# 2008 The Authors Journal compilation # 2008 Blackwell Munksgaard
Acta Neuropsychiatrica 2008: 20: 145–151 All rights reserved DOI: 10.1111/j.1601-5215.2008.00281.x
ACTA NEUROPSYCHIATRICA
Neuropsychiatric symptoms and immune activation in patients with genital herpes Vollmer-Conna U, Chen M, Lloyd A, Donovan B. Neuropsychiatric symptoms and immune activation in patients with genital herpes. Objective: Neuropsychiatric disturbances are common among patients with genital herpes simplex virus (HSV) infection. To date, no studies have examined the possible role of immune activation in the aetiology of these disturbances. The aim of this study was to examine the relationship between markers of immune activation and measures of emotional and somatic dysfunction among patients with symptomatic genital herpes. Methods: Twenty-two patients with documented genital herpes were assessed when herpetic lesions were present and when they were not. Each assessment included a clinical examination, self-reported symptom measures as well as a blood and urine collection. Markers of immune activation [neopterin and interleukin (IL)-6] in serum and urine were quantified by enzyme-linked immunoassay. These measures were also obtained from a group of healthy control subjects. Results: Urine, but not serum, levels of neopterin and IL-6 correlated significantly with measures of reported psychological distress and fatigue. These associations were not confined to periods of overt clinical lesions. Conclusions: HSV-related neuropsychiatric morbidity correlates selectively with regional, but not systemic, measures of immune activation. We hypothesise that communication between the local inflammatory site in the pelvis and the brain occurs through autonomic afferent pathways.
Introduction
Genital herpes is one of the most common sexually transmitted viral infections in the world, with an estimated prevalence of 10–50% in adult populations (1,2). Two closely related viruses – herpes simplex virus (HSV)-1 and HSV-2 cause the condition, although HSV-2 is more commonly associated with genital lesions and HSV-1 with orofacial lesions. Lesions may be preceded by systemic symptoms including fever, neuralgia and malaise (2,3). A unique biologic feature of HSV is the ability of this virus to invade and replicate in the host nervous system, to establish latent infection in lumbosacral sensory ganglia and possibly autonomic neurons including the paracervical and vagal ganglia (3,4). Latency in neurons enables the virus to evade immune detection, thus ensuring life-long infection of the host (5). Periodically, HSV may reactivate with viral particles travelling along sensory axons, giving rise to asymptomatic viral shedding or recurrent lesions (3,6). Significant emotional and somatic dysfunction has been documented for both
Ute´ Vollmer-Conna1, Marcus Chen2, Andrew Lloyd3, Basil Donovan2,4 1 School of Psychiatry, University of New South Wales, Sydney, New South Wales, Australia; 2 Sydney Sexual Health Centre at the Sydney Hospital, Sydney, New South Wales, Australia; 3Inflammatory Diseases Research Unit, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia; and 4The National Centre in HIV Epidemiology and Clinical Research, University of New South Wales, Sydney, New South Wales, Australia
Keywords: genital herpes; herpes simplex virus; immune activation; neuropsychiatric disturbance Dr Ute Vollmer-Conna, Department of Human Behaviour, School of Psychiatry, University of New South Wales, Sydney, NSW 2052, Australia. Tel: 161 (02) 9385 2945; Fax: 161 (02) 9385 2944; E-mail: [email protected]
primary as well as recurrent genital herpes episodes (2,7,8). The prevalent changes in the emotional state of these individuals may be a result of suffering from a painful, sexually restricting disorder, and the social stigma attached to the disease (7). However, there is a long-held notion among both patients and physicians that psychological distress may itself precipitate recurrences (9). Although this possibility has been extensively explored (10–14), empirical evidence linking psychological factors such as life stress, negative mood and personality to genital herpes recurrences remains inconclusive. Two longitudinal studies that monitored neuropsychiatric symptoms on a daily basis provide interesting insights into the temporal relationship between mood disturbance and recurrent herpes (11,13). Both studies reported a significant decrease in emotional wellbeing 3–4 days prior to the appearance of genital lesions: this was interpreted as supporting a role for psychological distress in the HSV reactivation. The close temporal relationship 145
Vollmer-Conna et al. between the increase in mood disturbance and the recurrence of herpes lesions alternatively suggests a neuropsychiatric prodrome associated with viral reactivation that may be mediated via an immunologically triggered sickness response. The term Ôsickness response’ refers to a set of stereotyped physical and behavioural changes (fever, hypersomnia, hyperalgesia, anorexia, mood and cognitive disturbances) that typically accompany acute infection and inflammation (15). Extensive research with animals established that the acute sickness response is mediated by the action of proinflammatory cytokines, such interleukin (IL)-1b, IL-6 and tumour necrosis factor-a, on central nervous system targets (16–18). For example, both the behavioural and neurochemical changes associated with acute infective illness can be induced in a dose-dependent fashion by systemic or intracerebral injection of these cytokines. Elaboration of this phenomenon has led to a major conceptual advance in our understanding of how sickness and recovery processes are organised in the brain, leading to a deeper appreciation of the physiological and psychological impact of infective illnesses (19). There is now increasing awareness that non-specific neuropsychiatric symptoms commonly accompany chronic infections and autoimmune diseases including hepatitis C virus infections (20) and in inflammatory bowel disease (21). Moreover, it has been speculated that neuropsychiatric conditions, including depression, fatigue syndromes and somatisation disorders may constitute abnormal variants of an immunologically triggered sickness response (22–24). The present study provides an initial examination of the role for immune activation in the manifestation of emotional and somatic symptoms reported by patients with genital herpes. As components of both the innate and adaptive immune systems are essential in the defence against herpes infections (5), we included two sensitive markers of cellular immune activation: neopterin, which is selectively induced by interferon g and the proinflammatory cytokine IL-6 (25–27). Both urinary and serum measures of these markers were included to distinguish regional (urogenital) from systemic immune activation.
