Abstract
Background
Only a few Helicobacter pylori-infected individuals develop severe gastric diseases and virulence factors of H. pylori appear to be involved in such clinical outcomes. Duodenal ulcer promoting gene A (dupA) is a novel virulence factor of Helicobacter pylori that is associated with duodenal ulcer development and reduced risk for gastric carcinoma in some populations. The aims of the present study were to determine the presence of dupA gene and evaluate the association among dupA and other virulence factors including cagA and vacA in Brazilian patients. Gastric biopsies were obtained from 205 dyspeptic patients (100 children and 105 adults). DNA was extracted and analyzed for the presence of H. pylori and its virulence factors using the polymerase chain reaction method.
Results
Patients with gastritis tested positive for H. pylori more frequently. The dupA gene was detected in 41.5% of them (85/205); cagA gene was found in 98 isolates (47.8%) and vacA genotype s1/m1 in 50.2%, s1/m2 in 8.3%, s2/m2 in 36.6%, s2/m1 in 0.5% and s1/s2/m1/m2 in 4.4%. We also verified a significant association between cagA and dupA genes [p = 0.0003, relative risk (RR) 1.73 and confidence interval [CI] = 1.3–2.3]. The genotypes s1/m1 were also associated with dupA gene (p = 0.0001, RR: 1.72 and CI: 1.3–2.2). The same associations were found when analyzing pediatric and adult groups of patients individually.
Conclusion
Ours results suggest that dupA is highly frequent in Brazilian patients and is associated with cagA gene and vacA s1/m1 genotype, and it may be considered an important virulence factor in the development of gastric diseases in adults or children.
Keywords: Helicobacter pylori, dupA, cagA, vacA, Gastroduodenal diseases, PCR
Background
Helicobacter pylori is a gram-negative spiral bacterium that colonizes the human stomach. It is estimated that approximately half of the world’s population is infected with it [1,2]. In 1994, the International Agency for Research on Cancer included H. pylori infection in group I carcinogens. In addition, several authors report that the chronic infection may induce gastritis, peptic ulcer, gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue lymphoma [3-5].
It is interesting to note that only approximately 20% of H. pylori-infected individuals develop severe gastric diseases, suggesting that clinical outcomes are determined by the interaction among bacterial virulence, host genetic susceptibility and environmental factors [2,6].
Virulence factors of H. pylori – including vacA, cagA and babA – play important roles in gastric diseases. The vacA gene is present in all H. pylori strains and it is composed of two main regions: the signal region (s1 or s2) and the middle region (m1 or m2), both contribute to variations in the vacuolating activity of different H. pylori strains. s1/m1 strains are the most cytotoxic, followed by the s1/m2 strains and the s2/m2 [7].
Moreover, cagA gene is present in approximately 60 to 70% of H. pylori strains and several epidemiological studies have revealed that its presence is correlated with a higher risk of developing peptic ulceration, gastric atrophy and gastric cancer [8,9].
Most recently, Lu et al.[10] described a novel virulence factor, a virbB4 homologue, called duodenal ulcer (DU) promoting gene (dupA). It is located in the “plasticity region” of H. pylori genome and is composed of two genes, jhp0917 and jhp0918, which form one continuous open reading frame by the insertion of a base T or C after the position 1385 in the jhp0917 3’ region [6,11].
Although the role of the dupA gene is unclear, Lu et al.[10] suggested that this gene is involved in cell division and chromosomal DNA transfer. The authors also reported that infections with dupA-positive strains increased the risk for duodenal ulcer, but they were protective against gastric atrophy, intestinal metaplasia and gastric cancer in the Japanese, Korean and Colombian patients.
The involvement of H. pylori virulence factors in gastric disease has been demonstrated around the globe. Therefore, the aim of the present study was to evaluate the presence of cagA, vacA and dupA genes in Brazilian patients (children and adults) and to analyze the relationship between virulence factors and gastroduodenal diseases.
Methods
Patients and samples
We analyzed 205 samples obtained from 100 pediatric patients (41♂/59♀ mean age 10.9 ± 3.7) and 105 adult patients (57♂/48♀ mean age 47.8 ± 14.3) with abdominal symptoms who had been submitted to upper gastrointestinal endoscopy in the Department of Gastroenterology of the Marília Medical School, Brazil. All subjects or their parents signed an informed consent form approved by the Research Ethics Committee of Sacred Heart University (process n. 068/12).
