Sensitivity and specificity of the Standard Q rapid diagnostic test for dengue fever in children in epidemic areas of Côte d´Ivoire

¹Laboratoire de Biologie et Santé, UFR Biosciences, Université Félix Houphouët Boigny, Abidjan, Côte d´Ivoire, ²Institut Pierre Richet, Institut National de la Santé Publique, Bouaké, Côte d´Ivoire, ³Service Laboratoire, Centre Hospitalier Universitaire de Bouaké, Bouaké, Côte d´Ivoire ⁴ Departement des Virus Epidémiques, Institut Pasteur de Côte d´Ivoire (IPCI), Abidjan, Côte d´Ivoire

^&Corresponding author
Yapo Marie-Ange Edwige Kadjo, Service Laboratoire, Centre Hospitalier Universitaire de Bouaké, Bouaké, Côte d´Ivoire

Introduction    

There are around 100 arboviruses that cause symptomatic infections of varying severity in humans. Among these, dengue fever is one of the most significant public health problems worldwide. It is estimated that more than 2.5 billion people are at risk of dengue virus (DENV) infection, with 96 million cases reported each year [1-3]. Dengue virus is an arthropod-borne flavivirus classified into four distinct serotypes (DENV-1 to DENV-4), which constitute an antigenic complex within the Flavivirus genus of the Flaviviridae family [4]. Currently, there is no vaccine or medication available to prevent or treat dengue fever [5]. Care for dengue patients is limited to supportive treatment, such as the administration of analgesics to relieve associated symptoms [6]. Early laboratory diagnosis of the virus allows appropriate clinical management and early referral to outpatient care to be initiated. Because it is not possible to clinically diagnose dengue with certainty, given that its symptoms are similar to those of other diseases such as malaria, tuberculosis and other arboviruses (e.g. West Nile fever and yellow fever). This step is an important part of public health monitoring. Laboratory confirmation of a dengue infection can be based on the isolation of the virus in a cell culture, identification of viral nucleic acids or antigens, or detection of virus-specific antibodies [7].

Detection of DENV NS1 antigen is performed by reverse transcription qPCR in patients during the period from the onset of fever until 5-6 days afterward. Anti-DENV immunoglobulins can be detected by serology from 3-5 days after the onset of fever until 6 weeks later for IgM and from one month to several years for IgG [8,9]. But virus isolation is an unsuitable diagnostic technique because it requires cell culture facilities with a long time and is less sensitive than two other methods [10]. In the context of the resurgence of dengue fever in many African countries, rapid diagnostic tests (RDTs) have become essential for facilitating virus detection to ensure prompt and appropriate patient care. These RDTs must be both highly sensitive and specific to the target virus, inexpensive, and accessible to hospitals. This will enable the immediate treatment of patients and the surveillance of epidemics [11]. These tests have demonstrated their effectiveness in combatting various diseases, such as malaria, particularly in countries where health systems lack the required laboratory equipment for biological diagnosis. The main benefits are that it's simple to use, doesn't require any electricity or equipment, and delivers results in a quick time frame of 15 to 30 minutes [12]. This study aims to evaluate the diagnostic performance of the Standard Q Duo test for the identification of NS1 antigens and IgM antibodies against the dengue virus in the Ivorian population. The specific objectives of the study were to determine the prevalence of dengue virus infection in both the general population and febrile individuals and to assess improvements in diagnostic performance for DENV between version 1 and version 2 of the Standard Q Duo RDT test.

Methods     

Study design: a cross-sectional study was conducted to evaluate the diagnostic performance of the Standard Q Duo test for the identification of dengue cases and determine the prevalence of dengue fever in the febrile population in Côte d´Ivoire.

Study setting and population: Côte d´Ivoire is a country in sub-Saharan Africa. It is bordered by Ghana to the east, Liberia and Guinea to the southwest, Mali and Burkina Faso to the north, and the Atlantic Ocean to the south. Administratively, it is divided into 31 regions and 14 districts. According to the 2025 population and housing census, its population was estimated to be 32.8 million people living in an area of 322,462 km², giving an average population density of 100.8 inhabitants per km². Around 52.5% of the population resides in urban areas, compared to 47.5% in rural areas. Children ≤14 years represent a significant proportion of the population, at 38% [13]. This study was conducted in two phases. The first was carried out between November 2015 and August 2016, and the second between July and August 2022. The first phase took place during a field consultation campaign in three cities across the country: Abidjan, Aboisso and Bouaké). Located in the south, Abidjan is the economic capital of the country. This city has increasingly faced multiple outbreaks of Aedes mosquito-borne arboviral diseases (e.g., dengue (DEN) and yellow fever (YF)) during the recent years, 2017-2024 [14]. Aboisso is situated in the South Comoé region of southeastern Côte d´Ivoire, from the border with Ghana. Bouaké is a city in central Côte d´Ivoire, and its geographical location makes it an important trade hub connecting the south and north of the country.

