Chagas disease, caused by the hemoflagellate protozoan Trypanosoma cruzi, remains a challenging medical, economic, and social burden in the Americas. According to the World Health Organization (WHO), over six million individuals are infected, and 75 million are living under the daily threat of infection (WHO, ).
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Chagas disease is characterized by two clinical phases: the acute and the chronic phases. During the acute phase, infected individuals usually exhibit high parasitemia and experience symptoms such as fever, anorexia, and tachycardia (Rangel-Gamboa et al., ). In the chronic phase, infected individuals may develop various conditions affecting the cardiovascular, digestive, or neurological systems (Echavarría et al., ).
Depending on the clinical stage, specific laboratory diagnostic tools can be employed to confirm T. cruzi infection. In the acute phase, direct T. cruzi detection can be achieved through parasitology techniques such as xenodiagnosis, or by employing molecular biology techniques. Several polymerase chain reaction (PCR) amplification-based assays have been tested to detect T. cruzi, with some becoming routine tests. These assays include real-time PCR (qPCR), digital droplet PCR (ddPCR) and loop-mediated isothermal amplification PCR (LAMP-PCR). During the chronic phase, parasitemia decreases and becomes intermittent, making the indirect detection of T. cruzi through the presence of antibodies against T. cruzi crucial. The most common serological techniques employed to detect specific T. cruzi Ig G are enzyme-linked immunosorbent assay (ELISA), complement fixation test, fluorescent antibody technique, hemagglutination test, radioimmunoprecipitation assay, and Western blot (Alonso-Padilla et al., ). This perspective aims to discuss the current use of PCR techniques for detecting T. cruzi DNA in Chagas disease studies and explore potential new directions for utilizing these techniques in endemic areas.
Traditional parasitological tests have been replaced by PCR, which has been proven to be more sensitive. However, some difficulties must be addressed to overcome unequal results due to sample volume, DNA extraction protocol or T. cruzi region of amplification (Junqueira et al., ; Virreira et al., ). For this reason, more conserved T. cruzi regions have been targeted, such as the satellite DNA and the variable region of kinetoplast DNA (kDNA) mini-circles (Schijman et al., ; Ramirez et al., ).
The introduction of qPCR has significantly improved molecular biology techniques. Automatization and standardization have allowed for the quantification of T. cruzi parasitic loads (Qvarnstrom et al., ).
Several efforts have been made to improve the sensitivity and specificity of qPCR. Table 1 provides a few examples of such research work.
Table 1Digital droplet PCR is a technique in which the amplification reaction is conducted for individual nucleic acid molecules in thousands of independent PCR reactions, previously divided into droplets from a sample. The advantage of ddPCR is its ability to provide absolute quantification without the need for a standard curve (Liu et al., ). However, it has some limitations, including the high cost of instruments, the requirement for well-trained personnel, and a tendency to yield false-positive results. When it comes to detecting T. cruzi DNA in patients’ blood samples, ddPCR does not outperform qPCR, with a sensitivity of 1 parasite/mL compared to 0.46 parasite/mL in qPCR (Ramírez et al., ). The LAMP-PCR protocol for T. cruzi DNA amplification requires only one temperature for the reaction to occur, thanks to Bacillus stearothermophilus. It employs colorimetric or fluorescent dyes for in situ detection (Alves, ). LAMP has demonstrated high sensitivity, cost-effectiveness, and speed. However, it has raised some concerns due to its propensity for high levels of non-specific amplification (Shrestha et al., ). To address these limitations, Argentinian researchers have introduced additional steps in the protocol, including more stringent sample preparation and specific kits. Schijman’s group found that their T. cruzi Lamp kit was as sensitive as qPCR (Besuschio et al., ; Muñoz-Calderón et al., ).
Early diagnosis and care are essential in congenital Chagas disease. Diagnosis in the early stages leads to the best outcomes for therapeutic success. However, this is challenging in the first months of life due to the transfer of maternal antibodies (Carlier and Truyens, ; Pecoul et al., ).
