Screening for Significant Refractive Error Using a Combination of Distance Visual Acuity and Near Visual Acuity

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Purpose To explore the effectiveness of using a series of tests combining near visual acuity (NVA) and distance visual acuity (DVA) for large-scale screenings for significant refractive error (SRE) in primary school children. Method Each participant underwent DVA, NVA and cycloplegic autorefraction measurements. SREs, including high myopia, high hyperopia and high astigmatism were analyzed. Cycloplegic refraction results were considered to be the gold standard for the comparison of different screening measurements. Receiver-operating characteristic (ROC) curves were constructed to compare the area under the curve (AUC) and the Youden index among DVA, NVA and the series combined tests of DVA and NVA. The efficacies (including sensitivity, specificity, positive predictive value, and negative predictive value) of each test were evaluated. Only the right eye data of each participant were analysed for statistical purpose. Result A total of 4416 children aged 6 to 12 years completed the study, among which 486 students had right eye SRE (SRE prevalence rate = 11.01%). There was no difference in the prevalence of high hyperopia and high astigmatism among different age groups. However, the prevalence of high myopia significantly increased with the age (χ² = 381.81, p<0.01). High hyperopia was the biggest SRE factor associated with amblyopia(p<0.01,OR = 167.40, 95% CI: 75.14∼372.94). The DVA test was better than the NVA test for detecting high myopia (Z = 2.71, p<0.01), but the NVA test was better for detecting high hyperopia (Z = 2.35, p = 0.02) and high astigmatism (Z = 4.45, p<0.01). The series combined DVA and NVA test had the biggest AUC and the highest Youden Index for detecting high hyperopia, myopia, astigmatism, as well as all of the SREs (all p<0.01). Conclusion The series combined DVA and NVA test was more accurate for detecting SREs than either of the two tests alone. This new method could be applied to large-scale SRE screening of children, aged 6 to 12, in areas that are less developed.

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Citation: Jin P, Zhu J, Zou H, Lu L, Zhao H, Li Q, et
al. (2015) Screening for Significant Refractive Error
Using a Combination of Distance Visual Acuity and
Near Visual Acuity. PLoS ONE 10(2): e0117399.
doi:10.1371/journal.pone.0117399

Academic Editor: Sanjoy Bhattacharya, Bascom
Palmer Eye Institute, University of Miami School of
Medicine;, UNITED STATES

Received: September 10, 2014

Accepted: December 21, 2014

Published: February 17, 2015

Copyright: © 2015 Jin et al. This is an open access
[article distributed under the terms of the Creative](http://creativecommons.org/licenses/by/4.0/)
[Commons Attribution License, which permits](http://creativecommons.org/licenses/by/4.0/)
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.

Data Availability Statement: All relevant data are
within the paper.

Funding: The research was funded by: 1. National
Natural Science Foundation of China for Young Staff
(Grant no. 81402695); 2. Three-year Action Program
of Shanghai Municipality for Strengthening the
Construction of the Public Health System (20112013)(Grant no. 2011-15); 3. Shanghai Municipal
Health Bureau (Grant no. 20114127 and 20134263).
The funders had no role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript.

RESEARCH ARTICLE
# Screening for Significant Refractive Error Using a Combination of Distance Visual Acuity and Near Visual Acuity

Peiyao Jin 1,2‡, Jianfeng Zhu 1‡, Haidong Zou 1,2, Lina Lu 1, Huijuan Zhao 3, Qiangqiang Li 3,
Xiangui He 1

1 Department of Preventative Ophthalmology, Shanghai Eye Disease Prevention and Treatment Center,
Shanghai, China, 2 Department of Ophthalmology, Shanghai First People’s Hospital, Shanghai Jiao Tong
University, Shanghai, China, 3 Department of Eye Disease Prevention and Control, Baoshan Center for
Disease Prevention and Control, Shanghai, China

- These authors contributed equally to this work.

- xianhezi@163.com

## Abstract

Purpose

To explore the effectiveness of using a series of tests combining near visual acuity (NVA)

and distance visual acuity (DVA) for large-scale screenings for significant refractive error

(SRE) in primary school children.

Method

Each participant underwent DVA, NVA and cycloplegic autorefraction measurements.

