These articles have been accepted for publication in the British Journal of Dermatology and are currently being edited and typeset. Readers should note that articles published below have been fully refereed, but have not been through the copy-editing and proof correction process. Wiley-Blackwell and the British Association of Dermatologists cannot be held responsible for errors or consequences arising from the use of information contained in these articles; nor do the views and opinions expressed necessarily reflect those of Wiley-Blackwell or the British Association of Dermatologists This article is protected by copyright. All rights reserved. Accepted Date: 06-Mar-2014 Article Type: Clinical and laboratory investigations
Herpes simplex virus reactivation as a trigger of mucous lesions in pemphigus vulgaris Running head: HSV reactivation as a trigger of PV M. Kurata,1Y. Mizukawa, 1 Y. Aoyama2, T. Shiohara1 1
Department of Dermatology, Kyorin University School of Medicine, Shinkawa, 6-20-2,
Mitaka, Tokyo 181-8611, JAPAN 2
Department of Dermatology, Okayama University Graduate School of Medicine,
Dentistry and Pharmaceutical Sciences, Okayama, Okayama 700-8558, JAPAN Correspondence and requests for materials should be addressed to Y.M. (E-mail: [email protected])
This work was supported by grants from the Ministry of Education, Sports, Science and Culture of Japan (T.S. and Y.M.) and from the Ministry of Health, Labor and Welfare of Japan (T.S.)
Conflict of interest: there is no conflict of interest.
Keywords: herpes simplex virus, pemphigus vulgaris, saliva, triggering factor
What’s already known about this topic? ・Although infectious agents have long been implicated in the induction or exacerbation of pemphigus vulgaris (PV), a convincing role for the agent in the etiology of PV has not been established.
This article is protected by copyright. All rights reserved.
What does this study add? ・ We successfully detected high levels of HSV DNA in the saliva samples from 6 of 16 patients with PV at the earliest stage, who had no episodes of herpes simplex. ・ These PV patients have run a more complex, intractable course refractory to conventional therapy. ・ Detection of HSV DNA in saliva will alert physicians to the possibility of greater risk of subsequently developing refractory PV.
Summary Background
Although infectious agents have long been implicated in the induction or
exacerbation of pemphigus vulgaris (PV), a convincing role for the agent in the etiology of PV has not been established.
Objectives Methods
To establish the association with PV and herpes simplex virus (HSV). We examined saliva for the presence of HSV DNA after onset of PV initially
localized to the oral lesions in addition to conventional serologic tests and immunohistochemistry.
Results We successfully detected high levels of HSV DNA in the saliva samples from 6 of 16 patients with PV at the earliest stage, who had no episodes of herpes simplex. The prevalence (37.5%) of detecting HSV DNA in the PV patients was lower than that of eczema herpeticum (56.5%), but comparable to that in patients with herpes labialis (30.0%).
Copy numbers of the HSV DNA were rather higher than those with herpes
labialis and with eczema herpeticum.
In general, detection of HSV DNA in saliva was
This article is protected by copyright. All rights reserved.
transient and restricted to the earliest phase of the disease.
In addition, anti-HSV IgG
titers in PV patients were significantly higher than those in patients with virologically confirmed HSV-induced disorders.
All salivary HSV DNA-positive PV patients had
run a more complex, intractable course refractory to conventional therapy. Conclusions
Detection of HSV DNA in saliva is a useful and noninvasive,
quantitative method for establishing the role of HSV in the pathogenesis of PV and for identifying individuals at greater risk for subsequently developing refractory PV.
Pemphigus vulgaris (PV) is the prototype of an autoimmune skin disease mediated by pathogenic autoantibodies against the appropriate desmogleins.1, 2
Although infectious
agents have long been implicated in the induction or exacerbation of PV, convincing role for the agent in the etiology of PV has not been established.
3-5
a
Earlier
case reports described the induction and exacerbation of PV following herpes simplex virus (HSV) infection.
6, 7
Based on these results, it can be suggested that HSV may
play a role in the earliest stages of the disease. etiological role in PV remains unknown. 8-11
However, whether HSV has an
In this regard, real-time polymerase chain
reaction (PCR) has greatly improved the sensitivity of detecting the virus in clinical specimens obtained from patients suspected of having HSV-triggered PV.
PCR for
HSV is mostly performed on specimens from skin lesions or peripheral blood,
8
but
recently real-time PCR has been used to diagnose HSV reactivation using saliva: the presence of HSV DNA in saliva of patients without apparent HSV lesions is thought to represent events of asymptomatic shedding of reactivated HSV in respective patients.12 These considerations led to this study of whether HSV DNA could be detected in saliva of patients with PV at the earliest stage.
This article is protected by copyright. All rights reserved.
