Stability of anticholinergic load in Australian community-dwelling older people: a longitudinal analysis.

BACKGROUND: It is recommended that anticholinergic medication is avoided in older people, especially those with cognitive impairment. OBJECTIVE: To investigate anticholinergic load (ACL) over time in older primary care patients with and without cognitive impairment. METHODS: Community-dwelling general practice patients at baseline (n = 1768), at year one (n = 1373) and a restricted cohort (with possible or definite cognitive impairment) at year two (n = 370) had medication regimens documented by a research nurse during a home visit. Anticholinergic medicines were categorized as levels 1-3 (low-high potency) and summed for each participant as a measure of their ACL. RESULTS: Most participants had no change in ACL over time, but there was some turnover in the anticholinergic medications used. The mean change in ACL was 0.012 ± 0.99 from baseline to 12 months and -0.04 ± 1.3 from baseline to 24 months. Cardiovascular drugs were the most commonly used level 1 anticholinergics, followed by antidepressants and opioids. Antidepressants and urologicals were the most commonly used level 3 anticholinergics. The rate of anticholinergic deprescribing was equivalent to the rate of anticholinergic initiation, and commonly involved the level 1 drugs warfarin, furosemide and temazepam, and the level 3 drugs amitriptyline and oxybutynin. People with dementia had a higher ACL at baseline and year one compared with other participants. CONCLUSION: ACL of community-dwelling older people was very stable over time. This may represent lost opportunities for deprescribing as well as potentially inappropriate prescribing, particularly in those with cognitive impairment.

An evaluation of the additional benefit of population screening for dementia beyond a passive case-finding approach.

OBJECTIVE: General practitioners (GPs) fail to identify more than 50% of dementia cases using the existing passive case-finding approach. Using data from the "Ageing in General Practice" study, we sought to establish the additional benefit of screening all patients over the age of 75 for dementia beyond those patients already identified by passive case-finding. METHOD: Patients were classified as "case-finding" (n = 425) or "screening" (n = 1006) based on their answers to four subjective memory related questions or their GP's clinical judgement of their dementia status. Cognitive status of each patient was formally assessed by a research nurse using the Cambridge Cognition Examination (CAMCOG-R). Patients then attended their usual GP for administration of the GP assessment of Cognition (GPCOG) dementia screening instrument, and follow-up care and/or referral as necessary in light of the outcome. RESULTS: The prevalence of dementia was significantly higher in the case-finding group (13.6%) compared to the screening group (4.6%; p < 0.01). The GPCOG had a positive predictive value (PPV) of 61% in the case-finding group and 39% in the screening group; negative predictive value was >95% in both groups. GPs and their patients both found the GPCOG to be an acceptable cognitive assessment tool. The dementia cases missed via case-finding were younger (p = 0.024) and less cognitively impaired (p = 0.020) than those detected. CONCLUSION: There is a very limited benefit of screening for dementia, as most people with dementia could be detected using a case-finding approach, and considerable potential for social and economic harm because of the low PPV associated with screening.

Screening for Dementia in Primary Care: A Comparison of the GPCOG and the MMSE.

BACKGROUND/AIMS: The General Practitioner Assessment of Cognition (GPCOG) is a brief cognitive test. This study compared the GPCOG to the Mini-Mental State Examination (MMSE), the most widely used test, in terms of their ability to detect likely dementia in primary care. METHODS: General practitioners across three states in Australia recruited 2,028 elderly patients from the community. A research nurse administered the GPCOG and the MMSE, as well as the Cambridge Examination for Mental Disorders of the Elderly Cognitive Scale-Revised that we used to define likely dementia. RESULTS: Overall, the GPCOG and the MMSE were similarly effective at detecting likely dementia. The GPCOG, however, had a higher sensitivity than the MMSE when using published cutpoints. CONCLUSION: The GPCOG is an effective screening tool for dementia in primary care. It appears to be a viable alternative to the MMSE, whilst also requiring less time to administer.

Predictors of agreement between general practitioner detection of dementia and the revised Cambridge Cognitive Assessment (CAMCOG-R).

