Dengue viruses (DENVs) and hantaviruses are global health threats accounting for 58.4 million annual dengue cases and as many as 0.2 million annual hantavirus infections respectively [1
]. With both dengue and hantavirus infection, the role of the vector is important namely the mosquito primarily Aedes aegypti
and rodents (Muridinae family e.g., rats and mice) respectively. Transmission of DENV and hantaviruses is supported by anthropogenic activities that increase population growth, urbanization, air travel and climate change.
Hantaviruses are single stranded (SS) negative-sense RNA viruses approximately 120–160 nm in diameter from the Hantaviridae virus family [3
]. Hantaviruses can be separated into two groups, Old World (Seoul (SEOV), Dobrava (DOBV), Puumala (PUUV) and Hantaan (HTNV)) and New World (Prospect Hill (PHV), Andes (ANDV) Sin Nombre (SNV), etc) based on the M segment (nucleotides 1987–2315) [3
]. DENV and hantaviruses are both RNA viruses, which infect similar human host cells. DENV infection can lead to dengue fever (DF), dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). Whilst hantavirus infection can lead to three main clinical diseases namely nephropathica epidemica (NE), haemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) [5
]. DENV and hantavirus infections have similar clinical symptoms making the differential diagnosis difficult without the use of clinical laboratory diagnostic testing. These symptoms can include fever, myalgia, arthralgia, nausea, vomiting, rash, headache, bleeding manifestations, abdominal pain, and jaundice. Their clinical symptoms are so similar that initially researchers thought DHF epidemics in SE Asia were due to epidemic haemorrhagic fever caused by hantaviruses [6
Dengue has been endemic in Barbados for over 30 years with the circulation of all four serotypes of DENV [9
]. DENV are transmitted year-round and dengue epidemics have occurred in 1995, 1997, 2001, and 2007 [11
]. Notably DHF in Barbados does not appear to be as prevalent as in Asian countries where dengue is more severe [18
]. Though dengue has been endemic in the Caribbean for several decades some of the host factors influencing dengue disease severity and hantavirus disease severity in the region remain unknown. DENV infection can impact on the clinical haematological profile resulting in leukopenia, thrombocytopenia, and haemorrhagic manifestations profile has been well established [19
]. The basis for assessing dengue disease severity using WHO 2009 guidelines is founded on research conducted on host responses during severe and non-severe dengue [23
Hantavirus transmission in Barbados has been detected since 2001 among febrile persons under investigation for acute DENV infection and in wild rats [25
]. Hantavirus infections have remained endemic in Barbados since then however the identity of the circulating strain(s) has proven elusive. Hantavirus infections are detected using Focus SelectDx™ hantavirus ELISA IgM and IgG kits which use a recombinant nucleoprotein (rNP) mixture for detecting antibodies to a broad range of hantavirus strains. including SEOV, HTNV, PUUV, DOBV, and SNV. In the Caribbean serological and molecular evidence of hantavirus transmission exists for Grenada, Trinidad and Cuba (Sin Nombre strain positive by RT-PCR) however no extensive epidemiological data on hantavirus infection exists for the Caribbean [27
]. Recent hantavirus outbreaks in adjacent regions including 4 fatal HPS cases observed in French Guiana (Maripa virus) in 2016 enhance the risk of new and more lethal hantavirus strains entering the Caribbean region via trade and travel [31
Lipopolysaccharide (LPS) or endotoxin is the immunologically relevant component of Gram-negative bacteria and elicits an immune response if present within the body [32
]. Gram-negative bacteria shed endotoxin during their normal growth [33
]. It is comprised of two segments namely a lipid portion (lipid A) and polysaccharide portion (O specific chain and core). Lipid A segment is the most highly conserved whilst the O specific chain is the most variable [34
]. The presence of bacterial endotoxin in serum or blood can arise through (a) active acute/systemic bacterial infection within the body or (b) its translocation (microbial translocation—MT) from the human gut colonized by large numbers of Gram-negative bacteria [34
]. Human host generated response in the clearance of LPS involves the production and binding of serum proteins including lipopolysaccharide binding protein (LBP), bacterial/permeability increasing protein and sCD14, host defence peptides, and chylomicrons [34
]. LBP and sCD14 can enhance the pathogenicity of endotoxin as they induce the production of inflammatory cytokines by monocytes [34
