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The lipid profile is important in the risk assessment for cardiovascular disease. The lipid profile includes total cholesterol, high-density lipoprotein (HDL)-cholesterol, triglycerides (TGs) and low-density lipoprotein (LDL)-cholesterol (LDL-C). LDL-C has traditionally been calculated using the Friedewald equation (invalid with TGs greater than 4.5 mmol/L and is based on the assumption that the ratio of TG to cholesterol in very- low-density lipoprotein (VLDL) is 5 when measured in mg /dL). LDL-C can be quantified with a reference method, beta-quantification involving ultracentrifugation and this is unsuitable for routine use. Direct measurement of LDL-C was expected to provide a solution with high TGs. However, this has some challenges because of a lack of standardisation between the reagents and assays from different manufacturers as well as the additional costs. Furthermore, mild hypertriglyceridaemia also distorts direct LDL-C measurements. With the limitations of the Friedewald equation, alternatives have been derived. Newer equations include the Sampson-National Institutes of Health (NIH) equation 2 and the Martin-Hopkins equation. The Sampson-NIH2 equation was derived using beta-quantification in a population with high TG and multiple least squares regression to calculate VLDL-C, using TGs and non-HDL-C as independent variables. These data were used in a second equation to calculate LDL-C. The Sampson-NIH2 equation can be used with TGs up to 9 mmol/L. The Martin-Hopkins equation uses a 180 cell stratification of TG/non-HDL-C to determine the TG:VLDL-C ratio and can be used with TGs up to 4.5 mmol/L. Recently, an extended Martin-Hopkins equation has become available for TGs up to 9.04 mmol/L.This article discusses the best practice approach to calculating LDL-C based on the available evidence.

The reference method for low-density lipoprotein-cholesterol (LDL-C) quantitation is β-quantification, a technically demanding method that is not convenient for routine use. Indirect calculation methods to estimate LDL-C, including the Friedewald equation, have been used since 1972. This calculation has several recognized limitations, especially inaccurate results for triglycerides (TG)4.5 mmol/l (400 mg/dl). In view of this, several other equations were developed across the world in different datasets.The purpose of this review was to analyze the best method to calculate LDL-C in clinical practice by reviewing studies that compared equations with measured LDL-C.We identified 45 studies that compared these formulae. The Martin/Hopkins equation uses an adjustable factor for TG:very low-density lipoprotein-cholesterol ratios, validated in a large dataset and demonstrated to provide more accurate LDL-C calculation, especially when LDL1.81 mmol/l (70 mg/dl) and with elevated TG. However, it is not in widespread international use because of the need for further validation and the use of the adjustable factor. The Sampson equation was developed for patients with TG up to 9 mmol/l (800 mg/dl) and was based on β-quantification and performs well on high TG, postprandial and low LDL-C samples similar to direct LDL-C.The choice of equation should take into the level of triglycerides. Further validation of different equations is required in different populations.

Antibodies have proven to be central in the development of diagnostic methods over decades, moving from polyclonal antibodies to the milestone development of monoclonal antibodies. Although monoclonal antibodies play a valuable role in diagnosis, their production is technically demanding and can be expensive. The large size of monoclonal antibodies (150 kDa) makes their re-engineering using recombinant methods a challenge. Single-domain antibodies, such as “nanobodies,” are a relatively new class of diagnostic probes that originated serendipitously during the assay of camel serum. The immune system of the camelid family (camels, llamas, and alpacas) has evolved uniquely to produce heavy-chain antibodies that contain a single monomeric variable antibody domain in a smaller functional unit of 12–15 kDa. Interestingly, the same biological phenomenon is observed in sharks. Since a single-domain antibody molecule is smaller than a conventional mammalian antibody, recombinant engineering and protein expression in vitro using bacterial production systems are much simpler. The entire gene encoding such an antibody can be cloned and expressed in vitro. Single-domain antibodies are very stable and heat-resistant, and hence do not require cold storage, especially when incorporated into a diagnostic kit. Their simple genetic structure allows easy re-engineering of the protein to introduce new antigen-binding characteristics or attach labels. Here, we review the applications of single-domain antibodies in laboratory diagnosis and discuss the future potential in this area.

The gold standard for measuring LDL-C is impractical for routine use while direct measurements have numerous shortcomings. This study investigated the correlation of predictive equations with currently used enzymatic assays in populations where these have historically proven unreliable to determine whether equations may be used interchangeably. No reference measure was available for comparison in this study.We examined two analytical datasets from different platforms to evaluate the correlation of predictive equations for LDL-C (Friedewald, Sampson-NIH2, Martin-Hopkins, Extended Martin-Hopkins, Hattori and Anandaraja) with direct LDL-C assays in a large paediatric (n = 7598) and an adult cohort with uncontrolled diabetes (n = 57165). Non-parametric statistics were used for comparison.In the paediatric cohort, the Sampson-NIH2 equation correlated best with the direct LDL-C assays with the most values falling within desirable bias (35.9-44%) and TEDifferent measurement platforms influence the results of predictive equations and directly measured LDL-C. We propose utilising the Martin-Hopkins equation as an alternative to dLDL-C assays in adults with diabetes and for screening purposes in paediatric populations to avoid underestimating cardiovascular risk.

