Confocal Laser Endomicroscopy in Gastrointestinal and Pancreatobiliary Diseases: A Systematic Review and Meta-Analysis

Confocal laser endomicroscopy (CLE) is an endoscopic-assisted technique developed to obtain histopathological diagnoses of gastrointestinal and pancreatobiliary diseases in real time. The objective of this systematic review is to analyze the current literature on CLE and to evaluate the applicability and diagnostic yield of CLE in patients with gastrointestinal and pancreatobiliary diseases. A literature search was performed on MEDLINE, EMBASE, Scopus, and Cochrane Oral Health Group Specialized Register, using pertinent keywords without time limitations. Both prospective and retrospective clinical studies that evaluated the sensitivity, specificity, or accuracy of CLE were eligible for inclusion. Of 662 articles identified, 102 studies were included in the systematic review. The studies were conducted between 2004 and 2015 in 16 different countries. CLE demonstrated high sensitivity and specificity in the detection of dysplasia in Barrett's esophagus, gastric neoplasms and polyps, colorectal cancers in inflammatory bowel disease, malignant pancreatobiliary strictures, and pancreatic cysts. Although CLE has several promising applications, its use has been limited by its low availability, high cost, and need of specific operator training. Further clinical trials with a particular focus on cost-effectiveness and medicoeconomic analyses, as well as standardized institutional training, are advocated to implement CLE in routine clinical practice.


Introduction
Confocal laser endomicroscopy (CLE) is an endoscopic modality that was developed to obtain very high magnification of the mucosal layer of the gastrointestinal (GI) tract [1], and it has the potential to enable histological diagnosis in real time [2]. CLE is based on tissue illumination using a lowpower laser and the subsequent detection of fluorescent light that is reflected back from the tissue through a pinhole [3]. The term "confocal" refers to the alignment of both illumination and collection systems in the same focal plane [4,5]. In brief, the laser light is focused at a selected depth in the tissue of interest and reflected light is then refocused onto the detection system by the same lens. Only the returning light that is refocused through the pinhole is detected. Any light that is reflected or scattered at other geometric angles from the illuminated object or any light that is refocused out of plane with the pinhole is excluded from detection. This dramatically increases the spatial resolution of CLE and enables cellular imaging and evaluation of tissue architecture at the focal plane to be performed during endoscopy [1].
To obtain confocal images, exogenous fluorescence agents can be administered either topically or systemically [6]. The most common topical contrast agents that are applied by a spraying catheter are acriflavine and cresyl violet [7], whereas the most widely used systemically administered fluorescent agent is intravenous fluorescein sodium. Fluoresceins are nontoxic, and their administration has been associated with only rare adverse events, including transient hypotension without shock (0.5%), nausea (0.39%), injection site erythema (0.35%), self-limited diffuse rash (0.04%), and mild epigastric pain (0.09%) [8].
There are two types of CLE: endoscope-based (eCLE) and probe-based (pCLE) endomicroscopy. To perform eCLE, a dedicated endoscope with a miniaturized confocal scanner integrated into the distal tip is employed [9]. This system was developed by Pentax (Tokyo, Japan) and enables simultaneous endoscopic imaging to be performed. Additionally, it frees the endoscopic working channel, and it can be used for targeted biopsies or combined enhancement techniques [9,10]. Images can be collected by sectioning down through the mucosa in 7 m increments to a depth of 250 m. Image stabilization is necessary to obtain good quality images [11]. The second system is pCLE (Cellvizio, Mauna Kea Technologies, Paris, France). In this system, confocal miniprobes can be passed down the accessory channel of any standard endoscope [12], providing rapid image capture and "stitching" of adjacent images to create a "mosaic image" in real time [11]. The advantages of pCLE are its versatility and the possibility of combining it with other imaging modalities such as virtual chromoendoscopy or magnification [9]. Image stabilization can be achieved by using a plastic cap on the endoscope tip. There are several types of probes that are characterized by different imaging depths, fields of view, and lateral resolutions (Supplementary Table S1 in Supplementary Material available online at http://dx.doi.org/10.1155/2016/4638683). More recently, a novel needle-based CLE (nCLE) miniprobe (AQ-Flex 19; Mauna Kea) has been developed which can pass through a 19G needle to perform endoscopic ultrasoundguided fine-needle aspiration (EUS-FNA) on solid organs and lymph nodes [13][14][15][16]. The probes can be reused after disinfection for as many as 10 to 20 examinations [1].
CLE can be applied to examine luminal structures, such as esophagus, stomach, and colon, and ductal structures, such as bile and pancreatic ducts. Ultimately, CLE can be used to optimize endoscopic diagnoses, thereby reducing unnecessary resections, avoiding repeated biopsies and unnecessary follow-up, and indirectly decreasing the risks and costs that are associated with repeated indiscriminative endoscopic exams. However, the interpretation of CLE images is still challenging. A standard classification system for distinguishing between normal and pathological GI conditions has only been developed for p-CLE devices; it was termed the Miami Classification, and it was based on a consensus that was reached among p-CLE users during a meeting that was held in Miami in 2009 [12]. Conversely, international recommendations and guidelines for other CLE systems have not yet been assessed.
The objective of the present systematic review is to summarize and analyze the current literature evaluating the sensitivity and specificity of this novel imaging modality (i.e., CLE) in detecting mucosal abnormalities. In assessing the available literature, we aimed to highlight the utility of CLE in the diagnosis of gastrointestinal and pancreatobiliary diseases, particularly in the screening or surveillance of dysplastic and neoplastic lesions, and to consider its potential future impact on daily clinical practice.

