Meditory (The Journal of Medical Laborator.
Volume 13 Issue 2 Page 123-134 December 2025 https://doi.
org/10.
33992/meditory.
DIAGNOSTIC VALIDITY ANALYSIS OF NS1-BASED RAPID TEST
COMPARED WITH RT-PCR FOR DENGUE DETECTION IN
BATAM
Dewi Inderiati1.
Yuli Yanti2.
Anita Sofia2.
Citra Amaniah Anhar1 1 Poltekkes Kemenkes Jakarta i.
Jl.
Arteri JORR Jatiwarna Pondok Melati.
Bekasi, 17415.
Indonesia 2 Balai Labkesmas Batam.
Kelurahan Sei Binti.
Kecamatan Sagalung.
Batam, 29434.
Indonesia Article History Received, 29th September 2025 Revised, 30th September 2025 Reviewed 7th November 2025 Posted, 14th December 2025 Editor Imam Faizal *Corresponding author Nama, e-mail:
Dewi Inderiari, e-mail:
biomedic@gmail.
Keywords Dengue.
NS1 rapid diagnostic test.
RT-PCR, diagnostic validity.
Batam Abstract Background: Dengue remains a significant health problem in Indonesia, including Batam.
Because its symptoms resemble other infections, early laboratory testing is crucial.
The non-structural protein one rapid diagnostic test (NS1 RDT) is widely used, but its accuracy depends on the day of illness, immune status, and viral serotype.
Objective: To evaluate the diagnostic validity of NS1 RDT compared to RT-PCR, assessing the level of agreement between the two methods, and analysing the influence of clinical phase and serotype on the results of the examination.
Methods: A retrospective analytic study used secondary data from 309 suspected dengue cases tested by NS1 RDT at primary care and confirmed by RT-PCR with serotyping at Batam Public Health Laboratory .
2Ae Diagnostic performance was calculated from 2y2 tables.
concordance was analysed with McNemar's test and Cohen's kappa.
Results: The NS1 RDT showed 75.
8% sensitivity, 81.
2% specificity, 62.
positive predictive value, 88.
9% negative predictive value, and 79.
overall accuracy, with moderate agreement with RT-PCR ( = 0.
NS1
positivity peaked on illness days 1Ae3 and varied by serotype, with the highest for DENV-3.
Conclusions: The NS1 RDT showed good diagnostic validity with moderate agreement and sensitivity and high specificity in early dengue disease screening compared to RT-PCR, so the diagnostic performance of the NS1 RDT can be used in local epidemiological testing in Batam.
Cite this Article Inderiati D.
Yanti Y.
Sofia A.
Citra Amaniah Anhar.
Diagnostic Validity Analysis of NS1Based Rapid Test Compared with RT-PCR for Dengue Detection in Batam.
Meditory J Med Lab.
:123Ae133.
A2025 The Author.
Published with license by Politeknik Kesehatan Denpasar.
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Inderiati D, et.
e-ISSN: 2549-1520, p-ISSN: 2338Ae 1159
INTRODUCTION
Dengue remains a significant public health problem in Indonesia, including Batam City.
Its clinical manifestations often resemble those of other acute infections, so early laboratory confirmation is essential to support an accurate diagnosis, guide appropriate therapy, and prevent clinical deterioration.
At the primary healthcare level, rapid diagnostic testing is necessary because clinical decisions must be made efficiently.
The laboratory results of non-structural protein 1 (NS.
antigen testing (RDT-NS.
can be influenced by the duration of illness, the number of days since the onset of fever, the infection status .
rimary or secondar.
, the viral serotype, and the brand or kit used .
Ae.
In general, the sensitivity of NS1 detection is higher during the early early fever phase, then may decline in subsequent phases or in secondary infections.
Studies in Indonesia have also demonstrated differences among NS1 RDT brands and variations in results according to circulating viral serotypes, underscoring the importance of local validation before widespread implementation.
Although several studies in Indonesia have evaluated the performance of NS1 RDTs, substantial variability has been reported in different regions due to differences in circulating serotypes, kit brands, and patient characteristics.
There is no data available from Batam, where the distribution of serotypes and transmission patterns differs from those in other provinces.
These regional differences highlight the need for local validation to ensure that the RDT NS1 performs diagnostic outcomes.
