The poor quality of diagnostics for T. cruzi infection is a major impediment to coping with a disease that affects as many as 20 million people. Without quality diagnostics, the statistic of the disease burden is at best a guess, the ability to conclusively identify who should be treated, or should be allowed to donate blood or tissues is greatly compromised and the effectiveness of interventions to limit transmission or drugs to treat those infected is impossible to determine with any certainty.
In the early stages of T. cruzi
infection, parasites can often be detected in blood. However, as immunity develops, even amplification techniques such as xenodiagnosis, hemaculture, and PCR, despite being repeated multiple times, routinely fail to detect infection 
. Consequently, determination of infection status is largely dependent on the consensus results of multiple tests with different formats (e.g. ELISA, indirect fluorescent antibody, indirect hemaglutination, complement fixation). However the unreliability of these tests is well documented 
. Many of these tests, including one recently licensed by the United States Food and Drug Administration for use as a blood screening test in the U.S. 
, use crude or semi-purified parasite preparations derived from parasite stages present in the insect vector but not in infected humans. Recently, a number of recombinant parasite proteins or peptides have also come into limited use for diagnosis 
A subject whose serum is consistently positive on multiple of the currently used tests is relatively easily determined to be infected. But the infection status of individuals positive on only one test (as used in blood bank screening) is unclear and detection of parasites in subjects who are negative using multiple conventional serologic tests 
or who are positive by alternative but not widely available serological tests 
is not uncommon. Furthermore, currently available tests are inadequate for monitoring treatment efficacy 
and thus may also give inaccurate measurements of the effectiveness of other interventions.
With these deficits in mind, we set out to identify parasite proteins that would more effectively detect T. cruzi infection and provide a tool for monitoring changes in infection status over time. Development of a repository of nearly 1500 T. cruzi genes cloned into Gateway entry vectors provided a relatively straightforward approach to producing a large number and diversity of T. cruzi proteins appropriate for high-throughput screening of diagnostics. Adding the targeted approach of selecting proteins documented for high level expression in trypomastigote and amastigote stages of T. cruzi allowed us to also focus on the proteins that would be predicted to elicit the strongest antibody response in infected humans. The Luminex-based multiplex bead array system permitted us to screen many proteins simultaneously with very low requirements for serum. The production of histidine-tagged proteins also made it relatively uncomplicated to attach the recombinant proteins to Luminex beads. This latter point is not trivial as the proteins could be coupled to the assay beads directly from the urea-based denaturing lysis buffer without the requirement of movement to a non-denaturing buffer, wherein many of the proteins precipitated. The strong response detected using proteins prepared in this way suggests either that natively folded proteins are not required for the detection of these antibodies, or that re-folding of the proteins attached to the Luminex beads during buffer exchange resulted in the formation of native conformational epitopes.
In addition to its utility for screening of a large number of proteins, the Luminex system also excels as a platform for multiplex analysis of antibodies to a relatively large set of targets. We were restricted in this work by the number of Luminex bead sets manufactured with Ni+2 and thus sought to identify a maximum of 16 independent T. cruzi
proteins that gave informative results from a large set of human sera. The ultimate panel selected by the screen included at least one protein previously identified as a potential diagnostic, the mitochrondrial HSP-70 
. It is possible that other proteins revealed in our screen have been studied previously. However since the identity of some of these previously assayed proteins is somewhat cryptic 
and few have been associated with annotated genes in the sequenced T. cruzi
genome, this possibility is difficult to evaluate. Also, over half of the antigens selected in our screen were among the 50 most abundant proteins in the trypomastigote and amastigote proteomes 
. Two hypothetical proteins and 2 proteins unique to T. cruzi
among the sequenced kinetoplastids, including 2 fragments from the very large and multicopy dispersed gene family protein, were among the proteins selected. Proteins that are unique to T. cruzi
could be particularly useful in a serological screen as they are absent from Leishmania
, one of the potentially confounding infections in terms of diagnosis of T. cruzi
. However the dispersed gene family fragments were among the worst performers in the large scale screen – with only 5–9% of all confirmed positive sera having detectable antibodies to these. Similarly, other gene family proteins, including trans-sialidases, mucins and mucin-associated proteins (MASPS) were part of the screen but failed to make even the initial selection cuts in our assays, presumably because only a small fraction of their diversity would be represented in the recombinant proteins screened.
A multiplex approach like the Luminex also provided a more detailed examination of responses than is possible using a single target consisting of either an individual protein or a protein/peptide mixture. Each individual was seen clearly to have a distinct pattern of responses to the protein panel and this pattern was impressively stable over time (several years). This is both interesting scientifically and serves as further validation of the quality and consistency of the data generated using this multiplex methodology. This heterogeneity of responses to pathogens among individuals appears to be more the norm than the exception, as similar results have been reported for individuals infected or immunized with viral (vaccinia), bacterial (Francisella tularensis
) and protozoal (Plasmodium falciparum
) pathogens 
. Thus serodiagnostics in general are likely to need to move toward multiplex assays, as single antigens that are recognized by all individuals infected by any pathogen appear to be rare 
The ability to simultaneously and independently assess antibody responses to multiple targets was instrumental to our success in addressing the issues of the detection of serological responses in subjects who are negative by conventional serology and the relatively rapid detection of changes in selected responses following drug treatment. The multiplex assay detected 100% of 121 samples consistently positive by conventional serology, and 100% of samples positive on 2 out of 3 conventional tests. In addition, however, we also detected antibodies specific for one or more recombinant proteins in 18 of 33 subjects judged as negative by conventional serology. Other investigators have documented cases of conventional seronegative subjects being seropositive on alternative tests or even parasite positive 
although these previous reports of “infected seronegatives” have been somewhat anecdotal – presumably because investigators rarely screen for parasites in seronegative subjects. However in some studies parasite-positive conventional seronegatives are very well documented. For example Picka et al. 
