ABSTRACT
One of the current greatest challenges of Chagas disease is the establishment of biomarkers to assess the efficacy of drugs in a short period of time. In this context, the reactivity of sera from 66 adults with chronic indeterminate Chagas disease (IND) for a set of four Trypanosoma cruzi antigens (KMP11, PFR2, HSP70, and 3973d) was analyzed before and after benznidazole treatment. The results showed that the reactivity against these antigens decreased at 9, 24, and 48 months after treatment. Moreover, the 42.4% and 68.75% of IND patients met the established standard criteria of therapeutic efficacy (STEC) at 24 and 48 months posttreatment, respectively. Meeting the STEC implied that there was a continuous decrease in the reactivity of the patient sera against the four antigens after treatment and that there was a substantial decrease in the reactivity for at least two of the antigens. This important decrease in reactivity may be associated with a drastic reduction in the parasite load, but it is not necessarily associated with a parasitological cure. After treatment, a positive PCR result was only obtained in patients who did not meet the STEC. The percentage of granzyme B+/perforin+ CD8+ T cells was significantly higher in patients who met the STEC than in those who did not meet the STEC (35.2% versus 2.2%; P < 0.05). Furthermore, the patients who met the STEC exhibited an increased quality of the multifunctional response of the antigen-specific CD8+ T cells compared with that in the patients who did not meet the STEC.
INTRODUCTION
Chagas disease, or American trypanosomiasis, is a chronic infection caused by the protozoan Trypanosoma cruzi (1, 2) that affects approximately 7 million people worldwide (3). This parasitic infection, which is recognized as a neglected tropical disease by the World Health Organization (4, 5), is endemic in the southern United States, Central America, and South America. However, in recent decades, the epidemiological profile of this infection has changed due to new patterns of migration, leading to the globalization of the disease (3).
This parasitic infection presents itself in two successive phases, namely, an acute and a chronic phase. Most patients remain in a clinically silent asymptomatic chronic stage called the indeterminate phase, which can lead to a symptomatic chronic phase that is primarily characterized by cardiac alterations (1, 6, 7). Treatment during the chronic phase of the disease is recommended, and benznidazole and nifurtimox are the drugs that are currently used for treatment (8–10). Benznidazole treatment was effective in controlling parasite persistence and the associated inflammation in experimental chronically infected rats; in this model, only the combination of benznidazole and antioxidants that were capable of modulating or delaying the onset of the oxidative insult and mitochondrial deficiency was shown to be beneficial in preventing cardiac pathology and the loss of left ventricular function in Chagas disease (11). However, it is difficult to determine the treatment efficacy given that the gold standard for evaluating efficacy (seronegativity using conventional serological tests) may take decades to be reached (12, 13). Thus, several authors have also suggested that although the treatment goal for infectious diseases is or should be pathogen elimination, there are other equally important therapeutic outcomes to be considered (10, 14). In this context, the control and reduction of the pathogen burden are well-recognized strategies for some infections, such as AIDS, which is now a classic example of a lethal infection that can be converted into a chronic controlled disease with the administration of appropriate treatments (10).
Moreover, the deficiency in biomarkers for the prediction of the parasitological outcome status and the potential cure of the disease represents a major hurdle for the development of new drugs for Chagas disease (15). Many efforts are being made to develop biomarkers for the follow-up of patients with Chagas disease. Serum biomarkers identified by surface-enhanced laser desorption ionization–time of flight (SELDI-TOF) mass spectrometry (such as MIPP1 alpha, C3a, apolipoprotein A1 [ApoA1], and C3 derivatives) have been proposed as confirmatory tests for Chagas disease (16). The use of the MultiCruzi assay, which is composed of 15 different antigens, allowed the selection of Ag3, which was detected with high titers by sera from most T. cruzi-infected individuals (17) and exhibited the highest titers in patients who had a positive PCR result when parasite detection was performed. Markers associated with cardiac dysfunction, such as NTproBNP, were also correlated with parasitemia and were elevated in patients with chronic Chagas cardiopathology (18).
During the development of reliable tools to evaluate the therapeutic efficacy of drugs for Chagas disease, several markers have been proposed and, in some cases, have been demonstrated to be effective for assessing the responses to specific treatments (19–23). Previous laboratory studies showed that the KMP11, PFR2, HSP70, and 3973 T. cruzi antigens are recognized with high specificity and sensitivity in the sera from patients in the chronic stage of the disease (21, 24). Likewise, a decrease in the reactivity against these antigens was detected shortly after benznidazole treatment (6 and 9 months) (21, 25). These results showed that the treatment of Chagas patients with benznidazole modifies the humoral response pattern against these specific antigens, exhibiting different kinetics depending on whether the patient is in the asymptomatic or symptomatic stage of the disease (21, 25). Relatedly, previous laboratory studies also showed that treatment leads to an improvement in the quality of the antigen-specific response of CD8+ T cells in asymptomatic chronic Chagas disease patients (26). This improved response was associated with a decrease in the frequency of CD8+ T cells coexpressing inhibitory receptors and an increase in the multifunctional capacity (cytokine and cytotoxic molecule expression) of these cells (26). These results are relevant because in T. cruzi infection, as in other chronic infectious diseases, it has been shown that persistent antigenic presence is associated with the gradual development of the dysfunction of CD8+ T cells, which is linked to the expression of several inhibitory receptors that might negatively regulate the function of antigen-specific T cells and compromise pathogen control (27–29). Therefore, the evaluation of the CD8+ T cell response in treated chronic Chagas disease patients could be another useful tool for monitoring treatment effectiveness. Thus, the purpose of the present study was to analyze the predictive value of four previously identified molecules (21, 25) for evaluating the therapeutic response in asymptomatic chronic Chagas disease patients as they may constitute a set of useful biomarkers. Thus, the reactivity of patient sera against these four molecules was evaluated at short (from 9 to 24 months) and long (up to 4 years) time intervals after benznidazole treatment. This study was also focused on analyzing the phenotype and functional capacity of the T. cruzi antigen-specific CD8+ T cells of the patients and evaluating whether patients who met the established therapeutic efficacy criteria had a specific functional profile of antigen-specific CD8+ T cells.
