What causes false positive Non-Structural Protein 1 (NS1) reports?

Causes of False Positive NS1 Reports in Dengue Testing

False positive results in dengue NS1 antigen detection tests are primarily caused by cross-reactivity with other flaviviruses, though this typically requires substantially high viral titers of the cross-reacting virus. 1

Cross-reactivity with Other Flaviviruses

• The most common cause of false positives in NS1 testing is cross-reactivity with other flaviviruses due to their similar antigenic profiles, particularly with:

  • Zika virus
  • West Nile virus
  • St. Louis encephalitis virus
  • Yellow fever virus 2, 1

• Cross-reactivity occurs because flaviviruses share structural similarities in their nonstructural proteins, including NS1 3

• Research shows that substantially high viral titers of Zika virus, Kunjin virus, and Yellow fever virus are required to produce false-positive results in commercial dengue NS1 rapid detection assays 1

Timing of Sample Collection

• NS1 antigen is detectable from day 1 of symptom onset up to 10 days thereafter, with highest sensitivity in the first week of illness 4, 5

• Testing outside this optimal window (especially after 7 days) may lead to unreliable results, including false positives 5

• As antibodies develop (typically 3-5 days after symptom onset for IgM), they may form immune complexes with NS1, potentially affecting test results 6

Test-Related Factors

• Different commercial NS1 detection assays have varying specificities and sensitivities:

  • Rapid tests generally have lower specificity than ELISA-based tests 1
  • ELISA-based tests have shown better specificity against cross-reactivity with other flaviviruses 1

• The quality and design of the antibodies used in the assay significantly impact specificity 7

Clinical and Epidemiological Factors

• Testing in areas with low prevalence of dengue but high prevalence of other flaviviruses may increase the likelihood of false positives due to the lower positive predictive value 2

• Previous flavivirus infections or vaccinations (e.g., yellow fever vaccine) can potentially lead to cross-reactive immune responses that may interfere with NS1 testing 2

Recommendations for Minimizing False Positives

• In areas where multiple flaviviruses co-circulate, consider using more specific ELISA-based NS1 tests rather than rapid tests 1

• For definitive diagnosis in ambiguous cases, consider confirmatory testing with:

  • Plaque reduction neutralization tests (PRNT) which are considered the reference standard for specificity 2
  • Nucleic acid amplification tests (NAAT) during the acute phase (first week) 2, 4

• Interpret NS1 results in conjunction with clinical presentation and epidemiological context 4

• When testing patients with previous flavivirus exposure, be aware of the increased risk of false positives and consider additional confirmatory testing 2