Clinical outcomes of prestripped, prestained, and preloaded Descemet's membrane endothelial keratoplasty ("P3 DMEK").

PURPOSE: Despite faster healing and reduced risk of rejection, some surgeons are hesitant to adopt Descemet membrane endothelial keratoplasty (DMEK) due to difficult intraoperative tissue preparation. Use of eye bank prestripped, prestained, and preloaded (p3) DMEK tissue can reduce the learning curve and risk of complications. MATERIALS AND METHODS: We conducted a prospective study including 167 eyes undergoing p3 DMEK and compared outcomes to a retrospective chart review of 201 eyes that underwent standard DMEK surgery. The primary outcomes were graft failure, detachment, and re-bubbling frequency. The secondary outcomes included baseline and postoperative visual acuity at months 1, 3, 6, and 12. Baseline and postoperative central corneal thickness (CCT) and endothelial cell counts (ECC) were collected. RESULTS: ECC decrease for p3 DMEK at 3, 6, and 12 months were 15.0%, 18.0%, and 21.0%, respectively. Forty (24%) of p3 DMEK and 72 (35.8%) of standard DMEK eyes had at least a partial graft detachment. There was no difference in CCT, graft failures, or re-bubble frequency. At 6 months, mean visual acuity was 20/26 and 20/24 for standard and p3 DMEK, respectively. Mean case time for p3 DMEK with phaco or p3 DMEK alone was 33 and 24 min, respectively. Mean case time for eyes undergoing DMEK with phaco or DMEK alone was 59 and 45 min, respectively. CONCLUSION: P3 DMEK tissue is safe and can provide excellent clinical outcomes that are comparable to standard DMEK tissue. Eyes undergoing p3 DMEK may have lower graft detachment and ECC loss.

A Randomized Trial of Topical Fibrinogen-Depleted Human Platelet Lysate Treatment of Dry Eye Secondary to Chronic Graft-versus-Host Disease.

Purpose: The purpose of the study was to evaluate, as a pilot trial, safety and tolerability of CAM-101 10% and 30% topical ophthalmic fibrinogen-depleted human platelet lysate (FD hPL) solution in patients with dry eye disease (DED) secondary to graft-versus-host disease (GvHD) after 6 weeks of treatment. Design: A phase I/II, pilot, prospective, multicenter, randomized, double-masked clinical trial. Participants: Patients with DED secondary to GvHD. Methods: Sixty-four adult patients were stratified by "symptom severity" (Ocular Surface Disease Index [OSDI], ocular discomfort Visual Analog Scale (VAS), ocular symptom frequency, and use of artificial tears) and then randomized 1:1:1 to CAM-101 (FD hPL) at 10% or 30% concentration or an electrolyte (Plasma-Lyte A) vehicle control, 1 drop in both eyes, 4 times daily, for 42 days. After 42 days, control patients were offered 42 days of open-label treatment with 30% FD hPL. Main Outcome Measures: Primary outcome safety measures were ocular and systemic adverse events and the number of patients in each group with clinically significant change from normal to abnormal in any ocular findings. Secondary outcomes were changes from baseline to day 42 in ocular discomfort, OSDI, fluorescein corneal staining, and lissamine green conjunctival staining relative to the vehicle control. The ocular symptom frequency was assessed on a 100-point VAS. Results: FD hPL 10% and 30% were safe and well tolerated. Relative to the vehicle control, significant decreases from baseline to day 42 were seen in the FD hPL 30% group with regard to ocular discomfort (mean decrease = -18.04; P = 0.018), frequency of burning/stinging (-20.23; P = 0.022), eye discomfort (-32.97; P < 0.001), eye dryness (-21.61; P = 0.020), pain (-15.12; P = 0.044), photophobia (-24.33; P = 0.0125), and grittiness (-20.08; P = 0.0185). Decreases were also seen for itching and foreign body sensation, though not statistically significant. Improvements were seen in tear breakup time (mean increase = 1.30 seconds; P = 0.082) and the investigator's global evaluation 4-point scale (mean decrease = -0.86; P = 0.026). Corneal fluorescein staining was not improved. The OSDI had a mean decrease of -8.88 compared to the vehicle, although not statistically significant. Conclusions: Fibrinogen-depleted human platelet lysate appears to be well tolerated, with no significant toxicity at concentrations of 10% and 30%. These initial data suggest some efficacy, especially for subjective outcome measures relative to baseline assessments and treatment with the vehicle, but larger studies are needed to confirm these effects.