RESULTS
Detection of specific antibodies against KMP11, HSP70, PFR2, and 3973d antigens in sera from chronic Chagas disease patients treated with benznidazole.Previous laboratory studies have demonstrated that the KMP11, PFR2, HSP70, and 3973 T. cruzi antigens were individually recognized with high specificity and sensitivity by sera from chronic Chagas disease patients and that the reactivity against these molecules was modified after treatment with benznidazole (21, 25). Here, we evaluated the dynamics of the reactivity against these molecules (KMP11, HSP70, PFR2, and 3973d) over time after benznidazole treatment, searching for the patterns of therapeutic efficacy and failure. Thus, the reactivity against the KMP11, HSP70, PFR2, and 3973d T. cruzi antigens was analyzed in 66 IND Chagas disease patients before (T0) and after 9, 24, and 48 months of benznidazole treatment (T9, T24, and T48, respectively) by enzyme-linked immunosorbent assay (ELISA). High titers of specific antibodies against the four molecules were detected in all sera at T0, with the exception of the sera from five patients whose titers against one out of the four molecules (or against two molecules in the case of one patient) were comprised, with optical density (OD) values between 0.21 and 0.316 with a 1/100 dilution of the sera. Specifically, two patients had low antibody titers against KMP11, and one of these patients also had a low antibody titer against PFR2, two patients had low titers against 3973d, and one patient had a low titer against the HSP70 antigen. As expected, the antibody levels detected before treatment in the patient sera against the KMP11, HSP70, PFR2, and 3973d antigens progressively decreased during the posttreatment follow-up (Fig. 1A). The decrease in reactivity against the four biomarkers, measured as a drop in the antibody level (OD values) against each one of the antigens and in each sample of each patient at any of the time points tested, was statistically significant when the values obtained at T9 (P < 0.0001), T24 (P < 0.0001), and T48 (P < 0.001) (Fig. 1A) were compared with the pretreatment value (T0), as deduced from the analysis of variance (ANOVA) Friedman test followed by Dunn’s post hoc test for multiple comparisons. As shown in Fig. 1A, the graphical representation with the 95% confidence intervals shows a continuous decrease in the antibody titer against each one of the antigens throughout all the time points posttreatment. Conversely, no downward trend in the antibody levels against the T. cruzi whole-cell lysate (measured by the Ortho T. cruzi ELISA system) was observed in the sera from IND patients at any time during the posttreatment follow-up period (Fig. 1B). In fact, a statistically significant increase in the antibody levels was detected at long-term time points after treatment (T48) relative to those based on the pretreatment OD values (Fig. 1B). When the same data shown in Fig. 1A related to the reactivity of each patient sera against each one of the four antigens (KMP11, HSP70, PFR2, and 3973d) at all the analyzed time points were represented as “before-after” plot, we observed a continuous and substantial drop in the antibody levels in many patients along the posttreatment follow-up (see Fig. S1A in the supplemental material). However, when the analysis was carried out as before, but using the commercially available Ortho T. cruzi ELISA for Chagas disease diagnosis, in those patients who had been analyzed with this test at most time points after treatment (40 patients at T0, T9, and T24 and 15 of them also at T48), no statistically significant differences in reactivity were observed along the time of follow-up (Fig. S1B). As shown in Table 1, the percentage of patients in whom there was a decrease in the level of antibodies against each one of the biomarkers ranged from 76% to 82% at T9, from 86% to 89% at T24, and from 91% to 97% at 48 months. When the decrease in the reactivity against the four biomarkers (BMKs) was evaluated based on a set of molecules (see Fig. S2 in the supplemental material), there was a drop in the reactivity in nearly half of the study population of IND Chagas patients at T9 (45.5%) compared with the pretreatment reactivity. Furthermore, the percentage of patients in whom the level of antibodies against the four BMKs decreased after treatment increased up to 68.2% and 81.3% at 24- and 48-months posttreatment, respectively (Fig. S2). These data were taken as an indication that the drop in reactivity against the 4 molecules within a short time posttreatment could be useful for treatment monitoring and the evaluation of the treatment impact.
Reactivity of the sera from asymptomatic Chagas disease patients against T. cruzi antigens before and after treatment with benznidazole. (A) The levels of antibodies (IgG) against KMP11, HSP70, PFR2, and 3973d were measured by ELISA in the sera from indeterminate (IND) patients before benznidazole treatment (T0) and at 9 (T9), 24 (T24), and 48 (T48) months after treatment. The reactivity of the sera was analyzed in 66 IND patients at T0, T9, and T24 and in 32 patients at T48. Sera were always tested in triplicate and at 1/100, 1/200, 1/400, 1/800, and 1/1600 dilution for each of the four molecules at T0. For the posttreatment follow-up, each patient’s serum was analyzed at two dilutions, which were selected based on the reactivity observed at T0. It was examined whether a decrease or an increase in the reactivity could be observed in response to treatment (OD values were between 0.45 and 2). The sera from the 66 patients were tested at different dilutions against each antigen (1/100 [5 assayed patients for KMP11, 1 for HSP70, and 2 for 3973d], 1/200 [13 assayed patients for KMP11, 4 for HSP70, 5 for PFR2, and 5 for 3973d], 1/400 [41 assayed patients for KMP11, 13 for HSP70, 4 for PFR2, and 1 for 3973d], 1/800 [7 assayed patients for KMP11, 48 for HSP70, 55 for PFR2, and 54 for 3973d], and 1/1600 [2 assayed patients for PFR2 and 4 for 3973d]). The P values were obtained using the Friedman with Dunn’s post hoc test, and the statistically significant differences are indicated (***, P < 0.0001 and ****, P < 0.0001). (B) Detection of the IgG antibody levels against the T. cruzi whole-cell lysate antigens in IND patients before treatment (T0) and at 9, 24, and 48 months posttreatment (T9, T24, and T48, respectively) using the Ortho T. cruzi ELISA system. The reactivity of the sera against T. cruzi whole-cell lysate antigens was analyzed in 62 IND patients at T0, 60 IND patients at T9, 45 IND patients at T24, and 16 IND patients at T48. Data in both graphs are expressed as the optical density (OD) value measured at 492 nm. Notches are extended for 1.58 × interquartile range (IQR)/square root of all values, as this gives a 95% confidence interval for comparing the medians. In this plot, the outliers are the observations that lie outside 1.58 × IQR, where IQR is the difference between the 75th and 25th quartiles. The P values were obtained using the Wilcoxon matched-pair test, and statistically significant differences are indicated (*, P < 0.05).
