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AIMS: In pursuit of enhancing prevention efforts for the notably high endemic Opisthorchis viverrini infection in lower Mekong sub-region countries, particularly Thailand and Laos, this cross-sectional study investigated the transmission potential of O. viverrini and other intestinal helminths along the Thailand-Laos border in seven Thai villages. METHODS AND RESULTS: Human and pet faecal samples, Bithynia snails and cyprinid fish were analysed for helminth infections. Additionally, a questionnaire survey assessed relevant risk factors among the human population. Two groups of helminth infections were detected in humans: foodborne infections (FBIs) including O. viverrini, minute intestinal flukes (MIFs), and Taenia spp., and soil-transmitted infections (STIs) including hookworm and Strongyloides stercoralis, with prevalence rates of 7.4%, 0.5%, 2.5%, 0.5%, 2.5% and 3%, respectively. Smoking was identified as a risk factor for O. viverrini infection [adjusted odds ratios (ORa) = 3.12, 95% confidence intervals (CI): 1.33-7.30, p = 0.009] and FBIs (ORa = 2.47, 95% CI: 1.14-5.33, p = 0.022), while male was a risk factor for FBIs (ORa = 2.62, 95% CI: 1.16-5.94, p = 0.021). In dogs, hookworm, Toxocara spp., Spirometra mansoni, Trichuris vulpis and Hymenolepis diminuta were identified with prevalence rates of 35.6%, 8.1%, 8.1%, 1.2% and 1.2%, respectively. In cats, hookworm, Toxocara spp., S. mansoni, Strongyloides spp., Platynosomum fastosum, MIFs and H. diminuta were identified with prevalence rates of 50%, 17.9%, 10.7%, 7.1%, 3.6%, 3.6% and 3.6%, respectively. Bithynia snails showed 2% virgulate and 0.7% unknown cercariae infections, while among 19 cyprinid fish species, only unknown metacercariae were found. CONCLUSIONS: Our findings underscore the necessity of an integrated approach following the One Health concept to effectively combat these parasitic diseases while addressing human, animal and environmental health.
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Cholangiocarcinoma (CCA) is a life-threatening disease that impacts patients worldwide. In Southeast Asian countries, the liver fluke Opisthorchis viverrini plays a major role in inducing carcinogenesis of the bile ducts. Due to its asymptomatic nature, O. viverrini infections are rarely treated, consequently leading to the development of advanced stages of CCA before diagnosis. Despite the current use of exosomal microRNAs (miRNA) as diagnostic biomarkers for the early detection of many types of cancer, the applications for miRNA remain limited with CCA. Circulating exosomes, membranous vesicles essential for intercellular communication, were found to contain unique miRNA. In this study, we conducted next-generation sequencing (Ion Torrent PGM) and bioinformatics to characterize and compare the contents of exosomal miRNA derived from the plasma of CCA patients, O. viverrini-infected patients, and healthy individuals, as well as to identify and validate key molecules as markers for screening the diagnosis of CCA and O. viverrini infection. The obtained results showed the success of using NGS technology in discovering exosomal miRNAs, specifically miR-194-5p and miR-192-5p, both of which were upregulated in the O. viverrini-infected group. Interestingly, miR-192-5p was upregulated while miR-194-5p was downregulated in CCA, suggesting their potential use as biomarkers for screening CCA and O. viverrini infection, especially in O. viverrini-endemic areas.