The Dry Eye Assessment and Management (DREAM) extension study - A randomized clinical trial of withdrawal of supplementation with omega-3 fatty acid in patients with dry eye disease.

PURPOSE: To determine effects of continued or discontinued use of omega-3 (ω3) fatty acid supplements through a randomized withdrawal trial among patients assigned to ω3 supplements in the first year of the DREAM study. METHODS: Patients who were initially assigned to ω3 (3000 mg) for 12 months in the primary trial were randomized 1:1 to ω3 active supplements or placebos (refined olive oil) for 12 more months. The primary outcome was change in the Ocular Surface Disease Index (OSDI) score. Secondary outcomes included change in conjunctival staining, corneal staining, tear break-up time, Schirmer test, and adverse events. RESULTS: Among 22 patients assigned to ω3 and 21 to placebo supplements, the mean change in OSDI score between month 12 and 24 was similar between treatment groups (mean difference in change -0.6 points, 95% confidence interval [CI], (-10.7, 9.5), p = 0.91). There were no significant differences between groups in mean change in conjunctival staining (difference in mean change -0.5 points; 95% CI (-1.2, 0.3)), corneal staining (-0.3 points; 95% CI (-1.2, 0.3)), tear break-up time (-0.8 s; 95% CI (-2.6, 0.9)) and Schirmer test (0.6 mm, 95% CI (-2.0, 3.2)). Rates of adverse events were similar in both groups. CONCLUSION: Among patients who received ω3 supplements for 12 months in the primary trial, those discontinuing use of ω3 for an additional 12 months did not have significantly worse outcomes compared to those who continued use of ω3. ClinicalTrials.gov number NCT02128763.

Screening for Harassment, Abuse, and Discrimination among Surgery Residents: An EAST Multicenter Trial.

Estimating the prevalence of harassment, verbal abuse, and discrimination among residents is difficult as events are often under-reported. The purpose of this study was to determine the prevalence of discrimination and abuse among surgical residents using the HITS (Hurt, Insulted, Threatened with harm or Screamed at) screening tool. A multicenter, cross-sectional, survey-based study was conducted at five academic teaching hospitals. Of 310 residents, 76 (24.5%) completed the survey. The HITS screening tool was positive in 3.9 per cent. The most common forms of abuse included sexual harassment (28.9%), discrimination based on gender (15.7%), and discrimination based on ethnicity (7.9%). There was a positive correlation between individuals who reported gender discrimination and racial discrimination (r = 0.778, n = 13, P = 0.002). Individuals who experienced insults were more likely to experience physical threats (r = 0.437, n = 79, P < 0.001) or verbal abuse (r = 0.690, n = 79, P < 0.001). Discrimination and harassment among surgical residents in academic teaching hospitals across the United States is not uncommon. Further research is needed to determine the impact of these findings on resident attrition.

Central Corneal Thickness Increase Due to Stromal Thickening With Diabetic Peripheral Neuropathy Severity.

