sign in sign up
<<
Home , Wart

Aus dem Centrum für Chronische Immundefizienz des

Universitätsklinikums Freiburg im Breisgau

The Characteristics of Viral Skin in the Immunocompromised Host ______

I N A U G U R A L - D I S S E R T A T I O N

zur Erlangung des Medizinischen Doktorgrades der Medizinischen Fakultät der Albert-Ludwigs-Universität Freiburg i. Br.

Vorgelegt 2018

von Svenja Abel

geboren in Emmendingen

Dekan: Prof. Dr. Norbert Südkamp Erstgutachter: Prof. Dr. Bodo Grimbacher Zweitgutachterin: Dr. Siobhan Burns Jahr der Promotion: 2019

Contents

Contents

Contents ...... I List of Figures ...... III List of Tables ...... V List of Abbreviations ...... VI 1 Introduction ...... 1 1.1 Skin Infections ...... 1 1.2 Common Viral Skin Infections ...... 1 1.2.1 Herpes ...... 1 1.2.2 Human ...... 4 1.3 Viral Infections and Immune Response ...... 5 1.3.1 Immune Control of Viruses ...... 5 1.3.2 Infections ...... 7 1.3.3 Varizella Zoster Infections ...... 8 1.3.4 HPV Infections ...... 9 1.4 Primary immunodeficiencies (PID) with a Susceptibility to HSV or HPV Infections ...... 10 1.4.1 Onset in Childhood ...... 10 1.4.2 PID Onset in Adulthood ...... 12 1.5 Treatment ...... 15 1.5.1 HSV Treatment ...... 15 1.5.2 VZV Treatment...... 18 1.5.3 HPV Treatment ...... 20 2 Study Aims ...... 25 3 Materials and Methods ...... 26 3.1 Recruitment of Participants ...... 26 3.2 Ethical Approval and Consent Forms ...... 26 3.3 Study Design ...... 26 3.3.1 Identification of Patients ...... 26 3.3.2 Approaching Patients ...... 27 3.3.3 Data Collection...... 27 3.3.4 Questionnaires ...... 29 3.3.5 Quality of Life Index ...... 30 3.3.6 Isolation of PBMC from Whole Blood ...... 30 3.3.7 HPV Phenotyping ...... 31 3.4 Data Analysis ...... 34 3.4.1 HPV Type Analysis ...... 34 I Contents

3.4.2 DLQI Analysis ...... 34 3.4.3 Blood Values Analysis ...... 35 3.4.4 Treatment ...... 35 4 Results ...... 37 4.1 Characteristics of Patients with Warts ...... 37 4.2 Characteristics of Patients with Herpes...... 38 4.3 HPV Type Analysis ...... 41 4.4 Dermatology Quality of Life Index (DLQI) Analysis...... 44 4.5 Blood Analysis...... 50 4.5.1 Immunodeficiency Panel ...... 51 4.5.2 T Cell Phenotyping ...... 55 4.5.3 B Cell Phenotyping ...... 55 4.5.4 Immunoglobulins ...... 57 4.6 Treatment ...... 59 4.6.1 Warts Patients...... 59 4.6.2 Herpes Patients ...... 63 5 Discussion ...... 68 5.1 Clinical Features of Patients ...... 68 5.1.1 Characteristics of Patients with Warts ...... 68 5.1.2 Characteristics of Patients with Herpes ...... 70 5.2 Immunology Features of Patients ...... 72 5.3 Dermatology Quality of Life Index (DLQI) ...... 76 5.3.1 Patients with warts ...... 76 5.3.2 Herpes Patients ...... 78 5.4 Treatment of Patients ...... 80 5.4.1 Wart Patient’s Treatment ...... 80 5.4.2 Herpes Patient’s Treatment...... 83 6 Summary and Conclusion ...... 87 7 Zusammenfassung ...... 88 8 References ...... IX 9 Appendix...... XXXVI 9.1 Blood Values Spreadsheet...... XXXVI 9.2 Questionnaire Dermatology Quality of Life Index (DLQI) ...... XL 9.3 Questionnaire Herpes Patients ...... XLII 9.4 Questionnaire Wart Patients ...... XLVIII 9.5 Eidesstattliche Versicherung ...... LIV 10 Acknowledgements...... LV

II List of Figures

List of Figures

Figure 1: β- and α-HPV type virus prevalence ...... 42 Figure 2: HPV genotype distribution by anatomical location ...... 43 Figure 3: A. DLQI scores for warts and herpes patients; B. DLQI scores for warts and herpes patients divided into groups ...... 45 Figure 4: Comparison between the herpes no outbreak cohort Q (1/1) and Q (1/2) groups and the herpes outbreak cohort ...... 46 Figure 5: DLQI for when patients answer, “not at all” ...... 48 Figure 6: DLQI for when patients answer, “a little” ...... 48 Figure 7: DLQI for when patients answer, “a lot” ...... 49 Figure 8: DLQI for when patients answer, “very much” ...... 49 Figure 9: DLQI for when patients answer, “not relevant” ...... 50 Figure 10: Comparing lymphocyte count of the warts cohort (divided into CVID and no-PID patients in plot B), DOCK8 and IL7Ra deficient patients and herpes cohort ...... 51 Figure 11: Comparing absolute CD4 count of the warts cohort (divided into CVID and no-PID patients in plot B), DOCK8 and IL7Ra deficient patients and herpes cohort ...... 52 Figure 12: Comparing absolute CD8 count of the warts cohort (divided into CVID and no-PID in plot B), DOCK8 and IL7Ra deficient patients and herpes cohort ...... 52 Figure 13: Comparing absolute CD19 count of the warts cohort (divided into CVID and no-PID patients in plot B), DOCK8 and IL7Ra deficient patients and herpes cohort ...... 53 Figure 14: Comparing absolute CD16+CD56 count of the warts cohort (divided into CVID and no-PID patients in plot B), DOCK8 and IL7Ra deficient patients and herpes cohort ...... 53 Figure 15: Comparing % of CD19+ B cells of the warts cohort (divided into CVID and no-PID patients in plot B), DOCK8 and IL7Ra deficient patients and herpes cohort ...... 54 Figure 16: Comparing % of CD16+ CD56 NK cells of the warts cohort (divided into CVID and no-PID patients in plot B), DOCK8 and IL7Ra deficient patients and herpes cohort ...... 54 Figure 17: Comparing % of CD4+ CD45RA+ (CD4 Naive) % CD4+ in the warts cohort (divided into CVID and no-PID patients in B), DOCK8 and IL7Ra deficient patients and herpes cohort ...... 55

III List of Figures

Figure 18: Comparing % of CD19+ B cells for the warts cohort (divided into CVID and no-PID patients in plot B), DOCK8 and IL7Ra deficient patients and herpes cohort ...... 56 Figure 19: Comparing % of CD27+ IgD+ (IgM Memory) cells % CD19+ cells for the warts cohort (divided into CVID and no-PID patients in plot B), DOCK8 and IL7Ra deficient patients and herpes cohort...... 56 Figure 20: Comparing % of CD27+ IgD- (Sw. Memory) cells % CD19+ cells for the warts cohort (divided into CVID and no-PID patients in plot B), DOCK8 and IL7Ra deficient patients and herpes cohort...... 57 Figure 21: Comparing IgA levels for the warts cohort (divided into CVID and no-PID patients in plot B), DOCK8 and IL7Ra deficient patients and herpes cohort ...... 58 Figure 22: Comparing IgM levels for the warts cohort (divided into CVID and no-PID patients in plot B), DOCK8 and IL7Ra deficient patients and herpes cohort ...... 58 Figure 23: Treatment modalities and their outcomes for the warts cohort ...... 59 Figure 24: CVID and no-PID patients. A. Outcome of the treatment; B. Outcome of the treatment ...... 60 Figure 25: CVID and no-PID patients. C. Outcome of the treatment; D. Outcome systemic acitretin treatment ...... 61 Figure 26: E. Outcome Cautery treatment for CVID and no-PID patients; F. Outcome CO2 treatment for CVID and no-PID patients ...... 62 Figure 27: HPV patients with warts ...... 62 Figure 28: The number of patients that took acute or prophylactic medication ...... 63 Figure 29: Medication success of the patients with acute medication ...... 64 Figure 30: Patients on prophylactic medication. A. How many take aciclovir, valaciclovir, or famciclovir by virus type; B. In what way was the treatment beneficial ...... 65 Figure 31: Frequency of outbreaks pre- and on prophylactic medication, by virus type ...... 66 Figure 32: A. Number of patients with outbreaks on prophylactic medication. B. Number of patients with outbreaks on prophylactic medication by virus type...... 67 Figure 33: How many patients have had Zostavax vaccination? ...... 67 Figure 34: Examples of patients with severe warts ...... 70 Figure 35: Warts from the DOCK 8 deficient and one CVID patient ...... 77

IV List of Tables

List of Tables

Table 1: Blood values measured on the patient’s blood samples ...... 28 Table 2: Reagents for the isolation of the PBMC from whole blood protocol ...... 30 Table 3: Reagents for cryopreserving PBMCs protocol ...... 31 Table 4: Reagents for DNA isolation from wart scrapings protocol ...... 31 Table 5: A.PCR reaction constituents and B. Cycling conditions ...... 33 Table 6: Primer pairs use to detect α-HPV types in the PCR method ...... 34 Table 7: Characteristics of patients with warts ...... 38 Table 8: Characteristics of patients with herpes ...... 40 Table 9: List of herpes virus difficulties and triggers ...... 41 Table 10: Trigger factors of recurrent herpetic (n=15/21) ...... 41 Table 11: HPV type distribution by patient ...... 43 Table 12: HPV type distribution by location ...... 44 Table 13: Meaning of DLQI scores ...... 46 Table 14: Comparison of individual DLQI question scores (min=0, max= 3)...... 47 Table 15: Blood values analysed in more detail with normal healthy control ranges...... 50 Table 16: Treatment regimens for cryotherapy and imiquimod ...... 60 Table 17: Treatment regime and number of outbreaks for each herpes patients ...... 65 Table 18: Laboratory comparison of CVID patients with warts reported in the literature ...... 74

V List of Abbreviations

List of Abbreviations

ADA adenosine deaminase BCIP 5-bromo-4cloro-3indolyphosphate BCPT B cell phenotyping CID combined immunodeficiency CTL cytotoxic T lymphocyte CVID common variable immunodeficiency DCs dendritic cells DLQI Dermatology Quality of Life Index DMSO dimethylsulfoxid DOCK8 dedicator of cytokinesis 8 EBV Epstein–Barr virus EV epidermodysplasia verruciformis EV-HPVs epidermodysplasia verruciformis human papilloma viruses FBC full blood count FCS filtered HI foetal calf serum HCR healthy control range HIES hyper-IgE syndromes HIV human immunodeficiency virus HPA hypothalamic-pituitary-adrenal HPV human papilloma virus HSCT hematopoietic stem cell transplantation HSE herpes simplex (HSV-1) induced encephalitis HSV HZ herpes zoster HZV herpes zoster virus IFN Ig immunoglobulin IgA immunoglobulin A IgD immunoglobulin D IgE immunoglobulin E IgM immunoglobulin M IL interleukin

VI List of Abbreviations

IP immunodeficiency panel JAK3 janus kinase-3 LOCVID Late onset common variable immunodeficiency MCV virus MDS myelodysplastic syndrome MHC major histocompatibility complex MonoMAC monocytopenia and m. avium complex NBT nitroblue tetrayolium NK natural killer NLRs nucleotide-binding oligomerization domain (NOD)-like receptors NOD nucleotide-binding oligomerization domain NTM nontuberculous mycobacteria PAMP pathogen-associated molecular patterns PAP pulmonary alveolar proteinoses PBMC peripheral blood mononuclear cells PBS phosphate-buffered saline PCR polymerase chain reaction pDCs plasmacytoid dendritic cells PHN post-herpetic neuralgia PID primary immunodeficiency PM-PCR broad-spectrum polymerase chain reaction PRRs pattern recognition receptors QoL quality of life RCT randomised control trial RFH Royal Free Hospital RHA reverse hybridization assay RIG retinoic acid-inducible gene RLRs retinoic acid-inducible gene (RIG)-like receptors SCID severe combined immunodeficiency STAT signal transducer and activator of transcription TCPT T cell phenotyping TLRs toll-like receptors TNF tumour factor UVR radiation

VII List of Abbreviations

VSI viral VZV WHIM warts, hypogammaglobulinemia, infections, myelokathexis WILD warts, immunodeficiency, lymphedema, anogenital ZAP zoster-associated pain ZEST zostavax efficacy and safety trial Zostavax zoster vaccine α-HPV α-human papilloma virus β-HPV β-human papilloma virus

VIII Introduction

1 Introduction

1.1 Skin Infections

Skin disease is one of the most common human medical conditions (Hay et al. 2014) and is a frequent reason for consultation in primary care and dermatology practice worldwide (Sladden und Johnston 2004). It permeates all cultures, is seen in all age groups, and affects between 30% and 70% of individuals, with even higher rates in high-risk sub-populations (Hay und Fuller 2011; Schofield et al. 2009). Young children are particularly vulnerable to infections, by both viral and bacterial pathogens while their innate and adaptive immune responses mature and acquire memory, as they are born with an immature immune system (Simon et al. 2015). This is reflected in the high prevalence of skin infections in childhood which are rarely seen the healthy adult population.

Schofield et al (2009), looking at skin conditions in the UK, state that skin infections, that can be sub-divided in bacterial, fungal and viral skin infections, represent the commonest group of skin problems for which patients visit their general practitioner. My dissertation will focus on skin infections of viral origin in adults, including cutaneous viral warts, Herpes Simplex Virus (HSV) and Herpes Zoster Virus (HZV) infections.

1.2 Common Viral Skin Infections

1.2.1 Herpes Viruses

There are more than eighty types of herpes viruses, of which eight are known to infect humans. These are members of the family and include: Herpes Simplex Virus type 1 (HSV-1); Herpes Simplex Virus type 2 (HSV-2); Varicella Zoster Virus (VZV); ; Epstein-Barr Virus; Human Herpes Viruses 6 and 7; and Kaposi’s sarcoma-associated herpesvirus (type 8) (Fatahzadeh und Schwartz 2007b). These viruses are ubiquitous, affecting urban and remote populations throughout the world in all age groups (Gupta et al. 2008).

1.2.1.1 Herpes Simplex Virus (HSV) HSV-associated infections are among the most widespread diseases. There are two serotypes of HSV, serotype 1 and serotype 2, and an estimated 60% to 95% of the adult population is infected by at least one of them (Brady und Bernstein 2004). In immunocompetent people, they can often cause a debilitating illness, which may have a physical and psychological impact, especially when recurrences are frequent (Simmons 2002).

1 Introduction

HSV-1 mainly causes orofacial lesions, whereas HSV-2 is predominantly associated with genital diseases (Arduino und Porter 2008). However, crossover infection via sexual practice is possible and more than 25% of infections are attributable to HSV-1 (Siegel 2002; Fatahzadeh und Schwartz 2007a).

For this dissertation, I will mainly concentrate on HSV-1 infections, which are endemic worldwide and whose seroprevalence increases gradually from childhood, reaching 70% to 80% in adolescence (Arduino und Porter 2006; Whitley und Roizman 2001).

Most primary infections with HSV are asymptomatic and therefore unrecognised (Arduino und Porter 2008; Siegel 2002). Only 13% to 30% of patients, usually children, will develop clinical manifestations of primary herpetic gingivostomatitis, the most common manifestation of primary HSV infection, which is characterised by vesiculoulcerative lesions in and around the oral cavity (Amir 2001).

HSV is transmitted by direct contact with infected secretions. HSV enters the host through abraded skin or mucocutaneous surfaces, invades epithelial cells and initiates intracellular replication. Subsequently, the virus enters axon endings of sensory that innervate the infected cell and travels retrograde to the trigeminal, cervical, lumbosacral, or autonomic ganglia of the host’s nervous system. There the virus replicates, is sequested from the host immune surveillance, and able to remain as a latent infection for the entire life of the infected person (Steiner et al. 2007; Gupta et al. 2008; Fatahzadeh und Schwartz 2007b).