Methods
The methodological approach included the definition of search strategies, the development of selection criteria, an assessment of study quality, and the abstraction of relevant data. The PRISMA statements checklist for systematic review reporting was followed.

Study Inclusion Criteria.
The study selection criteria were defined prior to initiating data collection to enable the proper identification of eligible studies for inclusion in the analysis.
The search was restricted to studies that were performed in humans and that were published in English. Prospective and retrospective clinical studies were both eligible for inclusion, and there were no limits based on trial duration. Review articles, case reports, commentaries, editorials, letters, and conference abstracts were not considered. Likewise, ex vivo studies were excluded. Endoscopic applications of CLE were only considered if they were being used to evaluate the following types of diseases/lesions: Barrett's esophagus; squamous cell carcinoma; esophagitis; gastroesophageal reflux disease; gastric polyps; gastric atrophy and reactive metaplasia, dysplasia, and neoplasia; Helicobacter pylori-related gastritis; inflammatory bowel disease (IBD); celiac disease; colonic neoplasm; colonic polyps; biliary duct disease; and benign or malignant pancreatic diseases. To be eligible for inclusion, a study must have included at least one of the following outcomes: sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), accuracy, description of CLE indications, or mucosal features found using CLE.

Literature Search Strategy.
A literature search was performed using the following online databases: MEDLINE (through PubMed), EMBASE, Scopus, and Cochrane Oral Health Group Specialized Register. A grey literature search was also performed by using the OpenGrey database. To increase the probability of identifying all relevant articles, a specific research equation was formulated for each database, using the following key words and/or MeSH terms: confocal laser endomicroscopy, CLE, endomicroscopy, gastrointestinal, esophagus, esophageal, bile duct, biliary, gastric, colon, colic, pancreatic, and pancreas. Additionally, reference lists from eligible studies and relevant review articles (not included in the systematic review) were cross-checked to identify additional studies.

Study Selection and Quality Assessment.
The titles and abstracts of the retrieved studies were independently and blindly screened for relevance by two reviewers (Alessandro Fugazza and Federica Gaiani). A full article was retrieved when the information in the title and/or abstract appeared to meet the objective of this review. To enhance sensitivity, records were removed only when both reviewers excluded the record at the title screening level. All disagreements were resolved through discussions with a third and fourth reviewer (Nicola de' Angelis, Michaël Lévy). Subsequently, two reviewers (Alessandro Fugazza, Federica Gaiani) independently performed a full-text analysis and quality assessment of the selected articles. The Cochrane criteria, which are described in the Cochrane Handbook for Systematic Reviews of Interventions [17], were applied for randomized clinical trials, and the Newcastle-Ottawa Scale (NOS) [18] was used for nonrandomized studies.