Background Many studies have assessed the predictive accuracy of serum osmolality equations. Different approaches for selecting a usable equation were compared using thirty published equations and patient data from a regional hospital laboratory. Methods Laboratory records were extracted with same-sample results for measured serum osmolality, sodium, potassium, urea and glucose analysed in a regional hospital laboratory between 1/1/2017–31/12/2018. Differences were analysed using Passing-Bablok and difference (Bland-Altman) analysis. Three approaches were compared: the shotgun approach, adjusting for bias, and deriving a novel equation using multivariate analysis. The criteria for success included bias ≤0.7%, a 230 – 400 mOsm/kg range, and osmolal gap (OG) 95% reference limits within ±10 mOsm/kg. Results The majority of equations produced proportionally negative-biased results. The shotgun approach identified two equations (EQ19, EQ6) with bias ≤0.7% but unworkable OG reference limits. The bias adjustment approach produced several equations with bias ≤ 0.7% and OG reference limits within or equivalent to ±10 mOsm/kg. A novel equation generated by us (1.89Na+ + 1.71 K+ + 1.08 Urea + 1.08 Glucose + 13.7) improved with the adjustment of bias and was not superior to the adjusted published equations. Conclusion Few published equations are immediately usable. Adjustment of bias derives several usable equations of which the best had OG ranges

This is the third in the series of historical articles dealing with developments in clinical pathology. Bence Jones proteins are immunoglobulin light chains found in excessive quantities in urine in multiple myeloma and are believed to be one of the first tumour markers ever discovered . Dr Henry Bence Jones is credited with the discovery of this protein in 1847 that bears his name and he can also be regarded as the first chemical pathologist/clinical chemist. Since then, numerous advances and refinements have been made in the measurement and detection of urine light chain proteins which have resulted in the current sensitive serum free light chain assays used today.

Background Chronic liver disease with conjugated hyperbilirubinaemia and failure to thrive can have multifactorial aetiologies. Investigations can be complex and difficult especially when obscured by a viral infection affecting liver function. Methods A 5 month old male infant was referred for investigation of chronic liver disease and a history of jaundice with multiple febrile episodes. Liver function tests were performed followed by a liver biopsy and microbiological workup for infectious disease. In addition, urine analysis of organic acids was also performed. Results There was marked conjugated hyperbilirubinaemia with markedly elevated hepatocellular enzymes and normal ductal enzymes. Proteinuria and near normal renal function suggested early renal impairment. There was also leukocytosis and bicytopenia. An extensive bacteriological investigation including TB workup was negative. CMV infection was confirmed by viral load and antibody reactivity. There was prolonged PT and PTT and high INR. The liver biopsy showed giant cell transformation of hepatocytes with mild cholestasis, portal and peri-cellular fibrosis with alpha-1-antitrypsin positive granules in the hepatocyte cytoplasm suggesting alpha-1-antitrypsin deficiency. Urine organic acids revealed significantly elevated mevalonolactone. Conclusions We confirmed the genetic diagnosis of mevalonic aciduria caused by MVK deficiency which had been masked by liver disease and the possible misdiagnosis of alpha-1-antitrypsin deficiency.

Background Functioning gonadotroph adenomas (FGAs) are rare, since most pituitary adenomas of gonadotroph origin are clinically silent or non-functioning. FGAs produce hormones that are adequate to result in clinical syndromes. Clinical case We describe a 38-year-old male who presented with pressure effects from a sellar mass manifesting as persistent and progressive headache. He also complained of swelling of the feet, excessive sweating and coarsening of facial features as well as increased libido. The patient also reported symptoms consistent with obstructive sleep apnoea. His shoe size remained the same and had no features of acromegaly. His visual fields appeared intact on informal assessment. He appeared well virilised and had bilateral macroorchidism, with both testes markedly larger than the largest measurement on the orchidometer (25ml). He was found to have elevated follicle stimulating hormone (FSH) 26.7 to 38.4 IU/L [1.3 -19.3] and testosterone levels from 28.1 to 44.3 nmol/L [6.1 - 27.1] with luteinising hormone (LH) levels being either inappropriately non-suppressed or high, 5.7 - 12.3 IU/L [1.2 - 8.6]. He also had erythrocytosis [Hb 19.5 to 21.7 g/dL (14.3 -18.3) and macroorchidism, owing to high gonadotropin levels. The rest of his pituitary axis appeared intact, including TFT, cortisol levels, and IGF-1, were all within normal limits. Although histology of the pituitary adenoma could not be obtained as the patient declined surgery, clinical and biochemical findings were supportive of FGA diagnosis. Conclusions FGAs are rare, can have none or subtle clinical features, and often present late with mass effects. Functional gonadotroph adenoma should be suspected in a patient with a sellar mass, with high or non-suppressed gonadotropins in the presence of elevated gonadal steroids. Clinical features such as macroorchidism and polycythaemia may be easily missed or misinterpreted. Surgical removal should be the therapy of choice, and if successful can lead to reversal of the biochemical and clinical abnormalities. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Increased healthcare demand has placed pressure on laboratory medicine to improve turnover and optimise efficiency using digitalisation, automation and artificial intelligence (AI). This will bring new challenges for the clinical laboratory. Laboratorians need to understand the utility of AI, its limitations and implementation. The management of big data requires ready access and accurate and contextual analysis. AI uses complex algorithms and data from medical and laboratory data to mimic human analysis and this requires accurate and reliable data. The role of AI in laboratory medicine is rapidly expanding owing to recognition of its potential to improve detection, laboratory workflows, decision support and reduce costs and increase efficiency. In this thematic issue on AI, we have attempted to present an overview of the uses of AI in laboratory medicine and advances in the digitalisation of pathology. Rakha et al 1 from the University of Nottingham and Google Health provide an erudite overview of current and future applications of AI in pathology. The applications of AI in pathology range from prognostic/predictive applications to workflow and diagnostic …

Apolipoprotein L1 (APOL1) is a protein encoded by the APOL1 gene, found only in humans and several primates. Two variants encoding two different isoforms exist for APOL1, namely G1 and G2. These variants confer increased protection against trypanosome infection, and subsequent African sleeping sickness, and also increase the likelihood of renal disease in individuals of African ancestry. APOL1 mutations are associated with increased risk of chronic kidney disease, inflammation, and exacerbation of systemic lupus erythematosus-associated renal dysfunction. This review serves to outline the structure and function of APOL1, as well as its role in several disease outcomes.

