B-cell antigen D8/17 is a marker of rheumatic fever susceptibility in Aboriginal Australians and can be tested in remote settings

It has long been observed that rheumatic fever (RF) appears to occur only in susceptible individuals. This is based on observations of a familial association,1 controlled studies2,3 and the fact that only a limited proportion of individuals exposed to rheumatogenic Group A streptococci subsequently develop RF.4,5

Various potential markers of susceptibility to RF have been investigated, including blood groups and human leukocyte antigen (HLA) subtypes, but findings have often been inconsistent.6-8 A non-HLA B-cell antigen known as D8/17 has been investigated in several population groups. A study of American and Caribbean people showed that, in healthy individuals, a mean of 6.12% of B cells were positive for D8/17, compared with 33.5% in patients with a past history of RF (P < 0.0001). Intermediate levels were seen in unaffected first-degree relatives of index cases, suggesting that D8/17 might be an inherited factor.9 Subsequently, D8/17 was found to be highly sensitive as a marker for previous RF in the United States, Mexico, Russia, Chile, and Israel.10,11 However, in north India it was less sensitive than other, locally derived B-cell markers.12-14

The World Health Organization recognises an urgent need to develop new strategies to control RF.15 The ability to identify a subgroup of individuals at increased risk of RF could help direct primary prevention programs.15

The D8/17 non-HLA B-cell antigen has not been studied in Australia, where Aboriginal people have among the world’s highest prevalence of rheumatic heart disease (RHD).16 Populations with the highest rates of RF and RHD often live far from laboratories with the technology to perform D8/17 assays. Assays for D8/17 expression described to date usually require testing of fresh whole blood within 1 or 2 hours of sample collection, which has made it difficult to assess D8/17 expression in people living in remote settings. We aimed to study the expression of the B-cell marker D8/17 in Australians with a past history of RF or RHD, their relatives and healthy people. We also studied some technical modifications to the D8/17 assay that might allow it to be used for people living in remote settings.

Participants were enrolled in three centres:

• a remote Aboriginal community in the Northern Territory;

• a regional tertiary referral hospital; and

• a tertiary paediatric centre in Melbourne.

Patients with acute RF, past RF (with or without RHD), healthy relatives of these patients and healthy volunteers unrelated to the patients were recruited. The diagnosis of previous RF was confirmed from medical notes. In most cases the Jones criteria17 were supplemented with an echocardiogram report consistent with RHD. The absence of a history of RF or RHD in relatives and controls was documented prospectively by interview with the individual, review of medical notes and cardiac auscultation. Relationship status was determined by a detailed discussion in the local language.

Ethical approval was obtained from the Human Research Ethics Committee of Territory Health Services and Menzies School of Health Research. Participants were educated about the study in the local language and written consent was obtained.

One hundred and six individuals were enrolled; all but 19 were Aboriginal (Box 1). Participants included 42 males and 64 females; the proportions of males and females did not differ significantly between the groups.

Box 2 and Box 3 show that D8/17 expression was different in the four groups (ANOVA, P < 0.0001) — patients with acute RF had the highest levels (mean, 83.7%; SD, 10.1), followed in descending order by patients with RHD or past RF (mean, 39.3%; SD, 11.8), relatives (mean, 22.5%; SD, 5.2) and controls (mean, 11.6%; SD, 7.3). Aboriginal or non-Aboriginal ethnicity appeared to have no effect on D8/17 expression. The difference in mean D8/17 level between first-degree relatives and second-degree relatives (22.5% [SD, 5.2] v 16.8% [SD, 8.7]) approached significance (t test, P = 0.08), so only first-degree relatives were included in the analysis. The differences of the means of all groups were significant and substantial (greater than 10%).

In the ROC analysis, patients with either acute RF or a history of past RF or RHD were defined as positive cases and were compared with healthy controls. Based on this analysis, a cut-off of greater than 22.1% of B cells positive for D8/17 yielded the greatest percentage of accurate results (95.4%), with a sensitivity of 95.1% and a specificity of 95.6%. The likelihood ratio of a positive test at this cut-off was 21.4. Thus, a patient’s likelihood of having the disease with a D8/17-positive B-cell level greater than 22.1% was 21.4 times the pretest probability. If a negative result (D8/17-positive B-cell level less than 22.1%) is obtained, the pretest probability is multiplied by 0.05.

Box 4A shows a good correlation between the results for the D8/17 analysis of blood stained immediately and that stained with a delay of about half a day (mean, 0.55 days; SD, 0.2) for 38 sample pairs (correlation coefficient [r] = 0.94). A Bland–Altman plot (Box 4B) confirmed that differences between the two methods were not related to the level of D8/17 positivity. There was poor correlation between the results of the D8/17 assay obtained from analysis of fresh whole blood compared with frozen PBMC, in 19 sample pairs (r = 0.32).