Data Extraction and Analysis.
The data that were extracted from the studies for inclusion in the systematic review were processed for qualitative and possibly quantitative analyses. The data were collected and summarized based on whether CLE was applied for gastrointestinal or pancreatobiliary diseases. Data that would enable us to perform "per patient," "per biopsy," and "per lesion" analyses were separately extracted whenever available. A "per patient" analysis was performed by comparing each patient's final CLE outcome against their histopathological diagnosis; similarly, "per biopsy" and "per lesion" analyses were conducted by comparing images produced by CLE with the results of histologic biopsies. All data extraction was performed by one author (Alessandro Fugazza) and was verified by a second author (Federica Gaiani), and a 100% rate of final agreement was maintained.
To perform quantitative analysis, data on sensitivity, specificity, PPV, NPV, and main findings were extracted from the eligible studies. If necessary and possible, outcome variables were calculated by the authors based on the data that were available in individually selected studies. Only on-site (i.e., real-time) data of CLE imaging analysis were considered. Meta-analyses were performed using MetaDisc (version 5.2; Clinical Biostatistics Unit, Hospital Ramon y Cajal, Madrid, Spain) [19]. Heterogeneity was assessed using 2 statistics.

Results
All database searches were performed without time limit until January 2015. Overall, the combined search identified 662 articles (after removing duplicates); of these, 348 were rejected based upon title and abstract evaluation. Out of the remaining articles, which underwent full-text evaluations, 212 were excluded because they either were not pertinent to the review questions, had nonrelevant study designs, or had language limitations. A final total of 102 articles were considered to be eligible for the systematic review and were evaluated by both qualitative and quantitative analyses. A flowchart of the study's identification and inclusion/exclusion processes is shown in Figure 1. . The studies that were included  were published between 2004 and 2015. They included 8  RCTs, 85 prospective studies, and 9 retrospective studies. Twenty-eight (27.4%) studies were multicentric, with a maximum of 8 centers included. The studies were conducted in 16 different countries. The included single-center studies were conducted in Asia ( = 33), Europe ( = 31), USA ( = 7), and Oceania ( = 3); the multicentric studies were conducted in Europe + USA ( = 15), Europe + Asia ( = 2), Europe ( = 5), and the USA ( = 6).

Study Characteristics
Overall, the 102 included studies enrolled a total of 6943 patients; the number of patients per study ranged from 4 to 1572, with a median sample of 67.4 patients. The median patient age was 56.9 years (range: 0.7 to 90 years). All of the patients that underwent CLE were administered intravenous fluorescein prior to the procedure.
The outcomes produced by CLE technology are hereafter classified by organ of application, including esophagus, stomach, pancreas, biliary tree, and colon.

Stomach and
Duodenum. The use of CLE in patients presenting with gastric disease has raised great interest, particularly in Asian countries, where these pathologies are highly prevalent. The primary applications of CLE with respect to the stomach and duodenum have included the detection of polyps and neoplastic lesions and the study of gastritis and metaplastic lesions [37,41,43,44,46,47,49,51,52,54,56]; the application of CLE has also shown utility in patients with celiac disease [60][61][62] and Helicobacter pylori-related gastritis [58] ( Table 2). The performance of this innovative technique has been evaluated both alone and in comparison or in addition to other methods, such as endoscopic ultrasound (EUS) and narrow band imaging (NBI); however, the majority of these studies had descriptive aims and focused on the utility of employing CLE in targeting biopsies [40,48]. With respect Table 1: Summary of the studies applying CLE for esophageal lesion screening and diagnosis ("per biopsy" or "per patient" analysis).
Author n/a n/a n/a n/a n/a Endoscopists with minimal experience in CLE can effectively use this technology for targeted biopsy, decreasing the need for intense tissue sampling without lowering the diagnostic yield in detecting dysplasia. n/a n/a n/a n/a n/a Scoring and quantification of CLE images may be useful for the differential diagnosis and determination of superficial invasion by squamous cell carcinoma.
Author n/a n/a n/a n/a n/a n/a CLE could be a new screening tool for early detection of malignancy. n/a n/a n/a n/a n/a n/a CLE observations of vascular architecture may be assistant in the identification of early gastroesophageal cancer.

