Abstract
Background
The significance of detection of Trypanosoma cruzi (T. cruzi) DNA in blood of antibody-positive patients for risk of development of Chagas heart disease is not well established. The objective of this study was to compare detection of T. cruzi DNA with known clinical and laboratory markers of Chagas cardiomyopathy (CC) severity.
Methods
This is a case-control study nested within a retrospective cohort developed in Brazil to understand the natural history of Chagas disease. The study enrolled 499 T. cruzi seropositive blood donors (SP-BD) and 488 frequency matched seronegative control donors (SN-BD) who had donated between 1996-2002, and 101 patients with clinically diagnosed CC. In 2008-2010 all enrolled subjects underwent a health questionnaire, medical examination, electro- and echocardiograms and PCR analyses. A blinded panel of three cardiologists adjudicated the outcome of CC. T. cruzi kinetoplast minicircle sequences were amplified by real-time PCR using an assay with a sensitivity of one parasites/20mL of blood. All testing was performed on coded samples.
Results
Rates of PCR detection of T. cruzi DNA were significantly (p=0.003) higher in CC patients and SP--BD diagnosed with CC (79/105 [75.2 %]) compared with SP-BD without CC (143/279 [51.3%]). Presence of parasitemia was significantly associated with known markers of disease progression such as QRS and QT interval duration, lower left ventricular ejection fraction, higher left ventricular index mass, and elevated troponin and NTpro-BNP levels.
Conclusion
T cruzi PCR positivity is associated with presence and severity of cardiomyopathy, suggesting a direct role of parasite persistence in disease pathogenesis.
Keywords: Trypanosoma cruzi (T. cruzi), PCR, cadiomyopathy
Introduction
Chagas disease is caused by the parasite Trypanosoma cruzi (T. cruzi), which infects up to 10 million people in Latin America causing approximately 12,500 deaths annually. The evolution from the time of infection to Chagas cardiomyopathy (CC) and/or the megacolon and megaesophagus syndromes occurs over 10 to 20 years in a slow but progressive fashion, although only in a proportion of chronically infected individuals.(1-4)
The pathogenic mechanisms of CC are not fully understood and the importance of parasitemia for disease progression is poorly defined (1-3, 5) The parasite can be detected in blood by classical methods such as hemoculture and xenodiagnosis, but the sensitivities of these techniques are low, they require fresh blood and are expensive and hence not practical for large scale research or clinical applications. Non-commercial PCR assays have been developed but have been difficult to optimize and standardize and discrepant results are common when techniques are compared.(6-7) The lack of a reliable assay to detect and quantify parasitemia is an obstacle to advancing our understanding of the impact of persistent parasitemia on the natural history of the disease as well as to characterization of parasite load for prognostic and therapeutic assessments.(6-7)
We recently developed a target-capture real-time PCR assay that can detect as few as one parasite per 20mL of processed blood, has a high degree of specificity and yields reproducible quantitative results across a wide dynamic range of parasite load.(8) We describe here the results obtained employing this assay to detect and quantify parasitemia in blood specimens from a well-characterized retrospective cohort of T cruzi seropositive blood donors (SP-BD), frequency matched seronegative control donors (SN-BD) and CC clinical cases developed as part of the National Heart, Lung and Blood Institute (NHLBI) Retrovirus Epidemiological Donor Study-II (REDS-II) .(9-10) The aim of this analysis was to compare the presence and the levels of parasitemia in blood and assess if parasitemia is associated with the development of Chagas cardiomyopathy and with clinical parameters and laboratory markers of severity of cardiac disease. In addition the study addressed the controversial issue of whether “sero-silent” T cruzi infections exist(11-13) by performing sensitive PCR analysis of samples from ∼500 matched SN-BD.
Methods
Ethics statement
The study follows the Declaration of Helsinki of the Ethical Principles for Medical Research Involving Human Subjects, was approved by the Brazilian National Ethical Committee (CONEP# 1312/2006) and all subjects gave written informed consent.