Periodically, the virus reactivates and is transported anterograde down the sensory nerves to the skin or mucosal surface. Lesions do not need to occur at the exact site of primary infection because the virus can exit at the ending of any branch of the axon (Gupta et al. 2008; Simmons 2002). Viral shedding during reactivation can result in the formation of lesions or might occur in the absence of clinically recognised symptoms (Gupta et al. 2008; Brady und Bernstein 2004). About 20% to 40% of patients, who are HSV-1 seropositive, will develop recurrent clinically recognised infections (Arduino und Porter 2008; Siegel 2002; Stock et al. 2001). The frequency of these recurrent infections varies between individuals, ranging from months to years with most patients suffering 2 or less outbreaks per year (Arduino und Porter 2008; Fatahzadeh und Schwartz 2007b). This typically presents as fever at the mucocutaneous junction of the face, commonly referred to as “cold sores” or (Fatahzadeh und Schwartz 2007a; Arduino und Porter 2008).

Prodromal symptoms, such as tenderness, pain, burning sensation or paraesthesia at the site of reactivation arise in 46% to 60% of herpes labialis patients (Arduino und Porter 2008) and in about 80% of patients with acute VZV infection (Wittek et al. 2010). These symptoms are thought to be due to early viral replication at sensory nerve endings and in the mucosa or (Simmons 2002).

2 Introduction

The triggers that induce HSV-1 reactivation from latency are poorly defined, but disease recurrence is often associated with exposure to a variety of internal and external factors. Typical triggers include emotional or physical stress, common cold, fever, menstruation, immunosuppressant or , corticosteroid administration and exposure to heat, cold, or sunlight (Arduino und Porter 2008; Fatahzadeh und Schwartz 2007b).

1.2.1.2 Varicella Zoster Virus (VZV) VZV, one of the eight human herpes viruses of the Herpesviridae family, causes varicella () during primary infection, establishes latency in sensory ganglia, and reactivates as herpes zoster (Abendroth und Arvin 2001; Heininger und Seward 2006). Varicella is predominantly a childhood disease that usually results in a mild to moderate illness in immunocompetent patients and is characterised by a diffuse vesicular with prodromal fever and/or malaise (Heininger und Seward 2006; Steiner et al. 2007). Varicella is a highly infectious disease, becoming symptomatic in 61% to 100% susceptible children (Seward et al. 2004). Before the implementation of the varicella vaccination program in 1995, more than 95% of individuals were infected before adolescence in most temperate climates (Heininger und Seward 2006).

During primary infection, VZV inoculates the epithelial cell of the nasopharyngeal mucosa through airborne viral droplets. Following local replication, the virus is spread to the tonsils and other regional lymphatic tissues from where infected T cells transport the virus via the bloodstream to the skin, causing the typical vesicular (Arvin 2008; Zerboni et al. 2014). This cell-associated viremia in the pathogenesis of primary VZV infection differs from the primary HSV infection in which the virus is restricted to localised mucocutaneous tissue (Abendroth und Arvin 2001). As in HSV infection, the VZV gains access to the sensory ganglia during primary infection where latent infection is established. Access to the ganglia is presumed to be gained through retrograde axonal transport from the infected skin and in addition by T cell viremia (Abendroth und Arvin 2001). During reactivation, VZV reaches the skin via anterograde axonal pathways, causing the characteristic unilateral dermatomal vesicular rash, that is accompanied by sensory disturbances and neuropathic pain (Abendroth und Arvin 2001).

The reactivation of VZV infection occurs in nearly one million people annually in the Unites States (Yawn et al. 2007) and a life time incidence between 25% and 30% of the population with an increase to 50% of those who are over 80 years of age (Yawn und Gilden 2013; Schmader und Dworkin 2008). Due to waning VZV cell-mediated immunity, older adults have the highest risk factor for the development of herpes zoster (Avin, 2005) but it also occurs more frequently in immunocompromised people who tend to have a rash that is more severe with prolonged duration (Harpaz et al. 2008).

3 Introduction

Recurrent herpes zoster infections in immunocompetent patients are uncommon with an estimate range from 1% to 5% (Gebo et al. 2005; Yawn et al. 2011). For example, Tseng et al (2012) assessed the incidence of herpes zoster recurrence and reported only 25 cases out of 5,180 unvaccinated immunocompetent individuals, aged 60 years or older over a follow up period of 4.25 years and Yawn et al (2011) report recurrence rates of 5.7% after 8 years follow up for immunocompetent people. However, recurrences are more common in immunocompromised patients with rates of 10% to 26% seen in human immunodeficiency virus (HIV) infected patients (Gebo et al. 2005). Moreover, Yawn et al (2011) reported an increased risk to 6.2% in people with decreased immune competency.

1.2.2 Human Papilloma Virus

Cutaneous viral warts are common skin infections caused by human papilloma virus (HPV), affecting the general population at some point in their life (Gibbs et al. 2002; Antonsson 2012). More than 150 different genotypes of HPV are currently described (Sterling et al. 2014; Antonsson 2012) which, based on defined variation of the viral DNA, can be classified into 5 genera: α-, β-, γ-, µ-, and ν-PV´s (Handisurya et al. 2009). The majority of cutaneotropic HPV types are found in the β-genus, but also in the γ- and α-genus, with a few in the µ- and v-genera (Bernard et al. 2010). Depending on the HPV genotype and location, HPV infections present as common warts (verruca vulgaris), plantar warts (verruca plantaris which are the equivalent of verruca vulgaris on the dorsal aspect of the hands or feet), flat warts (verruca plana) and genital warts (condyloma accuminata) (Sri et al. 2012; Sterling et al. 2014). The majority of common warts mainly involve hands and feet and are caused by HPV types 2, 27, 57 from the α-genus, 4 and 65 from the γ-genus and HPV type 1 from the µ-genus. Flat warts usually occur on the face and distal limbs and are primarily caused by HPV type 3, 10, and 28 from α-genus and HPV type 41 from the v-genus (Handisurya et al. 2009; Villiers et al. 2004).

Genital warts are also very common and caused again by different HPV types. However, I have not included them in my dissertation, as of all the patients I approached, only a IL17Ra- deficient patient suffered from genital warts.

Transmission of HPV occurs from one individual to another by direct contact or indirectly by fomites. HPV penetrates deeper epithelial cell layers through maceration or abrasions and infects basal keratinocytes, probably stem cells that are able to perform cell division (Lipke 2006; Handisurya et al. 2009). Clonally keratinocyte proliferations following infection causes epidermal thickening and hyperkeratinisation, which eventually results in a visible wart, weeks or even months later (Sterling et al. 2014).

4 Introduction

Cutaneous viral warts are common in childhood, but can occur at any age with an estimated prevalence of 7 to 10% of the general population. Small epidemiology studies have report that 5 to 20% of children and young adults, and 2 to 5% of adults have warts (Rübben 2011), with a peak incidence between the ages of 12 and 16 years (Bacelieri und Johnson 2005; Clifton et al. 2003).

The 1958 National Child Development Study of Great Britain suggested that in 90% of children, warts at the age of 11 years had cleared by the age of 16 years (Sterling et al. 2001). Other studies report spontaneous clearance rates of 23% at two months, 30% at 3 months and 65 to 80% by 2 years (Sterling et al. 2001; Leiding und Holland 2012) with similar finding in a more recent study by Bruggink et al (2013). This study found a prevalence of warts of 33% in 1,009 school children and report that in 52% of the children’s warts had cleared within 1 year with an even higher clearance rate of 90% when newly developed warts were excluded.

The rate of clearance is influenced by factors such as patient’s immune status, viral type, extent and duration of warts (Lipke 2006; Rübben 2011). There is no precise definition for recalcitrant cutaneous warts but a good rule is failure to respond after five treatments over a period of 6 months (Leung 2011).

Intact and functioning cellular immunity, including T cell and Natural Killer (NK) cell cytotoxicity, is essential for elimination of HPV (Gonçalves und Donadi 2004; Leiding und Holland 2012). Therefore, in patients whose warts are severe or recalcitrant, concern for immune defects is raised (Leiding und Holland 2012; Rübben 2011).

1.3 Viral Infections and Immune Response

1.3.1 Immune Control of Viruses

Virus infected cells successively induce several defence strategies which include firstly innate immune responses mediated by type I interferon (IFN) and cytokine production, secondly Natural Killer (NK) cell responses and finally cell mediated immunity, including cytotoxic T lymphocyte (CTL) responses and antibody production (Matsumoto et al. 2011).

Viral infections result in the generation of pathogen-associated molecular patterns (PAMP) such as viral genome nucleic acid and viral proteins which can be recognised by pattern recognition receptors (PRRs), as a means of sensing the presence of virus. Vertebra hosts encode several classes of PRRs including Toll-like receptors (TLRs), Retinoic acid-inducible gene (RIG)-like receptors (RLRs) and Nucleotide-binding oligomerisation domain (NOD)-like receptors (NLRs) (Janeway und Medzhitov 2002; Akira et al. 2006).

5 Introduction

TLRs are expressed on various immune cells, including macrophages, dendritic cells (DCs), NK cells, specific types of T cells, and also on nonimmune cells such epithelial cells and fibroblasts (Janeway und Medzhitov 2002; Pisegna et al. 2004). Of the 10 TLRs identified in humans TLR3, TLR7, TLR8, and TLR9 are involved in the recognition of viral nucleotides which triggers the activation of distinct intracellular pathways including production of proinflammatory cytokines and chemokines (Zhang et al. 2007a). In particular, type I (interferon α and β), are the key cytokines produced that not only inhibit viral replication but also mediate induction of both the innate immunity and the subsequent development of adaptive immunity to viruses (Iwasaki und Medzhitov 2004; Le Bon und Tough 2002; Theofilopoulos et al. 2005).

Recent studies have shown that DCs undergo maturation in response to interferon α and β (Le Bon und Tough 2002). Through this, IFN links the innate and the adaptive immune response as DC maturation includes, antigen processing, increased major histocompatibility complex (MHC) molecule expression and migration to the draining lymph node. Here, together with providing costimulatory signals, DCs present the processed antigen within the cleft of a MHC molecule to naive T cells thereby activating them to undergo clonal expansion and to differentiate into effector T cells. These are known as T helper (TH) cells or cytotoxic T lymphocytes (CTLs) for CD4+ and CD8+ T cells, respectively (Lanzavecchia und Sallusto 2001; Kaech et al. 2002; Steinman und Banchereau 2007). Once activated, CTLs can recognize the same viral peptide bound to class I MHC molecules on the surface of a virus- infected cell. Upon recognition, CTLs are capable of killing the target cell through the induction of apoptosis and are able of releasing antiviral cytokines that inhibit replication of the pathogen (Cullen und Martin 2008). While some specific CD4+ T cells are also capable of cytotoxic killing (Kaech et al. 2002) their most important role seems to be providing help to CD8+ T cells and B cells (Burton 2002). Apoptotic cells are cleared through phagocytosis as a result of membrane changes that enables phagocytes to recognise and remove these cells (Delves et al. 2014).

Interferons α and β also activate NK cells (Biron et al. 1999; Biron 1998; Trinchieri und Santoli 1978). NK cells play an important role in the innate immune response against viral infections through their rapid cytotoxic activity and their IFN-ɣ production (Lodoen und Lanier 2006). Macrophages for example respond to IFN-ɣ by increasing microbicidal activities and also by producing other cytokines such as IL-12 and DCs respond by enhancing their antigen presentation function (Sheng et al. 2013). IFN-ɣ also recruits and activate effector leukocytes, including cytotoxic T lymphocytes and CD4 + T helper type 1 cells (Lee und Biron 2010).

6 Introduction

Antibodies also play a role in the antiviral protection and their most important activity is the neutralisation of free virus particles (Burton 2002). Neutralisation, by the means of reducing viral infectivity through the binding to the surface of viral particles (virions), thereby preventing cell entry and subsequently viral replication (Dimmock 1995; Klasse und Sattentau 2002). Further, in binding to virions or infected cells, antibodies can provide a link between the pathogen and NK cells or the complement system, thereby inducing antibody- dependent cellular or complement-dependent cytotoxicity respectively (Delves et al. 2014). Usually, clearance of a virus does not require the action of neutralising antibodies. However, on re-exposure, neutralising antibodies provide an effective line of defence so that the viral infection can be contained by cellular and innate immunity without symptoms of disease (Burton 2002). In addition, most current work through virus specific antibody responses that attenuate infections and thus provide effective early protection (Plotkin 2008; Siegrist 2008) against Herpes Simplex Infections.

1.3.2 Herpes simplex Infections

1.3.2.1 Control and Clearance of Infection Coordinated responses by both, the innate and the adaptive immune system are required for a successful initial control of HSV replication in the sensory ganglia and for the establishment of latency (Khanna et al. 2004; Klenner et al. 2015; Sabatini und Yungbluth 2006). During the initial phase of infection antiviral activities of macrophages (Reading et al. 2006) and NK cells are known to be important in limiting virus replication and spread (Nandakumar et al. 2008). Macrophages participate in the control of viral spread by secretion of tumour necrosis factor- α (TNFα), interferon-γ (IFN-γ) and nitric oxide (NO) (Kodukula et al. 1999), which have been shown to inhibit HSV-1 replication (Feduchi und Carrasco 1991; Croen 1993). Evidence from human and murine studies suggest that NK cells play a relevant role in generating an effective adaptive immune response by producing effector cytokines such as IFN-ɣ and/or interleukin (IL)-15 and IL-12 and by aiding dendritic cells (DCs) in enabling effective antigen processing and presentation (Nandakumar et al. 2008).

Following primary infection, the cell-mediated immune response to HSV is also important for control and clearance of recurrent infections. Increased severity and prolonged duration of infection and the potential of systemic dissemination are seen in immunocompromised patients (Arduino und Porter 2008; Decman et al. 2005) and reported in immunosuppressed transplant or patients and in advanced HIV-infected individuals (CD4 count <200 cells/µL). This reflects the key role of T cells, especially CD4+ T cells (Aumakhan et al. 2010; Kusne et al. 1991) which are the main source of IFN-ɣ in the early phase of infection (Cunningham et al. 2006). IFN-γ induces MHC class II expression in infected epidermal cells, allowing recognition by CD4+ T cells and restores the HSV induced down-regulation of MHC

7 Introduction class I of infected cells, allowing recognition and cytotoxic activity by CD8+ T cells (Miklos et al. 1998; Cunningham et al. 2006).

1.3.2.2 Maintenance of Latency The mechanisms for establishment of latency, its maintenance and reactivation have not been fully understood. Recent evidence strongly supports the role of CD8+ T cells in maintaining HSV in a latent state (Decman et al. 2005; Khanna et al. 2004). It is suggested that CD8+ T cells are able to monitor and restrict HSV-1 gene expression and restrict replication of infectious virions trough production IFN or through the release of lytic granules (Khanna et al. 2004). Factors that influence T cell function may therefore predispose to HSV- reactivation. In fact, patients tend to suffer from reactivation when they are immunologically compromised or when exposed to emotional or physical stress (Arduino und Porter 2006). Stress as a trigger factor for reactivation can be explained as it causes activation of the hypothalamic-pituitary-adrenal (HPA) axis whose pathway ends with the release of glucocorticoid which has been shown to reduce T cell proliferation and increase T cell apoptosis (Newton 2000).

Both CD8+ but also CD4+ T cells have been found in the trigeminal ganglion during HSV-1 latency, however the role of CD4+ T cells in maintaining latency still needs to be clarified (Decman et al. 2005; Khanna et al. 2004).

1.3.3 Varizella Zoster Infections

1.3.3.1 Immune Response During Primary Infection The first response of the naive host, when VZV inoculates the respiratory mucosal during primary infection, is mediated by innate immune responses. This involves in particular NK cells and IFN-α (Abendroth und Arvin 2001). These responses are likely to be important to limit the initial spread of the virus within the host and to activate the VZV specific, adaptive immune system but are not efficient enough to prevent progression of the disease (Arvin 2008).

