Search

Abstract Background Transient elastography (TE) is an innovative, noninvasive technique to assess liver fibrosis by measuring liver stiffness in patients with chronic liver diseases. The purpose of this study has been to explore the accuracy of TE and clinical parameters in predicting the presence of esophageal/gastric varices in children with biliary atresia (BA) following portoenterostomy. Methods Patients with BA status post portoenterostomy and normal children were recruited. Splenomegaly and presence of EV/GV were determined by physical examination and endoscopy, respectively. Aspartate transaminase to platelet ratio index (APRI) was used as a serum fibrosis marker. TE was performed by using FibroScan. Data was expressed as mean ± SD. Results Seventy-three BA patients (male:female = 32:41; age 9.11 ± 5.64 years) and 50 normal controls (male:female = 19:31; age 11.00 ± 3.31 years) were enrolled. The liver stiffness score of BA patients was significantly higher than that of normal controls (27.37 ± 22.48 and 4.69 ± 1.03 kPa; p < 0.001). Patients with EV/GV had significantly higher liver stiffness score and APRI than those without EV/GV. As for EV/GV diagnosis, the areas under the receiver operating characteristic curve were 0.89 (95% CI 0.80 to 0.98) for TE and 0.87 (95% CI 0.78 to 0.96) for APRI, respectively. The sensitivity (and specificity) of TE (using a cut-off value of 12.7 kPa) and APRI (using a cut-off value of 1.92) in predicting EV/GV were 84% (77%) and 84% (83%), respectively, whereas the sensitivity (and specificity) of splenomegaly in predicting EV/GV were 92% (85%). Conclusions Transient elastography is a useful tool for predicting the presence of EV/GV. In addition, basic physical examination, routine biochemical and hematological tests, are still worthwhile and correlate well with the presence of EV/GV in patients with BA post portoenterostomy.

Introduction: Data on the relationship between bacterial translocation, hepatic encephalopathy (HE), and mortality are scarce. This study aimed to assess the association between bacterial DNA (bactDNA) translocation, inflammatory response, ammonia levels, and severity of HE in patients with cirrhosis, as well as the role of bactDNA translocation in predicting mortality., Methods: Cirrhotic patients without bacterial infection were prospectively enrolled between June 2022 and January 2023. Grading of HE was classified by the West Haven Criteria and Psychometric Hepatic Encephalopathy Score ≤ -5., Results: Overall, 294 cirrhotic patients were enrolled, with 92 (31.3%) and 58 (19.7%) having covert and overt HE, respectively. BactDNA translocation was detected in 36.1% of patients (n = 106). Patients with overt HE had more bactDNA translocation and higher serum lipopolysaccharide-binding protein (LBP), tumor necrosis factor-α, interleukin-6 (IL-6), and ammonia levels than those without HE. Patients with detectable bactDNA had higher white cell counts and serum LBP and IL-6 levels than those without. By contrast, bactDNA, serum LBP, and soluble CD14 levels were comparable between patients with covert HE and those without HE. The multivariate Cox regression analysis revealed that bactDNA translocation (hazard ratio [HR] = 2.49, 95% confidence interval [CI]: 1.22-5.11), Model for End-Stage Liver Disease score (HR = 1.12, 95% CI: 1.09-1.16), age (HR = 1.05, 95% CI: 1.000-1.002), and baseline IL-6 (HR = 1.001, 95% CI: 1.000-1.002) were independent factors associated with 6-month mortality., Discussion: Apart from hyperammonemia, bactDNA translocation is a possible factor associated with overt HE in cirrhotic patients. BactDNA translocation and IL-6 are independent factors associated with 6-month mortality., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology.)

Knowledge about the epidemiology of hepatitis D virus (HDV) is essential for effective screening and management. Our study aimed to update the prevalence of HDV infection among patients with hepatitis B virus (HBV) infection at hepatology clinics in Thailand. We enrolled HBV-infected patients from hepatology clinics at King Chulalongkorn Memorial Hospital, Bangkok, Thailand, between June 2022 and November 2023. Demographic, biochemical characteristics, and liver-related complications (LRC), including cirrhosis and hepatocellular carcinoma, were reviewed. The competitive enzyme and chemiluminescence immunoassays were used to detect anti-HDV antibodies. Real-time polymerase chain reaction (RT-PCR) was used to test for HDV RNA in anti-HDV-positive patients. The HDV genotype was identified in detectable HDV RNA samples. Of the 702 enrolled patients, four (0.6%) had positive and equivocal for both anti-HDV tests. Two (50.0%) of the four patients tested positive for HDV RNA and genotype 1 was identified; one had multiple risk factors. Anti-HDV seroprevalence was not significantly different between patients with and without LRC. In conclusion, HDV co-infection is less common in Thailand than globally. Additionally, our study identified genotype 1, the predominant HDV genotype worldwide, and observed co-infection even without LRC., (© 2023. The Author(s).)