Study design
This is case-control study nested within a retrospective cohort that enrolled 499 T. cruzi SP-BD identified by blood bank screening in 1996-2002 (255 from the city of São Paulo and 244 from the city of Montes Claros in the State of Minas Gerais, Brazil) and 488 SN-BD who were frequency matched to the SP-BD by site, donation date (year), age and gender. This retrospective blood donor cohort was supplemented by parallel enrollment and evaluation of 101 previously diagnosed cases of CC from the Heart Institute of University of São Paulo Medical School; inclusion criteria included a physician diagnosis of CC, confirmed T. cruzi seropositivity, no previous treatment with benznidazole, and no co-morbidities such as diabetes, hypertension or renal failure. These individuals were recruited by letter and telephone call using the blood center and hospital databases. From July 2008 to October 2010, recruited individuals (BD and CC patients) underwent standardized health questionnaires and medical evaluations including electrocardiogram (ECG), echocardiogram (ECHO), and phlebotomy with processing and cryopreservation of samples for subsequent batched blinded analyses of cardiac markers, PCR and other biomarkers in the United States (US; see below)..
Results of ECG and ECHO were reviewed blindly by centralized reading centers in the US. All data were centralized by the REDS-II Data Coordinating Center (Westat). A pre-defined set of abnormalities in the ECHO or ECG measurements triggered the expert panel composed of three Brazilian cardiologists to review cardiac findings blinded to the subject's serostatus(10). The expert panel was asked to reach a consensus regarding the following question “If this patient were seropositive for T cruzi how would you classify them: definite CC, probably CC, possible CC, or no CC. (10) The definition used by the expert panel were defined by an expert consensus and can be accessed at (http://circ.ahajournals.org/content/suppl/2013/02/07/CIRCULATIONAHA.112.123612.DC1.html). Further details of the cohort procedures and rates and clinical correlates of CC have been previously reported.(10) The local physician also received all test results and counseled the participants when necessary.
Blood processing and PCR procedures
At the time of interviews and medical examinations 20 mL of EDTA-anticoagulated blood was collected from each enrolled subject which was immediately mixed with an equal volume of 6M guanidine HCl-0.2 M EDTA solution. The guanidine-EDTA blood mixture was maintained at room temperature until boiled for 15 min, followed by vortexing and aliquoting (1.0 mL). Aliquots were frozen in Brazil at -20oC until shipped to the US REDS-II Central Laboratory (BSRI) on dry ice, followed by maintenance at -70oC.
The target-capture (TC) real-time (RT) PCR assay used in this study(8) was developed based on the PCR method described by Virreira M et al.(14) that targets kinetoplast minicircle T. cruzi DNA. The DNA extraction was improved through use of a TC step that employed magnetic beads coated with a T. cruzi-specific 20-mer capture oligonucleotide (CaptureTc_121 AAAAAAAAAAAAAAAAAAAAAAAAAAAATAATGTACGGGKGAGATGCATGA;CaptureTc_122 GGTTCGATTGGGGTTGGTGTAATATAAAAAAAAAAAAAAAAAAAAAAAAAA; CaptureTc-S35 AAAAAAAAAAAAAAAAAAAAAAAAAATAATGTACGGGKGAGATGCATG; CaptureTc-S36 GGGTTCGATTGGGGTTGGTGTAAAAAAAAAAAAAAAAAAAAAAAAA). Four replicate TC-RT-PCR assays were performed and results interpreted as positive if two to four of four replicates were found to have specific products and negative if one or zero replicates yielded specific product. As previously reported(8) this TC-RT-PCR assay can detect a single parasite spiked into a 20 mL blood sample with a mean cycle threshold (CT) of 35 cycles, relative to the cutoff CT value of 40 cycles. The sensitivity of the assay was validated by spiking a serial dilution of lysed T. cruzi parasites into 20mL of blood. The blood was heated at 95C, aliquoted and frozen at -20C . Each dilution was tested in quadruplicate. CT values were lower, reflecting increased kinetoplast target DNA levels before amplification, for samples with higher input parasite concentrations, with a linear increase in CT values with the serial dilutions, indicating capacity to quantify parasite load in clinical samples. Quantification of parasitemia was based on the difference between the CT of a well characterized positive run control and the average CT of the sample (n=4). The test was performed on coded samples so the testing laboratory (BSRI, San Francisco, CA, US) was blindly to the serological and clinical status of the donors and CC patients, with coded results including categorical positivity/negativity and parasite concentrations for positive samples sent to the data coordinating center (Westat) for decoding of results and analysis.
