Diagnostic approaches for dengue infection.

INTRODUCTION: Every year, a significant rise in dengue incidence observed is responsible for 10% of fever episodes in children and adolescents in endemic countries. Considering that the symptoms of dengue are similar to those of many other viruses, early diagnosis of the disease has long been difficult, and lack of sensitive diagnostic tools may be another factor contributing to a rise in dengue incidence. AREAS COVERED: This review will highlight dengue diagnostics strategies and discuss other possible targets for dengue diagnosis. Understanding the dynamics of the immune response and how it affects viral infection has enabled informed diagnosis. As more technologies emerge, precise assays that include some clinical markers need to be included. EXPERT OPINION: Future diagnostic strategies will require the use both viral and clinical markers in a serial manner with the use of artificial intelligence technology to determine from the first point of illness to better determine severity status and management. A definitive endpoint is not in the horizon as the disease as well as the virus is constantly evolving and hence many developed assays need to be constantly changing some of their reagents periodically as newer genotypes and probably too serotypes emerge.

Serological cross-reactivity among common flaviviruses.

The Flavivirus genus is made up of viruses that are either mosquito-borne or tick-borne and other viruses transmitted by unknown vectors. Flaviviruses present a significant threat to global health and infect up to 400 million of people annually. As the climate continues to change throughout the world, these viruses have become prominent infections, with increasing number of infections being detected beyond tropical borders. These include dengue virus (DENV), West Nile virus (WNV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV). Several highly conserved epitopes of flaviviruses had been identified and reported to interact with antibodies, which lead to cross-reactivity results. The major interest of this review paper is mainly focused on the serological cross-reactivity between DENV serotypes, ZIKV, WNV, and JEV. Direct and molecular techniques are required in the diagnosis of Flavivirus-associated human disease. In this review, the serological assays such as neutralization tests, enzyme-linked immunosorbent assay, hemagglutination-inhibition test, Western blot test, and immunofluorescence test will be discussed. Serological assays that have been developed are able to detect different immunoglobulin isotypes (IgM, IgG, and IgA); however, it is challenging when interpreting the serological results due to the broad antigenic cross-reactivity of antibodies to these viruses. However, the neutralization tests are still considered as the gold standard to differentiate these flaviviruses.

Host immune response against DENV and ZIKV infections.

Dengue is a major public health concern, affecting almost 400 million people worldwide, with about 70% of the global burden of disease in Asia. Despite revised clinical classifications of dengue infections by the World Health Organization, the wide spectrum of the manifestations of dengue illness continues to pose challenges in diagnosis and patient management for clinicians. When the Zika epidemic spread through the American continent and then later to Africa and Asia in 2015, researchers compared the characteristics of the Zika infection to Dengue, considering both these viruses were transmitted primarily through the same vector, the Aedes aegypti female mosquitoes. An important difference to note, however, was that the Zika epidemic diffused in a shorter time span compared to the persisting feature of Dengue infections, which is endemic in many Asian countries. As the pathogenesis of viral illnesses is affected by host immune responses, various immune modulators have been proposed as biomarkers to predict the risk of the disease progression to a severe form, at a much earlier stage of the illness. However, the findings for most biomarkers are highly discrepant between studies. Meanwhile, the cross-reactivity of CD8+ and CD4+ T cells response to Dengue and Zika viruses provide important clues for further development of potential treatments. This review discusses similarities between Dengue and Zika infections, comparing their disease transmissions and vectors involved, and both the innate and adaptive immune responses in these infections. Consideration of the genetic identity of both the Dengue and Zika flaviviruses as well as the cross-reactivity of relevant T cells along with the actions of CD4+ cytotoxic cells in these infections are also presented. Finally, a summary of the immune biomarkers that have been reported for dengue and Zika viral infections are discussed which may be useful indicators for future anti-viral targets or predictors for disease severity. Together, this information appraises the current understanding of both Zika and Dengue infections, providing insights for future vaccine design approaches against both viruses.

Effect of Blastocystis sp. in dengue patients-Increase in the treatment cost and exacerbation of symptoms.