Evidence from Indonesian cohort studies shows heterogeneous clinical presentations and shifts in dominant serotypes that may influence antigen and antibody detection in health care facilities .
Therefore, the selection of diagnostic tools and the interpretation of results should consider local epidemiology and referral capacities.
In terms of technological development, lateral-flow multiplex NS1 assays are being evaluated to enable rapid detection and serotype identification, particularly in resource-limited settingsAioffering potential to strengthen clinical decision-making and public health responses during case surges .
Prospective studies and diagnostic evaluations also indicate that test characteristics, including combined NS1/IgM/IgG assays, and test outcomes vary with illness duration and immune status, so combining tests (NS1 A RT-PCR early, serology after day .
often provides added value .
,4,.
Reference laboratories in Batam already utilize RT-PCR for confirmation and however, local studies that integrate RDT-NS1 and RT-PCR results while considering illness duration and serotype distribution remain limited.
This study evaluates the diagnostic validity of NS1 RDT compared to RT-PCR, assessing the level of agreement between the two methods, and analysing the influence of clinical phase and serotype on the results of the examination among suspected dengue patients in Batam.
The findings are expected to inform people about the development of a practical testing algorithm for primary healthcare and support local serotype surveillance.
MATERIALS AND METHODS
This study employed an analytical, observational, and retrospective design based on secondary laboratory data from the Public Health Laboratory (Labkesma.
in Batam.
The unit of analysis covered all suspected dengue cases in 2022Ae2024 that underwent an NS1 rapid antigen test (NS1 RDT) at first-level facilities and had RT-PCR confirmation at Labkesmas Batam.
A retrospective design was selected to assess the diagnostic validity of June Edition 2025 Inderiati D, et.
e-ISSN: 2549-1520, p-ISSN: 2338Ae 1159 RDT-NS1 against RT-PCR, as it reflects how tests are applied in routine healthcare practice .
Ae.
The study commenced with the development of the protocol and the obtaining of ethics approval.
then collected records from the laboratory information system, including minimal identifiers.
NS1 RDT using Fast Clear Q Ae Dengue Duo kit.
RT-PCR (RNA extraction using the QIAamp Viral RNA Mini Kit and serotype-specific primers for DENV1 to DENV-.
, the time since symptom onset .
ummarized as early fever, mid-course, and recover.
, and basic patient characteristics.
The team verified the completeness of the those that did not meet the eligibility criteria or lacked essential information were Eligible data were organized and prepared for analysis as planned .
Ae3,.
The sampling method employed was total sampling, meaning that all eligible records from the study period were included, resulting in 309 paired NS1 RDTAeRT-PCR results.
Total sampling is commonly used in service-based diagnostic studies because it does not subsample the data.
it provides a more representative picture of the referred population and helps reduce selection bias .
Ae.
This study was conducted through total sampling of secondary data on patients diagnosed with dengue fever by performing NSI RDT and RT PCR tests.
Patients diagnosed with infections other than dengue fever were not included, so that the data processed did not produce biased results.
After sample definition.
RDT-NS1 was treated as the index test .
eactive/nonreactiv.
In contrast.
RT-PCR served as the reference to determine the presence or absence of dengue virus and, when positive, to identify the serotype.
Following current guidance, the testing pathway is adapted to the clinical course: NS1 and/or RT-PCR are recommended early in the early fever phase, and IgM/IgG serology can be added later to increase diagnostic certainty and support surveillance.
Consistent with systematic evaluations.
NS1 results may vary with timing of testing, immune status .
rimary vs secondary infectio.
, viral serotype, and test brand/kit .
Ae5,7Ae.
The study protocol was reviewed and approved by the Research Ethics Committee of Poltekkes Kemenkes Jakarta i.
All analyzed data were de-identified, and data handling occurred after obtaining ethical clearance.
Data analysis progresses in stages.
Descriptive summaries presented age, gender, phase of illness .
arly fever, mid-course, recover.
RDT-NS1 results.
RT-PCR results, and serotype distribution.
Paired comparison of the two tests in the same subjects used McNemar's test for paired proportions and Cohen's kappa () for agreement, while the chisquare test was used for unpaired group comparisons .
g, across phases or serotype.
