*2.4.1.10. Automated equipment for virus counting*

There are redesigned flow cytometers to quantify a virus in solutions like The ViroCyt® Virus Counter (VC) 2100 (ViroCyt, Boulder, CO, USA) using a specific fluorescent dye for the envelope proteins and the other one for the nucleic acids that allows recognizing viral particles having both components in its structure. Then, it eliminates anything that represents one type of fluorescent [27].

same as 50% as a positivity criterion. In case of having only a serum sample, a primary infection is considered when the antibody IgG titer is ≤20, and a secondary infection is considered if the antibody titer is ≥1280. In both cases, the sample must be collected on 5–7 days. If there are paired samples, a primary infection is considered when the seroconversion in antibody titers from the acute and convalescent phase occurs, and a secondary infection is considered when the antibody titers increase four times or more between the acute and convalescent

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

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It was used in some studies to identify the infected people in an early stage of dengue infection. Thus, it was found 100% of sensitivity in people with secondary dengue infection after symptom onset-day 1. However, the results were not very favorable for primary infections [29].

This test is considered the gold standard to detect neutralizing IgG antibodies because they have high sensitivity but can have a cross-reaction among members of flavivirus group. It is based on the binding of antibodies present in the sample which contains a known virus load (working dilution). The mixture of both is incubated and inoculated in a cell line until forming lytic plaques that are observed when coloring the cell monolayer. All samples neutralizing and avoiding the forming of certain number of plaques (being the most commonly used for 50–90% reduction) are with the presence of neutralizing antibodies indicating exposure to dengue. Using 1 in 30 diluted serum samples allowed discriminating between DENV-1 and DENV-2 serotypes in collected serum in Cuba, before and after the 1981 epidemic caused by DENV-2. There are many different variants of a PRNT test that could produce a variation in the results when being compared with using different used reduction rates, different PRNT methods (solid or semi-solid), and different cell lines. The most used for dengue are VERO cells that are recommended by World Health Organization (WHO), and BHK-21 that are used by other laboratories like Pedro Kouri Institute of Cuba. The use of different genotypes can alter the antibody titer results. Kochel et al. [30] evaluated Peruvian samples infected with DENV-2 American genotype. Antibody titers were found in higher levels when using the same genotype rather than using the Asian genotype. PRNT can be used to differentiate dengue infection to yellow fever infection. It has been found that the lowest serum dilution capable of distinguishing between both infections is 1 in 5. In a dengue secondary infection, PRNT can have a cross-reaction with other serotypes. In infected populations with sequentially different serotypes of dengue, the highest antibody titer pertains to the first infection

It combines the PRNT test with immunofluorescence or ELISA to count fluorescent foci or spots and calculate their reduction rate in the samples. Among its advantages, this allows reducing the days to obtain the results and undertaking studies with native strains so that

phase, or in both serums [6].

*2.4.2.4. IgA ELISA test*

*2.4.3. Neutralization test*

*2.4.3.1. Plaque reduction neutralization test (PRNT)*

(the "original antigenic" sin phenomenon) (**Figure 4**) [5–7, 30–32].

*2.4.3.2. Focus reduction neutralization test (FRNT)*
