Jill Carlton
email: j.carlton@sheffield.ac.uk

Eva Kaltenthaler

Health related quality of life measures (HRQoL) in patients with amblyopia and strabismus: a systematic review

Keywords:

Health related quality of life measures (HRQoL) in patients with amblyopia and strabismus: a systematic review

Jill Carlton, Eva Kaltenthaler

The use of HRQoL measures in patients with amblyopia and/or strabismus is a developing area. Further research is necessary to determine the impact of issues such as diplopia and poor cosmesis upon patient groups, and to determine the influence of ethnicity and parental reporting in these patients.

Introduction

Health related quality of life (HRQoL) measures are increasingly used in health care.

Such measures help inform clinical decision making through evidence based practice, and can also be used in economic evaluations to help inform policy making decisions.

HRQoL is a concept that incorporates physical and functional status, emotional status and social functioning [START_REF] Schumacher | Assessment of quality of life in clinical trials[END_REF] and may be assessed using generic or disease or conditionspecific instruments. When considering the application of HRQoL instruments within ophthalmology, there are concerns that generic measures are not sensitive to the recognised symptoms of vision loss or emotional aspects of a given ophthalmic condition, such as strabismus.

A number of validated disease-specific or condition-specific instruments have been developed and applied in ophthalmology, both in research and clinical practice.

These include the Visual Function Index (VF-14); National Eye Institute Visual Function Questionnaire [START_REF] Mangione | Identifying the content area for the 51-item National Eye Institute Visual Function Questionnaire[END_REF][START_REF] Mangione | Psychometric properties of the National Eye Institute Visual Function Questionnaire (NEI-VFQ)[END_REF][START_REF] Mangione | Development of the 25-item National Eye Institute Visual Function Questionnaire[END_REF] (NEI-VFQ: 51 and 25 item questionnaires); Impact of Visual Impairment [START_REF] Keeffe | Description and measurement of handicap caused by vision impairment[END_REF] (IVI); and the Activities of Daily Vision Scale [START_REF] Mangione | Development of the Activities of Daily Vision Scale[END_REF] (ADVS), to name a few. Such instruments have been used: to describe the impact of a condition within a given population; as a measure to determine the success or suitability of treatment interventions; and to assess the impact of vision loss. The use of patientreported outcome measures within the specific field of amblyopia and/or strabismus is not as well established. The purpose of this study is to identify and discuss HRQoL instruments which may be used in the investigation and management of patients with amblyopia and/or strabismus. Following removal of duplicates, a total of 39 articles were applicable for this review.

Methods

Articles were rejected at title if they were not related to the subject area (n=4);

rejected at abstract if a vision-specific or disease-specific instrument was not used in the study (n=8); and rejected at full paper if the study subjects could not be described as "orthoptic patients" -that is, did not include subjects with a diagnosis of amblyopia and/or strabismus (n=21). These included assessment of HRQoL in patients with sensory impairment, ARMD, low vision, cataract, diabetic retinopathy, choroidal melanoma, and glaucoma. This left a total of eleven papers identified through the outlined search strategy subject to full review. These papers described the development or administration of four HRQoL measures; the Amblyopia and Strabismus Questionnaire (A&SQ); [START_REF] Van De Graaf | Amblyopia & Strabismus Questionnaire: design and initial validation[END_REF][START_REF] Felius | The Amblyopia and Strabismus Questionnaire: English translation, validation, and subscales[END_REF][START_REF] Van De Graaf | Amblyopia and Strabismus Questionnaire (A&SQ): clinical validation in a historic cohort[END_REF][START_REF] Van De Graaf | Construct validation of the Amblyopia and Strabismus Questionnaire (A&SQ) by factor analysis[END_REF] the Amblyopia Treatment Index (ATI); [START_REF] Cole | The amblyopia treatment index[END_REF][START_REF] Eye | Impact of patching and atropine treatment on the child and family in the Amblyopia Treatment Study[END_REF][START_REF] Holmes | Further validation of the Amblyopia Treatment Index parental questionnaire[END_REF] the Adult Strabismus Quality of Life Questionnaire (AS-20); [START_REF] Hatt | Development of a quality-of-life questionnaire for adults with strabismus[END_REF][START_REF] Hatt | Comparison of quality-of-life instruments in adults with strabismus[END_REF] and the Intermittent Exotropia Questionnaire (IXTQ). [START_REF] Hatt | Quality of life in intermittent exotropia: child and parent concerns[END_REF][START_REF] Hatt | Development and initial validation of quality-of-life questionnaires for intermittent exotropia[END_REF]Additional literature was identified through a clinical expert (n=3) and through references of the papers identified through the electronic search (n=2). The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) [18] flow diagram of study identification is shown in Figure 1. This additional searching was undertaken in order to identify any further papers which reported the psychometric properties of the instruments identified from the initial literature search. Instruments were assessed in terms of reliability, validity and responsiveness (see Table 2). It is possible to have a reliable measure that is not valid; however, a valid measure must also be reliable.

Explanations of reliability, validity and responsiveness are shown in Table 2. For the purpose of this paper, construct validity will be determined if compared to objective clinical measures such as visual acuity or binocularity; concurrent validity will be a comparison to an existing vision-specific HRQoL measure. • factor analysis is a method of determining the structure of an

Results

The literature search identified four disease-specific instruments that can used in the investigation and management of orthoptic patients; the Amblyopia and Strabismus Questionnaire (A&SQ), the Amblyopia Treatment Index (ATI); the Adult Strabismus Questionnaire (AS-20) and the Intermittent Exotropia Questionnaire (IXTQ).

Summary details of the instruments are shown in Tables 3 and4.

Table 3 Summary of HRQoL orthoptic-specific instruments The instruments identified all used factor analysis in their development. Factor analysis is a technique that can reduce the number of items (i.e. questions) within an instrument, by identifying redundant items. It may also be used to assess the structure of a domain (i.e. sub-group or theme, such as "cosmesis" or "ability"), by grouping items together that are related to each other. Factor analysis can be thought of as a confirmatory tool to assess the structure of a developed instrument or measure.

Instrument

Each instrument was shown to be reliable (in that internal consistency data was reported; see Table 2 for details); however, no test-retest data was reported for any instrument. Test-retest data would add to the evidence of reliability for the instrument.

Each instrument was also deemed to be valid; however, only the A&SQ and AS-20

were tested against another recognised HRQoL measure (the NEI-VFQ-25).

Amblyopia and Strabismus Questionnaire (A&SQ)]

The A&SQ was designed to assess the "decrease in quality of life" of patients with amblyopia and strabismus. [START_REF] Van De Graaf | Amblyopia & Strabismus Questionnaire: design and initial validation[END_REF] The instrument was designed by clinicians who listed problems that amblyopic and strabismic patients experience. These were categorised into five domains. All items in the questionnaire are measured on Likert-type rating scales, which are assumed to be linear and run from 0 to 100 (0 corresponding to the least favourable score and 100 corresponding to the most favourable score). The total A&SQ score is defined as the mean of all item scores.

The reliability of the A&SQ has been reported in terms of internal consistency. [START_REF] Van De Graaf | Amblyopia & Strabismus Questionnaire: design and initial validation[END_REF] Validity has been reported in terms of discriminatory validity, [START_REF] Van De Graaf | Amblyopia & Strabismus Questionnaire: design and initial validation[END_REF] concurrent validity [START_REF] Van De Graaf | Amblyopia & Strabismus Questionnaire: design and initial validation[END_REF] and construct validity [START_REF] Van De Graaf | Amblyopia and Strabismus Questionnaire (A&SQ): clinical validation in a historic cohort[END_REF][START_REF] Van De Graaf | Construct validation of the Amblyopia and Strabismus Questionnaire (A&SQ) by factor analysis[END_REF] (see Table 2). An English-language version has also been developed and has shown to be both reliable and valid. [START_REF] Felius | The Amblyopia and Strabismus Questionnaire: English translation, validation, and subscales[END_REF] Amblyopia Treatment Index (ATI) [START_REF] Cole | The amblyopia treatment index[END_REF][START_REF] Eye | Impact of patching and atropine treatment on the child and family in the Amblyopia Treatment Study[END_REF][START_REF] Holmes | Further validation of the Amblyopia Treatment Index parental questionnaire[END_REF] The ATI was developed following consultation with specialists and parents of children with amblyopia. A list of 20-items was created and the initial questionnaire was issued to parents of children participating in the Amblyopia Treatment Study (a multi-centre randomised controlled clinical trial designed to compare atropine and conventional patching treatment). Slight modifications/rewording of some of the questions were made depending upon whether the child was undertaking conventional patching treatment or atropine therapy for the management of their amblyopia. The ATI now consists of 18-items, scored on a 5-part Likert-type scale. The overall ATI score is the mean of all the item scores. The reliability of the ATI has been reported in terms of internal consistency. [START_REF] Cole | The amblyopia treatment index[END_REF][START_REF] Eye | Impact of patching and atropine treatment on the child and family in the Amblyopia Treatment Study[END_REF][START_REF] Holmes | Further validation of the Amblyopia Treatment Index parental questionnaire[END_REF] The validity of the ATI has been reported in terms of construct validity [START_REF] Eye | Impact of patching and atropine treatment on the child and family in the Amblyopia Treatment Study[END_REF] and the structure of the instrument has also been investigated. [START_REF] Cole | The amblyopia treatment index[END_REF][START_REF] Eye | Impact of patching and atropine treatment on the child and family in the Amblyopia Treatment Study[END_REF][START_REF] Holmes | Further validation of the Amblyopia Treatment Index parental questionnaire[END_REF] The AS-20 [START_REF] Hatt | Development of a quality-of-life questionnaire for adults with strabismus[END_REF][START_REF] Hatt | Comparison of quality-of-life instruments in adults with strabismus[END_REF] The AS-20 is a patient-derived instrument for use in patients with a diagnosis of strabismus. Subjects with strabismus were interviewed and statements or phrases were used to generate a 181-item questionnaire. A number of items were subsequently removed as they were found to be not applicable. Of the remaining items, two subscales were identified that were applicable to the strabismic population (psychosocial and function); with each subscale containing ten items. A 5-point Likert type scale is applied to each question. The overall AS-20 is given from 0 (worst HRQoL) to 100 (best HRQoL). The reliability of the AS-20, in terms of internal consistency, and validity, in terms of discriminative validity has been reported. [START_REF] Hatt | Development of a quality-of-life questionnaire for adults with strabismus[END_REF] The instrument has also been validated in terms of concurrent validity against the VFQ-25, and was found to be more sensitive than the NEI-VFQ-25 in detecting reduced HRQoL in adult subjects with strabismus. [START_REF] Hatt | Comparison of quality-of-life instruments in adults with strabismus[END_REF] Intermittent Exotropia Questionnaire (IXTQ) [START_REF] Hatt | Quality of life in intermittent exotropia: child and parent concerns[END_REF][START_REF] Hatt | Development and initial validation of quality-of-life questionnaires for intermittent exotropia[END_REF] The IXTQ is a patient and parent derived instrument developed to measure the impact of intermittent exotropia upon HRQoL. Children and parents of children with intermittent exotropia were interviewed, and the results analysed to identify items and themes associated with the diagnosis. [START_REF] Hatt | Quality of life in intermittent exotropia: child and parent concerns[END_REF] Three types of questionnaire were developed: child (addressing the child's QoL), proxy (parallel to the child questionnaire addressing parent's perception of the child's HRQoL), and parent (addressing the parent's own HRQoL). The child questionnaire was developed in two formats: (5 to 7 year olds, with a three-point Likert-type scale) and (8 to 17 years, with a five-point Likert-type scale). The final child (both versions) and proxy questionnaires contain 12 items. The parent questionnaire consists of three subscales; function (8 items), psychosocial (7 items) and surgery (2 items). The parent instrument is also measured on a 5-point Likert-type scale. The overall IXTQ score is calculated as the mean of all the item scores. The IXTQ has undergone initial validation, in terms of internal consistency reliability, [START_REF] Hatt | Development and initial validation of quality-of-life questionnaires for intermittent exotropia[END_REF] with further validity testing planned.

