Relevant studies were retrieved from Medline, PubMed, Embase, and Cochrane databases, with the last search being conducted in December 2021. A combination of search terms such as “Helicobacter pylori” or “H. pylori”, “gallstone disease”, “cholelithiasis”, “choledocholithiasis”, “cholangitis”, “gallbladder cancer”, “cholangiocarcinoma”, and “biliary tract cancer” was used. Manual retrieval of eligible studies from references mentioned in the review articles was also conducted. We will first draw the link between H. pylori and its relationship with the biliary system before delving into hepatobiliary condition-specific interactions with H. pylori.

For any infectious agent to cause an infection in the body, it must fulfill two fundamental principles—the ability to arrive at the said location and the ability to survive in that environment. Although predominantly a colonizer of the gastric mucosa, H. pylori has been detected in the biliary system, suggesting potential translocation and, by extension, the potential for pathogenicity. The two current prevailing theories suggest that H. pylori may enter bile either through retrograde reflux from the sphincter of Oddi or hematologically from the portal circulation [13,14].

After translocating to the biliary system, it is also important to consider how it can survive in a relatively alkaline environment. One possibility is that the reflux of bile from the duodenum into the stomach plays a role in selecting specific H. pylori strains that are resistant to bile salt [15]. Another possibility is that inflammation from biliary pathologies may inadvertently lower biliary pH leading to a more favorable environment for H. pylori [16]. This shows that H. pylori can reach and survive in the gallbladder. This provides both the means and the path for H. pylori, a known group 1 carcinogen to cause chronic inflammation and malignancy in the hepatobiliary system.

There are many techniques for detecting H. pylori, but no gold standard method exists [17]. The methods which directly demonstrate the presence of H. pylori in the biliary system include culture and histopathological examination of the gallbladder, bile culture, or culture of gallbladder mucosal scrapings. Indirect methods which confirm the presence of H. pylori include polymerase chain reaction (PCR) and serology. Depending on the method and type of sample used, detection rates vary. Table 1 [17-23] summarizes the advantages, disadvantages and accuracy of the current H. pylori detection techniques.

PCR has a high sensitivity in detecting H. pylori [24,25] by using primers of conserved genes. In addition, nested PCR (NPCR) has even higher specificity due to the amplification of a narrower sub-region [26]. This provides a much quicker and more specific way to detect the growth of H. pylori in samples as compared to histopathological analysis. However, the PCR method comes with typical flaws; it generates false-positive results due to non-specific primers [27,28]. NPCR further compounds the false positive rate because of its susceptibility to spray contamination [26,29].

Serological methods are relatively inexpensive, and most laboratories can perform them easily [30]. Furthermore, it is useful in situations where bacterial density is expected to be low [17]. However, serology-based diagnosis has been regarded as less reliable than testing gallbladder samples due to the cross-reactivity of antigens among the Helicobacter species themselves [31] as well as with Campylobacter organisms [32]. Furthermore, serology is not specific to H. pylori infection of the gallbladder as it only indicates the presence of H. pylori infection; and not necessarily within the gallbladder [33]. Thus, a clinical correlation of the symptoms present with the positive serology testing must be performed to determine if it is likely that H. pylori resulted in hepatobiliary infections.

Another method used to detect H. pylori in the gallbladder directly is histology. This is the most accurate and specific. The Giemsa stain is more routinely used among the different stains as it is simple and relatively inexpensive [23,34]. However, the sensitivity of histology is often affected by multiple factors, such as the site and pattern of colonization, previous antibiotic use, sample representativeness of the entire gallbladder, and pathology doctors’ diligence [17].

