Establishment of digital cutoff values for intraepithelial lymphocytes in biopsies from colonic mucosa with lymphocytic colitis.

BACKGROUND/INTRODUCTION: Lymphocytic colitis (LC) and the incomplete form (LCi) are common causes of chronic watery diarrhea. Endoscopy is often inconspicuous, and the diagnosis relies on histopathological assessment of colonic biopsies. Digital Image Analysis (DIA) eliminates interobserver variation. The aim of this study was to establish digital cutoff values for LC and LCi on CD3 stained slides. MATERIAL AND METHODS: One hundred and six patients with a hematoxylin and eosin (HE) diagnosis of normal colonic mucosa (N = 19), non-specific reactive changes (N = 24), LCi (N = 23) and LC (N = 40) were eligible for analysis. The number of intraepithelial lymphocytes (IELs) reached by DIA in the total surface epithelium and in hot spots of the biopsies was compared with the diagnostic category assigned by the pathologists based on HE stained slides. The digitalized slides were analyzed for number of IELs using Visiopharm Quantitative Digital Pathology software. All digitalized slides were examined manually to identify differences in the approach to the evaluation of the biopsies by the pathologists and DIA. RESULTS: The median IEL counts and interquartile range in the total surface epithelium were 3.6 (3.2-4.3), 4.4 (3.4-5.3), 19.8 (16.6-30.0) and 41.3 (37.0-47.8) in normal colon mucosa, mucosa with non-specific reactive changes, LCi and LC, respectively. Discrimination between normal mucosa and non-specific reactive changes was not possible. Digital cutoff values with the best separation between non-LC, LCi and LC were > 13 IELs/100 epithelial cells for LCi and > 36 IELs/100 epithelial cells for LC. These cutoff values resulted in an agreement between the pathologist's and DIA that was very good with a kappa value of 0.90. CONCLUSION: Despite differences among the approach of DIA and the pathologist's assessment of IELs in colonic mucosa DIA is able discriminate between the HE based diagnoses of the three subgroups non-LC, LCi and LC with high accuracy.

Is revision of cutoff values needed when using CD3 immunohistochemical staining in histopathologic diagnosis of lymphocytic colitis?

Lymphocytic colitis (LC) and LC incomplete (LCi) are common causes of chronic watery diarrhea. The diagnosis relies on clinical findings and histopathologic evaluation. The diagnostic criteria of LC are based on hematoxylin and eosin (HE) staining. However, supplementary immunohistochemical staining for highlighting the lymphocytes in borderline cases is now widely used. This change in diagnostics could lead to incorrectly diagnosing patients with LC and LCi if the present histologic criteria are used. The number of intraepithelial lymphocytes (IELs) was estimated and categorized in intervals based on HE- versus CD3-stained slides from patients with an HE diagnosis of normal colonic mucosa (n = 19), mucosa with nonspecific reactive changes (n = 24), LCi (n = 24), and LC (n = 40). The number of IELs was compared with clinical symptoms. Overall, the number of IELs was higher with CD3 stain compared with HE stain in 73% of cases, unchanged in 26% of cases, and lower in 1 case. The number of IELs detected was higher using the CD3 stain in 53%, 79%, 79%, and 75% of cases included as normal colonic mucosa, nonspecific reactive changes, LCi, and LC, respectively. Based on CD3 stain, 58% of the cases with nonspecific reactive changes fulfilled the HE criteria for LCi, and 79% of the cases with LCi fulfilled the HE criteria for LC. Automated image analysis of CD3-stained slides resulted in even higher numbers of IELs in all 4 diagnostic groups. Conclusively, our data support considering increased cutoff values for LCi and LC when assessed in CD3-stained specimens.

Genetic variants in the apoptosis gene BCL2L1 improve response to interferon-based treatment of hepatitis C virus genotype 3 infection.

