Dengue Virus Lab Diagnosis: A Comprehensive Guide for Healthcare Professionals

Introduction

Efficient and accurate laboratory diagnosis of dengue virus infection is paramount for a multitude of critical reasons. In clinical care, it enables early detection of severe cases, facilitates case confirmation, and aids in differential diagnosis from other infectious diseases presenting with similar symptoms. Beyond individual patient management, robust diagnostic capabilities are essential for effective surveillance activities, rapid outbreak control, advancing our understanding of dengue pathogenesis, supporting academic research endeavors, and accelerating vaccine development and clinical trials.

The laboratory diagnosis of dengue virus infection encompasses a range of sophisticated methods designed to detect the virus itself, its genetic material (nucleic acid), viral antigens, or the antibodies produced by the host in response to infection. Often, a combination of these techniques is employed to achieve a definitive diagnosis. During the initial 4–5 days following the onset of illness, the dengue virus can be found in various bodily fluids and tissues, including serum, plasma, circulating blood cells, and other tissue samples. This early phase, characterized by viremia, is ideal for diagnostic approaches such as virus isolation, nucleic acid detection, and antigen detection. Conversely, as the acute phase of infection subsides and the body’s immune response matures, serological assays become the preferred diagnostic method, focusing on the detection of dengue-specific antibodies.

The antibody response to dengue infection is intricately influenced by the host’s prior exposure to flaviviruses. In individuals experiencing a primary dengue infection – meaning they have not been previously infected with dengue or other flaviviruses, nor vaccinated against flaviviruses like yellow fever, Japanese encephalitis, or tick-borne encephalitis – the antibody response follows a characteristic pattern. This primary response is marked by a gradual increase in specific antibodies. Immunoglobulin M (IgM) antibodies are typically the first to emerge, becoming detectable in approximately 50% of patients between days 3 and 5 post-illness onset. This detection rate rises significantly to 80% by day 5 and reaches an impressive 99% by day 10 (See Figure 4.1). IgM levels peak around two weeks after symptom onset, subsequently declining to undetectable levels over a period of 2–3 months. Dengue-specific IgG antibodies, on the other hand, generally appear at lower titers towards the end of the first week of illness. IgG levels then increase slowly but persistently, remaining detectable for many months, and potentially even for life.

Figure 4.1: Timeline of dengue virus infections and diagnostic methods.

In contrast, secondary dengue infections, occurring in individuals with pre-existing dengue immunity from prior infection or flavivirus vaccination, elicit a markedly different antibody response. Antibody titers in secondary infections rise rapidly and exhibit broader reactivity across multiple flaviviruses. IgG is the dominant immunoglobulin isotype in these cases, reaching high levels even in the acute phase and persisting for extended periods, from 10 months to life. Notably, IgM levels in the early convalescent stage of secondary infections are significantly lower compared to primary infections and may even be undetectable in some cases, depending on the sensitivity of the diagnostic test employed. To accurately differentiate between primary and secondary dengue infections, clinical laboratories now frequently utilize IgM/IgG antibody ratios, which have proven more reliable than older methods like the haemagglutination-inhibition test (HI).

A diverse array of laboratory diagnostic methods has been developed to effectively support patient care and public health disease control initiatives. The selection of a specific diagnostic method is guided by several factors, including the intended purpose of testing (e.g., clinical diagnosis, epidemiological surveys, vaccine development), the resources and technical expertise available in the laboratory setting, cost considerations, and crucially, the timing of sample collection relative to the course of the illness.

Generally, diagnostic tests characterized by high sensitivity and specificity often necessitate more advanced technologies and specialized technical skills. Conversely, rapid diagnostic tests, while offering ease of use and speed, may compromise on sensitivity and specificity. Virus isolation and nucleic acid detection methods, while more labor-intensive and costly, are recognized for their superior specificity compared to antibody detection through serological methods. Figure 4.2 illustrates the general inverse relationship between the accessibility and ease of use of a diagnostic method and the level of confidence in the test results it provides.

Figure 4.2: Accessibility vs. Confidence in Dengue Diagnostic Tests.