Our findings indicate that the B-cell antigen D8/17 is a sensitive and specific marker of past RF in Australians and are consistent with those of researchers in the United States, Mexico, Russia, Chile and Israel.9-11,19,20 This adds to the growing evidence that D8/17 may be a universal marker of past RF in ethnically disparate populations. Negative findings by some US and north Indian researchers remain unexplained, but may reflect ethnic differences in the expression of B-cell antigens, selection of control patients, variation in antibody quality, or technical issues relating to the technique for selecting B cells.12-14,21

In previous reports, first-degree relatives have been shown to express D8/17 at intermediate levels, averaging 14.0% of B cells.9 Intermediate expression of D8/17 was also demonstrated in the relatives of patients with Sydenham’s chorea.20 These findings suggest that the B-cell marker is inherited, possibly along autosomal recessive lines. In our study, the absolute percentages of B cells positive for D8/17 in each category (patients with a past history of RF or RHD, first-degree relatives and healthy controls) reflect previous results and increase their reliability and generalisability.9

It is clear that D8/17 expression is not merely a marker of streptococcal infection, as it is not increased in poststreptococcal glomerulonephritis and uncomplicated streptococcal tonsillitis.9,22 However, post-streptococcal reactive arthritis is associated with elevated levels of D8/17, with D8/17 expression not conclusively differentiating between RF and poststreptococcal reactive arthritis in one small series.23

The functional significance of the D8/17 protein remains unknown. It does not match any known protein or HLA.20,24 The antibody to D8/17 has been shown to cross-react with cardiac muscle, skeletal muscle, smooth muscle cells of blood vessels and recombinant M6 streptococcal protein. Adsorption testing showed that B cells from a patient with RHD inhibited the binding of the D8/17 antibody to cardiac muscle. It was thus suggested that the D8/17 antigen may act as a streptococcal binding site on the B cells, and consequently become up-regulated after an infection, with B cells acting as antigen-presenting cells and influencing T-cell-specific cytotoxicity to heart and brain cells.22

For the first time, we evaluated D8/17 as a diagnostic test for RF or RHD by looking at its receiver operator characteristics, and found that a cut-off of 22.1% cells positive for D8/17 was the most accurate break point to distinguish between affected patients and healthy controls. Clearly, a positive test result is not diagnostic of RF or RHD, as 50% of unaffected first-degree relatives and 4% of healthy controls were found to be positive for D8/17. Rather, D8/17 may be seen as a marker of susceptibility to RF. The intermediate level of expression of D8/17 in unaffected first-degree relatives supports the conjecture that susceptibility is inherited rather than acquired. The consistency of the degree of elevation of D8/17 in first-degree relatives, but not controls, in a region where Group A streptococcal (GAS) infection is endemic suggests that inheritance, rather than environment or exposure to GAS, is the cause of the observed difference. Conclusive proof requires a prospective study of D8/17 expression in a high-risk population.

If D8/17 is an inherited marker of susceptibility to RF, it has a number of potential useful clinical applications. A cut-off point could be selected that could “rule out” past or current RF, which would be particularly useful in remote-living patients. In populations with a high incidence of RF, D8/17 could be used as a screening test to identify a group at increased risk of developing RF, who may be suitable for a primary prevention intervention, such as any future GAS vaccine.

A hypothesis is that D8/17, already expressed in excess in susceptible patients, is further augmented by the process that leads to RF. This suggests that D8/17 might be incorporated into the Jones criteria for the diagnosis of acute RF,17 either as an additional major manifestation, or as an obligatory feature in all diagnoses of acute RF. We are presently evaluating this in a larger, prospective study.

For clinical applicability, a diagnostic test must be readily available and cost effective. We looked at one way of reducing the overheads of this technically sophisticated test — storing specimens as frozen PBMC so that they could be tested in batches. We found that flow cytometry for D8/17 was not reliable when performed on frozen PBMC stored at – 80°C. However, we showed that a delay of up to half a day, such as that required to transport whole blood from a remote community to a central laboratory, had no significant effect on the accuracy of D8/17 analysis. As flow cytometry is not available in many locations where RF is common, further studies should investigate quantitative (rather than simple positive or negative) D8/17 assays that are cheap and practical in remote settings.

2 Percentage of D8/17-positive B cells in the four groups of participants

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RF = rheumatic fever. RHD = rheumatic heart disease. P < 0.0001 comparing all four groups by analysis of variance.

4 Comparison of immediate versus delayed staining

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A: Comparison of D8/17 analyses with immediate versus delayed staining.

B: Bland–Altman plot of immediate and delayed staining results. This plot is used to look for systematic differences when comparing two measurement techniques and to determine if the difference in results between the two techniques may be clinically important. In this case, the results with immediate and delayed staining were within 18% of each other on 95% of occasions.