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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
 ~ Acknowledgement
 ~  References
 ~  Article Figures
 ~  Article Tables

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  Table of Contents  
ORIGINAL ARTICLE
Year : 2015  |  Volume : 33  |  Issue : 4  |  Page : 560-564
 

Clinical significance of various diagnostic techniques and emerging antimicrobial resistance pattern of Helicobacter Pylori from Gastric Biopsy Samples


1 Department of Microbiology, Karnatak Lingayat Education University’s, Jawaharlal Nehru Medical College, Belgavi, Karnataka, India
2 Department of Pathology, Karnatak Lingayat Education University’s, Jawaharlal Nehru Medical College, Belgavi, Karnataka, India

Date of Submission11-Dec-2014
Date of Acceptance08-Apr-2015
Date of Web Publication16-Oct-2015

Correspondence Address:
M A Vagarali
Department of Microbiology, Karnatak Lingayat Education University’s, Jawaharlal Nehru Medical College, Belgavi, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.167349

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 ~ Abstract 

Background: There is no single technique that can meet the criteria in identification of Helicobacter pylori. The diagnosis is important asantimicrobial resistance is frequently observed and associated with treatment failure. The present study was conducted to evaluate diagnostic tests for identification of H pylori and to assess their antimicrobial resistance pattern.Materials and Methods: Biopsies of gastric tissue from 200 patients with disorders of the upper gastrointestinal tract were studied for detection of H pylori by various methods like culture, H and E staining and urease test. Antimicrobial susceptibility testing was carried out by Kirby Bauer's disc diffusion method. Results: Out of 200 patients, H pylori was detected by rapid urease test, H and E staining and culture in 26.5%, 14.5% and 2.5% cases respectively. H and E was taken as the gold standard. Sensitivity of urease test was 76.6% and of culture 13.3%. Specificity of urease was 81.7% in comparison with culture which showed 99.4% specificity. Metronidazole (05) showed high level of resistance followed by amoxicillin (03) and norfloxacillin (03). Tetracycline, erythromycin, levofloxacin and cotrimoxazole showed one resistance each to H pylori. Conclusion: H and E is taken as the gold standard according to CDC. Urease test is a better screening procedure than culture. H pylori resistance to metronidazole in our zone was highest. This is due to general and extensive use of metronidazole for other infectious diseases. Our study suggests need for a systematic approach to determine antibiogram of the strains before considering the drug regimens.


Keywords: Gastric biopsy, H and E staining, H. pylori, metronidazole, urease test


How to cite this article:
Vagarali M A, Metgud S C, Bannur H, Karadesai S G, Nagmoti J M. Clinical significance of various diagnostic techniques and emerging antimicrobial resistance pattern of Helicobacter Pylori from Gastric Biopsy Samples. Indian J Med Microbiol 2015;33:560-4

How to cite this URL:
Vagarali M A, Metgud S C, Bannur H, Karadesai S G, Nagmoti J M. Clinical significance of various diagnostic techniques and emerging antimicrobial resistance pattern of Helicobacter Pylori from Gastric Biopsy Samples. Indian J Med Microbiol [serial online] 2015 [cited 2018 Nov 17];33:560-4. Available from: http://www.ijmm.org/text.asp?2015/33/4/560/167349



 ~ Introduction Top


Helicobacter pylori (H. pylori) is commonly associated with chronic gastritis in more than 50% of the people worldwide. In developing countries, 70–90% of the population carries H pylori.[1],[2] Many methods, invasive and non-invasive have been developed for the diagnosis of H pylori infection.[3] Invasive methods requiring endoscopic evaluation include bacteriologic culture, histopathologic studies, smear examination like Grams and giemsa stain, rapid urease test and molecular studies. Non-invasive methods include serologic testing, urea breath testing, antigen detection in the stool.[4] In spite of available diagnostic methods to detect H pylori, there is no single technique that can meet the criteria for acceptable sensitivity and specificity in identification of the bacterium.[1] Therefore various diagnostic methods are recommended in a combination of two or more to meet diagnostic criteria.[5],[6]

Although culture is needed for antimicrobial susceptibility, it is difficult to isolate, requires an enriched transport medium, is expensive and time consuming.[4] Histopathology is considered as the standard method for the diagnosis of H pylori infection, but the reliability depends on the number and site of specimens collected.[7] A further limitation of uses of histopathology with regard to sensitivity and specificity is the quality of the biopsies. If the biopsy is too small, poorly oriented or inappropriately fixed or stained, detection of H pylori may not be possible.[4]

