2nd June 2017, Volume 130 Number 1456

Clinical utility of hypo and hyperpigmentation of skin in diffuse cutaneous systemic sclerosis

Kamal K Solanki,1,2 Cecil Hor,3 Winston SJ Chang,4 Christopher Frampton,5 Douglas HN White.1,2

1Rheumatology Department, Waikato Hospital, Hamilton, New Zealand, 2Waikato Clinical School, University of Auckland, New Zealand, 3Western Hospital, Melbourne, Australia, 4Ng Teng Fong Hospital, Jurong, Singapore, 5University of Otago, Christchurch, New Zealand.

Objectives

Cutaneous involvement is an early manifestation of systemic sclerosis (SSc). Localised areas of “salt and pepper skin” [S&P]) may develop. We hypothesise that S&P skin occurs frequently in dcSSc, which can be used in its early diagnosis and may correlate with joint contractures.

Methods

Sixty-five patients were recruited for this study which was approved by the Health and Disability Ethics Committee. The demographic profiles of SSc were ascertained from hospital records. These patients fulfilled the 2013 ACR/EULAR classification criteria. Patients were examined for skin pigmentary changes, mRSS, telengiectasias, calcinosis, arthritis and joint contractures and pruritis.

Findings

Sixty-five patients (59 female) were recruited with median age of 62.87 years. Forty-four had limited cutaneous systemic sclerosis, 16 diffuse cutaneous systemic sclerosis (dcSSc), five had scleroderma overlap syndrome.

Multivariate stepwise logistic regression indicated that mRSS severity and the presence of contractures were independently (p<0.05) associated with dcSSc. The strong positive association between S&P and mRSS severity may explain the non-significance of S&P in this analysis. If mRSS severity is not included in the logistic regression analysis, the presence of contractures and S&P (odds ratio=15.1) show significant (p<0.01) independent associations with the dcSSc subtype.

S&P skin and pruritis were similar in patients with Scl-70 and anti-RNA polymerase antibodies. Anti-centromere antibodies were negatively associated with the S&P (X2=7.89, p=0.005).

Conclusions

Our study demonstrates strong association of S&P skin with dcSSc (69%), increased risk of pruritis and contractures. Its presence can be used as another clinical tool to diagnose dcSSc in early stages. Observing for salt and pepper skin changes does not require much training. 

Using pluripotent stem cell-derived organoids for determining the cellular basis of hearing loss

B Forrester-Gauntlett,1,2 L Peters,2 B Oback.1

1AgResearch, Ruakura Research Centre, Hamilton, New Zealand, 2University of Waikato, School of Science, Hamilton, New Zealand.

Background

Mammalian auditory hair cells are very sensitive to damage from physical trauma, loud noises, infections and pharmaceutical drugs. Damage to the hair cells responsible for hearing is irreversible and will accumulate over time, resulting in age-related or noise-induced hearing loss. Susceptibility to damage and/or an impaired repair mechanism has been linked to genetic factors. One such gene is the grainyhead-like 2 (GRHL2) gene. Two family studies have identified mutations within GRHL2 that cause autosomal dominant non-syndromic sensorineural deafness (DFNA28). In addition, multiple mutations within this gene have been highly significantly associated with progressive hearing loss in genome wide association studies. The exact cellular mechanisms by which the hearing is lost is not yet known.

Objectives

The aim of this study is to elucidate the function of the GRHL2 gene, define its role in auditory sensory hair cell formation and maintenance, and to determine the cellular basis of DFNA28. This will be achieved by disrupting GRHL2 in pluripotent mouse embryonic stem cells (ESCs) using clustered regularly interspaced short palindromic repeats-associated enzyme Cas9 (CRISPR/Cas9) technology. These cells will then be induced to differentiate in an organotypic, three dimensional (3D) in vitro model system. Thus, this model provides an alternative to studying human hearing loss where progress has been hindered due to the location, accessibility, size and delicate nature of the sensory hair cells within in the temporal bone. Genome editing allows to precisely introduce genetic changes associated with human hearing loss into cells or animals.