Following inoculation and replication, the virus spreads to the tonsils and other regional lymphoid tissues with predominant infection of memory CD4+ T cells which express skin- homing protein markers that makes them more likely to travel to cutaneous epithelia (Ku et al. 2002; Zerboni et al. 2014). VZV has the capacity to down regulate MHC class I and II, preventing their recognition by CD4+ and CD8+ T cells. This way, infected T cells are protected from recognition by the host immune system and able to transport the virus to the skin for replication (Abendroth und Arvin 2001; Ku et al. 2005). Successful requires enough replication to create lesions at the skin surface containing large amount of virus. To have sufficient time for replication, VZV promotes survival of infected T cells by

8 Introduction reprogramming cell signalling to induce Signal Transducer and Activator of Transcription 3 (STAT3). This triggers upregulation of the anti-apoptotic protein survivin and inhibits the expression of IFN-α, which is needed to reduce viral replication (Sen et al. 2012). However, control of viral replication by innate immune responses is advantageous for the virus as life threatening infections would diminish opportunities for VSV transmission to other susceptive people and persistence in the host population. Thus, at the same time, epidermal cells adjacent to the infected cells show upregulation of STAT1, IFN-α and other innate cytokines to provide a barrier against an uncontrolled cell to cell spread of VZV (Ku et al. 2004; Zerboni et al. 2014).

1.3.3.2 Controlling VZV Latency VZV-specific cell-mediated immune responses are needed to clear primary infection, to limit the potential for reactivation and to prevent recurrent infections (Arvin 2008; Heininger und Seward 2006; Tseng et al. 2012). This has been shown indirectly by the increased severity of infection in children with cellular immunodeficiencies (Heininger und Seward 2006) and higher Zoster incidences in HIV infected patients, leukemic children (Gross et al. 2003; Gershon et al. 1997) and patients with lymphoproliferative malignancies (Arvin et al, 1980). In addition, VZV cell-mediated immunity wanes in elderly individuals in whom more frequent and more severe Zoster infections are seen (Steiner et al. 2007; Zerboni et al. 2014).

In contrast, the absence of serious primary varicella infection or exacerbations of Zoster in patients with hypogammaglobulinemia (Heininger und Seward 2006) demonstrate that humoral immunity is less important than cellular immunity for VZV reactivation and for disseminated Zoster (Gross et al. 2003).

1.3.4 HPV Infections

The period between HPV infection and the appearance of a lesion can vary from weeks to months (Stanley 2001) suggesting that the virus has effective methods to evade host immune mechanisms (Stanley 2001; Frazer 2009). HPV primarily infects basal keratinocytes and replication is restricted to intraepithelial sites, tightly coupled with the keratinocytes’ differentiation process. This intraepithelial life cycle and the fact that keratinocytes are cells destined for apoptosis and desquamation have the consequence that no inflammation accompanies viral infection; hence no danger sign alerts the immune system (Stanley 2001; Stanley 2009). In addition, there is no systemic viremia in natural infection, leaving the draining lymph nodes (where immune responses are initiated) with poor access to free virus particles (Stanley et al. 2008; Leiding und Holland 2012). Despite the virus’s efforts to evade host defences, most HPV infections are detected with time and an effective immune response is mounted (Antonsson 2012; Stanley 2009). Molecular and cellular mechanisms responsible for the elimination of HPV infection are not well understood. However, the

9 Introduction increased incidence, progression and prolonged duration of warts in immunosuppressed patients illustrate the critical importance of cellular and cytotoxic immune response provided by T cells and NK cells in the clearance and control of HPV infections (Stanley 2009; Frazer 2009). For example, up to 90% of immunosuppressed organ-transplanted patients develop warts within 15 years (Lindelöf et al. 2000) and HIV infected patients show an increased incidence of both of cutaneous and genital warts (Stanley 2009), which highlights the importance of CD4+ cells in controlling HPV infections. In addition, immunohistological studies of spontaneously regressing genital warts report massive infiltrate of mainly CD4+ but also CD8+ cells while non-regressing genital warts lack immune cells (Coleman et al. 1994). Therefore, when cutaneous warts are extensive, recurrent and/or recalcitrant to treatment, an underlying immunodeficiency should be considered (Leiding und Holland 2012; Rübben 2011).

1.3.4.1 Humoral Immunity Numerous studies have shown that HPV infections are usually followed by the development of serum neutralising antibody to the major capsid protein L1 (Stanley 2009). Antibodies generally bind on structural components of a microorganism and protects against re-infection by assisting phagocytosis and by neutralising infectivity (Frazer 2009). This immune response however takes approximately 6 to 18 months, with low levels of type-specific antibodies detectable in approximately 50–70% of infected individuals (Stanley et al. 2012). This suggested that generating neutralizing antibodies to virus capsid proteins would be an effective prophylactic vaccine strategy. Two HPV L1 VLP vaccines have been developed; ®, a bivalent HPV 16, 18 VLP vaccine and ® a quadrivalent HPV 16/18/6/11 vaccine. In clinical trials, these vaccines have shown high efficiency in protecting against both benign and neoplastic HPV related disease (Dochez et al. 2014; Erickson et al. 2014).

1.4 Primary immunodeficiencies (PID) with a Susceptibility to HSV or HPV Infections

1.4.1 Onset in Childhood

1.4.1.1 Epidermodysplasia Verruciformis Epidermodysplasia Verruciformis (EV) is a rare genodermatosis characterised by abnormal susceptibility to HPV infections (Handisurya et al. 2009). From early childhood patients suffer from life-long persistent cutaneous warts and frequently develop cutaneous squamous-cell (Orth 2008). Certain HPV types, referred as “EV-HPVs” are specifically associated with EV including HPV types 5, 8, 12, 14, 19, 20, 21, 24, 25, 36, 47, 9, 15, 17, 22, 23, 37, 38 and 49 (Orth 2008). They belong to the β-genus (β-HPVs) and patients are usually infected with multiple types (Orth 2008; Leiding und Holland 2012). Although EV-HPVs are 10 Introduction acquired early in infancy (Antonsson et al. 2003) and are highly prevalent in the general population (Boxman et al. 1997; Antonsson et al. 2000; Astori et al. 1998) the virus is only pathologic (causing symptomatic infections and malignant progression) in patients with EV or in those with other immunodeficiencies (Leiding und Holland 2012; Sri et al. 2012).

In the majority of cases, EV is caused by loss of function mutations in either the EVER1 or EVER2 gene (Orth 2008; Patel et al. 2010). The precise function of these genes remains unclear, but they play a role in regulating the zinc homeostasis in the cell and may thereby form a natural protective barrier to EV-HPV infection (Lazarczyk et al. 2009). EVER proteins in the endoplasmic reticulum of keratinocytes form a complex with zinc transporter-1 (ZNT-1) and it is hypothesised that the activity of this ZnT-1/EVER complex limits the access of viral proteins to cellular zinc storage, an iron which is a necessary cofactor for many viral proteins (Lazarczyk und Favre 2008). Disruption of the complex leads to higher zinc levels in the cytoplasma, allowing replication of EV-HPV and abnormal sensitivity to EV-HPV infections (Lazarczyk et al. 2008). However, EVER genes are not only transcribed in keratinocytes but also in CD4+ and CD8+ T lymphocytes, NK cells and B lymphocytes at high levels (Ramoz et al. 2002). It is therefore speculated that the deficiency of EVER proteins in these immune cells might contribute to the susceptibility and persistence of HPV infections due to impaired cell-mediated immune response (Lazarczyk et al. 2012).

1.4.1.2 Deficiency in Toll-Like Receptor 3 Pathways Deficiency in Toll-Like Receptor 3 (TLR3) is a member of the Toll-like receptor (TLR) family which plays an important role in pathogen recognition and activation of innate immune response (Schroder und Bowie 2005). TLR3 recognises double-stranded RNA (dsRNA), which is produced in the replication of most viruses (Alexopoulou et al. 2001) and induces a range of responses including production of type 1 interferons, proinflammatory cytokine and dendritic cell-mediated activation of NK cells and CTLs (Alexopoulou et al. 2001; Matsumoto et al. 2014; Schroder und Bowie 2005).

TLR3 has been detected in various tissues including the placenta, lung, liver, pancreas, heart, lymph node, spleen, and brain (Alexopoulou et al. 2001; Nishimura und Naito 2005; Zarember und Godowski 2002). Immune cells that contribute to an innate immune response that express TLR3 are dendritic cells, macrophages, and NK cells (Matsumoto et al. 2014; Pisegna et al. 2004; Wesch et al. 2006). In the brain TLR3 is expressed in neurons, microglia and astrocytes (Farina et al. 2005; Préhaud et al. 2005; Town et al. 2006). Here, TLR3 has vital role in natural immunity to HSV-1 as it has been shown that children born with deficiencies in the TLR3 pathway have a specific susceptibility to herpes simplex (HSV-1) induced encephalitis (HSE) due to decreased production of interferons in the central nervous

11 Introduction system which is crucial for protective immunity against primary HSV-1 infection (Casrouge et al. 2006; Guo et al. 2011; Perez de Diego et al. 2010; Zhang et al. 2007b).

1.4.1.3 Severe Combined Immunodeficiency Severe combined immunodeficiency (SCID) is a rare primary immunodeficiency, which is characterized by the dysfunction or absence of T cells, affecting both cell-mediated and humoral adaptive immunity. B cells and NK cells may be absent or present depending on the genetic defect (van der Burg und Gennery 2011). The deficiency predisposes those to severe, life-threatening infections and is caused by several identified genetic mutations

(Fischer 2000), most commonly, in the gene encoding the common gamma (γc) chain of the IL-2 receptor, adenosine deaminase (ADA) deficiency, and Janus kinase-3 (JAK3) mutations (Sri et al. 2012).

SCID patients classically present in infancy with chronic diarrhoea, failure to thrive, and severe recurrent bacterial, viral, or fungal infections including persistent mucocutaneous , adenovirus, CMV, EBV, rotavirus, respiratory syncitial virus, VZV, HSV, , influenza, and parainfluenza 3 (Leiding und Holland 2012).

The current treatment for SCID is hematopoietic stem cell transplantation (HSCT) early in life

(Gaspar et al. 2004). Despite successful HSCT, patients with γc and JAK3 forms of SCID may still develop severe, chronic cutaneous HPV infections, suggesting that immune reconstruction may be incomplete (Laffort et al, 2004). Laffort et al (2004) performed a retrospective analysis of severe chronic HPV disease and report cutaneous lesions in 50% of patients with either γc or JAK3 deficiency. Similar rates of 64% are reported by Gaspar et al (2004) and a more recent study with a smaller cohort showed 19% of HPV-affected patients (Kamili et al. 2014). In the two previous reports, no significant differences in transplant- specific or immune parameters could be demonstrated between the γc or JAK3-deficient patients with and without chronic HPV (Gaspar et al. 2004; Laffort et al. 2004) while Kamili et al (2014) showed a significant low NK cell count in PBMCs in their 4 patients with HPV disease. The etiology of severe HPV infection after transplantation in SCID patients with γc or

JAK3 mutation is still unknown. It has been proposed that dysfunctional γc or JAK3 dependent cytokine signalling in host keratinocytes may contribute to HPV susceptibility (Laffort et al. 2004) but further studies are needed to clarify this theory and I have excluded SCID patients from my dissertation.

1.4.2 PID Onset in Adulthood

1.4.2.1 Common Variable Immunodeficiency Common variable immunodeficiency (CVID) is the most common clinically symptomatic primary immunodeficiency characterised by impaired B cell differentiation with progressive

12 Introduction hypogammaglobulinemia due to defective immunoglobulin (Ig) production (Gathmann et al. 2009; Gathmann et al. 2013). The usual age of onset is 20 to 30 years of age (Ballow 2002; Cunningham-Rundles und Bodian 1999) and both adults and children have been described (Gathmann et al. 2014; Hammarström et al. 2000). CVID forms a heterogeneous group of disorders with a broad range of clinical manifestations including recurrent chronic infections, chronic lung disease, gastrointestinal disease, autoimmune disorders and an increased risk of lymphoproliferative, malignant, and granulomatous complications (Cunningham-Rundles und Bodian 1999; Chapel et al. 2008).

The diagnostic criteria for CVID require: serum IgG and IgA with or without IgM levels to be at least two standard deviation less than the mean for the patient’s age; poor or absent antibody response to immunisation; the diagnosis to be established after the 4th year of life; no evidence of profound T cell deficiency; and a secondary reason for low Ig levels must be excluded (Gathmann 2016).

Warts are uncommon in CVID, but a few cases with disseminated or unremitting warts are reported (Lin et al. 2009; Lynn et al. 2004; Reid et al. 1976; Uluhan et al. 1998). Firstly, a 19- year- old CVID patient with a 13-year history of recalcitrant on his feet, hands, face, neck, and chest (Reid et al. 1976). Then a 9-year-old boy with CVID, juvenile rheumatoid arthritis and multiple warts (Uluhan et al. 1998). And a third case presenting a 55-year-old with hypogammaglobulinemia and persistent warts for 4 years who was then diagnosed with intestinal lymphangiectasia (Lynn et al. 2004). All three cases revealed impaired T cell response to mitogen and T lymphopenia. The most recent case described an 18-year-old man with CVID who had normal T cell numbers but decreased T cell proliferation to antigen and extensive warts on his hands, feet and arms (Lin et al. 2009). This combination of decreased humoral and cell mediated immunity likely contributes to the susceptibility to warts in CVID patients (Leidig and Holland, 2013).

1.4.2.2 Dedicator of Cytokinesis 8 Deficiency Dedicator of cytokinesis 8 (DOCK8) deficiency is an autosomal recessive form of Hyper-IgE Syndromes (HIES). HIES are primary immunodeficiencies characterised by elevated IgE levels, eczema, recurrent staphylococcus aureus skin , frequent pulmonary infections, and eosinophilia (Freeman und Holland 2008). Autosomal dominant and recessive forms of these immunodeficiencies have been identified (Yong et al. 2012; Holland et al. 2007). Patients with the autosomal dominant form which are caused by mutations in signal transducer and activator of transcription 3 (STAT3) (Minegishi et al. 2007; Holland et al. 2007) may in addition develop unique skeletal, connective tissue and dental abnormalities. These unique features are rarely seen in the autosomal recessive form of HIES, and instead patients are susceptibility to severe cutaneous viral infections, including HPV, HSV, HPV,

13 Introduction molluscum contagiosum virus (MCV), and VZV, present frequently with atopic disease, neurologic abnormalities and show a predisposition to malignancies at a young age (Freeman und Holland 2010). This autosomal recessive form has been identified to be caused by homozygous and compound heterozygous mutations or deletions in the gene encoding DOCK8 resulting in a combined immunodeficiency (Engelhardt et al. 2009; Zhang et al. 2009), affecting B cells, NK cells, and various T cells (Aydin et al. 2015). Lymphopenia is common and progresses with age and specifically CD8 T cells of DOCK8-deficient individuals fail to activate, proliferate, and have low cytokine production (Zhang et al. 2009; Engelhardt et al. 2009). Also NK cell function is severely impaired (Mizesko et al. 2013). CD8 T cells and NK cells both are important components in the defence against virally infected cells and their impairment may contribute to the increased susceptibility of these patients to cutaneous viral infections (Lambe et al. 2011).

1.4.2.3 WHIM Syndrome Warts, hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome is a rare combined immunodeficiency characterised by susceptibility to infection with human papilloma virus, neutropenia and leukopenia and hypogammaglobulinemia related recurrent respiratory infections (Diaz und Gulino 2005; Gorlin et al. 2000). This autosomal dominant inherited disease is caused by gain of function mutations in the chemokine receptor CXCR4 gene leading to an increased response to its unique ligand CXCL12 which impairs haematopoiesis and trafficking of haematopoietic cells (Balabanian et al. 2005; Diaz und Gulino 2005; Gulino 2003).

HPV infections in WHIM patients manifests as extensive and persistent cutaneous warts and, in some adults, as intractable genital condyloma acuminata that often progresses to severe dysplasia and carcinoma (Balabanian et al. 2005; Diaz und Gulino 2005; Gorlin et al. 2000). The mechanism accounting for this susceptibility to infection with HPV however, is still unclear (Chow et al. 2010). Balabanian et al (2005) observed an increased CXCL12 expressions in HPV-infected (compared with healthy skin) and suggested that patients susceptibility to HPV infection may therefore be mediated by upregulation of CXCR4 (Balabanian et al. 2005). Further, analysis of myeloid and plasmacytoid dendritic cells (pDCs) in patients suffering with WHIM revealed a reduced number and function compared to healthy subjects. pDCs have an important role in the defence against viruses and their function and generation is partly dependent on CXCR4. Disturbed homeostasis of pDCs may therefore be enhancing the susceptibility to HPV infections in WHIM patients (Tassone et al. 2010).

