Comparison Between the SD BIOLINE Dengue Duo and Two InBios Prototype Dengue Immunochromatographic Tests

Dengue fever, caused by any of the four serotypes of dengue viruses (DENV1-4), is the most important arthropod-borne viral disease in the world. Although dengue diagnostic tests based on polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) have received the U.S. Food and Drug Administration (FDA) clearance, these assays require specialized equipment and highly trained personnel, and thus not suitable for resource-limited settings. Two immunochromatographic test (ICT) prototypes (traditional and multiplex formats) were recently developed by InBios International Inc. (Seattle, WA) to detect the presence of dengue viral non-structural protein 1 (NS1) antigen or anti-DENV IgG and IgM antibodies in serum. Here, we report a follow-up evaluation of two InBios ICT prototypes compared to the commercially available SD BIOLINE Dengue Duo rapid test (Abbott, Santa Clara, CA) with an additional panel of 130 well-characterized, de-identified clinical samples. This panel included 25 acute-convalescent pairs of DENV3, 20 pairs of DENV4, 10 acute non-DENV febrile samples and 10 normal human sera along with 20 Zika clinical samples. Both the InBios traditional format and the SD Duo test showed the same overall sensitivity (81.1%) and specificity (80.0%). When compared to the reference testing methods. The InBios traditional format had a better overall performance (94.8% sensitivity and 87.9% specificity on DENV3 samples; 100.0% sensitivity and 80.0% specificity on DENV4 samples) than the multiplex format (93.0% sensitivity and 28.6% specificity on DENV3 samples; 86.7% sensitivity and 40.0% specificity on DENV4 samples) when compared to the SD Duo test results. The InBios multiplex format had higher overall sensitivity (85.6%) but much lower specificity (55.5%). All three dengue rapid tests cross-reacted with Zika clinical samples resulting in false positive for dengue in 20% to 73.3% of the Zika positive samples. Both the InBios traditional format and the SD Duo test had better overall performance than the InBios multiplex format. These easy and simple rapid tests with fast turn-around time are ideal for use in resource-limited settings where dengue is endemic.

. There are four serotypes present of dengue virus (DENV1-4) and infection with any of these four serotypes can result in an asymptomatic infection or a symptomatic infection whose manifestation ranges from mild self-limiting fever to severe disease with high fever, hemorrhage, and shock [4]. Dengue fever ranks third on the list of the Global Ranked List of Infectious

Diseases Threats from the U.S. Military Infectious Disease Threats
Prioritization Panel (ID-TPP) due to its high risk and potential impacts to military operations [5]. Efforts to mitigate associated morbidity and mortality of dengue are a high priority for the U.S.
Unfortunately, even though a licensed dengue vaccine is available for use in individuals with previous dengue infection, this option is not available for dengue naïve populations including most of U.S. military personnel. Also, there is no specific antiviral therapeutic to prevent or treat dengue infection. Hence, early and prompt diagnosis of dengue infection is crucial for monitoring patients and for alerting health care providers to provide appropriate hospitalization and supportive therapy for patients in anticipation that they may progress to manifest the severe form of the disease. There are U.S. Food and Drug Administration (FDA)cleared diagnostic assays for dengue infection using polymerase chain reaction (PCR) and IgM enzyme-linked immunosorbent assay (ELISA) based assays [8,9]. However, these assays require specialized equipment and highly trained personnel that are not always readily available especially in resource-constrained areas such as in rural areas, developing countries, or military deployment missions.
InBios International Inc. (Seattle, WA) recently developed two immunochromatographic test (ICT) prototypes (traditional and multiplex formats) for the detection of the dengue viral nonstructural protein 1 (NS1) antigen or anti-DENV IgG and IgM antibodies in serum samples of dengue suspected patients. These rapid tests can give result within 20 minutes by naked eyes and also has the advantage of being able to be performed without any equipment.
Recently, an evaluation of two InBios ICT prototypes (traditional and multiplex formats) was performed by our group with 183 well-characterized clinical samples from a previously conducted multicenter clinical trial [10] because of the extensive historical performance data available for the SD Duo test [11][12][13][14]. The overall aim of this study was to choose the best candidate between the two InBios ICT prototypes that will be used in a multicenter clinical trial as part of the process to achieve the FDA clearance. All clinical samples were tested by an established traditional pathogen diagnostic algorithm at NAMRU-2 [15]. Basically, all of the clinical samples were first tested for influenza virus by a universal RT-PCR. If they were negative for influenza, they were then tested for malaria by the standard microscopy method, rickettsia by inhouse ELISA, dengue by PanBio ELISA kits (Abbott, Santa Clara, CA; former Alere Inc., Waltham, MA) (acute samples by IgM ELISA and convalescent samples by IgG ELISA), and pan-flaviviruses by using a universal primer set within a RT-PCR. If the sample was positive by PCR for pan-flaviviruses, it was then tested for dengue using serotype-specific PCR assays. For Zika clinical samples from NIDDL, the Trioplex real-time RT-PCR assay that was developed by the Centers for Disease Control and Prevention (CDC) (for the simultaneous detection of dengue, chikungunya, and Zika viruses) was used for Zika virus detection and IgM antibody capture ELISA followed by a Zika confirmatory plaque reduction neutralization test (PRNT) was used for the detection of antibodies to Zika [16].

Test Performance
The manufacturers' instructions were followed in performing  or composite sensitivity was calculated for those samples that were considered positive if any of the three analytes for that particular sample was positive. Binomial method was used to calculate the confidence intervals (CIs) for sensitivity and specificity.

Results
When compared to the standard clinical diagnostic results determined by traditional diagnostic assays (including PCR and IgM/IgG ELISA) performed at NAMRU-2 as the reference standard for those 110 dengue and non-dengue samples, both the InBios traditional format and SD Duo test showed the same overall sensitivity (81.1%) and overall specificity (80.0%). The InBios multiplex format had higher overall sensitivity (85.6%) but much lower overall specificity (55.5%) (

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Both InBios formats had identical sensitivity (92.9%) and specificity (94.4%) for NS1 detection on DENV3 samples (Table 2a) and had very high sensitivity (100.0%) and specificity (multiplex format 100.0%; traditional format 96.6%) for NS1 detection on DENV4 samples ( samples [11,21], our study showed that both InBios ICTs actually had high sensitivity on DENV4 samples that may be caused by a higher sensitivity of the monoclonal antibodies from InBios to DENV4. However, the detailed design, construction, and reagents of both ICT prototypes were proprietary to InBios. In conclusion, the InBios traditional dengue ICT is simple, easy to perform without needing sophisticated equipment, rapid, sensitive, and specific, supporting its use for a rapid and reliable diagnosis of dengue infection in resource-limiting settings. However, it should be noted that Zika positive serum samples did cross-react with this InBios traditional dengue ICT (20% and 47% for Zika PCR and antibody positive samples, respectively). Therefore, special caution needs to be taken when performing the test in an area where Zika is co-circulating or additional efforts should be placed by the company to minimize cross-reactivity with Zika samples.