Evaluation of Adult Chronic Chagas Heart Disease Diagnosis

37 Chagas disease caused by Trypanosoma cruzi is endemic in Latin America .T cruzi 38 presents heterogeneous populations and comprises two main genetic lineages, named T. 39 cruzi I and T. cruzi II. The diagnosis in the chronic phase is based on conventional 40 serological tests including IIF and ELISA, and in the acute phase based on parasitological 41 methods, including hemoculture. The objective of this study is to evaluate the diagnostic 42 procedures of Chagas disease in adult patients in the chronic phase using a PCR assay and 43 conventional serological tests including TESA-blot as the gold standard. Samples were 44 obtained from 240 clinical chronic Chagasic patients. The sensitivities using TESA-blot 45 were 70% for PCR using the kinetoplast region and 75% using nuclear repetitive region, 46 99% by IIF and 95% by ELISA. According to the serological tests results, we recommend 47 that researchers assess the reliability and sensitivity of the commercial kit Chagatest 48 ELISA recombinant, version 3.0 (Chagatest Rec v3.0; Wiener Lab®, Rosario, Argentina) , 49 due to the lack of sensitivity . Based on our analysis, we concluded that PCR cannot be 50 validated as a conventional diagnostic technique for Chagas disease. These data have been 51 corroborated by the low concordance values with serology tests. It is recommended that 52 PCR be used only as an alternative diagnostic support. Using the nuclear repetitive region 53 of T. cruzi, PCR could also be applicable for monitoring patients receiving etiologic 54 treatment. 55 56 Introduction 57 58 Chagas disease is a complex zoonosis caused by the parasite Trypanosoma cruzi. This 59 parasite can be genetically classified into two major lineages namely T. cruzi I that is 60 related to the South American northern countries while T. cruzi II is related to the South 61 American southern countries of America (46). Chagas disease is a chronic systemic disease 62 endemic to both South and Central America. T. cruzi is transmitted through the infected 63 dejections of triatomine bugs, by blood transfusion, congenital infection, laboratory 64 accidents, or by oral infection (44). Chagas disease constitutes a serious public health 65 problem in terms of both social and economic impact. The disease currently affects 15 66 million people and about 28 million are at risk of acquiring the infection. In America, 67 on A uust 0, 2017 by gest ht://jcm .sm .rg/ D ow nladed fom nearly 41,200 new cases occur each year along with an average of 12,500 deaths per year 68 (17). 69 Chagas disease presents two distinct clinical stages. The acute phase begins about one week 70 after initial infection and nearly 30% of the patients recall having had relevant symptoms 71 and signs during this period. During the chronic disease stage, the parasites are no longer 72 easily detectable in the bloodstream, but serological tests remain positive. Diagnosis of 73 Chagas disease is based on parasitological and serological methods. Infection can usually 74 be detected by microscopic examination or by parasitological tests such as hemoculture or 75 PCR (7, 28). There are several targets for the detection of T. cruzi by PCR. The variable 76 region of the minicircle kinetoplast (kDNA) and a repeat tandem sequence of nuclear DNA 77 (stDNA) of the parasite have been the most widely used regions as target sequences for 78 diagnosis via PCR (2, 7, 14, 32, 45). Serological diagnosis of T. cruzi infection is typically 79 performed using two of three individual tests according to availability (44). Enzyme Linked 80 Immunosorbent Assay (ELISA), Indirect Immunofluorescence (IIF) and Indirect 81 Hemagglutination (IHA) are often used. These three tests also referred to as the 82 conventional tests usually employ recombinant and/or crude antigenic T. cruzi preparations 83 (22). The major innovation in Chagas disease diagnosis with the detection of antibodies 84 against T. cruzi is the TESA-blot (Trypomastigote Excreted Secreted Antigens). This is an 85 immunoblot assay that has been widely used because of its high sensitivity and specificity 86 when compared with the conventional serological methods (38). The isolation and gene 87 cloning of this immunodominant peptide have been intended and ELISA tests based on 88 TESA antigens have been performed with high quality results (25) In addition, this test has 89 shown the presence of false negatives when comparing this technique with conventional 90 serology in a cohort from Bolivia (47). These reasons make TESA-blot one of the most 91 feasible and available tests for the diagnosis of Chagas disease (38, 39, 40). Recently, 92 TESA-blot has shown great usefulness in solving doubtful serology and cross-antigenicity 93 issues with related protozoan parasites in endemic regions (41). Because of these previous 94 reports, TESA-blot has been selected as the gold standard in several different reports due to 95 the high sensitivity and specificity of the test. 96 on A uust 0, 2017 by gest ht://jcm .sm .rg/ D ow nladed fom Among the conventional techniques used for serological diagnosis of Chagas disease are 97 ELISA because of its high sensitivity and IIF due to its specificity. However, it has been 98 observed that these tests can detect a certain number of false positives and false negatives. 