Early T. cruzi detection by qPCR can provide more accurate estimations of congenital cases. This could improve the early detection of cases, providing more accurate records on the number of infants born to Chagas disease mothers in endemic and non-endemic countries and allowing better estimation of case numbers. Early qPCR diagnosis tests have been done by Benatar et al. () (Table 1) however they recognized that improvements need to be made. A year earlier, the same Schijman group used the LAMP-PCR test to analyze 13 congenital Chagas disease individuals and found that LAMP-PCR was sensitive and specific, comparable to qPCR (Besuschio et al., ). In a meta-analysis by Candia-Puma et al. (), it was found that qPCR is the most effective among molecular diagnostic tools, particularly in acute cases.
Efforts have been made over the years to establish a standardized PCR protocol for monitoring the treatment of chronic patients, as serology alone is not accurate enough to validate treatment efficacy. In adults, antibodies against T. cruzi can remain detectable for six months to several years after treatment. Sulleiro et al. () detected the presence of T. cruzi by qPCR in 42% of untreated chronic patients, with almost 55% of a subgroup of them showing intermittent parasitemia.
However, PCR negativity does not guarantee that the infection has been cured. Although treatment can demonstrate excellent effectiveness in eliminating blood-stage parasites, its capacity to target tissue forms remains uncertain (Simón et al., ).
There is still no consensus about the usefulness of PCR as a predictive marker of disease progression. Sulleiro et al. () demonstrated that a positive qPCR is not necessarily associated with visceral abnormalities. However, Sabino et al. () observed that a positive qPCR is linked to Chagas cardiomyopathy and disease severity, contradicting the findings of a smaller study by Norman et al. ().
As shown in Table 1 more specific qPCR protocols have been developed; however, these tests are still recommended to be used in combination with serological tests, which could significantly improve Chagas disease treatment. This combination of tests can not only be useful for therapy indication, but also for monitoring, and control, as well as for surveillance of T. cruzi transmitters and control.
In , Candia-Puma et al. () performed a meta-analysis over the last 30 years. They observed that PCR and qPCR are not as good as the ELISA test, which proved to be the best diagnostic tool in acute and chronic Chagas disease. When they analyzed the molecular techniques, they found that these techniques have not been standardized. Despite its analytical validation, qPCR remains to be clinically validated to determine its practical usefulness (Duffy et al., ). Recent findings by Muñoz-Calderon et al. () have shown promising results for LAMP-PCR. Even with a small sample size, they suggested that LAMP could be used as indicator of treatment failure.
In endemic areas, dogs and cats are considered as good indicators of potential active T. cruzi transmission. In the acute phase, Curtis-Robles et al. () proposed the use of molecular methods to confirm infection. Additionally, molecular techniques could be useful for monitoring parasitemia during drug treatment of Chagas disease in dogs (Lana et al., ). However, in the chronic phase, dogs and cats generally show low and intermittent parasitemia (Eloy and Lucheis, ), which diminishes all diagnostic methodologies. This issue has also been found in animals in captivity, in , Ndao and colleagues conducted a study involving a colony of captive New World monkeys (Ndao et al., ). Their research revealed an interesting phenomenon: among the monkeys initially tested negative for T. cruzi using PCR, a subsequent round of testing showed that a small subset of these monkeys (n=5) became positive on both smear and PCR test. This observation raised concerns regarding the possibility of false negatives.
Due to the intermittent nature of parasitemia in the chronic stage, it is difficult to determine the best time to obtain accurate results. Other factors to be considered are the strain of the parasite and the clinical variability, which have been attributed to the high genetic diversity and multiclonality of natural populations of T. cruzi (Macedo and Pena, ). Depending on the geographical origin of the strain and the source of infection, PCR values can vary, as several authors have published. The behavior of the strain is an important factor since the pattern of the release of the infective forms into the bloodstream is not well established. The lineage of the parasite must also be considered. T. cruzi populations show high genetic diversity and are classified into six Discrete Typing Units (DTUs) named TcI to TcVI (Zingales et al., ). The vast regional diversity and the course of chronic infection might reflect complex interactions between the genetic variability of T. cruzi strains, host immunogenetics, and eco-epidemiological characteristics (Moreira et al., ).
