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Year : 2020  |  Volume : 63  |  Issue : 4  |  Page : 570-574
Gallbladder stone formation in Iraqi patients is associated with bacterial infection and HLA class II-DRB1 antigens

1 Depatment of Clinical Laboratory Science, Pharmacy College, Mustansiryah University, Baghdad, Iraq
2 Department of Pathology, Teaching Hospital of Kadhimiya, Baghdad, Iraq
3 Department of Surgery, Teaching Hospital of Al-Yarmouk, Baghdad, Iraq

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Date of Submission26-Oct-2019
Date of Decision22-Dec-2019
Date of Acceptance09-Feb-2020
Date of Web Publication28-Oct-2020


Background: Gallbladder stone is recently increased among the Iraqi society due to many risk factors such as bacterial infection and some HLA class II antigens. Aim(s): This study investigates the types of bacterial infection and HLA-DRB1 antigens' ratio that may be correlated with gallbladder stone formation. Setting and Design: The study included 45 patients and the same number of healthy individuals as a control group. Patients were with multiple gallstones. Gallstone bacterial culture was demonstrated to diagnose viable bacteria. HLA-DRB1 alleles' frequency was investigated using sequence-specific oligonucleotide probes (PCR-SSOP). Results: Irrespective of gallstone type and size, different types of living viable bacteria were isolated from the cores of the studied gallstones in 80% of the studied cases versus 20% of sterile gallstones. Gram-negative bacteria cultures were the dominant (89.3%), including Escherichia coli, Klebsiella spp., Proteus spp., Acinetobacter spp., and Enterobacter spp. Mixed infection of Gram-positive and negative bacteria was noted: Escherichia coli and Enterococus spp. and the others of Escherichia coli and Acitobacter spp., and Klebsiella spp. and Pseudomonas spp. Gram-positive bacteria cultures were also detected at lower rate (10.7%) including Staphylococci spp. The frequency of HLA-DRB1*03:01, HLA-DRB1*4:03, HLA-DRB1*13:22, and HLA-DRB1*15:10 alleles was significantly elevated in patients compared to the healthy control group. Conclusion: Results ensured the viability of the bacteria isolated from the core of gallstones and showed positive correlation between gallbladder stone and different bacterial infection. In addition, HLA-DRB1 alleles were significantly high in patients compared to healthy control group suggesting them as risk factors (P < 0.05).

Keywords: Gallstone, HLA Class II

How to cite this article:
Abdullah BH, Jassam SA, Hadi WA, Hameed B. Gallbladder stone formation in Iraqi patients is associated with bacterial infection and HLA class II-DRB1 antigens. Indian J Pathol Microbiol 2020;63:570-4

How to cite this URL:
Abdullah BH, Jassam SA, Hadi WA, Hameed B. Gallbladder stone formation in Iraqi patients is associated with bacterial infection and HLA class II-DRB1 antigens. Indian J Pathol Microbiol [serial online] 2020 [cited 2020 Nov 25];63:570-4. Available from: https://www.ijpmonline.org/text.asp?2020/63/4/570/299330

