The prevalence of mixed infection by iceA1 and iceA2 was also different in each study; it ranged from 1.9% [14] to 36.7% [40]. Overall, the rate of mixed infection was 13.9%. Although strains from mixed infections were excluded in several studies [23], [29], [43], [46], [49], most studies included these mixed genotypes as total denominator
http://plosone.org/article/info:doi/10.1371/journal.pone.0030354#article1.body1.sec3.p1

Dear Editor
Shioto et al recently published the comprehensive Meta analysis on Helicobacter pylori (H. pylori) iceA in which the valuable data about this genetic marker has been collected [1]. The main vague subject that scattered throughout this Meta analysis is about the definition of mixed infection on the basis of the simultaneous presence of iceA1 and iceA2 in a sample, which considered as infection by multiple strains. The main question is that is it truly mixed infection or it could be genetic diversity in the iceA locus. To answer this question, we examined biopsies which were taken from antrum of 48 patients. The biopsies were cultured on supplemented Brucella Agar with 7% sheep blood and antibiotics in 37⁰C, at microaerophilic conditions for 3-10 days. After biochemical and molecular identification (16s rRNA and glmM), 5-10 single colonies were arbitrarily selected from each sample. Pure cultures of single colonies were used for DNA extraction. Then PCR was carried out for evaluation of the presence of the iceA1 and iceA2 alleles for 258 isolates. Interestingly 95/258 (34.4 %) of our single isolates had the iceA1 and iceA2 simultaneously which undoubtedly reject the presence of mixed infection (Figure 1). Also in this Meta analysis the authors claimed that multiple genotypes indicate the presence of multiple strains because there is no full-sequenced strain containing both iceA1 and iceA2 genes in Genbank. For further analysis, we search for iceA gene sequences in Genbank and found the sequences of the iceA1 and iceA2 alleles that present in a single isolate (Helicobacter pylori B8; NCBI Reference Sequence: NC_014256.1)[2], which provide us another evidence in this regard. In conclusion, we suggest that because of the co-existence of iceA variants (iceA1 and iceA2) in a single isolate, this genetic marker cannot be a suitable criterion for the evaluation of mixed infection in H. pylori related diseases.
Sincerely
Farzam Vaziri, PhD, Department of Bacteriology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran
Masoud Alebouyeh, PhD , Gastroenterology and Liver Diseases Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran



Figure 1. Allelic polymorphism of iceA locus. Lane 1;DNA ladder mix, Lane 2; Negative control,Lane 3-6:show coexistence of iceA1+iceA2 alleles among single isolates of H. pylori

References:
1.Shiota S, Watada M, Matsunari O, Iwatani S, Suzuki R, et al. (2012) Helicobacter pylori iceA, Clinical Outcomes, and Correlation with cagA: A Meta-Analysis. PLoS ONE 7(1): e30354. doi:10.1371/journal.pone.0030354
2.Farnbacher M, Jahns T, Willrodt D, DanielR, HaasR, GoesmannA, KurtzS and RiederG.(2010)Sequencing, annotation, and comparative genome analysis of the gerbil-adapted Helicobacter pylori strain B8. BMC Genomics, 11:335 doi:10.1186/1471-2164-11-335