SYSTEMATIC REVIEW published: 31 January 2022 doi: 10.3389/fcvm.2021.787214 Association Between Trypanosoma cruzi DNA in Peripheral Blood and Chronic Chagasic Cardiomyopathy: A Systematic Review Pau Bosch-Nicolau 1,2 , Juan Espinosa-Pereiro 1,2 , Fernando Salvador 1,2 , Adrián Sánchez-Montalvá 1,2 and Israel Molina 1,2* 1 Tropical Medicine & International Health Unit Vall d’Hebrón - Drassanes, Infectious Diseases Department, PROSICS Barcelona, University Hospital Vall d’Hebron, Barcelona, Spain, 2 Medicine Department, Universitat Autònoma de Barcelona, Barcelona, Spain Chronic chagasic cardiomyopathy (CCC) is the most important complication of patients with Chagas disease (CD). The role of persistent detection of DNA in peripheral blood and its association to CCC is unknown. We performed a systematic review up to July 2021, including studies that reported ratios of CCC and PCR positivity among non-treated adult patients. We identified 749 records and selected 12 for inclusion corresponding to 1,686 patients. Eight studies were performed in endemic countries and 4 in non-endemic Edited by: Livia Passos, countries. Only two studies showed an association between CCC and Trypanosoma Brigham and Women’s Hospital and cruzi parasitemia by means of PCR detection. Six studies reported greater positive PCR Harvard Medical School, United States ratios among patients with CCC than in the patients with indeterminate chagas disease Reviewed by: (ICD) with no statistical significance. A significant risk of bias has been detected among Erik Josef Behringer, most of the studies. Therefore, while we performed a meta-analysis, wide inter-study Loma Linda University, United States heterogeneity impeded its interpretation. Marcelo Santos Da Silva, São Paulo State University, Brazil Conclusions: With the available information, we could not establish a correlation *Correspondence: between PCR-detectable parasitemia and CCC. Israel Molina imolina@vhebron.net Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42020216072, Specialty section: This article was submitted to identifier: CRD42020216072. General Cardiovascular Medicine, Keywords: Chagas disease, Chagas cardiomyopathy, T. cruzi, polymerase chain reaction, PCR a section of the journal Frontiers in Cardiovascular Medicine Received: 30 September 2021 INTRODUCTION Accepted: 28 December 2021 Published: 31 January 2022 Chagas disease (CD) is a protozoal disease caused by Trypanosoma cruzi, a zoonotic infection Citation: mainly found in endemic areas of the American continent. It affects about 8 million people Bosch-Nicolau P, Espinosa-Pereiro J, worldwide and because of globalization and international migrations during the last decades, it Salvador F, Sánchez-Montalvá A and has become a cause of concern in non-endemic countries (1). Molina I (2022) Association Between Chagas disease has an acute phase that generally runs its course asymptomatic or with Trypanosoma cruzi DNA in Peripheral rather unspecific symptomatology. Once patients overcome this phase, they enter a chronic Blood and Chronic Chagasic Cardiomyopathy: A Systematic phase, defined by the absence of trypomastigotes in the blood smear. Most people with T. Review. cruzi infection are diagnosed at this stage. Approximately 30–40% of chronically infected Front. Cardiovasc. Med. 8:787214. patients will develop visceral involvement comprising the chronic chagasic cardiomyopathy doi: 10.3389/fcvm.2021.787214 (CCC), the digestive form, or both during the following 10–30 years after infection (2). Frontiers in Cardiovascular Medicine | www.frontiersin.org 1 January 2022 | Volume 8 | Article 787214 Bosch-Nicolau et al. T. cruzi PCR and Chagas Cardiomyopathy CCC is considered the cause of at least 7,000 deaths every studies considering any PCR protocol whenever the same year and is the most common reason for performing heart procedure was maintained throughout the study. transplants in Latin America. The principal underlying causes