The specific diagnosis is important because clinical experience has demonstrated that H pylor i infection is not easy to cure.[8] (Triple therapy combining a proton pump inhibitor with two antibiotics like clarithromycin, metronidazole or amoxicillin, represents the regimen for eradication. Treatment failure occurs due to the resistant strains particularly metronidazole.[8] The resistance of H pylori to the antimicrobials is a growing problem. In developed countries, metronidazole resistance is found in 10–50% of adult H pylori infected patients. Whereas in developing countries, almost all strains are resistant to the antimicrobial agent.[9] Antibiotic resistance frequently causes failure of eradication of H pylori.[9]

Therapeutic regimens followed currently are mostly based on either insufficient data or obtained from other geographically un-related region. In the Indian population, the prevalence of H pylori resistance is very high to metronidazole (77.9%) followed by clarithromycin (44.7%), amoxicillin (32.8%) and ciprofloxacin showed least resistance (1–4%).[10] This study was done to evaluate the antimicrobial resistance pattern in this region.

Each of the above methods has advantages and disadvantages and none can be considered as a single gold standard. A combination of endoscopic biopsy based methods usually gives the most reliable diagnosis.[11] Thus this study is aimed to evaluate diagnostic methods like urease test, culture and Hematoxyline and Eosin (H and E) staining and to assess the antimicrobial resistance pattern.


 ~ Materials and Methods Top


Two hundred patients having disorders of the upper gastrointestinal tract were studied by video-endoscopy. Patients who had taken antibiotics, H2 blockers or proton pump inhibitors 24 hrs prior to endoscopy were excluded. Patients with dyspepsia but a normal endoscopic appearance were classified as non-ulcer dyspepsia. Others who had oedema or redness of the gastric mucosa which was histologically confirmed as gastritis were included in the chronic gastritis group. Informed consent and ethical clearance from the Institutional Ethical Committee were obtained.

Two biopsy specimens of gastric tissue from each patient were collected by gastroenterologist during endoscopy. One biopsy was transported to the microbiology laboratory within half to one hour in brain heart infusion broth (BHI) with glycerol. They were submitted for rapid urease test and culture. The other biopsy for histopathology examination was transported in 10% buffered formalin, routinely processed and embedded in paraffin. The slides were stained with routine H and E stain. The H pylori were identified as curved rods on the luminal surface of the gastric epithelial cells [Figure 1].
Figure 1: H and E Stain showing curved rods on the luminal surface of the gastric epithelial cells

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Culture of H pylori from biopsy sample: The biopsy specimen was inoculated onto Columbia blood agar plates supplemented with 10% sheep blood, Campylobacter growth supplement and Campylobacter selective supplement, incubated in CO2 jar with multiple wax candles lighted to create the micro-aerophilic atmosphere. While closing the jar each time, petroleum jelly was put on the side of the rim of the jar and then the jar was closed tightly. The jar was kept at 37°C for 5–7 days.[12] The bacterial colonies were identified on the basis of colony morphology, Grams staining, positive oxidase, catalase and urease reactions. Bacteria were subcultured into two different media columbia blood agar with supplements and brain heart infusion broth with 20% glycerol.

Rapid urease test

This was done using a urea broth prepared and standardised in our set up where the concentration of urea was increased to double the amount to increase the sensitivity of the test.

Antimicrobial susceptibility testing of the isolates

A total of 05 isolates of H pylori subcultured were tested for antibiotic susceptibility using Kirby-Bauer disc diffusion method on Muller-Hinton agar plate supplemented with 10% sheep blood. A standard inoculum of H pylori culture was suspended in BHI broth. The turbidity was adjusted equal to McFarland 3. The inoculum was seeded on to Mueller-Hinton blood agar plate using sterile cotton wool swab, antibiotic discs with the following drug contents: Metronidazole (5 µg), amoxicillin (10 µg), tetracycline (30 µg), erythromycin (15 µg), levofloxacin (5 µg), norfloxacin (5 µg), cotrimoxazole (10 µg) were placed on the plates. The plates were incubated at 37°C in CO2 jar for 3–4 days. The results were interpreted as per Clinical Laboratory Standards Institute (CLSI) 2011 guidelines.[13]


