Implementation of point-of-care HbA1C instruments into community pharmacies: Initial development of a pathway for robust community testing.

BACKGROUND: Point-of-care (POC) analysers in community settings can provide opportunistic and regular HbA1c monitoring. Community pharmacies in NHS Scotland are utilised by populations at greatest risk of type two diabetes (T2D). This study describes initial development of an HbA1c pathway using a POC analyser in community pharmacies. METHODS: The Abbott Afinion analyser was compared in (i) NHS Tayside's Blood Sciences Service and (ii) community pharmacies from four Scottish Health Boards. A side by side comparison with standard operating procedures for HbA1c quantification using 80 T2D patient venous samples. The machine was implemented into 11 community pharmacies and 144 samples obtained from patients for comparison to their recent laboratory HbA1c. Four focus groups examined themes around the intervention and an exit questionnaire was administered. RESULTS: Laboratory assessment verified the efficacy of the POC test machine. The value for level 1 quality control was 44 mmol/mol and the mean during testing 42.7 mmol/mol. The greatest percent coefficient of variation (cv) was within-run for both levels of quality control material, at a value of 1.63% and 1.62%, respectively. The analyser performed robustly within the pharmacy assessment, with a mean difference of 1.68 and a standard deviation of 0.71 (CV 0.423). Patients with T2D reported positive experiences of using a pharmacy. The focus groups identified an appreciation of the convenience of pharmacies and of the longitudinal relationships with pharmacy staff. CONCLUSION: POC HbA1c analysers can be successfully established in community pharmacies. The target patient group responded positively to the opportunity to use a pharmacy service.

Longitudinal Serological Analysis and Neutralizing Antibody Levels in Coronavirus Disease 2019 Convalescent Patients.

BACKGROUND: Understanding the longitudinal trajectory of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies is crucial for diagnosis of prior infection and predicting future immunity. METHODS: We conducted a longitudinal analysis of coronavirus disease 2019 convalescent patients, with neutralizing antibody assays and SARS-CoV-2 serological assay platforms using SARS-CoV-2 spike (S) or nucleocapsid (N) antigens. RESULTS: Sensitivities of serological assays in diagnosing prior SARS-CoV-2 infection changed with time. One widely used commercial platform that had an initial sensitivity of >95% declined to 71% at 81-100 days after diagnosis. The trajectories of median binding antibody titers measured over approximately 3-4 months were not dependent on the use of SARS-CoV-2 N or S proteins as antigen. The median neutralization titer decreased by approximately 45% per month. Each serological assay gave quantitative antibody titers that were correlated with SARS-CoV-2 neutralization titers, but S-based serological assay measurements better predicted neutralization potency. Correlation between S-binding and neutralization titers deteriorated with time, and decreases in neutralization titers were not predicted by changes in S-binding antibody titers. CONCLUSIONS: Different SARS-CoV-2 serological assays are more or less well suited for surveillance versus prediction of serum neutralization potency. Extended follow-up should facilitate the establishment of appropriate serological correlates of protection against SARS-CoV-2 reinfection.

Epidemiology of injury in English Professional Football players: A cohort study.

OBJECTIVE: To estimate the current incidence and location of injury in English professional football. DESIGN: Prospective cohort study conducted over one competitive season (2015/16). SETTING: Professional football players competing in the English Football League and National Conference. PARTICIPANTS: 243 players from 10 squads (24.3 ±â€¯4.21 per squad). MAIN OUTCOME MEASURE: Injury incidence, training and match exposure were collected in accordance with the international consensus statement on football injury epidemiology. RESULTS: 473 injuries were reported. The estimated incidence of injury was, 9.11 injuries/1000 h of football related activity. There was a higher incidence of injury during match play (24.29/1000 h) compared to training (6.84/1000 h). The thigh was the most common site of injury (31.7%), muscle strains accounted for 41.2% of all injuries. The hamstrings were the most frequently strained muscle group, accounting for 39.5% of all muscle strains and 16.3% of all injuries. Moderate severity injuries (8-28 days) were the most common (44.2%). CONCLUSIONS: Incidence of injury has increased over the last 16 years with muscle strains remaining the most prevalent injury. The hamstrings remain the most commonly injured muscle group.

The epidemiology of hyperprolactinaemia over 20 years in the Tayside region of Scotland: the Prolactin Epidemiology, Audit and Research Study (PROLEARS).

