This review and meta-analysis arise from the need to assess the optimal diagnosis of Chagas disease, especially during the chronic phase. Compared to direct diagnostic methods that depend on the observation and identification capacity of health care professionals, PCR has proven to be more sensitive and specific [5]. All the studies analysed confirm the low diagnostic efficacy of molecular techniques in chronic patients. However, these techniques constitute a basic tool in the acute phase. Above all, in cases of congenital transmission, PCR is considered as the reference diagnostic tool. In addition, PCR is essential in detecting reactivations in cases of immunosuppression, transplantation, and transfusion.
Most of the articles analysed focus on the diagnosis of the chronic phase. Regarding the acute phase, almost all the works analyse its congenital transmission. The articles that analyse the chronic phase assess the possible detection of the parasite in the phase of the disease in which parasitemia is minimal, when direct conventional methods are ineffective. The diagnostic possibilities of molecular methods in the acute phase are focused on nonendemic countries, especially on vertical transmission [42]. The presence of maternal antibodies in the foetus and the low parasitemia during the first months of life make direct and indirect diagnosis difficult. Thus, in neonates, the only way to obtain an accurate diagnosis is to confirm the presence of the parasite. For a serological test to be decisive, it should be carried out after 9 to 12 months of age, when it has been concluded that the antibodies against T. cruzi in the mother have disappeared. Early diagnosis in the acute phase is extremely important due to the high efficacy of treatment of close to 90% [43].
The quality of the studies was analysed using the QUADAS method. Most of the articles included are based on trials in the second phase of validation by calculating the predictive values in a comparative cross-sectional study in subjects with suspicion of the disease or examining routine laboratory methods; therefore, they present some bias in the choice of patients. In many of them, the availability of the reference test result information was not detailed before the index test was performed, nor were they clear in relation to the reference tests, especially in the case of chronic infection. All studies used reference serological methods, although it is necessary to perform other tests to differentiate between acute phase, chronic phase or a past infection. It is worth noting the lack of information in most of the articles, especially in the sampling and reference tests, especially in the cases of commercial methods.
The 32 selected studies exhibit high variability of results for the different molecular targets, primers, probes, extraction methods and amplification methods, making it difficult to standardize molecular techniques for the diagnosis of CD. In addition, most studies indicate the propensity for errors in clinical diagnosis. Analytical sensitivity is more consistent for kDNA-based PCR versus satDNA, depending on the doses present in the different genomes of DTUs [44]. The main problem presented by kDNA as a diagnostic target lies in the enormous number of false positives found in patients infected with T. rangeli, since the kinetoplast minicircles seem to be quite conserved within the genus; therefore, in areas where T. cruzi and T. rangeli are endemic, the use of satDNA PCR is recommended, as Tripanosoma has few copies of the satellite sequence [45].
Observationally, it can be determined that kDNA-focused molecular diagnostic methods are more sensitive and those based on satDNA are more specific. Multiple comparative studies have been carried out between the different PCR methods for the diagnosis of CD [23, 46]. The international study developed by PAHO and WHO in 2011 stands out [47], in which the diagnostic techniques used in 26 laboratories in 16 countries with dilutions of isolated strains, clinical samples and artificially infected samples are evaluated, defining satDNA and kDNA as the most effective targets. Many studies present inconclusive results that must be analysed on multiple occasions to determine the infection status. When the disease becomes chronic, time is not an entirely determining factor, but time could be vital in cases of congenital infections, oral infections, conditions of immunosuppression or for patients undergoing a transplant process.
After the statistical analysis of the diagnostic efficacy of the protocols that apply molecular techniques, it is concluded that they are recommendable for routine diagnosis in the acute phase. These are sufficiently specific and sensitive methods for application (84.9% cumulative sensitivity and 98.5% cumulative specificity). None of the different molecular techniques (cPCR, qPCR and LAMP) present a DOR lower than 10, indicating a very high capacity to discriminate between infected and healthy patients [48]. These results are explained by the close relationship between parasitemia and diagnostic efficacy for any direct diagnostic method. In cases of acute infection, parasitemia is relatively high, which makes it possible to detect the pathogen's DNA in most blood samples from patients.
The diagnostic effectiveness of molecular techniques in the chronic phase presents a low sensitivity (cumulative sensitivity of 67%, CI: 95%) for use as a routine diagnostic tool. This depends on the number of circulating parasites. Furthermore, in the chronic phase, parasitemia is low and intermittent. Therefore, in the processing of the sample from collection to the development of the test, there may be insufficiently high concentrations of parasitic DNA to be amplified. A possible solution to this limitation is the collection of several serial samples from the same patient at different times or an increase in the volume of blood drawn.
