Dr Anna Elizabeth Phillips
email: a.phillips05@ic.ac.uk

Catherine M Lowndes

Marie-Claude Boily

Geoff P Garnett

Kaveri Gurav

B M Ramesh

John Anthony

Stephen Moses

Michel Alary

Lowndes Cm

Boily Mc

Garnett Gp

Gurav

Ramesh Bm

Men who have sex with men and women in Bangalore, South India, and potential impact on the HIV epidemic

Keywords: HIV, India, MSM, Bridging behaviour, Bisexuality 2

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Approximately 2.5 million people in India were living with HIV in 2006, with the predominant mode of HIV transmission through sexual activity. [START_REF]2.5 million People in India living with HIV, according to new estimates[END_REF][START_REF]HIV Sentinel Surveillance and HIV Estimation 2007 -A Technical Brief[END_REF] At the national level, the overall HIV prevalence continues to suggest a concentrated HIV epidemic in India with, for example, a very high prevalence among men who have sex with men (MSM) (7.4%), which is more than 15 times the prevalence among ANC clinic attendees (0.48%). [START_REF]HIV Sentinel Surveillance and HIV Estimation 2007 -A Technical Brief[END_REF] Several studies in India have shown that MSM are a highly complex and diverse group, many of whom may have frequent sexual relations with women. [START_REF] Shinde | Male sex workers: are we ignoring a risk group in Mumbai, India[END_REF][START_REF] Singh | Long Distant Truck Drivers in India: HIV infection and their possible role in disseminating HIV into Rural Areas[END_REF][START_REF] Verma | Homosexual activity among rural Indian men: implications for HIV interventions[END_REF][START_REF] Hernandez | Sexual Behaviour Among Men Who have Sex with Women, Men, and Hijras in Mumbai, India -Multiple Sexual Risks[END_REF][START_REF] Gupta | Same-sex Behaviour and High Rates of HIV Among Men Attending Sexually Transmitted Infection Clinics in Pune, India[END_REF][START_REF] Setia | Men who have sex with men and transgenders in Mumbai, India: An emerging risk group for STIs and HIV[END_REF][START_REF] Kumar | Sexual Behaviour and HIV infection risks in Indian homosexual men: a cross-cultural comparison[END_REF] A study in Andhra Pradesh found that 42% of MSM were married and that 50% reported sexual relations with a woman in the past three months. [START_REF] Dandona | Sex behaviour of men who have sex with men and risk of HIV in Andhra Pradesh, India[END_REF] Some men might turn to men for sex due to difficulties accessing females. On the other hand, given the stigma associated with homosexual behaviour and the Indian penal code, which until recently cited sodomy as an offence [START_REF]Delhi High Court overturns ban on gay sex[END_REF], the pressure to deny male-with-male sex is pronounced, and many MSM marry women. [START_REF] Asthana | The social construction of male 'homosexuality' in India: implications for HIV transmission and prevention[END_REF] It has been reported that condom use by MSM with spouses tends to be low, even more so than with male partners, which suggests that through bisexual behaviour, men could link circuits of high risk male-with-male activity with the general female population. [START_REF] Schneider | HIV infection dynamics in rural Andhra Pradesh south India: A sexual-network analysis exploratory study[END_REF] Some studies have shown that the number of cases of women infected with HIV through heterosexual transmission within marriage is increasing in India, and that the behaviour reported by the husband was an important risk factor for infection among many of these women. [START_REF] Hawkes | Diverse realities: sexually transmitted infections and HIV in India[END_REF] The frequency of bisexual behaviour among MSM, coupled with low condom use, high HIV prevalence and increased transmission efficiency of anal sex, means that the contribution of men who have sex with men and women (MSMW) to the HIV epidemic, through transmission to their female partners, could be substantial. [START_REF] Caceres | HIV among gay and other men who have sex with men in Latin America and the Caribbean: a hidden epidemic?[END_REF] Despite high rates of marriage among MSMW, prevention efforts have tended to consider bisexual and homosexual men as a single group, with less attention given to understanding the impact of their sexual relations with women. This may be in part because bisexual men tend not to identify socially with the MSM community.

There is evidence from Latin America that MSMW are more likely than MSMO to engage in insertive rather than receptive anal sex with other men; they also have unprotected sex with their female partners. [START_REF] Tabet | HIV, syphilis and heterosexual bridging among Peruvian men who have sex with men[END_REF][START_REF] Hernandez | Sexual behaviour and status for Human Immunodeficiency Virus Type 1 among homosexual and bisexual males in Mexico City[END_REF][START_REF] Izazola-Licea | Sexual behaviour patterns and HIV risks in bisexual men compared to exclusively heterosexual and homosexual men[END_REF][START_REF] Muñoz-Laboy | Bisexual Latino men and HIV and sexually transmitted Infections risk: an exploratory analysis[END_REF] Some have argued that MSMW with a non-"gay" identity may be at higher risk of HIV infection because of a lack of peer support and limited access to prevention services that are available to MSM who are more open about their sexuality. [START_REF] Doll | Homosexually and non-homosexually identified men who have sex with men: A behavioural comparison[END_REF] In order to develop more effective preventive responses, we need to better understand sexual risk behaviour, and the extent to which bisexual behaviour serves as a bridge for infection into the heterosexual population. The definition of identity among men who have sex with men in India is very complex and is increasingly the focus of qualitative [12][21-22] and quantitative studies. [START_REF] Phillips | Sexual identity and its contribution to MSM risk behaviour in Bangalore, India: the results of a two-stage cluster sampling survey[END_REF][START_REF] Brahmam | Sexual practices, HIV and sexually transmitted infections among self-identified men who have sex with men in four high prevalence states of India[END_REF] Elsewhere, we have explored identities of MSM, including transgenders, and how their sexual behaviour is related to identity. [START_REF] Phillips | Sexual identity and its contribution to MSM risk behaviour in Bangalore, India: the results of a two-stage cluster sampling survey[END_REF] The objective of the current study was to disentangle the risk behaviour of men who have sex with men only versus those who reported sex with men and women, which has important implications for both the HIV epidemic and prevention interventions in India.

Firstly, we quantified differences in the characteristics and sexual risk of MSMW and men who have sex with men only (MSMO), and then explored differences in risk behaviour of MSMW with their female partners compared to that with their male partners.

METHODS

From July to August 2006, men/Hijras from Bangalore city were sampled using a two-stage cluster sampling method described in detail elsewhere. [START_REF] Phillips | Sexual identity and its contribution to MSM risk behaviour in Bangalore, India: the results of a two-stage cluster sampling survey[END_REF] In brief, following pre-survey mapping of cruising sites, time-location cluster (TLC) sampling was used for the selection of 'floating' populations in public-place cruising sites (locations where men seek other men for sex such as bus stops, train stations and public parks) to eliminate systematic bias of people with different levels of risk cruising at different days/times at a particular site. Conventional cluster sampling (CCS) was used for the sampling of Hammams (bath houses where transgendered persons sell sex to men) since the population under study is 'stable' in these locations. In total 12 Hammams and 112 public-place cruising sites were identified. Each of the 112 public-place cruising sites was divided into peak and off-peak periods and each cluster multiplied by the number of days sampled. A total of 5528 TLCs were defined, from which 77 were randomly selected. From a list of 12 Hammams, eight were chosen at random. Within each cluster the aim was achieve five interviews resulting in a total of 400 interviews. The objective of the survey was to measure changes in selected behaviour over two time periods in year 1 (2006), 3 and 5 of the intervention, calculating the sample size accordingly. [START_REF]Guidelines for repeated behaviour surveys in populations at risk of HIV[END_REF] A power calculation was carried out to ensure the available sample size would provide enough power.

In this example the outcome of interest was "ever reporting non-commercial sex with a man", between MSMW and MSMO. The alpha level was set at 0.05, corresponding to a type 1 error, with a 5% probability. This resulted in 88% power in the survey. [START_REF] Lachin | Introduction to Sample Size Determination and Power analysis for Clinical Trials[END_REF] Men were eligible to participate in the study if they had sex with a man at least once in their lifetime and were at least 18 years of age. In our analysis, men were classified as MSMW if they reported sex with a female within a year prior to interview. In this paper, the term "MSM" is used to describe a behavioural phenomenon that encompasses both men and male-to-female transgenders (Hijras).

Although Hijras do not socially and psychologically identify themselves as men, they are included in this study for two reasons. Firstly, the program through which respondents were recruited targets men and Hijras, thereby creating a degree of overlap between their social networks; and, secondly, many Hijras engage in receptive anal intercourse. [START_REF] Phillips | Sexual identity and its contribution to MSM risk behaviour in Bangalore, India: the results of a two-stage cluster sampling survey[END_REF] In India different categories of MSM, generally associated with different HIV-risk behaviours, are often used and interpreted as characterising different identities of MSM. Five categories of MSM identities, all of which have been described in previous publications [START_REF] Asthana | The social construction of male 'homosexuality' in India: implications for HIV transmission and prevention[END_REF][START_REF] Phillips | Sexual identity and its contribution to MSM risk behaviour in Bangalore, India: the results of a two-stage cluster sampling survey[END_REF][START_REF] Chakrapani | Secondary HIV prevention among Kothi-identified MSM in Chennai, India[END_REF] were defined by the local NGO at the outset of the study, and confirmed by a pilot study. The question asked in the interview was open-ended: 'How do you identify yourself?' with possible answers being {Hijra, Kothi, Double-Decker, Bisexual, Panthi and 'no identity'}.

