PRISMA 2020 main checklist.

Diagnosis of arbovirus infection or exposure by antibody testing is becoming increasingly difficult due to global expansion of arboviruses, which induce antibodies that may (cross-)react in serological assays. We provide a systematic review of the current knowledge and knowledge gaps in differential arbovirus serology. The search included Medline, Embase and Web of Science databases and identified 911 publications which were reduced to 102 after exclusion of studies not providing data on possible cross-reactivity or studies that did not meet the inclusion criteria regarding confirmation of virus exposure of reference population sets. Using a scoring system to further assess quality of studies, we show that the majority of the selected papers (N = 102) provides insufficient detail to support conclusions on specificity of serological outcomes with regards to elucidating antibody cross-reactivity. Along with the lack of standardization of assays, metadata such as time of illness onset, vaccination, infection and travel history, age and specificity of serological methods were most frequently missing. Given the critical role of serology for diagnosis and surveillance of arbovirus infections, better standards for reporting, as well as the development of more (standardized) specific serological assays that allow discrimination between exposures to multiple different arboviruses, are a large global unmet need.</div

A minimum standard for metadata regarding the methods used to confirm the virus exposure, determine the prior infection history and test for antibody cross-reactivity.

A minimum standard for metadata regarding the methods used to confirm the virus exposure, determine the prior infection history and test for antibody cross-reactivity.</p

Distribution of (sub)category scores and total reliability groups.

Scores per individual variable of each category and total reliability groups of all studies scored in the reliability scoring system. Color legend as in Fig 5. NR = information not reported (grey striped pattern), NS = information not sufficiently specified (grey dotted pattern). The number of studies (N) represents the number of subdivided data sets used to score each variable.</p

A minimum standard for metadata regarding the studied individuals.

A minimum standard for metadata regarding the studied individuals.</p

Supplemental information.

A–Additional information regarding the reliability scoring system. Information about scoring the three categories: diagnostic specificity, arbovirus background and study size, as well as the overall data quality score. Table A–Search strategy of three different databases. Three different databases that cover all scientific articles were used to search articles for this systematic literature search. We aimed to only select articles studying human arbovirus antibody cross-reactivity in serological assays. Reviews, meta-analyses, and case reports were excluded from the selection. Table B–Included articles with their references. The “Study_number” corresponds to the study numbers in the S1 Datafile. For all details about the results of scoring and the subdivided datasets, see S1 Datafile. Table C–Overview reliability scoring system. Variables scored in this reliability scoring system were classified in three main categories: Diagnostic specificity, arbovirus background and study size. Maximum number of points for type of test and confirmation was 40, whereas for all other variables this was 4, or 8 or 2 based on the weight of the variable. Only studies that received either 18 or 0 points in the type of test and confirmation variable, were further scored for the other serological variables of the diagnostic specificity score. The highest possible score of each variable correlates with the lowest bias by diagnostic specificity, arbovirus background and study size. This means that in this case, the diagnosis of study groups can be viewed as correctly determined and true, as well as the antibody cross-reactivity results presented by the study. Table D–Circulation of arboviruses per area used in scoring system. All arboviruses circulating in specific geographic areas, according to Cleton et al [70,74], additional literature [16,71–73,88–106], and CDC and WHO circulation maps, were used to calculate chances of present pre-exposure antibodies in residence and travel areas of study participants. For arboviruses that only recently circulate, the reported year of the start of circulation in particular areas was taken into account. For calculating the effect of possible pre-exposure antibodies on the antibody cross-reactivity results, DENV and ZIKV were considered as the same serogroup based on their high antigenic similarities. Table E–Classification of residence and travel areas per geographic area. All residence and travel areas of the literature search were classified in determined geographic regions that have a similar composition of arbovirus circulation based on Cleton et al [70,74] and CDC and WHO circulation maps. Table F–Category groups of sum of points. Total sum of points of each category was divided into four quartiles (Group A, B, C and D) to be able to equally weigh and compare the total scores of the different categories. Table G–Overview combinations A, B, C and D of the three categories scoring reliability. The table shows an overview of all the possible combinations and the found combinations (A, B, C and D) of the three main categories scoring reliability of the included studies (N = 1082). The table is ordered by showing the best possible reliable combination at the top of the table and the least possible reliable combination at the bottom of the table. Table H–Overview of total reliability groups based on category combination score. Total reliability groups were defined per category combination score of A, B, C and D for included studies (N = 1082). Total reliability group 1 is most reliable, whereas total reliability group 5 is the least reliable. Number of studies per total reliability group can be seen in the last column. Table I—A minimum standard for metadata regarding the studied individuals. Table J—A minimum standard for metadata regarding the methods used to confirm the exposure, determine the prior infection history and test for antibody cross-reactivity. Fig A–Three-dimensional plots of category scores and total reliability groups. Scores for all three categories (study size, diagnostic specificity and arbovirus background) in three-dimensional plots (N = 1082). Total reliability groups are depicted in colours ranging from group 1 (highest) to group 5 (lowest) (yellow, turquoise, dark blue, purple and pink, respectively). Frequency of a specific score is shown by size. (DOCX)</p