PURPOSE: To investigate the relationship between central corneal thickness (CCT) and diabetes disease severity among patients with diabetic peripheral neuropathy (DPN) compared with controls. METHODS: In this cross-sectional study, 34 participants were examined. DPN status was assessed by clinical examination, nerve conduction studies, and quantitative sensory testing. All participants underwent comprehensive eye examination that included intraocular pressure measured by Goldmann applanation tonometry. CCT was measured by ultrasound pachymetry, and the thickness of corneal layers was assessed by corneal confocal microscopy. Association of CCT and DPN was examined using ANOVA. RESULTS: Among the 34 participants, there were 9 controls, 16 patients with mild DPN, and 9 patients with severe DPN. CCT was significantly increased in the DPN groups compared with controls (P = 0.0003). Mean CCT among controls was 552.7 ± 29.2 µm compared with 583.4 ± 25.0 µm in the mild DPN group and 613.3 ± 28.8 µm in the severe DPN group. In addition, stromal thickness differed significantly between the 3 study groups (P = 0.045). Mean stromal thickness among controls was 439.5 ± 23.5 µm compared with 478.9 ± 37.5 µm in the mild DPN group and 494.5 ± 39.1 µm in the severe DPN group. CONCLUSIONS: This study demonstrates that CCT increases with DPN severity because of an increase in stromal thickness. CCT increase associated with DPN has important clinical implications including glaucoma progression, keratoconus susceptibility, and intraocular pressure assessment and should be accounted for when evaluating patients with diabetes.

The effect of humidity and temperature on visual outcomes after myopic corneal laser refractive surgery.

OBJECTIVE: To determine whether procedure room environmental conditions are associated with outcomes after myopic laser in situ keratomileusis (LASIK) or laser-assisted keratomileusis (LASEK). DESIGN: Retrospective chart review. PARTICIPANTS: Eight hundred sixty-three eyes of 458 consecutive patients at a university-based academic practice. METHODS: We reviewed the medical records of consecutive patients who underwent LASIK or LASEK over a 3-year period. Linear mixed regression models were used to investigate the association of laser room temperature and humidity with the outcomes of visual acuity and postoperative manifest spherical equivalent refraction. Repeated measures logistic regression models were used for the outcomes of diffuse lamellar keratitis (DLK) and need for enhancement surgery. RESULTS: Subjects were on an average 38.6 years old at the time of surgery (standard deviation [SD] =10.3) and the average spherical equivalent refraction of eyes was 3.8 diopters (SD =2.03). Regression models did not reveal a significant association between temperature and uncorrected distance visual acuity (UDVA) or corrected distance visual acuity (CDVA), or between humidity and UDVA (P>0.05 for all). However, increased humidity was associated with a small but statistically significant improvement in CDVA after LASIK at 1 day, 1 month, 3 months, and 1 year postoperatively (P<0.05 for all). There was no significant association between temperature and humidity and the need for enhancement, the incidence of DLK, or postoperative manifest refraction. CONCLUSION: While increased laser room humidity was consistently associated with small improvements in CDVA after myopic LASIK over time, variations in room temperature and humidity were not associated with UDVA, the need for enhancement, the incidence of DLK, or refraction after myopic LASIK or LASEK.

Discordant Dry Eye Disease (An American Ophthalmological Society Thesis).

PURPOSE: To improve understanding of dry eye disease and highlight a subgroup of patients who have a component of central sensitization and neuropathic pain contributing to their condition. METHODS: Prospective, cross-sectional, IRB-approved study comparing isolated dry eye disease (n=48) to fibromyalgia (positive control; n=23) and healthy (negative control; n=26) individuals with ocular surface examination, corneal confocal microscopy, quantitative sensory testing, and self-reported ocular symptoms and systemic associations. A subset of patients also underwent skin biopsy and/or brain neuroimaging. Dry eye patients were split into concordant (ie, those with dry eyes on examination) and discordant (ie, those with dry eye symptoms but normal examination) subgroups for further analysis. We hypothesized that on the systemic measures included, concordant patients would resemble healthy controls, whereas discordant patients would show evidence of centralized mechanisms similar to fibromyalgia. RESULTS: Schirmer test and Ocular Surface Disease Index (OSDI) scores indicated significant decreases in tear production (Schirmer: healthy, 18.5±8.2 mm; dry, 11.2±5.4 mm; fibromyalgia, 14.4±7.5; P<.001) and increases in self-reported dry eye symptoms (OSDI: healthy, 1.9±3.0; dry, 20.3±17.7; fibromyalgia, 20.3±17.1; P<.001) in the dry eye and fibromyalgia patients, compared to controls. The discordant subgroup had decreased corneal nerve density and decreased visual quality-of-life scores, similar to patients with fibromyalgia. Concordant patients were more similar to healthy controls on these measures. CONCLUSIONS: Individuals with discordant dry eye may have a central pathophysiologic mechanism leading to their eye pain symptoms, which could be an important factor to consider in treatment of chronic idiopathic dry eye.