The rapidly increasing rate of antibiotic resistance is of great concern. Approximately two million deaths result annually from bacterial infections worldwide. Therefore, there is a paramount requi...

Ebola is a dangerous virus transmitted by animals and humans and to date there is no curable agent for such a deadly infectious disease. In this study, pharmacoinformatics-based methods were adopted to find effective novel chemical entities against Ebola virus. A well predictive and statistical robust pharmacophore model was developed from known Ebola virus inhibitors collected from the literature. The model explained the significance of each of hydrogen bond acceptor and donor, and two hydrophobic regions for activity. The National Cancer Institute and Asinex (Antiviral library) databases were screened using the final validated pharmacophore model. Initial hits were further screened with a set of criteria and finally eight molecules from both databases were proposed as promising anti Ebola agents. Further molecular docking and molecular dynamics studies were carried out and it was found that the proposed molecules possessed capability to interact with amino residues of Ebola protein as well as retaining equilibrium of protein-ligand systems. Finally, the binding energies were calculated using molecular mechanics Poisson-Boltzmann surface area approach and all proposed molecules showed strong binding affinity towards the Ebola protein receptor. Communicated by Ramaswamy H. Sarma.

Objectives Low-density lipoprotein cholesterol (LDL-C) estimation is critical for risk classification, prevention and treatment of atherosclerotic cardiovascular disease (ASCVD). Predictive equations and direct LDL-C are used. We investigated the comparability between the Martin/Hopkins, Sampson, Friedewald and eight other predictive equations on two analysers, to determine whether the equation or analyser influences predicted LDL-C result. Methods In two unpaired datasets, 9,995 lipid profiles were analysed by the Abbott Architect and 4,782 by the Roche Cobas analysers. Non-parametric statistics and Bland Altman plots were used to compare LDL-C. Results On the Abbott analyser; the Martin/Hopkins, Sampson and Friedewald LDL-C were comparable (median bias ≤1.8%) over a range of 1–4.9 mmol/L. On the Roche platform, Martin/Hopkins LDL-C was comparable to Friedewald (median bias 0.3%) but not to Sampson LDL-C (median bias 25%). In patients with LDL-C Conclusions Performance of predictive equations is influenced by the choice of analyser for total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and TG. Replacement of the Friedewald equation with Martin/Hopkins estimation to improve quality of LDL-C results can be safely implemented across analysers, whereas caution is advised regarding the Sampson equation.

Background Molar pregnancy is a complication characterised by abnormal benign or malignant proliferation of trophoblastic cells resulting in markedly elevated β-hCG (human chorionic gonadotrophin) levels, an established marker for the presence of the disease. Owing to the structural homology between β-hCG and TSH, the raised β-hCG can result in secondary hyperthyroidism. Methods Two patients aged 20 (Case 1) and 31 years (Case 2) presented to the emergency department within a few days of each other complaining of vaginal bleeding associated with abdominal pain. Ultrasound evaluation, β-hCG and thyroid function tests were performed on both patients. Results Both had elevated β-hCG levels and ultrasound evidence of molar pregnancy and were diagnosed with gestational trophoblastic disease (GTD) associated with hyperthyroidism based on thyroid function test results. Case 1 had lower β-hCG levels and free T4 levels compared with Case 2 but clinical assessment of the former revealed severe illness and more complicated course with the development of a thyroid storm. Case 2 had β-hCG levels almost double those of Case 1, yet was stable and her levels decreased much faster, reaching and maintaining undetectable levels. Conclusions These cases demonstrate that the β-hCG levels do not always correlate with disease severity and prognosis in patients with GTD.

The renin–angiotensin system is mainly associated with the regulation of blood pressure, but recently many other functions of this system have been described. ACE2, an 805-amino acid monocarboxypeptidase type I transmembrane glycoprotein, was discovered in 2000 and has sequence similarity to two other proteins, namely ACE and collectrin. The ACE2 gene is located on Xp22 and is highly polymorphic. ACE2 is expressed in numerous tissues especially the lung alveolar epithelial cells, heart, kidney and gastrointestinal tract. Animal studies have found that ACE2 is central in diseases affecting almost all organ systems, among other cardiac, respiratory, renal and endocrine functions. ACE2 was identified as the cellular contact point for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the global pandemic (COVID-19), and is a potential drug target. SARS-CoV-2 infection has several effects on the renin–angiotensin system and conversely, regulation of this receptor may affect the progress of infection. We describe the genetics and functions of ACE2, explore its various physiological functions in the renin–angiotensin system and discuss its role in the pathophysiology of disease. ACE2 opposes the vasopressor ACE pathway of the renin–angiotensin system by converting angiotensin (Ang) I to Ang (1–9) and Ang II to Ang (1–7) which initiates the vasodilatory pathway. ACE2 may have a protective effect in the lung and kidney as knockout mice display susceptibility to acute respiratory distress and hypertensive nephropathy. Binding of SARS-CoV-2 and the subsequent fusion and downregulation of this pathway during SARS-CoV-2 infection may explain some of the unusual sequelae seen in COVID-19.

