Real-Time PCR in HIV/Trypanosoma cruzi Coinfection with and without Chagas Disease Reactivation: Association with HIV Viral Load and CD4+ Level

Chagas disease is endemic in Latin America and is caused by the flagellate protozoan T. cruzi. The acute phase is asymptomatic in the majority of the cases and rarely causes inflammation of the heart or the central nervous system. Most infected patients progress to a chronic phase, characterized by cardiac or digestive involvement when not asymptomatic. However, when patients are also exposed to an immunosuppressant (such as chemotherapy), neoplasia, or other infections such as HIV, T. cruzi infection may develop into a severe disease (Chagas disease reactivation) involving the heart and central nervous system. The current microscopic methods for diagnosing Chagas disease reactivation are not sensitive enough to prevent the high rate of death observed in these cases. Therefore, we propose a quantitative method to monitor blood levels of the parasite, which will allow therapy to be administered as early as possible, even if the patient has not yet presented symptoms

Results of xenodiagnosis^a, C-PCR^b and qRT-PCR^c in the three groups of patients^d (CO, CR, RE).

a<p>XD - Xenodiagnosis (% of nymphs positive per assay) was performed in 90 patients,</p>b<p>C-PCR (number of parasites) in 38 patients and</p>c<p>qRT-PCR in 91 patients (Ct for 53 positive samples and number of parasites for 91 patients = 91 samples),</p>d<p>groups of patients: chronic Chagas disease with (CO, 29 patients) and without HIV infection (CR, 57 patients) and reactivation of Chagas disease in HIV infected patients (RE, 5 patients).</p

Sensitivities of the qualitative PCR^a, xenodiagnosis^b and blood culture^c in Chagas disease with/without HIV infection.

a<p>qualitative S35/36 PCR,</p>b<p>xenodiagnosis (XD),</p>c<p>blood culture (BC),</p>d<p>groups of patients: chronic Chagas disease with (CO) and without HIV infection (CR) and reactivation of Chagas disease in HIV infected patients (RE). Xenodiagnosis was not performed on three samples from chronic chagasic patients. Blood culture was not performed on one sample from co-infected patient.</p

Standardization of qRT-PCR using the SYBR Green system.

<p><b>A)</b> Standard amplification curve generated by10 fold serial dilutions of DNA from blood spiked with <i>T. cruzi</i> epimastigotes DNA (from 5.10⁴ to 5.10⁻³ parasites/mL) and negative controls, threshold = 0.12, efficiency = 1.05. <b>C)</b> Linear regression curve and regression coefficient, <i>R</i>² = 0.986. <b>B)</b>. Melting curve analysis of amplicons of samples represented in A: Tm = 89.66±0.25°C.</p

C-PCR and qRT-PCR in the blood of patients' groups (CR, CO and RE).

<p><b>A)</b> Number of <i>T. cruzi</i>/mL (log₁₀) in blood by C-PCR. CR (<i>n</i> = 17); CO (<i>n</i> = 16) and RE (<i>n</i> = 5). The line represents the median. Comparative analysis among the groups showed a statistically significant difference (<i>P</i><0.001) by Kruskal–Wallis test. Comparing the groups, a statistically significant difference was observed between CR×CO, <i>P</i><0.001; CR×RE, <i>P</i><0.001; and CO×RE, <i>P</i>>0.05) (Dunn s Multiple Comparison test). <b>B)</b> Number of <i>T. cruzi</i>/mL (log₁₀) in blood by qRT-PCR. CR (<i>n</i> = 57); CO (<i>n</i> = 29) and RE (<i>n</i> = 5). Comparison of the qRT-PCR results observed in the three groups of patients with Chagas disease. The results revealed a statistically significant difference (<i>P</i><0.001) by Kruskal–Wallis test, and comparison between the groups showed statistically significant differences between CR×CO (<i>P</i><0.001), CR×RE (<i>P</i><0.001), and CO×RE, (<i>P</i><0.05) (Dunn's Multiple Comparison test).</p

Correlation between the number of parasites/mL of blood (qRT-PCR) and CD4⁺/CD8⁺ T cell ratio.

<p>Spearman correlation index (<i>r</i>_s) = −0.484, <i>P</i> = 0.007 by analysis of 30 samples from patients infected with HIV/<i>T. cruzi</i> with and without Chagas disease reactivation.</p

Amplification of T. cruzi DNA extracted from blood of four patients with HIV/T. cruzi coinfection (CO).

<p>Colored continuous lines represent CO patients (4.83–142.4 parasites/mL), and black line represents the positive control, 5×10⁻¹⁴ parasites/mL. <b>A)</b> Dashed lines represent the controls without Chagas disease, and the negative controls for the reagents and room of DNA application. <b>B)</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.</p

Correlation between number of parasites/mL of blood by competitive PCR (C-PCR) and real-time PCR (qRT-PCR).

<p>Correlation between number of <i>T. cruzi</i>/mL in 38 paired samples from patients infected with HIV/<i>T. cruzi</i> with or without Chagas disease reactivation. Spearman's correlation index (<i>r</i>_s) = −0.725, <i>P</i><0.001.</p