are Studies assessing the impact of treatment with benznidazole, sudden death from malignant arrhythmias and heart failure as a nifurtimox, or azole-derivative drugs were excluded. In addition, consequence of dilated cardiomyopathy (3). we excluded studies focusing on acute infections, pregnant Both host immune response and the persistence of infection women, children, or immunocompromised patients. are crucial on CCC progression (4). Host factors, such as genetic polymorphisms involved in the immune response have been Literature Search, Data Collection, and proposed as prognosis markers (5). Regarding the role of T. Reporting of Results cruzi persistence, several studies have evidenced that visceral We searched Medline, EMBASE, and LILACS databases. involvement is directly linked to the parasite presence on such Additionally, we tracked citations to relevant studies in Scopus organs in both human and animal models (6). Besides, an and the ISI Web of Knowledge for review purposes, and association between certain discrete typing units (DTU), which manually screened references lists of these studies. We adapted are used to group T. cruzi genetic diversity, and its virulence and the search strategy to the requirements of each database tissue tropism has been described (7). (Supplementary Material Appendix 1). There were no language Blood parasites can be detected by PCR. However, parasite or publication period restrictions. We conducted the last search dynamics is still a matter of debate. While in the acute phase, during July 2021. the presence of T. cruzi in the blood is constant, in the chronic One reviewer (PBN) screened the titles and abstracts resulting phase low level parasitemia is observed in a subset of patients from the search against inclusion criteria. We obtained a full-text (8). Different PCR assays have been developed to detect the copy of eligible references to finally decide on their inclusion. parasite DNA with initial difficulties in standardizing techniques A second reviewer (JEP) independently checked the eligibility to obtain reliable and comparable results (9). However, in decisions for accuracy. We discussed disagreements until a recent years, notable advances have been made with new decision was reached; and planned to involve a third investigator methodologies showing reliable results that have been used to if discrepancies remained. monitor the parasite load in patients with CCC and follow-up the Two researchers (PBN and JEP) extracted data from included effectiveness of etiologic treatments (10). Besides, the relationship studies using the standardized extraction forms. No pilot between parasitemia or the presence of parasitic DNA on study was conducted due to the low number of articles peripheral blood, and disease progression is controversial included. Whenever possible, article authors were contacted for (11, 12). unreported or additional data to minimize the missing data. Therefore, this systematic review has the objective of assessing For each study, we collected (when available): study design, the association between the presence of parasitic DNA of T. cruzi baseline characteristics, cardiac involvement, PCR method, on peripheral blood through PCR and CCC. PCR status of the included patients at the inclusion, and association measures as reported by authors of each study. MATERIALS AND METHODS We independently assessed the risk of bias for each study using a modified Quality In Prognosis Studies (QUIPS). Tool Study Design checklist (Supplementary Material Appendix 2), appropriate We conducted a systematic review following the standardized for prognostic factor review questions (15). guidance (13), and we adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Statistical Analysis statement using a flow diagram and following its checklist We described the population of each study and the frequencies of to ensure that all recommended information is captured positive PCR among ICD and