Dengue virus detection test

Dengue diagnostic testing molecular and serologic is available from several commercial reference diagnostic laboratories, state and local public health laboratories, and CDC. Consultation on dengue diagnostic testing can be obtained from CDC at Skip directly to site content Skip directly to page options Skip directly to A-Z link.

Section Navigation. NS1 is detectable during the acute phase of dengue virus infections. NS1 tests can be as sensitive as molecular tests during the first days of symptoms.

After day 7, NS1 tests are not recommended. A positive NS1 test result is indicative of a dengue infection but does not provide serotype information. Knowing the serotype of the infecting virus is not necessary for patient care; however, if serotype information is needed for surveillance purposes, the sample should be tested by NAT. Though studies show that NS1 can be found in whole blood or plasma, most NS1 tests have been developed and evaluated in serum samples. While combined testing with a NS1 and IgM antibody test can usually provide a diagnostic result during the first days of illness, a second, convalescent phase specimen should be obtained and tested for IgM when both antigen and antibody tests are negative.

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. Before day 5 of illness, during the febrile period, dengue infections may be diagnosed by virus isolation in cell culture, by detection of viral RNA by nucleic acid amplification tests NAAT , or by detection of viral antigens by ELISA or rapid tests.

Virus isolation in cell culture is usually performed only in laboratories with the necessary infrastructure and technical expertise. For virus culture, it is important to keep blood samples cooled or frozen to preserve the viability of the virus during transport from the patient to the laboratory.

The isolation and identification of dengue viruses in cell cultures usually takes several days. Nucleic acid detection assays with excellent performance characteristics may identify dengue viral RNA within 24—48 hours. However, these tests require expensive equipment and reagents and, in order to avoid contamination, tests must observe quality control procedures and must be performed by experienced technicians.

NS1 antigen detection kits now becoming commercially available can be used in laboratories with limited equipment and yield results within a few hours. Rapid dengue antigen detection tests can be used in field settings and provide results in less than an hour.

Currently, these assays are not type-specific, are expensive and are under evaluation for diagnostic accuracy and cost-effectiveness in multiple settings. Table 4. Summary of operating characteristics and comparative costs of dengue diagnostic methods 9. After day 5, dengue viruses and antigens disappear from the blood coincident with the appearance of specific antibodies.

NS1 antigen may be detected in some patients for a few days after defervescence. Dengue serologic tests are more available in dengue-endemic countries than are virological tests.

Specimen transport is not a problem as immunoglobulins are stable at tropical room temperatures. For serology, the time of specimen collection is more flexible than that for virus isolation or RNA detection because an antibody response can be measured by comparing a sample collected during the acute stage of illness with samples collected weeks or months later.

Results of rapid tests may be available within less than one hour. Reliance on rapid tests to diagnose dengue infections should be approached with caution, however, since the performance of all commercial tests has not yet been evaluated by reference laboratories A four-fold or greater increase in antibody levels measured by IgG ELISA or by haemagglutination inhibition HI test in paired sera indicates an acute or recent flavivirus infection.

However, waiting for the convalescent serum collected at the time of patient discharge is not very useful for diagnosis and clinical management and provides only a retrospective result. Dengue fever can easily be confused with non-dengue illnesses, particularly in non-epidemic situations. Depending on the geographical origin of the patient, other etiologies — including non-dengue flavivirus infections — should be ruled out.

These include yellow fever, Japanese encephalitis, St Louis encephalitis, Zika, and West Nile, alphaviruses such as Sinbis and chikungunya , and other causes of fever such as malaria, leptospirosis, typhoid, Rickettsial diseases Rickettsia prowazeki, R. Unfortunately, an ideal diagnostic test that permits early and rapid diagnosis, is affordable for different health systems, is easy to perform, and has a robust performance, is not yet available.

During outbreaks some patients may be seen presenting with fever with or without rash during the acute illness stage; some others may present with signs of plasma leakage or shock, and others with signs of haemorrhages, while still others may be observed during the convalescent phase. One of the priorities in a suspected outbreak is to identify the causative agent so that appropriate public health measures can be taken and physicians can be encouraged to initiate appropriate acute illness management.

