Zika virus (ZIKV) is a mosquito-borne flavivirus that belongs to the Flaviviridae Family. Although the majority of infections are asymptomatic or produce mild and self-limited clinical manifestations, such as rash, low-grade fever, and arthralgia, it can lead to severe neurological complications, exemplified by the Guillain-Barré syndrome and the congenital Zika syndrome [1
Diagnosis of Zika virus (ZIKV) infection is based on clinical, epidemiological, and laboratorial data. Viral RNA can be detected in the first 5–7 days following the onset of symptoms, and, after this period, IgM antibodies may be detected by serological assays [4
]. However, ZIKV has antigenic similarities with other flaviviruses, especially dengue virus (DENV), which can result in antibody cross-reactivity between the two [6
]. As ZIKV and DENV share similar transmission determinants [7
], serological diagnosis of ZIKV infection is challenging in areas where DENV is endemic.
Studies evaluating the performance of serological assays for ZIKV IgM detection are needed to help clinicians, surveillance personnel, and public health authorities to determine which assays should be used and how to interpret the tests results in order to guide proper clinical management, as well as prevention and control measures. However, few studies have used paired samples to determine the capacity of serological assays to differentiate between ZIKV and DENV infection in settings where both viruses cocirculate. This study aims to evaluate the accuracy of the Zika IgM antibody capture enzyme-linked immunosorbent assay from the Center for Disease Control and Prevention (CDC Zika MAC-ELISA) using a panel of sera samples from febrile patients with RT-PCR-confirmed Zika and dengue, and blood donors, from Bahia, Brazil. In addition, our study provides insights into the kinetics of IgM immune response against ZIKV over time, which help optimize the timing of test use.
2. Materials and Methods
2.1. Study Design and Sample Selection
To evaluate the sensitivity of the CDC Zika MAC-ELISA, we used a panel of acute- and convalescent-phase sera from 21 febrile patients with RT-PCR-confirmed ZIKV infection. To evaluate the test specificity, we used acute- and convalescent-phase sera from 60 RT-PCR-confirmed dengue patients and sera from 23 blood donors collected prior to the Zika epidemic in the region.
Acute- and convalescent-phase sera from 14 of the 21 RT-PCR-confirmed Zika patients were obtained from a long-term acute febrile illness (AFI) enhanced surveillance study that aimed to detect arboviral infections in a public emergency health unit of Salvador (2.9 million pop.), the capital of the Bahia state, Brazil. Details on the surveillance protocol were described previously [7
]. Briefly, self-reported data on clinical characteristics, including days of symptoms, and an acute-phase blood sample were collected at enrolment and, whenever possible, a convalescent-phase sample was collected ≥10 days later. We were able to obtain a convalescent-phase sample for 8 of the 14 Zika cases. Of note, all the 14 Zika cases were detected between May and July 2015, the period in which ZIKV transmission in Salvador peaked [7
Acute- and convalescent-phase sera from other 7 RT-PCR-confirmed Zika patients were collected during the investigation of a ZIKV outbreak in Campo Formoso, one of the 30 most populous cities from Bahia (~71,000 pop.), in April 2016. These cases underwent ZIKV RT-PCR diagnosis based on the presence of fever and/or maculopapular rash, associated with myalgia, arthralgia, pruritus, headache, or retro-orbital pain, that initiated ≤10 days before the day of interview. Whenever possible, convalescent-phase samples from these patients were collected repeatedly, at different follow-up times. Of these 7 Zika cases, four had two convalescent-phase samples collected and three had only one sample collected, totaling 11 convalescent-phase samples available from this group.
We evaluated the CDC Zika MAC-ELISA specificity in a group of 60 dengue cases selected among the RT-PCR-confirmed dengue cases detected during the AFI enhanced surveillance study previously described. These 60 dengue cases were the first 20 cases of DENV-1, DENV-2, and DENV-4 detected during the AFI surveillance study for which paired acute- and convalescent sera were available. Samples from these dengue cases were collected between February 2009 and May 2011, before the estimated introduction of ZIKV in Brazil, i.e., between late 2012 and early 2013 [12
]. We also tested the acute-phase sera available for 47 of these patients by ZIKV RT-PCR [15
] to discard a concomitant ZIKV infection; all of them tested negative. DENV-3 patients were not included in this assessment due to the low number of DENV-3 cases identified by our surveillance prior to ZIKV introduction in Brazil.
To assess the test specificity, we also included sera samples from 23 blood donors obtained in December 2013, prior the first ZIKV case detection in Bahia [11
]. All blood donors underwent regular health screening for donation, which included absence of symptoms, such as fever, in the 15 days prior to the donation.
All blood samples were kept refrigerated until centrifugation, and the obtained sera were stored at −20 °C and −80 °C for serological and molecular testing, respectively.