CCC participants. The association and findings are properly reported (14). The review protocol of PCR status and visceral involvement was extracted as the was registered in the PROSPERO database (registration ID amount of positive PCR among ICD participants and CCC number: CRD42020216072). participants, using as association measure the odds ratios (ORs) and 95% CI. Pooled OR was calculated using the Eligibility Criteria and Patient Population Mantel–Haenszel approach with a random effects model due We included clinical trials, controlled observational and cross- to the heterogeneity of each study population. Inter-study sectional studies in adult patients (>16 years old) with chronic heterogeneity was assessed with a restricted maximum likelihood CD reporting data on the results of the peripheral blood T. cruzi model and reported as the I 2 and tau statistics. We adhered PCR and CCC. to the methods recommended in the Cochrane Handbook for Eligible studies had to establish the chronic CD diagnosis Systematic Reviews of interventions whenever possible (16). through two different positive serological tests. Indeterminate Chagas disease (ICD) concerns patients diagnosed with a chronic RESULTS CD that presented with normal ECG and/or echocardiogram, regardless of the presence or absence of gastrointestinal disease. Our search retrieved 749 records. After removing duplicates CCC was defined as electrocardiographic or echocardiographic and reading titles and abstracts, we discarded 719 records as alterations not attributed to other conditions. We included they were in vitro experiments, were performed in animals, Frontiers in Cardiovascular Medicine | www.frontiersin.org 2 January 2022 | Volume 8 | Article 787214 Bosch-Nicolau et al. T. cruzi PCR and Chagas Cardiomyopathy FIGURE 1 | The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow diagram of included articles. CCC, chronic chagasic cardiomyopathy; PCR, polymerase chain reaction. corresponded to Trypanosoma species other than T. cruzi, from Bolivia and 1 from Japan. Patients’ ages ranged from 16 to included acute infection patients or treated patients, or did 81 years old, and most of them were women (58.8%; 885/1,505). not report visceral involvement. Of the remaining 30 reports, Chronic chagasic cardiomyopathy assessment was performed we proceeded to a full-text review and finally included 10 using ECG in all studies. In 4 (33.3%) studies, ECG was articles. The characteristics of excluded studies are reported combined with echocardiography, in 1 (8%) was combined in Supplementary Material Appendix 3 (8, 11, 17–34). After with chest radiography, and in 4 (33.3%), the 3 ancillary tests checking the reference lists of these studies, we identified two were performed. CCC classification was very heterogeneous and additional eligible studies. The complete eligibility process is included either predefined criteria by the authors (35–37, 40, 43) depicted in a PRISMA flowchart (Figure 1). or standardized classifications as Minnesota criteria (12, 38), Finally, we included 12 studies comprising 1,686 patients Kuschnir criteria (39, 44, 45), Rocha criteria (42), and New (12, 35–45), which are described in Table 1. Eight studies were York Heart Association (NYHA) classification (41). Among the cross-sectional studies, 3 were designed as prospective cohorts, included patients, 998 (59.2%) were classified as ICD and 688 and 1 was a case-control study. Countries where studies were (40.8%) as CCC. CCC proportion among different studies varied performed in endemic areas were as follows: 3/8 (37.5%) in Brazil, from 12.1% (40) in a study including young childbearing-aged 3/8 (37.5%) in Chile, 1 (12.5%) in Argentina, and 1 (12.5%) women to 80% (43) in a study including patients under the in Bolivia. Among them, 1 was performed in a rural site, 2 in suspicion of organ involvement in Latin-American people living urban facilities, and 5 combined patients living in both urban and in