Helicobacter ISSN 1523-5378 doi: 10.1111/hel.12161
REVIEW ARTICLE
Diagnosis of Helicobacter pylori Infection graud,*,† Emilie Besse de*,† and Philippe Lehours*,† Francis Me *Laboratoire de bacteriologie, Universite de Bordeaux, 33076, Bordeaux, France, †INSERM U853, 33076, Bordeaux, France
Keywords Endoscopy, histology, culture, molecular methods, urea breath test, serology. Reprint requests to: Francis Megraud, riologie, Universite de Laboratoire de Bacte Bordeaux, 146 rue Leo Saignat, 33076 Bordeaux Cedex, France. E-mail: francis.
Abstract The present manuscript focuses on the new information that was published in the field of diagnosis of Helicobacter pylori this past year. While there is little news about the invasive tests, more data are coming concerning the endoscopic features of H. pylori infection. Major efforts were also done to improve molecular detection of the mutations involved in antibiotic resistance. New antibodybased tests (stool antigen test or indirect antibody tests) were also developed.
[email protected]
Endoscopic Diagnosis There have been an increasing number of attempts to diagnose features of Helicobacter pylori infection during endoscopic examination. In 2 studies from Japan, conventional endoscopy was used. Kato et al. conducted a prospective study in 24 centers where indigo carmine contrast was used in 275 patients. This method was found very sensitive in both the corpus (94.3%) and the antrum (88.1%) while not specific (62.8 and 52.8%, respectively) in comparison with H. pylori diagnosis by histology and serology [1]. The second study by Watanabe et al. was not based on staining. They based their diagnosis on 11 specific endoscopic findings. The diagnostic yield was 88.9% for H. pylori eradicated patients. The importance of training was emphasized. Magnifying endoscopy (ME) may improve the diagnosis. The specificity of the patterns predicting H. pylori infection was significantly higher when i-scan (a newly developed image enhanced endoscopy system) was used (93.5%) compared to white light microscopy (80.6%), while sensitivity was the same [2]. Narrow band imaging (NBI) coupled with ME is now a commonly used technique. It was applied to detect intestinal metaplasia (IM). Savarino et al. evaluated semi-quantitatively the light blue crest appearance typical of IM in comparison with histological findings on 100 patients and obtained a sensitivity of 80% and a specificity of 96% [3]. The same technique was used for patients who received an eradication therapy. The surface maturation producing a “gastritis-like” appearance, even after endoscopic resection for early gastric
6
cancer (GC), may indicate a differentiated GC with low-grade atypia [4]. NBI-ME was also practical for prediction of H. pylori status after endoscopic resection for early GC with sensitivity of 79% and specificity of 52%, but with a substantial interobserver agreement [5]. A characteristic of gastric MALT lymphoma is “a tree-like” appearance of the mucosa. This finding completely disappeared after H. pylori eradication [6]. The need for proper training in NBI was also emphasized. A web-based video accessible through YouTube can be used. After 200 videos, sensitivity was good for IM but not for H. pylori gastritis [7].
Histology It has been a number of years since recommendations for histological assessment of H. pylori gastritis and other gastric mucosa changes have been published (Sydney system, OLGA, OLGIM). It is now time to evaluate how they are applied in routine practice. In the US, Lash & Genta reviewed a large number of biopsy sets (400,738) and found that 2 antral and 2 corpus biopsies in separate containers were available in only 3.9% of the cases. Compliance to the Sydney system led to significantly greater diagnostic yields than single-site sets (14.8 vs 6%), while incisura angularis samples yielded minimal additional diagnostic information [8]. Other authors from Canada also indicated that of 10,268 biopsies, only one region was sampled in 60% of the patients, mainly in the antrum (47%). Moreover, 47% of the patients were taking PPI at endoscopy
© 2014 John Wiley & Sons Ltd, Helicobacter 19 (Suppl. 1): 6–10
graud et al. Me
contributing to false negative results despite guidelines, for example those of the American Gastroenterology Association [9]. The Gastrointestinal Pathology Society in the US suggests that only hematoxylin and eosin staining is done as a first step and that the use of ancillary stains is appropriate only when biopsies show chronic gastritis without detectable H. pylori in hematoxylin and eosinstained sections [10]. In Europe, Leja et al. compared the interobserver variation of 2 expert pathologists and a general pathologist in the assessment of gastric premalignant lesions in 121 patients. The agreement was substantially higher for IM than for atrophy, both in the antrum and corpus. The level of agreement for the general pathologist was especially low for atrophy [11]. In China, it was shown that immunohistochemical detection of H. pylori in patients with GC is a factor of poor prognosis, with the survival rate being decreased by more than 9 months, that is, 25% [12].