Mixing Western notions of identity (homo-heterosexual) is unlikely to be recognized by Indian men unless they are of higher or middle class and have been exposed to Western gay men or environments [START_REF] Asthana | The social construction of male 'homosexuality' in India: implications for HIV transmission and prevention[END_REF]. Despite this, bisexual was a term recognized and used by respondents during the pilot study.

The interviewers were trained only to give examples of identities but not to probe the respondent or eliminate categories according to sexual behaviour.

Statistical Analysis

The association between sociodemographic characteristics and bisexual behaviour was examined using the Wald test for continuous variables and Pearson chi-square tests with Rao-Scott correction for categorical variables. [START_REF] Rao | On chi-squared tests for multiway contingency tables with cell proportions estimated from survey data[END_REF] Each outcome was related individually to the outcome, bisexual behaviour. A logistic regression model was created, adjusting for sociodemographic variables known to be important risk factors (or confounders) for bisexual behaviour based on the published literature.

In constructing the multiple regression models for the adjusted analyses, all important sociodemographic variables were included, with the variable with the smallest p-value chosen as the first independent variable in the multivariate model. There were no significant interaction terms and any missing data was excluded from the analysis. All statistical analyses took into account the survey design (clustering), in order to take into account the correlation between individuals when estimating variance and confidence intervals, using SVY commands in STATA, version 10. [START_REF]Introduction to Survey Analysis in Stata 9[END_REF]

RESULTS

In total, 357 of the 572 men approached agreed to be interviewed giving a participation rate of 62%. Figure 1 shows the extent of lifetime and current (past year) bridging behaviour by partner type. Over half of the sample (55%) reported sex with men and women in their lifetime; 45% reported sex only with men, and 3% reported sex only with men and Hijras. No men reported having had sex only with

Hijras.

Over the past year, 41% reported sex with men and women, of which 30% reported sex with men and women and 11% reported sex with all partner types -men, women and Hijra. On the other hand, 12% of those reporting lifetime bisexual behaviour reported sex only with men in the past year, while 2% reported sex only with men and Hijras (Figure 1).

In our analysis, men were classified as MSMW if they reported sex with a female (and male) within a year prior to interview. MSMO were defined as men who reported having sex with men only in the last year.

Sociodemographic Characteristics

Over a third of MSMW (38%) and MSMO (33%) were recruited into the survey in public parks, and 12% of MSMO were recruited in Hammams due to the higher proportion of Hijras in this group (Table 1). Those who reported bisexual behaviour in the last year were older on average than exclusive homosexuals. Age at first sex was significantly higher among MSMW than MSMO (19 vs.

years).

Most of the sample was Hindu and literate (71%) and this did not vary significantly between MSMW and MSMO. A third (34%) of all respondents had been married to a woman at some stage in their life.

There were some differences among identities. More masculine identities such as Panthis, Double-Deckers, and bisexuals, were more likely to have ever been married (38%, 27% and 63% respectively) compared with 22% of Kothis and no Hijra. More details on characteristics of different identities have been published elsewhere [START_REF] Phillips | Sexual identity and its contribution to MSM risk behaviour in Bangalore, India: the results of a two-stage cluster sampling survey[END_REF].

The majority of MSMW were currently married (68%), and most married MSMW (66%) lived with their wife. Among men not reporting sex with a woman in the last year (MSMO), 3% were currently married.

Slightly fewer MSMW originated from Bangalore (47% vs 52%) and had ever had sex with another man outside Bangalore (25% vs. 28%). Considerably more MSMW concealed their same-sex behaviour from their families than MSMO (96% vs. 74%). Significantly fewer MSMW than MSMO perceived themselves to be at risk of contracting HIV (32% vs. 46%). * MSMW refers only to men who had sex with men and women in the last year § May not add up to 100% as some respondents answered 'Other' ‡ A definition of 'family' was not given and may have been interpreted differently by different respondents. For example it may include wives, but be restricted to parents for others. ¤ These variables were all included in the multivariate analysis in Table 2 since significant differences in proportions were seen between MSMW and MSMO for they are also known to be important risk factors (or confounders) for bisexual behaviour based on the published literature. Living arrangements were not included in the final multivariate model as this variable did not remain significantly associated and widened the CIs.

Sexual identity and bisexual behaviour (Table 1)

HIV intervention programs in India have tended to assert MSM identities typically described as passive (receptive) or active (insertive) in terms of sexual role in anal sex [START_REF] Boyce | Conceiving kothis': men who have sex with men in India and the cultural subject of HIV prevention[END_REF]. In brief, Hijras are male-to-female transgenders; Kothis have feminine characteristics and may cross-dress but remain biologically male; Double-Deckers and bisexuals are more masculine or neutral than Kothis; and

Panthis have a masculine identity and may not self-identify as such although they are identified so by Kothis or Hijras. As previously described [START_REF] Phillips | Sexual identity and its contribution to MSM risk behaviour in Bangalore, India: the results of a two-stage cluster sampling survey[END_REF] and in the present study, self-reported sexual identity was strongly associated with having sex with women. Although MSMW frequently did not identify as bisexual: 43% self-identified as bisexual; 23% as Panthi, 12% as Double-Decker, 11% as Kothi, while 11% of MSMW did not self-identify with any group. No Hijra reported sex with women.

Sexual risk behaviour of MSMW and MSMO (Table 2)

As shown in Table 2, there was no significant difference between MSMW and MSMO in terms of the type of male partner they first had sex with, or reporting anal sex with known or unknown noncommercial partners.

Men who sold sex were less likely to be MSMW than those who did not sell sex; while those who had ever bought sex from males and Hijras were more likely to be MSMW. Men who reported ever having non-commercial (NC) male partners were more likely to be MSMW than those who did not.

Men who reported receptive anal intercourse were less likely to be MSMW than those who had insertive anal sex, with both known and unknown non-commercial male partners.

Results of the multiple logistic regression model are shown in Table 2. After controlling for sociodemographic characteristics, the association between sex with men and women, and buying sex from male and Hijra sex workers, remained significant, as did selling sex and receptive sex with known non-commercial partners. On the other hand, the relationships between MSMW and noncommercial sex with men, as well as type of anal sex with known and unknown non-commercial male partners, were confounded, and after controlling for sociodemographic variables, such as marital status and sexual identity, did not remain significant. 3) 0.002 § Does not add up to 100% as some respondents answered 'Other' ¤ The percentage of MSMW only reporting each behaviour * The reference category for the OR is MSMO with MSMW as the comparison group Þ Odds ratio adjusted for age, age at first sex with a male, cruising site, marital status and sexual identity ‡ These questions were only answered by those respondents who had ever had sex with known/unknown NC men and therefore th total does not add up to the total number of individuals in the sample

DISCUSSION

Men who have sex with men link circuits of high risk activity with the general population. Bisexual behaviour was commonly reported among men who seek sex with men in the public places sampled in this survey. The findings from this paper are consistent with other research findings from India, with proportions of MSM currently married to women ranging from 23% to 42%. [START_REF] Setia | Men who have sex with men and transgenders in Mumbai, India: An emerging risk group for STIs and HIV[END_REF][START_REF] Dandona | Sex behaviour of men who have sex with men and risk of HIV in Andhra Pradesh, India[END_REF] Some differences were found between sexual identity and behaviour reported in this study, compared to others. It has been previously reported in qualitative research in Chennai (India) that engaging in sex with women is a source of stigma within the Kothi community. [START_REF]Introduction to Survey Analysis in Stata 9[END_REF] In this study 11% of MSMW self-identified as Kothis and only 43% as bisexual. Selection of participants based on selfidentification as bisexual, rather than on the basis of current sexual practices, would thus have underestimated the true prevalence of bisexual behaviour.

MSMW reported lower rates of risky behaviour than MSMO, with lower proportions reporting selling sex to men, and receptive anal intercourse, in both unadjusted and adjusted analyses. This is likely a reflection of the many MSMO that are Kothis and Hijras, who have previously been shown to be more likely than other identities to sell sex. [START_REF] Phillips | Sexual identity and its contribution to MSM risk behaviour in Bangalore, India: the results of a two-stage cluster sampling survey[END_REF] However, a significant proportion of MSMW buy sex from sex workers (male, female and Hijra), and of those who reported insertive and receptive anal sex with unknown non-commercial partners, 60% were MSMW. It was not possible to examine the frequency of sex acts by type (insertive, receptive or insertive/receptive) as the question was not interview; however it would be interesting to look at this in future.

Condom use by MSMW with their wives was extremely low. A study carried out recently in Bangalore showed a high HIV-1 prevalence in both MSMO (20%) and MSMW (19%), so the risk of HIV transmission to the wives of MSMW is high. [START_REF]Integrated Behavioural and Biological Assessment among MSM-T in Karnataka, prepared[END_REF] In addition, many MSMW reported sex with FSWs as well, with 20% reporting anal sex with an FSW in the past year. Given high HIV prevalence among FSWs [START_REF] Ramesh | Determinants of HIV prevalence among female sex workers in four south Indian states: analysis of cross-sectional surveys in twenty-three districts[END_REF], this indicates a significant risk of HIV infection through sex with FSW.

