Available online at www.sciencedirect.com Diagnostic Microbiology and Infectious Disease 60 (2008) 43 – 49 www.elsevier.com/locate/diagmicrobio Virology Evaluation of the Panbio dengue virus nonstructural 1 antigen detection and immunoglobulin M antibody enzyme-linked immunosorbent assays for the diagnosis of acute dengue infections in Laos☆ Stuart D. Blacksell a,b,c,⁎, Mammen P. Mammen, Jr., d , Soulignasack Thongpaseuth a , Robert V. Gibbons d , Richard G. Jarman d , Kemajittra Jenjaroen c , Ananda Nisalak d , Rattanaphone Phetsouvanh a , Paul N. Newton a,b , Nicholas P.J. Day a,b,c a Wellcome Trust-Mahosot Hospital-Oxford Tropical Medicine Research Collaboration, Microbiology Laboratory, Mahosot Hospital, Vientiane, Lao PDR b Centre for Clinical Vaccinology and Tropical Medicine, Nuffield Department of Clinical Medicine, Churchill Hospital, University of Oxford, Oxford OX3 7LJ, United Kingdom c Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand d Armed Forces Research Institute of Medical Sciences, Bangkok 10400, Thailand Received 16 January 2007; accepted 23 July 2007 Abstract We evaluated 2 commercial enzyme-linked immunosorbent assays (ELISAs) for the diagnosis of dengue infection; one a serologic test for immunoglobulin M (IgM) antibodies, the other based on detection of dengue virus nonstructural 1 (NS1) antigen. Using gold standard reference serology on paired sera, 41% (38/92 patients) were dengue confirmed, with 4 (11%) acute primary and 33 (87%) acute secondary infections (1 was of indeterminate status). Sensitivity of the NS1-ELISA was 63% (95% confidence interval [CI], 53–73) on admission samples but was much less sensitive (5%; 95% CI, 1–10) on convalescent samples. The IgM capture ELISA had a lower but statistically equivalent sensitivity compared with the NS1-ELISA for admission samples (45%; 95% CI, 35–55) but was more sensitive on convalescent samples (58%; 95% CI, 48–68). The results of the NS1 and IgM capture ELISAs were combined using a logical OR operator, increasing the sensitivity for admission samples (79%; 95% CI, 71–87), convalescent samples (63%; 95% CI, 53–73), and all samples (71%; 95% CI, 65–78). © 2008 Elsevier Inc. All rights reserved. Keywords: Dengue; Serology; Point-of-care; Diagnosis; Immunochromatographic; Laos; NS1; IgM 1. Introduction The dengue virus is an important cause of acute febrile illness in much of the tropics, encompassing a spectrum of ☆ The study was funded by the Wellcome Trust of Great Britain. The Mahidol-Oxford Tropical Medicine Research Unit, Bangkok, Thailand, and the Wellcome Trust-Mahosot Hospital-Oxford Tropical Medicine Research Collaboration, Vientiane, Lao, PDR, has received test kits from Panbio for evaluation purposes. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or as reflecting the view of the US government. ⁎ Corresponding author. Wellcome Trust-Mahidol University-Oxford Tropical Medicine Programme, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok 10400, Thailand. Tel.: +66-23549172; fax: +66-2-3549169. E-mail address: [email protected] (S.D. Blacksell). 0732-8893/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2007.07.011 disease including dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. Patients with dengue virus infections present with symptoms and signs similar to those of other acute tropical febrile illnesses, necessitating laboratory tests for confirmation of the diagnosis (Gibbons and Vaughn, 2002; Shu and Huang, 2004; Teles et al., 2005). Serologic tests are most commonly used and rely on the detection of dengue virus-specific immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies. During the acute phase, the presence of IgM antibodies alone suggests primary infection, whereas detection of both IgM and IgG antibodies is suggestive of secondary or later infection (Shu and Huang, 2004). Rapid diagnostic tests using immunochromatographic or immunoblot technologies have been developed for point-of-care serologic testing (Vaughn et al., 44 S.D. Blacksell et al. / Diagnostic