Background
Viruses transmitted by arthropods (arboviruses) are the causative agents of some of the most significant infectious diseases emerging in the world. Among these arboviruses are the dengue virus (DENV), the chikungunya virus (CHIKV), and the Zika virus (ZIKV), which all circulate in the same tropical and subtropical geographical regions. These three arboviruses cause diseases with similar clinical presentations in the initial stages and in which the diagnoses are made clinically, particularly in countries where dengue is endemic. Dengue (the disease caused by DENV) is the most significant epidemiological arbovirus worldwide [
1]. The term Dengue, includes both non-apparent or silent forms of the disease, the non-severe clinical forms (dengue fever [DF]) and severe clinical forms (dengue hemorrhagic fever [DHF] and dengue shock syndrome [DSS]). Although this classification of Dengue is still in force, the World Health Organization (WHO) proposed some years ago a new classification of the disease based on severity levels (severe and non-severe dengue). In the new classification, the non-severe dengue group includes patients who develop a self-limiting illness with fever, anorexia, nausea, rash, body aches, leucopenia and positive tourniquet test, and those patients who also have warning signs of developing severe dengue such as abdominal pain, persistent vomiting, fluid accumulation, mucosal bleeding, lethargy, liver enlargement and increased hematocrit with a rapid decrease in platelet count. The severe dengue group includes patients with severe plasma extravasation that can lead to hypovolemic shock or respiratory distress due to the accumulation of fluid in the lung, massive bleeding, and organ involvement (liver, heart or nervous system) [
2]. An estimated 50 million cases of dengue fever occur annually, particularly in tropical and subtropical areas [
3]. In turn, chikungunya fever (caused by CHIKV) and the disease caused by ZIKV have joined dengue as emerging arboviruses that threaten global public health [
4,
5].
DENV and ZIKV belong to the family
Flaviviridae, genus
Flavivirus, whereas CHIKV belongs to the family
Togaviridae, genus
Alphavirus. All of these viruses possess a single-stranded, positive-sense RNA genome that encodes for both structural (forming part of the viral particle) and nonstructural (responsible for replication) proteins. The viral genome encodes three structural proteins, one of them being the envelope protein (E), which is the main inducer of neutralizing antibodies and is therefore responsible for classifying the virus into four serotypes: DENV-1, DENV-2, DENV-3, and DENV-4 [
6].
DENV, CHIKV, and ZIKV are all transmitted by the mosquito vectors
Aedes aegypti and
Aedes albopictus [
5,
7‐
9], causing clinical manifestations that are difficult to differentiate, including fever, myalgia, cephalalgia, arthralgia, conjunctivitis, and rash; thus, laboratory confirmation of cases is required [
9]. Since to that these viruses are transmitted by the same vector, the co-circulation is frequent and, moreover, there is an increasing possibility of co-infection in those regions where the vector is present [
10]. Additionally, the results of the analysis of the epidemics of CHIKV [
11,
12] and ZIKV [
13] in the Pacific, Asia, and more recently in the Americas have demonstrated an association between these viruses and adverse neurological, motor, and teratogenic events; even some authors have postulated that the clinical outcome could be affected with the co-circulation of the three agents [
14,
15].
Colombia is considered a hyper-endemic country for DENV, registering more than 147,000 cases of dengue in 2010 [
16]. The recent arrival of CHIKV in 2014 [
17,
18] and the subsequent entry of ZIKV [
19] into the country in 2015 have overwhelmed the Colombian health system [
20] and have demonstrated the enormous need to improve the clinical definition of the diseases and reduce diagnostic errors [
10]. Although the criteria for the clinical definition of patients with febrile syndrome because of DENV have been widely explored [
7,
16], the lack of sufficient facilities to make an accurate viral diagnosis before the arrival of the new arboviruses created a diagnostic gap that has jeopardized the health of patients, even more so when the confirmation of cases, both of CHIKV and ZIKV, is performed by clinical definitions in regions that have already confirmed the circulation of these viruses [
21,
22].
Furthermore, with the co-circulation of these three arboviruses (DENV, CHIKV, and ZIKV) in Colombia and different regions of Latin America, an increase of the number of reports of co-infections with different clinical outcomes was unusual [
23‐
26]. However, to date, no information is available that establishes the prevalence of co-circulation and co-infection for these arboviruses in Colombia. Therefore, the objective of this study was to molecularly confirm the presence of DENV, CHIKV, and ZIKV and their co-infections in a cohort of patients with febrile syndrome consistent with dengue in the Colombian-Venezuelan region. Our results molecularly confirm the co-circulation of DENV, CHIKV, and ZIKV in equal dominance and with a significant prevalence of co-infection among them.
