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Laboratory Tests For The Diagnosis Of Dengue Virus Infection

30 Nov 2007

Source: WHO/TDR


Philippe Buchy 1 , Sutee Yoksan 2 , Rosanna W Peeling 3 , Elizabeth Hunsperger 4

1Institut Pasteur in Cambodia, Virology Unit, 5 Monivong boulevard, PO Box 983, Phnom Penh, Cambodia
2Mahidol University, Centre for Vaccine Development, Bangkok, Thailand
3UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR), World Health Organization, Geneva, Switzerland
4Centers for Disease Control and Prevention (CDC), Dengue Branch, San Juan, Puerto Rico

Working paper for the Scientific Working Group on Dengue Research, convened by the Special Programme for Research and Training in Tropical Diseases, Geneva, 1–5 October 2006

Full text source: Scientific Working Group, Report on Dengue, 1–5 October 2006, Geneva, Switzerland, Copyright © World Health Organization on behalf of the Special Programme for Research and Training in Tropical Diseases, 2007,


Dengue, a mosquito-transmitted viral disease that produces variable symptoms, ranging from asymptomatic infection to life-threatening disease, is present in about 110 tropical and subtropical countries. As dengue is increasing in incidence, improved diagnosis, early detection of severe cases, and efficient medical management are of primary importance in all areas where dengue is endemic. Traditionally, dengue has been diagnosed by virus isolation or serological methods, but with recent advances in molecular techniques and in rapid detection technology, a range of novel diagnostic tests will soon be commercially available that will improve case management and aid disease control efforts.

The goal of this paper is to review the diagnostic tools that are currently available or in development and their potential role in case detection, identification of prognostic markers of severe disease, surveillance and outbreak investigations.

The immunological response to infection with dengue virus

The acquired immune response to infection with dengue virus consists of the production of IgM and IgG antibodies primarily directed against the virus envelope proteins. The immune response varies depending on whether the individual has a primary or a secondary infection [40] In general, serodiagnosis of dengue is dependent on the stage of the infection. Figure 1 depicts the general time-line of a primary infection from virus isolation/identification to detection of IgM and IgG.

Figure 1

General time-line of a primary infection with dengue virus, from identification and isolation of the virus to detection of IgM and IgG.

A primary infection with dengue is characterized by a slow and low-titre antibody response. IgM antibody is the first immunoglobulin isotype to appear. Anti-dengue IgG at low titre is detectable at the end of the first week of illness, increasing slowly thereafter. In contrast, during a secondary infection (a dengue infection in a host that had been previously infected by a dengue virus or other flavivirus) antibody titres rise extremely rapidly and antibody reacts broadly with many flaviviruses [10]. High levels of IgG are detectable even in the acute phase and they rise dramatically over the following 2 weeks. The kinetics of the IgM response are more variable. Since IgM levels are significantly lower in secondary dengue infections, some false-negative results in tests for anti-dengue IgM are observed during secondary infections. According to the Pan American Health Organization (PAHO) guidelines [27], IgM antibody is detectable by day 5 of illness in 80% of all dengue cases, and by day 6–10 of illness in 93–99% of cases, and may then remain detectable for more than 90 days. IgM antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) has become an important tool in the routine diagnosis of dengue; this technique has a sensitivity and specificity of approximately 90% and 98%, respectively, but only when used 5 or more days after the onset of fever. Different formats, such as capture ELISA, capture ultramicroELISA, dot-ELISA, AuBioDOT IgM capture and dipsticks, have been developed. Serum, blood on filter paper, and saliva, but not urine, can be used for detection of IgM if samples are taken within the appropriate time frame (5 days or more after the onset of fever) [37]. The different commercial kits available have variable sensitivity and specificity [8,3]. A further challenge in the diagnosis of dengue is the fact that anti-dengue IgM antibodies also cross-react to some extent with other flaviviruses, such as Japanese encephalitis, St Louis encephalitis and yellow fever.

A review of diagnostic methods for the detection of dengue infection

Techniques for virus isolation and identification

In the early stages of infection, isolation and identification of dengue virus is traditionally the only way to diagnose a current dengue infection. In this technique, serum from patients is applied to mosquito cell lines. After amplification of the virus in infected cells, the serotype is identified using monoclonal antibodies specific to each dengue serotype. This technique is only sensitive when there is a relatively high level of infectious particles in the serum. Dengue viraemia is short, typically starting 2 or 3 days before the onset of fever and lasting until day 4 or 5 of illness. Serum is the sample of choice for routine diagnosis by virus detection, although dengue virus can also be detected in plasma, leukocytes and in some tissues obtained at autopsy.