These three cities were chosen because cases of dengue fever have already been reported there. Additionally, their infrastructure (e.g. port areas, markets, and train stations) represents potential entry points for arboviruses into the country via maritime and land routes, given the high presence of Aedes vectors [14-16]. In the first phase, the study population was selected based on age. Only children (≤14 years) were eligible for enrollment during the consultation campaign in these three cities. This age group constitutes the largest proportion of the population (38%) and is at risk of arbovirus infection [13]. Children from eight neighborhoods were consulted for this study, and a blood sample was taken from each child (Table 1). They are a high-risk group for arboviral diseases and are most severely affected by dengue fever. And then, on the following inclusive criteria: being unvaccinated; not sleeping under a mosquito net; having a temperature of at least 37.5°C on the day of collection or within the previous 15 days; a subpopulation of 754 children was selected as the febrile population for dengue virus testing in the laboratory. In the second phase, a retrospective study was conducted on blood samples from this febrile population that had been stored at -80°C in the Biological Resource Centre (CeReB) of the IPCI biobank. Additionally, new blood samples were included. These were samples received at this laboratory from Abidjan during the 2022 dengue epidemic, which tested positive for DENV by RT-qPCR.

Variables: sociodemographic and clinical information were collected for each participant: age, gender, mobility, location of residence, temperature, certain clinical signs (e.g. headache, eye pain, or joint pain), any treatment administered, history of fever in the 15 days preceding the consultation, yellow fever vaccination status, and whether a mosquito net was used while sleeping. And concerning the biological tests, the participants' positive or negative status for the dengue virus was determined using RDTs and laboratory confirmation tests. These data were used to assess the diagnostic performance of the RDTs through the calculation of performance parameters: sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), and Youden index (Y) [17-19].

Data resource and measurement

Data collection: a clinical examination was performed on each child, and the above-mentioned variables were recorded on a survey form. Next for all participants, a rapid diagnostic test (the Standard Q Zika/Dengue/Chikungunya Quad Test) was performed to determine their status for Zika, dengue, and chikungunya viruses and the prevalence of these viruses in three study sites. 10µl of blood was collected using a microcapillary pipette and placed in the sampling well of the testing device. Two or three drops of the dilution reagent were added to the dilution well. A timer set for 15 min was started, and at the end of which the test result was read. The technician recorded the start and end time of each RDT performed in the data collection form. After, approximately 5 ml of venous blood was collected in an EDTA tube, then sent to the virology laboratory of the Pasteur Institute of Côte d´Ivoire (IPCI) in a cooler at + 4°C. These samples were then stored at -80°C at the Biological Resources Center (CeReB) of the IPCI Biobank.

Laboratory activities: viral RNA was extracted in 200 µL of each sample using the QIAamp Viral RNA extraction kit protocol (Qiagen Catalog # 52904) according to the manufacturer's recommendations. And then, real-time RT-PCR was used for the detection of dengue virus-specific viral antigen NS1-Ag in these extracts. The different processes (amplification and detection) were carried out in the same reaction tube using the real-time RT-PCR monoplex developed by the Center for Disease Control and Prevention (CDC) for the detection of the dengue virus [20]. The PCR master mix with a final volume of 25 µl was prepared with 12.5 µl of 2X buffer+8.25 µl of nuclease-free water+1.25 µl of each primer (F and R) 0.5 µl of probe+0.25 µl of RNAse (enzyme)+1 µl of RNA extract. The PCR cycling conditions were as follows: reverse transcription at 50°C for 10 minutes, initial denaturation at 95°C for 15 minutes, 40 extension cycles at 95°C for 15 seconds for denaturation, 95°C for 15 seconds for annealing, and 60°C for 1 minute for final elongation. The product was analyzed on an Appleid Biosystems LightCycler. Concerning the serological tests, they were performed according to the protocol WHO diagnostic guidelines for Africa [21]. The MAC-ELISA test was used to detect IgM immunoglobulins in the extracts. The different steps were sensitization, saturation, blood/antigen deposition, and detection. After, the second version, Standard Q Dengue Duo RDT, specific to dengue fever only, was provided by the manufacturer to verify the improvement in the performance of the test for the diagnosis of dengue fever. The same protocol was followed for RDTs according to the manufacturer. Also, the same procedure was carried out in the laboratory for this second phase of evaluation.