BACKGROUND: Dementia is a complex and variable condition which makes recognition of it particularly difficult in a low prevalence primary care setting. This study examined the factors associated with agreement between an objective measure of cognitive function (the revised Cambridge Cognitive Assessment, CAMCOG-R) and general practitioner (GP) clinical judgment of dementia. METHODS: This was a cross-sectional study involving 165 GPs and 2,024 community-dwelling patients aged 75 years or older. GPs provided their clinical judgment in relation to each of their patient's dementia status. Each patient's cognitive function and depression status was measured by a research nurse using the CAMCOG-R and the 15-item Geriatric Depression Scale (GDS), respectively. RESULTS: GPs correctly identified 44.5% of patients with CAMCOG-R dementia and 90% of patients without CAMCOG-R dementia. In those patients with CAMCOG-R dementia, two patient-dependent factors were most important for predicting agreement between the CAMCOG-R and GP judgment: the CAMCOG-R score (p = 0.006) and patient's mention of subjective memory complaints (SMC) to the GP (p = 0.040). A higher CAMCOG-R (p < 0.001) score, female gender (p = 0.005), and larger practice size (p < 0.001) were positively associated with GP agreement that the patient did not have dementia. Subjective memory complaints (p < 0.001) were more likely to result in a false-positive diagnosis of dementia. CONCLUSIONS: Timely recognition of dementia is advocated for optimal dementia management, but early recognition of a possible dementia syndrome needs to be balanced with awareness of the likelihood of false positives in detection. Although GPs correctly agree with dimensions measured by the CAMCOG-R, improvements in sensitivity are required for earlier detection of dementia.

Diagnosis and disclosure of a memory problem is associated with quality of life in community based older Australians with dementia.

BACKGROUND: Identification of factors associated with quality of life (QoL) in people having dementia will help develop strategies for maintenance and improvement of patient QoL. This study examined the predictors of QoL in a community-dwelling population aged 75 years and over, with or without dementia. METHODS: This was a cross-sectional study involving 169 GPs and 2,028 patients. Patients were interviewed to collect information on personal circumstances. Several instruments were administered including the WHOQOL-BREF (quality of life outcome measure), Geriatric Depression Scale, GPAQ (satisfaction with GP care), and the CAMCOG-R (cognitive function). Patients with a CAMCOG-R score < 80 were allocated to the dementia group. GPs provided an independent clinical judgment of cognitive function for each of their participating patients. RESULTS: The dementia group had significantly lower QoL scores in all four domains of the WHOQOL-BREF (all p ≤ 0.002). The GDS score was negatively correlated with all four domains in the non-dementia group and with physical, psychological, and environmental QoL in the dementia group (all p < 0.001). Satisfaction with GP communication was positively associated with psychological QoL in the dementia group and all domains in the non-dementia group. Participants in the dementia group who had been given a diagnosis of a memory problem had significantly higher physical (2.05, 95% CI 0.36 to 3.74) and environmental (2.18, 95% CI 0.72 to 3.64) QoL. CONCLUSIONS: Satisfaction with GP communication is associated with a higher QoL in their older patients. Diagnosis and disclosure of memory problems is associated with better QoL in people with dementia. Clinicians should not be deterred from discussing a memory diagnosis and plans for the future with patients.

Ageing in general practice (AGP) trial: a cluster randomised trial to examine the effectiveness of peer education on GP diagnostic assessment and management of dementia.