Kitabatake
Gheorghe et al. 2009 [40] Romania 11 patients 59.7 n/a n/a n/a n/a n/a CLE and endoscopic ultrasound are successfully associated for histological assessment, disease staging, and optimal therapeutic decision. n/a n/a n/a n/a 94.2 CLE demonstrates a high diagnostic accuracy for gastric epithelial neoplasia.
The use of CLE could possibly reduce the number of unnecessary biopsies and mistaken diagnoses before endoscopic submucosal dissection. n/a n/a n/a n/a n/a CLE allows functional imaging of mucosal barrier defects. Gastric intestinal metaplasia is associated with an impaired paracellular barrier irrespective of H. pylori eradication. n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a CLE with targeted biopsies is superior to white-light endoscopy with standard biopsies for the detection and surveillance of gastric intestinal metaplasia. n/a n/a n/a CLE can accurately show the histological severity of H. pylori infection-associated gastritis. n/a n/a CEL offers the prospect of diagnosis of celiac disease during ongoing endoscopy.
It also enables targeting biopsies to abnormal mucosa and thereby increasing the diagnostic yield, especially when villous atrophy is patchy in the duodenum.
stands for the number of patients enrolled in the study; n/a, not available or not applicable; pCLE, probe-based confocal laser endomicroscopy; CLE, confocal laser endomicroscopy; PPV, positive predictive value; NPV, negative predictive value; and BE, Barrett's esophagus. n/a n/a n/a n/a n/a CLE is reliable for real-time assessment of inflammation activity in UC by evaluation of crypt architecture, microvascular alterations, and fluorescein leakage.
Liu et al. 2011 [66] Canada USA 57 patients 46.6 n/a n/a n/a n/a n/a Epithelial gap density visualized by CLE is significantly increased in patients with IBD compared with controls. n/a n/a n/a n/a n/a CLE allows the analysis of the subsurface structure of lymphoid follicles, those surrounded by a red ring may represent an early marker of CD.

Moussata
Neumann et al. 2012 [70] Germany 72 patients 39 n/a n/a n/a n/a n/a CLE has the potential to significantly improve diagnosis of CD compared with standard endoscopy.
Turcotte et al.
2012 [71] Canada, USA 41 patients 41.1 n/a n/a n/a n/a n/a Increased epithelial gaps in the small intestine as determined by pCLE are a predictor for future hospitalization or surgery in IBD patients.

Musquer et
al. 2013 [72] France, USA 16 patients 35.5 n/a n/a n/a n/a n/a CLE allows quantitative analysis of colonic pit structure in healthy and CD patients. n/a n/a n/a n/a n/a Molecular imaging with fluorescent antibodies has the potential to predict therapeutic responses to biological treatment in CD and autoimmune or inflammatory disorders.
Buda et al. 2014 [74] Italy, Germany, and UK 38 patients 52 n/a n/a n/a n/a  n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a CLE demonstrates higher sensitivity than chromoendoscopy with similar specificity in differentiating colorectal polyps. n/a n/a n/a n/a n/a 89.6 The proposed software for automated classification of pCLE videos of colonic polyps achieves high performance, comparable to that of offline diagnosis of pCLE videos established by expert endoscopists. n/a n/a n/a n/a n/a n/a Imaging of blood vessels with CLE is feasible in normal and tumor colorectal tissue by using fluorescently labeled antibodies targeted against an endothelial marker. The method could be translated into the clinical setting for monitoring of antiangiogenic therapy.
Coron et al. 2012 [92] France USA 16 patients 13 lesions 62 n/a n/a n/a n/a n/a n/a n/a n/a n/a By p-CLE, it is possible to identify specific crypt architecture modifications associated with changes in cellular infiltration and vessels architecture, highlighting a good correspondence between p-CLE features and histology. Shahid   n/a n/a n/a n/a n/a 97.6 CLE has the potential to enable an immediate diagnosis of CRC and the degree of differentiation of CRC during ongoing endoscopy in vivo.

Liu et al. 2013 [100]
China 37 patients 37 lesions 70 n/a n/a n/a n/a n/a CLE could be used in molecular imaging with specific targeting of EGFR in colorectal neoplasia. n/a n/a n/a n/a n/a Based on CLE analysis of IBS patients with a suspected food intolerance, exposure to candidate food antigens caused immediate breaks, increased intervillous spaces, and increased IELs in the intestinal mucosa.   Figure 6). CLE has also been widely applied to describe IBD morphologic features, although it was not possible to perform a quantitative analysis of this application. In this particular indication, CLE appears to be a safe and feasible diagnostic tool for imaging bowel morphology, assessing disease activity, and predicting therapeutic responses.

Biliary Duct.
In biliary tract and pancreatic cysts, routine forceps are not accurate for sampling and pathology exam fails to address diagnosis. CLE with in situ diagnosis might be a valuable alternative. However, few studies have been carefully conducted.