Ocular surface disease in patients with diabetic peripheral neuropathy.

PURPOSE: To analyse clinical signs and symptoms of ocular surface disease in patients with diabetes mellitus (DM), based on severity of diabetic peripheral neuropathy (DPN). METHODS: This cross-sectional study included participants who were carefully phenotyped by a multidisciplinary team and categorised into groups based on severity of DPN. All study participants underwent ophthalmic evaluation and completed the Ocular Surface Disease Index (OSDI) and Visual Function Questionnaire (VFQ-25). RESULTS: The 34 study participants were healthy controls (n=9), patients with DM and mild or no DPN (n=16) and patients with DM and severe DPN (n=9). Tear osmolarity was increased, and corneal nerve fibre length was decreased, with increasing severity of DPN. In addition, patients with DM were found to have decreased Schirmer's test values when compared with healthy controls. No statistically significant differences were found between groups in OSDI, tear breakup time or corneal sensitivity. No statistically significant correlations were noted between the OSDI or VFQ-25 scores and clinical signs of dry eyes. CONCLUSION: This study demonstrates some increased clinical signs of ocular surface disease but not an increase in subjective symptoms of dry eyes, with increasing severity of DPN. Furthermore, no significant correlation was found between OSDI scores and clinical signs of dry eye. A periodic evaluation of the ocular surface is important for patients with DM, in addition to retinopathy screening, as they may be asymptomatic but have severe dry eye disease, which can lead to further ocular surface complications such as corneal ulceration. TRIAL REGISTRATION NUMBER: NCT01695629.

Prospective Randomized Trial Comparing Efficacy of Topical Loteprednol Etabonate 0.5% Versus Cyclosporine-A 0.05% for Treatment of Dry Eye Syndrome Following Hematopoietic Stem Cell Transplantation.

PURPOSE: To evaluate the safety and efficacy of topical loteprednol etabonate (LE) 0.5% compared with cyclosporine A (CsA) 0.05% for the prophylaxis and treatment of dry eye syndrome (DES) after hematopoietic stem cell transplantation (HSCT). METHODS: Seventy-five patients were randomized to LE (n = 76 eyes of 38 patients) or CsA (n = 74 eyes of 37 patients) pre-HSCT. Lissamine green and fluorescein staining, tear break-up time, tear osmolarity (Osm), Schirmer score (Sch), intraocular pressure, visual acuity, and Ocular Surface Disease Index were assessed pre-HSCT, 3, 6, 9, and 12 months post-HSCT. RESULTS: There were no differences in DES incidence (P = 0.22; log-rank test) or progression (P = 0.41; log-rank test) between the 2 treatment arms during the course of the study. Among eyes with no DES at enrollment, the Kaplan-Meier analysis yielded a 90% rate of DES development in cyclosporine-treated eyes and a 79% rate of DES development in LE-treated eyes by 12 months post-HSCT. The Kaplan-Meier analysis of eyes with DES at enrollment demonstrated a 38% rate of disease progression among cyclosporine-treated eyes and a 26% rate of disease progression among loteprednol-treated eyes by 12 months. No patient in either group had an elevation of 10 mm Hg or greater from baseline at any study visit, and no patients had their treatment discontinued for elevation in intraocular pressure. CONCLUSIONS: Pre-HSCT initiation of LE 0.5% appears to be safe and may be as effective as CsA 0.5% for the treatment and prophylaxis of DES following HSCT.

Long-term use of autologous serum 50% eye drops for the treatment of dry eye disease.