Microbiology and Infectious Disease 60 (2008) 43–49 1998), although these tests have generally demonstrated limited clinical utility due to poor sensitivity on admission samples (Blacksell et al., 2006). Recent studies have shown that the dengue virus nonstructural 1 (NS1) antigen, a highly conserved glycoprotein produced in both membrane-associated and secreted forms and abundant in the serum of patients in the early stages of dengue virus infection (Alcon et al., 2002; Dussart et al., 2006; Young et al., 2000; Xu et al., 2006), may be an appropriate marker of acute dengue virus infection. Here, we evaluate the diagnostic utility of a commercial dengue IgM antibody detection enzyme-linked immunosorbent assay (ELISA) and a commercial ELISA kit for the detection of dengue virus NS1 antigen in patients with a clinical diagnosis of dengue in the endemic setting of the Lao People's Democratic Republic (Laos). 2. Methods 2.1. Patient samples The study was conducted at Mahosot Hospital, Vientiane, Laos, between September 2004 and September 2005. Ethical clearance was granted by the Ethical Review Committee of the Faculty of Medical Sciences, National University of Laos, Vientiane, Laos, and by the Oxford University Tropical Ethics Research Committee, United Kingdom. Patients were recruited to the study if they gave informed written consent and if the responsible physician suspected dengue virus infection on clinical grounds based on the World Health Organization (1997) guidelines (an acute febrile illness with 2 or more of the following signs: headache, retro-orbital pain, myalgia, arthralgia, rash, hemorrhagic manifestations, and leukopenia). Venous blood samples were collected on the day of admission (admission specimen) and discharge from hospital (convalescent specimen). Serum was divided for immediate use and for storage at −80 °C. 2.2. Enzyme-linked immunosorbent assays Commercial ELISAs for the detection of dengue virus NS1 antigen (cat. no. E-DEN01A, lot no. 06200; Panbio, Brisbane, Australia, provided for evaluation purposes) and dengue virus IgM antibody capture ELISA (cat. no. E-DEN01M; Panbio) were performed according to the manufacturer's instructions. Results were calculated as “Panbio units” with results b9.0, 9.0–11.0, and ≥ 11.0 defined as negative, equivocal, and positive, respectively. Samples that initially returned an equivocal result were retested to confirm the result. 2.3. Dengue reference assays Dengue reference assays were performed at the Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, Thailand. Dengue virus infections were confirmed on an individual patient basis using the results of paired admission and convalescent specimens tested by the AFRIMS IgM and IgG antibody capture ELISAs using the following diagnostic interpretations as previously described (Innis et al., 1989; Vaughn et al., 1999). For paired specimens, admission samples with b15 U of dengue virus IgM antibodies rising to ≥30 U in the convalescent specimen (with dengue IgM antibodies greater than Japanese encephalitis virus [JEV] IgM antibodies) was considered evidence of an acute primary dengue virus infection. In the absence of IgM N40 U in the admission specimen, a 2-fold rise in IgG to a value ≥100 U was indicative of a secondary or later dengue virus infection. In addition, dengue virus primary and secondary infections were also determined when the ratio of anti-dengue virus IgM to IgG is N1.8 to b1.8, respectively. Specimens were considered negative for serologic evidence for a recent dengue virus infection if a paired specimen collected at least 7 days apart were absent of dengue-specific antibody as defined above. The reverse transcriptase–polymerase chain reaction (RT-PCR) was used to determine the serotype identity (Blacksell et al., 2006; Lanciotti et al., 1992). Both reference serology and RT-PCR assays were performed blinded to the results of dengue virus NS1 antigen or IgM capture ELISAs. All samples were labeled using a code that was devoid of personal identifiers. 