Considerations in Choosing Dengue Diagnostic Methods

Clinical Management of Dengue

Dengue virus infection presents with a wide spectrum of clinical manifestations, many of which are nonspecific and overlap with other febrile illnesses. Relying solely on clinical symptoms for diagnosis is therefore unreliable. Early laboratory confirmation of a clinical suspicion of dengue is highly valuable due to the unpredictable disease course. Some patients may rapidly progress from mild illness to severe dengue and potentially fatal outcomes. In such cases, timely and appropriate clinical intervention guided by laboratory diagnosis can be life-saving.

In the early febrile phase of illness, specifically before day 5, dengue infections can be effectively diagnosed using virological methods. These include virus isolation in cell culture, detection of viral RNA through nucleic acid amplification tests (NAATs), or detection of viral antigens using enzyme-linked immunosorbent assays (ELISA) or rapid diagnostic tests (RDTs). Virus isolation, typically conducted in specialized laboratories with appropriate infrastructure and expertise, requires meticulous sample handling. Blood samples intended for virus culture must be kept cooled or frozen during transport to maintain virus viability. The process of virus isolation and subsequent identification in cell cultures typically takes several days.

Nucleic acid detection assays, known for their excellent performance characteristics, offer the potential to detect dengue viral RNA within a shorter timeframe of 24–48 hours. However, these sophisticated tests necessitate expensive equipment and reagents and demand stringent quality control procedures and execution by highly experienced technicians to prevent contamination and ensure accurate results.

NS1 antigen detection kits have emerged as a valuable tool, becoming increasingly commercially available. These kits can be utilized even in laboratories with limited resources and provide results within a few hours. Rapid dengue antigen detection tests represent an even more accessible option, suitable for field settings and delivering results in under an hour. Currently, most commercially available NS1 antigen tests are not serotype-specific. Ongoing evaluations are crucial to comprehensively assess their diagnostic accuracy and cost-effectiveness across diverse clinical and epidemiological settings. Table 4.1 provides a summary of various dengue diagnostic methods, outlining their operating characteristics and comparative costs.

Table 4.1: Dengue diagnostic methods: characteristics and costs.

After day 5 of illness, coinciding with defervescence (fever resolution) and the emergence of dengue-specific antibodies, dengue viruses and antigens typically become undetectable in the bloodstream. While NS1 antigen may persist for a few additional days in some patients post-defervescence, serological tests become the mainstay of diagnosis in the later stages of illness. Dengue serological tests hold a significant advantage in dengue-endemic regions due to their greater availability compared to virological tests. Furthermore, specimen transport for serology is less demanding as immunoglobulins are stable at ambient tropical temperatures.

For serological diagnosis, the timing of specimen collection is more flexible than for virus isolation or RNA detection. The antibody response can be effectively assessed by comparing antibody levels in an acute-phase sample with a convalescent sample collected weeks or months later. However, the diagnostic accuracy of IgM ELISA tests can be reduced in secondary dengue infections due to lower or undetectable IgM responses in some cases. Rapid serological tests can provide results in under an hour, offering quick turnaround time. Nevertheless, caution should be exercised when relying solely on rapid tests for dengue diagnosis, as comprehensive evaluations of all commercially available tests by reference laboratories are still ongoing.

A definitive serological diagnosis of acute or recent flavivirus infection can be established by demonstrating a four-fold or greater increase in antibody levels between paired sera, measured by IgG ELISA or haemagglutination inhibition (HI) test. However, waiting for a convalescent serum sample collected at hospital discharge is often impractical for timely clinical management and provides only a retrospective diagnosis.

Differential Diagnosis of Dengue Fever

Dengue fever, particularly in non-epidemic settings, can be easily clinically confused with a range of other febrile illnesses. Accurate differential diagnosis is critical to ensure appropriate patient management and rule out other potentially serious conditions. Depending on the patient’s geographical origin and travel history, the differential diagnosis should consider other etiologies, including infections caused by other flaviviruses. These include yellow fever, Japanese encephalitis, St. Louis encephalitis, Zika virus, and West Nile virus. Furthermore, alphavirus infections, such as Sindbis and chikungunya, and other causes of fever like malaria, leptospirosis, typhoid fever, and Rickettsial diseases (Rickettsia prowazeki, R. mooseri, R. conori, R. rickettsii, Orientia tsutsugamushi, Coxiella burnetii, etc.), measles, enteroviruses, influenza and influenza-like illnesses, and various haemorrhagic fevers (e.g., Arenaviridae: Junin virus; Filoviridae: Marburg virus, Ebola virus; Bunyaviridae: hantaviruses, Crimean-Congo haemorrhagic fever virus, etc.) should be considered and excluded as necessary.