   Introduction Top

Gallbladder stone is one of the common gastrointestinal problems; it may cause different clinical conditions such as acute cholecystitis and jaundice or being asymptomatic and diagnosed accidentally. Gallstone is a fluctuated product; it may dissolve, grow in size, or even remodel its shape. Depending on the cholesterol ratio, they are chemically classified into three major groups: black pigment, brown pigment, and cholesterol stones besides the other categories including combined, mixed, and composite and complex. Brown pigment gallstone is mostly linked with a bacterial infection. The previous studies argued regarding the inducing factors that are involved in the formation of gallstone, including multi factors such as concentrated bile,[1] age, gender, hormonal therapy, obesity, and diabetes,[2] and genetic factors were suggested to play a crucial role, since 50%–70% of cholecystitis patients had positive family history.[3] However, the majority showed bacterial infection correlation, with gut organisms mainly E. coli and Bacteroides spp.[4] Immunological studies link some of Human leukocytes antigens (HLA) with gallstone formation such as: HLA-A-A3, HLA-A-A30, HLA-B5, HLA DRB1, and B-blood group; on contrary, HLA-B8, HLA-B14, HLA, and O-blood group are predisposed as genetic protectors of the cholecystitis.[1],[5] Recent studies referred to HLA-DRB1 as a key player in gallbladder stone formation in Iraqi society.[6] HLA class II are expressed on the cell membrane of immune cells such as macrophages, B lymphocytes, antigen-presenting cells, and dendritic cells,[6] and different DRB1 alleles are correlated with variety of disorders.[7] HLA class II genes code the immune-proteins. Those proteins play a key role in initiating the immune response; they are exclusively expressed on highly specialized cells as antigen-presenting cells, B cells, the epithelia of thymus, and specific endothelial cells.[8]HLA-DRB1 genes are cytogenetically located on the short arm of chromosome 6 position 21.32 and on the molecular location: base pairs 32, 439, 842 to 32, 445, 046.[9]HLA-DRB1 encode beta chain protein of DRB proteins; both molecules bind with another protein chain called alpha chain (DRA) to form the heterodimeric functional protein molecules of HLA-DRB1 proteins.[10] Hundreds of various alleles of HLA-DRB1 genes are nominated by the previous studies; each allele encodes a unique peptide that represents an immune antigen to initiate the immune response.[11] In our study, we aim to investigate the bacterial species that correlated to the formation of gallstone and test the HLA DRB1 alleles' frequency in Iraqi patients, to find out the burden of hereditary in this illness and its correlation with the environmental burden, which manifests itself with the bacterial infection.

   Patients and Methods Top

A total number of 45 patients with gallstone (s) were selected randomly from the outpatient clinic of Al-Yarmouk teaching hospital, and Medical City Hospital in Baghdad (surgery unit), and the same number of healthy individuals were comprised as a healthy control who were volunteers and had no family history of gallbladder stone, history of abdominal disorders, or recent illnesses. Patients included female and male patients. All patients were clinically diagnosed with symptomatic gallstone and had a history of pain in the upper quadrant and epigastric regions from the past 3 to 6 months; final diagnosis was based on the ultrasonographic examination that showed the presence of gallstone(s) combined with edematous gallbladder wall. Gallstones were obtained from all the patients through surgery. Later on, the stones were analyzed by a classical chemical method in which stones were collected and powdered in pestle and mortar. Patients and healthy control group were ethnically compatible, with an average age of 19–65 years.

Bacterial cultures

Gallbladder stones were surgically isolated from the gallbladder and transferred via transfer serial media to the Microbiology unit. The surface of stones was sterilized by washing stones with 70% ethanol for 5 min; the stone was bisected and the core was cultured for aerobic both Gram-negative and Gram-positive bacteria using the routine recommended media. The results of bacterial growth were grouped into three major groups: group A represents the positive growth, group B represents the negative growth (Sterile), and group C represents the contaminated plates (with fungal growth). Results were presented in percentage and t-student's test was used to analyze the statistical significance.

HLA class II-DRB1 genotyping

Genomic DNA was extracted from the collected whole blood samples using genomic Promega DNA extraction kit. The targeted locus was amplified via PCR multiplexer (Bio-Rad) using INNO-LiPA HLA-DRB1 Multiplex; 5 μl (10 ng) of the extracted genomic DNA product was mixed with 10 μl of amplification buffer, 10 μl of HLA-DRB1 multiplex primer solution, 1 μl of LiPA-Taq (2 U), and 24 μl of autoclaved deionized distilled water; the final volume of amplification mix was 45 μl. The cycling profile started with primary denaturation at 95°C/5 min, followed by 35 cycles of denaturation 95°C/30 s, annealing 58°C/20 s, and extending 72°C/20 s. It ended with elongation step for 72°C/10 min; PCR reaction was conducted using the thermal cycler multiplexer CFX96 Touch Real-Time PCR detection system-BioRad. HLA-DRB1 alleles were detected using a line probe assay via specialized kits of hybridization (INNO-LiPA HLA-DRB1 Plus from Fujirebio Europe®). Amplified DNA samples were introduced on the strips, and then strips were mounted on the tray of Auto-LiPA device. The strips were developed automatically and results were interpreted using LiRAS software (Fujirebio Europe®); test conditions were performed in accordance with the manufacturer's instruction (Hassan 2003).[12] One-way ANOVA test was used to analyze the distribution of targeted alleles in both patient and healthy control groups, the 95% confidence interval was employed, and all P value less than 0.05 was considered statistically significant.