Given the fluctuating levels of parasitemia observed in individuals with chronic Chagas disease, it might be useful to perform repeated examinations with blood taken at different times using reliable qPCR kits. However, this can be challenging with a limited budget (Seiringer et al., ).
Currently, it is still recommended that PCR and qPCR be validated with a serological test. The robustness of immunological techniques has been well established (Ferrer et al., ), with ELISA being widely recognized for its performance (Candia-Puma et al., ).
However, qPCR is not exempt from limitations such as a higher cost of consumables compared to conventional methods. It requires a thermal cycler coupled with an optical reading system to allow for interpretation and a high level of technical skill. In general, molecular techniques require expensive resources and equipment. While LAMP is a promising technique, it requires further testing by other research groups in endemic settings to assess accessibility, affordability, accuracy, and sensitivity. There is a need for more rapid tests that do not sacrifice sensitivity and can be used in both clinical settings and resource-poor field settings. Research efforts should focus on the development of new diagnostic methods including serological, molecular, and proteomics approaches.
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It is important to continue improving molecular tools with high-throughput instrumentation to provide more reliable and accurate results. However, affordability is essential in the neglected disease field. Simple technology and temperature-resistant reagents are mandatory. Techniques that can be implemented in the field without requiring sophisticated equipment and expensive reagents are needed. Identifying biomarkers for simple, easy-to-use tests is crucial. While some candidates have emerged, substantial efforts are still required to develop these kits and make them accessible in the field.
NB-L: Conceptualization, Formal analysis, Writing – original draft, Writing – review & editing. MN: Conceptualization, Formal analysis, Project administration, Writing – review & editing.
The author(s) declare that no financial support was received for the research, authorship, and/or publication of this article.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The author MN declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.
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Chagas disease, caused by infection with the protozoan parasite Trypanosoma cruzi (T. cruzi), is the neglected tropical disease exerting a highest burden in the Western hemisphere [1]. It is estimated that there are ~7 million people affected worldwide, the majority of them living in Latin America where the insect vectors that transmit the infection are endemic [1]. Besides, vector-independent transmission routes like blood-transfusion, organ transplant, and, very importantly, from mother-to-child have also been described [2]. These are of relevance in endemic and non-endemic countries (like Spain), where the disease impact has spread to in the last decades due to population movements [3].
Three clinical stages are distinguished in the progression of Chagas disease. First there is a short lasting acute phase that is mostly asymptomatic and goes unnoticed. It is followed by a long lasting chronic phase that may span for decades without showing any symptoms associated to the infection (indeterminate stage). About 40% of those chronically infected will end up developing life-threatening disruptions to the heart and/or digestive tract tissues (determinate stage); lesions that can lead to death if left untreated [2].
Chagas suspicion can be made by observation of its associated symptomatology, but serological confirmation following WHO/PAHO criteria is mandatory in order to establish a diagnosis [4, 5]. However, it is fundamental to achieve it before this is advanced as the anti-parasitic treatments available (benznidazole and nifurtimox), efficacious against the parasite, cannot revert the damage to the tissues [6]. Both drugs have a very good efficacy against the acute stage of the infection and are well tolerated by children [1, 2]. Unfortunately their efficacy diminishes in the chronic stage of the infection and the advent of adverse events linked to their long administration regimes is frequent in adults [7, 8]. Nevertheless, they are the only drugs available at present, and several studies have proved the benefits of their administration [6, 9–12]. But, despite the advantages of treatment, it is estimated that less than 1% of those infected eventually get access to it [13], due to a series of factors amongst which overarches the lack of a timely diagnosis [14]. Thereby, propelling a drive to this dramatic figure should be immediately addressed. A goal further supported by the acknowledged benefits of drug administration to women at childbearing age towards the prevention of congenital transmission [14].
