Comparison of Helicobacter pylori Detection Using Immunohistochemistry and Giemsa and Its Association with Morphological Changes in Active Chronic Gastritis

Background: the gaster. The most common aetiology of chronic gastritis is Helicobacter pylori (H. pylori) infection. Presence of morphology, H. pylori is known in 2 forms, which are rod-shaped and coccoid-shaped. Coccoid-shaped and evaluation of the sensitivity of coccoid-shaped of H. pylori are needed. Method: Cross-sectional study on 90 biopsy tissues of chronic gastritis patients in year 2015 and 2014, which included 30 Giemsa cases with positive H. pylori, 30 cases of active chronic gastritis with negative immunohistochemistry staining of H. pylori. Results: Conclusion: Immunohistochemistry staining in active chronic gastritis was more sensitive compared to Giemsa staining in detecting H. pylori, particularly the coccoid-shaped bacteria.


INTRODUCTION
Gastritis is one of the most common digestive tract problems.Worldwide, the incidence of gastritis is 1.8 -2.1 million, while in South East Asia, 583,635 per year.The incidence of gastritis in Indonesia is quite high, which is 247,396 cases from 238.452.952population. 1 or irritation of the gaster. 2Generally, the cause of chronic gastritis is the infection of Helicobacter pylori (H.pylori), a gram-negative bacteria, presence of autoimmune disease and reaction towards chemical and drugs. 3lobally, the prevalence of H. pylori varies.Infection is more common to be found in the developing countries compared to developed countries.Infection may affect all ages, starting from childhood to adulthood. 4H. pylori infection in developing countries can reach up to 25-30%, where 5-27% are found in early childhood and 50-60% are found in adults aged more than 60 years old. 5Based on the report from several studies, it was known that the prevalence of H. pylori infection in Indonesia varied.H. pylori infection in Dr. Mohammad Husein Palembang Hospital reached 46.7% in 2009, 24.3% in Tugurejo Semarang Hospital in year 2004-2010, 20.1% in Surakarta in year 1997. 6,7,8eanwhile, in Jakarta based on serologic examination in 150 primary school children, the obtained prevalence was 27%. 9 Along with the increased prevalence of H. pylori infection, various methods have been developed to detect it, either using invasive or non-invasive methods.Some known non-invasive methods include urea breath test, nitrogen excretion test, blood immunoglobulin (Ig) G and IgA serologic examination, and faecal H. pylori antigen test.On the other hand, invasive methods include microbial culture test, urease examination in biopsy tissue, histopathology, and polymerase chain reaction (PCR) of the biopsy tissue.Appropriate H. pylori diagnostic test is chosen based on the sensitivity equipment availability. 10Invasive detection of H. pylori is performed through gastric biopsy.Gastric from the antrum and corpus. 11H. pylori detection in gastric biopsy can be performed through Giemsa staining, immunohistochemistry staining, McMullen modification method, and silver staining method.Rotimi et al studied 63 gastric samples to detect H. pylori.Sensitivity of all the four methods being used were immunohistochemistry using H. pylori antibody (98.3%), McMullen modification (90%), Giemsa (86.7%), and silver staining (85%), respectively. 12ased on its morphology, H. pylori bacteria are known in two forms, rod-shaped and coccoid-shaped.Rod-shaped H. pylori has tendency to become coccoidshaped in several environmental conditions, such as: oxygen exposure, base pH, starvation, long-term treatment, and inadequate proton pump inhibitor (PPI) or antibiotic administration. 13,14Coccoid-shaped bacteria is hard to detect using Giemsa staining due to the difficulty in differentiating coccoid-shaped bacteria from artefact or other bacteria.Therefore, immunohistochemistry staining of H. pylori is needed.A different opinion from several researchers stated that the coccoid-shaped is a transformation form leading to degenerative state or death, while some others considered it as active and viable form. 13,14occoid-shaped H. pylori may also be caused by increased oxygen pressure and antibiotic administration.Coccoid-shaped H. pylori is form that cannot be cultured but is still alive and can be induced back to the virulent form (spiral).Coccoid-shaped H. pylori is thought to play role in bacterial transmission and Comparison of Helicobacter pylori Detection Using Immunohistochemistry and Giemsa and Its Association with Morphological Changes in Active Chronic Gastritis some are responsible in the recurrence of infection after antimicrobial treatment; however, the pathogenesis of coccoid-shaped H. pylori is still unclear and has not been much studied. 13,14In the study performed by She et al, there were 3 strains of coccoid-shaped H. pylori which changed from spiral-shaped due to exposure to metronidazole. 14In this study, we would like to know the correspondence between immunohistochemistry and Giemsa staining to detect H. pylori in chronic gastritis and to observe the morphological or histological difference of chronic gastritis with rod-shaped and coccoid-shaped H. pylori.Immunohistochemistry staining method becomes a consideration in increasing the sensitivity in the detection of H. pylori as it relies

