Abstract Chagas disease has a major impact on human health in Latin America and is becoming of global concern due to international migrations. Trypanosoma cruzi, the etiological agent of the disease, is one of the rare human parasites transmitted by the feces of its vector, as it is unable to reach the salivary gland of the insect. This stercorarian transmission is notoriously poorly understood, despite its crucial role in the ecology and evolution of the pathogen and the disease. The objective of this study was to quantify the probability of T. cruzi vectorial transmission to humans, and to use such an estimate to predict human prevalence from entomological data. We developed several models of T. cruzi transmission to estimate the probability of transmission from vector to host. Using datasets from the literature, we estimated the probability of transmission per contact with an infected triatomine to be 5.8×10−4 (95%CI: [2.6 ; 11.0]×10−4). This estimate was consistent across triatomine species, robust to variations in other parameters, and corresponded to 900–4,000 contacts per case. Our models subsequently allowed predicting human prevalence from vector abundance and infection rate in 7/10 independent datasets covering various triatomine species and epidemiological situations. This low probability of T. cruzi transmission reflected well the complex and unlikely mechanism of transmission via insect feces, and allowed predicting human prevalence from basic entomological data. Although a proof of principle study would now be valuable to validate our models' predictive ability in an even broader range of entomological and ecological settings, our quantitative estimate could allow switching the evaluation of disease risk and vector control program from purely entomological indexes to parasitological measures, as commonly done for other major vector borne diseases. This might lead to different quantitative perspectives as these indexes are well known not to be proportional one to another.

Author Summary Chagas disease is a parasitic disease affecting about 10 million people, often living in poor conditions, and the disease contributes to impede their development. As several other infectious diseases (malaria, dengue or sleeping sickness), it is transmitted by blood-feeding insect vectors. While most other human pathogens are directly injected with the vector's saliva, Chagas disease parasite is transmitted through the insect's feces that are deposited on the skin during bloodmeals, which seems to be a very inefficient process. The probability of such transmission to human has thus been very hard to estimate, although it is crucial to predict where people are at risk and design effective control strategies. Using mathematical models integrating data on vectors and humans collected across Latin America, we estimated that for several vector species transmission occurs in 1 over 900–4000 contacts with infected insects. We further showed that our estimate allows prediction of human infection rates in various ecological conditions. These models will provide health policy makers with improved indexes to better prioritize/evaluate of the outcomes of vector control programs.

Citation: Nouvellet P, Dumonteil E, Gourbière S (2013) The Improbable Transmission of Trypanosoma cruzi to Human: The Missing Link in the Dynamics and Control of Chagas Disease. PLoS Negl Trop Dis 7(11): e2505. https://doi.org/10.1371/journal.pntd.0002505 Editor: Richard Reithinger, RTI International, United States of America Received: November 20, 2012; Accepted: September 16, 2013; Published: November 7, 2013 Copyright: © 2013 Nouvellet et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This investigation received financial support from the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases (TDR)/International Development Research Center (IDRC), Project #A90276 and the European Union EU-FP7-PEOPLE-Intra-European Fellowship for career development (IEF) grant no. 253483. For part of this study, PN received funding from the European Union Seventh Framework Programme [FP7/2007–2013] under Grant Agreement no. 278433-PREDEMICS and thanks the MRC Centre for Outbreak Analysis and Modelling for support. This work was performed within the framework of the LABEX ECOFECT (ANR-11-LABX-0048) of Université de Lyon, within the program “Investissements d'Avenir” (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Vector-borne diseases represent one of the biggest challenges to current and future human wellbeing. They have severe impacts on many tropical and subtropical countries, where they are responsible for ∼10% of human deaths and contribute to impoverishment by imposing an annual burden of >50 millions of DALYs [1]. They also are an emerging threat for more developed countries as climate change and increasing international exchanges modify the geographic distributions of vectors and pathogens [2]. Vectorial transmission is traditionally thought to critically depend on the incubation period and the survival rate of the pathogen in the vector, and on the frequency of vector feeding on humans. This is well reflected in classical measures of transmission such as the vectorial capacity, entomological inoculation