Methods

Gene editing of GRHL2 was carried out using a commercial system, which consists of a pool of CRISPR/Cas9 guide RNAs (gRNAs) and homology-directed repair (HDR) plasmids targeting exons 2 and 3 within the GRHL2 gene. While gRNAs direct the Cas9 endonuclease to introduce double stranded breaks in genomic DNA, the HDR template mediates insertion of puromycin resistance and fluorescent reporter genes at the target loci. Following transfection, clonal cell strains were established by puromycin and reporter gene selection. End-point PCR was used to identify insertion of the HDR template within the targeted exons. Sequencing was carried out on the targeted exons to determine non-homologous end joining events. qPCR and western blot were used to quantify the abundance of GRHL2 mRNA and protein, respectively.

Findings

Wild-type and edited ESCs were cultured using an established 3D in vitro model system to create inner ear-like organoids (IEOs) containing sensory hair cells. Morphological differences were noted between wild-type and edited IEOs and are presently being characterised further.

Conclusions

In summary, CRISPR/Cas9 is an effective method for editing GRHL2 in ESCs and the 3D in vitro model system is an efficient tool for differentiating ESCs towards an inner ear-like morphology.

A cross-cultural comparison of general hospital specialists’ attitudes toward management of psychological/psychiatric problems

Inoka Wimalaratne,1 Graham Mellsop,2 David B Menkes.2

1Waikato District Health Board, Waikato Hospital, Hamilton, New Zealand, 2University of Auckland, Auckland, New Zealand.

Background

Psychiatric comorbidities are common in physical illness and significantly affect healthcare outcomes. Attitudes of general hospital doctors toward psychiatry are important in this regard; they influence quality of care and are shaped by cultural factors operating at multiple levels relevant to clinical interactions. Few studies have examined these attitudes and factors in the general hospital setting.

Objectives

To identify differences in attitudes toward management of psychological/psychiatric problems among general hospital specialists in relation to practice setting, individual cultural identity and other variables (gender, age, seniority, specialty).

Methods

A cross-sectional study is underway in several countries, including New Zealand, China, Netherlands, Brazil, Russia, Israel and Sri Lanka. Data are being collected by anonymous, self-administered questionnaires to senior medical staff of various disciplines working in general hospital settings (secondary and tertiary level hospitals). A sample of 100 respondents is sought from each country. Descriptive statistics will be recorded for questions and univariate comparisons will be performed using chi-squared or Fisher’s exact tests as appropriate.

Findings

Preliminary data are available from China and data collection is underway in other countries. The Chinese sample included 306 respondents, of whom half were males and a majority (59%) aged more than 30 years. Almost all respondents (99%) agreed that psychological factors play an important role in the course of physical illness; 97% thought dealing with patients’ emotional problems was part of general hospital doctors’ work and 86% agreed that general hospital doctors were responsible for emotional care of patients. However, just over half (52%) held the view that emotional care of patients by general hospital doctors was impractical under current conditions. The great majority (93%) agreed with routine assessment of patients’ psychological factors, 94% welcomed more contact with psychiatric services and 99% more help in providing psychological and social care. Respondents’ demographic characteristics or vocational status had only minimal influence on their attitudes. Female doctors were more likely to express concern about emotional care and psychological assessment of patients with chronic physical illness, while surgeons tended to confine themselves to physical assessment.

Conclusions

Preliminary results indicate widespread positive attitudes toward management of patients’ psychological/psychiatric problems among non-psychiatric doctors in China. However, results also suggest an urgent need for time and access to psychiatric services and professional support for these doctors. When available, data from other countries will enable cross-cultural comparisons and formulation of an agenda to address unmet psychological need in general hospitals.

Sensitivity of staphylococcus strains to Manuka Cyclopower

Julian Ketel,1 Lynne Chepulis,1 Ray Cursons,2 Linda Peters.2

1Department of Nursing and Health Studies, Toi Ohomai Institute of Technology, Tauranga & Rotorua, New Zealand, 2Laboratory of Molecular Genetics, University of Waikato, Hamilton, New Zealand.

Background

Staphylococcus aureus can be a significant threat to human life and wellbeing in community and acute healthcare settings and it has developed resistance to antibiotics to the extent that some strains are almost pan-resistant. Therefore, research and development of antimicrobials that can help to prevent S. aureus colonisation and/or infection is essential.

Manuka honey is known to be particularly antibacterial, largely due to its methylglyoxal content, and the development of antimicrobial resistance in S. aureus to Manuka honey has not been reported. Manuka honey has been complexed with alpha-cyclodextrin to create Manuka Cyclopower™ (MCP), creating a powdered formulation of methylglyoxal active Manuka honey that, for example, could be added to a cream base for use in the anterior nares.