Percentage of Chagas disease patients who showed a decrease in the antibody level against the Trypanosoma cruzi antigens after treatment
Establishment of the therapeutic efficacy criteria and the behavior of the serological BMK set.The aim of the study was to search for the serological patterns capable of evaluating the impact of treatments and to predict the therapeutic effect associated with efficacy or, by contrast, with failure. Based on the fact that three antigens in the BMK set are not present in humans, the first condition that we used to evaluate the therapeutic efficacy was whether there was a continuous drop in the reactivity against each of these biomarkers after benznidazole treatment (T9<T0; T24/T48<T9, and T24/T48<T0). It was also considered relevant that the drop had to be substantial; the decrease had to be by at least 40% for KMP11, PFR2, and 3973d and 30% for HSP70. A drop in reactivity of 40% was established as substantial based on the fact that the reactivity of the sera from IND Chagas patients against the 3973d antigen was 40% lower than that observed in the sera from symptomatic Chagas disease patients (24). In addition, the median reactivity to the 4 BMKs in sera from IND Chagas disease patients at 24 months after treatment was approximately 35% of that detected before treatment. For the HSP70 antigen, a decrease in the reactivity level of 30% was considered substantial based on the fact that a basal level of anti-HSP70 antibodies was detected in the sera from some healthy donors (30). As shown in Fig. 2, the independent analysis of the behavior of each BMK showed that there was a substantial drop in the reactivity against KMP11, HSP70, PFR2, and 3973d molecules in 41%, 39%, 24%, and 35% of the patients, respectively, (Fig. 2, white bars) at T24 and in 56%, 53%, 47%, and 44% of the patients, respectively, at T48. In a small number of patients, a slight increase in the reactivity against the 4 BMKs was observed at T9 posttreatment (≤20%) compared with that at baseline (T0). This slight increase may have been due to exposure to the antigenic fragments from nonviable parasites in response to benznidazole, as previously reported (24). When this increase was considered, the percentage of the patients who demonstrated the described seroconversion increased up to 44%, 41%, 30%, and 38% for the KMP11, HSP70, PFR2, and 3973d antigens, respectively, at T24 and 66%, 59%, 56%, and 53%, respectively, at T48 (Fig. 2, the black portion of the bars).
Number and percentage of Chagas disease patients whose reactivity against each of the four molecules met the STEC at 24 and 48 months posttreatment. Reactivity was measured against the KMP11, HSP70, PFR2, and 3973d molecules before treatment (T0) and at 9 (T9), 24 (T24), and 48 (T48) months after treatment. White bars represent the number and percentage of indeterminate (IND) patients whose sera reacted against each of the four assayed BMKs and met the following conditions: T9<T0, T24/T48<T9, and T24/T48<T0 at least 40% for KMP11, PFR2, and 3973d or 30% for HSP70. The black portion of the bars includes, in addition, those patients who do not present a change or presented a slight increase in the reactivity (below 20%) at 9 months after treatment, which means T9≥T0; this increase was always below 20% of the reactivity detected at T0.
Next, two criteria were established to measure the predictive value of the BMK set in regard to the therapeutic efficacy at 24 and 48 months after treatment. In both criteria, a drop in the reactivity against the four BMKs was required after treatment. The standard therapeutic efficacy criteria (STEC) was defined as a continuous decrease in the reactivity of the patient sera against each of the four biomarkers after treatment (T9≤T0 or T9 no more than 20% of T0; T24/T48<T9 and T24/T48<T0) together with a substantial drop in reactivity for at least two out of the four BMKs (>40% reduction in the reactivity for KMP11, PFR2, and 3973d or >30% reduction in the reactivity for HSP70) relative to that detected before treatment. A more restrictive set of criteria (RTEC) required a continuous drop in the reactivity against the four BMKs (as mentioned above) and a substantial drop in the reactivity (>40% reduction in the reactivity for KMP11, PFR2, and 3973d or 30% reduction in the reactivity for HSP70) for three out of the four BMKs.
Subsequently, a computer algorithm based on the patient sera reactivity (measured as OD values) against the four molecules before treatment and at two posttreatment times was designed to quickly and easily evaluate the behavior of the patients in terms of their reactivity against these molecules and their characterization achieving the STEC, RTEC, or therapeutic failure. These results are summarized in Table 2 and indicated that 42.4% (28 out of 66) of IND Chagas patients at T24 and 68.75% (22 out of 32) of the patients at T48 met the STEC. Interestingly, when the reactivity against the whole lysate of the parasite was evaluated after treatment in the various patient groups, patients who presented a drop in reactivity against the 4 BMKs and, consequently, met the STEC and those who did not meet the STEC, no differences were observed among the patients (see Fig. S3 in the supplemental material). This result demonstrates the limitation of the reactivity against T. cruzi whole-cell lysate for the follow-up of treated patients with Chagas disease. This is consistent with previously reported data that indicate that regression to negativity in response to treatment occurs earlier for particular antigens than for a large number of antigens and for antigens mixtures (31). This idea was also supported by the observation that a high proportion of the antibodies against T. cruzi that are detected by conventional serology are directed against Galα1→3Gal (a carbohydrate residue, galactosyl1α1→3galactose), which is widely distributed among microorganisms among the intestinal and pulmonary microflora, which will stimulate lymphocytes that were previously primed by T. cruzi Galα1→3Gal (32). This fact, together with the long life of these particular antibodies, may well explain the high titer of antibodies observed against T. cruzi whole-cell lysate antigens during the follow-up period and the particularly increased reactivity observed at T48, which otherwise corresponds to the reactivity of patients who did not meet the STEC (met the STEC, n = 6, P = 0.2188; did not meet the STEC, n = 10, P = 0.078) (data not shown). In addition, the differences in the level of the antibodies detected against T. cruzi whole-cell lysate antigens at T48 may be related to the number of patients that participated in the follow-up period, namely, 66 patients at T0, 60 at T9, 45 at T24, and 16 at T48.
Number and percentage of indeterminate chronic Chagas disease patients who met the therapeutic efficacy criteria after treatment based on computational analysis
On the other hand, the strictest condition (RTEC) was achieved by 15.2% (10 out of 66) of IND patients at 24 months (T24). With a longer period of time after treatment (T48), the percentage of patients who met the RTEC reached 40.6%, achieving a ratio of response similar to that obtained at T24 with the STEC (Table 2). The custom script that was designed to analyze the therapeutic efficacy as well as the data used in the present manuscript is available at https://bitbucket.org/IPBLN/chdpredictcon/.
