Chagas disease is a dangerous tropical illness caused by single-cell parasites known as Trypanosoma cruzi. In most cases, if not treated immediately, the infection becomes chronic: the immune system of the host greatly reduces the number of parasites present in the body yet fails to fully eradicate them. Current diagnostic approaches often fail to detect these low numbers of parasites. More broadly, without a reliable way to measure Trypanosoma cruzi levels, researchers and clinicians struggle to test new treatments as well as determine whether patients carrying more parasites tend to develop more severe disease.

In response, White et al. aimed to develop a new way of measuring parasitic loads. They based their approach on standard PCR (polymerase chain reaction), an experimental method that amplifies specific DNA sequences in proportion to their initial levels in a sample. This allows researchers to not only pinpoint the presence of the parasites, but also to assess their relative number However, the approach has limited sensitivity: if the amount of target DNA initially collected is too low, it may not be detected.

To bypass this limitation, White et al. adopted a ‘deep-sampling PCR’ approach and made several crucial changes. First, they collected several samples from the same patient, therefore increasing the overall sampling volume. Second, they fragmented the sample DNA before the amplification step in order to disperse the target DNA, thus increasing the chances of its detection in each PCR reaction. Third, they performed as many as 400 PCRs per sample. These modifications greatly improved sensitivity compared to usual approaches.

White et al. used their newly improved method to examine Trypanosomas cruzi levels in infected macaques over long periods. The results show that parasitic burden can be stable over a year, but vastly differs between individuals (with some having a million times more parasites than others). Similar findings were also observed in humans and dogs.

The method developed by White et al. is likely to be too labour intensive to be routinely used in diagnostic laboratories; however, it represents an important and immediate advance for researchers testing new compounds to treat Chagas disease.

Chagas disease, the result of infection with the protozoan Trypanosoma cruzi, is endemic to the Americas, where it is among the highest impact infectious diseases, and is also a major source of infection-related heart disease globally. Chagas disease is a result of the long-term persistence of T. cruzi primarily in muscle tissues, despite highly effective immune responses that generally control but fail to completely clear the infection in the majority of individuals. Although T. cruzi continuously alternates between replicating forms inside host cells and non-replicating forms in extracellular spaces, including the bloodstream where it can be acquired by blood-feeding triatomine insects, detection of parasites or parasite products in the blood is generally undependable, using even the most sensitive methods. Consequently, diagnosis of infection generally rests mainly on serological tests, which are often not fully reliable.

Positive serological tests reflect prior exposure but not necessarily active infection. Thus, determining the effectiveness of current treatments to clear the infection or whether some subjects have spontaneously resolved the infection (apparently rare, but anecdotally reported) and thus should not be treated, remains out of reach. The inability to routinely and sensitively detect active infection coupled with the undependable curative capabilities of current therapeutics and their substantial side effects, accounts for the estimates that less than 1% of T. cruzi-infected individuals receive anti-parasitic treatment (Cucunubá et al., 2017). Furthermore, the lack of sensitive methods to definitively establish cure hinders the identification and validation of improved therapies.

Amplification techniques such as PCR can specifically enrich very low quantities of pathogen DNA and have been extensively used to enhance the detection of T. cruzi DNA in the blood of infected hosts. Multiple, high copy number (>10⁵ copies per organism) targets for amplification have been identified and rigorous, highly specific amplification protocols have been developed and evaluated (Schijman et al., 2011). Nevertheless, it is generally agreed that these protocols, as currently employed, frequently fail to detect T. cruzi in infected hosts and thus are not reliable tests of the absence of infection. Multiple studies have attempted to address the sensitivity and specificity of the T. cruzi PCR methodology, for example, varying the target (there are two primary ones used, a kDNA and a genomic satellite sequence), primer and probe sequences, DNA storage and purification techniques (automated or not), and volume of blood drawn for DNA isolation (1–10 ml) (Sulleiro et al., 2019; Silgado et al., 2020). In general, none of these variations significantly alter test outcomes. What does alter the ability of PCR to detect T. cruzi infection is the number of independent blood collections done and the number of PCR determinations conducted for each sample (Parrado et al., 2019).