SREs, including high myopia, high hyperopia and high astigmatism were analyzed. Cyclo
plegic refraction results were considered to be the gold standard for the comparison of dif
ferent screening measurements. Receiver-operating characteristic (ROC) curves were

constructed to compare the area under the curve (AUC) and the Youden index among DVA,

NVA and the series combined tests of DVA and NVA. The efficacies (including sensitivity,

specificity, positive predictive value, and negative predictive value) of each test were evalu
ated. Only the right eye data of each participant were analysed for statistical purpose.

Result

A total of 4416 children aged 6 to 12 years completed the study, among which 486 students

had right eye SRE (SRE prevalence rate = 11.01%). There was no difference in the preva
lence of high hyperopia and high astigmatism among different age groups. However, the
prevalence of high myopia significantly increased with the age (χ² = 381.81, p<0.01). High
hyperopia was the biggest SRE factor associated with amblyopia p 0.01, OR = 167.40, 95%
CI: 75.14*372.94). The DVA test was better than the NVA test for detecting high myopia
(Z = 2.71, p<0.01), but the NVA test was better for detecting high hyperopia (Z = 2.35, p =
0.02) and high astigmatism (Z = 4.45, p<0.01). The series combined DVA and NVA test

PLOS ONE | DOI:10 1371/journal pone 0117399 February 17 2015 1 / 9

Competing Interests: The authors have declared
that no competing interests exist.

Combining Distance and Near Visual Acuity in Screening

had the biggest AUC and the highest Youden Index for detecting high hyperopia, myopia,
astigmatism, as well as all of the SREs (all p<0.01).

Conclusion

The series combined DVA and NVA test was more accurate for detecting SREs than either

of the two tests alone. This new method could be applied to large-scale SRE screening of

children, aged 6 to 12, in areas that are less developed.

Introduction

Uncorrected significant refractive error (SRE) is a universal and serious problem, particularly
in children. According to a recently published study, 7% of preschool children in Taiwan have
uncorrected SREs[1]. This not only leads to eyestrain, headaches and poor academic performance, but can also result in amblyopia[2]. Although screening programs for refractive errors
are carried out all over China, the outcomes of these screenings are affected by several factors.
Firstly, refractive error that is successfully detected by the screening programs is not always
taken seriously enough. Some parents and children neglect the advice of health care workers
because they believe that low grade refractive error is very common and harmless. However, if
SREs are detected, particularly in children, specific warnings regarding amblyopia risks may
help to increase awareness. Secondly, in some rural areas of China, a large number of children
under 6 years old do not attend preschool, thus screening programs are not accessible. According to the 2010 national census in China, 39% of the population of Shanghai immigrated from
other less developed provinces in China. It has been reported that the risk of finding an undiagnosed, significant eye disorder in a school entrant was more than 6 times greater for a child
who was not examined during the preschool years, and the risk of finding an amblyopic child
was more than 10 times greater[3]. Therefore, it is vital that primary school students in less developed areas are screened for SREs.

Cyclopledic autorefraction is a widely used method for diagnosing refractive error, however,
due to the length of time it takes to induce cyclopledia, it is seldom used for the screening of
large populations[4]. Currently, noncycloplegic autorefraction is used for refractive error
screening in most primary schools in urban areas of Shanghai[5]. However, in some less developed districts, an uncorrected distance visual acuity test (DVA) is the only screening method
used in schools[6]. Previous studies have reported that although the DVA test has a high sensitivity and specificity for detecting myopia, it cannot accurately detect hyperopia and astigmatism. Thus, the DVA test alone is not satisfactory for SRE screening[7,8]. The uncorrected near
vision (NVA) test is a simple and useful eye examination with many benefits. It is most commonly used for detecting presbyopia in older people[9]. A recently published study indicated
that the NVA test is suitable for the detection of amblyopia risk factors and for low vision examinations in children[10]. Therefore, in the current study, a method combining DVA and
NVA was used for the detection of SREs in children.

To the best of our knowledge, there have been few studies that have examined using a combination of NVA and DVA for SRE detection in schoolchildren. The purpose of the present
study was to explore the effectiveness of using a series combined test of NVA and DVA for
large-scale SRE screening in primary schools.