An interesting finding was that MSMO were younger than MSMW (median 28 vs. 33 years). This raises the question of whether some of the MSMO would go onto have sex with women, particularly through marriage. It would be worthwhile for future studies to examine this question in more detail, through for example examining the future intentions of young MSMO with regard to marriage or sex with other women.

A limitation of this study is that it included only men who seek sex with men in public places, and this may not reflect the population of MSM in the larger community. Furthermore, the overall participation rate was about 62%, which raises concerns over the representativeness of the study, and consequently the generalisability of the results. For example, many MSMW are unlikely to identify themselves as "MSM", as reflected by the few respondents who had informed their families that they had had sex with men, and the substantial proportion married to women: it is likely that such men would have been less amenable to study participation. Nevertheless the study does highlight the diverse range of homosexual and bisexual behaviours among MSM in India, which may have significant implications for the risk of HIV transmission to female sexual partners. Changing behaviour in this bridging population could slow the progression of the HIV epidemic. Previous studies have critically examined the use of indigenous sexual 'identities' that have been popularised by HIV-prevention programs in India [21][23]. Although the politics of such categories is not the focus of the current paper, it is noted that bisexual behaviour among MSM is correlated with identity, with nearly half of MSMW self-identifying as bisexual.

In this regard, at least two prevention efforts must occur: one that focuses on MSM who identify as such, and another that reaches out to men who have male and female partners, but who may not identify as MSM or bisexual, and who seek sex in locations such as public parks. The latter group could be better reached by outreach workers with more behaviourally bisexual (and generally more 13 masculine) MSM identities, such as Panthis and bisexuals, rather than the often used Kothi and Hijra outreach workers. [START_REF] Safren | A survey of MSM HIV prevention workers in Chennai, India[END_REF] It is also important to focus on increasing condom use rates with the female partners of MSMW, who are generally perceived as low risk. This may be difficult to achieve and sustain, however, because the desire for children may compete with the concern to protect partners from HIV infection.

Bridging to the general population will however continue to contribute to the HIV epidemic unless condom use with higher-risk partners becomes more consistent.

Word Count: 3214

Table 1 : Sociodemographic characteristics and behaviours of MSMW and MSMO

 1

		Total	MSMW *	MSMO	P-Value
	Characteristic	% (N=357)	% (N=146)	% (N=211)
	Cruising site where recruited:¤				0.031
	Public Park	35	38
	Public Toilet	19	21
	Bus station	18	16
	Hammam	8	2
	Other including train station, cinema etc	20	23
	Age (Median) ¤	(30)	(33)	(28)	<0.001
	18-19	6	3
	20-29	51	34
	30-39	27	40
	40-75	16	23
	Median age at first sex with a man (years) ¤ 18	19		0.007
	Religion §				0.656
	Hindu	71	72
	Islam	21	21
	Christian	8	6
	Literate: can read and write	71	74		0.265
	Marital Status ¤				<0.001
	Currently married	29	68
	Separated/Divorced/Widowed	5	3
	Never married (including all Hijra)	65	29
	Living Arrangements §				<0.001
	Living with Male Partner	6	2
	Living with Wife	28	66
	Living Alone/Friend/Hotel	25	13
	Living with Guru/Hijra	10	0
	Living with Family (no wife)	31	18
	Born in Bangalore	49	52		0.331
	Ever had sex with men outside Bangalore	27	25		0.382
	Family does not know respondent has sex				<0.001
	with men ‡	83	96
	Feel at risk of being infected with HIV	40	32		0.027
	Had an HIV test ever	44	42		0.523
	Self-reported sexual identity ¤				<0.001
	Hijra	13	0
	Kothi	26	11
	Panthi	15	23
	Double-Decker	15	12
	Bisexual	23	43
	No Identity	8	11

Table 2 : Sexual risk behaviour of bisexual (MSMW) versus homosexual (MSMO) men

 2

		Total N %	Univariate OR	Univariate	Adjusted OR Þ	Adjusted
	Characteristic		MSMW¤	(95% CI)*	P-Value	(95% CI)	P-value
	Male first had sex with §:
	Classmate/Friend/Colleague	195	42	1		1
	Relative	47	32	0.65 (0.32-1.31)	0.220	0.76 (0.20-2.84)	0.681
	Paying client/Male sex worker	23	65	2.58 (0.95-7.01)	0.062	3.80 (0.98-14.6)	0.053
	Stranger	79	38	0.84 (0.51-1.40)	0.507	0.66 (0.30-1.45)	0.296
	Ever sold sex to another man
	No	210	58	1		1
	Yes	147	17	0.15 (0.08-0.27) <0.001	0.34 (0.12-0.96) 0.041
	Ever had non-commercial (NC)
	male partners
	No	132	30	1		1
	Yes	224	48	2.16 (1.27-3.68) 0.005	1.13 (0.47-2.69) 0.787
	Ever had anal sex with known
	NC men
	No	24	58	1		1
	Yes	167	48	0.66 (0.28-1.54) 0.327	0.70 (0.08-5.97) 0.736
	Type of anal sex with known
	NC men ‡
	Insertive	64	70	1		1
	Receptive	61	28	0.16 (0.07-0.39)	<0.001	0.15 (0.03-0.69)	0.016
	Both insertive & receptive	42	43	0.34 (0.13-0.77)	0.013	0.44 (0.06-3.08)	0.398
	Ever had anal sex with
	unknown NC men
	No	14	43	1		1
	Yes	103	44	1.03 (0.36-2.96) 0.949	1.74 (0.28-10.7) 0.543
	Type of anal sex with unknown
	NC men ‡
	Insertive	32	59	1		1
	Receptive	47	30	0.29 (0.11-0.78)	0.015	0.02 (0.00-2.92)	0.116
	Both insertive & receptive	15	60	1.03 (0.34-3.07)	0.962	0.88 (0.63-1.24)	0.467
	Ever bought sex from male sex
	worker
	No	245	28	1		1
	Yes	112	69	5.61 (3.34-9.42) <0.001	4.06 (1.82-9.08) 0.001
	Ever bought sex from Hijra sex
	worker
	No	298	36	1		1
	Yes	58	67	3.81 (1.94-7.51) <0.001	5.47 (1.95-15.

Table 3 : Bisexual behaviour of men who had sex with men and women in the past year

 3

		MSMW
	Variable	% (N=146)
	Mean age at first sex with a woman (years)	22
	Female first had sex with §:
	Wife	37
	Relative	17
	FSW	23
	Classmate/Friend/Colleague	19
	First sexual partner
	Male	66
	Female	21
	Male and Female same year	13
	Type of sex with wife in the last month (N=103)
	Vaginal	95
	Anal	5
	Ever bought sex from a female sex worker (FSW)	42
	Bought sex from a FSW in the last year (N=62)	31
	Type of sex with FSW in the last year (N=45)
	Vaginal	100
	Anal	20
	Currently having sex with 'other' female partner	22
	§ Does not add to 100% as some respondents answered 'Other'

Consistent condom use and mean number of monthly sex acts by different types of partner

Consistent condom use was defined as 'always' using a condom with that partner or partner type. There were no significant differences in rates of consistent condom use between MSMO and MSMW with different types of male partners, whether commercial or non-commercial (Figure 2).

Condom use was particularly low with wives (only 2% always used a condom with wives), even though the highest average number of monthly sex acts took place with wives (average 10 acts per month). Of those who had ever bought sex from a male sex worker (MSW), condoms were used consistently by 64% and 65% of MSMW and MSMO respectively. This was higher than condom use with FSWs (56%).

MSMW reported significantly fewer sex acts with known clients (5 per month) than MSMO (12 per month; p=0.05). However, the mean number of monthly sex acts with both unknown and known non-commercial male partners was similar for MSMW (2 and 3 respectively) and MSMO (2 and 4 respectively). Both MSMW and MSMO who bought sex reported an average of 3 sex acts per month with MSW (p=0.82). As shown in Figure 2, inconsistent condom use did not necessarily correspond with the partner with whom they have the least number of sex acts.

Sexual behaviour patterns of MSMW with female partners (Table 3)

Among MSMW, mean age at first sex with a woman (22 years) was higher than mean age at first sex with a man (19 years). This is consistent with the majority of MSMW reporting that their first sexual partner was male (66%). Most men reported vaginal sex with their wives (95%) and FSWs (100%), and few reported anal sex with wives (5%), although this proportion was higher for anal sex with FSWs (20%). Consistent condom use was lower for anal sex with both wives (0%) and FSWs (33%) than for vaginal sex (see Figure 2: 2% and 56% respectively), although numbers reporting anal sex with women were low. Twenty-two percent of all MSMW were currently having sex with other female partners (besides cohabiting females and FSWs).

ACKNOWLEDGEMENTS & DISCLAIMERS

The authors thank the field team, as well as the men and transgenders who participated in this study, as well as Sangama and the Karnataka Health Promotion Trust (KHPT) for supporting and facilitating Competing Interest: None declared.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 STI and any other BMJPGL products and sub-licences such use and exploit all subsidiary rights, as set out in our licence http://group.bmj.com/products/journals/instructionsforauthors/licence-forms.


