Acknowledgment This report was written by PATH and supported in whole or part by a grant from the Bill & Melinda Gates Foundation. The views expressed herein are solely those of the authors and do not necessarily reflect the views of the Gates Foundation.

Suggested citation PATH. Diagnostic Gaps and Recommendations for Chagas: Assessment of User Needs, Use Cases, and the Diagnostic Landscape: Seattle: PATH; 2016.

Contact information Tala de los Santos Program Leader, Diagnostics PATH Email: [email protected]

About PATH PATH is the leader in global health innovation. An international nonprofit organization, we save lives and improve health, especially among women and children. We accelerate innovation across five platforms— vaccines, drugs, diagnostics, devices, and system and service innovations—that harness our entrepreneurial insight, scientific and public health expertise, and passion for health equity. By mobilizing partners around the world, we take innovation to scale, working alongside countries primarily in Africa and Asia to tackle their greatest health needs. Together, we deliver measurable results that disrupt the cycle of poor health.

For more information on PATH’s work in diagnostic technologies, visit http://sites.path.org/dx/.

Copyright © 2016, PATH. All rights reserved. The material in this document may be freely used for educational or noncommercial purposes, provided that the material is accompanied by an acknowledgment.

Cover photo: PATH/Mike Wang

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Contents

Acronyms ...... 4 Executive summary ...... 5 Introduction ...... 6 Diagnostic landscape ...... 8 Conclusions ...... 17 References ...... 19

Acronyms

DALY disability-adjusted life years DNA deoxyribonucleic acid ELISA enzyme-linked immunosorbent assay IB immunoblot IF immunofluorescence IFAT indirect immunofluorescent antibody test IHA indirect hemagglutination assay kDNA kinetoplast DNA LAMP loop-mediated isothermal amplification MSF Médecins sans Frontières NTD neglected tropical disease PCR polymerase chain reaction PPV positive predictive value rDNA recombinant DNA RDT rapid diagnostic test RIPA radioimmunoprecipitation assay TESA trypomastigote excretion–secretion antigens satDNA satellite DNA SAPA shed acute phase antigens SOP standard operating procedures WB western blot WHO World Health Organization

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Executive summary

Chagas disease is a neglected tropical disease caused by the protozoan Trypanosoma cruzi and spread through the triatomine bug. The World Health Organization estimates that nearly six million people are infected with T. cruzi in Latin American and seventy million people are at risk of infection, which can cause cardiac and intestinal complications. If untreated, infection is lifelong and can be life threatening. Morbidity and mortality from Chagas disease is a critical health problem affecting poor communities throughout Latin America.

The WHO has put forward goals for the interruption of transmission in the home (Latin America) and through blood transfusions (Latin America, Europe, and Western Pacific) by 2015, as well as the elimination of Chagas-carrying insects near homes in Latin America by 2020. The London Declaration on NTDs backed these goals with commitments from public and private institutions. However, according to the London Declaration 3rd Report, more concerted efforts to improve access to diagnosis and treatment are needed to achieve these goals.

PATH aims to catalyze engagement of the diagnostics industry and product development efforts in support of the London Declaration goals. As part of this work, PATH examined the Chagas diagnostics landscape to identify gaps in availability, accessibility, and use of appropriate diagnostic tools for Chagas. We conducted literature reviews and interviews with key stakeholders to identify use cases for Chagas diagnostics, understand current practices, and assess progress toward more robust diagnostics. The findings were used to analyze the current diagnostic landscape for Chagas and highlight current needs to improve access to diagnostics for prioritized use cases.