Culture Bessa et al. tested H. pylori growth under different conditions assessing the sessile (free living) versus adherent (biofilm) modes of growth. Serum supplementation favoured free-living organisms while the synthetic medium, Ham’s F-12, alone led to a larger biomass with an important polysaccharide matrix, which is also increased by subinhibitory concentrations of antibiotics [13]. For H. pylori isolation by culture, instead of using the usual agar plates, Peretz et al. inoculated blood culture bottles. The biopsies were manually diced with a scalpel, placed in 6 ml of fetal bovine serum, and transferred in a bottle (BactecTM Plus Anaerobic/F Medium, Beckton Dickinson, Franklin Lakes, NJ, USA). All 25 biopsies positive by the rapid urease test, as well as one of the 15 negative biopsies, were detected positive (based on the amount of CO2 released) in a short incubation time (average 31.6 h, extremes 26–32 h). Nevertheless, gram-positive cocci also grew in 7 samples. This method is interesting to decrease the delay of positivity [14]. Seo et al. tested cryopreservation ( 70 °C) of H. pylori in gastric biopsies for more than 10 years with success [15].
Molecular Methods Formaldehyde-fixed paraffin-embedded gastric tissue can be a good material to detect H. pylori by PCR provided that the fixation time is not too long. To give insight to the controversial issue of whether H. pylori is present or not in gastric tissue of patients
© 2014 John Wiley & Sons Ltd, Helicobacter 19 (Suppl. 1): 6–10
Diagnosis of H. pylori Infection
with GC, a study using scorpion real-time PCR was carried out in Iran and found that 78.4% of the specimens were positive [16]. The real-time PCR described by Oleastro et al. [17] was also applied to such specimens to detect H. pylori. The 16% discordance between immunochemistry (122+) and PCR (103+) was explained essentially by a false positivity of the former due to cross reactivity. Two of the 24 negative samples were indeed PCR positive. Clarithromycin resistance was then detected by melting curve analysis in half of the positive specimens [18]. In all, 52 of 105 (50%) PCR-positive samples demonstrated resistance mutations, and it was determined that a heterogeneous population of mutated and non-mutated organisms was present in 21.15% of samples. Another possibility is to use a peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method to look for H. pylori and its resistance to clarithromycin. However, in a prospective study, its sensitivity was only 80% for a specificity of 93.8%. The direct visualization of the bacteria within the biopsy is a positive characteristic of this technique [19]. The detection of H. pylori DNA can be improved by laser capture microdissection which allows targeting of selected areas where bacteria are present [20]. The GenoTypeHelicoDR (Hain Lifescience GmbH, Nehren, Germany), a reverse hybridization assay, was also used successfully to detect H. pylori and the mutations associated with clarithromycin and levofloxacin resistance of H. pylori in South Africa [21]. Of DNA extracted from isolated H. pylori strains, 15.38% were resistant to clarithromycin, A2147G mutation being the most prevalent. For fluoroquinolone, 10.26% were resistant with mainly N87K QRDR gyrA mutation. When compared to the results of clarithromycin resistance by Etest in 42 strains, surprisingly, real-time PCR using the TaqMan format detected the 3 most common point mutations in only 23 cases (54.8%) in the study by De Francesco et al. They found novel point mutations in a further 14 of 19 discordant cases, postulating the putative emergence of new mutations [22]. Typing has different applications. Recently, LPS glycotyping of H. pylori was proposed. A significantly higher proportion of a-1,6 glucan was detected in clarithromycin resistant versus susceptible strains [23]. Among the more classical typing methods, multilocus sequence typing could be applied to H. pylori DNA extracted from fecal specimens and give insight to the mode of transmission in families [24]. Finally, comparative genomics of East Asian and non-Asian H. pylori strains identified divergent genes
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Diagnosis of H. pylori Infection
which, like vacA and cagA, are rapidly evolving under positive selection [25].
Urea Breath Test (UBT) Few studies were carried out on UBT this year. When comparing the 14C-UBT using encapsulated (which was previously recommended) versus non-encapsulated 14Curea, Pathak et al. favoured the latter. They presented dynamic scintiscan images showing a possible incomplete resolution of the capsule in the stomach. They showed a better sensitivity, 97.2% versus 91.8%, respectively, after 15 minutes in a series of 100 dyspeptic patients [26].
