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Editor,—Following the publication of our paper,1 we would like to thank the above authors for their comments and respond in order to clarify our study methodology, interpretation of the data, the impact of the media, and comment on the directions of future work in this area.
The possibility of PCR contamination has been suggested by Franciosi and Koletzko and we agree that this is a potential problem in studies of this type. We guarded against this by utilisation of separate laboratory areas and pipettes for pre-PCR, PCR and post-PCR stages of the procedure, use of sterile bunged pipette tips, and inoculation of the positive control as a last step in the pre-PCR preparation. In each run, we used sterile distilled water and DNA extract from human ureter as negative controls, and we examined samples in duplicate. Throughout our study, duplicated samples consistently gave concordant results, and negative controls were consistently negative.1
Dr Koletzko suggests that the two separate nested PCR-ELISAs utilised in our study may have doubtful specificity as we did not sequence the products. We agree that amplicon sequencing is desirable not only to ensure specificity but in the present context would also provide additional data on the molecular epidemiology of thecagA gene which was detected in these cases. We believe our assays to be specific. For example, the binding of oligonucleotides of 20 or more bases to template DNA at 55°C has been shown to be 100% specific. And in one of our PCR-ELISAs, there were five such interactions.1 We agree with Dr Koletzko and other workers that it would be valuable to test other tissues from the same patients by the same method.
Regarding our controls, use of these cases is quite appropriate and to illustrate this we include additional relevant data in table 1. Dr Vieth says our controls have had no normal environmental contact, however, five of these eight had spent time in the home environment since birth. Regarding antibiotic treatment, only one control had received antibiotics for more than one day prior to death, and this is the case in which H. pylori was detected.
Dr Koletzko states that “the fact that H. pylori was not demonstrated in the stomach, trachea or lung by histology in any of the children must raise major concerns that the applied methods were not specific”. However, as pointed out by Dr Vieth, haematoxylin and eosin staining, although a routinely used stain in histopathology practice, may not be optimal for microscopic visualisation of H pylori.
In response to Dr Vieth's claim that our suggested role for interleukin-1β (IL-1β) in H pyloriinfection is “totally speculative”, we would like to point out that these mechanisms have been demonstrated in an animal model.2 3Also, proteins of H pylori are known to activate macrophages leading to production of IL-1β4-6 which is known to inhibit acid secretion by parietal cells and may actually be the most potent inhibitor of acid secretion discovered to date.7IL-1β gene polymorphisms associated with increased IL-1β production have recently been associated with an increased risk of gastric cancer.8 In addition, systemic and mucosal humoral recognition of the cagA protein has been linked with peptic ulceration,9 10 duodenal ulcer patients may more frequently harbour cagA+ H. pylori strains,6 10 and it has been shown that infection withcagA+ as compared withcagA− strains is associated with increased transcription of IL-1β.6 It is therefore interesting that 25 of 28 cases of H pyloriassociated SIDS in our study had a detectablecagA gene in their tissues,1which may provide further support for the proposed pathogenesis ofH pylori in SIDS and a contributory role for IL-1β.11
Dr Paul Beggs from Macquarie University in Australia points out the link between dwelling crowding and H pyloriinfection,12 13 which has been shown to be independent of socioeconomic status,14 and the need for research on the possible link between dwelling crowding and SIDS. We agree that “given the importance of SIDS and the growing body of evidence suggesting H pylori as a cause of SIDS, it would be pertinent for future studies to consider dwelling crowding in more detail”.
We feel that Wiklund and colleagues, and MacKay and colleagues (in separate letters) have misunderstood the proposed hypothesis. Wiklund states that total breakdown of ingested urea occurs in all normal infants without ammonia intoxication and that SIDS victims have undigested urea in their faeces. MacKay states that “it is difficult to imagine that an organism specifically adapted to the microaerophilic and acidic conditions of the gastric mucosa thriving well enough in the lung to produce toxic amounts of ammonia in infants that presumably had normal livers”. To reiterate, there are two parts to the hypothesis. First, interleukin-1β production in the H pylori infected stomach, and second, supply of ammonia to the systemic circulation11 (and not the hepatic circulation as MacKay implies). Therefore, faecal urea content is irrelevant and so is ammonia produced in the stomach as this will be detoxified by the liver.
Regarding comments in the media, these are clearly not under our control and we have always stated that our findings are preliminary and require confirmation.
In conclusion, we would encourage researchers to repeat our studies and those of Pattison and colleagues15-17 in order to clarify the proposed role of H pylori in SIDS. In the meantime, we re-emphasise accepted measures to reduce mortality from SIDS and suggest the following additional precautions, all of which constitute good personal hygiene and are therefore advisable even in the absence of such a link. First, to prevent the transfer of saliva from the mouths of carers to babies.Second, prompt disposal of vomitus, decontamination of soiled surfaces, and washing of soiled clothes/bedclothes, followed by hand washing, in order to minimise transmission to the baby via the gastro-oral route.Third, good general hand and personal hygiene. In addition, parents should be reassured that they do not need to do anything more than the above at present.
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