The H. pylori culture taken from bile or mucosal scrapings remains the definitive method for detecting the presence of H. pylori infection within the gallbladder. However, challenges arise in culturing viable H. pylori within the gallbladder. Studies that used frozen tissue to culture H. pylori have reported unsuccessful viable bacteria culturing from the gallbladder [35-37]. For example, in a study by Fox et al. [36], cultures taken from 46 subjects yielded no viable H. pylori, despite positive PCR results. This was attributed to the use of frozen specimens, which may have inadvertently undermined the viability of H. pylori. Furthermore, H. pylori is an oxygen-sensitive microaerophile that cannot survive under normal atmospheric oxygen tension. As such, this can further complicate and hinder the process of culturing and result in false negatives [38]. On the other hand, studies that directly inoculated tissue specimens onto a sterile culture medium could successfully culture H. pylori colonies from gallbladder mucosa [39,40]. This indicates that the presence of H. pylori DNA detected via PCR may not merely represent ‘dead’ material.

Given the present challenges to culture, it remains unknown whether H. pylori detection in the gallbladder, through other tests such as PCR and histology, represents an active invasion of the gallbladder or only enterohepatic circulation of the bacteria [33,41]. However, PCR technology remains promising and can become the gold standard in identifying H. pylori. There is also a need for better growth conditions for the culture of H. pylori from biliary samples as this would allow confirmation of the viability of H. pylori in the biliary system. Finally, microbial isolation via culture is inaccurate in patients treated with antibiotics based on a local antibiogram. For example, in a local audit of 262 acute cholangitis patients, only 95 patients (36.3%) had positive blood cultures [42].

Although less commonly known, H. pylori infection has been associated with cholelithiasis and cholecystitis. Various meta-analyses have examined the relationship between gallstones and H. pylori infection [43,44], and reported that patients with H. pylori infection of the gallbladder had a significantly higher risk of gallstones than the control group. Studies conducted by Zhang et al. [45] and Takahashi et al. [46] on the prevalence of gallstones following the eradication of H. pylori support this conclusion. In the study by Zhang et al. [45] involving 15,523 participants, authors reported that gallstone prevalence was significantly lower among H. pylori-eradicated patients compared with H. pylori-positive patients with no prior eradication (9.02% vs 9.47%; p < 0.0001). Takahashi et al. [46] found similar results with a sample size of 15,551 participants (6.08% vs 4.73%; p < 0.001). Thus, the possible role of H. pylori eradication in managing gallstone diseases should be investigated. Furthermore, based on the meta-analyses and studies that demonstrate a lower prevalence of gallstones in patients with prior H. pylori eradication, we can conclude that there is a possible link between H. pylori infection and gallstone disease. With H. pylori infection being easily treatable with antibiotics, it is important to recognize this association, especially in H. pylori endemic regions [47].

However, with the limitations of the current methods of diagnosis, it is difficult to determine if the H. pylori detected is truly from the gallbladder or the stomach. Neither urea breath tests nor serology can accurately detect H. pylori infection of the gallbladder. On the other hand, more specific detection methods, such as PCR require tissue samples and therefore are invasive. Consequently, they may not be practical unless the patient has a strong indication for cholecystectomy or invasive biliary tract procedures. We acknowledge that the reliability of these studies may be compromised by the method of detection.

In addition to being associated with a higher prevalence of gallstones, H. pylori infection can play a causal role in the pathogenesis of gallstones in three main ways. Firstly, H. pylori may act as a nidus for stone formation [38], providing a starting point for accumulating stones. Secondly, H. pylori infection of the gallbladder increases oxidative stress in the infected regions. Through the production of reactive oxygen species and reactive nitrogen species, which affects the absorptive and secretory function of the gallbladder, supersaturation of bile can occur, resulting in the formation of stones [39]. Lastly, H. pylori can increase the precipitation of calcium bilirubinate through its ability to produce urease. This enzyme increases the pH for calcium precipitation and induce enzymes that deconjugate bile [38]. However, these theories are limited by the inability to demonstrate active colonization of the gallbladder by H. pylori [39]. PCR remains the most used method for detecting H. pylori in gallbladder samples [48]. However it is unable to distinguish between live and dead bacteria, leading to the possibility of false-positive results [33]. Given that H. pylori can likely survive in the gallbladder, it is not unreasonable to suggest that this supports the possible presence of live H. pylori. However, most existing studies investigating the relationship between H. pylori infection and gallstones are cross-sectional and are therefore unable to establish a temporal relationship between gallstone formation and H. pylori infection [49].