rate or the basic reproductive number, which are central to empirical and theoretical literature on the ecology, evolution and control of vector-borne diseases [3], [4]. Also appearing in these standard measures is the parasite transmission efficacy from infected vectors to hosts, whose effects on vector-borne diseases has received less attention, and has frequently been assumed to be systematic, in particular for malaria transmission [5]. However, efficacy of vector transmission may become a key parameter when it takes on unusually low values, as even small variations could then have major effects on disease dynamics and the resulting prevalence in hosts [6]. The vast majority of causal agents of human vector-borne diseases, such as Plasmodium, Leishmania, dengue and other flaviviruses, are ‘salivarian’ pathogens. After entering the vector during a blood meal, the pathogens multiply inside the gut or haemolymph before spreading to the salivary glands to be directly injected to a human or a reservoir host during a subsequent blood meal. The probability of transmission from vector to a given host species is a complex process that depends on the size of the inoculate and on the within-host dynamics following inoculation. For some salivarian pathogens, the number of pathogens injected at the biting site can be measured [7], as well as the subsequent dynamics of the host-pathogen interactions [8]. Quantitative assessments of the overall resulting probability of transmission based on experimental infections gave values of 0.5 to ∼1 per bite for Plasmodium spp. and dengue virus, 0.3–0.6 for African trypanosome, 0.2–0.4 for Leishmania spp., and as low as 0.01–0.04 for the Japanese encephalitis virus [6], [9]. There also are pathogens for which this probability of transmission can be much lower as they are unable to reach the salivary glands of the vector. The so-called ‘stercorarian’ transmission, sometimes considered as the ancestor of salivarian transmission [10], occurs through the contact of vector's feces and the biting wound (or a mucosa). Successful transmission requires an extraordinary combination of somewhat unlikely events. An infected vector has to defecate sufficiently close to the biting site whilst or shortly after feeding, the infected feces must be brought to the bite wound by the host by scratching, and the pathogen then has to cross the skin of the host to initiate infection [11]. Trypanosoma cruzi is one of the rare parasites that has managed to establish an endemic human infection through this transmission route, and despite its presumably low probability of transmission from vector to human, it has become a major public health problem. It is indeed the etiological agent of American trypanosomiasis, also called Chagas disease, a widely distributed neglected tropical disease in Latin America, with an estimated 8–9 million infected persons and another 25–90 million at risk of infection [12]. Although maternal and oral transmissions have been documented [13], vectorial transmission remains the main cause of human infection. This protozoan kinetoplastid parasite is transmitted by a large diversity of hematophagous bugs of the Reduviidae family to multiple species of sylvatic and domestic mammalian hosts, and at least 20 species of triatomines are involved in transmission to humans [14]. Nonetheless, parasite transmission through these highly diverse vector and host communities remains poorly understood, mostly because the probability of stercorarian transmission can hardly be estimated from experimental infection due to the complexity and rareness of the process. Estimates thus rely on indirect approaches based on a combination of entomological and epidemiological studies at fine temporal and spatial scales. Given the difficulty to collect such integrative datasets, there are currently only three estimates of the probability of stercorarian transmission of T. cruzi to its hosts. This probability was found to be per contact with an infected vector for transmission to human [11], to guinea pigs, a typical domestic host in many Latin American regions [15], and to opossums, the likely ancestral mammalian host of T. cruzi [16]. Although these point estimates seem rather consistently low, their usefulness remains limited as there is no information on their confidence intervals, and no sensitivity analysis to uncertainties in the entomological and epidemiological raw data has been performed. A significant benefit of gaining a robust estimate of the probability of stercorarian transmission would be to establish a clear link between basic entomological data and the prevalence of T. cruzi infection in humans. Potentially, this could allow evaluating disease risk and vector control program in terms of parasitological rather than entomological indexes, as commonly done for other human vector borne diseases such as malaria [4] or dengue [17]. In this contribution we thus aimed at (i) providing a robust quantitative estimate of the probability of stercorarian transmission of T. cruzi to humans based on information available in the literature, and at (ii) determining if this estimate allows predicting the prevalence of the infection from basic entomological data.