14 Introduction

1.4.2.4 GATA2 Deficiency GATA2 deficiency is a rare immunodeficiency, separately described as monocytopenia and M. avium complex (MonoMAC) infection syndrome; dendritic cell, monocyte, B cell, and NK cell lymphoid deficiency (DCML deficiency), Emberger syndrome (familial myelodysplasia or leukaemia with lymphedem) and familial myelodysplastic and leukaemia syndrome. The transcription factor GATA2 is involved in embryonic and definitive haematopoiesis (Rodrigues et al. 2005; Vicente et al. 2012) as well as lymphatic and vascular development (Lim et al. 2012). GATA2 mutations lead to decreased or absent circulating monocytes, B cells, DCs and NK cells (Bigley et al. 2011; Vinh et al. 2010) causing a broad spectrum of clinical manifestations including susceptibility to HPVs, nontuberculous mycobacteria (NTM) and opportunistic fungal infections, predisposition to Myelodysplastic syndrome (MDS), acute or chronic leukaemia’s, pulmonary alveolar proteinoses (PAP) and lymphedema (Hsu et al. 2015; Spinner et al. 2014). Patients suffer with major infection, most commonly of viral origin in particular with severe with HPV infections causing recalcitrant warts followed by herpes family viruses’ infections including HSV, VZV and Epstein–Barr Virus (EBV) (Spinner et al. 2014; Vinh et al. 2010). Protection against HPV requires an effective CD4 T cells as well as NK cells response (Woodworth 2002). The combined monocytes and NK cell deficiency in this disease may explain the clinical severity of patient’s HPV infection (Mace et al. 2013). The reduced number and function of NK cells in GATA deficiency patients may also account for the susceptibility to herpesvirus infections (Vinh et al. 2010).

As illustrated in the PID above, although rare, there are many defects causing patients to have an increase disposition to HPV or Herpes infection. Considering the unusual manifestation of their skin infection, I wanted to screen the patients in this study for an underlying immunodeficiency.

1.5 Treatment

1.5.1 HSV Treatment

In patients who experience occasional clinical recurrences of HSV infections with minimal symptoms, lesions may be adequately managed with local anaesthetics and antiseptics to provide symptomatic relief (Sciubba 2002; Huber 2003). Topical antiviral therapy with creams or ointments based on aciclovir or related compounds are an additional treatment option. Randomised Control Trials (RCTs) of patients with recurrent HSV-1 infection have demonstrated significant but modest reduction in healing time and time of symptoms when topical treatment was commenced during the prodrome (Fatahzadeh und Schwartz 2007b; Rahimi et al. 2012).

15 Introduction

When patients suffer from severe frequent symptoms oral antiviral therapy is used for treatment and prophylaxis. Antiviral agents, including aciclovir, penciclovir, famciclovir, and valaciclovir, are all cyclic guanosine analogues which inhibit the viral DNA polymerase and viral replications and have been recommended for the treatment and prevention of recurrent herpes labialis lesions (Rahimi et al. 2012; Fatahzadeh und Schwartz 2007b).

1.5.1.1 Treatment of Acute Attacks

Aciclovir A few trials assessing the effect of oral aciclovir in non-genital HSV infections have shown modest treatment benefits but lack of impact to completely abort lesion development (Laiskonis et al. 2002).

In one double-blind trial, 174 immunocompetent patients were randomly assigned to oral administration of 400 mg aciclovir, 5 times daily for 5 days versus placebo within one hour of onset of prodromal symptoms (Spruance et al. 1990). In the aciclovir group the frequency of HSV culture-positive lesions was significantly lower and the lesion healing time and duration of lesion pain was reduced by 27% and 36% respectively. However, aciclovir did not prevent the development of lesions. An earlier study used a smaller dose of aciclovir (200mg 5 times a day) versus placebo, finding that healing time was similar in both groups with only 5% more patients healed by day 6 (Raborn et al. 1987).

The disadvantage of oral aciclovir is its poor bioavailability of only 10% to 20% and its short plasma life time, making frequent dosing necessary. As a result, it is increasingly being replaced by the newer and more convenient drugs valaciclovir and famciclovir (Brown et al. 2002).

Valaciclovir Valaciclovir is the L-valine ester prodrug of aciclovir which is absorbed from the gastrointestinal tract and rapidly and almost completely converted to acyclovir in the liver and intestines resulting in a 3- to 5-times increase in bioavailability (Brown et al. 2002; Arduino und Porter 2006; Tyring et al. 2002).

Valaciclovir has minimal adverse effects and minimal toxicity (Woo und Challacombe 2007) and studies have demonstrated the efficiency in treating cold sores when given in the early prodromal stage, but there is no evidence for the optimal dose (Chosidow et al. 2003; Laiskonis et al. 2002; Spruance et al. 2002).

One randomised, double-blind trial of 308 patients concentrated on the dosing and duration of valaciclovir in recurrent herpes labial (Laiskonis et al. 2002). Overall, there was no significant difference in the resolution and duration of pain and healing time between treatment regimens of either 1,000 mg twice daily for 1 day or 500 mg twice daily for 3 days.

16 Introduction

Although there was no placebo comparison, 44% of subjects reported abortion of lesions, especially if valaciclovir was taken in the prodromal stage (Laiskonis et al. 2002). Other studies have confirmed the benefit of systemic valaciclovir in shortening outbreaks and duration of pain (Spruance et al. 2003). Dosage regimens of 2,000 mg valaciclovir twice daily for 1 or for 2 days (Spruance et al. 2003) or a single dose of 500 mg, 1,000 mg or 2,000 mg (Chosidow et al. 2003) did not show any differences in frequency of aborted lesions compared with placebo (Chosidow et al. 2003; Spruance et al. 2003).

Famciclovir Another guanosine analogue is penciclovir, which has a similar mechanism of action to that of aciclovir with higher intracellular concentration and longer half-life in HSV infected cells (Brady und Bernstein 2004; Spruance et al. 2006). Penciclovir is poorly absorbed orally and is therefore only formulated for intravenous and topical use (Brady und Bernstein 2004; Woo und Challacombe 2007). Famciclovir, the oral prodrug of Penciclovir, is well absorbed from the gastrointestinal tract and has high bioavailability of approximately 60% (Gill und Wood 1996).

Spurance et al (2006) assessed 701 patients for the benefit of famciclovir as a single dose of 1,500 mg once a day or 750 mg twice a day for a single day compared to placebo in a randomised, double-blind trial for acute treatment. In time to healing of primary or of all vesicular lesions, there was no significant difference between the two famiclovir treatment groups but the healing time was significant shorter in the two famciclovir regimes compared with placebo.

1.5.1.2 Prophylactic Therapy For patients with severe or frequent fascial herpes infection, prophylactic suppression with oral antiviral medication may be warranted (Esmann 2001).

In a small double-blind, placebo-controlled, cross-over trial, 11 patients received oral aciclovir in a dose of 200 mg 4 times daily for up to 12 weeks. Only 2 patients developed lesions during treatment, compared with 9 during placebo treatment, indicating that aciclovir may be effective in suppressing non-genital HSV infections (Thomas et al. 1985). This was also seen in another small study of 20 patients with recurrent herpes labials infections, where long-term aciclovir prophylaxis (400 mg twice daily) reduced the number of frequently recurrent infections and prolonged the median time to the first clinical documented recurrence (Rooney et al. 1993).

A double-blind, randomised study using valaciclovir 500 mg once daily versus placebo for 16 weeks showed significant reduction in recurrences (60% versus 38%) and a significant longer mean time to first recurrence (Baker und Eisen 2003). In another study, a cross-over trial by Gilbert (2007), a 6-month prophylactic regimen of 1,000 mg valaciclovir a day, when

17 Introduction compared with an episodic regimen, had a lower incidence of herpes labialis. However, the difference was small, with a reduction of 0.10 episodes per patient per month (Gilbert 2007).

Only one trial has assessed prophylactic famciclovir versus placebo for prevention of herpes simplex labialis (Rahimi et al. 2012). Spurance et al (1999) tested 3 doses of famciclovir for treatment of ultraviolet radiation (UVR) induced herpes labialis; 243 patients were randomised to receive 125 mg, 250 mg, or 500 mg of famciclovir or placebo 3 times daily for 5 days following UVR exposure. Only in the famciclovir 500 mg treatment group a reduction in lesion healing time was observed but the drug was unsuccessful in preventing the development of new lesions in all treatment regimes. However, studies investigating short courses of prophylactic famciclovir in patients undergoing laser resurfacing, demonstrated promising results. Here, famciclovir prophylaxis markedly reduced or prevented herpes labialis infections (Wall et al. 1999; Bisaccia und Scarborough 2003; Alster und Nanni 1999).

Overall, there are no clinical trials directly comparing any of these antiviral agents. However, placebo-controlled trials have demonstrated that antiviral therapy with aciclovir, valaciclovir or famciclovir, reduce the healing time of HSV lesions and duration of pain, if treatment is initiated during the prodromal phase (Arduino und Porter 2006) and when administered prophylactically, aciclovir, valaciclovir and famciclovir have been shown effective in preventing herpes simplex lesions (Rahimi et al. 2012).

1.5.2 VZV Treatment

1.5.2.1 Symptomatic Therapy Primary VZV infection in children is a self-limited disease, and is usually managed symptomatically unless they are immunocompromised. Reactivation later in life however, is more likely to be treated. Here primary goal of the treatment in immunocompetent patients is to accelerate healing, reduce the severity and duration of acute and chronic pain, and limit complications. In immunocompromised patients, an additional therapeutic goal is the cessation of viral replication (Dworkin et al. 2007). Generally, patients will benefit from education, reassurance and social support while pharmacological treatment include the use of analgesics and antiviral therapy (Schmader und Dworkin 2008). My dissertation will focus on the antiviral therapy of herpes zoster.

Antiviral agents have been primarily recommended for the following six patient groups: patients aged greater than 50 years old; patients with moderate or severe pain; patients with moderate or severe rash; patients with face or eye involvement; patients with zoster complication; and immunocompromised patients (Dworkin et al. 2007; Harpaz et al. 2008). In addition, other patients with zoster might also benefit from antiviral therapy, although they have a lower risk for complications from zoster (Cohen et al. 2013).

18 Introduction

Three guanosine analogues, aciclovir, valaciclovir, and famciclovir, are approved by the Food and Drug Administration (FDA) for treatment of herpes zoster infection (Cohen et al. 2013; Harpaz et al. 2008). Clinical trials have shown that these agents, administered orally, reduce the duration of viral shedding and lesion formation, accelerate healing and the resolution of acute pain (Beutner et al. 1995; Tyring 1998; Tyring et al. 1995; Wood et al. 1996).

As current evidence from clinical trials have initiated antivirals within 72 hours of rash onset, treatment is recommended to start within this therapeutic window as early as possible.

There is no evidence for the efficacy of antivirals for late therapy (greater than 72 hours after rash onset). However, if new vesicles are still appearing at the time of presentation or complications of zoster are present, many experts recommend that treatment should be initiated. In the absence of complication, treatment is usually given for 7 days (Cohen et al. 2013; Harpaz et al. 2008; Schmader und Dworkin 2008; Whitley et al. 2010).

Oral aciclovir has been considered the “gold standard” of herpes zoster treatment (Tyring 2007). For example, in a large randomised, double-blind trial, patients were given 800 mg aciclovir orally, 5 times daily for 7 days within 47 hours of onset and had significantly faster rash healing and significant reduction in pain compared with placebo (McKendrick et al. 1986). In another large study, aciclovir, compared with placebo, reduced duration of virus shedding by 0.8 days (Huff et al. 1988). Also, in meta-analysis of 4 placebo-controlled trials, involving a total of 691 patients, oral aciclovir was found to significantly reduce pain duration and prevalence (Wood et al. 1996).

However, aciclovir’s clinical use is limited by its poor bioavailability and need for frequent daily dosing (800 mg, 5 times daily) compared with that of the second-generation antiviral agents valaciclovir and famciclovir which are administrated 1,000 mg and 500 mg respectively 3 times daily (Tyring 2007). Famciclovir or valaciclovir are safe, effective and more convenient alternatives and have been shown to be superior to aciclovir for reducing pain associated with herpes zoster in some studies.

For example, Beutner et al (1995) compared two different regimes of valaciclovir (1,000mg, 3 times daily for 7 or 14 days) with aciclovir (800 mg, 5 times daily for 7 days) in a randomised, double-blind study. Valaciclovir significantly accelerated the resolution of zoster-associated pain (ZAP) in both valaciclovir groups compared with aciclovir. However, cutaneous lesions resolved at similar rates with both drugs. Another double-blind RCT with a total of 545 patients, compared the efficacy and tolerability of famciclovir administered at 250 mg, 500 mg and 750 mg 3 times daily with aciclovir 800 mg 5 times daily. Here, famciclovir was as effective as aciclovir at all dose levels for the healing of cutaneous lesions and resolution of

19 Introduction acute pain. However, resolution of ZAP occurred at faster in patients treated with famciclovir compared with aciclovir (Degreef 1994).

Famciclovir and valaciclovir were compared in two RCTs for the treatment of herpes zoster in immunocompetent patients and were shown to be therapeutically equivalent, in terms of efficacy for accelerating healing and reducing acute pain (Shafran et al. 2004; Tyring 2000). While in a more recent study, a statistically significant earlier reduction in pain with famciclovir was found (Ono et al. 2012).

An additional nucleoside analogue used to treat herpes zoster is brivudin. This is licensed in varies countries and is 200 to 1,000 times more effective in inhibiting viral replication in-vitro than aciclovir or penciclovir. A restriction applies to immunosuppressed patients, children and pregnant women and it must not be used in combination with 5- (5-FU) because of a potentially fatal interaction (Clercq 2004). As none of the patients in this study were prescribed brivudin, I will not discuss the clinical evidence of this drug.

1.5.2.2 Prophylactic Therapy The majority of patients in the herpes cohort in this study were on prophylactic antiviral medication. To my knowledge there are no studies on prophylactic antiviral medication in patients with recurrent episodes of zoster.

1.5.2.3 Vaccine The herpes zoster vaccine (Zostavax) is approved in the European Union for the prevention of herpes zoster (HZ) and post-herpetic neuralgia (PHN) in adults aged greater than or equal to 50 years (Schmader et al. 2012; Oxman et al. 2005). This vaccine consists of live attenuated VZV which activates specific T cell production, therefore increasing existing immunity and avoiding reactivation of viral replication (Arvin 2005).

The prevention study, a double blind, placebo-controlled randomised trial of 38,546 adults 60 years of age or older demonstrated that the efficacy of the vaccine to prevent HZ is 64% in adults aged 60 to 69 years, and 38% in adults aged 70 years or above (Oxman et al. 2005). In addition, in vaccinated compared to unvaccinated patients who developed HZ, pain was significantly shorter in duration and less severe (Oxman et al. 2005). The Zostavax Efficacy and Safety Trial (ZEST) evaluated the efficacy of vaccination among adults aged 50 to 59. Compared with placebo, Zostavax reduced the risk of developing shingles by 70 percent (Schmader et al. 2012).

1.5.3 HPV Treatment

Although cutaneous viral warts are ubiquitous, no single therapy has been established to achieve complete remission in every patient (Lynch et al. 2014; Handisurya et al. 2009; Mulhem und Pinelis 2011).

20 Introduction

High quality evidence based studies for the optimal treatment are limited (Lipke 2006; Lynch et al. 2014). This is likely a result of confounding factors such as high rates of spontaneous regression (Lynch, 2014) and the large variety of treatment choice (Lipke 2006; Sri et al. 2012). In addition, the efficacy can vary widely depending on the wart location, type and duration of presence, method of application and skill of the technician, patient compliance, age, and immune status (Sri et al. 2012; Lipke 2006).

Treatment and cure of HPV infections is particularly challenging in immunocompromised patients (Leiding und Holland 2012). Multiple interventions are often required including destructive treatments, topical or systemic antiproliferative agents, and immunomodulators (Leiding und Holland 2012; Sterling et al. 2014).

1.5.3.1 Destructive Treatment

Cryotherapy Liquid nitrogen with a temperature of -196 ºC is the most commonly used cryotherapy agent (Lipke 2006; Sterling et al. 2014). As HPV can survive liquid nitrogen (Sterling et al. 2014; Mulhem und Pinelis 2011), the effect on wart regression may be through thermal necrosis of the virus infected keratinocytes or due to local inflammation that generates an effective cell- mediated immune response (Sterling et al. 2001).

