Simple Stain For Helicobacter PyloriCorrespondence Address :
Dr. D. Vijaya
Professor& HOD Microbiology
AIMS, B.G.Nagara 571448
Phone no; 94820 09120
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Several methods are available for the detection of Helicobacter pylori in the gastric antral biopsies. Most of these are technically demanding, slow and expensive with varying sensitivity. Aim of the present study was to find out the prevalence of Helicobacter pylori in various gastroduodenal diseases and to evaluate the advantages of Loefflerâ€™s methylene blue stain for crushed biopsy smear.
A total of 236 (male 176, female 60) randomly selected patients with endoscopically proved chronic duodenal ulcer (180), gastroduodenitis (42) and gastric ulcer (14) formed the study group. Four gastric antral biopsies were taken from each patient. Biopsies were subjected to histology, rapid urease test, Gramâ€™s and Loefflerâ€™s methylene blue (LMB) stains for crushed smears. Evidence of H. pylori infection was found in 62.71% considering histopathology as the gold standard. H. pylori was more common in males (65.90%) and in the age group of 31-40 years (77.50%). In duodenal ulcer, gastric ulcer and gastroduodenitis, 68.88%, 57.14% and 38.09% were positive for H. pylori respectively. A smear stained with LBM was found to be rapid, simple, reliable, and cost effective with good sensitivity and specificity for detecting H. pylori.
Helicobacter pylori, Loefflerâ€™s methylene blue stain
Helicobacter pylori is an important pathogen in humans causing chronic gastritis & playing a major role in the development of peptic ulcers & gastric cancer (1). The present study was undertaken to find out the presence of H.pylori in various gastroduodenal diseases & to evaluate the advantages of Loefflerâ€™s methylene blue (LMB) stain for crushed biopsy smears. The study was conducted in the department of Microbiology and Gastroenterology of B and L.C. Hospitals Bangalore. The study period was from July 2000 to December 2003.
A total of 236 randomly selected, symptomatic patients (male 176, female 60) in the age group of 16-65 years, endoscopically diagnosed as duodenal ulcer (180), gastroduodenitis (42) and gastric ulcer (14) formed the study group. Ethical clearance from the institution and consent of patients was taken prior to endoscopy.
Four endoscopic antral biopsy specimens from each case were transported in vials containing normal saline, urease medium & 10% formalin. From the specimens in normal saline, two crushed smears were made and fixed with methanol.Of this one smear was stained by Gramâ€™s using carbol fuchsin as counter stain (Table/Fig 1). 2nd smear was stained with LMB for 30 seconds (Table/Fig 2). Specimens in urease medium were incubated at 370C for 60 minute which turns pink if positive (2). Histopathology sections were stained by over night Giemsa stain,which is considered as the gold standard for the detection of H.pylori (Table/Fig 3).
Of the 236 cases studied 176 were males & 60 females. The age varied from 16-65 years. A total of 148 (116 males, 32 females) were positive for H.pylori infection, considering histopathology section stained by Giemsa as the gold standard.
In duodenal ulcer, gastric ulcer and gastroduodenitis, 68.88%, 57.14% and 38.09% were positive for H. pylori respectively.
Various techniques are available for detection of H.pylori, but the aim of modern research is to find a method, which is rapid, cost effective and accurately identifies the H. pylori. The sensitivity of the test depends on the number of bacteria present in the specimen. In experienced hands every test has good sensitivity & specificity (3).
Out of 236 cases 148 (62.71%) were positive and 88 (37.29%) negative for H.pylori. H.pylori was more common in males (65.90%) than females (53.66%). H.pylori positivity was found in all the age group (16-65 years), as exposure to H.pylori occurs early in India & is widespread (4). Maximum positivity was found in age group of 31-40 years (77.50%). In duodenal ulcer, gastric ulcer and gastroduodenitis, 68.88%, 57.14% and 38.09% were positive for H.pylori respectively. Nair D, Ayyagari in their studies have found similar prevalence of H.pylori in all three conditions mentioned (5),(6). This prevalence has not increased as seen from the report presented from this department in 1999 (7).
The rapid urease test has the advantage of being simple, inexpensive, rapid & positive in 60 minutes, as evidenced in other studies also (2),(8). False positives could occur in urease test due to its high sensitivity(100%).
Crushed smears stained with Gramâ€™s showed sensitivity of 98%, specificity of 100% can provide rapid diagnosis. This is in correlation with the study of VanHorn (9). No false positive will be found in an experienced hand. False negative may be due to, organisms hidden in proteinaceous material.
H.pylori take up deep blue and mucus faint blue colour in LMB stain and stood very well against the background. Staining time was only one minute. Smears stained with LMB showed high sensitivity and specificity, is similar to the study of others (10),(11). LMB staining is technically simple, rapid, reliable, inexpensive, with high positive and negative predictive value ,hence found to be excellent stain for the detection of H.pylori as it can pick up a very light load of infection, compared to technically demanding, expensive, slow histopathology and/or culture method (10),(11).
Authors are of the opinion that smear stained with LMB has high specificity (95.45% )and PPV(97.36), will be sufficient for the detection of H.pylori in an ordinary set up.
Date of Submission: Mar 28, 2012
Date of peer review: Apr 23, 2012
Date of acceptance: Apr 26, 2012
Date of Publishing: May 31, 2012
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