The predictive value of the STEC at 24 months after treatment was calculated based on the data obtained from the samples from 32 IND Chagas patients who were evaluated at both 24 and 48 months after treatment (see Fig. S4 in the supplemental material). Only 12 out of the 22 patients who obey STEC at T48 are contained in the 28 patients who obey STEC at T24. Moreover, the therapeutic efficacy criteria (STEC) were reached at 48 months (but not at 24 months posttreatment) in 10 out of 32 patients (Fig. S4). Since 12 out of the 14 patients met the STEC at both posttreatment times (24 and 48 months) and, consequently, two patients met the STEC at T24 but not at T48 months after treatment, the predictive value in terms of therapeutic efficacy at 24 months after treatment was 85.7% (Fig. S4). Therefore, the percentage of patients who met the STEC after a longer period of time after treatment (T48) was 68.75% (22 out of 32). On the other hand, eight patients did not meet the STEC at either 24 or 48 months, indicating a therapeutic failure rate of 31.25% (10 out of 32 patients).
Furthermore, the contribution of each of the BMKs to the variance that was observed in the reactivity of the patients after treatment was evaluated using principal-component analysis (PCA) among the patients who met the STEC and those who did not meet the STEC (Fig. 3). When a plot of the first (PC1) and second (PC2) components (those explaining the greatest variances) was also performed, taking into consideration the patients who met (red) and did not meet (blue) the STEC, important differences in the variances were observed between the patients who met the standard therapeutic efficacy criteria and those who did not meet the STEC (Fig. 3). The first principal component, accounting for 53% of the variance in the data, was significantly associated with the four variables (KMP11, PFR2, 3973d, and HSP70), and the principal components 2, 3, and 4, accounting for 22%, 17%, and 7% of the variance in the data, respectively, were associated with three out of the four variables, namely, KMP11, 3973d, and HSP70 for PC2; KMP11, PFR2, and 3973d for PC3; and KMP11, PFR2, and HSP70 for PC4 (Fig. 3). These data indicate the important contribution of each of the four molecules in the BMK set for predicting the therapeutic impact of treatment in Chagas disease patients.
Principal-component analysis (PCA) according to the behavior against each biomarker in the follow-up after treatment of indeterminate patients. PCA score plot for the values of changes in the reactivity (variance) of 66 IND patients against each of the four biomarkers (4 variables) between the pretreatment time and after 24 months of treatment. Red spots correspond to patients who met the STEC, and blue spots correspond to the patients who did not meet the therapeutic efficacy criteria. The table inside the figure contains the loadings of each variable to the four components (PC1, PC2, PC3, and PC4).
The presence of T. cruzi DNA in the peripheral blood was evaluated in 33 out of the 66 IND Chagas chronic patients in this study. Notably, none of the patients who met the STEC presented a positive PCR result at 9, 24, or 48 months after treatment (Table 3). Conversely, the presence of parasite DNA was detected by PCR in 4 IND patients who did not meet the therapeutic efficacy criteria based on the set of serological biomarkers (Table 3). Consistent with the data reported by other authors (33), the level of the antibodies against T. cruzi whole-cell lysates was slightly higher in patients who had a positive PCR result (7.12 ± 1.26) than in that in patients with a negative PCR result (6.34 ± 2.71); however, in this study, the observed difference was not statistically significant (P = 0.64).
Detection of T. cruzi by PCR in patients with chronic indeterminate Chagas disease pre- and postbenznidazole treatment
Functional profile of the T. cruzi antigen-specific CD8+ T cells associated with the therapeutic efficacy criteria.We next wanted to analyze the phenotype and functional capacity of the T. cruzi antigen-specific CD8+ T cells of the patients and if there were differential patterns between the patients who met the STEC and those who did not meet the STEC. Thus, the analysis of the multifunctional capacity of CD8+ T cells was performed in IND patients who met the STEC based on their humoral response (n = 10) and those who did not meet the STEC (n = 7) before and 24 months after benznidazole treatment. The functional activity of the antigen-specific CD8+ T cells isolated before and after benznidazole treatment was evaluated based on the expression and coexpression of Th1-type cytokines (interferon gamma [IFN-γ], interkeukin 2 [IL-2], and tumor necrosis factor alpha [TNF-α]) and/or cytotoxic molecules (granzyme B and perforin) following stimulation with total T. cruzi antigens (STcA). As shown in Fig. 4, the percentage of antigen-specific CD8+ T cells from the patients who met the therapeutic efficacy criteria which expressed 2, 3, and 4 molecules increased at 24 months posttreatment compared with that observed at the pretreatment time point. Specifically, the number of CD8+ T cells expressing 2 molecules increased from 31.8% to 37.6%, the CD8+ T cells expressing 3 molecules increased from 4.5% to 12.6%, and the CD8+ T cells expressing 4 molecules increased from 0.7% to 0.9% at pretreatment and 24-months posttreatment, respectively. Moreover, in those patients who met the therapeutic efficacy criteria, there was a marked increase in the CD8+ T cell population that exhibited a Th1-type cytotoxic profile, expressing IFN-γ (Th1-like cytokine), perforin, and granzyme B (cytotoxic molecules) from 3.1% (pretreatment) to 9.7% (posttreatment) (Fig. 4). However, the proportion of antigen-specific CD8+ T cells that expressed 2 or 3 molecules in the patients who did not meet the therapeutic efficacy criteria decreased after 24 months of treatment (from 13.1% to 8.9% expressing 2 molecules and from 12.2% to 10.6% 3 molecules, at pretreatment and at 24-months posttreatment, respectively). Moreover, the monofunctional CD8+ T cells from the patients who did not meet the therapeutic efficacy criteria represented the highest proportion of total antigen-specific CD8+ T cells both before (74.4%) and after treatment (79.2%) (Fig. 4). Interestingly, the functional pattern of antigen-specific CD8+ T cells before treatment from patients who met the STEC was completely different from that of the patients who did not meet the STEC in terms of both the multifunctional capacity and cytokine production pattern of the T cells. Thus, before treatment, those patients who did meet the STEC had a higher proportion of multifunctional antigen-specific CD8+ T cells that expressed granzyme B and perforin than that in patients who did not meet the STEC.
Functional profile of the Trypanosoma cruzi antigen-specific CD8+ T cells from treated Chagas disease patients that were associated with therapeutic success or failure. The multifunctional profile of antigen-specific CD8+ T cells from 17 indeterminate patients was analyzed after in vitro stimulation with STcA. Evaluation was carried out before (T0) and after 24 months of benznidazole administration (T24) in patients who met the therapeutic efficacy criteria (STEC) (n = 10) and those who did not meet the criteria (n = 7). The values are represented in a pie chart. The colors of the pie chart show the number of molecules associated with the cells (1 to 5). The pie chart arcs indicate the proportion of the pie that corresponds to the molecule or molecules that the cells were able to produce (IFN-γ, IL-2, TNF-α, granzyme B, and/or perforin).