Study Quality Assessment.
Based on the GRADE system, the overall quality of the evidence included in our analysis was judged as low (10 studies had a very low level of quality; 59 were low; 31 were moderate; and 2 were high). The risk of bias in the comparative randomized and nonrandomized studies was evaluated by two independent reviewers (Alessandro Fugazza, Federica Gaiani) based on Cochrane and NOS criteria. Specifically, two RCTs [55,77] were classified as having a low risk of bias, and 6 RCTs had a high risk of bias [22,25,27,28,31,54] (Table S2). Based on NOS criteria, 5 studies [29,52,62,70,74] were classified as having a low risk of bias, and 17 [36, 39, 48, 59-61, 63, 66-69, 71, 72, 78, 94, 106, 112] had a high risk of bias (Table S3).

Discussion
To the best of our knowledge, this is the first systematic review to analyze the diagnostic accuracy of CLE across all of the applications for which it has been used. A crucial difference between CLE and alternative endoscopic techniques is that CLE does not merely identify a lesion, but it also enables the discrimination of benign or malignant features via direct and immediate microscopic investigation, which can be performed simultaneously with endoscopic examination [119]. Despite this, CLE is still viewed as an expensive tool that requires standardized criteria for the diagnosis of select pathologies and needs specific training to interpret CLE images before it can be implemented as a routine diagnostic tool.

CLE in the Esophagus.
Several studies have validated the diagnostic and therapeutic role of CLE with respect to premalignant lesions and cancers of the upper GI tract. In a feasibility study, the use of pCLE demonstrated an up to 92% accuracy rate in detecting malignant and premalignant modifications of GI mucosa compared to conventional histopathology [120]. However, the primary application of CLE in esophageal tissue has been for the detection of high-grade dysplasia and cost-effective treatment strategies of BE. In particular, CLE has been used for follow-up of BE patients presenting with HG dysplasia and to define the lateral extent of neoplasias prior to therapy. It has been demonstrated that when using endomicroscopy for the surveillance of BE, the number of required biopsies can be significantly decreased by up to 87% [22], as this n/a n/a n/a n/a n/a pCLE of the CBD via the GastroFlex UHD miniprobe is feasible and may offer improved image quality over the standard CholangioFlex probe.   n/a n/a n/a n/a n/a nCLE is the pancreas is technically feasible. Konda     technique produces a higher diagnostic yield for the detection of neoplasia compared to random biopsy. This improved yield directly results from the need to sample only the suspicious areas that have been identified by CLE [22,31]. However, what the optimal surveillance biopsy procedure is in BE patients remains unclear. Current surveillance programs, such as the four-quadrant Seattle biopsy protocol, are relatively expensive and time-consuming [29]. The Preservation and Incorporation of Valuable Endoscopic Innovation (PIVI) initiative that was recently implemented by the American Society of Gastrointestinal Endoscopy (ASGE) [121] recommends that, before replacing the current Seattle protocol, a targeted imaging technique should have a per patient sensitivity of at least 90%, an NPV of at least 98%, and a specificity of at least 80% in the detection of high-grade dysplasia or early adenocarcinoma. The present meta-analysis demonstrated that CLE yields a "per biopsy" pooled sensitivity of 58% and a pooled specificity of 90%, which was slightly increased to 79% sensitivity in the "per patient" analysis. Therefore, based on the PIVI initiative requirements, using CLE for the surveillance of BE does not appear to be sensitive enough to replace the Seattle biopsy protocol. However, a recent multicenter RCT showed that combining CLE with highdefinition WLE surpassed the PIVI threshold, with a per patient sensitivity of 95%, an NPV of 98% and a specificity of 92% [31,122]. Thus, the combined use of CLE with highdefinition WLE or NBI may be considered a valuable diagnostic tool for premalignant and malignant lesions. Nevertheless, prospective medicoeconomic studies have yet to be conducted.