Genetic variation upstream of the apoptosis pathway has been associated with outcome of hepatitis C virus (HCV) infection. We investigated genetic polymorphisms in the intrinsic apoptosis pathway to assess their influence on sustained virological response (SVR) to pegylated interferon-α and ribavirin (pegIFN/RBV) treatment of HCV genotypes 1 and 3 infections. We conducted a candidate gene association study in a prospective cohort of 201 chronic HCV-infected individuals undergoing treatment with pegIFN/RBV. Differences between groups were compared in logistic regression adjusted for age, HCV viral load and interleukin 28B genotypes. Four single nucleotide polymorphisms (SNPs) located in the B-cell lymphoma 2-like 1 (BCL2L1) gene were significantly associated with SVR. SVR rates were significantly higher for carriers of the beneficial rs1484994 CC genotypes. In multivariate logistic regression, the rs1484994 SNP combined CC+TC genotypes were associated with a 3.4 higher odds ratio (OR) in SVR for the HCV genotype 3 (p=0.02). The effect estimate was similar for genotype 1, but the association did not reach statistical significance. In conclusion, anti-apoptotic SNPs in the BCL2L1 gene were predictive of SVR to pegIFN/RBV treatment in HCV genotypes 1 and 3 infected individuals. These SNPs may be used in prediction of SVR, but further studies are needed.

Influence of hepatitis C virus and IL28B genotypes on liver stiffness.

OBJECTIVE: Liver fibrosis has been associated with hepatitis C virus (HCV) genotype and genetic variation near the interleukin 28B (IL28B) gene, but the relative contribution is unknown. We aimed to investigate the relation between HCV genotypes, IL28B and development of liver stiffness. PATIENTS AND METHODS: This cross-sectional study consists of 369 patients with chronic hepatitis C (CHC). Liver stiffness was evaluated using transient elastograhy (TE). Factors associated with development of liver fibrosis were identified by logistic regression analysis. RESULTS: We identified 369 patients with CHC. 235 were male, 297 Caucasians, and 223 had been exposed to HCV through intravenous drug use. The overall median TE value was 7.4 kPa (interquartile range (IQR) 5.7-12.1). HCV replication was enhanced in patients carrying the IL28B CC genotype compared to TT and TC (5.8 vs. 5.4 log10 IU/mL, p = 0.03). Patients infected with HCV genotype 3 had significantly higher TE values (8.2 kPa; IQR, 5.9-14.5) compared to genotype 1 (6.9 kPa; IQR, 5.4-10.9) and 2 (6.7 kPa; IQR, 4.9-8.8) (p = 0.02). Within patients with genotype 3, IL28B CC genotype had the highest TE values (p = 0.04). However, in multivariate logistic regression, using various cut-off values for fibrosis and cirrhosis, only increasing age (odds ratio (OR) 1.09 (95% confidence interval (CI), 1.05-1.14 per year increment)), ALT (OR 1.01 (95% CI, 1.002-1.011), per unit increment) and HCV genotype 3 compared to genotype 1 (OR 2.40 (95% CI, 1.19-4.81), were consistently associated with cirrhosis (TE>17.1 kPa). CONCLUSIONS: Age, ALT and infection with HCV genotype 3 were associated with cirrhosis assessed by TE. However, IL28B genotype was not an independent predictor of fibrosis in our study.

Hepatitis C virus infection in the human immunodeficiency virus infected patient.

Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) share the same transmission routes; therefore, coinfection is frequent. An estimated 5-10 million individuals alone in the western world are infected with both viruses. The majority of people acquire HCV by injection drug use and, to a lesser extent, through blood transfusion and blood products. Recently, there has been an increase in HCV infections among men who have sex with men. In the context of effective antiretroviral treatment, liver-related deaths are now more common than Acquired Immune Deficiency Syndrome-related deaths among HIV-HCV coinfected individuals. Morbidity and mortality rates from chronic HCV infection will increase because the infection incidence peaked in the mid-1980s and because liver disease progresses slowly and is clinically silent to cirrhosis and end-stage-liver disease over a 15-20 year time period for 15%-20% of chronically infected individuals. HCV treatment has rapidly changed with the development of new direct-acting antiviral agents; therefore, cure rates have greatly improved because the new treatment regimens target different parts of the HCV life cycle. In this review, we focus on the epidemiology, diagnosis and the natural course of HCV as well as current and future strategies for HCV therapy in the context of HIV-HCV coinfection in the western world.

Factors associated with resolution and progression of HIV/hepatitis C virus infection.

