Current status
Laboratory diagnosis – current status
Yara de Miranda Gomes
Immunoparasitology Laboratory, Immunology Department, Aggeu Magalhães Institute/Fiocruz
Email: yara@cpqam.fiocruz.br
In the Chagas disease acute and chronic forms, the etiological diagnosis can be made by detecting the parasite through parasitological methods (direct or indirect) and by the antibodies presence in the serum, through serological tests, the most used being indirect immunofluorescence (IIF), indirect hemagglutination (IHA), and enzyme-linked immunosorbent assay(ELISA). More complex tests, such as the molecular test using polymerase chain reaction (PCR), despite its limitations due to the lack of standardized protocols, are indicated when serological tests present indeterminate results or to control the cure after antiparasitic treatment. In this case, PCR must be performed by recognized competence laboratories and performed by a specialist in the field.
Acute phase
In the Chagas disease acute phase, laboratory diagnosis is based on the observation of the parasite present in the infected individuals blood, through direct parasitological tests such as fresh blood examination, smears, and thick blood smears (Figure 1). The direct fresh test is more sensitive than the stained smear and should be the choice method for the acute phase. If these tests are negative, indirect parasitological methods should be used. Concentration tests (micro-hematocrit or Strout) have 80 to 90% positivity and are recommended in strong suspicion case of acute Chagas disease and the direct fresco test negativity. In symptomatic cases for more than 30 days, they should be the choice tests as parasitemia begins to decline (according to the First Brazilian Consensus on Chagas Disease of the Ministry of Health in 2005), revised in 2015 in the form of the Second Brazilian Consensus on Chagas Disease (Epidemiol. Serv. Saúde, Brasilia, 25 (núm. esp):7-86, 2016.
Chronic phase
In the disease chronic phase, direct parasitological diagnosis is compromised because of the parasitemia absence. The indirect parasitological methods (xenodiagnosis – or hemocultivation) that can be used, have low sensitivity (20-50%). Thus, the disease is usually diagnosed by detecting IgG that binds specifically to T. cruzi. Therefore, in the chronic phase, the diagnosis is essentially serological and should be performed using two tests of different methodological principles: one test of high sensitivity (ELISA with total antigen or semi-purified parasite fractions or the IFI) and another of high specificity (ELISA, using recombinant antigens specific to T. cruzi) or two serological tests involving distinct antigen preparations, both of which must be carried out concomitantly (Figure 2).
Vertical transmission
In congenital transmission suspected cases, it is important to confirm the mother’s serological diagnosis. If maternal infection is confirmed, a newborn parasitological examination should be performed. If positive, the child should undergo etiological treatment immediately. The children of chagasic mothers with negative parasitological examination or without examination should return between six and nine months for serological tests for IgG class anti-T. cruzi antibodies. If the serology is negative, vertical transmission is ruled out. Figure 3 shows the flow chart for diagnosis in vertical transmission suspected cases. Positive cases should be treated, considering the high cure rate at this stage. Because of the high number of false-negative results in congenital transmission, anti-T. cruzi antibody of classes IgM and IgA testing is not recommended.
History
History of Chagas disease diagnostic methods
Alejandro Luquetti Ostermayer*
Institute of Tropical Pathology and Public Health, Federal University of Goiás
Email: luquetti@ufg
* The author thanks Emeritus Professor Dr. Joffre Marcondes de Rezende for his suggestions on this text elaboration.
The Trypanosoma cruzi infection diagnosis, the Chagas disease causal agent, as in other infectious diseases, is based on three distinct parameters: the clinical manifestations, which, if present, allow the physician to suspect the infection; the epidemiological antecedents, which also induce the clinician to suspect; and the diagnostic methods, generally laboratory, which confirm or exclude the diagnostic suspicion in most situations. It will be up to the clinician, in possession of the above information, to decide whether the individual is infected or not. It is worth pointing out that infection by a certain agent is not synonymous with disease; many infections occur without clinically apparent disease. In T. cruzi infection, we must remember that more than half of those infected do not present heart disease, megaesophagus, or megacolon, the Chagas disease main manifestations. In these particular cases, the diagnosis is suggested by the epidemiological background and confirmed or excluded by the laboratory tests results.
Therefore, diagnostic methods are of particular importance in Chagas disease. Moreover, this infection presents itself in only two phases, distinct in terms of the infection chronology, the clinical manifestations, and the diagnostic methods. The acute, initial phase, with fever and non-specific symptoms (sometimes with Romaña’s sign or inoculation chagoma) is diagnosed by parasitological methods, due to the high parasitemia that defines this phase. In the chronic phase, which begins after the acute phase and which, as noted, is asymptomatic in more than half of the cases, the laboratory diagnosis is based on the indirect search for infection signs, that is, the presence of anti-T. cruzi antibodies.