Diagnostic indices for RDT-NS1 against RT-PCRAisensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracyAiwere calculated from a 2y2 table with 95% confidence intervals.
p < 0.
05 indicated statistical significance.
Reporting followed STARD 2015 recommendations for diagnostic accuracy studies .
RESULTS AND DISCUSSION
Characteristics of Subjects A total of 309 suspected dengue patients who met the inclusion criteria were analyzed, consisting of 168 males .
4%) and 141 females .
6%).
The age distribution showed an almost equal proportion of children (<15 year.
9% and young adults .
Ae45 year.
at 3%, whereas older adults (Ou45 year.
accounted for only 8.
Based on the clinical phase.
June Edition 2025 Inderiati D, et.
e-ISSN: 2549-1520, p-ISSN: 2338Ae 1159 most patients were in the early fever phase .
ay 1Ae.
2%), followed by the critical phase .
ay 4Ae.
0%) and the recovery phase .
ay Ou.
8%).
The most frequent fever days at sample collection were day 3 .
0%) and day 4 .
9%).
The complete demographic and clinical characteristics are presented in Table 1.
Table 1.
Demographic and Clinical Characteristics of Study Subjects Characteristic Sex Male Female Total Age group Children (<15 year.
Young adults .
Ae45 year.
Older adults (Ou46 year.
Clinical phase Early fever .
ay 1-.
Critical .
ay 4Ae.
Recovery .
ay Ou.
The predominance of male patients is consistent with reports from Indonesia demonstrating a higher dengue incidence in males than in females .
The age pattern, dominated by children and young adults, reflects national epidemiology, where younger populations remain more susceptible because of environmental exposure and an incompletely matured immune system .
The finding that most specimens were collected during the early fever phase supports the well-established evidence that NS1 antigen and viral RNA are most readily detectable early in the illness, when viremia peaks .
,6,.
Recent evaluations of commercial NS1 rapid diagnostic tests (RDT.
confirm that maximum sensitivity occurs within the first three days of fever .
, highlighting the importance of early sampling to enhance diagnostic Similar findings have been documented in other dengue-endemic Southeast Asian countries, underscoring the early fever phase as the optimal diagnostic window .
The NS1 antigen can remain detectable beyond the acute phase and into the early recovery period, meaning that some patients may still test positive during convalescence even as viremia declines .
This evidence supports the unique pattern observed in our dataset, where several individuals remained NS1-reactive even after entering the recovery phase.
Comparison and Agreement of RDT-NS1 and RT-PCR Results Analysis of the test results revealed differences in detection between the two diagnostic methods.
The RDT-NS1 assay yielded 110 positive cases .
6%), whereas RTPCR confirmed 91 positive cases .
4%).
Cross-tabulation showed 22 RT-PCRAepositive cases that were negative by RDT-NS1, as well as 41 RDT-NS1Aepositive cases that were negative by RT-PCR.
Detailed results are presented in Table 2.
June Edition 2025 Inderiati D, et.
e-ISSN: 2549-1520, p-ISSN: 2338Ae 1159 Table 2.
Comparison of Dengue Detection Results by NS1 RDT and RT-PCR
RT-PCR Result
RDT-NS1
Result Negative (%) Positive (%) Total (%) Negative Positive Total This difference demonstrates discordance in detection between RDT-NS1 and RTPCR, with RDT-NS1 producing more positive results overall.
Such discordance primarily reflects different detection targets and biomarker kinetics.
RT-PCR identifies viral RNA, which can persist longer and is detected with high analytical sensitivity, whereas RDT-NS1 detects the non-structural protein 1 (NS.
antigen, whose concentration varies with the phase of infection and the host immune status .
Variations in NS1 levels across clinical phases, differences in viral serotypes, and RNA degradation during transport and storage may also contribute to the inconsistent findings.
NS1 test results in primary infections can cause NS1 antigen levels in the blood to increase and remain stable during the acute phase, whereas in secondary infections, the body already has IgG antibodies that quickly bind to the NS1 antigen, making it more difficult to detect in RDTs .
Moreover, in secondary infections.
NS1 levels may decrease due to the formation of antigenAeantibody immune complexes, resulting in false-negative RDT-NS1 results despite detectable viral RNA.