Sample size: the data presented in this article are taken from a multidisciplinary study in Côte d´Ivoire and in Burkina-Faso ’Arborisk project’.´ During the implementation of the project, very few data were available on arboviral diseases in Côte d´Ivoire. Although some preliminary data suggested that DENV, CHIKV, FJ, and Zika viruses were circulating in the country, the prevalence of these infections in the population was unknown. Therefore, the sample size calculation could not be based on an estimated prevalence percentage for the target population of children aged 0 to 14 years. Consequently, an expected prevalence of at least 10% was considered, along with a sample size of 250 children per neighborhood, with a precision of 3%.

Data analysis: data were entered into an Excel database and analyzed using GraphPad Prism version 5.0.1. Sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), and Youden index (Y) were calculated for the two RDTs. Pearson´s chi-square test was used to compare the proportions of positive cases between study sites and between diagnostic tests. A p-value < 0.05 was considered statistically significant.

Ethical considerations: the Arborisk project, to which this work belongs, received approval from the French National Ethics Committee for Life Sciences and Health No. 040/MSLS/CNER-dkn. The study was conducted in accordance with the Declaration of Helsinki. The parents or legal guardians and the children included in this study received clear and accurate information about the study objectives, practical arrangements ((survey forms, blood sampling), data confidentiality management, and their right to refuse to participate in the study. Formal written consent was obtained from the parents or legal guardians and from subjects over 12 years of age before any participation in the study.

Results     

Report of the number of participants at each phase of study: a total of 2,093 children aged 0 to 14 years were included during the first phase of the study. Of the total participants, after the consultation, 1339 children were excluded from the remainder of the study because they were not eligible regarding the inclusion criteria. Therefore, only 756 children were identified as febrile participants, and laboratory analyses were performed on all of them. In the second phase, a random sampling was conducted among samples tested negative for NS1 antigen and IgM antibodies. Samples positive for NS1 antigen and/or IgM antibodies were retested using reference laboratory methods to verify sample quality. Overall, 146 samples were selected, including 102 negative (NS1 and IgM) and 44 positive (NS1 and/or IgM) samples. Due to the small number of positive samples, an additional 22 NS1-positive samples collected during the 2022 dengue outbreak were included for the evaluation of RDT2 (Figure 1).

Prevalence of dengue virus infection in both general and febrile populations: the RDT version 1 diagnosed 45 cases of dengue fever out of 2093 in all three cities, representing an overall prevalence of 2.15%. In the city of Abidjan, this test diagnosed 27 positive cases out of 813 children tested, corresponding to a prevalence of 3.32%. In Aboisso, the RDT diagnosed 14 positive cases out of 510 children tested, representing a prevalence of 2.75%. No significant difference was observed between these two cities (p = 0.340). In contrast, in Bouaké, 4 out of 770 children tested were diagnosed positive for dengue, representing a prevalence of 0.52%. This prevalence was significantly lower than those observed in Abidjan and Aboisso (p = 0.001) (Figure 2). Of the 754 children who constituted the febrile population, version 1 of RDT enabled the diagnosis of 45 positive cases of dengue, corresponding to a prevalence of 5.97%. The number of positive dengue cases diagnosed by this test was 27/245 children in Abidjan, 14/199 children in Aboisso, and 4/310 children in Bouaké, corresponding to prevalences of 11.02%, 7.04%, and 1.30%, respectively, in these three cities (Figure 2).

