Dental Pulp Vascular Response to Early Stages of Caries.

OBJECTIVES: During caries progression, dental pulp is increasingly pathologically affected. Since the accurate assessment of pulp is of vital importance in clinical decision-making, this study aimed to evaluate pulpal condition in the early stages of caries via laser Doppler (LD) flowmetry and histologic analysis and determine their agreement. METHODS: Fourteen patients with severe dental crowding were included. Prior to extractions and orthodontic treatment, dental pulp condition of 52 premolars was evaluated via LD flowmetry. Teeth were assessed for the presence of caries and lesions were graded according to the International Caries Detection and Assessment System (ICDAS). After extractions, teeth were split and histologically stained for endothelial cells with anti-von Willebrand factor and Movat pentachrome for collagen. Volume densities of vessels (Vvasc) and collagen were calculated. RESULTS: There was a significant negative correlation between LD flow and Vvasc of the dental pulp with ICDAS grade. Pulpal LD flow and Vvasc in teeth with the initial lesion were increased, decreasing with progressing stages of caries. A significant positive correlation between the the pulpal LD flow and Vvasc, and a negative correlation of LD flow with collagen fibre density were noted. CONCLUSIONS: Caries affects the physiology of the dental pulp, initially with increasing vascularity, and decreasing vascularity at later stges of caries progression. Collagen contents increase with grades of ICDAS. LD flow shows good agreement with the histologic constitution of the dental pulp. Use of clinical measurements of pulpal LD flow could provide a good noninvasive indication of pulpal vascular state and its health.

Could Capsule Endoscopy Be Useful in Detection of Suspected Small Bowel Bleeding and IBD-10 Years of Single Center Experience.

A retrospective study in patients who underwent video capsule endoscopy (VCE) between 2006 and 2016 was conducted in the Clinic for gastroenterology and Hepatology, University Clinical Center of Serbia. A total of 245 patients underwent VCE. In 198 patients the indication was obscure gastrointestinal bleeding (OGIB), with 92 patients having overt and the other 106 occult bleeding. The remaining 47 patients underwent VCE due to suspected small bowel (SB) disease (i.e., Von Hippel-Lindau syndrome, familial adenomatous polyposis, Peutz Jeghers syndrome, Crohn's disease, prolonged diarrhea, abdominal pain, congenital lymphangiectasia, protein-losing enteropathy, tumors, refractory celiac disease, etc.). VCE identified a source of bleeding in 38.9% of patients (in the obscure overt group in 48.9% of patients, and in the obscure occult group in 30.2% of patients). The most common findings were angiodysplasias, tumors, Meckel's diverticulum and Crohn's disease. In the smaller group of patients with an indication other than OGIB, 38.3% of patients had positive VCE findings. The most common indication is OGIB, and the best candidates are patients with overt bleeding; patients with IBD should be evaluated in this setting.

Assessment of dental pulp response to carries via MR T2mapping and histological analysis.

OBJECTIVES: The aim of our study was to assess the correlation between T2 relaxation times and their variability with the histopathological results of the same teeth in relation to caries progression. MATERIALS AND METHODS: 52 extracted permanent premolars were included in the study. Prior to extractions, patients underwent magnetic resonance imaging (MRI) scanning and teeth were evaluated using ICDAS classification. Pulps of extracted teeth were histologically analysed. RESULTS: MRI T2 relaxation times (ms) were 111,9 ± 11.2 for ICDAS 0, 132.3 ± 18.5* for ICDAS 1, 124.6 ± 14.8 for ICDAS 2 and 112. 6 ± 18.2 for ICDAS 3 group (p = 0,013). A positive correlation was observed between MRI T2 relaxation times and macrophage and T lymphocyte density in healthy teeth. There was a positive correlation between vascular density and T2 relaxation times of dental pulp in teeth with ICDAS score 1. A negative correlation was found between T2 relaxation times and macrophage density. There was a positive correlation between T2 relaxation time variability and macrophage and T lymphocyte density in teeth with ICDAS score 2. In teeth with ICDAS score 3, a positive correlation between T2 relaxation times and T2 relaxation time variability and lymphocyte B density was found. CONCLUSION: The results of our study confirm the applicability of MRI in evaluation of the true condition of the pulp tissue. CLINICAL RELEVANCE: With the high correlation to histological validation, MRI method serves as a promising imaging implement in the field of general dentistry and endodontics.

