The use of real time PCR for arboviruses diagnostics: integrative review

Christiane O. L. Licínio; Flávio M. Ayres
J. Bras. Patol. Med. Lab. 2021;57(1):1-9
DOI: 10.5935/1676-2444.20210048

Christiane O. L. Licínio; Flávio M. Ayres

Universidade Estadual de Goiás, Anápolis, Goiás, Brazil

Corresponding author

Christiane Oliveira Lima Licínio

First Submission on 3/20/2020
Last Submission on 3/27/2020
Accepted for publication on 6/26/2020
Published on 10/20/2021


Arboviruses are viral diseases transmitted by arthropods (arthropod-borne virus). Standing out dengue, zika virus, and chikungunya among the emergent and re-emergent arboviruses in recent years around the world. The similarity between the symptoms makes the clinical diagnosis ineffective, making difficult the prophylactic and preventive measures of new outbreaks. Molecular diagnosis using the real-time polymerase chain reaction (PCR) technique is one of the ways to diagnose such diseases. In this study, the literature on the diagnosis of arboviruses was compiled and evaluated. The objective was to answer the guiding question: Is the real-time PCR methodology effective in the diagnosis of arboviruses? Scientific articles of free access were searched in the databases Pubmed (50 articles) and Scielo (107 articles), between 2014 and 2019. The selection was done through the inclusion and exclusion criteria, only 20 articles remained. Among them, 85% cross-sectional studies, 10% systematic reviews, and 5% case studies. The period of publications was 50% in 2017, 35% in 2016, and 5% in 2014, 2015 e 2019, each. Regarding the viruses treated in the articles, 25% researched dengue and the same percentage for chikungunya, 20% researched about zika virus. The efficacy of the molecular diagnosis was published in 21% of the articles (sensitivity and specificity), 53% highlighted the limit of detection, 70% highlighted the absence of cross-reactions, and 80% highlighted the differentiation between viruses.

Keywords: molecular probes techniques; dengue virus; zika virus; chikungunya virus.


Las arbovirosis son enfermedades virales transmitidas por artrópodos (arthropod-borne virus). Dengue, zica y chikungunya se destacan entre los arbovirus emergentes y reemergentes en los últimos años en todo el mundo. La similitud de los síntomas de estas infecciones hace que el diagnóstico clínico sea ineficaz, dificultando las medidas profilácticas y preventivas para nuevos brotes. El diagnóstico molecular mediante la técnica de reacción en cadena de la polimerasa (PCR) en tiempo real es una de las formas de diagnosticar esas enfermedades. En este estudio se recopiló y evaluó la literatura sobre el diagnóstico de arbovirosis. Nuestro objetivo era responder a una pregunta orientadora: ¿la metodología de PCR en tiempo real es eficaz para diagnosticar arbovirosis? Se buscaron artículos científicos de acceso abierto en las bases de datos Pubmed (50 artículos) y Scielo (107 artículos), entre 2014 y 2019. La selección se realizó utilizando los criterios de inclusión y exclusión, quedando solo 20 artículos. Entre estos, el 85% fueron estudios transversales, el 10% fueron revisiones sistemáticas y el 5% fueron estudios de casos. El período de publicaciones fue del 50% en 2017; 35% en 2016; y 5% en 2014, 2015 y 2019, cada. En cuanto a los virus tratados en los artículos, el 25% de los estudios investigaron sobre el dengue; el 25% el chikungunya y el 20% el virus del Zica. La efectividad del diagnóstico molecular se publicó en el 21% de los artículos (sensibilidad y especificidad); el 53% destacó el límite de detección; 70%, ausencia de reacciones cruzadas; y el 80%, la diferenciación entre virus.

Palabras-clave: técnicas de diagnóstico molecular; vírus del dengue; vírus del zika; vírus del chikungunya.