The intrathoracic inoculation of mosquitoes (Ae. aegypti, Ae. albopictus, Toxorhynchites splendens, Tx. amboinensis) is the most sensitive system for the isolation of dengue virus, but because of the particular technical skill and special containment facilities required for direct inoculation of mosquitoes, cell culture is preferable for routine diagnosis. The mosquito cell line C6/36 (clone obtained from Ae. albopictus) has become the host cell of choice for routine isolation of dengue virus, although the Ae. pseudoscutellaris cell line AP61 has also been used successfully.[35,30] Mammalian cell cultures such as Vero cells, LLCMK2 and others have also been employed, with less efficiency.

Identification of the dengue virus is generally accomplished using immunofluorescence techniques with serotype-specific monoclonal anti-dengue antibodies on mosquito head squashes or infected cells.[9] Some strains are not easily identified because of a low concentration of virus. Plaque assay is the gold standard methodology for the quantification of dengue virus. An indirect immunofluorescence assay was proposed by Payne et al[28] as an alternative to this test. Flow cytometry has recently been reported as a useful method for the identification of dengue virus 1 (DEN-1), and allows the virus to be identified 10 hours earlier than with an immunofluorescence assay, using an anti-nonstructural glycoprotein (NS1) monoclonal antibody.[13]

Serological methods

MAC ELISA. Classic serological testing for dengue includes MAC-ELISA. This assay uses dengue-specific antigens from all four serotypes (DEN 1–4) for the capture of anti-dengue IgM-specific antibodies in serum samples. Most of the antigens used for this assay are derived from the dengue virus envelope protein. The limitations of this test include the specificity of these antigens and cross-reactivity with other circulating flaviviruses. These limitations have to be taken into account when working in regions where multiple flaviviruses co-circulate. IgM detection is not useful for the determination of dengue serotypes owing to cross-reactivity of the antibody, even during primary infections.

IgG ELISA. The classic IgG ELISA used for the detection of a past infection with dengue uses the same antigens as the MAC-ELISA. The assay is usually performed with multiple dilutions of the sera tested to determine an end-point dilution. This assay correlates with the haemagglutination assay used in the past. The higher the end-point dilution, the more robust the response obtained after the infection. In general, IgG ELISA lacks specificity within the flavivirus sero-complex groups; however, Cardosa et al [4] demonstrated that the IgG response to premembrane protein is specific to individual flaviviruses. No cross-reaction was observed when sera were tested from individuals infected with dengue virus or Japanese encephalitis virus. An excellent specificity of anti dengue-specific IgG was obtained by Baretto Dos Santos et al. [2] in an assay using a recombinant polypeptide located in the N-terminal portion of the envelope protein. Although the detection of specific IgG has been superseded in the diagnosis of acute infection, seroepidemiological studies are best carried out using ELISAs to detect specific IgG. IgG avidity ELISAs can be used to determine whether an infection is primary or secondary, and can be more useful than the haemagglutination inhibition test for this purpose.[21]

IgM/IgG ratio. The IgM/IgG ratio is also used to distinguish primary from secondary infections with dengue. A dengue virus infection has been defined as primary if the capture IgM/IgG ratio is greater than 1.2, or as secondary if the ratio is less than 1.2. This ratio testing system has been adopted by commercial vendors such as PanBio. However, Falconar et al. [7] have recently shown that the ratios vary depending on whether the patient has a serological non-classical or a classical dengue infection, and redefined the ratios, taking into consideration the four subgroups of classical infection with dengue. The adjusted ratios of greater than 2.6 and less than 2.6, established by these authors, correctly classified 100% of serologically classical dengue infections and 90% of serologically non-classical infections.

Plaque reduction and neutralization test (PRNT) and the microneutralization assay

PRNT is the most specific serological tool for the determination of dengue antibodies [5] and is used to determine the infecting serotype in convalescent sera. This assay measures the titre of neutralizing antibodies in the serum of the infected individual and determines the level of protection the individual had against the infecting virus. The assay is based on the principle of interaction of virus and antibody, resulting in inactivation of virus such that it is no longer able to infect and replicate in cell culture. Some of the variability found in this assay is attributable to differences in interpretation of the results. The cell lines and virus seeds used as well as the dilution of the sera accounts for these differences.