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Introduction

The HIV epidemic in sub-Saharan Africa continues relentlessly, with ~ 1.9 million new infections in 2007. [START_REF]UNAIDS AIDS epidemic update[END_REF] Over 80% of current HIV transmissions are through heterosexual intercourse, with women being more vulnerable due to transmission dynamics, socio-economic factors, and their limited control over safer sexual practices. 1,2, Female controlled HIV prevention methods are thus urgently needed for women in the developing world. The development of vaginal microbicides offers a potential new prevention strategy, but the results of effectiveness trials with nonoxynol-9, SAVVY, cellulose sulphate, and Carraguard have been disappointing. [START_REF] Cates | HIV prevention research: the ecstasy and the agony[END_REF] PRO 2000 is a microbicidal polyanionic naphthalene sulphonate polymer that has been formulated as a vaginal gel. [START_REF] Rusconi | Naphthalene sulfonate polymers with CD4-blocking and anti-human immunodeficiency virus type 1 activities[END_REF] In vitro and in animal models, PRO 2000 blocks infection by a range of HIV isolates, herpes simplex virus, Neisseria gonorrhoeae (NG) and Chlamydia trachomatis (CT) [START_REF] Rusconi | Naphthalene sulfonate polymers with CD4-blocking and anti-human immunodeficiency virus type 1 activities[END_REF][START_REF] Greenhead | Parameters of human immunodeficiency virus infection of human cervical tissue and inhibition by vaginal virucides[END_REF][START_REF] Weber | Chemical condoms' for the prevention of HIV infection: evaluation of novel agents against SHIV 89.6PD in vitro and in vivo[END_REF][START_REF] Bourne | The topical microbicide PRO 2000 protects against genital herpes infection in a mouse model[END_REF][START_REF] Spencer | Inhibition of Neisseria genital tract infection by leading-candidate topical microbicides in a mouse model[END_REF] .

Early safety studies showed PRO 2000 gel to be safe, well tolerated and acceptable. [START_REF] Van Damme | Phase I study of a novel potential intra-vaginal microbicide, PRO 2000, in healthy sexually inactive women[END_REF][START_REF] Mayer | Safety and tolerability of vaginal PRO 2000 gel in sexually active HIVuninfected and abstinent HIV-infected women[END_REF][START_REF] Smita | Phase I safety study of 0.5% PRO 2000 vaginal gel among HIV un-infected women in Pune, India[END_REF][START_REF] Morrow | The acceptability of an investigational vaginal microbicide, PRO 2000 Gel among women in a phase I clinical trial[END_REF] We conducted a phase II trial of two concentrations of PRO 2000 gel (0.5% and 2%) among sexually active women in Uganda. The primary objectives of the trial were to investigate product safety in terms of adverse events (AEs) both local, using clinical inspection and colposcopy, and systemic, using haematology, biochemistry and coagulation parameters. Secondary objectives included detection of unexpected inter-menstrual bleeding (IMB), effects on vaginal flora, and documentation of gel acceptability and adherence.

Methods

Screening, enrolment and follow-up

The study procedures were used in a previous study at the same site, and have been described elsewhere. [START_REF] Morrow | The acceptability of an investigational vaginal microbicide, PRO 2000 Gel among women in a phase I clinical trial[END_REF] Briefly, we recruited asymptomatic, sexually active women aged 18-45 years at St Francis Hospital, Nsambya, Kampala. Women were eligible if they were willing to undergo HIV counselling and testing, to use condoms and able to give informed consent. Exclusion criteria included pregnancy, intending to become pregnant, being within 6 weeks postpartum, abnormal colposcopy findings, untreated sexually transmitted infections (STIs), symptomatic bacterial vaginosis (BV), abnormal grade II haematology or biochemistry findings, coagulation disorders, cervical intraepithelial neoplasia grade II/III, post-coital or IMB in the preceding 3 months, pelvic inflammatory disease, latex allergy, and persistent abnormal vaginal discharge. HIV-seropositive women were eligible if they fulfilled all other entry criteria and were in an HIV-positive concordant relationship.

The study design is shown in figure 1. Informed consent (signed or thumb-printed for illiterate participants) was obtained separately for screening and enrolment procedures. Screening tests included HIV, STIs, haematology, biochemistry, and coagulation, and general and genital examinations, including colposcopy, were performed. All women with STIs were treated according to national treatment guidelines before enrolment, with partner notification.

Randomisation was performed in two phases: the first 100 women to either 0.5% gel, placebo gel or an observation (condom only) arm (phase 1); and the last 80 women to either to 2% gel, placebo gel or the observation arm (phase 2). The allocation was weighted so that at least 60 women received active gel in each phase. In both phases we included women in observation only arms who followed the same study schedule but without gel use. Our aim was to enrol between 60 and 65 women into the 0.5% and 2% arms respectively, which allowed for some loss to follow-up, 25 women into a placebo gel control groups, and a further 30 women into observation only arms.

Participants using gel were asked to use one applicator (approximately 2g of gel) intravaginally twice daily for 28 days with interruption during menses if preferred. They were instructed on applicator insertion and provided with sufficient supplies to last two days beyond their next clinic visit. Women were advised to wash their vagina prior to inserting the gel using plain water, as this is the normal local female hygiene practice. Women were provided with condoms and encouraged to use them at all times. An interim visit was conducted after 7 days gel use with a clinical examination only if genital symptoms were reported. Further follow-up visits were performed fortnightly until 6 weeks from enrolment. Adherence with gel administration was measured by structured face-to-face interviews, counting of returned and used applicators, and examination of diary cards at 14 & 28 days follow-up. [START_REF] Bakobaki | A rondomised controlled safety and acceptability trial of dextrin sulphate vaginal microbicide gel in sexually active women in Uganda[END_REF] Between phases 1 and 2 of the study, the medical staff who conducted the interviews and examination changed, in that a male clinician left and was replaced by two female clinicians.

Laboratory methods

NG and CT detection was by polymerase chain reaction (Amplicor, Roche Diagnostics) on endocervical specimens. Trichomonas vaginalis (TV) was detected by culture (TV-Inpouch, Biomed Diagnostics Inc.). Bacterial vaginosis was assessed by vaginal pH and Gram stained vaginal smears using Nugent's criteria. [START_REF] Nugent | Reliability of diagnosing bacterial vaginosis is improved by a standardised method of gram stain interpretation[END_REF] HIV testing was performed using a rapid test (Capillus HIV-1/HIV-2 rapid slide agglutination assay, Trinity Biotech). Positive results were confirmed with two independent EIAs (Recombigen HIV-1/HIV-2 EIA, Trinity Biotech, and Wellcozyme HIV Recombinant EIA, Murex Biotech).

Statistical Methods

Data management: Data were double entered and verified using an Access 1997 database (Microsoft Corporation, USA). The data were exported to STATA 9 (STATA Corporation, Texas, USA) using Stat Transfer (1986-2003 Circle Systems, Seattle, USA), for statistical analysis.

Sample size:

The sample size calculation was based on the procedure described by Fleming. [START_REF] Fleming | One sample multiple testing procedure for Phase II clinical trials[END_REF] The underlying assumptions were that a primary toxicity endpoint rate of 15% is unacceptable, and that a gel that results in a toxicity rate of 5% or less warrants further investigation; the study was powered to be able to distinguish between these two rates, for a type I error probability of 5% and a power of 80%, a total of 59 women would be required on active gel. The trial was not powered to make statistical comparisons between the trial arms.

Endpoints and analysis

The primary endpoints of the trial were (i) deep or extensive (>4 swab tips) ulceration of the genital mucosae, and (ii) the development of a clinically relevant coagulation abnormality. The secondary endpoints were Grade 3 clinical, laboratory, or unexpected vaginal bleeding events, Grade 1 unexpected vaginal bleeding (intermenstrual bleeding), development of abnormal vaginal flora, and acceptability of the gels.

All analyses were by intention to treat, at the 5% level of significance. The analysis of binary data was performed using chi-squared tests with the Mantel-Haenszel test controlling for possible confounders of treatment and phase where appropriate. Laboratory parameters at screening were compared with the week 4 visit results; comparisons were made using either a paired t-test or the Wilcoxon signed rank test, depending on the distribution of the data.

Ethical considerations

The Research Ethics Committees of Nsambya Hospital and the Uganda Virus Research Institute, the Uganda National Council of Science and Technology and St Mary's Hospital, London, UK, provided ethical approval of the study. The trial was overseen by a Data Monitoring Committee and a Trial Steering Committee. The participants were provided an equivalent of US$ 7.00 per visit to compensate for the time spent at the clinic and transport costs.

Results

A total of 320 women were screened between June 2003 and September 2004. 140 women were excluded. Reasons for exclusion were HIV positive with a serodiscordant partner (30), unlikely to comply with the protocol (34), no regular sexual partner (26), pregnancy (9), significant current medical illness (8), abnormal laboratory results (18), sexually inactive (3), recent history of IMB (4) and other gynaecological abnormalities (8).

A total of 180 (107 HIV negative; 73 HIV positive) participants were randomized during the two phases. The first phase included 100 women: 63 allocated to 0.5% gel, 15 to placebo (Plac-1) and 22 to observation only (Obs-1). In the second phase 80 women were randomised, 61 allocated to 2% gel, 10 to placebo (Plac-2), and 9 to observation only (Obs-2). Ten participants (3 on 0.5% gel, 6 on 2% and 1 in the observation group) were lost to follow-up. These participants contributed to the analysis only for periods prior to loss to follow-up. The reasons for loss were: 6 relocated, 2 withdrew consent, 1 was unable to adhere to the visit schedule and 1 was withdrawn by the investigator because the participant expected material gains. No participant discontinued due to AEs.