The year 2020 has seen a major and sustained outbreak of a novel betacoronavirus (severe acute respiratory syndrome (SARS)-coronavirus (CoV)-2) infection that causes fever, severe respiratory illness and pneumonia, a disease called COVID-19. At the time of writing, the death toll was greater than 120 000 worldwide with more than 2 million documented infections. The genome of the CoV encodes a number of structural proteins that facilitate cellular entry and assembly of virions, of which the spike protein S appears to be critical for cellular entry. The spike protein guides the virus to attach to the host cell. The spike protein contains a receptor-binding domain (RBD), a fusion domain and a transmembrane domain. The RBD of spike protein S binds to Angiotensin Converting Enzyme 2 (ACE2) to initiate cellular entry. The spike protein of SARS-CoV-2 shows more than 90% amino acid similarity to the pangolin and bat CoVs and these also use ACE2 as a receptor. Binding of the spike protein to ACE2 exposes the cleavage sites to cellular proteases. Cleavage of the spike protein by transmembrane protease serine 2 and other cellular proteases initiates fusion and endocytosis. The spike protein contains an addition furin cleavage site that may allow it to be ‘preactivated’ and highly infectious after replication. The fundamental role of the spike protein in infectivity suggests that it is an important target for vaccine development, blocking therapy with antibodies and diagnostic antigen-based tests. This review briefly outlines the structure and function of the 2019 novel CoV/SARS-CoV-2 spike protein S.

Background: Gestational trophoblastic disease (GTD) represents a group of tumours caused by abnormal proliferation of trophoblastic cells, including molar pregnancy. Elevated β-hCG levels are an established marker for the presence of the disease and useful for monitoring. Due to the shared structural homology of β-hCG and TSH, hyperthyroidism can occur. Clinical Cases: We present two patients with GTD associated with hyperthyroidisim. Case 1, a 20 year old female (G1P0) presented to the emergency department complaining of vaginal bleeding associated with abdominal pain. She was estimated to be 13 weeks. Laboratory evaluation were β-hCG 648 324 IU/L, TSH 0.06 (0.35 - 4.94 mIU/L, free T4 23.2 (9.0 - 19.0 pmol/L, Hb 8.0 (11.6 - 16.4 g/dL). Ultrasound revealed molar pregnancy. She underwent uterine evacuation, thereafter complicated with thyroid storm (Burch Wartofsky score = 45). Post- operative vitals were BP 192/112, pulse rate 120 bpm and temperature 360C. She was managed in high care on labetolol, carbimazole, lugol’s iodine and hydrocortisone. She was subsequently referred to Medical Oncology for further management. Histology sample obtained in theatre confirmed complete molar pregnancy. Her staging CT scan indicated the presence of small lung nodules, suggesting metastatic disease. The patient’s FIGO/WHO score was III: 2. At the time of preparing this study, she had already received 7 weeks of methotrexate intramuscularly and still had detectable β-hCG levels. Case 2 was a 31 year old female presented similarly. This was her second pregnancy (G2P1), 12 weeks by dates. Her vitals were BP 141/74, pulse rate 110 bpm and temperature 36oC. The Ultrasound revealed larger for gestational age uterus with cystic structures in utero. Her quantitative β-hCG was significantly elevated (> 1 500 000 IU/L) she was thyrotoxic [TSH ( Her staging CT scan showed no metastasis, and had a FIGO/WHO score of 1: 4 due to her pre-treatment hCG of >1.5 million IU/L. She received 7 cycles of intramuscular methotrexate from which she achieved and maintained suppressed β-hCG levels ( Conclusions: This study has demonstrated that the β-hCG levels may not always correlate with disease severity and prognosis. When comparing the two patients Case 1 had lower levels of β-hCG and of free T4 than Case 2, however was clinically more unwell, developed thyroid storm and had metastatic disease. Case 2 had hCG levels almost double those of Case 1, wsa stable and her levels decreased much quicker reaching undetectable levels

Background The objective of the study was to investigate the effectiveness of screening for hereditary galactosaemia with Benedict’s test and thin layer chromatography (TLC) in a tertiary laboratory from a developing country. Methods We retrospectively analysed the results of tests done in suspected galactosaemia patients including Benedict’s test, thin layer chromatography, GALT activity and DNA analysis. Results 878 paediatric patients were screened with Benedict’s test; the age range was 5 days to 19 years. 48% tested positive/trace on the Benedict’s test of which 52% of these had galactosuria evident on TLC. 22% of this sample had pathologically low GALT results on follow-up. 8 patients from the screened population were confirmed to have galactosaemia, in addition to 6 more patients diagnosed with galactosaemia without screening tests performed. Median ages at which the diagnoses were made in the screened and non-screened samples were 2 months and 6 months respectively. Confirmatory DNA testing was performed in 2 patients, whom were found to be heterozygous for S135L mutation. Conclusion Inadequate performance of Benedict’s test and TLC was demonstrated by false positive and false negative results leading us to conclude that screening test results require interpretation with caution.

In this study we searched for potential β-site amyloid precursor protein cleaving enzyme1 (BACE1) inhibitors using pharmacoinformatics. A large dataset containing 7155 known BACE1 inhibitors was evaluated for pharmacophore model generation. The final model (R = 0.950, RMSD = 1.094, Q