The diagnostic methods history can be divided into three periods, of different lengths. The first period, from discovery to 1960, is commented on below.The first diagnostic methods developed were parasitological methods. Carlos Chagas relied on the finding of T. cruzi in the child Berenice to state that this agent was responsible for the clinical picture. This was the disease acute phase, and the diagnosis today, more than 100 years later, continues to be made in the same way, with the T. cruzi direct detection in the peripheral blood (Figure 1).
In the trypanosomiasis chronic phase, in the first years after its discovery, laboratory diagnosis was made by inoculating blood from patients into guinea pigs. Chagas described the schizogonic parasitic forms presence in the infected guinea pigs lungs, which he thought were from T. cruzi. This finding became a diagnostic method in experimental infection of the guinea pig until 1913, when it was shown that it was actually another parasite, Pneumocystis carini, and the method was abandoned.
In 1914, Brumpt described another parasitological diagnosis modality, xenodiagnosis, which was initially little used. The first references to its use in the Chagas disease diagnosis are from Torrealba in Venezuela in 1934, Emmanuel Dias in Brazil, and Bacigalupo in Argentina. The method only came into routine use after it was standardized by Cerisola et al. in 1974.
The first antibody testing description dates back to 1913 and is due to Guerreiro and Machado. This was the complement fixation reaction, soon known as the Guerreiro and Machado reaction, which was the only serological test available for more than 50 years, and which was routinely performed for diagnosis until a few years. This test has undergone multiple modifications and standardizations, with Almeida and Fife’s contribution in 1976 standing out. The technical complexity, the use of several reagents that require daily standardization, and the reaction time, led to its abandonment as of 1995, mainly due to simpler tests existence. At that time, a technical opinion from the Ministry of Health recommended that it be replaced by other tests. (Rev Inst Med Trop São Paulo, 1996)
In this first period in the diagnostic knowledge evolution, until 1960, there were only two tests for more than 50 years. The second period, from 1960 to 1975, was one of great development, as will be reported below.
The hemocultivation, introduced as a parasitological diagnostic method since the 1940s, had far inferior results to xenodiagnosis, and was therefore not used. After the Chiari and Brener work in 1966 with successive refinements (reviewed in Chiari, 1992) it is still used today with results comparable to xenodiagnosis and the advantage of allowing the parasite isolation.
Inoculation in experimental animals has also long been used, but it is no longer employed as a diagnostic method due to operational difficulties, as well as its low sensitivity in the chronic phase.
Other tests have been used, such as circulating antigen detection, antigenuria, delayed hypersensitivity tests, but they have not been incorporated into the routine and are not used.
Numerous attempts to use serological tests using other methods, such as precipitation, latex agglutination, and flocculation tests, have failed, either because of low efficiency or high costs.
In 1962, Cerisola and collaborators described the indirect hemagglutination test (IHA) use for the infection serological diagnosis. This test, which is easy to perform and performs well, is still in use today, although it is less sensitive than the immunofluorescence and ELISA tests, which will be described later. For this reason, it is not recommended for the exclusion of blood donors.
In 1966, Camargo optimizes the use of the indirect immunofluorescence test, already described by Fife and Muschel. This highly sensitive test was used in the national serological survey, with more than one million samples from all over Brazil, which determined, quite accurately, the disease prevalence. Given its high sensitivity, it is ideal for epidemiological studies, as well as for diagnosis, although it presents cross-reactions, particularly with leishmaniases. It continues to be used to this day, simultaneously with IHA and ELISA, all three constituting the so-called conventional tests, with which there is extensive experience in all Latin American countries.
The direct agglutination test, perfected by Vattuone and Yanovsy with the systematic inclusion of the reducing agent 2 mercapto-ethanol, was used mainly in Argentina with good results, but its commercialization has been discontinued.In 1975, Voller and collaborators described the ELISA immunoenzymatic test on filter paper samples, a method that has been perfected and is currently used in the diagnostic routine of hemotherapy and diagnostic services, and there are several brands in Brazil approved by ANVISA (Table 1), with good performance.
Thus, in this second historical period in the diagnostics evolution, we see the methods development that form the diagnostics basis to this day.
In the third period, from 1976 to the present, advances in molecular biology have improved existing methods and developed new ones. In successive studies, attempts have been made to improve the antigens quality used until then, from the parasite total extracts (crude antigen), by antigens purified by various procedures, in an attempt to avoid cross-reactions observed with conventional diagnostic tests. Thus, in the 1980s, work was published using glycoproteins of 25 kDa, 90 kDa, and 72 kDa among others, with sera panels from patients with the disease different clinical forms. These reagents are not yet commercially available.