To close the gaps in the existing literature, we suggest conducting prospective studies in two areas to investigate the causal relationship between H. pylori infection and gallstones. Firstly, to validate the results of the existing cross-sectional studies, we propose long-term follow-up studies of H. pylori-positive, H. pylori-negative, and H. pylori-eradicated patients. Secondly, prospective studies investigating the impact of eradicating H. pylori on gallstone prevalence and recurrence can be conducted. In summary, despite the difficulty of establishing causality at this time, future prospective studies and advancements in the detection methods may provide insight into the relationship between H. pylori infection and gallstones.

Determining the exact relationship between H. pylori infection and gallstones is important due to its implications on gallstone prevention and H. pylori eradication regimes. Given that both H. pylori infection and gallstones are common diseases, it is important to determine if eradicating H. pylori can prevent gallstones [47]. This may also inform the decision for prophylactic treatment in close contacts and routine screening for H. pylori infection. Furthermore, it is unclear whether existing regimens for eradicating H. pylori from the stomach are adequate for eradicating H. pylori from the biliary tract. With the increasing rate of antibiotic resistance to H. pylori infections, the optimum therapeutic regime for eradicating H. pylori from the gallbladder should be established to prevent further reductions in the efficacy of eradication therapies [47]. Therefore, it is crucial to establish the exact relationship between H. pylori infection and gallstones to determine if adjustment to existing treatment protocols is required.

There currently exists no proven association between H. pylori infection and gallbladder polyps. This could be attributed to the benign nature of gallbladder polyps which do not spark attention to its possible risk factors. Two retrospective case-control studies have investigated the relationship between H. pylori infection and gallbladder polyps, and reported conflicting results [8,50].

Xu et al. [8] reported a positive correlation between H. pylori infection and gallbladder polyps in a study including 17,971 participants. The H. pylori infection group had significantly higher incidence of gallbladder polyps than that of the control group (odd ratio = 1.160, p = 0.033). The formation of gallbladder polyps is widely believed to be due to an underlying chronic inflammatory process involving the gallbladder mucosa [50-52]. This study thus highlighted the possibility of H. pylori infection triggering a local inflammatory process and thereby contributing to an increased incidence of gallbladder polyps. On the contrary, in a study conducted by Zhang et al. [50] involving 5,107 participants, no significant correlation between H. pylori infection and gallbladder polyps was found (p = 0.110). While both studies used abdominal ultrasonography for diagnosis and considered the possible confounding effect of certain variables, such as age, sex, and body mass index, before data analysis, the method of accounting for the impact of such variables was done in two different ways (adjusted odds ratio vs. case-control matching). Due to the conflicting results of the two studies, no conclusion can be derived about the correlation between H. pylori infection and gallbladder polyps. Gallbladder polyps are an important risk factor for gallbladder cancer [53,54]. Further research, especially prospective studies, is important in clarifying whether H. pylori infection has a cause-and-effect relationship with gallbladder polyps and by extension, whether H. pylori eradication can help in its prevention.

H. pylori has been implicated in the pathogenesis of biliary tract cancers [55,56]. In a case-control study of 156 bile samples, Boonyanugomol et al. [10] detected a significantly greater prevalence of H. pylori in the bile samples of cholangiocarcinoma (CCA) patients (66.7%) as compared to cholelithiasis patients (41.5%) and the control group (25.0%) by PCR (p < 0.05 in both comparisons). Significantly more inflammatory changes at the portal zones were seen in CCA patients who tested positive for H. pylori, indicating a possible role of H. pylori in a preceding inflammatory process before the development of CCA. A case-control study by Hassan et al. [55] also compared the gallbladder mucosal histology of non-infected gallbladders to H. pylori-infected gallbladders and reported that there was a significant increase in mucosal hyperplasia (p = 0.028) as a well as metaplasia and dysplasia (p = 0.049) amongst H. pylori-infected gallbladders compared to non-infected gallbladders. These changes have been identified as precursor lesions of gallbladder cancers [57]. Based on these case-control studies, there is a positive association between H. pylori infection and biliary tract cancers.