Inflammatory Bowel Disease: From Conventional Immunosuppression to Biologic Therapy.

BACKGROUND: Inflammatory bowel diseases (IBDs) are chronic, recurrent inflammatory diseases with partly understood etiology and pathogenesis. The course of IBD, both ulcerative colitis and Crohn's disease, is characterized by periods of relapse and remission with the possible occurrence of extraintestinal manifestations. SUMMARY: During the last decades, therapeutic goals in IBD evolved toward endoscopic remission and mucosal healing creating the need for early administration of disease-modifying agents (DMAs). DMAs include conventional immunosuppressants (thiopurines, methotrexate), biologic drugs (anti-TNF, anti-integrin, and anti-IL-12/23 monoclonal antibodies), and small molecules (JAK inhibitors, S1P receptor modulators). Patients with aggressive course of disease and risk factors for poor prognosis should be treated with biologic therapy early, while conventional immunomodulators should be used in those with milder course of disease in the absence of risk factors. KEY MESSAGES: Challenges in the treatment of IBD patients include the choice of effective yet safe drug and prevention or overcoming loss of response.

Expression of CD31, CD34, and smooth muscle actin (SMA) in endothelial cells of dental pulp vessels.

The dental pulp is a highly vascularized and innervated loose connective tissue surrounded by hard dental tissues - enamel and dentine. With the primary dentin formation and the closure of the root apex, the conditions in the dental pulp change and pulp tissue compliance are reduced. Endothelial cells of pulpal blood vessels are highly differentiated and are capable of adaptation to changes in the environment. We aimed to evaluate the phenotypic plasticity of endothelial cells of pulpal blood vessels in permanent premolars with open (N = 6) or closed root apex (N = 30). The pulp tissue was stained with hematoxylin-eosin (HE) for the histological analysis, and immunohistochemically for a cluster of differentiation 31 (CD31), a cluster of differentiation 34 (CD34), and for smooth muscle actin (SMA) to detect vessels with CD31, CD34, and SMA positive endothelial cells. We used the student's t-test and Pearson correlation test for the statistical analysis. We found a significantly higher percentage of the vessels with CD31 (P = 0.005) and CD34 (P = 0.000) positive endothelial cells in the group of teeth with closed root apex compared to the group with open apex. A significant positive correlation between SMA positive and CD31 positive vessels (P = 0.003) and between CD31 positive and CD34 positive vessels (P = 0.031) was also found. We conclude that the endothelial cells of dental pulp vessels express a small amount of CD31, but have a pronounced expression of SMA and CD34, which indicates their progenitor potential and contractile ability.

Successful pregnancies in a patient with Takayasu arteritis and antiphospholipid syndrome, maintained on infliximab corticosteroid-free regimen: case-based review.

Takayasu arteritis (TA) is a large vessel vasculitis affecting predominantly females below the age of 40. Patients with TA seem to be at increased risk for adverse pregnancy outcomes, resulting in mother or child complications. Although few studies analyzed the presence of antiphospholipid antibodies (APLA) in TA patients, an association between antiphospholipid syndrome (APS) and TA is rarely reported in the literature, mainly in the form of case reports. In fact, very few data regarding pregnancy outcomes in patients with TA and APS are available. An active form of Crohn's disease (CD) might be another risk factor strongly affecting the fertility rate. Here, we would like to present a 33-year-old woman with TA, double-positive APS and Crohn's disease (CD). The report is followed by the literature review of the association of APLA and/or APS with TA, focusing on analyzing the pregnancy outcomes. To our knowledge, this is the first case describing two successful, naturally occurring pregnancies, in a patient suffering from TA, APS and CD, and maintained on infliximab, azathioprine, and a corticosteroid-free regimen.

Differences between inflammatory cells infiltrated into tunica intima, media, and adventitia of ascending aortic aneurysms within diabetic and hypertensive patients.