METHOD
This study used cross-section design, performed in Department of Anatomical Pathology Faculty of Medicine University of Indonesia/Dr.Cipto Mangunkusumo Hospital (FMUI/CMH) in November 2015 to January 2016.Accessible population of this study was active chronic gastritis cases which were diagnosed in Anatomical Pathology Department FMUI in year 2014-2015 with topographic code C15, C16, and morphologic code H544 in accordance with standard.Samples of active chronic gastritis with positive H. pylori in Giemsa staining and active chronic gastritis with negative H. pylori in Giemsa staining, but had coccoid-shaped were obtained through consecutive method.Samples of non-active chronic gastritis were obtained through simple random sampling.Estimation of the sample size counted with formula (paired categorical) was 51 cases.
Search and exploration of cases were performed in Anatomical Pathology Department FMUI/CMH in January 2015 to September 2015, and if results were not adequate, samples were further taken from the previous years.Anatomical pathology examination form and slides were collected; subsequently, reevaluation towards active chronic gastritis H & E slides and Giemsa positivity were conducted.
were performed using visual analog scale.Later, the criteria were made and H. pylori (BC 7) antibody which was incubated for 1-2 hours with 1:50 dilution was examined.

Assessment of the results of H. pylori (BC 7 ) i m m u n o h i s t o c h e m i s t r y s t a i n i n g w a s
performed by researcher using light microscope.Immunohistochemistry staining evaluation was based on the presence of H. pylori staining in gastric mucosa.Staining results evaluation was performed by researchers together.Statistical analysis was performed using Chi-square test and if criteria had not alternative.These statistical tests were performed using SPSS 20 software.

RESULTS
Active chronic gastritis samples with Giemsa positive H. pylori and active chronic gastritis with Giemsa negative H. pylori but contained coccoidshaped were obtained through consecutive technique.Non-active chronic gastritis samples were collected through simple random sampling in one year period, which was January to December 2015, in each studied group, and if results were not adequate, samples were further taken from the previous years.This study evaluated 3 categories, which consisted of 30 cases with positive H. pylori with Giemsa, 30 cases of active chronic gastritis, and 30 cases of non-active chronic gastritis.Patients' age data distribution showed nonnormal distribution, which was median of age 51.50 years old, the youngest age was 7 years old and the eldest was 86 years old, with the age range of 79 years old and mean age of 49.08 years old.was found in 17.8% cases, severe atrophy in 5.6% cases and intestinal metaplasia was found in 6.7% cases.Evaluation was performed to 60 cases of active chronic gastritis which were divided into 2 categories; H. pylori in Giemsa staining but had coccoid-shaped, in the immunohistochemistry staining positive H. pylori was found in 16 cases (53.3%), but had coccoid-shaped H. pylori morphology (Figure 1 and 2).While, 30 cases which initially showed positive H. pylori with Giemsa staining, the immunohistochemistry staining also revealed positive results (Figure 3 and 4).
In this study, we also performed staining to 30 biopsy samples of non-active chronic gastritis with results of 1 sample which Giemsa staining was positive H. pylori, but the immunohistochemistry staining turned out to be negative H. pylori (Table 5).