With the advance in the molecular biology knowledge, from 1980 on recombinant proteins were isolated by several groups in Brazil, Argentina and the United States, obtaining good results according to each group. Studies with synthetic peptides have also been published (reviewed by Silveira and collaborators) In order to elucidate which ones would have greater applicability, the TDR (Tropical Diseases Research) program of the World Health Organization has promoted a multicenter study that also included some of the purified antigens. This study defined some of them as appropriate. Another study by Levin and collaborators confirmed some of these results, as did a third, larger study by Umezawa and collaborators.
After these studies several rapid tests were developed, some of them with the intention to verify the diagnosis in the field, with just one blood drop. One such test was validated in a multicenter study by Luquetti and collaborators. Later, a study coordinated by Doctors Without Borders compared the performance of eleven rapid tests available at the time, where some showed good sensitivity and specificity, with applicability in surveys and in emergency situations. (Sánchez-Camargo et al. 2014)
Other tests used were flow cytometry and western blot. Although the latter was marketed for a while (Tesa-blot®), neither are currently available.
In another approach, serological methods are being developed to detect specific antibodies directed against T. cruzi strains in an attempt to identify the Tc type (I to VI) predominant in each infected individual. Some results have already been published by Bhattacharyya et al. in 2014.
In another laboratory diagnosis phase, in the 1990s, studies were directed towards the nucleic acids amplification from the parasite itself, aiming at parasitological diagnosis by PCR amplification. Nowadays, it is possible to check for a one parasite presence in 20 mL of blood. However, because it is a method to verify the parasite presence, which may not be present in the chronically infected, a negative result has no diagnostic value. The sensitivity is superior to those of hemocultivation and xenodiagnosis, although there is concern about specificity when performed in routine services. In addition, it is not yet commercialized (reviewed by Luquetti and Rassi). See the following chapter.
A new commercial chemiluminescence test has been developed in the last decade, with excellent sensitivity, for application in hemotherapy services. Useful in centers with high demand, it is now widely used.
Although there are several serological tests that perform well, it is necessary that they are verified, batch by batch, and that laboratories maintain personnel with appropriate technical instructions. The Brazilian Ministry of Health has shown sensitivity in this regard, promoting commercialized reagents studies. In partnership with AIDS Management, it produced a manual and video (Telelab) on the disease diagnosis . Also in partnership with Biomanguinhos, the ANVISA has been developing an external quality program (EQA) since 2001, distributing three panels of serums per year, operating until today, for hemotherapy services in Brazil.
Despite all the advances that have been made, there is still a need for serological diagnosis using two different principle tests to ensure the infection presence (or its exclusion). However, for blood donor exclusion, as per WHO recommendations (ref. 2002), performing a single high-sensitivity test (ELISA or chemiluminescence) is sufficient, provided external quality control is in place.
New references
Luquetti AO, Alquezar AS, Moreira EF, Zapata MTG, Guimaraes MC, Gadelha F, Pereira JB, Arruda AHS & Nasser LF. Recomendações e conclusões da II Reunião do Comitê Técnico Assessor para o diagnóstico laboratorial da doença de Chagas, São Paulo, SP, 19-21/03/96. Rev Inst Med Trop São Paulo 38: 328, 1996.
Sánchez-Camargo CL, Albajar-Viñas P, Wilkins PP, Nieto J, Leiby DA, Paris L, Scollo K, Flórez C, Guzmán-Bracho C, Luquetti AO, Calvo N, Tadokoro K, Saez-Alquezar A, Palma PP, Martin M, Flevaud L. Comparative evaluation of 11 commercialized rapid diagnostic tests for detecting Trypanosoma cruzi antibodies in serum banks in areas of endemicity and nonendemicity. J Clin Microbiol 52: 2506-2512. 2014.
Bhattacharyya T, Falconar AK, Luquetti AO, Costales JA, Grijalva MJ, Lewis MD, Messenger LA, Tran TT, Ramirez JD, Guhl F, Carrasco HJ, Diosque P, Garcia L, Litvinov SV, Miles MA. Development of peptide-based lineage-specific serology for chronic Chagas disease: geographical and clinical distribution of epitope recognition. PLoS Negl Trop Dis 22;8: e2892, 2014.
Control of Chagas Disease. WHO Technical Report Series Nº. 905. Second Report of the WHO Expert Committee, World Health Organization, Geneva, 2002.
