Ethical Challenges in Clinical Research During the COVID-19 Pandemic.

The sudden emergence of the COVID-19 pandemic brought global disruption to every aspect of society including healthcare, supply chain, the economy, and social interaction. Among the many emergent considerations were the safety and public health of the public, patients, essential workers, and healthcare professionals. In certain locations, clinical research was halted-or terminated-in deference to the immediate needs of patient care, and clinical trials focusing on the treatment and prevention of coronavirus infection were prioritized over studies focusing on other diseases. Difficult decisions were made rapidly; flexibility and reconsideration were necessary not only because the intensity and severity of infection varied over time and by locale but also because knowledge of the disease and understanding of its treatment (and prevention) grew. Here we discuss the ethical challenges in decision-making and competing ethical tensions during the pandemic in an effort to advance future preparedness.

Capture-recapture to estimate completeness of tuberculosis surveillance in two communities in South Africa.

BACKGROUND: Reliable surveillance is essential for any tuberculosis (TB) control programme; however, under-registration of TB cases due to under-notification of patients on treatment or failure to initiate treatment has been well-documented internationally. OBJECTIVE: To determine the contribution of capture-recapture methods in estimating the completeness of bacteriologically confirmed pulmonary TB registration in two high-incident communities in South Africa. METHODS: Record linkage between the TB treatment register and two laboratory sputum TB result registers and three-source log-linear capture-recapture analysis. RESULTS: The number of bacteriologically confirmed pulmonary TB cases in the TB treatment register was 243, with an additional 63 cases identified in the two laboratory databases, resulting in 306 TB cases. The observed completeness of the TB treatment register was 79%. The log-linear model estimated 326 (95%CI 314-355) TB cases, resulting in an estimated completeness of registration of 75% (95%CI 68-77). CONCLUSION: Capture-recapture can be useful in evaluating the completeness of TB control surveillance and registration, including in resource-limited settings; however, methodology and results should be carefully assessed. Interventions are needed to increase the completeness of registration and to reduce the number of initial defaulters.

Accuracy and completeness of recording of confirmed tuberculosis in two South African communities.

BACKGROUND: Although tuberculosis (TB) treatment registers and laboratory records are essential tools for recording and reporting in TB control programmes, the accuracy and completeness of routinely collected data are seldom monitored. OBJECTIVE: To assess the accuracy and completeness of TB treatment register data in two South African urban communities using record linkage. METHODS: All cases of bacteriologically confirmed TB, defined as two smear-positive results and/or at least one culture-positive result, were included. Record linkage was performed between three data sources: 1) TB treatment registers, 2) the nearest central laboratory, and 3) the referral hospital laboratory. RESULTS: The TB treatment registers had 435 TB cases recorded, of which 204 (47%) were bacteriologically confirmed. An additional 39 cases recorded as non-bacteriological cases in the TB treatment registers were reclassified as bacteriologically confirmed, and 63 bacteriologically confirmed cases were identified from the laboratory databases that were not recorded in the TB treatment registers. The final number of bacteriologically confirmed TB cases was 306, giving an increase of 50%. CONCLUSIONS: The accuracy and completeness of the TB treatment register and central laboratory data were inadequate. A high percentage of bacteriologically confirmed cases from both laboratories were not recorded in the TB treatment registers. We are developing an electronic result management system to improve the management of laboratory results.

Application of stereolithographic custom models for studying the impact of biofilms and mineral precipitation on fluid flow.

Here we introduce the use of transparent experimental models fabricated by stereolithography for studying the impacts of biomass accumulation, minerals precipitation, and physical configuration of flow paths on liquid flow in fracture apertures. The internal configuration of the models ranged in complexity from simple geometric shapes to those that incorporate replicated surfaces of natural fractures and computationally derived fracture surfaces. High-resolution digital time-lapse imaging was employed to qualitatively observe the migration of colloidal and soluble dyes through the flow models. In this study, a Sphingomonas sp. and Sporosarcina (Bacillus) pasteurii influenced the fluid dynamics by physically altering flow paths. Microbial colonization and calcite deposition enhanced the stagnant regions adjacent to solid boundaries. Microbial growth and calcite precipitation occurred to a greater extent in areas behind the fabricated obstacles and less in high-velocity orifices.

Cloning of a human cDNA encoding a novel enzyme involved in the elongation of long-chain polyunsaturated fatty acids.

