Chronic pancreatitis (CP) is a progressive, fibro-inflammatory disease characterized by enzymatic autoactivation and subsequent fibrotic replacement of acinar cells. A significant proportion of patients develop pain, which may be due to many causes, including perineural inflammation, altered central processing of pain signals, parenchymal structural changes, and ductal obstruction. Currently there are no approved medical treatment options for CP-associated pain. NI-03 (camostat mesilate) is an orally administered serine protease inhibitor that reduces pancreatic enzyme activity and has been widely used for the treatment of CP-associated pain in Japan. The current study will assess the safety and efficacy of NI-03 for reduction of CP-associated pain in the USA.The current study consists of two phases. First, a phase I study will be performed to establish the pharmacokinetics and safety profile over a 1-week period following a single dose . Subsequently, a phase II study will be performed consisting of a double-blind, randomized, controlled trial (RCT). This RCT will evaluate the efficacy of each of the three doses of NI-03 given three times daily compared to placebo over 28 days. A 7-day, single-blind, run-in period will precede the double-blind phase to assess baseline pain characteristics. The primary efficacy outcome is the average of worst daily pain scores  over the terminal 7 days of the study period compared to baseline. Secondary efficacy outcomes include change in opioid dose and quality of life measures, and time to first rescue intravenous analgesic. Adverse events will be recorded.NI-03 has been used successfully and safely in Japan to treat CP-associated pain. The aim of the current study is to assess the safety and efficacy of NI-03 using a rigorous RCT in a population in the USA. This study may fill an important clinical gap to provide an effective medical treatment option for CP-associated pain.NCT02693093. Registered through the National Institutes of Health on 26 February 2016.ClinicalTrials.gov, The online version of this article (10.1186/s13063-019-3606-y) contains supplementary material, which is available to authorized users. Chronic pancreatitis (CP) is a progressive, fibro-inflammatory disease characterized by progressive inflammation resulting in loss of pancreatic endocrine and/or exocrine function . In the Surgical resection or drainage procedures and endoscopic therapy have been demonstrated to be effective for management of pain in the context of “large duct disease” , but effective therapies are needed for those without anatomic obstruction. While non-steroidal anti-inflammatory agents are commonly used in clinical practice, their efficacy has not been evaluated in human trials. A variety of medications have been examined in international studies including antioxidants, gabapentinoids, and serine protease inhibitors, with mixed results.Vitamins with antioxidant properties are hypothesized to reduce the oxidative stress within the pancreatic microenvironment leading to improvement in CP-associated pain due to perineural inflammation . Two larSerine protease inhibitors, or anti-proteases, are hypothesized to inhibit pancreatic enzyme activity thereby reducing hyperamylasemia, inflammatory symptoms, and pain . CamostaSeveral human studies have demonstrated the safety and efficacy of camostat mesilate, 200 mg three times daily, based on subjective complaints and objective findings in subjects treated for up to 8 weeks for AP and CP –21. ImpoThe effects of camostat mesilate on pancreatic function have also been examined. A small, double-blind RCT reported that short-term use of camostat mesilate improved maximal bicarbonate secretion, suggesting improved duct cell function compared to placebo . This stThe reported safety profile of this class of drugs is favorable. Reported adverse effects are rare (< 3%) and typically mild  , 14.The primary objective of this study is to assess the safety and efficacy of NI-03, a serine protease inhibitor, for the treatment of CP-associated pain compared to placebo. In this trial we will initially evaluate the safety and pharmacokinetics after administration of a single dose of three different dosages of NI-03 to confirm acceptability of a dosage (300 mg) that is higher than previously studied. Subsequently, we will perform a double-blind, randomized, placebo-controlled trial of these three dosages of NI-03 to assess the efficacy for treatment of CP-associated pain.Assess the pharmacokinetic and safety profiles following administration of a single dose of NI-03 .Evaluate the efficacy and safety profile of NI-03  administered three times daily for 28 days for CP-associated pain in a double-blind, randomized, placebo-controlled trial.The objectives of the study are to:Adults aged 18–85 years.Diagnosis of chronic pancreatitis supported by a combination of cross-sectional imaging, endoscopic ultrasound (EUS), endoscopic retrograde pancreatography (ERP), and/or assessment of pancreatic function Tables  and 2 22–24.Average baseline pain score ≥ 4/10 using a numeric rating scale (0–10) during the 7-day run in period.Stable analgesic regimen: if oral narcotic analgesics are utilized, the daily oral morphine equivalent (OME) dose should not exceed 100 mg.Ability to use contraception method from screening until 28 days after completion of the study medication.Ability to understand and provide written informed consent.The inclusion criteria are:Comorbid medical conditions: including clinically significant cardiovascular disease, active infection within 30 days of day 1, seizure within the past 12 months, pregnancy or planned pregnancy or active breast feeding, history of malignancy within 5 years of study enrollment, or HIV infection.Renal or hepatic dysfunction: including stage IV chronic kidney disease (estimated using the Cockcroft-Gault formula). Active, chronic hepatitis B infection (surface antigen positivity), chronic hepatitis C infection (including a detectable PCR level or undetectable levels in advanced fibrosis (histologic grade 3–4/4), or cirrhosis based on previous evaluation including biopsy, a noninvasive estimate of fibrosis, or radiographic features.Diagnosis of autoimmune pancreatitis: based on the International Consensus Diagnostic Criteria for Autoimmune Pancreatitis .Use of potentially confounding medications: including other experimental medications, recent change in selective serotonin reuptake inhibitor (SSRI)/serotonin-norepinephrine reuptake inhibitor (SNRI) dosage, systemic steroids, anti-epileptics, or antipsychotics.Potential confounding of pain assessment: including the presence of generalized pain syndrome prohibiting the differentiation of abdominal pain, major abdominal surgery or endoscopic intervention  within 90 days of enrollment.Substance abuse: including use of illegal substances, use of cannabinoids (subjects must have a 28-day wash-out period and negative drug test at screening and at day 29), or alcohol consumption exceeding 2 drinks per day (or 14 drinks per week).Miscellaneous: inadequate venous access, known hypersensitivity to NI-03 or one of its excipients, inability/unwillingness to comply with study restrictions, or blood donation or transfusion within 7 days of enrollment.The exclusion criteria are:The study drug, NI-03, is camostat mesilate and will be manufactured in tablet form by Nichi Iko (Japan). Placebo tablets are manufactured by Stason Pharmaceuticals, Incorporated . The drug will be placed into gelatin capsules in a manner that the tablet labeling is concealed and will be indistinguishable from placebo tablets. The encapsulated tablets will be distributed as a multiple dose card containing 24 capsules to account for three capsules per day and three extra capsules per week.We will evaluate drug safety and pharmacokinetics during the single-dose study. We will recruit approximately 6–8 subjects per dose  in this phase I study, giving a total of 18–24 subjects. After a single oral dose, blood samples will be drawn at 0.25, 0.5, 1, 2, 4, and 8 h. Samples will be collected, processed, and shipped according to standard operating procedures. Subjects will have a follow-up visit 1 week after the dose to evaluate subjects for adverse effects. These data will be submitted to the Food and Drug Administration (FDA) for formal safety evaluation before proceeding with further study.The phase II study will begin with a 7-day, single-blind, placebo run-in period. During this week, baseline characteristics will be assessed and subjects will have the opportunity to become familiarized with the pain rating scales. After the 1-week run in, subjects with an eligible baseline pain score will be randomized to one of four arms  for a 28-day treatment period  versus “High” (50–100 mg/day)). Subjects will be randomized by block. Each site has 6 reserved blocks, including 4 blocks of 4 units and 2 blocks of 8 units . Following randomization, investigators and participants will be blinded to treatment assignments for the remainder of the study.The primary outcome is change in average daily worst pain intensity score. Daily pain intensity will be scored using a numerical rating scale from 0 to 10. These values will be averaged over each week of the study, with the primary comparison between the placebo run-in period and the terminal 7 days of the treatment phase . Averaging pain scores over 1 week will adjust for those subjects with intermittent pain. Those with infrequent pain will be excluded if the average weekly pain score is < 4/10.Secondary outcomes include change from baseline in least pain score, average pain score, current pain score, change in average OME and gabapentin/pregabalin daily dosages, time to first intravenous analgesic use (as rescue therapy), and change in quality of life assessed by the Pancreatic Quality of Life Instrument (PANQOLI)  and painStudy recruitment will occur at 45 centers in the USA. Various methods will be used to identify potential participants, including surveillance of clinic schedules, notification of potential referring providers, and direct advertisement to potential participants using research match tools. Trained study personnel at each site will recruit subjects.Assuming a one-unit difference between each NI-03 dose and placebo, and a standard deviation (SD) of 1.6, the initial total sample size is calculated as 120 subjects. This will yield approximately 80% power at an overall two-sided type 1 error rate of 5% using Dunnett’s procedure. Assuming up to 5% of subjects will not qualify for the full analysis set due to incomplete data collection or failure to follow up, the total initial sample size is increased to 128 subjects. When approximately 50% of the subjects have completed the phase II study, an interim analysis will be performed to update the SD estimate primary endpoint, and the sample size will be recalculated. Data quality will also be reviewed during the interim analysis.All subjects who receive at least one dose of NI-03 and have baseline and post-baseline pain scores will be included in the dataset for analysis. Subjects’ data will be analyzed according to their assigned treatment . An additional per-protocol analysis is planned to evaluate only the data from subjects without protocol deviations -based repeated measures approach  analysis). The MMRM model will include treatment, stratification factor , center, baseline average daily pain intensity score, visit, and treatment by visit interaction as fixed-effect explanatory variables. The study center will be included in the model as a random effect. Additionally, analysis of covariance (ANCOVA) of the change from baseline to week 4 in the average daily worst pain intensity score, with the baseline score as the covariate, both treatment and stratification factor as fixed effects, and center as a random effect, will be assessed in a robustness analysis using the baseline observation carried forward (BOCF) approach.Regular monitoring of study sites will be performed by an independent contract research organization (CRO) throughout the study period with a focus on processes for handling, reporting, and archiving data. An additional data monitoring committee will not be used for this study as the CRO will be involved with auditing study procedures and completeness of case report forms. Data sharing among study investigators will occur via secure email. The final trial dataset will be available to