Brazilian Journal of Microbiology (2012): 903-908

ISSN 1517-8382

EVALUATION OF DIAGNOSTIC METHODS FOR THE DETECTION OF HELICOBACTER PYLORI IN GASTRIC

BIOPSY SPECIMENS OF DYSPEPTIC PATIENTS

Ivy Bastos Ramis*, Ernani Pinho de Moraes, Márcia Silveira Fernandes, Raul Mendoza-Sassi, Obirajara Rodrigues,
Carlos Renan Varela Juliano, Carlos James Scaini, Pedro Eduardo Almeida da Silva

Laboratório de Biologia Molecular, Universidade Federal do Rio Grande, Rio Grande, Rio Grande do Sul, Brasil.

Submitted: February 08, 2011; Approved: June 07, 2012.

ABSTRACT

Helicobacter pylori infects nearly 50% of the world’s population. This microorganism is accepted as the
most important agent of gastritis and as a risk factor for peptic ulcer disease and gastric adenocarcinoma.
Currently many diagnostic methods exist for detecting H. pylori, however they all have limitations, thus it is
recommend a combination of at least two methods. The aim of this study was to evaluate diagnostic
methods, such as in-house urease test, culture and Polymerase Chain Reaction (PCR), for the detection of
the H. pylori in gastric biopsy specimens of 144 dyspeptic patients, using as gold standard the association
between histology and rapid urease test. According to the gold standard used in this study, 48 (33.3%)
patients were infected with H. pylori, while 96 (66.7%) were classified as not infected. The in-house urease
test and the PCR were the most sensitive methods (100%), followed by culture (85.4%). However, the in-
house urease test and the culture were the most specific (100%), followed by PCR (75%). In conclusion, this
study showed that, in comparison with the combination of histology and rapid urease test, the in-house
urease test and the PCR presented 100% of sensitivity in the diagnosis of gastric infection by H. pylori,
while the in-house urease test and the culture reached 100% of specificity. These finding suggest that the
combination of two or more methods may improve the accuracy of the H. pylori detection.

Key words: Helicobacter pylori, histology, culture, urease test, Polymerase Chain Reaction.

INTRODUCTION This association between H. pylori and gastroduodenal

diseases demonstrates the need to diagnose the presence of
Helicobacter pylori is a Gram-negative, spiral and bacteria in dyspeptic patients. Traditionally, diagnostic
microaerophilic bacterium that is present in the human stomach methods for detecting H. pylori may be classified as invasive,
of approximately 50% of the world´s population (1, 8). This which require endoscopy to obtain biopsies of gastric tissues,
microorganism plays a significant role in the pathogenesis of and non-invasive. The invasive methods include histology,
chronic gastritis, peptic ulcer disease and gastric culture, urease test and molecular methods, while the non-
adenocarcinoma (1). invasive methods include urea breath testing, serology, stool

*Corresponding Author. Mailing address: Laboratório de Biologia Molecular, Universidade Federal do Rio Grande, Rua General Osório S/N, CEP: 96200-400,
Rio Grande, Rio Grande do Sul, Brazil.; Tel.: + 55 53 32330314 Fax.: + 55 53 32338863.; E-mail: ivynha_@hotmail.com