Parameters for evaluating the effectiveness of two versions of the Standard Duo rapid diagnostic tests

Diagnostic performance of the rapid diagnostic tests version 1: in (Figure 3) shows positive and negative results for dengue virus RDTs. Out of a set of 93 participants tested by RT-qPCR, 73 were positive for NS1-Ag antigen, and 20 were negative. Among the 73 positives for the antigen, 58 were detected as positive (true positive) by the RDT version 1, and all 20 negatives were actually detected as negative (true negative) by this RDT. The RDT version 1 showed a sensitivity of 79.45% and a specificity of 100% for NS1-Ag, a positive predictive value (PPV) of 100%, and a negative predictive value (NPV) of 57.10%, with a rate of 0% for false positives. The Youden index provided by this RDT was 0.79 for NS1-Ag of dengue. Of the 98 participants tested by the MAC-ELISA test, 78 were positive for IgM, and 20 were negative. Of this total, 45/78 were detected positives for IgM and 20 negatives by this RDT, which corresponds to 57.69% of sensitivity, 100% of specificity, a PPV of 100%, an NPV of 37.74% with a false negative rate of 62.26%, and a Youden index of 0.58 (Table 2).

Diagnostic performance of the rapid diagnostic tests version 2: a total of 174 participants were tested for the second phase. RT-qPCR was positive for 72 participants and negative for 102 participants. Rapid diagnostic tests version 2 detected 65 positives for the NS1 antigen and 102 negatives, corresponding to a sensitivity of 90.28% and a specificity of 100%. A positive predictive value (PPV) of 100%, a negative predictive value (NPV) of 58.62%, and a Youden index of 0.9 were recorded for this RDT. Of 171 samples tested by ELISA, 69 were positive, and 102 were negative. Among these, the RDT detected 45/69 positives for IgM antibody, and all 102 negatives were indeed found to be negatives. This RDT was allowed to have a sensitivity of 65.22%, a specificity of 100%, a positive predictive value (PPV) of 100%, and a negative predictive value (NPV) of 80.95% with a false negative rate of 19.05% and a Youden index equal to 0.65 (Table 3).

Discussion     

The result of the study revealed the circulation of dengue virus infection in the Ivorian population, particularly in Abidjan, Aboisso and Bouaké. The prevalence of DENV was 2.15% in the general population and 5.97% in the febrile population. Our results present an estimate of the prevalence of the dengue virus in Côte d´Ivoire during 2015 and 2016. During the implementation of the project, very little data were available on arboviral diseases in Côte d´Ivoire. Although some preliminary data suggested that DENV, chikungunya virus (CHIKV), FJ, and Zika viruses were circulating in the country, the prevalence of these infections in the population was unknown. Particularly in Abidjan, the prevalence was 11.02% in the febrile population, so 1 in 10 patients with a fever ≤ 37.5°C is likely to have dengue fever. This result is important information for health center staff to be aware of and consider when consulting patients. Our results showed a significantly equal prevalence between the populations of Abidjan and Aboisso using the Standard Duo RDT. These results are contrary to those of other studies, which have shown that Abidjan remains the city where the majority of dengue cases are recorded [22-25]. This could be explained by the easy access of the Abidjan population to health centers and laboratories equipped for the diagnosis of dengue fever.

Our results showed that version 1 of the RDT test was more sensitive in detecting the NS1 antigen (sensitivity (Se) = 79.45%, false negative rate (FNR) = 0.2%) and less sensitive in detecting IgM antibodies (Se = 57.69%, FNR = 62.26%) in patients with dengue fever. This result suggests that the test is more effective at diagnosing primary infections due to the presence of the NS1 antigen in patients being 0-6 days post fever onset [26]. The test displayed excellent specificity for detecting dengue, for both the NS1-Ag (specificity = 100%, FPR = 0%) and IgM (specificity = 100%, FPR = 0%). The PPV was 100% for both NS1-Ag and IgM detection. This suggests that if a patient tests positive for this RDT, they are almost certainly infected with dengue. The NPV was 57.10% and 37.74% for NS1-Ag and IgM detection, respectively, in patients without dengue. Consequently, the probability of a patient being free of dengue when the RDT is negative is between 37.74% and 57.10%. The accuracy of the RDT was determined by the Youden index (Y). The closer this index is to 1, the more reliable the test. The Youden index calculated for this RDT was 0.79 for NS1-Ag antigen detection and 0.58 for IgM detection. This suggests that the test is more effective at detecting the NS1 viral antigen in patients suspected of having dengue fever.