the Sponsor and co-authors of the study manuscript.Safety will be monitored through weekly contact: telephone calls during weeks 1 and 3 and office visits during weeks 2 and 4. All adverse events will be recorded. The investigator will determine both the severity of the event and relationship to study treatment, based on clinical judgment. Subjects may discontinue treatment if adverse events occur or they may be removed from treatment as determined by the investigator. Rationale for drug discontinuation will be reported. The investigator will not be un-blinded if a serious adverse event occurs. Every serious adverse event  will be reported to the lead investigator as soon as possible to determine causality. The event will be submitted expeditiously in the event of an adverse drug reaction. Medical care for study-related adverse events will be paid for by the study Sponsor.This phase I/II study will assess the safety and efficacy of NI-03 for the treatment of CP-associated pain in a double-blind, randomized, placebo-controlled trial. During the phase I study, a pharmacokinetic evaluation will occur after one dose, with safety assessments at 1 week. The subsequent phase II study will consist of a 1-week, single-blind, run-in period followed by a 4-week, double-blind treatment period to compare the change in average daily worst pain intensity scores. Key secondary outcomes will include changes in concurrent analgesic dose and quality of life scores. The purpose of this study is to fill an important clinical gap in providing an effective medical treatment option for CP-associated pain.The management of painful CP begins with abstinence from alcohol and tobacco and often includes assessment for local complications or pancreatic duct obstruction, which may lead to procedural or surgical interventions. Analgesia is commonly provided in keeping with the World Health Organization pain ladder for those in whom anatomic intervention is not clearly warranted. Unfortunately, these interventions involve generic management of chronic pain and do not target the underlying mechanisms leading to CP-associated pain or CP-related disease progression.Oral analgesic agents, including antioxidants and pregabalin, addressing the mechanisms of painful CP have been previously studied –7. HowevPain remains a common and frequently debilitating symptom for many patients with CP. Despite the associated morbidity, high-quality evidence-based treatment options remain limited. This study will evaluate the pharmacokinetics, safety, and efficacy of an orally available serine protease inhibitor, NI-03, for CP-associated pain. If efficacious, this will be a novel agent for treatment of symptoms among patients with CP in the USA.This trial was prospectively registered through the National Institutes of Health on 26 February 2016 (NCT02693093). Enrollment for the phase I study began in December 2015. Enrollment for the phase II study began in April 2017 and remains ongoing. Any amendments to the above protocol will be reported with the study publication.Additional file 1: Standard protocol items: recommendation for interventional trials (SPIRIT) checklist for the current study. (DOC 121 kb)


However, among nonalcohol drinkers, there was no association between smoking or secondhand smoking status and these risks. These findings suggest that women exposed to secondhand smoking at home or in the workplace might be at high risk of HR-HPV persistence when it is combined with alcohol drinking, even though neither active smoking nor secondhand smoking independently affects the risk.Tobacco smoking is established as a cofactor of human papillomavirus (HPV) for cervical cancer risk. However, the role of secondhand smoking in cervical carcinogenesis is controversial. We aimed to assess the association between secondhand smoking and high risk- (HR-) HPV persistence, a pivotal event in development of cervical cancer. In total, 9,846 women who underwent health-screening examinations from 2002 to 2011 at the National Cancer Center, Korea, were included. Secondhand smoking was defined as being exposed to secondhand smoke at home or in the workplace. Multivariate logistic regression analysis was used to estimate the odds ratios (OR) and 95% confidence intervals (CIs) for risks of HR-HPV infection at baseline , 1-year persistence , and 2-year persistence . Active smoking, secondhand smoking, and secondhand smoking in nonactive smokers had no association with these risks. Among alcohol drinkers, secondhand smoking in nonactive smokers had higher risks of HR-HPV infection at baseline (OR = 1.25, 95% CI = 1.05–1.48, Several studies, including a large meta-analysis and several cohort studies, have shown an association between cigarette smoking and cervical cancer or its precursor lesions –3. CigarIn the Republic of Korea, cigarette smoking among women is rare 7% in 2009), but the proportion of men aged ≥ 15 years who smoke is the second highest among the Organization for Economic Cooperation and Development (OECD) countries . Similar% in 2009Although most studies have investigated cervical cancer precursors, we have focused on the initiation of cervical carcinogenesis  in order to aid efforts at primary prevention of cervical cancer. In the present study, we aimed to assess the associations between smoking status  and the risks of HR-HPV infection at baseline, 1-year HR-HPV persistence, and 2-year HR-HPV persistence as well as the same associations among alcohol drinkers.This study was a part of the Korean Prospective Study for the Transition of Human Papillomavirus into Cervical Carcinoma , 15. AmoThis study received approval from the Institutional Review Board of the National Cancer Center, Korea (NCCNCS-11-433), and all of the study participants provided informed consent.supplement). All of the participants were asked if they had ever smoked a cigarette. Nonsmokers were defined as having smoked <100 cigarettes in their lifetimes . Smokers were defined as having smoked ≥100 cigarettes in their lifetimes (n = 825). Regarding SHS, all of the participants were asked, “Do you live with anyone in your household who regularly smokes cigarettes or other tobacco products (other than yourself)?” and “Do you work with anyone in your workplace who regularly smokes cigarettes or other tobacco products (other than yourself)?” (1) Exposure to SHS at home = No/Yes, how many family persons who smoked regularly, time of exposure to SHS; (2) exposure to SHS in the workplace = No/Yes, time of exposure to SHS. We did not account for exposure to SHS in public places. Secondhand smoking was defined as being exposed to SHS in either the home or the workplace, regardless of active smoking status, while non-SHS was defined as not being exposed to SHS at home or in the workplace. In addition, SHS in nonsmokers was defined as having never smoked in life but having been exposed to SHS.Information on smoking and secondhand smoking behaviors was collected using a standardized questionnaire . A chemiluminescent HPV DNA test was used to measure relative light units (RLU) with a probe designed for 13 types of HR-HPV. The test results were read as positive at concentrations of 1 pg/ml or greater than the RLU/cutoff ratio (RLU of specimen/mean RLU of two positive controls). The cytological grades for Pap smear reports were based on the Bethesda classification system . t-test were used to assess the differences in the distributions of categorical and continuous variables between groups, respectively. Multivariate logistic regression models were used to estimate odds ratios (ORs) and corresponding 95% confidence intervals (CIs). A multivariate logistic regression analysis was run to assess the associations between smoking status and risk of HR-HPV infection at baseline, 1-year persistence, and 2-year persistence after adjustment for confounding factors. We checked for confounding by calculating the Mantel-Haenszel summary measure for the effect of alcohol drinking status. After separating data for strata defined for alcohol drinkers and nonalcohol drinkers, a multivariate logistic regression analysis was again performed to assess the association between smoking status and risk. All of the analyses were adjusted for age and BMI continuously and for marital status, parity, menopausal status, oral contraceptive use, education level, and alcohol drinking status as well. Risk estimates were calculated for nonactive smoking, non-SHS smoking, and non-SHS smoking in nonactive smokers as reference categories. All of the analyses were performed with SAS 9.3  and the episensr packages in R language (version 3.3.2) [Descriptive statistics were used to summarize the overall distribution of characteristics by study group. The chi-square test andn 3.3.2) .2. Most of the subjects were married (97%) and had two or more children (87.3%). Approximately 60% of the subjects were postmenopausal women and 20% had used oral contraceptives (currently or in the past). Approximately one-half of the subjects had consumed alcoholic beverages (currently or in the past), and 8 and 37% were active cigarette smokers and secondhand smokers, respectively. At enrollment, the majority of the subjects (97%) had normal cytological results, but 186 (2.0%) had atypical squamous cells of undetermined significance (ASCUS), 58 (0.6%) had low-grade squamous intraepithelial lesions (LSIL), and 29 (0.3%) had high-grade squamous intraepithelial lesions (HSIL) or more severe abnormalities. There were wide variations in the distributions of age, BMI, menopausal status, education level, alcohol drinking status, and active smoking status. These confounding factors were adjusted in a subsequent multivariate logistic regression analysis.Of the 9,846 women, 980 (9.9%) were infected by HR-HPV at enrollment  and the risks of HR-HPV infection at enrollment, 1-year HR-HPV persistence, and 2-year HR-HPV persistence . Only alp = 0.038, homogeneity of the OR). The subjects were stratified by alcohol drinking status (nondrinkers/current or past drinkers), and the associations between smoking status and the risks of HR-HPV infection and its persistence were assessed among alcohol drinkers and nondrinkers, separately ( p for multiplicative interaction = 0.005), 1-year HR-HPV persistence , and 2-year HR-HPV persistence . Secondhand smoking in nonactive smokers also had higher risks of HR-HPV infection at baseline , 1-year HR-HPV persistence , and 2-year HR-HPV persistence . Among alcohol nondrinkers, active smoking had no association with these risks, but SHS had lower risks of HR-HPV infection at baseline  and 1-year HR-HPV persistence . SHS in nonactive smokers also had lower risks of HR-HPV infection at baseline  and 1-year HR-HPV persistence .Previously, alcohol drinking behaviors and their combination with high HR-HPV viral load were associated with risk of HR-HPV persistence , 15. Theparately . Among a Ours is the first report of an association between SHS and HR-HPV persistence and the combined effect of SHS and alcohol drinking on that risk. Whereas most of the previous studies focused on cervical cancer precursors [Our findings showed that the combination of exposure to SHS and alcohol drinking was associated with higher risks of HR-HPV infection and its persistence in women who had never smoked in their lives but had been exposed to SHS.ecursors , our preTobacco smoking has been established as an important risk factor in cervical carcinogenesis , 18–21 aAs these findings suggest, exposure to SHS might be related to the development of cervical neoplasia in Korea women. A previous study showed that alcohol drinking is associated with a higher risk of HR-HPV persistence in Korean women . It is cThere are plausible mechanisms explaining the impact of SHS on risk of HPV persistence. Tobacco includes carcinogens that could have a direct transformational effect on the cervix, leading to immune suppression and HPV persistence , 30. ActOur study has several limitations. First, although sexual behaviors are critical confounding factors of HPV infection , bias duIn conclusion, alcohol drinkers exposed to SHS at home or in the workplace had a higher risk of persistent HR-HPV infection than did alcohol drinkers not so exposed. This combined effect was observed also in women who had never smoked in their lives. Reducing both exposure to SHS and consumption of alcoholic beverages could be a prudent approach to the prevention of cervical cancer.


