1. IntroductionChagas disease (CD) is a neglected zoonosis with a high impact in Latin America [1]. It is caused by the parasite Trypanosoma cruzi [2], whose transmission is mediated by insects of the subfamily Triatominae (Hemiptera) [1]. Trypanosoma cruzi is subdivided into seven discrete typing units (DTUs): TcI (the most diverse and widely distributed in the Americas) to TcVI, and TcBat (associated with bats) [3]. Estimates indicate six million people infected and 30,000 annual vector-borne cases [4]. The endemic countries for CD have implemented control programs and cooperation between them to make efforts that allow the interruption of T. cruzi vector-borne transmission. However, there are more than a hundred native and non-native vector species in the Americas, some of them that can invade or reinfest. Therefore, it is difficult to reach the complete interruption of T. cruzi transmission by targeting its vectors, but knowing their behavior is vital for CD control [5].The Triatominae are classified into 5 tribes and 15 genera [6], but three are mainly related to transmission to humans: Triatoma (Laporte, 1832), Rhodnius (Stål, 1859) and Panstrongylus (Berg, 1879) [6]. The parasite triatomine feces transmit the parasite, which contains the infective forms of T. cruzi (metacyclic trypomastigotes), when they feed on the mammalian host and defecate later [2]. In Colombia, 26 triatomine species have been reported, but only 16 are considered to be naturally infected with T. cruzi [7,8]. Rhodnius prolixus (Stål, 1859) and Triatoma dimidiata (Latreille, 1811) are the main species and primary vectors transmitting the parasite [7,9]. However, in recent years, some species considered secondary vectors (Panstrongylus geniculatus (Latreille, 1811), Triatoma maculata (Erichson, 1848), Rhodnius pallescens (Barber, 1932), and Triatoma venosa (Stål, 1872)) were found to feed on human blood and be infected with sylvatic DTUs [10,11].In the last years, many triatomine species have become important for T. cruzi transmission due to the elimination of the primary vectors (e.g., R. prolixus) and reports of oral outbreaks of CD in endemic and non-endemic regions [7,10,12,13]. This situation implies some species could reach human dwellings and colonize because these structures can be adequate niches for triatomine species, increasing the risk of CD transmission [14]. Moreover, few studies have recently evaluated the parasitic load in vectors using molecular methods such as quantitative PCR (qPCR) instead of classic microscopy. One of them found 1.29 × 109 parasites in the Chilean vector Mepraia spinolai (Porter, 1934) [15]. The other was made for T. dimidiata in Boyacá, finding loads as large as 1.46 × 109 parasite equivalents [9]. These values indicate the high quantity of parasites that some species could harbor, and that this parameter should be more relevant, considering the importance of the parasite inoculum for disease development [12].Currently, more studies aiming to evaluate the parasitic load of naturally infected vectors are needed, which can be important for vector control programs to help target certain species that can influence the epidemiology of the disease. There is evidence of behavioral changes in the triatomines according to their infection status, such as a reduction in defecation time, improvement in host detection, a higher number of bites, reduction in feeding frequency, locomotor behavior, and fertility, among others [16,17,18,19,20,21,22,23,24]. Thus, there is an imminent risk for humans, and it is essential to track vector species and understand their ecology, behavior, and role in T. cruzi transmission. Hence, considering the relevance of the parasite inoculum for CD development, the vast number of triatomine species present in Colombia, and the need to prioritize some species for building efficient vector control programs. We aimed to describe a methodology to quantify parasite loads of T. cruzi and its ecoepidemiological implications in ten triatomine species collected in the field across 13 departments of Colombia that are endemic to CD. 4. DiscussionVector transmission of T. cruzi is still the most important transmission mechanism causing many infections annually [5]. In the last years, parasite load quantification has been evaluated using standard curves but mainly using human samples allowing the improvement of the detection of the parasite at the clinical level [32,33] using the qPCR assay targeting the tandem repeat satellite DNA of T. cruzi, proven to be an excellent method for parasite detection [34]. This tool could improve the analysis capacity, decision-making, management, and evaluation of the entomological surveillance programs in endemic zones, mainly because it can help to identify risk factors in potential transmission areas, track the vectors’ infection, and target epidemiologically important species [35].We implemented and standardized for the first time a qPCR to quantify the parasite loads of triatomine vectors, running a standard curve using metacyclic trypomastigotes of T. cruzi and colony non-infected specimens of R. prolixus as matrix. For the LOD of the standard curve, we found a value of 3.05 p-eq/mL, interpreted as the smallest number of parasites that the qPCR can detect, with an interval of −7.02 to 13.13 