Histomorphological changes in gall bladder diseases and its association with helicobacter infection

Correspondence: Dr. Geetika K.C., MD Department of Pathology, KIST Medical College, Imadole, Lalitpur Email Address: geetikakc@hotmail.com Background: Gall bladder diseases can be an incidental finding and when symptomatic, present with signs and symptom of cholecystitis and cholelithiasis. On histopathological examination however wide range of pathological changes are encountered including carcinoma. Several studies have been carried out to establish relationship of helicobacter infection with gallbladder diseases. This study tries to look at the frequency of risk factors, spectrum of histological changes and the relationship between different types of gallbladder diseases and helicobacter infection.


MATERIAL AND METHODS
This was a prospective study, conducted in department of pathology, Manipal Teaching Hospital, Pokhara, Nepal over the period of 24 months from October 2010 to October 2012.All the cases of cholecystectomy were included in the study.Informed written consent was obtained from all patients.Bile was aspirated peroperatively in a 5 ml syringe and microbiological culture done.The bile was then inoculated in blood agar and chocolate agar at 37 0 C for 72 hours along with Brucella agar with campylobacter supplement II (Butzler) was used for culture in microaerophilic environment.The cholecystectomy specimens fixed in 10% formalin was sent to department of pathology where it was processed routinely.The slides were examined microscopically after Hematoxylin and Eosin staining and Giemsa staining.Data were analyzed using SPSS.

RESULTS
A total of 500 cholecystectomy specimens were received in the study period.There were total of 390 (78%) female and 110 (22%) male with male to female ratio of 1:3.5.The peak age group involved by gall bladder diseases was 30 to 40 years followed by 40-50 years with the mean age was 44 years.
Helicobacter pylori was seen in 166 (33%) of cases.There was no statistical significance between any of these microscopic findings and Helicobacter infection except for malignancy which has a significant p value of 0.03 (<0.05).
have been reported in the gallbladder mucosa in various studies. 9,10art from cholecystitis and cholelithiasis other histopathological changes such as congenital anomalies, cholesterolosis, hydrops and mucocele, follicular cholecystitis, eosinophilic cholecystitis, xanthogranulomatous cholecystitis, ischemia and infraction may be seen.Also seen are non-neoplastic epithelial alterations, non-neoplastic tumour like lesions, noninvasive epithelial lesion and carcinoma.l All of these are usually incidental finding, including carcinoma. 11 mentioned earlier, Helicobacter species are seen in association with gallbladder diseases.Since the discovery of helicobacter pylori in 1984, by Marshal and Warren more than 30 different species of helicobacter have now been discovered. 124][15] The aim of this study was to to study the frequency of risk factors associated with cholecystitis, the histomorphological spectrum of gallbladder diseases and attempted to find out its association with Helicobacter infection.Eosinophilic cholecystitis was seen in 5 (1%) of the cases (fig.3).Detail histological findings are shown in Table 1.