The Saccharomyces cerevisiae protein ELO2p is involved in the elongation of saturated and monounsaturated fatty acids. Among several sequences with limited identity with the S. cerevisiae ELO2 gene, a consensus cDNA sequence was identified from the LifeSeq(R) database of Incyte Pharmaceuticals, Inc. Human liver cDNA was amplified by PCR using oligonucleotides complementary to the 5' and 3' ends of the putative human cDNA sequence. The resulting full-length sequence, termed HELO1, consisted of 897 bp, which encoded 299 amino acids. However, in contrast with the ELO2 gene, expression of this open reading frame in S. cerevisiae demonstrated that the encoded protein was involved in the elongation of long-chain polyunsaturated fatty acids, as determined by the conversion of gamma-linolenic acid (C(18:3, n-6)) into dihomo-gamma-linolenic acid (C(20:3, n-6)), arachidonic acid (C(20:4, n-6)) into adrenic acid (C(22:4, n-6)), stearidonic acid (C(18:4, n-3)) into eicosatetraenoic acid (C(20:4, n-3)), eicosapentaenoic acid (C(20:5, n-3)) into omega3-docosapentaenoic acid (C(22:5, n-3)) and alpha-linolenic acid (C(18:3, n-3)) into omega3-eicosatrienoic acid (C(20:3, n-3)). The predicted amino acid sequence of the open reading frame had only 29% identity with the yeast ELO2 sequence, contained a single histidine-rich domain and had six transmembrane-spanning regions, as suggested by hydropathy analysis. The tissue expression profile revealed that the HELO1 gene is highly expressed in the adrenal gland and testis. Furthermore, the HELO1 gene is located on chromosome 6, best known for encoding the major histocompatibility complex, which is essential to the human immune response.

Identification and characterization of an enzyme involved in the elongation of n-6 and n-3 polyunsaturated fatty acids.

The enzymes that are involved in the elongation of fatty acids differ in terms of the substrates on which they act. To date, the enzymes specifically involved in the biosynthesis of polyunsaturated fatty acids have not yet been identified. In an attempt to identify a gene(s) encoding an enzyme(s) specific for the elongation of gamma-linolenic acid (GLA) (18:3n-6), a cDNA expression library was made from the fungus Mortierella alpina. The cDNA library constructed in a yeast expression vector was screened by measuring the expressed elongase activity [conversion of GLA to dihomo-GLA (20:3n-6)] from an individual yeast clone. In this report, we demonstrate the isolation of a cDNA (GLELO) whose encoded protein (GLELOp) was involved in the conversion of GLA to dihomo-GLA in an efficient manner (60% conversion). This cDNA contains a 957-nucleotide ORF that encodes a protein of 318 amino acids. Substrate specificity analysis revealed that this fungal enzyme acted also on stearidonic acid (18:4n-3). This report identifies and characterizes an elongase subunit that acts specifically on the two Delta6-desaturation products, 18:3n-6 and 18:4n-3. When this GLELO cDNA was coexpressed with M. alpina Delta5-desaturase cDNA in yeast, it resulted in the conversion of GLA to arachidonic acid (20:4n-6) as well as the conversion of stearidonic acid to eicosopentaenoic acid (20:5n-3). Thus, this GLELO gene may play an critical role in the bio-production of both n-6 and n-3 polyunsaturated fatty acids.

cDNA cloning and characterization of human Delta5-desaturase involved in the biosynthesis of arachidonic acid.

Two human expressed sequence tag (EST) cDNA sequences with identity with Delta(5)- and Delta(6)-desaturases from a filamentous fungus, Mortierella alpina, were identified from the LifeSeq(R) database of Incyte Pharmaceuticals, Inc. (Palo Alto, CA, U.S.A.). An oligonucleotide complementary to the 3' EST cDNA sequences was used to screen human liver cDNA using rapid amplification of cDNA ends (RACE)-PCR. The amplified DNA fragment had 98% identity with a putative open reading frame (ORF) predicted from a human genomic sequence, and encoded 444 amino acids. Expression of this ORF in mouse fibroblast cells demonstrated that the encoded protein was a Delta(5)-desaturase, as determined by the conversion of dihomo-gamma-linolenic acid (C(20:3,n-6)) into arachidonic acid (C(20:4,n-6)). The human Delta(5)-desaturase contained a predicted N-terminal cytochrome b(5)-like domain, as well as three histidine-rich domains. A tissue expression profile revealed that this gene is highly expressed in fetal liver, fetal brain, adult brain and adrenal gland. A search of the existing databases led to localization of this ORF within a 14 kb interval flanked by the flap endonuclease-1 (FEN1) and vitelliform macular dystrophy (Best's disease; VMD2) loci of chromosome 11q12.

