The current investigation of the prevalence of dupA in clinical isolates from Australia and Sweden as well as from the three major ethnic groups (Chinese, Indians and Malays) resident in Malaysia and Singapore clearly shows that there is significant variability in the prevalence of dupA between not only geographical locations, but also between ethnic groups resident in the same country. In the present study the prevalence of dupA in H. pylori isolates collected from FD patients varied significantly between nationalities/ethnicities, with isolates from Swedish patients being significantly more likely to possess dupA (65.0%) than isolates from Australia (26.3%) or ethnic Chinese (28.9%), Indians (7.1%) or Malays (35.7%) resident in Malaysia or Singapore. Interestingly isolates from ethnic Indian FD patients were significantly less likely to possess dupA (7.1%) than isolates from any of the other groups. This difference in dupA prevalence between countries and ethnic groups is supported by several studies [15, 27] including the initial Lu et al. 2005  which identified that dupA was more prevalent in isolates from Columbian gastritis patients (39%) than in isolates from Japanese (14%) and Koreans (7%) gastritis patients. Furthermore, there is no significant difference in dupA prevalence reported previously for isolates from Chinese patients (25% and 35.3% respectively) [15, 28] and for isolates from ethnic Chinese Malaysians and Singaporeans reported in the present study (28.9%), despite different primer pairs being used, suggesting that the primer pair designed in this study is appropriate. Conversely, the prevalence reported in a north Indian population was considerably higher than that found in the ethnic Indian Malaysians in the present study (16/70 as compared with 3/42) . One possible explanation is that the Indian Malaysians are likely to be predominantly ethnic Tamil , originating from Southern India, rather than the north Indian Indeed, two studies in Brazil reported significantly different prevalences for dupA (92.3% vs. 62%) [14, 30].
We also demonstrated that the association between dupA and severe gastroduodenal disease was inconsistent between the Chinese and Swedish populations. In isolates from ethnic Chinese patients the prevalence of dupA was significantly higher in patients diagnosed with DU (62.5%) or GC (54.6%) as compared with those diagnosed with FD. This is in contrast to the observations of Lu et al. 2005  and Zhang et al. 2008  who reported a negative association with GC, but supports the observations by Argent et al. . In contrast, in the Swedish population there was no significant difference in the prevalence of dupA in isolates from patients diagnosed with DU, GC or FD, similar to findings reported from Brazil [14, 30]. Our observations are consistent with the reported variation in other H. pylori virulence factors such as the cagA and vacA genes , and further emphasise the fact that before a new virulence factor is associated with a specific disease outcome, studies must be undertaken in a range of geographic locations as well in different ethnic groups.
Lu et al. have reported that, with the exception of J99, JHP0917–JHP0918 forms a continuous open reading frame, dupA, due to the presence of a 1 bp C/T insertion in clinical isolates . In the present study, all clinical isolates sequenced possessed a continuous dupA gene, signified by the presence of the C/T insertion, a finding that is consistent with previous reports in other populations [13, 14, 32]. Our results also indicate that dupA is highly conserved over the region sequenced with an average partial sequence variation of only 1.9%. This indicates a high degree of conservation comparable to housekeeping genes such as atpA, ureI, efp, and ppa . We must acknowledge that as only 29 clinical isolates were sequenced and the sequences used for comparison represent less than 400 bp, the pairwise sequence variation for the entire gene and in a larger population may be higher than that described here. Interestingly in a recent study Douraghi et al. compared 3 regions of the dupA gene – JHP0917 (289 bp), JHP0918 (259 bp) and the junction region over JHP0917 and JHP0918 i.e. 'dupA' (216 bp) in 6 Iranian strains with that of 10 Brazilian and 3 Indian strains whose sequences had been deposited in GenBank . The reported sequence similarities in that study ranged from 86.1%–100% for JHP0917, 88–98.8% for JHP0918 and 93.4–99.5% for 'dupA, a finding that would support the high degree of conservation of this gene observed in our study. Surprisingly in the Douraghi et al. study none of the strains were compared for all 3 regions.
The high degree of sequence conservation reported and the finding that dupA is present in strains from different continents and in populations with different genetic backgrounds, suggests that dupA may confer a fitness advantage of some sort, which has resulted in the conservation of the gene and the sequence, throughout human migration. An alternate explanation, since dupA is located in the plasticity zone, is that the acquisition was a relatively recent phenomenon. Although there is, as yet, no specific evidence for either scenario, we believe the former explanation is more likely. Whole gene sequence analysis of dupA over an increased sample size is necessary to adequately investigate these points.
Although the initial study by Lu et al reported that dupA was associated with IL-8 induction in vitro in both knockout studies and in clinical isolates , in the present study we observed no significant difference in the level of IL-8 induction between strains with or without dupA. It is possible however that the strains used in the present study lacked the other essential components of the dupA type IV secretion system, however, as none of these components have yet been identified it is impossible to assess whether this is responsible for the lack of association with IL-8 induction in the present study. More comprehensive studies with strains lacking genes known to contribute to IL-8 induction such as cag PAI components and with oipA off would be beneficial in elucidating the contribution of dupA to IL-8 induction.