Japan. However, the last study included only 5 patients. rural contexts. Four studies included patients from non-endemic Cardiologic characteristics of patients with CCC are summarized countries: 3 from Spain where more than 90% of patients came in Supplementary Material Appendix 4. Frontiers in Cardiovascular Medicine | www.frontiersin.org 3 January 2022 | Volume 8 | Article 787214 Frontiers in Cardiovascular Medicine | www.frontiersin.org Bosch-Nicolau et al. TABLE 1 | Summary of the characteristics of the included studies. References Study Study site Included Age Sex Excluded CCC Clinical form PCR PCR results Association and design (excluded) comorbidities assessment technique* measure country Salomone Cross- Endemic 68 (23) 55 y (SD ± 12) 64% F ICD: Yes EKG + ICD: 27 Qualitative Global + PCR: OR 5.17 et al. (35) sectional (urban) ICD: 52 y (SD ± 9) 70% F CCC: EchoC (39.7%) CCC: c-PCR 14/68 (21%) ICD: (95%CI Argentina CCC: 59 y 59% F 41 (60.3%) 1 sample 2/27 (7.4%) CCC: 1.06–25.36) (SD ± 12) Kinetoplast 12/41 (29.2%) DNA Carrasco Cross- Endemic 38 (225) ICD: 40,57 y ICD: 62% M No EKG + ICD: 26 Qualitative Global + PCR: OR 3.20 et al. (36) sectional (rural) (SD ± 10.59) CCC: 62% M EchoC + (68.4%) CCC: c-PCR 18/38 (54.7%) (95%CI Chile CCC: –HF 68.4 y Thoracic 12 (31.6%) 1 sample ICD: 10/26 (36%) 0.76–13.46) (SD ± 12.9) –PM: X-Ray Kinetoplast CCC: 8/12 (66%) 54.1 y (SD ± 8.4) DNA –Altered EKG: 53.4 y (SD ± 19.1) Zulantay Prospective Endemic 30 (0) 33.2 y (R 18–50) 53.3% M Yes EKG ICD: 18 (60%) Qualitative Global + PCR: OR 1.12 et al. (37) cohort (urban and CCC: 12 c-PCR 17/30 (56.6%) (95%CI Chile rural) (40%) 1 sample ICD: 10/18 0.26–4.91) Kinetoplast (55.5%) CCC: 4 DNA 7/12 (58.3%) Borges- Cross- Endemic 12 (9) 48.6 y (R 16–82) 58,8% F ICD: No EKG ICD: 6 (50%) Qualitative Global + PCR: OR 0.40 Pereira et al. sectional (rural) ICD: 43.5 y CCC: 70% F CCC: CCC: 6 (50%) c-PCR 9/12 (75%) ICD: (95%CI (38) Brazil 64.4 y 42.9% F 1 sample 5/6 (83.3%) CCC: 0.03–6.18) Kinetoplast 4/6 (66.6%) DNA Murcia et al. Prospective Non 181 (0) 33 y (SD ± 11) No reported No reported EKG + ICD: 116 Qualitative Global + PCRs: OR 0.79 (39) Spain cohort endemic EchoC + (64%) CCC: c-PCR 123/181 (68%) (95%CI Thoracic 65 (36%) 1 sample ICD: 81/116 0.41–1.50) X-Ray Kinetoplast (69,8%) CCC: DNA 42/65 (64.6%) Sabino Cross- Endemic 485 (115) IND (PCR–): 48.4 y 53,6% M IND: Yes EKG + ICD: 279 Quantitative Global + PCR: OR 3.48 et al. (12) sectional (urban and (SD ± 10.1) IND 52,3% M EchoC (57.5%) CCC: rt-PCR 304/485 (62.7%) (95%CI T. cruzi PCR and Chagas Cardiomyopathy January 2022 | Volume 8 | Article 787214 Brazil rural) (PCR+): 49.1 y CCC: 59,4% 206 (42.5%) 1 sample ICD: 143/279 2.31–5.23) (SD ± 10.6) CCC M Kinetoplast (51.3%) CCC: (PCR–): 49.2 y DNA 161/206 (78.1%) (SD ± 6.3) CCC (PCR+): 47.8 y (SD ± 7.1) (Continued) Frontiers in Cardiovascular Medicine | www.frontiersin.org Bosch-Nicolau et al. TABLE 1 | Continued References Study Study site Included Age Sex Excluded CCC Clinical form PCR PCR results Association and design (excluded) comorbidities assessment technique* measure country Kaplinski Cross- Endemic 83 (337) ICD: 27 y 100% F No reported EKG IND: 73 Quantitative Global + PCR: OR 0.52 et al. (40) sectional (urban and (R 22–34) CCC: (87.9%) CCC: rt-PCR 36/83 (43.4%) (95%CI Bolivia rural) 32 y (R 24–39) 10 (12.1%) 1 sample ICD: 33/73 0.12–2.17) Kinetoplast (45.2%) CCC: DNA 3/10 (33.3%) Apt et al. Case- Endemic 200 (0) ICD: 50.5 y ICD: 79% F Yes EKG + ICD: 100 Qualitative Global +PCR: OR 1.05 (41) Chile control (urban and (R 20–77) CCC: CCC: 68% F EchoC (50%) CCC: c-PCR 145/200 (72.5%) (95%CI rural) 56.4 y (R 25–81) 100 (50%) 1 sample ICD: 72/100 (72%) 0.57–1.96) Kinetoplast CCC: 73/100 DNA (73%) Sánchez- Cross- Non 455 (316) 39 y (R 31–46.5) 68.2% F Yes EKG + ICD: 302 Qualitative Global +PCR: OR 1.13 Montalvá sectional endemic ICD: 37 y EchoC + (66.4%) CCC: rt-PCR 118/455 (25.9%) (95%CI et al. (45) (R 31–44) CCC: Thoracic 153 (43.6%) 1 