Cervical cancer disproportionately affects low-resource countries and is a significant health burden in South Africa. Pre-clinical studies have demonstrated numerous anti-cancer actions of vitamin D metabolites. Here, the anti-cancer action of the vitamin D precursor, cholecalciferol, was investigated in a high-grade cervical cancer cell line, SiHa. SiHa cell cultures were treated with a range of cholecalciferol doses (26 nM, 104 nM, 260 nM and 2600 nM) for 72 hours. Cell count and viability were assessed by crystal violet and trypan blue assays, respectively. Apoptotic cell death was investigated by flow cytometry, which measured mitochondrial membrane potential (∆Ψm), phosphatidylserine (PS) externalisation, effector caspase activation and the expression of DNA damage markers. Additionally, brightfield microscopy and transmission electron microscopy (TEM) were respectively used to characterise morphological and ultrastructural features of apoptosis. Expression of the vitamin D metabolising system (VDMS) – consisting of cholecalciferol activating (CYP2R1 and CYP27A1), calcidiol activating (CYP27B1) and calcidiol inactivating (CYP24A1) enzymes, and the vitamin D receptor (VDR) – was assessed by qPCR and Western blots. Data were analysed using a one-way ANOVA and Bonferroni post-hoc tests and p < 0.05 was considered statistically significant. Significant decreases in cell count (p = 0.011) and cell viability (p < 0.0001) were identified in SiHa cells treated with 2600 nM cholecalciferol. Furthermore, biochemical markers at 2600 nM treatment were significant for apoptosis, and included decreased ∆Ψm (p = 0.0145); increased PS externalisation (p = 0.0439); terminal caspase activation (p = 0.0025); and nuclear damage (p = 0.004). Moreover, biochemical apoptosis was corroborated by classical apoptotic features observed by brightfield microscopy and TEM. Additionally, a significant increase in CYP2R1 gene (p < 0.0001) and protein (p = 0.021) expression, and a converse significant decrease in CYP27B1 gene (p = 0.003) and protein expression (p = 0.031) were observed at 2600 nM cholecalciferol treatment. Furthermore, significant increases in VDR gene (p = 0.033) and protein (p = 0.04) expression, and CYP24A1 gene (p < 0.0001) and protein (p = 0.0274) expression were observed at 2600 nM cholecalciferol. In summary, high-dose cholecalciferol treatment of SiHa cervical cancer cells inhibits cell growth, induces apoptosis, and furthermore, upregulates CYP2R1 and VDR expression. Taken together, these findings suggest that autocrine activation of cholecalciferol to calcidiol may mediate VDR signalling of cell growth inhibition, and apoptosis in SiHa experimental cultures.

Background Healthcare budgets face constraints, and laboratories have developed strategies to adapt to the concomitant increase in workload. Some of the tests (7.4%) may be attributed to unnecessary repeat testing. Electronic gatekeeping has been implemented at selected laboratories in South Africa to limit unnecessary repeat testing. We performed a study of chemistry tests subjected to electronic gatekeeping to determine its effectiveness as a sustainable demand management tool. Methods A 22-month retrospective study of chemistry test requests at a Pretoria hospital was performed. Tests violating electronic gatekeeping rules were rejected upon registration before analysis, and cost-savings were estimated from electronic gatekeeping-held tests. The impact of electronic gatekeeping on the test requesting pattern of clinicians was derived from the percentage cost of electronic gatekeeping-held tests. Results The total savings generated from electronic gatekeeping test rejections amounted to $84,380. Greatest savings were generated from high-cost tests: glycated haemoglobin ($14,139), urea ($8661) and thyroid-stimulating hormone ($7514). The average number of electronic gatekeeping-held tests as a percentage of their total requested number over 22 months was 3.18%. Discussion The savings from electronic gatekeeping-held tests were not as dramatic as anticipated, but were modest and may have some impact in a cost-constrained setting. Electronic gatekeeping was concluded not to have a substantial effect on the clinician test requesting pattern, demonstrated by the largely unchanged monthly percentage of electronic gatekeeping-held tests. As a solitary demand management strategy, electronic gatekeeping does not appear to be as effective as anticipated or as demonstrated in other studies.

In this study, we searched for potential DNA GyrB inhibitors using pharmacophore-based virtual screening followed by molecular docking and molecular dynamics simulation approaches. For this purpose, a set of 248 DNA GyrB inhibitors was collected from the literature and a well-validated pharmacophore model was generated. The best pharmacophore model explained that two each of hydrogen bond acceptors and hydrophobicity regions were critical for inhibition of DNA GyrB. Good statistical results of the pharmacophore model indicated that the model was robust in nature. Virtual screening of molecular databases revealed three molecules as potential antimycobacterial agents. The final screened promising compounds were evaluated in molecular docking and molecular dynamics simulation studies. In the molecular dynamics studies, RMSD and RMSF values undoubtedly explained that the screened compounds formed stable complexes with DNA GyrB. Therefore, it can be concluded that the compounds identified may have potential for the treatment of TB.

Seasonal pseudohyperkalaemia has been described in colder northern hemisphere countries. The lower temperatures may inhibit red cell Na-K-ATPase allowing the efflux of potassium and higher measured levels. It has not been described in warmer subtropical climates.The aim was to determine if seasonal variation in serum potassium occurred in a temperate climate.We conducted a retrospective review of serum potassium results over two years in two South African provinces with different microclimates and seasonal temperatures. The study included patient samples from surrounding clinics and hospitals in Pretoria, Gauteng province, and in Durban, KwaZulu-Natal province, South Africa. Average temperature ranges were obtained from the South African weather service from the same period (June 2015-June 2017).A total of 91,420 results were analysed and we found a statistically significant difference between the January (summer) and June (winter) serum potassium levels (p .0001). These results demonstrate that the winter months in South Africa are associated with significantly higher measured potassium results.Seasonal pseudohyperkalaemia may be more widespread than realized and can occur in more temperate climates and laboratories should take the appropriate action when transporting samples as this could influence interpretation and clinical management.

The rapidly increasing rate of antibiotic resistance is of great concern. Approximately two million deaths result annually from bacterial infections worldwide. Therefore, there is a paramount requirement to develop innovative and novel antibacterial agents with new mechanisms of action and activity against resistant bacterial strains. For this purpose, a set of benzothiazole and

The University of Pretoria Vice Chancellor's post-doctoral fellowship and the National Research Foundation (NRF), South Africa Innovation post-doctoral fellowship schemes.