Methodologies used to diagnose T. cruzi infection largely depend on the disease stage status. In the acute phase, the levels of parasitemia enable direct detection of the parasite either by parasitological or by molecular biology techniques [15]. Parasitemia becomes low and intermittent in the chronic phase, but the acute infection triggered seroconversion and anti-T. cruzi specific immunoglobulins are detectable for years, so the chronic stage can be indirectly identified by serological methods. The most commonly used is the enzyme-linked immunosorbent assay (ELISA), of which there are many kits commercially available [16]. Their sensitivity and specificity are generally very good, but they work with serum or plasma samples which involve blood extraction by venous puncture and a centrifugation step to segregate sera or plasma from other blood components. Besides, plasma and/or serum samples require of cold storage, similarly to some of the components of the ELISA kits. Furthermore, ELISAs must be performed by technically trained personnel and their results preferentially read with a spectrophotometer. All these features are common to other conventional serological methods for Chagas disease diagnosis like indirect immunofluorescence (IIF). Unfortunately such conditions are often unattainable in vast areas highly endemic for the disease that only count with poorly equipped laboratories [14]. Moreover, under the recently confirmed WHO/PAHO recommendation on the agreement of two techniques with distinct antigen sets for conclusive T. cruzi diagnosis [4, 5], results turnaround of conventional diagnostics may delay for several weeks due to logistical and operational matters. Plus it usually involves more than one visit to the hospital, which is unfeasible for a population with low-resources that often live far away from it. As a consequence, many people who could be targeted for treatment remain undiagnosed, and untreated.
Fortunately there are several rapid diagnostic tests (RDTs) commercially available for the diagnosis of chronic Chagas disease [17]. These were developed to be used as point-of-care (PoC) diagnostics in the context of highly endemic settings that lack of the appropriate laboratory resources [18]. Various studies have previously described they can provide very good sensitivity and specificity, even working with tiny volumes of whole blood [19–22]. Furthermore, the combined use of two RDTs has already been suggested as a substitute of the current algorithm based on conventional serological methods [19, 23]. In the present study we demonstrate that the field use of RDTs by mobile teams could indeed substitute the use of conventional tests in the highly endemic Chaco region of Bolivia.
This work represents the next step of a previous study that described the combined use of RDTs as an alternative to the use of conventional serological methods for the diagnosis of chronic Chagas disease [19]. We aim at validating the implementation of rapid easy-to-use tests so that these can substitute conventional serology, an advancement that would be especially relevant towards the disease diagnosis in highly endemic regions with poorly equipped laboratories. In contrast to the study by Egüez et al., we have now taken the RDTs outdoors and used them in the form of field screening campaigns by mobile teams.
Similarly to what it was reported in the study in Sucre, the seroprevalence reported by this work in the Chaco region around the cities of Yacuiba and Villa Montes was higher than the average Chagas disease seroprevalence reported in Bolivia [24]. This may be due to the fact that the surveyed regions are highly endemic for the disease. Moreover, the offer of a free of charge T. cruzi infection diagnosis to the population could have had a call-in effect to those people in suspicion of having acquired the disease. In any case, the high seroprevalence rate observed (44.4% of the serum samples analyzed) was very similar to that reported before in studies performed with CSP and ELISAs in the close municipality of Carapari (Gran Chaco province) with people of all ages [22], as well as the rates reported with samples from pregnant adult women by Shah et al. in their field study with CDP and ELISAs in the municipality of Camiri (Cordillera province of the Bolivian Chaco) [21].
In our study, the agreement between the two main RDTs used was high (93.1%), although not as high as the agreement between the two main ELISAs (96.1%). The level of agreement of the RDTs was neither as good as what we had described before for the same kits in the Sucre study (100%) [19]. The lower agreement rate observed now might be due to eco-epidemiological differences of the surveyed areas of the Chaco region in comparison to Sucre peri-urban area, as well as to the fact that the rapid tests were performed outside the lab in a non-controlled environment. It addition, it must be indicated that due to the operational characteristics of the field campaigns, RDTs results were interpreted by a single operator. This may have biased the agreement of the tests due to a lack of independence.
