Commercially developed NS1 antigen capture assays have the potential to improve dengue diagnosis. However, understanding the clinical circumstances under which they are most useful is important in developing a diagnostic algorithm that maximizes the likelihood of a correct diagnosis using these tests. Routine diagnostics for dengue include using RT-PCR or viral isolation during the acute phase of the infection (0 to 5 DPO) and serology tests including IgM and IgG antibody detection in the convalescent phase of the infection (6 to 14 DPO). The NS1 assay has not been routinely incorporated into the current diagnostic scheme, and the manufacturer of the test kit suggests using the assay during the first 8 DPO; however, this suggestion requires validation. Therefore, the product's primary use has been for the acute phase of dengue infection (0 to 5 DPO), for which the highest and most consistent sensitivities have been observed in independent studies. These independent studies determined that the sensitivity of the NS1 assay by Bio-Rad ranged from 63 to 97% and that the specificity ranged from 98 to 100% (2
). In addition, previous studies performed in our laboratory noted differences in sensitivity among DENV serotypes (for DENV1, 93%; for DENV2, 82%; for DENV3, 87%; and for DENV4, 71%) (1
The study presented here revealed that the Bio-Rad kit was able to detect NS1 in 37% of the acute-phase samples positive for DENV (with positivity confirmed by seroconversion) that were incorrectly classified by PCR (false negatives). Because the sensitivity of PCR for acute-phase specimens has been shown to be as low as 80%, depending on the primer/probe sets and the assay used for detection, an additional assay capable of improving the sensitivity of the diagnostic algorithm for a single acute-phase sample would be extremely valuable (4
).With our current surveillance strategy, final classification of approximately 50 to 60% of samples remains unresolved as a result of failure to obtain a convalescent-phase sample for serological confirmation in cases in which the acute-phase sample is negative by PCR testing (unpublished CDC data). The ability to resolve the identity of the etiological agent from a single acute-phase sample is important for clinical triage for patients, as well as epidemiological investigations. In addition to being used for acute-phase samples, the NS1 assay may resolve difficult cases, including those of secondary DENV infection convalescent-phase samples that do not express detectable levels of IgM as well as late-convalescent-phase samples in which IgM titers have already declined (11
). This study was designed to determine the utility of this assay for these difficult sample types.
Because IgM antibody kinetics differ between primary and secondary flavivirus infections, serological diagnosis is complicated. The IgM response appears later in primary infections (>6 DPO) than secondary infections (<5 DPO) and has a longer duration for primary infection (14 to 90 days) than for secondary infection (<10 days). Moreover, it has been estimated that in up to 30% of secondary infections, an IgM response is never mounted at all (11
). Despite a precipitous drop in the sensitivity of the NS1 test for samples collected after 5 DPO (convalescent-phase samples), we found that 20% of samples from patients with secondary infections that failed to mount an IgM response detectable in the convalescent-phase sample still tested positive for NS1. This result illustrates the utility of an NS1 assay as an adjunct test in the diagnosis of DENV infection in patients with a positive history of DENV infection whose current status cannot be resolved by an IgM ELISA alone.
The NS1 assay may be useful for late-convalescent-phase samples to resolve cases in which apparent acute DENV infections cannot be confirmed by the current testing algorithm using IgM ELISA and PCR. Since the IgM response remains elevated for up to 90 DPO of a dengue infection, a positive IgM response is considered to indicate a recent flavivirus infection and not an acute infection. In other words, a positive NS1 response in this type of sample redefines the clinical diagnosis from a recent dengue infection to an acute dengue infection. Furthermore, a positive NS1 response further identifies the infection as a DENV infection versus a flavivirus infection because the NS1 assay is 98 to 100% specific for DENVs, in contrast to an IgM ELISA, which is between 70 and 90% specific depending on the ELISA format used (10
). Therefore, NS1 can be utilized for differential diagnosis in cases in which the antigen for the IgM ELISA cross-reacts with other anti-flavivirus antibodies and the infecting virus cannot be identified by conventional RT-PCR.
The Platelia DENV NS1Ag test allowed us to significantly improve upon the sensitivity of RT-PCR alone for the detection of DENV in acute-phase serum samples. Additionally, the improved sensitivity afforded by the Bio-Rad assay for the subset of secondary-infection-associated samples that do not demonstrate IgM seroconversion makes it a valuable addition to a DENV testing algorithm aimed at diagnosis of infection with a single clinical sample. The use of an algorithm based on a single clinical sample has the potential to reduce the number of indeterminate diagnoses due to failure to obtain a convalescent-phase sample. Moreover, since secondary dengue infections are likely to cause a more severe form of disease than primary infections, this alternative testing may prove valuable in the clinical setting, when early diagnosis and treatment can decrease mortality to <1% (17
). The excellent sensitivity and specificity of the assay, combined with its relatively low technological and resource requirements, make it an attractive alternative to RT-PCR for diagnosis of acute DENV infection in resource-poor areas with a potential for definitive point-of-care diagnosis in a single clinic visit.