In general, the major problem with direct diagnostic techniques, whether molecular or conventional, is their limited application in endemic areas. These are regions in which the vast majority of those infected are in the indeterminate chronic phase of the disease; in these cases, the PCR result is positive for between 50% and 90% of those infected [49].
In addition, the results present great variability depending on multiple factors, such as the patient's parasitemia, the volume and processing of the sample, the target of the technique or the characteristics of the population [49].
On the other hand, a negative result does not exclude infection, and a serological test would be unavoidable. The entire international community agrees that the most recommendable approach is the combination of both types of diagnosis [25].
The heterogeneity between studies was very high, both in the acute phase (acute: I2 sensitivity = 95.2% and I2 specificity = 64.9%, p < 0.001) and chronic phase (chronic: I2 sensitivity = 95.2% and I2 specificity = 64.9%; p < 0.001). This heterogeneity could be explained by different factors that imply a certain patient selection bias and the scarcity of comparative studies. Another determining factor that can explain these contrasts would be the different DTUs circulating in each geographical region in which the studies were carried out, as well as methodological errors in some protocols lacking amplification controls.
The analysis of the heterogeneity of the studies in the chronic phase showed significant relationships between two fundamental variables for the diagnosis of CD: use of the boiling bath and the addition of guanidine to the blood sample. Thus, the addition of guanidine buffer to the blood sample after its extraction and the bath in boiling water significantly increased the sensitivity of the technique. This result was confirmed by other meta-analyses carried out on molecular diagnostic techniques for CD, such as that of Brasil et al. [49].
This difference is based on the release of parasite DNA, both by guanidine and by the boiling bath. The addition of guanidine hydrochloride to the sample with EDTA, forming the so-called guanidine-EDTA-blood (GEB), homogenizes the parasite's DNA, inhibits DNases and facilitates sample preservation, even at room temperature. With this procedure, it is possible to detect 1 parasite in up to 10 mL of blood [50].
The boiling water bath has previously been described as an efficient physical method of separation of the DNA networks present in the kinetoplast of the parasite, increasing the homogeneity of the genetic material in the blood sample [50]. The low concentration of parasites in the samples from patients in the chronic phase makes it necessary to homogenize the genetic material to ensure that after collecting the volume for the extraction process, the highest possible concentration of free parasite DNA is achieved. The search for new methods capable of increasing the ability to dissociate and disperse DNA throughout the sample seems to be the way forward in clinical practice.
No significant differences were found in parameters such as age, location, endemicity, type of study, reference serological tests, or year of publication. The volume of sample processed for DNA extraction did not turn out to be a factor involved in the heterogeneity of the studies either, in the same way as the different extraction techniques (salting out, phenol‒chloroform method or commercial methods), the different sample conservation protocols, or the bath in boiling water.
It should be noted that no significant differences were observed between the different main molecular techniques (cPCR, qPCR and LAMP) or in their variants, such as nested PCR, hot-start techniques, multiplex PCR or duplex PCR. The different molecular targets do not appear to be determinant in the diagnosis of the disease either, although this result is not entirely clear, as there exist a vast majority of articles in which kDNA and satDNA detection techniques are assessed. We can conclude that both targets have similar diagnostic efficacy, but in the case of the other molecular targets such as H2DNAA, Tc24, pE13DNA or 18SrRNA, no conclusion could be drawn due to the scarcity of articles that analysed techniques focused on these genomic regions.
A large number of different primer sets have been defined in the different bibliographic records, but it was not possible to identify the different combinations as a source of heterogeneity. Furthermore, the differences between the articles that used different types of development of the results or development using fluorescence or turbidimetry techniques in real time were not significant.
Limitations of evidence
There is a specific probability that the observed heterogeneity comes from items not included in this analysis due to incomplete data in the included publications. In many studies, methodological characteristics, such as sample collection period, age or mean age of participants, sex distribution of the sample, rural or urban origin, clinical presentation of the disease, sample preservation conditions, period between compared tests, DTUs endemic to the different geographic areas at the time of the study, inhibition and reaction contamination controls, or the time between sample collection and processing, were not determined/specified/recorded. In addition, summary estimates of diagnostic efficacy were grouped into subgroups under conditions of high heterogeneity; therefore, we should be careful/cautious/conservative in the interpretation of the results.
Applicability of findings/interpretation
We recommend that health authorities standardize and optimize molecular diagnostic techniques for better diagnosis and control of this disease.