Molecular Diagnosis
Chagas disease Molecular Diagnosis
Constança Britto and Otacílio Moreira
Laboratory of Molecular Biology and Endemic Diseases, Oswaldo Cruz Institute/Fiocruz
Email: cbritto@ioc.fiocruz.br / otacilio@ioc.fiocruz.br
Accurate diagnostic tools for the Trypanosoma cruzi infection detection and parasitological markers identification response to treatment are considered priorities in research and development in the Chagas disease context. Timely diagnosis and treatment are strategic to prevent disease progression and the occurrence of functional limitations, disability, and impairment. According to the updated version of the Brazilian Consensus on Chagas disease, the etiological disease diagnosis should be made in all suspected cases, both in the acute and chronic phases. Depending on the disease stage, the T. cruzi infection diagnosis must follow defined criteria.
In the initial stage (acute phase), parasite trypomastigotes forms large number are observed circulating in the bloodstream, and the diagnosis can be obtained by direct on the peripheral blood microscopic examination. At this stage the detection of IgM anti-T. cruzi antibodies is also possible. Parasitological diagnosis in the acute phase (direct examination, xenodiagnosis and blood culture or hemocultivation) is based mainly on the parasite identification and its sensitivity is dependent on the parasitemia level. Concentration methods (Strout’s, micro hematocrit and leukocyte cream or buffy coat) are rapid and low cost, and are recommended as the diagnostic test of first choice for symptomatic acute phase cases with more than 30 days of evolution, due to the decline in parasitemia with time. Direct microscopic examination of peripheral blood (with or without staining) becomes important for parasite verification and morphological characterization, especially in geographical areas where Trypanosoma rangeli infection can coexist with T. cruzi; the former being non-pathogenic to man. The lack of diagnosis and treatment in the disease acute phase is the main cause for the Chagas disease chronic progression.
In the chronic phase, parasitemia levels are below the detection limit by microscopy and thus diagnosis is based mainly on the specific IgG anti-T. cruzi antibodies detection by conventional serological tests, or by in vitro amplification indirect parasitological methods of the parasite population (hemocultivation and xenodiagnosis) for the T. cruzi isolation and identification. Although specific, parasitological methods for chronic phase diagnosis have shown low sensitivities, thus implying no diagnostic value when the result is negative, according to the Brazilian Ministry of Health (2013). Xenodiagnosis has a limited sensitivity, detecting the parasite in only 20-60% of the chronically seropositive patients, depending on the endemic area under study, and consequently generating a significant number of false-negative results. Blood culture has not been used frequently in diagnosis, also because of its low positivity in the chronic phase of the infection. In addition, both biological amplification assays can select parasite subpopulations, leading to a distortion of the etiologic agent typing results and the epidemiological data generated. On the other hand, positive parasitological results are very useful, especially for monitoring specific treatment or in cases of inconclusive results by serology. According to Luquetti and Schmuñis (2010) and referenced by the Brazilian Ministry of Health (2013, 2014), serological diagnosis in the chronic phase should be performed with a highly sensitive test together with another of high specificity. Thus, it is recommended two serological tests use with different principles/methods and that have different antigenic preparations. Although, in general, these tests present sensitivity and specificity rates higher than 90%, even so, discordant results between conventional tests and a limited specificity can be observed in relation to infections with other trypanosomatids that circulate in the same geographical area as T. cruzi(Leishmania spp. and T. rangeli), which can lead to cross antigenicity and false-positive serological results, requiring subsequent confirmation. Non-conventional serological tests – those that employ recombinant antigens, for example – should preferably be used, together with another conventional test. Another problem linked to serological diagnosis refers to a patient’s clinical profile that may not be related to his or her humoral response, such as during the first weeks of infection (when the serological reaction is not yet observed) or after specific treatment (when an immune response can persist for years even if the treatment was successful). In this last condition, positive serology results are only indirect infection evidence with T. cruzi, considering that its occurrence does not depend on the parasite presence, but on the patient immunological memory.
Limitations in the parasitological laboratory tests sensitivity, especially when it comes to chronic phase diagnosis, reinforce the need for the application of a direct and more sensitive method that allows monitoring the parasite presence and confirming the disease etiology. In this sense, the Polymerase Chain Reaction or PCR has been increasingly employed for the T. cruzi DNA detection directly in the chronic patients blood, through the use of synthetic oligonucleotides or primers that amplify pathogen-specific DNA sequences, through the DNA exponential synthesis by the polymerase enzyme. The need for its execution in adequately equipped laboratories with exclusive space for its execution, in addition to personnel trained in the practice of molecular diagnosis, can be considered limiting factors with regard to the PCR testing use in outpatient or hospital situations, or even in the blood banks routine. Although in recent years, the PCR testing costs have been declining and equipment for rapid and automated molecular testing is becoming widely available.