The microneutralization assay is based on the same principle as PRNT; however, instead of counting the number of plaques per well, this assay uses a colorimetric measurement of virus-induced cell lysis to determine the end-point dilution. This assay was designed to use small amounts of reagents and to be suitable for the high-throughput testing of large numbers of samples. Some of the limitations of the assay include a poor correlation with PRNT results with samples from people with secondary infections.

Molecular methods

Reverse-transcriptase polymerase chain reaction (RT-PCR) and real-time RT-PCR

The PCR assay routinely used by some laboratories for the identification of dengue virus is the nested RT-PCR assay developed by Lanciotti et al. [16]. This comprises a two-step PCR reaction involving an initial reverse transcription and amplification step using universal dengue primers targeting a region of the virus genome (C-prM) followed by a second amplification that is serotype specific. The products of these reactions are separated by electrophoresis on an agarose gel, and the different-sized bands observed are compared with a standard marker for the relative molecular mass of nucleic acids. Dengue serotypes are identified by the size of their bands.

The real-time RT-PCR assay is a one-step assay system using primer pairs and probes that are specific to each dengue serotype. The use of a fluorescent probe enables the detection of the reaction products in real time without need for electrophoresis. Many real-time RT-PCR assays have been developed either as ‘singleplex’ (only detecting one serotype at a time) or ‘multiplex’ (able to identify all four serotypes from a single sample). The multiplex assays have the advantage that a single reaction can be used to determine all four serotypes without the potential for introduction of contamination during manipulation of the sample.[6,12] The fourplex real-time RT-PCR assays are often less sensitive than nested RT-PCR assay but are faster. An advantage of this assay is the ability to determine viral load in a given sample, which is believed to be important in determining the severity of dengue disease.[38]

Innovation in the development of dengue diagnostics

NS1 assays

The NS1 gene product is a glycoprotein produced by all flaviviruses and is essential for viral replication and viability. During viral replication, NS1 is localized to cellular organelles. The protein is secreted by mammalian cells, but not by insect cells. The secreted form of the protein is a hexamer composed of dimer subunits. Glycosylation of this protein is believed to be important for secretion. NS1 antigen appears as early as day 1 after the onset of fever and declines to undetectable levels after day 5–6. NS1 is also a complement-fixing antigen and it produces a very strong humoral response. Because this protein is secreted, many studies have been dedicated to the utility of NS1 as a tool for the diagnosis of infection with dengue virus. These studies focus on various aspects of diagnosis, including antigen-capture enzyme-linked immunosorbent assay (ELISA), and NS1-specific IgM and IgG responses.

In the last 6 years there have been several studies addressing the use of NS1 antigen and anti-NS1 antibodies as a tool for the diagnosis of dengue. An antigen-capture ELISA test was described, with sensitivities ranging from 4 to 1 ng/ml.[18,43] These studies identified a correlation between disease severity and the quantity of NS1 antigen in the serum. However, another study did not find this correlation and in fact could not differentiate between a primary and a secondary infection.[1] Recently, a serotype-specific monoclonal antibody-based NS1 antigen-capture ELISA that showed good serotype specificity has been developed.[42] Shu et al. [33] have standardized an NS1 serotype-specific IgG indirect ELISA to differentiate primary and secondary dengue virus infections and demonstrated a good correlation between anti-NS1 serotype-specific IgG (determined by ELISA) and PRNT results. The NS1 serotype-specific IgG ELISA worked reliably for the serotyping of dengue virus in convalescent-phase sera from patients with primary infection and in acute-phase sera from patients with secondary infection (which would detect the serotype that caused the first infection), but not so with convalescent-phase sera from patients with secondary infections. Because the results of these studies were varied, results correlating with IgM and IgG assays as well as disease severity and predictors of viraemia, further evaluation of this assay should be performed to determine the main differences between each study.

Commercial kits for the detection of NS1 antigen in serum samples are available. These assays do not differentiate between the serotypes. As NS1 antigen appears early in infection and before the appearance of antibody, such assays are useful for early case detection and for outbreak investigations. Evaluations of these assays should be performed to assess their utility and cost-effectiveness.