Table 1 shows baseline demographic characteristics and menstrual history by study arm. In general the groups were similar. The overall HIV prevalence was 40%, with a somewhat lower prevalence among those on 2% gel (33%) compared to those on 0.5% gel (46%). Approximately 85% of women reported 2-3 sexual acts per week. Six (2 on 0.5%, 2 on 2% and 2 in Obs-1) women reported a previous history of IMB or post coital bleeding at screening. Participants were tested for STIs at screening and at week 4. There were no significant differences in the frequencies of infection across different arms, or between HIV-positive and negative women.

Prevalence rates at screening were NG 1.1%, CT 3.3%, TV 6.1%, active syphilis (RPR & TPHA +ve) 8.3%, Although participants were treated at screening and advised re partner notification, a similar proportion of infections were detected at 4 weeks. The frequency in the two assays that can truly detect new or ongoing infection were NG 0.6%, TV 8.1%.

Primary and secondary endpoints

Only one woman developed ulceration > 4 swab tips in area (20 x 10 mm) in the trial, who was randomised to 0.5% PRO 2000 gel (1/63 women, 1.6%, 95% CIs 0.04-8.5%). This woman had ulceration < 4 swab tips in area at visit 4, the end of dosing, which progressed to >4 swab tips at final follow-up. No woman in the trial developed a clinically relevant coagulation abnormality, These rates are, for the 0.5% arm 0%, 97.5% CIs 0-5.7%, and for the 2% arm 0%, 97.5% CIs 0-5.9%.

Table 2 shows a summary of AEs reported. There were no clinical, laboratory, or unexpected vaginal bleeding serious (Grade 3) adverse events in the study. There were similar rates of AEs among all the study arms. During phase 1, IMB was reported by 7/63 (11.1%) women on 0.5% gel, 1/15 (6.7%) on Plac-1, and 2/22 (9.1%) in the observation arm. In phase 2 IMB was reported by 19/61 (31.1%) women on 2% gel, 2/10 (20.0%) on Plac-2 and 3/9 (33.3%) in the observation arm. All IMB episodes reported by women on active gel were mild except for one graded as moderate and all except one (from 0.5% gel group) were considered as possibly related to gel use.

One participant on 2% gel reported post coital bleeding. Six participants (four on 2%, one on 0.5% and one in Obs-1) had signs of vaginal bleeding on examination; of these, 3 did not report related symptoms. In no case could a bleeding source or concurrent epithelial lesion be identified. IMB events that were not observed by a clinician resolved spontaneously, and for gel recipients they resolved without discontinuation of gel use.

There was a borderline significant association between IMB and HIV status after adjusting for treatment arm and randomisation phase (Mantel-Haenszel odds ratio=2.14, 95% CI 0.99-4.65). In addition, there was a significant positive association between an abnormal colposcopy and HIV status (P=0.01) which remained so after controlling for treatment arm and randomisation phase (Mantel-Haenszel odds ratio= 3.16, 95% CI 1.38-7.23).

Analysis of the haematology and biochemistry values at screening and week 4 visits by study arms

showed no statistically or clinically significant changes. Median vaginal pH remained the same (pH=5.0) at both visits in phase 1 across all trial arms; in phase 2, pH was 6.0 in the 2% gel arm, 5.5 in the Plac-2 arm, and about 5.0 in the Observ-2 arm, all at both visits. There was no evidence that women with a Nugent score of <7 at baseline developed increased scores while using the gel (P=0.506).

Adherence with gel use

Overall gel adherence at weeks 2 & 4 was 69% (71% in the 0.5% gel, 60% in the Plac-1, 73% in the 2% gel and 50% in the Plac-2 arms, respectively). The main reasons reported for not using the gel were being away from home (11), spotting (5), forgotten (5), and ill health (3).

Six of 146 participants interviewed after 2 weeks and 5 of 144 interviewed after 4 weeks of gel use reported having found the gel difficult to apply, because the applicator was painful on insertion in the vagina. All but 1 woman reported that they would recommend the gel to others and would use it if it were effective and freely available.

Discussion

The study demonstrated that 0.5% and 2% PRO 2000 gel concentrations were acceptable and safe when applied intra-vaginally twice daily for 4 weeks by sexually active women in Uganda. Of the 180 women enrolled, only 7 missed the week 4 follow-up visit. Reasons for not using the gel were largely unavoidable: menstruation, being away from home and ill health.

There was no evidence of local or systemic toxicity related to gel use, and the gels fulfilled the protocol definitions for further study in large scale trials. Earlier studies conducted in sexually abstinent women had also demonstrated lack of systemic absorption of PRO 2000 after vaginal use. [START_REF] Lacey | Direct measurement of in vivo vaginal microbicide levels of PRO 2000 achieved in a human safety study[END_REF] Approximately 60% of the AEs were genital tract related and the remainder were due to common background morbidities. None of the AEs necessitated discontinuation of gel use, or expedited reporting to ethics or regulatory authorities.

The rate of abnormal colposcopies was low in both active gel arms (about 4%). The majority of these abnormalities were small vulval superficial lesions. Overall, the rate of reported IMB in both randomisation phases was 19%. We also noted that the reported rate during the 2% phase was significantly higher than in the 0.5% phase in all the comparison arms (Mantel-Haenszel odds ratio=3.78, 95% CI 1.62-8.84). However the clinical staff conducting the interviews changed from a male clinician to 2 female clinicians between phase 1 and phase 2. We believe it likely that female participants in Africa would feel more comfortable reporting genital symptoms to female clinicians, and we feel this is a plausible explanation for the higher reported rate of IMB in all arms during the 2% gel phase. Differences in patient communication of biomedical information with female or male physicians have been previously reported. [START_REF] Roter | Physician gender and patient-centred communication: a critical review of empirical research[END_REF] We found no influence of PRO 2000 on vaginal flora, whereas a reduction in BV has been reported in a phase I safety study in India. [START_REF] Smita | Phase I safety study of 0.5% PRO 2000 vaginal gel among HIV un-infected women in Pune, India[END_REF] The safety and tolerability of PRO 2000 gel observed in this study are similar to findings in earlier studies in sexually abstinent European women and sexually active HIV-uninfected and abstinent HIV-infected women exposed to 2% and 4% PRO 2000 gel. [START_REF] Van Damme | Phase I study of a novel potential intra-vaginal microbicide, PRO 2000, in healthy sexually inactive women[END_REF][START_REF] Mayer | Safety and tolerability of vaginal PRO 2000 gel in sexually active HIVuninfected and abstinent HIV-infected women[END_REF] We observed a high STI prevalence rate (~20%) at screening and although STIs were treated in participants and partner notification was conducted, the rate was similar at the week 4 visit. Due to CT PCR positivity carry over, and syphilis serology dynamics it is not possible to interpret the findings for those infections. However, the GC and TV data presumably represent a mixture of reinfections, new infections and treatment failures. These observations emphasise the need for regular STI screening and health education in microbicide clinical trials. Our data also suggests that PRO 2000 gel does not significantly interfere with the STI assays we used. Indeed the Amplicor CT/NG test includes an internal positive control which detects PCR inhibition, and only about 1% of the specimens collected at the screening visit and 2% of those collected at week 4 showed inhibition.

The high HIV prevalence in our study population was related to the presence of a busy HIV clinic on the hospital campus and does not reflect the background prevalence in the general population.

In summary, 0.5% and 2% concentrations of PRO 2000 gel were well tolerated in terms of both local and systemic toxicity by sexually active women both HIV negative and HIV positive when used twice daily for four weeks, and the gel was well accepted within the context of the trial. There was no evidence of systemic toxicity related to gel use. Almost all women stated that they would recommend the gel to others and would definitely use it if it became available. These results provided the justification to evaluate PRO 2000 at both concentrations in a large phase 3 trial to assess its effect on HIV transmission, the results of which will be announced in December 2009. 19 Trial Registry number: ISRCTN02518250 Andrew Nunn conducted the statistical analyses. All authors contributed to the preparation of the manuscript.

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 STI and any other BMJPGL products and sub-licences such use and exploit all subsidiary rights, as set out in our licence http://group.bmj.com/products/journals/instructionsfor-authors/licence-forms.

Competing interests: None declared.

Table 1 :

 1 Baseline characteristics of participants by arm

	Variable

Table 2 :

 2 Comparison of adverse events reported during gel use (N =number of participants) by arm

	, 4)

Acknowledgements

The authors would like to acknowledge the input and assistance of many staff at both the MRC Uganda/UVRI Research Unit on AIDS and St Frances Hospital, Kampala.

Disclaimer


Richard Harding
email: richard.harding@kcl.ac.uk

Fiona C Lampe

Sally Norwood

Heather Leake

Claudine Clucas

Martin Fisher

Margaret Johnson

Simon Edwards

Jane Anderson

Lorraine Sherr

Symptoms are highly prevalent among HIV outpatients and associated with poor adherence and unprotected sexual intercourse

Keywords: HIV, pain, symptoms, adherence, risk

KEY MESSAGES

• The burden of psychological and physical symptoms among HIV outpatients with treatment access is seldom researched yet essential to improved management and patient outcomes.

• Among 778 UK outpatients, the 7-day period symptom prevalence was high. Being on treatment was not associated with lower symptom distress. Psychological symptoms were associated with poorer adherence and sexual risk behaviour.