903
Ramis, I.B. et al. Detection of H. pylori in gastric biopsy

antigen testing and molecular methods (12). aimed to evaluate diagnostic methods, including an in-house
Although there are several available diagnostic methods urease test, culture and Polymerase Chain Reaction (PCR). The
for detecting infection with H. pylori, there is no single results were compared to the gold standard (histology and rapid
technique that can meet, on its own, the criteria for acceptable urease test) for H. pylori detection. This combination employed
sensitivity and specificity in identification of the bacterium. as a gold standard is the most frequently used in studies (10,
Therefore, diagnostic methods are recommended in a 16, 19).
combination of two or more to meet diagnostic criteria (14, 19,
25). MATERIALS AND METHODS
Histology is considered the standard method for the
diagnosis of H. pylori infection, providing additional and Patients and Samples
essential information on the status of the mucosa such as the A transversal study was performed with 144 dyspeptic
presence of acute or chronic inflammation, lymphoid patients submitted to upper gastrointestinal endoscopy between
aggregates, intestinal metaplasia and glandular atrophy. The October 2008 and March 2009 in the Integrated Center for
reliability of this method depends essentially on the number Gastroenterology at the Hospital of the Federal University of
and localization of specimens collected (12). Rio Grande, Rio Grande do Sul, Brazil. The study excluded
Culture permits testing of the sensitivity of H. pylori to those patients who presented recent use (within 15 days) of
antimicrobial agents used for its eradication and detection of antibiotics or non-steroidal anti-inflammatory drugs (NSAIDs),
factors and mechanisms related to pathogenicity of the those treated for H. pylori infection and those with
bacterium (14, 20). gastrointestinal bleeding in the last seven days. This study was
The urease test is simple and provides quick results (11); approved by the Research Ethics Committee (FURG —
however, this method is affected by the use of antimicrobial process number 23116.003335/2008-43). Informed consent
agents, proton pump inhibitors (PPIs) and bismuth-containing was obtained from all patients.
compounds, which inhibit urease activity (7). Furthermore, the Through endoscopy, 11 biopsy specimens were obtained
presence of other microorganisms that produce urease can lead from each patient. Of these, five were destined for histology
to false-positive results (19). (two from the gastric antrum, two from the gastric body and
Finally, molecular methods are widely used for the one from the angular incisure). The other six biopsy specimens
diagnosis of H. pylori infection as well as for analyses of were intended for the rapid urease test (one specimen from the
diversity, virulence and resistance patterns of these bacteria (4). angular incisure), the in-house urease test (one specimen from
However, the high degree of genomic plasticity between strains the angular incisure), culture (two specimens from the gastric
of H. pylori complicates the choice of target genes. Even antrum) and PCR (two specimens from the gastric antrum) (6,
nucleotide sequences that are highly conserved in different 13, 15, 26).
strains of the pathogen, such as urease A (ureA), urease C
(ureC) and 16S rRNA, may fail to detect the bacteria (19, 23). Histology
Each of the methods above has advantages and The biopsy specimens from each patient that were
disadvantages, and none can be considered as a single gold destined for histological examination, were fixed in formalin
standard. A combination of endoscopic biopsy-based methods and stained with Hematoxylin-Eosin (H&E) and Giemsa. The
usually gives the most reliable diagnosis (9). Thus, this study classification of gastritis was established in accordance with