The risk factors that are the most significant for the development of most cardiovascular diseases are arterial hypertension (AH), type 2 diabetes (DM), and inflammation. However, for the development of aortic aneurysms, DM is not one of them. Our study aimed to evaluate the difference between inflammatory infiltration in three individual layers of the ascending aortic aneurysm within diabetic and hypertensive patients. Forty-five patients aged 36 to 80 were divided into a group with diabetic patients without AH (group DM, N=8) and hypertensive patients without DM (group AH, N=37). For the histological analysis, aortic aneurysms were stained with hematoxylin eosin and Movat. We used immunochemical methods to detect pro- (M1), anti-inflammatory (M2) macrophages, T-helper, T-killer cells, B cells, and plasma cells. Statistical analysis was done by independent-samples Kruskal-Wallis test adjusted by Bonferroni correction for multiple tests (P<0.05). We found no difference in the volume density of collagen, elastin, vascular smooth muscle cells (VSMC), and ground substance between groups. In the DM group, there were significantly fewer M2, T-helpers, and T-killers in the media than in the intima and the adventitia (P<0.05). There were no significant differences in the number of M1, B, and plasma cells between all three vascular layers (P<0.05). In the AH group, there were significantly fewer B and plasma cells, T-helper, T-killer cells, M1, and M2 in the media than in the intima and adventitia (P<0.05). Our results conclude that the tunica media in the aneurismal wall of the AH group retained immune privilege. In contrast, in the DM group, all three layers were immune-privileged.

Autoimmune Hemolytic Anemia in Inflammatory Bowel Disease-Report of a Case and Review of the Literature.

A wide spectrum of extraintestinal manifestations (EIMs) can burden patients with inflammatory bowel disease (IBD). EIMs contribute fairly to morbidity and mortality rates in IBD patients. Moreover, EIMs in IBD patients are so frequent that some suggest that IBD should be approached as a systemic disorder. Anemia is very common in IBD patients. The two most common types of anemia in IBD, iron deficiency anemia and anemia of chronic disease, are extraintestinal complications. Autoimmune hemolytic anemia (AIHA) is a rare extraintestinal manifestation of IBD, more frequent in ulcerative colitis (UC) than in Crohn's disease (CD). In this case-based review of the literature, we present a 36-year-old female patient diagnosed with Crohn's disease (CD) and Coombs positive AIHA, complicated by pulmonary thromboembolism and successfully treated with anti-tumor necrosis factor (anti-TNF) therapy. The underlying pathophysiological mechanism of AIHA in IBD is unclear. Treatment options for AIHA in IBD patients before biologic therapy included corticosteroids alone or in combination with azathioprine (AZA), methotrexate, and surgical treatment (colectomy and/or splenectomy). Currently, biologic therapy is a promising therapeutic option, especially in corticosteroid refractory or corticosteroid-dependent IBD patients with AIHA.

Genetic Aspects of Micronutrients Important for Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD), Crohn's disease (CD) and ulcerative colitis (UC) are complex diseases whose etiology is associated with genetic and environmental risk factors, among which are diet and gut microbiota. To date, IBD is an incurable disease and the main goal of its treatment is to reduce symptoms, prevent complications, and improve nutritional status and the quality of life. Patients with IBD usually suffer from nutritional deficiency with imbalances of specific micronutrient levels that contribute to the further deterioration of the disease. Therefore, along with medications usually used for IBD treatment, therapeutic strategies also include the supplementation of micronutrients such as vitamin D, folic acid, iron, and zinc. Micronutrient supplementation tailored according to individual needs could help patients to maintain overall health, avoid the triggering of symptoms, and support remission. The identification of individuals' genotypes associated with the absorption, transport and metabolism of micronutrients can modify future clinical practice in IBD and enable individualized treatment. This review discusses the personalized approach with respect to genetics related to micronutrients commonly used in inflammatory bowel disease treatment.

Intestinal Ultrasonography as a Tool for Monitoring Disease Activity in Patients with Ulcerative Colitis.

Background: Ultrasonography is a noninvasive, inexpensive, and widely available diagnostic tool. In the last two decades, the development of ultrasound techniques and equipment has significantly increased the usage of intestine ultrasound (US) in the assessment of the gastrointestinal tract in patients with inflammatory bowel disease (IBD). Although current guidelines suggest routine utilization of US in patients with Crohn's disease, data regarding US usage in ulcerative colitis are still scarce. We aimed to assess the reliability of intestinal ultrasonography in the assessment of disease activity and extension of patients with ulcerative colitis. Methods: Fifty-five patients with a histologically confirmed diagnosis of ulcerative colitis, treated at University Clinical Center of Serbia in the period from 2019 to 2022 were included in this retrospective observational study. The data were obtained from the patient's medical records including history, laboratory, US, and endoscopy findings. US examined parameters were as following: bowel wall thickness (BWT), presence of fat wrapping, wall layer stratification, mesenteric hypertrophy, presence of enlarged mesenteric lymph nodes, and absence or presence of ascites. Results: Our results suggest that there is a strong correlation of BWT and colonoscopy findings regarding disease extension (r = 0.524, p=0.01, p < 0.05). Furthermore, our results have shown a statistically significant correlation of BWT with the Mayo endoscopic score (r = 0.434, p=0.01, p < 0.05), disease activity score (r = 0.369,p=0.01, p < 0.05), degree of ulcerative colitis burden of luminal inflammation (r = 0.366, p=0.01, p < 0.05), and Geboes index (r = 0.298, p=0.027, p < 0.05). Overall accuracy of US for disease extension and activity was statistically significant (p < 0.05). Conclusions: Our results suggest that US is a moderately accurate method for the assessment of disease activity and localization in patients with UC.