BACKGROUND: Dementia is increasing in prevalence as the population ages. An earlier rather than later diagnosis allows persons with dementia and their families to plan ahead and access appropriate management. However, most diagnoses are made by general practitioners (GPs) later in the course of the disease and are associated with management that is poorly adherent to recommended guidelines. This trial examines the effectiveness of a peer led dementia educational intervention for GPs. METHODS: The study is a cluster randomised trial, conducted across three states and five sites. All GPs will complete an audit of their consenting patients aged 75 years or more at three time points - baseline, 12 and 24 months. GPs allocated to the intervention group will receive two educational sessions from a peer GP or nurse, and will administer the GPCOG to consenting patients at baseline and 12 months. The first education session will provide information about dementia and the second will provide individualised feedback on audit results. GPs in the waitlist group will receive the RACGP Guidelines by post following the 12 month audit OUTCOMES: Primary outcomes are carer and consumer quality of life and depression. Secondary outcomes include: rates of GP identification of dementia compared to a more detailed gold standard assessment conducted in the patient's home; GP identification of differential diagnoses including reversible causes of cognitive impairment; and GP referral to specialists, Alzheimers' Australia and support services. A "case finding" and a "screening" group will be compared and the psychometrics of the GPCOG will be examined. SAMPLE SIZE: Approximately 2,000 subjects aged 75 years and over will be recruited through approximately 160 GPs, to yield approximately 200 subjects with dementia (reducing to 168 by 24 months). DISCUSSION: The trial outlined in this paper has been peer reviewed and supported by the Australian National Health and Medical Research Council. At the time of submission of this paper 2,034 subjects have been recruited and the intervention delivered to 114 GPs. TRIAL REGISTRATION: Australia and New Zealand Clinical Trials Register (ANZCTR): ACTRN12607000117415.

Impact of multiple low-level anticholinergic medications on anticholinergic load of community-dwelling elderly with and without dementia.

BACKGROUND: Elderly people, particularly those with dementia, are sensitive to adverse anticholinergic drug effects. This study examines the prevalence of anticholinergic medication, and anticholinergic load and its predictors, in community-dwelling elderly patients (aged 75 years and older) in Australia. METHODS: A research nurse visited the home of each participant (n = 1,044), compiled a list of current medications, and assessed participants' cognitive status using a subsection of the revised Cambridge Examination for Mental Disorders of the Elderly (CAMCOG-R). Anticholinergic load was determined for each patient using the Anticholinergic Drug Scale (ADS). RESULTS: Multivariate analysis identified several patient factors that were associated with higher anticholinergic burden, including polypharmacy (i.e. taking five or more medications) (p < 0.001), increasing age (p = 0.018), CAMCOG-R dementia (p = 0.003), depression (p = 0.003), and lower physical quality of life (p < 0.001). The dementia group (n = 86) took a significantly higher number of medications (4.6 vs. 3.9; p = 0.04), and had a significantly higher anticholinergic load (1.5 vs. 0.8; p = 0.002) than those without dementia (n = 958). Approximately 60% of the dementia group and 40% of the non-dementia group were receiving at least one anticholinergic drug. This difference was due to the higher proportion of dementia patients taking level 1 (potentially anticholinergic) (p = 0.002) and level 3 (markedly anticholinergic) (p = 0.005) drugs. CONCLUSIONS: There is considerable scope for the improvement of prescribing practices in the elderly, and particularly those with dementia. Importantly, level 1 anticholinergics have been identified as major contributors to the anticholinergic load in people with dementia. Longitudinal studies are required to determine the effects of increased and decreased anticholinergic load on cognitive function and other clinical outcomes for people with dementia.

Comparison of glucose metabolism in in vivo- and in vitro-matured tammar wallaby oocytes and its relationship to developmental potential following intracytoplasmic sperm injection.

Although marsupial oocytes undergo nuclear maturation in vitro, there is, at present, no indication of their developmental potential, largely owing to the lack of in vitro fertilisation and related technologies for marsupials. Glucose metabolism has proven a useful indicator of oocyte cytoplasmic maturation and developmental potential in several eutherian species. Therefore, the aims of the present study were to compare: (1) the rates of glycolysis and glucose oxidation in immature, in vitro-matured and in vivo-matured tammar wallaby oocytes; and (2) the metabolic rate of individual oocytes with their ability to form pronuclei after intracytoplasmic sperm injection. The rates of glycolysis measured in immature (2.18 pmol oocyte(-1) h(-1)), in vitro- matured (0.93 pmol oocyte(-1) h(-1)) and in vivo-matured tammar wallaby oocytes (0.54 pmol oocyte(-1) h(-1)) were within a similar range to values obtained in eutherian species. However, unlike the trend observed in eutherian oocytes, the glycolytic rate was significantly higher in immature oocytes compared with either in vivo- or in vitro-matured oocytes (P < 0.001) and significantly higher in in vitro-matured oocytes compared with in vivo-matured oocytes (P < 0.001). No relationship was identified between glucose metabolism and the developmental capacity of oocytes after intracytoplasmic sperm injection when assessed after 17-19 h. Oocytes that became fertilised (two pronuclei) or activated (one or more pronucleus) were not distinguished from others by their metabolic rates. Longer culture after intracytoplasmic sperm injection (e.g. blastocyst stage) may show oocyte glucose metabolism to be predictive of developmental potential; however, culture to the single-cell stage did not reveal any significant differences in normally developing embryos.