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Neoplasias dos Ductos Biliares , Colangiocarcinoma , Exossomos , MicroRNAs , Opistorquíase , Opisthorchis , Colangiocarcinoma/parasitologia , Colangiocarcinoma/sangue , Colangiocarcinoma/genética , Colangiocarcinoma/diagnóstico , MicroRNAs/sangue , MicroRNAs/genética , Animais , Exossomos/genética , Humanos , Opisthorchis/genética , Opistorquíase/complicações , Opistorquíase/parasitologia , Opistorquíase/sangue , Opistorquíase/diagnóstico , Neoplasias dos Ductos Biliares/parasitologia , Neoplasias dos Ductos Biliares/sangue , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/diagnóstico , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Masculino , Pessoa de Meia-Idade , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Perfilação da Expressão Gênica , Biologia Computacional/métodos , IdosoRESUMO
OBJECTIVES: This study aimed to assess the diagnostic potential of cell-free DNA (cfDNA) and cell-free miRNA (cf-miRNA) for distinguishing between Healthy, asymptomatic opisthorchiasis viverrini and cholangiocarcinoma in a preliminary manner. METHODS: In this study, 36 participants were enrolled into three health status groups: a healthy control group (HC), Opisthorchis viverrini-infected group (OV), and a cholangiocarcinoma group (CCA), each comprising 12 participants. Concentration measurements of cfDNA and cf-miRNA from plasma were conducted. Additionally, ultra-low-pass whole-genome sequencing (ULP-WGS) was employed to investigate DNA alterations. RESULTS: The study revealed a significant elevation in plasma cfDNA concentration in the cholangiocarcinoma (CCA) group compared to healthy controls (HC) and Opisthorchis viverrini-infected (OV) groups (P < 0.001). The cfDNA concentration demonstrated a sensitivity of 75.00% and specificity of 95.83% for differentiating cholangiocarcinoma, with a cut-off of > 30.50 ng/ml plasma. Likewise, the concentration of cf-miRNA in the CCA group significantly differed from that in the HC and OV groups, demonstrating a sensitivity of 83.33% and specificity of 95.83% with a cut-off set at > 70.50 ng/ml plasma. Furthermore, a positive correlation between plasma concentrations of cfDNA and cf-miRNA suggests a potential relationship between these two biomarkers. These findings indicated the diagnostic potential of cfDNA and cf-miRNA in distinguishing cholangiocarcinoma, emphasizing their role as promising biomarkers for further investigation and clinical applications. CONCLUSION: Elevated plasma concentrations of cfDNA and cf-miRNA could serve as potential diagnostic tools for distinguishing cholangiocarcinoma from other conditions. cf-miRNA was superior to cfDNA in terms of sensitivity.
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Neoplasias dos Ductos Biliares , Ácidos Nucleicos Livres , Colangiocarcinoma , MicroRNAs , Opistorquíase , Opisthorchis , Animais , Humanos , Opistorquíase/complicações , Opistorquíase/diagnóstico , MicroRNAs/genética , Colangiocarcinoma/diagnóstico , Colangiocarcinoma/genética , Biomarcadores , Ductos Biliares Intra-Hepáticos/patologia , Neoplasias dos Ductos Biliares/diagnóstico , Neoplasias dos Ductos Biliares/genéticaRESUMO
This study aimed to identify the recent risk factors for Opisthorchis viverrini infection and cholangiocarcinoma (CCA) to improve disease prevention. The participants were divided into the following 3 groups based on their health status: healthy control (nonOV and nonCCA), those with O. viverrini infection (OV), and those with CCA. A questionnaire was used to explore their lifestyle and behaviors. Multivariate logistic regression and backward elimination were used to identify the significant risk factors. The results showed that the significant risk factors for both O. viverrini infection and CCA were age>50 years (odd ratio (OR)=8.44, P<0.001, 95% confidence intervals (CI) 2.98-23.90 and OR=43.47, P=0.001, 95% CI 14.71-128.45, respectively) and raw fish consumption (OR=8.48, P< 0.001, 95% CI 3.18-22.63 and OR=3.15, P=0.048, 95% CI 1.01-9.86, respectively). A history of O. viverrini infection was identified as an additional risk factor for CCA (OR=20.93, P=0.011, 95% CI 2.04-215.10). This study provided an update on the risk factors for O. viverrini infection and CCA. Asymptomatic patients with O. viverrini infection, particularly those>50 years old, should be carefully monitored to prevent CCA.