The DENFRAME project

The DENFRAME project, funded by the European Economic Community in 2006, proposes to develop novel tools for the diagnosis of dengue based on the use of chemiluminescent optical-fibre biosensors to detect virions, genome and anti-dengue antibodies. Dengue-specific recombinant antigen and new monoclonal antibodies are also used to produce new ELISA tests for the detection of anti-dengue IgM, IgG, IgA and IgE in the serum and saliva of infected patients, in order to increase the sensitivity and specificity of these assays and to facilitate their standardization and automation.

Luminescence-based optical fibre biosensor

Luminescence-based techniques are becoming increasingly popular owing to their high sensitivity, low background, wide dynamic range and relatively inexpensive instrumentation. Luminometry is up to five orders of magnitude more sensitive than absorption spectroscopy and more than 1000 times more sensitive than fluorometry.[32] A state-of-the-art luminometer can detect as little as 0.6 pg of ATP or 0.1 fg of luciferase (approximately 1100 molecules), two common luminescent analytes.[36] Compared with fluorescence, luminometry does not need an excitation source or interference filters, luminescent analytes do not undergo photobleaching, and remains the technology that is most suitable for use on a DNA microarray, or ‘chip’. Successful assays have already been developed for monitoring of water [29] and diagnosis of bacterial [17] and viral [15] infections, thanks to proprietary immobilization technologies [19] and a photon-counting device based on a photomultiplier tube.

Monoclonal antibody mAb4E11

This antibody is directed against the DEN-1 virus. It binds domain III (residues 296–400) of the viral envelope glycoprotein E and neutralizes the virus.[31] This monoclonal antibody recognizes and neutralizes the four serotypes of the virus and does not cross-react with other flaviviruses.[23] The Fab and Fv fragments of mAb4E11 and domain EDIII can be expressed in Escherichia coli.[31]

Natural cytotoxicity receptor immunoglobulins (NCR-Igs)

Natural cytotoxicity receptors (NCRs) are expressed by natural killer (NK) cells. Different NCRs recognize viral haemagglutinins from different virus families, including orthomyxoviruses, paramyxoviruses, pox viruses and flaviviruses. NCR-Igs are recombinant molecules comprising the extracytoplasmic part of the NCR fused to the Fc portion of human IgG1.

Recombinant proteins

A recombinant soluble form of E glycoprotein (sE) of dengue virus can be expressed in Drosophila melanogaster S2 cell lines. S2 cell clones grow well in serum-free medium at room temperature, without the need for a carbon dioxide incubator.[24] sE secretion is easily induced by addition of copper sulfate. Large amounts of purified sE for each of the four serotypes of dengue virus can be easily produced (Després P, personal communication). D. melanogaster S2 cell clones expressing the domain III (EDIII) from glycoprotein E have been developed. Purified soluble EDIII is immunoreactive with neutralizing anti-E monoclonal antibodies.

An optical fibre immunosensor has been generated by modifying the fibre tip with recombinant proteins or with a phage-display library of selected epitopes/mimotopes. This methodology has been shown to be highly sensitive, allowing the detection of antibodies in a serum titer of as low as 1:2 621 440.[19] In the West Nile virus (WNV) model, the optical-fibre immunosensor was found recently to be at least 100 times more sensitive than the typical ELISA methodology, and allowed the assay time to be reduced to only 30 minutes (Marks RS, personal communication). This assay will be adapted for the detection of anti-dengue IgM, IgG, IgA and IgE. Other samples (whole blood, plasma and saliva) from patients infected with dengue will also be tested.

A biosensor based on modified optical fibres designed to detect the viral genome as well as an optical fiber tip modified with monoclonal antibodies or NCR-Igs for virions capture will be built and evaluated for the DENFRAME project.

Since the use of either monoclonal antibodies [26] or recombinant proteins [2,4,39] significantly improves the quality (sensitivity and specificity) of IgM- and IgG-specific ELISA assays, we propose using both approaches simultaneously. The use of recombinant antigen eliminates the problems and avoids the laborious procedures associated with the standardization of dengue virus antigen prepared in mouse brain or cell culture. By using ‘classical’ ELISA and MAC-ELISA approaches, new assays for the detection of anti dengue-specific immunoglobulins (IgM, IgG, IgA, IgE) using novel recombinant antigens and several appropriate antibodies are being developed. Serum, plasma and saliva from infected patients will be screened in parallel in different laboratories and results will be compared with the ‘gold standard’ diagnostic methods. Such ELISA tests can eventually be automated, thus allowing large-scale screening during dengue outbreaks or its use in sero-epidemiological studies.