Methods

A series of experiments were carried out to 1) determine the antibacterial effect of MCP against a strain of MRSA, and 2) to compare it to the source honey. Low levels (5–10% w/v) Manuka honey and MCP were incubated with MRSA for 18 hours.

Findings

When compared to the initial bacterial inoculum, 10% MCP was significantly (P<0.01) more bactericidal than the 10% Manuka honey, producing a 99.99% (log4) reduction, whereas Manuka honey produced an 81.32% (log2) reduction. Further, MCP displayed a bactericidal action whereas the honey appeared to be more bacteriostatic. Further research should be undertaken with a large number of S. aureus strains in vitro to assess whether MCP has any potential clinical value.

The efficacy and safety of antenatal glucocorticoids in late gestation (≥37 weeks) in women with diabetes in pregnancy

Carissa Murugesh,1 Sarah Waymouth,2 Manjula Ratnaweera,3 Jade AU Tamatea,1,3 Louise Wolmarans,1,3 Ryan G Paul.3

1Waikato Clinical Campus, University of Auckland, New Zealand, 2Obstetric Department, Waikato Hospital, Hamilton, New Zealand, 3Waikato Regional Diabetes Service, New Zealand.

Background

All major international guidelines recommend that antenatal glucocorticoids are administered before 35 weeks gestation to reduce neonatal morbidity and mortality. However, guidelines differ on the use of antenatal glucocorticoids in late gestation (≥37 weeks) due to discrepant data on their efficacy. In particular, it is not known whether antenatal glucocorticoids are effective and/or further increase hypoglycaemia in babies born to mothers with diabetes in pregnancy (DIP).

Objectives

To determine whether antenatal glucocorticoids reduce the rates of admission to the Neonatal Intensive Care Unit (NICU) and/or increase hypoglycaemia in babies born to women with DIP.

Methods

Data was retrospectively collected for 40 women with DIP who were administered antenatal glucocorticoids and delivered ≥37 weeks gestation at Waikato Hospital between 2011 to 2016. For a control group, data was also collected on 103 women with DIP who did not receive antenatal glucocorticoids and were matched for gestation at delivery, mode of delivery, glycaemic control and birth weight of the baby. Neonatal hypoglycaemia was defined as a blood glucose ≤2.5mmol/L within the first 24 hours post-delivery.

Results

Antenatal glucocorticoids did not significantly reduce admissions to NICU (15% versus 27%, P=0.19), or increase rates of neonatal hypoglycaemia (58% versus 42%; P=0.13), when compared with matched controls. Despite not increasing the incidence, antenatal steroids increased the severity of neonatal hypoglycaemia (mean [± S.E.M.] blood glucose 2.40±0.11mmol/L versus 2.65±0.07mmol/L; P<0.05).

Conclusions

Antenatal glucocorticoids do not appear to be effective and may increase the severity of neonatal hypoglycaemia when administered in late gestation to women with DIP. Our study suggests that caution is required until randomised control trials prove both the efficacy and safety of antenatal glucocorticoids in late gestation in women with DIP.

Pain and pupillary light reflex parameters under general anaesthesia

S McCabe, A Gaskell, J Sleigh.

Objectives

Many patients experience post-operative pain despite intraoperative analgesia. Pupil size is affected by sympathetic drive and may indicate adequacy of analgesia. This study investigates the relationships between painful stimulation during anaesthesia and pupillary light reflex parameters and whether any of the pupillary light reflex parameters at the end of surgery are predictive of early postoperative pain in the post-anaesthesia care unit.

Methods

Patients scheduled to undergo surgery requiring general anaesthesia were consented. Preoperative pain expectation and anxiety were recorded on an 11-point visual scale. Intraoperative EEG, ECG, pupillometry readings, vital signs and drug administrations were recorded throughout the surgical procedure. Pain and nausea levels and opioid requirements were measured in PACU. Pearson correlation coefficient was used for the analysis.

Results

Patients who received glycopyrolate or atropine intraoperatively (excluding glycopyrolate used in reversing muscle relaxation) were excluded from the analysis.

Expected pain correlated with maximum pain in PACU with each point increase in expected pain correlating with an increase in maximum PACU pain of 0.446 (p=0.0048), this explained 16.7% of variation. Preoperative anxiety did not correlate significantly with postoperative pain.

