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. 2008 Oct 8;2(10):e316.
doi: 10.1371/journal.pntd.0000316.

High throughput selection of effective serodiagnostics for Trypanosoma cruzi infection

Gretchen Cooley  1 , R Drew Etheridge, Courtney Boehlke, Becky Bundy, D Brent Weatherly, Todd Minning, Matthew Haney, Miriam Postan, Susana Laucella, Rick L Tarleton
Affiliations

High throughput selection of effective serodiagnostics for Trypanosoma cruzi infection

Gretchen Cooley et al. PLoS Negl Trop Dis. .

Abstract

Background: Diagnosis of Trypanosoma cruzi infection by direct pathogen detection is complicated by the low parasite burden in subjects persistently infected with this agent of human Chagas disease. Determination of infection status by serological analysis has also been faulty, largely due to the lack of well-characterized parasite reagents for the detection of anti-parasite antibodies.

Methods: In this study, we screened more than 400 recombinant proteins of T. cruzi, including randomly selected and those known to be highly expressed in the parasite stages present in mammalian hosts, for the ability to detect anti-parasite antibodies in the sera of subjects with confirmed or suspected T. cruzi infection.

Findings: A set of 16 protein groups were identified and incorporated into a multiplex bead array format which detected 100% of >100 confirmed positive sera and also documented consistent, strong and broad responses in samples undetected or discordant using conventional serologic tests. Each serum had a distinct but highly stable reaction pattern. This diagnostic panel was also useful for monitoring drug treatment efficacy in chronic Chagas disease.

Conclusions: These results substantially extend the variety and quality of diagnostic targets for Chagas disease and offer a useful tool for determining treatment success or failure.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Screening process for the high-throughput selection of diagnostic proteins for detection of T. cruzi infection.
Figure 2
Figure 2. SDS-PAGE gel of production of pooled protein.
Sets of 6–8 genes were moved in pools from pDONR entry plasmids into pDEST-PTD4 via a Gateway LR reaction (Invitrogen) and the resulting plasmids transformed into BL21(DE3)pLysS cells for protein production. Recombinant HIS-tagged proteins were purified on Co+2 affinity resin and the bound proteins analyzed by SDS-PAGE. Protein pools depicted in lanes 1–5 were generated from the pooling of 8 genes, while lanes 6, 7 and 8 were derived from 7, 6, and 6 genes respectively. Lane “S” contained molecular weight standards (BenchMark Prestained Protein Standard; Invitrogen). Overall, approximately 80% of genes yielded proteins when expressed as pools.
Figure 3
Figure 3. Reactivity of a representative set of proteins tested with individual sera.
A selection of 29 individual recombinant proteins was tested for the ability to bind IgGs present in the sera of 54 subjects. The sera are grouped as “uniformly positive” (reactive on all three conventional serological tests and a commercial assay kit), “inconclusive” (negative on at least one conventional serologic tests), “negative by conventional tests” (negative by all three conventional tests), and “known negative” (from residents of North America with very low chance of being infected based upon residency and travel history). Recombinant ovalbumin and T. cruzi lysate –coated beads were used as negative and positive controls, respectively. Horizonal bars in each box indicate mean fluorescence intensity (MFI) on a scale from 0 to 30,000 arbitrary light units. A number of the recombinant proteins either failed to discriminate between uniformly positive and known negative sera sets (e.g. 3, 16, 17, 22, 26, 27, 28) or showed no reaction with either set (e.g. 10). In contrast a number of proteins detected nearly the entire uniformly positive group, as well as some in the inconclusive and conventional negative groups but none in the known negative set (e.g. 4, 11, 14, 19 and 20 – stippled highlighting).
Figure 4
Figure 4. Reactivity of negative, borderline or discordant sera in the 16 protein multiplex assay.
Sera judged cumulatively as “seronegative” based upon conventional serology were grouped into negative but “no other evidence” of exposure (16 sera), those “born in an endemic area” (12), those with evidence of “heart disease” consistent with Chagas disease (5) and compared to (top) pools of strongly positive sera (high reactivity in all serological tests) and to (bottom) sera from subjects who were negative on at least one of the three conventional serologic tests (discordant positive). Reactivity in the conventional serological tests (HAI, ELISA and IFI) and the summary consensus of these tests (neg = below cut-off for all three tests; discord = positive on one of the three tests; discord + = positive on 2 of the 3 tests), as well as reactivity to the 16 recombinant protein sets and the T. cruzi lysate are shown. Cutoffs for a positive ELISA is an O.D.>0.200 and for IFA and HAI is a dilution>1/32 (a reaction at 1/16 is considered “reactive but negative” and <1/16 non-reactive (nr). The metric for reactivity of each serum for each protein is expressed as the number of standard deviations that the ratio of the MFI for T. cruzi protein to the MFI for GFP was above the average ratios of sixteen true negative sera run in the same assay. Values>4 S.D. above this “background” reactivity are considered reactive and are colored. The total number of reactive recombinant proteins for each serum is indicated in the right-most column. nd = not determined (insufficient numbers of beads detected in this sample).
Figure 5
Figure 5. Stability of serological responses over time.
The MFI of sera to a panel of 16 recombinant proteins (top 16 in Table 1), GFP negative control protein and T. cruzi lysate for a total of 18 measurements (bars) are shown for each serum. A) Reactivity of a set of 8 known negative sera. B) Stability of unique pattern of antigen activity for 6 seropositive subjects assayed at 4 time points over 12–21 months. Arrows in lower right panel (RD 07) indicate that detection of protein “8” (paraflagellar rod protein) which distinguishes the pattern of reactivity of serum RD07 from that of the similar RD09.
Figure 6
Figure 6. Effect of benznidazole treatment on serological responses in chronically infected subjects.
The MFI of sera to a panel of 16 recombinant proteins (top 16 in Table 1), GFP negative control protein and T. cruzi lysate for a total of 18 measurements (bars) are shown for each serum. A) Change in pattern of reactivity over 36 months post- benznidazole treatment in 4 subjects using both the multiplex serologic assay (left) and conventional serology (right). B) Sera from 2 benznidazole-treated subjects exhibiting no evidence of change in multiplex assay for 24 months post-treatment (left; PP044) and changes in reactivity to selected recombinant proteins (right; PP024).
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