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ORIGINAL ARTICLE
Year : 2014  |  Volume : 28  |  Issue : 1  |  Page : 9-13

Study of Helicobacter pylori infection and its antibiogram in gastroduodenal disorders in Regional Institute of Medical Sciences Hospital


1 Department of Microbiology, Regional Institute of Medical Sciences, Imphal, Manipur, India
2 Department of Surgery, Regional Institute of Medical Sciences, Imphal, Manipur, India

Date of Web Publication24-Jun-2014

Correspondence Address:
Prof. Gulamjat S Moirangthem
Head, Department of Surgery and Surgical Gastro-enterology & Minimal Access Surgery Unit, Regional Institute of Medical Sciences, Imphal - 795 004, Manipur
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-4958.135217

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  Abstract 

Objective: Isolation of Helicobacter pylori from biopsy samples taken from cases of gastroduodenal disorder, correlate isolation of H. pylori with urease test, direct modified Grams-stain and H. pylori antibody test, to assess antimicrobial susceptibility pattern of H. pylori isolates. Materials and Methods: Sixty patients with gastroduodenal disorders were included in the study. Urease test, direct modified Grams-stain, H. pylori antibody test, culture and antimicrobial susceptibility were performed as per standard guidelines. Statistical analysis was performed using Fisher's exact test. Results: Antral gastritis was the most common endoscopic findings accounting 43.3%. The study revealed direct Gram-stain positivity of 20%, urease test positivity 46.7%, H. pylori antibody test positivity of 65% and culture positivity rate of 3.3%. Correlation of isolation of H. pylori with Gram-stain found to be significant statistically (P < 0.037). The correlation of isolation with urease test and H. pylori antibody and H. pylori antibody test was statistically not significant. Antimicrobial susceptibility pattern of H. pylori isolates, showed high sensitivity to gentamycin, amoxiclav, tetracycline, clarithromycin, and ciprofloxacin. Higher resistance to metronidazole was observed. Conclusion: Urease test, direct smear, H. pylori antibody test in combination proved to be helpful in the diagnosis of H. pylori infection. Culture from gastric mucosa in infected subjects is low; this could be due to patchy distribution of organisms, prior administration of proton pump inhibitors and antibiotics. Antimicrobial susceptibility testing can be carried out in cases of refractory to treatment.

Keywords: Antibiogram, Gastroduodenal disorders, Helicobacter pylori


How to cite this article:
Chyne WW, Moirangthem GS, Gunjiganvi M, Devi KS. Study of Helicobacter pylori infection and its antibiogram in gastroduodenal disorders in Regional Institute of Medical Sciences Hospital. J Med Soc 2014;28:9-13

How to cite this URL:
Chyne WW, Moirangthem GS, Gunjiganvi M, Devi KS. Study of Helicobacter pylori infection and its antibiogram in gastroduodenal disorders in Regional Institute of Medical Sciences Hospital. J Med Soc [serial online] 2014 [cited 2020 Oct 30];28:9-13. Available from: https://www.jmedsoc.org/text.asp?2014/28/1/9/135217


  Introduction Top


Helicobacter pylori a Gram-negative bacteria, has been proved to be of overwhelming importance in the etiology of a number of common gastroduodenal diseases, such as chronic gastritis, peptic ulceration, and gastric cancer. [1] The prevalence of H. pylori infection ranges from 25% in developed countries to >90% in developing countries. H. pylori has been classified by the International Agency Research in Cancer as a Group I carcinogen and a definite cause of cancer in humans. [2] Several methods have been used diagnose H. pylori infection. Direct methods are dependent on endoscopic gastric biopsy specimen and include rapid urease test (RUT), smear microscopy, culture isolation, histopathology examination and molecular diagnosis by polymerase chain reaction. The indirect methods include urea breath test, serology for antigen and antibody detection. [3] H. pylori eradication regime includes proton pump inhibitor (PPI) and antibiotics such as metronidazole, amoxicillin, and clarithromycin. However, antibiotic resistance results in an increased failure rate of therapies. [4] Susceptibility testing of H. pylori has become increasingly important for the search of efficient antimicrobial combination that allows for eradication of this bacterium from the stomach. [5] This study proposes to determine the rate of H. pylori infection in cases of gastroduodenal disorders, to assess the susceptibility of H. pylori isolates in patients of gastroduodenal diseases and correlation of isolation H. pylori with direct modified Gram-stain, urease test, H. pylori antibody test.