Reported cure rates of cryotherapy for warts in randomised trials range from 0% to 69 % (Sterling et al. 2014). A 2012 meta-analysis of RCTs (Kwok et al. 2012), unexpectedly, found no statistically significant benefit of cryotherapy over placebo, which may reflect diversity in study design and the high number of spontaneous resolution (Lynch et al. 2014). This analysis also reported no significant differences in effectiveness in trials comparing cryotherapy with salicylic acid which appeared to be more effective than placebo, the latter suggesting that cryotherapy may also be adequately effective, like salicylic acid (Kwok et al. 2012).

Cryotherapy was only superior in one study, showing the highest overall cure rate of 39% (Bruggink et al. 2010). This three-arm RCT compared the effectiveness of cryotherapy, topical salicylic acid and wait-and-see approach for the treatment of common and plantar warts in 240 patients. Cryotherapy also was the superior treatment when focusing on common warts while no significant difference between treatment groups was seen amongst patients with plantar warts. This equal effectiveness for clearance of plantar warts with either cryotherapy or salicylic acid is also reported by Cockayne et al (2011) comparing both treatment modalities in 240 patients.

Salicylic acid

21 Introduction

Salicylic acid is and treats warts by slowly destroying the infected epidermis, thereby causing mild irritation which may stimulate local immunity (Stanley 2001; Sri et al. 2012).

In a meta-analysis by Kwok et al (2011), five studies of 333 patients were included showing salicylic acid to be more effective compared to placebo, with a 1.6 times greater chance of clearance. The same authors performed a pooled analysis of Salicylic acid of 16 studies with 813 patients showing a mean cure rate of 52% with a range of 0% to 87% compared to observed cure rates of 23% in the placebo arm. Previous data pooled from 6 placebo- controlled trials showed similar results with a cure rate of 75% in the salicylic acid treatment arm compared with 48% in the control group (Gibbs et al. 2002).

Carbon Dioxide (CO2) Laser Carbon dioxide laser is a treatment modality used to ablate cutaneous viral warts by causing non-selective thermal tissue destruction through the vaporisation of water (Lipke 2006; Lynch et al. 2014). No RCTs have been published on the efficacy of CO2 laser therapy (Lipke 2006) but in two case series a cure rate of 64% to 71% at 12 months is reported (Sloan et al. 1998; Street und Roenigk 1990). For periungual and subungual warts, which can be difficult to clear with other treatments, this method may be particular useful (Sterling et al. 2001). In one study, promising results were found in immunosuppressed patients with recalcitrant warts, where 12 of 13 patients experienced dramatic improvement after 1 to 3 treatment sessions and in six of these patients full remission was reported (Läuchli et al. 2003).

Surgical Removal by Curettage or Cautery Surgical removal of warts by curettage followed by cautery, is one of the oldest methods of treatment (Lynch et al. 2014; Mulhem und Pinelis 2011) and it still widely practice but no RCT has been published (Lipke 2006; Sterling et al. 2001). Successful removal is reported in 65% to 85% of patients but there is a high risk of scarring after these procedures and recurrence rates may be as high as 30% (Sterling et al. 2001; Mulhem und Pinelis 2011).

A variety of other destructive methods, with limited or conflicting evidence are available, including Yttrium/Aluminum/Garnet (YAG) laser, pulse dye laser, photodynamic therapy, , and trichoroacetric acid (Sterling et al. 2014).

The best approach to the treatment of severe or recalcitrant warts is still unclear. Therapies with antiproliferative agents such as 5-fluorouracil, intralesional , retinoids or and with contact allergens, intralesional Interferon, intralesional or candida antigens, cimetidine, zinc sulphate or imiquimod may be beneficial (Sterling et al. 2014; Sri et al. 2012; Lipke 2006). For the purpose of this study I will review the treatment modalities which have been prescribed in my patient cohort:

22 Introduction

1.5.3.2 Antiproliferative Agents (Retinoids) Retinoids disrupt epidermal proliferation and differentiation and so can thereby reduce the volume of the wart without a direct antiviral effect (Lipke 2006; Sterling et al. 2001; Sterling et al. 2014). Their main adverse-effect, both from topical and systemic administration, is skin dryness and irritation, which may influence the skin´s immune response and add to the drug’s immunomodulatory effect (Sterling et al. 2014).

There are only a limited number of small trials or case reports assessing the effectiveness of retinoids in the treatment of warts. For example, in a randomised controlled pilot study, 25 children with plantar warts were treated with 0.05% tretinoin cream once daily for 6 weeks. The author reported a clearance rate of 85% compared to 32% in the control group of 25 untreated children (Kubeyinje 2009). In another small study with 23 organ transplant patients, topical retinoids were applied. A reduction of 45% of the number of warts was seen after 3 months of treatment compared with 23% of the placebo treated arm, but the effect was less 3-month post treatment with 29% and 19% respectively (Euvrard et al. 1992). Gelmetti (1987) treated 20 children with extensive warts with a daily dose of 1mg per kg per day of etretinate orally for up to 3 months, of whom 16 showed complete regression. A more recent study assessed the benefit of low dose oral isotretinoin (0.5mg/kg/day) for 2 months in the treatment of recalcitrant facial plane warts in 26 patients; 19 showed a complete regression of whom 78% were still clear of warts 4-months post treatment (Al-Hamamy et al. 2012).

1.5.3.3 Immunomodulators

Imiquimod Imiquimod 5% cream is an immune modifying drug. Its mechanism of action is not completely understood but as an agonist of TLRs it may act by enhancing both innate and cell mediated immune responses by stimulating release and synthesis of cytokines, including IFN-α, IL-1, IL-6 and IL-12 and TNF-α (Harwood et al. 2005; Lipke 2006; Sterling et al. 2014). Imiquimod is an established treatment for anogenital warts and its approval has more recently been extended for the treatment of actinic and basal cell carcinoma (Wagstaff und Perry 2007).

Although there are no RCTs studying the effect of topical imiquimod on recalcitrant cutaneous warts (Bacelieri und Johnson 2005; Lipke 2006; Sterling et al. 2014), numerous open-label studies show different success rates but overall encouraging results in both immunocompetent (Micali et al. 2003; Grussendorf‐Conen und Jacobs 2002) and immunosuppressed (Harwood et al. 2005; Hengge et al. 2000) patients.

In a small study cure rates of 88.9% were observed when applied twice daily for a mean duration of 5.8 months (Grussendorf‐Conen und Jacobs 2002) with similar clearance rates of 80% in patient’s recalcitrant subungual and periungual cutaneous warts (Micali et al. 2003).

23 Introduction

In a larger cohort, in which imiquimod was applied once daily for 5 days per week, 50% clearance of warts was seen in 56% of 50 patients, of whom 15 were immunosuppressed (Hengge et al. 2000).

A more recent study assessed 16 weeks imiquimod compared to placebo (Petrolatum), with 20 patients in each treatment arm; imiquimod led to a complete resolution of warts in 40% and to a partial resolution in 30% of patients, while in the placebo group results of 0% and 20% were found respectively (Kim et al. 2013).

Slightly lower success rates were found by Harwood et al (2005) who assessed the therapeutic effect of imiquimod in 15 immunosuppressed patients of whom 36% experienced a more than 30% clearance of their persistent warts.

In addition, a review by Sterling et al (2014) states that numerous case reports suggest the benefit of imiquimod in both immunocompetent and immunocompromised patients.

Vaccine To date there are no published clinical trials investigating the quadrivalent HPV vaccine as a treatment modality for recalcitrant warts (Cyrus et al. 2015). However, case reports have described the resolution or substantial regression of recalcitrant warts following quadrivalent HPV vaccination, injected in 3 doses at 0, 2 and 6 months (Kreuter et al. 2010; Landis et al. 2012; Silling et al. 2014; Venugopal und Murrell 2010). These studies included a 59-year old healthy woman (Landis et al. 2012), a developmentally delayed otherwise healthy, 31-year- old man (Venugopal und Murrell 2010), a 41-year old woman with post chemotherapy developed secondary immunodeficiency (Silling et al. 2014) and a 41-year old woman diagnosed with warts, immunodeficiency, lymphedema, and anogenital dysplasia (WILD) syndrome (Kreuter et al. 2010). All patients presented with numerous, widespread, longstanding cutaneous warts on their hands or feet which had been treated unsuccessfully with numerous therapies prior to the vaccine. Spontaneous resolution of warts, independent of treatment, cannot be excluded in these patients, however, HPV vaccines have been proven to provide cross-protection against related HPV strains (Ault 2007) which may be a possible mechanism for resolution of their long standing, recalcitrant warts (Venugopal und Murrell 2010; Landis et al. 2012). Further research is needed to fully clarify the cross- protective properties of the quadrivalent HPV vaccines (Ault 2007) but also clinical trials investigating HPV vaccines as a treatment modality for recalcitrant warts (Sri et al. 2012).

24 Study Aims

2 Study Aims

This study looked at patients with severe viral skin infections, referred to immunology at the Royal Free Hospital (RFH) in London with the following aims:

 To describe the clinical and immunological features of these patients.  To determine whether these patients have an underlying PID.  To understand whether and how viral skin infections impact QoL in affected individuals.  To explore the treatment modalities and responses in these patients.

25 Materials and Methods

3 Materials and Methods

3.1 Recruitment of Participants

All patients over 18 years of age with a diagnosis of persistent cutaneous warts and/or recurrent herpes infections and treated by the Immunology team at the RFH in London were invited to participate in this study.

Additional inclusion criteria for the patients with warts were:

 More than 5 warts or warts greater than 2cm in diameter with a duration of over 2 years.  Periungual, palmar or plantar warts.  Not on immunosuppressive therapy.  HIV negative.  No Severe combined immunodeficiency (SCID)  No development of warts post chemotherapy  No development of warts post post transplantation

The inclusion criterion for the patients with recurrent herpes infections was more than 6 outbreaks of the herpes infection per year and/or severe herpes infections.

3.2 Ethical Approval and Consent Forms

All patients who consented to Version 8 of the project ‘The Investigation of the cause and the mechanism of Immunodeficiency diseases’ were eligible to take part in the study. This consent form includes the use of blood and skin samples and questionnaires. This project was approved by the Research Ethics Committee and the Research and Development office at the RFH National Health System (NHS) Foundation Trust in March 2015. In addition, patients who had medical photographs taken consented that their photographs may be used for teaching and in this dissertation.

3.3 Study Design

3.3.1 Identification of Patients

The departmental database was searched by David Guzman (IT, Immunology department, RFH, London) for patients with the words “warts”, “HSV”, “cold sores”, “VZV”, and/or “shingles” in their diagnosis. In addition, letters in the “Immunology share-drive” from 169 CVID patients attending the RFH Immunology Department (search date 5th May 2015), and

26 Materials and Methods letters from patients that attended the departmental Viral Skin Infection (VSI) Clinic from 24th October 2014 to 28th August 2015 were searched for the terms “warts”, “HSV”, “cold sores”, “VZV”, and/or “shingles”. To avoid missing patients, the clinic list which is available one week in prior to each clinic was screened for new patients or patients that were missed in the others searches.

3.3.2 Approaching Patients

Patients were invited to participate in the study on the day of their clinical appointment either by myself, by the departmental research nurse, or the research assistant.

3.3.3 Data Collection

When patients confirmed that they were happy to take part in the study, they were given (depending on their diagnosis) a questionnaire for patients with persistent warts or a questionnaire for patients with recurrent herpes infections (see section 3.3.4) to be completed while waiting to be seen by the doctor. Table 1 lists the blood values measured on the patient’s blood samples.

27 Materials and Methods

Table 1: Blood values measured on the patient’s blood samples

Immunoglobulin (Ig) Immunoglobulin Subclasses (Ig s/c) IgM IgG1 IgG IgG2 IgA IgG3

Immunodeficiency Panel (IP) T Cell Proliferation Essay Lymphocytes count Spontaneous Proliferation CD3 PHA stimulation CD4 a- CD3 stimulation CD8 a- CD3/CD28 stimulation CD19 NK cells CD4/CD8 Ratio % of CD3+ T cells % of Lymph % of CD4+ T cells % of Lymph % of CD8+ T cells% of Lymph % of CD19+ B cells % of Lymph % of CD16+CD56 NK cells % of Lymph

T Cell Phenotyping (TCID) Full Blood Count (FBC) % of CD3+ CD4+ (CD4 T Cells) Neutrophils % of CD3+ CD8+ (CD8 T Cells) Lymphocytes % of CD4+ CD5RO+ (CD4 Memory) % CD4+ Monocytes % of CD4+ CD45RO+ CXCR5+ (TFH) % CD4+ Eosinophils % of CD4+ CD45RO+ CXCR5+ (TFH) % Memory Basophils % of CD4+ CD45RO+ CXCR5+ (TFH) % CD8+ % of CD8+ CD27- CD28- (Late Ef) % CD8+ % of CD8+ CD27+ CD28- (Ef) % CD8+ % of CD4- CD8- (Double-ve T) % TCRaB+ % of CD4+ CD45RA+ (CD4 Naive) % CD4+ % of CD4+ CD45RA+ CD31+ (RTEs) % CD4+ % of CD4+ CD45RA+ CD31+ (RTEs) Naïve % of CD45RO+ CD127low CD25+ % CD4+ % of CD45RO+ CD127low CD25+ Memory

B Cell Phenotyping (CVID) Serum Level Of: % of CD19+ B Cells Tetanus Toxoid specific antibodies % of CD27- IgD+ (Naïve) % CD19+ Pneumococcal C.P. specific antibodies % of CD27+ IgD+ (IgM Memory) % CD19+ Hib specific antibodies % of CD27+ IgD- (Sw. Memory) % CD19+ HIV 1 & 2 Antibodies % of CD27+ IgD- (Sw. Memory) % PBL HSV 1 & 2 IgG Antibodies % of CD21- CD38- % CD19+ VZV IgG Antibody % of Transitional B cells, % of Plasmablasts

Autoimmunity Antinuclear antibodies (ANA)

Any missing values were requested by the attending physician, on the day of the appointment. In addition, patients were asked whether they were happy to give additional 30ml blood for biobanking.

28 Materials and Methods

For patients with persistent warts, scrapings of their warts were taken with a sterile disposable scalpel (Swann-Morton) and stored in a Dermapak Type 4 to be send to Dr. Karin Purdie (Cancer Research, UK Skin Tumour Laboratory Centre for Cutaneous Research, Blizard Institute Queen Mary University of London) for HPV typing. Medical photographs of the warts were taken at the RFH Medical Illustration Unit.

3.3.4 Questionnaires

Two questionnaires were designed by myself with help of Dr. Siobhan Burns and Professor Grimbacher; one questionnaire for patients with persistent warts and one for patients with recurrent herpes infections (see Appendix). The questionnaires were designed in order to get a precise understanding of how the patient´s viral infections present, how different treatment influences the infection, and to screen patients for other medical conditions or medications that may have an influence to the viral phenotype. The questionnaires were given to all consented patients with HPV or herpes virus infections visiting the RFH between October 2015 and April 2016.

3.3.4.1 Questionnaire for patients with recurrent herpes infection In questions 1 to 6 and 12 to 15 the patients were asked about the clinical presentation of their herpes infection, including onset of their symptoms, location, trigger, frequency of outbreaks before commencing treatment and complications.

In questions 7 to 11 patients were asked about their treatment including name of antiviral medication, the duration of the treatment, its dose and effectiveness.

In questions 16 to 24 patients were asked about their medical history, including diagnosis of primary immunodeficiency, autoimmune diseases, allergies, cancer and HIV, family history of recurrent herpes infection and drug history of immunosuppressive medication or antibiotics.

3.3.4.2 Questionnaire for patients with persistent skin warts In questions 1 to 3 patients were asked about the clinical presentation of their warts, including persistence, location and any lifestyle difficulty caused.

In question 5 patients were asked about their treatment, including topical and oral medication, HPV vaccine, its dose and effectiveness.

Questions 4 and 6 to 15 were similar to the question about the patient’s medical history in the questionnaire for patients with recurrent herpes infection. In addition, patients were asked if they had a bone marrow/HSCT as the persistence of chronic, severe HPV infections have been reported in a subset of SCID patients post transplantation (Laffort et al, 2004).

In question 18 patients were given the choice of consenting to the use of photographs of their warts in my work for illustration.