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Neoplasias dos Ductos Biliares , Colangiocarcinoma , Opistorquíase , Opisthorchis , Animais , Humanos , Pessoa de Meia-Idade , Opistorquíase/complicações , Tailândia/epidemiologia , Neoplasias dos Ductos Biliares/epidemiologia , Colangiocarcinoma/epidemiologia , Fatores de Risco , Ductos Biliares Intra-Hepáticos/patologiaRESUMO
Background and Aims: Hyperlipidemia is a risk factor for cardiovascular diseases. This study is aimed at investigating the effects of consuming omega-3-rich pork lard on the serum lipid profile and gut microbiome of the mice model. Methods and Results: We divided 23 C57BL/6NJ males (16-week-old) into 3 groups, and each group received either a control diet, a high-fat diet of coconut oil (coconut oil), or a high-fat diet of omega-3-rich pork lard (omega lard) for 28 days. Thereafter, fasting serum lipids and fecal microbiomes were analyzed. The serum cholesterol, triglyceride, and LDL levels of the omega lard-treated group were significantly reduced compared to the coconut oil-treated group (P < 0.05). However, the microbiome analysis revealed a significant increase in the abundance of Lachnospiraceae in the omega lard-treated group compared to the coconut oil-treated group (P < 0.05). Furthermore, Spearman's correlation analysis revealed that the increased serum lipid content was positively correlated with the abundance of Bacteroidaceae (P < 0.05) and negatively correlated with the abundance of Lachnospiraceae (P < 0.05). Conclusions: These findings suggested that omega-3-rich pork lard altered the serum lipid profile and gut microbiome in the mice model. Practical Application. The excellent protection offered by omega-3-rich pork lard against hyperlipidemia indicated that pork lard could be used as alternative cooking oil for health-conscious individuals. It could also be introduced as a functional ingredient for patients with hyperlipidemia.
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In Southeast Asian countries, nitrosamine compounds and the liver fluke Opisthorchis viverrini have long been identified as carcinogens for cholangiocarcinoma (CHCA). In order to effectively treat O. viverrini infections and prevent the development of CHCA, methods for disease detection are needed. This study aims to identify biomarkers for O. viverrini infection and CHCA. In the discovery phase, technical triplicates of five pooled plasma pools (10 plasma each) of healthy control subjects (noOVCCA), O. viverrini subjects (OV), and cholangiocarcinoma subjects (CCA), underwent solution-based digestion, with the label-free method, using a Thermo Scientific™ Q Exactive™ HF hybrid quadrupole-Orbitrap mass spectrometer and UltiMate 300 LC systems. The noOVCCA, OV, and CCA groups demonstrated different profiles and were clustered, as illustrated by PCA and heat map analysis. The STRING and reactome analysis showed that both OV and CCA groups up-regulated proteins targeting immune system-related proteins. Differential proteomic profiles, S100A9, and polymeric immunoglobulin receptor (PIGR) were specifically expressed in the CCA group. During the validation phase, another 50 plasma samples were validated via the PIGR sandwich ELISA. Using PIGR >1.559 ng/ml as a cut-off point, 78.00% sensitivity, 71.00% specificity, and AUC = 0.8216, were obtained. It is sufficient to differentially diagnose cholangiocarcinoma patients from healthy patients and those with Opisthorchiasis viverrini. Hence, in this study, PIGR was identified and validated as a potential biomarker for CHCA. Plasma PIGR is suggested for screening CHCA, especially in an endemic region of O. viverrini infection.
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Among the snail species acting as hosts for medically significant trematodes, only three taxa of Bithynia are responsible for transmitting the carcinogenic liver fluke Opisthorchis viverrini to humans in different geographical areas. Although B. siamensis goniomphalos is the primary species responsible for O. viverrini transmission in endemic areas, B. siamensis siamensis and B. funiculata remain potential hosts for transmission. This study objects to determine the susceptibility of B. siamensis siamensis and B. funiculata to O. viverrini to assess the risk of O. viverrini transmission in non-endemic areas. The snails of both species were first introduced to O. viverrini eggs, after which O. viverrini infection was investigated using specific PCR primers after a period of 1, 7, 14, 28, and 56 days post-infection (dpi). Opisthorchis viverrini infection in both B. siamensis siamensis and B. funiculata was high in the early period (1 and 7 dpi) while decreasing over time. It was also shown that the odds of susceptibility to O. viverrini infection in B. siamensis siamensis were 64.5% higher relative to the odds of susceptibility in B. funiculata (P < 0.05). Results of this study provide an early insight into the Bithynia-Opisthorchis relationship and thus have great potential to assess risk and raise awareness of opisthorchiasis in non-endemic regions, especially in regions endemic for B. siamensis siamensis.