Expression of cytotoxic molecules by CD8+ T cells from chronic IND Chagas disease patients is associated with the therapeutic efficacy.To analyze the cytotoxic profile of T. cruzi-specific CD8+ T cells from patients whose humoral response pattern against the set of 4 BMKs met and did not meet the STEC in more detail, we focused on the cytotoxic molecule expression pattern (granzyme B and perforin) of these cells before and after benznidazole treatment. The results (Fig. 5) showed that the patients who met the therapeutic efficacy criteria had a higher frequency of cells expressing cytotoxic molecules before treatment than that detected in patients who did not meet the therapeutic efficacy criteria. Thus, in patients who achieved therapeutic success, 30.1% of the CD8+ T cells produced granzyme B versus 4.5% of the cells from patients who did not meet the STEC. Additionally, 13.7% of the CD8+ T cells were Granzyme B+Perforin+ in patients who met the STEC versus 2% of the cells from patients who did not respond to treatment. Twenty-four months after treatment, the CD8+Granzyme B+Perforin+ cell population substantially increased in patients who met the STEC (from 13.7% to 35.2%). However, the percentage of CD8+Granzyme B+Perforin+ cells did not change after treatment in patients who did not meet the STEC (2% versus 2.2%). The observed difference in the percentage of CD8+ T cells that coexpressed granzyme B and perforin between the patients who met the STEC and did not meet the STEC was significantly different (P < 0.05; 35.2% compared with 2.2%, respectively). Moreover, patients who met the STEC showed a slight increase (5%) in the percentage of CD8+ T cells that expressed only granzyme B and a large decrease in the population of CD8+ T cells that produced only perforin after treatment (11.6% pretreatment to 0.7% posttreatment) (Fig. 5). The patients who did not meet the therapeutic efficacy criteria showed a marked increase in the percentage of CD8+ cells that were solely Perforin+ (14.8% to 39%) or Granzyme B+ (4.5% to 23.4%). In addition, the patients who did not meet the therapeutic efficacy criteria had a higher percentage of cells that did not express any of the cytotoxic molecules examined relative to that in the patients who did meet the therapeutic efficacy criteria (CD8+Granzyme B-Perforin−), both before (78.8% respect to 44.5%, respectively) and after treatment (35.4% respect to 29.1%, respectively) (Fig. 5).
Cytotoxic profile of Trypanosoma cruzi-specific CD8+ T cells from treated Chagas disease patients that were associated with therapeutic success or failure. The functional activity of CD8+ T cells was determined based on perforin and granzyme B expression after stimulation with STcA in cells from indeterminate patients who met or did not meet the therapeutic efficacy criteria (STEC) before (T0) and after 24 (T24) months of benznidazole treatment. The P values of the permutation test in the coexpression analysis are shown in the pie charts; *, P < 0.05. Production was measured after antigenic stimulation with STcA in the CD8+ T cells of 17 patients with chronic indeterminate Chagas disease before and after 24 months of benznidazole treatment (10 patients who met the STEC and 7 patients who did not meet the therapeutic efficacy criteria).
Expression of inhibitory receptors in CD8+ T cells from chronic IND Chagas disease patients is associated with therapeutic efficacy.The evaluation of the level of the expression of the inhibitory receptors PD-1, 2B4, and CD160 was carried out in the CD8+ T cell population of 20 chronic indeterminate Chagas disease patients (11 patients who met the STEC and 9 patients who did not meet the therapeutic efficacy criteria). The frequency of CD8+ T cells expressing these molecules (PD-1, 2B4, and CD160) was detected pretreatment (T0) and after 24 months of benznidazole administration (T24) (Fig. 6). A statistically significant decrease in the frequency of CD8+ T cells expressing the PD-1 and 2B4 receptors (CD8+PD-1+ and CD8+2B4+ T cells) was detected in the patients who met the therapeutic efficacy criteria, based on their reactivity against the 4 BMKs, at T24 compared with that at T0 (both P values of <0.05). In contrast, those patients who did not meet the therapeutic efficacy criteria did not show a statistically significant decrease in the frequency of CD8+ T cells expressing the PD-1 or 2B4 receptors at T24 compared with that at T0 (Fig. 6).
Expression of inhibitory receptors on CD8+ T cells from Chagas disease patients before and after treatment with benznidazole that was associated with therapeutic success or failure. The frequencies of CD8+ T cells that expressed PD-1, 2B4, or CD160 were detected before and after treatment in indeterminate Chagas disease patients who met the STEC (n = 11) and who did not meet (n = 9) the therapeutic efficacy criteria. The whiskers of the box plot represent the minimum and maximum values. The P values less than 0.05 are marked with an asterisk. The Wilcoxon rank-sum test was performed.
DISCUSSION
The lack of appropriate clinical and biomarker tools limits the direct measurement of the treatment efficacy for many neglected tropical diseases. In the case of Chagas disease, one of the greatest current challenges is the establishment of early markers that allow for the evaluation of the therapeutic efficacy of the available (benznidazole and nifurtimox) and newly proposed drugs (34, 35). Existing serological tests have high sensitivity and specificity and are able to diagnose chronic patients via the conversion to negative serology that is associated with parasitological cure (36). However, it can take years to decades after treatment for seronegativity to be achieved in the adult population and is, therefore, not an adequate endpoint for clinical trials (37). Thus, we hypothesized that a clinically relevant seroconversion could be measured as a continuous and substantial drop in the reactivity of patient sera against specific T. cruzi antigens and could be a useful tool for evaluating the effect of treatment. According to the target product profile (TPP), an ideal or acceptable biomarker of therapeutic response allows the detection of changes in the observed response over a short period of time (from 3 up to 24 months) after treatment (34). In this regard, in previous laboratory studies, a series of T. cruzi antigens, including KMP11, PFR2, HSP70, and 3973d, was described as therapeutic follow-up markers. These antigens were recognized by Chagas disease patient sera at any clinical stage of the disease. Furthermore, shortly after benznidazole treatment (6 to 9 months), a significant drop in the antibody titers against these antigens was observed (21, 25).