For optimal dengue diagnosis and differential diagnosis, a combined approach involving both virus/viral RNA/viral antigen detection and antibody response detection is preferable to relying on either approach alone. Table 4.2 provides guidance on the interpretation of dengue diagnostic tests in the context of differential diagnosis.

Table 4.2: Interpretation of dengue diagnostic tests for differential diagnosis.

Unfortunately, an ideal dengue diagnostic test that is rapid, affordable, easy to perform, highly accurate, and suitable for diverse healthcare settings remains an unmet need.

Dengue Diagnosis in Outbreak Investigations

During dengue outbreaks, the clinical presentation of patients can vary considerably depending on the stage of illness at presentation. Some individuals may present with fever with or without rash during the acute febrile phase, while others may exhibit signs of plasma leakage or shock, haemorrhagic manifestations, or be seen during the convalescent phase of the illness.

A primary objective in a suspected dengue outbreak is to rapidly identify the causative agent. This is crucial for implementing appropriate public health interventions and guiding physicians in initiating appropriate acute illness management strategies. In outbreak settings, the speed and specificity of diagnostic tests are often prioritized over test sensitivity. Samples collected from febrile patients can be tested using nucleic acid amplification methods in well-equipped laboratories. Alternatively, a broader range of laboratories, including those with less sophisticated infrastructure, can utilize ELISA-based dengue antigen detection kits for rapid diagnosis. If specimens are collected after day 5 of illness, commercial IgM ELISA or sensitive dengue IgM rapid tests can be used as initial screening tools to suggest a dengue outbreak. However, it is crucial to confirm presumptive positive results with more reliable serological tests performed in a reference laboratory with comprehensive arbovirus diagnostic capabilities. Serological assays also play a vital role in determining the geographic extent and magnitude of outbreaks.

Dengue Surveillance and Diagnostic Tools

Dengue surveillance systems are designed to proactively monitor and detect the circulation of dengue viruses within human and mosquito populations. Effective surveillance relies on diagnostic tools that are not only sensitive and specific but also affordable and practical for implementation in resource-limited settings. Laboratories tasked with dengue surveillance, typically national and/or regional reference laboratories, must possess the capacity to perform a wide spectrum of diagnostic tests. These include the methods discussed previously for dengue diagnosis, as well as tests to identify a broad range of other potential etiologies to accurately differentiate dengue from other febrile illnesses in surveillance efforts.

Dengue Diagnosis in Vaccine Trials

Vaccine trials are critical for evaluating the safety and efficacy of dengue vaccine candidates in human populations. Specific laboratory assays are employed in vaccine trials to measure correlates of protection, such as neutralizing antibodies. The plaque reduction neutralization test (PRNT) and microneutralization assays are commonly used for this purpose.

Following primary dengue infections in individuals without pre-existing flavivirus immunity, neutralizing antibodies measured by PRNT tend to be relatively or completely specific to the infecting dengue virus serotype. PRNT is considered the gold standard assay for quantifying neutralizing antibody titers in serum, providing a reliable measure of the level of protection against a specific dengue virus serotype. The principle of PRNT is based on the ability of neutralizing antibodies to inactivate the virus, preventing it from infecting and replicating in susceptible target cells.

After a secondary dengue virus infection, a more complex antibody response emerges. High-titer neutralizing antibodies are produced that exhibit cross-reactivity, often targeting at least two, and frequently all four, dengue virus serotypes, as well as non-dengue flaviviruses. This cross-reactivity is attributed to the activation of memory B-cells, which produce antibodies directed at conserved epitopes shared among dengue viruses. Interestingly, during the early convalescent stage following sequential dengue infections, the highest neutralizing antibody titer is often directed against the first infecting virus serotype rather than the most recent one. This phenomenon is known as “original antigenic sin” or antibody-dependent enhancement (ADE).

Despite its reliability, PRNT has limitations, primarily being labor-intensive and time-consuming. To address these limitations, several laboratories have developed high-throughput neutralization tests suitable for large-scale surveillance studies and vaccine trials. However, inter-laboratory variability in PRNT results has been observed. Standardization of PRNT protocols, including the use of standardized cell lines, virus strains, and incubation conditions, is crucial to minimize variability and ensure comparability of results across laboratories. Mammalian cell lines, such as VERO cells, are recommended for the production of seed viruses used in PRNT assays.