Other measurements
Resting 12-lead ECG's were recorded using the same model of machine at both sites (General Electric MAC 1200 electrocardiograph; GE Healthcare, Waukesha, WI) using standardized procedures. All ECGs were processed blindly by the central ECG laboratory (Epidemiological Cardiology Research Center, Wake Forest University, Winston-Salem, NC). ECGs were analyzed electronically, with manual over-reading by trained cardiologists to ensure quality control. ECGs were classified by Minnesota code criteria using variables that were derived from the median complex of the Marquette measurement matrix.(15-16)
ECHO studies were performed using a Sequoia 512 ultrasound instrument (Acuson, Mountain View, CA, USA) at Sao Paulo site and GE Vivid3 (GE Healthcare, Waukesha, WI) at Montes Claros site. Cardiac measurements were performed according to the guidelines of the American Society of Echocardiography.(17) Studies were recorded in digital format and all measurements were performed on digital loops using a Digisonics offline analysis station (version 3.2 software, Digisonics, Houston, Tex) at the Cardiovascular Branch, Echocardiography Laboratory, National Heart, Lung, and Blood Institute, Bethesda, Maryland, US. LV ejection fraction was calculated based on modified form of Simpson's biplane method. (17)
Troponin, CKMB and NT-proBNP levels in plasma were measured using US FDA cleared assays on the VITROS System (Ortho Clinical Diagnostics, Raritan, NJ, US).
Statistical Methods
Statistical analyses were conducted using SAS (SAS 9.2, Cary, NC), SPSS (SPSS for Windows 7, Chicago, USA) and R (Version 2.14). The associations between dichotomous ECG and Echocardiogram parameters and PCR status (+/-) were tested using logistic regression with case status (SN-BD, SP-BD, CC patients) included as covariates (appendix Table). The PCR and cardiac biomarkers differences between SP-BD with CC and SP-BD with no CC were compared using the Wilcoxon-Mann-Whitney test and Fisher's exact test (Table 2). Key ECG, Echo and laboratory parameters (BNP, Troponin) were compared between total PCR- and total PCR+ SP subjects, between SP-BD who tested PCR- vs PCR+ and between CC patients who tested PCR- vs PCR-, all using multivariate linear regression with case status included as covariates (Figures 1, 2, 3). Within all PCR positive samples, the Spearman correlation was calculated to compare the parasite concentrations and continuous ECG, ECHO and laboratory variables.
Table 2.
Results of TC-RT-PCR and cardiac biomarkers according to disease classifications based on expert panel review (ref).
