|Year : 2015 | Volume
| Issue : 3 | Page : 218-222
Comparison of different stains in imprint cytology with the conventional diagnostic tools in detection of Helicobacter pylori infection
Shashikant Adlekha1, Tandra Chadha2
1 Department of Pathology, Sree Narayana Institute of Medical Sciences, Ernakulam, Kerala, India
2 Department of Microbiology, Sree Narayana Institute of Medical Sciences, Ernakulam, Kerala, India
|Date of Web Publication||12-Jun-2015|
Dr. Shashikant Adlekha
Department of Pathology, Sree Narayana Medical college, Chalakka, Ernakulam, Kerala - 683 594
Source of Support: None, Conflict of Interest: None
Background: Helicobacter pylori (H. pylori) infection is one of the most common causes of gastrointestinal morbidity and mortality worldwide. Early diagnosis of infection is imperative for the cure of distressing symptoms and prevention of complications. Objective: The objective of the study is to evaluate imprint cytology of gastric biopsy specimens as a rapid and cost-effective diagnostic tool and its comparison with conventional diagnostic tests. Materials and Methods: Antral biopsies were collected from 130 patients and evaluated for H. pylori infection by imprint cytology and histopathological examination by different stains and rapid urease test (RUT). Histopathological features of gastric biopsy specimens were also assessed. Results: A total of 118 patients showed H. pylori infection by two or more methods. Giemsa histology showed highest sensitivity, specificity, positive predictive value, negative predictive value and Youden's Index (YI). Among imprint cytology stain methods, toluidine blue showed highest sensitivity and highest YI was obtained for PAP stain. Conclusion: Helicobacter pylori infection is associated with gastric mucosa changes like chronic active gastritis, atrophy, intestinal metaplasia, ulceration and carcinoma. Imprint cytology has high sensitivity and comparable predictive values to conventional diagnostic tools-histopathological examination and RUT in the detection of H. pylori infection.
Keywords: H . pylori, histopathology, imprint cytology, RUT
|How to cite this article:|
Adlekha S, Chadha T. Comparison of different stains in imprint cytology with the conventional diagnostic tools in detection of Helicobacter pylori infection. CHRISMED J Health Res 2015;2:218-22
|How to cite this URL:|
Adlekha S, Chadha T. Comparison of different stains in imprint cytology with the conventional diagnostic tools in detection of Helicobacter pylori infection. CHRISMED J Health Res [serial online] 2015 [cited 2020 Jul 6];2:218-22. Available from: http://www.cjhr.org/text.asp?2015/2/3/218/158679
| Introduction|| |
0Helicobacter pylori, a gram-negative flagellated bacilli has been implicated in causing a spectrum of gastrointestinal pathologies with dyspepsia as the main presenting symptom. ,
The organism is usually found under the mucus layer in the gastric pits in close apposition to gastric epithelial cells where it causes damage to the cells.  The pathological lesions range from mild chronic gastritis to gastric malignancies like adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. Studies have shown an association of 80% and 92% of H pylori infection with gastric carcinoma and MALT lymphoma respectively. ,,, H. pylori are considered the most common bacterial infection in the world with an estimated 75% of population in developing country being infected with organism even at early age and lower in the developed country (typically <40%) with a declining pattern worldwide.  The annual incidence of the H. pylori infection is 0.3-0.7% in the developed countries and it is 6-14% in the developing countries. 
Diagnosis of H. pylori infection is done by a wide array of invasive and noninvasive methods. Noninvasive tests include urea breath test, serological immunoglobulin G and immunoglobulin M detection, saliva and urinary antibody test and stool antigen test.  The invasive tests are endoscopy based tests which include histopathological examination, cytological examinations such as brush cytology and imprint cytology, rapid urease test (RUT) and polymerase chain reaction. Whereas invasive tests carry high sensitivity and specificity of >90%,  the role of noninvasive tests such as serology is limited in areas of high prevalence, because of non-distinction between previous and current infection. Two or more tests are commonly employed to increase the accuracy and prevent bias. This strategy leads to higher cost. ,,
The present study evaluated the predictive values of different stains in imprint cytology for detection of H. pylori in comparison with the conventional and most widely used methods-biopsy examination and RUT.