Molecular targets and procedures for detection of Trypanosoma cruzi DNA in blood by PCR
In order to select the best target sequence, it is important to gain knowledge about the parasite’s genome. Based on previous studies, such as that of Requena et al. (1996), the total DNA T. cruzi content ranges from approximately 120 to 330 phentograms (fg) and is distributed both in the nucleus and in the single mitochondria.
The T. cruzi mitochondrial genome, consisting of kinetoplast DNA or kDNA, represents about 20 – 25% of the total cellular DNA and is organized in a network structure, where thousands of small circular molecules, the minicircles, are concatenated representing 95% of the cell kDNA content. According to Degrave et al. (1988), the T. cruzi minicircle molecules have sizes of approximately 1,400 base pairs (bp), are present in thousands of copies (between 5,000 and 30,000 per kDNA network) and have a peculiar sequence organization, distributed in four smaller regions, with sizes ranging from 120 to 160 bp (dependent on the parasite strain), arranged at 90° angles along the circular molecule and exhibit a high level of intraspecies sequence conservation. These conserved regions are interspersed with larger regions, around 330 bp, that exhibit extreme sequence variability, even among the thousands of minicircles that make up the kDNA network of a single cell (Figure 1). Considering that each parasite has between 5,000 to 30,000 minicircles and that there are four copies of the conserved region per molecule, a total amount of up to 120,000 copies of these sequences per parasite can be assumed. Thus, these molecules present characteristics that make them ideal targets for PCR detection, taking into account that they are present in a high copy number per kDNA network, and that each minicircle contains four highly conserved DNA sequences regions present in all strains and isolates representative of the different T. cruzi genetic lineages (DTUs or Discrete Typing Units). The PCR strategy using kDNA as the amplification target, employs oligonucleotides (primers) designed for the minicircles conserved sequences, amplifying a specific 120 bp fragment (minicircle “conserved region”) or 330 bp fragment (minicircle “variable region”) (Figure 1). PCR designed for the kDNA target detects all T. cruzi DTUs. The excess human DNA presence does not interfere with the selective amplification process of the parasite DNA.
Regarding the T. cruzi nuclear genome, the first repetitive sequence described was a satellite DNA (satDNA) with 195 bp repeat units, arranged in tandem and distributed in most T. cruzi chromosomes, corresponding to 9% of the parasite total genome, according to Requena and collaborators. (1996). Through hybridization assays, these researchers were able to estimate the number of satDNA repeats at approximately 120,000 copies per parasite. Russomando and collaborators. (1992), using these nuclear satellite sequences found a high sensitivity and specificity of detecting the parasite directly in the serum of Chagas disease carriers. More recently, the same group reported the prenatal diagnostic system implementation using satDNA-targeted PCR for the congenital Chagas disease cases detection in endemic areas of Paraguay (Russomando and collaborators., 2005). In this context, the PCR sensitivity seems to be higher than that observed by microscopic examination, demonstrating its potential application in the early congenital infection diagnosis.
Therefore, due to the high number of conserved sequences copies distributed in the T. cruzi nuclear and mitochondrial genome, both the nuclear satDNA sequences and the minicircles that make up the kDNA network majority can be used as potential targets for the specific primers design for this parasite species detection by PCR.
In endemic countries, affected by Chagas disease, it has been common practice to collect clinical samples in rural areas, far from the laboratory that performs the diagnosis, and this routine can compromise the sample integrity to be analyzed. Thus, the need to collect clinical samples in rural areas promoted a simple procedure development for the collection, transport, and preservation of blood samples for subsequent PCR analysis, targeting T. cruzi kDNA for detection. Peripheral blood samples (10 mL) are collected in dedicated collection tubes containing EDTA as an anticoagulant and mixed in the same volume of lysis buffer (6M Guanidine-HCl/ 0.2M EDTA), which can remain at room temperature for up to 60 days without compromising the molecular assay final result. It was also suggested that the kDNA network structure disruption to release the thousands of minicircles, the amplification target molecules, was necessary to increase the parasite detection sensitivity directly in the chronic patients blood. To this end, Britto et al. (1993) described the physical cleavage method of the kDNA network by heat (boiling blood lysates at 100°C for 15 min), a rapid and very low-cost procedure that has been used routinely by different laboratories, contributing to the fact that PCR performed using this lysate has a detection limit of up to a single parasite present in 10 mL of collected blood. This sensitivity level has proven adequate for accurate parasite detection directly in the patients blood with chronic disease. Furthermore, PCR is undoubtedly a faster and more practical method than in vitroamplification indirect parasitological methods, such as xenodiagnosis and hemocultivation.