CLE in the Stomach and
Duodenum. CLE has been used for the description and detection of several gastric diseases, including polyps, metaplasia, and neoplastic lesions, as well as for the surveillance of gastric resections, Helicobacter pylorirelated gastritis, and celiac disease. However, knowledge surrounding the application of CLE to the stomach remains limited (although promising), and further clinical trials are needed to support the clinical impact of employing CLE in the diagnosis and management of total gastric atrophy. The usefulness of CLE is especially evident with regard to targeting biopsies of specific pathological mucosal areas. Indeed, a recent prospective study comparing NBI, chromoendoscopy (CE), and CLE for the diagnosis of atrophic gastritis found NBI to be equivalent to CE in classifying gastric pits, whereas CLE had higher sensitivity, specificity, and accuracy than CE [53]. Furthermore, with respect to the detection of gastric intestinal metaplasia (GIM), CLE demonstrated high diagnostic yields and a substantial superiority over conventional endoscopy; with CLE, the number of biopsies needed to confirm GIM was about one-third of what was needed compared to using WLE and standard biopsies [50,55] also suggesting a gain of time. This may be also the case in those patients at very high risk of early carcinoma (Lynch syndrome, CDH mutated individuals).
CLE has also been used for descriptive purposes; a blinded prospective study investigating gastric pit patterns provided an obvious distinction between normal mucosa, chronic inflammation, atrophy, and neoplastic mucosa and demonstrated a sensitivity and specificity for predicting gastric atrophy of 83.6% and 99.6%, respectively, whereas the corresponding values for predicting gastric cancer were 90.0% and 99.4% [123].
With respect to gastric polyps and neoplastic lesions, CLE has demonstrated several promising applications, including distinguishing between adenomas and hyperplastic polyps [42], the identification of microvascular patterns [39], followup after resection [45], and the diagnosis of cancer at different stages. The majority of studies have indicated that CLE has high diagnostic yields, and elevated interobserver agreement rates were evident in most cases [42]. However, CLE accuracy can be limited by the acquisition of good quality images [38], which may not be always possible. The present meta-analysis demonstrated that CLE yields remarkable pooled sensitivity and specificity, reaching values of 85% and 99%, respectively, in a "per patient" analysis.
Interestingly, CLE has also been applied to assess duodenal histology in patients with celiac disease. Employing CLE offers the prospect of diagnosing celiac disease during ongoing endoscopy and enables targeting biopsies to be performed in abnormal mucosa, thereby increasing diagnostic yield. However, although CLE appears to be sensitive and specific at detecting increased numbers of intraepithelial lymphocytes and villous atrophy, the evidence that CLE is effective for such applications remains scarce. More likely, CLE methodology would need to be improved before it could be routinely used in patients with celiac disease.
Overall, applying CLE to examine the stomach and duodenum demonstrated high sensitivity, specificity, accuracy, and positive and negative predictive values in comparison with both histopathology and other endoscopic techniques (e.g., WLE, NBI, and CE). However, these data are based on a limited number of publications and therefore caution should be used when interpreting their results.

CLE in the Colon.
The increasing incidence of colorectal cancers in parallel with the increased use of colonoscopy screening necessitates continued improvements in diagnostic accuracy and precocity. Therefore, the advent of CLE has been considered to be an important and valuable innovation for the management of colorectal neoplasms and inflammatory diseases [77].
Based on the present meta-analysis, the use of CLE in the detection of colorectal neoplasms and malignant foci in polypoid lesions is associated with a pooled sensitivity of 83% and a pooled specificity of 90%. This confirms the robust diagnostic power of CLE that has been observed in previous studies. Moreover, when compared to NBI, pCLE exhibited higher sensitivity (86% versus 64%) and similar overall accuracy (82% versus 79%), although it also exhibited lower specificity (78% versus 92%) [96]. Interestingly, the diagnostic accuracy of CLE does not appear to be influenced by operator expertise in the evaluation of confocal images; in contrast, learning how to perform CLE appears to involve only a short learning curve [102]. In addition to its role as a diagnostic tool, CLE is useful for evaluating the presence of residual tumor left behind following endoscopic treatment of colon polyps and does not require waiting for biopsy results. If there is an eventual need for reintervention via a complementary resection, it can therefore be carried out during a single endoscopic session [97].
CLE also demonstrated high applicability and superiority over standard endoscopy in the study of IBD. In particular, CLE can be used in the assessment of disease activity [70], in the prediction of relapse [71,74], and in the description of mucosal alterations such as epithelial gaps, all of which are useful toward enhancing the comprehension of new pathogenic features that develop in patients with IBD [66]. However, the detection of neoplastic transformations in the background of chronic inflammation in IBD patients remains highly challenging. Conversely, using CLE in patients with IBD is promising approach, as it offers the possibility of directly observing microvessels by immunostaining and therefore may serve as a foundation for the development targeted antiangiogenic therapies [91,101].
Applying CLE to patients GVHD, infectious colitis and irritable bowel syndrome has been less extensively studied; however, this technique has demonstrated good performance in these indications (100% specificity and less invasiveness in comparison with standard diagnosis technique), although its standards remain outside of current guidelines [103,106].