PATH identified four use cases for Chagas diagnostics: case diagnosis, congenital case detection, treatment monitoring, and screening of blood and organ donations. Our analysis found that access to and adoption of current tools remain a barrier to their effective use in Chagas disease control efforts in many endemic settings. This may be due in part to insufficient evidence to support their implementation. Additionally, because of the reliance on serologic tests and inadequate markers of active infection, current tools available to detect congenital infections and monitor treatment are not yet sufficient to fully support needs in these use cases. Based on these findings, we offer the following recommendations:

1. Establish more standardized policies and practices for Chagas diagnosis and treatment by generating evidence around the efficacy of current tools in diverse settings and building consensus around standards among global and regional stakeholders. 2. Develop a point-of-care diagnostic tool to detect congenital infections, which would enable early initiation of treatment and a clear linkage to postnatal care. 3. Develop a diagnostic tool to monitor treatment efficacy, which would inform clinical decision-making during treatment and support the development of better drugs.

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Introduction

Chagas disease, a neglected tropical disease (NTD) found primarily in Latin America, is responsible for a higher burden of morbidity and mortality than any other parasitic disease in the western hemisphere, including malaria.1 A disease caused by the protozoan Trypanosoma (T.) cruzi, Chagas is transmitted primarily though the feces of insects called triatomine bugs. Notable clinical symptoms are rare during the acute phase. However, if untreated, Chagas disease can enter a chronic phase during which symptoms may develop 10 to 30 years after the initial infection. The chronic phase can include cardiac and digestive complications, which lead to the morbidity and mortality associated with Chagas disease.

The prevalence of Chagas disease is estimated to be nearly six million people worldwide, primarily in Latin America, where over 70 million people are at risk (see Figure 1).2,3 The World Health Organization (WHO) estimates that there are about 50,000 new infections annually. Bolivia reports the highest estimated incidence at 4 percent per year.1 Each year, 10,000 deaths are attributed to Chagas disease.4 The epidemiology of Chagas disease is driven both by transmission in endemic areas and by large-scale migration of infected people from endemic rural villages to Latin American cities and countries outside of Latin America.5,6 Migration of infected people outside of endemic areas to countries that lack experience with and infrastructure for detection and treatment means that Chagas disease is becoming a global rather than a regional health problem.7

Figure 1. Global distribution of cases of Chagas disease. Data reported by WHO, Control of Neglected Tropical Diseases, 2006–2010.8

Estimates of disability-adjusted life years (DALYs) vary and there may be insufficient data to understand the overall burden of Chagas disease. Estimates of DALYs attributable to Chagas disease range from

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546,000 to over 800,000;4,9 however, these are likely to be underestimates as recognition of Chagas- attributable morbidity and mortality is challenged by the lag between the acute phase of infection and symptomatic disease.10 Accurate prevalence estimates are also difficult to achieve due to the disease’s spatial heterogeneity, quickly evolving temporal trends, limited data on disease progression, and absence of data outside of endemic countries.10

The 2012 WHO Neglected Tropical Disease Roadmap describes two goals for Chagas disease:11

1. By 2015: transmission within the home via Chagas-carrying insects in Latin America, and through blood transfusions in Latin America, Europe, and the Western Pacific, will have been interrupted. 2. By 2020: infestations of Chagas-carrying insects in areas surrounding homes will have been eliminated in Latin America.

Shortly after the release of the NTD Roadmap, 20 public and private institutions that support global health and international development—including pharmaceutical companies, donors, governments from endemic countries, nonprofit organizations, and others—joined the efforts to reach the 2020 goals for 10 of the 17 diseases, in a document known as the London Declaration on Neglected Tropical Diseases.12 The London Declaration represents a commitment from these institutions to sustain, expand, and extend programs that ensure the necessary supply of drugs and other interventions to achieve the NTD Roadmap goals for Chagas disease by 2020. The London Declaration 3rd Report noted that more concerted efforts to improve diagnosis and treatment are needed to achieve the NTD Roadmap goals.13

In response to this need, PATH conducted a diagnostic landscape analysis to identify gaps in the availability, accessibility, and use of appropriate diagnostic tools to support the control of Chagas disease in endemic settings. This analysis was informed by a review of literature and interviews with stakeholders in the Chagas community. The literature review included peer-reviewed publications, WHO policies and guidelines, country-level case studies, and a review of the product development landscape. Country-level policies were reviewed and compared to understand the diverse range of clinical diagnostic and treatment guidelines. The product development landscape was informed by diagnostic evaluations, materials from manufacturers, and input from key stakeholders in the Chagas community.