The microneutralization assay shares the same fundamental principle as PRNT. Various modifications of microneutralization assays exist. One common variation involves quantifying viral antigen production colorimetrically using labeled antibodies instead of plaque counting. Nucleic acid quantification using PCR can also be incorporated into microneutralization assays. Microneutralization assays were specifically designed to reduce reagent consumption and facilitate high-throughput testing of large sample numbers. In viral antigen detection-based microneutralization assays, the incubation period needs standardization to avoid measuring growth after multiple replication cycles, as virus spread is not restricted as in PRNT assays using semisolid overlays. Incubation periods are virus-specific due to variations in viral growth rates. Similar to PRNT, microneutralization assays performed on samples from individuals with secondary dengue infections may exhibit broad cross-reactivity across all four dengue virus serotypes.

In clinical drug trials for dengue therapeutics, confirmed etiological diagnosis is a prerequisite for patient enrollment. Table 4.2 outlines criteria for highly suggestive and confirmed dengue diagnoses relevant to drug trial inclusion. Table 4.3 summarizes the advantages and limitations of various dengue diagnostic methods in relation to their intended purpose.

Table 4.3: Advantages and limitations of dengue diagnostic methods.

Current Dengue Diagnostic Methods in Detail

Virus Isolation Techniques

Specimen collection for virus isolation is most effective during the viremic phase of dengue infection, typically within the first 5 days of illness onset. Suitable specimens for virus isolation include serum, plasma, peripheral blood mononuclear cells (PBMCs), and tissues collected at autopsy, such as liver, lung, lymph nodes, thymus, and bone marrow. Dengue virus is heat-labile, necessitating careful specimen handling to maintain virus viability. Specimens awaiting transport to the laboratory should be refrigerated or packed in wet ice. For short-term storage (up to 24 hours), temperatures between +4 °C and +8 °C are recommended. For prolonged storage, specimens should be frozen at -70 °C or stored in liquid nitrogen. Storage at -20 °C, even for short durations, is not advisable.

Cell culture is the most widely adopted method for dengue virus isolation. Mosquito cell lines, particularly C6/36 (derived from Ae. albopictus) and AP61 (derived from Ae. pseudoscutellaris), are the preferred host cells for routine dengue virus isolation. Since not all wild-type dengue viruses induce cytopathic effects in mosquito cell lines, cell cultures must be meticulously screened for evidence of infection. This is typically achieved using an antigen detection immunofluorescence assay, employing serotype-specific monoclonal antibodies and flavivirus group-reactive or dengue complex-reactive monoclonal antibodies. Mammalian cell lines, such as Vero, LLCMK2, and BHK21, can also be utilized but are generally less efficient for dengue virus isolation. Virus isolation followed by immunofluorescence assay confirmation typically requires 1–2 weeks and is contingent on proper specimen transport and storage to preserve virus viability.

In resource-limited settings where cell culture facilities are unavailable, alternative virus isolation methods can be employed. These include intracranial inoculation of clinical specimens into suckling mice or intrathoracic inoculation of mosquitoes. Infected newborn mice may develop encephalitis symptoms, although some dengue strains may not induce overt illness in mice. Virus antigen detection in mouse brain or mosquito head squashes is performed by staining with anti-dengue antibodies to confirm virus presence.

Nucleic Acid Detection Methods

Similar to virus isolation, specimens intended for nucleic acid detection require careful handling and storage to preserve RNA integrity, as RNA is heat-labile. The same storage and transport procedures recommended for virus isolation should be followed for nucleic acid detection specimens.

Reverse Transcription Polymerase Chain Reaction (RT-PCR)

Since the 1990s, reverse transcriptase-polymerase chain reaction (RT-PCR) assays have revolutionized dengue diagnostics. RT-PCR offers significantly improved sensitivity compared to virus isolation and substantially reduces turnaround time. In situ RT-PCR further expands diagnostic capabilities by enabling dengue RNA detection directly in paraffin-embedded tissue samples.

All nucleic acid detection assays, including RT-PCR, involve three fundamental steps: nucleic acid extraction and purification, amplification of the nucleic acid target, and detection and characterization of the amplified product. Viral RNA extraction and purification from clinical specimens were traditionally performed using liquid phase separation methods (e.g., phenol-chloroform extraction). However, silica-based commercial kits, available in bead or column formats, have largely replaced these methods due to their enhanced reproducibility, speed, and automation potential using robotic systems.