As previously documented, a decrease in patient serological titers against several specific parasite antigens is associated with a substantial reduction in the parasitic load and with an improvement in the clinical status of treated Chagas disease patients (38, 39). Therefore, the evaluation of the reactivity against these antigens could represent a useful serological marker system for monitoring the effectiveness of drug treatment in Chagas disease patients and a tool that could allow the early detection of therapeutic failure. Under these circumstances, the aim of this study was to establish the predictive value of the therapeutic efficacy of the KMP11, PFR2, HSP70, and 3973d molecules as a set of biomarkers and to determine the functional modifications induced in the CD8+ T cells from chronic Chagas disease patients after treatment. Thus, we evaluated and compared the reactivity of sera from chronic Chagas disease patients against these four T. cruzi antigens before treatment administration and at 24 months after benznidazole treatment. The marked decreases in the antibody levels against the four parasite-specific molecules may be associated with a drastic decline in the parasite load that was induced by the drug and could, thus, be connected with the therapeutic efficacy (25, 34). According to the TPP, a drop in the reactivity produced within a short period of time (up to 24 months) would meet the recommendation that is considered acceptable for the evaluation of anti-Trypanosoma therapy (34). Conversely, an increase in the antibody levels against any of the four parasite-specific molecules would be considered a therapeutic failure. To determine if there was a subsequent reversion in the specific response, the reactivity against the four molecules was also analyzed after a longer period of time (48 months).
As expected, the reactivity against each of the analyzed antigens continuously decreased after treatment in many IND patients, and this difference was statistically significant. However, changes in the reactivity against the total parasite antigens were not observed. The percentage of patients in whom the reactivity against each one of the antigens decreased (analyzed individually) was similar for each of the antigens and ranged from 86% (PFR2) to 89% (KMP11) at 24 months and from 91% (PFR2) to 97% (KMP11 and 3973d) after 48 months of follow-up (Table 1). However, when the drop in the reactivity against the four molecules (KMP11, HSP70, 3973d, and PFR2) was measured, 45.5% of the patients showed a decrease in the reactivity against the four antigens at T9, 68.2% showed a decrease at 24 months, and up to 81.3% showed a decrease at 48 months after treatment (Fig. S1). Previous reports have described a slight increase in the antibody titers against parasite molecules as a consequence of parasite lysis in response to treatment and the subsequent liberation of the lysed parasites into the bloodstream. This behavior has also been observed for the antigens analyzed in this study. When a slight increase in the patient sera reactivity (lower than 20%) against each of the antigens was permitted at a very short time point after treatment (T9), the percentage of patients exhibiting a continuous decrease in reactivity was slightly higher at 24 months after treatment and was greater at 48 months posttreatment than that when a slight increase in reactivity was not permitted (Fig. 2).
Two of the criteria of the therapeutic efficacy were established based on a necessary continuous decrease in the reactivity to the four BMKs at 24/48 months posttreatment and a particularly strong drop in the reactivity of at least for two (STEC) or three (RTEC) of the four BMKs. The drop was considered substantial when the decrease in reactivity was at least 40% for KMP11, PFR2, and 3973d and 30% for HSP70 compared with the reactivity at T0. Both criteria allowed a slight increase in the reactivity shortly after treatment (T9), which was no higher than 20% of the total reactivity value prior to treatment administration.
According to the established criteria of treatment success or therapeutic failure, a custom script was designed (available at https://bitbucket.org/IPBLN/chdpredictcon/). To run the program, the OD values obtained for the four biomarkers at three time intervals, namely, T0 and at least two times after treatment, were required. Focusing on the STEC criterion at 24 months posttreatment, 42.4% (28 out of 66) of IND Chagas patients met the therapeutic efficacy criteria, as they exhibited a continuous decrease in the reactivity against the four biomarkers after treatment relative to the reactivity that was measured before treatment (T0), alongside a substantial decrease in the reactivity against at least two out of the four biomarkers at T24 compared with T0. Furthermore, 68.75% of patients with chronic indeterminate Chagas disease showed evidence of therapeutic efficacy at 48 months after treatment, and this percentage was the overall rate of response to benznidazole that was estimated in this study. Interestingly, since 12 out of the 14 patients who met the therapeutic efficacy criteria at 24 months after treatment also met the criteria at a later time point (48 months), we could assert that the STEC had a predictive value of 85.7%. When the strictest therapeutic efficacy criteria were considered (RTEC), the therapeutic effectiveness value that was obtained at 48 months was similar to that detected at 24 months under the STEC conditions (40.6% versus 42.4%, respectively). These data indicate that more restrictive conditions (RTEC) result in the same predictive value of therapeutic efficacy as the STEC, although the use of the RTEC delays the detection of the response rate to treatment. Moreover, there was no correlation between the patients’ origin, years of residence in Spain, and whether the patients met or did not meet the STEC or RTEC (P = 0.141). Twenty-two patients had lived in Spain for 2 years when they were treated, 28 for 4 years and 16 for more than 4 years. Among these patients, 12 (54.5%), 11 (39.3%), and 5 (31.3%), met the STEC. The observed predictive value of the BMK set at an early time point is relevant, since other studies have shown complete or partial seroconversion after periods of time that were five times longer than those in the present study (19, 34, 40). Furthermore, as stated by the TPP, this set of four biomarkers meets the requirements to be considered an acceptable biomarker for evaluating potential therapeutic efficacy in chronic indeterminate Chagas disease patients. These conditions are based on the nature of the sample, namely, peripheral blood, which can be collected and processed everywhere at room temperature in a volume lower than 5 ml using a fast and common technique (less than 48 h) that requires three samples per patient (one before treatment and two posttreatment) (34). The important contribution of each of the four molecules for evaluating the impact of the treatment in Chagas disease patients, as demonstrated by PCA (Fig. 3), reinforces the use of the four molecules as a set of biomarkers. In addition, the use of the four molecules as a set is also supported by the fact that the reactivity against three (or two) out of the four antigens that were detected in five patients who had low titers of specific antibodies against one molecule of the set of 4 antigens (or against two molecules in the case of 1 patient) at T0 allowed for their classification as meeting the STEC (1 patient presented a decrease in reactivity) or not meeting the STEC (3 patients did not show changes in reactivity, and 1 patient experienced an increase in the reactivity at 48 months after treatment).