  Materials and Methods Top


A cross-sectional study was carried out in the Department of Microbiology, in collaboration with Surgical Gastroenterology and Minimal Access Surgery Unit, Department of Surgery. The duration of this study was for a period of 2 years from September 2008.

The study was carried out only after obtaining approval from Institutional Ethical Committee.

The study population was 60 patients with gastroduodenal diseases, irrespective of sex, religion, and socioeconomic condition who were determined at the surgery outpatient department (OPD) to have an indication for an endoscopy or those patients who were referred to surgical gastroenterology and minimal access surgery unit for endoscopy. Patients excluded from the study were those <18 years of age, those who refused to undergo upper gastrointestinal (GI) endoscopy and those who had a history of intake PPI, H 2 blockers, and antimicrobial drug intake within 1 week prior to this study.

Patients were enrolled in the study only after taking informed consent.

Three pieces of gastric tissues from the antrum was taken by using upper GI endoscope. The gastric tissue specimen was collected in a sterile vial containing normal saline. Specimens were within 1 h to the microbiology laboratory for isolation, diagnosis, and antimicrobial susceptibility testing.

Biopsy specimen were placed in sterile Petri dish and minced with sterile scalpel blades.

A part of the specimen was inoculated in brain heart infusion agar with 7% defibrinated sheep blood and skirrows supplement (trimethoprim 5 mg/L, vancomycin 10 mg/L, polymyxin B 2500 units/L) (HiMedia). [6] Plate were incubated at 37°C for 7-10 days in microaerophilic environment (5% oxygen, 10-15% carbon dioxide, 80-85% nitrogen) in a humid environment.

To another part of the specimen it was processed for urease test, the biopsy specimen was placed in Christensen's urea broth and was incubated at 37°C. Urease positive media changes the color from orange-yellow to pink, the change in color in minutes or hours was noted accordingly and to another part of the specimen a direct modified Grams-stain was performed on a heat fixed smear. In a modified Gram-stain, the counter stain safranin was replaced with carbol fuchsin.

The growth of H. pylori was confirmed by doing biochemical standard test (Mackie and McCartney). [7]

Antimicrobial Susceptibility Test

The antimicrobial susceptibility of H. pylori isolates was determined by disc diffusion method (Kirby-Bauer method). [8] The antibiotic discs that were tested included metronidazole, clarithromycin, amoxicillin, co-amoxiclav, tetracycline, furazolidone, gentamycin, and ciprofloxacin.

Helicobacter pylori antibody test was also performed in 20 patients by SD Bioline test device. The H. pylori antibody test (SD Bioline) is a rapid test for the qualitative detection of antibodies of isotypes (immunoglobulin G [IgG], IgM, and IgA). In the test procedure, 10 μL of serum/plasma or 20 μL of whole blood is added into the sample well, and three drops of the assay diluent is added. The result was interpreted at 10 min.

Above all data were recorded on a predesigned proforma. The findings were statistically analyzed using Fisher's test.


  Results Top


A total of 60 gastroduodenal disorders patients were taken up in the study. The ages of the patients are 18 years and above. Males constitute 68.3% and 31.7% were females, male:female is 2.1:1. The number of patients that have been treated with antiulcer drugs in the past accounts to 76.7%. Antral gastritis constitutes 43.4% of the endoscopic findings [Table 1].
Table 1: Distribution of endoscopic findings

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The laboratory findings of urease test, direct modified Gram-stain, culture and H. pylori test results are shown in [Table 2].
Table 2: Laboratory diagnosis

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Correlation/comparison of culture with direct modified Gram-strain, urease test and H. pylori antibody test are shown on [Table 3], [Table 4] and [Table 5], respectively.
Table 3: Correlation/comparison of culture with direct Gram-stain (n = 60)

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Table 4: Correlation/comparison of culture with urease test (n = 60)

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Table 5: Correlation/comparison of culture with H. pylori antibody test (n = 20)

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  Discussion Top


This study analyzed a cross-section of consecutive patients who underwent upper GI endoscopy. The age of the patients in this study was from 18 years and above, the percentage of age distribution was seen maximum in the age group of 48-57 years (28.3%) followed by 38-47 years (21.7%). Alazmi et al. [9] have reported the percentage of H. pylori infection in Kuwait and non-Kuwait patients was seen maximum in the age group 30-49 years (59.3%).