Stool Antigen Test (SAT) There are several SATs using either monoclonal or polyclonal antibodies and available as ELISAs on immunochromatographic tests (ICTs). Five of them were tested on 198 dyspeptic patients’ stool specimens in Turkey. The results are presented on Table 1. They show that the Premier Platinum HpSA Plus (Meridian Bioscience, Inc, Cincinnati, OH, USA) using monoclonal antibodies and an ELISA format is the only one providing >90% accuracy [27]. A new test, the Asan Easy Test H. pylori (Asan Pharma, Seoul, Korea) was also evaluated. It used monoclonal antibodies against the flagellin and provides a result within 15 minutes. Table 1 Performance of five stool antigen tests for the diagnosis of Helicobacter pylori
ELISA Premium Platinum HpSA Plus (Meridian Biosciences, Cincinnati, OH, USA) HP-Ag-EIA (Dia-Pro Diagnostic Bioprobes, Milan, Italy) ICT Immunocard STAT!HpSA (Meridian Biosciences) H. pylori fecal antigen test (Vegal Farmaceutica, Madrid, Spain) One-Step H. pylori Antigena (Acon Lab, San Diego, CA, USA)
Sen
Spe
PPV
NPV
ACC
92.2
94.4
93.3
93.6
93.4
48.9
88.9
78.6
67.6
71
68.9
92.6
88.6
78.1
81.8
78.9
87.0
83.5
83.2
83.3
86.7
88.9
86.7
88.9
88
Reference: Histology + Rapid Urease Test. ICT, immunochromatographic test; Sen, sensitivity; Spe, specificity; PPV, positive predictive value, NPV, negative predictive value, ACC, accuracy. a Polyclonal antibodies.
8
Its sensitivity was only 84.5% and its specificity was 96.2% when 266 patients were tested [28]. A nice review on the interest of the SAT for the management of H. pylori infection was published by Shimoyama [29]. Furthermore, H. pylori SAT (easy One-Step Test, Firstep Bioresearch, Taiwan) was added to the fecal occult blood tests used for colorectal cancer screening, in order to detect upper gastrointestinal (GI) lesions, mostly due to H. pylori, in a program in Taiwan. Of 31,721 participants, the prevalence of upper GI lesions was higher in those with a positive H. pylori SAT (34.6%) than in those with a positive guaiac-based test (24.7%) [30]. The same type of tests against H. pylori flagellin or urease was used to detect H. pylori in saliva in a Chinese study. The authors claim that saliva is a reservoir for H. pylori when these tests are positive despite a negative UBT. Indeed, the specificity of their test can be challenged given that the oral microbiome includes a number of species with potential cross-reacting antigens [31].
Antibody Tests A comparison of 29 commercial serological kits (17 ELISAs and 12 ICTs) was carried out in France. Depending on the type of analysis performed, 2 to 4 of the ELISAs presented an excellent performance. In contrast, only one of the 12 ICTs had an accuracy >90% [32]. A line assay using 6 recombinant proteins corresponding to virulence factors (CagA, VacA, GroEL, gGT, HcpC, and UreA) was developed in Germany. It was validated on a group of 600 patients (42% H. pylori positive by histology) and showed 97.6% sensitivity and 96.2% specificity, that is, an improvement on currently available serological tests [33]. The same group in collaboration with researchers in Iran was able to identify a H. pylori protein, FliD, essential in the assembly of the flagella. The recombinant FliD protein was tested on a group of 618 patients (51.4% H. pylori positive) with 97.4% sensitivity and 99% specificity using a line assay, and 97 and 96% by ELISA, respectively [34]. Other attempts to select antigenic proteins of potential diagnostic value were made (CafI, ureG, ureB), but have not been evaluated yet [35]. Interestingly, using Helicoblot 2.1 (Genelabs Diagnostics, Singapore), it was possible to identify a low molecular weight protein (35KDa) associated with a low risk of GC (OR = 0.4, 95% CI:0.1–0.9) and the VacA protein associated with a high risk of GC (OR = 2.7, 95 CI:1–7.1) among patients with GC (102) and dyspepsia (122) in Iran [36].
© 2014 John Wiley & Sons Ltd, Helicobacter 19 (Suppl. 1): 6–10
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A review on ICTs was also produced last year [37].
Non-Invasive Diagnosis of Premalignant Lesions Pepsinogen I and II and their ratio as predictors of atrophy were evaluated this year in Iran [38], Turkey [39], and Italy [40], but they were found to be insensitive predictors of these lesions. A toll-like receptor 4 was found helpful to differentiate between dysplasia and other precancerous lesions [39]. Both miR-106b and miR-21 were found as markers of increased risk for GC after H. pylori eradication [41].
Conclusion The progress in imaging techniques allows now to have a more accurate approach of the features associated to H. pylori infection. There are continuous attempts to improve the existing diagnostic methods or to evaluate their use in real life.
Acknowledgements and Disclosures Competing interest: The authors have no competing interests.