Stakeholders were identified through their roles in global and country-level Chagas management programs, academic research, participation in Chagas consultative meetings, and through referral from other stakeholders. A discussion guide was developed for stakeholder interviews that focused on several themes including disease progression and treatment, diagnostic use cases and user needs, and current barriers to achieving control of Chagas disease. Information from the literature review, product development landscape, and stakeholder interviews was compiled and used to:

 Identify use cases and understand current diagnostic practices associated with each use case.  Analyze progress toward robust diagnostics across serology, parasitology, and molecular diagnostic platforms.  Highlight priority use cases for product development and other interventions.

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Diagnostic landscape Disease course and transmission Chagas disease is caused by the protozoan T. cruzi. Six discrete typing units of T. cruzi have been identified and are geographically distributed within endemic areas (see Figure 2).14,15 The heterogeneity of the parasite is believed to contribute to the varying clinical manifestations of Chagas disease and the geographical differences in morbidity and mortality.16

Figure 2. Geographical distribution of Trypanosoma cruzi discreet typing units in human patients. Reported in WHO technical report on research priorities for Chagas disease, human African trypanosomiasis and leishmaniasis.17

Following T. cruzi exposure, the incubation period is one to two weeks.18 The acute infection stage lasts for 8 to 12 weeks and is characterized by nonspecific symptoms that include fever, malaise, sweating, muscular pains, irritation, anorexia, and sometimes vomiting and diarrhea.2,19 The majority of cases are not diagnosed and treated during the acute phase of infection.1 In the absence of treatment, the disease progresses into the chronic phase wherein parasites remain in the heart and digestive muscles but parasitemia may be intermittent and at levels not readily detectable by diagnostic tools including microscopy and polymerase chain reaction (PCR).

Heart disease is the most frequent and significant clinical presentation of chronic Chagas disease.2 Around one-third (30 to 40 percent) of those chronically infected will develop cardiac or digestive complications.20 These complications include myocarditis, arrhythmias, congestive heart failure, and gastrointestinal pathology such as megacolon and megaesophagus.2 The disease can progress to sudden death or heart failure caused by progressive destruction of the heart muscle.21 The 60 to 70 percent of

8 chronically infected people who never develop clinical signs remain seropositive for T. cruzi antibodies and are described as in the chronic indeterminate phase (see Figure 3).

Figure 3. Progression of Chagas disease.

Transmission in endemic areas is primarily vector-borne. T. cruzi infection is usually acquired in childhood and infection persists for life.22 Transmission can occur also occur vertically via congenital infection, through blood transfusions and organ transplants, and through oral routes via contaminated food and drink. Congenital infection represents 26 percent of new Chagas disease cases.23 Rates of congenital transmission vary. Congenital transmission has been observed in many as 10 percent of babies born to chronically infected mothers in Paraguay and as few as 1 to 2 percent of babies in other endemic countries.2,24

Diagnosis The clinical diagnosis of suspected Chagas disease is based on mild and nonspecific symptoms during the acute phase. Confirmatory laboratory diagnosis is primarily based on serologic testing. Secondary testing of parasite confirmation is challenging. Parasitemia is generally present during the acute phase but these cases often go undetected to due to mild symptoms.25 The chronic phase is characterized by a low parasite load. Diagnosis during the initial acute phase or early chronic phase provides a critical but time-limited opportunity to detect and successfully treat patients with anti-parasitic medicines. Early diagnosis can also prevent the potentially high expenditures associated with under diagnosis or misdiagnosis, which lead to a high morbidity burden, consequent mortality, and possible co-infections and co-morbidities during the chronic phase.

