Background
Malaria is caused by the protozoan parasite
Plasmodium and is transmitted by female
Anopheles mosquitoes. Although significant advances have been made towards its elimination in several previously endemic countries, malaria remains a significant public health concern [
1]. The World Malaria Report in 2018 estimated that the global burden of malaria comprised around 219 million reported cases and 435,000 deaths worldwide [
2]. Specifically, in Colombia, there was a decrease in the estimated number of malaria cases by more than 20% between 2016 and 2017 [
2]. Despite this, malaria remains one of the foremost public health concerns in some states in Colombia such as Nariño, which is located along the Pacific coast of the country. In 2017, 26% of malaria cases in Colombia came from Nariño where, unlike other regions,
Plasmodium falciparum is the most common species (96.3%) [
3].
More than 47
Anopheles species in five subgenera have been reported in Colombia [
4]. The majority of primary malaria vectors in Colombia belong to the subgenus
Nyssorhynchus, with
Anopheles (
Nys.)
nuneztovari, Anopheles (
Nys.)
albimanus and
Anopheles (
Nys.)
darlingi as the most important malaria vectors in areas of high malaria transmission [
5]. On the South Pacific coast, several species has been associated with malaria transmission with
An. albimanus is the main vector [
6,
7]. Previous studies reported that the
An. albimanus lineage circulating the Southern region may be different from the one found the in the Northern part of the country suggesting that two different lineages are circulating in the country [
7‐
9]. Interestingly, malaria prevalence in these sites is significantly different and further studies evaluating vector competence and susceptibility to both,
Plasmodium vivax and
P. falciparum [
7] as well as to measure potential changes in salivary content that could impact pathogen transmission [
10] are necessary.
Extensive entomological research has been done in the Nariño Department [
7,
11,
12]. This research suggests that mosquitoes from the subgenus
Anopheles,
Anopheles (
An.)
calderoni and
Anopheles (
An.)
punctimacula are also important malaria vectors in the area. However, these two species are often misclassified due to their high morphological similarities [
11]. However,
An. calderoni was found infected with both
P. vivax and
P. falciparum with an annual entomological inoculation rate (EIR) of 2.84 bites/human/year in Nariño between 2012 and 2013 [
11]. Also, a previous study reported EIR for
An. calderoni between 1.7 and 14.7 from 2009 to 2010, while EIR reported for
An. albimanus during the same period was found between 0.1 and 2.6 [
12]. Suggesting that
An. calderoni is a primary vector of malaria in Nariño. Furthermore, in the Tumaco city, located in the Narino Department), Ahumada et al., reported different malaria incidence in places where
An. albimanus and
An. calderoni were found in the 2011–2012 study. Specifically, they reported a high Annual Parasite Index (API) (73 cases/1000 inhabitant) in places where
An. calderoi is the predominant species compared to lower (27 cases/1000) where
An. albimanus was predominant [
7].
To design a proper vector control method, it is necessary to accurately determine human-vector interaction and the proportion of those vectors that are infected. Vectorial capacity (VC) and EIR are quantitative entomological indicators used to determine epidemiology of vector-borne diseases such as malaria. The VC is used as the measure of a mosquito population’s proficiency to transmit an infectious agent to a susceptible population [
13], while EIRs are useful to establish a direct estimation of transmission risk [
14,
15]. In the case of malaria, the EIR is the gold standard for measuring transmission intensity. EIRs are based on the number of mosquitoes captured and the proportion of mosquitoes infected with
Plasmodium [
16]. However, estimation of EIR is expensive and may be insufficient in areas of low or seasonal transmission [
17,
18]. Human Landing Collection (HLC) is currently the only mosquito catching method that can directly measure the biting rates of human-seeking mosquitoes. Unfortunately, it is only applicable to mosquitoes seeking human adults and results are difficult to extrapolate to children or to pregnant women that are the most vulnerable to malaria [
19]. Furthermore, during HLC, the human bait is exposed to the diseases transmitted by the landing mosquitoes posing ethical concerns on implementation of this technique [
20]. As an alternative, catching traps such as the CDC (Center for Disease Control) light trap and the bed net traps have been developed and the data collected is useful in estimating vector populations when the studies are properly controlled. However, these trapping methods often differ in the number of host-seeking mosquito population sampled [
21]. Still, in spite the high number of mosquitoes captured on these studies (up to 12,000 specimens) a few mosquitoes (up to 4 specimens) were found positive for
Plasmodium parasites even in their high abundance months [
11,
12]. So, the question remains on how much is people being exposed to mosquito bites and acquiring the parasite. Thus, it is important to design alternative methods able to reflect the vector-human contact and complement the data collected by mosquito trapping methods.