| Materials and methods|| |
A total of 130 patients undergoing upper gastrointestinal endoscopy in the hospital were enrolled in this study. Three different diagnostic methods were used- histology, imprint cytology and RUT. Three antral biopsy fragments were obtained from each patient and two samples sent for pathological examination in unfixed state and one sample being sent for RUT. Imprint smears were prepared from one fragment by keeping one biopsy fragment on a glass slide and gently touching it without pressing. Imprint slides were immediately stained for toluidine blue (T. blue), alcohol fixed and stained for H and E and papanicolaou (PAP) stains, air dried and stained for Giemsa stain. The imprinted and second biopsy specimens were fixed in 10% formalin and processed for three micrometer thick sections and stained with H and E and Giemsa stains. H. pylori classically appear as small curved or s-shaped structures [Figure 1], [Figure 2] and [Figure 3]. Occasional coccoid forms may be seen and are difficult to be interpreted by routinely used stains. Immunohistochemical staining may be employed for detection of these forms. On another biopsy fragment, RUT was performed by following method-Urea (2 g) was dissolved in 20 ml double distilled water. 20 drops of phenol red was added to the solution and pH was adjusted between 6.8 and 6.9 by adding a drop of N/10 HCl, if pH was greater or N/10 NaOH, if pH was less. Solution was having faint yellow tint at this stage. This was transferred to sterile vial each containing 2 ml in each vial. Biopsy material was added and the temperature was kept constant at 35-37°C. Test was considered positive, if color changed within 30 min and weekly positive, if the change occurred after 2 h.
|Figure 1: Imprint Cytological smear showing cluster of tiny curved rod like structures (Arrow head) amidst dirty smear background, Giemsa, ×1000|
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|Figure 2: Imprint Cytological smear showing scattered tiny curved rod like structures (Arrow head) amidst dirty smear background, Toluidine blue, ×1000|
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|Figure 3: Section shows small curved structures (Arrow head) in gastric foveolae, Giemsa stained section, ×1000|
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Histopathological changes of gastric mucosa were also assessed. Lymphoplasmacytic infiltrates without neutrophilic infiltration was regarded as chronic gastritis and with neutrophilic infiltration as chronic active gastritis. Atrophy of glands was regarded as atrophic gastritis and goblet cell metaplasia of glandular lining was regarded as intestinal metaplasia. Density of H. pylori was assessed according to visual analogue scale of updated Sydney grading system.  To increase the accuracy and prevent bias, positivity for two or more methods (by any of the stains, RUT) was considered as true positive. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of different methods were computed and compared.
Sensitivity = True positive/(True positive + False negative); Specificity = True negative/(True negative + False positive); PPV = True positive/(True positive + False positive); NPV = True negative/(True negative + False negative).
Youden's index (YI) = Sensitivity + Specificity − 100.
Informed consent was taken from each patient and the study was approved by scientific research committee of the institution.
| Results|| |
A total of 130 persons with dyspeptic symptoms were enrolled in the present study with 68 males and 62 females with mean age of 49 ± 9.7 years. Taking the criteria of two or more positive results to be positive (true positive), 118 out of 130 patients tested positive for H pylori infection. 94 patients tested positive for all the methods [Table 1]. 110 patients tested positive by histological methods (108 by H and E and 110 by Giemsa). RUT results showed 108 patients to be positive for H. pylori infection. Imprint cytology examination showed positivity for H. pylori infection in 107, 109, 98 and 94 patients by Giemsa, T. blue, PAP and H and E stains respectively. Histopathological assessment [Table 2] of 130 patients showed chronic active gastritis in 76 patients, chronic gastritis in 20 patients, chronic active gastritis with intestinal metaplasia in 12 patients, chronic follicular gastritis in 10 patients and ulcerative changes in 6 patients. Dysplastic changes were not seen in any patient. Normal mucosal study was seen in 6 patients.