As arboviroses são doenças virais transmitidas por artrópodes (arthropod-borne virus). Destacam-se dengue, vírus da zica e chikungunya entre as arboviroses emergentes e reemergentes nos últimos anos em todo o mundo. A semelhança dos sintomas dessas infecções faz com que o diagnóstico clínico seja ineficaz, dificultando medidas profiláticas e preventivas para novos surtos. O diagnóstico molecular por meio da técnica de reação em cadeia da polimerase (PCR) em tempo real é uma das formas de diagnosticar tais doenças. Neste estudo, foi compilada e avaliada a literatura sobre o diagnóstico das arboviroses. Nosso objetivo foi responder a uma pergunta norteadora: a metodologia PCR em tempo real é eficaz no diagnóstico das arboviroses? Foram pesquisados artigos científicos de livre acesso nos bancos de dados Pubmed (50 artigos) e Scielo (107 artigos), entre 2014 e 2019. A seleção foi realizada por meio dos critérios de inclusão e exclusão, restando apenas 20 artigos. Entre estes, 85% eram estudos transversais, 10%, revisões sistemáticas e 5%, estudos de caso. O período das publicações foi de 50% em 2017; 35% em 2016; e 5% em 2014, 2015 e 2019, cada. A respeito dos vírus tratados nos artigos, 25% dos estudos pesquisaram sobre dengue; 25%, sobre chikungunya e 20%, sobre o vírus da zica. A eficácia do diagnóstico molecular foi publicada em 21% dos artigos (sensibilidade e especificidade); 53% destacaram o limite de detecção; 70%, a ausência de reações cruzadas; e 80%, a diferenciação entre os vírus.

Palavras-chave: técnicas de diagnóstico molecular; vírus da dengue; vírus da zica; vírus da chikungunya.


Arbovirus is a nomenclature used to indicate a grouping of arthropod-borne viruses. Mosquitoes and ticks are examples of arthropods capable of transmitting, through the bite, viruses belonging primarily to three families: TogaviridaeFlaviviridae, and Bunyaviridae. The most important genus among these three families is Flavivirus, which has four members with great epidemiological importance: dengue (DENV), zika virus (ZIKV), yellow fever (YFV), and West Nile virus (WNV). From the Togaviridae family, the Alphavirus genus stands out, whose member of greatest epidemiological importance is chikungunya (CHIKV). All of these genera are enveloped, positive-sense, single-stranded ribonucleic acid (RNA) viruses(1).

These pathogens are responsible for major disease outbreaks around the world, especially in the last 20 years. The fact that they are transmitted by insects, which are capable of spreading over a large geographic extension, contributed to the occurrence of epidemics(2). There is no presence of arboviruses only on the Antarctic continent(3, 4). Vectors, vertebrate hosts, and climatic conditions are the main factors for arboviruses to spread so quickly(1). In addition, the high capacity for mutation and adaptation also influence the occurrence of large outbreaks(5).

Recent climate change, large agglomerations due to uncontrolled urbanization, precarious sanitary conditions, and the great human movement between continents contribute to the proliferation of arboviruses(1-3, 6). Therefore, these viruses are the protagonists of emerging and re-emerging diseases, which caused a significant number of deaths and economically impacted several countries over the years(3). Viruses are co-circulating in several countries and, in some cases, have the same vector(7).

Initially, arboviruses present as an acute febrile illness, followed by symptoms of arthralgia, myalgia, and thrombocytopenia(5, 8). This makes the clinical differential diagnosis precarious, that is, the onset of these symptoms only is inefficient to identify the pathogen causing the disease. Consequently, laboratory diagnosis, with high sensitivity and specificity, is essential for this approach(8). Although initial symptoms are common, some cases progress to complications after infection. As an example, hemorrhagic fever in cases of DENV, microcephaly, and Guillain-Barré syndrome in cases of ZIKV(5). Thus, the differentiation between arboviruses is important for patient management in order to avoid complications, as well as to assist in taking preventive measures to control the spread of the disease(9).

The laboratory diagnosis for arboviruses can be performed in two ways: indirectly, through the investigation of antibodies in the infected patient’s blood, or directly, through the investigation of the pathogen in the blood and other body fluids(1). The most common diagnosis for Flaviviruses is made by the enzyme-linked immunosorbent assay (Elisa), which screens for class M immunoglobulin (IgM) antibodies for the early stages of the disease. However, the use of this methodology leads to numerous cross-reactions between the various arboviruses(10); therefore, the main method for diagnosing arboviruses in the early stage of the disease is the reverse transcription reaction followed by the polymerase chain reaction (RT-PCR)(9).

Molecular diagnoses, such as RT-PCR and real-time PCR, are very sensitive and specific, as they reduce the occurrence of cross-reactivity and identify pathogens in the early stages of the disease(10). Several biological matrices can be used, such as urine, semen, amniotic fluid, and saliva, besides whole blood, serum, and plasma(1). The advantage of real-time PCR compared to traditional PCR is the quantification of the amplified material simultaneously with the amplification of the tested genetic material. This process reduces the time spent on the reaction and the possibility of cross contamination. In RNA amplifications, which have low stability in the molecule, speed reduces the incidence of false negative results(11).

