Intraocular Pressure Lowering Is Associated with an Increase in the Photopic Negative Response (PhNR) Amplitude in Glaucoma and Ocular Hypertensive Eyes

Abstract

Purpose.: The aim of our study was to determine whether IOP lowering in glaucomatous and ocular hypertensive (OHT) eyes leads to an improvement in the full-field photopic negative response (PhNR) of the electroretinogram.

Methods.: A prospective nonrandomized interventional cohort study was conducted. Patients with OHT or glaucomatous optic neuropathy were recruited, and photopic full-field electroretinograms (ERG) were performed at baseline and then repeated 1 to 2 months later. The change in PhNR amplitude was compared between those eyes that had a significant lowering in IOP (defined as >25% decrease from baseline or to a predetermined target IOP) during follow-up and those that did not.

Results.: From a cohort of 30 eyes, 18 eyes had a significant reduction in IOP during follow-up (n = 18) and 12 eyes had no significant change in IOP (<25% reduction in IOP, n = 12). A significant increase in PhNR amplitude and the PhNR/b-wave amplitude ratios was observed in the reduced IOP group, but not in the IOP stable group for the two flash intensities used (2.25 and 3.00 cd.s/m²).

Conclusions.: The full-field PhNR amplitude provides a potentially reversible measure of inner retinal function that improves after IOP lowering. Further study now is required to assess its use as a measure of optic nerve health in glaucoma patients.

Introduction

Glaucoma is a disease characterized by the loss of retinal ganglion cells (RGCs). Before the death of RGCs in glaucoma, it is feasible that there is a loss of RGC function, which may be reversible with the reduction of IOP. The ability to detect any improvement in RGC function as IOP is reduced is useful clinically. The photopic negative response (PhNR) is the negative response of the photopic electroretinogram (ERG) that follows the b-wave. The PhNR amplitude is associated with cone-related RGC function. Intravitreal injection of tetrodotoxin, a voltage-gated sodium (Na⁺) channel antagonist that blocks inner retinal cell function (principally of RGCs), completely eliminated the PhNR. ¹ Loss of PhNR also has been observed in a primate glaucoma model ¹ and in several clinical cohorts of glaucoma patients. ^2–10 Full-field PhNR amplitude is correlated with the mean deviation of the visual field and with retinal nerve fiber layer (RNFL) thickness, ⁴ suggesting that the PhNR amplitude of the full-field ERG provides a relative measure of the RGC function in human eyes. The improvement of RGC function with the lowering of IOP has been shown previously using the pattern electroretinogram (PERG). ^11–14 The PhNR, in contrast, has not been investigated extensively, although a prior cross-sectional study demonstrated that treated ocular hypertensive (OHT) patients had greater PhNR amplitudes than a group of untreated OHT patients. ⁷ To our knowledge, however, no studies have been conducted to assess the impact of IOP lowering on PhNR in the same eyes. Hence, the purpose of our study was to determine whether IOP lowering in glaucomatous or OHT eyes with elevated IOP alters PhNR amplitude. 

Methods

Patients

Patients with OHT and glaucoma were recruited from the glaucoma clinic at the Royal Victorian Eye and Ear Hospital, Melbourne. Only eyes with visual acuity 6/12 or better, clear media, and no coexisting retinal disease were chosen. The diagnosis of OHT was made on the basis of elevated IOP (>21 mm Hg), no glaucomatous optic neuropathy, and no visual field defect. The diagnosis of glaucoma was made on the basis of rim and RNFL loss with corresponding visual field defect. 

The research was conducted after ethical approval by the Royal Victorian Eye and Ear Hospital Ethics Committee, and under the tenets of the Declaration of Helsinki. Informed consent was obtained from all subjects after a full explanation of the procedures involved in the study. 