This study was approved by The Research Ethics Committee of Instituto Gonçalo Moniz, Fundação Oswaldo Cruz (number 3.363.703; CAAE: 55904616.4.0000.0040; 3 June 2019) and School of Medicine, University Federal of Bahia (UFBA) (number 1.657.324; CAAE 56910516.3.0000.5577; 1 August 2016). All adult subjects provided written informed consent and participants <18 years of age who were able to read provided written assent following written consent from their parent or guardian.
2.2. Laboratorial Tests
All acute-phase samples underwent RNA extraction using the Maxwell®
16 Total RNA Purification kit (Promega, WI, USA) or QIAmp®
Viral RNA Mini kit commercial kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. We then performed RT-PCR on each extract product using specific oligonucleotides to amplify DENV [17
] and ZIKV [15
] separately, and/or multiplex qRT-PCR for ZIKV and DENV [18
]. We also tested acute-phase sera samples with DENV NS1-, IgG- and IgM-ELISA (Abbott, Santa Clara, CA, USA; former Panbio Diagnostics, Brisbane, Australia) according to manufacturer’s instructions.
All acute- and convalescence-phase study samples were tested for the presence of IgM anti-ZIKV antibodies using the CDC Zika MAC-ELISA protocol (NIAID-NIH BEI Resources Repository, catalog no. NR-50449) [4
]. Briefly, 96-well high-affinity microtiter plates (Nunc MaxiSorp flat-bottom 96 well plates) were coated with goat anti-human IgM antibody diluted at 1:1000 (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA, catalog #01-10-03) and incubated at 4 °C overnight. The next day, plates were washed with phosphate buffer with 0.05% tween 20 (PBS-T), and nonspecific binding sites were blocked with PBS-T containing 5% nonfat dry milk for 1h at room temperature. Meanwhile, serum samples were incubated at 56 °C for two hours for arboviral inactivation. Plates were washed with PBS-T, and each inactivated serum samples and IgM anti-ZIKV positive and negative controls provided by CDC and diluted at 1:400 were added to four wells and incubated at 37 °C for 1 h. Plates were washed with PBS-T, and each Zika Vero E6 and Normal Vero E6 cell culture antigen were splitted in two of the four wells of patients′ and controls′ samples, and incubated at 4 °C overnight. The next day, plates were washed and horseradish peroxidase (HRP)-conjugated monoclonal antibody 6B6C-1 (Hennessy Research, Lenexa, KS, USA, catalog #DC153-100) diluted at 1:5000 was added to the plates and incubated at 37 °C for 1h. Plates were washed and blue tetramethylbenzidine (TMB) substrate was added and incubated protected from light, at room temperature (20–25 °C) for 10 min. The enzyme/substrate reaction was stopped by adding the stop solution (H2
) and incubated at room temperature for 5 min before reading using an optical density microplate reader (FilterMax F3, Molecular Devices, Sunnyvale, CA, USA) and SoftMax Pro 6.2 Software.
Results of the CDC Zika MAC-ELISA were determined by calculating the ratio of the optical density mean (OD) of the wells containing Zika Vero E6 cell culture antigen (P) divided by the normal Vero E6 OD mean (N) for each patient and control sample. Ratio values obtained for each patient were classified as negative when P/N < 2, inconclusive when P/N ranged from 2 to <3, and positive when P/N ≥ 3. Samples with inconclusive results were retested and the result of the repetition was considered final.
The samples from the RT-PCR-positive Zika cases from Campo Formoso were the first to be tested by the CDC Zika MAC-ELISA, and this testing was not blinded. However, all the remaining samples, including the acute- and convalescent-phase samples from Zika and dengue cases from Salvador and the blood donor samples, were randomly numbered for deidentification and blindness in the CDC Zika MAC ELISA tests.
2.3. Data Analysis
We used absolute and relative frequency or median and interquartile range (IQR) to characterize the RT-PCR-positive Zika cases (overall and by place of residency) and the RT-PCR-positive dengue cases, according to demographics and clinical manifestations. We calculated sensitivities and 95% confidence intervals (95% CI) amongst RT-PCR-positive Zika cases and specificities, and 95% CI amongst RT-PCR-positive dengue cases, as well as amongst blood donors. Sensitivity was calculated according to timing of serum sample collection, to determine the presence of antibody kinetics during infection. Thus, acute-phase samples were classified as early acute-phase (collected 0–4 days post symptoms onset (DPSO)), and late acute-phase (5–9 DPSO); convalescent-phase samples were classified into three periods: early convalescent-phase (12–102 DPSO), intermediate convalescent-phase (258–260 DPSO), and late convalescent-phase (722–727 DPSO). The CDC Zika MAC-ELISA specificity was also stratified by the infecting DENV serotype and by type of DENV infection (primary versus secondary). We excluded from the accuracy analysis samples with a final inconclusive MAC-ELISA result.