This prospective study of 16 sequential patients with PV and 66 controls was performed over a follow-up period of 2~6 years in Tokyo and Okayama, Japan.
Our study
provides evidence indicating that detection of HSV in saliva by real-time PCR at the earliest stage of the disease is a useful and noninvasive, quantitative method for establishing the role of HSV in the pathogenesis of PV.
Case reports Patient 1
A 47-year-old man presented with oral PV lesions at a time when HSV DNA was detected in saliva but not in the blood (Fig. 1). 13
PDAI (pemphigus diseases area index)
in the oral mucosa was 15, higher than that (3) in the skin.
He was treated
immediately with intravenous acyclovir (250mg 2 times daily for 7 days).
Two days
after antiviral treatment, HSV DNA was no longer detected in saliva but no improvement in his oral lesions was seen. (DSG) 3 titer dramatically increased.
Despite antiviral treatment, anti-desmoglein
Upon withdrawal of acyclovir, HSV DNA was
again detected in saliva, coincident with the exacerbation of oral PV lesions.
This
patient was started on a treatment regimen of prednisone (50mg/day) and he showed gradual improvement of the PV lesions.
Two weeks after starting prednisone therapy,
the highest HSV DNA levels were detected in saliva while anti-DSG3 and anti-DSG1 levels (data not shown) declined rapidly.
Because recent studies have shown that the
rebound in viral DNA levels occurred following withdrawal of anti-viral agents predominantly in patients with continued need for systemic immunosuppression,14 intravenous acyclovir was again added for 7 days and double-filtration plasmapheresis
This article is protected by copyright. All rights reserved.
(DFPP) followed by intravenous immunoglobulin (IVIg) was started with marked improvement.
Three days after starting acyclovir and DFPP, anti-DSG3 and
anti-DSG1 levels (data not shown) decreased markedly and HSV DNA was no longer detected for the following 3 years in saliva despite several flare-ups.
Despite a marked
change in HSV DNA levels in saliva during the course of the illness, alterations in anti-HSV IgG titers were not so remarkable.
To
confirm
that
HSV
infects
epithelial
cells
of
the
oral
PV
lesions,
immunohistochemical detection of HSV was performed in mucosal sections using rabbit polyclonal antibody directed to the HSV (DakoCytomation, Glostrup, Denmark). As shown in Fig. 2, HSV antigen was detected in the cytoplasm of epithelial cells in the oral mucosa, but not in the epithelial cells from HSV DNA negative cases.
The
epithelial cells revealed the viral cytopathic changes that would suggest an active herpesvirus infection.
Patient 2
A 56-year-old man with PV was also longitudinally studied (Fig. 3). were detected only in oral mucous area (PDAI:20).
His PV lesions
At his initial presentation, HSV
DNA was detected in saliva, coincident with an increase in anti-DSG 3 and anti-DSG 1 levels (data not shown). effect.
He was treated with oral prednisone 70 mg/day but with no
DFPP followed by IVIg was started with marked improvement.
After starting
prednisone and DFPP followed by IVIg, HSV DNA was not detected in saliva and a gradual decrease in anti-DSG 3 and anti-DSG 1 levels (data not shown) was noted. However, this combination therapy did not result in remission.
Although he received
This article is protected by copyright. All rights reserved.
two sessions of plasma exchange (PE) over a period of 14 days, the patient remained unable to control his diseases.
After starting cyclosporine A (300 mg/day), he
achieved partial remission associated with a reduction in anti-DSG levels and anti-HSV IgG titers.
During the follow-up period of 6 years, several flare-ups were observed
often after detection of HSV DNA in saliva (data not shown).
Materials and methods Detection of saliva HSV DNA and serum HSV IgG titers We examined saliva for the presence of HSV DNA at or near the time of the initial presentation in patients with PV and thereafter at various time points usually on a bimonthly basis.
This study was approved by the institutional review board at Kyorin
University School of Medicine. summarized in Tables 1 and 2.
The clinical characteristics of the patients are A total 66 saliva samples were sequentially obtained
from 16 PV patients (11 men and 5 women; age range 34-84 years; mean age, 56.6±3.7 years) with oral lesions: usually 5-6 saliva samples were obtained from each patient over a 2-6 year period (during the observation period from 2002 to 2013).
Results Longitudinal analysis of HSV DNA and HSV IgG titers HSV DNA was found in the saliva samples from 6, including patients 1 and 2, of 16 cases (37.5%) with PV, whose lesions were initially localized to the oral areas and who were diagnosed as PV based on clinical, histological and immunofluorescence criteria. HSV-DNA copy number was determined by real-time PCR analysis of saliva samples thus collected; participants were requested to spit 2 ml saliva; this was performed as
This article is protected by copyright. All rights reserved.
part of a standard operating procedure for detecting herpesviruses reactivation as previously described.