PURPOSE: The aim of this study was to describe the outcomes of 50% autologous serum (AS) eye drops after long-term use in a large cohort of patients with dry eyes. METHODS: A retrospective cohort study was conducted on all patients treated with 50% AS eye drops at our institution between June 2008 and January 2013. Records were reviewed for clinical history, systemic risk factors, dry eye etiology, patients' symptoms, and adverse events. Ocular surface evaluation included Schirmer testing with topical anesthesia, fluorescein staining, and ocular surface disease index. Data were reviewed at initial visit, 1 month, and every 3 to 6 months during treatment with AS. Paired t tests were performed to compare the progression of signs and symptoms of dry eye disease. RESULTS: A total of 123 eyes of 63 patients were evaluated with a mean follow-up of 12 months (range, 3-48 months). Corneal fluorescein staining (mean baseline, 1.77 ± 1.1) improved at the 3- to <6-month, 6- to <12-month, and final follow-up (mean: 1.2 ± 1.0, 1.3 ± 1.0, and 1.1 ± 1.1; P = 0.003, 0.017, and 0.0003, respectively). Schirmer scores (mean baseline, 6.6 ± 6.5 mm) improved at the 12- to 24-month follow-up (mean = 10.7 ± 11.4, P = 0.03), whereas ocular surface disease index scores (mean baseline, 54.1 ± 22.3) improved at the 3- to <6- and 6- to <12-month follow-up (mean: 49.5 ± 8.2 and 39.3 ± 21.4, P = 0.029 and 0.003, respectively). No complications were noted. CONCLUSIONS: Fifty percent AS eye drops seem to be a safe and effective long-term treatment for dry eye disease, especially in patients with severe disease who have exhausted all other conventional forms of treatment.

Differential reduction in corneal nerve fiber length in patients with type 1 or type 2 diabetes mellitus.

AIM: To examine the relationship between corneal nerve fiber length (CNFL) and diabetic neuropathy (DN) status in patients with type 1 or type 2 diabetes mellitus (DM). METHODS: In this cross-sectional study, we examined 25 diabetic patients without DN, 10 patients with mild DN, 8 patients with severe DN, and 9 controls without diabetes. DN status was assigned based on a combination of clinical symptoms, signs, and electrophysiological testing. Patients underwent corneal confocal microscopy (CCM) of the sub-basal nerve plexus. Post-hoc analysis of the CCM images was performed to quantify the average CNFL, and ANOVA was used to assess for differences in CNFL. RESULTS: All 25 subjects without DN had type 1 DM, and subjects with DN had type 2 DM. Participants with severe DN had significantly lower CNFL (12.5±6.1mm/mm(2)) compared to controls (20.7±2.2mm/mm(2)) (p=0.009). However, lower CNFL was also found in participants with type 1 DM who did not have DN (15.1±4.7mm/mm(2)) relative to controls (p=0.033). CONCLUSIONS: CCM of the sub-basal nerve plexus may be an indicator of early peripheral nerve degeneration in type 1 DM. Type of diabetes, in addition to degree of neuropathy, may influence the extent of corneal nerve damage.

Effect of tamsulosin on iris vasculature and morphology.

PURPOSE: To determine whether preoperative iris vasculature and morphology are altered in patients who have taken tamsulosin (Flomax). SETTING: Academic multispecialty practice. DESIGN: Case series. METHODS: Patients with current or past tamsulosin use and age- and sex-matched control patients were included. Anterior segment optical coherence tomography (AS-OCT) and iris fluorescein angiography were performed to measure iris vasculature and thickness before cataract surgery. Data collected at surgery included pupil diameter, clinical signs of intraoperative floppy-iris syndrome, and surgical complications. RESULTS: Tamsulosin was currently used by 16 patients and in the past by 4 patients; the control group comprised 10 patients. Pharmacologically dilated pupil diameter was statistically significantly smaller preoperatively and immediately postoperatively in the tamsulosin group than in the control group (P=.009 and P=.003, respectively). There was a statistically significant decrease in pupil size intraoperatively in the tamsulosin group (P=.05) but not in the control group (P=.3). Iris-vasculature parameters, specifically time to first vessel fill and percentage of vessel fill on iris fluorescein angiography, were not significantly different between the 2 groups. The AS-OCT measurements of iris morphology were not statistically significantly different between the groups. No surgical complications occurred. No fluorescein dye leakage, staining, or other vascular anomalies were observed. CONCLUSIONS: Although there were differences in pupil measurements and intraoperative iris behavior between patients who had been on tamsulosin and control patients, there were no significant differences in iris vasculature on iris fluorescein angiography or in iris morphology on AS-OCT.