Two samples from each patient were taken endoscopically from the gastric antrum, one for genotyping and detection of H. pylori by polymerase chain reaction (PCR) and the second for histopathological analyses. Only H. pylori positive patients were included in this study.
All patients were from the same socioeconomic level and had similar cultural habits. Regarding ethnic origins, approximately 40% were Caucasian, 45% were of Amerindian origin, and 15% were of mixed origin. The ethnic origins were determined by self-report and by family geographical origin.
DNA extraction and H. pylori isolation
DNA from gastric biopsies was extracted using the QIAamp® tissue kit (Qiagen, Germany) according to the manufacturer´s instructions. For detection of the H. pylori, PCR assays were performed using one set of oligonucleotides Hpx1/Hpx2 that amplifies a 150-bp fragment corresponding to 16S-rRNA from H. pylori (Table 1). In each experiment, positive (strain 26695) and negative (water) controls were included.
Table 1.
Primers and condition of amplification used in the study
| Primers | Primer sequence (5′-3′) | Amplification condition | Gene | Reference |
|---|---|---|---|---|
|
DupA1 |
CGTGATCAATATGGATGCTT |
35 cycles: 45 s, 94°C; 45 s, 52°C and 45 s, 72°C |
dupA |
Gomes et al.[6] |
|
DupA2 |
TCTTTCTAGCTTGAGCGA |
|||
| Cag1 |
ATGACTAACGAAACTATTGATC |
40 cycles: 1 min, 94°C; 1 min, 53°C and 1 min, 72°C |
cagA |
Rasmussen et al.[9] |
| Cag2 |
CAGGATTTTTGATCGCTTTATT |
|||
| Hpx1a |
CTGGAGARACTAAGYCCTCC |
40 cycles: 1 min, 94°C; 1 min, 59°C and 1 min, 72°C |
16S rRNA |
Scholte et al. [12] |
| Hpx2 |
GAGGAATACTCATTGCGAAGGCGA |
|||
| SA |
ATGGAAATACAACAAACACAC |
40 cycles: 45 s, 94°C; 45 s, 54°C and 45 s, 72°C |
vacA-s |
Atherton et al. [13] |
| SCa |
CCTGARACCGTTCCTACAGC |
Doorn et al. [14] |
||
| MAa |
CACAGCCACTTTYAATAACGA |
35 cycles at: 45 s, 94°C, 45 s, 55°C and 1 min, 72°C | vacA-m | Doorn et al. [14] |
| MB | CGTCAAAATAATTCCAAGGG |
Detection of cagA, vacA and dupA genes
The analysis of the presence of target genes, cagA, dupA and vacA genotypes was performed through PCR, using one set of oligonucleotides for each gene fragment (Table 1).
Genomic DNA was amplified by PCR for cagA according to Rasmussen et al.[9], van Doorn et al.[14], using a 232-pb fragment. The “s” and “m” regions of vacA were genotyped with the previously described primer sets SA/SC and MA/MB. The SA/SC primers amplified “s1” fragments of 176 bp and “s2” fragments of 203 bp. The “m1” fragments were 400 bp and the “m2” fragments were 475 bp [9,13-15] (Table 1).
To evaluate the presence of the dupA gene, we used the primers described by Gomes et al.[6] which amplifies a 197-bp fragment. It is important to note that these primers were constructed in well-conserved regions based on Brazilian strains.
Histopathology
Gastric mucosa biopsy specimens were fixed in 10% buffered formalin, embedded in paraffin, sequentially cut and stained with haematoxylin and eosin and Giemsa stain. The histological parameters were graded using the criteria described in the Sydney system for analysis of chronic inflammation, polymorphonuclear activity and intestinal metaplasia [16].
Statistical analysis
Associations among the virulence factors were evaluated by the two-tailed Chi-square test with Yates’ correction for each group of patients separately. Differences were considered significant when p value was less than 0.05. All statistical analyses were performed with SPSS version 20.0.