The psychometric hepatic encephalopathy score (PHES) is the gold standard for diagnosing minimal hepatic encephalopathy (MHE). Screening for MHE is frequently overlooked in clinical practice due to time constraints. Furthermore, the simplified animal naming test (S-ANT1) is a new simple tool for evaluating MHE in cirrhotic patients. The purpose of this study was to standardize the PHES in a healthy Thai population, assess the prevalence of MHE, and validate the S-ANT1 in detecting MHE in patients with cirrhosis. The study included 194 healthy controls and 203 cirrhotic patients without overt HE. Psychometric tests and the S-ANT1 were administered to all participants. Multiple linear regression was used to analyze factors related to PHES results, and formulas were developed to predict the results for each PHES subtest. In healthy controls, age and education were predictors of all five subtests. The PHES of the control group was −0.26 ± 2.28 points, and the threshold for detecting MHE was set at ≤ −5 points. The cirrhotic group had PHES values of −2.6 ± 3.1 points. Moreover, MHE was found to be present in 26.6% of cirrhotic patients. S-ANT1 had a moderate positive correlation with PHES (r = 0.44, p < 0.001). S-ANT1 < 22 named animals detected MHE with a sensitivity of 71.2%, specificity of 65%, and area under the receiver operating curve of 0.68 (p < 0.001). In conclusion, Thai PHES normative data have been developed to detect MHE in cirrhotic patients who do not have overt HE. The optimal cutoff for detecting MHE in Thai cirrhotic patients was PHES ≤ −5 points and S-ANT1 < 22 named.

Background: Heterologous prime-boost vaccination potentially augments the immune response against SARS-CoV-2 in liver transplant (LT) recipients. We investigated immunogenicity induced by different primary prime-boost vaccination protocols and the subsequent response to the booster vaccine among LT recipients. Methods: LT recipients, who received primary immunisation with ChAdOx1/ChAdOx1 or ChAdOx1/BNT162b2, were administered the third dose of mRNA-1273 three months following the primary vaccination. Blood samples were collected before and after primary vaccination and post-booster. The levels of receptor binding domain antibody (anti-RBD) and neutralising antibody (sVNT) and spike-specific T-cell responses were assessed. Results: Among the 89 LT recipients, patients receiving ChAdOx1/BNT162b2 had significantly higher anti-RBD titres, sVNT, and cellular response after primary vaccination than those receiving ChAdOx1/ChAdOx1 (p < 0.05). The antibody response decreased 12 weeks after the primary vaccination. After the booster, humoral and cellular responses significantly improved, with comparable seroconversion rates between the heterologous and homologous groups. Positive sVNT against the wild type occurred in >90% of LT patients, with only 12.3% positive against the Omicron variant. Conclusions: ChAdOx1/BNT162b2 evoked a significantly higher immunological response than ChAdOx1/ChAdOx1 in LT recipients. The booster strategy substantially induced robust immunity against wild type in most patients but was less effective against the Omicron strain.