Hepatitis B virus genotype C isolates with wild-type core promoter sequence replicate less efficiently than genotype B isolates but possess higher virion secretion capacity.

Infection by hepatitis B virus (HBV) genotype C is associated with a prolonged viremic phase, delayed hepatitis B e antigen (HBeAg) seroconversion, and an increased incidence of liver cirrhosis and hepatocellular carcinoma compared with genotype B infection. Genotype C is also associated with the more frequent emergence of core promoter mutations, which increase genome replication and are independently associated with poor clinical outcomes. We amplified full-length HBV genomes from serum samples from Chinese and U. S. patients with chronic HBV infection and transfected circularized genome pools or dimeric constructs of individual clones into Huh7 cells. The two genotypes could be differentiated by Western blot analysis due to the reactivities of M and L proteins toward a monoclonal pre-S2 antibody and slightly different S-protein mobilities. Great variability in replication capacity was observed for both genotypes. The A1762T/G1764A core promoter mutations were prevalent in genotype C isolates and correlated with increased replication capacity, while the A1752G/T mutation frequently found in genotype B isolates correlated with a low replication capacity. Importantly, most genotype C isolates with wild-type core promoter sequence replicated less efficiently than the corresponding genotype B isolates due to less efficient transcription of the 3.5-kb RNA. However, genotype C isolates often displayed more efficient virion secretion. We propose that the low intracellular levels of viral DNA and core protein of wild-type genotype C delay immune clearance and trigger the subsequent emergence of A1762T/G1764A core promoter mutations to upregulate replication; efficient virion secretion compensates for the low replication capacity to ensure the establishment of persistent infection by genotype C.

Hepatitis B virus core promoter mutations contribute to hepatocarcinogenesis by deregulating SKP2 and its target, p21.

BACKGROUND & AIMS: Clinical studies have associated hepatitis B virus core promoter (CP) mutations with an increased risk of hepatocellular carcinoma. The CP region overlaps with the HBV X (HBx) gene, which has been implicated in hepatocarcinogenesis. The cyclin kinase inhibitor p21WAF1/CIP1 is an important regulator of cell cycle progression and proliferation. We determined whether HBx mutants that result from mutations in the CP deregulate p21 and these processes. METHODS: We constructed a series of HBx mutants with changes in the CP region that correspond to A1762T/G1764A (TA), T1753A, T1768A, or a combination of these (combo) and expressed them, along with wild-type HBx under control of its endogenous promoter, in primary human hepatocytes (PHHs) and HepG2 cells. We then analyzed the effects of CP mutations on expression and degradation of p21 and the effects on cell cycle progression and proliferation. RESULTS: The combo mutant decreased levels of p21 and increased cyclin E expression in PHHs and HepG2 cells. The combo mutant, but not HBx with single or double CP mutations, accelerated p21 degradation in HepG2 cells. The combo mutant increased expression of S-phase kinase-associated protein 2 (SKP2) in PHHs and Huh7 cells. Silencing of SKP2 abrogated the effects of CP mutations on p21 expression. The kinetics of p21 expression correlated with changes in cell cycle distribution. The combo mutant accelerated cell cycle progression; p21 overexpression restored G1 arrest. CONCLUSIONS: HBx mutants with changes that correspond to a combination of CP mutations up-regulate SKP2, which then down-regulates p21 via ubiquitin-mediated proteasomal degradation. CP mutations might increase the risk of hepatocellular carcinoma via this pathway.

Occult and previous hepatitis B virus infection are not associated with hepatocellular carcinoma in United States patients with chronic hepatitis C.