Our results strongly suggested that the second version was more sensitive for detecting both NS1-Ag (Se = 90.28%) and IgM (Se = 65.22%). These data confirm that the second version of the TDR is more sensitive for diagnosing the dengue virus compared to the first version of the TDR. The specificity of the test remained at 100% for the detection of NS1-Ag and IgM. Compared to other dengue RDTs, for example, SD Duo NS1 (Sp=98.75%) [27], SD bioeasy Dengue Duo (Sp=79-98.75%) [28], and SD Bioline Dengue Duo NS1 (Sp >96%) [29], TDR Standard Duo test Q Dengue Duo showed greater specificity for the dengue virus. The PPV was also 100% for both NS1-Ag and IgM, indicating that if this RDT is positive, there is a 100% probability that the patient has dengue. The NPV values calculated for version 2 were higher than those for version 1: 58.62% and 80.95% for NS1-Ag and IgM detection, respectively. The limitations of this study include the choice of the study population. Within the framework of this study, we only considered children aged 0 to 14 years as a population at risk. The other fraction of the population aged 15 years and older was not surveyed. However, these individuals could also be people exposed to and infected by arboviruses, particularly DENV. Excluding this segment of the population could introduce sampling bias. The actual prevalence of DENV in the general population may differ from that obtained in our study, which will affect the results of the study findings and affect the generalizability of the study.

Conclusion     

This study revealed a significant enhancement in the sensitivity of the Standard Duo test between its initial and subsequent iterations. Consequently, this rapid diagnostic test is a useful and reliable tool for detecting the NS1 antigen of the dengue virus in febrile patients. It could be used as a routine point-of-care test in Côte d´Ivoire's primary healthcare system to enable the rapid and early diagnosis of primary infections and the monitoring of dengue epidemics.

Competing interests     

The authors declare no competing interests.

Authors' contributions     

Conceptualization: Akré Maurice Adja. Methodology: Akré Maurice Adja. Valery Edgard Adjogoua and Yapo Marie-Ange Edwige Kadjo: formal analysis. Akré Maurice Adja, Yapo Marie-Ange Edwige Kadjo: investigation. Sylla Yahaya, Yapo Marie-Ange Edwige Kadjo, Mintokapieu Didier Stéphane Kpan, Ghislain Tiemoko Gueu and Mélissa Ahou Koffi. Writing-original draft preparation: Yapo Marie-Ange Edwige Kadjo. Writing-review and editing: Akré Maurice Adja, Yapo Marie-Ange Edwige Kadjo, Valery Edgard Adjogoua, Mintokapieu Didier Stéphane Kpan and Sylla Yahaya. All authors have read and agreed to the final manuscript.

Acknowledgments     

The authors are grateful to the Epidemic Viruses Department of the Institut Pasteur de Côte d´Ivoire for their support in laboratory activities, the health authorities, local authorities and inhabitants of the study areas.

Tables and figures     

Table 1: number of children (≤14 years) participating in the study, recruited from the consultation campaign for dengue detection in the three cities (Abidjan, Aboisso and Bouaké) in Côte d´Ivoire, from November 2015 to August 2016 (N=2,093)

Table 2: evaluation of the Q Zika/Dengue/Chikungunya Quad test parameters for detecting NS1 antigen and IgM antibodies against DENV, compared to the reference methods of PCR and serology, in children (≤14 years) in three cities (Abidjan, Aboisso and Bouaké) in Côte d´Ivoire, from November 2015 to August 2016 (N=2,093)

Table 3: evaluation of the Test STANDARD Q Dengue DUO parameters for detecting NS1 antigen and IgM antibodies against DENV, compared to the reference methods of PCR and serology, in participants study in three cities (Abidjan, Aboisso and Bouaké) in Côte d´Ivoire, from July and August 2022 (N=174)

Figure 1: diagram showing the number of participants included and excluded from the study evaluating STANDARD DUO RDTs for dengue detection in three cities (Abidjan, Aboisso, and Bouaké) in Côte d´Ivoire, from November 2015 to August 2022

Figure 2: prevalence of dengue virus (DENV) in children (≤14 years) in the overall and febrile populations recruited as part of the dengue detection consultation campaign in three cities in Côte d´Ivoire (Abidjan, Aboisso and Bouaké) from November 2015 to August 2016 (N= 2,093)

Figure 3: negative and positive results for dengue virus RDTs during the study on for dengue detection in the three cities (Abidjan, Aboisso and Bouaké) in Côte d´Ivoire, from November 2015 to August 2016 (N=2,093)

References