Coinfection with hepatitis C virus (HCV) is common in human immunodeficiency virus (HIV)-infected individuals as a result of shared routes of transmission, and this coinfection represents a special challenge. For HIV-HCV-coinfected individuals, the burden of disease is largely related to their HCV diseases, including a faster progression to liver fibrosis, cirrhosis and liver-related deaths. In the present thesis we investigated factors associated with spontaneous resolution and progression of HIV-HCV coinfection. In study I, we identified the study cohort of 327 individuals with HIV-HCV coinfection and a rate of spontaneous HCV resolution of 23%. We showed that female sex, coinfection with hepatitis B virus and individuals exposed through injecting drug use (IDU) or homosexual contact (MSM) had an increased rate of spontaneous HCV resolution. We speculate that differences in resolution rate may be caused by immunological memory induced by repeatedly being exposed to low-dose inoculum of HCV. In study II, we found three single-nucleotide-polymorphisms (SNPs) in the interleukin 28B (IL28B) gene associated with spontaneous HCV resolution in 208 Europeans of Caucasian origin with HIV-HCV coinfection. Further, we showed that the IL28B CC genotype favourable of HCV resolution was associated with a higher HCV viral load (VL) than non-CC genotypes. These results may indicate an influence of IL28B in viral control. In study III, we conducted a survival analysis in the 264 HIV-HCV-coinfected individuals with chronic infection. We showed in a time-updated Poisson regression that HCV VL, HCV genotype 3 and IL28B CC genotype were predictors of increased mortality. This may indicate a need for closer observation in HIV-HCV-coinfected individuals with HCV genotype 3 and maybe even initiation of antiviral therapy.

Mannose-binding lectin gene, MBL2, polymorphisms are not associated with susceptibility to invasive pneumococcal disease in children.

BACKGROUND: Most children are transiently colonized with Streptococcus pneumoniae, but very few develop invasive pneumococcal disease (IPD). Host genetic variation of innate immunity may predispose to IPD. We investigated the effect of genetic variation in the mannose-binding lectin gene, MBL2, on susceptibility and disease severity of IPD in previously healthy children aged <5 years. METHODS: IPD cases were identified through national registries. DNA was obtained from the Danish Neonatal Screening Biobank. Pneumococcal serotypes were determined by Quellung reaction. The associations between MBL2 diplotypes and IPD susceptibility, serotypes, and outcome were investigated using logistic regression analysis. RESULTS: We included 372 cases with meningitis, 907 with bacteremia, and 1263 age- and sex-matched controls; 2372 individuals were successfully genotyped and assigned MBL2 diplotypes. The median age in our combined case series was 13 months. Children with defective diplotypes were not at higher risk for meningitis than children with other diplotypes (odds ratio [OR], 0.85; 95% confidence interval [CI], .56-1.28). Similar results were found for bacteremia (OR, 0.89; 95% CI, .68-1.15) as well as for all cases (OR, 0.87; 95% CI, .70-1.09). There was no association with susceptibility to recurrent IPD (n = 12) for children with defective diplotypes compared with cases with a single episode (OR, 0.53; 95% CI, .07-4.13) and with all controls (OR, 0.46; 95% CI, .06-3.56). There was no association between diplotypes and mortality or between diplotypes and pneumococcal serotypes. CONCLUSIONS: Defective MBL2 polymorphisms did not predict increased IPD susceptibility in children born in Northern Europe.

Hepatitis C viral load, genotype 3 and interleukin-28B CC genotype predict mortality in HIV and hepatitis C-coinfected individuals.

OBJECTIVE: We hypothesized that hepatitis C virus (HCV) load and genotype may influence all-cause mortality in HIV-HCV-coinfected individuals. DESIGN AND METHODS: Observational prospective cohort study. Mortality rates were compared in a time-updated multivariate Poisson regression analysis. RESULTS: We included 264 consecutive HIV-HCV-coinfected individuals. During 1143 person years at risk (PYR) 118 individuals died [overall mortality rate 10 (95% confidence interval; 8, 12)/100 PYR]. In multivariate analysis, a 1 log increase in HCV viral load was associated with a 30% higher mortality risk [adjusted mortality rate ratio (aMRR): 1.30 (1.10,1.54)] when adjusted for sex, age, HIV exposure group, CD4 cell count, HIV RNA, HCV genotype and interleukin (IL)-28B genotype. Further, HCV genotype 3 vs. 1 [aMRR: 1.83 (1.12, 2.98)] and HIV RNA [aMRR: 3.14 (1.37,7.17) for undetectable vs. just detectable HIV RNA] were independent predictors of mortality, whereas a higher CD4 cell count was associated with a 41% reduction in mortality rate per 50 cell increase between 0 and 200 cells/µl [aMRR: 0.59 (0.48, 0.72)] and a 10% reduction for increases above 200 cells/µl [aMRR: 0.90 (0.82-0.98)]. IL28B) CC genotype was associated with 54% higher mortality risk [aMRR: 1.54 (0.89, 3.82] compared to TT genotype. CONCLUSION: High-HCV viral load, HCV genotype 3 and IL28B genotype CC had a significant influence on the risk of all-cause mortality among individuals coinfected with HIV-1. This may have consequences for the management of HIV-HCV-coinfected individuals.