CLE in the Biliary Duct.
With respect to the biliary duct, the addition of CLE to histological examination results in a significant increase in diagnostic reliability [114]. Malignant pancreatobiliary strictures are difficult to diagnose, and up to 30% of patients with cholangiocarcinoma have negative sampling [111]. This is because, unlike other tumors, the majority of cholangiocarcinomas grow along bile duct walls rather than radially forming a mass [124]. Currently, biliary strictures are staged using a combination of endoscopic ultrasound and advanced imaging techniques, such as computed tomography (CT), magnetic resonance imaging (MRI), or EUS, whereas endoscopic retrograde cholangiopancreatography (ERCP) is typically used for tissue sampling, including biopsy and cytological brushing. However, the current sensitivity of each of these methods is quite low, ranging from 20% to 60% [125][126][127].
The present meta-analysis demonstrated that combining CLE with ERCP yields high sensitivity (90%) in the assessment of biliary strictures. This supports the ASGE guidelines and demonstrates that CLE is a useful tool for differentiating benign from malignant biliary strictures in patients with biliary neoplasia [128].

CLE in the Pancreas.
The accurate diagnosis of pancreatic cystic neoplasms continues to be problematic despite technological improvements [117]. The application of an optical needle biopsy by using nCLE may significantly improve the discrimination of mucinous and nonmucinous cysts. Based on 2 studies that were included in the meta-analysis, CLE yields a pooled sensitivity of 68% and pooled specificity of 90%, which might be suboptimal and influenced by the high heterogeneity that was observed between the studies. However, the current standard diagnostic techniques, such as imaging, EUS, fluid analysis (e.g., chemistry, tumor markers), and cytology [14,118], which when combined lead to a correct diagnosis in not more than 79% of cases, are also far from being satisfactory diagnostic procedures [129].
It should be noted that the literature regarding the use of nCLE for the diagnosis of pancreatic cystic neoplasms is scarce [14,117] and is mainly focused on serous cystadenoma [118]. Further studies are needed to improve nCLE diagnostic power and accuracy.

Study Limitations and Future
Directions. The present systematic review and meta-analysis attempted to summarize the current literature on the applications of CLE in gastrointestinal and pancreatobiliary diseases. Although a considerable number of studies were retrieved overall, the total evidence per organ is rather scarce and is often too low to draw definitive conclusions. Moreover, high heterogeneity was observed in many of our pooled data analyses, which indicates that caution is required when interpreting their results. Finally, the included studies were primarily conducted in specialized centers; thus, CLE outcomes cannot be generalized to tertiary care centers or nonspecialized institutions.
Despite these limitations, the present systematic review and meta-analysis highlights the novel and unique advantages of using CLE to provide real-time histological examination during diagnostic and therapeutic procedures. Future clinical trials should aim to improve the diagnostic accuracy of CLE in all of its possible applications, to institutionalize training programs to standardize the interpretation of CLE images, and to reduce procedure related costs and limitations to increase its application. By improving and implementing CLE techniques during routine clinical practice, we believe that in the near future CLE not only will become part of the diagnostic arsenal of the gastroenterologist/endoscopist but also may find application in related fields such as minimally invasive surgical techniques.

Conclusions
In gastrointestinal and pancreatobiliary diseases, endoscopyassociated new technologies should offer the possibility to make clear diagnosis when routine procedures make it difficultly be cost-effective with clear impact on the choice of endoscopy versus surgical therapies for macroscopic lesions and achieve early detection of malignancies in those individuals with very high risk of cancer development. CLE is one of these new technologies able to address the challenge. The overall sensitivity, specificity, accuracy, and predictive values of CLE are favorable and were often found to be superior in comparison with standard endoscopy plus histopathology. However, the widespread use of CLE remains limited by its low availability, high costs, and need for trained personnel. Moreover, there is a need for further clinical trials, including medicoeconomic evaluations, to assess the applicability and implementation of CLE in routine clinical practice, as currently very few such studies exist.

Conflict of Interests
None of the authors have a conflict of interests to disclose in relation to the present systematic review.