These findings emphasize that RDT-NS1 is a valuable rapid screening test.
however, its results should be interpreted cautiously, considering the clinical context and timing of specimen collection.
To evaluate the significance of these differences, statistical analyzes were performed in Table 3.
Table 3.
Statistical Analysis of RDT-NS1 and RT-PCR Results Statistical test Value p-Value Chi-square < 0.
McNemar Kappa The Chi-square test demonstrated a significant difference between RDT-NS1 and RTPCR .
< 0.
, confirming the discordance between the two methods.
The Kappa coefficient of 0.
537 indicates moderate agreement, suggesting that, although a correlation exists, the two assays are not entirely consistent .
Furthermore, the significant McNemar test .
= 0.
provides additional evidence that the observed difference is not due to random variation, but reflects the intrinsic limitations of RDT-NS1 when used as a stand-alone diagnostic tool .
These observations are consistent with recent studies reporting that RDT-NS1 sensitivity varies and tends to be lower than RT-PCR, particularly in later disease phases or during secondary infections .
Consequently.
RDT-NS1 is best suited for early screening, whereas RT-PCR remains essential as the gold standard for confirmatory June Edition 2025 Inderiati D, et.
e-ISSN: 2549-1520, p-ISSN: 2338Ae 1159 diagnosis .
Integration of both methods in a diagnostic algorithm is therefore essential to improve diagnostic accuracy and support timely and appropriate patient management .
Diagnostic Validity of RDT-NS1 The diagnostic validity analysis presented in Table 4 shows that RDT-NS1 achieved a sensitivity of 75.
8%, a specificity of 81.
2%, a positive predictive value (PPV) of 62.
7%, a negative predictive value (NPV) of 88.
9%, and an overall accuracy of 79.
6% when compared with RT-PCR as the gold standard.
Table 4.
Diagnostic Validity of RDT-NS1 Compared with RT-PCR Parameter Value (%) Sensitivity Specificity Positive predictive value (PPV) Negative predictive value (NPV) Accuracy DT-NS1 demonstrated good for screening and capability in detecting dengue A sensitivity of 75.
8% indicates that most dengue cases can be detected, although some infections may remain undetected .
esulting in false negative.
Variability in sensitivity is influenced by the clinical phase, viral serotype, and host immune status .
In secondary infections.
NS1 antigen levels may decrease due to the formation of antigenAe antibody complexes, which can lead to false-negative results .
The high specificity .
2%) and high NPV .
9%) indicate that a negative RDT-NS1 result can reliably exclude dengue infection.
This observation is consistent with previous reports showing that NS1 assays provide high confidence in ruling out infection .
Nevertheless, patients with strong clinical manifestations should undergo additional testing, such as RT-PCR, to ensure diagnostic certainty.
Conversely, the moderate PPV .
7%) and the overall accuracy of 79.
6% suggest that positive RDT-NS1 results should be confirmed with RT-PCR for final diagnostic These findings reinforce the role of RDT-NS1 as an effective rapid screening tool, while highlighting that it cannot fully replace RT-PCR as the gold standard for definitive laboratory confirmation.
,8,.
Furthermore.
NS1-negative or non-reactive results do not exclude dengue infection.
Several guidelines, including those from WHO .
and PAHO .
, emphasize that NS1 sensitivity declines substantially after the early febrile phase, resulting in a higher likelihood of false-negative results after day 3Ae5 of Therefore, patients with a negative NS1 result should undergo RT-PCR testing during the early phase, or IgM/IgG serology during later phases, when clinical suspicion remains high.
These considerations further support the role of RDT-NS1 as an initial screening tool rather than a standalone confirmatory diagnostic method.
Clinical Phase and Viral Serotype Analysis of clinical phases based on the day-of-fever categories (Table .
showed that the highest proportion of RDT-NS1 positives occurred in the early fever phase .
2%),
followed by the critical phase .
0%) and the recovery phase .
1%), with no statistically June Edition 2025 Inderiati D, et.
e-ISSN: 2549-1520, p-ISSN: 2338Ae 1159 significant difference .
= 0.
For RT-PCR, positive results were also more frequent in the early fever phase .
7%) and critical phase .
8%), but again the difference was not significant .
= 0.