MA Islam and TS Pillay were funded by the University of Pretoria Vice Chancellor’s post-doctoral fellowship and National Research Foundation (NRF), South Africa Innovation Post-doctoral fellowship schemes.

Acquired immunodeficiency syndrome (AIDS) is a life-threatening disease which is a collection of symptoms and infections caused by a retrovirus, human immunodeficiency virus (HIV). There is currently no curative treatment and therapy is reliant on the use of existing anti-retroviral drugs. Pharmacoinformatics approaches have already proven their pivotal role in the pharmaceutical industry for lead identification and optimization. In the current study, we analysed the binding preferences and inhibitory activity of HIV-integrase inhibitors using pharmacoinformatics. A set of 30 compounds were selected as the training set of a total 540 molecules for pharmacophore model generation. The final model was validated by statistical parameters and further used for virtual screening. The best mapped model (R = 0.940, RMSD = 2.847, Q(2) = 0.912, se = 0.498, Rpred(2) = 0.847 and rm(test)(2) = 0.636) explained that two hydrogen bond acceptor and one aromatic ring features were crucial for the inhibition of HIV-integrase. From virtual screening, initial hits were sorted using a number of parameters and finally two compounds were proposed as promising HIV-integrase inhibitors. Drug-likeness properties of the final screened compounds were compared to FDA approved HIV-integrase inhibitors. HIV-integrase structure in complex with the most active and final screened compounds were subjected to 50 ns molecular dynamics (MD) simulation studies to check comparative stability of the complexes. The study suggested that the screened compounds might be promising HIV-integrase inhibitors. The new chemical entities obtained from the NCI database will be subjected to experimental studies to confirm potential inhibition of HIV integrase.

Pharmacoinformatics approaches are widely used in the field of drug discovery as it saves time, investment and animal sacrifice. In the present study, pharmacore-based virtual screening was adopted to identify potential HIV-protease ligands as anti-HIV agents. Pharmacophore is the 3D orientation and spatial arrangement of functional groups that are critical for binding at the active site cavity. Virtual screening retrieves potential hit molecules from databases based on imposed criteria. A set of 30 compounds were selected with inhibition constant as training set from 129 compounds of dataset set and subsequently the pharmacophore model was developed. The selected best model consists of hydrogen bond acceptor and donor, hydrophobic and aromatic ring, features critical for HIV-protease inhibitors. The model exhibits high correlation (R=0.933), less rmsd (1.014), high cross validated correlation coefficient (Q(2)=0.872) among the ten models examined and validated by Fischer's randomization test at 95% confidence level. The acceptable parameters of test set prediction, such as R(pred)(2)=0.768 and r(m(test))(2)=0.711 suggested that external predictivity of the model was significant. The pharmacophore model was used to perform a virtual screening employing the NCI database. Initial hits were sorted using a number of parameters and finally seven compounds were proposed as potential HIV-protease molecules. One potential HIV-protease ligand is reportedly confirmed as an active agent for anti-HIV screening, validating the current approach. It can be postulated that the pharmacophore model facilitates the selection of novel scaffold of HIV-protease inhibitors and can also allow the design of new chemical entities.

This is the second in the series of historical articles dealing with developments in clinical pathology. As one of the most commonly measured analytes in pathology, the assessment of glucose dates back to the time of the ancient Egyptians. It was only in the 19th century that advances in chemistry led to the identification of the sugar in urine being glucose. The following century witnessed the development of more chemical and enzymatic methods which became incorporated into the modern analysers and point-of-care instruments which are as ubiquitous as the modern day cellphones. Tracking the milestones in these developments shows the striking paradigms and the many parallels in the development of other clinical chemistry methods.

As in all branches and specialties in medicine, educational activities and conferences are an essential part of training and continuous professional development and this is also mandated by statutory licensing bodies. Traditionally, industry has supported many educational activities in medicine. These have included support for congresses, workshops and other meetings. Many annual congresses and conferences rely heavily on support from both the pharmaceutical and medical technology industries. In the case of pathology, the predominant support is from the medical technology companies, as would be expected. MedTech Europe is the trade association that represents the medical technology industries including the manufacturers of diagnostic instruments and medical devices. MedTech Europe was formed in October 2012 from an alliance of European Diagnostic Manufacturer's Association, representing the European in vitro diagnostic industry and Eucomed representing the medical devices industry. MedTech Europe recently introduced a code of practice1 for companies, which is being implemented in 2018 with a transition period in 2017. After the 2017 transition period, member companies can no longer provide financial or in kind support to individual pathologists to cover costs of attendance at congresses with the exception of third-party-organised procedure training meetings or if a pathologist (or scientist) is engaged by a member of …

Background Liver dysfunction is common and often unrecognized. Liver biopsy is the gold standard in the assessment of liver fibrosis, but has disadvantages. We assessed the diagnostic accuracy of serum prolidase enzyme activity (SPA) in predicting the presence and degree of liver fibrosis, as compared with liver biopsy. Further, we evaluated the effect of hemolysis on measured SPA levels. Methods We undertook a prospective case control study. Thirty eight outpatients without apparent liver illness and 20 patients with liver pathology scheduled to undergo liver biopsy had their SPA levels measured. Results Patients undergoing liver biopsy had higher SPA levels (361 (268) IU/l [median (interquartile range)]) compared with controls (169 (160) (P < 0.001)). A SPA cutoff value of 200 IU/l yielded a sensitivity of 89%, specificity of 59%, an odds ratio of 11.5, negative predictive value of 92%, and a positive predictive value of 50%. Hemolysis causes an apparent increase in SPA levels. Conclusion Higher SPA levels in patients undergoing liver biopsies compared with controls may reflect the presence of liver fibrosis. SPA levels could not be used to stage the degree of fibrosis. SPA measurement may be useful in the diagnostic workup of suspected liver disease.

