Performance of Dengue Diagnostic Tests in a Single-Specimen Diagnostic Algorithm

The Journal of Infectious Diseases MAJOR ARTICLE Performance of Dengue Diagnostic Tests in a SingleSpecimen Diagnostic Algorithm Elizabeth A. Hunsperger,1 Jorge Muñoz-Jordán,1 Manuela Beltran,1 Candimar Colón,1 Jessica Carrión,1 Jesus Vazquez,1 Luz Nereida Acosta,1 Juan F. Medina-Izquierdo,1 Kalanthe Horiuchi,2 Brad J. Biggerstaff,2 and Harold S. Margolis1 1 Dengue Branch, Division of Vector-Borne Diseases, Centers for Diseases Control and Prevention (CDC), San Juan, Puerto Rico; and 2Office of the Director, Division of Vector-Borne Diseases, CDC, Fort Collins, Colorado (See the editorial commentary by Peeling and Olliaro on pages 828–9.) Dengue is an important public health problem worldwide, with an estimated 96 million cases annually in tropical and subtropical countries [1, 2]. Infection with each of the 4 dengue virus (DENV) serotypes (DENV-1–4) may cause dengue, and there are no vaccines or drugs to prevent or treat the disease. The clinical presentation of dengue is similar to that of other acute febrile illnesses (AFIs; eg, malaria, leptospirosis, measles, influenza, and chikungunya); therefore, early laboratory diagnosis should improve clinical management and public health surveillance. Historically, laboratory diagnosis of dengue required demonstration of anti-DENV immunoglobulin M (IgM) seroconversion in 2 serum samples, one collected during the first 5 days of illness and a second collected after 5 days [3]. However, the long interval between specimens made collection difficult, was Received 11 December 2015; accepted 25 January 2016; published online 16 March 2016. Presented in part: American Society for Tropical Medicine and Hygiene Conference, Atlanta, Georgia, 12 November 2012. Poster 1097; Third International Conference on Dengue and Dengue Hemorrhagic Fever, Bangkok, Thailand, 22 October 2013. Correspondence: E. A. Hunsperger, 1324 Calle Canada, San Juan, Puerto Rico 00920 (enh4@ cdc.gov). The Journal of Infectious Diseases® 2016;214:836–44 Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US. DOI: 10.1093/infdis/jiw103 836 • JID 2016:214 (15 September) • Hunsperger et al not useful for patient-care decisions, and introduced an inherent bias in surveillance data. Patients with dengue or other AFIs usually seek medical attention within several days of fever onset [4, 5], providing the opportunity to diagnose dengue by evaluating a single serum specimen obtained at this time. DENV viremia occurs for 3–5 days prior to fever onset and continues for approximately 5 days into the febrile illness, and it can be detected by molecular assays specific for DENV RNA or immunoassays targeting DENV nonstructural protein 1 (NS1) antigen [6]. In addition, an antiDENV IgM response becomes detectable by IgM-capture immunoassays 3–5 days after onset of fever, peaks 6–10 days after fever onset, and may persist for up to 90 days [7, 8]. We conducted a retrospective study, using serum specimens prospectively collected from patients with clinically suspect dengue identified 1–10 days after onset of illness to determine the performance of single-specimen diagnostic testing versus that of IgM anti-DENV seroconversion in paired specimens. Results were analyzed to determine which tests provided the best overall diagnostic result on each day after onset of illness among persons with primary and secondary DENV infection. These results were used to design a diagnostic testing algorithm based on when a specimen was obtained after illness onset. Background. Anti–dengue virus (DENV) immunoglobulin M (IgM) seroconversion has been the reference standard for dengue diagnosis. However, paired specimens are rarely obtained, and the interval for this testing negates its usefulness in guiding clinical case management. The presence of DENV viremia and appearance of IgM during the febrile phase of dengue provides the framework for dengue laboratory diagnosis by using a single serum specimen. Methods. Archived paired serum specimens (n = 1234) from patients with laboratory-confirmed dengue from 2005 through 2011 were used to determine the diagnostic performance of real-time reverse transcription polymerase chain reaction (RT-PCR), for detection of DENV serotypes 1–4, and enzyme-linked immunosorbent assays (ELISAs), for detection of DENV nonstructural protein 1 (NS1) antigen and anti-DENV IgM. Results. During 1–3 days after illness onset, real-time RT-PCR and NS1 antigen testing detected 82%–69% and 90%–84% of cases, respectively, as viremia levels declined, while anti-DENV IgM ELISA detected 5%–41% of cases as antibody appeared. Over the 10-day period of the febrile phase of dengue, the cumulative effect of using these 3 types of tests in a diagnostic algorithm confirmed ≥90% of dengue cases. Conclusions. The use of molecular or NS1 antigen tests to detect DENV and one to detect anti-DENV IgM in a single serum specimen collected during the first 10 days of illness accurately identified ≥90% of dengue primary and secondary cases. Keywords. dengue virus; diagnostics; enzyme-linked immunoassay; dengue virus nonstructural protein 1 (NS1); NS1 antigen detection; anti-dengue virus IgM; dengue virus molecular diagnostics. METHODS Clinical Specimens Ethics Statement This study was reviewed by the CDC Human Research Protection Office and determined to be exempt from US human subjects’ protection regulations ( protocol 6273). Specimens used in this study were deidentified according to CDC Institutional Review Board approved protocol 6279. Dengue Diagnostic Testing Procedures Selected specimens were previously tested according to the diagnostic algorithm in use for each surveillance system (PDSS and EDSS) and included anti-DENV IgM. Specimens in the final panel were reevaluated according to the manufacturer’s instructions by means of the following 6 tests: the CDC DENV-1– 4 Real-Time RT-PCR (RT-PCR) Assay [11], performed in the multiplex mode according to the package insert and used to detect DENV RNA (limit of detection, 1 × 103 genome copy equivalents); 2 DENV NS1 antigen-capture assays, the Dengue Early ELISA (Panbio, Melbourne, Australia) and the DENV Detect NS1 ELISA (InBios, Seattle, WA); and 3 anti-DENV IgMcapture ELISAs, the CDC MAC ELISA (serum dilution, 1:400) [7], the Dengue Virus IgM Capture DxSelect ELISA (Focus Diagnostics, Cypress, CA), and the DENV Detect IgM Capture ELISA (InBios). Testing was performed in a Clinical Laboratory Improvement Amendment–certified laboratory, and results were presented according to day after illness onset. Analytical Plan Analysis of Test Sensitivity Anti-DENV IgM seroconversion detected by the CDC MAC ELISA, defined as a negative test result for the acute-phase specimen (obtained 1–5 days after illness onset,) and a positive test result for the convalescent-phase specimen (obtained 6–10 days after illness onset), was used as th (...truncated)