UNLABELLED: Previous studies have suggested that prior exposure to hepatitis B virus (HBV) infection may increase the risk of development of hepatocellular carcinoma (HCC) in patients with chronic hepatitis C. The aim of this study was to compare the prevalence of previous or occult HBV infection in a cohort of hepatitis B surface antigen-negative patients with histologically advanced chronic hepatitis C in the United States who did or did not develop HCC. Stored sera from 91 patients with HCC and 182 matched controls who participated in the Hepatitis C Antiviral Long-term Treatment against Cirrhosis (HALT-C) Trial were tested for hepatitis B core antibody (anti-HBc), hepatitis B surface antibody, and HBV DNA. Frozen liver samples from 28 HCC cases and 55 controls were tested for HBV DNA by way of real-time polymerase chain reaction. Anti-HBc (as a marker of previous HBV infection) was present in the serum of 41.8% HCC cases and 45.6% controls (P=0.54); anti-HBc alone was present in 16.5% of HCC cases and 24.7% of controls. HBV DNA was detected in the serum of only one control subject and no patients with HCC. HBV DNA (as a marker of occult HBV infection) was detected in the livers of 10.7% of HCC cases and 23.6% of controls (P=0.18). CONCLUSION: Although almost half the patients in the HALT-C Trial had serological evidence of previous HBV infection, there was no difference in prevalence of anti-HBc in serum or HBV DNA in liver between patients who did or did not develop HCC. In the United States, neither previous nor occult HBV infection is an important factor in HCC development among patients with advanced chronic hepatitis C.

Sensitivity and accuracy of an updated line probe assay (HBV DR v.3) in detecting mutations associated with hepatitis B antiviral resistance.

BACKGROUND/AIMS: Early detection of antiviral drug-resistant mutations enables prompt initiation of rescue therapy. The aim of this study was to determine the accuracy and sensitivity of a new line probe assay in the detection of antiviral drug-resistant HBV mutations. METHODS: One-hundred samples from 54 patients with virologic breakthrough during entecavir, lamivudine or adefovir treatment and 21 samples from 21 nucleoside-naïve patients were tested by direct sequencing and an updated line probe assay (Innogenetics, HBV DR v.3) which incorporates probes that can detect mutations at 11 positions of the reverse transcriptase region of the HBV polymerase gene. RESULTS: Complete concordance between line probe and sequencing results was observed for 90/121 samples (74.3%) and 1291/1331 amino acid positions (96.9%). Testing of follow-up samples and clonal analysis of discordant samples confirmed the presence of mutations where line probe assay but not direct sequencing detected mutations. HBV DR v.3 assay consistently detected mutations present in > or = 5% of the virus population when HBV DNA concentration was > or = 4 log10copies/mL. CONCLUSIONS: The updated version of the line probe assay (HBV DR v.3) has high concordance with direct sequencing in detecting antiviral drug-resistant mutations but its sensitivity in detecting mutations at some positions needs to be improved.

Impact of the hepatitis B virus genotype on pre- and post-liver transplantation outcomes.

Emerging data suggest that the hepatitis B virus (HBV) genotype and the precore and core promoter variants impact the outcome of orthotopic liver transplantation (OLT) for hepatitis B. The aim of this study was to determine if there is a correlation between HBV genotype, precore and core promoter variants, and pre- and post-OLT outcomes. Serum samples from patients participating in the National Institutes of Health HBV-OLT study were tested for HBV genotype and precore and core promoter variants. A total of 123 patients were studied: 43% were Asians, 46% were Caucasians, and 8% were African Americans. HBV genotypes A (35%) and C (35%) were the most prevalent, followed by genotypes D and B. Precore and core promoter variants were detectable in 44% and 90% of patients. Patients with genotype C were more likely to have hepatocellular carcinoma (HCC) at listing (P < 0.001). Waitlist mortality was highest among patients with genotype D, while posttransplant mortality was highest among patients with genotype C. Precore or core promoter variants did not correlate with pre- or post-OLT survival. In conclusion, in this US patient population, patients with genotype C were more likely to have HCC at the time of transplant listing and to die after transplant than patients with non-C genotypes. Patients with genotype D had the highest posttransplant survival, but this was offset by higher waitlist mortality. Our study suggests that HBV genotypes but not precore or core promoter variants may have an impact on pre- and post-OLT outcomes of hepatitis B patients.