Results
Histopathology analyses were performed in 168/205 (82%) patients (99 adults and 69 children) and they revealed 153 patients with chronic gastritis, 13 patients with normal gastric mucosa and just two patients with duodenal ulcer. These results suggest an association between chronic gastritis and the presence of H. pylori.
cagA, vacA and dupA status
Overall, the cagA gene was obtained in 98/205 (47.8%) samples. If we considered age, cagA gene was detected in 51/105 and 47/100 samples of adults and pediatric patients respectively (Table 2).
Table 2.
Frequency of genes cagA and dupA and genotypes of vacA in H. pylori strains isolated from 105 adults and 100 children with gastric chronic and normal gastric mucosa
| Genes |
N (%) |
Samples from N (%) |
Histopathology |
||||
|---|---|---|---|---|---|---|---|
|
Chronic gastritis |
NGT |
||||||
| Total | Adults | Children | Adults | Children | Adults | Children | |
|
cagA + |
98 (47.8) |
51 (48.6) |
47 (47) |
47 (30.7) |
26 (16.9) |
1 (7.7) |
4 (30.75) |
|
cagA – |
107 (52.2) |
54 (51.4) |
53 (53) |
48 (31.4) |
32 (21) |
1 (7.7) |
7 (53.85) |
|
dupA + |
85 (41.4) |
48 (45.7) |
37 (37) |
44 (28.7) |
26 (16.9) |
1 (7.7) |
2 (15.4) |
|
dupA – |
120 (58.6) |
57 (54.3) |
63 (63) |
51 (33.3) |
32 (21) |
1 (7.7) |
9 (69.2) |
|
s1/m1 |
103 (50.2) |
58 (55.2) |
45 (45) |
54 (35.3) |
26 (16.9) |
2 (15.4) |
4 (30.75) |
|
s2/m2 |
75 (36.6) |
33 (31.4) |
42 (42) |
28 (18.4) |
26 (16.9) |
0 (–) |
4 (30.75) |
|
s1/m2 |
17 (8.3) |
10 (9.5) |
7 (7) |
10 (6.5) |
4 (2.7) |
0 (–) |
2 (15.4) |
|
s2/m1 |
1 (0.5) |
0 (–) |
1 (1) |
0 (–) |
0 (–) |
0 (–) |
0 (–) |
|
s1/s2/m1/m2 |
9 (4.4) |
4 (3.9) |
5 (5) |
3 (1.9) |
2 (1.4) |
0 (–) |
1 (7.7) |
| Total | 205 (100) | 105 (100) | 100 (100) | 153 (100) | 13 (100) | ||
NGT: normal gastric tissue.
Regarding the vacA gene, the most virulent allelic combination s1/m1 was found in 58 (55.2%) of the H. pylori strains, the s2/m2 was detected in 33 (31.4%) and the other genotypes, s1/m2 and s1/s2/m1/m2 were verified in 14 (13.4%) samples from adults. In children, we isolated 45 (45%) strains of the common vacA genotype s1/m1 and 42 (42%) strains of vacA s2/m2. Thirteen percent (13 strains) had vacA genotype s1/m2, s2/m1 and s1/s2/m1/m2 (Table 2).
The dupA gene was considered positive when harboring the jhp0917 and jhp0918 genes. dupA was detected in 48 (45.7%) and in 37 (37%) H. pylori strains from adults and children, respectively (Table 2).
When children and adults were compared, the dupA gene was more frequent in strains from adults, but this difference was not statistically significant (p = 0.2610). Similar results were observed for cagA gene (p = 0.932) and genotypes of vacA (p = 0.1414).
Association of dupA gene with cagA and vacA genes
When the total gastric samples were collectively analyzed (adults and children), the presence of dupA was associated with cagA positive strains (p = 0.0003; RR = 1.73; CI = 1.3–2.3). A similar association was verified between the dupA gene and the s1/m1 genotypes of vacA gene (p = 0.0001; RR = 1.72; CI = 1.3–2.2). As expected, the virulent allelic combination s1/m1 was associated with cagA positive (p = 0.0001; RR = 3.52; CI = 2.5–4.99). When children and adults were analyzed separately, a similar association was observed (data not shown).
It is worthwhile to emphasize that the combination of dupA positive, cagA positive and s1/m1 genotype was found in 47 strains of H. pylori suggesting a possible relationship among the main virulence factors.