A 33-year-old woman presented for the first time at the age of 9 years with recurrent seizures and was subsequently treated for epilepsy for 2 years. At 11 years of age, it was noted that the patient had hypocalcemia with an increased parathyroid hormone (PTH)4 measured with use of an intact PTH assay. Serum calcium was 5.73 mg/dL [1.43 mmol/L; reference interval (RI) 8.82–10.42 mg/dL (2.2–2.6 mmol/L)]; serum phosphate was 10.42 mg/dL [3.36 mmol/L; RI 1.86–4.34 mg/dL (0.6–1.4 mmol/L)]; and PTH was 319.68 pg/mL [33.9 pmol/L; RI 8.49–68.84 pg/mL (0.9–7.3 mmol/L)]. Vitamin D (Total 25-OH; 25-OH D2 and D3) concentrations, magnesium concentrations, liver and renal function tests were all normal. No other endocrine abnormalities such as thyroid or gonadotropin resistance were detected. The patient also was of short stature and was overweight. However, she became overweight after the age of 13 years (body mass index at 13 years was 22.8 kg/m2), and the short stature was not significant, as she was at the lower end of the RI for height at 1.55 m. There was no cognitive impairment. Positive Chvostek sign was observed but not Trousseau sign. The patient was placed on active vitamin D (alfacalcidol) and calcium supplements. The patient was followed up for 13 years; selected laboratory results are presented in Table 1. Although the patient was managed on calcium supplements and active vitamin D and followed up at a tertiary endocrine clinic, serum phosphate concentrations remained persistently high (Table 1). This was attributed to intermittent noncompliance and unavailability of active vitamin D. The patient had also been prescribed calcium carbonate as a phosphate binder. View this table: Table 1. Trends of calcium, phosphate, and parathyroid hormone concentrations over 13 years of follow-up.a ### QUESTIONS TO CONSIDER 1. What are the causes of hypocalcemia with an increased PTH concentration? 2. What is the explanation for …

Salivary cortisol has been used to monitor hydrocortisone replacement in patients with Addison’s disease (AD). Since salivary cortisol is metabolised to salivary cortisone, it may be an adjunctive analyte to assess adequacy of hydrocortisone replacement in patients with AD. We aimed to characterise the exposure of salivary cortisol and cortisone in patients and healthy controls. We measured salivary cortisol and cortisone by liquid chromatography-tandem mass spectrometry and constructed a day curve (08:00 until 24:00 h) with 16 time points in 25 AD patients taking their usual hydrocortisone dose and in 26 healthy controls. The median (interquartile range) area under the curve (AUC) for cortisol was not different for patients, compared with controls [55.63 (32.91–151.07) nmol*min*l –1 vs. 37.49 (27.41–52.00) nmol*min*l –1 ; p=0.098, respectively], whereas the peak cortisol C max was higher in patients [32.61 (5.75–146.19) nmol/l vs. 8.96 (6.96–12.23) nmol/l; p=0.013], compared with controls. The AUC for cortisone [23.65 (6.10–54.76) nmol*min*l –1 vs. 227.73 (200.10–280.52) nmol*min*l –1 ; p≤ 0.001, respectively], and peak cortisone C max was lower in patients than in controls [11.11 (2.91–35.85) nmol/l vs. 33.12 (25.97–39.95) nmol/l; p=0.002]. The AUC for salivary cortisol and salivary cortisone were not correlated with any measures of hydrocortisone dose. The time-course and AUC of salivary cortisol were similar between Addison’s patients and healthy controls. Patients had substantially lower salivary cortisone AUC, compared to healthy controls. Salivary cortisol AUC and pharmacokinetics were not related to hydrocortisone dose and thus are not likely useful markers for the adequacy of hydrocortisone replacement.

BackgroundUncertainty exists whether glucocorticoid receptor (GCR) polymorphisms play a role in steroid-related side effects in Addison's disease (AD) patients on hydrocortisone. The polymorphismsBcll and N363S appear to increase sensitivity to cortisol, while the ER22/23EK polymorphism has been associated with resistance to cortisol.MethodOne hundred and forty seven AD patients, and gender, and ethnicity-matched controls were recruited in South Africa. Three polymorphisms in the GCR were studied, using PCR followed by restriction fragment length analysis. Associations with BMI, lipids, glucose and inflammatory markers were investigated.ResultsIn both patients and controls, theBcll polymorphism occurred more frequently in whites than in other ethnic groups studied but was not associated with any of the metabolic parameters tested. The ER22/23EK polymorphism was associated with an increased BMI in both patients (29.4 vs 24.7 kg/m2) and control subjects (26.3 vs 24.2 kg/m2). The ER22/23EK polymorphism was also associated with lower LDL cholesterol in control subjects (3.46 vs 3.93 mmol/l) and in patients (3.52 vs 4.10 mmol/l). N363S was associated with increased BMI in controls 29.9 kg/m2vs wild type 24.8 kg/m2. Median hydrocortisone doses were greater in patients heterozygous for either ER22/23EK 30.0 mg or N363S 25.0 mg polymorphisms than in wild type patients 20.0 mg (both comparisons).ConclusionAlterations in lipids, BMI and hydrocortisone dose were associated with two polymorphisms. Further larger studies are warranted to corroborate these findings.