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© 2014 John Wiley & Sons Ltd, Helicobacter 19 (Suppl. 1): 6–10
Diagnosis of H. pylori Infection
7 Lash JG, Genta RM. Adherence to the Sydney System guidelines increases the detection of Helicobacter gastritis and intestinal metaplasia in 400738 sets of gastric biopsies. Aliment Pharmacol Ther 2013;38:424–31. 8 Dias-Silva D, Pimentel-Nunes P, Magalh~ aes J, Magalh~ aes R, Veloso N, Ferreira C, Figueiredo P, Moutinho P, Dinis-Ribeiro M. The learning curve for narrow-band imaging in the diagnosis of precancerous gastric lesions by using web-based video. Gastrointest Endosc 2014;79:910–20. 9 El-Zimaity H, Serra S, Szentgyorgyi E, Vajpeyi R, Samani A. Gastric biopsies: the gap between evidence-based medicine and daily practice in the management of gastric Helicobacter pylori infection. Can J Gastroenterol 2013;27:e25–30. 10 Batts KP, Ketover S, Kakar S, Krasinskas AM, Mitchell KA, Wilcox R, et al. Appropriate use of special stains for identifying Helicobacter pylori: recommendations from the Rodger C. Haggitt Gastrointestinal Pathology Society. Am J Surg Pathol 2013;37: e12–22. 11 Leja M, Funka K, Janciauskas D, Putnins V, Ruskule A, Kikuste I, et al. Interobserver variation in assessment of gastric premalignant lesions: higher agreement for intestinal metaplasia than for atrophy. Eur J Gastroenterol Hepatol 2013;25:694–9. 12 Li G, Wang Z, Wang Z, Xu J, Cui J, Cai S, et al. Gastric cancer patients with Helicobacter pylori infection have a poor prognosis. J Surg Oncol 2013;108:421–6. 13 Bessa LJ, Grande R, Di Iorio D, Di Giulio M, Di Campli E, Cellini L. Helicobacter pylori free-living and biofilm modes of growth: behavior in response to different culture media. Apmis 2013;121:549–60. 14 Peretz A, On A, Koiefman H, Brodsky D, Isakovich N, Glyatman T, et al. BACTEC FX system as a tool for culturing gastric biopsies and Helicobacter pylori diagnosis. Eur J Clin Microbiol Infect Dis 2013;32:1541–3. 15 Seo JH, Youn HS, Goo MJ, Jun JS, Lim JY, Park CH, et al. Recovery of Helicobacter pylori from gastric tissue cryopreserved for more than 10 years. Helicobacter 2013;18:167–8. 16 Naserpour Farivar T, Johari P, Najafipour R, Farzam A, Nasirian N, HajManouchehri F, et al. The relationship between gastric cancer and Helicobacter pylori in formaldehyde fixed paraffin embedded gastric tissues of gastric cancer patients-scorpion real-time PCR assay findings. Pathol Oncol Res 2014;20:113–7. 17 Oleastro M, Menard A, Santos A, Lamouliatte H, Monteiro L, Barthelemy P, et al. Real-time PCR assay for rapid and accurate detection of point mutations conferring resistance to clarithromycin in Helicobacter pylori. J Clin Microbiol 2003;41:397– 402. 18 Schmitt BH, Regner M, Mangold KA, Thomson RB Jr, Kaul KL. PCR detection of clarithromycin-susceptible and -resistant Helicobacter pylori from formalin-fixed, paraffin-embedded gastric biopsies. Mod Pathol 2013;26:1222–7. 19 Cerqueira L, Fernandes RM, Ferreira RM, Oleastro M, Carneiro F, Brandao C, et al. Validation of a fluorescence in situ hybridization method using peptide nucleic acid probes for detection of Helicobacter pylori clarithromycin resistance in gastric biopsy specimens. J Clin Microbiol 2013;51:1887–93. 20 Rabelo-Goncalves EM, Sgardioli IC, Lopes-Cendes I, Escanhoela CA, Almeida JR, Zeitune JM. Improved detection of Helicobacter pylori DNA in formalin-fixed paraffin-embedded (FFPE) tissue of patients with hepatocellular carcinoma using laser capture microdissection (LCM). Helicobacter 2013;18:244–5. 21 Tanih NF, Ndip RN. Molecular detection of antibiotic resistance in South African isolates of Helicobacter pylori. Gastroenterol Res Pract 2013;2013:259457.