KCNJ2, FBN2, SLC38A3 and TNNI1. Overall, our study provides additional insights into post-transcriptional gene regulation of malformed hearts of TOF patients.MicroRNAs (miRNAs) play an important role in guiding development and maintaining function of the human heart. Dysregulation of miRNAs has been linked to various congenital heart diseases including Tetralogy of Fallot (TOF), which represents the most common cyanotic heart malformation in humans. Several studies have identified dysregulated miRNAs in right ventricular (RV) tissues of TOF patients. In this study, we profiled genome-wide the whole transcriptome and analyzed the relationship of miRNAs and mRNAs of RV tissues of a homogeneous group of 22 non-syndromic TOF patients. Observed profiles were compared to profiles obtained from right and left ventricular tissue of normal hearts. To reduce the commonly observed large list of predicted target genes of dysregulated miRNAs, we applied a stringent target prediction pipeline integrating probabilities for miRNA-mRNA interaction. The final list of disease-related miRNA-mRNA pairs comprises novel as well as known miRNAs including miR-1 and miR-133, which are essential to cardiac development and function by regulating  The symptoms vary from life-threatening at one extreme to somewhat benign. One severe malformation is Tetralogy of Fallot (TOF). This heart defect is the most common cyanotic one with a prevalence of ~0.3 per 1.000 live births, accounting for 7–10% of all CHDs3. TOF is characterized by four structural abnormalities, namely a narrowing of the right outflow tract (pulmonary stenosis), a ventricular septal defect (VSD), a displacement of the aorta to the right side over the VSD (an overriding aorta) and a hypertrophy of the right ventricle4. Like the majority of CHDs, TOF is most probably caused by an interplay of multiple subtle genetic, structural genomic or epigenetic alterations with environmental stimuli9. Moreover, etiologies of CHDs also include the disruption of developmentally transcriptional regulation, which requires precise spatiotemporal control of gene expression8. Here, post-transcriptional regulation by microRNAs (miRNAs) has emerged as an important regulator in development and maintaining function of the human heart10.Over the last century, cardiovascular diseases have slowly overtaken infectious diseases as the leading cause of death worldwide. Among various subdivisions, congenital heart diseases (CHDs) feature prominently and contribute appreciably to the mortality rates11. Studies have been performed on tissue-specific and circulating miRNAs as novel diagnostic and prognostic biomarkers14. Recently, altered expression levels of circulating miRNAs were found in blood of TOF patients after surgical repair being mainly surgical related. These miRNAs differ compared to those seen in the right ventricle (RV) of the patients15 and show limited correlation with cardiac functional indices assessed by echocardiography16. Studies of cardiac tissue of TOF range from single candidate approaches to microarray studies21 up to whole small RNA transcriptome analysis using high-throughput sequencing22. Overall, these studies show a broad range of altered miRNAs in TOF with heterogeneous results between them.MicroRNAs are small, non-coding RNAs that non-precisely complementary bind to the 3′ untranslated regions of target mRNA, which results in translational repression or mRNA degradationHere, we profiled genome-wide the whole transcriptome in cardiac tissues. We analyzed the relationship of miRNAs and mRNAs in RV tissue of a homogeneous group of non-syndromic TOF patients compared to normal right and left heart controls. To narrow down the large list of putative functional target genes of differentially expressed miRNAs, the expression is correlated to mRNA expression profiles of cardiac tissue from the same group of patients and controls. By this, we add a novel layer of data as this comparison was so far limited.Small RNA sequencing of RV tissue of 22 isolated TOF patients (TOF-rv) as well as tissue from RV and LV of four normal hearts  serving as controls . Among the 111 up-regulated miRNAs in TOF, we only found three miRNAs , which harbor decreased reads counts from infant TOF hearts to the mid-age adult heart of NH-08. For the 61 down-regulated miRNAs, there are five miRNAs  with continuous increased read counts from infant TOF hearts to the mid-age adult normal heart. Whether differential expression of these miRNAs between TOF and normal heart (only 5 out of 172) is age- or disease-related cannot be clearly determined and therefore, we did not exclude these miRNAs from further analysis.23. Approximately 25% of the differentially expressed miRNAs (43 out of 172) overlaps with results from other studies based on right ventricular tissue of TOF patients versus normal hearts, with only one miRNA altered in all studies, namely miR-222-3p . Interestingly, miR-223-3p is also down-regulated in the TOF-rv compared to NH-rv, which is characterized by a hypertrophic cardiac RV mass and increased ventricular pressure that is in line with the comparison of NH-rv and NH-lv. This miRNA regulates the Glucose Transporter 4 (Glut4) protein expression and cardiomyocyte glucose metabolism27 and TargetScanHuman28 and linking to 972 mRNAs, which are also significantly differentially expressed in the right ventricle of these patients and controls7. Note that the already published mRNA-seq data7 were generated from cardiac tissue of the same TOF and normal hearts. Following a stringent approach, we only selected negatively correlated miRNA-mRNA pairs , which corresponds to the main regulatory mechanism of miRNAs29. This prediction and filtering approach resulted in 344 pairs comprising 235 genes (protein-coding mRNAs) and only 11 miRNAs from 10 families , miR-140-5p & KLF9 (Kruppel Like Factor 9) and miR-1 & KCNJ2 (Potassium Voltage-Gated Channel Subfamily J Member 2), which are also included in public databases such as DIANA/miRTarBase30. GJA1 and KCNJ2, both targeted by miR-1, are two ion channel genes, which are known to play a major role in cardiac disease and development31.The set of 172 significantly differentially expressed miRNAs between TOF-rv and NH-rv was further selected for target prediction using MIRZA-G32. The results are miRNA-gene pairs, which are most likely to occur given the whole expressed miRNA (n = 657) and mRNA  background. Here, cases that are uniquely predicted in either TOF patients or normal heart controls are most interesting. This holds true for 19 pairs in the TOF patients (comprising 5 miRNAs and 18 genes) and 10 pairs in the controls (comprising 3 miRNAs and 10 genes) es) Fig. . The fines) Fig. .33, which revealed three novel miRNA candidates. Moreover, all three miRNAs are significantly differentially expressed in the right ventricle of TOF patients compared to normal heart controls     Fig.  revealedor) Fig. , which aor) Fig. . The taror) Fig.  of the n34. Furthermore, our study design enabled us to find several miRNAs that are well-known for non-congenital heart diseases35 to be also altered in TOF patients.Post-translational regulation of gene expression by non-coding RNAs plays an important role in multiple cellular pathways and diseases. Here, genome-wide small RNA sequencing of cardiac biopsies from isolated TOF patients and healthy unaffected individuals was performed to investigate the role of miRNAs in the normal and malformed human heart. Deep sequencing revealed mostly up-regulated miRNAs in TOF compared to normal heart controls, which is in line with other heart failure studies22. In general, we do not observe significant sex-specific differences of miRNA expression profiles in our TOF cohort. For miR-1, the fold change of mean normalized read counts between male and female TOF hearts is 1.16, although there is a difference of approximately 800k reads . For miR-133b, the fold change is 1.95 but there is only a difference of 471 reads in total . However, both miRNAs are significantly down-regulated in TOF versus normal heart, which also holds true for a gender-specific analysis. In comparison to the normal heart, which is the subject of this study, sex-specific differences of miRNA expression profiles can be ignored as a balanced number of male and female TOF hearts  and normal hearts  is compared.Recently, sexual differences in miRNA expression of TOF hearts was shown, with miR-1 and miR-133 accounting for the most variance between sexes31 and miR-29a (involved in the control of cardiac fibrosis)31 as well as the up-regulated miR-206 36. Interestingly, these miRNAs were not found to be altered in the already existing array- or sequencing-based studies based on right ventricular tissue of TOF patients24. This is surprising and might be explained by technical aspects such as platform (array versus sequencing), normalization strategy and read count statistic. In particular, the last two aspects are important for highly expressed miRNAs such as miR-1 with more than one million reads for each sample. In general, the overlap with other TOF studies, which are already very heterogeneous in respect to their altered miRNAs, is limited with 25%, and only miR-222-3p overlaps all studies 40. In particular, the cardiac miR-21 is up-regulated with age in mice, and overexpression of Argonaut proteins synergistically induced miR-21. In general, major changes occurred later in life, from middle- to old-aged mice, and half of the candidate miRNAs were clustered40. A miRNA analysis on human skeletal muscle biopsies of young (~31 y) and older adults (~73 y) also revealed age-related differences , and dysregulated miRNAs are related to genes associated with cell cycle, inflammation and stress41. For ethical reasons, it is highly difficult to have high quality cardiac samples of young children. This is further reflected by the limited availability of normal cardiac datasets from young children in general. The only solution to overcome this bottleneck is a conservative analysis and consideration of different aspects. In our study, we compared infant TOF hearts (0–3 y) to young/mid-age adult normal hearts. Our dysregulated miRNAs are not clustered , miR-133b with a component of connective tissue microfibrils  or a sodium-dependent glutamine transporter , and miR-133b or miR-140-5p with the slow skeletal inhibitory subunit of the troponin complex . Of note, we showed that the sarcomeric gene TNNI1 is also affected by DNA methylation changes co-localized with novel, differential splicing events in these TOF patients5.To further reduce the list of significantly expressed miRNAs and to find candidates with impact on gene expression in TOF, target prediction incorporating significantly expressed mRNAs of the same patient and control cohort was performed. Combining miRNA and mRNA expression has been shown to be efficient to identify the top candidates out of a huge number of putative targets for each miRNA5. The network comprises mutated CHD genes and significantly differentially expressed and methylated genes or miRNAs (based on the 344 miRNA-mRNA pairs) in the TOF patients. In summary, genome-wide miRNA and mRNA expression profiles of TOF patients and normal heart controls were investigated. We found several known and few novel altered miRNAs with respectively altered target genes in the right ventricle of TOF patients. Our data suggest disease-relevant miRNA-mRNA pairs that are open to further investigations in heart tissue as well as circulating in the blood.The overlap of differentially expressed miRNA and mRNA expression profiles could dramatically reduce the huge number of putative mRNA targets potentially disease-relevant genes. However, translational repression and target mRNA degradation by miRNAs can only be one small aspect to fully understanding a complex phenotype like TOF. MiRNAs are part of larger co-regulation and interaction networks, which are influenced by multiple factors such as genomic variations, DNA methylation, other non-coding RNAs such as piwi-interacting RNAs or even alternative splicing events as in the case of TNNI1. This is also reflected by miRNA extension to a multilevel interaction network in TOF Fig. 5. The ne44. Moreover, the number of samples derived from male and female individuals is almost equally distributed for both TOF patients and controls  was selected based on our previous evaluations45. The quality of the sequencing data was checked using FASTQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). All samples passed sequence quality.The total RNA was isolated and prepared for small RNA sequencing according to manufacturer’s protocol . Sequencing libraries were generated and purified DNA was used directly for sequencing. 