Strabismus and amblyopia -should these issues be considered together in the same measure?

and/or amblyopia upon HRQoL have not been examined. The identified measures did not disclose their study populations in terms of ethnicity; the country of origin for the study is shown in Table 4. Whilst the items included in any of the developed measures may not have differed should a racially heterogeneous group have been used, the impact of strabismus and/or amblyopia upon different groups has not been fully investigated. Socio-economically deprived groups or ethnic minority groups may report different HRQoL implications of strabismus and/or amblyopia.

Discussion

Within the field of treating patients with a diagnosis of amblyopia and/or strabismus, the application of HRQoL measures is a relatively new area. However, the need for such instruments has become recognised, with the development of four new measures over recent years. These instruments are important in that they address the specific HRQoL issues that patients with amblyopia and/or strabismus face; issues that would not be detectable using generic HRQoL instruments, such as the Health Utilities Index [START_REF] Horsman | The Health Utilities Index (HUI ® ): concepts, measurement properties and applications[END_REF] (HUI) or the Euroqol Quality of Life questionnaire [START_REF] Euroqol Group | Euroqol -a new facility for the measurement of healthrelated quality of life[END_REF] (EQ-5D). HRQoL instruments in orthoptics (the speciality of investigating and managing patients with binocular disorders) are likely to determine the impact of amblyopia and/or strabismus, such as the A&SQ, [START_REF] Van De Graaf | Amblyopia & Strabismus Questionnaire: design and initial validation[END_REF][START_REF] Felius | The Amblyopia and Strabismus Questionnaire: English translation, validation, and subscales[END_REF][START_REF] Van De Graaf | Amblyopia and Strabismus Questionnaire (A&SQ): clinical validation in a historic cohort[END_REF][START_REF] Van De Graaf | Construct validation of the Amblyopia and Strabismus Questionnaire (A&SQ) by factor analysis[END_REF], ATI [START_REF] Cole | The amblyopia treatment index[END_REF][START_REF] Eye | Impact of patching and atropine treatment on the child and family in the Amblyopia Treatment Study[END_REF][START_REF] Holmes | Further validation of the Amblyopia Treatment Index parental questionnaire[END_REF] and AS-20. [START_REF] Hatt | Development of a quality-of-life questionnaire for adults with strabismus[END_REF][START_REF] Hatt | Comparison of quality-of-life instruments in adults with strabismus[END_REF] The IXTQ [START_REF] Hatt | Quality of life in intermittent exotropia: child and parent concerns[END_REF][START_REF] Hatt | Development and initial validation of quality-of-life questionnaires for intermittent exotropia[END_REF] also addresses specific issues that patients with intermittent exotropia experience. It is possible that other strabismic conditions have their own specific issues that impact upon HRQoL; and to that end other condition-specific measures may be required to be developed in the future. Further research on sub-groups of patients is necessary to determine whether this is the case.

The appropriateness of condition-specific, or disease-specific, measures in determining HRQoL is a contentious issue. Whilst it is acknowledged that generic measures are unlikely to be sensitive enough to detect the implications of vision-loss, poor ocular alignment or diplopia, the question as to which of the condition-specific measures should be taken as the "gold standard" has yet to be answered. Further studies are required to compare the appropriateness of the AS-20, A&SQ, ATI and (if appropriate) the IXTQ against each other; and a well-validated comparator (such as the VFQ-25). Further research is also required to investigate any differences between self-reported HRQoL, and proxy reported HRQoL in orthoptic patients, specifically the differences between parental and self-reported HRQoL components. It is possible that the HRQoL implications of strabismus and/or amblyopia are not the same for both adult and child patients. Should this be the case then amendments to existing measures or development of new measures are necessary in order to quantify the HRQoL implications of strabismus and/or amblyopia for children. Exploration of the impact of ethnicity upon HRQoL in orthoptic patients is also required.

The use of HRQoL instruments need not be confined to clinical trials or research.

Their application within everyday clinical practice should also be encouraged. The information provided from a HRQoL measure may assist in the clinical decisionmaking process of which treatment to prescribe and when. All of the questionnaires described can be used without licence, and therefore do not add any significant financial burden to the patient's investigation, aside from the extra time taken to administer the instrument. Their inclusion in patient investigations can be considered justified as another tool to facilitate the implementation of evidence-based practice.

The Corresponding Author has the right to grant on behalf of all authors, and does grant on behalf of all authors, an exclusive licence (or non-exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd, and its Licensees to permit this article (if accepted) to be published in British Journal of Ophthalmology and any other BMJPGL products and to exploit all subsidiary rights, as set out in our licence.

A

  systematic literature review was undertaken during November 2009. A specific search strategy was employed to identify HRQoL instruments that may be used in the investigation and management of patients with amblyopia and/or strabismus. The electronic databases of AMED (Allied and Complementary Medicine: 1985 to November 2009); British Nursing Index and Archive (1985 to October 2009); Ovid Medline In-Process and Other Non-Indexed Citations and Ovid Medline (1950 to present); and PsycINFO (1806 to November Week 1 2009) were searched. Details of the search strategy (i.e. keywords used) are shown in

  instrument in terms of domains or subscales. It can be used to identify redundant or duplicate items. It may also be used to determine domain structure. Some papers refer to this as a measure of internal validity. Responsiveness • the extent to which the instrument can detect in patients known to have a change in their physical condition.

  -based; LB = literature-based; PB = patient-based DV = discriminant validity; CV = construct validity; CCV = concurrent validity; IC = internal consistency; R = responsiveness; TRR = testretest reliability

  -item questionnaire to be completed by the patient Tested on 3 groups (current amblyopic and strabismic patients; healthy controls; historical cohort) Also administered 12-item Short-Form (SF-12) and the VFQ-25 Assessed clinical validity of A&SQ by correlating domains to orthoptic parameters of amblyopic patients treated 30-35yrs Current DRT of amblyopic eye correlated to four domains (P<0.01) (fear of losing better eye; distance estimation; diplopia; compared with clinical outcomes of VA, diplopia and asthenopia; and disability questionnaire of 6items on specific health, daily functioning, social interaction, concerns about the future, self-image, and job-related difficulties contact and cosmetic problems) Current degree of binocular vision correlated to two domains (P<0.01) (distance estimation; social contact and cosmetic problems) ASQE correlated with disability questionnaire r=-0.76, P<0.001 Cronbach's α = fear of losing better eye (0.80); distance estimation (0.90); visual disorientation (0.82); diplopia (0.82)factor analysis to compare correlations among 26-item responses contact and cosmetic problems (0.92) Spearman rank correlation coefficient of ASQE total score and level of unilateral acuity loss (-0.23, P<0.01); diplopia assessment (-0.29, P<0.001); and asthenopia assessment (-0.45, P<0.001) Determined that the hypothesised 5 domains of the A&SQ are valid. Report that the domain of distance estimation ought to be separated into 2 domains (questionnaire developed from clinical experience and literature evidence issued 4 weeks after treatment started Used factor analysis to identify three treatment-related factors; adverse effects (5 items), compliance (4 items) and social stigma (2 items) Internal consistency Cronbach's α = 0.82 for adverse effects; 0.81 for compliance; 0factor analysis to identify three treatment-related factors; adverse effects (8 items), compliance (5 items) and social stigma (3 items) Internal consistency Cronbach's α = 0.85 for adverse effects; 0.85 for compliance; 0-item questionnaire issued. Subsequent removal of 132 items leaving 49 items for factor analysis Final questionnaire (20-items) issued in second phase, containing two domains (psychosocial and function) Completed AS-20 and VFQ-25 Compared results with normal VFQ-25 data and normal AS-20 data Overall Cronbach's α = 0.94 (psychosocial 0.95; function 0.94) Significantly lower scores for strabismus patients (56) compared with controls (98; P<0.001); and patients with other eye conditions (88, P<0.001) Of strabismus patients, scored below normal on AS-20 than VFQ-25 (90% cf 29%, child versions developed (5-7 yrs and 8-17 yrs); proxy version and parent version Questionnaires issued to patient and control groups Child questionnaire Cronbach's α =0.93 Proxy questionnaire Cronbach's α =0.97 Parent questionnaire overall Cronbach's α = 0.92 (psychosocial =0.94; function =0.94; surgery =0.91) DRT = Dutch Reading Test

Figure 1 PRISMA

 1 Figure 1 PRISMA Flow Diagram of Study Identification

Table

  . No language restrictions were applied to the search.

