**2.6. The future of laboratory tests and its connection with dengue studies**

done nor using sophisticated equipment. Within its limitations, it is not possible to identify the circulating serotype in a virus, the band intensity is not related to the antibody titers nor the actual NS1 antigen in the sample, cross-reactions with other flavivirus are common, a negative result in these tests does not exclude that the patient has been exposed to the virus, and some samples present high titers of rheumatoid factor affecting the results. The positive

**Figure 6.** Indirect immunofluorescence test performed on C6/36 cells. In (A), uninfected red cells are observed and in (B), infected green cells are observed (a photo taken on April 24, 2018 in the serology laboratory with the authorization of the

Studies to evaluate antivirals against dengue, vaccines, plant extracts, etc. mainly use the mouse model and nonhuman primates (NHPs). However, each of them presents a series of constraints that impair research developments. The infections of the mouse model are used for pre-clinical development of vaccines. In this model, a neurotropic disease can occur; however, this does not usually happen in humans. Some mice like C57BL/6, BALB/c and A/J enable viral replication, but A/J and BALB/c mice develop paralysis. Some studies show that C57BL/6 mice can have hemorrhages when being infected with a DENV2 strain 16681 using a virus with a titer of 3 × 109 PFU/mL. Moreover, an infection and endothelial cell damage as well as hemorrhages in tissues can occur without showing signs of disease. The DENV replication in mice is low, but suckling mice inoculated by an intracranial via at high DENV doses can induce neurological illnesses and paralysis that is used to measure the effectiveness of the vaccines. They can also be used to test a virus neurovirulence or an attenuation to produce vaccines. NHPs are used to research an immune reaction against DENV, an evaluation of candidates' vaccines, a replication kinetic, etc. This model has a major constraint because it does not produce signs of clinical disease. Nonetheless, DENV can infect some cells in the body. An inoculation with 10<sup>5</sup> PFU DENV concentrations enables a lower viral replication in humans, and it is restricted to lymphoid-rich tissues producing lymphadenopathy, lymphocytosis, and leukopenia. Rhesus macaques can produce antibodies against DENV and a similar viral load to the humans. The inoculation of high titer DENV via intravenous in rhesus macaques can produce hemorrhage. The physiology of pigs is similar to the human one. Yucatan miniature swine models present

samples for these results should be verified with other alternative methods [28].

**2.5. The animal models for dengue virus**

U.S. Naval Medical Research Unit Six authorization (NAMRU-6)).

70 Dengue Fever - a Resilient Threat in the Face of Innovation

Throughout a dengue research, the use of laboratory tests play a fundamental role in identifying the virus (serotype, genotype and lineage), its genetic material, viral proteins, the presence of antibodies against the virus or assessing the patient's condition, and for instance, hematocrit, platelet count, white blood cell count, blood count, etc. together with patient's signs and symptoms and some epidemiological criteria, it allows categorizing the disease for dengue with or without warning signs or severe dengue. This classification assists in deciding what therapy to choose and preventing from a severe dengue development. Besides the above mentioned techniques in this chapter, there are laboratory techniques which are not commonly used at present because they are expensive and/or they require sophisticated equipment but they could be used for DENV studies. Among them, there is the transmission electron microscopy (TEM) which is a gold standard technique for the absolute quantification of particles. However, it does not allow differentiating an infectious virus from a noninfectious one. The high performance liquid chromatography (HPLC) where the virus load is quantified through the UV analysis of fractions produced during HPLC. Flow cytometry (FC) can be used to quantify viral proteins being present on the surface of the infected cells. The next-generation whole genome sequencing is the most advanced sequencing technology which allows learning the complete and detailed genome sequence of an organism in a short period of time (days), though its cost is still quite higher to some researches [27, 38].

Dengue researches in the future will try to respond to different problems, needs, and gaps in knowledge like:

The production and improvement of the vaccine against DENV like Dengvaxia® (CYD-TDV), produced by Sanofi Pasteur, is a prophylactic, tetravalent, live attenuated recombinant viral vaccine which uses a 3-dose vaccination schedule, and it is recommended for people ranged from 9 to 45, or 9 to 60 year old (depending on the license) [39]. Nonetheless, it is necessary to continue with searches to develop a vaccine which can be used from the first year of life enabling to achieve the maximum immunity with a single dose and can be used in different endemic areas obtaining the same immunity level regardless the disease prevalence.