Background: Heterogeneity of liver function and tumor burden in intermediate-stage hepatocellular carcinoma (HCC) results in different outcomes after transarterial chemoembolization (TACE). Easy albumin-bilirubin (EZ-ALBI), a simplified albumin-bilirubin (ALBI) score, has recently been proposed as a new prognostic score for HCC. This study aimed to validate the EZ-ALBI score and evaluate the impact of dynamic changes in patients with intermediate-stage HCC undergoing TACE., Methods: All patients with HCC treated with TACE at King Chulalongkorn Memorial Hospital, Bangkok, Thailand, between January 2015 and December 2019 were prospectively enrolled. Intermediate-stage HCC was defined as Barcelona Clinic Liver Cancer (BCLC) stage B or unresectable single HCC with size > 5 cm in BCLC stage A. EZ-ALBI and ALBI scores were calculated and stratified into three different grades. Overall survival (OS) and prognostic factors were assessed using the Kaplan-Meier curve and Cox proportional hazard model. Decision analysis curves were used to evaluate the clinical utility of the predictive scores., Results: Among 672 patients with HCC treated with TACE, 166 patients with intermediate-stage HCC who met the eligibility criteria were enrolled. The median OS of all patients in the cohort was 21 months. A good correlation between the EZ-ALBI and ALBI scores was observed (correlation coefficient 1.000, p < 0.001). The baseline EZ-ALBI grades 1, 2, and 3 were 24.5%, 70%, and 5.5%, respectively. EZ-ALBI grade can stratify patients with significantly different prognoses (p = 0.002). Baseline EZ-ALBI grade 2, 3, and serum alpha-fetoprotein > 20 ng/ml were significantly associated with OS [hazard ratio (HR) 2.20 (95% confidence interval [CI] 1.24-3.88, p = 0.007), 3.26 (95% CI 1.24-8.57, p = 0.016), and 1.77 (95% CI 1.10-2.84, p = 0.018), respectively]. Following TACE, 42 (29.6%) patients had a worsening EZ-ALBI grade. However, the EZ-ALBI grade migration was not significantly correlated with OS. EZ-ALBI and ALBI score provided improved discriminatory ability (Harrell's concordance index 0.599 and 0.602, respectively) and better net benefit compared with Child-Turcotte-Pugh and Model for End-stage Liver Disease scores., Conclusions: The baseline EZ-ALBI score demonstrated good predictive performance for survival and a strong correlation with conventional ALBI scores. Both the EZ-ALBI and ALBI scores outperformed other prognostic models in patients with intermediate-stage HCC receiving TACE. However, the dynamic change in the EZ-ALBI grade after TACE was not associated with postprocedural survival., (© 2022. The Author(s).)

Background: Hepatitis B virus (HBV) pregenomic RNA (pgRNA) has gained increasing attention owing to its role in replication of covalently closed circular DNA (cccDNA) in HBV. This marker has the potential to be used in clinical programs aimed to manage HBV infections. However, several reports on HBV pgRNA levels in clinical cases have conflicting results. RNA is easily degraded when exposed to heat and other environmental stressors. However, the stability of HBV pgRNA, during blood sample collection before the standard automated quantification, has never been estimated. This study aimed to demonstrate the effect of two different temperature conditions and storage durations on the stability of HBV pgRNA., Method: Blood from forty patients with chronic hepatitis B infection, who also showed evidence of active HBV DNA replication, was collected and processed within 2 h of collection. Plasma from each patient was divided and stored at 4 °C and 25 °C (room temperature) for six different storage durations (0, 2, 6, 12, 24, and 48 h) and subsequently transferred to -80 °C for storage. The effect of multiple cycles of freezing and thawing of plasma at -20 °C or -80 °C was evaluated using samples from ten patients. Quantification of pgRNA from the samples was performed simultaneously, using the digital polymerase chain reaction (dPCR) method. The differences in pgRNA levels at baseline and each time point were compared using generalized estimating equation (GEE). A change greater than 0.5 log 10 copies/mL of pgRNA is considered clinically significant. Statistical analyses were conducted using Stata 16.0., Results: The mean HBV pgRNA level in the initially collected plasma samples was 5.58 log 10 copies/mL (ranging from 3.08 to 8.04 log 10 copies/mL). The mean pgRNA levels in samples stored for different time periods compared with the initial reference sample (time 0) significantly decreased. The levels of pgRNA for 6, 12, 24, and 48 h of storage reduced by -0.05 log 10 copies/mL (95% confidence interval (CI) -0.095 to -0.005, p  = 0.03), -0.075 log 10 copies/mL (95% CI [-0.12 to -0.03], p  = 0.001), -0.084 log 10 copies/mL (95% CI [-0.13 to -0.039], p  =  < 0.001), and -0.120 log 10 copies/mL (95% CI [-0.17 to -0.076], p  =  < 0.001), respectively. However, these changes were below 0.5 log 10 copies/mL and thus were not clinically significant. Compared with the samples stored at 4 °C, there were no significant differences in pgRNA levels in samples stored at 25 °C for any of the storage durations (-0.01 log 10 copies/mL; 95% CI [-0.708 to 0.689], p  = 0.98). No significant difference in the levels of pgRNA was observed in the plasma samples, following four freeze-thaw cycles at -20 °C and -80 °C., Conclusion: The plasma HBV pgRNA level was stable at 4 °C and at room temperature for at least 48 h and under multiple freeze-thaw cycles. Our results suggest that pgRNA is stable during the process of blood collection, and therefore results of pgRNA quantification are reliable., Competing Interests: The authors declare there are no competing interests., (©2021 Rattanachaisit et al.)