DISCUSSION
Gastritis cases in this study were found in patients aged 7 years old to 86 years old with the peak incidence found in 51-60 years old age group with the mean age of 49 years old.This is in concordance with the literature which state that the incidence of chronic gastritis may happen in all age group from childhood to adulthood. 4The results of this study was not so different from the study conducted by Dhakwa et al which revealed that the average age of gastritis patients were 41.5 years old.This was also in agreement with the study performed by Kalebi et al which concluded that the mean age was 43 years old with variation of 18-86 years old. 14,15The incidence of gastritis is higher in female (57.8%) as compared to male.This was in accordance with the study done by Al Ammar et al which reported 58.19% female, and different from the study conducted by Capelle et al that found 55% chronic gastritis incidence were male. 27,16ere was no atrophy in 6 cases (6.7%), mild atrophy in 57 (63.3%), moderate atrophy in 22 cases (24.4%), and severe atrophy in 5 cases (5.6%).Metaplasia was found only in 6 cases (6.7%).Hashemi et al found gastric histopathological appearance of normal mucosa in 8.7%, inactive chronic gastritis in 37.7%, active chronic gastritis in 47.1%, atrophy changes in 25%, and intestinal metaplasia in 8.9%.Zhang et al reported gastric histology appearance of patients with chronic gastritis (non-ulcer dyspepsia) H. pylori infection was mucosa in 36.8%, and intestinal metaplasia in 37.0%. 17,18ctive chronic gastritis cases with negative H. pylori Giemsa staining, but with positive H. pylori in immunohistochemistry staining were found in 53.3% cases with coccoid-shaped morphology.Study performed by Tajalli et al towards 54 samples found that the positivity of H. pylori with immunohistochemistry method was as many as 43 cases (79.63%), while the positivity with Giemsa method was as many as 24 cases (44.44%) and 18 (33.33%)with H & E staining.The results of this study revealed that classical method was not sensitive enough to identify H. pylori particularly the coccoid-shaped. 43Key et al detected 37% H. pylori with H & E staining, 55% with Giemsa staining, 62% with Warthin starry, 66% with immunohistochemistry and 45% were detected using PCR.Immunohistochemistry staining was positive in all cases where H. pylori was detected using other methods. 19udy performed by Orhan et al revealed that with immunohistochemistry method, low-density coccoid-shaped H. pylori could be observed easily.Positive H. pylori with immunohistochemistry staining were found in 3 from 10 cases of negative urea breath test (UBT).This study concluded that compared to Giemsa and UBT in detecting H. pylori infection. 20Immunohistochemistry examination statistical test was performed towards the coccoidshaped H. pylori difference between the two (Appendix 1).
Results of statistical test showed that there was H. pylori in active and non-active chronic gastritis with Giemsa staining.Statistical test was also performed towards H. pylori in active and non-active chronic gastritis towards immunohistochemistry staining; fisher exact test was performed and found that there was H. pylori in active and non-active chronic gastritis towards immunohistochemistry staining (Appendix 2-3). Urease examination of biopsy tissue (campylobacter like organism test/CLO test), Monteiro et al obtained sensitivity value of 83%, specificity 96.4%. 25istopathology method, Monteiro et al found with polymerase chain reaction (PCR) of the biopsy 25   Rotimi et al (year 2000) evaluated 63 gastric samples to detect H. pylori.Observation results using combination test, culture, serology, and histology) found that from interobserver consensus the best method from all the four methods were H. pylori antibody (98.3%), silver staining (85%), respectively. 12is study also evaluated Giemsa and immunohistochemistry staining in non-active chronic gastritis from 30 samples with the result that 1 case with positive H. pylori in Giemsa staining but the immunohistochemistry staining showed negative H. pylori results; this is possible that the positive result in Giemsa staining was a false positive result.

CONCLUSION
Morphological appearance of active chronic gastritis in positive H. pylori and negative H. pylori in immunohistochemistry staining has significant difference with the degree of inflammation.Morphological appearances of active chronic gastritis in the mucosa that are infected with coccoid-shaped and rod-shaped H. pylori were not significantly different; probably this coccoid-shaped is the active form of H. pylori.Immunohistochemistry staining to detect H. pylori is more sensitive compared to the Giemsa staining, particularly in the coccoid-shaped.False positive H. pylori was found in Giemsa staining in non-active chronic gastritis case.The results of this study may be extended to study further about coccoidshaped H. pylori, both in terms of coccoid bacteria activity or even the morphological changes it caused.Immunohistochemistry examination is recommended to be used in diagnosing H. pylori in active chronic gastritis, particularly the coccoid-shaped.

Table 4 . Evaluation Results of Active Chronic Gastritis Based on H. pylori in Immunohistochemistry Staining Immunohistochemistry p Rod-shaped H. pylori Coccoid- shaped H. pylori
Comparison of Helicobacter pylori Detection Using Immunohistochemistry and Giemsa and Its Association with Morphological Changes inActive Chronic Gastritis 2= 14/14 = 100%.
Study conducted by Yakoob et al on 176 cases of chronic gastritis concluded that there was and activity of H. pylori.Aggregated lymphoid was