p-eq/mL. This was calculated with a 95% confidence interval, which along with the R2 value of 0.990 and the efficiency of 91.4% (Figure 2) provided us with good reliability, analytical sensitivity, and good qPCR performance for T. cruzi quantification. A similar study in Brazil for field-collected triatomines found high sensitivity of the qPCR with a LOD of 0.41 parasites in the gut [36]. Accordingly, with the methodology implemented, we obtained 150 of 182 positive insects for T. cruzi with parasite loads ranging from hundreds to millions of equivalent parasites per milliliter, with significant differences among certain species that will subsequently be discussed. Our results showed that our qPCR was highly sensitive in evaluating the parasite load, an important parameter to further evaluate the vector capacity of triatomines epidemiologically relevant in CD endemic zones.Regarding the infection and parasite loads, as expected, we found P. geniculatus, R. prolixus, and T. dimidiata were the species with the highest quantities of parasites (Figure 3). This implies Colombia has excelling vectors for Chagas disease, even though some other species ranked between a hundred and a thousand parasites such as R. pallescens, E. mucronatus, T. maculata, T. venosa, Ps. arthuri, and R. colombiensis (Figure 3). Moreover, P. geniculatus, R. pallescens, and R. prolixus were the species collected in more departments, showing their broad distribution in the country (Figure 4 and Figure S2). However, the ranges some triatomine species have is interesting, even though their role as vectors is still unknown, such as Psammolestes arthuri [8], and other species considered secondary vectors such as E. mucronatus and R. pallescens [7,10,37,38,39]. In the case of T. venosa, it presented many parasites in a narrower range. Still, in recent years, it is becoming important as a secondary vector due to its domiciliation along T. dimidiata following the elimination of R. prolixus in Boyacá, Colombia [9,11]. The above suggests that the secondary vectors could have similar parasite loads to those of the primary vectors and play an essential role in the parasite’s transmission cycle. Therefore, along with the evidence of domiciliation processes of some of these species, we highlight the need to include them in the vector control programs to evaluate their vector capacity and more aspects of their biology and ecology.Considering the above, we found there was statistically significant differences among the parasite load values (ppT. cruzi transmission [40,41], and the parasite loads might be associated with the presence of reservoirs such as Didelphis marsupialis and dogs, which have high infection rates and seroprevalence [40,41]. In Colombia’s Caribbean, the main vector species are R. pallescens and T. maculata. The first one is associated with the palm tree Attalea butyracea [41], and the second was found to mediate the peridomestic transmission [40]. Here, for Bolivar and Magdalena, we found R. pallescens and R. prolixus, respectively. However, the ecoepidemiological situation for R. prolixus in Magdalena (specifically Sierra Nevada de Santa Marta) is different because it is found intradomiciliary, with high infection rates and feeding on humans [42]. Currently, in Boyacá, the situation is slightly different because the vector control programs achieved the elimination of the main vector R. prolixus in some municipalities [13], but T. dimidiata is gaining strength as a vector following the elimination of R. prolixus in Boyacá, maintaining the transmission cycle of T. cruzi and promoting the circulation of different genotypes of the parasite into the domestic cycle [9]. In Meta (Orinoco region), the situation for R. prolixus is the similar to Magdalena, except that there is no evidence of the domiciliation process [43]. Therefore, we can hypothesize that the possible differences the test produce for parasite loads might be due to the ecoepidemiological differences among the species and ecotopes per department and feeding sources as well.In terms of the results of the parasite loads per species, we found statistically significant differences among P. geniculatus with Ps. arthuri, R. colombiensis, and R. pallescens (Figure 3). Studies have found that P. geniculatus presents one of the highest rates of T. cruzi infection; a wide range of blood meals including humans; and also the presence of many DTUs (TcI-TcV), which is evidence of domiciliation processes due to attraction by artificial light [10]. This species has a long-term defecation time post-feeding (almost an hour after), but what matters is the quantity of parasites they could eject considering their infection rates, as we evidenced here. This characteristic might be important for oral transmission because it has been observed they return to their shelter after feeding and then defecate, probably on food, but no studies have been conducted to determine this [44]. We also found DTU TcI, except from one sample of P. geniculatus, which was identified as TcII (Table S2).Rhodnius pallescens is considered a secondary vector that has a wide variety of blood sources, along with other important species such as P. geniculatus and R. prolixus, and also includes high infection rates (Figure S1). This species is the main vector in Panamá, whose