OBJECTIVE: To estimate the prevalence and incidence of hyperprolactinaemia. Hyperprolactinaemia is a common problem in endocrine practice, but its epidemiology has not been accurately established. STUDY DESIGN: A population-based retrospective follow-up study in Tayside, Scotland (population 400,000), from 1993 to 2013. PATIENTS: Record linkage technology (biochemistry, prescribing, hospital admissions, radiology, mortality and maternity data) was used to identify all patients with a serum prolactin measurement. From these, cases were defined as those with a prolactin greater than 1000 mU/L (47·2 ng/ml) or at least three prescriptions for a dopamine agonist. MEASUREMENTS: Number of prevalent and incident cases of hyperprolactinaemia per calendar year by age, sex and cause of hyperprolactinaemia. RESULTS: A total of 32289 patients had a serum prolactin assay undertaken, of which 1301 had hyperprolactinaemia not related to pregnancy: 25·6% patients had pituitary disorder, 45·9% were drug-induced, 7·5% had macroprolactin and 6·1% had hypothyroidism, leaving 15·0% idiopathic. Over the 20 years, there was a fourfold increase in the number of prolactin assays performed, and prevalence of hyperprolactinaemia was initially 0·02%, but rose to 0·23% by 2013. Overall incidence was 13·8 cases per 100000 person-years (20·6 in 2008-13) and was 3·5 times higher in women than in men. The highest rates were found in women aged 25-44 years. Drug-induced causes tripled during the 20 years. CONCLUSIONS: Rising prevalence of hyperprolactinaemia is probably due to an increased ascertainment and increased incidence of psychoactive drug-related causes. Rates are higher in women than in men but only before the age of 65 years.

A comparative study of methylglyoxal metabolism in trypanosomatids.

The glyoxalase system, comprising the metalloenzymes glyoxalase I (GLO1) and glyoxalase II (GLO2), is an almost universal metabolic pathway involved in the detoxification of the glycolytic byproduct methylglyoxal to d-lactate. In contrast to the situation with the trypanosomatid parasites Leishmania major and Trypanosoma cruzi, this trypanothione-dependent pathway is less well understood in the African trypanosome, Trypanosoma brucei. Although this organism possesses a functional GLO2, no apparent GLO1 gene could be identified in the T. brucei genome. The absence of GLO1 in T. brucei was confirmed by the lack of GLO1 activity in whole cell extracts, failure to detect a GLO1-like protein on immunoblots of cell lysates, and lack of d-lactate formation from methylglyoxal as compared to L. major and T. cruzi. T. brucei procyclics were found to be 2.4-fold and 5.7-fold more sensitive to methylglyoxal toxicity than T. cruzi and L. major, respectively. T. brucei also proved to be the least adept of the 'Tritryp' parasites in metabolizing methylglyoxal, producing l-lactate rather than d-lactate. Restoration of a functional glyoxalase system by expression of T. cruzi GLO1 in T. brucei resulted in increased resistance to methylglyoxal and increased conversion of methylglyoxal to d-lactate, demonstrating that GLO2 is functional in vivo. Procyclic forms of T. brucei possess NADPH-dependent methylglyoxal reductase and NAD(+)-dependent l-lactaldehyde dehydrogenase activities sufficient to account for all of the methylglyoxal metabolized by these cells. We propose that the predominant mechanism for methylglyoxal detoxification in the African trypanosome is via the methylglyoxal reductase pathway to l-lactate.

Trypanothione-dependent glyoxalase I in Trypanosoma cruzi.

The glyoxalase system, comprizing glyoxalase I and glyoxalase II, is a ubiquitous pathway that detoxifies highly reactive aldehydes, such as methylglyoxal, using glutathione as a cofactor. Recent studies of Leishmania major glyoxalase I and Trypanosoma brucei glyoxalase II have revealed a unique dependence upon the trypanosomatid thiol trypanothione as a cofactor. This difference suggests that the trypanothione-dependent glyoxalase system may be an attractive target for rational drug design against the trypanosomatid parasites. Here we describe the cloning, expression and kinetic characterization of glyoxalase I from Trypanosoma cruzi. Like L. major glyoxalase I, recombinant T. cruzi glyoxalase I showed a preference for nickel as its metal cofactor. In contrast with the L. major enzyme, T. cruzi glyoxalase I was far less fast-idious in its choice of metal cofactor efficiently utilizing cobalt, manganese and zinc. T. cruzi glyoxalase I isomerized hemithio-acetal adducts of trypanothione more than 2400 times more efficiently than glutathione adducts, with the methylglyoxal adducts 2-3-fold better substrates than the equivalent phenylglyoxal adducts. However, glutathionylspermidine hemithioacetal adducts were most efficiently isomerized and the glutathionylspermidine-based inhibitor S-4-bromobenzylglutathionylspermidine was found to be a potent linear competitive inhibitor of the T. cruzi enzyme with a K(i) of 5.4+/-0.6 microM. Prediction algorithms, combined with subcellular fractionation, suggest that T. cruzi glyoxalase I localizes not only to the cytosol but also the mitochondria of T. cruzi epimastigotes. The contrasting substrate specificities of human and trypanosomatid glyoxalase enzymes, confirmed in the present study, suggest that the glyoxalase system may be an attractive target for anti-trypanosomal chemotherapy.