These results highlight that detection of T. cruzi DNA in infected hosts is frequently a problem of sampling. Specifically, when pathogen numbers are low, collection of a high number of test samples that are extensively subsampled may be required in order to have an opportunity to detect a pathogen. For T. cruzi, this sampling problem is strongly supported by the remarkable study done in the 1970s by Cerisola in Argentina (Cerisola, 1977) in which 30 untreated subjects submitted to approximately monthly xenodiagnosis with 80 triatomines each month for >2 years. This study showed that some individuals had multiple bugs positive at every sample point (thus, presumably a high parasite load) while others were more variable (strongly positive some months but not others), and a few were only very occasionally positive (in the most extreme case, as few as 2 positive bugs out of >1000 fed over 24 months).

In the current study, we have used humans, non-human primates (NHPs), and dogs, all with naturally acquired T. cruzi infections, to demonstrate that serial sampling of blood and exhaustive PCR of optimally prepared DNA from that blood, can confirm even the most difficult to detect infections with T. cruzi. The ability to obtain relative quantification of parasite load in these naturally infected hosts reveals the wide range of parasite load among infected subjects in a population (>6 log₁₀) and the conditions under which parasite control can change, both slowly in the early years of infection and dramatically when overall health changes. Deep-sampling PCR, while laborious, offers the first true test of cure for Chagas disease and should also assist in the determination of associations between parasite load, immune response parameters, and the risk for disease development in chronically infected hosts.

Without question, the inability to detect and quantify T. cruzi in blood or other samples from individuals with suspected infection, is the key impediment to identifying candidates for treatment, determining if the treatment has been effective in resolving the infection, and validating potential new treatments. Additionally, without the ability to routinely quantify parasite load in humans and other naturally infected hosts, there are multiple, sometimes contentious issues in Chagas disease that will remain unresolved, among these, the relative role of parasite and host genetics in establishing the parasite burden in this persistent infection, the stability of parasite load over time, and particularly important, the relationship between parasite burden and the presence and severity of clinical disease.

Quantifying T. cruzi in blood and other tissues in hosts after the early peak of infection is challenging because the immune response to T. cruzi infection is normally extremely effective in tightly limiting parasite numbers within a few months of the initial infection. The bulk of T. cruzi in vertebrate hosts at any point in time are intracellular, mostly in various muscle types throughout the body. However, to maintain and to transmit the infection, T. cruzi must also exit host cells (following a 4- to 5-day period of parasite multiplication) and infect new host cells or on occasion, be ingested by blood-feeding triatomines. In addition to intact parasites, the blood is likely to contain DNA released by parasites that are killed by immune effector mechanisms and thus may be available for detection, either free in plasma or bound to blood cells (Lam et al., 2021; Thompson et al., 2024). In short, even in hosts with very low-level/well-controlled infections, the potential always exists for T. cruzi DNA to be present in the blood, albeit often at extremely low amounts. And when present in such low quantities, the chances of detection, even with highly optimized PCR protocols, is random and highly prone to sampling error. This is not a situation unique to T. cruzi infection. For example, detection of SARS-CoV-2 (Waked et al., 2020) and HIV (Rutsaert et al., 2018) using PCR is also prone to failure when employing single amplification reactions. The ability of replicate PCR reactions to overcome this limitation has been discussed in depth in PCR optimization reviews (Taylor et al., 2019) and one study (Stowers et al., 2010) reported that by averaging hundreds of replicate reactions one can vastly extend the concentration range over which PCR can provide reliable detection of DNA that is in low abundance. However, the use of hundreds of replicate PCR reactions as conducted here has been rarely, if ever, used to routinely validate the presence of any infectious agent in blood.

It is well documented that T. cruzi-positive hosts give variable results in PCR tests, and this variability increases with decreasing parasites or parasite DNA in a sample. This is primarily due to subsampling error (Taylor et al., 2019) and this error functions at two levels when screening blood for the presence of pathogen DNA. First, a blood sample is a subsample of the entire blood volume of a potential host; e.g. a 10-ml blood sample is ~0.2% of the total volume of blood in an average adult human. The existence of this subsampling error in T. cruzi infection is well illustrated by the fact that two replicate blood samples from macaques collected using the same needle stick can yield a substantial number of positive PCR reactions in one sample, and zero positive reactions in the other (Figure 5). Simply collecting samples at different times, whether a few days (Figure 5) or a few months (Figure 3) apart, does not necessarily solve this problem. When parasite numbers are very low, the chance that a small subsample of the total blood will contain parasite DNA is random irrespective of when it is collected. The only solution is to obtain more or larger blood samples so as to increase the chances of collecting a fragment of the parasite DNA that is in the circulation. This same subsampling error acting at the level of whole parasites was evident in the xenodiagnosis studies of Cerisola, wherein it was rare to obtain parasite-positive bugs when they fed on certain subjects who apparently had very low levels of parasites circulating in the blood (Cerisola, 1977).