Table 5.
RDT-NS1 and RT-PCR Results by Clinical Phase
Number of positives Clinical phase
RDT-NS1 RT-PCR RDT-NS1 RT-PCR
Early fever .
ays 1Ae.
Critical .
ay 4Ae.
Recovery .
ay Ou.
Total Although not statistically significant, the declining detection pattern in the later phases is consistent with the pathophysiology of dengue infection, in which viremia and antigenemia typically peak during the early fever phase and then decrease as the immune system clears the virus.
NS1 RDT is used to detect the NS1 protein, which is a dengue virus antigen secreted in the blood of patients and is effective mainly in the early fever phase up to the 7th day of fever .
iremia phas.
, while RT PCR detects genetic material from the dengue virus even at the onset of infection .
efore fever symptoms appea.
until traces of the dengue virus are still actively replicating in the patient's body .
,6,.
The absence of statistical significance is likely related to study design limitations, particularly the uneven distribution of samples, with very few specimens from the recovery phase .
= .
, resulting in low statistical power.
Additional factors, such as individual variability in immune responses and possible inaccuracy in fever-day recording, may also have been contributed .
Serotype analysis (Table .
revealed that the highest RDT-NS1 positivity was observed for DENV-3 .
8%), while the lowest occurred with DENV-2 .
0%).
Two cases of coinfection with DENV-2 and DENV-4 were identified.
A Chi-square test showed a significant association between viral serotype and RDT-NS1 results .
< 0.
Table 6.
RDT-NS1 Positivity by Dengue Virus Serotype RDT-NS1 Results Viral serotype Total
Negative Positive DENV-1
Positive DENV-2
DENV-3
DENV-4
DENV- Coinfection Total These results indicate that RDT-NS1 sensitivity is strongly influenced by viral Variations in epitope structure and NS1 protein expression affect antigen antibody binding affinity in different kits .
In this study, the highest sensitivity was observed for DENV-3, demonstrating superior detection capability for this serotype.
DENV-1 also June Edition 2025 Inderiati D, et.
e-ISSN: 2549-1520, p-ISSN: 2338Ae 1159 showed consistently high NS1 detection, representing the second-highest sensitivity in our This finding is consistent with recent kinetic studies reporting that primary DENV1 infections exhibit higher circulating NS1 levels and 100% detection during the first three days of illness, markedly higher than DENV-2 .
In contrast, the lower sensitivity for DENV-2 increases the likelihood of false-negative results, meaning that individuals infected with DENV-2 may be misclassified as uninfected, particularly in areas where DENV-2 predominates, representing a critical diagnostic limitation.
Moreover.
RDT-NS1 cannot detect coinfections, highlighting the advantage of RT-PCR for identifying mixed infections .
The predominance of DENV-3 in this study differs from several previous reports showing that DENV-2 has often been more prevalent in various regions of Indonesia .
Such serotype shifts may be influenced by epidemiological cycles, population immunity, and human mobility.
Batam, as an island city with high population movement, facilitates the circulation of diverse viral strains, which may drive rapid changes in serotype patterns .
supports the presence of dynamic serotype circulation in this area.
Biologically.
DENV3 has been shown to produce higher NS1 concentrations and higher viremia levels during acute infection compared with other serotypes, which may enhance detectability and increase its apparent positivity in clinical settings .
Epidemiologically.
DENV-3 has been associated with periodic resurgence and faster spread in populations with waning crossprotective immunity from prior DENV-1 or DENV-2 exposure, contributing to transient dominance during certain transmission cycles .
The contrast with earlier data from Batam supports the presence of dynamic serotype circulation in this area.
CLINICAL IMPLICATION
During the first 1Ae3 days of fever, the NS1 rapid test can help doctors screen for suspected dengue early.
If the NS1 result is negative, it is usually reassuring that dengue is If the NS1 result is positive, clinicians should still consider the person's symptoms and the local situation .
or example, ongoing dengue cases in the are.
and, when needed, confirm with PCR.
If clinical suspicion remains high despite a negative NS1 result, or if the sample was not collected in the early fever phase.
PCR should be prioritized.
Although NS1-RDT may still detect antigen up to day 4Ae5 of illness, its sensitivity decreases substantially after the peak of viremia, making it unsuitable as a standalone diagnostic tool in the mid to late clinical phase .