| Group | N | PCR+ N (%) |
Parasite Concentration Log/mL Median (Interquartile range) | NT-proBNP pg/mL Median (Interquartile range) | Troponin pg/mL Median (Interquartile range) | CKMB ng/mL Median (Interquartile range) |
|---|---|---|---|---|---|---|
| 1. CC patients | 101 | 82 (81.2) | 1.77(0.16-5) | 746 (357-2223) | 0.022(0.013-0.040) | 1.06(0.64-1.79) |
| 2. SP-BD with CC | 105 | 79 (75.2) | 0.31 (0-2.32) | 75(39-186) | <0.012(<0.012-0.021) | 0.85(0.54-1.27) |
| 3. SP-BD inconclusive for CC | 61 | 35 (57.4) | 0.08(0-1.77) | 56(40-103) | <0.012(<0.012-<0.012) | 0.78(0.51-1.44) |
| 4. SP BD without-CC | 279 | 143 (51.3) | 0.05(0-1.63) | 39(23-65) | <0.012(<0.012-<0.012) | 0.80(0.48-1.26) |
| 5. SP-BD treated with Benznidazole | 48 | 13 (27.1) | 0 (0-0.01) | 46(23-76) | <0.012(<0.012-<0.012) | 0.77(0.44-1.02) |
| 6. SN-BD | 483 | 0(0.0) | NA | 38(23-65) | <0.012(<0.012-<0.012) | 0.68(0.42-1.17) |
SP= seropositive; SN= seronegative; BD= blood donors, CC= Chagas cardiomyopathy,
Statistical significance of comparisons of SP-BD with CC versus SP-BD without CC: PCR positivity p=0.003; parasite concentration p=0.08; NT-proBNP p<0.0001; Troponin p=0.003; CKMB p=0.48.
Figure 1.
Box plots of ECG variables (QRS and corrected QT intervals). In the left all seropositive subjects were combined according to PCR status. In the right each case group (SP-BD and CC patients) were analyzed separated.
Figure 2.
Box plots of ECHO variables (Ejection Fraction and Left Ventricular index mass) In the left all seropositive subjects were combined according to PCR status. In the right each case group (SP-BD and CC patients) were analyzed separated. .
Figure 3.
Box plots of Troponin and Pro-BNP level. In the left all seropositive subjects were combined according to PCR status. In the right each case group (SP-BD and CC patients) were analyzed separated.
Results
Of the 499 SP-BD, 488 SN-BD and 101 CC patients who participated in the study, TC-RT-PCR results could be obtained for 493 (98.8%), 483 (99.0%) and 101 (100%) subjects, respectively. Of the 493 SP-BD, 48 had history of previous treatment with Benzonidazole and were excluded of the analyses due to treatment-induced clearance of parasitemia (see below). Table 1 summarizes the demographics and ECG and ECHO results from the SN-BD, SP-BD and CC patients according to their PCR status. The CC patients presented more severe cardiac disease with all ECG and ECHO parameters significantly different from the SP-BD group irrespective of PCR status Within the SP BD the following ECG abnormalities were significantly more common among PCR+ as compared to PCR- subjects: complete right bundle branch block (p<0.001), pathological q waves (p=0.02), major Isolated ST-T abnormalities (p=0.01), and left anterior hemi block (p=0.02).
Table 1.
Clinical, laboratory, ECG and echocardiogram findings for each study group (seronegative control blood donors (SN-BD), seropositive blood donors (SP-BD) and diagnosed Chagas cardiomyopathy (CC patients) according to their PCR status.
| Variables | SN-BD PCR- N=483 |
SP-BD PCR- N=188 |
SP-BD PCR+ N=257 |
CC Patients PCR- N=19 |
CC Patients PCR+ N=82 |