• Clinical care should routinely assess multidimensional symptoms as these are controllable and may improve outcomes such as treatment response, transmission and quality of life.

ABSTRACT Objectives

There is a paucity of data reporting the prevalence and burden of pain and symptoms among HIV patients in the era of antiretroviral therapy (ART). This study aimed to measure symptom prevalence and determine associations with key variables: demographics, treatment status, adherence and risk behaviours.

Design

Cross-sectional self-completion questionnaire in five HIV outpatient clinics in London and South East of the UK.

Methods

Consecutive patients were invited to participate, responding to clinical and behavioural variables including the Memorial Symptom Assessment Schedule (Short-form). Four multivariable models examined the relationship between dependent variables of 1) psychological, 2) physical, 3) global symptom burden scores, 4) number of symptoms and key independent variables. 778 patients participated, response rate 77% of all patients approached.

Results

Both physical and psychological symptoms were highly prevalent: in the previous seven days, 70.8% experienced lack of energy, 69.9% worry, 53.6% diarrhoea, 53.5% sexual dysfunction, and 53.2% pain. In multivariable analysis, both unprotected sexual intercourse with a partner of unknown status, and poorer ART treatment adherence were significantly and independently associated with psychological symptom burden. Lower educational achievement was significantly associated with increasing physical, psychological and global symptom burden, and with higher number of symptoms. Being on ART was not associated with any symptom distress measure.

Conclusions

In the era of treatment, patients continue to experience high prevalence and burden of psychological and physical symptoms, which are not associated with treatment status. Attention to these distressing problems is essential and may enhance quality of life, enhance adherence and minimise risk behaviour.

Symptoms are highly prevalent among HIV outpatients and associated with poor adherence and unprotected sexual intercourse

BACKGROUND

HIV disease is associated with highly prevalent and distressing symptoms from the point of diagnosis [START_REF] Selwyn | Palliative care for AIDS: challenges and opportunities in the era of highly active anti-retroviral therapy[END_REF] . A significant proportion of patients receiving antiretroviral therapy (ART) experience drug toxicities such as peripheral neuropathy and gastrointestinal problems(2), requiring drug discontinuation or treatment change within, and between, classes of ART(3). However, the vast majority of data on pain and symptom prevalence has been reported from the pre-ART era on patients with AIDS-defining illnesses, and it has been difficult to quantify the relative contribution of treatment to the presence and severity of symptoms [START_REF] Breitbart | Pain in ambulatory AIDS patients. I: Pain characteristics and medical correlates[END_REF].

The World Health Organisation (WHO) advocates pain and symptom control as an essential component of HIV clinical care (5). However, the evidence suggests inadequate clinical attention to pain and symptom assessment and management, as clinicians detect three-fold fewer HIV-related symptoms than their patients report [START_REF] Justice | Clinical importance of provide-reported HIV symptoms compared with patient-report[END_REF], and patients perceive that their symptoms remain untreated (7;8).

In order to maximise clinical benefit from ART, adherence must be optimised and movement between classes of drugs minimised so that future treatment options can be retained. In order to achieve this, ART-related symptomatology must be taken into account. Further, clinicians need to be able to identify prevalent symptoms, and which patients may experience symptom distress, as well as non-clinical potential ramifications of symptom distress such as risk behaviour and adherence variation. Currently, not only is there a lack of focus on pain and symptom prevalence in the era of ART, but these variables have not been assessed in relation to the key clinical and behavioural components of care such as treatment adherence and behavioural risk reduction.

Investigation into prevalence and correlates of symptoms is required to inform clinical practice and thereby improve clinical outcomes and quality of life(1;9;10).

METHODS

Design and recruitment

This study aimed to measure the prevalence and distress related to physical and psychological symptoms, and to determine the relationship between these and demographic variables, ART use and adherence, risk behaviour and HIV status disclosure.

This cross-sectional questionnaire study was conducted in five outpatient clinics in London and the South East of England during 2005/6. Consecutive attending patients were approached and invited to participate (n=1007). The inclusion criteria (HIV positive adults with sufficient English language and cognitive/physical ability to self-complete) were fulfilled by 904 patients (90%) who received a questionnaire, of which 778 were completed and returned (77% of all patients, 86% of those eligible to receive a questionnaire). Multi-centre UK ethical approval was granted by the Royal Free Hospital Clinical Ethics Committee ref 05/Q1907/26.

Data collection

The questionnaire comprised of four core components of items that were selected to meet the aims of the study in line with the existing literature.

Firstly, a demographic component ( addressing age, gender, ethnicity, employment, country of birth, sexuality, and education).

Second a behavioural component (unprotected sexual intercourse with a partner of unknown status in the previous three months; treatment optimism and infectiousness optimism on a standardised scale [START_REF] Elford | High-risk sexual behaviour increase among London gay men between 1998 and 2001[END_REF]; and disclosure of HIV status to others based on Kalichman 2003(12)).

Third, treatment items (current treatment status was recorded as naïve, on first regimen, switched regimen or stopped; treatment adherence based on a seven day self report recall on three levels: fully adherent, partially adherent or non-adherent, a composite measure according to whether i) all doses were taken, ii) at the correct time and iii) in line with dietary requirements(13)).

Fourth, symptoms were measured using the Memorial Symptom Assessment Scale Short Form, a standardised scale reporting on symptom prevalence in the previous seven days, with three subscale indices of Physical Symptom Distress (MSAS-Phys), Psychological Symptom Distress (MSAS-Psych) and Global Distress Index (MSAS-GDI) [START_REF] Chang | The memorial symptom assessment scale short form (MSAS-SF)[END_REF]. Each of these 3 subscales has a possible score range of 0-4. This well-validated multidimensional instrument captures the presence and distress of 26 physical and 6 psychological symptoms. Total number of symptoms (out of a possible 32) was also ascertained. Although it was originally validated among cancer patients, it is commonly applied in a range of progressive conditions, and has the advantage of enabling comparison between patient populations.

Analysis

Descriptive statistics were used to report the entire sample characteristics.

Then, the following analysis was conducted on the full sample.

Univariate linear regression analyses were conducted to test the association of various factors with the following four dependent variables: 1) physical symptom distress subscale, 2) psychological symptom distress subscale, 3), global symptom distress subscale and 4) number of symptoms. Each of these 4 dependent variables formed a separate model in the subsequent multivariable analyses described below. Independent variables entered in univariate analysis for the whole sample were: age, gender/sexuality, ethnicity, UK-born or not, education, unprotected intercourse, disclosure, and current treatment status. For each model gender/sexuality, ethnicity, education, and treatment were collapsed to two levels: gay male/heterosexual, university educated/not, Caucasian/not Caucasian, and on ART treatment/not on treatment.

Subsequently, for the subsample of patients currently on ART treatment, the analysis was repeated for those patients only with additional treatment-related independent variables entered: treatment switching as two levels (switched/not switched), and adherence entered as a three level variable: fully adherent/partially adherent/non-adherent. Current treatment status was not entered into these models.

Following each univariate regression, multivariable regressions models were constructed. Independent variables from the univariate analyses above were entered stepwise into the multivariable model if significant at the 25% level [START_REF] Altman | Practical Statistics for Medical Research[END_REF]. Cases with missing data were excluded from the multivariable models .

The multivariable analysis was conducted first on the entire sample, then for the subsample currently on ART.

RESULTS

Sample characteristics

The sample mean age was 40.4 years (SD=8.6). The majority identified as gay or bisexual males (n=496, 65.7%), with n=183 (24.2%) female and n=76 (10.1%) heterosexual male (missing=23). The majority were Caucasian (n=513, 67.3%), with n=188 Black (24.7%), n=23 Asian (3.0%), and n=38

(5.0%) mixed/other (missing n=16). The majority were UK born (396, 50.9%).

Educational attainment was reported as follows: no qualifications (n=67, 9.0%), exams attained at age 16 n=190 (25.4%), exams attained at age 18 n=154 (20.6%), university degree or above n=336 (45.0%), missing n=31.

Treatment variables

Most patients were currently on ART (524, 67.4%), with 155 (19.9%) treatment naïve (the remaining 99 having stopped treatment). Adherence in the preceding seven days was as follows: fully adherent 41.5%, partially adherent 36.1%, non-adherent 22.4%. HIV status had not been disclosed to anyone by 47 respondents (6%).Unprotected sexual intercourse in the previous three months with a person who respondents were not sure was also HIV-infected was reported by 85 of the sample (10.9%).

With respect to treatment switching, of those on ART, 161 (32.7%) had not switched from their first treatment regimen, 135 (27.4%) had switched once, 196 (39.8%) reported multiple switches, (32 missing)

INSERT TABLE 1 HERE

Symptomatology

The mean number of reported symptoms was 17.9 (SD=12.1), the mean (standard deviation) symptom distress subscale indices were physical distress 0.81 (0.7), psychological distress 1.34 (1.0) and global distress 1.16 (0.8).

The prevalence and distress of the ten most commonly reported symptoms are described in Table 1. Manifestations of fatigue were most common, namely lack of energy (70.8%) and feeling drowsy/tired (67.5%). Problems related to psychological distress included difficulty sleeping (61.8%), difficulty concentrating (60.7%), feeling worried (69.9%), feeling sad (66.3%) and feeling irritable (56.6%). Other common physical symptoms were diarrhoea (53.6%), problems with sexual activity (53.5%) and pain (53.2%).