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Combining Distance and Near Visual Acuity in Screening

Methods

Setting and participants

The present study is part of a trial, which aims to establish archives of refractive development of
children in Shanghai, China. One ophthalmologist, three optometrists, and two public health doctors conducted the study. In total, 7 primary schools from the Baoshan district, a suburban district
of Shanghai, were included. Students in grades 1 to 5, aged 6 to 12 years participated in the study.
Those students who didn't consented to, or were not suitable for cyclopaedia, were excluded.

This study was conducted according to the tenets of the Declaration of Helsinki and was approved by the institutional review board of the Shanghai Eye Disease Prevention and Treatment Centre. Written informed consent was received from the parents of all children who were
enrolled in the study.

Screening procedure and study outcomes

The screening procedure contained several items. Firstly, DVA and NVA were examined monocularly in each student. A professional ophthalmologist then conducted a detailed ophthalmic
examination, including a slit lamp examination and intraocular pressure (IOP) measurement.
Lastly, if a student consented to, and was suitable for cyclopledia (i.e. no allergy to tropicamide,
anterior chamber angle was not narrow and IOP was not high), he/she underwent cyclopledic
autorefraction conducted by an optometrist.

Trained vision-screening technicians examined DVA and NVA, using the Standard Logarithmic Distance Visual Acuity E Chart (Weheng wh01, GuangZhou WeiShiKang) and the
Standard Logarithmic Near Visual Acuity E Chart (Wehen 8620-37630978), which are the
most commonly used visual acuity charts in China. This procedure has been thoroughly described in a previous paper[5]. DVA and NVA were performed monocularly, without optical
aids, at 5 m and 40 cm respectively. The right eye was always examined first. Students were required to identify each optotype within 5 seconds and they were not allowed to squeeze their
eyes or squint during the examination.

One drop of 0.5% tropicamide (Wuxi Shanhe Group, Wuxi, China) was instilled to both
eyes, every 5 minutes (5 drops total), to induce cyclopledia. Tropicamide is the most commonly
used cycloplegic agent in China and many researchers have indicated that it works well, particularly in subjects with dark irises[11,12]. Autorefraction measurements were obtained using a
table-mounted KR-8800 (Topcon, Tokyo, Japan) after adequate cyclopledia was achieved. Adequate cyclopledia was defined as pupil dilation > 6 mm and an absent light reflex. Spherical
equivalent refraction (sphere+1/2 cylinder) was used to classify refraction status.

High myopia, high hyperopia, and high astigmatism, were analyzed in this study and were
defined as spherical equivalent degree �−3.0 dioptres (D), spherical equivalent degree � +4.5 D,
and cylinder degree � 2.0 D, respectively[1,4]. Cyclopledic refraction results were considered
to be the gold standard when comparing among different screening measurements. The definition of amblyopia was taken from the Refractive Error Study in Children (RESC) surveys. Amblyopia was considered to be a best-corrected visual acuity � 20/40, with no apparent organic
lesion, if one or more of the following criteria were met: (1) esotropia, exotropia, or vertical tropia at 4-m fixation or exotropia or vertical tropia at 0.5 m, (2) anisometropia of 2.00 SED or
more, or (3) bilateral ametropia of at least +6.00 spherical equivalent degrees.

Statistical analysis

Two staff members independently input all data into an Epidata 3.1 database. The general
characteristics of all participants were analyzed using SAS (version 9.3, SAS Institute, Cary,

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Combining Distance and Near Visual Acuity in Screening

NC, USA). Categorical variables were analysed by the Chi-square test. Cochran-Armitage
Trend Test was used to determine the trend among age groups. Stepwise multiple logistic
regression analysis was used to determine whether SREs were associated with amblyopia (inclusion p value = 0.05 and exclusion p value = 0.05). Since DVA, NVA, and cyclopledic autorefraction results were closely related between the right and the left eyes (Spearman correlation =
0.81, 0.71, and 0.91 for DVA, NVA and cyclopledic autorefraction respectively, p<0.001), the
right eye was chosen for data analysis. Receiver-operating characteristic (ROC) curves were
constructed to compare the area under the curve (AUC) and the Youden index between DVA,
NVA and the series combined tests of DVA and NVA by MedCalc (version 13). The best
screening criterion was defined as the point closest to the top left-hand corner of the ROC
curve[13]. The efficacies (including sensitivity, specificity, positive predictive value and negative predictive value) of each test were evaluated. Results were presented as the mean ± standard deviation for continuous variables and as rates (proportions) for the categorical data. All
reported p values are two-sided. p<0.05 was considered statistically significant.