	14. 11 or 12 or 13
	15. 10 and 14
	Table 1	Search strategy employed
	1. orthoptic
	2. strabismus
	3. amblyopia
	4. visual acuity
	5. vision
	6. loss
	7. impairment
	8. 5 and 6
	9. 5 and 7
	10. 1 or 2 or 3 or 7 or 8 or 9
	11. questionnaire
	12. quality of life
	13. cosme*

Table 2

 2

		Assessment of HRQoL measures
		Definition	Other comments
	Reliability	• "ability of a measure to	• can be over time or between
		reproduce the same value	methods of administration[19] may be
		on two separate occasions	considered in terms of internal
		when there has been no	consistency (the extent to which all
		change in health".[19]	items measure the same concept or
			test-retest reliability (the extent to
			which the results of the instrument
			compare if the test is administered to
			the same subject on more than one
			occasion when there has been no
			demonstrable change of health status)
	Validity	• the extent to which a	• may be considered in terms of
		measure reflects the	content validity ("degree to which the
		concept that it is intended	instrument is reflective of aspects
		to measure	important to the patients and disease
			of interest"); construct validity "how
			well a measure correlates with other
			indicators of similar or related
			constructs"); concurrent validity ("the
			extent to which an instrument
			correlates to other measures of the
			same or similar construct"); and

Table 4

 4 Summary of research results of published studies using orthoptic-specific HRQoL instruments

	Reference	Sample	Description of study	Results
	A&SQ
	Van de Graaf et al.,	n=68 current amblyopic	Clinical experts identified problems patients	Average scores for SF-12, VFQ-25 and
	Netherlands	and strabismic patients	with strabismus and amblyopia experience	A&SQ were 47.16, 93.66, 95.76 for controls;
	2004[7]	n=53 healthy controls		47.13, 92.40, 83.31 for historical cohort; and
		n=174 patients from	Complaints categorised into themes (loss of	45.77, 79.16, 67.52 for patients respectively.
		historical cohort	depth perception; diplopia and visual
			disorientation; appearance)	Cronbach's α = fear of losing better eye
				(0.93); distance estimation (0.76); visual
			From the themes, 5 domains were developed	disorientation (0.87); diplopia (0.78); social
			(distance estimation; visual disorientation;	contact and cosmetic problems (0.83)
			fear of losing better eye; diplopia;
			appearance)

Acknowledgements:

Competing Interests: Nil

Funding: This work is produced by Jill Carlton under the terms of Personal Development Award research training fellowship issued by the NIHR. The views expressed in this publication are those of the author(s) and not necessarily those of the NHS, The National Institute for Health Research or the Department of Health.

What is known about this topic?

The use of HRQoL measures in health-care is becoming increasingly common, and can be used to help inform clinical decision-making and policy. The HRQoL implications of strabismus and/or amblyopia have been documented previously; however, quantifying the impact of these conditions has been scarcely reported.

What this study adds:

This study identifies existing HRQoL measures that can be used in the investigation and management of patients with amblyopia and/or strabismus.

Copyright licence statement:

The presence of strabismus can be associated with three main HRQoL issues: poor cosmetic appearance, diplopia and loss of stereopsis. The psychosocial implications of strabismus have been well documented. [START_REF] Satterfield | Psychosocial aspects of strabismus study[END_REF][START_REF] Menon | Study of the psychosocial aspects of strabismus[END_REF][START_REF] Olitsky | The negative psychosocial impact of strabismus in adults[END_REF][START_REF] Archer | Social and emotional impact of strabismus surgery on quality of life in children[END_REF] The impact of amblyopia upon a person's HRQoL has not been as adequately explored. It is possible that the impact upon HRQoL may be related more to the treatment of amblyopia, rather than the condition itself. To investigate the impact of amblyopia upon HRQoL, it would be appropriate to use the ATI; this instrument was specifically designed to quantify the impact of amblyopia on the child and the family. On a similar note, to investigate the impact of strabismus alone on HRQoL, the AS-20 could be used. An argument against the A&SQ as a measure is that if decreased HRQoL was found, could the detriment to HRQoL be attributed to amblyopia or strabismus, or indeed both. Whilst it may not be necessary to make such a differentiation, this disadvantage of the A&SQ as a HRQoL measure should be acknowledged.

Practical issues

All of the measures described are short instruments and as such can be easily administered, and can be considered as low burden for the patient/parent (see Table 3).

Each instrument involves a five-point Likert-type scale, with a three-point scale used for the child-respondent version aged 5-7 years in the IXTQ measure. Only the IXTQ contains a child-respondent version. It has been recognised that proxy reporting of HRQoL can differ from self-reported HRQoL, particularly paediatric HRQoL. [START_REF] Matza | Assessment of health-related quality of life in children: A review of conceptual, methodological, and regulatory issues[END_REF] Parents have been shown to be more able to judge physical HRQoL components compared to emotional or social components. [START_REF] Eiser | Can parents rate their child's health-related quality of life? Results of a systematic review[END_REF] It is possible that the HRQoL implications of strabismus and amblyopia are different depending upon whether the child or adult perspective is taken. Ethnic differences in the impact of strabismus


Bhaskar Gupta

Temilade Adewoyin

Sheryl-Kay Patel

Sobha Sivaprasad
email: sobha.sivaprasad@nhs.net

Ms Sobha

MS, FRCOphth Sivaprasad Dm

COMPARISON OF TWO INTRAVITREAL RANIBIZUMAB TREATMENT SCHEDULES FOR NEOVASCULAR AGE-RELATED MACULAR DEGENERATION

Keywords:

Ranibizumab

Neovascular age

Introduction

Ranibizumab (Lucentis), a humanized antibody fragment that inhibits vascular endothelial growth factor (VEGF-A), is widely used for the treatment of neovascular age-related macular degeneration (NV-AMD). The objective of this study was to compare the outcomes of two different treatment protocols: loading dose (LD) and a pro re nata (PRN) dosing schedule from baseline.

Methods

This retrospective chart review was conducted at King's College Hospital, London, UK. Consecutive patients were identified using the "Ranibizumab in NV-AMD" database. These patients had treatment-naive choroidal neovascularization (CNV) secondary to AMD, received ranibizumab therapy and had completed 12 months of follow-up. Baseline examination included visual acuity (ETDRS letters), slit-lamp biomicroscopy, fluorescein angiography, and qualitative and quantitative assessment of central macular characteristics on optical coherent tomography (OCT). Intravitreal ranibizumab (0.5 mg/0.05 ml) was given to all patients at baseline. Patients on LD regimen received two further consecutive monthly intravitreal ranibizumab injections independent of clinical findings. Further injections were determined by the same retreatment criteria as patients on PRN schedule from baseline.

The main outcome variables in the two treatment groups were visual acuity and central macular thickness at different time points.

Results

The LD group contained 47 patients and the PRN group contained 31 patients.

There were no significant differences between groups in the mean changes in visual acuity or central macular thickness. Visual acuity was similar in both groups at 6 INTRODUCTION Ranibizumab (Lucentis) is a humanized antibody fragment designed to bind and inhibit all vascular endothelial growth factor (VEGF-A) isoforms. [START_REF] Rosenfeld | Ranibizumab for neovascular age-related macular degeneration[END_REF] It is currently indicated for use in neovascular age-related macular degeneration (NV-AMD) based on the MARINA and ANCHOR trials, which showed that monthly intravitreal injections of the drug stabilized visual acuity in 90-95% of patients irrespective of lesion subtype and improved visual acuity in 1 in 3 cases 2;3 . In 2006, the United States Food and Drug Administration approved the use of ranibizumab for the treatment of NV-AMD. Following analyses of clinical efficacy and cost-effectiveness of the drug for this condition, the National Institute of Clinical Excellence (NICE) in the UK approved the use of this drug as a loading dose (LD) consisting of three initial injections at monthly intervals followed by OCT (optical coherence tomography) guided re-treatment [START_REF] Fung | An optical coherence tomography-guided, variable dosing regimen with intravitreal ranibizumab (Lucentis) for neovascular age-related macular degeneration[END_REF] . Many patients were initiated on this therapy as soon as the drug was licensed and before the NICE appraisal was completed. These patients received a pro re nata (PRN dosing schedule) based on an OCT-guided re-treatment criteria from the start of the therapy. The objective of this study is to compare the outcomes of the two different protocols: Loading dose (LD) versus PRN dosing schedule from baseline (LD versus PRN).

Methods

This retrospective chart-review was conducted at King's College Hospital. The Institutional Review board and the Clinical Effectiveness Department of the hospital approved the project. The PRN cohort was defined as the cohort before the implementation of the NICE guidance and the LD cohort included consecutive patients soon after the changes of treatment regimen was made to comply with the guidance. All patients were treatment-naive NV-AMD that were initiated on ranibizumab therapy and have completed at least 12 months follow-up. Eyes with subfoveal choroidal neovascularisation (CNV) secondary to AMD of any lesion subtype with lesion size of less than 12 disc areas and visual acuity between 24 and 73 ETDRS (Early Treatment Diabetic Retinopathy Study) letters were included.

Exclusion criteria included patients with CNV secondary to causes other than AMD, lesions previously treated with laser photocoagulation, intravitreal triamcinolone, intravitreal bevacizumab or photodynamic therapy.

Baseline examination

Each patient underwent best corrected visual acuity measurement with ETDRS charts at 2 metres and slit-lamp biomicroscopy. Fundus fluorescein angiography was done to assess the lesion characteristics. Baseline qualitative and quantitative assessment of central macular characteristics were measured by OCT (Stratus III OCT, Carl Zeiss, Dublin, CA) utilizing 6 diagonal 6-mm radial line scans and fast macular thickness scans respectively. Intravitreal ranibizumab (0.5mg/0.05ml) was given to all patients at baseline.

Re-treatment criteria

Follow-up assessments of visual acuity were done by trained personnel in a busy clinic setting and refraction was not repeated. Biomicroscopy and OCT examinations were done at each monthly visit. Fundus fluorescein angiography was repeated only if an increase in lesions size or new haemorrhage was noted on slit lamp biomicroscopy. Patients on LD regimen received two further consecutive monthly intravitreal ranibizumab injections independent of clinical findings. Further injections were determined by the same re-treatment criteria as patients on PRN schedule from baseline.

Criteria for retreatment included persistence 1) recurrence of any subretinal fluid or intra-retinal fluid on OCT in a previously dry macula; 2) increase or new sub-retinal fluid (SRF) and/or intraretinal fluid (IRF) on OCT; 3) decrease of five letters in visual acuity associated with fluid on OCT; 4) new subretinal or intraretinal haemorrhage and/or angiographic evidence of increase in lesion size.

The presence of pigment epithelial detachments (PED) was not considered as a retreatment criterion.

Outcome measures

Main outcome variables in the two treatment groups were visual acuity and central macular thickness at different time points. One month was defined as an interval of 30 10 days. Last observation carried forward method was applied to replace missing values to the last measurement. The influence of baseline lesion characteristics on final visual outcome was analyzed using a linear regression model. Statistical significance was set at p<0.05 for all analyses.

Results

Patient Demographics

Seventy-eight eyes of 78 consecutive patients that met the inclusion criteria were included in this study. There were more females (n=50) than males (n=28).

Average age of patients was 80.67 years (range 67-91). Mean duration of symptoms was 3.28 months (range 1 week -12 month). Average time interval from point of first diagnosis (optician or general ophthalmologist) to treatment was 5.3 weeks (range 0-24). The main reasons for the delay to initiate treatment were delay in referral from one hospital to another and approval of funding of the drug before the recommendations of NICE guidelines in 2008. The baseline characteristics of the two groups are shown in Table 1.

Table 1. Baseline characteristics of the patients in the two groups.