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22 De Francesco V, Zullo A, Giorgio F, Saracino I, Zaccaro C, Hassan C, et al. Change of point mutations in Helicobacter pylori rRNA associated with clarithromycin resistance in Italy. J Med Microbiol 2014;63(Pt 3):453–7. 23 Altman E, Harrison BA, Chandan V, Slinger R. Lipopolysaccharide glycotyping of clarithromycin-resistant and clarithromycinsusceptible Canadian isolates of Helicobacter pylori. Can J Microbiol 2014;60:35–9. 24 Osaki T, Okuda M, Ueda J, Konno M, Yonezawa H, Hojo F, et al. Multilocus sequence typing of DNA from faecal specimens for the analysis of intra-familial transmission of Helicobacter pylori. J Med Microbiol 2013;62(Pt 5):761–5. 25 Duncan SS, Valk PL, McClain MS, Shaffer CL, Metcalf JA, Bordenstein SR, et al. Comparative genomic analysis of East Asian and non-Asian Helicobacter pylori strains identifies rapidly evolving genes. PLoS ONE 2013;8:e55120. 26 Pathak CM, Kaur B, Bhasin DK, Mittal BR, Sharma S, Khanduja KL, et al. Comparison of encapsulated versus nonencapsulated (14) C-urea breath test for the detection of Helicobacter pylori infection: a scintigraphy study. Helicobacter 2014;19:116–23. 27 Korkmaz H, Kesli R, Karabagli P, Terzi Y. Comparison of the diagnostic accuracy of five different stool antigen tests for the diagnosis of Helicobacter pylori infection. Helicobacter 2013;18:384–91. 28 Jekarl DW, An YJ, Lee S, Lee J, Kim Y, Park YJ, et al. Evaluation of a newly developed rapid stool antigen test using an immunochromatographic assay to detect Helicobacter pylori. Jpn J Infect Dis 2013;66:60–4. 29 Shimoyama T. Stool antigen tests for the management of Helicobacter pylori infection. World J Gastroenterol 2013;19:8188–91. 30 Lee YC, Chiu HM, Chiang TH, Yen AM, Chiu SY, Chen SL, et al. Accuracy of faecal occult blood test and Helicobacter pylori stool antigen test for detection of upper gastrointestinal lesions. BMJ Open 2013;3:e003989. 31 Yee KC, Wei MH, Yee HC, Everett KD, Yee HP, Hazeki-Talor N. A screening trial of Helicobacter pylori-specific antigen tests in saliva to identify an oral infection. Digestion 2013;87:163–9. 32 Burucoa C, Delchier JC, Courillon-Mallet A, de Korwin JD, Megraud F, Zerbib F, et al. Comparative evaluation of 29
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commercial Helicobacter pylori serological kits. Helicobacter 2013;18:169–79. Formichella L, Romberg L, Bolz C, Vieth M, Geppert M, Gottner G, et al. A novel line immunoassay based on recombinant virulence factors enables highly specific and sensitive serologic diagnosis of Helicobacter pylori infection. Clin Vaccine Immunol 2013;20:1703–10. Khalifeh Gholi M, Kalali B, Formichella L, Gottner G, Shamsipour F, Zarnani AH, et al. Helicobacter pylori FliD protein is a highly sensitive and specific marker for serologic diagnosis of H. pylori infection. Int J Med Microbiol 2013;303:618–23. Khalilpour A, Santhanam A, Wei LC, Saadatnia G, Velusamy N, Osman S, et al. Antigenic proteins of Helicobacter pylori of potential diagnostic value. Asian Pac J Cancer Prev 2013;14:1635–42. Karami N, Talebkhan Y, Saberi S, Esmaeili M, Oghalaie A, Abdirad A, et al. Seroreactivity to Helicobacter pylori antigens as a risk indicator of gastric cancer. Asian Pac J Cancer Prev 2013;14:1813–7. Karakus C, Salih BA. Comparison of the lateral flow immunoassays (LFIA) for the diagnosis of Helicobacter pylori infection. J Immunol Methods 2013;396:8–14. Hosseini M, Amoueian S, Attaranzadeh A, Montazer M, Soltani G, Asadollahi K, Abangah G. Serum gastrin 17, pepsinogen I and pepsinogen II in atrophic gastritis patients living in NorthEast of Iran. J Res Med Sci 2013;18:225–9. Yakut M, Ormeci N, Erdal H, Keskin O, Karayel Z, Tutkak H, et al. The association between precancerous gastric lesions and serum pepsinogens, serum gastrin, vascular endothelial growth factor, serum interleukin-1 Beta, serum toll-like receptor-4 levels and Helicobacter pylori Cag A status. Clin Res Hepatol Gastroenterol 2013;37:302–11. Masci E, Pellicano R, Mangiavillano B, Luigiano C, Stelitano L, Morace C, et al. GastroPanel(R) test for non-invasive diagnosis of atrophic gastritis in patients with dyspepsia. Minerva Gastroenterol Dietol 2014;60:79–83. Shiotani A, Murao T, Kimura Y, Matsumoto H, Kamada T, Kusunoki H, et al. Identification of serum miRNAs as novel non-invasive biomarkers for detection of high risk for early gastric cancer. Br J Cancer 2013;109:2323–30.