 ~ Results Top


A total of 200 patients [152 males and 48 females; 3.12:1] with various upper gastrointestinal disorders were included in the study [Table 1]. H pylori was detected by rapid urease test in 54 (27%) cases where urease was positive in 46 (24.21%) cases with chronic gastritis and 8 (80%) cases with peptic ulcer, by histopathology slides stained with H and E in 29 (14.5%) cases where 23 (12.11%) cases had chronic gastritis and 06 (60%) cases had peptic ulcer and culture in 5 (2.5%) cases where 01 (0.53%) case had chronic gastritis and 04 (40%) had peptic ulcer as shown in [Table 2]. H and E was taken as the gold standard. Sensitivity of urease test was 76.6% and of culture 13.3%. Specificity of urease was 81.7% in comparison with culture which showed 99.4% specificity [Table 3].
Table 1: Sex distribution with endoscopic diagnosis

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Table 2: Detection of Helicobacter pylori in gastric biopsy samples by different methods in various conditions (N = 200)

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Table 3: Sensitivity and specificity of different methods for identification of Helicobacter pylori

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Out of five isolates subjected to antimicrobial susceptibility testing, metronidazole (5) showed high level of resistance followed by amoxicillin (3) and norfloxacillin (3). Tetracycline, erythromycin, levofloxacin and cotrimoxazole showed one resistance each to H pylori. A total of three strains were multi-drug resistant (MDR). They showed resistance to metronidazole, amoxicillin and norfloxacillin [Table 4].
Table 4: Antimicrobial resistance pattern of H pylori (N = 05)

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Five metronidazole resistant strains were found in the patients with duodenal ulcer, gastric ulcer and chronic gastritis. Three of the strains were resistant to amoxicillin and Norfloxacin in patients with duodenal ulcer.


 ~ Discussion Top


In our study multiple tests were used for the detection of H pylori in gastric biopsy specimens. The presence of H pylori in gastric biopsies was detected by urease test, H and E stain and culture in 26.5%, 14.5% and 2.5% cases respectively. The overall positivity of rapid urease test correlates with another study, where out of 81 samples studied by rapid urease test 35 (43.21%) were positive.[14] In another study done by Akanda et al., rapid urease test and H and E staining detected H. pylori in 56.4% and 45.6% cases respectively.[4] The specificity of urease test is 81.7%. In another study the specificity of RUT was 60%.[15] In the present study, the sensitivity and specificity of urease test in the demonstration of H pylori is higher than culture. It is cheap, easy to perform and available in most laboratories.

In the present study, H. pylori was isolated in five out of 200 patients (2.5%) with a sensitivity of 13.3% [Table 2]. Similar isolation rate has been reported in another study (4.2%).[15] Studies from India have shown low rates of isolation. Ayyagari et al., reported 23.9% isolation rate.[16] In another study 8/92 (8.7%) samples were positive by culture with a sensitivity of 8.69%.[17] In contrast, the Indian studies reported a sensitivity which ranged from 1.09–63%.[16],[18] The sensitivity of our culture is in accordance with these results. The low rate of isolation may be due to the fastidious nature of H pylori and a number of factors that are hard to control, such as patchy distribution of the organism on the gastric mucosa, loss of viability of the organisms during transportation etc. All these factors together, result in low sensitivity and a low negative predictive value.[18] Thus the need persists for a high H pylori recovery rate from gastric biopsy specimens.

The presence of H. pylori in gastric biopsy was detected by H and E in 14.5%. In another study out of 65 samples processed for H and E, a total of 56 (86.15%) showed the changes associated with chronic gastritis.[17] CDC recommends that histopathology [H and E] should be taken as gold standard. In comparison with H and E, urease test is better screening test when compared to culture.

Out of total 27% cases detection by urease test, 24.21% were chronic gastritis. So it can be used as one of the rapid test and can be used as bed side investigation. Gastritis can be having varied aetiology when urease test is used it will pinpoint at the diagnosis of H pylori infection since Urease test has got 76.6% sensitivity. Thus urease test can be used for early diagnosis of dyspepsia. Culture has high specificity, which can be used as adjunct.

Patients were treated with H pylori kit containing Pantoprazole 40 mg, clarithromycin 500 mg and amoxicillin 750 mg for 14 days then symptomatic improvement was assessed followed by maintenance dose of Pantoprazole 40 mg for 3 months. For gastric ulcer cases Pantoprazole 40 mg was put for 3 weeks then followed by antibiotic treatment after the symptomatic improvement.