After boiling the blood lysates, DNA can be purified from a small aliquot (300 µL) by methodologies based on organic solvents (phenol-chloroform) or by the commercial kits use containing silica columns or magnetic beads. After performing PCR targeting the parasite kDNA or satDNA, samples that are negative for the T. cruzi DNA presence should be tested for the possible reaction inhibitors presence and to control the recovered DNA integrity, through another PCR assay with primers derived from a specific human sequence (such as the ß-globin gene, ß-actin, or RNAse P). This step becomes crucial to rule out false-negative diagnostic results.
PCR as a complementary diagnostic tool and to monitor parasitemia in response to trypanocide treatment
In the Chagas disease chronic phase, PCR can be used as a complementary tool to confirm the etiologic diagnosis in cases where serological tests provide inconclusive or discordant results. It is worth pointing out that a negative PCR test does not rule out the infection possibility, taking into consideration the small and intermittent number of circulating parasites during the disease chronic phase; however, a positive test has an absolute diagnostic value. A PCR result confirmed as true negative indicates the parasite DNA absence at the time of testing, i.e. the blood sample collected for PCR does not contain the parasite. Therefore, the PCR accuracy may be compromised by an unknown behavior of T. cruzi parasitemia in the chronic phase, where detectable parasitemia periods are not predictable. An alternative solution to this limitation would be to collect blood samples serially, in order to increase the probability of identifying parasite DNA in at least one of the samples.
Several studies have reported the qualitative PCR application for the T. cruzi detection in a range of biological samples obtained from mammalian hosts and insect vectors. In the available commercial kits absence, with only one described in 2009 by Deborggraeve and collaborators, but still with limited access (T. cruzi OligoC-TesT), the methods, protocols and operating procedures should follow the recommendations presented by Schijman et al. (2011), for standardizing the PCR clinical use in Chagas disease. This need arises from the extreme variability in sensitivity levels generated by different protocols, variability that depends not only on the epidemiological characteristics of the populations studied, but also on the blood collected volume, the method selected for DNA extraction, the choice of target DNA sequences and primers, reagents and thermal cycling conditions.
The Schijman and collaborators research. (2011) resulted from an international collaborative study conducted from 26 laboratories with prior PCR experience for Chagas, representing 16 countries. The ability of different PCR protocols to detect T. cruzi DNA was evaluated by analyzing three samples panels: genomic DNA serial dilutions from three parasite genetic strains [DTUs I, IV, and VI] (panel A), blood samples “contaminated” with different concentrations of T. cruzi (panel B), and blood samples from seropositive patients and uninfected controls (panel C). For the panel consisting of T. cruzi DNA samples at different concentrations, 48 PCR protocols were compared and in the others, this number was reduced to 44, 28 for the kDNA target, 13 for the nuclear satellite DNA (satDNA) and the remaining protocols corresponded to other targets with lower copy number, as 18S rDNA (18S ribosomal DNA) and 24Sα rDNA (24Sα ribosomal DNA), spliced leader genes intergenic region (SL-DNA) and the mitochondrial gene for cytochrome oxidase subunit II (CO II-DNA).
In the panels A and B analyses, the protocols that employed commercial kits for DNA extraction (containing columns with silica resin) instead of solvents (phenol-chloroform) and satellite DNA as the amplification target, showed better specificity. However, in panel A, PCR-kDNA was more sensitive for detecting TcI DNA (DTU I). Regarding panel B, tests targeting kDNA also demonstrated higher sensitivities, detecting up to 5 x 10-3 parasites/mL. After panels A and B analysis, 16 methods were selected as “Good Performance”, demonstrating high specificities and consistency, being able to detect up to 10 fg/mL of each DNA stock representative of three T. cruzi DTUs (TcI, TcVI and TcIV) (panel A) and 5 parasites/mL in artificially contaminated blood (panel B). The mean values for sensitivity, specificity, and accuracy, obtained from the clinical samples (panel C) analysis from the 16 methods considered to perform well, varied considerably. Of these, only four PCR protocols revealed the best performing parameters across all sample panels, three of which were for satDNA and one for kDNA. These tests were able to detect up to 10 fg/mL of each T. cruzi DNA stock (TcI, TcVI and TcIV); 0.5 parasite/mL in the reconstituted blood samples; sensitivities between 83.3 – 94.4%; specificities of 85 – 95% and 86.8 – 89.5% accuracy. As for the performance of the four methods in the clinical samples panel analysis (panel C), sensitivities of 63 – 69%, 100% specificity, and accuracy of 71.4 – 76.2 % were obtained compared to the patients serological diagnosis. In this study, targets directed at ribosomal DNA gene sequences, miniexon or cytochrome oxidase subunit II were not sufficiently sensitive and appeared not to be suitable for the Chagas disease molecular diagnosis in clinical routine. However, these parasite targets have been widely used for genotyping T. cruzi DTUs.