Many laboratories utilize nested RT-PCR assays for dengue virus detection. Nested RT-PCR typically employs universal dengue primers targeting the conserved C/prM region of the dengue genome for initial reverse transcription and amplification. This is followed by a nested PCR amplification step using serotype-specific primers to identify the infecting dengue serotype. Multiplex RT-PCR, which combines serotype-specific oligonucleotide primers for all four dengue serotypes in a single reaction tube, offers an attractive alternative to nested RT-PCR. The amplified products from RT-PCR are separated by electrophoresis on agarose gels. DNA bands of different molecular weights, visualized using ethidium bromide dye and compared to standard molecular weight markers, indicate the presence and serotype of dengue virus. In this assay format, dengue serotypes are distinguished based on the size of their amplified DNA bands.

Compared to virus isolation, RT-PCR methods exhibit sensitivity ranging from 80% to 100%, influenced by factors such as the genomic region targeted by primers, the RT-PCR approach (e.g., one-step vs. two-step RT-PCR), and the subtyping method employed (e.g., nested PCR, blot hybridization, restriction site-specific PCR, sequencing). To minimize false-positive results due to nonspecific amplification, primers should target genomic regions highly specific to dengue viruses and not conserved in other flaviviruses or related viruses. False-positive results can also arise from amplicon contamination from previous PCR runs. Stringent laboratory practices, including physical separation of pre- and post-PCR areas and adherence to rigorous decontamination protocols, are essential to prevent contamination.

Real-Time RT-PCR Assays

Real-time RT-PCR assays represent a significant advancement in nucleic acid detection, offering rapid viral RNA quantification. These one-step assay systems utilize serotype-specific primer-probe sets for each dengue serotype. Fluorescent probes enable real-time detection of reaction products within specialized PCR instruments, eliminating the need for post-amplification electrophoresis. TaqMan and SYBR Green technologies are commonly employed in real-time RT-PCR assays. TaqMan real-time PCR offers high specificity due to the sequence-specific hybridization of the probe. However, the sensitivity of primers and probes is dependent on their homology to the target viral gene sequence and may not detect all dengue virus strains. SYBR Green real-time RT-PCR simplifies primer design and utilizes universal RT-PCR protocols but may be theoretically less specific than TaqMan assays.

Real-time RT-PCR assays are available in both singleplex (detecting one serotype at a time) and multiplex (detecting all four serotypes in a single reaction) formats. Multiplex assays offer the advantage of identifying all four serotypes simultaneously, reducing the risk of contamination associated with multiple sample manipulations. While faster, multiplex real-time RT-PCR assays are currently generally less sensitive than nested RT-PCR assays. A key advantage of real-time RT-PCR is its ability to quantify viral load in clinical samples, providing valuable insights into dengue pathogenesis and disease severity.

Isothermal Nucleic Acid Amplification Methods

Isothermal amplification methods provide alternatives to PCR-based nucleic acid detection, eliminating the need for thermal cycling instruments. Nucleic acid sequence-based amplification (NASBA) is an isothermal, RNA-specific amplification assay. NASBA begins with reverse transcription, converting single-stranded RNA into double-stranded DNA, which then serves as a template for RNA transcription. Amplified RNA detection can be achieved through electrochemiluminescence or real-time fluorescence using molecular beacon probes. NASBA has been adapted for dengue virus detection, achieving sensitivity comparable to virus isolation in cell culture. NASBA holds promise for dengue diagnostics in field studies and resource-limited settings.

Loop-mediated isothermal amplification (LAMP) methods have also been developed for dengue virus detection. However, comprehensive performance comparisons of LAMP to other nucleic acid amplification methods are still needed to fully define their role in dengue diagnostics.

Antigen Detection Assays

Historically, dengue antigen detection in acute-phase serum was challenging, particularly in secondary infections, due to the formation of virus-IgG antibody immunocomplexes. However, advancements in ELISA and dot blot assays targeting the dengue envelope/membrane (E/M) antigen and the non-structural protein 1 (NS1) have overcome this limitation. These newer assays can detect high concentrations of these antigens, often present as immune complexes, in patients with both primary and secondary dengue infections, up to nine days after illness onset.