In such cases, the rapidity and specificity of diagnostic tests is more important than test sensitivity. Samples collected from febrile patients could be tested by nucleic acid methods in a well-equipped laboratory or a broader spectrum of laboratories using an ELISA-based dengue antigen detection kit.

If specimens are collected after day 5 of illness, commercial IgM ELISA or sensitive dengue IgM rapid tests may suggest a dengue outbreak, but results are preferably confirmed with reliable serological tests performed in a reference laboratory with broad arbovirus diagnostic capability. Serological assays may be used to determine the extent of outbreaks.

Dengue surveillance systems aim to detect the circulation of specific viruses in the human or mosquito populations. The diagnostic tools used should be sensitive, specific and affordable for the country. Vaccine trials are performed in order to measure vaccine safety and efficacy in vaccinated persons. The plaque reduction and neutralization test PRNT and the microneutralization assays are commonly used to measure protection correlates.

Following primary infections in non-flavivirus immunes, neutralizing antibodies as measured by PRNT may be relatively or completely specific to the infecting virus type 11 , This assay is the most reliable means of measuring the titre of neutralizing antibodies in the serum of an infected individual as a measure of the level of protection against an infecting virus.

The assay is based on the principle that neutralizing antibodies inactivate the virus so that it is no longer able to infect and replicate in target cells.

After a second dengue virus infection, high-titre neutralizing antibodies are produced against at least two, and often all four, dengue viruses as well as against non-dengue flaviviruses. This cross reactivity results from memory B-cells which produce antibodies directed at virion epitopes shared by dengue viruses.

During the early convalescent stage following sequential dengue infections, the highest neutralizing antibody titre is often directed against the first infecting virus and not the most recent one. The disadvantages of PRNT are that it is labour-intensive. A number of laboratories recently developed high through-put neutralization tests that can be used in large-scale surveillance studies and vaccine trials. Variable results have been observed in PRNTs performed in different laboratories.

Variations can be minimized if tests are performed on standard cell lines using the same virus strains and the same temperature and time for incubation of virus with antibody. Input virus should be carefully calculated to avoid plaque overlap. The microneutralization assay is based on the same principle as PRNT. Variable methods exist. In one, instead of counting the number of plaques per well, viral antigen is stained using a labelled antibody and the quantity of antigen measured colorimetrically.

The test may measure nucleic acid using PCR. The microneutralization assay was designed to use smaller amounts of reagents and for testing larger numbers of samples. In viral antigen detection tests the spread of virus throughout the cells is not limited because, in PRNTs using semisolid overlays, the time after infection must be standardized to avoid measuring growth after many cycles of replication.

Since not all viruses grow at the same rate, the incubation periods are virus-specific. As with standard PRNTs, antibodies measured by micromethods from individuals with secondary infections may react broadly with all four dengue viruses. In drug trials, patients should have confirmed etiological diagnosis see Table 4. Advantages and limitations of dengue diagnostic methods 9. Specimens for virus isolation should be collected early in the course of the infection, during the period of viraemia usually before day 5.

Virus may be recovered from serum, plasma and peripheral blood mononuclear cells and attempts may be made from tissues collected at autopsy e. Because dengue virus is heat-labile, specimens awaiting transport to the laboratory should be kept in a refrigerator or packed in wet ice. Cell culture is the most widely used method for dengue virus isolation. Since not all wild type dengue viruses induce a cytopathic effect in mosquito cell lines, cell cultures must be screened for specific evidence of infection by an antigen detection immunofluorescence assay using serotype-specific monoclonal antibodies and flavivirus group-reactive or dengue complex-reactive monoclonal antibodies.

Virus isolation followed by an immunofluorescence assay for confirmation generally requires 1—2 weeks and is possible only if the specimen is properly transported and stored to preserve the viability of the virus in it. An isothermal nucleic acid sequence-based amplification NASBA assay was optimized to amplify viral RNA of all four dengue virus serotypes by a set of universal primers and to type the amplified products by serotype-specific capture probes.

The NASBA assay involved the use of silica to extract viral nucleic acid, which was amplified without thermocycling.