Symptomatic profile of the patients before and after the treatment was assessed and there was endoscopic improvement after the treatment. Metronidazole resistant cases were put on quadruple treatment with proton pump inhibitor and three antibiotics.

Many H pylori strains show resistance to one or more antimicrobial agents in vitro and this may be the cause of eradication failure.[19] Noncompliance of the patient and location of the bacterium which is beneath the gastric epithelium, are involved in the treatment failure. Antibiotic resistance varies widely by geographic location. Metronidazole is an important antimicrobial used in the treatment of H pylori infection.[20] Determination of antibiotic susceptibility, particularly to metronidazole is very essential.[21] In our study, antibiotic susceptibility testing was carried out by disc diffusion method. It is a good alternative for determining antibiotic susceptibility of H pylori, particularly to metronidazole.[21],[22]

In our study all the strains [05] were resistant to metronidazole. In another study H pylori resistant rate was 77.9% to metronidazole, 32.8% to amoxicillin.[10] Metronidazole resistance was high in Lucknow, Chennai, Hyderabad (68%, 88.2% and 100% respectively) and moderate in Delhi (37.5%) and Chandigarh (38.2%), ciprofloxacin and tetracycline resistance was the least ranging from 1.0–4%.[10] This is in accordance with our study.

The prevalence rate of metronidazole resistance is higher in developing countries.[8] This is due to general and extensive use of metronidazole in developing countries for other infectious problems, such as protozoal, genital and dental infections.[8],[16] In our study, metronidazole has highest resistance rate. primary resistance of H pylori to metronidazole is consistent with earlier research conducted in several areas.[9]

There is lot of geographical variation in resistance pattern. indiscriminate use of drugs in different areas might be the cause for high metronidazole resistant H pylori.[23]

We found three of the strains resistant to amoxicillin. Frequent use of this drug for other infections like respiratory conditions in our area may contribute to resistance.

In our zone, we got emerging resistance with norfloxacin (03). The reason for high resistance with norfloxacin is due to its widespread usage in the treatment of urinary tract infections.[23]

Although norfloxacin is not a drug of choice in the treatment of H pylori infection, drug combination used in the H pylori kit may be considered as an alternative in cases of resistance to first line drugs.[23] one resistance was seen with other drugs like tetracycline, levofloxacin, cotrimoxazole and erythromycin. The wide discrepancy in the antibiotic susceptibility pattern of H pylori, suggest the need for a systematic approach to determine antibiogram of the strains before considering the drug regimens.

Limitations of the study

The present study had certain limitations. The results of this study depicted low culture positives in controlled conditions existing. Further long term studies need to be conducted comparing the different cultivation methods and molecular techniques.


 ~ Acknowledgement Top


We gratefully thank Dr Santosh Hazare, Professor of gastroenterology for providing endoscopic gastric biopsy samples to carry out the study.

 
 ~ References Top

1.
Ramis IB, de Moraes EP, Fernandes MS, Mendoza-Sassi R, Rodrigues O, Juliano CR, et al. Evaluation of diagnostic methods for the detection of Helicobacter pylori in gastric biopsy specimens of dyspeptic patients. Braz J Microbiol 2012;43:903-8.  Back to cited text no. 1
    
2.
Nimri LF, Matalka I, Bani Hani K, Ibrahim M. Helicobacter pylori genotypes identified in gastric biopsy specimens from Jordanian patients. BMC Gastroenterol 2006;6:27.  Back to cited text no. 2
    
3.
Entzeng J, Lin YL, Chung SM, Chu YT. Comparison of four diagnostic methods for Helicobacter pylori. Tzu Chi Med J 2005;17:339-43.  Back to cited text no. 3
    
4.
Akanda MR, Rahman AN. Comparative study of different methods for detection of Helicobacter pylori in gastric biopsies. Dinajpur Med Col J 2011;4:1-6.  Back to cited text no. 4
    
5.
Krogfelt KA, Lehours P, Megraud F. Diagnosis of Helicobacter pylori infection. Helicobacter 2005;10:5-13.  Back to cited text no. 5
    
6.
Megraud F, Lehours P. Helicobacter pylori detection and antimicrobial susceptibility testing. Clin Microbiol Rev 2007;20:280-322.  Back to cited text no. 6
    