2.4. Non-dengue serology The diagnosis of JEV infection was confirmed by the detection of specific IgM antibodies by the AFRIMS JEV IgM capture ELISA (Burke and Nisalak, 1982). Sera were screened for the presence of Chikungunya virus antibodies using the hemagglutination inhibition (Clarke and Casals, 1958) method at a 1:10 dilution. The presence of antibodies against Orientia tsutsugamushi (scrub typhus) and Rickettsia typhi (murine typhus) were assessed with an indirect micro-immunofluorescence assay (Robinson et al., 1976) using a 4-fold (or greater) rising titer suggesting acute infection (Coleman et al., 2002). 2.5. Analysis Diagnostic accuracy was calculated for the dengue virus NS1-ELISA and IgM capture ELISAs relative to the final patient diagnosis (i.e., dengue-positive or dengue-negative) based on the results of reference serology. To estimate assay accuracy, NS1-ELISA and IgM capture ELISA results that were confirmed to be of equivocal status by retesting were regarded as negative. Diagnostic accuracy indices of sensitivity, specificity, negative predictive values (NPVs), and positive predictive values (PPVs) with exact 95% confidence intervals (CIs), interquartile (IQR) ranges of days of fever, and area under the receiver operating characteristic curves (AUROCCs) were calculated using Stata/SE 8.0 (Stata, College Station, TX). Statistically significant differences (P b 0.05) in the Panbio unit results S.D. Blacksell et al. / Diagnostic Microbiology and Infectious Disease 60 (2008) 43–49 45 Table 1 Summary of samples, results, and diagnostic accuracy scores for the NS1 and IgM detection ELISAs Assay Results (Panbio unitsa) Samples Sample timing n Days with Non-dengue fever cases (median (median [IQR]) [IQR]) NS1 ELISA Admission 92 5 (4–7) Convalescent 92 9 (7–12) All samples 184 7 (5–10) IgM ELISA Admission 92 5 (4–7) Convalescent 92 9 (7–12) All samples 184 7 (5–10) Combined Admission 92 5 (4–7) Convalescent 92 9 (7–12) All samples 184 7 (5–10) a b c 4.4 (3.7–5.2) 4.3 (3.8–4.8) 4.4 (3.7–4.9) 3.5 (2.9–4.9) 5.1 (2.9–16.6) 3.9 (2.9–7.5) NA NA NA Diagnostic accuracy (95% CI) Dengue cases Sensitivity (median [IQR]) Specificity PPVb NPVc 14.2 (5.0–28.1) 3.6 (3.2–4.4) 4.6 (3.4–15.4) 9.5 (3.7–28.8) 14.1 (3.7–36.6) 12.6 (3.7–34.4) NA NA NA 100 100 100 94.4 (89.8–99.1) 72.2 (63.1–81.4) 83.3 (78.0–88.7) 94.4 (84.8–99.1) 72.2 (63.1–81.4) 83.3 (78.0–88.7) 100 100 100 85.0 (77.7–92.3) 59.5 (49.4–69.5) 68.4 (61.7–75.1) 90.9 (75.7–98.1) 61.5 (51.6–71.5) 75.0 (68.7–81.3) 79.4 (71.2–87.7) 60.0 (50.0–70.0) 68.4 (61.6–75.1) 70.8 (61.6–80.1) 70.9 (61.6–80.2) 70.9 (64.3–77.4) 86.4 (74.4–93.4) 73.6 (64.6–82.6) 89.4 (74.6–86.1) 63.2 (53.4–73.0) 5.3 (0.7–9.8) 34.2 (27.4–41.1) 44.7 (34.6–54.9) 57.9 (47.8–68.0) 51.3 (44.1–58.5) 79.0 (62.7–90.5) 63.2 (53.3–73.0) 71.1 (64.5–77.6) b9.0, negative; 9.0–11.0, equivocal; ≥11.0, positive. Positive predictive value. Negative predictive value. NS1-ELISA and IgM capture ELISA results for the diagnosis of dengue? To answer this question, the results of the NS1-ELISA and IgM capture ELISA were combined using Boolean operators (i.e., AND, OR, NOT) to derive a final cumulative result for each patient sample. A positive combined result was assigned to those patient samples that were positive for NS1-ELISA and/or the IgM capture ELISA (i.e., either or both assays). The combined patient sample results were compared to the final patient diagnosis based on the reference assay results to calculate accuracy scores. and positivity rates were calculated using Student t test and McNemar test, respectively. 2.6. Practical assessment of diagnostic utility To examine and compare the true diagnostic utility of the NS1-ELISA and IgM capture ELISA in a clinical setting, the following 3 questions were posed: 1) In a patient presenting with suspected acute dengue virus infection, how accurate are the NS1-ELISA and IgM capture ELISA for making the diagnosis of dengue? To answer this question, the NS1-ELISA and IgM capture ELISA results for the admission sample were compared with the final patient diagnosis (based on paired reference serology). 