This study shows that benznidazole treatment in Chagas disease patients alters the humoral response pattern against specific antigens. The purpose of therapeutic treatment is to completely eliminate parasites (39). Therefore, therapeutic failure is defined as the persistence of the parasite after treatment, which is detected using different methods, such as PCR (10). PCR analyses allowed the detection of 4 treated patients with a positive PCR result, which is considered unequivocal evidence of therapeutic failure. Interestingly, these four patients did not meet the STEC and, based on their humoral response against the set of four molecules, were classified as patients with therapeutic failure. Consistent with the reliability of this set of biomarkers to detect therapeutic failure, none of the patients who met the STEC/RTEC had a positive PCR result after treatment. In this context, it is important to highlight that a negative PCR result does not imply parasite absence because low parasitemia or fluctuations in the parasite load may give false-negative results. In contrast, a positive PCR proves the presence of the parasite and, in treated patients, is associated with therapeutic failure. The results obtained using the set of 4 biomarkers that classified the patients as having a positive response to treatment or no response to treatment were consistent with the PCR results that were obtained. On the other hand, it has been reported that the persistent presence of the parasite can lead to a proportion of the CD8+ T cell population that experiences immunological exhaustion, which, in turn, might impair the capacity for infection control, allowing the disease to persist (28, 41, 42). Thus, decreased parasitemia could restore the functionality of the host immune system to some extent to control the infection (43, 44). A recent study carried out in chronic indeterminate Chagas disease patients treated with benznidazole showed that treatment also induces an improvement in the quality of the CD8+ T cell response against parasite antigens, characterized by an increased production of cytokines (IL-2, IFN-γ, and TNF-α) and cytotoxic molecules (granzyme B and perforin) (26). Therefore, the evolution of the multifunctional profile of antigen-specific CD8+ T cells and their association with the serological changes linked to therapeutic efficacy or to therapeutic failure were also assessed in this study. Interestingly, these results showed that the multifunctional antigen-specific capacity of the CD8+ T cells from patients who met the therapeutic efficacy criteria improved after treatment. Conversely, the antigen-specific CD8+ T cells from patients who did not meet the therapeutic criteria mostly showed a monofunctional capacity, which is related to poor disease control (28). The cytotoxic capacity of CD8+ T cells also improved after treatment in patients who met the therapeutic efficacy criteria; the frequency of T. cruzi antigen-specific CD8+ T cells expressing granzyme B and perforin at the same time (CD8+Granzyme B+Perforin+ T cells) was higher after treatment than that of the patients who did not meet the therapeutic efficacy criteria, who maintained a much smaller proportion of CD8+Granzyme B+Perforin+ T cells before and after treatment. Interestingly, prior to treatment administration, the patients who met the standard therapeutic efficacy criteria presented a greater multifunctional T cell profile and a stronger cytotoxic capacity than that in patients who did not met the STEC. Thus, before treatment, the patients who met the STEC presented a higher percentage of CD8+ T cells independently expressing granzyme B and perforin and coexpressing granzyme B and perforin than that in the patients who did not meet the STEC (Fig. 4 and 5). Moreover, treatment improved the cytotoxic capacity of the antigen-specific CD8+ T cells, as strong and statistically significant differences were observed among those patients who met the STEC and those who did not meet the STEC. As shown in Fig. 5, the percentage of antigen-specific CD8+ T cells that coexpressed granzyme B and perforin in treated patients who met the STEC was 35.2% and was 2.2% in those who did not meet the STEC. These data indicate that the functional capacity of the CD8+ T cells of the patients whose humoral response against specific antigens of the parasite decreased after treatment is different and stronger in terms of the cytotoxic capacity than that of patients whose humoral response did not decrease after treatment. Moreover, the data also indicate that treatment induces an important improvement in the cytotoxic capacity of the CD8+ T cells in the patients whose humoral response decreased after treatment, which was expected to compromise parasite survival and, consequently, to help to control the infection.
Cytotoxic T cells, which express both granzyme B and perforin, have been reported to induce cell death through the classic apoptosis pathway. During the apoptotic process, the cell undergoes phagocytosis by macrophages so that the leakage of potentially toxic cytoplasmic material is avoided (45). In contrast, patients who did not meet the therapeutic efficacy criteria showed a 35% increase in the proportion of CD8+ T cells expressing perforin but not granzyme B. Interestingly, it has been reported that perforin activity without granzyme B induces cell death in a less efficient manner (45), and myocardial dysfunction is susceptible to a perforin-dependent process, which could be a key factor in cardiomyocyte injury and cardiac dysfunction (46). The increase in the multifunctional capacity after treatment accompanied by the decrease in the inhibitory receptor expression observed in the patients who met the established therapeutic efficacy criteria is critical for improving the control of chronic infectious diseases. It has been reported that the gradual increase in the dysfunction of CD8+ T cells is linked to the pathological severity of T. cruzi infection (28). The development of a dysfunctional T cell response is characterized by the elevated expression of inhibitory receptors and an impaired ability to simultaneously produce multiple molecules, such as cytokines and cytotoxic mediators (multifunctional profile) (28), which might compromise infection control and, thus, favor the progression of the disease (47).
In summary, our results show that the biomarker set comprising KMP11, HSP70, PFR2, and 3973d and the application of the criteria established in this study could be useful tools for monitoring the efficacy of benznidazole treatment in chronic Chagas disease patients. Patients who showed treatment efficacy based on this set of serological biomarkers appeared to demonstrate improved multifunctional antigen-specific CD8+ T cell responsiveness. Interestingly, treated patients who met the STEC presented a cellular functionality pattern that differed from that of patients who did not meet the STEC. These data demonstrate that the CD8+ T cell status of patients allows the prediction of treatment effects.
MATERIALS AND METHODS
Human samples.Serum samples from 66 adult IND patients were collected at the Hospital Virgen de la Arrixaca (Murcia, Spain) and Hospital Virgen de las Nieves (Granada). Chagas disease was diagnosed after an analysis using two conventional serological tests, namely, an ELISA (Bioelisa Chagas Biokit, Spain) and an indirect immunofluorescence assay (Inmunofluor Chagas, Biocientífica, Argentina), following the WHO criteria (48). The enrolled patients had not received any treatment for Chagas disease before their inclusion in this study. All patients were adults in the indeterminate chronic phase of the disease and were between 22 and 53 years of age. Sixty-five of the patients were from Bolivia, and one was from El Salvador. All of the patients were residents of Spain, a country where Chagas is nonendemic, and 86.4% of them had resided in Spain for at least 2 years. Subsequently, the patients were treated with benznidazole (5 mg/kg of body weight per day for 60 days) and clinically followed up for 48 months. Samples from all patients (n = 66) were collected and analyzed before treatment (day 0, T0) and at 9 and 24 months posttreatment (9 and 24 months post day 0); samples were collected from 32 patients at 48 months posttreatment, as the other 34 patients did not attend the medical consultation at 48 months posttreatment (48 months post day 0).
Thirty-milliliter blood samples from 20 IND patients were collected in tubes with EDTA. Peripheral blood mononuclear cells (PBMCs) were purified from the blood samples by Ficoll (Lymphoprep; Alere Technologies AS, Oslo, Norway) density gradient centrifugation and cryopreserved in inactivated fetal bovine serum (iFBS) with 10% dimethyl sulfoxide in liquid nitrogen until further use.