© 2014 John Wiley & Sons Ltd, Helicobacter 19 (Suppl. 1): 6–10
REVIEW ARTICLE
Diagnosis of Helicobacter pylori Infection graud,*,† Emilie Besse de*,† and Philippe Lehours*,† Francis Me *Laboratoire de bacteriologie, Universite de Bordeaux, 33076, Bordeaux, France, †INSERM U853, 33076, Bordeaux, France
Keywords Endoscopy, histology, culture, molecular methods, urea breath test, serology. Reprint requests to: Francis Megraud, riologie, Universite de Laboratoire de Bacte Bordeaux, 146 rue Leo Saignat, 33076 Bordeaux Cedex, France. E-mail: francis.
Abstract The present manuscript focuses on the new information that was published in the field of diagnosis of Helicobacter pylori this past year. While there is little news about the invasive tests, more data are coming concerning the endoscopic features of H. pylori infection. Major efforts were also done to improve molecular detection of the mutations involved in antibiotic resistance. New antibodybased tests (stool antigen test or indirect antibody tests) were also developed.
[email protected]
Endoscopic Diagnosis There have been an increasing number of attempts to diagnose features of Helicobacter pylori infection during endoscopic examination. In 2 studies from Japan, conventional endoscopy was used. Kato et al. conducted a prospective study in 24 centers where indigo carmine contrast was used in 275 patients. This method was found very sensitive in both the corpus (94.3%) and the antrum (88.1%) while not specific (62.8 and 52.8%, respectively) in comparison with H. pylori diagnosis by histology and serology [1]. The second study by Watanabe et al. was not based on staining. They based their diagnosis on 11 specific endoscopic findings. The diagnostic yield was 88.9% for H. pylori eradicated patients. The importance of training was emphasized. Magnifying endoscopy (ME) may improve the diagnosis. The specificity of the patterns predicting H. pylori infection was significantly higher when i-scan (a newly developed image enhanced endoscopy system) was used (93.5%) compared to white light microscopy (80.6%), while sensitivity was the same [2]. Narrow band imaging (NBI) coupled with ME is now a commonly used technique. It was applied to detect intestinal metaplasia (IM). Savarino et al. evaluated semi-quantitatively the light blue crest appearance typical of IM in comparison with histological findings on 100 patients and obtained a sensitivity of 80% and a specificity of 96% [3]. The same technique was used for patients who received an eradication therapy. The surface maturation producing a “gastritis-like” appearance, even after endoscopic resection for early gastric
6
cancer (GC), may indicate a differentiated GC with low-grade atypia [4]. NBI-ME was also practical for prediction of H. pylori status after endoscopic resection for early GC with sensitivity of 79% and specificity of 52%, but with a substantial interobserver agreement [5]. A characteristic of gastric MALT lymphoma is “a tree-like” appearance of the mucosa. This finding completely disappeared after H. pylori eradication [6]. The need for proper training in NBI was also emphasized. A web-based video accessible through YouTube can be used. After 200 videos, sensitivity was good for IM but not for H. pylori gastritis [7].
Histology It has been a number of years since recommendations for histological assessment of H. pylori gastritis and other gastric mucosa changes have been published (Sydney system, OLGA, OLGIM). It is now time to evaluate how they are applied in routine practice. In the US, Lash & Genta reviewed a large number of biopsy sets (400,738) and found that 2 antral and 2 corpus biopsies in separate containers were available in only 3.9% of the cases. Compliance to the Sydney system led to significantly greater diagnostic yields than single-site sets (14.8 vs 6%), while incisura angularis samples yielded minimal additional diagnostic information [8]. Other authors from Canada also indicated that of 10,268 biopsies, only one region was sampled in 60% of the patients, mainly in the antrum (47%). Moreover, 47% of the patients were taking PPI at endoscopy
© 2014 John Wiley & Sons Ltd, Helicobacter 19 (Suppl. 1): 6–10
graud et al. Me
contributing to false negative results despite guidelines, for example those of the American Gastroenterology Association [9]. The Gastrointestinal Pathology Society in the US suggests that only hematoxylin and eosin staining is done as a first step and that the use of ancillary stains is appropriate only when biopsies show chronic gastritis without detectable H. pylori in hematoxylin and eosinstained sections [10]. In Europe, Leja et al. compared the interobserver variation of 2 expert pathologists and a general pathologist in the assessment of gastric premalignant lesions in 121 patients. The agreement was substantially higher for IM than for atrophy, both in the antrum and corpus. The level of agreement for the general pathologist was especially low for atrophy [11]. In China, it was shown that immunohistochemical detection of H. pylori in patients with GC is a factor of poor prognosis, with the survival rate being decreased by more than 9 months, that is, 25% [12].