sample ICD: 76/302 0.72–1.75) Spain 42 y (R 36–49) X-Ray Satellite DNA (25.2%) CCC: 42/153 (27.4%) D’Ávila et al. Cross- Endemic 91 (0) ICD: 44 y ICD: 65.2% F No reported EKG + ICD: 23 Quantitative Global + PCR: OR 1.13 5 (42) Brazil sectional (urban and (SD ± 10.3) CCC: CCC: 33.8% EchoC + (33.8%) CCC: rt-PCR 65/91 (71.4%) (95%CI rural) 54 y (SD ± 10.3) F Thoracic 68 (66.2%) 1 sample ICD: 16/23 0.40–3.17) X-Ray Satellite DNA (69.6%) CCC: 49/68 (72%) Salvador Prospective Non 38 (16) 36 y (R 22–55) 75.6% F No reported EKG + ICD: 27 (71%) Qualitative Global + PCR: OR 1.21 et al. (44) cohort endemic Thoracic CCC: 11 rt-PCR 16/38 (42.1%) (95%CI Spain X-Ray (29%) 1 sample ICD: 11/27 0.29–4.98) Satellite DNA (40.7%) CCC: 5/11 (45.4%) Imai et al. Cross- Non 5 (12) 57.6 y (R 49–68) 60% F No EKG + ICD: 1 (20%) Qualitative Global +PCR: 3/5 OR 0.33 (43) Japan sectional endemic EchoC CCC: 4 (80%) rt-PCR (60%) ICD: 1/1 (95%CI 1 sample (100%) CCC: 2/4 0.01–12.82) Satellite DNA (50%) T. cruzi PCR and Chagas Cardiomyopathy January 2022 | Volume 8 | Article 787214 CCC, chronic chagasic cardiomyopathy; c-PCR, conventional polymerase chain reaction; EchoC, echocardiography; ECG, electrocardiogram; ICD, indeterminate chagas disease; R, range; RR, risk ratio; rt-PCR, real time polymerase chain reaction; SD, standard deviation; y, years; PM, pacemaker; HF, heart failure. *PCR methodology: specifying PCR technique (conventional PCR or real time PCR) and target of the used primer (kinetoplast DNA or nuclear satellite DNA). Bosch-Nicolau et al. T. cruzi PCR and Chagas Cardiomyopathy All the included studies used a PCR for DNA parasite a positive PCR rate that favored patients with ICD (38–40, detection in peripheral blood. However, many different protocols 43). were used for its determination. All studies used a single When we meta-analyzed the OR, pooled results showed that venous blood sample for PCR determination. Six studies (50%) the estimated OR for positive T. cruzi PCR of patients with CCC used a conventional PCR method (35–39, 41) while the other compared to patients with ICD was 1.36 (95% CI: 0.87–2.12) half used real-time PCR method (12, 40, 42–45). Only four (Figure 2). However, we found significant heterogeneity among studies used a quantitative method to report PCR results studied variables in the meta-regression analysis. To diminish (12, 40–42) while the rest of the studies used a qualitative variability, we grouped studies by patient’s countries of origin and method. There is a wide variation regarding primers used. by PCR technique with the same results. Thus, no conclusions Most studies (8/12; 66.3% used a real time PCR method can be achieved from the meta-analysis. based on the amplification of a genomic DNA sequence of the Kinetoplast. In the other 4 studies, satellite DNA amplification was used (42–45). Parasite detection in patients DISCUSSION with ICD ranged from 7.4 (35) to 100% (43). When considering patients with CCC, parasite detection varied from 27.4 (45) to In this review, despite 8 out of 12 studies reporting higher 78.1% (12). ratios of positive PCR among patients with CCC, we could not The results of the risk of bias assessment using the QUIPS scale find a correlation between parasitemia by means of PCR and are shown in Supplementary Material Appendixes 5, 6. Most CCC. When we analyzed the characteristics of the included studies included a representative population with CD. However, studies, in only 2 studies, the proportion of PCR positivity was 2 studies were considered at an overall high risk as one included significantly greater within CCC (12, 35). These studies were only childbearing-aged women (40), and the other included performed in an endemic region and their specific objective was patients under suspicion of organ involvement (43). As per study to determine the correlation between parasitemia and CCC. It attrition, most of the studies were classified as low risk of bias has been suggested that