**2.3. Primary and secondary dengue infections and dengue diagnostic**

Although most infections are asymptomatic or subclinical, a set of symptoms starts after a dengue infection elapses the 4–10-day incubation period. A four-fold increase of the IgG antibody titers in matched serums measured by ELISA IgG test or hemagglutination inhibition indicates recent flavivirus infection. When people are infected with the virus on the first time, dengue infections are known as a primary infection in which a viral load and the relevant antibody formation (IgM, IgG and IgA) are triggered. In a primary infection, the titer of IgM is generally much higher and more specific than in a secondary infection. Some studies consider that an infection is primary if the IgM/IgG relation is higher than 1.2 with diluted samples at 1:100 or 1.4 using diluted serum at 1:20. When people are previously exposed to any serotype or flavivirus, or even after a vaccine (i.e., yellow fever vaccine), dengue infections are secondary and the IgM/IgG relation is lower than 1.2 or 1.4. In secondary infections, the IgG is detected in the highest levels and even on the acute phase. It remains higher for 10 months and even lifelong in order to consider a person being infected with dengue virus (DENV), the following laboratory test interpretations need to be followed:

technique in order to extract plasma RNA so that the original technique is used for cell or tissue samples being more cost effective than the kit commercial one. It was found that 34 samples out of 47 were positive by using the Chomczynski-Sacchi method, and the remaining

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The RT-PCR techniques and their different variants are converted into one of the main tools for diagnosing DENV and other arbovirosis. Less time to process the results, being able to identify the circulating serotypes of the virus, presenting the highest sensitivity and specificity levels are among its advantages. This type of test has the benefit of obtaining rapid results while identifying circulating serotypes of dengue. The RT-PCR technique is the extraction of a RNA sample followed by a reverse transcription process, the actual PCR (nested or not), and the last screening in gel obtaining a qualitative result. The technique developed by Lanciotti and his collaborators or the variant developed by Harris and his collaborators, which have been used for many years, is recommended by the Pan American Health Organization (PAHO/OPS). The technique developed by Lanciotti starts with converting RNA into DNA by using a reverse transcriptase enzyme. Then, the PCR is carried out when primers are used to amplify prM genes and C virus areas to continue with a specific primer-nested PCR for each virus serotype. Harris and his collaborators developed a multiplex RT-PCR from Lanciotti and his collaborators' method that uses five pairs of primers (four specific pairs for serotypes and one pair of the region of the capsid gene). There are many different variants in this technique having different sensitivity levels that are used in research laboratories. One of the main disadvantages is the possible existence of a false positive due to the contamination produced by amplicons during the reverse transcription of the genetic material, and it is necessary to consider that a negative result in these tests does not rule out DENV infection or other arboviruses, and the analysis must be complemented by serological findings (**Figure 1**) [9, 11].

27 samples were positive by using the kit commercial method [8–10].

*2.4.1.3. RT-PCR multiplex to DENV and different microorganism identifications*

the following results for DENV like DENV-1 (Hawaii) 2.7 × 101

Chikungunya (CHIKV), Zika (ZIKV), Yellow Fever (YFV) and DENV are arboviruses with the highest prevalence in the American continent. They are transmitted by the same vector *Aedes aegypti y Aedes albopictus* facilitating its cocirculation in some areas of the region. Because of these arboviruses, the affected patients develop similar symptoms but its clinical management and its possible results as the aftermath of the disease, and mortality rates are different. They can even produce coinfection with other microorganisms making a proper identification necessary in an early stage of the disease (acute phase). There are commercial and standardized testing in a laboratory allowing qualitative identification of DENV, ZIKV, and CHIKV in serum, plasma, and even some urine samples [12]. Other commercial testing like FilmArray Global fever panel has the capacity of detecting genetic material in viruses, bacteria and protozoa (nine viruses like YFV, DENV, ZIKV, WNV, CHIKV, among others, six bacteria and four protozoa) in whole blood (EDTA), with automated equipment. A study to determine a detection limit for microorganisms using FilmArray Global fever panel found

, DENV-2 (New Guinea C)

*2.4.1.2. Reverse transcription polymerase chain reaction (RT-PCR)*


## **2.4. Laboratory test**

The laboratory tests can be interchangeably used in different researches, both basic and applied ones. We can classify them into direct methods that allow virus detection or part of its structure and indirect methods which identify a reaction produced by the presence of DENV in the organism.

#### *2.4.1. Direct detection methods*

#### *2.4.1.1. Viral RNA extraction*

The genetic material extraction has a key role for PCR tests so that the quality of a product extracted can vary depending on the type of sample being used, and the extraction method applied will directly affect the test sensitivity. The dengue RNA can be recovered from serum, blood, urine, plasma samples and other organs. However, the viral load in blood is much higher (7.9 × 102 –1.9 × 10<sup>5</sup> PFU/mL) in comparison with saliva and urine samples (1 × 101 –5 × 101 PFU/mL). The RNA extraction can be done by guanidine thiocyanate and trizol methods and by the use of commercial kits like Qiagen kit (QIAamp® RNA Viral mini kit), etc. RNA extraction techniques in serum/plasma samples from patients using QIAamp® UltraSens Virus Kit (Qiagen Inc., Valencia, USA) were compared to the modified Chomczynski-Sacchi extraction technique in order to extract plasma RNA so that the original technique is used for cell or tissue samples being more cost effective than the kit commercial one. It was found that 34 samples out of 47 were positive by using the Chomczynski-Sacchi method, and the remaining 27 samples were positive by using the kit commercial method [8–10].