Prevalence and significance of occult hepatitis B in a liver transplant population with chronic hepatitis C.

Occult hepatitis B virus (HBV) infection is defined as the detection of HBV deoxyribonucleic acid (DNA) in the serum or liver tissue of individuals who test negative for hepatitis B surface antigen (HBsAg). We undertook a prospective study to evaluate the significance and course of occult HBV in patients with hepatitis C virus (HCV) cirrhosis undergoing orthotopic liver transplantation (OLT). A sensitive real-time polymerase chain reaction assay was utilized to test for serum HBV DNA at enrollment and for hepatic HBV DNA within the explant liver. Patients were followed with serum HBsAg and HBV DNA post-OLT. A total of 56 patients with HCV cirrhosis were enrolled between October 2002 and July 2004; of these, 44 underwent OLT. The overall prevalence of occult HBV based on positive serum HBV DNA was 16 of 56 (28%), and based on positive hepatic HBV DNA ("occult HBV liver") was 22 of 44 (50%). The presence of serum hepatitis B core antibody (anti-HBc) and a past history of injection drug use correlated with occult HBV.Explant-proven hepatocellular carcinoma (HCC) was found in 13 of 22 (59%) patients with occult HBV liver compared to 8 of 22 (36%) patients without occult HBV liver (P » 0.36, odds ratio 2.5; confidence interval 0.76­8.54 [corrected]. Post-OLT, no cases of HBV reactivation were noted, and there was no significant association between occult HBV and recurrent HCV. In conclusion, occult HBV is far more prevalent in patients with end-stage HCV than would be expected from its prevalence in the general population. Occult HBV infection is strongly associated with the presence of anti-HBc, history of injection drug use, and explant-proven HCC.

Presence of intrahepatic (total and ccc) HBV DNA is not predictive of HBV recurrence after liver transplantation.

Previous studies reported that hepatitis B virus (HBV) deoxyribonucleic acid (DNA) can be detected in livers of patients who received transplants for hepatitis B despite the absence of serological markers of HBV recurrence. Quantification of HBV DNA was not performed and presence of covalently closed circular (ccc) DNA was not analyzed in most studies. We aimed to quantify total and ccc HBV DNA in explant liver and post-orthotopic liver transplantation (OLT) biopsies and to correlate the values with HBV recurrence post-OLT. Frozen liver tissue from 34 patients (9 with explant liver only, 9 with explant liver and post-OLT liver biopsies, and 16 with post-OLT biopsies only) in the National Institutes of Health HBV-OLT study was examined using real-time polymerase chain reaction (PCR). Among the 18 patients with explant liver, 7 were hepatitis B e antigen (HBeAg)-positive, 8 had detectable serum HBV DNA, and 10 received antiviral therapy prior to OLT. Total and ccc HBV DNA was detected in explant livers of 17 and 16 patients, respectively. Of the 10 patients who received antiviral therapy pre-OLT, serum HBV DNA was undetectable in 8 at transplantation but 7 had detectable total and ccc HBV DNA in their explant liver. Of the 25 patients with post-OLT biopsies, total HBV DNA was detected in 83% and ccc DNA in 17% of 47 biopsies, although only 2 patients had HBV recurrence. In conclusion, total and ccc HBV DNA could be detected in explant livers of most patients despite antiviral therapy pre-OLT. Total but not ccc HBV DNA could be detected in post-OLT liver biopsies of most patients despite undetectable serum HBV DNA and hepatitis B surface antigen (HBsAg). Our findings suggest that occult HBV reinfection occurs in most HBV patients after OLT and continued administration of appropriate prophylactic therapy is important in preventing overt HBV recurrence.