36 bp single-end read sequencing was performed using the Illumina’s Genome Analyzer. The sequencing and probability output files were converted to FASTQ format using MAQ46. The parameters were set as follows: -m 20 (maximum number of best matches), -pa  and -sL 18 (seed length for miRNA of length 19–25 nt). In addition, reads can have at most one error in the seed sequence to be robust towards possible sequencing errors and sequence variations. As MicroRazerS is prefix-based, meaning that the read mapper searches for the longest contiguous match starting at the first read base, no adapter trimming is required. Moreover, this mapping process is robust to possible sequencing errors, which especially occur at the 3′ end of reads46.After initial quality check, the reads were mapped to the human reference genome (hg19) using MicroRazerSMapped reads are annotated based on their overlap to known genomic annotations including precursor and mature miRNAs, transfer RNAs (tRNA), C/D box & H/ACA small nucleolar RNAs (snoRNAs), cajal body-specific RNAs (scaRNAs), ribosomal RNAs (rRNAs), small cytoplasmic RNAs (scRNAs), small nuclear RNAs (snRNAs), miscellaneous other RNAs (miscRNAs), mitochondrial tRNAs (Mt-tRNAs), piwi-interacting RNAs (piRNAs), mRNAs and repeats. Annotations are obtained from the miRBase v20 (GRCh37) and UCSC database . If a read overlapped to known mature/precursor miRNAs it was assumed that the read was a sequencing product of this miRNA and the read was added to its read count. All other small RNA classes were annotated in the same manner and ordered as mentioned above. Reads which could not be overlapped with any known annotations were declared as unknown sequences.47. After removing outliers (TOF-09 and NH-03) based on multi-dimensional scaling, a negative binomial (gamma-Poisson) model was applied to miRNAs with a minimal tag count of more than 50 over all analyzed samples. An exact test was conducted to test differential expression appropriate for the negative binomially distributed miRNA read counts48. The observed p-values from gamma-Poisson tests of TCC normalized miRNA read counts follow a normal distribution  and mRNAs (n = 972) in right ventricular tissue of TOF patients and normal heart controls. Note that the mRNA-seq data from the same TOF and normal hearts were already published by us and the data analysis including differential expression are described elsewhere7. As MIRZA-G combines all miRNA-mRNA pairs in one single pair, the same approach was applied to all TargetScan pairs. TargetScanHuman was adapted to report all miRNAs belonging to a miRNA family. All transcript scores for one gene were combined to one gene score. The score was taken from best score over all transcripts. The scores of MIRZA-G ranged between 0 and 3.6 and for TargetScan between −3 and −0.02. No additional thresholds were applied to the scores. The predicted miRNA-mRNA pairs were further filtered applying a probabilistic miRNA-mRNA interaction signature (ProMISE) approach32 using all expressed miRNAs  and mRNA  > 1 in at least one individual) in TOFs and/or controls. Only pairs with a ProMISE score > 0.004 were used (~90% quantile in NH-rv and ~87% quantile in TOF-rv). Over-representation analysis for GO terms and biological pathways based on genes identified in miRNA-mRNA pairs after ProMISE filtering was performed using ClueGO 49.To predict miRNA targets, the tools MIRZA-G33. Briefly, all read sequences with a mapped read length of less or equal than 25 nt (longer sequences are unlikely to represent mature miRNA sequences) as well as a sequence count of more than 25 (removing noise) which are not annotated to known miRNAs or other small non-coding RNAs were used for novel miRNA prediction. Within the miRDeep approach, each potential miRNA precursor sequence was assessed after folding into a hairpin structure using the RNA folding algorithm from the ViennaRNA package50. Furthermore, miRDeep searches for potential cleavage sites of Drosha and Dicer, and uses phylogenetic conservation and a filtering of other known small non-coding RNA species to improve the predictions. The stability of potential precursors sequences is tested using Randfold v2.051. In summary, each potential miRNA precursor sequence was scored based on its read signature, secondary structure , cleavage, conservation and overlap to known small non-coding RNAs. The novel miRNA candidates were further subjected to differential expression analysis along all known miRNAs . For each novel miRNA, target prediction was performed based on TargetScanHuman28 using differential expressed mRNAs in TOF-rv versus NH-rv and filtering by an aggregated PCT score > 0.8, a context++ score ≤0.4 and a percentile >8552. For the sets of target genes, on over-representation analysis for GO terms and pathways was performed using ClueGO49 and ConsensusPathDB53 .The fold- and scoring-based approach of the miRDeep package was used to identify novel miRNAsSupplementary information


In a setting with low HIV-tuberculosis incidence, is repeated testing for latent tuberculosis infection (LTBI) cost-effective for managing individuals with HIV who have negative LTBI test results at baseline?In this decision analytical model using a cost-effectiveness analysis, based on 3130 HIV-positive individuals in Hong Kong, China, a strategy of baseline LTBI testing followed by up to 3 subsequent annual tests could avert a similar proportion of new tuberculosis infections while incurring a lower cost compared with annually repeated testing. The strategy did not meet the willingness-to-pay threshold but would likely be cost-effective if the threshold were raised.The findings suggest that less intense subsequent LTBI testing strategies may be effective and are likely cost-effective. This decision analytical model analyzes the cost-effectiveness of latent tuberculosis infection testing for individuals in Hong Kong with HIV who had negative latent tuberculosis infection test results at baseline. With immune recovery following early initiation of antiretroviral therapy (ART), the risk of tuberculosis (TB) reactivation among individuals with HIV could be reduced. The current strategy of annual latent TB infection (LTBI) testing should be revisited to increase cost-effectiveness and reduce the intensity of testing for individuals.To analyze the cost-effectiveness of LTBI testing strategies for individuals in Hong Kong with HIV who had negative LTBI test results at baseline.This decision analytical model study using a cost-effectiveness analysis included 3130 individuals with HIV in Hong Kong, China, which has an intermediate TB burden and a low incidence of HIV-TB coinfection. A system dynamics model of individuals with HIV attending a major HIV specialist clinic in Hong Kong was developed and parameterized by longitudinal clinical and LTBI testing records of patients during a 15-year period. The study population was stratified by age group, CD4 lymphocyte level, ART status, and right of abode. Alternative strategies for LTBI testing after a baseline test were compared with annual testing under different coverages of ART, LTBI testing, and LTBI treatment scenarios in the model. An annual discounting rate of 3.5% was used in cost-effectiveness analysis.Proportion of new TB cases averted above base case scenario, discounted quality-adjusted life-years gained (QALYG), incremental cost, and incremental cost-effectiveness ratios in 2017 to 2023.A total of 3130 patients with HIV  with 16 630 person-years of follow-up data from 2002 to 2017 were analyzed. Of these, 94 patients  developed TB. Model estimates of cumulative number of TB cases would reach 146 by 2023, with the annual number of new TB diagnoses ranging from 6 to 8. For patients who had negative LTBI test results at baseline, subsequent LTBI testing strategies were ranked by ascending effectiveness as follows: (1) no testing, (2) test by risk factors, (3) biennial testing for all, (4) up to 3 tests for all, and (5) annual testing for all. Applying a willingness-to-pay threshold of $50 000 per QALYG, none of the subsequent testing strategies were cost-effective. Test by risk factors and up to 3 tests for all were cost-effective only if the willingness-to-pay threshold was increased to $100 000 per QALYG and $200 000 per QALYG, respectively. More new TB cases would be averted by expanding LTBI testing and/or treatment coverage.Changing the current testing strategy to less intense testing strategies is likely to be cost-effective in the presence of an increased coverage of baseline LTBI testing and/or treatment. A study in Canada observed a decrease in TB incidence in HIV-positive individuals following increasing ART coverage from 2003 to 2012.6 In the United Kingdom, LTBI screening of patients with HIV based on risk factors, such as low CD4 lymphocyte level, country of origin, and duration of ART, is recommended.7 A study in Belgium concluded with similar doubts about the need for regular LTBI testing in HIV-positive individuals in places with a low TB burden.8 In the United States, in accordance with guidelines published in 2016,9 HIV-positive individuals belong to a high-risk category that warrants LTBI screening without subcategorization by risk.The risk of tuberculosis (TB) disease development among HIV-positive patients has been reported to be 26-fold higher than among HIV-negative individuals.10 compared LTBI testing and treatment strategies for nonlocal residents with and without medical comorbidities. The study concluded that LTBI screening for HIV-positive individuals not born in the United States was cost-effective.10 In the United Kingdom, universal LTBI testing was considered expensive, and there was a need for an alternative.11Different strategies of LTBI testing following negative baseline test results have not been fully evaluated. The associations between LTBI screening, baseline LTBI prevalence, impact of immunological recovery on rate of TB reactivation and LTBI detection, and other factors are complex. Mathematical modeling is a useful approach to analyze the association of LTBI testing strategies with the incidence of TB. To our knowledge, the number of modeling studies addressing this issue to date has been very limited. An LTBI cost-effectiveness analysis in the United States12 At a time when immediate ART initiation is being implemented locally and internationally, this study aimed to compare the cost-effectiveness of different LTBI testing strategies for HIV-positive individuals after baseline screening.We hypothesized that annually repeated LTBI testing for HIV-positive individuals with a negative baseline LTBI test result is not cost-effective in Hong Kong, China, a city with an intermediate TB burden and a TB incidence rate among the HIV-positive population of 0.13 per 100 person-years (PYs).12 Patients with positive LTBI test results  are treated with isoniazid for 9 months.3The study population is a cohort of HIV-positive individuals attending the Integrated Treatment Centre, the major HIV specialist clinic in Hong Kong. After diagnosis of HIV, LTBI testing  is performed at baseline, then annually until diagnosis with TB or a positive LTBI test.Empirical clinical data for patients diagnosed with HIV between January 2002 and June 2017 were accessed retrospectively. Data fields included baseline demographics , baseline HIV status , clinical status , first AIDS-defining illness, ART initiation date, TB diagnosis and treatment records, LTBI testing dates and results, LTBI treatment, and longitudinal CD4 lymphocyte, CD8 lymphocyte, and