2) In a patient who has been recently acutely ill with dengue-like symptoms and is now recovering (such as a returning traveler from a dengue virus endemic region) (Wichmann and Jelinek, 2004), how accurate are the NS1-ELISA and IgM capture ELISA for making the diagnosis of dengue? To answer this question, the NS1-ELISA and IgM capture ELISA results for the convalescent sample were compared with the final patient diagnosis (based on paired reference serology). 3) In a patient presenting with suspected acute dengue virus infection, how accurate is the combination of 3. Results 3.1. Patient samples and reference diagnosis Ninety-two patients were recruited into the study and 41% (38/92) were diagnosed with dengue virus infection (Table 1) as defined by AFRIMS diagnostic criteria. All 4 dengue serotypes were represented (Table 2). Both admission and convalescent samples from all 92 patients were tested by NS1-ELISA and IgM capture ELISA (Table 1). The median IQR number of days between admission and convalescent sera was 6 (5–7). Of the 92 patients without a reference diagnosis of dengue, 12 were diagnosed as having scrub typhus (13%), 4 with murine Table 2 Sensitivity of the dengue virus NS1 antigen and IgM antibody detection ELISAs by infection status and serotype for admission samples ELISA format NS1 IgM a % Positivity (n positive/n true dengue cases × 100) Disease classification (n = 38)a Serotype (n = 25) Acute primary Acute secondary or later DEN-1 DEN-2 DEN-3 DEN-4 75.0 (3/4) 75.0 (3/4) 60.1 (20/33) 39.4 (13/33) 77.8 (7/9) 22.2 (2/9) 60.0 (3/5) 25.0 (2/5) 0 (0/2) 50.0 (1/2) 66.7 (6/9) 33.3 (3/9) One dengue confirmed case was of indeterminate infection status. 46 S.D. Blacksell et al. / Diagnostic Microbiology and Infectious Disease 60 (2008) 43–49 typhus (4%), 1 with JEV (1%), and 1 with Streptococcus pyogenes septicemia (1%). Chikungunya virus antibodies were not detected, and no diagnosis was available for 39% (36/92) of patients. Clinical information for 87 patients has recently been reported elsewhere (Blacksell et al., 2007). 3.2. Diagnostic utility in a clinical setting 1) How accurate are the NS1-ELISA and IgM capture ELISA for the diagnosis of acute dengue? The sensitivity of the NS1-ELISA on admission specimens was 63% (24/38) with 100% specificity (Table 1). For the IgM capture ELISA, the sensitivity for admission samples was 45% (17/38) and specificity was 94% (51/54). There was no significant difference in admission sample results between the 2 ELISAs for the distribution of results (P = 0.52, McNemar test) and receiver operating curve analysis (P = 0.51) (AUROCC: NS1-ELISA = 0.783 and IgM capture ELISA = 0.751) (Fig. 1). 2) How accurate are the NS1-ELISA and IgM capture ELISA for the diagnosis of later-presentation dengue? The sensitivity of the NS1-ELISA and IgM capture ELISA using convalescent samples were 5% (2/38) and 58% (22/38) respectively, and were significantly different (P b 0.00005, McNemar test) (Table 1). The NS1-ELISA and IgM capture ELISA were 100% and 72% (39/54) specific, respectively. 3) How accurate is the combination of NS1-ELISA and IgM capture ELISA results for the diagnosis of acute dengue? The results of the NS1 and IgM capture ELISAs were combined using a logical OR to give a final cumulative result that increased the sensitivity of admission (79%, 30/38) and convalescent (63%, 24/38) samples. The sensitivity using the cumulative result when all samples were tested was 71% (54/76) and specificity was 83% (90/108) (Table 1). All samples positive in both the NS1-ELISA and IgM capture ELISAs (n = 11) were taken on admission after 4–8 days of fever (Fig. 2). 