Treatment Benznidazole and nifurtimox the two drugs currently available to treat Chagas disease. Treatment is effective in only 60 percent of cases and the efficacy of treatment declines as the infection persists.26 Treatment is successful only in 10 to 20 percent of chronic cases.26 In addition to limited efficacy, there is substantial risk of serious side effects including digestive disturbances such as epigastric and abdominal pain, nausea and vomiting, anorexia, hematological disorders, and weight loss.27,28 Stakeholders report a high rate of patient noncompliance with the drug regimen due to its long duration (6 to 9 months) and side effects. Potentially toxic and complex due to the length of the regimen, the treatment is better tolerated and more effective in children, with reports of over 70 percent efficacy and over 90 percent efficacy in treating congenital infections if the treatment is given within the first year of life.26

In 2007, Bayer agreed to donate nifurtimox; the treatment is distributed through the WHO-Bayer Nifurtimox Donation Program.29 However, according to interviewed stakeholders, these drugs are not widely available in endemic countries, and a waiver is needed to access the drugs in the United States as it is not currently approved by the US Food and Drug Administration (FDA). The efficacy of current drugs

9 for Chagas treatment and the development of new drugs is problematic as it is difficult to accurately test whether a cure has been achieved.30 This is acts as a barrier to both patient case management and the development of new drug regimens.

Historically, due to limited treatment efficacy, individuals with chronic Chagas disease were not treated but monitored for the development of symptoms and complications. More recently, some practitioners and researchers are advocating for more widespread treatment of chronic cases as stakeholders assert that this can slow the progression of more severe complications such as cardiomyopathy. Described by stakeholders as a “paradigm shift” in Chagas treatment options, the expanded willingness to treat and potentially re-treat chronically infected cases will require a clear understanding of treatment outcomes.

Use cases This analysis identified four use cases for Chagas diagnostics: case diagnosis, congenital case detection, treatment monitoring (including test of cure and drug development), and the screening of blood and organ donations.

Figure 4. Use cases for Chagas disease diagnostics.

Congenital case Treatment Screening of blood Case diagnosis detection monitoring and organ donations

Case diagnosis Active surveillance for Chagas case diagnosis is limited outside of more research-oriented activities and targeted health interventions in hyper endemic areas.31 Currently, case diagnosis is most commonly achieved through passive surveillance and affected individuals are identified when they seek care at health facilities. Depending on the location and capacity of the health center, diagnostic testing may be performed by the health care providers either with or without laboratory support. Results are used to inform the initiation of treatment, considerations for antenatal care, and monitoring the progression of cardiac and digestive complications.

Detectable levels of parasitemia are most common during the acute phase. Thus, infection detection methods such as microscopy or PCR can more readily detect the presence of parasites in a blood sample taken during this phase. However, diagnosis during the acute phase is uncommon as there is often a lack of clinical signs and symptoms specific to acute Chagas infection. Additionally, the relatively mild and nonspecific clinical signs associated with acute infection may not prompt care-seeking and even for those that do enter the health system, confirmatory testing may not be considered as the diagnostic methods required are expensive and complex.

Thus, identification of the vast majority of cases occurs during the chronic stage of infection. At this stage, serologic tests are commonly used for diagnosis. There are currently multiple serological tests

10 commercially available including tests intended for use at the point-for-care such as rapid diagnostic tests (RDTs), some of which are reported to have good performance (94 to 99.5 percent sensitivity and 94 to 96 percent specificity).32,33 Earlier diagnosis during the chronic phase of Chagas can improve treatment outcomes and may limit the progression of associated cardiac and gastrointestinal complications.