Predictive values of different methods when computed and compared [Table 3], showed highest sensitivity of 93.65% for Giemsa histology followed by, in descending order-T. blue (92.91%), H and E histology (92.18%), RUT (92.18%), Giemsa imprint (91.47%), PAP imprint (85.80%) and H and E imprint (83.09%). Specificity of all the methods was 100%, except for Giemsa imprint and and T. blue imprint stains with a specificity of 92.30%. Similarly, T. blue imprint and Giemsa imprint showed PPV of 99.15% and all other methods showed PPV of 100%. NPV was highest for Giemsa histology (60%), followed in descending order by 57.14% for T. blue-imprint cytology, 54.54% for H and E histology and RUT-54.54%, Giemsa imprint of 52.14%, PAP imprint of 37.5% and H and E imprint of 33.3%. YI when calculated was highest for Giemsa histology (94%), followed by in descending order-H and E histology-92 and RUT-92, PAP imprint-86, T. blue imprint-85, Giemsa imprint-84 and H and E imprint-83.
| Discussion|| |
0Helicobacter pylori infection is associated with varying degree of inflammation and architectural distortion in different individuals. This variability is not only accounted by the variation in bacterial load/density, but also relies on the immunogenicity of host/patient. In 2% of cases, H. pylori infection leads only to mild chronic gastritis or almost unremarkable mucosal change. , H. pylori infection diagnosis entails different methods each with different advantages and limitation. The most popular and widely used method is the histopathological examination of antral biopsies, employing H and E stain .  Histopathological examination is not only very sensitive and specific method of diagnosing H. pylori infection, but also provides valuable information regarding the mucosal architectural distortion and atypia if any. It is considered the gold standard for diagnostic tests. The major limitation is that it is time-consuming and expensive. Among cytology methods, brush cytology and imprint cytology are equally sensitive methods of diagnosing H. pylori infection. Imprint cytology is much easier to perform, and limitations of brush cytology such as imprecise procedure leading to unsuitable cytological examination are overcome. Imprint cytology and RUT are faster, and results are available when the patient is still in the endoscopy unit. This results in the early initiation of anti-H. pylori treatment. Imprint cytology by rapid stains such as T. blue, Diff-quick, and commercially available rapid H and E and PAP leads to much earlier detection, in comparison to 2 h time of RUT.
In the present study, we evaluated the predictive values of different methods. Giemsa biopsy has highest sensitivity, specificity, PPV, NPV and YI. Comparable sensitivity, specificity, PPV and NPV were seen for T. blue imprint and Giemsa imprint methods. There was significant difference noted in predictive values between histology, RUT and PAP and H and E stain for imprint cytology method. Various studies have compared the predictive values of different stains in diagnosing H. pylori infection. Tajalli et al.  reported higher predictive values for T. blue in H. pylori detection. Misra et al.  reported equal sensitivity and specificity of imprint cytology as that of biopsy examination. Preparing imprint cytology and subsequent biopsy slides did not deter the quality of biopsy, as evaluated by Misra et al.  When evaluated in terms of YI, highest YI was found for Giemsa histology. For imprint cytology, highest YI was seen for PAP stain followed by T. blue stain. YI validates a technique by taking both sensitivity and specificity into account. Low NPV noted in this study for different methods can be attributed to false negative cases reported. Low bacterial load and multifocality of the bacteria can lead to false negative cases in imprint smears, as sparse H. pylori are difficult to interpret amidst the dirty background of smears. Even in histopathology examination, specimen processing can lead to false negative result due to partial loss of area in or beneath the surface mucosal layer, especially in set up of low bacterial density. , This is in concordance with the present study findings, as all false negative cases seen in different methods, had low H. pylori density.
| Conclusion|| |
Imprint cytology is a rapid inexpensive method of diagnosing H. pylori infection. It has comparable predictive values to biopsy and RUT. Cytological smear examination and biopsy should be used in conjunction, as rapid diagnosis and architectural assessment of gastric mucosa is essential for effective management of the patient.
| References|| |
Suerbaum S, Michetti P. Helicobacter pylori
infection. N Engl J Med 2002;347:1175-86.