Notes: LD = Loading dose; PRN = pro re nata; CNV = choroidal neovascularization.

Visual outcome

Baseline mean visual acuity at baseline was 48 ± 15.25 ETDRS letters in the LD group and 44.48 ±15.41 ETDRS letters in the PRN group. The proportions of patients that gained vision (≥15 letters) was 29.8% in the LD group compared to 12.9% in the PRN group (p=0.01). There were no statistical differences in the proportions that stabilized vision (loss of less than 15 letters) or lost vision (loss of 15 or more letters) in the two groups at 12 months (table 2). There were also no significant differences in mean change in visual acuity at any time-point during the 12 months (figure 1). At the end of 12 months, the LD group gained a mean of 4

Central Macular Thickness (CMT)

There was no significant differences in CMT at any time points (table 3).The influence of baseline lesion characteristics (SRF, IRF, PED, macular volume) on the initial visual acuity and final visual acuity post treatment was analysed using a stepwise linear regression model with SRF, IRF and PED handled as categorical variables (table 4). None of the baseline characteristics were found to have any influence on outcome post treatment between the two groups. Notes: OCT = optical coherence tomography; SRF = subretinal fluid; IRF = intraretinal fluid; PED = pigment epithelial detachment; IOL = intra-ocular lens.

Treatment frequency

The mean number of ranibizumab injections was 4.5 in the PRN group and

Sub-group analyses

The patients in the PRN group were classified into those that required less than or equal to 3 injections or more than 3 to assess predictive factors that could determine lesions that require less number of injections (Table 6). There were no significant differences in baseline features that could predict the need for less number of injections. In the PRN group: 9.68% patients received only 1 injection, 64.52% received 2 injections and 25.80% received 3 injections in the first 3 months. Reanalysis of the visual outcome after excluding the patients who received 3 injections in the PRN cohort did not reveal and significant difference. In the LD group, only 12.77% of the eyes received a total of 3 injections only.

Discussion

We compared two treatment regiments with ranibizumab for NV-AMD. The LD received a loading dose of 3 ranibizumab injections followed by an as-needed dosing schedule. The PRN group received ranibizumab injections as-needed from baseline.

The re-treatment criteria for the as-needed part of the arms were derived from the PrONTO study that showed that an OCT-guided regiment resulted in a mean gain of +9.3 letters with a mean injection frequency of 5.6 [START_REF] Lalwani | A variable-dosing regimen with intravitreal ranibizumab for neovascular agerelated macular degeneration: year 2 of the PrONTO Study[END_REF] . Our study showed that the mean gain in vision in the whole cohort was +4.2 letters with a mean injection frequency of 5.41 injections in 12 months. Although the dosing schedules were different in the two groups, the overall re-treatment criteria after 3 months were similar in the two groups. We also believe that we had a lower threshold to treat compared to the ProNTO study at 12 months because we did not consider any minimum numerical changes in central macular thickness and treatment was initiated if any fluid was present on OCT. Despite that, the overall result of the cohort showed that the mean gain in visual acuity was only approximately half that obtained in the PrONTO study.

Caution should be expressed when we compare our cohort with the PrONTO study because the PrONTO cohort were not treatment-naïve. In fact, the study better reflects the visual gain achieved by the SUSTAIN study of +3.6 letters at 12 months with a mean injection rate of 5.7 at 12 months [START_REF] Kirchhof | Visual Acuity and Central Retinal Thickness Response Profiles and Treatment Patterns in Patients With Neovascular AMD Treated With Ranibizumab in the SUSTAIN Study. On Behalf of SUSTAIN study Group[END_REF] . The SUSTAIN study evaluated the outcome of 3 loading doses of ranibizumab followed by as-needed OCT-guided dosing regimen on 531 patients with NV-AMD. Similarly, our results also mirror the outcomes of the lucentis monotherapy arm of the MONT BLANC study that showed a mean gain of 4.4 letters with a mean injection rate of 5.1 at end of 12 months. So our study mirrors the results of the SUSTAIN and MONT BLANC studies and demonstrates that a PRN dosing schedule with a mean of 5 injections annually will only result in a gain of approximately one ETDRS line of vision in real-life settings [START_REF] Neuner-Jehle | Clinical Pharmacogenetic Analysis of Patients With Neovascular AMD Treated With Ranibizumab in the EXCITE Trial[END_REF] .

This study also showed that nearly three times as many patients in the LD group gained vision compared to the PRN group at 12 months. In fact, on further analyses of frequency of injections, the time point of maximum gain of vision in both groups correlates with greatest frequency of injections given. The LD had the maximum mean gain in vision of +7.7 letters at 3 months and these results do not differ significantly with the results obtained in the SUSTAIN study of +5.8

respectively. The PRN group had their maximum visual gain at 6 months (+6.8 letters) and this correlates to the maximum number of injections given to the PRN group in this period. However, the maximum gain in the PRN group is less than the LD group suggesting that 3 loading doses are required to achieve maximum potential gain of vision.

The visual outcomes at different time points were not dependent on any baseline features of the subfoveal lesion or OCT characteristics. Although the sample size was small for sub-group analyses of the PRN group, there were no obvious predictive factors that determined the groups of patients that required less than 3 injections based on the re-treatment criteria used in this study.

Sub-group analyses of the ANCHOR and MARINA studies indicated that the baseline visual acuity, lesion size and age were the important predictors of final visual outcome. This study did not show any of these factors to be relevant probably due to the smaller sample size but more importantly, it may be due to the fact that the injection frequency may be the most important predictor of final visual outcome.

Our study substantiates the study by Dadgostar et al [START_REF] Dadgostar | Evaluation of injection frequency and visual acuity outcomes for ranibizumab monotherapy in exudative age-related macular degeneration[END_REF] However, the mean change in visual acuity in the other eye was -1.5 ETDRS letters suggesting that the report is an accurate estimate of changes in visual acuity. Recent publications of several studies of real-life outcomes of OCT-guided ranibizumab therapy show significant differences in outcomes indicating that re-treatment based on changes in visual acuity and OCT may not be sufficient to obtain optimal results.

A recent study showed that other tests of visual function such as microperimetry may be a superior tool compared to visual acuity [START_REF] Squirrell | Visual Outcome After intravitreal Ranibizumab For Wet Age-Related Macular Degeneration: A Comparison Between Best-Corrected Visual Acuity and Microperimetry[END_REF] . Similarly, this study was based on Stratus OCT (Ziess, Germany) and now with the availability of new spectral domain OCT, patients in the same clinic settings are requiring more injections suggesting that we may have under-treated patients previously using time domain OCT.

Despite the significant increase in the number of hospital visits for patients, increased work-load for retinal specialists and consequent increase in economic burden to healthcare providers, this study and the review of published studies show that improvement of visual acuity is best achieved with a loading dose of 3 injections and higher rate of injections. These findings should be validated in further randomised clinical trials.

Disclosure: Sobha Sivaprasad has received research and travel grants from Novartis, Pfizer and Allergan.

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non-exclusive for government employees) on a worldwide basis to the BMJ Group and co-owners or contracting owning societies (where published by the BMJ Group on their behalf), and its Licensees to permit this article (if accepted) to be published in [BJO] and any other BMJ Group products and to exploit all subsidiary rights, as set out in our licence."

) 9 /

 9 20(45.0%) Notes: PRN = pro re nata; N = number; SD = standard deviation; DA = disc area; OCT = optical coherence tomography; CMT = central macular thickness; SRF = subretinal fluid; IRF = intraretinal fluid; PED = pigment epithelial detachment; CNV = choroidal neovascularisation.. Complications Complication included retinal pigment epithelial tear in 7 eyes (4 in LD group and 3 in PRN group) and one eye in the PRN group developed acute anterior uveitis which was successfully treated with a course of topical steroids and cycloplegic drops. All the eyes with retinal pigment epithelial tear had pigment epithelial detachment at baseline presentation. There were no cases of endophthalmitis or retinal detachment.

FIGURE 1 :

 1 FIGURE 1: Mean change in visual acuity at each follow up visit in the two groups:

FIGURE 2 :Fig. 1 :Fig 2 :

 212 FIGURE 2: Number of injections in each treatment groups: loading dose (LD) versus

Table 2 . Visual outcome (ETDRS letters) in the two groups.

 2 the PRN group gained 4.03 letters. The maximum mean gain in visual acuity was noted at the end of 3 months in LD of +7.7 letters while the PRN group reached a maximum of + 6.2 letters at 6 months.

	.44

Table 3 . Comparison of change in central macular thickness in the two treatment groups over time.

 3

	Treatment
		1 mth	2 mth	3 mth	6 mth	9 mth 12 mth
	Group
	LD	-48.5	-73.0	-56.0	-44.0	-33.0	-37.5
	PRN	-51.0	-28.0	-20.0	-31.5	-33.5	-43.5
	P value	0.61	0.08	0.06	0.74	0.81	0.62
	Notes: LD = loading dose of 3 injections; PRN = pro re nata

Table 4 .

 4 Regression analysis investigating the influence of baseline lesion characteristics on final visual outcome.

			Standard		95%	95%
	Variables	Coefficient		P value
			Error		Lower	Upper
	Sex	6.825	3.578	0.0609	-0.321	13.970
	Age (years)	-0.106	0.238	0.6576	-0.580	0.369
	Laterality	-0.656	3.711	0.8602	-8.067	6.755
	Symptom
		-1.335	0.900	0.1429	-3.134	0.463
	duration (months)
	Lesion type	-1.136	1.676	0.5006	-4.484	2.212
	Baseline visual
		0.953	0.135	0.2000	0.683	1.223
	acuity
	OCT central
		0.0461	0.0348	0.1903	-0.0235	0.1156
	thickness
	OCT central
		1.756	1.992	0.3814	-2.223	5.734
	volume
	SRF	-13.692	6.887	0.0510	-27.446	0.063
	IRF	0.757	3.785	0.8422	-6.803	8.317
	PED	3.367	3.626	0.3565	-3.874	10.609
	Lesion size (disc
		-6.006	3.961	0.1343	-13.917	1.904
	area)
	Lens status:
		0.515	5.740	0.9288	-10.948	11.978
	Phakia/ IOL

Table 5 .

 5 6.00 in the LD group. Overall 422 injections were performed for this cohort during the 12 month period, with a mean number of injections of 5.41. The distribution of injections every 3 months in the two groups is shown in table 5. The mean numbers of injections received by the two groups at different time points are shown in figure2. Relation of injection frequency with each regiment.