Recording of PhNR

All PhNR recordings were conducted by an observer who was masked to the diagnosis and IOP status. The PhNRs were elicited by a brief red flash stimuli of 2.25 and 3.00 cd.s/m² delivered through a Ganzfeld sphere on a blue background of 20 cd/m². These settings were similar to those reported previously by other groups. ¹⁵ An Espion system (ColorDome Diagnosys LLC, Redwood City, CA) was used for stimulus generation and data acquisition. Pupils were dilated with tropicamide 0.5% and patients were light adapted to background light for 5 minutes before recording ERGs. Recordings were made with Dawson, Trick, and Litzkow (DTL) fiber electrodes in the fornix of the eye following topical anesthesia with proparacaine 0.5%. The ground electrode was attached to the forehead and the reference electrode was attached to the lateral canthus. At least 3 reliable recordings were obtained for each stimulus intensity. The lower light intensity stimulus was tested before testing at the higher light intensity. The PhNR was measured after the a-, b-, and i-wave in each case. The PhNR amplitude was recorded from the baseline to the trough of the negative response following the i-wave (Fig. 1). The a-wave amplitude was measured from the baseline to the trough of the a-wave. The b-wave amplitude was measured from the trough of the a-wave to the peak of the b-wave as described by Machida et al. ⁴ The PhNR amplitude also was expressed as a ratio of the PhNR to b-wave amplitude to reduce intersession variability and increase the specificity of the effect to the inner retinal function. ¹⁶ The photopic full-field ERG was repeated 1 to 2 months later by the same masked observer. 

Figure 1

 

Diagram of a full-field ERG trace, showing a-wave (a), b-wave (b), i-wave (i), and photopic negative response (PhNR) amplitudes.

Visual Field Analyses

The Humphrey Field Analyser (Model 740; Carl Zeiss Meditec, Inc., Dublin, CA) central 24-2 threshold (white-on-white) test was performed on all patients before pupil dilation. The mean deviation (MD) represented the mean depression of the visual field sensitivity compared to age-matched controls. 

Quantitative Assessment of RNFL Thickness

Average RNFL thickness was measured with the Cirrus optical coherence tomograph (OCT; Cirrus HD-OCT 4000; Carl Zeiss Meditec, Inc.) after full dilation of pupils. 

Statistical Analysis

The significance of the differences within a subgroup was determined by Student's two-tailed t-test for paired data and differences between groups were determined by Student's t-test for unpaired data. The variances within each group were not significantly different between groups. For comparisons between three groups the one-way ANOVA with Tukey's multiple comparison test was used. A P value <0.05 was considered statistically significant. 

Results

We recruited 24 eyes with open angle glaucoma (OAG), 4 eyes with chronic primary angle closure glaucoma (PACG), and 5 OHT eyes (total 33 eyes). Study participants ranged in age from 37 to 83 years (with a mean ± SD age of 67 ± 12 years). Eyes were grouped into 2 groups on the basis of whether or not IOP reduced by >25% or to predetermined target IOP during follow-up (Fig. 2). Treatment was added or changed for 25 eyes. Antihypertensive drops (latanoprost 0.005%, bimatoprost 0.03%, timolol 0.25%, brimonidine 0.2%/timolol 0.5% [Combigan], and/or brinzolamide 1%) were added in 11 (44%) eyes, drops (brinzolamide 1%) added, and selective laser trabeculoplasty (SLT) in 1 (4%) eye, SLT alone in 1 (4%) eye, trabeculectomy in 9 (36%) eyes, insertion of a glaucoma drainage device in 1 (4%) eye, bleb needling in 1 (4% ) eye, and removal of intraluminal suture from a glaucoma drainage device in 1 (4%) eye. Successful lowering of IOP by >25% or to predetermined target IOP occurred in 20 eyes. Two patients had IOP lowered in both eyes by similar levels and one patient had stable IOP in both eyes. Only 1 eye of each patient was incorporated in each group, with the selected eye being the one with the least severity of glaucoma (on basis of mean deviation of visual field). In addition, we recorded the PhNR in 20 eyes of 20 individuals (aged 29–77 years, mean 56 ± 12 years) who had no ophthalmic disease to determine the characteristics of a normal PhNR. 

Figure 2

 

Flow chart showing the establishment of the two groups for analysis; the IOP reduced group (n = 18 eyes) and the IOP stable group (n = 12 eyes).

The IOP reduced group (18 eyes of 18 patients) all had IOP reduction by >25% (mean reduction 47%, range 25%–84%) except for 1 eye that attained a predetermined target IOP with an 18% reduction in IOP. The IOP stable group (12 eyes of 12 patients) had eyes that did not have significant lowering of IOP (<25% reduction, mean reduction 6%, range −14%–+5%) with treatment (5 eyes), together with eyes that did not have a change in treatment and did not have a change in IOP (7 eyes). Nine patients had one eye included in each subgroup. No patients had cataract extractions during the period of follow-up. The patient characteristics are shown in Table 1. The ages of patients and severity of glaucoma, in terms of vertical cup-to-disc ratio (CDR), average RNFL thickness, and MD, were similar in both groups. 