|---|---|---|---|---|---|
| Clinical | Mean±SD | ||||
| Age | 49.5(10.1) | 48.4(10.1) | 49.1(10.6) | 49.2(6.3) | 47.8(7.1) |
| Diastolic blood pressure, mmHg | 77.3(14.6) | 77.1(13.5) | 77.4(13.6) | 84.1(12.3) | 79.3(12.4) |
| Systolic blood pressure, mmHg | 126.7(18.7) | 126.2(17.6) | 127.1(18.9) | 125.9(16.5) | 117.7(16.8) |
| Total cholesterol, mg/dL | 204.5(51.3) | 198.0(43.6) | 200.6(39.7) | 183.3(39.5) | 178.5(41.8) |
| Low-density lipoprotein cholesterol, mg/dL | 126.0(38.7) | 122.5(38.3) | 121.9(35.8) | 112.0(36.1) | 107.3(33.3) |
| High-density lipoprotein cholesterol, mg/dL | 48.3(13.0) | 49.6(13.0) | 50.8(13.6) | 47.3(11.0) | 50.1(18.9) |
| Triglycerides, mg/dL | 150.2(99.0) | 133.1(81.0) | 142.0(98.1) | 122.3(72.6) | 113.2(56.7) |
| Glycemia mg/dL | 92.7(26.9) | 89.0(20.3) | 90.9(23.0) | 87.5(14.1) | 85.0(11.9) |
| Body mass index, kg/m2 | 27.7(4.5) | 26.6(3.6) | 26.7(4.1) | 27.8(4.6) | 25.2(4.2) |
| N(%) | |||||
| Male | 236 (48.9) | 94 (50) | 139 (54.1) | 12 (63.2) | 48 (58.5) |
| Sao Paulo site | 239 (49.5) | 105 (55.9) | 146 (56.8) | 19 (100) | 79 (96.3) |
| NYHA Functional Class > I | 10 (2.1) | 4 (2.2) | 8 (3.2) | 2 (13.3) | 17 (26.2) |
| ECG | |||||
| Complete right bundle branch block | 4 (0.8) | 13 (7) | 59 (23.2) | 6 (31.6) | 38 (46.3) |
| Pathological q waves | 8 (1.7) | 1 (0.5) | 11 (4.3) | 3 (15.8) | 18 (22) |
| Major Isolated ST-T abnormalities | 11 (2.3) | 8 (4.3) | 29 (11.4) | 5 (26.3) | 16 (19.5) |
| Frequent ventricular premature beats | 5 (1) | 3 (1.6) | 8 (3.1) | 3 (15.8) | 10 (12.2) |
| Atrial fibrillation | 1 (0.2) | 0 (0) | 2 (0.8) | 0 (0) | 3 (3.7) |
| Pacemaker rhythm | 0 (0) | 3 (1.6) | 2 (0.8) | 4 (21.1) | 17 (20.7) |
| Left anterior hemi block | 11 (2.3) | 19 (10.2) | 46 (18.1) | 6 (31.6) | 35 (42.7) |
| Echocardiogram | |||||
| Abnormal wall motion score index (>1) | 16 (3.4) | 14 (7.6) | 33 (13.1) | 17 (100) | 69 (95.8) |
| Abnormal EE' ratio (>15) | 62 (12.8) | 33 (17.6) | 47 (18.3) | 8 (42.1) | 40 (48.8) |
| Reduced LVEF (< 50%) | 18 (3.8) | 12 (6.4) | 30 (11.7) | 17 (100) | 68 (90.7) |
| LVED (Mean±SD) | 45.1(5.7) | 45.8(5.4) | 46.6(6.1) | 61.5(5.2) | 61.8(8.2) |
LVEF =left ventricular ejection fraction. LVED = left ventricular end diastolic dimension. NYHA= New York Heart Association
We evaluated if TC-RT-PCR-positivity rates and levels of parasitemia were associated with clinical and laboratory parameters that are known to be related with severity of CC (fig 1-3). The figures present the comparison of PCR + vs PCR- for all seropositive subjects (BD and patients) as well as for SP-BD and CC patients separately. Positive T. cruzi PCR results in the combined group were associated with longer durations of the QRS and QTc (corrected QT using Bazett's formula) intervals (fig. 1), lower LV ejection fractions and higher LV mass index values (fig. 2), and higher troponin and NTpro-BNP blood levels (fig 3). All of these parametesr were also associated with PCR positivity when SP-BD were analyzed separately. With the exception of troponin, none of these parameters were associated with PCR status when CC patients were evaluated separately.
