METHODS: This prospective cross-sectional study consecutively recruited 494 patients with suspected dengue from a health clinic in Malaysia. Both RDTs were performed onsite. The evaluated ELISA and reference tests were performed in a virology laboratory. The reference tests comprised of a reverse transcription-polymerase chain reaction and three ELISAs for the detection of dengue NS1 antigen, IgM and IgG antibodies, respectively. The diagnostic performance of evaluated tests was computed using STATA version 12.
RESULTS: The sensitivity and specificity of ViroTrack were 62.3% (95%CI 55.6-68.7) and 95.0% (95%CI 91.7-97.3), versus 66.5% (95%CI 60.0-72.6) and 95.4% (95%CI 92.1-97.6) for SD NS1 ELISA, and 52.4% (95%CI 45.7-59.1) and 97.7% (95%CI 95.1-99.2) for NS1 component of SD Bioline, respectively. The combination of the latter with its IgM and IgG components were able to increase test sensitivity to 82.4% (95%CI 76.8-87.1) with corresponding decrease in specificity to 87.4% (95%CI 82.8-91.2). Although a positive test on any of the NS1 assays would increase the probability of dengue to above 90% in a patient, a negative result would only reduce this probability to 23.0-29.3%. In contrast, this probability of false negative diagnosis would be further reduced to 14.7% (95%CI 11.4-18.6) if SD Bioline NS1/IgM/IgG combo was negative.
CONCLUSIONS: The performance of ViroTrack Dengue Acute was comparable to SD Dengue NS1 Ag ELISA. Addition of serology components to SD Bioline Dengue Duo significantly improved its sensitivity and reduced its false negative rate such that it missed the fewest dengue patients, making it a better point-of-care diagnostic tool. New RDT like ViroTrack Dengue Acute may be a potential alternative to existing RDT if its combination with serology components is proven better in future studies.
METHODS: In this study undertaken between April and May 2015, a total of 277 adult participants were recruited from households across three localities in the Sungai Segamat subdistrict in Segamat district. Sera were tested for immunoglobulin G (IgG) (Panbio® Dengue Indirect IgG ELISA/high-titer capture) and immunoglobulin M (IgM) (Panbio®) antibodies. The plaque reduction neutralization test (PRNT) was conducted on random samples of IgG-positive sera for further confirmation. Medical history and a recall of previous history of dengue were collected through interviews, whereas sociodemographic information was obtained from an existing database.
RESULTS: The overall seroprevalence for DENV infection was 86.6% (240/277) (95% CI: 83-91%). Serological evidence of recent infection (IgM/high-titer capture IgG) was noted in 11.2% (31/277) of participants, whereas there was evidence of past infection in 75.5% (209/277) of participants (indirect IgG minus recent infections). The PRNT assay showed that the detected antibodies were indeed specific to DENV. The multivariate analysis showed that the older age group was significantly associated with past DENV infections. Seropositivity increased with age; 48.5% in the age group of <25 years to more than 85% in age group of >45 years (P
METHODS: Recombinant PPDK (rPPDK) was expressed, purified and evaluated by Western blot. In parallel, recombinant galactose-and-N-acetyl-D-galactosamine inhibitable lectin (Gal/GalNAc lectin) was produced and tested similarly. The protein identity was confirmed by analysis using MALDI-TOF/TOF. A lateral flow dipstick (LFD) test using rPPDK was subsequently developed (rPPDK-LFD) and evaluated for serodiagnosis of ALA.
RESULTS: rPPDK was expressed as soluble protein after 4 hours of induction with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 30°C. Purification using nickel-nitrilotriacetic acid (Ni-NTA) resin yielded 1.5 mg of rPPDK from 1 L of culture with estimated molecular mass of 98 kDa on SDS-PAGE. Western blots using sera from patients with ALA, healthy individuals and other diseases probed with anti-human IgG4-HRP showed the highest sensitivity (93.3%) and specificity (100%); as compared to blots using IgG and IgG1 as secondary antibodies. Moreover, rPPDK showed better specificity when compared to rGal/GalNAc lectin. In the development of the LFD test, the optimum amount of rPPDK was 0.625 μg per dipstick and the optimum working concentration of colloidal gold conjugated anti-human IgG4 was optical density (OD) 5 (1.7 μg of anti-human IgG4). Evaluation of rPPDK-LFD using ALA patients and controls serum samples showed 87% diagnostic sensitivity and 100% specificity.
CONCLUSION: The developed rPPDK-LFD showed good potential for rapid diagnosis of ALA, and merit further multicentre validation using larger number of serum samples.
OBJECTIVES: To characterize the formation of anti-vatreptacog alfa ADAs in hemophilia patients with inhibitors.
METHODS/PATIENTS: This was a post hoc analysis of adept(™) 2. Immunoglobulin isotype determination, specificity analysis of rFVIIa cross-reactive antibodies, epitope mapping of rFVIIa single mutant analogs and pharmacokinetic (PK) profiling were performed to characterize the ADAs.
RESULTS: Immunoglobulin isotyping indicated that the ADAs were of the immunoglobulin G subtype. In epitope mapping, none of the rFVIIa single mutant analogs (V158D, E296V or M298Q) contained the complete antibody epitope, confirming that the antibodies were specific for vatreptacog alfa. In two patients, for whom PK profiling was performed both before and after the development of ADAs, vatreptacog alfa showed a prolonged elimination phase following ADA development. During the follow-up evaluation, the rFVIIa cross-reactivity disappeared after the last vatreptacog alfa exposure, despite continued exposure to rFVIIa as part of standard care.
CONCLUSIONS: Results from the vatreptacog alfa phase III trial demonstrate that the specific changes made, albeit relatively small, to the FVIIa molecule alter its clinical immunogenicity.