Methods Subjects
Patients with primary or recurrent genital herpes were recruited through the Sydney Sexual Health Centre, a public sexual health service in Sydney, Australia. Eligible subjects were required to have had 146
microbiologically confirmed genital herpes, either by viral culture from a genital swab or, if this was not possible, positive serology for HSV-2. Patients were excluded if they had any other infectious, autoimmune, neoplastic or psychiatric conditions. Patients taking antiviral drugs were also excluded. Participating patients were asked to visit the clinic on two occasions: once when they experienced genital lesions, and again when they did not have any genital lesions, as indicated both by self-report and the absence of lesions on clinical examination. At both visits, patients completed selfreport instruments assessing mood states and clinical symptoms, underwent a clinical examination and provided a blood and a urine sample. Control subjects with no history of HSV infection and with a similar age and sex distribution as the patient sample were recruited by advertisement from the University of New South Wales. Controls were included in the study if they had no reported history of genital herpes. Serological testing for herpes was not undertaken on the controls. The control subjects were interviewed, completed the self-report instruments and provided a blood and urine sample. Written informed consent was obtained from all participants. The study was approved by the institutional Human Research Ethics Committee. Questionnaires
Subjects were asked to complete the Somatic and Psychological Health Report (SPHERE) (28), which produces two validated subscales (SOMA and PSYCH) reflecting somatic (fatigue, pain) and psychological distress, respectively. Current (last 24 h) mood states were also assessed using the fatigue, anger, confusion, depression and anxiety subscales of the Profile of Mood States (POMS) questionnaire (29). The Brief Disability Questionnaire (BDQ) (30), documented impairment in daily activities in the areas of self-care, work and leisure activities. A clinical symptom record was used to assess for the presence of patient-reported symptoms: genital pain, itch, dysuria, neuralgia, blisters, ulcers or crusts on genitals, general malaise, fatigue and fever. Measures of immune activation
Blood and urine samples were collected from participants between 12 AM and 4 PM and processed within 1 h of collection before freezing serum and urine in aliquots at minus 80°C. As neopterin is light sensitive, specimens were collected, processed and stored under light-protected conditions. Neopterin
Neuropsychiatric symptoms and immune activation content in sera and urine samples was determined by competitive enzyme immunoassay (ELItest; Brahms, Berlin, Germany) in accordance with the manufacturer’s instructions. Urinary neopterin concentrations were related to creatinine concentrations measured in the same urine specimen by the Jaffe rate method. Absorbance readings were taken at 520 nm (SYNCHRON LX-20; Beckman Coulter, Sydney, Australia). This served as an internal reference standard to compensate for physiological variations in urinary concentrations. Urinary neopterin levels were thus expressed as the ratio: mmol neopterin/mol creatinine. The concentrations of IL-6 in sera and urine samples were quantified by solid-phase enzymelinked immunosorbent assay in accordance with the manufacturer’s instructions [Quantikine and Quantikine-HS (range: 0.156–10 pg/ml); R & D Systems, Minneapolis, MN, USA]. Using this assay system, separate assessment of creatinine concentrations is not required for urinary IL-6 determination. Statistical analyses
The SPSS for Windows (version 13) statistical package was used for data analysis. ANOVA with Bonferroni correction for multiple testing was used to assess differences between groups and over time (repeated measures). Spearman correlations were used to assess the relationship between the severity of symptoms and concentrations of immunological proteins. The appropriate number of subjects for this study had been estimated from effect sizes previously obtained for correlations between physical and psychological symptoms and IL-6 concentrations in acute infections (15). On this basis, the present study had an estimated power of 80% to detect significant (a ¼ 0.05) correlations with a magnitude of r ¼ 0.46.
Results Sample characteristics
Thirty-three patients with genital herpes were recruited into the study, of which 22 (67%) completed all assessment components. There were no significant differences in those who completed the study and those who did not in terms of age [mean age 35 (SD: 11) vs. 32 (SD: 6) years, respectively, p ¼ 0.42]; the proportion experiencing an initial episode of genital herpes (36 vs. 30%, p ¼ 0.64); the severity of reported somatic and emotional symptoms at baseline (all p ¼ 0.21); or the concentrations of immune markers at baseline (all
p ¼ 0.39). However, the gender distribution differed, with more females among those lost to follow-up (70% vs. 32%). The data did not suggest any significant gender-dependent differences in either the severity of somatic and emotional symptoms (all p ¼ 0.51) or concentrations of immune markers (all p ¼ 0.38). The 22 patients who completed the study included 15 males and 7 females. There were eight control subjects, including five males and three females [mean age: 34 years (SD: 6)]. Of the 22 patients with herpes, 8 (6 males; 2 females) presented with their first episode of genital herpes, while 14 (9 males; 5 females) presented with a recurrent episode of herpes. Among those with a history of recurrent herpes, the median interval since the first reported episode was 6.5 years (interquartile range 1.5–11 years) with the median number of recurrences reported per year being 10 (interquartile range 3.5–12 years). All had declined to take suppressive antiviral therapy. Eighteen patients (82%) reported at least one of the following non-genital symptoms at the time they were experiencing genital lesions: fatigue, malaise, headaches and muscle or joint pain. Pain on passing urine (dysuria) or difficulty passing urine was reported by seven patients (33%). Twenty patients (91%) had genital HSV-2, while two (9%) had genital HSV-1. The median interval between the two clinic visits for participants was 28 days (interquartile range 14–45 days). Neuropsychiatric symptoms