Within all PCR positive subjects the parasite concentration in blood samples was weakly but significantly correlated with LV ejection fraction (rho = -0.11, p = 0.04) and troponin levels (rho = 0.15, p = 0.007). (see appendix figure 4)
Table 2 summarizes the comparison of parasitemia and clinical diagnosis of CC In this analysis we have included the SP-BDs who reported usage of benznidazole (n=48) prior to recruitment into the study as a separate group. TC-RT-PCR was positive in 81% of the CC patients and 75% of the SP-BD classified as having CC; these rates were similar and significantly higher than the 51% rate of detection of T. cruzi DNA among untreated SP-BD who did not have CC (p=0.003 for comparison of CC SP-BD [75%] versus non-CC SP-BD [51%]; and p<0.0001 for comparison of CC patients [81%] versus non-CC SP-BD [51%]). Furthermore, T. cruzi DNA concentrations in blood of TC-RT-PCR positive subjects appeared to be higher among patients with CC (median 1.8 parasites/20 mL) and SP-BD with CC (median 0.3 parasites/mL), as compared to non-CC SP-BD (median 0.05 parasites/mL), although the latter difference (0.3 v. 0.05) was non-significant (p value = 0.08). The benznidazole-treated SP-BD had a TC-RT-PCR-positivity rate of 27%, significantly lower than the 51% rate among non-CC non treated SP-BD (p= 0.003).
None of the 483 samples from SN-BD yielded a positive TC-RT-PCR result on blinded testing with the seropositive samples, indicating high specificity of the TC-RT-PCR assay and absence of sero-silent T. cruzi infections in the location, age and gender matched control donors
Discussion
The lack of a reliable test for T. cruzi detection in blood has been an obstacle to investigating the importance of parasite persistence in the pathogenesis of Chagas disease(5). By testing large volume blood samples (20 mL) from a well characterized retrospective cohort of seropositive and control blood donors and clinical CC patients using a sensitive and quantitative PCR assay, we found an association between the presence of T. cruzi parasites in blood with a diagnosis of CC as well as with parameters of disease severity.
Our data highlights the difficulty of using a cross-sectional sample to detect an association between parasitemia: the sample size needed to be large, and it was important to include subjects representing the full spectrum of infection and disease in the analyses (SP-BD without CC, less severe cardiomyopathy represented by the SP-BD diagnosed as having CC during the study[74% with LEVF >50%], and severe cases represented by previously diagnosed CC patients[85% with LEVF<50%]). By analyzing the groups separately, the association between detection of T. cruzi DNA detection and CC was only significant among the SP-BD group, suggesting that in the late stage of the disease, as reflected in the CC patient group, parasite persistence is no longer driving the progression of the clinical/laboratory markers.
Norman et al analyzed 246 seropositive Chagas patients from Instituto deSalud Carlos III (Madrid, Spain), and could not find an association between PCR positivity and cardiomyopathy (18). However, only 20% of the samples tested positive. The smaller sample size and low positive rate probably precluded them from detecting the associations between parasitemia and disease status and severity observed in our study.
There is only one small prospective cohort study of 56 patients in Argentina who had PCR results at baseline and were followed for approximately 2.5 years(19). Progression to cardiomyopathy was detected in 12 patients (21.4%) and the relative risk for disease progression was 4.09-fold higher, (95%CI 1.60 to 9.85) among the patients who tested PCR positive at baseline.(19)
The etiology of myocardial damage that occurs in at least 30-40% of patients infected by T. cruzi is not fully understood(20). The parasite is barely detected in the heart by conventional microscopy, which has led some investigators to suggest that the inflammatory process was a consequence of autoimmunity and not a direct response to the presence of the parasite in the heart.(21)
The importance of parasite persistence in the pathogenesis of Chagas disease was suggested by previous experimental studies and clinical studies with small sample sizes. Schijman et al. demonstrated an association of parasite level and myocardial inflammation as detected by PCR on heart tissue from 21 Chagas patients.(22) This finding was confirmed by Benvenuti et al. who analyzed heart biopsies from 29 individuals for T. cruzi DNA and found 25 positive by PCR (immunohistochemistry failed to demonstrate T. cruzi antigens in any of the samples resulting in speculation by critics that the PCR positivity could represent detection of residual DNA from dead parasites) (23). In a murine model of T. cruzi infection Zhang & Tarleton used an in situ PCR technique to demonstrate correlations between the persistence of parasites and the presence of disease in heart muscle tissue. (24) In a clinical trial with unblinded, non-random assignment, Viotti et all have shown reduced progression of Chagas among Benznidazole treated patients, supporting the hypothesis that parasite persistence is a key element in the pathogenesis of the chronic form of the disease.(25)
Although our study presents data supporting the importance of parasite persistence in the pathogenesis of cardiac disease, we recognize that parasite persistence, as detected in blood, does not fully explain the etiology of CC nor is it highly predictive of CC. Approximately 20% of the individuals with cardiomyopathy were PCR negative and 50% of seropositive donors without CC tested PCR-positive, and hence the predictive value of parasite detection in blood for disease progression is limited. Furthermore, the PCR load was only weakly associated with markers of disease severity.