	At the time of last follow up, 15/47 (31.91%) eyes in LD group and 11/31 (35.48%) in
	the PRN group were still active as defined by the criteria for re-treatment stated
	above.
		0 -3	4 -6	7 -9	10 -12	Total
		months	months	months	months	Injection
						286
	LD (n =	141 (mean	46 (mean	50 (mean	49 (mean
						(mean 6)
	47)	3)	0.87)	1.06)	1.04)
	PRN (n =	53 (mean	32 (mean	23 (mean	30 (mean	136 (mean
	31)	1.77)	1.03)	0.78)	0.97)	4.5)
	Total	194	77	72	79	422
	p-value	P = 0.63	P = 0.66	P = 0.64	P = 0.65	P=0.61

Mean number of injections: 5.41 for the whole cohort. Note: LD = loading dose; PRN = pro re nata.

Table 6 .

 6 Sub-group analysis comparing those in the PRN group who required ≤ 3 injections and > 3 injections.

	Characteristics	< 3 Injections	> 3 Injections
	N (%)	11 (35.4%)	20 (64.6%)
	Age in years: mean ± SD	80.27 ± 4.77	82.18 ± 6.16
	Range	77 -91	74 -97
	Male	4	8
	Female	7	12
	Predominantly classic CNV	2	6
	Minimally classic CNV	4	6
	Occult CNV	5	8
	Mean lesion size ± SD (in DA)	2.47 ± 1.41	2.88 ± 2.48
	Range	0.87 -5.78	0.79 -11.79
	≤ 4 DA: n (%)	9/11 (81.8%)	18/20 (90.0%)
	>4 DA: n (%)	2/11 (18.2%)	2/20 (10.0%)
	Mean Baseline VA	42.4 ± 13.3	46.0 ± 15.6
	Mean VA at 3 month	49.7 ± 18.2	54.35 ± 17.7
	Mean VA at 6 month	47.8 ± 19.6	52.4 ± 18.3
	Mean VA at 9 month	46.2 ± 19.9	53.7 ± 19.5
	Mean VA at 12 month	43.2 ± 23.8	53.1 ± 20.0
	OCT CMT

  that indicated that visual improvement after ranibizumab is related to the frequency of injections received and not to the resolution of fluid by OCT. Michalova et al[START_REF] Michalova | Ranibizumab treatment for neovascular age-related macular degeneration: from randomized trials to clinical practice[END_REF] demonstrated results similar to the MARINA and ANCHOR study on 185 patients with a mean injection rate of 9 injections in 12 months. Although the baseline mean visual acuity was better (57.6 ±15.5 ETDRS letters) in that study compared to this study, the higher rate of injections may also explain the difference in visual outcome between the studies.There are two published studies that used a PRN dosing schedule from baseline. Both Rothenbuehler et al[START_REF] Rothenbuehler | Effects of ranibizumab in patients with subfoveal choroidal neovascularization attributable to age-related macular degeneration[END_REF] and Querques er[START_REF] Querques | Ranibizumab for exudative age-related macular degeneration: 24 months outcomes from a single-center institutional setting[END_REF] and associates reported visual acuity comparable to MARINA, ANCHOR and PrONTO studies. Despite the same treatment protocol, the mean number of injections in Rothenuehler study was 5.6 ± 2.9 at 12 months and that of Querques was 5.10 ± 2.5 but our PRN group required a mean of 4.5± 2.0 injections only. The limitations of our study are the retrospective nature and the lack of standardized visual acuity measurements.


MD Raffaele Parrozzani

Daniela Lazzarini

Ernesto Alemany-Rubio

Francesca Urban

MD Edoardo Midena
email: edoardo.midena@unipd.it

Topical 1% 5-Fluoruracil in Ocular Surface Squamous Neoplasia: a Long-term Safety Study

Keywords: Ocular surface, neoplasia, drugs, cornea, conjunctiva

Background/aims: The aim of this study was to evaluate long-term corneal toxicity of topical chemotherapy with 1% 5-Fluoruracil (5-FU) as a sole or adjuvant treatment of ocular surface squamous neoplasia (OSSN).

Methods: Forty-one consecutive cases of OSSN were included in this prospective study. Patients underwent topical chemotherapy with 1% 5-FU four times/day for 4 weeks (one course). Adjunctive courses were were repeated until clinical and cytological tumor regression.

Clinical confocal microscopy (CCM) (Confoscan 4.0, Nidek Co Ltd, Gamagori, Japan) was used to check for 5-FU long-term corneal toxicity.

Results: Mean follow-up was 89.7±14.4 months (range, 63-122 months). Twenty-two patients (53.7%) underwent topical 5-FU as a sole treatment, whereas nineteen patients (46.3%) as adjuvant and/or debulking therapy. Mean number of 5-FU cycles was 1.9 (range, 1-5 cycles).

Three tumors (7,3%) treated with 5-FU alone recurred during follow-up. Recurrences were successfully treated with additional 5-FU courses. CCM showed no long-term difference between treated eye and fellow (control) eye in: endothelial cells count, pleomorphism and polymegatism, anterior stromal keratocyte density, sub-basal nerve plexus fibre number, density, and beadings and central cornea epithelium thickness (p=n.s.).

Conclusion:

Topical 5-FU, as a sole or combined therapy, must be considered a long-term safe and effective treatment for patients affected by OSSN.

INTRODUCTION

Ocular surface squamous neoplasia (OSSN) is a spectrum of disease with the potential of visual loss and local morbidity. [START_REF] Lee | Ocular surface squamous neoplasia[END_REF] OSSN are traditionally seen in Caucasian older male or in lighter skinned populations in tropical countries. [START_REF] Lee | Ocular surface squamous neoplasia[END_REF][START_REF] Basti | Ocular surface squamous neoplasia: a review[END_REF] Risk factors for this disease include: ultraviolet light exposure, petroleum products, cigarette smoking, human papillomavirus and human immunodeficiency virus infection. [START_REF] Napora | Factors associated with conjunctival intraepithelial neoplasia: a case control study[END_REF] The term OSSN encompasses corneo-conjunctival intraepithelial neoplasia (CIN) and invasive squamous cell carcinoma (SCC). [START_REF] Nolan | Application of impression cytology to the diagnosis of conjunctival neoplasms[END_REF] These lesions are commonly seen in the exposed corneo-conjunctival areas and described as being slightly elevated, variably shaped, relatively sharply demarcated from the surrounding normal tissues, accompanied by feeding blood vessels and having from pearly gray to reddish gray appearance. [START_REF] Lee | Ocular surface squamous neoplasia[END_REF] Surgical excision with adjunctive cryotherapy was formerly considered the treatment of choice. [START_REF] Tunc | Intraepithelial and invasive squamous cell carcinoma of the conjunctiva: analysis of 60 cases[END_REF] However, recurrence rates after surgery are significant, even when free surgical margins were achieved. [START_REF] Tunc | Intraepithelial and invasive squamous cell carcinoma of the conjunctiva: analysis of 60 cases[END_REF] Topical chemotherapy has become a valuable therapeutic option for the treatment of ocular surface tumors in the last decade. 5-Fluoruracil (5-FU), Mitomicyn C (MMC) and Interferon α2b (INFα) have been introduced, and are currently widely used, as both adjuvant therapy (to prevent tumor recurrence) and primary treatment of OSSN. [START_REF] Midena | Treatment of conjunctival squamous cell carcinoma with topical 5-fluorouracil[END_REF][START_REF] Karp | Treatment of conjunctival and corneal intraepithelial neoplasia with topical interferon alpha-2b[END_REF][START_REF] Frucht-Pery | Mitomycin C treatment for conjunctival-corneal intraepithelial neoplasia: a multicenter experience[END_REF] Although many reports suggest a shortterm safety of topical chemotherapy with 1% 5-FU, only few data were reported on a long-term base. [START_REF] Midena | Treatment of conjunctival squamous cell carcinoma with topical 5-fluorouracil[END_REF][START_REF] Midena | Conjunctival squamous cell carcinoma treated with topical 5-fluoruracil[END_REF][START_REF] Poothullil | Topical medical therapies for ocular surface tumors[END_REF] Moreover, morphologic and morphometric analysis of corneal layers was never performed in-vivo, at microscopic level, after topical chemotherapy. The aim of this study was to evaluate long-term corneal toxicity of topical chemotherapy with 1% 5-FU, as a sole or adjuvant treatment of OSSN, using Clinical Confocal Microscopy (CCM).

MATERIAL AND METHODS

This study complied with the tenets of the Declaration of Helsinki and was approved by the IRB of our Institution. Each patient was recruited from those referred to the Ophthalmic Oncology Unit of our Departments and underwent a baseline full ophthalmologic examination. Clinical and demographic characteristics were collected, including: age, gender, tumor location (cornea, conjunctiva, corneo-conjunctival) and tumor largest basal diameter. Adjunctive tumor clinical features such as nodularity, mutifocality, prominent vascularization (presence of macroscopically evident tumor vessels or conjunctival feeder vessels) and fornix involvement were also reported. [START_REF] Basti | Ocular surface squamous neoplasia: a review[END_REF] Tumor clinical aspect was documented by anterior segment photography. Cytologic analysis (scraping technique) was obtained from all patients and reported as low-grade dysplasia (cells with enlarged nuclei, hyperchromasia and irregular contour of the nuclear membrane with increased nuclear/cytopasmic ratio) or high-grade dysplasia (pleomorphism of the nucleus with dyskeratotic cells. [START_REF] Lee | Ocular surface squamous neoplasia[END_REF] The presence of syncytial sheath, nucleoli and infiltration of inflammatory cells was reported as invasive SCC. [START_REF] Lee | Ocular surface squamous neoplasia[END_REF] Each patient was treated with 1% 5-FU as a sole or in a surgical combined approach. Our standard protocol consists in topical instillation of 1% 5-FU prepared in aqueous solution and administered four times a day during the course of 4 weeks (one course). [START_REF] Midena | Treatment of conjunctival squamous cell carcinoma with topical 5-fluorouracil[END_REF][START_REF] Midena | Conjunctival squamous cell carcinoma treated with topical 5-fluoruracil[END_REF] Patients were instructed about possible 5-FU local side effects. After instillation of the eyedrops the inferior lacrimal punctum was briefly occluded and an eye ointment was applied on the inferior eyelid skin to minimise skin contact with the drug. A new medication bottle was used for one entire course and than returned to the pharmacy. Adjunctive courses were administered after three months of free interval and repeated until clinical and cytological tumor regression. Patients treated with topical chemotherapy as a combined treatment underwent wide surgical excision followed by cryotherapy to the cut edges of the conjunctiva. Alcohol epitheliectomy was performed for corneal involvement. Large tissue defects were managed by amniotic membrane reconstruction of the ocular surface. Topical chemotherapy was administered at least three weeks after surgery, following our standard protocol. [START_REF] Midena | Treatment of conjunctival squamous cell carcinoma with topical 5-fluorouracil[END_REF][START_REF] Midena | Conjunctival squamous cell carcinoma treated with topical 5-fluoruracil[END_REF] Follow-up was performed monthly until clinical resolution, after 1, 3, and 6 months and every 6 months thereafter. Follow-up included full ophthalmologic examination, slit lamp biomicroscopy and anterior segment photography. Scraping cytology was also performed after 1 and 6 months of complete clinical tumor regression. Follow-up was longer than 5 years.