#### *2.4.1.2. Reverse transcription polymerase chain reaction (RT-PCR)*

**2.3. Primary and secondary dengue infections and dengue diagnostic**

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

following laboratory test interpretations need to be followed:

suggestive case [5–8].

*2.4.1. Direct detection methods*

*2.4.1.1. Viral RNA extraction*

–1.9 × 10<sup>5</sup>

**2.4. Laboratory test**

in the organism.

higher (7.9 × 102

Although most infections are asymptomatic or subclinical, a set of symptoms starts after a dengue infection elapses the 4–10-day incubation period. A four-fold increase of the IgG antibody titers in matched serums measured by ELISA IgG test or hemagglutination inhibition indicates recent flavivirus infection. When people are infected with the virus on the first time, dengue infections are known as a primary infection in which a viral load and the relevant antibody formation (IgM, IgG and IgA) are triggered. In a primary infection, the titer of IgM is generally much higher and more specific than in a secondary infection. Some studies consider that an infection is primary if the IgM/IgG relation is higher than 1.2 with diluted samples at 1:100 or 1.4 using diluted serum at 1:20. When people are previously exposed to any serotype or flavivirus, or even after a vaccine (i.e., yellow fever vaccine), dengue infections are secondary and the IgM/IgG relation is lower than 1.2 or 1.4. In secondary infections, the IgG is detected in the highest levels and even on the acute phase. It remains higher for 10 months and even lifelong in order to consider a person being infected with dengue virus (DENV), the

• When a sample taken from the acute phase is positive for dengue due to the PCR test, viral isolation, and IgM serocoversion in matched serum samples, the IgG seroconversion in matched serum samples or the fourfold increase of IgG titer is considered confirmed cases. • When a positive IgM occurs in a single serum sample or a positive IgG in a single sample with hemagglutination inhibition titer is the same or higher than 1:1280, it is considered a

The laboratory tests can be interchangeably used in different researches, both basic and applied ones. We can classify them into direct methods that allow virus detection or part of its structure and indirect methods which identify a reaction produced by the presence of DENV

The genetic material extraction has a key role for PCR tests so that the quality of a product extracted can vary depending on the type of sample being used, and the extraction method applied will directly affect the test sensitivity. The dengue RNA can be recovered from serum, blood, urine, plasma samples and other organs. However, the viral load in blood is much

PFU/mL). The RNA extraction can be done by guanidine thiocyanate and trizol methods and by the use of commercial kits like Qiagen kit (QIAamp® RNA Viral mini kit), etc. RNA extraction techniques in serum/plasma samples from patients using QIAamp® UltraSens Virus Kit (Qiagen Inc., Valencia, USA) were compared to the modified Chomczynski-Sacchi extraction

PFU/mL) in comparison with saliva and urine samples (1 × 101

–5 × 101

The RT-PCR techniques and their different variants are converted into one of the main tools for diagnosing DENV and other arbovirosis. Less time to process the results, being able to identify the circulating serotypes of the virus, presenting the highest sensitivity and specificity levels are among its advantages. This type of test has the benefit of obtaining rapid results while identifying circulating serotypes of dengue. The RT-PCR technique is the extraction of a RNA sample followed by a reverse transcription process, the actual PCR (nested or not), and the last screening in gel obtaining a qualitative result. The technique developed by Lanciotti and his collaborators or the variant developed by Harris and his collaborators, which have been used for many years, is recommended by the Pan American Health Organization (PAHO/OPS). The technique developed by Lanciotti starts with converting RNA into DNA by using a reverse transcriptase enzyme. Then, the PCR is carried out when primers are used to amplify prM genes and C virus areas to continue with a specific primer-nested PCR for each virus serotype. Harris and his collaborators developed a multiplex RT-PCR from Lanciotti and his collaborators' method that uses five pairs of primers (four specific pairs for serotypes and one pair of the region of the capsid gene). There are many different variants in this technique having different sensitivity levels that are used in research laboratories. One of the main disadvantages is the possible existence of a false positive due to the contamination produced by amplicons during the reverse transcription of the genetic material, and it is necessary to consider that a negative result in these tests does not rule out DENV infection or other arboviruses, and the analysis must be complemented by serological findings (**Figure 1**) [9, 11].

