Residues Distal to the Active Site Contribute to Enhanced Catalytic Activity of Variant and Hybrid β-Lactamases Derived from CTX-M-14 and CTX-M-15

A variety of CTX-M-type extended-spectrum β-lactamases (ESBLs), including hybrid ones, have been reported in China that are uncommon elsewhere. To better characterize the substrate profiles and enzymatic mechanisms of these enzymes, we performed comparative kinetic analyses of both parental and hybrid CTX-M enzymes, including CTX-M-15, -132, -123, -64, -14 and -55, that are known to confer variable levels of β-lactam resistance in the host strains. All tested enzymes were susceptible to serine β-lactamase inhibitors, with sulbactam exhibiting the weakest inhibitory effects. CTX-M-55, which differs from CTX-M-15 by one substitution, A⁷⁷V, displayed enhanced catalytic activity (k_cat/K_m) against expanded-spectrum cephalosporins (ESCs). CTX-M-55 exhibits higher structure stability, most likely by forming hydrophobic interactions between A⁷⁷V and various key residues in different helices, thereby stabilizing the core architecture of the helix cluster, and indirectly contributes to a more stable active site conformation, which in turn shows higher catalytic efficiency and is more tolerant to temperature change. Analyses of the hybrids and their parental prototypes showed that evolution from CTX-M-15 to CTX-M-132, CTX-M-123, and CTX-M-64, characterized by gradual enhancement of catalytic activity to ESCs, was attributed to introduction of different substitutions to amino acids distal to the active site of CTX-M-15. Similarly, the increased hydrolytic activities against cephalosporins and sensitivity to β-lactamase inhibitors, clavulanic acid and sulbactam, of CTX-M-64 were partly due to the amino acids that were different from CTX-M-14 and located at both the C and N termini of CTX-M-64. These data indicate that residues distal to the active site of CTX-Ms contributed to their enhanced catalytic activities to ESCs.     

What should men living with haemophilia need to know? The perspectives of Canadian men with haemophilia

Haemophilia is an inherited bleeding disorder affecting approximately 3000 Canadian men (Walker 2012). To manage their disease effectively individuals must be knowledgeable about the disease, bleed prevention strategies, treatment approaches, and complications. Data on individuals’ knowledge levels are scarce. The availability of such data could lead to better educational strategies for disease management. The aim of this study was to determine current knowledge levels, needs and gaps among Canadian individuals with haemophilia to facilitate optimal disease management. A survey was disseminated to adult males with haemophilia at three Haemophilia Treatment Centres (HTCs) in Canada. Self‐reported current knowledge levels and knowledge seeking were measured. Survey respondents reported highest levels of knowledge in the following areas: identifying and treating a bleed, haemophilia and physical activity, travel, career issues and genetics. Lower levels of knowledge were reported in the areas of sexual activity, product safety, information about factor, haemophilia and ageing, advocacy, timing of prophylactic infusions, and new or alternative therapies. Treating a bleed was the most commonly sought information, followed by information about factor, product safety, identifying a bleed and other health care issues. There was a positive correlation between knowledge seeking and severity of disease. HTC attendance was associated with knowledge seeking, and HTCs were the most frequented knowledge source, followed by the Canadian Haemophilia Society website. Canadian men were well informed; the HTC's role in knowledge sharing was recognized. Timing of infusions, sexual activity and ageing are areas which should be targeted in knowledge sharing.     

Combination therapy of interferon and nucleotide/nucleoside analogues for chronic hepatitis B

Chronic hepatitis B is one of the leading causes of cirrhosis and hepatocellular carcinoma globally. At present, seven drugs, including two interferons and five oral nucleos(t)ide analogues (NAs), have been approved for the treatment of chronic hepatitis B. Interferon works by immunomodulation, but is successful in less than a third of treated patients and is a relatively weak antiviral. NAs directly suppress the hepatitis B virus but have limited durability. Based on current data, combination of NA and interferon results in greater viral suppression but does not translate to off‐treatment sustained response. Concomitant or sequential treatment also does not make a difference. Combining telbivudine and interferon also runs the risk of severe peripheral neuropathy. On the other hand, interferon switch or additional therapy in patients well controlled with NAs appears to improve the durability of off‐treatment response. This article reviews current data on interferon and NA combination and discusses potential future developments.     

Are we getting the critical view? A prospective study of photographic documentation during laparoscopic cholecystectomy

Background

At laparoscopic cholecystectomy, most surgeons have adopted the operative approach where the ‘critical view of safety’ (CVS) is achieved prior to dividing the cystic duct and artery. This prospective study evaluated whether an adequate critical view was achieved by scoring standardized intra-operative photographic views and whether there were other factors that might impact on the ability to obtain an adequate critical view.