Symptom and disability scores for patients with genital herpes – when lesions were present and when they were not – and for the control subjects are shown in Table 1. Patients reported significantly higher levels of emotional disturbance and functional impairment when experiencing genital lesions than when they were lesion-free. Compared with the controls, the somatic and emotional symptoms reported by the patients with herpes were significantly more severe across all measures when genital lesions were present. Even when lesions were absent, the symptom and disability ratings from herpes patients, while significantly improved, remained higher than for the controls for all but two measures of mood. The scores obtained for mood states over Ôthe past 24 hours’ from control subjects were comparable to published norms using the same version of the questionnaire (27). Similarly, among the controls, ratings on both subscales of the SPHERE were consistent with scores typically obtained from health samples (SOMA: ,3; PSYCH: ,2) (26). 147
Vollmer-Conna et al. Table 1. Severity of emotional and somatic disturbance reported by patients with genital herpes (n ¼ 22) with and without genital herpes lesions, in comparison to healthy control subjects (n ¼ 8) Subject groups and time points, mean scores (SD) Symptom measures POMS Fatigue Anger Confusion Depression Anxiety SPHERE SOMA PSYCH BDQ
Lesions present
14.5 15.6 7.5 21.1 11.4
(8.6) (9.9) (5.0) (12.6) (8.1)
10.1 (3.6) 9.8 (3.7) 5.2 (5.9)
Lesion-free
8.3 6.6 3.5 9.9 5.9
(7.2) (6.5) (5.5) (11.2) (8.8)
9.5 (3.0) 7.8 (3.1) 3.3 (4.7)
Controls
Lesions vs. lesion-free
Lesions vs. controls
Lesion-free vs. controls
(3.0) (2.1) (1.5) (1.3) (1.4)
0.001 0.001 0.01 0.001 0.005
0.001 0.001 0.04 0.001 0.001
0.04 0.009 0.35 0.002 0.46
0.8 (0.7) 0.4 (0.5) 0.1 (0.4)
0.03 0.007 0.01
0.001 0.001 0.004
0.001 0.001 0.01
3.6 2.4 4.7 1.9 4.1
A consistent correlation between the scores obtained from the patients at the two study visits was evident for somatic complaints (SOMA: r ¼ 0.46, p ¼ 0.03; fatigue: r ¼ 0.8, p , 0.001) and disability (BDQ: r ¼ 0.54, p ¼ 0.009), but not for any of the measures of mood disturbance (POMS anger, confusion, anxiety or depression or PSYCH of SPHERE). The only significant difference in the severity of symptoms and functional impairment reported by patients experiencing an initial episode of herpes and those reported by patients with recurrent herpes was for somatic complaints [mean SOMA scores 12.9 (SD: 3.4) vs. 9.6 (SD: 3.3) respectively; effect size ¼ 0.45; p ¼ 0.04]. Immune measures
Serum neopterin levels did not differ between patients and control subjects, or did they differ between patients experiencing initial and recurrent herpes episodes, whether lesions were present or not (data not shown). Data on serum concentrations of IL-6 are not shown as these were below the detectable range of the assay in 41 of 44 (93%) of samples. Serum samples in which IL-6 could be
B
A 300
Statistical comparisons (p values)
detected were all obtained from subjects with initial herpes episodes (3 of 8, 38%), but with a very modest mean concentration (3.9 pg/ml). Figure 1 illustrates the urinary levels of neopterin and IL-6. As no statistically significant difference was found between subjects with initial and recurrent herpes, these data were aggregated across all 22 patients. Urinary neopterin levels were significantly higher among patients with herpes compared with controls when genital herpes lesions were present (p ¼ 0.01), and approached significance when they were absent (p ¼ 0.054). Levels of urinary IL-6 showed considerable individual variation, consequently, the apparent difference between levels of urinary IL-6 in patients with herpes and controls did not reach statistical significance (p ¼ 0.057). The levels of urinary neopterin and IL-6 measured in our control samples were consistent with reference values published for healthy individuals of similar age, with a mean urinary neopterin among participants of 137 mmol neopterin/mol creatinine (SD: 40) compared with 125 mmol neopterin/mol creatinine (SD: 34) among healthy individuals (29) and a mean urinary IL-6 of 0.81 pg/ml (SD: 0.85) vs. 1.25 pg/ml (SD: 2), respectively (30).
5
4 200 3
2 100 1 0 Lesions
148
No lesions
Controls
0
Lesions
No lesions
Controls
Fig. 1. Mean levels of urinary (a) neopterin (b) and IL-6 in 22 patients with genital herpes measured during active lesions and at follow-up with no lesions, and in 8 healthy control subjects. Error bars show standard errors of the mean.
Neuropsychiatric symptoms and immune activation Associations between immune activation and neuropsychiatric symptoms
Urinary levels of both neopterin and IL-6 correlated significantly with several measures of physical and emotional symptoms. These correlations were significant whether genital lesions were present or not (Table 2). At both study visits, urinary IL-6 correlated significantly with reports of dysuria or difficulty passing urine (r ¼ 0.6, p ¼ 0.009; and 0.46, p ¼ 0.03). Serum markers of immune activation did not correlate with any of the measures of somatic or emotional symptoms (data not shown). Similarly, there was no correlation between the symptom measures and concentrations of any immune markers among the control subjects.