Polyunsaturated fatty acid-specific elongation enzymes.

We have isolated a novel gene (GLELO) from Mortierella alpina and its homologue (CEELO1) from Caenorhabditis elegans and demonstrate the involvement of their encoded proteins in the elongation of C(18) polyunsaturated fatty acids.

Constraints facing the female medical practitioner in private family practice in the Western Cape.

OBJECTIVES: To assess the existence and extent of employment-related problems facing female family practitioners in the context of a rapidly growing number of female doctors in South Africa. SUBJECTS AND METHODS: A descriptive survey was conducted using bilingual questionnaires. These were posted to all 280 female family practitioners in private practice in the Western Cape. RESULTS: Of the 280 questionnaires posted 169 were returned, but 45 of these were missampled. A response rate of 53% was obtained. The largest age category was 30-39 years. Of those not in solo practice, 68 (75%) were able to negotiate the terms of their working hours, 13 (19%) negotiated sick leave on commencing work, and only half had paid leave. Vacation leave was negotiated by 34 (50%), while only 6 (9%) discussed maternity leave with employers or colleagues. Of the 124 practices included in the survey, 6 (5%) had formal arrangements to cope with maternity leave. One hundred and seven respondents (86%) felt there was a need for maternity leave guidelines in the private sector in South Africa. Regarding practice-related problems, 33 female family practitioners (27%) reported some incidents of sexual harassment by patients. Despite these constraints, 88 respondents (71%) planned to continue working in this field. CONCLUSION: Definite obstacles exist in private family practice with regard to working conditions, in particular the lack of national regulations regarding maternity leave and the absence of legislation on pregnancy discrimination. This has important implications for the inclusion of female doctors in group practices and managed health care organisations--private primary health care of the present and future!

Distribution of S(-)-zacopride-insensitive [125I]R(+)-zacopride binding sites in the rat brain and peripheral tissues.

Increasing evidence indicates that the 5-HT3 receptor antagonist R(+)-zacopride labels an additional site in brain tissue that is not sensitive to 5-HT (non-5-HT R(+)-zacopride site, R(+)-site). Since the levels of R(+)-sites in the brain are relatively low, the present studies explored the use of [125I]R(+)-zacopride to label the R(+)-site; the incorporation of an [125I] atom considerably increasing the specific activity of the radioligand relative to [3H]R(+)-zacopride that has been utilised previously. Competition experiments with [125I]R(+)-zacopride (1.0 nM) binding to rat whole brain homogenates, in the presence of the 5-HT3 receptor antagonist granisetron (1.0 microM), identified that R(+)-zacopride and prazosin bound to two sites (pIC50: 7.59 and 5.28, respectively, for R(+)-zacopride; 6.75 and 4.42, respectively, for prazosin) whereas S(-)-zacopride and mianserin possessed relatively low affinity (pIC50: 4.37 and 3.80, respectively) while (-)sulpiride and 5-HT failed to compete for [125I]R(+)-zacopride binding at concentrations up to 10 microM. Autoradiographic radioligand binding studies using [125I]R(+)-zacopride (0.5 nM) identified a heterogeneous distribution of specific binding sites (defined by unlabelled R(+)-zacopride, 1.0 microM) throughout the rat brain. In the presence of a saturating concentration of granisetron (1.0 microM), highest levels of specific [125I]R(+)-zacopride, binding sites (defined by R(+)-zacopride, 1.0 microM; R(+)-site), were detected in the olfactory tubercle, thalamus, corpus callosum, colliculus, dorsal and median raphe nucleus, spinal cord and the pons (8.0-13.0 fmol/mg). Moderate densities of R(+)-sites were located in the striatum, nucleus accumbens, substantia nigra, ventral tegmental area, globus pallidus, septal nuclei, frontal cortex and cerebellum (2.0-7.9 fmol/mg). In the hippocampus, amygdala and cortical areas. R(+)-site levels were low but detectable (0.1-1.9 fmol/mg). [125I]R(+)-zacopride labelled R(+)-sites were also detected in some rat peripheral tissues, for instance kidney cortex, adrenal gland and liver (2.4-6.8 fmol/mg). The present results indicate that specific non-5-HT [125I]R(+)-zacopride sites are heterogeneously distributed throughout the rat brain and are expressed in various peripheral tissues.