Methods

One hundred consecutive patients undergoing a laparoscopic cholecystectomy were studied. At each operation, two photographs were taken. Two independent experienced hepatobiliary surgeons scored the photographs on whether a critical view of safety was achieved. Inter-observer agreement was calculated using the weighted kappa coefficient. The Cochran–Mantel–Haenszel test was used to analyse the scores with potential confounding clinical factors.

Results

The kappa coefficient for adequate display of the cystic duct and artery was 0.49; 95% confidence interval (CI) 0.33 to 0.64; P = 0.001. No bias was detected in the overall scorings between the two observers (χ² 1.33; P = 0.312). Other clinical factors including surgeon seniority did not alter the outcome [odds ratio (OR) 0.902; 95% confidence interval 0.622 to 1.264].

Conclusion

Heightened awareness of the CVS through mandatory documentation may improve both trainee and surgeon technique.     

Long‐term hepatitis B surface antigen (HBsAg) kinetics during entecavir treatment in Korean patients—Functional cure unlikely

The aim of this study was to investigate the on‐treatment kinetics of quantitative HBsAg during entecavir therapy to predict the treatment period needed to achieve functional cure. From a cohort of 1009 CHB treatment‐naïve patients who were started on entecavir, the kinetics of quantitative HBsAg decline was assessed in 410 patients by a linear mixed model. The difference in the kinetics of quantitative HBsAg was determined based on the HBeAg positivity, HBeAg seroclearance and presence of baseline liver cirrhosis. Among the 410 patients, 213 patients (52.0%) were HBeAg‐positive and 217 patients (66.1%) were male with a median age of 48 years. During a median follow‐up of 53.5 months, the quantitative HBsAg level showed a slow but consistent decrease. The expected log qHBsAg levels as a function of time during entecavir treatment in HBeAg(+) and HBeAg(?) patients were 3.4773‐0.0039 × Months and 3.1853‐0.0036 × Months, respectively. The estimated time to clearance of quantitative HBsAg in our study was greater than 74.1 years in HBeAg‐positive patients and 73.5 years in HBeAg‐negative patients. The calculated time to achieve functional cure is lifelong without regard to HBeAg seroclearance or presence of liver cirrhosis. The mathematical modelling from a long‐term follow‐up of chronic hepatitis B patients on entecavir shows that HBsAg clearance requires decades of treatment. Thus, lifelong therapy is inevitable in entecavir‐treated patients to achieve functional cure.     

The efficacy of hepatitis B treatments in achieving HBsAg seroclearance: A systematic review and meta‐analysis

Current therapies for chronic hepatitis B (CHB) include nucleos(t)ide analogues (NAs) and interferon (IFN), but their relative efficacy as monotherapy or in combination has not been examined systematically for HBsAg loss (functional cure). Hence, we systematically reviewed the evidence for HBsAg loss in CHB patients treated with IFN, NA or the combination. We searched PubMed, EMBASE and abstracts from EASL, Asia Pacific Association for study of the Liver and American Association for the Study of Liver Disease for randomized controlled trials of CHB patients, comparing NA, IFN or the combination. The Cochrane Risk of Bias tool v2.0 and GRADE method were used. Analyses were stratified by NA genetic barrier, cirrhosis, type of combination therapy, HBeAg, treatment naivety, IFN dosage/duration and outcome duration. Sensitivity analysis was performed for selected strata, and HBsAg loss was measured at the end‐of‐study (EOS), end‐of‐treatment (EOT) or end‐of‐follow‐up (EOF). Effects were reported as risk differences (RD) with 95% confidence intervals (CI) using a random‐effects model. Forty‐five studies were included, all with low risk of bias. For HBsAg loss at EOS, when comparing combination vs IFN, RD = 1%, 95%CI‐1%, 2%; combination vs NA, RD = 5%, 95%CI 3%,7%; IFN vs NA, RD = 3%, 95%CI 2%,5%. Subgroup analysis showed a significant effect of standard IFN dose vs nonstandard; IFN duration ≥48 weeks vs <48 weeks, and loss of efficacy >2 years of follow‐up. Similar findings were seen in HBsAg seroconversion, but only three studies reported HBsAg seroreversion. In conclusion, IFN monotherapy/combination had a small but significant increase in HBsAg loss over NA, associated with standard dose of IFN and ≥48 weeks of therapy, although this effect faded over time.