Principle of scoring the reliability of cross-reactivity signals.

True cross-reactivity is defined as the combination of a high diagnostic certainty (diagnostic specificity score), a low chance of bias by possible previous arbovirus exposures (arbovirus background score) together with a large study size (study size score). Study data sets considered reliable regarding the serological cross-reactivity signals were selected for analyzing cross-reactivity.</p

Overview of studies providing information on multi-antigen antibody-reactivity testing.

Overview of all the combinations for which antibody measurements were done. Colours depict what the exposure had been for individuals sampled for serology testing (specified in the legend and the inner circle of the figure, ordered by serogroup), and the labels above each bar describe the virus antigens included in (cross-)reactivity panels. Total N is 1082 studies. Barmah forest (B), Semliki Forest (Se), Venezuelan equine encephalitis (V), Phlebovirus fever (P), Japanese encephalitis (J), Mammalian tick-borne (M), Yellow fever (Y), Japanese encephalitis, Mammalian tick-borne, Yellow Fever (JMY), Dengue (D), Spondweni (Sp).</p

Reliability scoring system categories and variables used for scoring.

Principles of scoring are based on either WHO or CDC recommendations [65–69], literature or were defined by the study team. For details see Supplemental Information A in S1 Appendix.</p

Overview of scoring results and total reliability of studies.

Overview of scoring results of each category (diagnostic specificity, arbovirus background, study size) and total reliability for all included studies (N = 1082) ordered by publication date (oldest publication (left) to the most recent (right)). Scores of variables are divided into highest (red), high (orange), middle (dark blue), low (light blue) and lowest (light grey), whereby a high score indicates a low chance of bias and a low score a high chance of bias. The possible options for scoring can vary between variables (see legends per variable). Studies scoring highest on type of test and confirmation (studies with vaccinees or patients confirmed by PCR) are not evaluated for the other variables of the diagnostic specificity score weighing the serological diagnostic evidence (NA, dark grey). Total scores of each category are shown in four groups ranging from high to low: A (dark green), B (light green), C (yellow), D (light grey). The total composite reliability score of the studies is shown at the bottom ranging from highest (Group 1) to lowest (Group 5) in yellow, turquoise, dark blue, purple and pink, respectively.</p

Reliability of arbovirus cross-reactivity results mapped to antigenic distance.

Calculated percentage of cases exposed to different arboviruses for which (multi-)(cross-)reactive antibodies were found to viruses listed at the bottom of the panels. The heading of each panel depicts the arbovirus(es) of exposure. Coloured circles indicate the scoring from group 1 (highest) to 5 (lowest) (yellow, turquoise, dark blue, purple and pink, respectively). The transparency of the circles corresponds to the different options of calculated cross-reactivity percentages. The blue overlays depict which arboviruses tested for cross-reactivity are in the same serogroup as the arbovirus of exposure, whereas the dark grey overlays show which arboviruses are within the genus of the arbovirus of exposure. *YFV, TBEV, JEV represents all different combinations of two of these viruses or all.</p