A second opportunity for subsampling error under low target conditions occurs when an aliquot of the total blood DNA obtained in a blood draw is used for PCR amplification. In the case of the ~5-ml macaque blood samples used in the bulk of the assays in this study, from 1/100th to 1/250th of the total recovered DNA was used for each replicate PCR reaction. In a number of animals, the entire blood DNA sample was exhausted doing replication PCR reactions without a positive reaction while in most animals, some DNA remained after nearly 200, sometimes all negative, amplification reactions/sample. We show that fragmentation of DNA significantly decreases the subsampling error at this level, presumably by breaking the DNA containing one or more target satellite DNA regions into smaller fragments that are then more widely dispersed in the total blood DNA. DNA fragmentation led to an increased frequency of positive replicate PCR reactions and generally more consistent Cq values between these aliquots. Fragmentation increased amplification consistency and the sensitivity of the PCR protocol by one order of magnitude and should be incorporated as a standard step for amplification of satellite DNA target in T. cruzi even when deep-sampling is not used. However ultimately, the limiting factor in the consistent detection of T. cruzi in blood is the total amount of blood collected and processed for fragmentation and amplification. If there is no parasite DNA in a particular sample, no amount of fragmentation or number of amplification reactions will detect it. Also, in addition to sampling errors, the inefficiency of PCR to amplify very low abundance targets, particularly among a complex DNA mixture as exists in blood (termed the Monte Carlo effect; Karrer et al., 1995; Bustin and Nolan, 2004) may also contribute to the failure to detect target T. cruzi DNA in some samples despite its presence.

In addition to providing much greater sensitivity for detecting active infection relative to conventional single or low replicate (2–3/sample) PCR analysis, conducting 100s of replicate PCR reactions on fragmented blood DNA also provides information on the relative abundance of parasites in the blood of infected hosts. Most implementations of PCR for T. cruzi have a limit of consistent detection of ~10^–3 PE/assay, which equates to ~0.5 parasites/ml of blood in our assays. Combining sample DNA fragmentation and deep-sampling extends the range of quantification to at least 10^–6 PE per aliquot, or ~0.00025 parasites/ml (1 parasite per 4 l) of blood.

This considerably increased sensitivity of infection detection and relative quantitation made possible through the use of deep sampling PCR reveals the previously undocumented range of parasite burden between individuals with chronic T. cruzi infections. Although best demonstrated in the frequently sampled macaques in this work, both humans and dogs also exhibit a range of parasite burdens exceeding five orders of magnitude when compared to a standard curve (e.g. from Cq values of ~25 in the highest macaque and dog fragmented samples to <1% frequency of positive PCR reactions in some members of all three species). The similarity in the ranges of parasite load in the three species examined in this study again reinforces the similarities between T. cruzi infection in these species and the appropriateness of dogs and NHPs as models of the human infection (Tarleton et al., 2024).

In addition to the sampling error discussed above, the variation in the number of satellite DNA repeats per genome among T. cruzi isolates (Schijman et al., 2011) makes absolute quantitation of parasite load virtually impossible. However, such quantitation is not necessary in order to conclude that individuals with long-established T. cruzi infections control these infections to vastly different degrees. This ability to more precisely quantify parasite load should now allow investigations into the potential immune mechanisms that might be responsible for this variable control and assessment of the potential correlation between relatively stable parasite load and the chances of having or developing clinical disease – which is detected in <50% of long-term infected subjects.