904
Ramis, I.B. et al. Detection of H. pylori in gastric biopsy

the Sydney System (21). incisure was incubated immediately after collection in 1 mL of
urea broth (Isofar, BR). This broth was prepared according to
Culture the manufacturer’s instructions and stored at 4 °C until the time
After collection, biopsy specimens intended for culture of use. The test was considered positive when, after 24 hours of
and obtained from the gastric antrum were kept in 1 mL Brain incubation at 25 °C, there was a change in the color of the
®
Heart Infusion (BHI; Acumedia , USA) Broth with 20% broth from yellow/orange to pink/purple.
glycerol and refrigerated (4 to 8 °C) for a maximum of 4 hours
(19). Extraction of DNA
Subsequently, this broth was vortexed vigorously for 2 The DNA was extracted from biopsy specimens from the
min. Two hundred microliters were added to selective medium gastric antrum using DNAzol® Reagent (InvitrogenTM, USA)
Columbia Agar (Oxoid, UK) and supplemented with 7% sheep and 10 μg/μL of Proteinase K (Promega, USA). The biopsies
blood (Newprov, BR) and with a selective mixture for isolating were separated from the broth and resuspended in 100 μL
Helicobacter sp. (Cefar, BR) containing: 6 µg/mL vancomycin, Proteinase K and 500 μL DNAzol® Reagent. The mixture was
20 µg/mL nalidixic acid and 2 µg/mL amphotericin B. The incubated at 55 °C for 3 hours, and after this period, another
medium was incubated at 37°C in an anaerobic jar (JA 0400; 500 μL of DNAzol® Reagent were added. After centrifugation
Permution, BR) under microaerophilic conditions (5 to 15% O2 at 14,000× g for 10 min, the supernatant was collected, and 500
and 10% CO2), using an atmospheric generator (Microaerobac; μL cold absolute ethanol was added followed by centrifugation
Probac, BR), for a period of 4 to 10 days. Bacterial growth was at 12,000× g for 10 min, after which the supernatant was
monitored every 48 hours (7, 19). discarded. The DNA pellet was washed two times with 800 μL
When growth in the culture medium was observed, 75% ethanol, air dried and resuspended in 50 μL of 8 mM
microscopy was performed. When straight or curved Gram- NaOH. The DNA was stored at – 20 °C until used.
negative bacilli were observed, the following tests for DNA extraction from the culture was performed after 48
identification of H. pylori were performed: catalase, oxidase, hours of bacterial growth. The colonies were collected and
and urease. Identification was also carried out by PCR resuspended in 500 μL of 1× TE buffer (10 mM Tris–HCl, 1
amplification of a ureA fragment (7, 15). mM EDTA — pH 8.0). The suspension was centrifuged at
10,000× g for 5 min, and the supernatant was discarded. The
Rapid Urease Test total DNA from the clinical isolates was then extracted
From each patient, a biopsy specimen from the angular according to the protocol for extraction by DNAzol® Reagent
incisure was incubated immediately after collection in Pre- and preserved in 50 μL of 8 mM NaOH at – 20 °C.
Made Broth (TUPF; Laborclin, BR) for the urease test. This PCR
test detects the presence of H. pylori urease. The test was In this study, ureA was used for the detection of H. pylori.
considered positive when the color of the solution changed The total DNA of the clinical isolates and the biopsy specimens
from yellow/orange to pink/purple within 2 hours of incubation was amplified with the primers UREA1 (5´ - GCCAATGGTA
at 25 °C. AATTAGTT – 3´) and UREA2 (5´ - CTCCTTAATTGTTTTT
AC – 3´) (Invitrogen®, USA). These primers amplify a
In-House Urease Test fragment of 394 bp of ureA (5). PCR was performed as
From each patient, a biopsy specimen from the angular described by Rota et al. (22).

905
Ramis, I.B. et al. Detection of H. pylori in gastric biopsy

Statistical analysis (33.3%) patients were infected with H. pylori, while 96

A previous description of the sample was performed, (66.7%) were classified as not infected. It should be
calculating means and standard deviations for continuous data emphasized, however, that the gold standard chosen may have
and proportions for categorical data. For validation purposes, introduced errors in patient classification as infected or not
the numbers of positive and negative results for each test were infected. Among patients that were H. pylori-positive, 47,9%
calculated with their respective proportions. Sensitivity (Se), (23/48) had endoscopic diagnosis of erosive gastritis, and
specificity (Sp), positive predictive value (PPV), negative 52,1% (25/48) had enanthematous gastritis.
predictive value (NPV), and 95% confidence intervals (95%
CI) were calculated for each of the testing methods, using as Comparative study among different diagnostic methods
the gold standard a positive result in both histology and rapid According to the results of the methods of histology, rapid
urease test, as previously stated. Analyses were performed in urease test, in-house urease test, PCR and culture, the presence
Stata 9.2. of H. pylori, respectively, were identified in 75.7% (109/144),
33.3% (48/144), 33.3% (48/144) 50% (72/144) and 28.5%
RESULTS (41/144) of patients.
Considering the combination of histology and rapid urease
Determination of infection with H. pylori test as a gold standard, the in-house urease test and PCR were
Of the 144 patients undergoing upper gastrointestinal the most sensitive methods (100%), followed by culture
endoscopy, 99 were female, and 45 were male. Their ages were (85.4%). The in-house urease test and culture were the most
between 14 and 80 years (mean age 46.5, SD 15.5 years). specific (100%), followed by PCR (75%).
Based on the gold standard used in this study (correlation The PPV, NPV and diagnostic accuracy of each method
between the results of histology and rapid urease test), 48 are noted in Table 1.