Results

A total of 5214 participants were recruited for this study, among which 4416 completed all examinations and were included in the analysis. Children were excluded if they were unwilling to
accept cyclopledia (n = 778), were unsuitable for cyclopaedia (n = 18) or were uncooperative
during the exams (n = 2).

Prevalence of SREs in different age groups

The mean age of included participants was 8.21±1.73 years old and 54.53% were male (n =
2408). Overall, 486 students with right eye SRE were diagnosed by cyclopledic autorefraction
(SRE prevalence rate = 11.01%), including 6 with high hyperopia and astigmatism, 30 with
high myopia and astigmatism, 32 with high hyperopia alone, 347 with high myopia alone and
71 with high astigmatism alone. The prevalence of each disease in different age groups were
shown in Table 1. Although there were no differences in the prevalence of hyperopia and astigmatism among different age groups, the prevalence of myopia significantly increased with increasing age (χ² = 381.81, p<0.01), and increased fastest in children from 9 years old to 11
years old (partition of Chi-square analysis, p<0.01). Although the prevalence of SREs increased
with the age (χ² = 272.21, p<0.01), the prevalence of amblyopia was stable among different age
groups (Table 1). Among 60 students with amblyopia, 22 had high myopia (including 2 had

Table 1. The prevalence of significant refractive error in different age groups: number (rate).

age All 6 7 8 9 10 11 12 p

Number of children 4416 929 815 798 814 601 232 227

High Hyperopia* 38 (0.86) 13 (1.40) 4 (0.49) 5 (0.63) 7 (0.86) 5 (0.83) 4 (1.72) 0 (0.00) 0.39

High Myopia § 377 (8.54) 6 (0.65) 20 (2.45) 48 (6.02) 69 (8.48) 98 (16.31) 67 (28.88) 69 (30.40) <0.01

High Astigmatism ¥ 71 (1.61) 18 (1.94) 19 (2.33) 7 (0.88) 16 (1.97) 7 (1.16) 1 (0.43) 3 (1.32) 0.08

All SREs 486 (11.01) 37 (3.98) 43 (5.28) 60 (7.52) 92 (11.30) 110 (18.30) 72 (31.03) 72 (31.72) <0.01

Amblyopia 60 (1.36) 15 (1.61) 10 (1.23) 8 (1.00) 10 (1.23) 8 (1.33) 4 (1.72) 5 (2.20) 0.70

Note: *High hyperopia: spherical equivalent degree � +4.5 dioptres, including 6 students with high hyperopia and astigmatism, and 32 students with high

hyperopia alone.
§High myopia: spherical equivalent degree �−3.0 dioptres, including 30 with high myopia and astigmatism, and 347 with high myopia alone.
¥High Astigmatism: cylinder degree � 2.0 D with no significant spherical degree

doi:10.1371/journal.pone.0117399.t001

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Combining Distance and Near Visual Acuity in Screening

high myopia and high astigmatism), 16 had high hyperopia (including 1 had high hyperopia
and high astigmatism), and 5 had high astigmatism alone. The remaining 17 students were suffering from other diseases such as strabismus and congenital cataract. Stepwise multiple logistic
regression analysis indicated that high hyperopia was the biggest SRE factor associated with
amblyopia(p<0.01, OR = 167.40, 95%CI: 75.14*372.94), followed by high astigmatism
(p<0.01, OR = 17.44, 95%CI: 6.25*48.68), and high myopia(p<0.01, OR = 14.27, 95%CI:
7.51*27.11). There was no difference in the prevalence of high hyperopia, high myopia, high
astigmatism or amblyopia between males and females in any age group (p>0.05).