As not all chronic cases of Chagas go on to have severe clinical manifestations, clinicians are particularly interested in the potential of a disease prognostic. This prognostic would be used to understand who might progress to the chronic symptomatic phase and prioritize treatment accordingly. However, at this time there is little evidence to support the feasibility of this type of prognostic including the availability of validated biomarkers.34

Congenital case detection Congenital case detection involves the screening of neonates born to seropositive mothers. As treatment of newborns during this period is highly effective at resolving the infection, congenital case detection is becoming more common in many national diagnosis and treatment policies.35 Despite the relatively high rate of facility births in some endemic countries, use of lab-based diagnostic methods remains limited. This is due to issues with access to more complex testing in remote areas as well as timing: laboratories may not be open at the time of delivery when the mother and child are still on-site and available for diagnosis and follow-up.

Ideally, diagnostic methods for congenital case detection would provide timely results and be broadly accessible to a wide spectrum of target end users such as nurses, midwives, or other health workers involved with antenatal and postnatal care. Unfortunately, low-complexity serologic detection methods, including the commercially available RDTs, are of limited use early after birth due to the persistence of maternal antibodies. Thus direct detection of parasite infection is often required to detect congenital cases and initiate treatment until an infant is at least 10 months of age and the maternal antibodies have disappeared. At the time of birth, direct parasite detection using cord blood (either by microscopy or PCR) has demonstrated good performance, but this method is often limited to use in more well-resourced health care settings. Thus, the complexity of current methods available for use after birth invites significant loss to follow-up and misses an opportunity to initiate treatment at a time when it is highly effective.

Treatment monitoring Treatment monitoring, including test of cure and tests to support drug development, represent overlapping use cases with potentially distinct end users and contexts. As antibodies remain for years and direct detection of parasites may not be feasible, the efficacy of treatment is not well understood. Currently, research tools, including sensitive PCR methods and multiplex immunoassays against Chagas antigens, are being evaluated for application in monitoring treatment.36 However, there is little consensus as to how to define cure or treatment success.

This has several important implications. For one, it may be more difficult to motivate people to undergo the complex and toxic treatment when the outcomes will remain unknown. Clinical decision-making is also complicated by the lack of a clear diagnostic indicator for response to treatment. This in turn further limits health care providers’ willingness to re-treat or treat with an alternative drug since treatment failure

11 is difficult to accurately ascertain using available diagnostic methods. Finally, clinical trials to support drug development needed to product better treatment and treatment regimens may be hindered without reliable and reproducible ways to monitor treatment efficacy.37

Blood and organ donation screening Globally, screening of blood and organ donation is the most widespread use of Chagas diagnostics.38 Strategies to protect the blood and organ donation supply vary between endemic and non-endemic settings. In endemic settings, regulations require that all blood and organ donations are screened for the presence of T. cruzi antibodies while in non-endemic areas, screening policies are based on considerations of risk and cost-effectiveness .39 40 In both endemic and non-endemic settings, blood screening is generally conducted within central laboratory facilities often using automated lab-based methods that allow for high-throughput testing.41

Current diagnostic tools There are several current tools as well as tools in development to support the use cases outlined above. See Table 1 for an overview of the diagnostic landscape.

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Table 1. Overview of the diagnostic landscape for Chagas disease.