7.
Hirschl AM, Makristathis A. Methods to detect Helicobacter pylori: From culture to molecular biology. Helicobacter 2007;12:6-11.  Back to cited text no. 7
    
8.
Khashei R, Shojaei H, Adibi P, Shavakhi A, Aslani MM, Naser AD. Genetic diversity and drug resistance of Helicobacter pylori strains in Isfahan, Iran. Iranian J Basic Med Sci 2008;11:174-82.  Back to cited text no. 8
    
9.
Pandya HB, Agravat HH, Patel JS, Sodagar NR. Emerging antimicrobial resistance pattern of Helicobacter pylori in central Gujarat. Indian J Med Microbiol 2014;32:408-13.  Back to cited text no. 9
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10.
Thyagarajan SP, Ray P, Das BK, Ayyagari A, Khan AA, Dharmalingam S, et al. Geographical difference in antimicrobial resistance pattern of Helicobacter pylori clinical isolates from Indian patients: Multicentric study. J Gastroenterol Hepatol 2003;18:1373-8.  Back to cited text no. 10
    
11.
Dzieranowska-Fangrat K, Lehours P, Megraud F, Dzierzanowska D. Diagnosis of Helicobacter pylori infection. Helicobacter 2006;11:6-13.  Back to cited text no. 11
    
12.
Salim SM, Mandal J, Parija SC. Isolation of Campylobacter from human stool samples. Indian J Med Microbiol 2014;32:35-8.  Back to cited text no. 12
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13.
Clinical Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Twenty First Informational Supplement. CLSI document M100-S21. Wayne: CLSI, 2011.  Back to cited text no. 13
    
14.
Subbukesavaraja V, Balan K. Comparative study of invasive methods for diagnosis of Helicobacter pylori in humans. Int J Curr Microbiol App Sci 2013;2:63-8.  Back to cited text no. 14
    
15.
Sharma M, Mehta P, Vohra P. Comparative evaluation of different diagnostic techniques available for diagnosis of Helicobacter pylori. Int J Sci Res Publ 2012;2:1-5.  Back to cited text no. 15
    
16.
Ayyagari A, Ray P, Kochhar R, Bhasin D, Siddishi ER, Singh K, et al. Evaluation of different methods for detection of Helicobacter pylori in patients with gastric disease. Indian J Med Res 1990;91:126-8.  Back to cited text no. 16
    
17.
Kaore NM, Nagdeo NV, Thombare VR. Comparative evaluation of the diagnostic tests for Helicobacter pylori and dietary influence for its acquisition in dyspeptic patients: A rural hospital based study in central India. J Clin Diagn Res 2012;6:636-41.  Back to cited text no. 17
    
18.
Akbar DH, Eltahawy AT. Helicobacter pylori infection at a university hospital in Saudi Arabia: Prevalence and comparison of the diagnostic modalities and the endoscopic findings. Indian J Pathol Microbial 2005;48:181-5.  Back to cited text no. 18
    
19.
Alarcon T, Domingo D, Lopez-Brea M. Antibiotic resistance problems with Helicobacter pylori. Int J Antimicrob Agents 1999;12:19-26.  Back to cited text no. 19
    
20.
Osato MS, Reddy R, Reddy SG, Penland RI, Graham DY. Comparison of the Etest and the NCCLS-approved agar dilution method to detect metronidazole and clarithromycin resistant Helicobacter pylori. Int J Antimicrob Agents 2001;17:39-44.  Back to cited text no. 20
    
21.
Chaves S, Gadanho M, Tenreiro R, Cabrita J. Assessment of metronidazole susceptibility in Helicobacter pylori: Statistical validation and error rate analysis of breakpoints determined by the disk diffusion test. J Clin Microbiol 1999;37:1628-31.  Back to cited text no. 21
    
22.
Xia H, Keane CT, Beattie S, Omorain CA. Standardisation of disk diffusion test and its clinical significance for susceptibility testing of metronidazole against Helicobacter pylori. Antimicrob Agents Chemother 1994;38:2357-61.  Back to cited text no. 22
    
23.
Loivukene K, Maaroos HI, Kolk H, Kull I, Labotkin K, Mikelsaar M. Prevalence of antibiotic resistance of Helicobacter pyloriisolates in Estonia during 1995-2000 in comparison to the consumption of antibiotics used in treatment regimens. Clin Microbiol Infect 2002;8:598-603.  Back to cited text no. 23
    


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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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