In order to obtain information about the kinetics of the Zika IgM antibodies detected by the CDC Zika MAC-ELISA, we graphically plotted the median and IQR of the P/N ratio values obtained for the Zika cases at the different time points of sample collection (early acute, late acute, early convalescent, intermediate convalescent, and late convalescent). We also plotted the same graph with individual ratios per Zika positive case.
We evaluated the performance of the CDC Zika IgM-MAC-ELISA for Zika diagnosis, aiming to determine its sensitivity at different sampling time points during the disease course. We also evaluated the test specificity on sera from RT-PCR-positive dengue cases and from blood donors from a dengue endemic setting, which is particularly important given DENV and ZIKV cocirculation, the clinical similarity in the signs and symptoms they cause [7
], and the potential for immunological cross-reactions due to the close genetic and antigenic relationship between them [19
]. Furthermore, the panel of DENV samples was obtained from well characterized patients regarding clinical and laboratory data, allowing a detailed evaluation to be made of the assay specificity (i.e., according to infecting DENV serotype and presence of DENV antibodies at acute-phase sample collection).
As expected due to the short time elapsed from onset of symptoms, the CDC Zika IgM MAC-ELISA sensitivity for acute-phase samples obtained in the first four days was very poor (12.5%), but it increased after 5 days of illness (75.0%), and was high (90.9%) for samples obtained early during convalescence (12–102 DPSO). It is noteworthy that our findings on the kinetics of ZIKV IgM antibody response, as revealed by the serial sampling of RT-PCR-confirmed Zika patients at different time points, are in agreement with those of other studies that showed that ZIKV IgM antibodies starts to be detected four or five days post infection and maintain detectable levels for at least twelve weeks [5
The test also showed high specificity when applied to the acute- and convalescent-phase sera of RT-PCR-confirmed dengue cases (100.0% and 93.2%, respectively) and to blood donor sera (100.0%). Lower specificity was found in the subgroup of convalescent-phase sera from DENV-1 cases, but it was not substantially different from those observed for DENV-2 and DENV-4 cases. The specificity was very similar for samples of primary and secondary DENV infection cases. Of note, none of the five acute-phase samples that were RT-PCR and IgM positive for DENV returned a positive result in the CDC Zika IgM MAC-ELISA, suggesting that the observed early DENV IgM response did not cross-react on the Zika IgM MAC-ELISA, or was not strong enough to be detected. On the other hand, the unique acute-phase sample from a ZIKV RT-PCR-positive case that was also positive for DENV by RT-PCR and IgM-ELISA also tested positive in the CDC Zika IgM MAC-ELISA. This finding may be due to DENV and ZIKV antibody cross-reaction, but the limited number of samples fulfilling this condition hampers a proper conclusion.
The CDC assay has been evaluated before [21
]. In general, the evaluations showed that its accuracy is high, but the results were somewhat varied. One study, that, like ours, used specimens from RT-PCR-positive patients to evaluate the test sensitivity at different time points, had similar findings: 34.6% for samples collected at 1–4 DPSO (similar to our early acute-phase group), 74.3% at 5–10 DPSO (similar to our late acute-phase group), and 83% at 11–34 DPSO (similar to our early convalescence-phase group) [21
]. However, specificity was not determined. Another study evaluated the CDC Zika IgM MAC-ELISA and the combined commercial Euroimmun anti-Zika Virus IgM and IgG ELISA assays (Euroimmun AG, Lübeck, Germany) against PRNT results and found sensitivities of 100% and 83.3%, respectively, and specificities of 47.1 and 81.2%, respectively [22
Some studies [23
] used the CDC Zika IgM MAC-ELISA as the standard method for comparison with commercial ELISAs. Granger and colleagues compared the InBios ZIKV Detect IgM capture ELISA (InBios MAC-ELISA; InBios International, Inc., Seattle, WA, USA) and the Euroimmun anti-Zika Virus IgM ELISA (Euroimmun ELISA; Euroimmun AG, Lübeck, Germany) to the CDC Zika IgM MAC-ELISA. Their overall agreement was 90.7% and 51.9%, respectively, which might suggest that the CDC and the Inbios tests had better performance than the Euroimmun test [23
]. Safronetz and colleagues evaluated four Zika IgM ELISA methodologies in comparison to the CDC Zika IgM MAC-ELISA [24
]. Although the study did not aim to evaluate the performance of the CDC Zika IgM MAC-ELISA, the data provided allowed us to estimate the CDC MAC-ELISA sensitivity for 30 samples that were PRNT-positive for ZIKV and 10 samples that were RT-PCR-positive for ZIKV (100% and 20.0%, respectively). It also allowed us to estimate the specificity for 10 samples that were PRNT-positive for DENV and for 25 samples that were RT-PCR-negative for ZIKV (0.0% and 100%, respectively). Three of the four evaluated ZIKV IgM ELISAs had low sensitivities against the CDC test, and only the InBios Zika Virus Detect MAC-ELISA showed a performance comparable to the CDC ELISA.