Discussion The lack of a quantitative estimate of the probability of T. cruzi transmission to human through the feces of the vector has hindered the development of approaches that integrate ecological and epidemiological information on Chagas disease. These approaches have had an impressive influence in mitigating several vector-borne diseases including malaria [47], dengue [48] or leishmaniasis [49], and would help better understand the complex features of the transmission of T. cruzi and compare it with other vector-borne diseases. Based on data from the literature we built here epidemiological models to derive 6 estimates of this probability of transmission, all being of the order of 10−4–10−3 per contact. This primarily illustrates the paradox of Chagas disease; despite the ‘milli-transmission’ of the parasite from vectors to humans, the disease affects millions of people across the Americas. The quantitative knowledge of its transmission probability also opens new perspectives for the study of the disease, with key implications for both parasite evolution and public health policy. 1. The ‘milli-transmission’ of Trypanosoma cruzi to humans: Possible causes The probability of transmission of T. cruzi from triatomine vectors to humans was found to be very small, per contact with infected vector (95% CI ), relatively consistent across the different study systems, with point estimates ranging from to , and in agreement with the only other point estimate available in the literature [11]. This narrow range of probabilities was observed in spite of marked differences in vector density, vector species (taxonomic, ecologic and behavioural differences), prevalence of infection in humans and vectors, resulting in very different epidemiological situations. Such a broad consistency was confirmed by our sensitivity analyses, which further supported that estimates are robust to changes in the entomological and epidemiological raw data used for their calculation. These estimates of parasite transmission to human are similar to what has been calculated for guinea pigs [15], but differ substantially with the probability of stercorarian transmission to opossums that was estimated to be 10–100 times larger [17]. This suggests a reduced adaptation of T. cruzi to domestic hosts compared to its likely ancestral and sylvatic host, which is consistent with the much shorter period of coevolution between T. cruzi and humans. Indeed, estimates suggest around ∼10000 years of coevolution of T. cruzi with humans, compared to ∼80 millions years with the opossum [50]. Such a low probability of transmission does not mean that humans are of secondary importance or even ‘dead ends’ in term of parasite transmission, as suggested by the ∼40% prevalence of infection in humans observed in 9000-years old mummies [51] as well as in today's highly endemic areas [45]. In fact, the potential amplification and dilution effects [52] that human and other domestic hosts could have on the populations of T. cruzi still remain to be properly quantified (but see [53]). A low probability of vectorial transmission of T. cruzi was expected, given the succession of unlikely events required to occur and the many parameters involved. However, the narrow range of probabilities was more surprising given that all of these parameters could potentially affect parasite transmission quite dramatically. This suggests that these parameters combine in an independent way to produce an almost universal efficacy of transmission of T. cruzi from vectors to humans. While more accurate data may allow refining our estimate of the probability of transmission of T. cruzi to human, potentially detecting species specificity, the residual variations in the probability of transmission are expected to have little impact on the prevalence of infection in humans. Indeed, we were able to predict rather accurately the prevalence in humans from infected vector density, the frequency of human blood meal, and a unique probability of vectorial transmission. Triatomine vectorial capacity is thus primarily dependent upon vector density and feeding frequencies on specific hosts, a conclusion which is consistent with the key influence of those parameters on the spread and persistence of the disease [6]. As vector demography has been documented for a variety of triatomine species and entomological context [21], [53], [54], [55], the emerging eco-epidemiology of Chagas disease would benefit from a substantial improvement of our knowledge on vector feeding ecology. The emergence of methods based on the use of metagenomics [56] and stable isotopes, which potentially allow identifying vector trophic networks [57], should shortly allow tackling the transmission of T. cruzi in the context of host communities, as it has already been done successfully for the transmission of plague [58]. The very low probabilities of transmission of T. cruzi from vector to vertebrate hosts raises an obvious evolutionary question: why has T. cruzi not evolved from a stercorarian to a salivarian mode of transmission while closely related species such as T. rangeli [59] and T. brucei [60] or Leishmania [61] have been able to do so? A first line of explanation could lie in a lower ‘evolvability’ of T. cruzi [62]. However, there is no evidence that T. cruzi has a lower mutation rate compared to other kinetoplastids [63], and T. cruzi does experience reproduction and recombination [64] as demonstrated for other related taxa [65], [66], [67]. A lower ‘evolvability’ could also result from specific features in T. cruzi genotypes to phenotypes mapping function, which may be evaluated by mutagenesis and artificial selection experiments [62]. A second line of explanations that could explain T. cruzi ineffective mode of transmission are the costs associated with the migration of the parasite across the midgut, the escape of the immune response in the haemocoel, and the invasion of the salivary glands, which all may exert selective pressure to restrict the parasite to the gut. Those costs have not been identified yet, although