Serology Parasitology Molecular

Biomarker Host antibody to T. cruzi antigens Whole parasite DNA Formats RDT, enzyme-linked immunosorbent assay (ELISA), indirect Microscopy, PCR, isothermal amplification chemistries immunofluorescent antibody test (IFAT), indirect xenodiagnoses, blood hemagglutination assay (IHA), Immunofluorescence (IF), culture Western Blot (WB), Radioimmunoprecipitation Assay (RIPA); Immunoblot (IB) Diagnostic Exposure Infection Infection measurement Stage of product Commercialized Research and development Research and development development Example • OnSite Chagas Ab Rapid test (CTK Biotech) • T. cruzi OligoC-Test (Coris Bioconcept) products • WL Check Chagas (Wiener Lab) • Loop-mediated isothermal amplification • Chagas Instantes (Silanes) (LAMP) assay (FIND/Eiken) • TrypanosomaDetect Rapid Test (InBios, Inc.) • Chagas Quick Test (Cypress Diagnostic) • Chagas Stat-Pak assay (Chembio) • Immu-Sure Chagas (T. cruzi) (Millennium Biotech) • SD Chagas Ab Rapid (Standard Diagnostics) • Simple Chagas WB (Operon) • Serodia Chagas (Fujirebio) • ImmunoComb II Chagas Ab (Orgenics) Description • Detects host antibodies against parasite antigens • Directly detects the • Detects parasite nucleic acid in blood or • Antibodies usually detectable within a few days of presence of parasite in biopsy infection and can persist for life patient’s blood or biopsy • PCR and LAMP based assays developed Biomarkers include: T. cruzi trypomastigote excretion– • Provides a definitive Biomarkers include: T. cruzi minicircle secretion antigens (TESA); shed acute phase antigens diagnosis of infection or kinetoplast DNA (kDNA) and the 195-bp (SAPA); recombinant antigens representing cytoplasmic and treatment failure satellite DNA (satDNA), 18S recombinant flagellar proteins (TcF, FP, etc.) • Sensitivity can be DNA (rDNA), 24S rDNA improved by repeated sampling and/or parasite concentration

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Serology Parasitology Molecular

Proposed target • Congenital case detection (late) • Congenital case detection • Congenital case detection (early) use • Case diagnosis (early) • Treatment monitoring/test for cure • Screening blood/organ products • Treatment • Drug development monitoring/test for cure • Xenomonitoring • Drug development Advantages • Approved for use by WHO • Good sensitivity during • Direct detection of infection • Field-deployable formats acute phase of infection • Potentially easier use and higher • Good performance has been demonstrated in recent • Provides definite throughput than traditional parasitology multi-center evaluations (sensitivity >95%; 94%–99% diagnosis of current • Ease of test interpretation reported depending on kit) infection • Ability to perform additional • Historical and current use characterization/typing • Low-cost formats available • Research methods available for further characterization and typing Limitations • Current WHO recommendations require use of multiple • Limited sensitivity • Limited sensitivity serologic tests particularly in the chronic • No international standard operating • No recognized gold standard phase procedure (SOP) • Limited utility in post-treatment monitoring (low positive • Multiple tests needed to • Limited commercial availability predictive value due to long-lived antibodies) confirm negatives (low • Not WHO-approved • Limited utility in congenital infection (until 6 to 9 months negative predictive value) • All available methods are complex, time- of age) • All available methods are consuming, and labor-intensive • Possible strain-specific performance variation complex, time- • Multiple tests needed to confirm • Antibodies long-lasting (limited positive predictive value consuming, and labor- negatives (PPV) in post-treatment monitoring) intensive

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Serology Specific antibodies to T. cruzi are usually detectable within a few days of infection and can persist for life. Commonly used serological tests include the rapid immunochromatographic lateral flow tests (RDTs), indirect immunofluorescent antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA), and indirect hemagglutination assay (IHA). Most of these tests use lysates of the epimastigote form of the parasite as target antigens, but several recent products use recombinant forms of parasite antigens. Evaluation of the performance of serologic test products, including multiple RDTs, indicate relatively good performance with sensitivity ranging from 96.5 to 100 percent and specificity from 87 to 98.9 percent.32,42 However, specificity of some products, particularly those using whole parasite lysates, may be affected by cross-reactivity with host antibody responses to other related pathogens, particularly the causative agent of visceral and cutaneous leishmaniasis, which may co-exist in the same geographic regions as Chagas disease.43

Additionally, the reduced reactivity of different tests against geographically distributed strains of T. cruzi has also been expressed as a concern both from a policy perspective and in the choice of what products are preferred for use within a specific endemic country. WHO recommendations state that: “No single serological assay has sufficiently high sensitivity and specificity to be relied on alone and therefore, serum specimens should be tested by at least two assays based on different antigens or principles.”44 Stakeholders also report a tension between the accuracy of serological tests and their ease of use versus more accurate tests that require lab support.