Timing and ultrastructure of events following intracytoplasmic sperm injection in a marsupial, the tammar wallaby (Macropus eugenii).

The aim of the present study was to determine the timing of oocyte activation, sperm decondensation and pronucleus formation after intracytoplasmic sperm injection (ICSI) in the tammar wallaby and to determine the fate of sperm structures at an ultrastructural level. Metaphase II-stage tammar wallaby oocytes were injected with spermatozoa and cultured for 1 (n = 15), 2 (n = 24), 4 (n = 30), 6 (n = 14), 8 (n = 32), 10 (n = 25), 12 (n = 29) or 19 h (n = 12). Oocytes were assessed using light, fluorescence and electron microscopy. The timing of oocyte activation and sperm decondensation after ICSI in the tammar wallaby is relatively similar to that of some eutherian species. Resumption of meiosis II was observed from 1 h and the first female pronucleus was seen 6 h after ICSI. Most oocytes (88%) possessed a female pronucleus by 10 h. Intact acrosomes persisted with intact sperm heads up to 2 h after ICSI. At 10 h, 80% of oocytes possessed a male pronucleus. The sperm tail had undergone considerable degeneration by 10 h after ICSI, including breakdown of the fibrous sheath dense fibres. The identification of sperm tail and midpiece remnants adjacent to pronuclei confirms that the events observed in wallaby oocytes after ICSI are not due to parthenogenetic activation.

Immunocontraception of Eastern Grey kangaroos (Macropus giganteus) with recombinant brushtail possum (Trichosurus vulpecula) ZP3 protein.

This study examined the potential of a recombinant marsupial zona pellucida 3 protein as a contraceptive vaccine for the Eastern Grey kangaroo, a marsupial that is locally overabundant in several regions of eastern Australia. First, a pilot study using porcine zona pellucidae (PZP) demonstrated that ZP proteins, primarily the ZP3 component of PZP, are highly immunogenic in the grey kangaroo and produce a long-lasting humoral response to a single immunisation, as found in other marsupials. Immunisation with 300 microg of a non-glycosylated recombinant brushtail possum ZP3 (recBP-ZP3) protein in complete Freund's adjuvant produced a similar, significant and sustained antibody response, and none of the immunised kangaroos (n=7) produced offspring during the following breeding season compared with four out of the six control animals. An epitope analysis of the B-cell response to recBP-ZP3 using a brushtail possum ZP3 identified numerous B-cell epitope regions clustered around the N- and C-terminal regions of the protein. Two regions of interest for further fertility vaccine development based on their immunogenicity and fertility trials and functional studies in other species were found to be immunogenic. These results suggest that immunocontraception based on targeting the ZP3 protein within the zona pellucida may be an effective strategy for fertility reduction in Eastern Grey kangaroos.

The immune response and fertility of koalas (Phascolarctos cinereus) immunised with porcine zonae pellucidae or recombinant brushtail possum ZP3 protein.