viral load measurements at follow-ups. Patients with HIV with a history of TB, with concurrent active TB disease, and younger than 18 years at HIV diagnosis were excluded.CHEERS) reporting guideline.Data access approval from the Department of Health, Hong Kong Special Administrative Region Government in compliance with the Personal Data (Privacy) Ordinance and ethical approval of the Joint Chinese University of Hong Kong–New Territories East Cluster Clinical Research Ethics Committee (CREC) were obtained. Individual consent was waived because the data were deidentified and had been collected retrospectively. The analysis was conducted according to Consolidated Health Economic Evaluation Reporting Standards  with 95% CIs assuming Poisson distribution. Descriptive statistics were displayed to summarize the characteristics of patients. The test for calculating incidence was 2-sided, with statistical significance set at .05.13 and optimizing an HIV testing program in Canada.14System dynamics modeling with elements of stocks, flows, feedback loops, and time delays was applied. In the model, a stock represents accumulations, and a flow is the rate of change in a stock. The methodology was used for understanding nonlinear behavior of complex systems over time, an approach that had been adopted in studying the associations of tobacco exposure with TB15 For patients who had negative LTBI test results at the first test, subsequent annual LTBI tests were performed. Cases that had positive LTBI results in subsequent tests would be offered LTBI treatment. Model parameters and equations are shown in the eAppendix, eTable 1,20 eTable 2, and eFigures 2-6 in the We developed the model in R version 3.5.1 (The R Foundation) to simulate the flow of HIV-positive individuals with LTBI progressing to TB reactivation and HIV-positive individuals without LTBI. In the base case model, the annual number of LTBI cases was the function of new HIV diagnoses and prevalence of LTBI for HIV-positive individuals by right of abode. After HIV diagnosis, LTBI might continue to be undetected before the first LTBI test  and stored these states in stocks, some of which change over time at different rates of flow , discounted quality-adjusted life-years gained (QALYG), and incremental cost-effectiveness ratios (ICERs) in 2017 to 2023. The annual number of TB cases diagnosed in the clinic from 2002 to 2015 was used for model validation.3 For active TB cases, the costs of TB treatment and monitoring as well as multidrug resistant TB treatment and monitoring were estimated.18 The costs for routine HIV care were not included, as every patient was in care. The utility of HIV-positive patients without TB was assumed as 1. Loss of utility was 0.17 for newly developed, active TB cases with CD4 lymphocyte level of 200/μL or higher, 0.298 for newly developed, active TB cases with CD4 lymphocyte levels less than 200/μL, and 0.32 for multidrug-resistant TB regardless of CD4 lymphocyte level in the previous year.20 The annual inflation rate was assumed to be 2.3%,23 and the annual discount rate was assumed to be 3.5% for cost and quality-adjusted life-years in the analysis. Testing strategies were ranked by effectiveness, with comparisons made between each strategy and the next best alternative strategy for an ICER calculation after the exclusion of the strongly dominated and extendedly dominated strategy.25 A strategy was considered strongly dominated when it cost more but had negative QALYG compared with an alternative strategy. If the strategy had a higher ICER than a more effective strategy, it was considered extendedly dominated. A strategy was likely cost-effective if the ICER was below the willingness-to-pay (WTP) threshold, $50 000 per QALYG.26Cost for TST was estimated for LTBI testing. For patients with positive LTBI test results, the costs of TB testing, LTBI treatment, and LTBI monitoring were included in the analysis  for patients who had negative LTBI test results at baseline. These strategies were compared under different scenarios of coverage rates  .To examine the impact of new TB infections after HIV diagnosis, we developed an expanded model by including new TB infections after HIV diagnosis and baseline LTBI testing  were male, 2796 of 3123  local residents, 2450 (78.3%) ethnic Chinese, and 2800 (89.5%) younger than 50 years at diagnosis of HIV. Among all patients without or before TB diagnosis, 2697 (86.2%) had initiated ART, 99 (3.2%) had been diagnosed with diabetes, and 373 (11.9%) had been diagnosed with AIDS-defining illness. After HIV diagnosis, 2412 (77.1%) had been tested for LTBI, of whom 631 (26.2%) had positive test results. Of patients with positive LTBI tests, 504 (79.9%) initiated treatment. A total of 94 patients were diagnosed with TB during the 15-year observation period.In the cohort of patients diagnosed with HIV between 2002 and 2013 (n = 2079), the overall TB incidence was 0.67  per 100 PYs. The incidence was 1.26  per 100 PYs from HIV diagnosis to ART initiation, decreasing to 0.37  per 100 PYs after ART initiation. The TB reactivation rates for those with positive LTBI test results or TB diagnoses after their HIV diagnosis are shown in eTable 3 in the In the base case model, the cumulative estimated number of TB cases from 2002 to 2008 resembled the cumulative number of diagnosed TB cases in the clinic, despite some discrepancies after 2008 . Model eComparing LTBI testing strategies, strategy D (up to 3 subsequent tests) and strategy E  were estimated to avert the highest proportion of new TB cases above the base case scenario , while sCompared with no subsequent LTBI testing in each coverage scenario, offering subsequent LTBI testing by different criteria (strategies B-E) resulted in more QALYG but a higher cost. Biennial testing and current annual testing strategies were extendedly dominated, as their ICERs were higher than more effective alternative strategies. With a $50 000 per QALYG WTP threshold, no testing strategy was likely cost-effective . If the Compared with the status quo scenario, the relative positions of LTBI testing strategies on the ICER plane were similar for different scenarios of coverage for LTBI testing and treatment   after baseline testing was likely to be cost-effective. The risk-based testing strategy and the strategy using up to 3 subsequent tests were cost-effective if the WTP threshold was increased to $100 000 per QALYG and $200 000 per QALYG, respectively. The effect of expanding LTBI treatment coverage alone was small. To be cost-effective, the concurrent expansion of coverage for both LTBI testing and treatment is essential.27 or those who might have become infected with TB after the first test. From our sensitivity analysis, the impact of detecting new TB infections after HIV diagnosis on the epidemic curve projection was very small. In Hong Kong, nonlocal infections  were also not significantly associated with positive subsequent LTBI tests.21 To identify patients who had false-negative test results in previous LTBI tests, targeted subsequent LTBI tests by risk factor (strategy B), which cost less than annual testing strategy, might be an option. The risk-based testing strategy is currently recommended by the National Institute for Health and Care Excellence and the British HIV Association in the United Kingdom; this strategy involves testing HIV-positive individuals based on specific criteria, such as CD4 lymphocyte levels, country of origin, and ART duration.11 The 2 UK strategies were cost-effective compared with no testing, although the timing of LTBI testing was not stated clearly.11 A risk-based testing strategy has also been recommended in Belgium.28In clinical practice, repeated LTBI testing is theoretically useful for identifying individuals who might have received false-negative results in the previous tests owing to defective immune status5 It is possible to be receiving ART and experience immune recovery within 3 years with annual LTBI tests. If all 3 subsequent tests are negative, the chance of a false-negative result could be very low. While this strategy was effective , it cost more than the no testing and risk-based testing strategies. It would be cost-effective only if the WTP threshold were increased to $200 000 per QALYG. The results illustrated the small but definite benefits of targeted and time-limited LTBI following baseline screening.Our study results support another testing strategy, strategy D, which involves repeating a small number of LTBI tests  for HIV-positive individuals who had a negative baseline result. Currently, HIV-positive individuals begin ART within a relatively short period after diagnosis.10 showing that a confirm-negative testing strategy  was cost-effective ($63 000 per QALYG) among HIV-positive individuals. However, strategy A had the lowest QALYG among testing strategies. It was least effective in identifying a high proportion of LTBI cases followed by treatment. In clinical and public health contexts, the selection of a testing strategy requires a careful balance between effectiveness and cost-effectiveness.Among the testing strategies, the strategy of requiring no repeated LTBI testing (strategy A) was cost-saving and likely cost-effective. The results were consistent with a US study that analyzed the cost-effectiveness of baseline LTBI testing and treatment among residents not born in the United States,30 In our study, 100% coverage of baseline LTBI testing and treatment without subsequent LTBI tests could avert at least 30% of new TB cases compared with the base case scenario. This highlighted the importance of the first LTBI test for reducing TB incidence, which is in line with our analysis of clinical data and a cohort study in Spain.29 The cost does not necessarily increase with expanded coverage for LTBI testing. Compared with the base case scenario, expanding LTBI testing coverage in strategy D  could decrease cost and increase QALYG, as shown in our sensitivity analyses. On the other hand, full ART coverage could avert an additional 3% to 4% of new TB infections above the base case scenario. The small marginal change was mainly because of the already high baseline ART coverage (80% of patients).A higher LTBI testing rate followed by higher treatment coverage is important for increasing effectiveness. With more testing, more LTBI cases would receive treatment, which could reduce the risk of TB reactivation substantially.31 This was mainly because of its insignificant association with TB disease development in our analysis on the clinical data.21 Overall, only 4% of HIV-positive individuals in the data set had been diagnosed with diabetes. Age group and progression were included in the model structure, but age was not associated with TB reactivation risk in the model. This could again be because of the insignificant association between the 2 variables.21 As a short-term modeling study, the impact of age in the relatively young study population (98% of whom were younger than 65 years) is anticipated to be small. To assess the impact of risk-based testing strategy, analysis based on local empirical clinical data to examine the main risk factors of TB disease development is needed.Our study had limitations. First, our model does not account for new TB infections occurring after HIV diagnosis. In places with higher chances of acquiring a TB infection after HIV diagnosis, the expanded model in the sensitivity analysis might be a more suitable reference (eFigure 8 in the This study suggests that the current annually repeated LTBI testing strategy for HIV-positive individuals in Hong Kong could be improved. Repeated testing is excessive because of the generally low risk of TB reactivation and infection, as shown by a high number of repeatedly negative LTBI test results. By offering a less intense subsequent LTBI testing strategy and expanding baseline LTBI testing and treatment, either risk-based testing or a limited number of yearly tests could lower cost and increase QALYG. However, the coverage of baseline LTBI testing and treatment should be increased to achieve the best health outcomes with maximum cost-effectiveness.