29 Materials and Methods

3.3.5 Dermatology Quality of Life Index

The Dermatology Life Quality Index (DLQI) is one of the most widely used dermatology- specific quality of life (QoL) outcome measure (Basra et al. 2008), used for many different skin conditions including in patients with warts (Lewis und Finlay 2004).

The DLQI consists of 10 questions measuring the impact the had on the patient´s QoL over the last week. The questions touch aspects such as symptoms, emotions, daily and social activities, sport, work, personal relationships and side-effects of treatment. Each question has five possible answers: ‘‘not at all’’, ‘‘a little’’, ‘‘a lot’’, ‘‘very much’’, and “not relevant”, scored as 0, 1, 2, 3 and 0 respectively, giving a minimum score of 0 for no impairment and a maximum score of 30, representing maximum impairment on QoL (Lewis und Finlay 2004).

3.3.6 Isolation of PBMC from Whole Blood

The reagents needed for this protocol are shown in Table 2.

Table 2: Reagents for the isolation of the PBMC from whole blood protocol

Reagent Volume Cell culture Phosphate Buffered Saline (PBS), pH7.4 (Sigma Aldrich) For dilution Histopaque® 1077 (Sigma Aldrich) 15 ml Trypan blue exclusion For staining

Peripheral Blood Mononuclear Cells (PBMC) were isolated by Histopaque® density gradient centrifugation. Whole blood was diluted 1:1 with phosphate-buffered saline (PBS) and then 20ml to 35ml of diluted cell suspension was layered over 15ml of Histopaque® in a 50ml conical tube. The suspension was centrifuged at 2,300 RPM for 25 minutes at 20°C in a swinging-bucket rotor without brake. The top layer of plasma was aspirated off leaving the PBMC layer (lymphocytes, monocytes, and thrombocytes) undisturbed at the interphase. The PBMC layer was transferred to a new 50ml falcon tube, making sure that the Histopaque® layer was left undisturbed. The 50ml falcon tube was filled with PBS, mixed and centrifuged at 1,800 RPM for 10 minutes at 20°C. Afterwards the supernatant was poured off. To remove platelets, the PBMCs were washed in 50ml of PBS and centrifuged at 1,300 RPM for 10 minutes at 20°C. The supernatant was then removed without disturbing the PBMC pellet. This procedure was then repeated to ensure all platelets were removed. Finally, the PBMCs were stained with trypan blue exclusion, counted using a Haemocytometer and frozen within 24 hours for storage.

30 Materials and Methods

3.3.6.1 Cryopreserving PBMCs The reagents needed for this protocol are shown in Table 3.

Table 3: Reagents for cryopreserving PBMCs protocol

Reagent Volume Filtered HI Foetal Calf Serum (FCS) (Gibco) 900 µl Dimethylsulfoxid (DMSO) (Sigma Aldrich) 100 µl

In preparation for cryopreserving PBMCs, fresh filtered HI foetal calf serum (FCS) was defrosted and cryotubes and dimethylsulfoxid (DMSO) were placed on ice at 4oC. The PBMCs were pelleted at 1500 RPM for 5 minutes and the supernatant discarded. Then the cells were re-suspended with 900µl of FCS and 100µl of DMSO and mixed in a cryotube which was placed immediately on ice. To achieve a rate of cooling very close to -1°C/minute, the cryotube was transferred to “Mr. Frosty” and placed in -80oC freezer for 24 hours. After 24 hours at -80oC the cryotube was transferred to liquid nitrogen for storage.

3.3.7 HPV Phenotyping

3.3.7.1 DNA Isolation

The reagents needed (supplied by Qiagen QIAamp DNA micro kit, Catalog No. 56304) are shown in Table 4.

Table 4: Reagents for DNA isolation from wart scrapings protocol

Reagents Volume Buffer ATL 180 µl Proteinase K 20 µl Buffer AL 200 µl (96-100%)- not provided in the kit 200 µl Buffer AW1 (concentrate) 500µl Buffer AW2 (concentrate) 500µl Buffer AE 100 µl

DNA isolation from wart scrapings was carried out in four steps (see flowchart) with the Qiagen QIAamp DNA micro kit (QIAGEN GmbH, Hilden, Germany). To lyse the sample DNA, the scrapings were transferred to a 1.5ml micro-centrifuge tube. Immediately, 180µl ATL Buffer was added and equilibrated to room temperature (15 to 25°C), then mixed with 20µl proteinase K by pulse-vortexing for 15 seconds. Then the 1.5ml tube was placed in a thermomixer and incubated at 56°C overnight until the sample was completely lysed. To allow optimal binding of sample DNA to the QIAamp MinElute column membrane, first 200µl Buffer A, then 200µl ethanol were added (each time mixed by pulse-vortexing for 15 seconds) and incubated for 5 minutes at room temperature. Then the lysate was transferred onto a QIAamp MinElute column (in a 2ml collection tube) where DNA was adsorbed onto a

31 Materials and Methods silica-gel membrane as the lysate is drawn through by centrifugation. The lysate was centrifuged at 800 RPM for 1 minute and to wash away any residual contaminants, first 500µl Buffer AW1, then 500µl Buffer AW2 was added to the QIAamp MinElute column and each time centrifuged at 8,000 RPM for 1 minute. To completely dry the membrane, the QIAamp MinElute column was centrifuged a last time at 14,000 RPM for 3 minutes. After each centrifuging the column was placed in a clean 2ml collection tube and the collection tube with the flow-through was discarded. Afterwards the QIAamp MinElute column was placed in a clean 1.5ml micro-centrifuge tube. To elute the bound DNA from the column, 20 to 100µl Buffer AE was applied to the center of the membrane, incubated at room for 1 minute and centrifuged at 14,000 RPM for 1 minute.

3.3.7.2 β-HPV Typing To genotype patient’s warts for β-HPV the broad spectrum PCR (PM-PCR) in combination with a reverse hybridization assay (RHA) (PM-PCR RHA method) was used. This PM-PCR RHA method is highly sensitive, specific and reproducible for the detection of β-HPV DNA and reliable in identifying β-HPV genotypes in fresh patient material (Koning et al. 2006). The kit was obtained from Dissay B.V. (a Hi-tech biotech-company based in Rijswijk, the Netherlands) which allows fast and simultaneous identification of 25 β-HPV types, namely HPV types 5, 8, 9, 12, 14, 15, 17, 19, 20, 21, 22, 23, 24, 25, 36, 37, 38, 47, 49, 75, 76, 80, 92, 93 and 96 types (Koning et al. 2006).

This method consists of a PCR reaction to amplify the sample DNA, using biotinylated primers that target the relatively well conserved E1 open reading frame of HPVs, generating a biotinylated product of 117 base pair length from the E1 region. This reaction is followed by RHA which allows simultaneously identification of 25 β-HPV genotypes (Koning et al. 2006).

The PCR was carried out according to the manufactures protocol. Table 5 shows the reagents and cycling parameters used in this protocol.

32 Materials and Methods

Table 5: A.PCR reaction constituents and B. Cycling conditions

A. PCR Reagents B. Cycling Conditions Reagents Concentration Action Temperature Time Isolated DNA 10 µl Taq Activation 94 °C 9 minutes MgCl 2.5 mM 2 35 cycles: 1x GeneAmp PCR Buffer II Diluted from 10x concentration Denaturation 94 °C 30 seconds

dNTP 0.2 mM Annealing 52 °C 45 seconds Elongation 72 °C 45 seconds AmpliTaq Gold DNA 1.5 U Final Elongation 72 °C 5 minutes PM primer mix 10 µl

The RHA was performed following the kit protocol. Briefly, 10µl of Denaturation solution was transferred into each test trough in which 10µl 3B-Buffer and 10µl of amplified PCR product was added and incubated at 20 to 25°C for 5 minutes. Then 2ml prewarmed Hybridization Solution (45mM Na citrate, 450mM NaCl and 0.1% sodium dodecyl sulfate) and a nylon membrane strip, carrying probes for 25 HPV types were added to the denaturated samples and incubated for 60 *minutes in a shaking water bath at 50°C to allow hybridization. Afterwards the strips were washed three times with 2ml stringent wash solution. Twice for 20 seconds and the third time for 30 minutes incubated in a shaking water bath at 50°C. For the colour development, the strips were put through two cycles of being rinsed and then incubated at 20 to 25°C for 30 minutes on a shaker. During the first cycle the strips were rinsed twice for 1 minute with 2ml of Rinse solution (concentrated rinse solution 1:5 in water) before incubated in 2ml of alkaline phosphate-streptavidin conjugate. During the second cycle the strips were rinsed twice for 1 minute with 2ml Rinse solution (concentrated conjugate, 1/100 on conjugate Diluent), and once for 1 minute with 2ml of substrate buffer. Then substrate solution (5-bromo-4cloro-3indolyphosphate and nitro blue tetrazolium (BCIP/NBT) was added into each test trough before incubation took place. The reaction was stopped by two cycles of 3 minutes incubation in 2ml distilled water at 20 to 25°C. The strips were dried and visually interpreted. If the PCR amplimers were homologous to any of the probes of the 25 HPV types, biotinylated DNA would bound to them. This was visually seen as purple bands on the strips caused by the streptavidin-conjugated alkaline phosphatise, given to bind the biotin, converting BCIP/NBT chromogen to give a purple colour.

3.3.7.3 α-HPV Typing To genotype patient’s warts for cutaneous α-HPV a modified degenerate nested PCR approach, first described by Harwood et al (1999) was used. This PCR method is highly sensitive and has the capacity to detect a broad spectrum of cutaneous, mucosal and EV HPV types (Harwood et al. 1999). PCR amplification and sequence analysis were performed exactly as described in the original paper by Harwood et al (1999). Here, to increase the

33 Materials and Methods sensitivity and specificity for detection of the cutaneous α-HPV types, the specific primer pairs were used (Table 6).

Table 6: Primer pairs use to detect α-HPV types in the PCR method

Primer Pair For Detection of HPV CN3F-CN3R 3, 10, 28, 29, 77 CN2F-CN2R 2, 27, 57 CN1F-CN1R 1, 41, 63 HVP2-B5 detection of HPV from all groups with exception of 4, 48, 50, 60, 65

3.4 Data Analysis

All data collation and analysis was carried out using IBM SPSS Statistics (release 24.0.0.0) software (except for one Microsoft Excel graph - Figure 2). The following sections summarise the methodology used to create the various tables and graphs in SPSS in this study (with instructions on where to ‘click’ in SPSS in brackets).

All tables were created either through the custom tables dialog in SPSS Statistics (main ribbon > Analyze > Tables > Custom Tables), and the graphs either through the main chart builder dialog (main ribbon > Graphs > Chart Builder), the legacy chart builder (main ribbon > Graphs > Legacy Dialogs) or directly from tables in the output viewer.

3.4.1 HPV Type Analysis

Figure 1: Opened the custom table dialog and created a table with all β- and α-HPV types in rows and the default count statistic in the column, excluding the no value (Categories and Totals > Exclude no value); in the output viewer selected the table and created a bar graph (right click > Create Graph > Bar).

3.4.2 DLQI Analysis

Figure 3 and Figure 4: Opened the custom table dialog and created a table with the DLQI scores in the rows and warts/herpes outbreak/herpes no outbreak in the columns and then selected the required summary statistics for the scores (Summary Statistics > Count/Maximum/Minimum/Median/Mean); created a simple boxplot of scores against warts and herpes patients.

Figure 5 to Figure 9: Opened the custom table dialog and created a table with all the DLQI questions (Q1 to Q10) in rows and warts/herpes outbreak/herpes no outbreak in the columns, excluding the different answers for each graph in turn (Categories and Totals > Exclude not at all/a little/a lot/very much/not relevant); in the output viewer selected the table,

34 Materials and Methods transposed rows and columns (main ribbon > Pivot > Transpose Rows and Columns), and then created a bar graph (right click > Create Graph > Bar).

3.4.3 Blood Values Analysis

Figure 10 to Figure 22: Opened the custom table dialog and created a table with the blood values in the rows and the warts and herpes groups in the columns, and then selected the required blood values statistics (Summary Statistics > Count/Maximum/Minimum/Median/Mean); created a simple boxplot of the warts and herpes groups against the blood values.

3.4.4 Treatment

Figure 28: Opened the custom table dialog and create a table with the only acute medication and prophylactic medication in the rows and the required category in the column (Count statistic only or Virus Type with Count statistic), then selected the required variable for the row category (Categories and Totals > exclude ‘no’); in the output viewer selected the table, and created a bar graph (right click > Create Graph > Bar).

Figure 29: Created a simple bar graph with acute medication success on the x-axis and the default count statistic on the y-axis.

Figure 30A: Created a clustered bar graph with virus type on the x-axis and the default count statistic on the y-axis and clustered on prophylactic medication type.

Figure 30B: Opened the custom table dialog and created a table with symptoms less severe, lesion smaller, duration shorter and frequency outbreaks less in the columns and the default count statistic in the row, then changed the category position to column labels in rows; in the output viewer selected the table, and created a bar graph (right click > Create Graph > Bar).

Figure 31: Opened the custom table dialog and created a table with outbreaks (pre- and/or on prophylactic medication) in rows and the default count statistic in the column, then selected the required additional statistics (Summary Statistics > Count/Maximum/Minimum/Median/Mean); created another table with outbreaks (pre- and/or on prophylactic medication) in rows and virus type in the column and then selected the required statistics for the outbreaks (Summary Statistics > Count/Maximum/Minimum/Median/Mean); created a simple boxplot using the legacy chart builder dialog (summaries of separate variables) of the frequency of outbreaks pre- and on prophylactic medication; created simple boxplots using the main chart builder with the frequency of outbreaks pre- and on prophylactic medication on the y-axis and virus type on the x-axis.

35 Materials and Methods

Figure 32: Created a simple bar graph with outbreaks on prophylactic medication on the x- axis and the default count statistic on the y-axis; created a clustered bar graph with virus type on the x-axis and count on the y-axis, clustered on outbreaks on prophylactic medication.

Figure 33: Created a simple bar graph with patients on Zostavax medication on the x-axis and the default count statistic on the y-axis.

Figure 23: Opened the custom table dialog and created a table with the treatment modalities in the rows and the outcomes in the columns with default count statistics; in the output viewer selected the table, transposed rows and columns (main ribbon > Pivot > Transpose Rows and Columns), then created a bar graph (right click > Create Graph > Bar).

Figure 24: Created clustered bar graphs using the main chart builder dialog with PID on the x-axis and the default count statistic on the y-axis and clustered on the required treatment modality.

36 Results

4 Results

The departmental data base search revealed 121 patients with the words “warts”, “HSV”, “cold sores”, “VZV”, and/or “shingles” in their diagnosis. In addition, in letters from patients that attended the VSI Clinic and in patient´s lists of the VSI clinics, another six and eight patients were found respectively. Out of these 135 possible candidates, 27 patients were to date already discharged, 14 patients were symptom free, 38 patients did not attend their appointment, 14 patients did not meet the inclusion criteria, and three patients did not consent to take part. Of the 39 patients that finally consented to this study, 18 presented with persistent warts and 21 with recurrent herpes infections.

4.1 Characteristics of Patients with Warts

The characteristics of the cohort of patients with warts are shown in Table 7. This cohort consisted of eleven (61%) male and seven (39%) female patients. Nine patients had CVID (warts CVID group), one was diagnosed with a DOCK8-deficiency, one with IL7Ra deficiency (very unusual late presentation), and no primary immunodeficiency (no-PID) was found in the seven remaining patients by blood values (n=7) and whole genome sequencing analysis (n=3). The age of the patients ranged from 26 to 67 years (mean of 46 years), with only two patients under the age of 34 and with a slightly higher mean in the warts CVID group (50 years) compared to the warts no-PID group (43 years). Only one patient (patient with ID number 10), a CVID patient, had a family history of warts. Six (33%) patients had plantar warts only, five (28%) patients had common warts (mainly hand warts) only, one patient (6%) had plane warts and six patients (33%) had a combination of different types. The duration of the patient’s warts had a large range of three to 40 years (mean 19 years) with the majority of patients (83%) having had their warts for more than five years. Four patients (22%) had less than five warts, five patients (28%) had five to 20 warts and nine patients (50%) had more than 20 warts with the DOCK8- and IL17Ra-deficiency patients showing the highest number and severity of warts. Warts caused inconvenience (pain or irritation during daily living activities such as walking and driving) in ten patients (56%), while eight patients (44%) denied any inconvenience.