Therefore, it is important to understand how H. pylori may result in biliary tract cancers as this will help to develop effective treatment. The ability of H. pylori to produce pro-oncogenic molecules such as CagA and VacA, in tandem with its promotion of a chronic inflammatory state, can result in an increased production of free radicals and the dysregulation of various proliferation pathways, like the nuclear factor kappa B (NF-κB) and JAK/STAT transcription pathway. Boonyanugomol et al. [10] had shown that the CagA gene was significantly higher in patients with CCA than with cholelithiasis (36.2% to 9.1%, p < 0.05), suggesting that it could be involved in the pathogenesis of CCA. CagA pathogenicity island is essential in the internalization of H. pylori in cholangiocytes [10]. This can lead to the activation and induction of various carcinogenic cascades. Moreover, an increased cell turnover through the H. pylori-induced inflammatory response can also result in an increased mutation rate. These chronic inflammatory processes predispose patients to the development of both gallbladder cancer and CCA [58,59].

A recent study by Wang et al. [60] identified H. pylori proteins with potential involvement in gallbladder cancer pathogenesis using a bioinformatics approach. Briefly, the UniProt database containing the entire H. pylori proteome was used to predict which H. pylori proteins may potentially target the nucleus of host cells. Through the localization of specific protein sequences called Nuclear Localisation Signal, it was possible to determine which of the 1,552 H. pylori proteins possessed nuclear targeting activity. Leveraging on this novel approach, future studies may employ similar techniques to identify proteins involved in the pathogenesis of other H. pylori implicated gallstone diseases. Furthermore, this opens new avenues for targeted therapy.

While these studies employed sensitive serological, PCR, and histopathological diagnostic methods, they are cross sectional, and thus a causal relationship between an H. pylori infection and biliary tract cancer could not be concretely determined. As a group, although biliary tract cancer are rare cancers, there is some evidence that it is rising [61]. Currently, surgery is the only chance of cure for biliary tract cancer, and there is a high recurrence rate even with adjuvant chemotherapy [62]. Given the poor prognosis of biliary tract cancer, further clarification on whether the eradication of H. pylori will reduce its prevalence is important [63]. The recommended test for H. pylori infection is a urea breath test [64]. While a significant proportion of patients who had a positive urea breath test also tested positive for H. pylori in their bile or gallbladder tissue by PCR, there is no evidence that a urea breath test would be able to prove whether H. pylori has been eradicated from the biliary tree [65]. Many such studies exclude patients with prior H. pylori treatment. Still, we believe that various non-invasive and invasive H. pylori tests could be compared in this group of patients to investigate their utility for a test of cure of H. pylori infection in the biliary tree.

It has been postulated that an infectious etiology such as H. pylori can result in primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) [66,67]. Nilsson et al. [25] reported a significant association between H. pylori infection with PSC and PBC. In a histological analysis of liver biopsies, 20/24 had PCR positivity for the Helicobacter genus, compared to 1/23 (p < 0.001). In this group of 20, 5 of 9 of the PSC patients and 4 of 11 of the PBC patients tested positive for H. pylori specific primers. Conversely, Boomkens et al. [68] did not detect any differences in the prevalence of PSC or PBC between the H. pylori positive and the control group (p = 0.783). While Nilsson’s control group included biopsies of healthy cadaveric livers, Boomkens’ results may have been less reliable as the choice of control may be a possible confounding factor as hepatitis B cirrhosis is associated with a concurrent H. pylori infection [69].

The etiology of PSC and PBC is elusive and with limited effective management strategies, outcomes are sub-optimal and this is an area of research interest [70]. We suggest conducting prospective studies on the causal relationship between H. pylori infections and PSC and PBC and long-term follow-up studies to study the effects of H. pylori eradication on the prognosis and outcomes of PSC and PBC (Fig. 1).

Table 2 summarizes the recent studies investigating the potential association between H. pylori infection and the presence of biliary diseases [8,14,25,39,45,46,50,55,65,68,71-85].