P < .001). These findings suggest a need for improvement in adherence among patients with employer-sponsored insurance.We used administrative claims data from 2014 on people with employer-sponsored health insurance to assess the proportion of patients taking antihypertensive medications, rates of nonadherence to these medication regimens, and out-of-pocket costs paid by patients. We performed multivariate logistic regression analysis to examine the association between out-of-pocket costs and nonadherence. Results indicated that patients filled the equivalent of 13 monthly prescriptions and paid $76 out of pocket over the calendar year; the likelihood of nonadherence increased as out-of-pocket costs increased (adjusted odds ratios ranged from 1.04 to 1.78;  Only 40% to 74% of patients treated for hypertension take medications as prescribed. Patients’ out-of-pocket costs for medications can affect medication adherence. We found a substantial proportion of people aged 35 to 64 with employer-sponsored health insurance to be nonadherent to antihypertensive medications (41%). People paid an average $6 for a 30-day supply of medication in 2014, and those paying higher out-of-pocket costs had a greater likelihood of being nonadherent.The results highlight room for improvement in medication adherence among patients with employer-sponsored health insurance, even if facing small out of pocket costs. Hypertension is a leading risk factor for cardiovascular disease, and only 40% to 74% of people with diagnosed hypertension are adherent to prescribed medication –3. NonadWe used 2014 administrative claims data from the IBM MarketScan Commercial Database (IBM Corp), which provided de-identified health care claims data for enrollees and their dependents in employer-sponsored health insurance plans. We limited the study sample to adults aged 35 to 64 who were continuously enrolled with prescription drug coverage in 2014 . We focused on 2014 after using the IBM Treatment Pathways online tool (IBM Corp), which reported stable trends of proportions of patients with any hypertension diagnosis (25%−26%) and proportions of patients filling an antihypertensive medication prescription (29%−30%) in 2010 through 2014.International Classification of Diseases, 9th Revision (ICD-9), diagnosis codes (401.x–405.x) on at least 2 outpatient or 1 inpatient or emergency department claim, and we identified cardiovascular disease events resulting in an inpatient or emergency department visit by using ICD-9 codes from the Center for Medicare and Medicaid Services’ Chronic Conditions Warehouse for ischemic heart disease, acute myocardial infarction, cerebrovascular disease, and heart failure   to analyze data.In 2014, 22% of adults aged 35 to 64  were diagnosed with hypertension . Among tPatients filled an average of 13 monthly antihypertensive medication prescriptions during the calendar year and paid on average $5.78 out of pocket per 30-day supply and $76 annually. Total costs (patient and insurer) for antihypertensive medications were $17 per 30-day supply and $229 annually. People who used branded medications had the highest out-of-pocket costs ($13.60 per 30-day supply) and highest total costs ($40.73 per 30-day supply). Patients in health maintenance organizations had higher out-of-pocket costs that those in other types of insurance plans. Residents of rural areas also had higher out-of-pocket costs than those paid by those in urban areas, but they used less expensive medications.About 90% of patients incurred out-of-pocket costs for medications, but 83% paid less than $10 per 30-day supply of antihypertensive medications. However, 30% of patients paid the full costs of their medications .We calculated the unadjusted and multivariate-adjusted odds ratios for the association between nonadherence and patient characteristics . The likThe likelihood of nonadherence was greatest among patients who used branded antihypertensive medications and those living in the South and was smallest among those with a hypertension diagnosis in 2014 and patients aged 55 to 64. When we restricted the sample to patients with a hypertension diagnosis as a sensitivity analysis, the associations were similar in magnitude and significance.We found that nonadherence to antihypertensive medication regimens was common and was most common among patients with higher out-of-pocket costs. A 2016 study estimated that patients with commercial insurance paid about $4.13 in copayments per antihypertensive medication prescription filled in 2014, slightly lower than the out-of-pocket cost estimate reported in our study, which provides a more comprehensive estimate that includes copayments, coinsurance, and deductibles .Numerous experimental and quasi-experimental studies have found a causal relationship between lowering patients’ out-of-pocket costs and reducing medication nonadherence . Our stuOur study findings show that there is room for improving adherence to antihypertensive medications among patients with employer-sponsored insurance and that patients who faced higher out-of-pocket costs had a greater likelihood of being nonadherent.


Chronic disease and opioid-related hospitalizations in the United States are increasing. We analyzed nationally representative data on patients aged 18 years or older from the 2011–2015 National Inpatient Sample to assess the association between opioid-related hospitalization and chronic diseases. We found that most patients with opioid-related hospitalization were white, aged 35–54 years, in urban hospitals, and had 2 or more comorbid conditions. Patients with 2 or more chronic conditions accounted for more than 90% of opioid-related hospitalizations in all years. The results suggest a need for targeted interventions to prevent opioid misuse in patients with multiple chronic conditions. Patients who take addictive pain relievers often have chronic conditions.From 2011 to 2015, more than 90% of US opioid-related hospitalizations were among patients with 2 or more chronic diseases. The trend in opioid-related hospitalization among patients with multiple chronic diseases is increasing and noteworthy.Multiple chronic diseases are associated with opioid-related hospitalizations, indicating that prevention of opioid-related abuse and overdose among patients with multiple chronic conditions requires alternatives to opioid prescription, when possible.Patients with chronic conditions such as cancer, stroke, asthma, and obesity often experience chronic pain and have a higher likelihood of receiving one or more opioid prescriptions . In thisIn this cross-sectional study, we analyzed data on patients aged 18 years or older from the National Inpatient Sample (NIS) from January 1, 2011, to September 30, 2015. Claims occurring from October through December of 2015 were not included because of the transition from the International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM) to the 10th revision (ICD-10-CM) coding system on October 1, 2015. We included arthritis, spinal disease, asthma, liver disease, stroke, cancer, and obesity because these diseases are most likely to be prescribed opioids in the internal and family medicine specialties. Family medicine (21.8%) and internal medicine 17.6%) reportedly write the most opioid prescriptions, followed by physical medicine/rehabilitation, anesthesiology/pain, hematology/oncology, and neurology –6. Relat% reporteopioid-related hospitalization (specific ICD-9-CM codes are available in www.hcup-us.ahrq.gov/db/nation/nis/trendwghts.jsp) into the original NIS 2011 data. We analyzed the trends in opioid-related hospitalizations by type of chronic disease for 2011–2015 and used the Pearson χ2 test to test for significance. Significance was set at P < .05.We combined opioid dependence and unspecified use, adverse effects of opioids, opioid abuse, and opioid poisoning to create a single variable, The highest total number of opioid-related hospitalization cases was in 2014  . From 20The prevalence of opioid-related hospitalizations significantly increased among patients with cancer, stroke, obesity, asthma, liver disease, and arthritis across all years . The mosWe found that more than 90% of opioid-related hospitalizations were among patients with 2 or more chronic diseases and that the trend in opioid-related hospitalization among patients with chronic diseases is increasing. This finding is salient given that 1 in 4 US adults are living with 2 or more chronic diseases . Both thUsing alternative approaches to pain management is one public health strategy for addressing the opioid crisis  and is cThis study has several limitations. Because we analyzed data from 2011 to 2015, our findings are not generalizable to more recent years. Our analysis was cross-sectional and examined the associations between opioid-related hospitalization and chronic diseases without controlling for covariates, which prevents us from drawing causal conclusions about the direction of the observed relationship. Furthermore, the nature of the data did not allow us to explore why the associations were not significant in some years. Opioid prescriptions for patients with arthritis have increased in recent years, so that could be an explanation for the strong correlation we found . FinallyDespite these limitations, our findings show an increasing trend in opioid-related hospitalization among patients with chronic diseases and a high prevalence of opioid-related hospitalization among patients with multiple chronic conditions. The observed association warrants further research to determine causal inference. Results from this study can help clinicians develop strategies to prevent opioid misuse in patients with multiple chronic conditions and inform strategies for addressing both the opioid epidemic and rising rates of chronic disease.