Microsphere-based immunoassay (MIA)

The traditional serodiagnostic methods use the MAC-ELISA and IgG-ELISA as principal tests. Depending on the sample, this testing is followed by a confirmatory PRNT for positive samples; however, this confirmation can only take place in laboratories with this capability. The MAC-ELISA is a 2-day test that requires about 4 hours of a technician's time. Therefore, a more rapid yet equally sensitive, single test to replace the dengue MAC-ELISA would be of benefit.

Microsphere-based immunoassays (MIAs) are becoming increasingly popular as a serological option for the laboratory diagnosis of many diseases (Kellar et al., 2001). This technology is based on the covalent bonding of antigen or antibody to microspheres or beads. The detecting instrument is a simplified flow cytometer. The lasers simultaneously identify the microsphere sets (bead sets) and measure the fluorescence associated with the reaction. This methodology is particularly attractive because it is faster than the MAC-ELISA and can be used to identify many different antibody responses to multiple viruses. MIAs have the potential to be especially useful in arbovirus serology because tests for infection by viruses of the same genus can share similar formats.

At the Centers for Disease Control and Prevention (CDC), USA, dengue-specific antigens are being developed that could be combined with the current WNV and St Louis Encephalitis (SLE) platform previously developed by Johnson et al.[11] In this assay system, unique beads will contain covalently-bonded flavivirus-reactive antibody. The dengue-specific antigen is allowed to bind to these beads and the sample from the patient is then mixed with the dengue antigen-coated beads. Using flow cytometry, the microspheres or beads are sorted to identify a sample as WNV, SLE or DEN1–4 in a single reaction.

Biosensor technology using mass spectrometry

Rapid advances in biosensor technology using mass spectrometry have led to the development of powerful systems that can provide rapid discrimination of biological components in complex mixtures. The mass spectra that are produced can be considered to be a specific fingerprint or molecular profile of the bacteria or virus analysed. The software system built into the instrument identifies and quantifies the pathogen in a given sample by comparing the resulting mass spectra with those in a database of infectious agents, and thus allows the rapid identification of many thousands of types of bacteria and viruses. Additionally, these tools can recognize a previously unidentified organism in the sample and describe how it is related to others previously encountered. This could be useful in determining not only dengue serotypes, but dengue genotypes during an outbreak. The infectious-agent identification kits can be designed to meet specific needs and come in a 96-well format. Processing the samples involves four main steps of DNA extraction, PCR amplification, mass spectrometry and computer analysis of results.

DNA extraction

The profile of the pathogen can be identified from human or animal clinical samples (blood, throat swabs, skin wipes, hair). Before amplification of any genetic material present, a lysis/purification protocol removes PCR inhibitors and concentrates nucleic acids.


The sample is applied to a microtitre plate containing a pair of broad-range PCR primers. The broad-range primers are designed to amplify the DNA/RNA of an individual viral family or families, typically resulting in a mixture of amplicons of about 100 base pairs in length that reflects the complexity of the original mixture of virus present in the starting sample.

Analysis by electrospray mass spectrometry

The PCR products are desalted and electrosprayed into a mass spectrometer, the fundamental component of the system. The mass spectrometer measures the precise weight of each nucleic acid present. Signals, which appear as peaks, are obtained for each of the amplified regions and for a calibrant molecule.

Signal processing and identification of organisms

Signal-processing software is used to process the spectral signals to determine the mass of each of the PCR products present with sufficient accuracy such that the base composition of adenosines, guanosines, cytidines, and thymidines can be established. Using the unique ‘fingerprint’ represented by combined base compositions from multiple PCR reactions, it is possible to identify the organisms present in the starting sample.

This technology has been successfully used to identify 24 bacterial contaminants in food.[22] One of the major advantages of this system is the ability to identify the pathogen in a relatively short period of time compared with standard methods, and to develop an arbovirus-testing algorithm that could be specifically designed for the needs of each country. In addition, identification of the agent responsible for an outbreak of an emerging infectious disease could be determined more rapidly than by conventional methods.