Corneal confocal microscopy

In-vivo corneal multi-layer analysis was performed using clinical confocal microscope Confoscan 4.0 (Nidek Co Ltd, Gamagori, Japan) equipped with a 40X immersion objective lens (Zeiss Inc, Oberkochen, Germany) and the Z-ring adapter system designed for full thickness examination of the cornea. Inclusion criteria were: patients affected by OSSN, treated with 1% 5-FU (as a sole or combined therapy), with follow-up longer than 5 years. Exclusion criteria were: any coexisting chronic cornea-conjunctival disease, unilateral cataract surgery, other mono-or bilateral intraocular surgery or refractive surgery, unilateral chronic use of preserved eyedrops, glaucoma therapy or other topical therapy. Confocal microscopy examination was performed according to a standard procedure. [START_REF] Brugin | Central Corneal thickness Z-Ring Corneal Confocal Microscopy Versus Ultrasound Pachimetry[END_REF][START_REF] Midena | Corneal diabetic neuropathy: a confocal microscopy study[END_REF][START_REF] Midena | Confocal microscopy of sub-basal nerve plexus: a quantitative and qualitative analisys in healthy and pathologic eyes[END_REF] Endothelial cell density (no. of cells/mm 2 ), pleomorphism (percentage of hexagonal cells) and polymegatism (percentage of oversized cells) were automatically calculated selecting the best-focused image (Fig 1A). Anterior stromal keratocyte density (cells/mm 2 ) was manually calculated selecting the first focused image of corneal stroma (Fig 1B). [START_REF] Brugin | Central Corneal thickness Z-Ring Corneal Confocal Microscopy Versus Ultrasound Pachimetry[END_REF][START_REF] Midena | Corneal diabetic neuropathy: a confocal microscopy study[END_REF][START_REF] Midena | Confocal microscopy of sub-basal nerve plexus: a quantitative and qualitative analisys in healthy and pathologic eyes[END_REF] Epithelium thickness was defined as the distance between the last focused image of the subbasal nerve plexus and the last focused image of the corneal epithelium, as described by Li et al. [START_REF] Li | Epithelial and corneal thickness measurements by in vivo confocal microscopy through focusing (CMTF)[END_REF] Subbasal nerve plexus was evaluated choosing the best focused image of this structure. [START_REF] Midena | Confocal microscopy of sub-basal nerve plexus: a quantitative and qualitative analisys in healthy and pathologic eyes[END_REF] Nerve fiber density (μm/mm 2 ), number of fibers (fibers/field) and number of beadings (beadings/100μm) were quantified. Nerve fibers were traced using Neuron J program © , an image processing computer tool, to outline nerve fibers from each corneal sub-basal nerve plexus frame (Fig 1C). [START_REF] Midena | Confocal microscopy of sub-basal nerve plexus: a quantitative and qualitative analisys in healthy and pathologic eyes[END_REF] Nerve fiber density was calculated for each image as the total length of the nerves (in micrometers) divided by the area of the image (0.132 mm 2 ) and expressed as micrometers per square millimetre (μm/mm 2 ). [START_REF] Midena | Confocal microscopy of sub-basal nerve plexus: a quantitative and qualitative analisys in healthy and pathologic eyes[END_REF] Nerve fibers number was calculated as the total number of principal nerve trunks seen in the selected (best focused) image. [START_REF] Midena | Confocal microscopy of sub-basal nerve plexus: a quantitative and qualitative analisys in healthy and pathologic eyes[END_REF] Number of beadings was manually calculated as the number of well-defined hyper-reflective points per unit of length (100μm) in a single nerve fiber, randomly selected from the best-focused fibers (Fig 1D). [START_REF] Midena | Confocal microscopy of sub-basal nerve plexus: a quantitative and qualitative analisys in healthy and pathologic eyes[END_REF] Fellow eye was also evaluated and considered as a control.

Statistical analysis

Data about corneal endothelium density, pleomorphism and polymegatism, keratocyte density, number and density of nerve fibers, beadings and epithelial thickness in treated eyes were compared to the data obtained in fellow eyes using Student' s paired t-test.

RESULTS

Forty-one consecutive patients with a mean age at treatment of 65.5 ± 10.2 years (range, 44-88) were included. Demographic data, tumor characteristics and cytologic results are reported in Table 1. 5-FU is a cell cycle-specific pyrimidine analogue with cytotoxic properties due to its effect on both DNA and RNA. [START_REF] Abraham | The clinical applications of fluorouracil in ophthalmic practice[END_REF] It was first synthesized in 1957 by Dushinski et al. and was found to have significant activity against tumours of the gastrointestinal tract, head, neck and breast. [START_REF] Abraham | The clinical applications of fluorouracil in ophthalmic practice[END_REF][START_REF] Dushinski | The synthesis of 5 fluoro pyrimidines[END_REF] This drug was originally known for its widespread use as an anticancer agent, but its ability to reduce fibroblastic proliferation and subsequent scarring has made it an important adjunct in ocular and periorbital surgery. [START_REF] Abraham | The clinical applications of fluorouracil in ophthalmic practice[END_REF] 5-FU acts on rapidly proliferating epithelial cells and was therefore used as a topical treatment for pre-invasive OSSN as early as 1986. [START_REF] De Keizer | Topical application of 5-fluorouracil in premalignant lesions of cornea, conjunctiva and eyelid[END_REF] Many reports confirm the efficacy of topical chemotherapy with 5-FU as a sole or combined treatment of OSSN. [START_REF] Napora | Factors associated with conjunctival intraepithelial neoplasia: a case control study[END_REF][START_REF] Midena | Treatment of conjunctival squamous cell carcinoma with topical 5-fluorouracil[END_REF][START_REF] Midena | Conjunctival squamous cell carcinoma treated with topical 5-fluoruracil[END_REF][START_REF] Yeatts | 5-Fluorouracil for the treatment of intraepithelial neoplasia of the conjunctiva and cornea[END_REF][START_REF] Yamamoto | Successful treatment with 5-fluorouracil of conjunctival intraepithelial neoplasia refractive to mitomycin-C[END_REF] Moreover, only transient, low to mild and self-limited side effects were reported. [START_REF] Midena | Treatment of conjunctival squamous cell carcinoma with topical 5-fluorouracil[END_REF][START_REF] Midena | Conjunctival squamous cell carcinoma treated with topical 5-fluoruracil[END_REF][START_REF] Poothullil | Topical medical therapies for ocular surface tumors[END_REF][START_REF] Brugin | Central Corneal thickness Z-Ring Corneal Confocal Microscopy Versus Ultrasound Pachimetry[END_REF][START_REF] Midena | Corneal diabetic neuropathy: a confocal microscopy study[END_REF][START_REF] Midena | Confocal microscopy of sub-basal nerve plexus: a quantitative and qualitative analisys in healthy and pathologic eyes[END_REF][START_REF] Li | Epithelial and corneal thickness measurements by in vivo confocal microscopy through focusing (CMTF)[END_REF][START_REF] Meijering | Design and validation of a tool for neurite tracing and analysis in fluorescence microscopy images[END_REF][START_REF] Buus | Microscopically controlled excision of conjunctival squamous cell carcinoma[END_REF][START_REF] Yeatts | 5-Fluorouracil for the treatment of intraepithelial neoplasia of the conjunctiva and cornea[END_REF][START_REF] Abraham | The clinical applications of fluorouracil in ophthalmic practice[END_REF][START_REF] Dushinski | The synthesis of 5 fluoro pyrimidines[END_REF] Poothullil et al, reviewing the use of topical medical therapies for ocular surface tumors, reported punctuate keratitis and hyperemia as the only side effects of topical 5-FU. Other side effects included small epithelial defects, their accompanying discomfort and erythema of the eyelid skin. [START_REF] Poothullil | Topical medical therapies for ocular surface tumors[END_REF] Topical corticosteroids were usually applied to improve tolerance during the treatment, and rarely (never in our experience) patients discontinued therapy because of 5-FU side effects. [START_REF] Midena | Treatment of conjunctival squamous cell carcinoma with topical 5-fluorouracil[END_REF][START_REF] Midena | Conjunctival squamous cell carcinoma treated with topical 5-fluoruracil[END_REF][START_REF] Poothullil | Topical medical therapies for ocular surface tumors[END_REF] Although many reports suggest a short-term safety of topical chemotherapy with 5-FU, there are no data on a long-term base. [START_REF] Midena | Treatment of conjunctival squamous cell carcinoma with topical 5-fluorouracil[END_REF][START_REF] Midena | Conjunctival squamous cell carcinoma treated with topical 5-fluoruracil[END_REF][START_REF] Poothullil | Topical medical therapies for ocular surface tumors[END_REF] In-vivo evaluation of the ocular structures at high magnification (to distinguish microscopic cell details) has always been a challenge for ophthalmic clinicians and researchers, but microscopic studies have, until recently, been limited to ex-vivo investigation. Confocal microscopy was introduced in the clinical practice as a non-invasive tool to observe at high magnification the structure of human cornea and conjunctiva in-vivo. [START_REF] Brugin | Central Corneal thickness Z-Ring Corneal Confocal Microscopy Versus Ultrasound Pachimetry[END_REF][START_REF] Midena | Corneal diabetic neuropathy: a confocal microscopy study[END_REF][START_REF] Midena | Confocal microscopy of sub-basal nerve plexus: a quantitative and qualitative analisys in healthy and pathologic eyes[END_REF] CCM analysis extends, in-vivo, the principles of biomicroscopy to the microscopic range, scanning tissue layer by layer, by changing the plane of focus of the detector source. [START_REF] Brugin | Central Corneal thickness Z-Ring Corneal Confocal Microscopy Versus Ultrasound Pachimetry[END_REF][START_REF] Midena | Corneal diabetic neuropathy: a confocal microscopy study[END_REF][START_REF] Midena | Confocal microscopy of sub-basal nerve plexus: a quantitative and qualitative analisys in healthy and pathologic eyes[END_REF] Confocal microscopy was previously used to analyze in detail corneal pathological changes secondary to ocular surface disease [START_REF] Wakamatsu | Conjunctival in vivo confocal scanning laser microscopy in patients with Siogren syndrome[END_REF], refractive surgery [START_REF] Midena | Long-term effects on corneal keratocytes of mitomycin C during photorefractive keratectomy: a randomized contralateral eye confocal microscopy study[END_REF], chronic use of topical therapy [START_REF] Whitson | Assessment of corneal epithelial integrity after acute exposure to ocular hypotensive agents preserved with and without benzalkonium chloride[END_REF], systemic disease [START_REF] Midena | Corneal diabetic neuropathy: a confocal microscopy study[END_REF] and systemic pharmacological therapy [START_REF] Falke | The microstructure of cornea verticillata in Fabry disease and amiodarone-induced keratopathy: a confocal laser-scanning microscopy study[END_REF] with promising results.