Discussion
This study provides the first evidence for a link between regional immune activation and neuropsychiatric manifestations in HSV. Furthermore, it confirms previous reports of significant emotional dysfunction and disability among individuals experiencing frequent episodes of symptomatic herpes. Perhaps surprisingly, initial episodes of genital herpes were only marginally more likely to be associated with higher levels of morbidity than recurrent episodes, with the only significant difference relating to somatic complaints. The high level of neuropsychiatric disturbance in patients with recurrent herpes in our sample may reflect a selection bias as individuals with infrequent or mild recurrences are less likely to attend a specialist medical clinic. Indeed, the frequency of recurrences was high in our sample (average recurrences 10 per year). Our findings are consistent with previous research showing that emotional dysfunction is most profound in individuals dealing with an
initial diagnosis of herpes and in those with frequent and severe recurrences (7,8). Interestingly, while reported neuropsychiatric symptoms were more severe at the time genital lesions were present in the patient group, they did not return to a level equivalent to that of control subjects when lesions were absent. It could be argued that this result simply reflects the greater emotional vulnerability of subjects dealing with a stigmatising genital infection. However, the data did not reveal a pattern of continuing emotional disturbance in individuals as there were no substantial correlations between measures of mood states taken at different times. A possible, alternative explanation is that some patients may have experienced subclinical reactivation of the virus at the time they were assessed as being lesion-free. Subclinical shedding of HSV has been shown to be particularly common among individuals with recently acquired genital herpes and among those with frequent recurrences (6). However, as we did not perform serial swabbing to detect subclinical shedding, we cannot be certain whether this occurred or not. Serum levels of neopterin in patients with herpes lesions were equivalent to those of healthy controls, and serum concentrations of IL-6 could only be detected in a minority of patients with initial herpes episodes. In this latter group, this systemic activation may have contributed to more severe somatic complaints as has been shown previously in subjects with acute infective illness (19). Overall, however, there was little evidence of systemic immune activation in this group. By contrast, despite marked interindividual variation, urinary parameters of immune activation were substantially elevated in the herpes patients. Moreover, data analysis revealed significant correlations between concentrations of immunological proteins in urine and
Table 2. Correlations between urinary markers of immune activation and measures of emotional and somatic disturbance in subjects with genital herpes (n ¼ 22) with and without genital herpes lesions Lesions present (Spearman's r and p values) Neopterin Symptom measures POMS Fatigue Anger Confusion Depression Anxiety SPHERE SOMA PSYCH BDQ
Lesion-free (Spearman's r and p values)
IL-6
Neopterin
IL-6
r
p
r
p
r
p
r
p
0.37 0.32 0.18 0.04 0.13
0.08 0.14 0.43 0.98 0.57
0.60 0.50 0.36 0.40 0.48
0.003 0.01 0.09 0.06 0.02
0.51 0.33 0.51 0.46 0.31
0.01 0.13 0.01 0.03 0.16
0.47 0.32 0.36 0.43 0.41
0.02 0.14 0.10 0.04 0.06
0.16 0.01 0.23
0.48 0.99 0.30
0.45 0.45 0.31
0.03 0.03 0.16
0.17 0.53 0.47
0.45 0.01 0.03
0.27 0.53 0.09
0.22 0.01 0.68
149
Vollmer-Conna et al. reported levels of disturbed mood, fatigue and somatic complaints. Such associations were evident not only when genital lesions were present but also at lesion-free follow-up. These findings support the hypothesis that aspects of emotional dysfunction in HSV may be mediated by an immunologically triggered sickness response. The selective elevation of immunological proteins in the urine of patients with genital herpes suggests that the immune activation is localised within the urogenital region, rather than being a systemic immune response involving the circulation and distant regional lymphoid tissue. Similar dissociations between levels of immunoproteins (including IL-6) in serum and urine samples have been reported in renal inflammatory disease (31) and in urinary tract infections (32) and are thought to reflect derivation of inflammatory mediators from local cellular sources including dendritic cells, tissue macrophages and epithelial cells participating in local immune reactions. The significant correlations between urinary levels of IL-6 and dysuria in our sample support the expansion of an inflammatory response to adjacent tissue sites (such as the bladder and urethra) beyond the actual genital lesions. Studies on mice exposed to HSV intravaginal have clearly documented the spread of virus to multiple neural sites including the paracervical ganglia whose postganglionic neurons innervate the uterus, vagina, urinary bladder and rectum (4,33). Viral reservoirs have been demonstrated not only in sensory neurons at the dorsal root ganglia, but in autonomic ganglia in the walls of the rectum and urinary bladder, which in turn have been associated with faecal and urinary retention (4). In humans, genital HSV infection has similarly been reported to cause autonomic dysfunction in the pelvis, including urinary retention and constipation (34,35). The current results suggest that the brain can be alerted to the presence of a localised disturbance in the body to which it responds by inducing changes in emotion and behaviour. Increasing evidence supports a role for primary autonomic (vagal) afferents in relaying information about peripheral immune activation directly to the brain where such signals trigger secondary cytokine systems that organise the sickness response (17,36,37). Similarly, recent evidence from functional anatomy and neuroimaging studies has detailed a sympathetic neural pathway innervating essentially all tissues of the body and are sensitive to changes, not only in visceral sensations, but in all physiological systems including local metabolism and immune activity (38,39). These interoceptive sensations are first projected to autonomic and homeostatic centres and then, in higher animals, processed 150
further in limbic and cortical regions where they prompt adjustments in emotions and motivated behaviour thought to be crucial to the maintenance of homeostasis and body integrity (37). It is conceivable that ongoing activation or sensitisation of such Ôsickness response’ systems within the brain may precipitate the development of neuropsychiatric syndromes including depression, fatigue syndromes and somatisation disorders (19,22). Limitations of this study
We did not perform serological screening for herpes among the control subjects and therefore cannot be certain that they did not in fact have genital herpes. Nonetheless, these subjects constituted an appropriate control group for the experience of overt and clinically apparent HSV infection or reactivation. The assessment of control subjects was undertaken on only one occasion. Although the scores obtained from self-report measures and the values for the immunological markers are consistent with published norms for the age group, our data do not control for potential fluctuation in outcomes on repeated assessment. Because of the limited number of subjects in this initial study, possible associations between immune markers and other measures of emotional dysfunction may not have been demonstrated. Finally, as there is a likelihood of selection bias, the extent to which these findings can be generalised to patients with less severe genital herpes is unknown. Conclusions
This study is the first to document an association between markers of immune activation and neuropsychiatric disturbances in patients with genital herpes. Moreover, this association appears to predominantly involve regional, rather than systemic, inflammatory responses, which again has not been documented previously. Replication of these findings with a larger sample and a longitudinal design with more frequent immune and symptom assessments and the addition of periodic HSV viral detection would also provide clearer insights into the temporal sequence of events and the potential importance of subclinical viral reactivation as a potential driver of neuropsychiatric disturbances in HSV. Acknowledgements We thank participating clinicians, laboratory staff and patients of the Sydney Sexual Health Centre for their important contributions to this research. The laboratory component of this study was supported by a grant from GlaxoSmithKline.