NS1 glycoprotein is produced by all flaviviruses and is secreted from infected mammalian cells. NS1 elicits a robust humoral immune response, making it an attractive target for early dengue diagnosis. Numerous studies have focused on utilizing NS1 detection for early dengue diagnosis. Commercial NS1 antigen detection kits are now widely available, although most currently do not differentiate between dengue serotypes. Ongoing evaluations by laboratories worldwide, including the WHO/TDR/PDVI laboratory network, are crucial to comprehensively assess their performance characteristics and clinical utility in diverse settings.

Fluorescent antibody assays, immunoperoxidase assays, and avidin-biotin enzyme assays are also employed for dengue antigen detection. These methods can detect dengue virus antigen in acetone-fixed leukocytes and in snap-frozen or formalin-fixed tissues collected at autopsy, aiding in dengue diagnosis in various clinical and research contexts.

Serological Tests for Dengue Diagnosis

IgM Antibody-Capture ELISA (MAC-ELISA)

The IgM antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) is a widely used serological test for dengue diagnosis. In MAC-ELISA, total IgM antibodies present in patient sera are captured by anti-human IgM antibodies (specific to the μ chain of human IgM) coated onto microplates. Dengue-specific antigens, encompassing one or more dengue serotypes (DEN-1, -2, -3, and -4), are then allowed to bind to the captured dengue-specific IgM antibodies. Bound dengue antigens are subsequently detected using monoclonal or polyclonal dengue antibodies conjugated with an enzyme. This enzyme catalyzes a reaction that transforms a colorless substrate into a colored product, which is quantified spectrophotometrically by measuring optical density.

Serum is the preferred specimen type for MAC-ELISA. However, blood collected on filter paper and saliva can also be used, while urine is generally not suitable. Sample collection timing is critical for IgM detection, with optimal sensitivity achieved when samples are taken five or more days after fever onset. Serum specimens can be tested at a single dilution or across multiple dilutions. Antigens used in MAC-ELISA are typically derived from dengue virus envelope proteins, often sourced from virus-infected cell culture supernatants or suckling mouse brain preparations. MAC-ELISA exhibits good sensitivity and specificity, particularly when used five or more days after fever onset. Various commercial MAC-ELISA kits (both ELISA and rapid test formats) are available, but their performance can vary in terms of sensitivity and specificity. Evaluations by the WHO/TDR/PDVI laboratory network have indicated that ELISA-based MAC-ELISAs generally perform better than rapid MAC-ELISA tests.

Cross-reactivity with other circulating flaviviruses, such as Japanese encephalitis virus, St. Louis encephalitis virus, and yellow fever virus, is generally not a significant issue with MAC-ELISA. However, false-positive results have been reported in sera from patients with malaria, leptospirosis, and past dengue infections. These limitations should be considered when interpreting MAC-ELISA results, especially in regions where these pathogens co-circulate with dengue. It is recommended to evaluate MAC-ELISA tests against a panel of sera from patients with relevant diseases prevalent in a specific region before widespread implementation. IgM antibodies detected by MAC-ELISA are broadly cross-reactive, even following primary dengue infections, making serotype determination using IgM assays generally unreliable. However, recent studies have described MAC-ELISA modifications (See Figure 4.3) that may enable serotype determination, but further validation is needed.

Figure 4.3: MAC-ELISA principle.

IgG ELISA for Dengue Diagnosis

The IgG ELISA is another valuable serological assay used to detect recent or past dengue infections. When paired acute and convalescent sera are tested, IgG ELISA can differentiate between recent and past infections. IgG ELISA utilizes similar dengue antigens as MAC-ELISA. A modified IgG ELISA, known as E/M-specific capture IgG ELISA (GAC-ELISA), can detect IgG antibodies for up to 10 months post-infection. Indirect IgG ELISA, coated with E/M antigen, can detect IgG antibodies for life, indicating past dengue exposure. A four-fold or greater increase in IgG antibody titers between acute and convalescent paired sera, measured by IgG ELISA, is indicative of a recent dengue infection. IgG ELISA results generally correlate well with the haemagglutination-inhibition (HI) test. An ELISA inhibition method (EIM) for detecting IgG dengue antibodies has also been developed and used for serological diagnosis and dengue surveillance. EIM is based on the competition between dengue-specific IgG antibodies in the patient sample and conjugated human IgG anti-dengue antibodies for binding to dengue antigens.