Many laboratories manage point-of-care services either in their hospital or in their wider locality in association with primary care commissioners. As a result, they tend to be asked to contribute to assessment of the acceptability of new point-of-care systems from the point of view of accuracy and comparability to current systems. This is particularly the case for glucose testing strips, which currently make up 53.7% of the total global point-of-care testing (POCT) market.1 In testing whether systems are acceptable for clinical use, it is usual for ‘pragmatic’ testing regimes to be used. A fully correct ISO 15197:2013 evaluation2 is difficult because it requires collection of capillary blood samples from volunteers with a wide range of blood glucose concentrations, meaning that many healthy individuals and many patients with diabetes mellitus need to be recruited. It is not only difficult to find suitable volunteers but it is also difficult to simultaneously test a wide range of POCT analysers because multiple lancet punctures are necessary as one puncture site rarely stays open long enough for multiple blood drops to be collected. Moreover, there is inherent variability in capillary blood samples collected from fingerprink sites, compounding the problem even further.3 Finally, the use of live volunteers means that regulatory approval for the study would be required from a Research …

Background : Transcription factor 7-like 2 gene (TCF7L2), fat mass and obesity-associated gene (FTO), and ectonucleotide pyrophosphatase/phosphodiesterase gene (ENPP1) are known risk loci for type 2 diabetes (T2DM) mostly in European populations. Objectives : To assess the association of these genes with T2DM risk in a South African mixed-ancestry population. Methods : Five hundred and sixty six participants were genotyped for ENPP1-rs997509 and -rs1044498, FTO-9941349 and -rs3751812, TCF7L2-rs12255372 and -rs7903146 polymorphisms using Taqman genotyping assays and validated by automated sequencing to assess the association of the polymorphisms with cardiometabolic traits. Results : In logistic regression models adjusted for age, sex, body mass index (BMI) and insulin resistance, minor allele of rs997509 was associated with a higher risk of prevalent T2DM under a recessive model [odd ratio 4.60 (95% confidence interval: 1.07 to 19.86); p = 0.040].Under additive model, the rs7903146 [1.43 (1.00 to 2.04); p= 0.053] and rs9941349 [1.43 (1.00 to 2.04); p = 0.052] minor alleles showed marginally significant associations with a high risk of T2DM. However, only the rs7903146 alleles (p=0.011) and genotypes (p=0.025) distributions were statistically significantly different between diabetic and non-diabetic individuals. Conclusion : Our findings demonstrate that ENPP1, TCF7L2, and FTO may predispose to T2DM in the mixed-ancestry population. Keywords : Type 2 diabetes, genetics, ENPP1, TCF7L2, FTO, Africa

Several naturally occuring mutations in the human insulin gene are associated with diabetes mellitus. The three known mutant molecules, Wakayama, Los Angeles and Chicago were evaluated using molecular docking and molecular dynamics (MD) to analyse mechanisms of deprived binding affinity for insulin receptor (IR). Insulin Wakayama, is a variant in which valine at position A3 is substituted by leucine, while in insulin Los Angeles and Chicago, phenylalanine at positions B24 and B25 is replaced by serine and leucine, respectively. These mutations show radical changes in binding affinity for IR. The ZDOCK server was used for molecular docking, while AMBER 14 was used for the MD study. The published crystal structure of IR bound to natural insulin was also used for MD. The binding interactions and MD trajectories clearly explained the critical factors for deprived binding to the IR. The surface area around position A3 was increased when valine was substituted by leucine, while at positions B24 and B25 aromatic amino acid phenylalanine replaced by non-aromatic serine and leucine might be responsible for fewer binding interactions at the binding site of IR that leads to instability of the complex. In the MD simulation, the normal mode analysis, rmsd trajectories and prediction of fluctuation indicated instability of complexes with mutant insulin in order of insulin native insulin insulin Chicago insulin Los Angeles insulin Wakayama molecules which corresponds to the biological evidence of the differing affinities of the mutant insulins for the IR.

Using salivary cortisol (SC) measurements, cortisol exposure in Addison's disease patients on hydrocortisone replacement was determined and compared with healthy controls. Cortisol pharmacokinetics was assessed in 31 patients with Addison's disease on replacement hydrocortisone doses (median daily dose 20 mg; range 5-50 mg) and 30 healthy control subjects. Saliva samples (n=16) were collected between 08:00 and 00:00 h in 1 day, using a passive drool technique. Cortisol exposure was evaluated by noncompartmental approach. In the patients, cortisol exposure was significantly higher than in controls: median inter-quartile range (IQR) peak cortisol (C(max)) 174.5 (59.3-837.0) vs. 6.50 (4.7-19.3) nmol/l, p=0.0001; area under the curve (AUC) 390.1 (177.1-928.9) vs. 21.4 (14.6-28.4) minutes*nmol/l, p=0.0001, trough cortisol level (C(min)) 0.49 (0.49-0.96) vs. 0.49 (0.49-0.49) nmol/l, p=0.02, occurring at 480.0 (0.1-660.0) vs. 405.0 (180.0-570.0) min, p=0.56. First peak cortisol was 174.5 (53.0-754.7) vs. 6.27 (3.90-8.47) nmol/l, p=0.0001 and second peak cortisol 18.90 (5.22-76.9) vs. 3.12 (1.76-4.79) nmol/l, p=0.0001. The time to first peak cortisol differed between the 2 groups, 30 (30-75) vs. 0.1 (0.1-30) minutes; p=0.0001. At doses studied, hydrocortisone replacement therapy results in cortisol pharmacokinetics being markedly different from endogenous cortisol profiles in healthy control subjects. Addison's disease patients had significantly higher SC levels compared to healthy control subjects.