Panda et al have recently analyzed the effect of topical MMC on corneal endothelium in 25 patients affected by OSSN using endothelial specular microscopy. These Authors reported that topical 0.04% MMC eyedrops do not significantly affect corneal endothelium. However they suggested that long-term data are mandatory. [START_REF] Panda | Effect of topical mitomycin C on corneal endothelium[END_REF] Moreover, this study has analyzed only endothelium and not the other corneal layers. To the best of our knowledge the present study is the first focusing on long-term safety of topical chemotherapy using full thickness clinical confocal microscopy. Our data, obtained in-vivo in a fully non-invasive way, shows no long-term corneal toxicity in eyes treated with topical chemotherapy with 1% 5-FU. Using clinical confocal microscopy we have confirmed that 5-FU is selective for neoplastic cells, demonstrating no long-term pathological change in corneal endothelium, epithelium, stroma and subbasal nerve plexus structures. These data, paired to clinical outcome of patients, suggest that topical chemotherapy with 1% 5-FU should be considered a long-term safe and effective method as a sole or combined treatment of selected OSSN.

Acknowledgment: none

Competing interest: none declared

Funding: none

Licence for Publication

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd to permit this article (if accepted) to be published in BJO and any other BMJPGL products and sublicences such use and exploit all subsidiary rights, as set out in our licence (http://group.bmj.com/products/journals/instructions-for-authors/licence-forms).

Fig 1 .

 1 Fig 1.Corneal confocal microscopy analysis (using ConfoScan 4.0) in a patient treated with 1% 5-FU. Endothelial cells were automatically selected, marked and analyzed by the software (A). Anterior stromal keratocyte density was manually calculated by the operator marking each one on-focus keratocyte (B). Nerve fibers were analyzed using Neuron J program © (C). Number of nerve beadings was manually calculated as the number of welldefined hyper-reflective points per unit of length (100μm) in a single nerve fibre.

Table 1 : demographic data, tumor characteristics and cytologic results.

 1

	# GENDER EYE AGE	LOCATION	LBD NODULARITY	VASCULARITY	MULTIFOCALITY FI CYTOLOGY
	1	M	RE	CC	5					LGD
	2	F	RE	CONJUNCTIVA	6					HGD
	3	M	LE	CC	5					SCC
	4	M	LE	CC	7					SCC
	5	F	RE	CORNEA	8		+			HDD
	6	F	LE	CC	14	+	+			LGD
	7	M	RE	CC	25			+	+	SCC
	8	F	LE	CONJUNCTIVA	12	+	+			SCC
	9	M	LE	CC	15					LGD
	10	M	LE	CC	6					SCC
	11	M	RE	CC	7					HGD
	12	F	RE	CONJUNCTIVA	7					SCC
	13	M	LE	CC	12		+			LGD
	14	F	LE	CC	14		+			HGD
	15	F	RE	CC	3					LGD
	16	M	RE	CC	11	+				SCC
	17	M	LE	CONJUNCTIVA	7					HGD
	18	M	LE	CC	7	+	+			SCC
	19	M	LE	CC	18			+	+	LGD
	20	M	LE	CC	5					HGD
	21	F	LE	CC	7					SCC
	22	M	LE	CONJUNCTIVA	6					HGD
	23	M	LE	CC	4					HGD
	24	F	LE	CONJUNCTIVA	3					SCC
	25	M	RE	CC	7	+				SCC
	26	M	RE	CC	15	+	+	+	+	HGD
	27	F	RE	CC	3					SCC
	28	F	LE	CC	17					HGD
	29	M	RE	CC	6					HGD
	30	M	LE	CONJUNCTIVA	9	+	+			SCC
	31	M	RE	CONJUNCTIVA	11	+				SCC
	32	M	RE	CONJUNCTIVA	4					SCC
	33	M	LE	CC	12	+	+			SCC
	34	M	RE	CC	6					HGD
	35	F	RE	CONJUNCTIVA	7					SCC
	36	F	RE	CC	13		+			LGD
	37	M	RE	CC	3					SCC
	38	F	LE	CC	3					HGD
	39	F	LE	CONJUNCTIVA	5					SCC
	40	M	RE	CC	14					HGD
	41	M	LE	CC	16	+	+		+	SCC
	*LBD= Largest basal diameter; FI=Fornix involvement; M=Male; F=Female; RE= Right eye;
	LE=Left eye; CC= Cornea and conjunctiva; LGD= Low-grade dysplasia; HGD= High-grade
	dysplasia; SCC= Squamous cell carcinoma.

double treatment (debulking + adjuvant) in seven cases

(17.1%)

. Histologic examination of all excided lesions confirmed baseline cytological diagnosis. Complete regression was achieved in all treated patients. Three patients (7.3%), treated with 5-FU as a sole therapy, developed early recurrences during follow-up. These patients were successfully re-treated with additional courses of chemotherapy. Mean 1% 5-FU courses number before definitive regression was 1.9 (range, 1-5). Mean follow-up was 89.7 ± 14.4 months (range, 63-122). Average age of disease onset, mean chemotherapy courses, mean follow-up and recurrences rate related to baseline cytology are reported in Table

2

.

Table 2 : Clinical and demographic characteristics related to baseline cytology.

 2

		Low-grade	High-grade	Squamous cell	Total
		dysplasia	dysplasia	carcinoma
	No. Of eye	7 (17.1%)	14 (34.1%)	20 (48.8%)	41
	Eyes (RE/LE)	3/4	7/7	9/11	19/22
	Gender (female/male)	3/4	5/9	7/13	15/26
	Average age of onset	64.4±9.1 years	64.4±10.6 years	68.7±10.3 years	65.5±10.2 years
	Mean treatment courses	1.7 (Range, 1-3)	1.8 (Range, 1-5)	2.0 (Range, 1-5) 1.9 (Range, 1-5)
	Recurrences rate	0%	7.1%	10%	7.3%
	Mean follow-up	88.2±14.6 months 90.1±14.9 months 89.9±14.3 months 89.7±14.4 months

Confocal microscopy analysis of both eyes was successfully conduced in all patients. No significant differences were observed between treated eye and fellow/control eye in: endothelial cells density, endothelial pleomorphism and polymegatism, mean keratocyte density, mean corneal nerve density, mean subbasal nerve fibers number/fields, mean nerve beadings number and mean epithelial thickness. Confocal microscopy analysis results are reported on Table 3.

DISCUSSION

The main treatment modality for OSSN remains wide surgical excision with cryotherapy (with accurate histological assessment of surgical margins) and amniotic membrane reconstruction for large tissue defects. [START_REF] Karp | Treatment of conjunctival and corneal intraepithelial neoplasia with topical interferon alpha-2b[END_REF][START_REF] Frucht-Pery | Mitomycin C treatment for conjunctival-corneal intraepithelial neoplasia: a multicenter experience[END_REF][START_REF] Midena | Conjunctival squamous cell carcinoma treated with topical 5-fluoruracil[END_REF][START_REF] Poothullil | Topical medical therapies for ocular surface tumors[END_REF] However, the long-term local recurrence rate remains high, ranging from 15% to 52%, even when surgical margins of primary excision are free from malignant cells. [START_REF] Lee | Ocular surface squamous neoplasia[END_REF] The recurrence rate associated with surgery is probably due to the difficulty in establishing clinically tumor free margins. [START_REF] Karp | Treatment of conjunctival and corneal intraepithelial neoplasia with topical interferon alpha-2b[END_REF] Unless an intraoperative frozen-section technique is used, residual tumor cells may remain. [START_REF] Tunc | Intraepithelial and invasive squamous cell carcinoma of the conjunctiva: analysis of 60 cases[END_REF][START_REF] Buus | Microscopically controlled excision of conjunctival squamous cell carcinoma[END_REF] Moreover, epithelial tumors of the conjunctiva can be multifocal or diffuse, rendering focal treatments less effective in eradicating these lesions. [START_REF] Lee | Ocular surface squamous neoplasia[END_REF][START_REF] Yeatts | 5-Fluorouracil for the treatment of intraepithelial neoplasia of the conjunctiva and cornea[END_REF] These data prompted us to introduce topical chemotherapy, as a sole or combined treatment, in a routine clinical setting. [START_REF] Midena | Treatment of conjunctival squamous cell carcinoma with topical 5-fluorouracil[END_REF][START_REF] Midena | Conjunctival squamous cell carcinoma treated with topical 5-fluoruracil[END_REF]


Dr Patrick J Horner
email: paddy.horner@bristol.ac.uk

David Taylor-Robinson

The association of Mycoplasma genitalium with balano-posthitis in men who have non-gonococcal urethritis

Keywords: Mycoplasma genitalium, balanoposthitis, balanitis, non-gonococcal urethritis

Objective: To determine whether Mycoplasma genitalium is associated with balanitis and/or posthitis in a previous study of the role of M. genitalium in men with acute non-gonococcal urethritis (NGU).

Methods:

In a previous study of men with acute NGU, the existence of balanitis and/or posthitis was recorded. Chlamydia trachomatis, M. genitalium and ureaplasmas were sought in urethral swabs and urine by using a direct fluorescent antibody test and in-house polymerase chain reaction, an in-house polymerase chain reaction and a culture method, respectively. Men were treated with doxycycline or erythromycin.

Results: M. genitalium was associated significantly (p=0.01) with balanitis and/or posthitis in 114 men with acute NGU. This association persisted when there was control for C. trachomatis and urethral discharge (p=0.021, OR 4.1, CI 1.2 -13.5). C. trachomatis and ureaplasmas were not associated with balanitis and/or posthitis.

Conclusion:

Detection of M. genitalium in men with acute NGU was associated significantly with balanitis and/or posthitis. The association is biologically plausible and may have a role in HIV-1 transmission and susceptibility.

Inflammation of the glans penis (balanitis) and inflammation of the prepuce (posthitis) frequently occur together (balanoposthitis).

Balanitis alone is seen occasionally in circumcised men. However, primary infections of the glans and prepuce with discharge and urethral involvement may occur, due to, for example, herpes simplex virus or Candida albicans and sometimes it may be difficult to ascertain whether a primary infection of the urethra (gonococcal or non-gonococcal) has led to balanoposthitis.