]. The cytokine production in the presence of endotoxin is key in the pathogenesis of the clinical syndromes of shock and multiple organ failure [40
]. Several cytokines are released in response to the presence of endotoxin including tumour necrosis factor (TNF), interleukin (IL)-1, IL-6, IL-8, IL-10 [34
DENV and hantavirus infections in humans target monocytes and tissue macrophages which are in highest concentration in the gut-associated lymphoid tissue (GALT). DENV and hantavirus replication in these monocytes and macrophages can lead to the movement of soluble factors across the intestinal lining resulting in increased endotoxin/LPS levels in the blood and increased severity of disease [41
]. LPS can inhibit DENV infection of macrophages/monocytes but only if present prior to DENV adsorption [43
]. LPS induction of flaviviral disease severity has been previously observed in West Nile virus (WNV) infections [44
]. Bacterial endotoxin contributes to the penetration of WNV from the blood into the central nervous system (CNS) causing a mild WNV infection to become a severe lethal encephalitis [44
]. Human immunodeficiency virus (HIV) also targets monocytes and high endotoxin levels are associated with HIV replication in the GALT due to MT [45
]. DENV replication in intestinal macrophages may lead to a pro-inflammatory environment in which the epithelial cells are disrupted allowing the passage of soluble factors from the gut into the blood [47
]. Some hantavirus infections can have clinical presentations like acute appendicitis suggesting replication in the human intestine [48
]. The detection of hantavirus antigen in the gut epithelium [49
] raised the possibility of MT occurring during acute hantavirus infection but a correlation of MT and disease severity was later confirmed in Argentine HPS patients [42
]. To date no studies have been reported on the role of serum endotoxin in hantavirus or dengue infection and no host markers and their association with disease severity has been investigated among hantavirus patients in the Caribbean.
We aimed to investigate the role of serum endotoxin levels (a) in hantavirus disease severity, (b) in dengue disease severity in the Caribbean using 2009 WHO dengue guidelines and (c) and their correlation with clinical parameters among severe dengue (SD) and hospitalised non-severe dengue (non-SD) patients.
A significant association of mean serum endotoxin and hantavirus infection in addition to hospitalization was observed for the first time ever among hantavirus cases. The mean serum endotoxin levels in hantavirus cases was higher than among non-hospitalized controls. Severe gastrointestinal (GI) symptoms including abdominal pain, nausea and vomiting were previously found in a high percentage of patients with PUUV infection [56
]. This was also observed in this study among hospitalised hantavirus cases suggesting the likelihood of MT in hantavirus infected patients and disease severity. With regards to hantavirus infection the detection of hantavirus antigen using immunohistochemistry (IHC) staining in the biopsied intestines (lamina propria) from Puumala (PUUV) infected patients and similar hantavirus tropism for monocytes and dendritic cells like DENV are suggestive of hantavirus replication in the GALT and microbial translocation [49
]. It is not known whether hantaviruses set the stage for a secondary bacterial infection or are the cause of inflammation [49
]. One study investigating the role of serum biomarkers in hantavirus infection and disease severity showed intestinal fatty acid binding protein (I-FABP) and interleukin 6 (IL-6) cytokine levels were associated with disease severity and poor outcome among Argentine HPS patients [42
]. Recent evidence of hantavirus infection via the intestinal route has been advanced defying conventional thought of hantavirus infection via infectious aerosols opening a new frontier of hantavirus research [59
]. Further studies on the role of MT in hantavirus infection, its role in disease severity in HFRS and HPS is thus warranted.
In this endotoxin study no statistically significant difference in the frequency of gastrointestinal related symptoms was observed between hospitalised and non-hospitalised patients indicating the similarity of clinical presentation regardless of hospitalisation. Though if vomiting is analysed separately there is a statistically significant difference between hospitalised and non-hospitalised patients [52
]. This does however underscore the importance of serum endotoxin levels to distinguish between hospitalised and non-hospitalised hantavirus patients adding support for the role of endotoxin in viral infections and disease severity via MT.