The study aims to investigate the effect of modern technology in the trigger of migraine headaches in high school students by using generalized estimating equation analysis. The prospective time series study was conducted in one hundred and forty-five smartphones using high school students in Chiang Mai Province who each completed a headache diary giving a total of 12,969 data entries. The smartphone output power was measured and recorded by a smartphone application. Smartphone use, sleep quality, anxiety, and depression also were assessed. Results revealed that the prevalence of repeated headache be 13.4% and migraine 16.9%. The migraine had the strongest association with handfree device use and internet use, followed by age and anxiety. Furthermore, the strongest effect of smartphone output power in triggering a migraine was found at ≤ 1.79×10-5 and 1.80-1.99×10-5 mW ranges. Meanwhile, Lag&#95;6 of daily smartphone output power exposure produced the migraine effect in a reverse dose-response manner. The smartphone electromagnetic radiation was a primary migraine trigger. The study results led to the recommendation to avoid triggers by use of smartphone talking with hand-free devices to prevent a recurrent migraine.

The World Health Organization aims to eliminate HCV infection worldwide by 2030. A targeted HCV screening policy is currently unavailable in Thailand, but a decrease in HCV infection has been observed in the country. However, a previous study showed that there was a higher HCV seroprevalence in adults aged between 30-64 years in the Phetchabun province (15.5%), as compared to the Khon Kaen province (3.6%). It was hypothesized that young adults had a lower rate of HCV seropositivity; this was determined by the age distribution of anti-HCV in Phetchabun and with the identification of high seroprevalence birth cohorts. In order to compare the provincial findings to the national level, anti-HCV birth cohorts were further analyzed in Khon Kaen (averaged-HCV prevalence) as well as the Thai data set that was derived from the previous literature. Thai individuals aged between 18-30 years residing in Phetchabun (n = 1453) were recruited, tested for the presence of anti-HCV antibodies and viral RNA and completed questionnaires that were designed to identify HCV exposure risks. Data was collected and compiled from previously published articles (n = 1667, age 30-64 years). The HCV seropositivity in Phetchabun by age group (18-64, at 5-year intervals) and the birth year were tabulated parallel to the Khon Kaen data set (n = 2233) in conjunction with data from the national survey 2014 (n = 5964) representing the Thai population. Factors such as age, male gender, agricultural work, blood transfusion, intravenous drug use and having a tattoo were associated with anti-HCV positivity in Phetchabun. HCV seroprevalence was less than 4.0% (ranging from 0.0-3.5%) from the age of 18-34 years. A dramatic increase of 15.1% was found in adults aged greater than or equal to 35 years, whereas, the age group in Khon Kaen and the national population with increasing prevalence of HCV were older (≥40). The HCV seropositivity cohort accumulated for those born between 1951-1982 accounted for 71.4-100.0% of all seropositive individuals. Subsequently, new cases occurred sporadically. This finding provides evidence that there is a disproportionately high HCV seroprevalence among people born before 1983 (or aged ≥35). This cohort should be targeted for priority screening as part of the national HCV screening policy. Incorporating this birth cohort with other risk factors could improve HCV diagnostic rates, resulting in overall improvements in parallel to those given by novel antiviral treatment., Competing Interests: The authors have declared that no competing interests exist.