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Acta Neuropsychiatrica 2008: 20: 145–151 All rights reserved DOI: 10.1111/j.1601-5215.2008.00281.x
ACTA NEUROPSYCHIATRICA
Neuropsychiatric symptoms and immune activation in patients with genital herpes Vollmer-Conna U, Chen M, Lloyd A, Donovan B. Neuropsychiatric symptoms and immune activation in patients with genital herpes. Objective: Neuropsychiatric disturbances are common among patients with genital herpes simplex virus (HSV) infection. To date, no studies have examined the possible role of immune activation in the aetiology of these disturbances. The aim of this study was to examine the relationship between markers of immune activation and measures of emotional and somatic dysfunction among patients with symptomatic genital herpes. Methods: Twenty-two patients with documented genital herpes were assessed when herpetic lesions were present and when they were not. Each assessment included a clinical examination, self-reported symptom measures as well as a blood and urine collection. Markers of immune activation [neopterin and interleukin (IL)-6] in serum and urine were quantified by enzyme-linked immunoassay. These measures were also obtained from a group of healthy control subjects. Results: Urine, but not serum, levels of neopterin and IL-6 correlated significantly with measures of reported psychological distress and fatigue. These associations were not confined to periods of overt clinical lesions. Conclusions: HSV-related neuropsychiatric morbidity correlates selectively with regional, but not systemic, measures of immune activation. We hypothesise that communication between the local inflammatory site in the pelvis and the brain occurs through autonomic afferent pathways.
Introduction
Genital herpes is one of the most common sexually transmitted viral infections in the world, with an estimated prevalence of 10–50% in adult populations (1,2). Two closely related viruses – herpes simplex virus (HSV)-1 and HSV-2 cause the condition, although HSV-2 is more commonly associated with genital lesions and HSV-1 with orofacial lesions. Lesions may be preceded by systemic symptoms including fever, neuralgia and malaise (2,3). A unique biologic feature of HSV is the ability of this virus to invade and replicate in the host nervous system, to establish latent infection in lumbosacral sensory ganglia and possibly autonomic neurons including the paracervical and vagal ganglia (3,4). Latency in neurons enables the virus to evade immune detection, thus ensuring life-long infection of the host (5). Periodically, HSV may reactivate with viral particles travelling along sensory axons, giving rise to asymptomatic viral shedding or recurrent lesions (3,6). Significant emotional and somatic dysfunction has been documented for both
Ute´ Vollmer-Conna1, Marcus Chen2, Andrew Lloyd3, Basil Donovan2,4 1 School of Psychiatry, University of New South Wales, Sydney, New South Wales, Australia; 2 Sydney Sexual Health Centre at the Sydney Hospital, Sydney, New South Wales, Australia; 3Inflammatory Diseases Research Unit, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia; and 4The National Centre in HIV Epidemiology and Clinical Research, University of New South Wales, Sydney, New South Wales, Australia
Keywords: genital herpes; herpes simplex virus; immune activation; neuropsychiatric disturbance Dr Ute Vollmer-Conna, Department of Human Behaviour, School of Psychiatry, University of New South Wales, Sydney, NSW 2052, Australia. Tel: 161 (02) 9385 2945; Fax: 161 (02) 9385 2944; E-mail: [email protected]
primary as well as recurrent genital herpes episodes (2,7,8). The prevalent changes in the emotional state of these individuals may be a result of suffering from a painful, sexually restricting disorder, and the social stigma attached to the disease (7). However, there is a long-held notion among both patients and physicians that psychological distress may itself precipitate recurrences (9). Although this possibility has been extensively explored (10–14), empirical evidence linking psychological factors such as life stress, negative mood and personality to genital herpes recurrences remains inconclusive. Two longitudinal studies that monitored neuropsychiatric symptoms on a daily basis provide interesting insights into the temporal relationship between mood disturbance and recurrent herpes (11,13). Both studies reported a significant decrease in emotional wellbeing 3–4 days prior to the appearance of genital lesions: this was interpreted as supporting a role for psychological distress in the HSV reactivation. The close temporal relationship 145
Vollmer-Conna et al. between the increase in mood disturbance and the recurrence of herpes lesions alternatively suggests a neuropsychiatric prodrome associated with viral reactivation that may be mediated via an immunologically triggered sickness response. The term Ôsickness response’ refers to a set of stereotyped physical and behavioural changes (fever, hypersomnia, hyperalgesia, anorexia, mood and cognitive disturbances) that typically accompany acute infection and inflammation (15). Extensive research with animals established that the acute sickness response is mediated by the action of proinflammatory cytokines, such interleukin (IL)-1b, IL-6 and tumour necrosis factor-a, on central nervous system targets (16–18). For example, both the behavioural and neurochemical changes associated with acute infective illness can be induced in a dose-dependent fashion by systemic or intracerebral injection of these cytokines. Elaboration of this phenomenon has led to a major conceptual advance in our understanding of how sickness and recovery processes are organised in the brain, leading to a deeper appreciation of the physiological and psychological impact of infective illnesses (19). There is now increasing awareness that non-specific neuropsychiatric symptoms commonly accompany chronic infections and autoimmune diseases including hepatitis C virus infections (20) and in inflammatory bowel disease (21). Moreover, it has been speculated that neuropsychiatric conditions, including depression, fatigue syndromes and somatisation disorders may constitute abnormal variants of an immunologically triggered sickness response (22–24). The present study provides an initial examination of the role for immune activation in the manifestation of emotional and somatic symptoms reported by patients with genital herpes. As components of both the innate and adaptive immune systems are essential in the defence against herpes infections (5), we included two sensitive markers of cellular immune activation: neopterin, which is selectively induced by interferon g and the proinflammatory cytokine IL-6 (25–27). Both urinary and serum measures of these markers were included to distinguish regional (urogenital) from systemic immune activation.
