Detection of Immune-Complex-Dissociated Nonstructural-1 Antigen in Patients with Acute Dengue Virus Infections

Abstract

Accurate and timely diagnosis of dengue virus (DEN) infections is essential for the differential diagnosis of patients with febrile illness and hemorrhagic fever. In the present study, the diagnostic value of a newly developed immune-complex dissociated nonstructural-1 (NS-1) antigen dot blot immunoassay (DBI) was compared to a commercially available DEN antigen detection kit (denKEY Blue kit; Globio Co., Beverly, Mass.) and a reverse transcription-PCR (RT-PCR) kit. Serial serum or plasma samples (n ‫؍‬ 181) obtained from 55 acute DEN-infected patients were used. In samples obtained from 32 of these 55 DEN-infected patients, viral RNA could be detected by RT-PCR. DEN antigen was detected in only 10 of these 55 patient samples by using the denKEY kit. When these samples were treated with acid to release the immune-complex-associated NS-1 antigen for detection by DBI, 43 of these 55 patients were found to be positive for DEN NS-1 antigen. In nondissociated samples, 22 of these patients were found to be positive by the DBI. In the presence of DEN-specific immunoglobulin M antibodies, both viral RNA and DEN (NS-1) antigen could be detected. The number of positive samples identified by RT-PCR and DBI from these patients with primary DEN infections varied between 28 and 78%. In secondary DEN infections, the number of samples that tested positive by the DBI after immune-complex dissociation (DIS-DBI) was 25% higher than the number of samples that tested positive by RT-PCR and was 35% higher than that determined by nondissociated antigen (NDIS-DBI) detection. We conclude that the denKEY kit has limited diagnostic value for acute DEN infections compared to the RT-PCR and the NDIS-DBI and DIS-DBI methods. We clearly demonstrate that in secondary DEN infections the dissociation of NS-1 immune complexes is essential for early diagnosis of DEN infections.Dengue virus (DEN) is one of the most widespread mosquito-borne human pathogens worldwide, accounting for more than 50 million infections per year (10). Mosquitoes of the Aedes species are responsible for transmitting the four serotypes of DEN (DEN1 to DEN4) to humans. Infection with DEN may be asymptomatic or may cause a variety of symptoms ranging from mild dengue fever (DF) to the more severe form of dengue hemorrhagic fever (DHF) with or without shock (dengue shock syndrome [DSS]) (17). In areas where DEN is endemic, DHF has become an increasingly important cause of pediatric morbidity and mortality since it was first described half a century ago (17). Accurate diagnosis of DEN infections is therefore essential.The diagnostic methods of choice for the identification of DEN infections have been the plaque reduction neutralization assay and/or virus isolation from patient serum samples by using mosquito cell lines (17, 18). However, both of these assays are laborious to perform and a period of at least 7 days is required to obtain accurate diagnostic results using them. Recently, several enzyme-linked immunosorbent assays (ELISAs) have become commercially available for the...

Evaluation of Nonstructural 1 Antigen Assays for the Diagnosis and Surveillance of Dengue in Singapore

“…Our observation of a reduced sensitivity of NS1 assays for secondary dengue infections is in agreement with previous findings (Koraka et al 2003, Kumarsamy et al 2007, Blacksell et al 2008. The possibility of anti-NS1 antibodies resulting from a previous infection in quenching the NS1 antigen of a current infection has been discussed to explain this observation (Valdes et al 2000, Libraty et al 2002, Koraka et al 2003.…”

“…The possibility of anti-NS1 antibodies resulting from a previous infection in quenching the NS1 antigen of a current infection has been discussed to explain this observation (Valdes et al 2000, Libraty et al 2002, Koraka et al 2003. Nevertheless, NS1 assay was found to have around 50% sensitivity among secondary dengue infections in Singapore, where approximately 45% of dengue cases are secondary infections (Low et al 2006).…”

“…The antibody assays ideally require appropriately timed paired serum samples for the confirmation of an acute dengue infection (Pan American Health Organization 1994, Guzman and Kouri 1996, Vorndam and Kuno 1997. Over the past decade, several studies (Young et al 2000, Huang et al 2001, Alcon et al 2002, Koraka et al 2003, Xu et al 2006 have shown that detecting dengue nonstructural 1 (NS1) protein could be a promising and effective alternative for the early diagnosis of dengue.…”

Dengue NS1 Antigen: A Tool in Early Detection of Dengue Virus Infection

“…12 A study conducted by Koraka et al in 2003 showed that antigen detection was low in endemic area due to formation of immune complexes following a secondary dengue infection and inappropriate temperature storage of samples. 13 So it is important that samples are collected, stored and transported properly to maintain the quality and stability of samples tested.…”

Dengue Rapid Diagnostic Tests; Evaluation of Utility in a Tertiary Care Hospital

“…Dengue NS1 antigen and Dengue specific IgA antibody based assays have been shown to be useful during early febrile days. [5][6][7][8][9][10][11][12][13][14][15][16][17] ELISA and ICT based NS1 assay have been used as a diagnostic tool during febrile phase till defervescence (1 -9 days of fever onset) however, due to presence of virus -IgG immunocomplexs this assay has shown to be less sensitive in secondary infection as compared to primary infections 18,19 and may give false negative results in dengue endemic and hyperendemic areas. Studies comparing sensitives and specificities of IgA and NS1 reported dengue IgA to be more sensitive during early febrile phase of both primary and secondary dengue infection.…”