15
Statistical analyses were performed using Mann-Whiney
U-test or Student’s t test or Dunnett’s test for comparison between each group. Correlation coefficients were determined by using the Spearman correlation test. p
Herpes simplex virus reactivation as a trigger of mucous lesions in pemphigus vulgaris Running head: HSV reactivation as a trigger of PV M. Kurata,1Y. Mizukawa, 1 Y. Aoyama2, T. Shiohara1 1
Department of Dermatology, Kyorin University School of Medicine, Shinkawa, 6-20-2,
Mitaka, Tokyo 181-8611, JAPAN 2
Department of Dermatology, Okayama University Graduate School of Medicine,
Dentistry and Pharmaceutical Sciences, Okayama, Okayama 700-8558, JAPAN Correspondence and requests for materials should be addressed to Y.M. (E-mail: [email protected])
This work was supported by grants from the Ministry of Education, Sports, Science and Culture of Japan (T.S. and Y.M.) and from the Ministry of Health, Labor and Welfare of Japan (T.S.)
Conflict of interest: there is no conflict of interest.
Keywords: herpes simplex virus, pemphigus vulgaris, saliva, triggering factor
What’s already known about this topic? ・Although infectious agents have long been implicated in the induction or exacerbation of pemphigus vulgaris (PV), a convincing role for the agent in the etiology of PV has not been established.
This article is protected by copyright. All rights reserved.
What does this study add? ・ We successfully detected high levels of HSV DNA in the saliva samples from 6 of 16 patients with PV at the earliest stage, who had no episodes of herpes simplex. ・ These PV patients have run a more complex, intractable course refractory to conventional therapy. ・ Detection of HSV DNA in saliva will alert physicians to the possibility of greater risk of subsequently developing refractory PV.
Summary Background
Although infectious agents have long been implicated in the induction or
exacerbation of pemphigus vulgaris (PV), a convincing role for the agent in the etiology of PV has not been established.
Objectives Methods
To establish the association with PV and herpes simplex virus (HSV). We examined saliva for the presence of HSV DNA after onset of PV initially
localized to the oral lesions in addition to conventional serologic tests and immunohistochemistry.
Results We successfully detected high levels of HSV DNA in the saliva samples from 6 of 16 patients with PV at the earliest stage, who had no episodes of herpes simplex. The prevalence (37.5%) of detecting HSV DNA in the PV patients was lower than that of eczema herpeticum (56.5%), but comparable to that in patients with herpes labialis (30.0%).
Copy numbers of the HSV DNA were rather higher than those with herpes
labialis and with eczema herpeticum.
In general, detection of HSV DNA in saliva was
This article is protected by copyright. All rights reserved.
transient and restricted to the earliest phase of the disease.
In addition, anti-HSV IgG
titers in PV patients were significantly higher than those in patients with virologically confirmed HSV-induced disorders.
All salivary HSV DNA-positive PV patients had
run a more complex, intractable course refractory to conventional therapy. Conclusions
Detection of HSV DNA in saliva is a useful and noninvasive,
quantitative method for establishing the role of HSV in the pathogenesis of PV and for identifying individuals at greater risk for subsequently developing refractory PV.
Pemphigus vulgaris (PV) is the prototype of an autoimmune skin disease mediated by pathogenic autoantibodies against the appropriate desmogleins.1, 2
Although infectious
agents have long been implicated in the induction or exacerbation of PV, convincing role for the agent in the etiology of PV has not been established.
3-5
a
Earlier
case reports described the induction and exacerbation of PV following herpes simplex virus (HSV) infection.
6, 7
Based on these results, it can be suggested that HSV may
play a role in the earliest stages of the disease. etiological role in PV remains unknown. 8-11
However, whether HSV has an
In this regard, real-time polymerase chain
reaction (PCR) has greatly improved the sensitivity of detecting the virus in clinical specimens obtained from patients suspected of having HSV-triggered PV.
PCR for
HSV is mostly performed on specimens from skin lesions or peripheral blood,
8
but
recently real-time PCR has been used to diagnose HSV reactivation using saliva: the presence of HSV DNA in saliva of patients without apparent HSV lesions is thought to represent events of asymptomatic shedding of reactivated HSV in respective patients.12 These considerations led to this study of whether HSV DNA could be detected in saliva of patients with PV at the earliest stage.
This article is protected by copyright. All rights reserved.
This prospective study of 16 sequential patients with PV and 66 controls was performed over a follow-up period of 2~6 years in Tokyo and Okayama, Japan.
Our study
provides evidence indicating that detection of HSV in saliva by real-time PCR at the earliest stage of the disease is a useful and noninvasive, quantitative method for establishing the role of HSV in the pathogenesis of PV.