This study also confirms across a broader range of individuals, the relative stability of parasite load over time, an expected phenomenon that was also apparent in the xenodiagnosis studies by Cerisola, 1977. A surprising finding was that a subset of macaques infected for 2–3 years and presenting a relatively higher parasite burden exhibited a significant decline in parasite load over the 1-year survey period. This result suggests that immune control mechanisms not only continue to confine parasite load but can in some cases also drive that load lower over time during the chronic phase of infection. However, this is not the pattern in all individuals, as some macaques with equally short-term infections have already restricted parasite numbers to nearly undetectable levels while some with longer-term infections maintain relatively higher parasite burden. It is also noteworthy that parasite control can be quickly lost when the health status (and presumably immune status) changes, as in the case of macaque P4, similar to what is observed in humans with suppressed immunity (Lattes and Lasala, 2014). These findings emphasize that even subjects controlling T. cruzi burden to very low levels should be considered for anti-parasite treatment as the immune control of the infection can be quickly lost.

The original goal of this study was to develop a ‘test of cure’ for use in the evaluation of clinical trials of anti-T. cruzi compounds. Previous candidate compounds used in clinical trials have failed to provide sterile cure and these failures were relatively easy to detect without a highly sensitive PCR protocol as described here (Tarleton, 2023a; Tarleton, 2023b). With the progression of highly promising new compounds, including one that provided 100% sterile cure in NHPs with naturally acquired T. cruzi infection (Padilla et al., 2022), an assay that detects success rather than only failure is needed. The combination of DNA fragmentation and deep-sampling of replicate blood samples should fulfill that need. Operationally, this would not require any special equipment but does necessitate extreme care in sample preparation and assay execution. Also, conducting hundreds of PCR reactions on the DNA from 10 or more high volume (e.g. 10 ml or greater) blood samples from each subject will be expensive, time-consuming and labor-intensive. It is also worthy of note that in cases of treatment failure, the apparent abundance of T. cruzi DNA in the blood often return to pre-treatment levels (Parrado et al., 2019), so selection of subjects with more readily detectable pre-treatment parasite loads for such trials would make it relatively easy to detect treatment failures and provide greater confidence that those that remain negative following deep-sampling are indeed cured.

There may also be opportunities for additional improvements in PCR-based detection of T. cruzi. One of the limitations of this study is that we have so far only examined samples from a small number of human subjects. However, those samples fall into the same pattern of wide-ranging parasite loads as the macaque and dog samples so there is little reason to think that more extensive testing of human samples will reveal any surprises. Additional targets for PCR amplification have been identified (Kann et al., 2020; Kann et al., 2023) and could be multiplexed in a single reaction to perhaps achieve greater sensitivity. We also may not have exhausted the limits of DNA fragmentation for dispersing target DNA in a blood sample in order to further reduce subsampling errors or explored the tolerance for increased loading of fragmented DNA for each PCR reaction. Improvements in any of these areas could reduce the number of replicate PCR reactions without compromising sensitivity. Finally, new technologies such as UltraPCR, which allows for higher DNA loading, extensive multiplexing, and the generation of >30 million individual PCR reaction per tube (Lai et al., 2023; Shum et al., 2022) could greatly reduce the number of replicate PCR assays that are needed for high sensitivity detection. Such developments could make the PCR-based detection of all T. cruzi-infected subjects possible, if not routine.

All data generated or analyzed during this study are included in the primary or supplemental data files provided in the manuscript. Tabulated raw data are provided in source data files.

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Author details

1. Brooke E White

   Center for Tropical and Emerging Global Disease, Athens, United States

   Contribution

   Investigation, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

2. Carolyn L Hodo

   Michale E. Keeling Center for Comparative Medicine and Research, The University of Texas MD Anderson Cancer Center, Bastrop, United States

   Contribution

   Resources, Investigation

   Competing interests

   No competing interests declared

3. Sarah Hamer

   Department of Veterinary Integrative Biosciences, School of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, United States

   Contribution

   Resources

   Competing interests

   No competing interests declared

4. Ashley B Saunders

   Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, United States

   Contribution

   Resources

   Competing interests

   No competing interests declared

5. Susana A Laucella

   Research Department, Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben", Buenos Aires, Argentina. Chagas Disease Unit, Hospital Interzonal General de Agudos Eva Perón, Buenos Aires, Argentina

   Contribution

   Resources

   Competing interests

   No competing interests declared

6. Daniel B Hall

   Department of Statistics, University of Georgia, Athens, United States

   Contribution

   Formal analysis

   Competing interests

   No competing interests declared

7. Rick L Tarleton

   1. Center for Tropical and Emerging Global Disease, Athens, United States
   2. Department of Cellular Biology, University of Georgia, Athens, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing

   For correspondence

   tarleton@uga.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9589-5243

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