patients in endemic regions are prone since basal characteristics including PCR and organ assessment to parasite re-exposure, contributing to higher rates of CCC were performed at the inclusion and, in consequence, data were and parasitemia among patients with CD (45). In the same line, available for all participants included in the studies. Regarding in a study performed in a non-endemic country, the positive outcome measurement (PCR), all the studies specified their PCR ratio decreased in patients with longer periods since the protocol and almost all included control samples and maintained first arrival (8). Also, geographic distribution should play a role the same procedure in all samples. As per CCC assessment, in CCC development and parasitemia burden. T. cruzi genetic six studies were rated at low risk of bias since they included diversity is unequally distributed among different countries, and blind ECG assessment and/or double assessment by different some authors have reported a cardiac tropism of some discrete investigators (12, 37, 38, 40, 41, 45). Conversely, two studies typing units (DTUs) (7). In this review, included patients came that neither used a standardized classification nor reported their mostly from 4 different countries and only one of them reported ECG or echocardiographic findings were considered at high DTUs determination (42). risk of bias (35, 43). Considering study confounders, all the Age was higher among patients with CCC in studies studies but one was rated as having a moderate to high risk performed in endemic countries recruiting the general of bias. Most of the studies did not consider cardiovascular population (range 49.2–68 years) than in non-endemic countries risk factors and other possible heart diseases or performed a (range 33–42 years). Regardless of the age per se having been stratified data analysis. Finally, result presentation and statistical described as a risk factor for CCC and its mortality, results analysis were classified at a moderate to high risk of bias in from different reports are inconsistent (46). On one hand, study a wide group of studies. Most of them were designed for participants in their second to the fourth decade of life may have another purpose and we retrieved the specific information from not yet developed target organ damage, or it is too incipient, as their results. it takes on average 20–30 years after the acute infection. On the An association between CCC and T. cruzi parasitemia by other hand, older people often associate other cardiovascular means of PCR detection was found in 2 studies (12, 35). They risk factors and this could lead to a misinterpretation of the CCC all were performed in endemic regions and found a greater assessment (47). In fact, only three studies were excluded from PCR positivity between patients with CCC and ICD with ORs their analysis due to other cardiovascular risk factors (12, 41, 45). of 5.17 (CI 1.06–25.36) and 3.48 (CI 2.31–5.23). In one study, Male sex has been related to CCC development and higher although a risk ratio (RR) of 4.45 was reported, it included mortality rates (48). Although, this predisposition was described both cardiac and digestive forms on the analysis, and when OR as an independent risk factor from parasitemia (20). Among the was calculated with only CCC patients, we could not achieve included studies, the male proportion ranged from 31.8 to 66.2%. statistical significance (36). None of the non-endemic studies As none of the included studies conducted stratified analysis by identified an association between parasite DNA detection in sex or age, results should be interpreted with caution. peripheral blood and CCC. Six studies reported greater positive Classically, CCC assessment has involved ECF, chest X-ray, PCR ratios among patients with CCC than in patients with and clinical status, as they are easy to collect and widely available. ICD although differences did not achieve statistical