One concern is that different recombinant proteins and target antigens are used across available serologic assays. In an effort to help address this concern and provide further evidence to advocate use of a single test for serologic diagnosis, Médecins Sans Frontières (MSF) coordinated a multi-center evaluation of 11 commercially available rapid serologic diagnostic tests (RDTs).32 The evaluation included 10 national reference labs in Argentina, Brazil, Colombia, Costa Rica, Mexico, the United States, France, Spain, and Japan. Results of the study indicated that 8 of the RDTs evaluated had similar high performance when tested on samples from different endemic regions. Though the performance of these tests is lower than reported by their manufacturers, the authors concluded that the results of this evaluation provide further evidence of the potential to use a single RDT to diagnose Chagas disease in endemic countries. Plans to further validate these findings are currently underway including field evaluation studies.32

Parasitology While diagnostic methods based on the direct detection of the parasite may perform relatively well if used during the acute phase of infection, the performance and utility of these methods become very limited during the chronic phase. Parasitemia rapidly decreases after the acute phase, even in the absence of treatment, and is often intermittent during the chronic phase. This limits the sensitivity of microscopy- based methods, particularly if only a single sampling is conducted that may miss time-points where more parasites are present within the blood. The complexity of parasitology-based methods further complicates the accuracy and reproducibility of these methods as the proficiency of the person performing testing may impact the result.

Pre-processing methods have been developed to try to improve sensitivity of microscopy-based methods. Xenodiagnosis, which involves amplification of the infection through transmission of the parasites from

15 the patient to the triatomine vector where it then is allowed to further proliferate, may increase sensitivity over microscopy alone. However, xenodiagnosis often lacks adequate sensitivity during the chronic phase, and the complexity of performing xenodiagnosis as well as its acceptability among patients severely restricts its use.

Other parasite concentration methods have been developed to try and improve performance. The microhematocrit method is the most widely used technique in Latin American health facilities. In this method, fresh blood sealed in four to six heparinized microhematocrit tubes is centrifuged, followed by light microscopic examination for parasites within the recovered buffy coat layer. However, this and other concentration methods add an additional level of complexity and cost to parasitology-based testing and yet still may not provide adequate sensitivity, particularly if testing is performed on a single blood specimen.44

Molecular methods There is growing interest in the application of sensitive molecular methods for amplification of parasite nucleic acid, such as PCR, for use in Chagas diagnosis. Currently, use of these methods remains limited to research and referral-level diagnostic testing for informing routine clinical care, due in part to the cost and complexity associated with performing these tests. Moreover, the vast majority of available methods are laboratory-developed assays as opposed to internationally accepted standard protocols or off-the-shelf commercial products. Several stakeholders confirmed that there are a significant number of PCR-based assays developed by different labs with variable performance in use within different countries. Additionally, similar to traditional parasitological methodology, the performance of molecular methods can also be limited by low and intermittent parasitemia within peripheral blood sampling. Thus, to confirm a negative result, serial sampling may be required to ensure an infection is not missed.

Improved diagnosis of congenital infection is one of the more promising near-term applications for molecular diagnostics in Chagas, particularly if lower-complexity molecular tests become available for use in antenatal care across lower tiers of the health system in endemic countries. Molecular methods may also have utility for monitoring treatment efficacy, as evidenced by their continued use during clinical trials for drug development, and may provide utility in monitoring for parasitemia following reactivation of disease during immunosuppression. However, the lack of a universal standard method is of particular concern in testing for a cure and in clinical trials for the development of new drugs, as results obtained using the variety of different assay procedures may not be directly comparable.

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Conclusions

Given current diagnostic tools and practices, gaps are evident in case diagnosis, congenital case detection, and treatment monitoring. There may be sufficient diagnostic tools available for case diagnosis but challenges with regard to standardized policies and practices for diagnosis and treatment exist, including consensus around a gold standard serological method to evaluate other tests. New diagnostic tools are needed to fill gaps in congenital case detection and treatment monitoring (see Table 2). PATH proposes the following recommendations to the Chagas research community, as summarized in Figure 5.