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Opistorquíase , Opisthorchis , Humanos , Animais , Opistorquíase/epidemiologia , Opistorquíase/veterinária , Caramujos , Tailândia/epidemiologiaRESUMO
Hyperlipidemia is a condition where the blood shows an elevated level of lipid, such as cholesterol and triglyceride. It is considered a risk factor for all coronary artery death globally. Association of microbiome with non-communicable diseases (NCDs) including hyperlipidemia has been reportedly associated. In this study, we hypothesize that the change in microbiome is correlated to the change in serum lipid level, which resulted by increasing dietary fat consumption. The 32 male, 14-week-old, C57BL/6N were divided into 4 groups, each group received control diet, 10%, 20%, and 40% kcal fat diet prepared from purified pork lard, respectively for 28 days. Fasting serum lipids and fecal microbiome were then analyzed. The group of animals assigned to 40% kcal fat showed significantly increased serum cholesterol, LDL, and HDL (p < 0.05). Microbiome analysis revealed the abundance of Muribaculaceae and Saccharimonadaceae were significantly decreased (p < 0.05). On the contrary, the abundance of Clostridia_UCG014, Akkermansiaceae, Bacteroidaceae, Oscillospiraceae, and Erysipelotrichaceae were significantly increased (p < 0.05). Spearman correlation indicated that the abundance of Akkermansiaceae and Bacteroidaceae were positively associated with the increased of serum cholesterol and LDL (p < 0.05), while the abundance of Muribaculaceae, Clostridia_UCG-014, and Saccharimonadaceae were negatively associated (p < 0.05). These results suggest that dietary fat have ability to manipulated microbiome with relative to elevation of serum lipid profile.
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Microbioma Gastrointestinal , Microbiota , Animais , Gorduras na Dieta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos EndogâmicosRESUMO
The human liver fluke Opisthorchis viverrini (Ov), the primary risk factor for cholangiocarcinoma (CHCA), is a parasite endemic to southeast Asian countries. With no effective treatments for CHCA currently available, early diagnosis and treatment of Ov infection remains the only practical method for the prevention of CHCA. In this study, plasma phosphoproteomes of patients in the non-Ov infection, non-cholangiocarcinoma subject group (non-OVCCA), the asymptomatic Ov infected group (OV), and the CHCA group (CCA), were investigated to identify potential biomarkers for Ov infection and CHCA. The AKT signalling pathway was found to be up-regulated. Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform (PIK3CB), an upstream signalling molecule, was selected as a potential biomarker and evaluated using indirect enzyme-linked immunosorbent assay (ELISA). Results demonstrated evidence that levels of PIK3CB in both the OV group and CCA group was statistically different compared to the non-OVCCA group (P < 0.01). However, the levels of PIK3CB between the OV group and the CCA group were found not to be statistically different. Sensitivity and specificity for OV using OD450 cut-off at >1.570 was 76 and 72%, respectively. For CCA, sensitivity and specificity using OD450 cut-off at >1.398 was 68 and 76%, respectively. Application of indirect ELISA detecting plasma PIK3CB will be of great benefit for screening of opisthorchiasis and CHCA.
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Neoplasias dos Ductos Biliares , Colangiocarcinoma , Opistorquíase , Opisthorchis , Animais , Neoplasias dos Ductos Biliares/diagnóstico , Neoplasias dos Ductos Biliares/epidemiologia , Neoplasias dos Ductos Biliares/metabolismo , Ductos Biliares Intra-Hepáticos/metabolismo , Ductos Biliares Intra-Hepáticos/parasitologia , Ductos Biliares Intra-Hepáticos/patologia , Biomarcadores/análise , Domínio Catalítico , Colangiocarcinoma/diagnóstico , Colangiocarcinoma/etiologia , Colangiocarcinoma/metabolismo , Humanos , Opistorquíase/diagnóstico , Opistorquíase/epidemiologia , Opistorquíase/parasitologia , Fosfatidilinositóis/metabolismoRESUMO
The purpose of this study was to investigate in vitro drug release kinetics and to develop diffusion model of curcumin loaded Pluronic F127/Oleic acid(OA)-Fe3O4 nanoparticles. The prepared superparamagnetic nanoparticles by co-precipitation technique were characterized by the average size, size distribution, crystallinity, colloidal stability and magnetic property. The release of curcumin was triggered by an acidic environment in pH 5.0 of phosphate buffer saline. Release data of various curcumin loading (15, 25 and 30 ppm) were fitted using non-linear first-order, second-order, Higuchi and Korsmeyer-Peppas model. All the curcumin release mechanism followed Korsmeyer-Peppas model with n values less than 0.45 indicating the Fickian diffusion of curcumin from the prepared nanomicelles. The dynamic of controlled drug release of dilute curcumin loading was well described by a combination of diffusion and first-order release rate. The corresponding diffusion coefficient and kinetic rate were 9.1 × 10-7 cm2â min-1 and 6.51 × 10-7 min-1, which were used as controlled release to achieve the desired curcumin constant release rate in the delivery system.
