Several studies, mainly from sub-Saharan Africa and Southeast Asia, report malaria coinfections with other pathogens such as DENV , CHIKV [17,18], HAV , HBV [10,19], LEP [20,21], HIV , helminths  and other febrile illnesses . In Latin America, however, reports of coinfections in malaria patients are limited [10,25,26,27,28]. To the best of our knowledge, there are no reports about the interactions of these pathogens in coinfections in Venezuela, despite multiple infections may complicate malaria and lead to failure when it comes to treatment responsiveness. High prevalence of malaria coinfection was found in this study (34.2%), even higher than reported in Brazil (20%) ; but lower than that found in a recent study in India (60%) . Thus, physicians should be suspicious of coinfection in malaria cases with inadequate treatment response or atypical manifestations.
The prevalence of malaria coinfection with DENV (24/161; 14.9%) was much higher than the found in a cross-sectional study in hospitalized patients with the acute febrile syndrome in the Brazilian Amazon (44/1578; 2.8%)  or in another study in Mumbai (16/156; 10.25%) , or during a dengue outbreak in India (27/367; 7.4%) . In contrast, in Pakistan, the prevalence found was higher (26/78; 33.3%) , as well as in India (29/66; 44%), . Thus, the prevalence of coinfection may fluctuate, depending on local endemicity and the sensitivity of the diagnostic methods used. In these studies, the prevalence was estimated based on hospitalized and non-hospitalized patients; therefore, it could not be extrapolated to the community level. We found that DENV coinfection was significantly associated with somnolence and splenomegaly. In agreement with our findings, a study in French Guiana showed worse clinical outcome, with a higher risk of severe thrombocytopenia and anemia in DENV coinfection than in patients with only malaria . Other studies have reported a markedly low platelet count  or high elevation of transaminases in the DENV coinfection group , however, we did not find these paraclinical alterations in our study. A study in Peru indicated Plasmodium/DENV coinfection was not associated with worse disease , similar to another study in India were the coinfection with DENV serotype 4 (DENV-4), even was associated with mild malaria. . Differences in DENV serotypes or Plasmodium spp may explain the differences of the results.
The second most frequent coinfection was HAV (19/161, 11.8%), higher than that found by Klein et al. (10/222, 1.7%) in children from sub-Saharan Africa . This high incidence could be due to a deteriorated water system in Venezuela  added to low vaccination rates . In this study, neurological manifestations (stupor and seizures) were associated with HAV coinfection. In contrast, no significant alterations were found in the clinic and in the liver function of the coinfected patients, as in the study carried out in sub-Saharan Africa . The age group studied could explain this; however, limited information is available on this coinfection. On the other hand, the prevalence of coinfection with HBV (10/161, 6.2%) in our study was similar to that found in Nigeria (11/166, 6.6%)  but higher than documented by Braga et al.  in western Brazilian Amazon (4.2%). In the same study, patients with coinfection presented no clinical differences from those with malaria only, and similar to our findings, nor showed any association with classic signs of a hepatic disorder. In another study, HBV coinfection was more likely to be asymptomatic (OR: 120.13, p<0.0001), even Plasmodium parasitemia inversely correlated with plasma HBV DNA levels (R = −0.6; p = 0.0003) . In contrast, other studies revealed that coinfection amongst individuals significantly affected the hematological and liver parameters [34, 35]. Our result should be interpreted with caution due to the limited number of coinfected patients evaluated. We found no malaria/HCV coinfection cases, although coinfection is possible , our finding may be explained by the low prevalence of HCV previously reported in Venezuela .
CHKV coinfection was found in 9/161 patients (5.5%), a prevalence lower to that found in Tanzania (8/112, 7.14%) , and reported in Kenyan Children (15/158, 9.4%) . In contrast, two extensive studies in India  and Senegal  found low coinfection prevalence (15/1564, 1.3%) and (3/13845, 0.02%), respectively. The observed variations in the prevalence of CHKV between different studies may be attributed to study site location including seasonal variations, targeted age groups, agricultural activities, and time [41-43]. Our findings on LEP coinfection (6/161, 3.7%) contrasts with those reported in South India (48/222, 22%)  and Thailand (15/193, 7.7%) , incidence of LEP in these regions could explain these differences. Although a previous study of Leptospira found a high prevalence (80.6 %) of Leptospirosis in Bolivar city, this was documented in a group of febrile patients highly suspected of leptospirosis . Other LEP coinfection cases have been documented  and have even been associated with severe sepsis . We found an association between LEP coinfection with aminotransferases elevation and thrombocytopenia previously described [47, 48]. In Latin America, LEP/malaria coinfections are rarely reported, but high clinical suspicion must prevail since a late diagnosis could increase morbidity and mortality. Thus, for patients with severe malaria presenting with fever, thrombocytopenia, and altered liver and kidney function diagnosis [49, 50] and empirical treatment for this coinfection should be considered.
Interestingly, simultaneous coinfection with DENV/HAV was found in 4/11 (36.4%) patients with malaria. To date, there are few case reports of this concurrent mixed infection . Thus, we consider they likely occur more frequently than reported in the available literature mainly in developing countries. Other coinfections with two or three pathogens could be explained by overlapping breeding sites for mosquito vector species, especially in malaria, DENV, and CHKV [52, 53]. Additionally, outbreaks of febrile illnesses are often associated with rainy seasons in the tropics .
A high frequency (42.9%) of complicated disease was found and complications were more likely in coinfected patients compared to patients without coinfections, suggesting that coinfection with another pathogen could exacerbate the clinical course of malaria. Nevertheless, due to the small sample size, further investigation is needed to confirm this observation. Similar results have been found for patients with P. vivax and DENV coinfection who had a higher chance of presenting severe disease than those mono-infected with dengue . In contrast, Andrade et al found (among 636 Brazilian patients) that HBV infection was associated with a decreased intensity of malaria infection among individuals in the study . In order to determine an appropriate correlation between coinfections, prospective studies should be designed that include a larger number of patients, however controlling real life variables remains a challenge in Venezuela.
This study has some limitations. Although we evaluated both specific IgG and IgM antibodies of possible coinfections, cross-reactivity cannot be ruled out due to chemical similarities of the antibodies investigated as a consequence of the polyclonal activation induced by Plasmodium spp infection [54, 55], just as it happens with other highly prevalent infectious including that caused by Epstein Barr virus . Another limitation was the absence of comparison between acute and convalescent sera from the same patient, and the inability to perform molecular tests to evaluate the coinfection. Nonetheless, this a frequent real-life situation regarding resources and poor settings where tests for follow-up of recovered patients are usually not collected and molecular diagnostic studies are limited. Another limitation is that all the enrolled individuals were febrile patients, then studies enrolling asymptomatic individuals should be performed in the future to evaluate the real burden of coinfections in malaria. Finally, the small number of coinfected patients along with the even smaller frequencies for some confections and a number of highly prevalent diseases that were not explored (Chagas disease, Tuberculosis, leishmaniasis, HIV, Syphilis), also represented a limitation.