Comparison of the vitality tests used in the dental clinical practice and histological analysis of the dental pulp.

In dentistry, indirect diagnostic methods such as electrical sensibility testing and pulse oximetry are used to assess the status of the pulp. Our study aimed to determine the correlation between hemoglobin oxygen saturation and vascular volume density (Vvasc). We also wanted to examine an electrical sensibility test and the volume density of myelinated nerve fibers (Vnerv). Twenty-six intact permanent premolars were included in the study. For histological analysis, the pulp tissue was stained with hematoxylin-eosin and immunohistochemically for von Willebrand factor and S100 to detect blood vessels and myelinated nerve fibers, respectively. The stereological analysis was used to determine the Vvasc and Vnerv. Statistical analysis was done using the Pearson correlation test and Welch's ANOVA test. Histological analysis showed that the pulp tissue was strongly vascularized and innervated. A significant positive correlation was found between Vvasc and hemoglobin oxygen saturation levels (p=0.030). A significant negative correlation was found between Vnerv and the lowest electrical voltage that patient felt (p=0.033). According to the maturity of the dental apex, teeth were divided into a group with open (N=6, OA group) and closed apex (N=20, CA group). We found that pulps in the CA group had higher Vnerv than the OA group (p=0.037). In contrast, there were no significant differences in Vvasc of the pulp tissue (p=0.059), oxygen saturation (p=0.907), or electrical voltage (p=0.113) between both groups. We can conclude that the measurement of pulse oximetry and electrical sensibility test reflect the morphology of healthy pulp tissue independently of the maturity of the dental apex.

Quantitative behavioral genetic and molecular genetic foundations of the approach and avoidance strategies.

Two studies examined genetic and environmental influences on traits proposed by the revised Reinforcement Sensitivity Theory (rRST) of personality. Both quantitative and molecular behavioral genetic methods were applied considering the effects of COMT, DRD2, HTR1A and TPH2 single nucleotide polymorphisms (SNPs). Study one included 274 monozygotic and 154 dizygotic twins for the quantitative behavioral study; and in study two there were 431 twins for the molecular genetic study. The Reinforcement Sensitivity Questionnaire was used to assess basic personality traits defined by the rRST. Univariate biometric modeling suggested that genetic influences accounted for 34-44% of variance of Behavioral Approach System (BAS), Behavioral Inhibition System (BIS) and Fight-Fligh-Freeze System. Molecular genetic analyses proposed the significant main effect of COMT SNP on the BAS and TPH2 SNP on the BIS, and pointed out epistatic effects of COMT x DRD2 on BAS and HTR1A x TPH2 on Fight. Results demonstrated substantial heritability for all rRST constructs, as well as for differences in the molecular genetic basis of both approach-related and avoidance-related dimensions.

Profiling of Circulatory Elements Reveals Alteration of Essential and Toxic Trace Metals in Crohn's Disease.

The status of essential and toxic trace metals in the blood of Crohn's disease (CD) patients is unexplained. This study aimed to provide the first elemental profiling of the most recognized essential elements (Mn, Cu, Zn, Se) and selected toxic trace elements (As, Cd, Pb, and U) in sera and cell lysate (CL) samples of CD patients (n = 84). The results were compared with sex- and age-matched samples from the control group (CG). CD sera contained significantly higher levels of Mn, As, Cd, Pb, and U than did CG sera. An identical pattern, with the added inclusion of Cu (also higher in CD patients than in the CG), was obtained for CL samples. However, the most important finding was hypermanganesemia, which indicates that Mn could act as a toxic trace metal in CD. As, Cd, and U were the most significant toxic elements that showed antagonistic effects on the extrusion of essential Mn and Cu. Circulatory system screening markers for CD are hereby proposed (Mn/Cu, Mn/As, and Mn/Pb ratios). These three metal ratios were strongly and significantly correlated with F-Calprotectin levels, and deserve consideration as new markers of CD. The target metals and metal ratios should be taken into consideration as novel initiating and/or modifying factors for CD.

