Comparison of two dengue NS1 rapid tests for sensitivity, specificity and relationship to viraemia and antibody responses

Dengue is a major public health problem in tropical and subtropical countries [1, 2]. Each of the four serotypes of dengue virus (DENV-1 to DENV-4) is able to cause disease. Clinically apparent disease varies in severity from mild undifferentiated fever through to more severe syndromes, such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). DHF is a vasculopathy characterized by capillary leakage and haematological deregulation, which in severe cases can result in life-threatening hypovolemic shock (DSS). There are no licensed vaccines or specific therapies for dengue, and patient management relies on good supportive care.

There are several reasons why early and accurate diagnosis of dengue is important. First, an early and accurate diagnosis can assist in patient management by directing clinical attention to the appearance of major warning signs of severe or even life threatening complications, e.g. rapidly rising hematocrit, poor peripheral perfusion. Second, an accurate dengue diagnosis prevents unnecessary and possibly expensive antibiotic usage. Third, prompt diagnosis of index cases can facilitate vector control activities in the community so as to mitigate further transmission. Lastly, the expanded use of accurate dengue diagnostics provides important data on the epidemiology and health burden of dengue and in doing so can inform and guide public health policy, particularly as dengue vaccines and anti-virals make their way through development pipelines.

Commercial ELISA tests that detect the DENV NS1 protein in plasma/sera have provided a new avenue for diagnosing dengue [3–10]. The detection of NS1 on rapid lateral flow point-of-care tests offers an even faster route to a presumptive dengue diagnosis [11]. As more point-of-care rapid diagnostic tests (RDT) for dengue, particularly those targeting NS1, reach the marketplace their prices will likely drop to the point they are affordable for use in even resource limited health-care settings. The example of RDTs for malaria that target HRP2 provides a useful example of a point-of-care antigen-detection test where a large number of manufacturers in the market have resulted in cheaper tests such that they are being promoted in even the most resource limited settings [12].

The purpose of the current study was to compare the sensitivity and specificity of 2 commercially available, lateral-flow dengue RDTs (Bio-Rad NS1 Ag Strip and SD Dengue Duo) in a panel of plasma samples from dengue patients with different viral serotypes and viraemia levels. The SD Dengue Duo is distinguished from the Bio-Rad NS1 Ag Strip in that it also tests for DENV IgM and IgG.

In the present study we showed that two different commercially available lateral flow RDTs, the Bio-Rad NS1 Ag Strip and SD Dengue Duo, have similar sensitivities (61.6% vs 62.4% respectively) for the detection of NS1 in plasma from RT-PCR positive patients. The factors negatively influencing the detection of NS1 included the presence of DENV-reactive IgG in the test sample and the presence of secondary infection. The inclusion of the IgM test in the SD Dengue Duo provides for greater sensitivity (75.5%) without compromising specificity. The reasonable sensitivity and specificity of the SD Dengue Duo suggests it warrants additional prospective evaluations.

There is, as yet, no dengue RDT that could be considered as providing a definitive diagnosis of dengue. At best, current tests provide a presumptive diagnosis for a patient with a dengue like illness. A recent, TDR/WHO sponsored multi-centre evaluation of anti-dengue virus IgM rapid tests highlighted the very modest specificity of several commercially available IgM rapid tests, particularly in samples from patients with other infections, with malaria being a major confounder [16]. As acknowledged by the authors of the evaluation study, specificity issues must be considered in the context of the setting in which a particular test is to be used [16]. For example, in SE Asia there is no Yellow Fever virus transmission and in many urban settings there is no malaria transmission that could serve to limit the accuracy of a dengue RDT. The challenge then is to identify the best test for a particular setting and this is best done by accumulating more systematic data across a range of patient populations in different health care settings. Decision-tree based algorithms, as suggested for selecting malaria rapid tests, could be helpful in this regard for dengue RDTs [17].

To the best of our knowledge this is the first side-by-side assessment of different NS1 RDTs. Our finding that they were equally sensitive for NS1 detection is encouraging and suggests other points of difference should be considered, e.g. ease of use, cost and stability at room temperature. The strength of the SD Dengue Duo is that the DENV IgM and IgG test windows provides an additional diagnostic investigation that complements NS1 detection. The inclusion of the DENV-IgM result in this current evaluation improved sensitivity without a reduction in the very high specificity associated with NS1 detection (100%). The inclusion of the IgG test result modestly further improved sensitivity. Caution is needed however as a positive IgM or IgG result alone could also represent infection anytime in the previous few months and should therefore be considered a presumptive diagnosis. The potential for reduced specificity was highlighted in that one patient with no laboratory acute evidence of dengue had a positive IgG test result alone. Nevertheless the use of IgM and IgG test parameters in a NS1 RDT is rational as it will likely provide improved presumptive diagnostic coverage towards the end of the acute illness when NS1 levels are declining but the DENV-specific IgM and IgG titres are climbing rapidly. The sensitivity and specificity of the IgM and IgG test components of the SD Dengue Duo have been described previously as part of the TDR/WHO assessment of dengue RDTs [16].

The presence of DENV-reactive IgG in the test sample, a relatively low viraemia and secondary dengue were the major factors associated with a negative NS1 finding on both tests. The bias of these RDTs towards patients with higher viraemia levels is probably a positive feature of these tests in that they are likely biased towards patients at risk for complications during their illness [18]. Somewhat surprisingly we did not replicate our previous observations in a different patient population that NS1 sensitivity by Bio-Rad rapid test was higher in the first 3 days of illness that at later times [11]. Differences exist between the two studies and these include- a) the first study included adults and b) the first study was done prospectively in real time with fresh plasma rather than frozen stored plasma. It is possible these factors might account for the differences between the study findings. It may also reflect chance differences associated with relatively small sample sizes. Similarly, we found greater NS1 sensitivity in samples where there was a measurable DENV-reactive IgM level, a finding inconsistent with our previous observations using ELISA based NS1 detection where the presence of IgM was not associated with higher or lower sensitivity [11]. The different findings might reflect chance differences in the patient population or differences between ELISA versus RDT.

A weakness of this study was that it was performed using a panel of stored plasma specimens and was heavily biased towards DENV-1, the most common serotype in circulation in Viet Nam since 2006. Similarly, assay performance and interpretation were performed by experienced lab analysts and not by clinicians "at the bedside". Different results may also have been obtained if an outpatient population, rather than a hospitalized set of patients, were used to generate the assessment panel used here. The evaluation panel is also biased in that all samples from dengue patients were RT-PCR positive. Despite these limitations, the current study provides a baseline in terms of sensitivity and specificity of these two RDTs for Vietnamese dengue patients and highlights virological and immunological factors associated with assay performance. Further prospective evaluations of both tests are warranted.

Our findings suggest that the NS1 components of each test are specific tools for diagnosing acute dengue, though the sensitivity of both is influenced by the level of viraemia and host humoral immune response. The addition of an IgM/G component to the SD Dengue Duo significantly improved diagnostic sensitivity above NS1 testing alone.