Specificity of the trypanothione-dependent Leishmania major glyoxalase I: structure and biochemical comparison with the human enzyme.

Trypanothione replaces glutathione in defence against cellular damage caused by oxidants, xenobiotics and methylglyoxal in the trypanosomatid parasites, which cause trypanosomiasis and leishmaniasis. In Leishmania major, the first step in methylglyoxal detoxification is performed by a trypanothione-dependent glyoxalase I (GLO1) containing a nickel cofactor; all other characterized eukaryotic glyoxalases use zinc. In kinetic studies L. major and human enzymes were active with methylglyoxal derivatives of several thiols, but showed opposite substrate selectivities: N1-glutathionylspermidine hemithioacetal is 40-fold better with L. major GLO1, whereas glutathione hemithioacetal is 300-fold better with human GLO1. Similarly, S-4-bromobenzylglutathionylspermidine is a 24-fold more potent linear competitive inhibitor of L. major than human GLO1 (Kis of 0.54 microM and 12.6 microM, respectively), whereas S-4-bromobenzylglutathione is >4000-fold more active against human than L. major GLO1 (Kis of 0.13 microM and >500 microM respectively). The crystal structure of L. major GLO1 reveals differences in active site architecture to both human GLO1 and the nickel-dependent Escherichia coli GLO1, including increased negative charge and hydrophobic character and truncation of a loop that may regulate catalysis in the human enzyme. These differences correlate with the differential binding of glutathione and trypanothione-based substrates, and thus offer a route to the rational design of L. major-specific GLO1 inhibitors.

Crystallization and preliminary X-ray analysis of Leishmania major glyoxalase I.

Glyoxalase I (GLO1) is a putative drug target for trypanosomatids, which are pathogenic protozoa that include the causative agents of leishmaniasis. Significant sequence and functional differences between Leishmania major and human GLO1 suggest that it may make a suitable template for rational inhibitor design. L. major GLO1 was crystallized in two forms: the first is extremely disordered and does not diffract, while the second, an orthorhombic form, produces diffraction to 2.0 A. Molecular-replacement calculations indicate that there are three GLO1 dimers in the asymmetric unit, which take up a helical arrangement with their molecular dyads arranged approximately perpendicular to the c axis. Further analysis of these data are under way.

A trypanothione-dependent glyoxalase I with a prokaryotic ancestry in Leishmania major.

Glyoxalase I forms part of the glyoxalase pathway that detoxifies reactive aldehydes such as methylglyoxal, using the spontaneously formed glutathione hemithioacetal as substrate. All known eukaryotic enzymes contain zinc as their metal cofactor, whereas the Escherichia coli glyoxalase I contains nickel. Database mining and sequence analysis identified putative glyoxalase I genes in the eukaryotic human parasites Leishmania major, Leishmania infantum, and Trypanosoma cruzi, with highest similarity to the cyanobacterial enzymes. Characterization of recombinant L. major glyoxalase I showed it to be unique among the eukaryotic enzymes in sharing the dependence of the E. coli enzyme on nickel. The parasite enzyme showed little activity with glutathione hemithioacetal substrates but was 200-fold more active with hemithioacetals formed from the unique trypanosomatid thiol trypanothione. L. major glyoxalase I also was insensitive to glutathione derivatives that are potent inhibitors of all other characterized glyoxalase I enzymes. This substrate specificity is distinct from that of the human enzyme and is reflected in the modification in the L. major sequence of a region of the human protein that interacts with the glycyl-carboxyl moiety of glutathione, a group that is conjugated to spermidine in trypanothione. This trypanothione-dependent glyoxalase I is therefore an attractive focus for additional biochemical and genetic investigation as a possible target for rational drug design.