populations are associated with Attalea butyracea palms but present sporadic active dispersion towards rural dwellings [45]. Moreover, it has been found with a T. cruzi infection rate of 72% in individuals collected inside the houses [45]. In Colombia, R. pallescens presents infection rates of 45%, and similar to Panamá, it is a sylvatic species with risk of intrusion in domestic ecotope and it has been involved in oral transmission cases [10,46]. Here, we report a wide range of parasite loads for this species, as far as 9 million parasites (Figure 3 and Figure 4), which can be important for oral outbreaks, as have been hypothesized previously [12,46]. Rhodnius colombiensis is another sylvatic triatomine associated with palms (Attalea butyracea) that gained epidemiological importance due to its high infection rates with T. cruzi and their frequency in invading human dwellings [47]. The parasite loads we found range between 100 and 5000 parasites, which can be important considering its high capacity to transmit T. cruzi, specifically TcI sylvatic (TcId) [47]. This DTU is the most frequent found in isolations from oral outbreaks in Colombia [48,49], implying there is an association among the sylvatic transmission cycle that probably can be attributed to secondary vectors [12]. On the other hand, Psammolestes arthuri is a sylvatic triatomine that feed on birds and is frequently found in bird nests [50]. It was recently found to be naturally infected with T. cruzi with an infection rate of 70%, with mixed infection with many DTUs and feeding on humans with a frequency of 26%, which implies that although its behavior remains ornithophilic, it may be feeding on other animals potentially infected with the parasite and therefore contributing to the maintenance of the parasite’s cycle [8].The differences observed above might be related to the geographical region, but better sampling is needed to reach a conclusion. The specimens of P. geniculatus were collected in some departments with a domestic ecoepidemiological situation (Boyacá, Santander), in contrast to Ps. arthuri collected in Casanare, R. colombiensis in Tolima, and R. pallescens in the northside (Bolivar, Antioquia, Cesar) (Figure 4). T. dimidiata could be in a similar situation with the same three sylvatic species mentioned previously. An additional and specific characteristic in this case is that T. dimidiata was the second species with the highest and wider range of parasite load behind P. geniculatus, even surpassing the main vector R. prolixus (Figure 3). This might indicate that the behavior of species with a domestic ecoepidemiological situation is somehow making them become infected with higher loads of parasites, which increase the potential risk of Chagas for humans. This is relevant considering that the interruption of vector-borne transmission can be better in areas where vectors present domiciliated or peri-domiciliated behavior, such as what happened, for instance, in vector control programs for T. infestans in Brazil, Chile, Uruguay, Paraguay, and Argentina. In Brazil, the programs have been working since 1975, and by 1993, they were able to report a reduction of infestation by T. infestans of 86% [51]. After this stage, most Southern Cone countries entered a phase of surveillance that consisted of monitoring infestation and focal spraying, but nowadays, the challenge is the sustainability of these programs and to deal with the control failures reported in Bolivia and Argentina as a result of triatomines’ resistance to insecticides components [51]. The Southern Cone model was implemented and adapted for the Andean countries and Central America initiatives, where the programs are underway [51].For the other species evaluated, T. maculata in Colombia is considered as secondary, with high percentage of infection of 67% just behind R. prolixus and P. geniculatus, feeding mainly on human blood [10]. We found a parasitic load slightly higher than that from T. venosa, R. colombiensis, and Ps. arthuri, but unfortunately, we were only able to analyze two specimens. In Venezuela, it was found that human dwellings could constitute an adequate niche for this species to survive, suggesting a potential domiciliation process [14]. Moreover, along with the results obtained previously, this species’ behavior suggests the existence of new transmission scenarios that together with their presence in the peridomestic ecotope underline the need to evaluate the potential risk this species could have [10]. A study conducted in Brazil evidenced the potential of this species as a vector by evaluating its infestation capacity, short developmental cycle, and defecation time [52]. Once again, this implies the need to start the surveillance of this species once considered sylvatic [7].Triatoma dispar is a sylvatic triatomine found in Antioquia, Cauca, Chocó, Nariño, Valle del Cauca, and Huila [7]. Here, we analyzed two specimens from Chocó that were infected with parasite loads of 55,000 and 7 million parasites, respectively. In Ecuador, it has been found in the peridomicile, which probably could represent an epidemiological risk for T. cruzi transmission according to its ecological characteristics [53]. In Colombia, there is no information regarding its ecological behavior, and therefore more studies are needed to unravel the possible importance this species could have in the transmission cycle of the parasite and if it could represent a risk for humans in the future. The results of parasite load of this species were the narrowest and one of the lowest we found. Even though the quantities of parasites we found are not conclusive, it is important to keep studying their importance for T. cruzi transmission. 