Following the detection by culture of Mycoplasma genitalium organisms in specimens from men with non-gonococcal urethritis (NGU) [START_REF] Tully | A newly discovered mycoplasma in the human urogenital tract[END_REF] and the development a decade later of a polymerase chain reaction (PCR) to detect the organisms sensitively, we undertook a study of the aetiology of NGU 2,3, [START_REF] Horner | Do all men attending departments of genitourinary medicine need to be screened for non-gonococcal urethritis?[END_REF] . In this, Chlamydia trachomatis and M. genitalium were independently and significantly associated with the disease. We also observed that some of the men had balanitis and/or posthitis. M. genitalium can infect vaginal epithelial cells, in vitro. [START_REF] Mcgowin | Intracellular Mycoplasma genitalium infection of human vaginal and cervical epithelial cells elicits distinct patterns of inflammatory cytokine secretion and provides a possible survival niche against macrophage-mediated killing[END_REF] These are stratified non-keratinized squamous epithelial cells [START_REF] Graney | Anatomy and physical examination of the female genital tract[END_REF] which suggests that M. genitalium might, therefore, be capable of infecting skin epithelia, particularly the foreskin which is poorly keratinised. [START_REF] Macdonald | Prevention of HIV transmission in the UK: what is the role of male circumcision?[END_REF] This prompted us to look at the association of M. genitalium with balanoposthitis in men we had studied before with urethritis. We also felt it was worthy of analysis in view of the importance of foreskin as a co-factor in HIV transmission, the ability of sexually transmitted infections to increase the transmission of HIV and the recent association of M. genitalium with HIV infection 8,9,10 .

PATIENTS AND METHODS

As detailed before Other information, such as age, ethnicity, number of sexual partners and time since last voiding urine, was recorded by the physician. The patient was examined and the occurrence of balanitis and/or posthitis and whether a discharge was present before or after urethral massage was recorded. Balanitis and/or posthitis was/were diagnosed if there was erythema of the glans and/or prepuce; no distinction was made between balanitis and balanoposthitis on the study proforma. Men with Neisseria gonorrhoeae, a herpes simplex virus infection or a urinary tract infection were excluded. Men were treated with doxycycline, 200 mg, given initially and then 100 mg per day for 13 days, or enteric-coated erythromycin, 500 mg, given four times per day for 14 days, if they were intolerant of doxycycline. Details of specific treatment for balanitis and/or posthitis were not available. A total of 86 men re-attended at least once 10-92 days after receiving treatment.

As detailed previously 2,3,4 , C. trachomatis was diagnosed using a direct fluorescent antibody test (Syva,UK) in which detection of one or more elementary bodies was regarded as a positive result on a urethral smear and/or the centrifuged deposit from a first-pass urine (FPU) specimen. An in-house C. trachomatis PCR was also undertaken at presentation on a FPU deposit from those patients who returned for follow-up. Men were considered C.

trachomatis-positive if any test was positive and C. trachomatis-negative if at least one specimen was available for examination by any test and this was negative. Results were not available for 6 men. Forty-five (42%) of 108 evaluable men were C. trachomatis-positive. A PCR was used on the centrifuged deposit from a FPU specimen to detect M. genitalium, as described before 2,3,4 . Results were not available for 4 men. Thirty-one (28%) of 110 evaluable men were M. genitalium-positive. At follow-up only men who tested M. genitaliumpositive at presentation or who had chronic urethritis were tested using the M. genitalium PCR. [START_REF] Horner | Role of Mycoplasma genitalium and Ureaplasma urealyticum in acute and chronic nongonococcal urethritis[END_REF] Ureaplasmas were sought by culture using a FPU specimen 3 . Results were not available for 5 men. Thirty-three (30%) of 109 evaluable men were ureaplasma-positive.

Speciation of the ureaplasmas was not undertaken as at the time of the study the greater significance of one or other of the two separate ureaplasma species was not appreciated.

Statistical analysis: Univariate associations were explored using Fisher's Exact test with SPSS for windows version 15 and odds ratios with 95% confidence intervals using EpiInfo version 6. Binary logistic regression was used for multivariate analyses, undertaken with SPSS for windows version 15. As detailed previously the data were not complete for every individual recruited [START_REF] Horner | Role of Mycoplasma genitalium and Ureaplasma urealyticum in acute and chronic nongonococcal urethritis[END_REF] .

RESULTS

Details of urethral discharge were available for 113 men; of these, 96 (85%) had a discharge, of whom 29 (30%) were unaware since it was present only on examination after urethral massage, 51% (57 of 112) had dysuria, 56% (63 of 113) had penile irritation and/or genital discomfort and 18% (19 of 103) balanitis and/or posthitis. Of the 113 evaluable men with details of urethral discharge available, 17 (15%) did not complain of discharge, dysuria or penile irritation and only had an urethral discharge on examination.

The presence of balanitis and/or posthitis was not associated with discharge, dysuria or penile irritation and/or discomfort (data not shown). The presence of C. trachomatis was not associated with balanitis and/or posthitis. In complete contrast, the presence of M. genitalium was associated significantly with balanitis and/or posthitis. Ureaplasmas were not associated with balanitis and/or posthitis (Table ).

We demonstrated previously 2,3,4 that M. genitalium and C. trachomatis were associated with discharge. Because of this we undertook a logistic regression analysis to control for discharge and for the presence of the other micro-organism. The detection of M. genitalium continued to be associated with balanitis and/or posthitis (p=0.021, OR 4.1, CI 1.2 -13.5)

and discharge (p=0.07, OR 7.7, CI 0.86-69.9). At follow-up examination, four men had balanitis and/or posthitis. Of these, two had had M. genitalium originally, including one man in whom balanitis and/or posthitis was/were not diagnosed at presentation; both were M.

genitalium-negative at follow-up.

DISCUSSION

The primary aim of the original study 2,3,4 had been to determine the relationship between M. genitalium and NGU. In this analysis we demonstrate that M. genitalium is associated with balanitis and/or posthitis in men with acute NGU and that this is independent of the presence of an urethral discharge.

The strength of the study is that it is a well characterised group, with detailed microbiological investigations undertaken blinded to clinical presentation. Weaknesses are that, despite including 114 men the study is still small and no distinction was made on the study proforma between balanitis and/or posthitis. Also whether any of the men with acute urethritis were circumcised was not recorded and the result cannot be extrapolated to balanitis and/or posthitis in men who are clinically asymptomatic and in whom urethritis is not present. In future studies it will be important to have balanitis and/or posthitis as the primary focus and to ascertain whether the association is with one or the other or both and consider the relation of micro-organisms to the disease in men who also have urethritis and in those who do not.

We are not aware of other studies which have investigated the association of M. genitalium with balanoposthitis. We suspect that this is because nobody has looked for it as information on the presence of balanoposthitis is not routinely collected in studies of men with acute NGU. Because patients with balanitis and/or posthitis also had urethritis, the association of M. genitalium might be with NGU and only spuriously with balanitis and/or posthitis. It was, therefore, interesting to find that an apparent real association exists between M. genitalium and balanitis and/or posthitis which is independent of the presence of urethral discharge. This is emphasised by the apparent lack of association found for C. trachomatis.

Furthermore, biologically, this makes sense since C. trachomatis organisms infect mucosal columnar epithelial cells and not squamous epithelial cells, and there is evidence that M.

genitalium can infect non-keratinous squamous epithelial cells. 5, [START_REF] Graney | Anatomy and physical examination of the female genital tract[END_REF] We originally failed to confirm a univariate association of M. genitalium with balanitis and/or posthitis following logistic regression analysis in the extended study of all men with urethritis while controlling for co-infection, sexual lifestyle and clinical disease. [START_REF] Horner | Do all men attending departments of genitourinary medicine need to be screened for non-gonococcal urethritis?[END_REF] The additional men with urethritis consisted of clinically asymptomatic men, whose recruitment as potential controls began later in the study, but were found to have urethritis on Gram-staining. We also included both clinically symptomatic and asymptomatic men who had a negative urethral smear but evidence of urethritis on Gram-staining a first pass urinary thread which had > 10 PMNLs per HPF. [START_REF] Horner | Do all men attending departments of genitourinary medicine need to be screened for non-gonococcal urethritis?[END_REF] Seven of these additional 55 men had balanitis and/or posthitis, of whom none was infected with M. genitalium. For the multi-variate analysis, 70 (41%) of the 169 men could not be included because of missing data, including 11 (42%) of 26 men with balanitis and/or posthitis. We believe that the exclusion of such a large number of individuals from the multivariate analysis probably introduced unknown bias(es) related to data collection and provided an unreliable result. In addition, 4 clinically asymptomatic men without urethritis (controls) from the original study were M. genitalium-positive and none had balanitis and/or posthitis. Although this may have been due to a lower load, it could also be because of the small number of M. genitalium-positive men studied in the control group leading to a type 2 statistical error. These results imply that i) men with balano-posthitis should be asked about symptoms of discharge, dysuria and penile irritation and examined for the presence of a discharge by undertaking urethral massage 2,3,4, [START_REF] Edwards | National Guideline on the Management of Balanoposthitis[END_REF] . In this study, 15% of men with acute NGU did not have symptoms of urethritis but did have a discharge present on examination before or after urethral massage. If these symptoms and/or sign are present, an urethral smear examination should be undertaken to exclude urethritis. If urethritis is present our results suggest that M.

genitalium may be the cause and patients should be offered treatment accordingly. Currently testing for urethritis is only recommended in men with a circinate balanitis. [START_REF] Edwards | National Guideline on the Management of Balanoposthitis[END_REF] It is possible that men in this study did indeed only have circinate balanitis but, from clinical experience, this condition is uncommon and is usually associated with reactive arthiritis [START_REF] Edwards | National Guideline on the Management of Balanoposthitis[END_REF] and we believe this is unlikely. This requires further study. ii) the presence of balano-posthitis could be an important co-factor in increasing both the risk of HIV transmission and acquisition following exposure in men with M. genitalium infection.

We believe that the association of M. genitalium with balanitis and/or posthitis merits further investigation, particularly the potential to facilitate HIV transmission and acquisition. M.

genitalium is common (6.6%) in MSM who remain at high risk of acquiring HIV 10, [START_REF]Sexually Transmitted Infections and Men who have Sex with Men in the UK: 2008 Report[END_REF] and is also a common cause of urethritis in men from sub-Saharan Africa. 9

Word Count: 1931

Key Messages

• Balano-posthitis is associated with M. genitalium in men with acute NGU.

• In men with balano-posthitis symptoms of urethritis and the presence of an urethral discharge should be sought and an urethral smear undertaken if present.

• The association of M. genitalium with balano-posthitis merits further investigation particularly the potential to facilitate HIV transmission and acquisition.

3,4,11

This research was funded by Medical Research Council Programme Patrick Horner contributed equally to the study design, drafting and revising the manuscript.