Table 1. 

 

Patient Characteristics

	IOP Stable (n = 12 Eyes)	IOP Reduced (n = 18 Eyes)	Significance
Age, y	69 ± 2	67 ± 3	0.7329
Vertical CDR	0.76 ± 0.05	0.78 ± 0.04	0.8122
Av RNFL thickness, μm	74.4 ± 5.1	72.5 ± 3.3	0.7293
Visual field MD, dB	−6.54 ± 2.05	−6.66 ± 1.80	0.9637
Change in IOP, mm Hg	−0.3 ± 0.7	−14.2 ± 2.7	<0.0001*
Follow-up, m	1.5 ± 0.3	1.6 ± 0.2	0.7492

 Data shown as mean ± SEM. Statistical significance by two-tailed Student's t-test for unpaired data with Welch's correction.

*  A significant difference between groups.

The mean baseline IOP of the IOP stable group was 15.5 ± 2.1 (median 14.5, range 6–28) mm Hg and the follow-up IOP in this group was 15.3 ± 1.9 (median 15.0, range 6–24) mm Hg. The change of IOP in the IOP stable group was a mean of −0.3 ± 0.7 (median −1, range −4–+4, P = 0.7184) mm Hg. The IOP in the reduced IOP group was decreased significantly by a mean of −14.2 ± 2.7 (median −10, range −3 to −52, P < 0.0001) mm Hg from a mean of 27.2 ± 2.4 (median 25, range 17–62) mm Hg to a mean of 13.1 ± 1.0 (median 14, range 6–20) mm Hg. The methods by which the IOP was reduced in the IOP reduced group are shown in Table 2. The relationship between the change in IOP during follow-up and the average IOP for each eye in each group is shown in Figure 3 (the outlier with a 52 mm Hg reduction in IOP was excluded). 

Figure 3

 

Scatter plot showing the relationship between the change in IOP during follow-up and the average IOP for each eye in each group (after the outlier with a 52 mm Hg IOP reduction was excluded).

Table 2. 

 

Methods of IOP Reduction in the IOP Reduced Group

Method of IOP Reduction	n of Eyes, n = 18	Mean IOP Reduction, mm Hg
Addition of drops	7 (39%)	−7.4 ± 1.3
Trabeculectomy	7 (39%)	−21.0 ± 5.8
Addition of drops + SLT	1 (6%)	−8.0
SLT	1 (6%)	−9.0
Bleb needling	1 (6%)	−14.0
Removal of intraluminal suture from glaucoma drainage device	1 (6%)	−25.0

 Data shown as mean ± SEM.

Electrophysiology

The mean latencies of the PhNR peak amplitude were 76.8 ± 1.1 and 77.2 ± 1.0 ms at stimulus intensities of 2.25 and 3.00 cd.s/m², respectively. To determine the range of normal PhNR amplitudes, we additionally investigated a group of 20 eyes of 20 control patients with no ophthalmic disease (mean IOP of 16.0 ± 0.9 mm Hg and mean vertical CDR of 0.37 ± 0.04), with recorded mean PhNR amplitudes of 27.4 ± 3.5 and 26.7 ± 2.7 μV, and mean PhNR/b-wave amplitude ratios of 0.319 ± 0.035 and 0.311 ± 0.035 at stimulus intensities of 2.25 and 3.00 cd.s/m², respectively (Table 3). The eyes with glaucoma (28 eyes) had mean PhNR amplitudes of 12.7 ± 1.6 and 14.4 ± 1.7 μV, and mean PhNR/b-wave amplitude ratios of 0.134 ± 0.016 and 0.146 ± 0.015 at 2.25 and 3.00 cd.s/m² stimulus intensities, respectively. The OHT eyes (5 eyes) had mean PhNR amplitudes of 18.5 ± 2.8 and 20.3 ± 4.1 μV, and mean PhNR/b-wave amplitude ratios of 0.269 ± 0.051 and 0.270 ± 0.050 at 2.25 and 3.00 cd.s/m² stimulus intensities, respectively. The a- and b-wave amplitudes were similar in all three groups at both stimulus intensities (one-way ANOVA with Tukey's multiple comparison test). 

Table 3. 