Figure 5. Proposed diagnostic recommendations to the Chagas research community.

2) Develop a diagnostic tool to detect congenital 1) Establish infections Access and standardized Product 3) Develop a guidelines for the adoption development diagnostic tool to use of current tests monitor treatment efficacy

1. Establish more standardized policies and practices for Chagas diagnosis and treatment by generating evidence around efficacy of current tools in diverse settings and building consensus around these standards among global and regional stakeholders. Based on substantial agreement among stakeholders and supported by the MSF evaluation, currently available RDTs are likely to provide sufficient performance to be used for routine case detection.32 Other barriers to access and adoption may need to be addressed to ensure their uptake and integration into health systems. Lack of consensus on guidelines, including recommended platforms and products, and lack of access were cited as key factors to explain the limited use of these RDTs. The lack of consensus may be attributed to differing views on the geographical variation in the performance of these RDTs, variation within national programs and policies, and the lack of a clear global standard for diagnosing Chagas disease. Underpinning some of this disagreement may be perspectives on the importance of different T. cruzi strains. Access issues may stem from the lack of consensus around test performance and regulatory and price barriers to the widespread distribution of these tests. Further exploration into these access and adoption barriers is warranted.

2. Develop a point-of-care diagnostic tool to detect congenital infections, which would enable early initiation of treatment and a clear linkage to postnatal care. New tools for congenital infection are needed, and the potential impact of these tools is compelling. Congenital infections represent a significant portion of new Chagas infections, and detection and treatment at birth offers the greatest chance of treatment success. Thus better tools could ensure that effective treatment decisions are made during early care of the infant. Due to the presence of maternal

17 antibodies, a test of active infection is needed. Stakeholders specifically requested a point-of-care test that would enable immediate intervention and prevent loss to follow-up. This test could be integrated into postnatal care settings and other routine newborn care policies and practices.

3. Develop a diagnostic tool to monitor treatment efficacy, which would inform clinical decision- making during treatment and support the development of better drugs. New tools are also needed for treatment monitoring. Current point-of-care and lab-based tools used to monitor treatment or test of cure are problematic, and there remains a lack of standard methods to define when the infection is cured. The need for new drugs with better efficacy and less toxicity is particularly clear but the development of new drugs requires a reliable test of cure. Currently, drug trials are using different PCR protocols that may produce results that are not comparable. Given the shift toward more routine treatment of chronic cases, tools that inform clinical decision-making during treatment will be critical. Providers would be able to choose to re-treat or treat with a different drug based on initial treatment outcomes. The development of new, less toxic drugs with a shorter regimen could prompt better treatment compliance. The overlapping nature of these use cases means a new tool or method could transform diagnosis and improve clinical decision-making, drug development efforts, and treatment policies.

Table 2. Summary of proposed product development recommendations for Chagas diagnostics.

Use case Congenital case detection Treatment monitoring Specimen Blood Blood Marker Active infection; antigen, molecular Active infection or exposure; antigen, molecular, or antibody Context of use Primary care and referral-level facilities Primary care and referral-level facilities Value A point-of-care tool that overcomes current A diagnostic tool not reliant on intermittent proposition barriers to congenital case detection would parasites or antibodies that could be used enable the immediate diagnosis of neonates to monitor treatment efficacy in individuals when they are still linked to the health and inform clinical management decisions. system and facilitate treatment of infected This tool could also support drug infants at birth when drugs are highly development efforts effective and less toxic

Current diagnostic tools and practices are unlikely to achieve the goals for the control of Chagas disease set forth by the WHO NTD Roadmap and the London Declaration. Existing diagnostics fail to fill critical use cases and are limited by lack of access and uptake. To achieve global goals for the control of Chagas disease, new tests and consensus around more standardized policies for the diagnosis and treatment of Chagas disease are still needed.

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