5. ConclusionsWe highlight that the parasitic loads and DTUs found in species considered secondary must begin to be spotlighted as they might be gaining importance in terms of the parasite’s transmission, mainly due to anthropogenic processes such as deforestation and urbanization that disturb the triatomines ecology and activities that promote the active dispersal of specimens into or near the dwellings [7,10]. Moreover, some of them could represent an imminent risk for oral transmission, as they could reach the dwellings and contaminate food or beverages. Similarly, as happened with R. colombiensis, the vectorial capacity for secondary and sylvatic species should be evaluated to determine if this species has a reliable epidemiological relevance for Chagas disease transmission and if there is a correlation between parasite load and vector capacity. Therefore, after R. prolixus, the vector control programs in Colombia must focus now on T. dimidiata and T. venosa and begin the surveillance of other species such as T. maculata, T. dispar, and R. pallescens, considering their parasite load, infection rates, and the importance they have as a vector in other countries of the region such as Venezuela, Panamá, and Brazil.We had some limitations in our study. For instance, (i) we had an unequal number of samples per species to analyze due to DNA availability (volume) that could have affected the standard deviations in the used statistics, including the lack of sampling effort; (ii) limited geographic representability (only 13 of 31 departments) and sample bias; (iii) we only used one marker for genotyping; and (iv) of the 182 specimens evaluated, only 4 were nymphs. For the standard curve, we only used R. prolixus, but we propose our standardized qPCR to evaluate other species and not only adults but more developmental stages of the individuals as well. However, for the DNA extraction, we suggest using only the rectal ampulla and not the whole abdomen section as we did, considering it is where the metacyclic trypomastigotes are present and that the triatomine gut could contain some components that might affect the qPCR reaction [54,55,56]. Additionally, because other infective forms of the parasite might be present in the rectal ampulla, a new approximation based on gene expression specifically for metacyclic trypomastigotes could be implemented. For future studies, we suggest improving the genotyping of T. cruzi using more genes and evaluating the presence of other Trypanosoma species, which might explain the lack of amplification of some samples in our study. Moreover, even though we provide a close-up of the parasite transmission, we did not evaluate if there was an effective metacyclogenesis, and therefore further studies must include gene markers solely expressed in metacyclic trypomastigotes as an approximation and then evaluate them in the insects’ guts. Lastly, future studies should then estimate the vector capacity and the impact of parasite loads on the transmission.

Our results confirm the suggestions for secondary species and their upcoming relevance for T. cruzi transmission in endemic regions for Chagas disease in Colombia. Moreover, we reiterate the need for serological studies to obtain a true picture of the risk the humans are exposed to, as well as vector-specific studies to decipher their potential as T. cruzi vectors. To conclude, our parasite load qPCR standardization had great sensitivity, efficiency, and can be useful in future studies directed to contribute with this important issue exposed here. Moreover, we hope this standardized technique can be applied across the country and the region. Additionally, there is still a lack of information about secondary vectors and their ecoepidemiology, and thus we encourage the scientific community to continue deciphering and studying this species with wider sampling and real-time data. Furthermore, we observed that in certain departments where few studies have been conducted, some species could have a huge role for the parasite’s transmission, as is the case for T. dispar in Chocó, where the only two individuals we analyzed presented high parasite loads. We also propose not to only focus on the main species such as R. prolixus (considering its potential to reinfest and/or recolonize) and T. dimidiata (which is gaining importance after R. prolixus elimination). We hope the data in the future will be obtained using the methodology exposed here and that they will significantly improve the entomological surveillance and vector control programs, as they could provide more information regarding vector infection and the risk factors that it entails for disease development, especially in CD-endemic areas. The above with the aim to continue understanding the vectors’ behavior and ecology that could lead to a decrease in the CD cases in the Americas, as well as making the vector control programs successful and sustainable.