In this study, percentage of distribution among males (68.3%) was higher than females (31.7%) accounting for male:female of 2.1:1. Similar result was reported by Young et al. [10] that the percentage of distribution was higher in males (62.1%) as compared to females (37.9%) with male:female 1.6:1

Antral gastritis was the most common endoscopic findings in this study constituting 26/60 (43.3%), followed by duodenal ulcer and gastric ulcer. Similar results were reported by Yakoob et al. [11] in which the most common endoscopic findings in their study was gastritis. However, Nair et al. [12] in their study stated that duodenal ulcer constitute the maximum endoscopic findings 74/136 (54.4%).

In this study, 23.3% (14/60) of the patients give a history of not taking antibiotics, H 2 blocker or PPIs, while 76.7% (46/60) used either antibiotics, H 2 blocker and PPI (>1 week back or months or years) before presentation to the OPD or Surgical Gastroenterology a Minimal Access Surgery Unit. Yakoob et al. [11] in their study found that 52% (57/109) were not on any medication, while 48% (52/109) used PPI before presentation to the OPD.

Several diagnostic tests have been used to document the presence of H. pylori in gastric mucosa. In this study, H. pylori was identified with the use of direct Gram-stain, culture and urease test. H. pylori antibody test was also used to document the presence of H. pylori. Antimicrobial susceptibility testing was also done on strains, which could be isolated from culture.

In this study, direct Gram-stain was positive in 20% (12/60). Similar findings have been reported by Nichols et al. [13] in which Gram-stain was positive in 20.6%. However, Oyedeji et al. [14] and Vijaya and Chadrashekhar [15] in their study found a higher percentage of positive results by Gram-stain that is 61.4% and 68%, respectively. While Berry and Sagar [16] showed a percentage of 8.28% positive Gram-stain (microscopy).

The RUT is the most frequently used test for the diagnosis H. pylori infection in routine GI endoscopy practice. In this study, urease test is positive in 28/60 (46.7%), which was consistent with the study of Yakoob et al. [11] which gives a result of 40% (by pontodry RUT). However, Vijaya and Chadrashekhar [15] give a higher percentage of urease test positive (74/100) accounting to 74%. While Nichols et al. [13] give a low percentage of 18.5% positive results by urease test, Berry and Sagar [16] in their study found only 10.9% positive by urease test.

Yakoob et al. [11] stated that previous or past treatment with PPI before endoscopy reduced the positivity of urease test from antral biopsies for H. pylori detection. In patients who had already taken previous treatment with PPI, biopsy specimen may contain low bacterial density of viable cells giving a negative urease test.

In this study, culture is positive in 3.3%, whereas Malik et al. [17] and Oyedeji et al. [14] have reported in 22.5% and 19.3% respectively.

Malik et al. [17] stated that the low isolation of the organisms have been attributed to patchy distribution of organisms and use of metronidazole by patients for protozoal infestations whose incidence is quite high in India.

Gupta et al. [18] stated that failure to culture is because of the fastidious nature of the organism and in some cases due to overgrowth of organisms like Proteus, especially in the presence of hypochlorhydria. Failure of culture may also result from sampling error of the specimen or delay in plating the material, other factors include swallowed local anesthetics, simethicone, prior treatment with bismuth, antibiotics or H 2 receptor antagonist and contamination of biopsy forceps with disinfectants. Though culture is highly specific it generally produces the highest number of false-negative results.