IgG ELISA can be used to detect IgG antibodies in serum, plasma, and blood samples stored on filter paper. Furthermore, IgG ELISA can aid in differentiating between primary and secondary dengue infections. However, IgG ELISA generally lacks specificity within the flavivirus serocomplex groups, meaning cross-reactivity with antibodies to other flaviviruses may occur. Antibody avidity, which reflects the binding strength of antibodies to their target antigen, can be assessed to differentiate primary and secondary dengue infections in specialized laboratories. Antibodies produced soon after infection typically exhibit lower avidity compared to antibodies produced months or years later. Antibody avidity assays are not widely used in routine dengue diagnostics and are not commercially available.

IgM/IgG Ratio for Dengue Diagnosis

The ratio of dengue virus E/M protein-specific IgM to IgG antibodies can be a useful tool in distinguishing between primary and secondary dengue virus infections. IgM capture and IgG capture ELISAs are the most common assays used to determine this ratio. In some laboratories, a dengue infection is classified as primary if the IgM/IgG optical density (OD) ratio is greater than 1.2 (using patient sera at 1/100 dilution) or 1.4 (using patient sera at 1/20 dilutions). Conversely, an infection is classified as secondary if the IgM/IgG OD ratio is less than 1.2 or 1.4. This algorithm has been adopted by some commercial test vendors. However, it is important to note that IgM/IgG ratios may vary between laboratories, highlighting the need for improved standardization of test performance and ratio cut-off values.

IgA Antibody Detection in Dengue

Detection of serum anti-dengue IgA antibodies using anti-dengue virus IgA capture ELISA (AAC-ELISA) often becomes positive one day after IgM detection. IgA titers typically peak around day 8 after fever onset and then decline rapidly, becoming undetectable by day 40. Studies have not found significant differences in IgA titers between patients with primary and secondary dengue infections. Although IgA levels are generally lower than IgM levels in both serum and saliva, combined testing for both IgA and IgM may provide additional information to aid in interpreting dengue serology. However, IgA testing for dengue is not routinely used and requires further evaluation to determine its clinical utility in routine dengue diagnostics.

Haemagglutination-Inhibition (HI) Test

The haemagglutination-inhibition (HI) test is a classical serological assay for dengue diagnosis. The HI test is based on the principle that dengue antigens can agglutinate red blood cells (RBCs) from ganders or trypsinized human type O RBCs. Anti-dengue antibodies present in patient sera can inhibit this agglutination. The potency of this inhibition is quantified in the HI test. Serum samples are pre-treated with acetone or kaolin to remove nonspecific inhibitors of haemagglutination and then adsorbed with gander or trypsinized type O human RBCs to remove nonspecific agglutinins. Each batch of antigens and RBCs is carefully optimized. Optimal pH conditions for dengue haemagglutinins vary, requiring the use of multiple pH buffers for each serotype. Ideally, the HI test requires paired sera collected during the acute phase (e.g., upon hospital admission) and convalescent phase (e.g., at hospital discharge) or paired sera collected with an interval of more than seven days. The HI assay does not differentiate between infections caused by closely related flaviviruses (e.g., dengue virus and Japanese encephalitis virus or West Nile virus) or between immunoglobulin isotypes. The antibody response in primary dengue infection is characterized by low antibody levels in acute-phase serum (drawn before day 5) followed by a slow increase in HI antibody titers. In contrast, secondary dengue infections elicit a rapid rise in HI antibody titers, typically exceeding 1:1280. Convalescent sera from patients with primary dengue infections generally exhibit HI antibody titers below this threshold.

Figure 4.4: Haemagglutination-inhibition assay principle.

Haematological Tests in Dengue Diagnosis

Platelet count and haematocrit values are commonly measured during the acute phase of dengue infection as part of routine clinical assessment. These haematological tests should be performed using standardized protocols, reagents, and equipment to ensure accurate and reliable results.

A decrease in platelet count below 100,000 per μL can be observed in dengue fever but is a hallmark feature of dengue haemorrhagic fever (DHF). Thrombocytopaenia (low platelet count) typically develops between day 3 and day 8 following illness onset.

Haemoconcentration, indicated by an increase in haematocrit of 20% or more compared to convalescent values, is suggestive of hypovolaemia resulting from increased vascular permeability and plasma leakage, a key pathophysiological feature of severe dengue.