Culture Bessa et al. tested H. pylori growth under different conditions assessing the sessile (free living) versus adherent (biofilm) modes of growth. Serum supplementation favoured free-living organisms while the synthetic medium, Ham’s F-12, alone led to a larger biomass with an important polysaccharide matrix, which is also increased by subinhibitory concentrations of antibiotics [13]. For H. pylori isolation by culture, instead of using the usual agar plates, Peretz et al. inoculated blood culture bottles. The biopsies were manually diced with a scalpel, placed in 6 ml of fetal bovine serum, and transferred in a bottle (BactecTM Plus Anaerobic/F Medium, Beckton Dickinson, Franklin Lakes, NJ, USA). All 25 biopsies positive by the rapid urease test, as well as one of the 15 negative biopsies, were detected positive (based on the amount of CO2 released) in a short incubation time (average 31.6 h, extremes 26–32 h). Nevertheless, gram-positive cocci also grew in 7 samples. This method is interesting to decrease the delay of positivity [14]. Seo et al. tested cryopreservation ( 70 °C) of H. pylori in gastric biopsies for more than 10 years with success [15].
Molecular Methods Formaldehyde-fixed paraffin-embedded gastric tissue can be a good material to detect H. pylori by PCR provided that the fixation time is not too long. To give insight to the controversial issue of whether H. pylori is present or not in gastric tissue of patients
© 2014 John Wiley & Sons Ltd, Helicobacter 19 (Suppl. 1): 6–10
Diagnosis of H. pylori Infection
with GC, a study using scorpion real-time PCR was carried out in Iran and found that 78.4% of the specimens were positive [16]. The real-time PCR described by Oleastro et al. [17] was also applied to such specimens to detect H. pylori. The 16% discordance between immunochemistry (122+) and PCR (103+) was explained essentially by a false positivity of the former due to cross reactivity. Two of the 24 negative samples were indeed PCR positive. Clarithromycin resistance was then detected by melting curve analysis in half of the positive specimens [18]. In all, 52 of 105 (50%) PCR-positive samples demonstrated resistance mutations, and it was determined that a heterogeneous population of mutated and non-mutated organisms was present in 21.15% of samples. Another possibility is to use a peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method to look for H. pylori and its resistance to clarithromycin. However, in a prospective study, its sensitivity was only 80% for a specificity of 93.8%. The direct visualization of the bacteria within the biopsy is a positive characteristic of this technique [19]. The detection of H. pylori DNA can be improved by laser capture microdissection which allows targeting of selected areas where bacteria are present [20]. The GenoTypeHelicoDR (Hain Lifescience GmbH, Nehren, Germany), a reverse hybridization assay, was also used successfully to detect H. pylori and the mutations associated with clarithromycin and levofloxacin resistance of H. pylori in South Africa [21]. Of DNA extracted from isolated H. pylori strains, 15.38% were resistant to clarithromycin, A2147G mutation being the most prevalent. For fluoroquinolone, 10.26% were resistant with mainly N87K QRDR gyrA mutation. When compared to the results of clarithromycin resistance by Etest in 42 strains, surprisingly, real-time PCR using the TaqMan format detected the 3 most common point mutations in only 23 cases (54.8%) in the study by De Francesco et al. They found novel point mutations in a further 14 of 19 discordant cases, postulating the putative emergence of new mutations [22]. Typing has different applications. Recently, LPS glycotyping of H. pylori was proposed. A significantly higher proportion of a-1,6 glucan was detected in clarithromycin resistant versus susceptible strains [23]. Among the more classical typing methods, multilocus sequence typing could be applied to H. pylori DNA extracted from fecal specimens and give insight to the mode of transmission in families [24]. Finally, comparative genomics of East Asian and non-Asian H. pylori strains identified divergent genes
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which, like vacA and cagA, are rapidly evolving under positive selection [25].
Urea Breath Test (UBT) Few studies were carried out on UBT this year. When comparing the 14C-UBT using encapsulated (which was previously recommended) versus non-encapsulated 14Curea, Pathak et al. favoured the latter. They presented dynamic scintiscan images showing a possible incomplete resolution of the capsule in the stomach. They showed a better sensitivity, 97.2% versus 91.8%, respectively, after 15 minutes in a series of 100 dyspeptic patients [26].
