Exploring Dengue Virus Infections with Metabolomics

Photo Credit: MedicalRF.com/Getty Images

Researchers from Colorado State University have demonstrated the viability of exploratory metabolomics and hydrophilic interaction liquid chromatography (HILIC)–mass spectrometry (MS) in identifying biomarkers associated with dengue virus (DENV) infections, such as dengue fever (DF), dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS).¹

Epidemic DF and dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS) are an enormous public health concern throughout tropical and subtropical countries. There were approximately 100 million diagnosed cases in 2010, but potentially 300 million go unreported because of diminished disease symptoms or substandard clinical data collection.^2,3 Furthermore, the latest available data suggests that around half the world’s population are at risk of contracting dengue virus.

“In part, this is a consequence of the world’s population growth and the consequential development of new habitational zones in areas that were previously uninhabited or wild, and that may not have full sanitation and clean water services available,” explained Natalia Voge of Colorado State University and lead author of the study.

Global warming may also be contributing to the increase risk of DENV infections,^4,5 enabling Aedes aegypti mosquitos to expand habitually and further increase the frequency of human vector interaction. These challenges are further compounded by diagnostic and treatment limitations, as no method of determining virulence is available. Around 20% of DF cases will progress to the more serious form of DHF with 3-5% developing into the potentially fatal DSS.

“Currently, there is no prognostic algorithm or ‘early diagnosis’ that can accurately, and in a timely manner, predict which patients will have a mild DF case, from the patients that are at first clinically diagnosed as dengue fever but could, in a matter of hours, develop haemorrhagic symptoms that could progress to a potentially fatal case of dengue shock syndrome,” said Voge.

Clinic accessibility is a major barrier to effective care in many areas heavily affected by dengue infections with accurate initial diagnosis being crucial. However, efforts to develop novel diagnostics and treatments have been hampered by the serological and metabolomic complexity of dengue disease. Combating dengue hinges on disease metabolites being identified to act as markers in diagnostic tests and potential targets for pharmaceutical treatments.

Using HILIC–MS researchers performed an exploratory metabolomic study to characterize the serum metabolome of patients experiencing the various dengue disease outcomes and identified 191 metabolites differentiating DF patients from non-dengue (ND) patients in Nicaraguan samples. A further 83 metabolites differentiated DHF/DSS and DF outcomes. In Mexican samples, 306 metabolites differentiated DF from ND and 37 differentiated DHF/DSS and DF outcomes.

The structural identities of 13 metabolites were later confirmed using tandem mass spectrometry (MS–MS) including isotypes of vitamin D3. “Vitamin D3 in its active form (1-25 OH vit D3) is critical for endothelial cell homeostasis, and in our studies that metabolite was found to be downregulated in patients with severe dengue; this was a very exciting moment, because in DHF and DSS the endothelial cells are the most affected cells that cause plasma leakage leading to severe symptoms,” said Voge.

“Another very interesting metabolite and its proposed pathways are the lysophosphatidylcholines 16:0 and 18:1,” she continued. “These single fatty acid chain lipids are involved in the alteration of membrane structures and can mediate acute inflammation and regulate pathophysiological events in the vasculature and may alter the homeostasis of the vascular endothelium, causing endothelial cell instability, barrier dysfunction, and vascular leakage, which is a major component of DSS.”

Despite these interesting findings, a lot of work is still required to develop improved diagnostic and prognostic capabilities. “Our analysis was performed in small sample sizes (101 patients from Mexico, 88 from Nicaragua), and further analysis needs to be done with larger sample numbers to quantify the exact abundance of the metabolites affected [by MRM platform]; also clinical trials that will be done in Nicaragua still need to take place to corroborate that these biomarkers can accurately predict severe dengue in the field,” explained Voge.

The researchers involved in this project are currently analyzing a further set of 500 serum samples after funding for the project was extended. Increasing the number of candidate biomarkers is a key aim of that project. The end goal is to develop a point of care diagnostic test that is effective, capable of being used at patient bedsides, and also cost effective for the developing nations in which dengue disease places the heaviest burden. - L.B.

References

1. N.V. Voge et al.,PLoS Negl. Trop. Dis. 10(2), (2016).
2. S. Bhatt et al.,Nature 469, 504–507 (2013).
3. D.S. Shepard et al.,PLoS Negl. Trop. Dis. 8(11), (2014).
4. S.Hales et al., Lancet 360, 830–834 (2002).
5. E.Van Kleef et al., TropIKA.net Available from http://journal.tropika.net/scielo.php?script=sci_arttext&pid=S2078-86062010005000001&lng=en&nrm=iso (2009).