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

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 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

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

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 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

Quantitative PCR as a marker for preemptive therapy and its role in therapeutic control in Trypanosoma cruzi/HIV coinfection.

BackgroundTrypanosoma cruzi and HIV coinfection can evolve with depression of cellular immunity and increased parasitemia. We applied quantitative PCR (qPCR) as a marker for preemptive antiparasitic treatment to avoid fatal Chagas disease reactivation and analyzed the outcome of treated cases.MethodologyThis mixed cross-sectional and longitudinal study included 171 Chagas disease patients, 60 coinfected with HIV. Of these 60 patients, ten showed Chagas disease reactivation, confirmed by parasites identified in the blood, cerebrospinal fluid, or tissues, 12 exhibited high parasitemia without reactivation, and 38 had low parasitemia and no reactivation.ResultsWe showed, for the first time, the success of the timely introduction of benznidazole in the non-reactivated group with high levels of parasitemia detected by qPCR and the absence of parasites in reactivated cases with at least 58 days of benznidazole. All HIV+ patients with or without reactivation had a 4.0-5.1 higher chance of having parasitemia than HIV seronegative cases. A positive correlation was found between parasites and viral loads. Remarkably, treated T. cruzi/HIV-coinfected patients had 77.3% conversion from positive to negative parasitemia compared to 19.1% of untreated patients. Additionally, untreated patients showed ~13.6 times higher Odds Ratio of having positive parasitemia in the follow-up period compared with treated patients. Treated and untreated patients showed no differences regarding the evolution of Chagas disease. The main factors associated with all-cause mortality were higher parasitemia, lower CD4 counts/μL, higher viral load, and absence of antiretroviral therapy.ConclusionWe recommend qPCR prospective monitoring of T. cruzi parasitemia in HIV+ coinfected patients and point out the value of pre-emptive therapy for those with high parasitemia. In parallel, early antiretroviral therapy introduction is advisable, aiming at viral load control, immune response restoration, and increasing survival. We also suggest an early antiparasitic treatment for all coinfected patients, followed by effectiveness analysis alongside antiretroviral therapy