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Curcumina , Nanopartículas , Neoplasias , Curcumina/química , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Humanos , Cinética , Nanopartículas Magnéticas de Óxido de Ferro , Nanopartículas/químicaRESUMO
BACKGROUND: Patients infected with a parasite often develop opisthorchiasis viverrini, which often progresses into cholangiocarcinoma (CCA) due to the asymptomatic nature of the infection. Currently, there are no effective diagnostic methods for opisthorchiasis or cholangiocarcinoma. OBJECTIVE: The aim of this study was to identify the host-responsive protein that can be developed as a diagnostic biomarker of opisthorchiasis and cholangiocarcinoma. METHODS: Plasma samples were collected from non-OVCCA, OV, and CCA subjects, and the proteomes were investigated by LC-MS/MS. Venn diagrams and protein network prediction by STITCH were used to identify the potential biomarkers. The level of candidate protein, the plasma checkpoint protein 1 (Chk1), was measured by indirect enzyme-linked immunosorbent assay (ELISA). RESULTS: Chk1 was present in the center of the protein network analysis in both the OV and CCA groups. In addition, the plasma Chk1 levels were significantly increased in both groups (P< 0.05). The sensitivity of the opisthorchiasis viverrini and cholangiocarcinoma was 59.38% and 65.62%, respectively, while the specificity of both was 85.71%. CONCLUSION: Chk1 was identified by differential plasma proteomes and was increased in O. viverrini-infected and cholangiocarcinoma-derived plasma samples. Higher levels of plasma Chk1 levels may serve as a potential diagnostic biomarker for opisthorchiasis and cholangiocarcinoma.
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Neoplasias dos Ductos Biliares , Colangiocarcinoma , Opistorquíase , Opisthorchis , Animais , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos/metabolismo , Ductos Biliares Intra-Hepáticos/parasitologia , Ductos Biliares Intra-Hepáticos/patologia , Biomarcadores/metabolismo , Colangiocarcinoma/patologia , Cromatografia Líquida , Humanos , Opistorquíase/diagnóstico , Opistorquíase/metabolismo , Opistorquíase/parasitologia , Espectrometria de Massas em TandemRESUMO
Opisthorchis viverrini infection, opisthorchiasis, is a food-borne trematodiasis that is the main cause of cholangiocarcinoma, a bile duct cancer, in the Lower Mekong sub-region of Lao PDR, Cambodia, Vietnam, and Thailand. Despite extensive research on opisthorchiasis, the eradication of this disease has yet to be achieved. One of the major reasons for this failure is due to the multi-host life cycle of the parasite, which requires complex medical and public health interventions to eradicate. Another reason is due to a lack of knowledge of not only the interactions between the parasite and the human immune system, but also the interactions between the parasite and its various hosts during its complicated life cycle. Recent advances in various high-throughput omics technologies has allowed for the identification of key biomolecules crucial to the processes of parasitic transmission, and the identification of novel drug and/or vaccine targets. In this paper, omics studies dealing with O. viverrini host-parasite biology will be reviewed. In particular, there will be a focus on the strategies O. viverrini uses to trigger, evade, and manipulate the host's defense systems. Recently-identified biological molecules with potential as targets for interventions will also be reviewed. The results obtained from these omics approaches to analyzing O. viverrini and host interactions will be of great importance in the future when developing effective and sustainable medical and public health models for the prevention and control of opisthorchiasis and opisthorchiasis-induced CCA.
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Neoplasias dos Ductos Biliares/etiologia , Neoplasias dos Ductos Biliares/prevenção & controle , Colangiocarcinoma/etiologia , Colangiocarcinoma/prevenção & controle , Genômica , Metabolômica , Opistorquíase/prevenção & controle , Proteômica , Animais , Humanos , Opistorquíase/parasitologiaRESUMO
AIM: The potential of biomarkers in detecting early cholangiocarcinoma (CCA) is facilitated by examining CCA-associated proteins from primary studies. One such protein is mucin 5AC (MUC5AC) but inconsistency of reported associations between its expression/serum levels and CCA prompts a meta-analysis to obtain more precise estimates. METHODS: A literature search yielded 17 included articles where multiple data in some raised the number of studies to 22. We calculated pooled odds ratios (OR) and 95% confidence intervals from negative and positive readings of MUC5AC levels. Data were subgrouped by ethnicity, detection method, sample source, and cancer type. RESULTS: Outcome in the overall analysis was non-significant but those in the subgroups were. Thus, significant associations (P < 0.001) indicating high MUC5AC levels were found in three subgroups: (i) Thai (OR 8.32) and (ii) serum (OR 4.52). Heterogeneity of these two outcomes (I2 = 90-93%) was erased with outlier treatment (I2 = 0%) which also modulated the pooled effects (OR 2.48-2.59). (iii) Immunoblot (OR 2.61) had low initial heterogeneity (I2 = 2%). Robustness and significant tests for interaction (Pinteraction = 0.01-0.02) improved MUC5AC associations with CCA in the Thai population. CONCLUSIONS: Our pooled effect findings target the biomarker potential of MUC5AC to the Thai population.