 

Comparison of PhNR Amplitude and PhNR/b-Wave Amplitude Ratios for Control, OHT, and Glaucoma Eyes

	Stimulus Intensity, cd.s/m2	Control Eyes (n = 20 Eyes)	OHT Eyes (n = 5 Eyes)	Glaucoma Eyes (n = 28 Eyes)
PhNR amplitude, μV	2.25	27.4 ± 3.5	18.5 ± 2.8	12.7 ± 1.6
	3.00	26.7 ± 2.7	20.3 ± 4.1	14.4 ± 1.7
PhNR/b-wave amplitude ratio	2.25	0.319 ± 0.035	0.269 ± 0.051	0.134 ± 0.016
	3.00	0.311 ± 0.035	0.270 ± 0.050	0.146 ± 0.015

 Data shown as mean ± SEM.

The PhNR Amplitude and the PhNR/b-Wave Amplitude Ratio Increased with Reduction of IOP

The reduction of IOP by >25% or to a predetermined target IOP in glaucomatous and OHT eyes (IOP reduced group, 18 eyes) significantly increased the PhNR amplitude by a mean +5.9 ± 2.4 μV (from 11.5 ± 1.7–17.4 ± 1.9 μV, mean 51.8% increase, P = 0.023) and +5.3 ± 2.3 μV (from 13.2 ± 2.1–18.5 ± 2.4 μV, mean 40.2% increase, P = 0.035) at 2.25 and 3.00 cd.s/m² stimulus intensities, respectively (Table 4). An example of the improvement in PhNR amplitude following reduction of IOP is shown in Figures 4A, 4B. The reduction of IOP also increased the PhNR/b-wave amplitude ratio by a mean +0.065 ± 0.026 (from 0.121 ± 0.020–0.186 ± 0.018 μV, mean 53.8% increase, P = 0.023) and +0.051 ± 0.016 (from 0.135 ± 0.020–0.187 ± 0.020 μV, mean 37.7% increase, P = 0.0046) at 2.25 and 3.00 cd.s/m² stimulus intensities, respectively (Table 4, Fig. 5). There was no significant change in a-wave (P = 0.25 and P = 0.44 for 2.25 and 3.00 cd.s/m² stimulus intensities, respectively) and b-wave (P = 0.89 and P = 0.72 for 2.25 and 3.00 cd.s/m² stimulus intensities, respectively) amplitudes following reduction of IOP. In addition, we found a significant correlation between the degree of reduction in IOP and the magnitude of increase in PhNR/b-wave ratio at the 2.25 cd.s/m² stimulus intensity (r = 0.429, P = 0.020, n = 29, Fig. 6A) and at the 3.00 cd.s/m² stimulus intensity (r = 0.544, P = 0.0023, n = 29, Fig. 6B) after the outlier with a 52 mm Hg reduction in IOP was excluded. 

Figure 4

 

Full-field ERG traces at a stimulus intensity of 3.00 cd.s/m² of a 63-year-old gentleman with OAG. (A) before trabeculectomy (pretreatment) and (B) after trabeculectomy (posttreatment) showing an increase in PhNR amplitude following a 16 mm Hg reduction of IOP.

Figure 5

 

PhNR/b-wave amplitude ratios pre- and posttreatment at 2.25 and 3.00 cd.s/m² stimulus intensities in the group that had reduction of IOP (mean ± SEM, n = 18 eyes). *P < 0.05 versus pretreatment 2.25 cd.s/m². ††P < 0.01 versus pretreatment 3.00 cd.s/m². Student's two-tailed paired t-test.

Figure 6

 

Scatter plots showing the relationship between IOP reduction and change in PhNR/b-wave ratio at stimulus intensities (A) 2.25 cd.s/m² (r = 0.429, P = 0.020, n = 29) and (B) 3.00 cd.s/m² (r = 0.544, P = 0.0023, n = 29) after the outlier with a 52 mm Hg IOP reduction was excluded.

Table 4. 