Discussion
Our results regarding the simultaneous co-circulation of DENV, CHIKV, and ZIKV and their co-infections clearly demonstrate the critical epidemiological situation in the tropical and subtropical regions of the Americas and the increasing need to perform differential diagnosis in patients with acute febrile syndrome. All of the patients involved in our study met the basic criteria for dengue or acute febrile illness (temperature ≥ 37.8 °C for less than seven days; articular, retroocular, and/or extremities pain; rash; hemorrhage) [
7], but despite this, only 52.2% of the patients could be diagnosed by molecular techniques for any of the three arboviruses analyzed (DENV, CHIKV, or ZIKV) and the 47.78% was negative. Acute febrile syndrome is a common feature of several infections endemic arbovirus countries and although Dengue in the most common cause of “Febrile Syndrome” in Colombia [
37], other etiological agents, such as bacterial (Leptospira spp., Rickettsia spp., Salmonella spp. and Brucella spp) [
38,
39], viral (influenza virus, hepatitis A virus, B and C, Venezuelan equine encephalitis virus, Mayaro virus and Hantavirus) [
40] or parasitic (Plasmodium vivax, P. falciparum) [
41], could be the cause for 47.78% of acute febrile syndromes. In ongoing studies we are using multiple approaches to try to explain and to reveal the etiological agent for those cases (data not shown). On the other hand, we believe that false negative results are less likely because all the participants enrolled had to be in acute stage of the disease, that is to say, fever no more than 7 days. Moreover, the samples were stored at the adequate temperature (− 70 °C) and the quality of RNA was confirmed by the ratio of absorbance at 260 nm and 280 nm (measured by spectrophotometry), being in all cases > 2.0.
As it can be observed in the Fig.
2, there was in increase in cases from December through February. This increase can be due to the arrival of ZIKV to Colombia, because the first case was reported in October 2015 and the higher peak of the outbreak was observed from December through February [
19]. Moreover, the increase too can be due to those months which coincide with holiday season in Colombia when the people who live in regions with low temperature (where there is not presence of
Aedes) move to regions with higher temperature (with presence of
Aedes), increasing the possibilities of transmission of infection.
The prevalence of DENV in the population evaluated was 21.02%, with an attack rate of 41 cases per 100,000 population. These data are in stark contrast with other studies conducted in the country that have reported the presence of DENV in approximately 30% of patients with febrile syndrome [
42] and a cumulative incidence of cases ranging between 36.5 and 268.7 cases/100,000 inhabitants in different regions of Colombia [
43,
44]. This low DENV detection rate can be explained by an increase in the number of cases of the other arboviruses studied, as it has been previously reported in India, where the prevalence of DENV decreased with the increase in cases of CHIKV [
45,
46].
As could be seen in Table
2, there is an age-related decline in DENV disease. This phenomenon has been widely reported for Colombia with the highest incidence in individuals 5–14 years of age [
16]. This epidemiological behavior could be explained by the high endemicity of the country, on which individuals acquires DENV Infection at young age with the subsequent immunity. For example, in Medellín (City located in a central region of Colombia) the 70% of the younger people (up to 21 years old) have antibodies against DENV due to primary infections acquired in childhood [
47]. This same endemic epidemiological status could be expected in the next years for CHIKV and ZIKV in several Colombian municipalities due to the similarity of DENV, ZIKV and CHIKV transmission cycle which bring on the possibility of endemic establishment of those arboviruses [
48].
Although an end to the Chikungunya epidemic in Colombia had already been officially declared for the time period in which the sampling was performed, the prevalence obtained (29.94%) showed that CHIKV had been established as one of the most significant arboviruses in the diagnostic panel, possibly establishing endemic cycles [
4]. Additionally, our data show an estimated 58.38 cases/100,000 inhabitants, which is considerably higher than the average attack rate established for Colombia during the epidemic period of 19.1 cases/100,000 inhabitants (range between 4.5 and 283.1) [
49]. These figures are important because although it is well known that mortality due to CHIKV is considerably less than that occasioned by DENV [
1], the chronic sequelae of CHIKV infection may have a greater long-term impact, affecting up to 25% of infected patients [
12]. This chronic sequelae is mainly associated with “post-chikungunya chronic inflammatory rheumatism” and thus has a significant impact on the future quality of life of the population [
11].
Our sampling began in the month of August 2015, a time period in which the entry of ZIKV into Colombia had not yet been reported. According to reports from the National Institute of Health [
19], the first cases of ZIKV that were identified in our study occurred in the month of November 2015. The prevalence of ZIKV was found to reach 18.47%, with an attack rate of 36 cases/100,000 inhabitants, which is considerably lower than that reported for the entire department of Norte de Santander of 655 cases/100,000 inhabitants [
19]. Notably, the official data represent the total number of patients diagnosed for a clinical picture compatible with infection by ZIKV whereas our results present the number of laboratory-confirmed cases, which typically are presented in lesser number [
8]. These differences are a result of the difficulties in the molecular diagnosis of ZIKV because of the low viral loads present in the serum of patients [
24,
36]. In agreement with the findings reported, in our study, the amplification “threshold cycle” of the qPCR was greater than 29.09.