#### *2.4.1.3. RT-PCR multiplex to DENV and different microorganism identifications*

Chikungunya (CHIKV), Zika (ZIKV), Yellow Fever (YFV) and DENV are arboviruses with the highest prevalence in the American continent. They are transmitted by the same vector *Aedes aegypti y Aedes albopictus* facilitating its cocirculation in some areas of the region. Because of these arboviruses, the affected patients develop similar symptoms but its clinical management and its possible results as the aftermath of the disease, and mortality rates are different. They can even produce coinfection with other microorganisms making a proper identification necessary in an early stage of the disease (acute phase). There are commercial and standardized testing in a laboratory allowing qualitative identification of DENV, ZIKV, and CHIKV in serum, plasma, and even some urine samples [12]. Other commercial testing like FilmArray Global fever panel has the capacity of detecting genetic material in viruses, bacteria and protozoa (nine viruses like YFV, DENV, ZIKV, WNV, CHIKV, among others, six bacteria and four protozoa) in whole blood (EDTA), with automated equipment. A study to determine a detection limit for microorganisms using FilmArray Global fever panel found the following results for DENV like DENV-1 (Hawaii) 2.7 × 101 , DENV-2 (New Guinea C)

*2.4.1.5. Viral isolation in cell lines*

level 2 is needed [18].

[6, 9, 17].

[19, 21].

good results for VEE isolation [19–22].

Viral isolation in cell cultures or mosquitoes followed by the virus detection using indirect immunofluorescence is considered as gold standard [8, 17]. In order to carry out the DENV viral isolation and as a general rule to any virus, it is necessary to consider the following:

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• To know the isolated virus (virus characteristics, replication, transmission mechanism, etc.) • To know which biosafety level a virus can be performed. In the case of DENV, biosafety

• To determine which cell line to use and to be able to isolate the virus, it is essential to identify the most sensitive cell line from mosquitoes and mammals, and its use for the isolation and DENV propagation, being the most sensitive cell lines of mosquitoes as follows:

• C6/36: they are easy cell propagation, highly sensitive to DENV infection, and culti-

• C6/36 HT (hot temperature): they can be spread at 34°C and have a bigger sensitivity to detect DENV. This cell line maintains its high sensitivity only for some weeks due to its higher temperatures, so some researchers suggest adapting at 34°C, a C6/36 cell

• TRAS-284-SF cell has the main benefit of not requiring the use of fetal bovine serum (FBS) as a culture medium, and it presents a higher sensitivity to the DENV isolation

• Knowing the viral isolation technique that provides better results to isolation and virus propagation. The standard method is based on the virus propagation in a sensitive cell line for inoculating a previous diluted sample in a cell culture medium. After the infection process, the cultures are placed on incubation for the binding of the virus to the cell; subsequentially, it is placed on a means of maintenance with the essential nutrients to maintain the live cultures for a period of time that can be 13–15 days. Then, the infected cells are recovered, and the virus presence is determined by an immunofluorescence process, ELISA, molecular techniques, and others. Bottles, tubes, 6–96 well culture plates, and others are used in order to sustain the binding to cell cultures. A modified shell vial technique allows the recovery of a higher number of YFV, SLV, WNV, ILHV, GCV, OROV, MAYV and DENV isolations. This technique follows the same steps as a standard method, but after inoculating cells, the cultures are centrifuged to velocities between 1800 and 2200 rpm. This technique can also be used to isolate DENV coinfections. However, it seems not to have

• The sample type to be used. The sample type to be used and its proper preservation until the processing time are extremely important to isolate a virus. The most used dilutions to a viral isolation vary from 1:5 to 1:20. A very concentrated dilution of the sample could generate a toxic effect in the cells. On the other hand, a much diluted sample could cause the inability of isolating the virus because of having a low concentration virus in the inoculum

vated at 28°C that are obtained from salivary glands of *Aedes albopictus*.

line growing at 28°C, and using an alternative method.

**Figure 1.** Reverse transcriptase semi-nested reactions, using primers targeted to the C/PrM genomic region as described by Lanciotti et al. (a photo taken on April 24, 2018 in the molecular biology laboratory with the authorization of the U.S. Naval Medical Research Unit Six (NAMRU-6).

3.6 × 101 , DENV-3 (H87) 1.6 × 103 and DENV-4 (H241) 7.6 × 101 . The main advantage of this test is time reduction to obtain results (about an hour), discriminating the amount of pathogens, and minimizing cross-contamination problems so that all reactions are carried out among a closed system. The cost of the product and a machine analyzing only a sample at a time is within its limiting [13].

#### *2.4.1.4. Real-time PCR*

It uses conventional RT-PCR principles, and it combines with fluorochromes like SYBR Green or TaqMan probes with fluorochromes capable of producing proportional fluorescence to the DNA copy samples. The strengths of this test are the same as the conventional RT-PCR, it also reduces time when releasing the results as well as the cross-contamination risk post PCR, the levels of sensitivity and specificity are higher than the conventional RT-PCR, and overall it allows quantifying the genetic material. There are different commercial kits in the market to diagnose DENV, and its sensitivity and specificity levels vary when they are compared among them [14, 15]. The CDC elaborated a real time RT-PCR in order to diagnose four serotypes in serum or human plasma samples using an ABI 7500 FAST DX thermo-cycler of Applied Biosystems and hydrolysis dual-marker TaqMan probes, and it is the first RT-PCR approved by Food and Drug Administration (FDA) to detect DENV [16].
