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By Medifit Biologicals

 

DENGUE TESTING

DENGUE TESTING 1

INTRODUCTION

Efficient and accurate diagnosis of dengue is of primary importance for clinical care (i.e. early detection of severe cases, case confirmation and differential diagnosis with other infectious diseases), surveillance activities, outbreak control, pathogenesis, academic research, vaccine development, and clinical trials.

Laboratory diagnosis methods for confirming dengue virus infection may involve detection of the virus, viral nucleic acid, antigens or antibodies, or a combination of these techniques. After the onset of illness, the virus can be detected in serum, plasma, circulating blood cells and other tissues for 4–5 days. During the early stages of the disease, virus isolation, nucleic acid or antigen detection can be used to diagnose the infection. At the end of the acute phase of infection, serology is the method of choice for diagnosis.

Dengue virus infection produces a broad spectrum of symptoms, many of which are non-specific. Thus, a diagnosis based only on clinical symptoms is unreliable. Early laboratory confirmation of clinical diagnosis may be valuable because some patients progress over a short period from mild to severe disease and sometimes to death. Early intervention may be life-saving.

 

LABORATORY INVESTIGATIONS:

 

  1. VIRUS DETECTION

Dengue virus can be isolated by the inoculation of diagnostic samples into mosquitoes, cell culture (using mosquito cell lines, such as C6/36 and AP61 or mammalian cell lines, such as Vero and LLC-MK2 cells) or intra-cerebral inoculation of suckling mice. Whole blood, serum or plasma is collected from patients during the acute phase of the disease or from tissues in fatal cases (dengue virus has also been isolated from liver, spleen, lymph nodes and other tissues). There is evidence that the virus isolation rates from whole blood are considerably higher than the isolation rates from serum or plasma. For virus serotype identification, immunofluorescent assays using serotype-specific monoclonal antibodies (mAbs) are used.

 

  1. VIRAL RNA DETECTION

Dengue viral RNA can be detected using a nucleic acid amplification test (NAAT) on tissues, whole blood or sera taken from patients in the acute phase of the disease. Various protocols have been developed that identify dengue viruses using primers directed to serotype-specific regions of the genome.

Nested PCR techniques improve the sensitivity of detection because the initial amplification product is used as the target for a second round of amplification. However, it is crucial that laboratories performing nested PCT take every precaution to prevent false-positive results that can occur as a result of contamination. In situ PCR can be carried out on tissue slides.

  1. ANTIGEN DETECTION

3.1. NS1-based assays.

A simplified method of diagnosis during the acute stage of dengue infection compared to viral isolation or nucleic acid detection is the detection of viral antigens in the bloodstream; however, antigen detection in the acute stage of secondary infections can be compromised by pre-existing virus–IgG immunocomplexes.

New developments in enzyme-linked immunosorbent assay (ELISA) and rapid immunochromographic assays that target non-structural protein 1 (NS1) have shown that high concentrations of this antigen can be detected in patients with primary and secondary dengue infections up to 9 days after the onset of illness12. NS1 is synthesized by all flaviviruses and is secreted from infected mammalian cells. The presence of secreted NS1 (sNS1) in the bloodstream stimulates a strong humoral response. Many studies have investigated the utility of sNS1 detection as a diagnostic tool during the acute phase of a dengue infection. A serotype-specific mAb-based NS1 antigen-capture ELISA has recently been developed and shows good serotype specificity. This test can differentiate between primary and secondary dengue virus infections. There is a good correlation between NS1 serotype-specific IgG as determined by ELISA and plaque reduction neutralization test (PRNT) results, but the performance and utility of these NS1-based tests require additional evaluation.

DENGUE TESTING 2

3.2. Immunohistochemistry.

Dengue antigens can be visualized in tissue sections using labelled mAbs that are visualized with markers such as fluorescent dyes, enzymes or colloidal gold. These tests can be undertaken on frozen tissue or paraffin-embedded slides from fatal cases.

