Longitudinal Study of Patients With Chronic Chagas Cardiomyopathy in Brazil (SaMi_Trop Project)
Longitudinal Study of Patients With Chronic Chagas Cardiomyopathy in Brazil (SaMi_Trop Project)
BACKGROUND: Chagas Disease (ChD) remains as one of the most neglected diseases in the world,
 with 8-10 million infected people and only one marginally effective therapeutic. The lack of
 good biomarkers for active infection or clinical end-points poses a problem for assessing the
 performance of new drugs or therapeutic interventions. Among the biomarkers, several studies
 showed that Brain Natriuretic Peptide (NT-ProBNP) is accurate maker of left ventricular
 systolic and diastolic dysfunction.

 OBJECTIVE: Our long term goal is to establish The Sao Paulo-Minas Gerais Tropical Medicine
 Research Center (SaMi-Trop) as a Center of Excellence for Neglected Infectious Disease
 Research in Brazil. The Specific Aims are to begin that process by focusing on Trypanosoma
 cruzi infection with the goal of finding an array of biomarkers that correlate with parasite
 persistence and Chagas cardiac disease status that can be used to infer risk of disease
 progression and death as well used as markers of cure (parasite eradication) or clinical
 efficacy (stabilize or reverse cardiac damage) of novel drugs

 METHOD: The investigators established a prospective cohort of 1,959 patients with chronic
 Chagas cardiomyopathy (CCC). The study is being conducted in 21 cities of the northern part
 of Minas Gerais state in Brazil, and includes a follow up of at least two years (baseline and
 24 months) . The evaluation included collection of socio-demographic information, social
 determinants of health, health-related behaviours, comorbidities, medicines in use, history
 of previous treatment for Chagas Disease (ChD), symptoms, functional class, quality of life,
 blood sample collection and ECG.
RESEARCH STRATEGY

 SIGNIFICANCE

 The Sao Paulo-Minas Gerais Tropical Medicine Research Center (SaMi-Trop) consists of a
 network of collaborating scientists in the States of Minas Gerais and São Paulo which has
 been established for the purpose of developing and conducting research projects on neglected
 infectious diseases in Brazil, with an initial focus on Chagas Disease biomarker, discovery
 and validation. The SaMi-Trop represents a major challenge for focused research in neglected
 diseases, with knowledge that can be applied in primary health care.

 Chronic Chagas Cardiomyopathy (CCC) is the most important clinical presentation of Chagas
 Disease (ChD), eventually affecting approximately 20% to 30% of those in the chronic phase of
 the disease. CCC comprises a wide range of manifestations, including heart failure,
 arrhythmias, heart-blocks, sudden death, thromboembolism and stroke [1]. Clinical
 presentation typically varies widely according to the degree of myocardial damage and most
 patients present a mild form of heart disease, frequently characterized only by the presence
 of asymptomatic abnormalities on the ECG or in other complimentary exams [2]. Nonetheless,
 when heart failure and/or severe arrhythmias manifest, the prognosis is ominous, with high
 and premature mortality rates, typically in adult male patients,1 but also in the elderly
 [3]. Indeed, when compared to patients with idiopathic cardiomyopathy, patients with CCC have
 poorer survival, irrespective of other clinical and echocardiographic parameters [4].

 Thus, the clinical course of Chagas heart disease is variable and identifying patients who
 are at risk of progressive CCC and dying remains a challenge. Several longitudinal studies
 performed in patients in the chronic phase of ChD demonstrated that many individual
 characteristics predict an unfavorable prognosis. This subject was systematically reviewed a
 few years ago and concluded that most studies were described in insufficient detail and/or
 lacked minimal standards in reporting results. Results have been somewhat inconsistent in
 different studies, and the best combination of clinical and laboratory variables predictive
 of mortality has not yet been established [5].

 A potentially helpful score to predict death was developed to permit the stratification of
 risk in Chagas heart disease [6]. Six independent prognostic factors were identified, and
 each was assigned a number of points: New York Heart Association class III or IV (5 points),
 evidence of cardiomegaly on radiography (5 points), left ventricular systolic dysfunction on
 echocardiography (3 points), non-sustained ventricular tachycardia on 24-hour Holter
 monitoring or stress testing (3 points), low QRS voltage on electrocardiography (2 points),
 and male sex (2 points) [6. Patients were classified in three risk groups according to the
 final score: low risk (0 to 6 points), intermediate risk (7 to 11 points), and high risk (12
 to 20 points). In the original study, the 5-year mortality rates for these three groups were
 2 percent, 18 percent, and 63 percent, respectively.6 Although the score was successfully
 validated in independent cohorts, [6,7] some important issues may arise. The derivation of
 the score is complex and it is not easy to calculate during the medical consultation. To be
 calculated, the patient must be submitted to several exams and procedures, including clinical
 examination, ECG, Chest x-ray, echocardiogram and Holter monitoring or stress testing. Most
 of these tests are not available in the rural areas, where most of ChD patients live.

