Trypomastigotes of Trypanosoma cruzi express a set of surface glycoproteins known, collectively, as Tc-85. A monoclonal antibody to these proteins, named H1A10, inhibits (50-90%) in vitro parasite interiorization into host cells, thus implicating these glycoproteins in the infection process. Two DNA inserts, a genomic DNA fragment and a full-length cDNA encoding the H1A10 epitope, have now been cloned and characterized. Results show that both have high sequence identity with all reported members of the gp85/trans-sialidase gene family, although the H1A10 epitope exists only in the Tc-85 subset of the family. The epitope has been mapped by competition of antibody binding to a Tc-85 recombinant protein with peptides having sequences predicted by the Tc-85 DNA sequence, which contains also putative N-glycosylation sites and COOH-terminal glycosylphosphatidylinositol anchor insertion sites, as expected, since an N-glycan chain and a glycosylphosphatidylinositol anchor have been characterized previously in the Tc-85 subset. The protein encoded by the full-length cDNA insert binds to cells and in vitro to laminin, but not to gelatin or fibronectin, in a saturable manner. For the first time it was possible to assign a defined ligand to a sequenced glycoprotein belonging to the gp85 family. This fact, together with the reported binding of family members to cell surfaces, reinforces the hypothesis that this family encodes glycoproteins with similar sequences but differing enough as to bind to different ligands and thus forming a family of adhesion glycoproteins enabling the parasite to overcome the barriers interposed by cell membranes, extracellular matrices, and basal laminae.

The trypomastigote specific surface antigens of Trypanosoma cruzi are encoded by a supergene family which includes the TSA family. The TSA family is characterized by the presence of a 27-bp tandem repeat array in the coding region. Here, we report the characterization and analysis of the three TSA family members in the Esmeraldo strain of the parasite. In this strain 2 distinct telomeric members are expressed abundantly as 3.7-kb mRNAs, while the remaining member is located at an internal chromosomal site and is expressed at less than 2% of the level seen for the telomeric members. Based on hybridization to DNA separated by PFGE, 3 chromosomes of sizes 1.8 Mb, 0.98 Mb, and 0.90 Mb each contain one of the telomeric members. In addition, the two smaller chromosomes also contain the single internal member. Since both chromosomes contain similar TSA family members, and vary only slightly in size, we suggest that they are homologues. Comparisons of the nucleotide sequences of the different members of the family show that the internal gene differs from the telomeric genes primarily in sequences found 3' of the repeat array. These comparisons also reveal that the three genes are analogous, supporting the hypothesis that short segments between the family members are exchanged by gene conversion events. We propose that similar conversion events between members of different gene families may generate some of the diversity found within the supergene family.

Background

New cervical cancer screening guidelines recommend primary human papillomavirus (HPV) testing for women age 30-65 years. Healthcare organizations are preparing to de-implement the previous recommended strategies of Pap testing or co-testing (Pap plus HPV test) and substitute primary HPV testing. However, there may be significant challenges to the replacement of this entrenched clinical practice, even with an evidence-based substitution. We sought to identify stakeholder-perceived barriers and facilitators to this substitution within a large healthcare system, Kaiser Permanente Southern California.

Methods

We conducted semi-structured qualitative interviews with clinician, administrative, and patient stakeholders regarding (a) acceptability and feasibility of the planned substitution; (b) perceptions of barriers and facilitators, with an emphasis on those related to the de-implementation/implementation cycle of substitution; and (c) perceived readiness to change. Our interview guide was informed by the Consolidated Framework for Implementation Research (CFIR). Using a team coding approach, we developed an initial coding structure refined during iterative analysis; the data were subsequently organized thematically into domains, key themes, and sub-themes using thematic analysis, followed by framework analysis informed by CFIR.

Results

We conducted 23 interviews: 5 patient and 18 clinical/administrative. Clinicians perceived that patients feel more tests equals better care, and clinicians and patients expressed fear of missed cancers ("…it'll be more challenging convincing the patient that only one test is…good enough to detect cancer."). Patients perceived practice changes resulting in "less care" are driven by the desire to cut costs. In contrast, clinicians/administrators viewed changing from two tests to one as acceptable and a workflow efficiency ("…It's very easy and half the work."). Stakeholder-recommended strategies included focusing on the increased efficacy of primary HPV testing and developing clinician talking points incorporating national guidelines to assuage "cost-cutting" fears.

Conclusions

Substitution to replace an entrenched clinical practice is complex. Leveraging available facilitators is key to ease the process for clinical and administrative stakeholders-e.g., emphasizing the efficiency of going from two tests to one. Identifying and addressing clinician and patient fears regarding cost-cutting and perceived poorer quality of care is critical for substitution. Multicomponent and multilevel strategies for engagement and education will be required.

Trial registration

ClinicalTrials.gov, # NCT04371887.