Ethics.These protocols were approved by the ethics committees at the Hospital Virgen de la Arrixaca (no. MTR-02/2014) and Consejo Superior de Investigaciones Científicas (no. 094/2016). Written informed consent was obtained from all individuals prior to their inclusion in the study.
Trypanosoma cruzi antigens.The T. cruzi recombinant proteins KMP11, HSP70, and PFR2 were overexpressed and purified as previously described (21). The peptide 3973d, formed from two molecules of the 3973 peptide linked by two glycine molecules (FGQAAAGDKPSLGGFGQAAAGDKPSL), was synthesized using a simultaneous multiple peptide solid-phase method (24).
ELISA measurement and data processing.ELISAs were performed in triplicate at different serum dilutions as previously described (21). Briefly, plates were coated with 0.5 μg of each antigen diluted in carbonate buffer (pH 9.6) for the recombinant proteins and in phosphate buffer (pH 7.4) for 3973d and stored in a dry atmosphere at –20°C until use. Subsequently, the wells were washed twice with 200 μl of phosphate-buffered saline (PBS)-0.05% Tween 20 and incubated for 1 h with blocking solution (5% nonfat dried milk powder in PBS). Sera were assayed at 1/100, 1/200, 1/400, 1/800, and 1/1600 dilutions in triplicate at T0 and incubated with the antigen for 2 h at 37°C. The dilution at which each antigen was assayed posttreatment corresponded to the linear part of the curve after the titration analysis at T0. Positive and negative serum controls were included in all plates. A custom Practical Extraction and Reporting Language (Perl) script was designed to process the reactivity data of the patients against the antigens that were assayed to establish the therapeutic efficacy criteria in the treated patients. The program was designed to include the optical density (OD) data from at least three samples, one of which was obtained prior to treatment administration.
PCR amplification.DNA extraction from the peripheral blood samples and PCR procedures were performed as previously described (49).
Isolation of soluble Trypanosoma cruzi antigens for T cell stimulation assays.Soluble T. cruzi antigens (STcA) were obtained as previously described (21). STcA isolation was performed at a ratio of 1:1 for the amastigote/trypomastigote parasite forms. The protein concentration of the extract was determined by a micro bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Waltham, MA, USA), and the protein profile was analyzed by SDS-PAGE followed by Coomassie blue staining.
Intracellular detection of cytokines and cytotoxic molecules by flow cytometry assays in PBMCs after stimulation with STcA.A total of 1 × 106 PBMCs/ml of medium was cultured with STcA (1 μg/ml) or with medium alone (basal response) in the presence of 1 μg/ml anti-CD28 (clone CD28.2) and anti-CD49d (clone 9F10) (BD Biosciences, San Diego, CA). During the last 10 h of culture, brefeldin A (1 μg/ml) and monensin (0.7 μg/ml) (BD Pharmingen) were added. After incubation, PBMCs were stained with LIVE/DEAD fixable aqua (Invitrogen, Eugene, OR) and with the following surface antibodies (Abs): CD3-Pacific Blue (clone UCHT1) and CD8-APC-H7 (clone SK1) (BD Biosciences). Then, the cells were fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences) for 20 min at 4°C for intracellular staining with the following Abs: IFN-γ-PE-Cy7 (clone B27), IL-2-APC (clone MQ1-17H12), TNF-α-Alexa Fluor 488 (clone MAb11), granzyme B-PE-CF594 (clone GB11) (BD Biosciences), and perforin-PE (clone B-D48) (Abcam, Cambridge, UK). Data from at least 100,000 lymphocytes were acquired according to forward scatter/side scatter (FCS/SSC) parameters for each condition by a FACSAria III flow cytometer (BD Biosciences). Data files were subsequently analyzed using FlowJo 9.3.2 software. The positivity for each marker was determined using fluorescence minus one (FMO) and isotype staining controls.
Evaluation of the inhibitory receptor expression in CD8+ T cells by flow cytometry assays.PBMCs from chronic indeterminate Chagas disease patients were cultured at a concentration of 1 × 106 PBMCs/ml with STcA (1 μg/ml), anti-CD28 (clone CD28.2), and anti-CD49d (clone 9F10) (BD Biosciences, San Diego, CA). After an overnight incubation, PBMCs were stained with LIVE/DEAD fixable aqua (Invitrogen, Eugene, OR) and with the following surface Abs: CD3-Pacific Blue (clone UCHT1), CD8-APC-H7 (clone SK1), 2B4-FITC (clone 2-69), CD160-Alexa Fluor 647 (clone BY55) (BD Biosciences), and PD-1-PE (clone J105) (Thermo Scientific). Data from the cells were acquired and analyzed in a manner similar to that for the flow cytometry assay, as previously described in the Materials and Methods.
Statistical analysis.The Prism statistical package version 6.0 (GraphPad Software, CA, USA) was used to perform statistical analyses. Shapiro-Wilk normality test was used to evaluate the type of data distribution. Consequently, nonparametric tests were used for the evaluation of the data. Thus, Friedman (with Dunn’s post hoc test), and Wilcoxon signed-rank tests were applied. A Fisher’s exact chi-square test was used to evaluate whether there was an association between the time of residence in Spain and the criteria of treatment efficacy. An additional analysis was carried out to compare the coexpression pie charts using 10,000 permutations with SPICE software (version 5.3). Differences were considered significant at a P value of <0.05. Principal-component analysis (PCA) was performed and plotted using Minitab software (version 17.1). In addition, the notched box-plots were constructed using the R statistical software (version 3.5).
ACKNOWLEDGMENTS
We thank A. López-Barajas and Celia Benitez from IPBLN-CSIC for their technical assistance with the recombinant protein purification and the ELISA tests, respectively, and L. Murcia (HVA, Murcia) for performing the PCR.
This work was financially supported by grants SAF2016-81003-R, SAF2016-80998-R, and RTC-2014-2130 from the Programa Estatal I + D+i (MINECO); the Network of Tropical Diseases Research RICET (RD16/0027/0005 and RD16/0027/0016); and FEDER.
This publication is part of the PhD thesis of the student Elena Pérez-Antón at the University of Granada in the Biomedicine Program.
FOOTNOTES
- Received 20 November 2018.
- Returned for modification 21 January 2019.
- Accepted 18 July 2019.
- Accepted manuscript posted online 29 July 2019.
Supplemental material for this article may be found at https://doi.org/10.1128/AAC.02436-18.
- Copyright © 2019 American Society for Microbiology.
