Several studies, mainly from sub-Saharan Africa and Southeast Asia, report Plasmodium spp. co-infections or co-circulation with other pathogens, such as DENV [16], CHIKV [17, 18], HAV [9], HBV [10, 19], LEP [20, 21], human immunodeficiency virus [22], intestinal helminths [23], and other febrile diseases [24]. However, in Latin America, these reports are limited [10, 25–28]. To the best of our knowledge, despite the multiple infections that may complicate malaria and lead to failure in treatment responsiveness, there are no reports on the interactions of these pathogens in co-infections in Venezuela, the most malaria-endemic region in Latin America. This study found a high prevalence of CoRE with other pathogens among malaria patients (34.2%), even higher than that reported in Brazil (20%) [25], but lower than that found in a recent study in India (60%) [29]. Thus, physicians should suspect co-infection in malaria cases with inadequate response to treatment or atypical clinical manifestations.
The prevalence of CoRE with DENV (14.9%) was higher than that found in a large cross-sectional study in hospitalised patients with acute febrile syndrome in the Brazilian Amazon (2.8%) [25], and in a study conducted in Mumbai (India) (10.3%) [16], but than in New Delhi (India) (44%) [29] and Pakistan (33.3%) [30]. Thus, the prevalence of co-infection or co-circulation may fluctuate, depending on the local endemicity even within the same country [16, 29], as well as on the sensitivity of the diagnostic methods used. In these studies, prevalence was estimated based on hospitalised and non-hospitalised patients; therefore, it could not be extrapolated to the community level.
Malaria CoRE with other pathogens do not induce a predictable clinical pattern. While we found that CoRE with DENV was significantly associated with somnolence and splenomegaly, a study conducted in French Guiana showed a worse clinical outcome, with a higher risk of severe thrombocytopenia and anaemia [28], or induced low platelet counts [30], or elevated transaminases [16]. More surprising is that a study in Peru indicated that Plasmodium/DENV co-infection was not associated with disease worsening [26], whereas in another study in India DENV serotype-4 co-infection was associated with even mild malaria [29]. Among the multiple factors that might influence the clinical outcome of the infection, one could hypothesise that differences in DENV serotypes or Plasmodium species, previous exposure to either pathogen or to both, age and gender, and other epidemiological conditions may explain the discrepancies among the studies.
The second most frequent CoRE was HAV (11.8%), higher than that found in children from sub-Saharan Africa (1.7%) [9]. This high incidence could be due to a deteriorated water system in Venezuela [31] added to low vaccination rates [32], as confirmed by the very high percentage of individuals (88%) in which HAV-specific IgG was not detected. The age group studied could explain this fact; however, the information on this co-infection is limited. On the other hand, the prevalence of CoRE with HBV (6.2%) in our study was similar to that found in Nigeria (6.6%) [19], but higher than that documented by Braga et al. [27] in western Brazilian Amazon (4.2%). In the same study, patients with co-infection showed no clinical differences from those with only malaria, and similar to our findings also showed no association with classic signs of a liver disorder. In another study, HBV co-infection was more likely to be asymptomatic, even Plasmodium parasitaemia correlated inversely with plasma HBV DNA levels [10]. In contrast, other studies revealed that co-infection amongst individuals significantly affected haematological and liver parameters [33, 34]. Our result should be interpreted with caution due to the low sample size. Although co-infection is possible, we found no CoRE with HCV cases [35]. Our finding may explain the low HCV prevalence previously reported in Venezuela [36].
CoRE with CHIKV was found in a lower proportion (5.5%) than that found in Tanzanian (7.1%) [17] and Kenyan children (9.4%) [18]. In contrast, two extensive studies in India [37] and Senegal [38] found a low co-infection prevalence (1.3% and 0.02%; respectively). The observed variations in CHIKV prevalence among different studies may be attributed to epidemiological and geographical factors [39–41]. Our findings on CoRE with LEP (3.7%) contrast with those reported in southern India (22%) [21] and Thailand (7.7%) [42], likely due to the higher incidence of LEP in these regions. A high prevalence (80.6%) of leptospirosis among febrile patients with high suspicion of LEP has been reported in Ciudad Bolivar [43]. Other LEP co-infection cases have been documented [20] and have even been associated with severe sepsis [44]. We found an association between CoRE with LEP with elevated aminotransferases and thrombocytopenia, as previously described [45, 46]. In Latin America, LEP/malaria co-infections are rarely reported, but high clinical suspicion should prevail as late diagnosis could increase morbidity and mortality. Thus, in patients with complicated malaria presenting with fever, thrombocytopenia, and alterations in liver and kidney function, the diagnosis [47, 48] and empirical treatment for LEP co-infection should be considered.
Interestingly, simultaneous CoRE with DENV/HAV was found in four malaria patients. There are few case reports of this concurrent mixed infection [49]. Thus, those are likely to occur more frequently than reported in the available literature, mainly in developing countries. Other CoRE with two or three pathogens could be explained by overlapping breeding sites of mosquito vector species, especially in malaria, DENV, and CHIKV [11, 50]. Additionally, febrile disease outbreaks are often associated with rainy seasons in the tropics [8].
A high frequency (42.9%) of complicated malaria was found and complications were more likely in CoRE patients compared to no CoRE ones, suggesting that CoRE with another pathogen could exacerbate the malaria clinical course. Nevertheless, further investigations are needed to confirm this observation due to the small sample size. Similar results have been found in patients with P. vivax/DENV co-infection, who were more likely to have severe disease than those mono-infected with DENV [25]. In contrast, Andrade et al. [10] found that HBV infection was associated with a lower intensity of malaria infection. In order to determine authentic association between co-infections, prospective studies should include a larger number of patients, although control of real-life variables remains a challenge in Venezuela.
The limitations of this study include the lack of double testing for viral infection due to economic reasons. Although we evaluated specific IgM antibodies, cross-reactivity cannot be ruled out due to polyclonal activation induced by Plasmodium infection [51, 52], as occurs with other highly prevalent infectious diseases, including that caused by Epstein Barr virus [53]. Another limitation was the absence of comparison between acute and convalescent sera from the same patient and the inability of molecular testing to confirm co-infection, since in some cases specific IgM remains positive for weeks after the acute phase [54–56]. Nonetheless, this is a frequent real-life situation regarding resources and poor settings where testing for follow-up of recovered patients is often not performed and where molecular diagnostic studies are restricted. Enrolment of only febrile individuals also constitutes a limitation; then future studies should include asymptomatic individuals to assess the real burden of co-infections in malaria. Finally, the small number of CoRE patients, along with even smaller frequencies of some co-infections and several highly prevalent diseases that were not explored (e.g., Chagas disease, tuberculosis, leishmaniasis, human immunodeficiency virus, or syphilis), also represented a limitation.