Methods Subjects
Patients with primary or recurrent genital herpes were recruited through the Sydney Sexual Health Centre, a public sexual health service in Sydney, Australia. Eligible subjects were required to have had 146
microbiologically confirmed genital herpes, either by viral culture from a genital swab or, if this was not possible, positive serology for HSV-2. Patients were excluded if they had any other infectious, autoimmune, neoplastic or psychiatric conditions. Patients taking antiviral drugs were also excluded. Participating patients were asked to visit the clinic on two occasions: once when they experienced genital lesions, and again when they did not have any genital lesions, as indicated both by self-report and the absence of lesions on clinical examination. At both visits, patients completed selfreport instruments assessing mood states and clinical symptoms, underwent a clinical examination and provided a blood and a urine sample. Control subjects with no history of HSV infection and with a similar age and sex distribution as the patient sample were recruited by advertisement from the University of New South Wales. Controls were included in the study if they had no reported history of genital herpes. Serological testing for herpes was not undertaken on the controls. The control subjects were interviewed, completed the self-report instruments and provided a blood and urine sample. Written informed consent was obtained from all participants. The study was approved by the institutional Human Research Ethics Committee. Questionnaires
Subjects were asked to complete the Somatic and Psychological Health Report (SPHERE) (28), which produces two validated subscales (SOMA and PSYCH) reflecting somatic (fatigue, pain) and psychological distress, respectively. Current (last 24 h) mood states were also assessed using the fatigue, anger, confusion, depression and anxiety subscales of the Profile of Mood States (POMS) questionnaire (29). The Brief Disability Questionnaire (BDQ) (30), documented impairment in daily activities in the areas of self-care, work and leisure activities. A clinical symptom record was used to assess for the presence of patient-reported symptoms: genital pain, itch, dysuria, neuralgia, blisters, ulcers or crusts on genitals, general malaise, fatigue and fever. Measures of immune activation
Blood and urine samples were collected from participants between 12 AM and 4 PM and processed within 1 h of collection before freezing serum and urine in aliquots at minus 80°C. As neopterin is light sensitive, specimens were collected, processed and stored under light-protected conditions. Neopterin
Neuropsychiatric symptoms and immune activation content in sera and urine samples was determined by competitive enzyme immunoassay (ELItest; Brahms, Berlin, Germany) in accordance with the manufacturer’s instructions. Urinary neopterin concentrations were related to creatinine concentrations measured in the same urine specimen by the Jaffe rate method. Absorbance readings were taken at 520 nm (SYNCHRON LX-20; Beckman Coulter, Sydney, Australia). This served as an internal reference standard to compensate for physiological variations in urinary concentrations. Urinary neopterin levels were thus expressed as the ratio: mmol neopterin/mol creatinine. The concentrations of IL-6 in sera and urine samples were quantified by solid-phase enzymelinked immunosorbent assay in accordance with the manufacturer’s instructions [Quantikine and Quantikine-HS (range: 0.156–10 pg/ml); R & D Systems, Minneapolis, MN, USA]. Using this assay system, separate assessment of creatinine concentrations is not required for urinary IL-6 determination. Statistical analyses
The SPSS for Windows (version 13) statistical package was used for data analysis. ANOVA with Bonferroni correction for multiple testing was used to assess differences between groups and over time (repeated measures). Spearman correlations were used to assess the relationship between the severity of symptoms and concentrations of immunological proteins. The appropriate number of subjects for this study had been estimated from effect sizes previously obtained for correlations between physical and psychological symptoms and IL-6 concentrations in acute infections (15). On this basis, the present study had an estimated power of 80% to detect significant (a ¼ 0.05) correlations with a magnitude of r ¼ 0.46.
Results Sample characteristics
Thirty-three patients with genital herpes were recruited into the study, of which 22 (67%) completed all assessment components. There were no significant differences in those who completed the study and those who did not in terms of age [mean age 35 (SD: 11) vs. 32 (SD: 6) years, respectively, p ¼ 0.42]; the proportion experiencing an initial episode of genital herpes (36 vs. 30%, p ¼ 0.64); the severity of reported somatic and emotional symptoms at baseline (all p ¼ 0.21); or the concentrations of immune markers at baseline (all
p ¼ 0.39). However, the gender distribution differed, with more females among those lost to follow-up (70% vs. 32%). The data did not suggest any significant gender-dependent differences in either the severity of somatic and emotional symptoms (all p ¼ 0.51) or concentrations of immune markers (all p ¼ 0.38). The 22 patients who completed the study included 15 males and 7 females. There were eight control subjects, including five males and three females [mean age: 34 years (SD: 6)]. Of the 22 patients with herpes, 8 (6 males; 2 females) presented with their first episode of genital herpes, while 14 (9 males; 5 females) presented with a recurrent episode of herpes. Among those with a history of recurrent herpes, the median interval since the first reported episode was 6.5 years (interquartile range 1.5–11 years) with the median number of recurrences reported per year being 10 (interquartile range 3.5–12 years). All had declined to take suppressive antiviral therapy. Eighteen patients (82%) reported at least one of the following non-genital symptoms at the time they were experiencing genital lesions: fatigue, malaise, headaches and muscle or joint pain. Pain on passing urine (dysuria) or difficulty passing urine was reported by seven patients (33%). Twenty patients (91%) had genital HSV-2, while two (9%) had genital HSV-1. The median interval between the two clinic visits for participants was 28 days (interquartile range 14–45 days). Neuropsychiatric symptoms