Correlates of spontaneous clearance of hepatitis C virus in a Danish human immunodeficiency virus type 1 cohort.

BACKGROUND: Around a quarter of individuals infected with hepatitis C virus (HCV) are spontaneously able to clear the virus. Correlates of spontaneous HCV clearance are not well established and the aim of this study was to characterize factors associated with spontaneous HCV clearance in a human immunodeficiency virus (HIV)-co-infected cohort. METHODS: We analyzed 327 anti-HCV-positive HIV-1-infected patients using multivariate logistic regression. HCV clearance was defined as the presence of anti-HCV with undetectable HCV RNA from at least 2 measurements more than 6 months apart. RESULTS: We included 327 HIV-1-infected individuals, predominantly of Caucasian race; 112 (34%) were females, 258 (79%) were injecting drug users (IDU), 25 (8%) were men who have sex with men (MSM), and 20 (6%) were hepatitis B surface antigen (HBsAg)-positive. Seventy-six (23%; 95% confidence interval (CI) 18-28) had cleared their HCV infection and 251 (77%; 95% CI 72-82) had a chronic infection. The clearance rate in HBsAg-positive individuals was 65%. Being female, HBsAg-positive, or belonging to HIV exposure groups IDU and MSM predicted higher HCV clearance rates (adjusted odds ratio (aOR) 1.8, 95% CI 1-3.2; aOR 7.6, 95% CI 2.7-21; aOR 5.2, 1.2-23.5; and aOR 10.2, 95% CI 1.8-58, respectively). Race, acquired immunodeficiency syndrome (AIDS), and antiretroviral therapy were not associated with HCV clearance. CONCLUSIONS: The HCV clearance rate in this HIV-1 cohort was 23%. MSM and IDUs may have higher clearance rates due to their repeated exposure to low-dose HCV, leading to immune memory. Our data suggest an interaction of hepatitis B virus and HCV that influences the outcome of acute HCV infection.

Low level of regulatory T cells and maintenance of balance between regulatory T cells and TH17 cells in HIV-1-infected elite controllers.

BACKGROUND: A subgroup of HIV-1-infected individuals, elite controllers, have spontaneous viral control and offer an exceptional opportunity to study virological and immunolocigal factors of possible involvement in control of HIV-1 infection. METHODS: The frequencies of Tregs and TH17 cells was evaluated and correlated to markers of disease progression in peripheral blood mononuclear cells from 3 different groups of individuals infected with HIV-1: treatment-naive viremic individuals, individuals on successful highly active antiretroviral therapy, and elite controllers. In addition, a group of HIV-1-negative individuals were included. RESULTS: We demonstrate that elite controllers have lower levels of Tregs compared with HIV-1-infected viremic individuals, but that the low Treg level does not differ between individuals with HIV-1 control, whether natural or therapy induced. We also show that T-cell activation and proliferation both correlate to the level of Tregs. Finally, the TH17/Treg ratio was similar in Elite Controllers and uninfected controls, whereas in viremic and treated HIV-1-infected individuals, the TH17/Treg ratio was lower compared with uninfected controls. CONCLUSIONS: We show that one feature of spontaneous HIV-1 control is a maintained balance between regulatory T cells and TH17 cells.

[Tuberculosis in the ankle of a Danish-born man]. / Tuberkulose i ankelleddet hos en danskfødt mand.

Since the 1990s, we have witnessed an increase in the incidence of extrapulmonary tuberculosis in Denmark among foreigners as well as Danes. This case history describes a Danish man with an infection in his right ankle colonised with Mycobacterium tuberculosis. The man was treated with surgical revision and antituberculous drugs, and arthrodesis of the right ankle and heel was performed. This case is described to draw attention to the possibility of extrapulmonary tuberculosis when presented with infections in bones and joints.