Future Directions in Dengue Diagnostic Test Development

Microsphere-based immunoassays (MIAs) are gaining prominence as a promising serological diagnostic option for various infectious diseases, including dengue. MIA technology involves the covalent attachment of antigens or antibodies to microspheres or beads. Detection is achieved using lasers to elicit fluorescence at varying wavelengths. MIAs offer advantages in terms of speed and throughput compared to traditional MAC-ELISA and have the potential for multiplexing serological tests, enabling simultaneous detection of antibody responses to multiple viruses in a single assay. MIAs can also be adapted for direct virus detection.

Rapid advancements in biosensor technology, particularly mass spectrometry-based biosensors, are leading to the development of powerful systems for rapid pathogen identification. Mass spectra generated by these systems serve as unique “fingerprints” or molecular profiles of bacteria and viruses. Sophisticated software analyzes these mass spectra, comparing them to a comprehensive database of infectious agents to identify and quantify pathogens in a given sample. This approach allows for rapid identification of thousands of bacterial and viral species. Furthermore, these tools can potentially identify previously unknown organisms and classify their relationship to known pathogens. In dengue diagnostics, mass spectrometry could be valuable for rapid dengue serotype and genotype identification, particularly during outbreak investigations. Mass spectrometry-based pathogen identification kits are available in 96-well plate formats and can be customized to meet specific diagnostic requirements. The workflow typically involves sample processing for DNA extraction, PCR amplification, mass spectrometry analysis, and computer-aided data interpretation.

Microarray technology offers another powerful approach for high-throughput pathogen detection and characterization. Microarrays enable simultaneous screening of a sample for numerous nucleic acid fragments corresponding to different viruses. Genetic material is typically amplified prior to hybridization to the microarray. Amplification strategies can target conserved viral sequences or utilize random priming approaches. Short oligonucleotides on the microarray slide provide high sequence specificity, while longer DNA fragments offer increased tolerance for mismatches, enhancing the ability to detect genetically diverse viral strains. Laser-based scanners are commonly used to detect hybridized fragments labeled with fluorescent dyes. Microarrays hold significant potential for multiplexed detection of dengue virus and other co-circulating arboviruses, as well as other pathogens causing dengue-like illness, in a single assay.

Other emerging diagnostic technologies are under development and evaluation, including luminescence-based techniques. Luminescence-based assays are attracting increasing interest due to their high sensitivity, low background signal, wide dynamic range, and relatively cost-effective instrumentation.

Quality Assurance in Dengue Laboratory Diagnosis

Many laboratories, particularly in resource-limited settings, utilize in-house developed dengue diagnostic assays. A significant challenge with in-house assays is the lack of standardization in protocols, making inter-laboratory comparison and data aggregation difficult. National reference laboratories and specialized diagnostic centers play a crucial role in organizing quality assurance programs. These programs are essential to ensure the proficiency of laboratory personnel in performing dengue diagnostic assays accurately and reliably and to produce reference materials for quality control of commercial test kits and in-house assays.

For nucleic acid amplification assays, stringent quality control measures are paramount to prevent contamination of patient samples and reagents. Implementing robust controls and participating in proficiency-testing programs are essential to maintain a high level of confidence in the accuracy and reliability of nucleic acid-based dengue diagnostic results.

Biosafety Considerations in Dengue Diagnostics

The collection and processing of blood and other clinical specimens in dengue diagnostics pose a potential risk of exposure to infectious materials for healthcare workers and laboratory personnel. To mitigate this risk, strict adherence to safe laboratory practices is mandatory. These practices include the consistent use of personal protective equipment (PPE), such as gloves, gowns, and face shields, and the use of appropriate leak-proof containers for specimen collection and transport. Comprehensive guidelines on safe laboratory techniques are detailed in the World Health Organization’s Laboratory biosafety manual.

Organization of Dengue Laboratory Services

In dengue-endemic countries, strategic organization of laboratory services is crucial to effectively meet patient needs and support national dengue control strategies. Adequate resource allocation and comprehensive training programs for laboratory personnel are essential components of a well-functioning dengue diagnostic laboratory network. Table 4.4 proposes a model for the organization of laboratory services for dengue diagnosis. Table 4.5 provides examples of good and bad practices in dengue laboratory diagnosis to guide the development and improvement of dengue diagnostic services.

Table 4.4: Model for organizing dengue laboratory services.

Table 4.5: Good and bad practices in dengue lab diagnosis.

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