Symptom and disability scores for patients with genital herpes – when lesions were present and when they were not – and for the control subjects are shown in Table 1. Patients reported significantly higher levels of emotional disturbance and functional impairment when experiencing genital lesions than when they were lesion-free. Compared with the controls, the somatic and emotional symptoms reported by the patients with herpes were significantly more severe across all measures when genital lesions were present. Even when lesions were absent, the symptom and disability ratings from herpes patients, while significantly improved, remained higher than for the controls for all but two measures of mood. The scores obtained for mood states over Ôthe past 24 hours’ from control subjects were comparable to published norms using the same version of the questionnaire (27). Similarly, among the controls, ratings on both subscales of the SPHERE were consistent with scores typically obtained from health samples (SOMA: ,3; PSYCH: ,2) (26). 147
Vollmer-Conna et al. Table 1. Severity of emotional and somatic disturbance reported by patients with genital herpes (n ¼ 22) with and without genital herpes lesions, in comparison to healthy control subjects (n ¼ 8) Subject groups and time points, mean scores (SD) Symptom measures POMS Fatigue Anger Confusion Depression Anxiety SPHERE SOMA PSYCH BDQ
Lesions present
14.5 15.6 7.5 21.1 11.4
(8.6) (9.9) (5.0) (12.6) (8.1)
10.1 (3.6) 9.8 (3.7) 5.2 (5.9)
Lesion-free
8.3 6.6 3.5 9.9 5.9
(7.2) (6.5) (5.5) (11.2) (8.8)
9.5 (3.0) 7.8 (3.1) 3.3 (4.7)
Controls
Lesions vs. lesion-free
Lesions vs. controls
Lesion-free vs. controls
(3.0) (2.1) (1.5) (1.3) (1.4)
0.001 0.001 0.01 0.001 0.005
0.001 0.001 0.04 0.001 0.001
0.04 0.009 0.35 0.002 0.46
0.8 (0.7) 0.4 (0.5) 0.1 (0.4)
0.03 0.007 0.01
0.001 0.001 0.004
0.001 0.001 0.01
3.6 2.4 4.7 1.9 4.1
A consistent correlation between the scores obtained from the patients at the two study visits was evident for somatic complaints (SOMA: r ¼ 0.46, p ¼ 0.03; fatigue: r ¼ 0.8, p , 0.001) and disability (BDQ: r ¼ 0.54, p ¼ 0.009), but not for any of the measures of mood disturbance (POMS anger, confusion, anxiety or depression or PSYCH of SPHERE). The only significant difference in the severity of symptoms and functional impairment reported by patients experiencing an initial episode of herpes and those reported by patients with recurrent herpes was for somatic complaints [mean SOMA scores 12.9 (SD: 3.4) vs. 9.6 (SD: 3.3) respectively; effect size ¼ 0.45; p ¼ 0.04]. Immune measures
Serum neopterin levels did not differ between patients and control subjects, or did they differ between patients experiencing initial and recurrent herpes episodes, whether lesions were present or not (data not shown). Data on serum concentrations of IL-6 are not shown as these were below the detectable range of the assay in 41 of 44 (93%) of samples. Serum samples in which IL-6 could be
B
A 300
Statistical comparisons (p values)
detected were all obtained from subjects with initial herpes episodes (3 of 8, 38%), but with a very modest mean concentration (3.9 pg/ml). Figure 1 illustrates the urinary levels of neopterin and IL-6. As no statistically significant difference was found between subjects with initial and recurrent herpes, these data were aggregated across all 22 patients. Urinary neopterin levels were significantly higher among patients with herpes compared with controls when genital herpes lesions were present (p ¼ 0.01), and approached significance when they were absent (p ¼ 0.054). Levels of urinary IL-6 showed considerable individual variation, consequently, the apparent difference between levels of urinary IL-6 in patients with herpes and controls did not reach statistical significance (p ¼ 0.057). The levels of urinary neopterin and IL-6 measured in our control samples were consistent with reference values published for healthy individuals of similar age, with a mean urinary neopterin among participants of 137 mmol neopterin/mol creatinine (SD: 40) compared with 125 mmol neopterin/mol creatinine (SD: 34) among healthy individuals (29) and a mean urinary IL-6 of 0.81 pg/ml (SD: 0.85) vs. 1.25 pg/ml (SD: 2), respectively (30).
5
4 200 3
2 100 1 0 Lesions
148
No lesions
Controls
0
Lesions
No lesions
Controls
Fig. 1. Mean levels of urinary (a) neopterin (b) and IL-6 in 22 patients with genital herpes measured during active lesions and at follow-up with no lesions, and in 8 healthy control subjects. Error bars show standard errors of the mean.
Neuropsychiatric symptoms and immune activation Associations between immune activation and neuropsychiatric symptoms
Urinary levels of both neopterin and IL-6 correlated significantly with several measures of physical and emotional symptoms. These correlations were significant whether genital lesions were present or not (Table 2). At both study visits, urinary IL-6 correlated significantly with reports of dysuria or difficulty passing urine (r ¼ 0.6, p ¼ 0.009; and 0.46, p ¼ 0.03). Serum markers of immune activation did not correlate with any of the measures of somatic or emotional symptoms (data not shown). Similarly, there was no correlation between the symptom measures and concentrations of any immune markers among the control subjects.