Table 1. Validation of in-house urease test, culture and ureA PCR in comparison with the combination of histology and rapid
urease test
Combination of Histology Urease Culture ureA PCR
and Rapid Urease Test Pos. Neg. Pos. Neg. Pos. Neg.
Pos. 48 0 41 0 48 24
Neg. 0 96 7 96 0 72
% 95% CI % 95% CI % 95% CI
Se. 100.00 98.96 - 100.00 85.42 74.39 - 96.44 100.00 98.96 - 100.00
Sp. 100.00 99.48 - 100.00 100.00 99.48 - 100.00 75.00 65.82 - 84.18
PPV 100.00 98.96 - 100.00 100.00 98.78 - 100.00 66.67 55.08 - 78.25
NPV 100.00 99.48 - 100.00 93.20 87.86 - 98.55 100.00 99.31 - 100.00
Se.: sensitivity; Sp.: specificity; PPV: positive predictive value; NPV: negative predictive value; 95% CI: 95% confidence intervals.

DISCUSSION least two methods based on different principles be used to

detect colonization by H. pylori (15). In this study, the results
Currently many diagnostic methods exist for detecting H. of the in-house urease test, culture and PCR were compared to
pylori infection. Each method has its own merits and the gold standard (histology and rapid urease test) for
drawbacks in terms of indication, sensitivity, specificity and bacterium detection.
cost (18). Thus, it is recommended that a combination of at The gold standard used in this study is frequently used by

906
Ramis, I.B. et al. Detection of H. pylori in gastric biopsy

authors because the rapid urease test allows rapid detection of less sensitive than for ureC, a 26-kDa species-specific antigen
H. pylori, and histology enables identification of bacteria and gene, and 16S rRNA gene detection.
evaluation of the type and intensity of inflammation of the The false-positive results found by PCR may have
gastric mucosa (10, 16, 24). However, it is known that in the occurred because of sample contamination by PCR products or
rapid urease test, false-negative results may occur because of inadequate disinfection of the endoscopes (13). The advantage
irregular distribution of bacteria in the gastric mucosa or the of the PCR is that it allows detection of specific genes relevant
use of antimicrobials or PPIs. On the other hand, the to pathogenesis and specific mutations associated with
contamination of biopsy with saliva can cause false-positive antimicrobial resistance in addition to detection of the
results because bacteria from the oral flora can produce urease microorganism (19).
(7, 8, 19). It was previously shown histologically that the The culture showed sensitivity and specificity of 85.4%
presence of structures similar to H. pylori could cause false- and 100%, respectively. These values are similar to those
positive results (19). Thus, the gold standard chosen may have found by other authors, who reported high specificity and
not been the most appropriate even though it is the most significant fluctuation in sensitivity. The false-negative results
commonly used by authors. of this method may have occurred due to the absence or low
In this work, the choice of collection site of the biopsies in density of bacterium in the biopsy specimens, use of
the stomach for histology, rapid urease test, in-house urease antimicrobials and PPIs, inappropriate conditions of transport
test, PCR and culture was based on previous studies (6, 13, 15, or loss of viability of the microorganism due oxygen exposure
26). However, the irregular distribution of the H. pylori in the (12, 13, 19).
gastric mucosa could influence on the results obtained. However, the main advantage of culture is that, in
The in-house urease test reached 100% sensitivity and addition to detecting H. pylori, it allows the testing of antibiotic
specificity in the diagnosis of gastric infection by H. pylori. sensitivity, which can be very useful in some patients who are
This method has been widely used because it is inexpensive not responding to treatment (2).
and easy to perform (19); however, does not provide In conclusion, this study showed that, in comparison with
information on the intensity of inflammation (7). the combination of histology and rapid urease test, the in-house
The urease enzyme, produced by H. pylori, seems to be urease test and the PCR presented 100% of sensitivity in the
necessary for the survival of this microorganism in the acidic diagnosis of gastric infection by H. pylori, while the in-house
gastric environment, suggesting a strong selective pressure to urease test and the culture reached 100% of specificity. These
maintain the amino acid sequence of this enzyme, resulting in finding suggest that the combination of two or more methods
the observed conservations of the DNA sequence among may improve the accuracy of the H. pylori detection.
strains (5). The ureA is a species-specific gene present in all
samples of H. pylori (3). REFERENCES
In this study, PCR was performed by ureA detection. This
method presented sensitivity of 100% and specificity of 75%. 1. Blaser, M.J.; Parsonnet, J. (1994). Parasitism by the "slow" bacterium
Helicobacter pylori leads to altered gastric homeostasis and neoplasia. J.
A result similar to this was reported by Smith et al. (24). In
Clin. Invest. 94 (1), 4–8.
contrast, Lu et al. (17) compared PCR methods for the 2. Brooks, H.J.L.; Ahmed, D.; McConnell, M.A.; Barbezat, G.O. (2004).
detection of H. pylori in gastric biopsy specimens and observed Diagnosis of Helicobacter pylori infection by polymerase chain reaction:
that the amplification of ureA, although highly specific, was is it worth it? Diagn. Microbiol. Infect. Dis. 50 (1), 1-5.