Stool Antigen Test (SAT) There are several SATs using either monoclonal or polyclonal antibodies and available as ELISAs on immunochromatographic tests (ICTs). Five of them were tested on 198 dyspeptic patients’ stool specimens in Turkey. The results are presented on Table 1. They show that the Premier Platinum HpSA Plus (Meridian Bioscience, Inc, Cincinnati, OH, USA) using monoclonal antibodies and an ELISA format is the only one providing >90% accuracy [27]. A new test, the Asan Easy Test H. pylori (Asan Pharma, Seoul, Korea) was also evaluated. It used monoclonal antibodies against the flagellin and provides a result within 15 minutes. Table 1 Performance of five stool antigen tests for the diagnosis of Helicobacter pylori
ELISA Premium Platinum HpSA Plus (Meridian Biosciences, Cincinnati, OH, USA) HP-Ag-EIA (Dia-Pro Diagnostic Bioprobes, Milan, Italy) ICT Immunocard STAT!HpSA (Meridian Biosciences) H. pylori fecal antigen test (Vegal Farmaceutica, Madrid, Spain) One-Step H. pylori Antigena (Acon Lab, San Diego, CA, USA)
Sen
Spe
PPV
NPV
ACC
92.2
94.4
93.3
93.6
93.4
48.9
88.9
78.6
67.6
71
68.9
92.6
88.6
78.1
81.8
78.9
87.0
83.5
83.2
83.3
86.7
88.9
86.7
88.9
88
Reference: Histology + Rapid Urease Test. ICT, immunochromatographic test; Sen, sensitivity; Spe, specificity; PPV, positive predictive value, NPV, negative predictive value, ACC, accuracy. a Polyclonal antibodies.
8
Its sensitivity was only 84.5% and its specificity was 96.2% when 266 patients were tested [28]. A nice review on the interest of the SAT for the management of H. pylori infection was published by Shimoyama [29]. Furthermore, H. pylori SAT (easy One-Step Test, Firstep Bioresearch, Taiwan) was added to the fecal occult blood tests used for colorectal cancer screening, in order to detect upper gastrointestinal (GI) lesions, mostly due to H. pylori, in a program in Taiwan. Of 31,721 participants, the prevalence of upper GI lesions was higher in those with a positive H. pylori SAT (34.6%) than in those with a positive guaiac-based test (24.7%) [30]. The same type of tests against H. pylori flagellin or urease was used to detect H. pylori in saliva in a Chinese study. The authors claim that saliva is a reservoir for H. pylori when these tests are positive despite a negative UBT. Indeed, the specificity of their test can be challenged given that the oral microbiome includes a number of species with potential cross-reacting antigens [31].
Antibody Tests A comparison of 29 commercial serological kits (17 ELISAs and 12 ICTs) was carried out in France. Depending on the type of analysis performed, 2 to 4 of the ELISAs presented an excellent performance. In contrast, only one of the 12 ICTs had an accuracy >90% [32]. A line assay using 6 recombinant proteins corresponding to virulence factors (CagA, VacA, GroEL, gGT, HcpC, and UreA) was developed in Germany. It was validated on a group of 600 patients (42% H. pylori positive by histology) and showed 97.6% sensitivity and 96.2% specificity, that is, an improvement on currently available serological tests [33]. The same group in collaboration with researchers in Iran was able to identify a H. pylori protein, FliD, essential in the assembly of the flagella. The recombinant FliD protein was tested on a group of 618 patients (51.4% H. pylori positive) with 97.4% sensitivity and 99% specificity using a line assay, and 97 and 96% by ELISA, respectively [34]. Other attempts to select antigenic proteins of potential diagnostic value were made (CafI, ureG, ureB), but have not been evaluated yet [35]. Interestingly, using Helicoblot 2.1 (Genelabs Diagnostics, Singapore), it was possible to identify a low molecular weight protein (35KDa) associated with a low risk of GC (OR = 0.4, 95% CI:0.1–0.9) and the VacA protein associated with a high risk of GC (OR = 2.7, 95 CI:1–7.1) among patients with GC (102) and dyspepsia (122) in Iran [36].
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A review on ICTs was also produced last year [37].
Non-Invasive Diagnosis of Premalignant Lesions Pepsinogen I and II and their ratio as predictors of atrophy were evaluated this year in Iran [38], Turkey [39], and Italy [40], but they were found to be insensitive predictors of these lesions. A toll-like receptor 4 was found helpful to differentiate between dysplasia and other precancerous lesions [39]. Both miR-106b and miR-21 were found as markers of increased risk for GC after H. pylori eradication [41].
Conclusion The progress in imaging techniques allows now to have a more accurate approach of the features associated to H. pylori infection. There are continuous attempts to improve the existing diagnostic methods or to evaluate their use in real life.
Acknowledgements and Disclosures Competing interest: The authors have no competing interests.
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