3.3. Comparison of admission and convalescent sample results Sample timing (admission and convalescent specimens) influenced the sensitivity of both ELISAs. For the 38 dengue-confirmed cases, the median Panbio unit result for the NS1-ELISA was significantly higher for admission than for convalescent samples (14.2 vs 3.6, P b 0.0005, Student t test) and the sensitivity was significantly different (P b 0.00005, Student t test) between acute and convalescent samples. The NS1-ELISA sensitivity for patients sampled during the first 2 days of fever was 100% (3/3 of true dengue infection patient samples were positive) (Fig. 3), whereas after 3–4, 5–6, and N7 days, it was 57% (4/7), 36% (9/25), and 24% (10/41), respectively. The IgM capture ELISA demonstrated a nonsignificant (P = 0.1, paired Student t test) increase in median Panbio unit results (9.5 versus 14.1) between admission and convalescent samples in confirmed dengue cases. For the non-dengue cases, the median Panbio unit results for both assays were all below the b9 units positivity cutoff. The Fig. 1. AUROCC analysis that compares the accuracy of the dengue virus NS1-ELISA and IgM capture ELISA. S.D. Blacksell et al. / Diagnostic Microbiology and Infectious Disease 60 (2008) 43–49 47 Fig. 2. Individual and dual positivity for dengue virus NS1-ELISA and IgM capture ELISA compared with days of fever for dengue virus-confirmed samples (n = 76 samples). IgM capture ELISA showed no significant (P = 0.33, McNemar test) difference in positivity between admission (45%, 17/38) and convalescent samples (58%; 22/38) in confirmed dengue cases. 3.4. The influence of infecting dengue virus serotype Dengue serotype information was available for 66% (25/38) of the dengue cases, with all 4 dengue serotypes Fig. 3. Overall positivity for dengue virus NS1-ELISA and IgM capture ELISA compared with days of fever for dengue virus-confirmed samples (n = 76 samples). 48 S.D. Blacksell et al. / Diagnostic Microbiology and Infectious Disease 60 (2008) 43–49 represented (Table 2). The IgM capture ELISA detected all serotypes, but the NS1-ELISA was negative for the 2 patients with dengue virus serotype 3 infections. 3.5. The influence of infection status The majority (87%; 33/38) of dengue patients were serologically classified as acute secondary or later infections, with 4 patients (11%) classified as having acute primary infections with 1 patient having indeterminate infection status (Table 2). For admission specimens, the NS1-ELISA detected 75% (3/4) of the acute primary and 60% (20/33) of the acute secondary (or later) infections. The IgM capture ELISA detected the same proportion (75%) of acute primary infections and 39% (13/33) of the acute secondary (or later) infections. This was not significantly different (P = 0.18 acute primary, P = 0.13 acute secondary, McNemar test) from those of the NS1-ELISA results. 4. Discussion Adequate test sensitivity is essential for the accurate laboratory diagnosis of dengue virus infection (Wichmann et al., 2006). Results of this study demonstrate that a commercial NS1 antigen ELISA, especially when used together with a dengue virus IgM capture ELISA, is sufficiently sensitive and specific to be clinically informative in an endemic setting. Sample timing is an important consideration in the serologic diagnosis of dengue virus infections. During an acute primary dengue virus infection, the detection of dengue IgM antibodies commences approximately 5 days after onset of fever and 3 days after defervescence (Vaughn et al., 1997). A sample collected in the febrile phase of dengue virus infection may have insufficient detectable IgM antibody and hence result in a false-negative serologic diagnosis, whereas a second sample collected during the convalescent phase may improve the accuracy of diagnosis. In contrast, NS1 antigens are detectable in serum from 1 to 9 days after the onset of fever (Alcon et al., 2002; Huang et al., 1999). In this study, we posed 3 practical diagnostic questions that relate to clinical presentation, which in turn dictates sample availability and timing. On acute presentation, the NS1 ELISA demonstrated higher sensitivity and specificity scores than the IgM capture ELISA, although these increased values were not statistically significant. The IgM capture ELISA first detected antibodies at day 3 after fever