The prevalence of hepatitis C virus (HCV) infection has been decreasing globally, but the growing effects of HCV-related morbidity and mortality remain of concern. Advances in curative medicine, involving direct-acting antivirals (DAAs), have led many countries to aim to eradicate HCV. Information on epidemiology and disease burden is essential for national policy development. Thus, this study aimed to determine the HCV-related hepatic disease burden in areas of Thailand with high and average HCV prevalence in order to extrapolate the viral burden across Thailand. Patients previously diagnosed as positive for anti-HCV antibodies were recruited to assess chronic HCV infection (CHC) status, liver function, HCV-RNA level and hepatic fibrosis. The number of patients eligible for Universal Health Coverage (UC) scheme and the approximately required expenditure on interferon (IFN)-based treatment were estimated. In areas of both high (12%) and average (2%) HCV viremic prevalence, over half of the patients (52.2% to 62.5%) had advanced liver fibrosis (F3 and F4). A striking percentage of patients with F4 (38.9%) were found in the high-prevalence area, while comparable proportions of advanced liver fibrosis presented in the two areas and disease burden peaked at 50-59 years. Under the current UC program treatment scenario, 78-83% of CHC patients with stage F2-F4 fibrosis were eligible for treatment. The estimated expenditure required for overall CHC treatment across the whole country was 1,240 million USD at this current status, but the declining cost of generic DAA-based therapy may reduce the requirement to <90 million USD. This study provides information on the estimated number of CHC patients, liver disease burden and expenditure requirements for Thailand. To eliminate HCV by 2030, proactive government strategies raising public health to minimize transmission and emphasizing targeted screen-and-treatment programs, novel therapeutic guideline development for decentralizing treatment, and effective budget allocation are urgently needed.

The core antigen of the hepatitis C virus (HCV Ag) presents an alternative marker to HCV RNA when screening patients for HCV viremia. This study sought to evaluate the utility of HCV Ag as a marker to assess active HCV infection in individuals residing in an HCV-endemic area. From 298 HCV-seropositive individuals evaluated for the presence of anti-HCV antibody, HCV Ag and HCV RNA, anti-HCV antibody was detected in 252 individuals (signal-to-cutoff ratios ≥5), HCV RNA was detected in 222 individuals (88%), and HCV Ag was reactive (≥3 fmol/L) in 220 individuals (87%). HCV genotype 1, 3, and 6 were identified. HCV Ag significantly correlated with HCV RNA irrespective of HCV genotype and/or HBV co-infection (log HCV RNA = 2.67 + 0.95 [log HCV Ag], R 2  = 0.890, p  < 0.001). To predict HCV viremia (HCV Ag ≥ 3 fmol/L), the accuracy, sensitivity, specificity, positive predictive value, and negative predictive value were 99%, 99%, 100%, 100% and 97%, respectively. We concluded that HCV Ag was a good surrogate marker for HCV RNA and could be used to diagnose active HCV infection in a resource-limited setting. As a result, a cost-effective strategy for screening and identifying active HCV carriers using HCV Ag detection would enable more patients access to efficacious and increasingly affordable direct-acting antivirals (DAAs) for the treatment of HCV infection., Competing Interests: The authors declare there are no competing interests.

Improved awareness of the hepatitis C virus (HCV) transmission has contributed to the overall decline in the HCV infection rate in some developing countries including Thailand. Chronic HCV infection in some rural Thai communities, however, presents a challenge in the efforts to treat and manage HCV-related diseases. Published and unpublished studies have suggested an unusually high incidence of HCV infection in a Thai province of Phetchabun compared to elsewhere in Thailand. To determine the magnitude of HCV infection and identify potential factors contributing to the higher rate of HCV infection in this province, we performed a population-based study in Phetchabun (n = 1667) and the neighboring Khon Kaen province (n = 1410) where HCV prevalence is much lower. Individuals between 30 and 64 years old completed detailed questionnaires designed to identify HCV risk factors and provided blood samples for anti-HCV antibody screening. The anti-HCV seropositive rates were 15.5% (259/1667) in Phetchabun and 3.6% (51/1410) in Khon Kaen. Positive samples were subsequently genotyped for HCV core gene sequence and assessed for the hepatitis B virus surface antigen (HBsAg) and human immunodeficiency virus antigen/antibody (HIV Ag/Ab). More individuals in Phetchabun possessed the combined presence of HBsAg (5.0%) and HIV Ag/Ab (0.4%) than those in Khon Kaen (3.9% HBsAg and 0.0% HIV Ag/Ab). While male gender, intravenous drug use (IVDU) and tattoos were significant HCV risk factors in both provinces (p <0.05), education less than high school and agriculture-related occupation were additionally associated with HCV in Phetchabun. HCV genotypes 6, 3, and 1 were identified in similar frequency in both provinces. We estimated that prevalence of HCV seropositivity and viremic carriers were higher in Phetchabun (143 and 111 per 1000) than in Khon Kaen (34 and 22 per 1000). Finally, we derived a simple risk factor-based scoring system as a useful preclinical tool to screen individuals at risk of chronic HCV infection prior to intervention. Knowledge gained from this study will assist in HCV screening and promote access to anti-viral treatment in high-risk groups.