907
Ramis, I.B. et al. Detection of H. pylori in gastric biopsy

3. César, A.C.G.; Cury, P.M.; Payão, S.L.M.; Liberatore, P.R.; Silva, A.E. 16. Lage, A.P.; Godfroid, E.; Fauconnier, A.; Burette, A.; Butzler, J.P.;
(2005). Comparison of histological and molecular diagnosis of Bollen, A.; Glupczynski, Y. (1995). Diagnosis of Helicobacter pylori
Helicobacter pylori in benign lesions and gastric adenocarcinoma. Braz. infection by PCR: comparison with other invasive techniques and
J. Microbiol. 36 (1), 12-16. detection of cagA gene in gastric biopsy specimens. J. Clin. Microbiol.
4. Cirak, M.Y.; Akyön, Y.; Mégraud, F. (2007). Diagnosis of Helicobacter 33 (10), 2752-2756.
pylori. Helicobacter. 12 (1), 4–9. 17. Lu, J-J.; Perng, C-L.; Shyu, R-Y.; Chen, C-H.; Lou, Q.; Chong, S.K.F.;
5. Clayton, C.L.; Kleanthous, H.; Coates, P.J.; Morgan, D.D.; Tabaqchali, Lee, C-H. (1999). Comparison of five PCR methods for detection of
S. (1992). Sensitive detection of Helicobacter pylori by using Helicobacter pylori DNA in gastric tissues. J. Clin. Microbiol. 37 (3),
polymerase chain reaction. J. Clin. Microbiol. 30 (1), 192–200. 772-774.
6. Coelho, L.G.V.; Zaterka, S., Representantes Indicados pela Federação 18. Malik, G.M.; Mubarik, M.; Kadla, S.A. (1999). Helicobacter pylori
Brasileira de Gastroenterologia; Núcleo Brasileiro para o Estudo do Infection in Endoscopic Biopsy Specimens of Gastric Antrum:
Helicobacter. (2005). II Consenso Brasileiro sobre Helicobacter pylori. Laboratory Diagnosis and Comparative Efficacy of Three Diagnostic
Arq. Gastroenterol. 42 (2), 128-132. Tests. Diagn. Ther. Endosc. 6 (1), 25-29.
7. Datta, S.; Chattopadhyay, S.; Chowdhury, A.; Santra, A.; Saha, D.R.; 19. Mégraud, F.; Lehours, P. (2007). Helicobacter pylori detection and
Ramamurthy, T.; Bhattacharya, S.K.; Berg, D.E.; Nair, G.B.; antimicrobial susceptibility testing. Clin. Microbiol. Rev. 20 (2), 280–
Mukhopadhyay, A.K. (2005). Diagnosis and genotyping of Helicobacter 322.
pylori by polymerase chain reaction of bacterial DNA from gastric juice. 20. Ndip, R.N.; Mackay, W.G.; Farthing, M.J.G.; Weaver, L.T. (2003).
J. Gastroenterol. Hepatol. 20 (8), 1253-1259. Culturing Helicobacter pylori from clinical specimens: review of
8. Dunn, B.E.; Cohen, H.; Blaser, M.J. (1997). Helicobacter pylori. Clin. microbiological methods. J. Pediatr. Gastroenterol. Nutr. 36 (5), 616–
Microbiol. Rev. 10 (4), 720-741. 622.
9. Dzieranowska-Fangrat, K.; Lehours, P.; Mégraud, F.; Dzierzanowska, D. 21. Price, A.B. (1991). The Sidney system: histological division. J.
(2006). Diagnosis of Helicobacter pylori Infection. Helicobacter. 11 (1), Gastroenterol. Hepatol. 6 (3), 209-222.