Autoradiographic distribution of [3H]-(S)-zacopride-labelled 5-HT3 receptors in human brain.

Autoradiographic binding studies using the 5-HT3 (5-hydroxytryptamine3) receptor radioligand, [3H]-(S)-zacopride (0.5 nM), identified a heterogeneous distribution of specific binding sites (defined by granisetron, 1 microM) throughout the human brain. Highest radiolabelled 5-HT3 receptor densities were detected in discrete nuclei within the brainstem (nucleus tractus solitarius, area postrema, spinal trigeminal nerve nucleus; 50-200 fmol/mg tissue equivalent) with more modest levels of expression in the forebrain (e.g. hippocampus, nucleus accumbens, putamen, caudate; 4-17 fmol/mg tissue equivalent). Within the hippocampal formation, radiolabelled 5-HT3 receptors were differentially distributed with highest levels in the granule cell layer of the dentate gyrus. Saturation studies with [3H]-(S)-zacopride (0.05-16 nM; non-specific binding defined by granisetron, 10 microM) binding to homogenates of human putamen indicated that [3H]-(S)-zacopride (0.05-16 nM; non-specific binding defined by granisetron, 10 microM) binding to homogenates of human putamen indicated that [3H]-(S)-zacopride labelled an apparently homogenous population of binding sites (Bmax = 72 + 7 fmol mg-1 protein, pKd = 8.69 +/- 0.09, Hill coefficient = 0.99 +/- 0.06, mean +/- SEM, n = 4). The pharmacological profile of [3H]-(S)-zacopride binding to homogenates of putamen indicated the selective labelling of the human variant of the 5-HT3 receptor. The marked differences, however, in the pharmacology (e.g. low affinity for D-tubocurarine) and relative distribution (e.g. presence of 5-HT3 receptors in the human extrapyramidal system) of 5-HT3 receptors in the human forebrain when compared with other species further necessitates caution in predicting clinical responses based on data generated in animal models of disease.

Barriers to compliance with OBRA'90 regulations in community pharmacies.

OBJECTIVE: To identify which barriers have been most significant to community pharmacists in their ability to comply with the Omnibus Budget Reconciliation Act of 1990 (OBRA'90) regulations during its first year of implementation. METHODS: Mailing of a two-page survey in April 1994. SETTING: Four hundred randomly selected community pharmacies in the Commonwealth of Massachusetts. PARTICIPANTS: Of 400 pharmacies surveyed, 156 surveys were completed and returned for analysis. RESULTS: Barriers that were considered most significant to pharmacies surveyed in their ability to implement OBRA'90 regulations were excessive workload, lack of financial compensation, and patients' attitudes. Of least significance were inadequate knowledge about drugs, inadequate references, and store layout. Almost half the responding pharmacists indicated that OBRA'90 regulations had not affected or changed their practice, one-quarter of the pharmacists believed their practice was less rewarding after OBRA'90, and about one-fifth believed it was more rewarding. CONCLUSIONS: Community pharmacists in Massachusetts are making an attempt to comply with OBRA'90, but there are specific barriers that are affecting their ability to do so. The OBRA'90 regulations appear to have had little impact on the practice of most community pharmacies. Community pharmacy management needs to examine (1) expanded roles of supportive personnel to give pharmacists more time to spend counseling patients, (2) reimbursement mechanisms for cognitive service, and (3) approaches to educating patients about these changes in the pharmacy profession.

Regulation of glutamate release by presynaptic kainate receptors in the hippocampus.

Most reported actions of kainate are mediated by AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) receptors. Here we report that, unlike AMPA which stimulates, kainate elicits a dose-dependent decrease in L-glutamate release from rat hippocampal synaptosomes and also depresses glutamatergic synaptic transmission. Brief exposure to kainate inhibited Ca(2+)-dependent [3H]L-glutamate release by up to 80%. Inhibition was reversed by kainate antagonists but not by the AMPA-selective non-competitive antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466). A corresponding reversible kainate-evoked depression of NMDA (N-methyl-D-aspartate) receptor-mediated excitatory postsynaptic currents (e.p.s.cs) was observed when AMPA receptors were blocked by GYKI 52466. The synaptic depression was preceded by a brief period of enhanced release and a small inward current was also observed. The effects of kainate were unaffected by metabotropic glutamate (mGlu), GABAA, GABAB, glycine and adenosine receptor antagonists. These results indicate that glutamate release can be modulated directly by kainate autoreceptors.