Anemia as a Problem: GEH Approach.

BACKGROUND: Anemia is present in almost 5% of adults worldwide and accompanies clinical findings in many diseases. Diseases of the gastrointestinal (GI) tract and liver are a common cause of anemia, so patients with anemia are often referred to a gastroenterologist. SUMMARY: Anemia could be caused by various factors such as chronic bleeding, malabsorption, or chronic inflammation. In clinical practice, iron deficiency anemia and the combined forms of anemia due to different pathophysiological mechanisms are most common. Esophagogastroduodenoscopy, colonoscopy, and the small intestine examinations in specific situations play a crucial role in diagnosing anemia. In anemic, GI asymptomatic patients, there are recommendations for bidirectional endoscopy. Although GI malignancies are the most common cause of chronic bleeding, all conditions leading to blood loss, malabsorption, and chronic inflammation should be considered. From a gastroenterologist's perspective, the clinical spectrum of anemia is vast because many different digestive tract diseases lead to bleeding. Key Messages: The gastroenterological approach in solving anemia's problem requires an optimal strategy, consideration of the accompanying clinical signs, and the fastest possible diagnosis. Although patients with symptoms of anemia are often referred to gastroenterologists, the diagnostic approach requires further improvement in everyday clinical practice.

Jaundice as a Diagnostic and Therapeutic Problem: A General Practitioner's Approach.

BACKGROUND: Jaundice is a common clinical finding in clinical practice of hepatologists and general practitioners. It occurs when serum bilirubin levels exceed 3 mg/dL. SUMMARY: In this review, we summarize the pathophysiological mechanism of jaundice, clinical approach to the patient with jaundice, and laboratory and imaging techniques. Clinical presentation of jaundice manifests through yellow skin and sclera coloration. Evaluation of every patient includes detailed medical history and examination. In the laboratory, evaluation of enzymes of hepatic inflammation as well as cholestatic enzymes with serum bilirubin must be included. Additional laboratory analysis and imaging modalities are needed in order to differentiate jaundice etiology. Moreover, imaging is available and needed in further evaluation, and treatment is dependent on the underlying cause. KEY MESSAGES: In this review, we will outline the pathophysiological mechanism of jaundice, clinical approach to the patient with jaundice, and diagnostic and treatment approach to these patients.

Chronic Abdominal Pain: Gastroenterologist Approach.

BACKGROUND: Abdominal pain is a common symptom of gastroenterology examination. Chronic abdominal pain is present for >3 months. SUMMARY: Gastroesophageal reflux disease (GERD) is one of the most common gastrointestinal diseases encountered by both gastroenterologists and general practitioners. GERD is usually a chronic disease presented with a set of symptoms including heartburn and/or regurgitation, and less commonly epigastric pain. Epigastric pain syndrome is characterized by the following symptoms: epigastric pain and/or burning. It does not necessarily occur after meal ingestion, may occur during fasting, and can be even improved by meal ingestion. Duodenal ulcers tend to cause abdominal pain that is localized in the epigastric region and commence several hours after eating, often at night. Hunger provokes pain in most of the cases and decreases after meal. Gastric ulcer pain occurs immediately after eating, and consuming food increases pain. Pain is localized in the epigastrium and can radiate to the back. Abdominal pain in irritable bowel syndrome is related to defecation. A typical symptom of chronic pancreatitis is pain that radiates to the back. In Crohn's disease, inflammation causes pain. Key Messages: Pain can occur at different locations with diverse intensity and propagation and is often associated with other symptoms. For any gastroenterologist, abdominal pain is a big challenge.

Diarrhea as a Clinical Challenge: General Practitioner Approach.