To evaluate the potential contraceptive effect of immunisation with zona pellucida antigens, 50 free-ranging koalas were immunised with either porcine zonae pellucidae (PZP), recombinant brushtail possum ZP3 (recBP-ZP3) or buffer, in complete Freund's adjuvant. A single booster immunisation in incomplete Freund's adjuvant was administered 3-5 months later. Where possible animals were recaptured, reproductive status was assessed and blood was collected at 1-3-month intervals for the next 33 months. Forty-three koalas were recaptured at least three times allowing reliable assessments of their fertility. Fourteen animals were observed never to have a pouch young. Of the remaining 29 animals the reproductive productivity of PZP treated females was reduced compared with control and recBP-ZP3 treated females, in terms of both total number of young produced, and failure to produce further young in females of proven fertility. One month after the initial immunisation, serum antigen-specific antibody titres were higher in animals immunised with PZP or recBP-ZP3 compared to controls, and reached a plateau by 4 months. Antibody against the relevant immunising antigen was also detected in ovarian follicular fluid, uterine fluid and vaginal secretions. Epitope analysis suggested that immune responses other than antibodies directed against the ZP3 amino acid sequence were responsible for mediating infertility. The results demonstrate that the fertility of female koalas can be compromised by immunisation against zona pellucida antigens. However, unlike in the eastern grey kangaroo and the brushtail possum, immunisation with bacterial recombinant brushtail possum ZP3 did not compromise fertility in the koala.

Towards a ZP-based contraceptive for marsupials: comparative analysis and developmental expression of marsupial ZP genes.

Fertility control in the form of a zona pellucida (ZP)-based immunocontraceptive has shown potential as a humane form of control for overabundant marsupials including the brushtail possum and macropods. Further refinement and development of a ZP-based vaccine requires detailed knowledge of the protein structure and expression in order to ensure maximum efficacy and specificity. Sequencing and comparative analysis of the ZP3 protein from three marsupial orders in this study found a high overall level of conservation; within order Diprotodontia, the ZP3 protein is 86.9-98.9% identical. ZP3 identity falls to 56.6-57.2%, when the grey, short-tailed opossum (a Didelphimorphian) is compared to dasyurid and diprotodontan marsupials. This is similar to its amino acid identity with ZP3 from eutherian species (50.7-52.8%). Comparison of a 21 amino acid epitope in marsupial ZP3 that has shown contraceptive effects, reveals 95-100% identity between the four macropodid species, 81-86% amino acid identity between brushtail possum and the macropods and 67-71% identity between the diprotodontans and the fat-tailed dunnart (a dasyurid). This is comparable to the level of identity between related eutherian mammals. The expression pattern of three ZP genes during brushtail possum and tammar wallaby pouch young development was examined by RT-PCR. This analysis of ZP gene expression has confirmed that ZP mRNA transcription begins in the ovary during pouch young development by about 51 days of age. The presence of ZP transcripts at this stage in pouch young development suggests that marsupial ZP gene transcription begins before the onset of follicular development.

Fertilization following intracytoplasmic sperm injection of in vivo and in vitro matured oocytes from an Australian marsupial, the tammar wallaby (Macropus eugenii).

Conventional in vitro fertilization (IVF) techniques have been unable to produce normal embryos in any Australian marsupial, largely owing to problems with the early stages of sperm-oocyte binding. This study has used intracytoplasmic sperm injection (ICSI) of in vivo and in vitro matured tammar wallaby oocytes to bypass these processes and achieve fertilization in vitro. The fertilization rate (i.e. development to the two-pronuclei stage) of in vivo and in vitro matured oocytes following ICSI and sham injection was assessed at 17-19 h after injection. Fertilization occurred in 48% (45/93) of in vivo matured oocytes that were injected with spermatozoa. Significantly fewer sham-injected oocytes (6/82, P < 0.005) and uninjected control oocytes (5/84, P < 0.005) formed two pronuclei. In a direct comparison, the numbers of in vivo and in vitro matured oocytes that formed two pronuclei after ICSI were 22/28 (78.6%) and 23/40 (57.6%), respectively, which are not significantly different. There was also no significant difference in the nuclear response of in vivo and in vitro matured oocytes to sham injection. The numbers of oocytes forming a single pronucleus after sham injection were 10/24 (41.7%) and 24/37 (64.9) for in vivo and in vitro matured oocytes, respectively. Immature germinal-vesicle-stage oocytes were unable to decondense sperm injected during ICSI or to form pronuclei. These results demonstrate that both in vitro and in vivo matured tammar wallaby oocytes can be fertilized by ICSI. The success of ICSI not only offers the opportunity for fundamental analysis of marsupial fertilization but could, in conjunction with development of appropriate culture conditions and embryo transfer technologies, contribute to increased production of offspring from rare or valuable marsupials.