reported on one subject who was negative by up to 5 replicates of 4 different conventional serological tests yet was positive by a combined hemaculture-PCR approach. The multiple examples of the failure of conventional serology to detect infection, in combination with the well-documented unreliability of parasitological tests, supports the conclusion that individuals who are seropositive in our multiplex assay are likely to be infected with T. cruzi
. This conclusion is further supported by on-going studies demonstrating T. cruzi
–specific T cell responses in subjects who are negative by conventional serology but positive in our multiplex assays (Postan, et al. in preparation). Without more sensitive parasitological tests we cannot conclusively determine if the subjects who are negative by conventional serology but positive in our multiplex assay are infected or possibly “exposed” but not still infected with T. cruzi
. And without additional extensive validation, we cannot exclude the possibility that other infections or immunological conditions resulted in some of the multiplex positive responses, although standard clinical analysis failed to detect other complicating infections in these subjects. However, especially for subjects who have antibodies to up to 8 different recombinant T. cruzi
proteins and were born in endemic areas and/or have evidence of heart disease, it is reasonable to conclude that they are indeed infected with T. cruzi
despite their negative results with conventional serologic assays. Overall these studies support the already documented conclusion that current serological tests can misdiagnose infection – perhaps to a significant extent.
A second issue we addressed using the multiplex serological assay for T. cruzi infection was that of efficacy of therapeutic treatment. Because most subjects are negative by parasitological assays prior to treatment (making a negative result after treatment uninformative) and remain positive by conventional serology for extensive periods of time after treatment, assessing whether treatment actually achieved cure, has been problematic. When combined with other evidence of treatment failures and the adverse effects of the drugs, the absence of a method to detect treatment efficacy has resulted in a very low rate of treatment in chronic Chagas disease. This absence of a reliable and timely test for treatment efficacy is also a major impediment to the development and testing of new drugs – an area that has been at a virtual standstill for decades.
Herein we show that the multiplex assay using the selected set of recombinant proteins can detect significant changes in antibody levels, in some cases as early as the first post-treatment assay point (2 months post-treatment completion). These changes are not evident in all cases – an outcome that is not surprising given that treatment failure is common 
. Our ability to assess responses to multiple targets on an individual basis appears to be crucial to the success of detection of serologic changes following treatment, as similar changes are not consistently observed using conventional serologic tests. Previous studies have suggested that various recombinant antigens may provide better assessment of treatment efficacy relative to conventional serology 
. Further studies of a large set of treated subjects, using both multiplex serology and cellular immune responses support the hypothesis that these immunological markers are effective indicators of treatment success or failure (Laucella, in preparation).
We define a set of diagnostic targets and an assay approach that we believe is a significant improvement upon current diagnostic tests for T. cruzi infection both for more consistently detecting infection and for assessing the effectiveness of treatment. Additional validation of these targets and the general methodology will require analysis of a larger set of subjects, a process that is currently on-going. Herein we have also not addressed the question of whether the antigens we identify would be useful throughout the wide endemic range for T. cruzi. Heterogeneity among different parasites strains in distinct regions could present a challenge. However here again this is a concern that a multiplex assay might rather easily address – it seems unlikely that all 16 proteins in our pool, most of which are abundant housekeeping proteins, would vary substantially among parasites in various regions. Furthermore, we have intentionally used a mixture of T. cruzi strains from geographically distinct regions as the source of genes encoding the proteins used in these studies, with the goal of capturing some of the heterogeneity that may exist in these proteins among various parasite isolates.
The problem of infection confirmation by detection of parasites or parasite products is likely to continue to be a roadblock to full acceptance of the results of this test, or any other, when they conflict with conventional serologic tests – despite the proven inadequacy of these “standard” tests. Currently there is no methodology that allows for the consistent detection of parasites or their products in chronically infected hosts. The well-documented failure of various PCR-based approaches indicates that even highly abundant T. cruzi
sequences are insufficient to document active infection in the majority of individuals, in addition to confirming the very low level of parasite persistence in most individuals. If the million-fold amplification afforded by PCR is unable to consistently reveal persistent T. cruzi
infection, it also seems that detection of other parasite “biomarkers” will be equally inadequate. One advantage of using host biomarkers such as pathogen-specific antibodies and T cells is that they are naturally and endogenously self-amplified in the course of immune recognition of the infection. Downsides of the Luminex system for multiplex analysis include the reagent expense as well as the requirement for specialized equipment to “read” the results. However, other multiplex platforms such as protein microarrays could be more cost conservative and require less infrastructure 
. Also, our results suggest that the number of proteins in the analysis could be reduced without substantial loss of sensitivity, and the possibility exists for additional improvements in sensitivity by the inclusion of T. cruzi
proteins previously validated by others or that could be detected in additional screens like that described herein. At a minimum, these results begin to lay the groundwork for the removal of one of the major impediments to the development and effective implementation of treatments for T. cruzi