  1. Serological methods

The acquired immune response to dengue virus infection consists of the production of immunoglobulins (IgM, IgG and IgA) that are mainly specific for the virus envelope (E) protein. The intensity of the response varies depending on whether the individual has a primary or secondary dengue infection. During a primary dengue infection, the IgM response is typically higher titre and more specific than during secondary infections. The titre of the IgG response is higher during secondary infection than during primary infection

It is difficult, if not impossible, to use serology to identify dengue serotypes following a recent infection because the antibodies produced following a primary dengue infection often demonstrate some degree of cross-reactivity with other dengue virus serotypes. Antibodies formed following secondary dengue infections are strongly cross-reactive within the dengue group and also usually crossreact with other flaviviruses.

4.1. IgM-based assays.

The detection of dengue-specific IgM is a useful diagnostic and surveillance tool. IgM is initially detectable between 3 to 5 days post onset of fever in ~50% of hospitalized patients and has a sensitivity and specificity of ~90% and 98%, respectively, when assays are undertaken five days or more after the onset of fever. Dengue-specific IgM is expressed earlier than dengue-specific IgG. In one study in Puerto Rico, by day 5 of illness, most patients (80%) with dengue infections that were subsequently confirmed by HAI on paired serum samples or by virus isolation had detectable IgM in acute-phase serum. Nearly all patients (93%) developed detectable IgM 6 to 10 days after the onset of fever, and 99% of patients tested between 10 and 20 days after onset had detectable IgM.

The sensitivity and specificity of IgM-based assays is strongly influenced by the quality of the antigen used and can vary greatly between commercially available products. ELISA-based IgM assays have become an invaluable tool for the surveillance of dengue. Many ELISAs use dengue E protein antigens from all four dengue virus serotypes. This ensures that the assay is capable of identifying any dengue infection regardless of the serotype. However, because IgM circulates for up to three months or longer, its presence might not be diagnostic of a current illness. To diagnose a current dengue infection, the demonstration of a seroconversion (four fold or greater changes in antibody titres) in paired sera is required.

In areas where dengue is not endemic, IgM-based assays can be used in clinical surveillance for viral illness or for random, population-based serological surveys, with the likelihood that any positive results detected indicate recent infections (within the past 2 to 3 months). The M antibody capture ELISA (MAC-ELISA) is based on detecting IgM in serum using human-specific IgM that is bound to the solid phase. MAC ELISAs are frequently run as a non-quantitative, single dilution test and positive results are commonly reported as a ‘recent flavivirus infection’.

Rapid IgM-based dengue diagnostic tests have been developed as a quick and easy method for use at point of care or bedside, and exist in different formats including particle agglutination and lateral flow immunochromatographic strips, with or without plastic cassettes. Most of these tests use recombinant antigens from all four dengue virus serotypes and the results are available within 15 to 90 minutes. Four rapid IgM-based kits have been evaluated. Their sensitivities ranged from 21%–99% and their specificities from 77%–98%, compared with gold standard laboratory-based ELISAs. False-positive results were present in patients with malaria or previous dengue infections. The ELISA format shows greater sensitivity in detecting dengue-specific antibodies than the rapid tests, but the rapid tests are field friendly, with the results available in a short timeframe.

IGG-BASED ASSAYS.

Dengue-specific IgG-based assays can be used for the detection of past dengue infections and current infections if paired sera are collected within the correct time frame to allow the demonstration of seroconversion between acute and convalescent serum samples. Assays are usually carried out using multiple dilutions of each serum tested to determine an end-point titre.

IgG avidity assays can be used to determine whether an infection is a primary or a secondary infection, based on the principle that the avidity of IgG is low after primary antigenic challenge but matures slowly within the weeks and months after infection. Thus, these assays can be more useful than the HAI test for this purpose. The IgG-based ELISA exhibits the same broad cross-reactivity with other flaviviruses as the HAI test; therefore, it cannot be used to identify the infecting dengue virus serotype. However, it has a slightly higher sensitivity than the HAI test.

By Medifit Biologicals

www.medifitbiologicals.com