Limited data are available from real-world practices in Europe describing prevailing treatment patterns and outcomes in relapsed/refractory multiple myeloma (RRMM), particularly by cytogenetic risk. A retrospective medical record review was conducted in 200 RRMM patients in France. From first relapse, patients were assessed on second-/third-line treatments, progression-free survival (PFS), overall survival (OS), and healthcare utilization. Fifty-five high risk and 113 standard risk patients were identified. Overall, 192 patients (96%) received second-line therapy after relapse. Lenalidomide-based regimens were most common (>50%) in second line. Hospitalization incidence in high risk patients was approximately twice that of standard risk patients. From Kaplan-Meier estimation, median (95% CI) second-line PFS was 21.4  months . Among second-line recipients, 47.4% were deceased at data collection. Median second-line OS was 59.4  months . The prognostic importance of cytogenetic risk in RRMM was apparent, whereby high (versus standard) risk patients had decidedly shorter PFS and OS. Frequent hospitalizations indicated potentially high costs associated with RRMM, particularly for high risk patients. These findings may inform economic evaluations of RRMM therapies. Multiple myeloma (MM) is a malignancy of clonal plasma cells. Worldwide, MM accounts for an estimated 0.8% 114,000) of all new cancer cases annually and 0.9% 63,000) of all cancer deaths annually 14,000 of. In Euro,000 of aIn Europe, autologous stem cell transplant (SCT) is recommended as the standard of care for patients less than 65 years old  with newly diagnosed MM, which should be preceded by induction therapy aimed at quickly achieving clinical response prior to transplantation –13. PatiDespite advancements in induction and maintenance therapies leading to improved response rates and OS, virtually all patients with MM eventually relapse and die from disease progression . FollowiTo date, little data from routine clinical practice in Europe have been generated to describe prevailing treatment patterns, clinical outcomes, and disease-related healthcare utilization in MM patients after they have relapsed or become refractory to treatment. Furthermore, the extent to which treatment selections, outcomes, and resource use vary according to baseline cytogenetic risk has not been widely explored for RRMM patients in real-world practice settings. Such information may not always comport with what might be expected regarding standards of care, patterns of treatment, and outcomes based on leading academic and clinical research. An assessment of whether, and to what extent, these patterns in real-world settings vary with expectations based on prevailing trial-based guidelines may help inform clinicians and other providers in the ongoing provision of optimal care. This information may also help inform future health technology, economic, and other regulatory assessments of existing and novel RRMM therapies.A retrospective medical record review was conducted in 200 patients with RRMM in France. Patients were selected from the caseloads of 40 hematology/oncology providers practicing across France in a variety of settings: academic, university-affiliated hospitals (35%), nonacademic general hospitals (42.5%), cancer-specialized hospitals (15%), and private community hospitals and clinics (7.5%). For providers with more than 5 patients meeting the study inclusion criteria, selection of 5 patients for the review was based on randomly selected first letters of patients' last names. All patients were aged at least 18 years at initial MM diagnosis and were first diagnosed with RRMM between January 1, 2009, and December 31, 2011. The case identification window terminated in 2011 on the basis of a median survival expectation of 3 to 6 years for RRMM patients receiving active therapy and the need in a retrospective study for an adequate potential follow-up period over which to observe relevant events. All patients were required to have a complete medical record on their treatment history related to MM, beginning with the initial MM diagnosis until death or date of last medical record entry, whichever occurred first. Patients were excluded from the study if they were diagnosed with another concurrent malignancy prior to first diagnosis of RRMM, with the exception of hematologic malignancies secondary to MM , and adequately treated nonmelanoma skin cancer or in situ neoplasm. All data collection was performed with the formal ethical approval of national competent authorities in France for the conduct of human subjects research. and (2) occurrence of disease progression while on or at any time after completion of first-line therapy. The RRMM diagnosis date  defined the study index date. Patients were further classified by cytogenetic risk, as follows: (1) high risk: cytogenetic abnormalities del(17p), t(4:14), or t; (2) standard risk: all patients with known cytogenetics not classified as high risk; and (3) unknown risk: patients with unknown cytogenetics. To obtain an adequate sample of high risk patients for selected stratified analyses, a soft quota was imposed on patient selection to ensure that between 20% and 30% of the final study sample had high risk disease.For purposes of case selection, the participating providers were asked to define RRMM by (1) treatment with a first-line (induction) regimen of chemotherapy with or without SCT and with or without other postinduction/SCT therapiesFrom date of first relapse, patients were retrospectively assessed for second- and third-line treatment regimens received, treatment duration, reasons for discontinuation, and MM-related healthcare utilization, as well as OS and progression-free survival (PFS) from initiation of second-line treatment. PFS was calculated as time from second-line treatment initiation until the earliest of clinical progression during treatment, switch to new therapy line, or death (during or after treatment) if no additional treatment lines were initiated. All analyses were descriptive and exploratory in nature. OS and PFS were descriptively analyzed using the Kaplan-Meier method.As shown in As shown in Clinical progression during second-line treatment occurred for 47.4% of the 192 patients initiating second-line treatment; 84.9% of patients had experienced a progression event by the time of their last available medical record over a median total follow-up duration of 52 months. Based on Kaplan-Meier estimation, the median  PFS from second-line treatment initiation was 21.4  months for all patients combined ; median Treatment selections in the relapse setting for patients with MM depends on several parameters such as age, performance status, comorbidities, the number of prior treatment lines, available remaining treatment options, and interval since completion of last therapy. The European Medicines Association (EMA) has approved lenalidomide in combination with dexamethasone, as well as bortezomib either alone or in combination with pegylated doxorubicin, as recommended therapy for RRMM based on results of numerous trials –39. CurrSecond-line treatment duration was generally less than 1 year, indicating an unmet need in relapsed MM patients in light of the primary reasons cited for discontinuation . Second- and third-line treatment durations were longer for oral lenalidomide-based regimens  as compared with intravenous bortezomib regimens (median: 6 cycles in both second- and third-line). Additional research is needed to further explore whether oral treatments in the relapse setting confer a potential treatment persistence benefit due to the simpler administration route.In our study sample, nearly all patients (192 of 200 [96%]) received at least one line of additional chemotherapy  after first relapse. Of these patients, only 114 (59.4%) went on to receive third-line therapy. This proportion is somewhat lower than expected. Among the 86 second-line initiators who did not receive third-line treatment, 53 (61.6%) died before a third-line could be initiated, including 23 patients who died while on second-line therapy and 30 patients who died after completion of second-line and before a next line could be started. Of the remaining patients who did not initiate a third-line therapy, 16 were still on their second-line treatment at last follow-up and 17 were censored (having not yet initiated a third-line treatment at last available follow-up). Thus, death was the predominant reason for the observed lack of third-line treatment, but censoring (loss to follow-up) was also a substantial factor.MM-related hospitalizations and emergency department visits were common after first relapse, indicating a potentially high cost burden borne by patients with RRMM. Hospitalization incidence was nearly 50% higher for patients with high risk cytogenetics as compared with those with standard risk. The introduction of additional, more efficacious treatment options for RRMM that reduce the incidence of hospitalizations related to disease progression or complications may therefore present cost savings to health systems. Additional research in the area of MM-related costs and cost-effectiveness is needed to formally assess the economic impact of newer therapies.Recent trial-based estimates of PFS and OS after MM relapse are wide-ranging and dependent upon specific patient characteristics, including treatment regimen administered, number of previous regimens received, and baseline risk category, among others factors. A recent review article by Nooka et al.  highlighOS in both MM generally and in RRMM specifically has seen a marked improvement from the year 2000 onward . One stuOur analysis was subject to several limitations. First, as is inherent to retrospective medical record reviews, assessments of disease progression were not protocol driven, particularly with regard to the timing of evaluations of disease progression. It is possible that the participating physicians, some of whom likely do not frequently participate as investigators in clinical trials, assessed clinical progression at less frequent intervals than would otherwise be required in a clinical trial, and progression events may have been identified somewhat later than they would have otherwise. In this case, PFS estimates may have been upwardly biased. For this reason, findings regarding the endpoints of PFS may not be directly comparable to those observed in clinical trials. Second, while measures were taken to randomize patient selection for the medical record review, the physicians and patients selected for study inclusion still represent a practical nonrandomized sample. Our study findings therefore may not be generalizable to the overall RRMM population and the noted comparisons to previous clinical trials should be made with caution based on the differing and generally more stringent inclusion criteria used in the interventional studies reviewed. Our study is also be limited by its relatively small sample size, which may further reduce generalizability of the results. Third, the criteria used for cytogenetic risk classification were based on consensus definitions contemporaneous to the study entry window (2009-2011) for the cohort studied here . Risk clTo date, only limited data from real-world clinical settings in Europe have been generated describing current practice patterns, outcomes, and healthcare utilization in MM patients in the relapse/refractory setting. Our study found that treatment practices in real-world settings to manage relapsed MM, starting with second-line treatment, generally align with ESMO guidelines in that lenalidomide/dexamethasone was the most commonly reported second-line regimen (53.7% of patients). Furthermore, the importance of initial MM risk classification as a prognostic factor in RRMM was apparent in this retrospective review, whereby (as in previous studies) patients with high risk disease had decidedly less favorable PFS and OS than patients with standard risk. Finally, based on frequent hospitalization and emergency department visits in the relapse setting, our study demonstrates the potentially high cost burden associated with RRMM and the additional cost burden that may be incurred by patients with high risk MM as compared with standard risk. Taken together, findings from this study address important literature gaps in RRMM and may help inform future economic  evaluations of novel RRMM therapies.