Ethical approval

This study was approved by the Scientific and Ethical Committee of College of Pharmacy- Mustansiriyah University and the Ethical Committee of Teaching Hospital of College of Medicine, Nahrain University.

   Results Top

Results showed interesting findings regarding clinical characteristics and medical history data of studied cases; starting with the gender of patients, gallbladder stones were seen in females more than male with the incidence of 71% of female vs. 29% in male (P < 0.05). Interestingly, 78% of female patients were on oral contraception treatment compared to 22% of female patients not on contraception pills. In addition, obesity was observed in 52% of the patient group compared to 18% overweight in healthy control group (P < 0.05) [Table 1].
Table 1: Clinical characteristics of gallbladder stone patients

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The living bacteria in the core of gallbladder stone

Results of bacterial growth showed both Gram-negative and Gram-positive growth. The Gram-negative bacteria were shown more frequently (96%) than Gram-positive bacteria (4%). Escherichia coli was seen in 37% of the studied cases, while Klebsiella spp. was diagnosed in 21% of patients, Proteus spp. was found in 15% of the samples, Enterobacter spp. grew in 13% of samples, and Pseudomonas spp. and Acinetobacter spp. were observed in 7% and 5% of the samples, respectively. Staphylococci aureus was indicated in 4% of the cases as the only Gram-positive bacteria, P < 0.05 [Figure 1] and [Table 2].
Figure 1: The percentage of frequency of bacteria grouped according to their species. Escherichia coli and Klebsiella spp. were the highest risk factors of gallbladder formation, *= P < 0.05

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Table 2: Prevalence of bacterial growth isolated from gallbladder stones

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The HLA Class II association with gallbladder stone

DRB1 alleles were typed in both gallbladder stone patients and the healthy control group, via DNA analysis method (PCR-SSOP). The frequency of targeted alleles was grouped as in [Table 2]. Results showed that the frequency of allele HLA-DRB1*03:01 in patient group was statistically significantly higher than the healthy control group [14 (31.2%) vs. 2 (4%), P < 0.05]. Similarly, elevated levels of HLA-DRB1*4:03 in patient group were statistically significantly higher than the healthy control group [7 (15.5%) vs. 1 (2.2%)]. HLA-DRB1*13:22 were significantly higher in patients 13 (28.8%) compared to healthy control group 6 (13.3%). Furthermore, HLA-DRB1*15:10 was significantly raised in patients 9 (20%) against control 1 (2.2%), [Table 3] and [Figure 2].
Table 3: Frequencies of human leukocytes antigen (HLA.DRB1) alleles in patients with gallbladder stone and the healthy control groups

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Figure 2: The frequencies of human leukocytes antigen (HLA-DRB1) alleles. The bar chart shows the comparison between the frequency of HLA-DRB1 alleles in patients with gallbladder stone (black bars) and the healthy control groups (gray bars). In patient group, a significant increase was seen in the following alleles: HLA-DRB1*03:01, *04:03, *07:01, and *15:01. *= P < 0.05

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   Discussion Top

Interesting findings were shown through this study; according to the medical history and clinical characteristics, the highest incidence of gallbladder stone was seen in females with the incidence ratio of 71%. This supports Novacek findings,[11] who proposed that elevated levels of estrogen increased the secretion of biliary cholesterol and agreed with Hassan,[12] Imran et al.,[13] and Alishi et al.[14] who studied the Middle Eastern society, particularly in Iraq and the Kingdom of Saudi Arabia.

Our results showed that females were at a higher risk of gallbladder stone formation compared to males and obesity was observed in 52% of the patient group compared to 18% overweight in healthy control group, P < 0.05 [Table 1]. Nevertheless, results revealed a strong correlation between oral contraceptive and gallbladder formation; 78% of female patients were on (estrogen-progesterone) long-term intake birth control, and similar results were shown by the previous study that pointed to the linkage of high levels of estrogen and high incidence of gallbladder stone.[15]