Expression in Escherichia coli and immunological characterization of three zona pellucida proteins (ZP1, ZP2, and ZP3) from a marsupial, the brushtail possum (Trichosurus vulpecula).

The brushtail possum (Trichosurus vulpecula) zona pellucida (ZP) is composed of three major glycoproteins, designated ZP1, ZP2, and ZP3 based on their size and homology with eutherian ZP proteins. These proteins are candidate antigens for the development of an immunocontraceptive vaccine to control the fertility of the brushtail possum in New Zealand, where it is an introduced pest. In order to further their immunological and functional characterization, recombinant possum ZP proteins were produced in Escherichia coli (E. coli) strain JM109, M15, SG13009, or BL21 codon plus. Each of the proteins produced possessed a N-terminal six histidine tag (His)(6) to facilitate purification and consisted of amino acid (aa) residues 18-471 of possum ZP1, aa residues 40-311 of ZP2 (ZP2-N), aa residues 305-634 of ZP2 (ZP2-C), and aa residues 23-342 of ZP3. Immunoblot using anti-RGS(His)(4) antibodies and polyclonal rabbit anti-porcine ZP antibodies detected major bands at 54 kDa for ZP1, 32 kDa for ZP2-N, 39 kDa for ZP2-C, and 40 kDa for ZP3. Immunization of male and female rabbits with ZP2-N, ZP2-C, and ZP3 purified on Ni-NTA resin under denaturing conditions generated antibodies reactive with recombinant ZP proteins on Western blot and with native ZP proteins in possum ovarian sections using immunofluorescence. Antibodies generated against ZP1 in the same way were reactive with recombinant ZP proteins on Western blot only. The recombinant possum ZP proteins and specific antibodies produced in this study give an indication of the antigenic relationship of the possum ZP proteins and are vital tools for future studies of sperm-ZP binding in marsupials and for the evaluation of ZP-based contraceptive vaccines in possums and other marsupials.

In vitro and in vivo maturation of oocytes from gonadotrophin-treated brushtail possums.

The time course of nuclear maturation of oocytes was examined in brushtail possums, Trichosurus vulpecula. Oocytes were recovered from ovarian follicles > 2 mm in diameter after pregnant mares' serum gonadotrophin/porcine luteinizing hormone (PMSG/LH) treatment (in vivo matured) or 72 hr after PMSG treatment (in vitro matured). Oocytes recovered from small (< 2 mm) and large (> 2 mm) follicles were also assessed for their ability to mature in vitro. Staining with the DNA-specific dye Hoechst 33342 was used to assess the stage of nuclear development by fluorescence microscopy. The process of nuclear maturation progressed rapidly in vivo, as oocytes collected at 20-27 hr post-LH all had a GV, but by 28-29.5 hr post-LH approximately a third of eggs were MII. By 30-hr post-LH, more than 70% of oocytes had reached MII stage and all ovulated eggs were MII. In vitro, all oocytes were at germinal vesicle stage at the start of culture. After 24 hr of culture, 67% of oocytes had progressed to metaphase I/anaphase I of meiosis. After 36 hr, 25% of oocytes had completed maturation to metaphase II, increasing to 52% after 48 hr. Maturation of oocytes after 48 hr in culture was unaffected by the presence or absence of granulosa cells, PMSG or LH/porcine follicle stimulating hormone (FSH). More oocytes from large follicles (55%) completed maturation by 48 hr than from small follicles (15%). The potential of oocytes to mature after 48 hr in culture was dependent on the follicle harvested having reaching a critical diameter of 1.5 mm.