37 Results

Table 7: Characteristics of patients with warts

All (n=18) CVID only (n=9) No-PID only (n=7) Defined PID (n=2) 1 2

Sex No. % No. % No. % Female 7 38.9 3 33.3 3 42.9 Female Male 11 61.1 6 66.7 4 57.1 Male Age 18-34 years 2 11.1 0 0 1 14.3 31 > 34 years 16 88.9 9 100 6 85.7 47 Range 26- 37-67 26-60 67 Mean 46.3 50.3 43.4 Family History Yes 1 5.6 1 11.1 0 0 No 17 94.4 8 88.9 7 100 No No Location of warts Plantar warts*1 only 6 33.3 3 33.3 3 42.9 Common warts*2 only 5 27.8 4 44.5 1 14.25 Plane warts*3 only 1 5.6 0 0 1 14.25 Combination of warts 6 33.3 2 22.2 2 28.6 Plantar, Common, common plane & & plane genital Duration of warts < 6 months 0 0 0 0 0 0 6 to 24 months 0 0 0 0 0 0 > 2 years 3 16.7 1 11.1 2 28.6 > 5 years 15 83.3 8 88.9 5 71.4 7 35 Exact duration of warts Range (years) 3-40 3-40 3-26 Mean (years) 18.8 15.3 13.4 Number of warts < 5 warts 4 22.2 4 44.5 0 0 5 to 20 warts 5 27.8 2 22.2 3 42.9 > 20 warts 9 50.0 3 33.3 4 57.1 >20++ >20++ Warts cause inconvenience (pain or irritation during daily living activities) Yes 10 55.6 2 22.2 5 71.4 Yes No 8 44.4 7 77.8 2 28.6 No 1 DOCK8 Deficiency; 2 IL17-receptor α deficiency; *1 warts on soles of the feet; *2 warts in other location than soles of the feet, mostly hand warts; *3 warts on the face

4.2 Characteristics of Patients with Herpes

The characteristics of the herpes cohort are shown in Table 8. The herpes cohort consisted of five male (24%) and 16 female (76%) patients. Ten patients were diagnosed with HSV, seven with VZV, and in four patients a definite diagnosis between HSV and VZV was not made. Note that herpetic lesions were not always swabbed (only five patients were swab positive for HSV and none for VZV) so that diagnosis was based on clinical judgement in most patients and VZV patients may have been mislabelled HSV patients. None of these

38 Results patients were diagnosed with a PID. The age of the patients ranged from 20 to 74 years (mean 44 years) with a lower mean in the HSV group (38 years), a higher mean in the VZV group (52 years) and the same mean in the VZV or HSV group (44 years). A family history of recurrent herpes infection was known in five patients (24%). The age of onset was much lower in the HSV group (mean 22 years) compared to the VZV and HSV or VSV groups (mean 32 to 35 years). Ten patients (48%) experienced an event that set off their herpes infection (see Table 9) and 15 patients (71%) reported trigger factors which cause herpetic infection reactivation, with physical or emotional stress being the most common (67%), followed by other infections and menstruation (27%) (see Table 10). Seventeen patients (81%) took prophylactic antiviral medication, while the remaining four patients (19%) only took antiviral medication during an acute episode, commencing when first symptoms were felt. Prior to commencing treatment, patients experienced a mean of 14 episodes per year (range 3 to 30 episodes), that reduced to a mean of 4.5 episodes per year (range 0 to 24) after being on prophylactic antiviral medication. Eighteen patients (86%) experienced prodromal symptoms (70% of the HSV and 100% of the VZV group). Genital herpes was experienced most frequently (52%), closely followed by herpes orolabialis (48%) and facial herpes (38%), with the majority of reactivations occurring at the same location (48%) or two different locations (33%). Herpes caused inconvenience in 18 patients (86%), and complications in seven patients (33%) (see Table 9).

39 Results

Table 8: Characteristics of patients with herpes

All (n=21) Only HSV (n=10) Only VZV (n=7) VZV or HSV (n=4)

Sex No. % No. % No. % No. % Female 16 76.2 8 80.0 5 71.4 3 75.0 Male 5 23.8 2 20.0 2 28.6 1 25.0 Age Range (years) 20-74 20-65 31-74 33-50 Mean (years) 43.7 37.9 52 43.7 Family History Yes 5 23.8 3 30.0 2 28.6 0 0 No 16 76.2 7 70.0 5 71.4 4 100.0 Age of onset of recurrent HSV infections Mean (years) 39.3 22.2 34.7 32.3 Range (years) 2-69 2-49 20-69 18-44 Event that set off the Herpes infections Yes 10 47.6 4 40.0 3 42.9 3 75.0 No 11 52.4 6 60.0 4 57.1 1 25.0 Trigger factors causing herpetic infection reactivation Yes 15 71.4 7 70.0 6 85.7 2 50.0 No 6 28.6 3 30.0 1 14.3 2 50.0 Antiviral medication

Prophylactic 17 81.0 8 80.0 5 71.4 4 100.0 Acute 4 19.0 2 20.0 2 28.6 0 0 Number of episodes per year pre-medication (n=20***) Range 2-30 2-30 3-24 12-24 Mean 9.2 8.5 9.1 17 Number of episodes per year (pre-prophylactic medication; n=16***) Range 3-30 4-30 3-24 12-24 Mean 14.3 12.4 14.6 17 Current episodes per year (with prophylactic medication, n=16***) Range 3-24 1-24 0-1 0-2 Mean 4.5 9.7 0.4 0.5 Prodromal symptoms *(e.g. tingling, pain, burning, or itching at the site of reactivation) Yes 18 85.7 7 70.0 7 100.0 4 100.0 No 3 14.3 3 30.0 0 0 0 0 Location of reactivation Orolabial 10 47.6 9 90.0 1 14.3 0 0 Facial 8 38.1 4 40.0 4 57.1 0 0 Genital 11 52.4 4 40.0 3 42.9 4 100.0 Herpetic 0 0 0 0 0 0 0 0 Herpes Keratitis 5 23.8 2 20.0 3 42.9 0 0 Other 2 9.5 1 10.0 1 14.3 0 0 Number of different reactivation locations** 1 only 10 47.6 3 30.0 3 42.9 4 100.0 2 7 33.4 4 40.0 3 42.9 0 0 3 4 19.1 3 30.0 1 14.2 0 0 Herpes virus cause inconvenience (pain, irritation or difficulties during daily living activities) Yes 18 85.7 7 70.0 7 100.0 4 100.0 No 3 14.2 3 30.0 0 0 0 0 Herpes virus complications Yes 7 33.3 3 30.0 2 28.6 2 50.0 No 14 66.7 7 70.0 5 71.4 2 50.0 ** one location refers to orolabial or facial or; ***one patients was excluded from this analysis as he did not provide data about number of episodes pre-prophylactic treatment

40 Results

Table 9: List of herpes virus difficulties and triggers

List of difficulties that patients suffer List of events that set off the due to Herpes virus infection Herpes infection Reduced confidence Chickenpox at age 21 Feeling depressed Stress due to new job Not able or even not allowed to go to work Helicobacter Pylori infection Not feeling like having sex or kissing Work stress Feeling stuck at home Kidney pathology Not able to get out of bed Tonsillitis Lack of stamina Unprotected sex Difficulty to concentrate Periods of being very stressed Difficulty to function normally Holiday in Hungary Overall tiredness Meningitis Managing pain afterwards Premenstrual syndrome Exercising

Table 10: Trigger factors of recurrent herpetic infection (n=15/21)

Trigger Factor Number % Physical or emotional stress 10 66.7 Other infections 4 26.7 Sun exposure 3 20 Menstruation 4 26.7 Change of season 1 6.7 Fatigue 1 6.7

4.3 HPV Type Analysis

Of the 18 patients, 14 provided wart scrapings for HPV typing and six of these provided scrapings from two different sites. Scrapings were taken from a cluster of warts, as warts were too close together to take separate specimens. β-HPV DNA was detected in all wart scrapings while α-HPV DNA was only detected in 70% of wart scrapings. When a patient provided wart scrapings from two anatomical sites, the different HPV types were only counted once for that individual.

The most common β-HPV types were 93 (8 patients), 8 and 24 (7 patients), and 23 (6 patients), with 12, 14, 20, 21, 25, 47 and 75 not identified in any scrapings (Figure 1). The most common α-HPV types were 27 (5 patients), 57 (4 patients), and 28 (3 patients) (Figure 1).

41 Results

Figure 1: β- and α-HPV type virus prevalence

The HPV type distribution by patients is shown in Table 11. In the β-HPV type analysis, multiple types were identified in all patients (except for one patient with only a single β-HPV), ranging from 2 to 8 with a mean of 5 different β-HPV types. In the α-HPV type analysis, two patients had two different α-HPV types in one location (HPV 28 and 57 in the hand sample; 27 and 28 in the plantar foot sample), one patient had two different α-HPV types in two different locations (HPV 28 in the face sample and HPV 27 in the plantar foot sample), seven patients had one single α-HPV type, and the remaining four patients (29%) were negative for α-HPV type DNA.

42 Results

Table 11: HPV type distribution by patient

Patient ID Diagnosis Site β-HPV type(s) α-HPV type(s) 1 CVID plantar foot, periungual 8, 23, 24, 76, 92,93 (n=6) 2 2 CVID plantar foot 9, 19, 23, 37, 96 9 (n=5) 27 3 CVID hand 22, 24, 80 (n=3) 28, 57 4 CVID plantar foot 8, 23, 92, 93 (n=4) 27 5 CVID periungual 8, 15, 17, 19, 49, 76, 93, 96 (n=8)

6 CVID plantar feet 5, 9, 15, 22, 23, 24, 93 (n=7) 27, 28 7 CVID hand 8, 19, 24, 37, 38, 49, 96 (n=7) 27 8 CVID left knee, plantar foot 8, 24, 36, 38, 49, 80, 92, 93 (n=8) 57 10 CVID plantar foot 38, 93 (n=2) 57 12 No-PID face, plantar foot 17, 24, 76, 93 (n=4) 27, 28 14 No-PID right hand, plantar foot 22, 23, 24 (n=4) 57 16 No-PID plantar foot 92

17 No-PID periungual, hand 17, 24 (n=2)

19 No-PID plantar foot 8, 23, 76, 93 (n=4)

In the HPV type distribution by anatomical location (Figure 2 and Table 12), scrapings from the same patient but different sites were counted separately. The most common HPV types in the foot warts were β-HPV 93, 23 and 92 and α-HPV 27 (3) and HPV 57 (2). In the hand warts β-HPV 8 and 24 and α-HPV 27, 28 and 57 was most common and in the periungual warts β-HPV 8, 76 and 93. In the one knee sample β-HPV types 8, 24, 36, 38, 49, 80, 92 and 93 and α-HPV 5 were found and the one face sample β-HPV 24, 76, 93 and α-HPV 28. In addition, β-HPV 5 and 9 were only found in foot warts and β-HPV 36 only in the knee wart.

Figure 2: HPV genotype distribution by anatomical location

43 Results

Table 12: HPV type distribution by location

Patient ID Diagnosis Site β-HPV type(s) α-HPV type(s) 1 CVID plantar foot 8, 23, 24, 76, 92 2 1 CVID periungual 8, 23, 76, 92, 93 2 2 CVID plantar foot 9, 19, 23, 37, 96 27 3 CVID hand 22, 24, 80 57, 28 4 CVID plantar foot 8, 23, 92, 93 27 5 CVID periungual 8, 15, 17, 19, 49, 76, 93, 96

6 CVID plantar foot left 5, 9, 22, 23, 24, 93 27, 28 6 CVID plantar foot right 5, 9, 15, 22, 23, 24, 93

7 CVID hand 8, 19, 24, 37, 38, 49, 96 27 8 CVID plantar foot 38, 92 57 8 CVID knee 8, 24, 36, 38, 49, 80, 92, 93 57 10 CVID plantar foot 38, 93 57 12 No-PID face 24, 76, 93 28 12 No-PID plantar foot 17, 93 27 14 No-PID hand 23, 24 57 14 No-PID plantar foot 22, 23, 24 57 16 No-PID plantar foot 92

17 No-PID hand 8

17 No-PID periungual 24

19 No-PID plantar foot 8, 23, 76, 93

4.4 Dermatology Quality of Life Index (DLQI) Analysis

All 39 patients (21 herpes, 18 warts) completed the DLQI. In the herpes cohort, all patients completed the DLQI in a week in which their herpes virus was not apparent (herpes no outbreak cohort), 10 of the 21 patients completed a second DLQI in a week of a herpes virus infection outbreak (herpes outbreak cohort), and the other 11 patients either did not have an outbreak during the data collection period or forgot to complete a second DLQI during that time.

The “herpes outbreak cohort” had a significantly higher mean score of 15, compared to the warts and herpes “no outbreak cohorts”, with mean scores of 3 and 2 respectively (Figure 3A). Dividing the patients into groups, the no-PID and CVID wart groups had similar low mean scores of 4 and 2 respectively, while in the outbreak groups, the VZV outbreak had a significantly higher mean score of 22, compared to the HSV outbreak group with a mean score of 13 (Figure 3B).

44 Results

Figure 3: A. DLQI scores for warts and herpes patients; B. DLQI scores for warts and herpes patients divided into groups

The herpes no outbreak patients that completed one questionnaire Q (1/1) had a lower mean score of 1, compared to the herpes no outbreak patients that completed a second questionnaire Q (1/2) with a mean score of 3 (Figure 4), suggesting a there is a subtle difference between the groups of no effect at all and small effect on the patient’s life, respectively (see Table 13).

45 Results

Figure 4: Comparison between the herpes no outbreak cohort Q (1/1) and Q (1/2) groups and the herpes outbreak cohort

Table 13: Meaning of DLQI scores

Meaning of DLQI Scores * Score Effect on Patient’s Life DLQI 0-1 No effect at all on patient’s life Band 0 2-5 Small effect on patient’s life Band 1 6-10 Moderate effect on patient’s life Band 2 11-20 Very large effect on patient’s life Band 3 21-30 Extremely large effect on patient’s life Band 4 * Hongbo et al (2005) banding system to aid the interpretation of DLQI scores

Table 14 shows a comparison of individual DLQI scores for the different cohorts. Overall, the herpes outbreak cohort had the highest score in all of the 10 questions. In the warts and the herpes outbreak cohorts, the highest scoring questions related to unpleasant feelings of embarrassment and self-consciousness (Question 2) and to physical symptoms (Question 1). In the herpes no outbreak Q (1/2) group, the highest effect was on work or study (Question 7), followed by emotional and physical symptoms (Question 1 and 2). The herpes

46 Results no outbreak Q (1/1) and Q (1/2) groups scored equally high for physical symptoms (Question 1), but in all other questions, the herpes no outbreak Q (1/2) group report a larger effect on their quality of life. The DOCK8-deficient patient felt that the treatment (Question 10) had the most effect on his quality of life, a question which had low scores in the other groups, and also suffered equally with physical symptoms (Question 1) and working life (Question 7). The IL17Ra-deficient patient had low scores for all questions indicating that no aspect of daily life in particular was affected.

Table 14: Comparison of individual DLQI question scores (min=0, max= 3)

Herpes Herpes no Herpes no DOCK8 IL17Ra Warts outbreak outbreak outbreak deficiency deficiency Q 2/2** Q 1/2 ** Q 1/1* Q Attribute Mean Mean Mean Mean

1 Physical symptoms 0.69 1.9 0.4 0.45 2 0 2 Emotions*** 0.9 2.4 0.5 0.09 1 0 3 Daily activities 0.31 1.3 0 0 1 0 4 Clothing 0.06 1.1 0.3 0.18 1 1 5 Social and leisure 0.25 1.8 0.2 0.18 1 1 6 Sport, exercise 0.19 1.2 0.1 0 1 0 7 Work, study 0.13 1.7 0.7 0.18 2 1 Personal 8 0.43 1.4 0.1 0.09 1 1 relationships 9 Sexual relationships 0 1.7 0.3 0.09 0 1 10 Treatment 0.06 0.5 0.3 0.09 3 0 * Patients who only completed one questionnaire in a no outbreak week ** Patients who completed two questionnaires, one in a no outbreak and one in an outbreak week *** Emotions including feelings of self-consciousness and/or embarrassment

Figure 5 to Figure 9 show the number of patients broken down by group that answered each DLQI question with “not at all” (scored as 0), “a little” (scored as 1), a lot (scored as 2), very much (scores as 3), “not relevant” (scored as 0).