Oluwasola AO, Ola SO, Saliu L, Solanke TF. Helicobacter pylori
infection in South Nigerians: A serological study of dyspeptic patients and healthy individuals. West Afr J Med 2002;21:138-41.
Malfertheiner P, Megraud F, O'Morain C, Bazzoli F, El-Omar E, Graham D, et al.
Current concepts in the management of Helicobacter pylori
infection: The Maastricht III Consensus Report. Gut 2007;56:772-81.
Marshall BJ. Helicobacter pylori
. Am J Gastroenterol 1994;89 8 Suppl: S116-28.
Faraker CA. Diagnosis of Helicobacter pylori
in gastric brush and biopsy specimens stained by Romanowsky and immunocytochemical methods: Comparison with the CLOtest. Cytopathology 1996;7:108-19.
Correa P. Is gastric carcinoma an infectious disease? N Engl J Med 1991;325:1170-1.
Eidt S, Stolte M, Fischer R. Helicobacter pylori
gastritis and primary gastric non-Hodgkin's lymphomas. J Clin Pathol 1994;47:436-9.
Sasidharan S, Ghayethry B, Ravichandran M, Latha LY, Lachumy SJ, Leng KM, et al
. Prevalence of Helicobacter pylori
infection among patients referred for endoscopy: Gender and ethnic differences in Kedah, Malaysia. Asian Pac J Trop Dis 2012;2:55-9.
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. JCDR 2012;6:636-41.
Graham DY, Sung JY. Helicobacter pylori. Sleisenger and Fordtran's Gastrointestinal and Liver Disease. In: Feldman M, Friedman LS, Brandt LJ, editors. Pathophysiology, Diagnosis, Management. 7 th
ed. Philadelphia: WB Saunders Co; 2006. pp. 1049-66.
Andersen LP, Kiilerick S, Pedersen G, Thoreson AC, Jørgensen F, Rath J, et al.
An analysis of seven different methods to diagnose Helicobacter pylori
infections. Scand J Gastroenterol 1998;33:24-30.
Trevisani L, Sartori S, Ruina M, Caselli M, Abbasciano V, Grandi E, et al.
Touch cytology. A reliable and cost-effective method for diagnosis of Helicobacter pylori
infection. Dig Dis Sci 1997;42:2299-303.
Greenberg PD, Koch J, Cello JP. Clinical utility and cost effectiveness of Helicobacter pylori
testing for patients with duodenal and gastric ulcers. Am J Gastroenterol 1996;91:228-32.
Dixon MF, Genta RM, Yardley JH, Correa P. Classification and grading of gastritis: The updated Sydney system. Am J Surg Pathol 1996;20:1161-81.
Bayerdörffer E, Oertel H, Lehn N, Kasper G, Mannes GA, Sauerbruch T, et al.
Topographic association between active gastritis and Campylobacter pylori
colonisation. J Clin Pathol 1989;42:834-9.
Prasad S, Mathan M, Chandy G, Rajan DP, Venkateswaran S, Ramakrishna BS, et al.
Prevalence of Helicobacter pylori
in southern Indian controls and patients with gastroduodenal disease. J Gastroenterol Hepatol 1994;9:501-6.
Tajalli R, Nobakht M, Mohammadi-Barzelighi H, Agah S, Rastegar-Lari A, Sadeghipour A. The immunohistochemistry and toluidine blue roles for Helicobacter pylori
detection in patients with gastritis. Iran Biomed J 2013;17:36-41.
Misra SP, Dwivedi M, Misra V, Gupta SC. Imprint cytology - A cheap, rapid and effective method for diagnosing Helicobacter pylori
. Postgrad Med J 1993;69:291-5.
Misra SP, Misra V, Dwivedi M, Singh PA, Gupta SC. Diagnosing Helicobacter pylori
by imprint cytology: Can the same biopsy specimen be used for histology? Diagn Cytopathol 1998;18:330-2.
Debongnie JC, Donnay M, Mairesse J. Gastrospirillum hominis (" Helicobacter heilmanii
"): A cause of gastritis, sometimes transient, better diagnosed by touch cytology? Am J Gastroenterol 1995;90:411-6.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]