Malaria is acquired when
Plasmodium spp. sporozoites are injected into human skin through the bite of a female
Anopheles along with the mosquito salivary proteins [
22]. Previous studies have shown that a significant number of mosquito salivary proteins are immunogenic and able to induce antibody responses, mainly IgG isotype. These antibodies can reflect the intensity of human exposure to mosquito bites and represent good indicators of the risk of infection with
Plasmodium spp. [
23‐
27]. Thus, the use of salivary gland and saliva antigens has been previously validated as an indirect proxy to determine mosquito bite exposure. Significant higher IgG antibody levels against
An. albimanus and
An. darlingi salivary proteins have been observed in people with active malaria infection in Central and South America when compared to uninfected people living in the same region [
23,
28]. A similar pattern has been observed in areas where
Anopheles (
Cel.)
gambiae and
Anopheles (
Cel.)
stephensi are among the most important vectors. A significant number of these studies were performed evaluating IgG responses against the
An. gambiae salivary protein gSG6, a highly conserved protein among
Anopheles species from the Subgenus
Cellia and
Anopheles [
29]. The peptide, gSG6-P1, was designed from the original
An. gambiae gSG6 sequence. IgG responses specific to this salivary peptide has been validated as a biomarker of human exposure not only in Africa but also in Asia and South America [
24,
27,
30]. Although there are no known species of the subgenus
Cellia in South America, the responses observed against the gSG6-P1 peptide could be hypothesized to result from the presence of mosquitoes belonging to the subgenus
Anopheles such as
Anopheles pseudopunctipennis and
An. punctimacula and
An. calderoni [
31].
Consequently, it is necessary to characterize a broader panel of biomarkers able to identify the risk of disease more closely in areas with a great diversity of Anopheles mosquitoes. Future studies are planned to identify exposure markers that include not only the primary malaria vectors but also markers for the majority of the circulating species playing an important role in malaria transmission in Latin America, even when these vectors species are in a smaller proportion. Since the use of salivary gland extract as antigen to indirectly measure exposure to mosquito species circulating in a region has been validated by several groups the main objective of this work was to measure IgG antibodies in humans living in an area where low-density P. falciparum infections are frequent. Thus, human IgG responses to Anopheles salivary gland extracts (SGE) were used to measure potential associations with low-density infections by P. falciparum and malaria risk. Additionally, it was evaluated whether gSG6-P1 peptide continues as a useful marker to detect exposure in areas where mosquitoes from the sub-genus Anopheles are important vectors of malaria in Colombia.
Discussion
The intensity of malaria transmission has been traditionally evaluated using the EIR, which is defined by the number of infected bites received per human per unit of time; nevertheless, this strategy has shown limitations in low endemic settings for malaria [
25,
36]. As a result, alternative methods to estimate human exposure to
Anopheles bites have been proposed, including the detection of IgG responses to
Anopheles SGE and salivary peptides. The purpose of the present study was to explore the possibility of using whole SGE from different
Anopheles species as tool to detect IgG antibodies in humans that could be used as indirect estimation of exposure to
Anopheles bites in a malaria-endemic area in Colombia where there is an important proportion of asymptomatic infections. Based in previous reports suggesting at least two
An. albimanus lineages in Colombia [
7,
9,
10], the SGE from two
An. albimanus strains were used to try to capture potential differences in immunogenicity of salivary proteins from colony mosquitoes isolated from different geographical regions and with differences in the colonization time. Specifically, this study includes the comparison of salivary gland content immunogenicity between the CTG strain, a recently colonized strain, that could potentially resemble more closely responses to “wild mosquito antigens” in the area, to the immunogenicity displayed by the STE strain, isolated in Central America in 1974.
Anopheles albimanus has been reported as one of the main malaria vectors in Nariño displaying EIR up to 2.6 in recent studies. Consistent with previous studies,
An. albimanus SGEs (STE and CTG) were associated with the infectious status, where people with active
Plasmodium infection presented significantly higher IgG antibody levels against the salivary proteins. This study also showed that people with higher antibody levels against STE, CTG and gSG6-p1 have between 2 and 4 times more probability of suffering a malaria infection. These results agree with previous findings in Haiti were the IgG antibody levels against
An. albimanus SGE were higher in patients with clinical malaria than those in uninfected people living in the same region [
23]. These studies suggest that the IgG antibody response against
An. albimanus SGE is associated with
Plasmodium exposure and highlights the relevance of using whole salivary content in the form of SGE as potentially useful antigen to measure risk of infection in areas of low and seasonal transmission. Interestingly, the relationship between parasitaemia and IgG antibodies against
Anopheles antigens was significant when using the antigen from the CTG strain and not the STE, suggesting that the antigens contained on the SGE from the CTG may be more closely related to the one the study subjects are exposed in the field. However, no association was found between antibodies levels against
An. darlingi SGE and malaria infection. This could be explained due to the low abundance (or probable absence) of
An. darlingi mosquito previously reported in areas where samples were collected [
6,
7]. Still, the observed antibody response against the
An. darlingi SGE may be explained by a potential cross reactivity between salivary proteins present in mosquitoes from the subgenus
Nyssorhynchus, which
An. darlingi belongs to.