INSERT TABLES 2 and 3 HERE

Multivariable analysis

For the full sample (n=778), Table 2 shows univariable associations and Table 3 shows multivariable regressions. In the multivariable models, lack of university-level education was significantly associated with poorer outcomes in all symptom distress indices; regression coefficients (b) for university-level versus other education were as follows: b=-0.111, p=0.004 for physical distress, b=-0.096, p=0.017 for psychological distress, b=-0.101, p=0.007 for global distress, b=-0.090, p=0.021 for total number of symptoms. Identifying as Caucasian was associated with lower global distress (b=-0.076, p=0.040).

Having disclosed HIV status was significantly associated with lower number of symptoms (b=-0.090, p=0.021). Reporting unprotected intercourse in the previous three months with a person not known to be also HIV positive was associated with greater psychological symptom distress (b=0.080, p=0.047).

Interestingly, currently being on ART was not significantly associated with any of the symptom measures.

Tables 4 and5 show the analysis restricted to patients currently on ART (n=524) (see Tables 4 and5). In multivariable models (Table 5), poorer ART adherence was significantly associated with both psychological (b for trend across adherence categories=0.126, p=0.001) and global symptom distress (b=0.125, p=0.006). Having ever switched treatment was significantly associated with higher physical symptom distress (b=0.135, p=0.003), higher psychological symptom distress (b=0.126, p=0.006), higher global symptom distress (b=0.152, p=0.001), and a greater number of symptoms (b=0.132, p=0.013). Being UK-born and university education were also independently associated with lower physical symptom burden.

INSERT TABLES 4 and 5 HERE

DISCUSSION

The data, among out-patient HIV clinic attendees, reveal high seven-day prevalence, and associated distress, of burdensome symptoms. Psychological problems and pain were of particular concern. A symptom prevalence grid comparing evidence for end-stage patients reported on three of the most prevalent symptoms also reported in this study [START_REF] Solano | A comparison of symptom prevalence in far advanced cancer, AIDS, heart disease, chronic obstructive pulmonary disease (COPD) and renal disease[END_REF]. Prevalence in our sample was at the top end of the reported range for both diarrhoea and fatigue, and in the mid-range for pain, compared to patients with advanced malignant, renal and pulmonary disease. Therefore, the symptom control needs of HIV outpatients, including those on treatment, are comparable to those with advanced malignant and non-malignant progressive diseases. This high prevalence of distressing symptoms should be taken into account to inform assessment and management within HIV clinical services.

The associations between symptom prevalence/burden and demographic, clinical and behavioural variables have identified significant relationships that should be taken account of to inform symptom assessment, adherence maximisation, and risk reduction. The associations with being non-UK born/not Caucasian may be a reflection of the tendency for this population to be diagnosed with more advanced disease. It may also be that persons born outside the UK, where HIV stigma is more prevalent, are less likely to disclose their infection and therefore experience a high symptom burden. The association between having switched regimen and greater number of symptoms may be due to those with more symptoms being more likely to wish to change treatment, having to take regimens with a higher side-effect burden, or simply having more advanced disease.

It is of particular note and importance that the data have shown that currently being on ART was not associated with having fewer symptoms or lower symptom distress, strongly indicating the continuing need for clinical attention to symptoms after initiating ART. Longitudinal studies are needed to identify causal relationships between initiating/maintaining specific treatment regimens and both physical and psychological symptomatology.

There are several potential limitations to our findings. Firstly, although the response rate was high (77% of all clinic attendees, 86% of eligible attendees), a small number of patients did not meet the inclusion criteria (i.e. ability to self-complete, in line with ethical review requirements). The period of recruitment (three months) would have enabled most patients to be approached in outpatient settings. The cross sectional nature of the study can identify associations, but cannot identify causal relationships. Although the response rate is high, we do not have data to compare prevalence of symptoms between responders and non-responders. Additional data in future studies on the relationship between biological markers of disease (CD4 count, viral load) and symptomatology would add further understanding of the aetiology of these problems. We would also advocate further studies to investigate these relationships in resource-poor settings. Lastly, due to the early provision of needle exchange in the UK, we have a very small population of people infected via intravenous drug use. Further study is warranted in countries where this population constitutes a greater proportion of HIV patients.

The body of evidence on the symptom control needs of people living with HIV disease in the ART era has been limited, and has not previously been investigated in relation to other clinical and behavioural variables. There are many barriers to effective symptom control for people with HIV disease, including the lack of awareness of symptoms associated with HIV disease, patient belief that pain should be expected following an HIV diagnosis, and a focus on purely virological outcomes [START_REF] Harding | Access and equality in HIV/AIDS palliative care: a review of the evidence and responses[END_REF]. Greater clinical attention must be paid to the high prevalence of distressing psychological symptoms experienced by the majority of people living with HIV, as a systematic review of the evidence demonstrates that symptoms can be effectively managed [START_REF] Harding | Does palliative care improve outcomes for patients with HIV/AIDS? A systematic review of the evidence[END_REF].

It is essential that quality management of HIV disease routinely assesses these distressing problems, so that key outcomes of risk behaviours and adherence may be optimally influenced. An essential step toward holistic management of the patient is to ensure that care protocols routinely ask about the full range of problems that the patient may experience. Physician-led identification of problems is likely to lead to improved identification and management of problems than by expectations that patients will present their physical and psychological symptoms. Effective treatment strategies should be implemented through integrated patient management between HIV clinicians, mental health providers, and symptom management specialists.

Generalist palliation of problems should be a core skill among all clinicians, with clear referral guidelines to, and knowledge of, local symptom control and palliation teams. In terms of management strategies for control of the highly prevalent range of psychological problems (and manifestations such as fatigue), systematic reviews of the evidence are urgently required to identify the most effective approaches. As HIV prognosis has greatly improved and a chronic disease model prevails, interventions that are appropriate to longerterm coping are required. It is clear that despite advances in universal treatment, the patient burden of disease remains high, and outcomes are unlikely to be improved without careful attention to the patient experience of disease and a clinical focus beyond virology.

*

  entered into multivariable model$ tests for trendTable 5 -Associations of factors with symptom measures: multivariable models only for those on treatment n=524

Table 1 -

 1 The ten physical and psychological symptoms with highest 7-day prevalence and their associated distress (MSAS-SF) (n=778)

	% who
	% of
	patients
	reporting
	symptom

reported symptom but did not rate distress Level of distress (as % of all patients)

	Physical Symptom			Not at all	A little bit	Somewhat Quite a bit	Very much
	Lack of energy	70.8%	4.5%	10.8%	19.8%	12.3%	12.1%	10.8%
	Feeling drowsy/tired	67.5%	4.9%	10.7%	19.8%	9.8%	12.3%	10.0%
	Difficulty sleeping	61.8%	4.8%	13.5%	10.9%	9.5%	12.1%	10.9%
	Difficulty concentrating	60.7%	4.0%	16.6%	15.2%	10.3%	9.1%	5.5%
	Diarrhoea	53.6%	2.7%	17.6%	12.6%	7.6%	7.5%	5.4%
	Problems with sexual activity	53.5%	3.6%	15.7%	8.1%	6.6%	7.1%	12.2%
	Pain	53.2%	3.1%	18.0%	12.0%	5.9%	8.6%	5.4%
	Psychological	% of	% who		How often
	Symptom	patients	reported
		reporting	symptom
		symptom	but did
			not rate distress	Rarely Occasionally Frequently	Almost constantly
	Worried	69.9%	5.4%	8.4%	25.4%	21.5%	9.1%
	Sad	66.3%	4.8%	11.8%	26.9%	16.7%	6.2%
	Irritable	56.6%	3.1%	10.4%	22.4%	16.3%	4.2%

Table 2 -

 2 Associations of factors with symptom measures: univariable analysis whole sample n=778

	Dependent
	Variable

Table 3 -

 3 Associations of factors with symptom measures: multivariable analysis models for whole sample n=778

	Dependent
	variable

*= sig at the 5% level, † = sig at the 1% level

Table 4 -

 4 Associations of factors with symptom measures: univariable analysis only for those on treatment n=524

	Dependent
	Variable

ACKNOWLEDGEMENTS

We wish to acknowledge the contribution of Gilly Arthur and Sarah Zetler. Also research assistance from Amanda Jayakody, research nurses at participating clinics, and all survey respondents. We are grateful to Lucy Bradley for manuscript management. This research was assisted with an unrestricted educational grant from GlaxoSmithKline, with input from the Adherence Strategy Group. The Authors state they have no conflict of interests.

WORD COUNT: 2236

FL is the study statistician; SN and CL were the study research assistants and co-ordinated data collection and data handling.

26

*= sig at the 5% level, †= sig at the 1% level, $=test for trend

CONFLICT OF INTERESTS

The authors have no conflict of interests.

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 STI and any other BMJPGL products and sub-licences such use and exploit all subsidiary rights, as set out in our licence http://group.bmj.com/products/journals/instructionsfor-authors/licence-forms".

CONTRIBUTIONS OF AUTHORS:

LS&RH designed the study and were involved at all stages; HLD MF MJ SE and JA commented on design and participated in data collection and write up;


Laura Jane Scott
email: laura.jane.scott@ggc.scot.nhs.uk

William F Carman

Andrew J Winter

A New Multiplex Real-time PCR Test for HSV 1 / 2 and Syphilis-An evaluation of its impact in the laboratory and clinical setting

Keywords: Syphilis, Herpes simplex virus, real-time PCR, multiplex, ulcer, Treponema pallidum

This manuscript describes the clinical utility and impact of testing for HSV and syphilis in a single sample using a single test.