Receiver operating characteristic curve analysis

The ROC curves and the best cutoff points of DVA, NVA and series combined vision tests are
shown in Table 2. The AUC differences among different screening methods were tested, and
the significance levels are shown in Table 3. The AUC of the DVA test was larger than that of
the NVA test when detecting high myopia in children; however, the NVA test had a much larger AUC than that of the DVA test when detecting high hyperopia and astigmatism (Tables 2
and 3). The series combined test of DVA and NVA had the biggest AUC when detecting high
hyperopia, myopia, astigmatism, as well as all SREs (Fig. 1, Tables 2 and 3). The Youden Index,
which describes the accuracy of a test, also confirmed that DVA is better than NVA for high
myopia screening and that NVA is better than DVA for detecting high hyperopia and astigmatism. Furthermore, the Youden Index of the series combined test was higher than that for the
DVA or NVA test alone for detecting high hyperopia, myopia and astigmatism, as well as for
detecting all SREs (Tables 2 and 3). The sensitivities and specificities for the detection of each
disease by each test are listed in Table 2. The positive predictive value (PPV) and the negative
predictive value (NPV) of the series combined test for detecting all SREs were 58.25 and 97.85,

Table 2. The receiver-operating characteristic (ROC) curve efficacies and cutoff points of near vision acuity (NVA), distance vision acuity (DVA)
and the series combined test.

Disease Method AUC* criterion YI § Se. ¥ Sp. ＄ PPV ※ NPV #

High hyperopia (N = 38) DVA 0.77 �20/32 0.50 89.5 60.6 1.93 99.85

NVA 0.85 �20/25 0.60 81.6 78.9 3.24 99.80

Combined 0.86 DVA�20/32 NVA �20/25 0.64 78.9 84.8 4.30 99.78

High myopia (N = 377) DVA 0.95 �20/50 0.79 97.1 81.7 33.15 99.67

NVA 0.91 �20/25 0.70 85.9 84.3 33.86 98.47

Combined 0.97 DVA�20/63 NVA �20/20 0.79 87.8 90.7 46.95 98.76

High astigmatism (N = 107) DVA 0.77 �20/32 0.48 86.9 61.3 5.28 99.47

NVA 0.85 �20/25 0.63 83.2 79.9 9.30 99.48

Combined 0.87 DVA�20/25 NVA �20/25 0.64 81.3 83.1 10.65 99.44

All SREs � (N = 486) DVA 0.91 �20/40 0.70 91.2 76.8 32.69 98.60

NVA 0.91 �20/25 0.71 85.2 86.2 43.26 97.92

Combined 0.95 DVA�20/32 NVA �20/25 0.76 83.5 92.6 58.25 97.85

Note: *AUC: area under the curve
§YI: Youden Index
¥Se.: Sensitivity
＄Sp.: Specificity
※PPV: positive predictive value
#NPV: negative predictive value
�SREs: significant refractive errors

doi:10.1371/journal.pone.0117399.t002

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Combining Distance and Near Visual Acuity in Screening

Table 3. The significance level (P) and Z statistics of the differences between distance vision acuity (DVA), near vision acuity (NVA) and
combined examinations: P (Z).

AUC* DVA NVA Combined

High hyperopia (N = 38) DVA 0.77 - 0.02 (2.35) <0.01 (2.79)

NVA 0.85 0.02 (2.35)              - 0.68 (0.41)

Combined 0.86 <0.01 (2.79) 0.68 (0.41)

High myopia (N = 377) DVA 0.95 - <0.01 (2.71) 0.86 (0.17)

NVA 0.91 <0.01 (2.71) - <0.01 (3.08)

Combined 0.97 0.86 (0.17) <0.01 (3.08)

High astigmatism (N = 107) DVA 0.77 - <0.01 (4.45) <0.01 (4.62)

NVA 0.85 <0.01 (4.45) - 0.56 (0.58)

Combined 0.87 <0.01 (4.62) 0.56 (0.58)
All SREs � (N = 486) DVA 0.91 - 0.58 (0.55) <0.01 (7.77)

NVA 0.91 0.58 (0.55) - <0.01 (9.23)

Combined 0.95 <0.01 (7.77) <0.01 (9.23) -

Note: *AUC, area under the curve
�SREs, significant refractive errors

doi:10.1371/journal.pone.0117399.t003

respectively. This indicated that, with referral criteria of DVA�20/32 and NVA�20/25, almost
60% of referred children, and only 2% of excluded children were actual SRE patients.

Discussion

The results of this study indicate that the prevalence of high myopia significantly increases
with age; however, there was no difference in the prevalence of high hyperopia and astigmatism
among different age groups. High hyperopia was the biggest SRE factor associated with amblyopia. DVA examination is a commonly used screening method, but has shortfalls in detecting
high hyperopia and astigmatism. NVA examination is much better for detecting high hyperopia and astigmatism and could be a good complement to DVA examination. The series combined test of DVA and NVA enables better detection of all SREs than either examination alone.