Previous studies suggest that
An. calderoni is a primary malaria vector in Narino [
11]. This may explain the current findings showing a high IgG response against gSG6-P1 peptide in samples from infected compared to uninfected people. These findings agree with a previous study in Colombian volunteers suggesting that the concentration of gSG6-P1 antibodies is significantly correlated with malaria infection status and that people with clinical malaria presented significantly higher levels of IgG anti-gSG6-P1 antibodies than healthy controls [
24]. Although,
Anopheles species from the subgenus
Nyssorhynchus are the main vectors of malaria in Colombia, at least six species from the sub-genus
Anopheles have been described as potential malaria vectors in the region [
37,
38]. Three of these species (
An. calderoni,
An. pseudopunctipennis and
An. punctimacula) are present along the Pacific coast, the main area where
P. falciparum is transmitted in Colombia [
3]. Although Arcà et al. reported that gSG6 had no degree of identity with orthologous proteins from vectors in Central and South America, and therefore serological data previously published about the usefulness of the gSG6-P1 peptide in Colombia [
24] should be interpreted with caution [
29], previous work also showed that a deduced gSG6 from the New World species
An. freeborni and
An. quadrimaculatus (from the subgenus
Anopheles) had between 67 and 71% of degree of identity with the gSG6 from Old World
Anopheles species [
39]. In the same way, Pollard et al. suggested that the antibodies to the gSG6-P1 peptide in the Colombian population may represent exposure to
An. punctimacula, which is a member of the
Anopheles subgenus or could hypothetically represent exposure to minor vectors in the country [
31]. Thus, the current results suggest that the gSG6-P1 peptide could be a useful marker for malaria risk in areas of Colombia where mosquitoes belonging to subgenus other than
Nyssorhynchus are present.
When comparing IgG levels against
An. albimanus among villages, it was observed that SGE from both STE and CTG, were higher in infected than uninfected people in all villages except California. This is interesting because California is an area with urban characteristics, unlike Tangareal which is a sub-urban area and Robles and Candelillas which are rural areas. To evaluate further, the multilevel analysis demonstrated that independent of site, both age and, anti-Pf-MSP IgG levels were associated not only with IgG antibody levels against the CTG strains of
An. albimanus but also against the gSG6-P1. Suggesting the importance of using a panel of exposure biomarkers (mosquito antigens) and concurrent entomological data to accurately evaluate risk especially in areas where several
Anopheles species are implicated in malaria transmission. Also, the current model described in this study revealed a negative association between age and IgG antibodies against all
Anopheles antigens. Similar trend has been observed in other studies measuring antibody responses against mosquito salivary antigens and has been associated with the development of tolerance against certain mosquito allergens [
35,
40,
41].
Recent studies revealed important differences in salivary content in arthropods collected in the field when compared to the same species maintained in a colony [
33]. Also, a previous study suggests the possibility of two
An. albimanus lineages circulating two geographically distant regions of Colombia. Thus, the aim of this study was to determine if the risk of infection can be affected by the salivary content of mosquitoes from the same species but from different origins. So, a recently colonized strain (CTG) and a long-term established laboratory colony (STE) each isolated from a distinct geographical region (Colombia and El Salvador) to account for potential changes in IgG responses based on salivary content were used. As the results indicate, the SGE from the CTG strain showed significant association with the Pf-MSP1 and not with the SGE from STE suggesting potential differences. Determination and confirmation of these differences are subject of further studies aimed to characterize salivary gland content of the two
An. albimanus lineages circulating in Colombia and comparing those to
An. albimanus isolates from other countries. This is important since the use of salivary antigens as vaccines for malaria are undergoing [
42] and characterization of the main immunogenic salivary proteins of the main vectors circulating in endemic areas are important for the success of such vaccine.
This study has several limitations. First, because this study was cross-sectional, association with the anti-Anopheles IgG levels should be interpreted with caution as they do not imply causality. Second, due to the lack of a symptomatic group, it was not possible to determine the risk factors for this kind of infection and to explore the differences in the anti-Anopheles IgG levels between uninfected, asymptomatic (both, submicroscopic and microscopic infections) and symptomatic groups. Also, the lack of concurrent entomological data is a significant limitation. Since this study did not included mosquito collection or other concurrent entomological surveillance, the current results should be interpreted as an indirect measurement of disease risk (currently calculated by OR) until further determination of the specific mosquitoes circulating in an area where these antibodies are measured. A future study phase will include to complete the serological data with entomological data to further validate the findings of this study. Despite these limitations, these results are useful to identify new potential biomarkers for malaria risk in Colombia.
Acknowledgements
The authors thank the Department of Entomology at Kansas State University, the Vector Control Unit at the Nariño Department of Health Institute and the Epidemiology Group in Public Health Faculty at Universidad de Antioquia for their support in this research. The authors also thank Dr. Audrey Lenhart and Dr. Franck Remoue for their valuable comments to improve the manuscript. A special thanks to the people from California, Tangareal, Robles, and Candelillas in Tumaco for allowing and participating in this study, and to Flor Portocarrero, Zully Toloza, Yuri Pino and Leidy Gonzáñel for their hard work and commitment in the fieldwork.
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