Comparative assessment of commercial enzyme-linked immunosorbent assay & rapid diagnostic tests used for dengue diagnosis in India

Dengue, a mosquito-borne viral disease caused by infection with any of the four serotypes of dengue virus (DENV 1-4), is a major public health problem worldwide and is endemic/hyperendemic in India12. DENV infection is diagnosed by detection of the virus (virus isolation and immunofluorescence assay), viral RNA [reverse transcriptase-polymerase chain reaction (RT-PCR)] and viral antigen (NS1) or anti-DENV IgM antibodies (immunoassays) in acute-phase patient serum3. On account of NS1/IgM dynamics during infection, variations among serotypes and occurrence of secondary infections, dengue diagnosis remains complicated and challenging. Timely diagnosis is important for clinical management of patients, surveillance and prevention/control of epidemics.

Immunoassays for NS1 and IgM offer a convenient format for dengue diagnosis, and several enzyme-linked immunosorbent assay (ELISA) and rapid diagnostic tests (RDTs) are commercially available. Performance of such diagnostic kits has been assessed globally using well-characterized serum panels456789. In a multicountry evaluation by the World Health Organization and others45678, eight NS1/IgM ELISAs and seven RDTs were assessed, of which two NS1 and four IgM ELISAs were approved for patient diagnosis while RDTs were recommended only for outbreak confirmation in resource-restricted countries45678. India was not part of this assessment.

The present study was aimed at evaluation of currently used diagnostic assays in the identification of DENV infections caused by recently circulating strains in Indian population. Such an evaluation would require a well-characterized panel of recently collected serum samples. In the absence of such an exclusive panel, testing of a large number of samples from suspected dengue patients was done using different kits and the results were compared with a reference test. One of the WHO-approved ELISAs67_, Panbio ELISA was included as a reference test. As this reference test itself is not 100 per cent specific and sensitive67, an unbiased Bayesian latent class analysis (BLCA) was carried out for determination of accuracy of diagnostic tests in the absence of a gold standard1011.

Material & Methods

This study was conducted at the department of Communicable Diseases, Interactive Research School for Health Affairs (IRSHA), departments of Medical Microbiology and Immunohaematology & Blood Transfusion, Bharati Medical College and Research Center, Pune, India, during the 2016 dengue season (July-November) in Pune, India. A total of 809 patients suspected to have DENV infection were included. Blood sample (4-5 ml) was collected within one week of symptom onset; exact day was recorded only for 300 patients. 434 patients were referred by a private tertiary care hospital, Bharati Hospital and Research Centre and tested at the hospital's laboratory (accredited by National Accreditation Board for Testing and Calibration Laboratories) using Dengue Day 1 Test (JM-RDT, J.Mitra and Co. Pvt. Ltd., New Delhi, India). The remaining 375 patients were tested at two private laboratories using SD BIOLINE Dengue Duo (SDB-RDT, Standard Diagnostics, Inc., Republic of Korea). For 127 of these patients, a repeat ELISA test was requested by the clinicians and accordingly was carried out at the respective laboratories using J. Mitra Dengue NS1 Ag MICROLISA (JME-NS1) and J. Mitra Dengue IgM MICROLISA (JME-IgM).

Left-over serum samples from these patients (n=809) were collected and brought to the laboratory at department of Communicable Diseases, IRSHA, and stored in at least two aliquots at −80°C, till testing. These samples were retested using JME-NS1, JME-IgM and the Panbio Dengue Early (NS1) ELISA (PBE-NS1) and Panbio Dengue IgM Capture ELISA (PBE-IgM) (Alere Inc., Australia). For comparing RDTs, 375 samples which were tested previously by SDB-RDT, were retested by using JM-RDT. However, the 434 samples tested earlier by JM-RDT could not be retested using SDB-RDT due to insufficient sample quantity.

Patients positive for NS1 and/or IgM (Panbio ELISA) were diagnosed as dengue-positive. Based on quantity, samples of 192 dengue-positive patients were subjected to Panbio Dengue IgG Capture ELISA (Alere Inc., Australia), for differentiating primary and secondary infections. As per manufacturer's instructions, samples showing Panbio units >22 were categorized as indicative of secondary infection. Among these, patients with IgM/IgG absorbance ratio of >1.2 were classified as primary dengue, and those with ratio <1.2 as secondary dengue, as per the WHO criteria12.

Detection of dengue virus (DENV) RNA in NS1-discordant samples: Twenty one samples showing discordant results in NS1 ELISA were subjected to detection of DENV RNA using RT-PCR, as described previously13.

Serotyping of NS1-positive samples: Fifty two NS1-positive serum samples serotyped previously13 were included for assessing efficiency of the diagnostic tests in detecting different serotypes.

The study was approved by the Ethics Committee (No. IEC/2017/04) of the Bharati Vidyapeeth (Deemed to be University), Pune.

Results

Detection of dengue virus (DENV) infection among suspected dengue patients: Table I describes results of individual tests for NS1 and IgM. For each patient sample, detection of NS1 and/or IgM was considered as the evidence for dengue-positive diagnosis.