onset with the peak of detectable antibodies at 7 days, but the NS1-ELISA detected NS1 antigen from day 1 onward followed by a plateau of NS1 positivity in true dengue virus infection patients over subsequent days, making it an appropriate test for the diagnosis of dengue virus infection in the acutely febrile patient. The high specificity of the NS1-ELISA demonstrated in this study (100%) is also a major asset in the tropical setting where many other illnesses serologically cross-react with other flaviviruses, notably JEV (Teles et al., 2005). The diagnostic sensitivity was improved for admission samples if the results of the 2 assays were combined in a logical “OR” manner, although as expected there was a marginal (6%) drop in specificity and in PPV (9%). Comparison of the NS1 antigen and IgM capture ELISA results by days of fever showed periods of exclusive positivity for one target analyte (NS1 antigen-early infection: IgM antibody-later infection), with an overlap of dual positivity at 4–8 days of fever. If an acute specimen tests negative for NS1 antigen, despite a clinical suspicion of dengue, the specimen should be tested for the presence of dengue IgM antibodies. Recent studies have reported higher sensitivity results for both the NS1 antigen (Xu et al., 2006; Kumarasamy et al., 2006; Dussart et al., 2006) and Panbio IgM capture ELISA (Cuzzubbo et al., 1999; Groen et al., 2000) than those presented here. The lower sensitivity described here may be attributable to the stringent patient diagnosis reference comparator, using paired sera, and the high proportion of secondary dengue virus infections. The use of individual sample (admission or convalescent alone) results that are assigned a dengue reference status after testing with imperfect reference comparators (i.e., PCR, IgM ELISA, Haemagglutination Inhibition) can artificially inflate sensitivity scores, especially in the case of admission samples. That the majority (87%) of the patient samples in this study were acute secondary infections, with only a small number (11%) of acute primary dengue infection cases, may also have affected the accuracy results. The NS1-ELISA had increased sensitivity for detection of acute primary dengue virus infections compared to secondary infections, consistent with a recent assessment of a commercial NS1 ELISA in Malaysia (Kumarasamy et al., 2006). The high proportion of secondary infections in our cohort may also account for the lower than expected sensitivity of the IgM capture ELISA in later samples, as the IgM antibody response may be “blunted” after high IgG titer secondary or later infections (Chanama et al., 2004; Schilling et al., 2004). These results illustrate the utility of the NS1-ELISA in dengue endemic settings where multiple serotypes circulate and there is a high incidence of secondary or later infections. However, the apparent inability of the NS1-ELISA to detect dengue virus serotype 3 infections is a concern, although there were insufficient number of patients (n = 2) infected with this serotype to determine the true nature of this potential limitation. While the sensitivity results presented here are lower than those of other evaluations, these probably reflect the real-life sensitivity of the assays. Acknowledgments We are very grateful to all the patients who participated in this study, the doctors, nurses, and staff of the Microbiology Laboratory and Mahosot Hospital, especially Douangdao Soukaloun, Simmaly Phongmany, Sengmanivong Khounnorath, Khonesavanh Luangxay, Bouachanh Rasachak, S.D. Blacksell et al. / Diagnostic Microbiology and Infectious Disease 60 (2008) 43–49 Vimone Soukhaseum, Valy Keolouangkot, Konkam Sisouk, Mayfong Mayxay, Anisone Changthongthip, Olay Lattana, Manivanh Vongsouvath, Viengmone Davong, Phonelavanh Phouminh, Sonbandit Duangsy, Sengmani Symanivong, Viengmala Sihalath, Kai-amporn Keopaseuth, the pediatric and medical wards, and the AFRIMS staff for the characterization of the specimens. 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