chagas disease reactivation (median 1428.90 T. cruzi/mL), followed by patients with HIV/T. cruzi infection without reactivation (median 1.57 T. cruzi/mL) and patients with Chagas disease without HIV (median 0.00 T. cruzi/mL). Spearman's correlation coefficient showed that xenodiagnosis was correlated with blood culture, C-PCR and qRT-PCR. A stronger Spearman correlation index was found between C-PCR and qRT-PCR, the number of parasites and the HIV viral load, expressed as the number of CD4(+) cells or the CD4(+)/CD8(+) ratio.

Conclusions: qRT-PCR distinguished the groups of HIV/T. cruzi coinfected patients with and without reactivation. Therefore, this new method of qRT-PCR is proposed as a tool for prospective studies to analyze the importance of parasitemia (persistent and/or increased) as a criterion for recommending pre-emptive therapy in patients with chronic Chagas disease with HIV infection or immunosuppression. As seen in this study, an increase in HIV viral load and decreases in the number of CD4(+) cells/mm(3) and the CD4(+)/CD8(+) ratio were identified as cofactors for increased parasitemia that can be used to target the introduction of early, pre-emptive therapy.

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, R ² = 0.986. B). Melting curve analysis of amplicons of samples represented in A: Tm = 89.66±0.25°C.

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).

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 (r _s) = −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 (r _s) = −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 (r_s) = 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.