In this study, antimicrobial susceptibility test, which was done on the two strains (3.3%) isolated by culture, showed a high sensitivity to amoxicillin, gentamycin, amoxiclav, tetracycline, clarithromycin, ciprofloxacin, and high resistance to metronidazole. One isolate showed resistance to furazolidone. As the number of isolate is low, we could not conclude the antibiotic resistance pattern of H. pylori. Dharmalingam et al. [4] in their study reported a 100% sensitivity to tetracycline, 99.1% sensitive to amoxicillin and ciprofloxacin, 89.1% sensitive to clarithromycin, while 89.1% of the isolates showed resistance to metronidazole.

Sharma et al. [19] in their study found a 66% resistance to metronidazole, their study stated that metronidazole being cheaper ad readily available is used for self-medication and prescribed very commonly for GI disorders like amoebiasis. The high frequency of resistance to metronidazole may be due to repeated exposure of H. pylori to this drug.

In this study, antibody test was carried out in 20 cases, out of which 13 were positive accounting to 65%.

Rahman et al. [20] in their study found a positivity rate of 71.9% by H. pylori rapid test immunochromatography.

Serology is technically simple, causes very little discomfort to the patients, an inexpensive means of confirming H. pylori exposure in patients where endoscopy is not indicated and useful in population screening for epidemiological research. [15]

In this study, the correlation of culture with direct modified Gram-stain showed that the proportion of direct modified Gram-stain test positivity was higher among those, which are culture positive and the difference was statistically significant (P < 0.037).

While the correlation of culture with urease test and H. pylori antibody test showed that the proportion of urease test and H. pylori antibody test positivity was higher among those which are culture positive; however, the finding is not statistically significant. This is because of low diagnostic yield by culture. In this study, the low isolation of the organism by culture is attributed due to the previous treatment with PPI, H 2 blocker and antibiotics. Besides, many patients were treated for other infections other than H. pylori infection with antibiotics such as amoxiclav, gentamicin, tetracycline, furazolidone, and metronidazole. The patchy distribution of the organism can reduce the diagnostic yield from infected patients.

The correlation of direct modified Gram-stain and urease test showed that GNB curved bacilli were detected more among the cases, which were urease positive as compared to those which are urease negative and the difference was statistically significant (P < 0.000).


  Conclusion Top


Urease test, direct smear (modified Gram-stain) and H. pylori antibody test in combination, proved to be helpful in establishing the diagnosis of H. pylori infection. H. pylori culture from gastric mucosa in infected subjects was low, the low isolation of the organism by culture was attributed due to the previous treatment with PPI, H 2 blocker and antibiotics, patchy distribution of the organisms in gastric mucosa and also because of the fastidious nature of the organism. Though culture is highly specific it generally produces the highest number of false negative results. In this study, antimicrobial susceptibility test revealed high sensitivity to amoxicillin, gentamicin, amoxiclav, tetracycline, clarithromycin, ciprofloxacin and high resistance to metronidazole. Antral gastritis was the commonest endoscopic finding to be associated with H. pylori infection (43.3%), followed by chronic duodenal ulcer (18.3%) and gastric ulcer (16.7%).

 
  References Top

1.Primrose JN. Stomach and duodenum Helicobacter pylori. In: Williams NS, Bulstrode CJ, Connell PR, editors. Bailey and Love's Short Practice of Surgery. 25 th ed. London: Edward Arnold; 2008. p. 1051-2.  Back to cited text no. 1
    
2.Akbar DH, Eltahawy AT. Helicobacter pylori infection at a university hospital in Saudi Arabia: Prevalence, comparison of diagnostic modalities and endoscopic findings. Indian J Pathol Microbiol 2005;48:181-5.  Back to cited text no. 2
    
3.Ricci C, Holton J, Vaira D. Diagnosis of Helicobacter pylori: Invasive and non-invasive tests. Best Pract Res Clin Gastroenterol 2007;21:299-313.  Back to cited text no. 3
    
4.Dharmalingam S, Rao UA, Jayaraman G, Thyagarajan SP. Relationship of plasmid profile with the antibiotic sensitivity pattern of Helicobacter pylori isolates from peptic ulcer disease patients in Chennai. Indian J Med Microbiol 2003;21:257-61.  Back to cited text no. 4
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5.Piccolmini R, Bonaventure GD, Catamo G, Carbone F, Neri M. Comparative evaluation of E test, agar dilution and broth microdilution for testing susceptibilities of Helicobacter pylori strains to 20 antimicrobial agents. J Clin Microbiol 1997;35:1842-6.  Back to cited text no. 5
    