 A simple score, based on widely available and low-cost tests, which could stratify the risk
 of death without complex calculations, might be useful in the primary care setting, where
 echocardiograms and Holter monitoring are not widely available. Easily obtained variables,
 such as clinical features, BNP levels and ECG measurements, are good candidates as biomarkers
 for developing a new simple predictive rule. Moreover, new biomarkers in blood, related
 mainly to inflammation and the immune response, such as TNF-alpha, adhesion molecules and
 other cytokines, may be determined to be potent prognostic factors and may be useful in the
 future for further improvement in risk stratification in CCC.

 METHOD

 Design

 Non-concurrent prospective open cohort study, with minimal follow-up of two years (baseline
 and 24 months).

 Outcome

 Primary: death Secondary: hospitalization due to cardiac conditions; new ECG abnormalities

 Study Population

 Participants will be selected from patients evaluated by the Tele Minas Saude (TMS), the
 state Tele Health System. Since the TeleHealth System is currently acting in support of the
 Primary Care system, these patients are under the care of primary care physicians of the
 Health Family Strategy, a public primary care program that has a high coverage in the state
 of Minas Gerais.

 The inclusion criteria are to be a client of the TMS TeleHealth System, be >18 years old,
 have a positive history for T cruzi infection or Chagas disease, have an abnormal ECG and to
 consent to participating in the research program.

 The exclusion criteria include pregnancy or breast feeding, and any severe condition with
 ominous prognosis that indicates a life expectancy of less than two years.

 Sample size for patient accrual

 Considering the minimal number of events per variable acceptable in a proportional hazards
 regression analysis of 10 events per variable,8 and maximal number of studied variables of
 ten, the number of events would be 100 in the whole study. Since the prediction model has to
 be developed and validated and the whole sample will be divided in two, the number of events
 should be 200. For a 2-year follow-up period and annual mortality rate of 5% in CCC (10% in 2
 years), the calculated sample size is 2,000 subjects.

 Since the TMS Telehealth system performs 25,000 ECGs each month (300,000 a year), that ECG
 abnormalities typical of CCC are observed in 5% of these exams (1,250), and projecting that
 10% of these patients would be recruited and agree to participate in this research program,
 the calculated sample size of 2,000 patients will be recruited in a 16 months' accrual
 period.

 Follow-up

 Follow-up will be conducted on a yearly basis, with standardized interviews and new ECG exams
 performed after two years of baseline. Vital status and hospitalization will also be
 ascertained by linkage of the study database with mortality and hospitalization databases of
 the Ministry of Health.

 Recruitment, initial evaluation and measurements

 Patients will be recruited from among those that regularly attend the public primary care
 service of State of Minas Gerais in places served by TMS Telehealth System. This system
 provides teleconsultation and Electrocardiogram services (TeleECG), including interpretation
 of all ECGs by a cardiologist in the University Hospital. A standardized simple form is
 completed before the transmission of the ECG data to the University hospital and the question
 "Do you know if you have Chagas disease?" will be added to this questionnaire. Those who
 answer yes to this question and have abnormal ECG will be selected. A second visit of the
 selected patients will be scheduled for performing a short questionnaire (including the
 evaluation of the functional class) and for collecting blood samples. Blood samples will be
 sent to a central laboratory for T. cruzi serology and BNP measurements, which will be done
 by standard techniques. Other blood samples, including plasma for protein and antibody
 studies and stabilized whole blood lysates for T cruzi PCR (Polymerase Chain Reaction), DNA,
 will be stored at -80o C to enable batched testing for other biomarkers.

 Electrocardiogram services (TeleECG)

 The ECG performed in the town of origin of the patients will be sent to be assessed by the
 TMS Telehealth System specialists. The Telecardiology activities were developed using a
 digital electrocardiograph installed in the towns, at the Basic Health Units of the public
 healthcare system. The examination was always carried out after being requested by a
 physician. The ECG will be sent using communication software developed by the TMS Telehealth
 System and will be analyzed by a on-duty cardiologist. The report will be sent to the Primary
 Care facility and the ECG tracings will be transferred to ECG reading center. In this ECG
 Reading Center, a software developed by Prof. Peter Mac Farlane, from Glasgow University,
 will analyzes and codify the ECG (by the Minnesota Code) and compare to the previous ECG,
 according to a validated algorithm [9]. All abnormal codes will be reviewed by an experienced
 cardiologist.