Evaluation of diagnostic tests for dengue

UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR)

Background. The mission of the Diagnostics Research and Development unit within TDR is to promote and facilitate the development, evaluation and deployment of diagnostic tools for the control of tropical diseases.

Accurate diagnostic tests have a key role in patient management and the control of most infectious diseases. Unfortunately, in many developing countries clinical care is often critically compromised by the lack of regulatory controls on the quality of these tests. As a result, in many developing countries diagnostic tests are sold and used without evidence of effectiveness. There is an urgent need for evaluation of commercially available diagnostic tests for dengue.

Identifying priorities. TDR, in collaboration with the Pediatric Dengue Vaccine Initiative (PDVI), convened a meeting of experts to determine the ideal test specifications for diagnostic tools for case management, epidemiological surveillance or vaccine efficacy trials. An inventory of antigen- and antibody-detection tests for the diagnosis of dengue was compiled. A consensus was reached that the highest priority was to evaluate assays for the detection of anti-dengue IgM, in either a rapid test or ELISA format. The next priority was the evaluation of antigen detection tests, such as the NS1 assays, for early case detection.

The group also identified the need to establish a bank of well-characterized specimens from different endemic areas for facilitating test evaluations.

Establishing a dengue specimen bank/evaluation network. TDR/PDVI identified a network of laboratories in Latin America and Asia that have the capacity and expertise to perform diagnostic evaluations as well as participate in the collection of well-characterized specimens. A reference laboratory has been identified in each region to which specimens from participating laboratories in the region are sent and stored.

In preparation for the evaluation, standardized evaluation protocols are developed and panel composition is determined according to the type of evaluation required. Evaluation panels are drawn from the two regional specimen banks and are validated by the reference laboratories. The two regional reference laboratories collaborate in the formation of final panel that will be used for the evaluations.

Table 1

TDR/PDVI dengue specimen bank/evaluation sites

Selected site

WHO Region

South-East Asia


Reference centre

Dr Sutee Yoksan

Dr Elizabeth Hunsperger

Centre for Vaccine Development, Mahidol University, Bangkok, Thailand

Dengue Branch, Centers for Disease Control and Prevention (CDC), San Juan, Puerto Rico

Evaluation laboratories

Dr Vinh Chau Nguyen

Dr Susana Vázquez

Hospital for Tropical Medicine (Cho Quan Hospital), Ho Chi Minh City, Viet Nam

Instituto Medicina Tropical ‘Pedro Kouri’, Havana, Cuba

Dr Philippe Buchy

Dr Pedro Vasconcelos

Institut Pasteur in Cambodia, Phnom Penh, Cambodia

Instituto ‘Edvandro Chagas’, Belem, Brazil

Dr Shamala Devi Sekaran

Dr Delia Enria

Department of Medical Microbiology, Faculty of Medicine, University of Malaya. Kuala Lumpur, Malaysia

Instituto Nacional Enfermedades Virales Humanas ‘Dr Julio I Maiztegui’ Pergamino, Argentina

a Names of the principal investigators are given in italic type

Engagement with industry. A letter was sent from TDR to manufacturers of commercially available dengue IgM tests describing the evaluation process, to explore their interest in participating in the evaluation. Companies that indicated interest were sent a copy of the standard WHO Confidentiality and Material Transfer Agreement that describes in detail their obligations in providing a sufficient number of tests for the evaluation and explains that they would be given courtesy review of the evaluation results before publication. Companies can visit the evaluation sites and send queries but are not in a position to alter or block the publication of the evaluation results. All results will be available to WHO Member States. They will also be posted on the TDR website and published in peer-reviewed journals. Companies that agree to the terms of the evaluation will be included in the evaluation. Table 2 describes the operational characteristics of the IgM tests that are currently under evaluation.