The recent study manifested the existence of live bacterial cultures isolated from the core of gallbladder stones, and the results of bacterial growth included: Gram-negative bacterium which was the most frequent (96%) than Gram-positive bacteria (4%), similar to a study demonstrated in KSA pointed to the involvement of bacterial infection in gallbladder stone formation.[16] We found that Escherichia coli was predominantly seen in 37% of the studied cases, agreed with Liu et al. results in a 16S rRNA-based microbiome study, which diagnosed E. coli in the microbial flora of gallbladder stone patients.[17] Furthermore, Klebsiella spp. live cultures were seen in 21% of our patients, similar to Kose et al. findings proved that Klebsiella spp. was associated with cholesterol gallbladder stone based on bacteria cultivation and 16S rRNA profiling.[18] The recent study also detected Salmonella spp. in 15% of the studied samples. That was expected due to the high rate of typhoid fever in unvaccinated areas out of Baghdad. Moreover, the previous studies referred to the involvement of Salmonella typhi in gall bladder infection and stones.[19]Enterobacter spp. cultures were detected in 13% of our samples; however, the incidence was lower than the previous studies that showed slightly higher rate of infections caused by Enterobacter spp.[17],[19] Pseudomonas spp. isolates were observed in 7% of our samples, agreed with Abeysuriya et al. findings that pointed to Pseudomonas aeruginosa as a major cause of bacterial infection of gall bladder and bile stone formation.[19] We isolated a live Acinetobacter spp. isolates from 5% of the studied samples; Acinetobacter baumannii complex was prescribed as one of the gallbladder-stone associated flora.[18] In addition, Staphylococci aureus was indicated in 4% of our cases as the only Gram-positive bacteria [Figure 1] and [Table 2]. A recent study reported that coagulase-negative Staphylococcus spp. caused 15% of colitis and gall bladder infection.[20] Geographic, nutrition, medication, and ethnicity were previously described as risk factors of gallstone.[12],[13] Western societies were the most affected with the prevalence rate of 5.9% to 21.9% followed by Asian societies with prevalence rated between 3.1% and 10.7%.[20] Similarly, the genetic burden of gallstone formation was recently studied in families, identical twins, and linkage studies implied the correlation of genetic factors with gallstone formation.[21] Gallstone formation is a multifactorial condition; however, the most recent study referred to genetic alteration in the ABCB4 transporter that leads to lack of function to cause low phospholipid-associated cholelithiasis syndrome and bile saturation with cholesterol.[22] The involvement of immune system in the formation of gallstone is still not fully unveiled; however, the previous studies focused on biliary epithelial cells' role in forming cholesterol gallstone, via expressing toll-like receptors (TLR2, TLR4), MyD88, and nuclear factor κ B that were involved in innate immune response.[23],[24] Moreover, biliary epithelial cells were shown to interfere with inflammatory cells by expressing HLA-class I and HLA-class II.[25] In our study, we investigated the frequency of HLA-class II DRB1 alleles conducting lined probing, the most recent technology in immunogenetic diagnosis. A prominent frequency of allele HLA-DRB1*03:01 was noted in patient group compared to that of healthy control group, the difference was statistically significant [31.2% vs. 4%, P < 0.05]. Similarly, we found elevated levels of HLA-DRB1*4:03:7 (15.5%) in patient group compared to those (2.2%) of healthy group. HLA-DRB1*13:22 were significantly higher in patients (28.8%) compared to healthy control group (13.3%). Furthermore, HLA-DRB1*15:10 was significantly raised in patients (20%) against control (2.2%) [Table 3] and [Figure 2], suggesting mentioned alleles might be considered for gallstone formation associated risk factors. Nonetheless, HLA-DRB1 was mentioned to be elevated in cholecystitis patients[1] as well as HLA-DR expression was considered a major key player in gallbladder dysplasia, carcinoma, and metastatic carcinoma (Tamiolakis et al. 2003).[26] Overall, the data obtained from our work suggested bacterial infection as a significant risk factor of gallstone formation and also a strong correlation between gallstone formation and the following alleles: HLA-DRB1*03:01, HLA-DRB1*4:03, HLA-DRB1*13:22, and HLA-DRB1*15:10, which were significantly raised in patients compared to healthy control group.

   Conclusion Top

In the majority of cases, gallstone formation is a multifactorial disease, genetic mutations, and environmental interactions. The noticeable increase in the number of registered cases every year and the serious health complications caused by gallstones both wealth more attention than it is given.

Financial support and sponsorship

This work was funded by the Ministry of Higher Education and the Ministry of Health -IRAQ.

Conflicts of interest

There are no conflicts of interest.

   References Top

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Correspondence Address:
Baydaa H Abdullah
Mustansiriyah University, College of Pharmacy, Baghdad
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/IJPM.IJPM_841_19

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


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