The accuracy of the CDC Zika IgM MAC-ELISA and another MAC-ELISA methodology (CNDR MAC-ELISA, Centro Nacional de Diagnóstico y Referencia (CNDR) of the Nicaraguan Ministry of Health) were also determined against a composite reference that included results from RT-PCR and serological methods for ZIKV, and RT-PCR for DENV in a panel samples from a pediatric cohort [25
]. Their sensitivities were 70.1% and 94.5%, and their specificities were 82.8% and 85.6%, respectively. According to the authors, the different performance may be related to the diverse antiflavivirus monoclonal antibodies used in the ELISAs, which were less specific in the CDC Zika IgM MAC-ELISA than in the CNDR MAC-ELISA.
Of note, we have previously used the same panel of samples (except for the RT-PCR-positive ZIKV samples from Campo Formoso) to evaluate the Euroimmun anti-Zika Virus IgM ELISA (Euroimmun ELISA; Euroimmun AG, Lübeck, Germany) [26
], which allowed a direct test-to-test comparison to be made between the results of this and the previously published study. The sensitivity for the CDC and the Euroimmun tests on the same acute-phase samples from RT-PCR-confirmed Zika patients were 7.1% (1/14) and 0.0% (0/14), respectively (McNemar p
value: 0.48), and on the same early convalescent-phase samples were 100.0% (8/8) and 12.5% (1/8) (McNemar p
value: 0.01), respectively (Table A1
in Appendix A
). Both tests had high specificities, but for the convalescent-phase samples of RT-PCR-confirmed Dengue patients, the specificity of the Euroimmun test was nonstatistically higher than the CDC test (98.3% (58/59) vs. 93.2% (55/59), respectively, McNemar p
value: 0.18). Altogether, the CDC Zika IgM MAC-ELISA was superior to the Euroimmun anti-Zika Virus IgM ELISA.
Although the CDC Zika IgM MAC-ELISA presented satisfactory results, 10 (5.5%) of the initially 183 tested samples presented inconclusive results and needed to be repeated; after retesting 9 of them, 6 (3.3%) remained inconclusive. These 6 samples were from 5 RT-PCR-confirmed Zika cases (1 acute-phase and 4 convalescent-phase samples from intermediate and late periods) and from 1 RT-PCR-confirmed dengue case (acute-phase). The inconclusive results may be due to background reactivity or to the inherent interlaboratory variability associated with the CDC Zika IgM MAC-ELISA preparation, as the assay requires site-specific optimization of the dilutions used for select reagents, such as ZIKV antigen, goat anti-human IgM or conjugated secondary antibody [4
]. However, the low frequency of invalid results and the good test performance indicated that the CDC Zika MAC-ELISA is reproducible and reliable.
Despite its high accuracy, the time of execution of CDC Zika IgM MAC-ELISA is an inherent limitation. As an in-house method, it requires covering the plate with IgM antibodies and overnight incubation, taking a total of two or three days to obtain the test results. Thus, in the context of large outbreaks, when several thousand samples need to be tested, this assay may not be practical, and standardization and evaluation of ready-to-use commercial methods for ZIKV serological diagnosis remain necessary.
This study has some limitations. It has been suggested that the PRNT should be used as the reference method for confirmation of the presence of anti-Zika antibodies [4
]. Although we lacked PRNT results, we classified positive and negative samples for ZIKV and DENV according to patient RT-PCR results during the acute-phase of disease, which ensured a more accurate distinction between these flaviviruses compared to serologic diagnosis. Second, although we increased the number of samples from RT-PCR-confirmed Zika patients in comparison to our previous study evaluating the Euroimmun IgM ELISA [26
], we still had a relatively small number of samples from Zika cases, and might lack power for comparisons, particularly when stratifying them according to the time of sample collection. Third, we did not evaluate the test specificity of control samples obtained after introduction of ZIKV in Brazil. Theoretically, prior ZIKV exposure may modify the likelihood of false-positive results. In addition, the immune response of individuals who had sequential flavivirus infections in the past (i.e., DENV infection following ZIKV infection or ZIKV infection following DENV infection) may differ from that of naïve individuals, possibly altering the performance of ZIKV serological tests. Further studies evaluating the accuracy of flavivirus serological tests may benefit from the inclusion of control samples obtained in the period following ZIKV introduction.