Table 2

Dengue IgM tests under evaluation


ELISA tests

Rapid tests

Country of origin

Australia, USA, UK

Australia, Japan, India, Republic of Korea, USA

Antibodies detected

IgM/IgG or IgM only

IgM/IgG or IgM only

Solid phase

Antigen adsorbed into plastic wells sold as 8-well strips x 12 set into a plastic tray

Nitrocellulose strips sold as dipsticks or encased in plastic cassettes

Specimen type


Whole blood, sera or plasma

Number of tests, by package

96- well or 192



Purified virus or recombinant antigen

Purified dengue antigen or recombinant antigen, four serotypes

Volume of sample required

1–10 µl

1–5 µl

Supplies required but not provided

ELISA reader, pipettes

Some may require a micropipette

Time taken to obtain results

1–4 hours

15–90 minutes

Price per kit (US dollars)

Depends on volume of order

Depends on volume of order

Storage (°C)

2–8 °C

2–8 °C or 4–30 °C

ELISA, enzyme-linked immunosorbent assay; UK, United Kingdom; USA, United States of America

Evaluation of dengue IgM tests

Tests under evaluation

The operational characteristics of the IgM tests under evaluation are described in Table 2.

Composition of the IgM evaluation panel

A panel of 350 well-characterized serum specimens will be used for the performance evaluation. The specimens will be assembled by the reference laboratories from archived collections from geographically diverse areas, provided by the evaluating laboratories. These samples will be validated by the reference laboratories and assigned panel codes.

The serological performance panel is intended to offer a comprehensive assessment of the performance of existing diagnostic laboratory tests, to ensure that such tests are specific to dengue viruses and do not give false positive results when tested against sera from patients infected with a related flavivirus or with other etiological agents causing acute febrile illness. The inclusion of dengue sera at different titres is intended to determine whether the kits being evaluated are capable of detecting sera with medium and low titres of antibody.

To evaluate sensitivity, a total of 200 serum specimens from patients with primary and secondary infections and by different serotypes are tested, as described in Table 3.

Table 3

Proposed composition of a serological panel to be used for the evaluation of the performance of diagnostic tests for dengue

No. of samples


Source of sera

Primary infection—serotypes 1, 2, 3, 4

Dengue types

Secondary infection—serotypes 1, 2, 3, 4

200 combined

Primary infection—high, medium, low titre

Antibody titre

Secondary infection—high, medium, low titre

Up to 50

Cross-reactive flavivirus (acute or convalescence)

West Nile virus, yellow fever, Japanese encephalitis, St Louis encephalitis, tick-borne flavivirus

Up to 100

Syndromic—acute febrile illnesses

Leptospira, malaria, scrub typhus, mayaro, enterviruses

Up to 25

Interference panel

Rheumatoid arthritis, myeloma, hypergammaglobulinaemia, medications (e.g. steroids)



Negative for dengue antibodies

For the evaluation of specificity, the panel contains 150 samples of sera that are negative for dengue, as described in Table 4, including those from patients infected with other flaviviruses, with acute febrile illness attributable to other causes, with clinical conditions such as rheumatoid arthritis that may interference with the assay causing false positives.

Prospective sample collection for serum banking

Since a reasonably large amount of serum is needed to meet the panel requirements, guidelines were developed for the collection process. Institutional review board (IRB) approval details were summarized as well as possible sources to locate specific panel components. A simple questionnaire and tracking records for specimen management will be developed, which should include a unique specimen number, date and site of specimen collection, demographic and epidemiology information. After receiving informed consent from the blood donor, samples can be collected by venepuncture or by plasmaphoresis. The sera should be coded in such a way that they cannot be traced to the donor.

Reference standard test

The CDC ELISA and the Armed Forces Research Institute of Medical Sciences (AFRIMS) ELISA are the reference methods for the IgM test evaluation and the results for all test kits will be compared to those for these assays.

Summary of the evaluation of scheme

A schematic of the evaluation scheme is shown in Figure 2. The roles and responsibilities of the reference laboratories, the network laboratories and industry are outlined for every stage of the evaluation process.

Figure 2

Schematic for laboratory-based evaluation of dengue diagnostics

Conclusions and recommendations

To improve case management, surveillance, outbreak investigations and to ensure the success of dengue vaccine trials, quality diagnostic tools are essential.. However, current diagnostic tools available for dengue are not practical for point-of-care use or during the febrile phase of the disease. Many tools are commercially available but their performance and operational characteristics have not been widely evaluated. More novel diagnostic techniques need to be developed for patient management. The goal of a new diagnostic tool would combine antigen (e.g. NS1 antigen) and IgM/IgG detection in a single test and ideally prognostic markers of disease severity would be paired with etiologic diagnosis. The recommended new tools, reference material collection and specimen banks discussed within this document address these needs.

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