Dengue: Characteristics and Laboratory Diagnosis

Dengue is the most prevalent urban arthropod-borne disease in the Americas and, along with other arboviruses, represents a major global public health problem. Infections are more recurrent in underdeveloped or developing countries, and there is a high likelihood of underreporting cases due to a lack of efficient epidemiological surveillance. In Brazil, according to the latest epidemiological report monitoring arbovirus cases in the country, nearly 1.5 million probable cases were reported in the year 2022, representing a 162.5% increase compared to 2021.

Dengue is a febrile disease that can be transmitted through vectors, vertically, and through blood transfusions. The vector-borne transmission is the most common form, and vertical transmission is very rare. The disease can range from mild to severe, with most cases not having critical clinical evolution. However, it can be fatal when there is clinical aggravation. It can affect individuals of any age, gender, or race. Nevertheless, the elderly, pregnant women, children, and individuals with chronic diseases such as diabetes, sickle cell anemia, hypertension, asthma, and previous infections with other virus subtypes are more susceptible to severe forms of the disease. Additionally, the clinical signs of other urban arboviruses like Zika and Chikungunya are generally similar, making it challenging to diagnose and manage the disease properly, which can lead to severe cases and deaths.

 

SYMPTOMS

The infection can be asymptomatic in some individuals while causing classical febrile symptoms in others. The incubation period of the disease is 4 to 10 days after contact with the virus, and acute symptoms appear after this period, lasting 2 to 7 days. Typically, the disease presents initially with fever above 38°C, headache, retro-orbital pain, myalgia, arthralgia, vomiting, diarrhea, nausea, and swollen lymph nodes. In most affected individuals, there are maculopapular rash lesions on the face, trunk, and limbs. After the febrile phase, between the 3rd and 7th day of symptom onset, warning signs may emerge, such as persistent bloody vomiting, continuous severe abdominal pain, mucosal bleeding, postural hypotension, lethargy, fluid accumulation, and hepatomegaly. These signs suggest clinical worsening and subsequent complications related to plasma leakage leading to shock, severe bleeding, respiratory distress, and severe organ involvement, particularly in the kidneys and liver. Severe dengue is one of the main causes of death in children in some Asian and Latin American countries, mainly due to the difficulty of early association of clinical manifestations with the disease, leading to the aggravation of the clinical condition.

 

VIRUS

The causative agent of the disease is the dengue virus (DENV), which has four circulating subtypes in the Americas: DENV-1, DENV-2, DENV-3, and DENV-4 with distinct lineages and genotypes. It is classified as an arbovirus because its replicative cycle occurs partially in arthropods and is capable of being transmitted through these insects. In this context, the mosquito species Aedes aegypti and Aedes albopictus are important vectors of the virus to humans. Mosquito infestations, especially of Aedes aegypti, are common in areas with high population density, providing a food source and breeding sites for females. These infestations mainly occur in the summer when there is more intense rainfall and high temperatures, factors that contribute to the hatching of eggs. Another factor is related to infrastructure problems in some areas, such as a lack of adequate water supply, which results in the storage of water in large containers without sufficient sealing and becomes targets for arthropods.

Belonging to the Flavivirus genus and the Flaviviridae family, the dengue virus is a single-stranded positive-sense RNA virus. Arboviruses are enveloped and spherical, with a single open reading frame (ORF) that is translated into a single polyprotein. During replication, the viral protein is cleaved, resulting in 10 mature proteins. The N-terminal of the polyprotein encodes the structural proteins of the capsid (C), membrane (prM/M), and envelope (E). The rest encodes the other 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) (figure 2). The NS1 protein plays an important role in viral replication and is strongly involved in stimulating the immune response against DENV.

 

Fonte: BMC, 2022

 

HUMORAL IMMUNE RESPONSE

All human beings are susceptible to infection by the dengue virus. However, infection with one serotype results in a permanent acquired immune response (homologous). On the other hand, protection induced by cross-immunity (heterologous) is temporary, partially active against other serotypes of the virus. In the case of primary infection, IgM antibodies (Type M Immunoglobulins) are rapidly produced and can be detectable in immunological tests from the sixth day of disease symptoms. On the other hand, for secondary infections by another serotype, IgG antibody titers increase rapidly, and IgM production is lower. It is important to emphasize that IgM antibodies reach their peak two weeks after the onset of symptoms, decreasing in the subsequent weeks. However, in primary infections, despite the lower levels, IgM antibodies can be detectable in serological tests for up to 3 months. Moreover, some individuals show a delayed antibody production, detectable only after the 10th day of symptoms.