 Data analysis

 The standardized mortality ratio (SMR) analysis and a survival analysis will be conducted.
 The outcome death is death. Also, a survival analysis considering the secondary outcomes -
 hospitalization due to cardiac conditions and new ECG abnormalities - will be run. The
 development and validation of the prognostic model will follow current recommendations,
 including the division of the sample in derivation and validation sample, and the use of
 modern methods to evaluate the performance of the model [10,11]. Additionally, descriptive
 spatial analysis using geographic distribution for the SMR and a Bayesian spatial model for
 small area data will be run.

 Innovation

 Beyond the relevance of the main goal of this study, it has many unique and innovative
 aspects, related to the use of knowledge and infra-structure developed for other projects
 conducted by our research group:

 1. The possibility of testing new biomarkers detected in other branches of this study or in
 the NIH-sponsored REDS-II/III Chagas disease study.

 2. The use of the infrastructure and the logistics of the TMS program, a state-wide
 TeleHealth System coordinated by the University Hospital in the Brazilian state of Minas
 Gerais

 3. The use of the methodology of linkage between our databases and administrative and
 mortality databases from the Health Ministry to complement the follow-up of the selected
 patients

 4. A standardized codification system of the ECGs, using the facilities of the ECG Reading
 Center of the long-term longitudinal study on the health of 15,000 adult public servants
 from Brazilian Universities (ELSA-Brazil).

 New biomarkers ChD has a complex physiopathology and many immunological, inflammatory,
 autonomic and micro-vascular processes have a role in the development or the progression of
 the disease [12]. The investigators have described increased levels of macrophage
 inflammatory protein (MIP-1alpha) and Tumor necrosis factor-alpha (TNF-alpha) in ChD
 patients, [13-15] and that those patients with more severe forms have more elevated levels
 [13]. Recently, in the ChD study (REDS-II), were studied 600 ChD patients and 500 controls
 searching for biomarkers related to the disease and CCC. In a preliminary analysis, several
 biomarkers including high sensitivity troponin, myoglobin, soluble vascular cell adhesion
 molecule 1 (sVCAM-1), soluble intracellular cell adhesion molecule (sICAM) and
 myeloperoxidase showed abnormal patterns in T cruzi seropositive and parasitemic donors and
 CCC patients. These and other biomarkers, including those identified in the transcriptome
 analysis are candidates to be studied in the whole cohort of 2,000 CCC patients in TMS
 program or, alternatively, using nested case-control design.

 Tele Minas Saude (TMS) Program, a state-wide public TeleHealth System

 The TMS program is a partnership between the Minas Gerais State Health Department and the
 State Research Agency (FAPEMIG) with six public universities of Minas Gerais: Universidade
 Federal de Minas Gerais (UFMG), Universidade Federal do Triângulo Mineiro (UFTM),
 Universidade Estadual de Montes Claros (UNIMONTES), Universidade Federal de Juiz de Fora
 (UFJF), Universidade Federal de São João del-Rei (UFSJ) and Universidade Federal de
 Uberlândia (UFU), forming the Teleassistance Network of Minas Gerais. Created from a
 telehealth pilot project in cardiology (2006), later expanded to other specialties and
 villages and transformed into permanent telehealth service, subsidized by the state
 government in 2009 [16-18].

 The objective is to support infrastructure projects of state public health, initially
 deployed to support the Primary Health Care and Family Health Teams [16]. At that stage, had
 as main objective to assist professionals in their daily work to improve access to
 specialized services, reducing and qualifying referrals of patients to secondary and tertiary
 care. The main activities offered are a) duty of teleconsultation offline in various medical
 specialties, nursing, dentistry, physiotherapy, psychology, pharmacy and nutrition, b) duty
 in telecardiology to analyze electrocardiograms (ECGs) and provide teleconsultations online
 for critical clinical cases c) technical support online and offline, d) permanent education
 through workshops and training and e) continuous monitoring of system operation in the
 municipalities.

 So far, the program was implemented in 608 municipalities in the state corresponding to 732
 telehealth sites. The primary care units in the villages were equipped with computers,
 electrocardiograph, digital printer and digital camera. Universities were equipped with
 workstations and videoconferencing equipment. Since 2006, over 20,000 teleconsultations were
 conducted and 600,000 ECGs analyzed by experts. These telehealth activities prevented the
 displacement of patients in 78% of the cases [16].

 Linkage of databases The Brazilian Health System is characterized by coexistence of a
 universal public health system (70-80% of coverage) and a supplementary private network.
 Mortality data from all Brazilians and from hospitalizations in the public health system are
 stored in large databases owned and managed by the Health Ministry. Investigators developed a
 methodology for searching these databases for specific subjects using probabilistic linkage
 methods. The probabilistic strategy we developed has high accuracy, with a sensitivity of
 90.6% and specificity 100% [19]. This method will be used in a complimentary way to direct
 follow-up.