We would like to highlight that we decided to use three different rapid assays, based on distinct antigen sets, in order be able to head-to-head compare their performance to that of the ELISAs algorithm (the “gold-standard”). In fact, the use of a third RDT (WL-Check, Wiener) was needed to untie 47 discordant results. We are aware that an algorithm relying on three RDTs might arguably not be a good option in the terrain due to its cost. Notwithstanding, we also individually compared the performance of each of the two main RDTs with the outcome of the ELISAs algorithm. Remarkably, both RDTs did independently provide a very good performance (see Table 3). CDP provided the highest sensitivity of the two (98.7%, versus 97.7% of CSP); whereas CSP had a very high specificity (97.4%). Notably, CSP yielded a DE of 97.5%, which means that it was capable of correctly classifying almost 98 of each 100 tested subjects. These figures would support the use of CSP by itself as a conclusive diagnosis of chronic T. cruzi infection. However, following current recommendation, a confirmation of the positively screened cases would be required with another test based on another antigenic set [4, 5]. The use of a conventional test to confirm a positive RDT outcome is already in place in Bolivia [32], and it was recently suggested in a meta-analysis article that reviewed the performance of several RDTs [33]. But we must emphasize that the necessity of a conventional assay to confirm a result obtained with an easy-to-use RDT would leave us in the same position where we are now. Such conventional confirmatory test will have to be made in an equipped laboratory by trained personnel, features that are often unavailable in highly endemic areas. Besides, the person would probably have to wait for several weeks to get a results turnaround with the consequent risk of being loss to follow-up. Thereby, confirmation of a seropositive output with another RDT would be much more desirable in this context. Even more, taking into consideration the very good performance obtained using just one diagnostic test in a highly endemic region like the one studied here, it would make full sense to adapt the current chronic Chagas disease diagnosis policy to the regional epidemiological reality.
In light of the results obtained by Egüez et al. [19] and in this study with any of the two main RDTs using a small volume of whole blood as sample, the use of a RDT could be applied as secondary confirmative tool, similarly to what is already done for the diagnosis of HIV infections [34]. The referenced document even contemplates “[…] the potential of HIV self-testing to increase access to and coverage of HIV testing […]” [34]. Such procedure could also be considered for Chagas disease, particularly when using CSP (Chembio Inc., Medford, USA) because it self-contains the required elements to conduct the diagnosis (like a blood-drop dispenser to add the sample into the diagnostic cassette). Operational robustness and easy-to-use are very important features to consider when working in the field. Unfortunately, this would not be the case for CDP (InBIOS Inc, Seattle, USA) as it is now, since it lacked the dispenser and was operationally more complicated to use.
Although we have obtained very good results using RDTs in Sucre [19] and in the Bolivian Chaco (this study), RDTs have been previously described to have limitations to their performance depending on the geographic region where they are used [35]. For instance a poorer performance in regions with lower T. cruzi prevalence (Arequipa area in Peru versus Santa Cruz area in Bolivia) has been described [35]. This would be explained by a potential direct relationship between RDTs sensitivity and the levels of anti-T. cruzi immunoglobulins in the studied population [35]. It could as well be due to the genetic background of the predominant parasite strains: the RDTs performing better in those regions with circulating parasites antigenically closer to the strains mirrored to design the kits [35]. Other feature that might influence the RDTs performance could be the occurrence of cross-reactivity events when evaluating them with samples from Leishmania spp. or T. rangeli infected subjects. This is particularly relevant in case RDTs had to be used in regions where these parasites co-exist with T. cruzi. To our knowledge, studies dealing with this potential issue have not been published yet, although the opposite, cross-reactivity of a Chagas disease positive case with an immunochromatographic rapid test to diagnose Leishmaniasis has been reported before [36]. The presence of leishmaniasis in the regions studied herein is very low as only 4 cases were registered during – (communication of SEDES Tarija, Ministry of Health, Bolivia).
In summary, the results obtained in this work would support the combined use of RDTs, in agreement with current recommendation, for delivering a conclusive diagnosis of Chagas disease in the region. We have shown that such tests can be used by mobile teams in the form of screening campaigns relying on a little volume of finger pricked whole blood as sample. Even though further studies are yet required to address the use of RDTs in areas with lower prevalence of T. cruzi and/or co-circulation of closely related pathogens, the results from this work encourage the use of RDTs as an alternative to conventional serological methods in the Bolivian Chaco and other areas with similar epidemiological characteristics.
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