Increasing incidences of dengue have become a global health threat with major clinical manifestation including high fever and gastrointestinal symptoms. These symptoms were also expressed among Blastocystis sp. infected individuals, a parasite commonly seen in human stools. This parasite has been previously reported to replicate faster upon exposure to high temperature. The present study is a hospitalized-based cross-sectional study involved the collection of faecal sample from dengue patients. Stool examination was done by in vitro cultivation to isolate Blastocystis sp. Growth pattern of all the positive isolates were analyzed to identify the multiplication rate of Blastocystis sp. isolated from dengue patients. Distribution of Blastocystis sp. among dengue patients was 23.6%. Dengue patients who were positive for Blastocystis sp. infection denoted a significantly higher fever rate reaching 38.73°C (p<0.05) compared to the non-Blastocystis sp. infected patients (38.44°C). It was also found that Blastocystis sp. infected patients complained of frequenting the toilet more than five times a day (p<0.05) compared to those who were non-Blastocystis sp. infected. At the same time, the duration of hospitalization was significantly longer (p<0.05) for Blastocystis sp. infected dengue patients compared to the non-Blastocystis sp. infected patients. Besides, Blastocystis sp. isolated from dengue patients (in vivo thermal stress) showed a higher growth rate compared to the non-dengue isolated which was exposed to high temperature (in vitro thermal stress). Our findings suggest that presence of Blastocystis sp. during dengue infection could trigger the increase of temperature which could be due to highly elevated pro inflammatory cytokines by both parasitic and virus infection. This could justify why the temperature in Blastocystis sp. infected dengue patients is higher compared to the non-Blastocystis sp. infected patients. Higher temperature could have triggered a greater parasite multiplication rate that contributed to the aggravation of the gastrointestinal symptoms.

In vitro and in vivo thermal stress induces proliferation of Blastocystis sp.

Blastocystis sp. is known to be the most commonly found intestinal protozoan parasite in human fecal surveys and has been incriminated to cause diarrhea and abdominal bloating. Binary fission has been widely accepted as the plausible mode of reproduction for this parasite. The present study demonstrates that subjecting the parasites in vitro to higher temperature shows the proliferation of parasite numbers in cultures. Transmission electron microscopy was used to compare the morphology of Blastocystis sp. subtype 3 isolated from a dengue patient having high fever (in vivo thermal stress) and Blastocystis sp. 3 maintained at 41 °C (in vitro thermal stress) and 37 °C (control). Fluorescence stains like acridine orange (AO) and 4',6'-diamino-2-phenylindole (DAPI) were used to demonstrate the viability and nuclear content of the parasite for both the in vitro and in vivo thermal stress groups of parasites. Blastocystis sp. at 37 °C was found to be mostly vacuolar whereas the in vitro thermal stressed isolates at 41 °C were granular with electron dense material seen to protect the granules within the central body. Parasites of the in vivo thermal stressed group showed similar ultrastructure as the in vitro ones. AO and DAPI staining provided evidence that these granules are viable which develop into progenies of Blastocystis sp. These granular forms were then observed to rupture and release progenies from the mother cells whilst the peripheral cytoplasmic walls were seen to degrade. Upon exposure to high temperature both in vitro and in vivo, Blastocystis sp. in cultures show higher number of granular forms seen to be protected by the electron dense material within the central body possibly acting as a protective mechanism. This is possibly to ensure the ability to survive for the granules to be developed as viable progenies for release into the host system.

High lipid storage in vacoular forms of subtype 6 Blastocystis sp. in ostrich.

BACKGROUND: Blastocystis sp., a widely prevalent intestinal protozoan parasite is found in a wide range of animals, including humans. The possibility of zoonotic transmission to human from birds especially ostriches led us to investigate on the cross infectivity of Blastocystis sp. isolated from the ostrich feces as well as the phenotypic and subtype characteristics. There is a need to investigate this especially with the rising number of ostrich farms due to the growing global ostrich industry. FINDINGS: 100% of the ostriches were found to be positive for Blastocystis sp. using the in-vitro cultivation method. Transmission electron microscopy revealed high electron dense material in the central body of the vacoular forms. The membrane layer of the ostrich isolate was significantly (p = 0.003) thicker as compared to human isolate. Sudan staining revealed that this was lipid accumulation. We provide evidence for the first time, the existence of subtype 6 which has been previously reported only in pigs and cattle. Cysts, ranging from 3.0 to 7.0 µm in diameter caused experimental infection in Sprague Dawley rats implicating that Blastocystis sp. isolated from ostriches exhibits low host specificity. CONCLUSION: The study for the first time demonstrates that Blastocystis sp. subtype 6 do exist in ostriches and show high lipid storage in the vacuoles of the parasites. The study further provides evidence for potential zoonotic transmission in ostrich farms as Blastocystis subtype 6 can infect rats and the same subtype have been previously reported in humans.