6.Destura RV, Labio ED, Barrett LJ, Alcantara CS, Gloria VI, Daez ML, et al. Laboratory diagnosis and susceptibility profile of Helicobacter pylori infection in the Philippines. Ann Clin Microbiol Antimicrob 2004;3:25.  Back to cited text no. 6
    
7.Collee JG, Marr W. Test for the identification of bacteria. In: Collee JG, Duguid JP, Fraser AG, Marmion BP, editors. Mackie and McCartny Practical Medical Microbilogy. 13 th ed. London: Churchill Livingstone; 1989. p. 152-3.  Back to cited text no. 7
    
8.McNulty C, Owen R, Tompkins D, Hawtin P, McColl K, Price A, et al. Helicobacter pylori susceptibility testing by disc diffusion. J Antimicrob Chemother 2002;49:601-9.  Back to cited text no. 8
    
9.Alazmi WM, Siddique I, Alateeqi N, Al-Nakib B. Prevalence of Helicobacter pylori infection among new outpatients with dyspepsia in Kuwait. BMC Gastroenterol 2010;10:14.  Back to cited text no. 9
    
10.Young KA, Akyon Y, Rampton DS, Barton SG, Allaker RP, Hardie JM, et al. Quantitative culture of Helicobacter pylori from gastric juice: the potential for transmission. J Med Microbiol 2000;49:343-7.  Back to cited text no. 10
    
11.Yakoob J, Jafri W, Abid S, Jafri N, Abbas Z, Hamid S, et al. Role of rapid urease test and histopathology in the diagnosis of Helicobacter pylori infection in a developing country. BMC Gastroenterol 2005;5:38.  Back to cited text no. 11
    
12.Nair D, Uppal B, Kar P, Gondal R, Mathur MD. Immune response to Helicobacter pylori in gastroduodenal disorders. Indian J Med Microbiol 1997;15:33-6.  Back to cited text no. 12
    
13.Nichols L, Sughayer M, DeGirolami PC, Balogh K, Pleskow D, Eichelberger K, et al. Evaluation of diagnostic methods for Helicobacter pylori gastritis. Am J Clin Pathol 1991;95:769-73.  Back to cited text no. 13
    
14.Oyedeji KS, Smith SI, Arigbabu AO, Coker AO, Ndububa DA, Agbakwuru EA, et al. Use of direct Gram stain of stomach biopsy as a rapid screening method for detection of Helicobacter pylori from peptic ulcer and gastritis patients. J Basic Microbiol 2002;42:121-5.  Back to cited text no. 14
    
15.Vijaya D, Chadrashekhar N. Urease test, grams stain, histology and serology in diagnosis of Helicobacter pylori in duodenal ulcer cases. J Acad Clin Microbiol 2004;6:7-10.  Back to cited text no. 15
    
16.Berry V, Sagar V. Rapid urease to diagnose Helicobacter pylori infection. JK Sci 2006;8:86-9.  Back to cited text no. 16
    
17.Malik A, Singhal S, Mukharji U. Evaluation of ELISA based antibody test against H. pylori in cases of non ulcer dyspepsia. Indian J Med Microbiol 1999;17:137-9.  Back to cited text no. 17
    
18.Gupta P, Pandey LB, Behra RN, Rai MK, Chandra R. Role of ELISA in H. pylori detection and its correlation with urease test. Indian J Pathol Microbiol 2003;46:511-4.  Back to cited text no. 18
    
19.Sharma S, Prasad KN, Chamoli D, Ayyagari A. Antimicrobial susceptibility pattern & biotyping of Helicobacter pylori isolates from patients with peptic ulcer diseases. Indian J Med Res 1995;102:261-6.  Back to cited text no. 19
    
20.Rahman SH, Azam MG, Rahman MA, Arfin MS, Alam MM, Bhuiyan TM, et al. Non-invasive diagnosis of H. pylori infection: Evaluation of serological tests with and without current infection marker CIM. World J Gastroenterol 2008;14:1231-6.  Back to cited text no. 20
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]


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