Today, it is acknowledged that the immunity against dengue after a natural infection can be for a lifetime [5]; however, some studies indicate that a dengue reinfection may occur under certain conditions [40].

The relation between chronic diseases and dengue has been studied as risk factors in order to determine the severity of dengue disease in individuals. In recent decades, the increase of chronic diseases and being one of the main causes of death in today's world require to continue and deepen the study of these diseases as well as its association with the dengue disease [5].

**Acknowledgements**

**Conflict of interest**

**Disclaimer**

**Author details**

**References**

17-05-2018]

331328015005

www.who.int/iris/handle/10665/44504

Juan Samuel Sulca Herencia

Address all correspondence to: juan.sulca4@unmsm.edu.pe

Universidad Nacional Mayor de San Marcos, Lima, Peru

The authors declare no conflict of interest.

I would like to thank to the U.S. Naval Medical Research Unit Six (NAMRU-6) and the virology and molecular biology laboratory of the Faculty of Biology at the National University of San Marcos for contributing with the photos of the laboratory tests mentioned in this chapter.

Laboratory Tests Used in the Diagnostic and Research of Dengue Virus: Present and Future

http://dx.doi.org/10.5772/intechopen.80519

73

The views expressed in this laboratory test used in the diagnostic and research of dengue virus: present and future are those of the author and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the U.S. Government.

[1] World Health Organization. Dengue and Severe Dengue [Internet]. 2018. Available from: http://www.who.int/news-room/fact-sheets/detail/dengue-and-severe-dengue [Accessed:

[2] Forshey BM, Guevara C, Laguna-Torres VA, Cespedes M, Vargas J, et al. Arboviral etiologies of acute febrile illnesses in Western South America, 2000-2007. PLoS Neglected

[4] Donis JH. Evaluación de la validez y confiabilidad de una prueba diagnóstica. Avances en Biomedicina. 2012;**1**(2):73-81. Available from: http://www.redalyc.org/articulo.oa?id=

[5] WHO. Dengue guias para el diagnóstico, tratamiento, prevención y control [Internet]. Ginebra: World Health Organization; 2009 [cited 17 May 2018]. Available from: http://

Tropical Diseases. 2010;**4**(8):e787. DOI: 10.1371/journal.pntd.0000787 [3] Gordis L. Epidemiology. 5th ed. Elsevier Saunders: Barcelona; 2014. p. 391

In endemic areas, dengue studies in blood banking are essential especially in epidemic outbreaks so that when there is a large amount of asymptomatic DENV, the virus may be transmitted through blood transfusions [41].

Individuals with inapparent dengue virus infection are considered dead-end hosts for transmission because they do not present high enough levels of viremia to infect mosquitoes; nonetheless, some studies show that asymptomatic people for dengue can transmit the virus to mosquitoes when bitten, although having a lower average of viremia, increasing the risk of disease spread in different areas [42]. Therefore, it is important to carry out studies with individuals presenting inapparent dengue virus infections aimed at breaking the transmission cycle and avoiding disease spread.

Wildlife mammals with genetic material and antibody against DENV like bats have been found, but their participation in the transmission cycle have not been approved yet [43].

Because of not having found an animal model able to replicate the dengue disease, it is necessary to continue searching for different animal models which can support the study of this disease so that the use of humanized mice becomes an attractive area of research [36, 37].

The development of new drugs and the vegetal compound assessment with antiviral activity against DENV are needed because there is no specific treatment for this disease yet.

The use of mathematical models to predict the pace of dengue spread based on clinical data and laboratory results may support the prognosis of this disease.

DENV has a type of RNA genome, with a dependent RNA polymerase without a corrective activity being able to accumulate genetic material changes leading to a false positive increase in PCR tests [11]. Thus, it is required to evaluate the necessity of primer redesigning which allows detecting a larger amount of DENV circulating strains worldwide as well as its standardization and assessment.

DENV coinfections along with other microorganisms circulating in the same endemic areas like DENV/*plasmodium* Sp. may be frequent depending on the disease prevalence in the area, especially if they are transmitted by the same vector in cases like DENV, CHIKV and ZIKV. Some studies have shown infections of *Aedes aegypti* with MAYV causing a concern about a possible natural transmission of such disease in urban areas [22, 44].
