Introduction and Background of the Project

1.  Introduction

Chagas disease, caused by the parasite Trypanosoma cruzi, affects >7 million people, mainly in the Americas. There are two drugs - benznidazole and nifurtimox – but access to treatment is hindered by poor access to diagnosis. This is usually achieved at the chronic stage of the infection, when the parasite presence can be detected by serological methods. These encompass conventional tools like enzyme-linked immunosorbent assays (ELISAs), as well as more innovative and easier-to-use point-of-care (POC) rapid diagnostic tests (RDTs). The current algorithm entails the agreement of two of them for confirmed diagnosis; if discordant, a third test is needed. This multiplicity of tests derives from the ample diversity of the parasite. As a result, diagnosis costs double (or thrice). Even, there are regions with under-represented parasite information where currently available tools have a very low performance.

Building on publicly available data and bioinformatics resources, in-house generated scripts, and a geographically diverse collection of clinical samples, we propose to design a universal RDT to streamline detection of chronic T. cruzi infections. Such a tool would generalize diagnosis in vast endemic regions with ill-equipped laboratories. Particularly, it could be a major asset for the screening of mothers-to-be, gating their newborns to diagnosis of T. cruzi vertical transmission.

2.  Project objective

Our ultimate objective is to achieve a prototype RDT to detect chronic T. cruzi infections that renders a very good performance regardless of the geographic origin of the clinical samples to analyze. For that, we envisage the following specific objectives:

(1) To sequence and assemble the whole genome of underrepresented T. cruzi isolates from Mesoamerica.

(2) To analyze, with the assistance of computational methods, all available T. cruzi protein sequences for prioritization of diagnostic antigens.

(3) To address the reactivity against those selected antigens by arraying them with plasma/serum samples originating from multiple Chagas disease endemic countries.

(4) To print the ultimately prioritized antigens on immunochromatographic (IC) strips for their analytical evaluation, and qualification, as a prototype POC RDT.

3.  Project design

To reach the goal of analytically validating a new RDT prototype that hypothetically works in all regions, we will first need to obtain the whole genome sequences of under-represented isolates circulating in Central America and Mexico, where it is acknowledged that currently available tools have a poor performance. Then, responding to the second specific objective, we will analyze all the sequences with the aid of computational methods in order to identify the antigens, or epitopes within them, of diagnostic interest. In search of the antigenic sequences that could encompass a pan-American, or regionally tailored, serological tool, we will consider evolutionary conservation as a major feature.

For evaluating the prioritized peptide sequences derived from the computational analysis, we will use a collection of samples from T. cruzi-infected individuals, and geographically matched non-infected controls. For the sake of time, this collection will be retrospective, and it will encompass two sets of samples: one for the exploratory part of the project, and another for the validation of the sequences.

We will address the reactivity of the samples over the distinct peptide sequences by means of peptide microarrays. These will render the necessary throughput to be able to check hundreds of peptides. Those supporting the widest geographical coverage and yielding the highest signal will be selected. Finally, the best will be taken to generate the RDT prototype in the shape of a lateral flow assay.

How can your partnership (project) address global health challenges?

T. cruzi infections have an impact on individual and global public health: >7 million people are infected, mainly in the Americas, where most of the 10,000 deaths per year from Chagas disease occur. The “silent” evolution of the infection hinders its clinical diagnosis, highlighting the necessity of sensitive and specific microbiological detection methods. Bearing in mind there are two anti-parasitic drugs available (benznidazole and nifurtimox), the lack of simple and reliable diagnostics constitute a major barrier for tackling the disease impact. Unavailability of suitable and affordable diagnostics leads to a very low access to diagnosis, hence to treatment.

In alignment to the WHO 2021-2030 roadmap for NTDs, our project aims to provide the prototype of a widely effective POC serological diagnostic for the detection of chronic T. cruzi infections. The reason for pursuing POC characteristics relate to the fact there are still vast areas highly endemic to Chagas disease where equipped referral laboratories are scarce. The test “universality” should mean the use of a single tool to achieve confirmatory diagnosis of the infection. At least two major advantages would derive from that: a reduction of costs and simplified distribution; hence a more sustainable supply chain.

What sort of innovation are you bringing in your project?

The uniqueness of the present proposal is that, departing from mass sequencing data analysis, it will move an innovative tool from in silico data to an immunochromatographic prototype. If results accompany, taking into consideration operational validation, licensing, and launch, the market availability forecast could fit within four years upon project completion. Such a tool would contribute to generalize access to diagnosis, including that of mothers-to-be, a key step towards timely detecting congenital Chagas disease and contribute to PAHO EMTCT framework.

Role and Responsibility of Each Partner

Nagasaki University (NU) will coordinate and manage the project. It will be responsible for the whole genome sequencing and assembly of T. cruzi isolates from Central America. Moreover, NU will lead the computational analysis of all newly sequenced genomes and those already publicly accessible towards the prioritization of the peptides/antigens to be synthesized for their subsequent experimental validation.

Tulane University (TU) will contribute to the sequencing of representative isolates from Mexico and will lead the experimental validation of all selected antigens. It will be responsible for the synthesis of the peptides, their array and evaluation against a collection of samples from chronically T. cruzi-infected and non-infected subjects originating from multiple countries in the Americas. Ultimately, TU will manage the printing of the prioritized antigens onto improved immunochromatography strips for their analytical evaluation on a lateral flow assay mode.

The Barcelona Institute for Global Health (ISGlobal) will have an advisory role on Chagas disease diagnostics. Additionally, it will grant access to its collection of samples from chronically T. cruzi-infected individuals and matched controls.

Others (including references if necessary)