BACKGROUND: Diarrhea is defined as the passage of loose stools and increase in stool frequency, weight, or volume. Diarrhea is an important health issue since it accounts for 2.5 million deaths in the world each year. SUMMARY: Diarrhea can be acute, persistent, or chronic. Acute diarrhea (AD) is usually infectious, caused by viruses, less frequently by bacteria and parasites. The majority of cases of AD are self-limiting and do not require diagnostic workup. The use of diagnostic tests in AD should be limited to patients with signs of severe dehydration, bloody stools, persistent fever and those suffering from immunodeficiencies using immunosuppressive therapy or to cases of suspected nosocomial infection. These patients should be referred to gastroenterologists or infectious disease specialists. Therapy in AD consists of early oral refeeding, antidiarrheal medications, antibiotics, and probiotics. Chronic diarrhea (CD) has diverse etiology. The majority of patients have self-limiting symptoms or functional gastrointestinal disorders. Patients with blood in stool, weight loss, clinical and laboratory signs of anemia, and palpable mass in the abdomen (red flag symptoms) need urgent gastroenterology referral. Therapy in CD is possible when the underlying cause of symptoms is identified. KEY MESSAGES: The general practitioner should identify high-risk patients with AD and/or red flag symptoms for urgent gastroenterology referral.

Inflammatory cells in the ascending aortic aneurysm in patients with type 2 diabetes versus patients with hypertension.

Aortic aneurysms occur relatively frequently in the ascending thoracic aorta, but are rarely seen in patients with type 2 diabetes. Our aim was to evaluate inflammatory cell infiltration in the ascending aortic aneurysm wall in patients with diabetes without arterial hypertension (DM2 group, N=6) versus hypertensive non-diabetic patients (AH group, N=34). For histologic analysis, the sections were stained with hematoxylin-eosin and Movat pentachrome. The immunohistochemical staining was used to analyze the infiltration of pro-inflammatory (CD68) and anti-inflammatory macrophages (CD163), T helper (CD4) and T killer cells (CD8), and B (CD79a) and plasma cells (CD138) in all three layers of aneurysms of both groups. The statistical significance of the differences between groups was evaluated by ANOVA and the Welch test. In comparison to the AH group, the DM2 group developed less severe infiltration of pro-inflammatory macrophages (P=0.004) and B cells (P=0.025) in the tunica intima, and tunica media (P=0.049, P=0.007, respectively), and fewer plasma cells in the tunica media (P=0.024) and tunica adventitia (P=0.017). We found no significant differences in the number of T helper, T killer cells, and anti-inflammatory macrophages and in the amount of collagen and elastic fibers, ground substance, and smooth muscle cells in all three layers of the vessel wall. Except in tunica adventitia of DM2 group, there were more collagen fibers overall (P=0.025).  Thus, we conclude that the histological structure of the aneurysm in diabetics without hypertension is almost the same as in hypertensive patients without diabetes. Diabetics had significantly less inflammatory infiltration in all three layers of the vessel wall, and more collagen fibers in tunica adventitia.

Comparative Phylogeography and Integrative Taxonomy of Ochlerotatus caspius (Dipera: Culicidae) and Ochlerotatus dorsalis.

Given that accurately identifying pathogen vectors is vital for designing efficient mosquito control programs based on the proper surveillance of the epidemiologically important species, it has been suggested the complementary use of independently evolving genes and morphometric traits as a reliable approach for the characterization and delimitation of related species. Hence, we examined the spatial distribution of COI mtDNA and ITS2 rDNA variation from the historical perspective of Ochlerotatus caspius (Pallas, 1771) and O. dorsalis (Meigen, 1830), while simultaneously testing the utility of the two markers in integrative species delimitation when combined with phenotypic character analyses of larvae and adults. Despite the striking difference in haplotype diversity (high in COI mtDNA, low in ITS2 rDNA), no evident phylogeographic structure was apparent in the Palearctic O. caspius. The Holarctic O. dorsalis species was subdivided into two highly distinctive COI mtDNA phylogroups which corresponded to the Nearctic and Palearctic regions. Strong support for the independence of the two allopatric evolutionary lineages suggested that geographical barrier and climatic changes during Pleistocene caused vicariance of the ancestral range. COI mtDNA reliably distinguished O. caspius and O. dorsalis, while ITS2 rDNA yet again lacked the proper resolution for solving this problem. An integrative approach based on the larval and adult morphological traits have varying taxonomic applications due to their differential diagnostic values. Thus, by the implementation of an integrative taxonomic approach, we successfully detected species borders between the two epidemiologically relevant species and uncovered the presence of cryptic diversity within O. dorsalis.