Considering the emergence and re-emergence of the DENV, CHIKV, ZIKV, and YF arboviruses in recent years and the need to identify and differentiate these viruses, the objective of this review was to compile and analyze the scientific literature regarding the diagnosis of arboviruses. The focus of the research was limited to the effectiveness of the molecular diagnosis used to identify and differentiate viruses, in particular on sensitivity, specificity, occurrence of cross-reactivity, and limit of detection.



This article is an integrative review that was carried out following the steps below: 1. selection and identification of the theme – formulation of the guiding question; 2. establishment of inclusion and exclusion criteria; 3. definition of information (articles) through research in databases; 4. evaluation and categorization of information; 5. interpretation of the results obtained; and 6. presentation of results.

The guiding question was: Is the real-time PCR methodology effective in the diagnosis of arboviruses? The PubMed and Scielo databases were used to search for open access scientific articles, in English and Portuguese. The period of publication was delimited from January 2014 to July 2019 in order to select the most current and relevant articles on the topic, since this subject has been more disseminated from 2014 on. MESH and its equivalent in Portuguese, DECS, were used to find the descriptors “real-time PCR”, “arbovirus”, and “molecular diagnosis”. The Boolean operator “AND” was used in the PubMed database search, and “OR” in the Scielo database. There was no restriction on the design of the article.

Publications were selected by title and abstract. Repeated articles and texts that did not contain information about the diagnosis of the disease or that were not about arboviruses were excluded. The exclusion criteria also applied to articles with approaches outside the aforementioned arboviruses.

The selection of articles was carried out in February 2020. Fifty articles in the PubMed database and 107 articles in the Scielo database were found (we did not find duplicate articles). After applying the inclusion and exclusion criteria, we selected 32 articles, however, nine were not open access and three did not meet the objectives. Therefore, the research was completed with 20 articles. Figure shows the flow of steps carried out in the research.


FIGURE – Flowchart of search and selection steps of the analyzed articles



Of the 20 articles selected, 85% were cross-sectional studies, 10% systematic reviews, and 5% were case studies. Most articles were written in 2017 (50%) and 2016 (35%). 2019, 2015, and 2014 had the same number of publications (5%), as described in Table 1.



Regarding the viruses of the study, Table 2 shows that 25% of the articles reported exclusively on DENV, the same percentage on CHIKV, and 20% on ZIKV. The percentage of articles that searched for all three viruses at the same time was also 20%. The search on DENV and CHIKV, and DENV and ZIKV in the same article was 5% each disease group. No article mentioned the YF.



Table 3 summarizes the results of the studies regarding the parameters investigated for the effectiveness of the test in question. 21%of the articles reported numerical data for sensitivity and specificity. The limit of detection was published in 53% of the articles. Differentiation between viruses was mentioned in 80% of the selected texts, and the absence of cross-reactivity in 70%.




Arboviruses have become a public health problem in almost every country in the world. Rapid spread, availability of vectors, lack of effective treatment, and prevention make epidemics frequent and increasingly virulent. The adaptive capacity of viruses as a result of genetic variations greatly contributes to their emergence in new geographic regions and to the frequency of outbreaks in regions where they are already established(12).

Arboviruses were much debated in Brazil due to the occurrence of ZIKV and CHIKV outbreaks in 2015 and the recurrence of DENV for several years. There were few publications on the diagnosis of arboviruses between 2014 and 2015, before the outbreak, and between 2018 and 2019, after the outbreak. Most publications were between 2016 and 2017. It was also possible to verify that articles from cross-sectional studies are the majority, which characterizes the predominance of observational articles.

Among arboviruses, the Flaviviruses (DENV, ZIKV, and YF) and Alphaviruses (CHIKV) stand out as the pathogens that cause acute febrile illness, with evolution to neurological febrile or hemorrhagic complications(12). As they are cocirculating in the same geographic area, the differential diagnosis is even more difficult(13). The consequences of this cocirculation are not yet fully known and can be a worsening factor in the evolution of infections(12). There are reports of co-infection in the same patient with DENV-ZIKV, DENV-CHIKV or ZIKV-CHIKV(14).

In addition to cases of coinfection, there is a concern with reinfection, especially in cases of DENV. Among arboviruses, it is the disease with the highest number of cases and the highest morbidity and mortality. The virus has four serotypes, each of which can cause a distinct infection(1). At each reinfection by a different serotype, the antibodies are not able to neutralize the virus and cause disease amplification mediated by antibodies [antibody-dependent enhancement (ADE)](12, 15). Therefore, a second DENV infection is even more serious than the first, with high-level viremia and several inflammatory markers released into the bloodstream(12).