99 This makes it necessary to search for diagnostic tests that provide more reliable results (6). 100 As a routine test for the diagnosis of Chagas disease, the WHO (World Health 101 Organization) recommends immunological techniques according to the type of diagnosis, 102 and a minimum of two positive serological tests are required for considering a patient to be 103 infected with T. cruzi. Nevertheless, in some cases there is a need to implement other 104 techniques for the diagnosis of T. cruzi. Because of the number of copies and organization 105 of the kinetoplast and nuclear repetitive DNA of T. cruzi a PCR assay has been developed 106 (29, 42). Comparative studies of PCR, hemoculture and serology showed that individuals 107 with positive hemoculture and with positive serology had a detection rate of 36.5%. When 108 using PCR, the detection of infection was 83.5%. These results demonstrate the higher 109 sensitivity of PCR compared with hemoculture (14). There is great variability within the 110 results obtained by PCR, xenodiagnosis and hemoculture that makes PCR a controversial 111 tool of choice for the accurate diagnosis of Chagas disease. 112 The specificity of serological techniques has been questioned because of the cross113 antigenicity between T. cruzi and parasites of related protozoan diseases, particularly 114 leishmaniasis and infection with T. rangeli (6, 39). This questioning arises because these 115 techniques use crude or partially purified parasite extracts, which can cause false-positive 116 results. In order to avoid false positive results, recombinant antigens and/or synthetic 117 peptides have been used with success (19, 27, 30, 39, 40). These problems may be 118 overcome by using recombinant antigens containing specific T. cruzi epitopes that elicit an 119 immune response in the majority of Chagasic patients (13, 15, 22, 39). Therefore, 120 parasitological tests are still extremely necessary to detect T. cruzi, especially in those 121 patients with doubtful serology and to determine treatment response. 122 The present study is a substudy from the BENEFIT population recruited in Colombia (23) 123 and the objective was to evaluate the serological diagnostic tests of ELISA, IIF and TESA124 blot compared to PCR amplification of the variable region of kinetoplast DNA (kDNA) and 125 the nuclear repetitive DNA region (stDNA) of T. cruzi in clinical and serologically 126 on A uust 0, 2017 by gest ht://jcm .sm .rg/ D ow nladed fom ascertained Chagasic patients from Colombia. We also aimed to optimize the procedure of 127 DNA parasite amplification by selecting the most suitable DNA extraction method. 128 Similarly, values of sensitivity, specificity, positive predictive value, negative predictive 129 value and Kappa Index were calculated. TESA-blot was used as the gold standard due to 130 the characteristics of this test that were mentioned above. 131 132 Materials and methods 133 134 Sample collection 135 A total of 240 clinical chronic Chagasic patients and 20 negative controls were included in 136 the study. We employed the inclusion and exclusion criteria required for the BENEFIT 137 (BENznidazol Evaluation For Interrupting Trypanosomiasis) project (23). Every patient 138 presented clinical heart failure evidenced by echocardiogram and positive serology . A 10 139 mL blood sample was collected from all patients and control subjects. A 10 mL blood 140 sample was collected from all patients and control subjects. Blood samples were mixed 141 with an equal volume of 6 M guanidine HCl/0.2 M EDTA solution immediately after 142 sample collection. The guanidine-EDTA blood (GEB) mixture was then maintained at 143 room temperature and later stored at 4°C until the DNA extraction. A 2 mL blood sample 144 was also collected for serum collection and analysis. The serum aliquots were then stored at 145 4°C until the serological tests were performed. The 20 individual control subjects were 146 from a non-endemic area, and they all tested negative for Chagas disease by IIF, ELISA, 147 PCR and TESA-blot. All of the control subjects also showed no clinical signs of heart 148