significance Echography, a non-invasive examination, has been progressively (36, 37, 41, 42, 44, 45). The remaining 4 studies reported studied in CD and its performance has been introduced in Frontiers in Cardiovascular Medicine | www.frontiersin.org 6 January 2022 | Volume 8 | Article 787214 Bosch-Nicolau et al. T. cruzi PCR and Chagas Cardiomyopathy FIGURE 2 | Evaluation of the association between PCR detection between patients with CCC and ICD. CI, confidence interval; OR, odds ratio. different CCC classifications (49). ECG abnormalities are usually (12, 40–42) but parasitemia levels were not related to any clinical seen before the patient develops a malignant arrhythmia or outcome. Parasitemia levels are usually low in chronic patients heart failure (50). However, echocardiographic alterations as with CD compared with parasitemia levels in patients with acute diastolic dysfunction can appear before electrical abnormalities CD. Different studies have analyzed the correlation between develop and may be used as an early marker of CCC (45). quantitative results of T. cruzi in peripheral blood and organ In this review, the CCC assessment was very heterogeneous. damage with inconsistent results (41, 50). Parasite detection While 3 studies performed only an ECG and 1 study included a ratios ranged widely within the included studies. In patients with chest X-ray, 8 of 12 studies used echocardiography to complete ICD, the detection ranged from 7.4 (35) to 100% (43) while in the cardiac evaluation. CCC classifications were disparate, patients with CCC varied from 27.4 (45) to 78.1% (12). Besides while 5 authors used a non-standardized classification (35– the previously described factors, parasite dynamics in blood have 37, 40, 43), 7 used different standardized criteria (12, 38, not been thoroughly characterized. Intermittent parasitemia is 39, 41, 42, 44, 45). Conduction system alterations as right- constant in the chronic phase of CD but its periodicity, triggers, bundle block and left anterior hemiblock prevailed over other determinants, or reservoirs are still unknown (8). Thus, although arrhythmias or contraction abnormalities in most of the studies the internal variability is probably acceptable among all studies as (Supplementary Material Appendix 3). Two studies (39, 42) did they used the same method throughout, lack of sensitivity could not provide cardiological findings of their participants, and only result in low positive ratios precluding differences detection. 4 studies (41, 42, 44, 45) provided the distribution of disease This systematic review has several limitations. As a neglected severity. As a result, between-study comparisons of CCC severity tropical disease, scarce investment over time has limited the are not feasible. generation of high-quality evidence (51). A significant risk of Current PCR-based methods for T. cruzi detection have bias has been detected among most of the studies resulting from shown high sensitivity and specificity compared with classical small sample sizes, differences among study designs, differences methods, such as blood culture or xenodiagnosis (8). However, not reporting on basal characteristics, different CCC assessments, sample collection, conservation, sample volume, DNA extraction and PCR methods along with the fact that most of the studies method, and primers used may influence its performance (9). were not even designed for this purpose. To improve studies As a result, in recent years, many initiatives have pursued the comparability, we have grouped them by the main confounding standardization of these techniques (33). In the present review, factors: country of origin and quality of PCR technique but earlier studies used conventional PCR methods preferably using variability remained. Therefore, while we have performed a as molecular targets of the kinetoplast DNA sequences. On meta-analysis, wide inter-study heterogeneity (I2 64%, p < 0.01) the other hand, later studies