LABORATORY DIAGNOSIS

Acute infections caused by the dengue virus are diagnosed through laboratory tests, along with clinical observations, mainly by antigen detection, and in late cases, by serology with detection of IgG and IgM antibodies. Although molecular viral detection methods are highly specific, most locations with a high incidence of the disease lack the infrastructure to apply them routinely.

 

Direct Tests

Detection of the virus NS1 protein (antigen) (ELISA or Rapid Test)

• Important marker during viremia;
• High sensitivity (during the correct detection window);
• Sample type: Blood, serum, or plasma;
• Best detection window: febrile onset period;
• Execution time: less than 24 hours.

 

Viral Research – Cell Inoculation Isolation

• Gold standard method (most specific);
• Allows differential identification of serotypes (DENV-1, DENV-2, DENV-3, and DENV-4);
• Sample type: Blood, visceral fragments (postmortem), and cerebrospinal fluid;;
• Best detection window: viremia period (up to the 5th day of symptom onset);
• The clinical sample should be preserved; if the test is not performed quickly, it should be stored at -70ºC;
• Execution time: approximately 7 days.

Viral Genome Research by Reverse Transcription Polymerase Chain Reaction (RT-PCR)

• High sensitivity and specificity;
• Allows detection of viral nucleic acids;
• Sample type: Blood, visceral fragments (postmortem), and cerebrospinal fluid;
• Best detection window: viremia period (up to the 5th day of symptom onset);
• The clinical sample should be preserved; if the test is not performed quickly, it should be stored at -70ºC;
• Execution time: 24 to 72 hours.

 

Serological tests

IgM antibody research (MAC ELISA or RAPID TEST)

• Detection of late infection (after the 6th day of symptom onset);
• High sensitivity (during the correct detection window);
• Sample type: Blood, serum, or plasma;
• Best detection window: late febrile period until approximately the 14th day of symptom onset;
• Interpretation should consider epidemiological information, observing the circulation of other Flaviviruses in the region.
• For infections during the first week of symptoms, an antigen test or RT-PCR should be added;
• Execution time: less than 24h.

 

Plaque Reduction Neutralization Test (PRNT) for IgM Detection

 

• High sensitivity and specificity;
• Detection of specific neutralizing antibodies against the dengue virus;
• Confirmatory test;
• Execution time: 5 days;
• The test may not be useful in areas with high circulation and for travelers with long-term exposure to arboviruses;
• Relatively expensive test.

Postmortem

Anatomopathological study followed by viral antigen research by immunohistochemistry (IHQ).

 

COMPLEMENTARY NON-SPECIFIC TESTS

Hematocrit

Platelet count

Albumin dosage

 

TREATMENT AND PREVENTION

In most cases of the disease, there is spontaneous cure within 10 days, but severe cases of dengue require medical support. Typically, patients receive constant hydration to maintain adequate body fluid volume.

For disease prevention, it is necessary to control the Aedes aegypti vector through public awareness regarding the elimination of breeding sites and individual protective measures in disease transmission areas to prevent mosquito bites.

 

Author: MsC. Angélica Cunha.

 

REFERENCES

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Dengue. OPAS. Available at: <https://www.paho.org/pt/topicos/dengue> Accessed: 2023.

Using amino acids co-occurrence matrices and explainability model to investigate patterns in dengue vírus proteins. BMC Bioinformatics.

Available at: <https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-022-04597-y>. Accessed: 2023.
 

SOUZA et. al. Serologic Tests for Dengue Virus. Centers for Disease Control and Prevention. 2019.

Available at: <https://www.cdc.gov/dengue/healthcare-providers/testing/serologic-tests.html>

TOMAS et.al. Dengue Plaque Reduction Neutralization Test (PRNT) in Primary and Secondary Dengue Virus Infections: How Alterations in Assay Conditions Impact Performance. Am J Trop Med Hyg. 2009.

 

Tissue Tests for Dengue Virus.  Centers for Disease Control and Prevention. 2019. Available at: <https://www.cdc.gov/dengue/healthcare-providers/testing/tissue-tests.html>

 

CDC DENV-1-4 rRT-PCR Multiplex and Trioplex rRT-PCR Assays, 2020. Centers for Disease Control and Prevention. Available at: <https://www.cdc.gov/dengue/healthcare-providers/testing/molecular-tests/assays.html>

 

Collaborative Protocol. Dengue management, clinical suspicion, diagnosis, and treatment. SUS BH. 2022.

 

Bhaskar et al., J. Prevalence, patterns, and factors associated with bleeding tendencies in dengue. Infect Dev Ctries. 2015. 9(1):105-110.

 

Dengue. Brasil. Available at: <https://www.gov.br/saude/pt-br/assuntos/saude-de-a-a-z/d/dengue> Accessed: 2023.