DISCUSSION
Gallbladder diseases are four times more common in female than male and are found to increase in frequency with age. 3 By 6 th decade of life every 4 th women is found to be affected by gallbladder disease. 3The present study showed a female predominance accounting to 390 (78%) of cases with male to female ratio of 1:3.5.The peak age group among both male and female affected by gallbladder disease in general was 30 -40 years.The age range was from 10 to 94 years.Similar finding were observed in other studies. 16,17ny organisms have been isolated worldwide in bile.The route of infection are ascending and descending infections.
The organisms isolated are Escherichia coli, Streptococci, Klebsiella species, Enterobacter species, Proteus species, Citrobacter species, Staphylococcus species, Pseudomonas, Salmonella typhi, Clostridium perfringes, Bacteroides fragilis and others.In our study all of the organisms isolated were aerobic bacteria.E. coli was the most common organism found in 36 (15%) cases.According to the order of frequency, Enterococcus 13 (5%), Actenobacter 11 (5%) and Klebsiela 11 (4%) were other organisms isolated.Also seen were few cases of Streptococcus 8 (3%) and Pseudomonas 7 (3%).In the study done by Brook et al 18 48% of the species were aerobic bacteria, 3% anaerobic bacteria and 49% were mixed.E. coli was the most common organism isolated, followed by Klebsiela.
No helicobacter colonies were grown under microaerophilic condition in a Brucella agar with Campylobacter supplement, in any of our cases.Similar findings were observed in a studty done Roe et al 19 and Bohr et al. 19 However Mishra et al 20 in their study found 13 (44.4%)positive cases and 11 (55.6%)negative cases for Helicobacter pylori culture.The lack of growth in our study could be because of coexistence of other aerobic organisms along with various other factors.Chronic cholecystitis with dystrophic calcification was seen in 3 (1%) of the cases and along with cholesterolosis in 1 (0.2%) of the cases.In a study done in Massachusetts General Hospital, 21 0.16% of cholecystectomy cases were calcified gallbladder, which was similar to our study finding.
No dysplasia or malignancy was noted along with calcified gallbladder.
In our study reactive atypia was noted in 36 (7%) of the cases.In the study done by Bracia22 regenerative/reactive changes were seen in 73% of cases which was very much higher than our finding.In the present study, metaplasia was seen in 14 (3%) of cases and intestinal type was more 13 (2%) compared to 1 (0.2%) of pyloric metaplasia.Pradhan et al, 23 found intestinal metaplasia in 20 (5.26%) of cases which was more than our study.Polyp was seen in 2 (0.4%) of our study cases.
Dysplasia was seen in 6 (1%) cases.It was graded into mild, moderate and severe and each group had 2 (0.4%) cases each.In the study of Mittal et al, 24 dysplasia was seen in 1 (0.08%) of cases.Pradhan et al 23 however found dysplasia in 15 (3.95%) of cases which was higher than our study finding.Adenoma was seen in 5 (1%) of cases in our study.It was seen in 10 (0.8%) cases in combination with metaplasia in study done by Mittal et al. 24 Gall bladder carcinoma was seen in 7 (1%) of cases in current study with a significant p value of 0.03 when correlated with helicobacter infection.Pradhan et al 23 had 10 (2.63%) cases and Parajuli et al 25 had 7 (2.5%) cases of malignancy.
Helicobacter species have long been identified in gallbladder and biliary tract.However, its definite role and relation in the gallbladder diseases have not been well defined.In the meta-analysis published by Zhou et al 26 in 2011, showed that the Helicobacter detection rate in gallbladder to range from 3.02% to 80%.The detection rate varied with the type of methods used.In our study, Helicobacter was positive in 166 (33%) of cases and negative in 334 (67%) of cases.
Parajuli et al 25 found 50 (19%) of their cases to be positive for Helicobacter infection in H&E and WSS.Variable findings were observed in other studies. 20,27,28 all the categories we had helicobacter negative cases more than positive.However helicobacter prevalence was more in eosinophilic cholecystitis and carcinoma cases.Acute cholecystitis had 3 (1%) and chronic cholecystitis had 117 (23%) prevalence of helicobacter infection.Karagin PH et al 16 saw in their study that 4 out of 50 (8%) were acute cholecystitis with helicobacter infection (higher than our study) and 3 out of 50 (6%) cases of chronic cholecystitis had helicobacter infection (lower than our study).All the carcinomas detected in our studies were incidental findings.
The rate of incidental carcinoma ranges from 0.14% to 6.1% worldwide. 29According to the WHO 2008 data gallbladder carcinoma incidence was 1.7% in South East Asia. 30It was seen in 1.3% of male and 2% females. 30licobacter infection has been reported in various articles in association with gallbladder carcinoma.In the present study, Helicobacter was seen in 5 (72%) out of 7 malignant cases.Both the higher stage (T2) tumours were positive for Helicobacter.The p value obtained between helicobacter infection and gallbladder carcinoma was 0.03 which was significant and odds ratio of 5.15 (95% CI of 0.98-26.86).
In other studies over the world various helicobacter species were specifically identified with relative risk of gallbladder carcinoma ranging from 2.6 to 9.9 % and 95%CI of 0.6 to 70.5. 20,25,27

CONCLUSION
Chronic cholecystitis was the most common histopathological diagnosis encountered.Helicobacter infection was prevalent in variety of gallbladder pathology and had significant association with gallbladder carcinoma.However, definite relation between the helicobacter infection and gallbladder diseases cannot be concluded

Figure 4 :
Figure 4: Gross of gallbladder carcinoma with prominent nodule.

Figure 1 :
Figure 1: Gross of cholesterolosis with yellow streaks of cholesterol deposition.

Table 1 : Histopathological diagnosis in gall bladder specimen and Helicobacter status HISTOPATHOLOGICAL DIAGNOSIS Helicobacter positive
H e l i c o b a c t e r negativeTotal