preferred real-time PCR (rt-PCR) impedes a robust interpretation of its results. A patient-level techniques toward satellite or minicircle DNA sequences which meta-analysis could improve the consistency and robustness of showed better sensitivity and specificity results and allowed the results and provide evidence to better inform physicians quantification (9). Only four studies used a quantitative method treating people living with CD. Frontiers in Cardiovascular Medicine | www.frontiersin.org 7 January 2022 | Volume 8 | Article 787214 Bosch-Nicolau et al. T. cruzi PCR and Chagas Cardiomyopathy In conclusion, with the current information, we could manuscript writing. JE-P has contributed to this study through not establish a correlation between PCR-detectable parasitemia abstract and article revision and final article review. FS, and CCC. Better prospective studies with a defined follow- AS-M, and IM contributed through study design and article up, representative population, homogeneous criteria, and reviewing. All authors contributed to the article and approved the standardized methods for PCR detection are needed to answer submitted version. this question. FUNDING DATA AVAILABILITY STATEMENT AS-M was supported by a postdoctoral grant Juan Rodés The original contributions presented in the study are included (JE18/00022) from the Instituto de Salud Carlos III through the in the article/Supplementary Material, further inquiries can be Spanish Ministry of Economy and Competitiveness. directed to the corresponding author/s. SUPPLEMENTARY MATERIAL AUTHOR CONTRIBUTIONS The Supplementary Material for this article can be found PB-N has majorly contributed to this study through study online at: https://www.frontiersin.org/articles/10.3389/fcvm. designing, abstract and articles revision, data analysis, and 2021.787214/full#supplementary-material REFERENCES by PCR is associated with Chagas cardiomyopathy and disease severity. Eur J Heart Fail. (2015) 17:416–23. doi: 10.1002/ej 1. Basile L, Jansá JM, Carlier Y, Salamanca DD, Angheben A, Bartoloni A, et al. hf.220 Chagas disease in European countries: the challenge of a surveillance system. 13. Riley R, Moons K, Snell K, Ensor J, Hooft L, Altman D, et al. A guide to Eurosurveillance. (2011) 16:19968. doi: 10.2807/ese.16.37.19968-en systematic review and meta-analysis of prognostic factor studies. BMJ. (2019) 2. Pérez-Molina J, Molina I. Chagas disease. Lancet. (2018) 364. doi: 10.1136/bmj.k4597 391:82–94. doi: 10.1016/S0140-6736(17)31612-4 14. 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[No association between persistence of the parasite and this article, or claim that may be made by its manufacturer, is not guaranteed or electrocardiographic evolution in treated patients with Chagas disease]. Rev Med Chil. (2005) 133:1153–60. doi: 10.4067/S0034-988720050010 endorsed by the publisher. 00004 38. Borges-Pereira J, Sarquis O, Zauza P, Britto C, Lima M. [Epidemiology Copyright © 2022 Bosch-Nicolau, Espinosa-Pereiro, Salvador, Sánchez-Montalvá of Chagas disease in four rural localities in Jaguaruana, State of Ceará: and Molina. This is an open-access article distributed under the terms of the Creative seroprevalence of infection, parasitemia and clinical characteristics]. Rev Commons Attribution License (CC BY). The use, distribution or reproduction in Soc Bras Med Trop. (2008) 41:345–51. doi: 10.1590/S0037-868220080004 other forums is permitted, provided the original author(s) and the copyright owner(s) 00005 are credited and that the original publication in this journal is cited, in accordance 39. Murcia L, Carrilero B, Muñoz M, Iborra M, Segovia M. Usefulness of PCR for with accepted academic practice. No use, distribution or reproduction is permitted monitoring benznidazole response in patients with chronic Chagas’ disease: a which does not comply with these terms. Frontiers in Cardiovascular Medicine | www.frontiersin.org 9 January 2022 | Volume 8 | Article 787214