 

Change in PhNR Amplitude and PhNR/b-Wave Amplitude Ratios for Eyes That Had IOP Reduced Compared with Eyes with Stable IOP

	Stimulus Intensity, cd.s/m2	IOP Stable (n = 12 Eyes)	IOP Reduced (n = 18 Eyes)	P
Change in PhNR amplitude, μV	2.25	−2.4 ± 2.6	+5.9 ± 2.4	0.027*
	3.00	−3.4 ± 1.7	+5.3 ± 2.3	0.010*
Change in PhNR/b-wave amplitude ratio	2.25	−0.036 ± 0.029	+0.065 ± 0.026	0.016*
	3.00	−0.034 ± 0.018	+0.051 ± 0.016	0.0014*

 Data shown as mean ± SEM. Statistical significance by two-tailed Student's t-test for unpaired data.

*  A significant difference between groups.

The PhNR amplitude of eyes with stable IOP (12 eyes) did not change significantly during follow-up with a mean change of −2.4 ± 2.6 (from 17.9 ± 2.5–15.5 ± 3.0 μV, P = 0.37) and −3.4 ± 1.7 μV (from 18.4 ± 2.6–15.0 ± 2.6 μV, P = 0.065) at stimulus intensities 2.25 and 3.00 cd.s/m², respectively (Table 4). The PhNR/b-wave amplitude ratio also did not change significantly in the IOP stable group, with a mean change of −0.036 ± 0.029 (from 0.219 ± 0.028–0.183 ± 0.033 μV, P = 0.23) and −0.034 ± 0.018 (from 0.208 ± 0.029–0.174 ± 0.026 μV, P = 0.079) at stimulus intensities 2.25 and 3.00 cd.s/m², respectively. There was no significant change in a-wave (P = 0.84 and P = 0.67 for 2.25 and 3.00 cd.s/m² stimulus intensities, respectively) and b-wave (P = 0.83 and P = 0.77 for 2.25 and 3.00 cd.s/m² stimulus intensities, respectively) amplitudes in the group with stable IOP. 

During follow-up, the change in PhNR amplitude was significantly greater following IOP reduction compared to the group that had stable IOP when assessed at both stimulus intensities (2.25 cd.s/m² P = 0.027, 3.00 cd.s/m² P = 0.010, Table 4). In addition, the change in the PhNR/b-wave amplitude ratio was significantly greater in the reduced IOP group compared to the group that had stable IOP when assessed at both stimulus intensities (2.25 cd.s/m² P = 0.016, 3.00 cd.s/m² P = 0.0014, Table 4, Figs. 7A, 7B). 

Figure 7

 

Box-and-whisker plots showing (A) the change in PhNR/b-wave amplitude ratio at 2.25 cd.s/m² stimulus intensity in the group that had stable IOP (n = 12 eyes) and the group that had reduced IOP (n = 18 eyes), and (B) the change in PhNR/b-wave amplitude ratio at 3.00 cd.s/m² stimulus intensity in the group that had stable IOP (n = 12 eyes) and the group that had reduced IOP (n = 18 eyes). Boxes represent 25% to 75% percentiles about the median (horizontal line), and bars at the end of whiskers represent maximum and minimum values.

Discussion

The data from our study showed that the PhNR amplitude increased with reduction of IOP in eyes with glaucoma and OHT. A mean reduction of IOP of 14 mm Hg (median reduction 10 mm Hg) in eyes with glaucoma and OHT led to a mean increase in the PhNR amplitude of 51.8% (P = 0.023) and 40.2% (P = 0.035) at stimulus intensities 2.25 and 3.00 cd.s/m², respectively. The PhNR/b-wave amplitude ratio also increased by a mean 53.8% (P = 0.023) and 37.7% (P = 0.0046) following reduction of IOP when measured at 2.25 and 3.00 cd.s/m² stimulus intensities, respectively. In addition, we found a possible correlation between the magnitude of IOP reduction and improvement in PhNR. This finding requires further investigation, since other groups studying IOP reduction and improvement in PERG have found no such correlation. ^12–14  

A previous cross-sectional study alluded to the ability of recovery of RGC function by measuring the PhNR, with the finding that untreated OHT patients had significantly lower PhNR amplitudes than a separate group of treated OHT patients. ⁷ Our study has the advantage of measuring the PhNR in the same eyes pre- and posttreatment to lower IOP. It is possible that improvement in PhNR amplitude reflects an improvement in RGC function, which may be compromised in a population of RGCs under conditions of elevated IOP. However, the slow implicit time of the PhNR raises the possibility that it is glial-mediated ¹ and it is possible that the improvement in PhNR amplitude following reduction of IOP is due to improved glial function (perhaps by more effective buffering of potassium, producing larger currents within glial cells). However, the implicit time of the PhNR is faster than the RGC-derived N95 component of the PERG, suggesting that the major component of the PhNR still may be from RGCs. The mechanisms underlying the observed improvement in the electrophysiologic parameter must be analyzed further. 