Discussion
This study provides the first evidence for a link between regional immune activation and neuropsychiatric manifestations in HSV. Furthermore, it confirms previous reports of significant emotional dysfunction and disability among individuals experiencing frequent episodes of symptomatic herpes. Perhaps surprisingly, initial episodes of genital herpes were only marginally more likely to be associated with higher levels of morbidity than recurrent episodes, with the only significant difference relating to somatic complaints. The high level of neuropsychiatric disturbance in patients with recurrent herpes in our sample may reflect a selection bias as individuals with infrequent or mild recurrences are less likely to attend a specialist medical clinic. Indeed, the frequency of recurrences was high in our sample (average recurrences 10 per year). Our findings are consistent with previous research showing that emotional dysfunction is most profound in individuals dealing with an
initial diagnosis of herpes and in those with frequent and severe recurrences (7,8). Interestingly, while reported neuropsychiatric symptoms were more severe at the time genital lesions were present in the patient group, they did not return to a level equivalent to that of control subjects when lesions were absent. It could be argued that this result simply reflects the greater emotional vulnerability of subjects dealing with a stigmatising genital infection. However, the data did not reveal a pattern of continuing emotional disturbance in individuals as there were no substantial correlations between measures of mood states taken at different times. A possible, alternative explanation is that some patients may have experienced subclinical reactivation of the virus at the time they were assessed as being lesion-free. Subclinical shedding of HSV has been shown to be particularly common among individuals with recently acquired genital herpes and among those with frequent recurrences (6). However, as we did not perform serial swabbing to detect subclinical shedding, we cannot be certain whether this occurred or not. Serum levels of neopterin in patients with herpes lesions were equivalent to those of healthy controls, and serum concentrations of IL-6 could only be detected in a minority of patients with initial herpes episodes. In this latter group, this systemic activation may have contributed to more severe somatic complaints as has been shown previously in subjects with acute infective illness (19). Overall, however, there was little evidence of systemic immune activation in this group. By contrast, despite marked interindividual variation, urinary parameters of immune activation were substantially elevated in the herpes patients. Moreover, data analysis revealed significant correlations between concentrations of immunological proteins in urine and
Table 2. Correlations between urinary markers of immune activation and measures of emotional and somatic disturbance in subjects with genital herpes (n ¼ 22) with and without genital herpes lesions Lesions present (Spearman's r and p values) Neopterin Symptom measures POMS Fatigue Anger Confusion Depression Anxiety SPHERE SOMA PSYCH BDQ
Lesion-free (Spearman's r and p values)
IL-6
Neopterin
IL-6
r
p
r
p
r
p
r
p
0.37 0.32 0.18 0.04 0.13
0.08 0.14 0.43 0.98 0.57
0.60 0.50 0.36 0.40 0.48
0.003 0.01 0.09 0.06 0.02
0.51 0.33 0.51 0.46 0.31
0.01 0.13 0.01 0.03 0.16
0.47 0.32 0.36 0.43 0.41
0.02 0.14 0.10 0.04 0.06
0.16 0.01 0.23
0.48 0.99 0.30
0.45 0.45 0.31
0.03 0.03 0.16
0.17 0.53 0.47
0.45 0.01 0.03
0.27 0.53 0.09
0.22 0.01 0.68
149
Vollmer-Conna et al. reported levels of disturbed mood, fatigue and somatic complaints. Such associations were evident not only when genital lesions were present but also at lesion-free follow-up. These findings support the hypothesis that aspects of emotional dysfunction in HSV may be mediated by an immunologically triggered sickness response. The selective elevation of immunological proteins in the urine of patients with genital herpes suggests that the immune activation is localised within the urogenital region, rather than being a systemic immune response involving the circulation and distant regional lymphoid tissue. Similar dissociations between levels of immunoproteins (including IL-6) in serum and urine samples have been reported in renal inflammatory disease (31) and in urinary tract infections (32) and are thought to reflect derivation of inflammatory mediators from local cellular sources including dendritic cells, tissue macrophages and epithelial cells participating in local immune reactions. The significant correlations between urinary levels of IL-6 and dysuria in our sample support the expansion of an inflammatory response to adjacent tissue sites (such as the bladder and urethra) beyond the actual genital lesions. Studies on mice exposed to HSV intravaginal have clearly documented the spread of virus to multiple neural sites including the paracervical ganglia whose postganglionic neurons innervate the uterus, vagina, urinary bladder and rectum (4,33). Viral reservoirs have been demonstrated not only in sensory neurons at the dorsal root ganglia, but in autonomic ganglia in the walls of the rectum and urinary bladder, which in turn have been associated with faecal and urinary retention (4). In humans, genital HSV infection has similarly been reported to cause autonomic dysfunction in the pelvis, including urinary retention and constipation (34,35). The current results suggest that the brain can be alerted to the presence of a localised disturbance in the body to which it responds by inducing changes in emotion and behaviour. Increasing evidence supports a role for primary autonomic (vagal) afferents in relaying information about peripheral immune activation directly to the brain where such signals trigger secondary cytokine systems that organise the sickness response (17,36,37). Similarly, recent evidence from functional anatomy and neuroimaging studies has detailed a sympathetic neural pathway innervating essentially all tissues of the body and are sensitive to changes, not only in visceral sensations, but in all physiological systems including local metabolism and immune activity (38,39). These interoceptive sensations are first projected to autonomic and homeostatic centres and then, in higher animals, processed 150
further in limbic and cortical regions where they prompt adjustments in emotions and motivated behaviour thought to be crucial to the maintenance of homeostasis and body integrity (37). It is conceivable that ongoing activation or sensitisation of such Ôsickness response’ systems within the brain may precipitate the development of neuropsychiatric syndromes including depression, fatigue syndromes and somatisation disorders (19,22). Limitations of this study
We did not perform serological screening for herpes among the control subjects and therefore cannot be certain that they did not in fact have genital herpes. Nonetheless, these subjects constituted an appropriate control group for the experience of overt and clinically apparent HSV infection or reactivation. The assessment of control subjects was undertaken on only one occasion. Although the scores obtained from self-report measures and the values for the immunological markers are consistent with published norms for the age group, our data do not control for potential fluctuation in outcomes on repeated assessment. Because of the limited number of subjects in this initial study, possible associations between immune markers and other measures of emotional dysfunction may not have been demonstrated. Finally, as there is a likelihood of selection bias, the extent to which these findings can be generalised to patients with less severe genital herpes is unknown. Conclusions
This study is the first to document an association between markers of immune activation and neuropsychiatric disturbances in patients with genital herpes. Moreover, this association appears to predominantly involve regional, rather than systemic, inflammatory responses, which again has not been documented previously. Replication of these findings with a larger sample and a longitudinal design with more frequent immune and symptom assessments and the addition of periodic HSV viral detection would also provide clearer insights into the temporal sequence of events and the potential importance of subclinical viral reactivation as a potential driver of neuropsychiatric disturbances in HSV. Acknowledgements We thank participating clinicians, laboratory staff and patients of the Sydney Sexual Health Centre for their important contributions to this research. The laboratory component of this study was supported by a grant from GlaxoSmithKline.
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