Despite the excellent satDNA and kDNA targets performance in the Chagas disease molecular diagnosis, it has been shown that both can amplify T. rangeli DNA (genetically very similar to T. cruzi) when present in high concentration. Even so, for kDNA, the amplified fragments from T. rangeli can be differentiated from T. cruzi by presenting distinct sizes on agarose gels, as demonstrated by Moreira et al. (2017).
The PCR protocols evaluation is the initial step towards technical improvement and the development of an international Standard Operating Procedure (SOP) for T. cruzi targeted PCR. As discussed by Schijman et al. (2011), methods that have demonstrated “Good Analytical Performance” could be recommended as a complementary diagnostic tool under the following conditions: 1) post-treatment follow-up of patients to investigate treatment failure and determine parasitological response to treatment; 2) congenital Chagas disease diagnosis in newborns, for whom serological tests are inadequate due to the maternal anti-T. cruzi antibodies presence; 3) Chagas disease reactivation identification after organ transplantation in recipients infected by T. cruzi, in immunosuppression conditions; 4) Chagas disease reactivation differential diagnosis in patients coinfected with HIV/T. cruzi, and 5) oral transmission suspected cases. In addition, an important prospect for the development of a highly sensitive and specific PCR assay is its potential application in routine blood banking for donor screening to solve the problem of false-positive serology results and the consequent uncontaminated blood disposal. Nowadays, the costs of PCR testing are rapidly decreasing and equipment for rapid and automated molecular assays is becoming widely available.
There is now a consensus that all individuals infected with T. cruzi should be treated, with the exception of those who have developed decompensated chronic heart failure. Treatment for Chagas disease has been considered a promising medicinal approach for patients in the acute, indeterminate, or chronic phase, except when they have extremely severe symptoms of heart disease or digestive tract involvement. Although more effective trypanocidal drugs are needed, treatment with benzonidazole (or nifurtimox) is reasonably safe, but its efficacy in the late chronic phase is still questionable due to a reliable system absence capable of monitoring therapeutic regimens and for establishing criteria for controlling patient cures. The negative serology demonstration or a persistent and progressive decline in serologic test titers is considered a cure indicator in the monitoring of trypanocidal treatment for Chagas disease. The reduction in serology titers occurs gradually, especially in the long-term chronic phase, usually over a period of 20 to 25 years after the treatment start, according to Rassi et al., 2012. Although it is not mandatory to perform parasitological tests to demonstrate cure, positive parasitological tests at any time during monitoring indicate therapeutic failure.
As discussed by Portela-Lindoso & Shikanai-Yasuda (2003) and reviewed by Britto (2009), PCR is an alternative option to indirect methods (xenodiagnosis and blood culture) for parasitological evaluation to indicate treatment failure. Studies have shown PCR greater sensitivity for determining parasite persistence in chronically treated individuals who were monitored for long periods after treatment. Again, it should be emphasized that the molecular methodology does not allow us to infer the success (efficacy) of the treatment, considering that even the generation of repeatedly negative results does not necessarily indicate a parasitological cure. With this scenario, quantitative real-time PCR assays become more adequate in demonstrating a significant decrease in parasite load, which is expected in a successful therapeutic scheme.
Quantitative Real-Time PCR (qPCR)
Since 2000, the methodology for the specific genes and sequences detection has been improved with the development of different quantitative real-time PCR (qPCR) systems, an approach based on the fluorogenic probes use (“TaqMan®” or others) or fluorescent dyes with affinity to intercalate non-specifically to the DNA molecule (“SYBR green® or others”), for the amplification reaction real-time measurement. The real-time PCR test, either qualitative (presence and absence assay) or quantitative, is recommended for both the infection diagnosis and the response measurement to etiologic treatment in clinical practice and in the new drugs testing for Chagas disease. Identifying whether a patient or experimental animal has been cured of T. cruzi infection is a critical question, whether evaluating the first-line drugs efficacy (benzonidazole or nifurtimox) or testing new chemotherapeutic compounds and immunotherapies.