Discussion
H. pylori has a large degree of genomic and allelic diversity, particularly in the “plasticity region” of the genome, which is composed of the dupA gene described by Lu et al.[10]. First, dupA gene was associated with an increased risk of duodenal ulcer and protection against gastric cancer in Japan and Korea, but these results are controversial and there can be variables depending mainly on the geographic areas studied.
Analyzing the Brazilian population, we detected the dupA gene in 85 (41.5%) patients, of which 48 (23.4%) adults and 37 (18.1%) children. Gomes et al.[6] found different values and registered the presence of dupA gene in 89.46 and 100% of adult and pediatric patients, respectively. Although we employed the primers described by Gomes et al.[6], such discordant results may be explained by the different geographic areas of Brazil, studied population, method for molecular analysis and lost or rearrangement in the plasticity zone.
Arachchi et al.[17] studying the Indian population, reported the presence of dupA in 37.5% of patients with duodenal ulcer (DU) and 22.86% in patients with functional dyspepsia. In addition, Zhang, et al.[18] detected dupA gene in 35.3% of the Chinese population. Both authors revealed that the prevalence of the dupA was significantly higher in strains from duodenal ulcer confirming the original results, which showed that dupA was associated with DU and can be a virulence marker to this specific disease. However, the present study reported a similar prevalence of dupA in patients with gastritis.
Recently, Imagawa et al.[19] showed that patients infected with dupA-positive strains have a gastric acid output significantly higher than dupA-negative patients. In addition, Abadi et al.[20] observed higher acid resistance of the dupA-positive strains which suggest that these strains are adapted to a stomach with high gastric acid output. Together, these results may explain the associations between the dupA gene and an increased risk for DU formation.
Regarding the detection of cagA gene, our results corroborate previous studies in Western countries, with 47.8% of cagA-positive patients, without significant differences between adults and children [6,9,15,21,22]. Other interesting result was the association between the presence of dupA and cagA genes in adults, children and when both groups were analyzed simultaneously. Arachchi et al.[17] and Gomes et al.[6] also found similar results; however, these authors reported this association only in patients with DU and in adults.
vacA genotypes were also investigated in the present study, and an important association was found between s1/m1 genotype and the presence of dupA gene, whereas H. pylori dupA-negative strains were associated with genotype s2/m2 of vacA gene. Similar results were reported by Arachchi et al.[17], but with the alleles s1 and m1 separately. Interestingly, strains of H. pylori with the combination dupA positive, cagA positive and s1/m1 were found in 47 (23%) patients. To the best of our knowledge, only Arachchi et al.[17] had described the same association, but they found only three patients with functional dyspepsia infected with strains dupA positive, cagA positive and s1/m1. The present results reveal an important association and suggest a possible relationship among the main virulence factors and the development of severe gastric disease.
Our work has a few limitations limitations. The studied patients had only gastritis and it would be interesting to conduct the same research on patients with duodenal ulcer and gastric cancer. It is important to note that dupA gene was detected using PCR methods, showing the presence of genes jhp0917 and jhp0918. Queiroz et al.[11] suggest that the presence of jhp0917 and jhp0918 genes may not be enough to characterize an intact and maybe functional dupA and that an analysis of dupA gene with other molecular biology techniques is important.
Conclusions
dupA is highly frequent in Brazil and it can be considered an important virulence marker. However, further studies are necessary to state that this is a specific-disease marker. In addition, the association among the major virulence markers in adults and children represents new information that can be associated with the prognosis of patients with severe gastric disease.
Ethics committee approval
Informed consent was obtained from all patients or their legal guardians for publication of the present study. The present study was approved by the Research Ethics Committee of Sacred Heart University (process n. 068/12).
Competing interests
The authors declare that there are no competing interests.
Authors’ contributions
WNP, MAF, LMZ and JPBX performed the sample collection, DNA extraction and molecular analysis. RWdeL, WAO and ACN participated in the study design, performed the statistical analysis and provided technical support and scientific discussions. SLMP and LTR conceived the study, participated in its design, coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
Contributor Information
Weendelly Nayara Pereira, Email: weendelly.biomed@yahoo.com.br.