• Using this single test leads to the detection of unexpected cases of syphilis where the suspected aetiological agent was HSV, and also improves the detection rates of primary syphilis.

• The test is likely to reduce costs and turn around times as testing is performed at a single location for both pathogens and only one sample is needed.

Introduction

It is important to be able to diagnose herpes simplex virus (HSV) and Treponema pallidum (syphilis) infections as they can be clinically indistinguishable. Primary syphilis can progress to more serious secondary and tertiary sequelae 1 , and can be difficult to diagnose, particularly in the early stages. Serological testing can be insensitive during early syphilis infection and therefore may miss cases of primary infection [START_REF] Wheeler | Dark ground microscopy and treponemal serological tests in the diagnosis of early syphilis[END_REF] . Dark ground microscopy (DGM) is not available at all clinical locations, is highly user-dependent and is unsuitable for use on rectal and oral sites [START_REF] Larsen | Laboratory diagnosis and interpretation of tests for syphilis[END_REF] . Polymerase Chain Reaction (PCR) is a sensitive and specific test for the diagnosis of primary syphilis [START_REF] Bruisten | Diagnosing genital ulcer disease in a clinic for sexually transmitted diseases in Amsterdam, The Netherlands[END_REF]5 and can be used on both oral and rectal sites 6 .

HSV has been routinely diagnosed by PCR at the West of Scotland Specialist Virology Testing Centre (WoSSVC) since 1997, while Syphilis PCR testing became available in Scotland at the Specialist Bacterial STI Reference Laboratory (SBSTIRL) in May 2006. In order to diagnose each infection, separate swabs need to be taken, and to ensure a swab for syphilis is obtained; a high degree of clinical suspicion is required. Sending the swabs to different locations leads to delays in turnaround times and patient treatment because of batching and transport delays. It may also lead to infections not being diagnosed because swabs, particularly for syphilis, are not taken. We decided to develop a multiplex PCR test for the detection of HSV1, HSV2 and syphilis at the WoSSVC to overcome these difficulties.

Methods:

Test Development

A real-time PCR test was developed targeting the 47kDa membrane gene of T. pallidum. The primers and probe used were; Forward 5'-GGATAGTTTTTCTGCACGTAAGGTAA, Reverse 5'-ACCCACCGTGTCTACCACAAG and Probe 5' VIC-

CAGCATGGAGAGCCCGCACG-tamra at concentrations of 100µM (primers) and 20µM (probe) using standard taqman conditions [START_REF] Gunson | Simultaneous Detection and Quantitation of Cytomegalovirus, Epstein-Barr Virus, and Adenovirus by Use of Real-Time PCR and Pooled Standards[END_REF] . This test was then multiplexed with the existing HSV 1/2 real-time PCR test in place at the WoSSVC which uses the fluorophores FAM and Cy5 (previously published [START_REF] Scoular | Polymerase chain reaction for diagnosis of genital herpes in a genitourinary medicine clinic[END_REF] ).

Prior to the clinical evaluation, the performance of the new multiplex was validated in the laboratory using a number of different panels. Firstly a dilution series of HSV1 and HSV2

was tested using the HSV1 and 2 duplex assay and the newly developed multiplex assay to ensure that the HSV PCR component was not affected by the addition on the syphilis primers and probe. Following this, the specificity of the multiplex assay was assessed by testing a panel containing other viruses and bacteria. A dilution series of a clinical sample containing T. pallidum (provided by the SBSTIRL and previously tested by an alternative reference assay) was also tested to ensure that the newly developed multiplex achieved the same end point detection limit for syphilis testing as the reference test already in place in the SBSTIRL.

Quality assurance panels were also tested to assess the robustness and reproducibility of the newly designed multiplex assay in the laboratory.

All samples were extracted using the virus kit on the MDX extractor (Qiagen) and tested using the new triplex real-time PCR assay using UGD platinum supermix (Invitrogen) on the ABI 7500 (Applied Biosystems).

Patients and samples

Clinical samples were obtained from patients presenting to the Sandyford Initiative with symptoms of genital ulceration or suspected early syphilis, in cases where an ulcer swab would usually have been taken. During initial clinical use (6 months), 692 samples (including genital, oral and anorectal swabs) were received.

All samples were processed as outlined above. All positive syphilis PCR samples were sent to the SBSTIRL for confirmatory PCR before reporting. The T. pallidum PCR test used at the reference laboratory targets the polA gene and has been previously published [START_REF] Koek | Specific and sensitive diagnosis of syphilis using a real-time PCR for Treponema pallidum[END_REF] . The PCR results were then aligned with DGM and serological results and analysed. Serological screening was performed using a combined IgG/IgM kit (Newmarket).

Results:

The initial validation showed that the HSV components of the newly developed multiplex assay had the same endpoint detection limits as the HSV 1 and 2 duplex assay already in place in the WoSSVC. The syphilis component was also shown to have the same endpoint detection limit as the syphilis reference test in place at the SBSTIRL. No cross reactions were observed when the sensitivity panel was tested and the multiplex produced reproducible results in the ongoing EQA.

The results of the 692 clinical samples are shown in The 16 syphilis PCR positive samples were from 15 patients and were all confirmed by the SBSTIRL. Of the 15 positive patients; 12 had a blood sample taken for serological testing on their first visit; of which 10 were positive (sensitivity 83.3% compared to PCR). The remainder, who were either sero-negative on their first visit or had no blood taken, all had positive syphilis serology on follow-up serological testing.

Only six of the 15 PCR positive patients were tested by DGM yielding three positives (50% sensitivity). During the study period, all cases of primary syphilis, diagnosed by the non-PCR tests (DGM/ serology) were reviewed to check if any of them had been missed by the PCR. The reviews showed that the PCR did not miss any cases.

Discussion:

The HSV1/HSV2/T. pallidum PCR had better detection rates for primary syphilis than both DGM and serology. In addition, the T. pallidum component of the new multiplex test was shown to perform well in comparison to the PCR already in place at the SBSTIRL. Less than half of the patients with PCR-proven primary syphilis had DGM testing performed, illustrating the difficulty of relying on this alone. DGM at the Sandyford is performed by a highly-experienced full-time laboratory biomedical scientist, but even here sensitivity was just 50% compared to PCR.

The multiplex PCR test is targeted at symptomatic patients so can be used as a valuable front line test to ensure primary syphilis infection is not missed or misdiagnosed; and that serological follow-up and patient review is carried out. In two of the syphilis positive patients during this study period, follow up and blood sampling was only initiated due to the positive PCR result. In one case, a young heterosexual woman with documented previous HSV2 was unexpectedly found to be T. pallidum positive when an HSV2 recurrence had been suspected.

Previously, separate swabs had to be taken and transported to the reference laboratory for syphilis testing. As syphilis and HSV testing is now performed using a single sample at a local laboratory, it is likely that the turnaround time and costs have been reduced as well as the number of misdiagnoses.

The multiplex assay covers the huge majority of diagnosable causes of genital ulceration in the UK. Additional targets might eventually include C. trachomatis (LGV serovars) which now generally presents as severe proctitis, although some classical genital ulceration is seen [START_REF] Sethi | Lymphogranuloma venereum presenting as genital ulceration and inguinal syndrome in men who have sex with men in London, UK[END_REF] , and H ducreyi 12 , which remains very rare in the UK. However, care should be taken to ensure that there is no loss of sensitivity when increasing PCR targets 13 .

Conclusions:

Multiplex PCR testing for HSV1, HSV2 and T. pallidum on ulcer swabs can be incorporated into routine laboratory workflows, improving detection rates and reducing turnaround times and costs, ultimately reducing the risk of onwards transmission of primary syphilis and development of sequelae.

table 1 Table 1 : PCR Results for GUM samples

 11

	PCR RESULT	TOTAL	%
	negative	401	57.95
	HSV1	139	20.09
	HSV2	136	19.65
	SYPHILIS	15	2.17
	HSV1 AND
	SYPHILIS	1	0.14
	GRAND TOTAL	692

Orle KA, Gates CA, Martin DH, Body BA, Weiss JB 1996. Simultaneous PCR detection of Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus types 1 and 2 from genital ulcers. J Clin Microbiol. ;34(1):49-54

Kingston M, French P, Goh B, Goold P, Higgins S, Sukthankar A, Stott C, Turner A, Tyler C, Young H; Syphilis Guidelines Revision Group 2008, Clinical Effectiveness Group. 2008 . UK National Guidelines on the Management of Syphilis 2008. Int J STD AIDS.;19(11):729-40.

Gunson RN, Bennett S, Maclean A, Carman WF. 2008. Using multiplex real time PCR in order to streamline a routine diagnostic service. J Clin Virol;43(4):372-5.

Acknowledgements:

Thanks to Dr Helen Palmer and the staff at the SBSTIRL for providing validation material and a confirmatory service and Heather Carre at NIBSC for her help.

Word Count: 1213

Evaluation of multiplex real-time PCR for detection of Haemophilus ducreyi, Treponema pallidum, herpes simplex virus type 1 and 2 in the diagnosis of genital ulcer disease in the Rakai District, Uganda. Sex Transm Infect.;85(2):97-101.