Enzyme-linked immunosorbent assay (ELISAs): Of the 809 samples tested, PBE-NS1 and JME-NS1 detected NS1 in 158 (19.5%) and 208 (25.7%) samples, respectively. For IgM detection, 231 (28.6%) and 144 (17.8%) samples were tested positive, respectively, by PBE-IgM and JME-IgM. Based on NS1 and/or IgM positivity, 312 (38.6%) and 275 (34.0%) patients were diagnosed as dengue positive, respectively, using PB and JM ELISAs.

Rapid diagnostic tests: JM-RDT identified 181 of 809 (22.4%) samples as NS1 reactive, 89 (11.0%) as IgM positive and 226 (27.9%) patients were diagnosed as dengue positive. SDB-RDT detected 101 (26.9%) and 76 (20.3%) of 375 as positive, respectively, for NS1 and IgM, and 143 (38.1%) patients were classified as dengue positive (Table I).

Discussion

This study compared performance of two RDTs and one ELISA used for dengue diagnosis in Pune, India. One of the WHO-approved kits, Panbio-ELISA, was selected as best possible option for dengue diagnosis for reference-based analysis (RBA). To overcome the bias introduced by the use of an imperfect reference test, the data were also analyzed using BLCA, which allowed comparative evaluation of different diagnostic tests, even in the absence of a gold standard. During the 2016 dengue season in Pune, 38.6 per cent of 809 suspected dengue patients seeking diagnosis were confirmed as dengue-positive by Panbio ELISAs, reflecting the degree of dengue positivity in Pune, and thus providing an opportunity to compare the performance of different diagnostic tests in a field setting. Importantly, all four DENV serotypes were circulating13, facilitating analysis of serotype-specific performance.

First, the two ELISAs were compared (PBE & JME). RBA revealed JME to be less specific for NS1 and less sensitive for IgM and combined diagnosis that was also reflected in its PPV (NS1) and NPV (IgM). However, with BLCA, PBE was only 84 per cent sensitive for NS1 and 86 per cent specific for IgM, while its PPV for IgM and combined diagnosis appeared to be low (60 and 77%, respectively). Consequently, BLCA-estimated sensitivity of JME for all markers appeared to be higher than that determined by RBA. For combined diagnosis, PPV and NPV for Panbio ELISAs (JME) were 77 per cent (90%) and 98 per cent (100%), respectively. Thus, when BLCA was used, the performance of JME appeared to be superior. It is to be noted that a substantial proportion of JME-NS1 alone positives (~60%) and PBE-IgM alone positives (~30%) were positive, respectively, for IgM (PBE) and NS1 (JME). This suggested true positivity of these discordant samples leading to higher sensitivity of JME-NS1 than PBE-NS1 and that of PBE-IgM than JME-IgM. However, in view of the possibility of non-specific reactions67 and absence of additional tests for confirmation, the validity of these findings remains debatable. These findings emphasize a definite need to test for both NS1 and IgM markers for accurate diagnosis, irrespective of the day of sample collection.

Evaluation of the RDTs employing RBA and BLCA showed comparable results by both analyses for JME-RDT (all 3 markers) and SDB-RDT (NS1 and IgM). Combined diagnosis for SDB-RDT improved substantially when BLCA was used (94% sensitive, 95% specific). The performance of SDB-RDT for combined diagnosis seemed comparable to ELISAs, as per BLCA. Earlier Indian studies employing limited number of stored serum samples have noted good agreement of JM-RDT and SDB-RDT with ELISAs for NS1 detection16171819. While confirming these observations, our study showed low sensitivity of the IgM component of both the RDTs. These results were in concurrence with previous reports of poor diagnostic performance of SDB and other RDTs for IgM detection in dengue-endemic regions20212223. The need to improve sensitivity of the IgM component of RDTs is obvious.

Another important issue in dengue diagnosis is the efficiency in detecting primary and secondary infections. Contrary to the earlier reports of higher sensitivity of NS1 tests in primary infections724252627, we did not detect any significant difference in NS1 positivity during primary and secondary dengue. Although IgM levels are generally low in secondary infections28, higher IgM sensitivity was recorded during secondary infections by both ELISAs and RDTs. Similar observations have been reported previously7 and warrant further investigation to understand the basis for this higher IgM positivity.

Identification of infections by all four DENV serotypes is a major requirement for dengue diagnostic tests. Both ELISAs detected all the four serotypes, although JME-NS1 was more efficient than PBE-NS1. Despite being from the same manufacturer, JM-RDT detected lower proportion of DENV serotypes (except DENV-3) than JME-NS1, probably reflecting influence of the format. Serotype-specific evaluation on a larger sample size would be required for further confirmation of these findings.

In summary, our study confirmed superiority of ELISAs over RDTs and requirement for both NS1 and IgM testing for accurate diagnosis. The results emphasize an urgent need for substantial improvement in RDTs.