The majority of hepatitis C virus (HCV) infection results in chronic infection, which can lead to liver cirrhosis and hepatocellular carcinoma. Global burden of hepatitis C virus (HCV) is estimated at 150 million individuals, or 3% of the world's population. The distribution of the seven major genotypes of HCV varies with geographical regions. Since Asia has a high incidence of HCV, we assessed the distribution of HCV genotypes in Thailand and Southeast Asia. From 588 HCV-positive samples obtained throughout Thailand, we characterized the HCV 5' untranslated region, Core, and NS5B regions by nested PCR. Nucleotide sequences obtained from both the Core and NS5B of these isolates were subjected to phylogenetic analysis, and genotypes were assigned using published reference genotypes. Results were compared to the epidemiological data of HCV genotypes identified within Southeast Asian. Among the HCV subtypes characterized in the Thai samples, subtype 3a was the most predominant (36.4%), followed by 1a (19.9%), 1b (12.6%), 3b (9.7%) and 2a (0.5%). While genotype 1 was prevalent throughout Thailand (27-36%), genotype 3 was more common in the south. Genotype 6 (20.9%) constituted subtype 6f (7.8%), 6n (7.7%), 6i (3.4%), 6j and 6m (0.7% each), 6c (0.3%), 6v and 6xa (0.2% each) and its prevalence was significantly lower in southern Thailand compared to the north and northeast (p = 0.027 and p = 0.030, respectively). Within Southeast Asia, high prevalence of genotype 6 occurred in northern countries such as Myanmar, Laos, and Vietnam, while genotype 3 was prevalent in Thailand and Malaysia. Island nations of Singapore, Indonesia and Philippines demonstrated prevalence of genotype 1. This study further provides regional HCV genotype information that may be useful in fostering sound public health policy and tracking future patterns of HCV spread.

Background: Transient elastography (TE) is an innovative, noninvasive technique to assess liver fibrosis by measuring liver stiffness in patients with chronic liver diseases. The purpose of this study has been to explore the accuracy of TE and clinical parameters in predicting the presence of esophageal/gastric varices in children with biliary atresia (BA) following portoenterostomy., Methods: Patients with BA status post portoenterostomy and normal children were recruited. Splenomegaly and presence of EV/GV were determined by physical examination and endoscopy, respectively. Aspartate transaminase to platelet ratio index (APRI) was used as a serum fibrosis marker. TE was performed by using FibroScan. Data was expressed as mean ± SD., Results: Seventy-three BA patients (male:female = 32:41; age 9.11 ± 5.64 years) and 50 normal controls (male:female = 19:31; age 11.00 ± 3.31 years) were enrolled. The liver stiffness score of BA patients was significantly higher than that of normal controls (27.37 ± 22.48 and 4.69 ± 1.03 kPa; p < 0.001). Patients with EV/GV had significantly higher liver stiffness score and APRI than those without EV/GV. As for EV/GV diagnosis, the areas under the receiver operating characteristic curve were 0.89 (95% CI 0.80 to 0.98) for TE and 0.87 (95% CI 0.78 to 0.96) for APRI, respectively. The sensitivity (and specificity) of TE (using a cut-off value of 12.7 kPa) and APRI (using a cut-off value of 1.92) in predicting EV/GV were 84% (77%) and 84% (83%), respectively, whereas the sensitivity (and specificity) of splenomegaly in predicting EV/GV were 92% (85%)., Conclusions: Transient elastography is a useful tool for predicting the presence of EV/GV. In addition, basic physical examination, routine biochemical and hematological tests, are still worthwhile and correlate well with the presence of EV/GV in patients with BA post portoenterostomy., (© 2011 Chongsrisawat et al; licensee BioMed Central Ltd.)