For hospitalised hantavirus patients gastrointestinal related, respiratory symptoms and thrombocytopenia were all statistically significant clinical symptoms observed when compared with non-hospitalised hantavirus patients. Respiratory symptoms can be observed in a significant number of HFRS patients mimicking HCPS thus observing a higher frequency of respiratory symptoms among hospitalised hantavirus patients is not unusual [60
]. Platelets play a significant role in VHFs and their disease severity and for hantaviruses the intensity of platelet β3 integrin factor is higher patients with more severe HFRS disease [61
]. The statistically significant association of thrombocytopenia among with hospitalised hantavirus patients is thus not surprising. A statistically significantly higher proportion of females was observed among hantavirus patients selected in this study. This is also reflected in the overall population infected by hantavirus studies in Barbados during 2008–2016 [52
]. The ratio of males to females in the general population of Barbados is effectively 1:1 however the ratio of hantavirus infected females to males is almost 2:1. Qualitative research among hantavirus patients is needed to determine the cause(s) for this disparity between sexes. Possible theories could include the higher percentage of females working in the sugar cane harvesting industry, higher exposure occupational risk working in sugarcane fields and the higher hantavirus seroprevalence among rodents in sugarcane fields and urban areas.
The correlation of serum endotoxin levels with dengue severity observed in our study appears to be consistent with the 2009 WHO dengue guidelines for dengue severity. A correlation of dengue disease severity and LPS levels exist where DENV replication in intestinal macrophages leads to disruption of intestinal integrity and passage of endotoxin from the gut to the blood [47
]. The presence of LPS in the blood can permit LPS mediated enhancement of DENV replication and synergistic IFN-α production with consequent modulation of disease progression [41
]. A similar effect of LPS and cytokines on dengue disease severity was observed among patients in Brazil with the occurrence of MT and activation of monocytes [62
Other studies have provided evidence for DENV infection, localized intestinal effects of DENV infection and disease severity. Gastrointestinal bleeding has been identified as a positive associated factor for DSS lending further support to the association of serum endotoxin and dengue disease severity via MT and disruption of the gut epithelium [21
]. Gastrointestinal bleeding was associated with DSS rather than other types of bleeding including mucosal and cutaneous and is likely expected if microbial translocation and GI epithelial damage occurs with dengue infection [21
]. In addition, some studies found abdominal pain or tenderness to be a predictor of severe dengue [63
]. This clinical sign may be indicative of the site of viral replication in the GALT. An analysis of 306 fatal cases of dengue to highlight clinical presentations as warning signs of fatal DHF showed massive gastrointestinal bleeding was a major observation [65
]. Studies using intestinal fatty acid binding protein (I-FABP) as a biomarker of intestinal injury showed a correlation of dengue disease severity and elevated I-FABP levels providing further evidence of DENV replication and MT [66
]. Elevated endotoxin levels are present in severe dengue cases compared with non-severe dengue and uninfected controls [67
DENV infection can impact on the clinical haematological profile resulting in leukopenia, thrombocytopenia, and haemorrhagic manifestations profile has been well established [19
]. The parameters identified in our study with significant differences between severe dengue and hospitalized dengue patients were platelet count, PTT and PT. However, these differences were prior to the Bonferroni correction, which reduced the significance due to the very small sample sizes. The parameters analysed were from the 1st day of clinical presentation to the physician and have previously been useful in identifying severe dengue cases. Data from a prospective observational study of Vietnamese children (5–15 years; n
= 2,301) show that daily platelet counts permit accurate discrimination of patients who develop DSS [68
]. A rapid drop in platelet count or thrombocytopenia is a warning sign for dengue severity in 2009 WHO dengue guidelines [69
]. Our findings agree with previous studies on coagulation factors as risk factors for DSS development [21
]. Some conflicting results have been obtained. One Thai study examining clinical laboratory findings collected within 72 h of fever onset from a prospective cohort of children found platelet count along with WBCs, haematocrit and percent monocytes were 97% sensitive to predict DSS patients [71
]. More studies support platelet counts as predictors of dengue severity [72
]. Conversely another study in Thailand found no significant difference in admission haematology laboratory data between both young DHF patients with and without shock concluding that admission haematology data is unable to predict shock [73
]. Our results differ from the latter study and may be due to age, study design, different population genetics and viral genetics of DENVs circulating in Asia.