The electrical stability of the myocardium is dependent on the dynamic balance between sympathetic and parasympathetic influences on the heart, which is reflected by heart rate variability (HRV). Reduced HRV is a proposed predictor of sudden death caused by ventricular tachyarrhythmias in cardiac patients. However, the link between individual differences in HRV and ventricular tachyarrhythmic risk in populations without known pre-existing cardiac conditions is less well explored. In this study we investigated the extent to which individual differences in resting state HRV predict susceptibility to spontaneous and pharmacologically-induced ventricular arrhythmias in healthy rats. Radiotelemetric transmitters were implanted in 42 adult male Wild-type Groningen rats. ECG signals were recorded during 24-h resting conditions and under β-adrenoceptor pharmacological stimulation with isoproterenol and analyzed by means of time- and frequency-domain indexes of HRV. No significant association was found between individual differences in resting measures of HRV and spontaneous incidence of ventricular arrhythmias. However, lower resting values of HRV predicted a higher number of ventricular ectopic beats following β-adrenergic pharmacological stimulation with isoproterenol (0.02 mg/kg). Moreover, after isoproterenol administration, one rat with low resting HRV developed sustained ventricular tachycardia that led to death. The present results might be indicative of the potential utility of HRV measures of resting cardiac autonomic function for the prediction of ventricular arrhythmias, particularly during conditions of strong sympathetic activation, in populations without known cardiac disease. Heart rate variability (HRV) refers to the beat to beat variation in the heart rate generated primarily by sympathetic and parasympathetic  influences on sinoatrial node activity. In humans, there are large individual differences in resting measures of HRV . A largThe investigation of the link between individual differences in HRV and risk of potentially life-threatening ventricular arrhythmias in a healthy population may be more easily addressed using animal models. For example, similar to what has been observed in humans, we have previously shown that several psychosocial factors (including chronic stress) can decrease resting HRV in rodents, with negative consequences on the electrical stability of the ventricular myocardium . CriTherefore, given the generally accepted view of a protective role of the vagal input to the heart against vulnerability to ventricular arrhythmias ,34, in tForty-two adult male wild-type Groningen (WTG) rats (Rattus norvegicus) were considered for this investigation, which was based on a secondary analysis of data from a previous study including 44 WTG rats . Two ratAt 15 weeks of age, rats were implanted, under tiletamine hydrochloride + zolazepam hydrochloride anesthesia , with radiotelemetric transmitters  for recordings of ECG (sampling frequency 1000 Hz) and locomotor activity  signals. The transmitter body was placed in the abdominal cavity; one electrode was fixed to the dorsal surface of the xyphoid process and another electrode was placed in the anterior mediastinum close to the right atrium, according to a previously described procedure . This elAfter recovery from surgery, ECG and LOC signals were recorded for 60 s every 60 min for ten consecutive days, with the animals left undisturbed in their home cages . ECG andThe pharmacological challenge with isoproterenol occurred the day after the end of 24-h baseline recordings and was conducted before assigning rats to the two experimental conditions described in the original study . On the 2; 0.75–2.5 Hz) parameters of vagally-mediated HRV were then quantified from 1-min ECG epochs using ChartPro 5.0 software . For spectral (frequency-domain) analysis of HRV, the power spectrum was obtained with a fast Fourier transform-based method . Mean HR and HRV values were calculated for each of the last three days of baseline recordings and then further averaged to obtain 24-h mean values. In addition, mean HR and HRV values were calculated for the 30-min pre-isoproterenol injection and 30-min post-isoproterenol injection periods.Initially, each raw ECG signal was visually inspected to ensure that all R-waves were correctly detected. Those parts of ECG recordings which exhibited recording artifacts were discarded without substitution and excluded from further analysis. Heart rate  and time-  and frequency-domain  of an analyzed ECG recording) and the pharmacological challenge with isoproterenol  was determined and quantified off-line based on the Lambeth Conventions (II) for the study of experimental arrhythmias  Figure .p < 0.05. Data were first checked for normality of variables and for violations of statistical assumptions of Linear Regression Models. As 24-h RMSSD and HF values were not normally distributed   included the incidence of SVA during 24-h recordings as the predictor. Step 2 (model 2) added 24-h HRV values as the predictor.All statistical analyses were performed using SPSS 24 software package . Statistical significance was set at ctively) , these vAs depicted in In humans, normal aging is associated with changes in the autonomic control of sinoatrial node activity. Specifically, a progressive impairment in cardiac parasympathetic influences, which is reflected by increased resting HR and reduced HRV, has been observed with age ,42. Thist = 2.4, p < 0.05) (2 = 0.10). Because lnRMSSD and lnHF-HRV were strongly correlated (As reported in  < 0.05) . Critica < 0.05) , Model 1 < 0.05) , Model 2rrelated , only lnrrelated . Remarkarrelated , suggestThe effect of sympathetic stimulation on global ventricular electrophysiology has been studied extensively in animal models ,53 and hThe present results demonstrate the existence of a negative relationship between HRV measures of resting vagal tone and vulnerability to ventricular arrhythmias under β-adrenoreceptor pharmacological stimulation in a non-selected population of young adult rats with no documented cardiac conditions. This might be indicative of the utility of HRV measures of resting cardiac autonomic functioning for the prediction of potentially life-threatening ventricular arrhythmias not only in the clinical setting, but also in normal populations. It must be acknowledged, however, that the episode of sudden death that occurred in one animal with low resting HRV remains, to some extent, anecdotal and particular caution must be taken in interpreting these findings. It will thus be important to replicate these results in other species of rats and/or older rats. However, the present results may provide the basis for future studies, using non-selected populations of rats with large individual differences in vagally-mediated HRV, to investigate the pathophysiology that underlies the generation of ventricular arrhythmias and explore whether adverse remodeling of the heart can be revealed prior to the onset of the cardiac disease phenotype in normal individuals with low resting HRV.


Steroid 11-beta-hydroxylase deficiency is a relatively rare form of congenital adrenal hyperplasia (CAH). We describe the case of a 46,XX child, reared as a male, who first presented to us at the age of three years with features of peripheral precocity and hypokalemic hypertension. Based on the clinical and biochemical profile, a diagnosis of 11-beta-hydroxylase deficiency CAH was established, and physiological glucocorticoid replacement was begun. Both hypertension and hypokalemia improved with glucocorticoid supplementation, and at eight years of age, antihypertensives were successfully withdrawn. Regression of left ventricular hypertrophy was also noted at this time. In keeping with the male gender identity, the child underwent hysterectomy, oopherectomy and breast reduction surgery at 13 years of age. We conclude that both hypertension and end-organ damage due to 11-beta-hydroxylase CAH may get reversed following optimal glucocorticoid treatment. Detailed genital examination at birth may help in early diagnosis of this rare disorder, thereby preventing the deleterious consequences of longstanding mineralocorticoid excess. CYP11B1 gene located on chromosome 8q24.3, which codes for the 11-beta-hydroxylase enzyme. The enzyme catalyzes the conversion of 11-deoxycortisol to cortisol and 11-deoxycorticosterone (DOC) to corticosterone in zona fasciculata of the adrenal cortex. An enzyme defect leads to low cortisol and elevated adrenocorticotrophic hormone (ACTH), with consequent adrenal hyperplasia and shunting of steroid precursors into the androgen synthesis pathway, resulting in hyperandrogenism. Accumulation of DOC and other steroid precursors results in low renin hypertension, a finding seen in two-thirds of patients at the time of diagnosis [Congenital adrenal hyperplasia (CAH) due to 11-beta-hydroxylase deficiency (online Mendelian inheritance in man (OMIM) #202010) is an autosomal recessive disorder of glucocorticoid biosynthesis, characterised by features of androgen and mineralocorticoid excess. It is the second most common form of CAH after 21-hydroxylase deficiency and accounts for 5-8% of all CAH cases [A 3-year-old child was brought to us with the complaints of the passage of urine from the undersurface of the phallus and downward phallic curvature since birth. The parents also noted the abnormal appearance of pubic hair and excessive aggressiveness in the child since the age of two years. The child was fifth in birth order and was born out of non-consanguineous parentage. There was no history of a salt-wasting crisis, antenatal maternal virilisation or intake of any hormonal preparation by the mother. The child was raised as a male by the family and was given a name consistent with the same. In retrospect, the parents admitted that the child was darker in complexion compared to other siblings, and the darkening increased further two months before the presentation. There was no family history of early neonatal deaths, ambiguous genitalia or infertility. On examination, the child had a supine blood pressure of 144-154/90-94 mmHg . Examination of genitals revealed local hyperpigmentation, enlarged phallus with ventral curvature, penoscrotal hypospadias, fused labioscrotal folds, and single urethral opening (Prader 4 virilisation)  with midparental height of 169 cm for a male child (between 25th and 50th centile) and 156 cm for a female child (less than 5th centile).Investigations revealed hypokalemia, primary adrenal insufficiency with elevated sex steroids, and elevated 11-deoxycortisol (Table Karyotype (GTG banding) was 46,XX. Bone age by Greulich and Pyle method was advanced at 11 years and six months. Imaging of abdomen and pelvis revealed enlarged hyperplastic bilateral adrenal glands, normal Mullerian structures, normal bilateral ovaries, and opening of the genital tract into the urethra. 2D echocardiography revealed concentric left ventricular hypertrophy (LVH). A dilated fundus examination was, however, normal.Differential diagnosis for hypertension with hypokalemia includes primary hyperaldosteronism , CAH , syndrome of inappropriate mineralocorticoid excess due to mutation in gene encoding 11-beta-hydroxysteroid dehydrogenase type 2 (11β-HSD2) enzyme, ectopic Cushing syndrome due to overwhelming of 11β-HSD2 enzyme, DOC producing tumour, glucocorticoid resistance syndrome, renal artery stenosis and Liddle syndrome ,4. BasedThe child was started on hydrocortisone supplementation with stress dosage education; hypokalemia was corrected with oral potassium supplements and hypertension controlled using a combination of amlodipine and spironolactone. On regular outpatient visits, the child’s growth velocity, bone age, blood pressure, and potassium were monitored. We found that the child could be gradually weaned off potassium supplements with the maintenance of normal serum potassium levels. The antihypertensive requirement also reduced gradually, leading to gradual withdrawal and complete cessation of all antihypertensives by the age of eight years. A 2D echocardiography repeated at this time revealed regression of LVH. The child was raised as a male by the family and had a male gender identity, which was confirmed with psychological tests. The child was subsequently lost to follow-up and again presented to us at the age of 13 years with complaints of progressive bilateral breast enlargement over the past four years (Figure There was no complaint of cyclical abdominal pain or hematuria. At this stage, the child was re-admitted and underwent definitive hysterectomy and bilateral salpingo-oophorectomy. This was followed by subcutaneous mastectomy and chordee correction. The child is now planned for a second stage surgery for hypospadias correction, following which testosterone replacement therapy will be initiated.The diagnosis of CAH, provided by the window of abnormal genitalia at birth, was missed in this child, accounting for late presentation with severe hypertension and end-organ damage. Most females with virilising CAH are diagnosed at birth with ambiguous genitalia, while the diagnosis is delayed in males, who have normal genitalia at birth and present in early childhood with precocity ,6. RarelKhattab et al. described the largest series of 11-beta-hydroxylase CAH, with 108 patients from 11 countries . In thisBoth hypertension and hypokalemia resolved with optimal glucocorticoid replacement in our patient. The severity of hypertension in 11-beta-hydroxylase CAH varies from severe refractory cases requiring bilateral adrenalectomy to milder cases, improving with steroid supplementation. Glucocorticoid therapy results in feedback inhibition of pituitary ACTH secretion, thus improving hypertension and its end-organ manifestations. Similar to the index patient, Onyiriuka et al. and Isiavwe et al. described the remission of hypertension and end-organ damage with glucocorticoid supplementation . Notably11-beta-hydroxylase deficiency CAH is a rare autosomal recessive disorder of glucocorticoid biosynthesis, which presents with features of mineralocorticoid and androgen excess. Delayed diagnosis, as in our case, may put such individuals at an increased risk of end-organ damage due to uncontrolled hypertension. A detailed genital examination and directed investigations at birth may go a long way in preventing these undesirable consequences. Hypertension and even end-organ damage in the setting of 11-beta-hydroxylase CAH may reverse with appropriate glucocorticoid replacement therapy. It is important to remember that patients with 46,XX CAH and severe androgen excess may identify themselves as males, highlighting the importance of psychological tests to confirm gender identity before embarking upon surgical intervention.