The use of methodologies to estimate the absolute circulating T. cruzi levels in infected individuals would make it possible to describe a possible correlation between parasite load and disease progression, besides being of great use for the parasite populations molecular characterization, prognostic evaluation, and therapeutic efficacy. However, it has not yet been established whether, even in the parasitological cure absence (parasite elimination), the reduction of the parasite load would lead to an improvement in clinical symptoms in individuals with heart disease who have Chagas disease in the chronic phase and who have undergone anti-T. cruzi chemotherapy. Furthermore, in immunosuppression cases, Chagas disease reactivation accurate diagnosis, followed by early treatment, is indicated (Almeida et al., 2011). Thus, quantitative molecular methods (qPCR) for the parasite load estimation (de Freitas et al., 2011; Duffy et al., 2013; Moreira et al., 2013; Ramírez et al., 2015; Melo et al., 2015) are highly recommended, as they make it possible to accurately track the reactivation status by measuring the significant increase in the circulating parasites number, and may result in greater benefits for the patients clinical management. However, for quantitative assays, (cut-off) levels have yet to be defined to help characterize reactivation and enable early therapeutic action.
Real-time PCR (qPCR) strategies were initially proposed as tools to quantify parasite load in clinical samples from T. cruziinfected patients who received etiologic treatment, with the goal of providing a therapeutic response surrogate marker, considering the long course to negative serological tests, in chronically treated patients. To date, few qPCR strategies have been developed for the T. cruzi DNA detection and quantification in patients with Chagas disease, but their application in clinical practice requires prior studies for analytical and clinical validation. In this context, the World Health Organization (WHO), the Pan American Health Organization (PAHO) and other international entities, such as the Drugs for Neglected Disease Initiative (DNDi) and Doctors Without Borders (MSF), have joined efforts to approve studies aimed at obtaining standard operating procedures for the nucleic acids recovery and amplification from patients peripheral blood samples with Chagas disease (Schijman et al., 2011; Ramírez et al., 2015). For this purpose, between November 2007 and December 2011, coordinated international activities were carried out, with the participation of representatives from molecular diagnostic laboratories, located in several endemic and non-endemic countries for Chagas disease, including Latin America, USA and Europe. These activities allowed us to simultaneously evaluate different real-time PCR strategies and compare the results obtained against clinical samples panels from individuals in the participating countries, as well as T. cruzi DNA samples in different concentrations, representative of the different parasite DTUs.
The study by Ramírez et al. (2015) was conducted in an attempt to establish standard operating procedures for quantifying T. cruzi parasite load in blood samples, using two of the four methods ranked among the best in the previous international study evaluating PCR protocols, described by Schijman et al. (2011). The performance of two multiplex real-time PCR strategies (simultaneous detection and quantification of more than one target in the same reaction), using TaqMan probes directed to kDNA and satDNA, was evaluated in independent reactions, including in each assay an extrinsic heterologous control, as internal amplification control (IAC). The IAC corresponds to a recombinant plasmid containing a plant sequence (Arabidopsis thaliana aquaporin), used as quality control of the entire procedure from DNA extraction to qPCR assays, and was originally described by Duffy et al. (2009). To do this, a normalized amount of IAC is added to each sample to be quantified before DNA extraction. The parameters evaluated for the analytical validation of the two TaqMan qPCR assays were: 1) Selectivity (inclusivity – ability to detect the target pathogen from different T. cruzi DTUs; exclusivity – other closely related trypanosomatids absence detection); 2) Dynamic range – set of measurement values obeying specified limits; 3) Limit of Detection (LOD) – lowest concentration that the method can reliably detect the target pathogen presence or absence; 4) Accuracy – the closeness of the agreement between the independent test/measurement results obtained according to the stipulated conditions; 5) Limit of Quantification (LOQ) – the lowest concentration at which the method has acceptable precision and accuracy to perform the measurement.
A higher analytical sensitivity uniformity was demonstrated for qPCR-kDNA in the analysis among different T. cruzi DTUs than that observed for qPCR-satDNA, due to the latter method being less sensitive for some TcI and TcIV strains, indicating a lower copy number of the satellite nuclear sequences in their genomes. That is, the copy number of the target satDNA can vary greatly depending on the T. cruzi strain.
The analytical parameters comparison for both qPCR methods, suggests a higher sensitivity of the kDNA target for the detection and quantification of samples with low parasite loads, but with lower specificity than satDNA (mainly in relation to T. rangeli detection). However, clinical samples analyses showed high concordance, in sensitivity and parasite loads terms, determined by both qPCR tests (satDNA and kDNA). In this paper, the authors discuss the appropriate application of each method depending on the epidemiological and/or T. cruzi infection clinical scenarios (Ramírez et al., 2015). Regardless of the target selected, qPCR assays can be sensitive enough to detect a single parasite presence from a 5 mL blood sample. However, to perform quantitative qPCR assays, the laboratory must be equipped with a real-time thermal cycler and the corresponding software. This should be considered for laboratories with a low budget that do not routinely perform real-time PCR tests and therefore do not have such equipment.
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