Mariane Avante Ferraz, Email: mariane17_10@hotmail.com.
Luanna Munhoz Zabaglia, Email: luannamunhoz@gmail.com.
Roger William de Labio, Email: roger@famema.br.
Wilson Aparecido Orcini, Email: wilson.orcini@usc.br.
João Paulo Bianchi Ximenez, Email: joaopaulo.ximenez@yahoo.com.br.
Agostinho Caleman Neto, Email: neto_caleman@yahoo.com.br.
Spencer Luiz Marques Payão, Email: slmpayao@famema.br.
Lucas Trevizani Rasmussen, Email: lucas_rasmussen@hotmail.com.
Acknowledgements
The authors would like to thank the State of São Paulo Research Foundation (FAPESP) for the funding of this research (grant number 20012/18333-3).
References
- Marshall BJ. Helicobacter pylori. Am J Gastroenterol. 1994;89(8 Suppl):S116–S128. [PubMed] [Google Scholar]
- Liu YE, Gong YH, Sun LP, Xu Q, Yuan Y. The relationship between H. pylori virulence genotypes and gastric diseases. Pol J Microbiol. 2012;61(2):147–150. [PubMed] [Google Scholar]
- Humans IW. GotEoCRt. Schistosomes, liver flukes and Helicobacter pylori. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. IARC Monogr Eval Carcinog Risks Hum. 1994;61:1–241. [PMC free article] [PubMed] [Google Scholar]
- Wang MY, Chen C, Gao XZ, Li J, Yue J, Ling F, Wang XC, Shao SH. Distribution of Helicobacter pylori virulence markers in patients with gastroduodenal diseases in a region at high risk of gastric cancer. Microb Pathog. 2013;59–60:13–18. doi: 10.1016/j.micpath.2013.04.001. [DOI] [PubMed] [Google Scholar]
- Yamaoka Y. Mechanisms of disease: Helicobacter pylori virulence factors. Nat Rev Gastroenterol Hepatol. 2010;7(11):629–641. doi: 10.1038/nrgastro.2010.154. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gomes LI, Rocha GA, Rocha AM, Soares TF, Oliveira CA, Bittencourt PF, Queiroz DM. Lack of association between Helicobacter pylori infection with dupA-positive strains and gastroduodenal diseases in Brazilian patients. Int J Med Microbiol. 2008;298(3–4):223–230. doi: 10.1016/j.ijmm.2007.05.006. [DOI] [PubMed] [Google Scholar]
- Yamaoka Y. Pathogenesis of Helicobacter pylori-related gastroduodenal diseases from molecular epidemiological studies. Gastroenterol Res Pract. 2012;2012:371503. doi: 10.1155/2012/371503. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Karlsson A, Ryberg A, Dehnoei MN, Borch K, Monstein HJ. Association between cagA and vacA genotypes and pathogenesis in a Helicobacter pylori infected population from South-eastern Sweden. BMC Microbiol. 2012;12:129. doi: 10.1186/1471-2180-12-129. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rasmussen LT, de Labio RW, Neto AC, Silva LC, Queiroz VF, Smith MAC, Payão SLM. Detection of Helicobacter pylori in gastric biopsies, saliva and dental plaques of dyspeptic patients from Marília, São Paulo, Brazil: presence of vacA and cagA genes. J Venom Anim Toxins incl Trop Dis. 2012;18(2):180–187. doi: 10.1590/S1678-91992012000200008. http://dx.doi.org/10.1590/S1678-91992012000200008. [DOI] [Google Scholar]
- Lu H, Hsu PI, Graham DY, Yamaoka Y. Duodenal ulcer promoting gene of Helicobacter pylori. Gastroenterology. 2005;128(4):833–848. doi: 10.1053/j.gastro.2005.01.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Queiroz DM, Rocha GA, Rocha AM, Moura SB, Saraiva IE, Gomes LI, Soares TF, Melo FF, Cabral MM, Oliveira CA. dupA polymorphisms and risk of Helicobacter pylori-associated diseases. Int J Med Microbiol. 2011;301(3):225–228. doi: 10.1016/j.ijmm.2010.08.019. [DOI] [PubMed] [Google Scholar]