 ECG Reading Center ECG interpretation is subjected to substantial intra- and inter-observer
 variability. To overcome this problem, a standardized ECG code was developed 50 years ago 20
 and has been widely used in epidemiological studies. The visual classification of the ECG
 using the Minnesota code, nonetheless, is time consuming. The investigators created a ECG
 Reading Center for the Elsa study, a 10-year longitudinal cohort study of 15,000 public
 employees of Brazilian Universities. For this ECG Reading Center, was installed a software
 developed by Prof. Peter MacFarlane, from Glasgow University, that analyzes, codifies and
 compares the ECG with previous tracings, according to a validated algorithm [9]. This system
 will be used in the present project.

 Preliminary Studies

 Diagnostic and prognostic value of BNP In 2002, investigators described, in a study published
 by The Lancet, that blood levels of BNP are inversely correlated to the systolic left
 ventricular (LV) ejection fraction, the most used index of LV function in ChD [21]. In
 patients with abnormal ECG and/or chest X-Ray, BNP elevation had a positive predictive value
 of 80% and a negative predictive value of 97% for the detection of patients with depressed
 left ventricular ejection fraction [21]. This results were confirmed in the following years
 by several other studies by our research group [22-26]. A diagnostic strategy including ECG
 and BNP performed better than the classical approach with ECG and chest X-Ray [24]. Moreover,
 BNP was also a marker of ventricular arrhythmia [26] and diastolic dysfunction [22,27].

 More recently, was described that BNP levels were strong predictors of the risk of death [28]
 and in a cohort of 1,398 indwelling elderly from the city of Bambuí, in the state of Minas
 Gerais, Brazil [28]. The hazard ratio for death was 1.27 for each unit of log-transformed BNP
 level (95% confidence interval (CI: 1.11, 1.45) among infected persons, independent of
 potentially confounding factors. Infected persons with baseline BNP levels in the top
 quartile had a risk of death twice that of persons in the bottom quartile (hazard ratio =
 2.07, 95% CI: 1.29, 3.32). In the same population, was found that BNP alone or in association
 with atrial fibrillation had prognostic value for stroke mortality in T. cruzi chronically
 infected older adults [29].

 Electrocardiographic measures and prognostic models The ECG is the single most important exam
 in CCC. Numerous epidemiological studies have shown that patients with a normal ECG have an
 excellent medium-term survival [30,31]. The greater the number and severity of ECG
 alterations registered in a same tracing, the more advanced the myocardial damage possibly
 is, and the worse the prognosis should be [12]. We have shown that the QRS duration is
 moderately correlated with the LV ejection fraction [32] and that, during the follow-up of
 these patients, the increase in the duration of the QRS complex and the appearance of new
 electrocardiographic alterations may help in identifying patients with a significant decrease
 (of 5% or more) in left ventricle ejection fraction [33].

 Based in the well-known prognostic value of LV ejection fraction and the presence of
 ventricular tachycardia, [5] was proposed an alternative approach to the well established
 Rassi's score, [34,35] considering only three risk factors: left ventricular ejection
 fraction < 50%, ventricular tachycardia at either stress testing or Holter monitoring, and
 QRS > 133 ms at ECG (or filtered QRS > 150 ms at signal-averaged ECG).34,35 Low risk group
 has zero or one risk factor (5-year mortality: 1%), intermediate risk, two factors (20%
 mortality), and high risk, all three factors (50% mortality). This simplified prognostic
 score had an excellent performance in predicting death in a study with 74 months of follow up
 (c statistic 0.92) and it may be an attractive alternative to the established six-factor
 score.

 More recently, the investigators showed that T-wave amplitude variability (TWV), a
 sophisticated ECG-derived measure of repolarization variability, is independently related to
 the risk of death in ChD [36]. They have also studied the prognostic value of several new
 echocardiographic measures [37-39] which could be of physiopathological and clinical
 relevance, but are not possible to be used for ambulatory patients in the rural area.

 Kaplan-Meier curves demonstrating mortality in Chagas disease patients Classified according
 to the presence prolonged of QRS > 133 ms at ECG; Classified according to a risk score in
 which each of the following factors is worth one point: ventricular tachycardia at either
 stress testing or Holter monitoring, left ventricular ejection fraction < 0.50, QRS > 133 ms
 at ECG. Low risk group has zero or one risk factors, intermediate risk, two factors, and high
 risk, all three factors.

 Summarizing, although we and others investigators have extensively studied prognostic markers
 in Chagas disease and CCC, there is no definite simple, low-cost prognostic model available
 to be used in the primary care setting. Standard ECG and BNP levels are good candidates to be
 selected in this simplified predictive model.
Eligibility Criteria: to be a client of the TMS TeleHealth System, have a positive history
 for T cruzi infection or Chagas disease, have an abnormal ECG and to consent to
 participating in the research program.

 Exclusion Criteria: The exclusion criteria include pregnancy or breast feeding, and any
 severe condition with ominous prognosis that indicates a life expectancy of less than two
 years.