Many researches are focused on identifying whether, as in the case of a DENV reinfection, cocirculation and co-infection with ZIKV can cause ADE in secondary infections by this pathogen(16). There are many similarities between DENV and ZIKV, which causes a large number of cross-reactions in antibodies. Some researchers have raised the possibility that ZIKV is a fifth serotype of DENV(17), as their E protein genetic sequence is shared in a proportion between 54% and 59%(15). Stettler et al. (2016)(18) showed that there was cross-reactivity of the immune system between a previous DENV infection and a secondary ZIKV infection in the antibodies against the viral envelope domains I and II (EDI/II), causing a low neutralization of the ZIKV.

Two publications refer to co-infections. One of them reports the case of a 28-year-old female patient who was diagnosed with co-infection with DENV and CHIKV, in Fortaleza, Ceará, Brazil. A severe hematologic complication was proven in the patient due to the coinfection; initially, she presented only the nonspecific symptoms of arboviruses (fever, myalgia, and arthralgia). The patient developed acquired thrombotic thrombocytopenic purpura (TTP), antibody-mediated thrombocytopenia, a serious complication of CHIKV infection(19). The other publication, from 2019, reports the case of a pre-symptomatic blood donor. Three days after the blood donation, she showed characteristic symptoms and returned to the donation site. In the investigation, RNA of ZIKV and DENV were detected simultaneously in blood plasma collected during donation(20).

The articles selected in this integrative review had objectives such as product development, comparative analysis between methodologies and, less frequently, the review of studies on the diagnosis of arboviruses. In 20% of them, the three most epidemiologically important viruses of arboviruses were investigated, as seen in Table 2. Article 12 identified 2.6% of cases of ZIKV-DENV coinfection in a hospital in Recife, Brazil, during the 2015 outbreak, highlighting the need to distinguish the viruses to improve sanitary controls.

The importance of diagnosing arboviruses is not restricted to differentiating which agent is the cause of the infection, it is also crucial in studies investigating severe cases of diseases, such as in the associations between ZIKV and neurological disorders. In addition, diagnosis is essential in seroprevalence studies and surveillance of new epidemics(17), as well as in alternative transmission routes(21).

Molecular diagnosis of arboviruses is a widely used tool in the acute stage of infection, in which viremia is at its apex. There are several methodologies for direct pathogen research, and the real-time PCR technique demonstrates many advantages compared to others available on the market. This is due to the ease of execution of the methodology, the lower value of reagents and equipment, and the lower risk of contamination, both for the operator and the sample(9).

The real-time PCR technique is a variation of traditional PCR; uses fluorescent probes that monitor the amplification of genetic material throughout the reaction. This process is called real time, as amplification and quantification occur simultaneously(22). It is a fast, specific, and sensitive technique that can detect more than one pathogen in the same reaction (multiplex qPCR), as long as there are different fluorescent dyes between the reagents(23).

The primers used for amplification are designed according to the pathogen being investigated. The development of these primers with the help of bioinformatics has increased the sensitivity and specificity of the reagents. This is due to greater identification of genomic regions of pathogens, differentiating them from other individuals of their family or genus(23).

The parameters used to determine the effectiveness of an assay are sensitivity and specificity. Sensitivity refers to the test being truly positive, that is, when the individual actually has the investigated disease and the test is positive. Specificity is related to the fact that the test is truly negative when there is no disease and the result is negative. The low sensitivity of an assay produces false-negative results, and the low specificity produces false-positive results(24).

The limit of detection and occurrence of cross-reactivity are derived from these parameters. The first parameter refers to the minimum amount of an analyte, in this case, the viral RNA – capable of detecting a particular sample. The articles analyzed showed that the real-time PCR technique is very sensitive. For each gene [deoxyribonucleic acid (DNA)] or gene expression (RNA) detection, there is a limit of detection that varies from less than 100 to 5.3 copies/sample run; this last amount is considered very low and borderline in the detection of viral RNA(25).

Real-time PCR primers are designed according to the genome of the virus under investigation. In differentiating between viruses, there is the use of distinct fluorescent reagents in different primers, highlighting each virus. In cross-reactions, the primers bind in different regions of the virus, allowing the distinction between phylogenetically similar viruses, such as ZIKV and DENV.


Real-time PCR methodology is effective in diagnosing arboviruses. It is able to differentiate DENV, ZIKV, and CHIKV viruses, with low occurrence of cross-reactivity and low nonspecific reaction. Additionally, the technique is able to identify the pathogen even at low-level viremia, which is important for early diagnosis of the disease. Diagnosis is fast, sensitive, and specific, which makes it a highly reliable diagnostic tool.


This work was carried out with the support of the Fundação de Amparo à Pesquisa do Estado de Goiás (Goias Research Foundation) – process number 201810267000572.


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