Normal platelet counts range from 150,000 to 450,000 platelets/µL or 150–450 platelets × 109
. The action of DENV on platelets and their production have been investigated and reviewed [62
]. During acute infection DENV reduces platelets either by decreasing platelet production or increased platelet destruction [62
]. DENV can decrease platelet production by inhibition of bone marrow progenitor cell growth [62
]. DENV also reduces platelet count through direct destruction of platelets by (1) directed binding of anti-dengue NS1 antibodies to platelets leading to complement-mediated lysis [76
], (2) high levels E-selection on endothelial cells promoting platelet adhesion and clearance [78
], (3) direct binding of DENV to platelets via lectin receptors such as CLEC-2 and DC-SIGN [74
] and disseminated intravascular coagulation (DIC) [79
] and increased apoptosis due to binding of DC-SIGN and caspases [80
Red blood cell distribution width-coefficient of variation (RDW-CV) is a measure of the variation of the red blood cell volume. This parameter is capable of change due to the storage conditions of the blood as it has been identified as showing statistically significant changes [81
]. Changes in RDW-CV can lead to anaemia (lowering of RBCs) where DENV infection leads to haemolysis and bleeding reducing the volume of RBCs [82
]. With SD/DHF patients, the risk of bleeding would be higher than those that are non-severe dengue patients.
Some studies have recommended the use of platelet transfusions where low platelet levels are observed i.e., 10–20 × 109
without haemorrhage or 50 × 109
with bleeding or haemorrhage. However, evidence exists that show platelet transfusions are not as effective as expected but can lead to several complications including no significant difference in the development of severe bleeding and time to bleeding cessation, a higher occurrence of pulmonary oedema, longer hospitalisation stays [84
]. Considering this platelet transfusions have been advised against by others [84
This study has some limitations including small sample sizes and possible selection bias. A larger sample size would allow more confidence in the correlations observed however these observations are in agreement with other published studies [42
]. The risk of endotoxin contamination was reduced by using depyrogenated glassware, endotoxin free LAL reagent water and tips. Serum tubes were sampled and tested for the presence of endotoxin (<0.080 EU/mL detection limit) (negative controls) and no detectable endotoxin was found. In addition, glucan blocking buffer Glucashield™ was used to prevent non-specific activation of the clotting cascade of LAL reagent by glucans that may have been present. Whole blood contains natural factors which bind endotoxin reducing its detection. The sample size of this study could have been larger to yield more robust data however the study was limited by the maximum number of severe dengue and hospitalised hantavirus cases, matching controls and availability of sufficient volume of patient sera. Future prospective studies on endotoxin should incorporate the monitoring of serum/plasma cytokines to evaluate its co-effect(s) with the immune response. Other limitations and sources of bias do exist for this study including incomplete clinical referral and hantavirus and dengue diagnostic testing which increases the potential to miss mild cases and non-symptomatic dengue and hantavirus cases.
The cytokine production in the presence of endotoxin is key in the pathogenesis of the clinical syndromes of shock and multiple organ failure [40
]. DENV NS1 protein functions like LPS in septic shock effecting vascular leakage in the endothelium [87
]. The action of elevated serum endotoxin and DENV NS1 levels can possibly exacerbate clinical illness during DENV infection. The role of serum endotoxin and host cytokines on disease severity in other forms of hantavirus clinical presentations other than HPS such as NE and HFRS should be investigated to further understand the host response to hantavirus infections.
This present study represents the first ever study showing an association of serum endotoxin and hantavirus infected patients. In addition, it represents the first evidence of serum endotoxin and its association with dengue severity in the Caribbean. The implications of our studies suggest that viral mediated MT may be one mechanism that enhances both dengue and hantavirus disease severity during acute infection. This also sheds light on other VHF infections such as Ebola virus (EBOV), Marburg virus (MARV), Crimean Congo haemorrhagic fever virus (CCHFV), severe fever with thrombocytopenia syndrome virus (SFTSV), Lassa Fever virus (LFV), and other bunyavirus and filovirus infections and the role serum endotoxin and MT play in disease severity. Further research identifying possible therapeutic targets (e.g., LBP, NS1, CD14) to attenuate disease severity by reducing the effect of MT should be explored.