- Scholte GH, van Doorn LJ, Quint WG, Lindeman J. Polymerase chain reaction for the detection of Helicobacter pylori in formaldehyde-sublimate fixed, paraffin-embedded gastric biopsies. Diagn Mol Pathol. 1997;6(4):238–243. doi: 10.1097/00019606-199708000-00008. [DOI] [PubMed] [Google Scholar]
- Atherton JC, Peek RM Jr, Tham KT, Cover TL, Blaser MJ. Clinical and pathological importance of heterogeneity in vacA, the vacuolating cytotoxin gene of Helicobacter pylori. Gastroenterology. 1997;112(1):92–99. doi: 10.1016/S0016-5085(97)70223-3. [DOI] [PubMed] [Google Scholar]
- van Doorn LJ, Figueiredo C, Rossau R, Jannes G, van Asbroek M, Sousa JC, Carneiro F, Quint WG. Typing of Helicobacter pylori vacA gene and detection of cagA gene by PCR and reverse hybridization. J Clin Microbiol. 1998;36(5):1271–1276. doi: 10.1128/jcm.36.5.1271-1276.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lobo Gatti L, Agostinho F Jr, De Labio R, Balbo Piason F, Carlos da Silva L, Fagundez de Queiroz V, Peres CA, Barbieri D, De Arruda Cardoso Smith M, Marques Payao SL. Helicobacter pylori and cagA and vacA gene status in children from Brazil with chronic gastritis. Clin Exp Med. 2003;3(3):166–172. doi: 10.1007/s10238-003-0021-0. [DOI] [PubMed] [Google Scholar]
- Fonseca TL, Moraes EP, Juliano CR, Silva AM, Scaini CJ, Mendoza-Sassi RA, Silva PE. Detection of Helicobacter pylori by phenotypic and genotypic methods. Dig Dis Sci. 2010;55:1643–1648. doi: 10.1007/s10620-009-0928-8. [DOI] [PubMed] [Google Scholar]
- Arachchi HS, Kalra V, Lal B, Bhatia V, Baba CS, Chakravarthy S, Rohatgi S, Sarma PM, Mishra V, Das B, Ahuja V. Prevalence of duodenal ulcer-promoting gene (dupA) of Helicobacter pylori in patients with duodenal ulcer in North Indian population. Helicobacter. 2007;12(6):591–597. doi: 10.1111/j.1523-5378.2007.00557.x. [DOI] [PubMed] [Google Scholar]
- Zhang Z, Zheng Q, Chen X, Xiao S, Liu W, Lu H. The Helicobacter pylori duodenal ulcer promoting gene, dupA in China. BMC Gastroenterol. 2008;8:49. doi: 10.1186/1471-230X-8-49. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Imagawa S, Ito M, Yoshihara M, Eguchi H, Tanaka S, Chayama K. Helicobacter pylori dupA and gastric acid secretion are negatively associated with gastric cancer development. J Med Microbiol. 2010;59(Pt 12):1484–1489. doi: 10.1099/jmm.0.021816-0. [DOI] [PubMed] [Google Scholar]
- Abadi AT, Taghvaei T, Wolfram L, Kusters JG. Infection with Helicobacter pylori strains lacking dupA is associated with an increased risk of gastric ulcer and gastric cancer development. J Med Microbiol. 2012;61(Pt 1):23–30. doi: 10.1099/jmm.0.027052-0. [DOI] [PubMed] [Google Scholar]
- Gatti LL, Modena JL, Payão SL, Smith M de A, Fukuhara Y, de Oliveira RB, Brocchi M. Prevalence of Helicobacter pylori cagA, iceA and babA2 alleles in Brazilian patients with upper gastrointestinal diseases. Acta Trop. 2006;100(3):232–240. doi: 10.1016/j.actatropica.2006.08.014. [DOI] [PubMed] [Google Scholar]
- Gonçalves MH, Silva CI, Braga-Neto MB, Fialho AB, Fialho AM, Queiroz DM, Braga LL. Helicobacter pylori virulence genes detected by string PCR in children from an urban community in northeastern Brazil. J Clin Microbiol. 2013;51(3):988–989. doi: 10.1128/JCM.02583-12. [DOI] [PMC free article] [PubMed] [Google Scholar]