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Biomarcadores Tumorais/sangue , Colangiocarcinoma/diagnóstico , Mucina-5AC/sangue , Mucina-5AC/metabolismo , Colangiocarcinoma/patologia , HumanosRESUMO
Cholangiocarcinoma (CCA) is the bile duct cancer which constitutes one of the important public health problems in Thailand with high mortality rate, especially in the Opisthorchis viverrini (a parasite risk factor for CCA) endemic area of the northeastern region of the country. This study aimed to identify potential biomarkers from the plasma peptidome by CCA patients. Peptides were isolated using 10 kDa cut-off filter column and the flow-through was then used as a peptidome for LC-MS/MS analysis. A total of 209 peptides were obtained. Among these, 15 peptides were concerned with signaling pathways and 12 related to metabolic, regulatory, and biosynthesis of secondary metabolite pathways. Five exclusive peptides were identified as potential biomarkers, i.e. ETS domain-containing transcription factor ERF (P50548), KIAA0220 (Q92617), phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform isoform 1 (P42338), LP2209 (Q6XYC0), and casein kinase II subunit alpha (P19784). Three of these biomarkers are signaling related molecules. A combination of these biomarkers for CCA diagnosis is proposed.
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Neoplasias dos Ductos Biliares/sangue , Ductos Biliares Intra-Hepáticos/metabolismo , Biomarcadores Tumorais/sangue , Colangiocarcinoma/sangue , Opistorquíase/complicações , Opisthorchis/isolamento & purificação , Fragmentos de Peptídeos/sangue , Adulto , Idoso , Animais , Neoplasias dos Ductos Biliares/parasitologia , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos/parasitologia , Ductos Biliares Intra-Hepáticos/patologia , Estudos de Casos e Controles , Colangiocarcinoma/parasitologia , Colangiocarcinoma/patologia , Cromatografia Líquida , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Opistorquíase/epidemiologia , Opistorquíase/parasitologia , Prognóstico , Fatores de Risco , Espectrometria de Massas em Tandem , Tailândia/epidemiologiaRESUMO
This study was conducted to investigate the plasma phosphoproteome and differential plasma phosphoproteins in cases of of Opisthorchis viverrini (OV)-related cholangiocarcinoma (CCA). Plasma phosphoproteomes from CCA patients (10) and non-CCA subjects (5 each for healthy subjects and OV infection) were investigated using gel-based and solution-based LC-MS/MS. Phosphoproteins in plasma samples were enriched and analyzed by LC-MS/MS. STRAP, PANTHER, iPath, and MeV programs were applied for the identification of their functions, signaling and metabolic pathways; and for the discrimination of potential biomarkers in CCA patients and non-CCA subjects, respectively. A total of 90 and 60 plasma phosphoproteins were identified by gel-based and solution-based LC-MS/MS, respectively. Most of the phosphoproteins were cytosol proteins which play roles in several cellular processes, signaling pathways, and metabolic pathways (STRAP, PANTHER, and iPath analysis). The absence of serine/arginine repetitive matrix protein 3 (A6NNA2), tubulin tyrosine ligase-like family, member 6, and biorientation of chromosomes in cell division protein 1-like (Q8NFC6) in plasma phosphoprotein were identified as potential biomarkers for the differentiation of healthy subjects from patients with CCA and OV infection. To differentiate CCA from OV infection, the absence of both serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit beta isoform and coiled-coil domain-containing protein 126 precursor (Q96EE4) were then applied. A combination of 5 phosphoproteins may new alternative choices for CCA diagnosis.