Opioid concentrations correlated significantly with several pupillary light reflex parameters under general anaesthesia, although notably not with pupil size. Contraction percentage increased with increasing opioid concentrations, while constriction and dilation velocities decreased with increasing opioid concentrations.

Pupillary parameters did not consistently change at the time of noxious stimuli (intubation, formation of pneumoperitoneum or incision). The maximum and minimum pupil sizes at the final intraoperative reading correlated with pain scores 15 minutes after admission to PACU, with each mm of increased diameter corresponding to an increased pain score of 1.32 (p=0.046) and 1.48 (p=0.048) respectively.

Conclusions

Pupillary light reflex parameters did not consistently change with noxious stimulation. There are many potential confounding factors, including the variable intensity of the stimuli, and the possibility of other painful stimuli or medications affecting pupillary reflexes. While absolute pupil sizes at the end of surgery did correlate with pain levels post-operatively they explained less than 10% of variation in reported pain levels at 15 minutes, and are thus in isolation unlikely to be of significant clinical utility in prompting additional analgesia at the end of surgery to reduce postoperative pain

Metastatic behaviour and outcomes of breast cancer subtypes

Melissa Edwards, Rachel Shirley, Jack Treloar, Jenni Scarlett, Ross Lawrenson, Ian Campbell, Marion Kuper-Hommel.

Background

Breast cancer patients that develop distant metastases can exhibit significant variation in the clinical course of the disease, such as the pattern of spread of metastases. This study aims to explore associations between breast cancer subtype and clinical course, including organ-specific metastases.

Methods

This research involves a retrospective observational study of data collected on 361 patients identified using the Waikato Breast Cancer Registry. Patients included were those diagnosed with early stage breast cancer between 2006–2015, who had developed metastatic disease. Tissue arrays and immunohistochemochemical staining for estrogen and progesterone receptors and HER2 were used to divide these breast cancers into the molecular subtypes of Luminal A (ER pos and PR pos, HER2 neg), Luminal B1 (ER+ or PR+, HER2 neg), Luminal B2 (ER pos and/ or PR pos, HER2 pos), HER2 enriched (ER neg & PR neg & HER2pos) and Basal-like (ER neg & PR neg & HER2 neg) for comparison.

Results

A total of 361 patients developed metastatic disease, and were subsequently included in the study. 44.3% of these were aged 50 or under at initial diagnosis, while 55.7% were over 50. 73.4% of patients were of New Zealand European ethnicity, 21.1% were of Māori ethnicity and 2.77% were of Pacific ethnicity. Of these patients, 36.3% had metastatic disease on their first presentation.
The luminal A subtype comprised 38.1% of these patients, while luminal B1 formed 22.4%, luminal B2 formed 7.2%, HER2 enriched formed 18.6%, and the basal-like subtype formed 13.6%.
Bone was the most common site of metastases in all subtypes except basal-like, where it was lung metastases.

In those of the Luminal A subtype that did not have metastatic disease on initial presentation, median time from first presentation to development of metastases was 30.3 months. The median overall survival time of this group after the development of metastases was 21.8 months. In this group, 71.5% of individuals were aged 50 or younger.
In those of the Luminal B1 subtype that did not have metastatic disease on initial presentation, median time from first presentation to development of metastases was 25.4 months. The median overall survival time of this group after the development of metastases was 9.6 months. In this group, 76.5% of individuals were aged older than 50.
In those of the Luminal B2 subtype that did not have metastatic disease on initial presentation, median time from first presentation to development of metastases was 32.7 months. The median overall survival time of this group after the development of metastases was 18.7 months. In this group, only 50% were of New Zealand European ethnicity.

In those of the HER2-enriched subtype that did not have metastatic disease on initial presentation, median time from first presentation to development of metastases was 24.7 months. The median overall survival time of this group after the development of metastases was 13.8 months.
In those of the Basal-like subtype that did not have metastatic disease on initial presentation, median time from first presentation to development of metastases was 22.5 months. The median overall survival time of this group after the development of metastases was 8.8 months. In this group, 87.8% of patients were older than 50.

Conclusion

Multiple aspects of the clinical course of the disease are noticeably influenced by the molecular subtype of breast cancer involved. The molecular subtype status of the tumours offer greater information on likely sites of metastases. The significant variation in overall survival times between subtypes, as well as in the timing of appearance of the first metastases, might also lend themselves to more individualised approaches to monitoring breast cancers and tumours that take subtype into account.