Case reports Patient 1
A 47-year-old man presented with oral PV lesions at a time when HSV DNA was detected in saliva but not in the blood (Fig. 1). 13
PDAI (pemphigus diseases area index)
in the oral mucosa was 15, higher than that (3) in the skin.
He was treated
immediately with intravenous acyclovir (250mg 2 times daily for 7 days).
Two days
after antiviral treatment, HSV DNA was no longer detected in saliva but no improvement in his oral lesions was seen. (DSG) 3 titer dramatically increased.
Despite antiviral treatment, anti-desmoglein
Upon withdrawal of acyclovir, HSV DNA was
again detected in saliva, coincident with the exacerbation of oral PV lesions.
This
patient was started on a treatment regimen of prednisone (50mg/day) and he showed gradual improvement of the PV lesions.
Two weeks after starting prednisone therapy,
the highest HSV DNA levels were detected in saliva while anti-DSG3 and anti-DSG1 levels (data not shown) declined rapidly.
Because recent studies have shown that the
rebound in viral DNA levels occurred following withdrawal of anti-viral agents predominantly in patients with continued need for systemic immunosuppression,14 intravenous acyclovir was again added for 7 days and double-filtration plasmapheresis
This article is protected by copyright. All rights reserved.
(DFPP) followed by intravenous immunoglobulin (IVIg) was started with marked improvement.
Three days after starting acyclovir and DFPP, anti-DSG3 and
anti-DSG1 levels (data not shown) decreased markedly and HSV DNA was no longer detected for the following 3 years in saliva despite several flare-ups.
Despite a marked
change in HSV DNA levels in saliva during the course of the illness, alterations in anti-HSV IgG titers were not so remarkable.
To
confirm
that
HSV
infects
epithelial
cells
of
the
oral
PV
lesions,
immunohistochemical detection of HSV was performed in mucosal sections using rabbit polyclonal antibody directed to the HSV (DakoCytomation, Glostrup, Denmark). As shown in Fig. 2, HSV antigen was detected in the cytoplasm of epithelial cells in the oral mucosa, but not in the epithelial cells from HSV DNA negative cases.
The
epithelial cells revealed the viral cytopathic changes that would suggest an active herpesvirus infection.
Patient 2
A 56-year-old man with PV was also longitudinally studied (Fig. 3). were detected only in oral mucous area (PDAI:20).
His PV lesions
At his initial presentation, HSV
DNA was detected in saliva, coincident with an increase in anti-DSG 3 and anti-DSG 1 levels (data not shown). effect.
He was treated with oral prednisone 70 mg/day but with no
DFPP followed by IVIg was started with marked improvement.
After starting
prednisone and DFPP followed by IVIg, HSV DNA was not detected in saliva and a gradual decrease in anti-DSG 3 and anti-DSG 1 levels (data not shown) was noted. However, this combination therapy did not result in remission.
Although he received
This article is protected by copyright. All rights reserved.
two sessions of plasma exchange (PE) over a period of 14 days, the patient remained unable to control his diseases.
After starting cyclosporine A (300 mg/day), he
achieved partial remission associated with a reduction in anti-DSG levels and anti-HSV IgG titers.
During the follow-up period of 6 years, several flare-ups were observed
often after detection of HSV DNA in saliva (data not shown).
Materials and methods Detection of saliva HSV DNA and serum HSV IgG titers We examined saliva for the presence of HSV DNA at or near the time of the initial presentation in patients with PV and thereafter at various time points usually on a bimonthly basis.
This study was approved by the institutional review board at Kyorin
University School of Medicine. summarized in Tables 1 and 2.
The clinical characteristics of the patients are A total 66 saliva samples were sequentially obtained
from 16 PV patients (11 men and 5 women; age range 34-84 years; mean age, 56.6±3.7 years) with oral lesions: usually 5-6 saliva samples were obtained from each patient over a 2-6 year period (during the observation period from 2002 to 2013).
Results Longitudinal analysis of HSV DNA and HSV IgG titers HSV DNA was found in the saliva samples from 6, including patients 1 and 2, of 16 cases (37.5%) with PV, whose lesions were initially localized to the oral areas and who were diagnosed as PV based on clinical, histological and immunofluorescence criteria. HSV-DNA copy number was determined by real-time PCR analysis of saliva samples thus collected; participants were requested to spit 2 ml saliva; this was performed as
This article is protected by copyright. All rights reserved.
part of a standard operating procedure for detecting herpesviruses reactivation as previously described.
15
Statistical analyses were performed using Mann-Whiney
U-test or Student’s t test or Dunnett’s test for comparison between each group. Correlation coefficients were determined by using the Spearman correlation test. p