Although it is not currently possible to speak of susceptibility by gender, our results are in complete agreement with various studies showing that the ratio of cases between women/men is generally at a ratio of 2/1 or 3/1 in DENV, CHIKV, or ZIKV infections. This relationship has been previously reported in Colombia for DENV infections [
16], during the CHIKV outbreak [
18], and recently for ZIKV [
19], as well as being previously reported internationally during other events, both endemic in the case of DENV [
50,
51] and epidemic in the case of CHIKV and ZIKV [
52,
53]. This difference in the presentation of infection in women could be the result of socio-cultural practices that lead to greater medical consultation by women or more actual risk associated with the domestic behaviors of women and the domestic behavior of
A. aegypti [
54], which favor an increased risk of exposure.
Importantly, our study also found a higher prevalence of infection by ZIKV among women aged 20–40 years than has been reported for the rest of the country [
19] and for other countries in the region during the current outbreak [
53]. Taking into account the possible adverse effects associated with infection by ZIKV during pregnancy [
55,
56], it is important to focus on prevention efforts in this age group to reduce unwanted pregnancies and to increase surveillance of such adverse events in pregnant women infected with ZIKV and their babies [
57].
The co-circulation of the four serotypes of DENV has been previously reported in the country [
16], and our results are consistent with those reports because we found the circulation of serotypes − 1, − 2, and − 3, although with a higher prevalence of DENV-2 (90.9%). However, the analysis of the sequenced samples demonstrated that the DENV-2 Asian/American genotype predominated, which is consistent with reports in recent years in the northern region of Colombia [
16]. Genotype V was identified for DENV-1, which is also representative of most of the strains of this serotype isolated in Colombia and generally in the Americas [
58,
59]. Finally, genotype III was found in the DENV-3 strains, which has been the predominant genotype in Colombia since its introduction in 2002 [
60]. In the case of the samples positive for CHIKV, sequencing revealed the presence of the Asian strain, the same strain that has been previously reported in Colombia [
18,
61,
62]. Finally, although no sequencing of the samples positive for ZIKV was performed because the real-time PCR primers amplify a specific region of the Asian/Caribbean strain [
31], we can presume that these samples belong to this lineage, which is the same that has been previously reported in the country [
62‐
64]. Subsequent phylogenetic analyses are necessary to understand the molecular epidemiology of DENV, as well as of CHIKV and ZIKV. In other studies, ongoing, we are evaluating the molecular epidemiology (by sequencing) of those positive samples to identify the evolution level and adaptation of those virus strains in the region.
The co-circulation of DENV, ZIKV, and CHIKV is a chance occurrence that has triggered alarms in all tropical and subtropical regions of the world, not only because of the difficulty of making an accurate clinical diagnosis, but because of the potential epidemiological effects of co-infections. Although some cases of co-infection among these arboviruses have recently been reported in Colombia [
23,
25], our study is the first cross-sectional study that demonstrates the extent of the co-circulation and the possibilities of co-infection of these three arboviruses in Colombia, demonstrating that although there is an apparent dominance of infection by Chikungunya, the three agents co-circulate simultaneously in the evaluated region. A similar context has been previously described in India, where the simultaneous co-circulation of DENV, CHIKV, and ZIKV was demonstrated in equal proportions during 2010 [
46] and subsequently during 2013 [
65], eliminating the possibility of displacement of any of the viruses [
66], as has been shown between different genotypes of DENV, both in the Americas [
67] and in Asia [
68].
The reported prevalence of DENV/CHIKV co-infection varied from very low (2.8%) [
9] to more than 10% [
65,
69,
70], which is consistent with the findings in our study (7.64%). However, although there are very few studies that present the prevalence of DENV/ZIKV or CHIKV/ZIKV co-infections, our results demonstrate a higher prevalence of DENV/ZIKV co-infection (6.37%) than that reported in Pernambuco, Brazil (2.6%) [
26] or in Nicaragua (1.7%) [
70] and a lower prevalence of CHIKV/ZIKV co-infection (5.10%) than that recently reported in Bahia, Brazil (13.3%) [
24], but a similar prevalence of CHIKV/ZIKV to that reported in Nicaragua (4.6%) [
70]. Finally, this is the first study that reports the prevalence of triple co-infection (1.9%) in South America, but the result is consistent with the recently reported prevalence in Central America (1.7%) [
70].
The co-circulation and co-infection of these arboviruses has particular importance when one considers that diseases such as Guillain-Barré syndrome (GBS), which has an autoimmune component, are usually triggered by an infectious process [
71]. GBS has been associated with infection by DENV [
72], CHIKV [
73], and ZIKV [
74] in our country, which could increase the risk of developing the syndrome, the mortality rates, and epidemiological complications. Notably, recent publications with clinical and histopathologic evidence of patients co-infected with DENV/CHIKV demonstrate that there may be an association between mortality and co-infection with these arboviruses [
75]; moreover, recent articles have demonstrated, both in vitro and in vivo, antibody-dependent potentiation in patients with anti-DENV antibodies who acquire the ZIKV infection [
76,
77] or vice versa [
78].