molecular studies are progressively unravelling the interaction between T. cruzi and its vector [14], [68], and insights could be gained by comparative analysis of vector-parasite interactions of the various kinetoplastids [14]. Comparisons with T. rangeli, a closely related and sympatric parasite that shares hosts and vectors with T. cruzi [69], should be especially informative as it is able to colonize the haemocoel and reach the salivary glands of its vector [59]. Finally, the selective pressure on T. cruzi may be too weak given its potential for direct transmission which is known to be of evolutionary and epidemiological importance in opossum [16], [70] as well as in human either because of oral or maternal transmission [13]. Our quantitative assessment of the probability of transmission of T. cruzi offers new opportunities to tackle these key eco-evolutionary questions, as it allows quantifying standard epidemiological measures such as R 0 or related quantities [3], [4], [71] which have been consistently missing in the epidemiology of Chagas disease [6], [ but see 72], while they are central tenets of the study of the epidemiological dynamics of malaria [4], dengue [17] and others human and livestock vector-borne diseases [73]. 2. Shifting the assessment of Chagas disease risk and control programs from entomological to parasitological indexes Our study demonstrates that the quantitative estimates of the probability of T. cruzi transmission from vector to humans allow expressing infection risk in terms of human incidence or prevalence, rather than in terms of purely entomological indexes such as vector presence/absence [74] or abundance [75]. Importantly, the entomological data used to make those predictions are basic estimates of vector abundance and infection rates that can inferred from typical entomological collections achieved by trained personnel or even low-cost studies based on community participation [76]. Although a proof of principle study is necessary to validate the proposed models' predictive ability in an even broader range of ecological settings, this approach offers a much more affordable way than large-scale serological surveys to estimate human prevalence over geographic areas and obtain better descriptions of the global and local burden of the disease. Such prevalence/incidence data would be more straightforward and explicit to interpret at all levels of public health systems for the design of epidemiological surveillance and vector control operations. In addition, the risk expressed in incidence or prevalence would likely differ from that expressed in vector presence/absence or abundance because, according to the catalytic model [18], the relationship between these variables is non-linear and follows a cumulative exponential distribution. At low vector densities the risk of human transmission increases rapidly with vector abundance, and is thus likely to be underestimated by the sole measure of vector abundance, while at large vector densities, human incidence and prevalence reach a plateau, so that variations in vector abundance have little or no influence on the already high transmission to human. Our model can also profoundly help the assessment of the efficacy of vector control interventions, which is traditionally measured in terms of reduction in vector abundance or vector presence (infestation index) [53], [77], [78]. Typically, current guidelines for vector control in several endemic countries aim at reducing triatomine presence below the somewhat arbitrary level of 5% of the houses of a community [79], based on the assumption that this may be sufficient to dramatically reduce or even interrupt parasite transmission to humans. The modelling developed here opens the possibility to convert a reduction of vector abundance into a variation in the actual level of parasite transmission to humans, allowing to rapidly define more rational target/threshold levels of infestation for vector control. Again, one expects that for high vector densities, very large reduction in vector populations will be needed to reduce human prevalence, while at low vector abundance, small reductions in vectors could result in significant decrease in human prevalence. Nonetheless, even small residual populations of bugs due to incomplete treatment [80], development of insecticide resistance [81] or infestation by non-domiciliated vectors [21], [55], [82], [83], [84], [85] will be sufficient to maintain an active transmission of T. cruzi to humans, which clearly appeals for the use of highly sensitive tools for entomological surveillance following the ‘action’ stage of control program [86]. In conclusion, we provided estimates of the probability of T. cruzi transmission from vector to human, which were shown to be highly consistent across vector species and epidemiological conditions. Such a new quantitative knowledge could allow expanding purely entomological indexes, which are typically calculated for triatomines, into parasitological measures (such as R 0 , the so-called force of infection and resulting incidence and prevalence), as routinely done for other human vector-borne diseases such as malaria or dengue. This offers the possibility to develop a better understanding of the ecology and evolution of one of the rare stercorarian human parasites. This also is of primary interest in public health, as parasitological measures provide a more straightforward evaluation of the disease risk and a better description of the outcomes of vector control program in terms of human infection rather than vector abundance. Studies specifically designed to validate our models' predictive ability in an even broader range of entomological and ecological settings would now be worth performing to strengthen the proposed approach, and to allow for its use in a large scale operational/policy setting.

Author Contributions Conceived and designed the experiments: PN ED SG. Performed the experiments: PN ED SG. Analyzed the data: PN ED SG. Contributed reagents/materials/analysis tools: PN ED SG. Wrote the paper: PN ED SG.