The reversibility of RGC dysfunction with IOP lowering also has been investigated using the PERG. ^11–14 The PERG has been shown to reflect the activity of RGCs, and improves following IOP reduction with topical, oral, or surgical treatment. ^11–14 However, compared to the PERG, the PhNR is easier to record as it does not require refractive correction and a long foveal fixation. ¹ The full-field PhNR also does not have the same level of requirement for excellent visual acuity and clear media. ³ In addition, a- and b-waves can be recorded simultaneously with the PhNR. 

Previous studies have investigated the PhNR amplitude and the PhNR/b-wave amplitude ratio of glaucomatous eyes with full-field and focal ERGs. ^2–10 Although the focal PhNR has been shown to be more sensitive than the full-field PhNR at detecting early and intermediate glaucoma, the full-field PhNR performed similarly to the focal PhNR for eyes with advanced glaucoma. ¹⁰ Focal ERG tests have the disadvantage that they cannot be performed reliably in the setting of cataract or vitreous opacities and require subjects to gaze at a fixation target for at least one minute without eye movements during recording for each retinal area. ⁹ The full-field ERG is simple to perform for patients and is not affected to the same degree by media opacities. Furthermore, the intersession variability of PhNR recording is greater with focal ERGs than full-field ERGs. ¹⁰ Hence, the full-field ERG was performed in our study. 

There was some intersession variability of the PhNR responses, but this was similar for the IOP reduced and IOP stable groups. The PhNR/b-wave amplitude ratio was measured in addition to the PhNR amplitude in our study, since the PhNR/b-wave amplitude ratio has been shown to have less intersession variability than the PhNR amplitude alone. ¹⁶ It has been shown previously that the PhNR/b-wave amplitude ratio has better sensitivity and specificity than the PhNR amplitude alone in detecting glaucomatous eyes. ³ The a- and b-wave amplitudes were not significantly different during follow-up in our study, indicating that the reversible change in the electrophysiology signal is derived largely from the inner retina. 

This was a pilot study to determine whether the full-field PhNR is reversed by IOP reduction. As such, there are a number of potential limitations with our study. It is not powered adequately to determine whether there is a difference in the degree of PhNR recovery with severity of glaucoma. Larger numbers of patients with a range of different severities of glaucoma are required to address this issue. Our study allowed IOP reduction by various methods, involving topical treatment, laser, and surgery. It is possible that different methods of IOP reduction could influence the full-field ERG or the degree of recovery of the PhNR amplitude by different magnitudes. This variable may be reduced in the comparison between stable and IOP reduced eyes, since 5 of 12 eyes (42%) of the stable group also had topical and surgical treatment, but this failed to reduce the IOP from baseline and did not affect the PhNR amplitude significantly. The control group was included in this study to show average PhNR and PhNR/b-wave amplitudes in the normal population. However, the control group was not age-matched and did not have the PhNR repeated, and as a result was not used in any statistical analysis to compare to the patient group. The test–retest variability was assessed in the stable IOP group, which showed very little change in PhNR amplitude and PhNR/b-wave ratio between visits compared to the group that had a significant lowering of IOP. All glaucoma and OHT patients were retested within 1 to 2 months, similar to other groups investigating the change in PERG with IOP lowering. ¹² This time interval was thought to be long enough to allow for a detectable change in electrophysiology. However, it is possible that the PhNR amplitude may have increased further over a longer term following IOP reduction. The time-period of recovery of the PhNR amplitude following intervention to reduce IOP would be an interesting future investigation. 

In summary, we have shown that the PhNR amplitude is improved after IOP reduction in glaucomatous and OHT eyes. Further work is now required to determine whether this reversible component of the ERG has use in determining whether IOP-lowering treatment has been effective for a particular eye of an individual. 

Acknowledgments

We thank Bang Bui at the University of Melbourne for reviewing the manuscript. 

Footnotes

 Disclosure: N. Niyadurupola, None; C.D. Luu, None; D.Q. Nguyen, None; K. Geddes, None; G.X.V. Tan, None; C.C.W. Wong, None; T. Tran, None; M.A. Coote, None; J.G. Crowston, None