In total, 11.01% of the children were diagnosed with SREs in the right eye and only the prevalence of high myopia increased with age. This finding is consistent with a previous study conducted in China[14]. In our study, the prevalence of high hyperopia, myopia and astigmatism
were 0.86%, 8.54%, and 2.42%, respectively. The prevalence of high myopia among participants
in the current study was much higher compared to previous research conducted in preschool
children in Taiwan[1], in which the prevalence of high hyperopia, myopia and astigmatism
were 1.0%, 0.6%, and 5.1%, respectively. It is likely that the higher prevalence of myopia observed in the current study is due to the older age of participants. The astigmatism prevalence
in the current study was lower than that reported in preschool children in Taiwan, though the
result is consistent with a recently reported study, in which the prevalence of astigmatism was
found to be lower in older children[15,16].

DVA is the most widely used method of refractive error screening in China and worldwide

[17]. DVA is highly accurate for detecting significant myopia (AUC = 0.95); however, it is not
accurate when screening for high hyperopia and astigmatism (AUC<0.8). The NVA test provides much better accuracy when screening for high hyperopia and astigmatism (AUC = 0.85),
but is an ineffective method for detecting myopia. Former epidemiological surveys have reported a high prevalence of both astigmatism and hyperopia in Chinese children[18,19], which

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Combining Distance and Near Visual Acuity in Screening

Fig 1. The receiver operating characteristic (ROC) curve of distance visual acuity (DVA), near visual acuity (NVA) and the series combined test of
distance and near visual acuity (DVA combined NVA) in detecting significant refractive errors.

doi:10.1371/journal.pone.0117399.g001

are also known risk factors for the development of strabismus and amblyopia[20,21]. Our result indicated that among common SRES, high hyperopia was the most important cause of amblyopia. Thus, combining DVA and NVA in SRE screenings is important and necessary.
Moreover, the series combined NVA and DVA test largely increased the specificity without significantly lowering the sensitivity. Since the prevalence of SREs is not low in primary school
students, increasing the specificity could largely decrease unnecessary referrals, thus reducing
the cost. During the screening, children with a DVA less than 20/32 would have an additional
NVA examination, and children with an NVA less than 20/25 would be suspected of having an
SRE. These children and their parents would then be notified. Parents who do not realize the
importance of uncorrected refractive error in their children would be given information, and
warned about the potential for the development of strabismus and amblyopia, which would
likely increase compliance with necessary treatments.

Although the series combined test of DVA and NVA was not as good as noncycloplaegic
autorefraction and photorefraction for detecting low refractive errors[5,22], its accuracy was
satisfactory for SRE screening. Furthermore, noncycloplaegic autorefraction and photorefraction require expensive equipment and experienced operators, while DVA and NVA tests are
easier to carry out and are much less expensive. School-teachers can be trained to administer

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Combining Distance and Near Visual Acuity in Screening

DVA and NVA tests and since the examinations are done quickly and cause no discomfort,
students and their parents easily accept them. Unlike noncycloplaegic autorefraction and
photorefraction equipment, DVA and NVA equipment is small and easily portable, which is
extremely valuable when travelling to regions that lack convenient transportation. These characteristics make the series combined DVA and NVA test appropriate for SRE screening in less
developed areas.

There were some limitations to the current study. Firstly, the referral criteria of the series
combined test might not effectively apply to other populations. Secondly, with different cycloplegic agents, the diagnosis of SRE might be a little biased in different population. Because although tropicamide was proved to be a useful cycloplegic agent in Asian population, some
former studies indicated that the cycloplegic effect of tropicamide was much less than that of
cyclopentolate or atropine in Caucasian population[23]. Thirdly, since the study population
was not randomly selected, the representativeness of the result could be affected.

In conclusion, the series combined test of DVA and NVA had better accuracy when screening for SREs than either of the tests alone. This new method could be used for large-scale SRE
screening of children, aged 6 to 12 years, who reside in less developed areas.

Author Contributions

Conceived and designed the experiments: XH JZ H. Zou. Performed the experiments: XH LL
H. Zhao QL. Analyzed the data: PJ XH. Contributed reagents/materials/analysis tools: PJ.
Wrote the paper: PJ XH.

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