WHO .
and PAHO .
recommend that from day 4 onward.
NS1 testing be combined with RT-PCR or IgM/IgG serology to improve diagnostic accuracy and avoid false-negative results .
From about day 5.
IgM/IgG antibody tests increasingly contribute to diagnostic confidence.
A practical testing sequence for primary healthcare settings is therefore: NS1 testing during the early febrile phase, followed by PCR confirmation and serotyping, and subsequently IgM/IgG serology several days later.
LIMITATIONS
This study has several limitations.
First, we utilized existing laboratory records, which resulted in some clinical information being incomplete, such as whether the infection was primary or recurrent, co-morbidities, or medications that could have influenced the We attempted to account for potential confounding, particularly the timing of June Edition 2025 Inderiati D, et.
e-ISSN: 2549-1520, p-ISSN: 2338Ae 1159 sample collection throughout the illness course and viral serotype, through subgroup however, residual confounding may remain.
Second, all samples were drawn from a single-city referral network and a single reference laboratory.
therefore, the results may not be representative of all healthcare settings.
Third, testing occurred at different times and places (NS1 at referring facilities.
RT-PCR at the reference laborator.
, so discrepancies may have arisen due to delays between sample collection and transport.
Fourth.
NS1 brand/lot differences were not distinguished, and quantitative measures .
g, viral load/C.
were unavailable, which prevented the assessment of inter-kit variation or the relationship between viral level and test outcomes.
Fifth, the number of patients in the recovery phase was relatively small, which reduces the precision of some estimates .
g, sensitivit.
Nevertheless, these findings provide a useful real-world picture to guide a practical testing pathway, including early NS1.
RT-PCR for confirmation/serotyping, and serology from approximately day 5.
CONCLUSIONS
This study This study demonstrates that the NS1 rapid diagnostic test provides valid diagnostic performance for early detection of dengue, particularly within the first three days of illness, when viremia is highest.
However, its validity decreases after the early febrile phase, and negative results cannot reliably exclude dengue in patients presenting later in the course of illness.
Therefore.
NS1-RDT should be used as an initial screening tool, with RT-PCR serving as the reference standard for confirmation, especially in cases with ongoing clinical suspicion or atypical presentations.
Integrating NS1 testing with PCR and serology offers a balanced diagnostic approach that aligns with current WHO and PAHO recommendations and supports improved clinical decision-making in dengue-endemic CONFLICT OF INTEREST The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
diagnostic manufacturers or vendors influenced the study design, data collection, data analysis, decision to publish, or manuscript preparation.
the authors alone are responsible for the content of this article.
AUTHOR CONTRIBUTIONS
Dewi Inderiati conceived and designed the study, coordinated ethical clearance, data access, and project administration, supervised methodology and overall conduct, and served as corresponding author.
Yuli Yanti coordinated the retrieval of laboratory records, curated the dataset, verified NS1AeRT-PCR pairing and serotype data, performed formal statistical analyses with DI, and drafted the original manuscript.
Anita Sofia contributed to laboratory procedures and record verification, supported data curation, and critically revised the Methods.
Results, and Discussion with emphasis on serotype-related Citra Amaniah Anhar provided methodological oversight, advised on analytics and visualization, contributed to clinical/public-health interpretation, and June Edition 2025 Inderiati D, et.
e-ISSN: 2549-1520, p-ISSN: 2338Ae 1159 supervised quality assurance.
All authors contributed to writing, review & editing, approved the final version, and agree to be accountable for all aspects of the work.
ACKNOWLEDGMENTS
Institutional support was provided solely by Polytechnic of Health Jakarta i.
Ministry of Health.
Indonesia, offering internal resources such as database and laboratory record data .
rom Labkesmas Bata.
, and collaboration with primary healthcare facilities for the NS1 testing process.
FUNDING
This research received no external funding from public, commercial, or non-profit
DECLARATION OF ARTIFICIAL INTELLIGENCE USE
The author utilized ChatGPT 5 (OpenAI) during the preparation of this work, specifically to aid in the English language drafting of the manuscript.
The author reviewed and edited the final submission thoroughly, accepting full responsibility for the content's accuracy and integrity.
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