Evaluation of a Dengue NS1 Antigen Detection Assay Sensitivity and Specificity for the Diagnosis of Acute Dengue Virus Infection

<div><p>Background</p><p>Currently, no dengue NS1 detection kit has regulatory approval for the diagnosis of acute dengue fever. Here we report the sensitivity and specificity of the InBios DEN Detect NS1 ELISA using a panel of well characterized human acute fever serum specimens.</p><p>Methodology/Principal Findings</p><p>The InBios DENV Detect NS1 ELISA was tested using a panel composed of 334 serum specimens collected from acute febrile patients seeking care in a Bangkok hospital in 2010 and 2011. Of these patients, 314 were found to have acute dengue by either RT-PCR and/or anti-dengue IgM/IgG ELISA. Alongside the InBios NS1 ELISA kit, we compared the performance characteristics of the BioRad Platelia NS1 antigen kit. The InBios NS1 ELISA Ag kit had a higher overall sensitivity (86% vs 72.8%) but equal specificity (100%) compared to the BioRad Platelia kit. The serological status of the patient significantly influenced the outcome. In primary infections, the InBios NS1 kit demonstrated a higher sensitivity (98.8%) than in secondary infections (83.5%). We found significant variation in the sensitivity of the InBios NS1 ELISA kit depending on the serotype of the dengue virus and also found decreasing sensitivity the longer after the onset of illness, showing 100% sensitivity early during illness, but dropping below 50% by Day 7.</p><p>Conclusion/Significance</p><p>The InBios NS1 ELISA kit demonstrated high accuracy when compared to the initial clinical diagnosis with greater than 85% agreement when patients were clinically diagnosed with dengue illness. Results presented here suggest the accurate detection of circulating dengue NS1 by the InBios DENV Detect NS1 ELISA can provide clinicians with a useful tool for diagnosis of early dengue infections.</p></div

Sensitivity of InBios and Bio-Rad Assays stratified by date after onset of illness in (A) All infections and in (B) Primary and (C) Secondary infections.

<p>Serum samples (total samples, n = 314; primary, n = 51; secondary, n = 260) were tested using the InBios and Bio-Rad NS1 kits. Sensitivity was plotted against day post-onset of illness. p-values were calculated using McNemar's Chi-square test. NA – not applicable. Unable to do statistical analysis when value equals 0. NS – not significant.</p

Sensitivity of InBios and Bio-Rad assays based on clinical diagnosis and serological diagnosis.

a<p>Dengue Fever clinical diagnosis based on SEARO WHO guidelines <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003193#pntd.0003193-World1" target="_blank">[22]</a>.</p>b<p>Includes patients clinically diagnosed with DHF with plasma leakage, DSS and deaths based on SEARO WHO guidelines <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003193#pntd.0003193-World1" target="_blank">[22]</a>.</p>c<p>Total number of Dengue Fever equals 159, but 1 case could not be classified as primary or secondary infection.</p>d<p>Total number of Dengue Hemorrhagic Fever equals 141, but 1 case could not be classified as primary or secondary infection.</p><p>Sensitivity of InBios and Bio-Rad assays based on clinical diagnosis and serological diagnosis.</p

Summary of study population.

a<p>IQR – interquartile range.</p>b<p>Represents subjects confirmed as dengue positive by serological testing only. The infecting serotype was unable to be determined since they were negative by RT-PCR.</p>c<p>Represents subjects confirmed as dengue positive by RT-PCR only. Serological studies were not positive and primary or secondary infection could not be determined.</p>d<p>Dengue Fever clinical diagnosis based on SEARO WHO guidelines <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003193#pntd.0003193-World1" target="_blank">[22]</a>.</p>e<p>Includes patients clinically diagnosed with DHF with plasma leakage, DSS and deaths based on SEARO WHO guidelines <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003193#pntd.0003193-World1" target="_blank">[22]</a>.</p>f<p>No diagnosis was given in 8 cases. Other clinical diagnoses included one each of bronchitis, gastritis, viral gastroenteritis, viral-induced thrombocytopenia, query rickettsial infection and nonspecific viral infection.</p><p>Summary of study population.</p

Overall performance characteristics of the InBios and Bio-Rad assays compared to reference standard^a.

a<p>The composite reference standard included samples that were positive either by serology and/or RT-PCR.</p>b<p>CI – confidence interval.</p>c<p>PPV – positive predictive value.</p>d<p>NPV – negative predictive value.</p>e<p>Sensitivity = (true positives)/(total positive by reference standard).</p>f<p>Specificity = (true negatives)/(total negative by reference standard).</p>g<p>Diagnostic Accuracy = (true positives+true negatives)/(total number of samples).</p>h<p>PPV = (true positives)/(total positive by InBios or Bio-Rad assay).</p>i<p>NPV = (true negatives)/(total negative by InBios or Bio-Rad assay).</p><p>Overall performance characteristics of the InBios and Bio-Rad assays compared to reference standard^{<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003193#nt107" target="_blank">a</a>}.</p

Sensitivity of InBios and Bio-Rad assays based on serotype.

<p>Sensitivity of InBios and Bio-Rad assays based on serotype.</p

Incidence of CHIKV infections and seroprevalence of CHIKV PRNT in different age groups.^a

<p>^aBased on 853 subjects with both enrollment and 12-month blood collections.</p><p>^bCHIKV PRNT titer at enrollment using 80% plaque reduction.</p><p>CHIKV = chikungunya virus; n = number; CI = confidence interval; PRNT = plaque reduction neutralization test.</p><p>Incidence of CHIKV infections and seroprevalence of CHIKV PRNT in different age groups.<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003764#t002fn001" target="_blank">^a</a></p

Clinical presentation of symptomatic CHIKV infections.^a

<p>^aBased on 20 symptomatic CHIKV infections and on clinical parameters present at any one of the acute, 2, 5, or 8 day visits.</p><p>CHIKV = chikungunya virus; n = number.</p><p>Clinical presentation of symptomatic CHIKV infections.<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003764#t004fn001" target="_blank">^a</a></p

Incidence of CHIKV infections among individuals with negative baseline CHIKV PRNT in different age groups.^a

<p>^aBased on 614 subjects with both enrollment and 12-month blood collections who had negative baseline CHIKV PRNT titer.</p><p>^bCHIKV PRNT titer at enrollment using 80% plaque reduction.</p><p>CHIKV = chikungunya virus; n = number; CI = confidence interval; PRNT = plaque reduction neutralization test.</p><p>Incidence of CHIKV infections among individuals with negative baseline CHIKV PRNT in different age groups.<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003764#t003fn001" target="_blank">^a</a></p

Phylogenetic tree showing five chikungunya viruses (CHIKVs) characterized in the study.

<p>The tree was constructed by neighbor-joining methods (1,000 bootstrap replications) using envelope protein-1 (E1) nucleotide sequences (1,320 bp) of 46 CHIKV strains; the five from the study are designated in bold. Bootstrap support values are shown for major nodes. Scale bar indicates nucleotide substitutions per site. Genotypes are indicated on the right. The sequences were named according to virus/country/strain/year of collection or isolation. GenBank accession numbers are shown in parentheses.</p