One of the limitations of our study was that PCR was performed at only one time point, so it is possible that the CC cases that tested PCR negative may have intermittent parasitemia and could test positive if PCR were performed on second or third sample collections..
It is important to acknowledge that our study was based in Brazil where Tc-II genotype of T cruzi is responsible for the majority of the cases, so the findings may not be generalized to individuals infected by Tc-I genotype that is more prevalent in Central America(5). Different strains of the T. cruzi have also been associated with distinct clinical outcomes in experimental and clinical models(20) Another limitation of our study was that PCR was performed at the time of the diagnosis of cardiomyopathy, and not at the time of blood donation, so we cannot infer how well PCR results would predict subsequent disease development.
An interesting finding of our study was that individuals with prior treatment with benznidazole had a 50% lower rate of PCR positivity as compared to SP-BD without CC. As previously reported, the rate of cardiomyopathy, however, was the same in this group as compared to the non-treated group of seropositive former blood donors (11/48 versus 105/445 p= 1.0)(10). Since we did not have data on the timing of drug treatment relative to the diagnosis of cardiomyopathy, we cannot make any inference on whether treatment decreases the incidence of CC. A large clinical trial (BENEFIT) is currently under way to clarify the role of trypanocidal therapy in preventing cardiac disease progression and death; importantly this study includes PCR analysis so will be able to further inform the predictive value of detection of blood parasitemia for disease progression and response to therapy.(5, 27). In the study of Norman et all, negative results on PCR was associated with Benznidazole treatment(18). Recently two studies have shown the usefulness of PCR for monitoring response to trypanocidal treatment.(28-30)
Finally, we did not detect any PCR positive samples from the 483 SN-BD who were matched to the SP-BD with respect to location and demographics which was intended to enhance the risk of exposure to T. cruzi. Although SN-BD had a lower risk exposure to T. cruzi when compared to SP-BD, 51% of them had lived in a rural area and 24% had a relative with Chagas disease (10). Several previous publications have reported so-called serosilent T. cruzi infections, raising concerns over the sensitivity of serological assays for diagnostics and donor screening (11-13). Our results are reassuring since they demonstrate that contemporary donor screening and diagnostic assays are highly sensitive in detecting antibodies in most if not all infected individuals.
In conclusion, our data provide evidence supporting a direct role of parasite persistence in chronic Chagas disease pathogenesis.
Supplementary Material
Figure 4 appendix Spearman correlation of parasite level versus ECHO variables(Ejection Fraction and Left Ventricular Index Mass), Biomarkers (Troponin and Nt-ProBNP) and ECG variables (QRS duration and corrected QT interval).
Acknowledgments
Supported by the NHLBI Retrovirus Epidemiology Donor Study-II (REDS-II), International Component, Contract HHSN268200417175C.
Footnotes
The authors have no conflict of interest.
The results of this study were partially presented at the American Association of Blood Bank(AABB) meeting in 2011 and at the American College of Cardiology meeting in 2013
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Supplementary Materials
Figure 4 appendix Spearman correlation of parasite level versus ECHO variables(Ejection Fraction and Left Ventricular Index Mass), Biomarkers (Troponin and Nt-ProBNP) and ECG variables (QRS duration and corrected QT interval).