6–13. 22. Rota, C.A.; Pereira-Lima, J.C.; Blaya, C.; Nardi, N.B. (2001). Consensus
10. Fabre, R.; Sobhani, I.; Laurent-Puig, P.; Hedef, N.; Yazigi, N.; and variable region PCR analysis of Helicobacter pylori 3’ region of
Vissuzaine, C.; Rodde, I.; Potet, F.; Mignon, M.; Etienne, J.P.; Braquet, cagA gene in isolates from individuals with or without peptic ulcer. J.
M. (1994). Polymerase chain reaction assay for the detection of Clin. Microbiol. 39 (2), 606-612.
Helicobacter pylori in gastric biopsy specimens: comparison with 23. Singh, V.; Mishra, S.; Rao, G.R.K.; Jain, A.K.; Dixit, V.K.; Gulati, A.K.;
culture, rapid urease test and histopathological tests. Gut. 35 (7), 905- Mahajan, D.; McClelland, M.; Nath, G. (2008). Evaluation of Nested
908. PCR in detection of Helicobacter pylori targeting a highly conserved
11. Granstrom, M.; Lehours, P.; Bengtsson, C.; Mégraud, F. (2008). gene: HSP60. Helicobacter. 13 (1), 30–34.
Diagnosis of Helicobacter pylori infection. Helicobacter. 13 (1), 7–12. 24. Smith, S.I.; Oyedeji, K.S.; Arigbabu, A.O.; Cantet, F.; Megraud, F.; Ojo,
12. Hirschl, A.M.; Makristathis, A. (2007). Methods to detect Helicobacter O.O.; Uwaifo, A.O.; Otegbayo, J.A.; Ola, S.O.; Coker, A.O. (2004).
pylori: From Culture to Molecular Biology. Helicobacter. 12 (2), 6-11. Comparison of three PCR methods for detection of Helicobacter pylori
13. Kisa, O.; Albay, A.; Mas, M.R.; Celasun, B.; Doganci, L. (2002). The DNA and detection of cagA gene in gastric biopsy specimens. World J.
evaluation of diagnostic methods for the detection of Helicobacter pylori Gastroenterol. 10 (13), 1958–1960.
in gastric biopsy specimens. Diagn. Microbiol. Infect. Dis. 43 (4), 251- 25. Thijs, J.C.; Van Zwet, A.A.; Thijs, W.J.; Oey, H.B.; Karrenbeld, A.;
255. Stellaard, F.; Luijt, D.S.; Meyer, B.C.; Kleibeuker, J.H. (1996).
14. Krogfelt, K.A.; Lehours, P.; Mégraud, F. (2005). Diagnosis of Diagnostic tests for Helicobacter pylori: a prospective evaluation of their
Helicobacter pylori infection. Helicobacter. 10 (1), 5–13. accuracy, without selecting a single test as the gold standard. Am. J.
15. Kullavanijaya, P.; Thong-Ngam, D.; Hanvivatvong, O.; Nunthapisud, P.; Gastroenterol. 91 (10), 2125–2129.
Tangkijvanich, P.; Suwanagool, P. (2004). Analysis of eight different 26. Woo, J.S.; El-Zimaity, H.M.T.; Genta, R.M.; Yousfi, M.M.; Graham,
methods for the detection of Helicobacter pylori infection in patients D.Y. (1996). The Best Gastric Site for Obtaining a Positive Rapid Urease
with dyspepsia. J. Gastroenterol. Hepatol. 19 (12), 1392-1396. Test. Helicobacter. 1 (4), 256-259.

All the content of the journal, except where otherwise noted, is licensed under a Creative Commons License

908