This demonstrates, as shown in Table 14, that patients gave the highest scoring answers to impact on emotions (Question 2), followed by physical symptoms and work/study related questions (question 1 and 7), but also there were some high scoring answers for impact on daily activities, social and leisure, and sexual activity (questions 3, 5 and 9). The herpes outbreak cohort gave the highest scoring answers and were also the least represented in the zero scoring answers The warts cohort gave the second highest scoring answers, while only four patients out of the “herpes no outbreak” cohort gave high scoring answers. None of the 39 patients found impact on physical symptoms (question 1) or emotions (question 2) “not relevant”.

47 Results

Figure 5: DLQI for when patients answer, “not at all”

Figure 6: DLQI for when patients answer, “a little”

48 Results

Figure 7: DLQI for when patients answer, “a lot”

Figure 8: DLQI for when patients answer, “very much”

49 Results

Figure 9: DLQI for when patients answer, “not relevant”

4.5 Blood Analysis

Of the blood values that were measured in each patient (see section 3.3.3), the absolute CD4, CD8, and CD16+CD56 count and the values that were low in five or more patients were analysed in more detail. These values are shown in Table 15, together with the normal healthy control range (HCR) for each. The results are presented in two plots: plot A showing the warts, DOCK8-deficient, IL7Ra-deficient, and herpes patients, and plot B showing the same as subset as in A but with the wart-patients subdivided into CVID and no-PID patients (Figure 10 to Figure 22).

Table 15: Blood values analysed in more detail with normal healthy control ranges

Immunodeficiency Panel (IP) HCR Range CVID B Cell Phenotyping (BCPT) HCR Range Lymphocyte Count 1.0 - 2.8 x 10^9/L % of CD19+ B Cells 4.9 - 18.4 % 7.4 - 32.5 (% Absolute CD4 Count 0.3 - 1.4 x 10^9/L % of CD27+ IgD+ (IgM Memory) CD19+) 6.5 - 29.1 (% Absolute CD8 Count 0.2 - 0.9 x 10^9/L % of CD27+ IgD- (Sw. Memory) CD19+) Absolute CD19 Count 0.10 - 0.50 x 10^9/L Absolute CD16+CD56 Count 0.09 - 0.6 x 10^9/L % of CD19+ B Cells 6 - 19 % of Lymph % of CD16+CD56 NK Cells 7 - 31 % of Lymph T Cell Phenotyping (TCPT) HCR Range Immunoglobulins (Ig) HCR Range

% of CD4+ CD45RA+ (CD4 IgA 0.7 - 4.0 g/L 33 - 66 % CD4+ Naive) IgM 0.40 - 2.30 g/L

50 Results

4.5.1 Immunodeficiency Panel

The mean values of the lymphocyte subsets of the immunodeficiency panel were within the HCR in the herpes and warts cohort (see Figure 10A to Figure 16A). However, when CVID and no defined PID patients were analysed separately, the majority of CVID patients were below the lower limit or just above the lower limit of the HCR for lymphocytes, absolute CD4 count, absolute CD8 count, absolute CD19 count, absolute CD16+ CD56 count and for % of CD19+ B cells (Figure 10B to Figure 15B). Interestingly, with regards to the percentage of CD16+ CD56 NK cells, the majority of no-PID patients had values close to the lower limit of the HCR (Figure 16B).

The DOCK8-deficient patient was low with the lymphocyte count, absolute CD4 count, and at the lower end of the HCR in absolute CD8 count, in the absolute CD19 count, and absolute CD16+CD56 count, but the percentage of B cells and NK cells were within the HCR (Figure 10B to Figure 16B).

The IL7Ra-deficient patient was low in lymphocyte count, absolute CD4 count, absolute CD8 count, and absolute CD19 count.

Figure 10: Comparing lymphocyte count of the warts cohort (divided into CVID and no-PID patients in plot B), DOCK8 and IL7Ra deficient patients and herpes cohort

51 Results

Figure 11: Comparing absolute CD4 count of the warts cohort (divided into CVID and no-PID patients in plot B), DOCK8 and IL7Ra deficient patients and herpes cohort

Figure 12: Comparing absolute CD8 count of the warts cohort (divided into CVID and no-PID in plot B), DOCK8 and IL7Ra deficient patients and herpes cohort

52 Results

Figure 13: Comparing absolute CD19 count of the warts cohort (divided into CVID and no-PID patients in plot B), DOCK8 and IL7Ra deficient patients and herpes cohort

Figure 14: Comparing absolute CD16+CD56 count of the warts cohort (divided into CVID and no-PID patients in plot B), DOCK8 and IL7Ra deficient patients and herpes cohort

53 Results

Figure 15: Comparing % of CD19+ B cells of the warts cohort (divided into CVID and no-PID patients in plot B), DOCK8 and IL7Ra deficient patients and herpes cohort

Figure 16: Comparing % of CD16+ CD56 NK cells of the warts cohort (divided into CVID and no-PID patients in plot B), DOCK8 and IL7Ra deficient patients and herpes cohort

54 Results

4.5.2 T Cell Phenotyping

For the percentage of CD4+ CD45RA CD4 naive T cells, the herpes cohort had a mean value close to the lower limit of the HCR, whereas the warts cohort had a mean below the lower HCR limit (Figure 17A). Subdividing the warts cohort, showed that the CVID group had low numbers of naive CD4 T cells. CVID patients had a mean of 18 percent while the CD4 naive cell count in the no-PID patients were well within the HCR (33-66%). The DOCK8- and the IL7Ra-deficient patient also showed a low percentage of CD4 naive cells with 7 percent and 24 percent respectively.

Figure 17: Comparing % of CD4+ CD45RA+ (CD4 Naive) % CD4+ in the warts cohort (divided into CVID and no-PID patients in B), DOCK8 and IL7Ra deficient patients and herpes cohort

4.5.3 B Cell Phenotyping

Overall, analysing the herpes and the warts cohort, the percentage CD19+ B cells, the percentage of CD27+ IgD+ IgM memory cells, and the percentage of CD27+ IgD- switched memory cells revealed normal values, however the warts cohort was close to the lower limit of the HRC in all three cell types (Figure 18 to Figure 20). This tendency was caused by the CVID patients which had a median below the HCR with regards to the percentage of B cells and all CVID patients, apart from one, had reduced percentages of Switch memory cells (Figure 20B). The percentage of IgM memory cells were at the lower end in CVID patients but notable also in the herpes cohort (Figure 19B).

The DOCK8- and the IL7Ra-deficient patient revealed normal percentage of B cells, of IgM memory cell and off switched memory cells, however the DOCK8-deficient patient revealed

55 Results values at the very low end for the percentage of IgM memory cells and for the percentage of switched memory B cells.

Figure 18: Comparing % of CD19+ B cells for the warts cohort (divided into CVID and no-PID patients in plot B), DOCK8 and IL7Ra deficient patients and herpes cohort

Figure 19: Comparing % of CD27+ IgD+ (IgM Memory) cells % CD19+ cells for the warts cohort (divided into CVID and no-PID patients in plot B), DOCK8 and IL7Ra deficient patients and herpes cohort.

56 Results

Figure 20: Comparing % of CD27+ IgD- (Sw. Memory) cells % CD19+ cells for the warts cohort (divided into CVID and no-PID patients in plot B), DOCK8 and IL7Ra deficient patients and herpes cohort

4.5.4 Immunoglobulins

As expected, the CVID group had low or no IgA and IgM, while the no-PID group and the herpes group showed mean values within the HCR (Figure 21 and Figure 22). IgG levels were not analysed as they were within the normal range, which in the CVID cohort was confounded by the fact that all patients received immunoglobulin replacement therapy. The immunoglobulins in the IL7Ra-deficient patient were normal with regards to IgG, IgA, and IgM. The Dock8-deficient patient’s IgM was on the lower side of the HCR, with normal IgG, and with high IgA.

57 Results

Figure 21: Comparing IgA levels for the warts cohort (divided into CVID and no-PID patients in plot B), DOCK8 and IL7Ra deficient patients and herpes cohort

Figure 22: Comparing IgM levels for the warts cohort (divided into CVID and no-PID patients in plot B), DOCK8 and IL7Ra deficient patients and herpes cohort

58 Results

4.6 Treatment

4.6.1 Warts Patients

The patients of the warts cohort used six different treatment modalities (Figure 23): cryotherapy, salicylic acid, imiquimod, systemic acitretin, cautery, and the CO2 laser. Two patients used a combination of therapies: Imiquimod with salicyclic acid and imiqimod with systemic acitretin.

Figure 23: Treatment modalities and their outcomes for the warts cohort

Cryotherapy was the most frequently used treatment (12 patients). This modality was curative in only one CVID patient and two patients (one CVID and one no-PID) had temporary benefits (reduced the number and size of warts) but only for a short period of time (Figure 24A). Treatment regimens were not uniform (see Table 16), ranging between weekly and monthly treatment, suggesting that some patients carried out cryotherapy at home while others were treated by their dermatologist. The patients with curative or temporally benefits had cryotherapy at the dermatologist.

Salicylic acid was the second most frequently used treatment (11 patients). Seven CVID and four no-PID patients applied salicylic acid daily or 3 times a week for several months, but none of the patients gained any benefit (Figure 24B).

59 Results

Figure 24: CVID and no-PID patients. A. Outcome of the cryotherapy treatment; B. Outcome of the salicylic acid treatment

Imiquimod was used by 10 patients, and was the most successful treatment; three CVID and seven no-PID patients used imiquimod, showing a nearly complete resolution in three no-PID patients and temporary benefit in one no-PID patient (Figure 25C). Patients applied imiquimod 3 times a week for 3 months to over 2 years (Table 16).

Table 16: Treatment regimens for cryotherapy and imiquimod

Cryotherapy Treatment Regime Imiquimod Treatment Regime Liquid nitrogen cream for 6 weeks Three times a week for 6 months Once per month for 1 year Three times a week for 4 months Once a week for 8 years Three times a week for 12 weeks Twice over 1 year Three times a week for 8 months Three times a week for 6 months + 6 months Three times a week for 2 years together with systemic acitretin Three powerful one at the doctor** Multiple times resulting in reduction of number Twice every 6 month for 1 year* Once a day for 12 weeks Two to three times Several times Five times in 3 month Multiple times As needed over 2 years * *cured wart temporarily ** cured one of patient´s biggest warts at one finger

The three patients with curative benefits were: patient 1 applied imiquimod for 8 months and felt an improvement after around 4 months; patient 2 used imiquimod for more than 2 years in the morning with salicylic acid at night and for the first 18 months, no new warts developed but following this, the warts started to improve dramatically; patient 3 was treated with imiquimod cream for 6 months with limited effect but after starting the combination of acitretin

60 Results

20g daily and imiquimod there was a dramatic improvement in recalcitrant warts within 6 months (the warts at his fingers and the foot-arch mosaic warts had completely resolved, while some residual heel warts remained).

Systemic acitretin treatment was used by three CVID and four no-PID patients, and was successful in one no-PID patient (Figure 25D). One of the CVID patients took 25mg of acitretin daily for 6 months and saw a reduction in size and softening of the warts, but they recurred when the treatment was stopped. The two other CVID patients stopped acitretin treatment after 1 month due to side effects and lack of improvement. One of the no-PID patients reported temporal reduction in wart size on acitretin but stopped the treatment due to side effects, and the two other no-PID patients stopped the Acitretin due to lack of improvement. The patient for whom systemic acitretin was successful took it in combination with Imiquimod, as mentioned above.

Figure 25: CVID and no-PID patients. C. Outcome of the imiquimod treatment; D. Outcome systemic acitretin treatment

Four patients had surgical removal of warts by cautery (Figure 26E). All of these patients reported successful removal but recurrence. CO2 Laser was unsuccessfully used in two patients (Figure 26F) and two patients had the HPV vaccine (Gardasil) but no improvement of their warts was observed (Figure 32).

61 Results

Figure 26: E. Outcome Cautery treatment for CVID and no-PID patients; F. Outcome CO2 laser treatment for CVID and no-PID patients

Figure 27: HPV vaccine patients with warts

The DOCK8-deficient patient was treated with different topical applications and freezing with limited benefit. He had received the CO2 laser treatment for warts on his right hand and noted that this improved the warts to a certain extent but only for a period of time. He was being treated with acitretin 25 mg once daily, fucidin ointment 30%, soak for his feet and applied imiquimod cream 3 days week. He felt that especially acitretin helped as it kept the warts on his feet soft. None of the different modalities however resolved his warts.

62 Results

No detailed wart treatment regime of the IL7Ra-deficient patient was available. The patient reported to have tried salicylic acid with no benefit and had received laser therapy for the multifocal genital neoplasia several times.

4.6.2 Herpes Patients

Out of the 21 herpes patients, 17 (81%) were treated with prophylactic antiviral medication and the remaining four patients (19%) took antiviral medication only during an acute episode; of the latter group, two had HSV and two had VZV (Figure 28). Two of these patients (one with HSV and one with VZV) were treated with 2g valaciclovir for 1 to 2 days, initiated when prodromal symptoms were felt and continued with 1g per day for 5 days if a lesion developed, while the other two patients were treated with aciclovir, but the exact treatment regime was not known.

Figure 28: The number of patients that took acute or prophylactic medication

Of the four patients taking acute treatment, two patients (one on aciclovir and one on valaciclovir) reported treatment success (Figure 29), including reduced severity, lesion size and duration of outbreak. One patient on aciclovir denied any benefit, and one patient on valaciclovir had just commenced treatment so no outcome could be documented. The mean number of episodes in these four patients was 4 per years (Table 8), but none reported a reduced outbreak frequency since commencing the acute medication.

63 Results

Figure 29: Medication success of the patients with acute medication

In the prophylactic medication group, eight patients had HSV, five patients had VZV and in four patients a clear decision between HSV and VZV could not be made (Table 8 and Figure 30A). In the HSV group, four were treated with aciclovir, three with valaciclovir and one with famciclovir. In the VZV group, three were treated with aciclovir and two with Valaciclovir. In the HSV or VZV group, one was treated with aciclovir and three with valaciclovir

Figure 30A).

Out of the patients that were treated with prophylactic aciclovir, five patients took a daily dose of 800mg, one 1600mg, one 200mg and one patient did not provide details, but most of these patients did not provide information about their treatment regime during an outbreak (Table 17). Out of the patients who were treated with prophylactic valaciclovir, six patients took a daily dose of 1g and two patients took 500mg and during an outbreak these doses were doubled (Table 17). Prior to prophylaxis, the aciclovir group patients had a mean of 8 outbreaks per year compared to 20 in the valaciclovir group, indicating that patients with a higher number of outbreaks were treated with valaciclovir. Reduction in outbreaks was similar in both groups with 78 percent in the aciclovir group and 71 percent in the valaciclovir group (see notes of Table 17).

64 Results

Table 17: Treatment regime and number of outbreaks for each herpes patients

Number of Number of Doses Patient Prophylactic Prophylactic Outbreaks Outbreaks During Notes ID Medication Doses with Pre- Outbreak Medication Medication 23 Aciclovir 800mg Not known 1 4 25 Aciclovir 1600mg Not known 0 Not known Av. 8 per year 28 Aciclovir Not known Not known 5 5 pre-medication 36 Aciclovir 800mg 3200mg 4 4 and av. 1.75 per year with 39 Aciclovir 800mg Not known 1 4 medication (78% reduction in 42 Aciclovir 800mg Not known 0 24 outbreaks) 44 Aciclovir 200mg 4g 1 3 37 Aciclovir 800mg Not known 2 12 26 Valaciclovir 1g 2g 12 12 30 Valaciclovir 1g 2g 24 30 Av. 20 per year 32 Valaciclovir 500mg 1g 10 20 pre-medication and av. 5.75 per 38 Valaciclovir 1g 2g 0 18 year with 40 Valaciclovir 1g 1.5g 0 24 medication (71.25 % 33 Valaciclovir 1g 2g 0 20 reduction in outbreaks) 29 Valaciclovir 1g Not known 0 12 31 Valaciclovir 500mg 1g 0 24 24 Famciclovir 500mg 750mg 12 12

Of the 17 patients on prophylactic medication, since commencing treatment, three patients reported smaller lesions, four shorter outbreaks, five less severe symptoms, but the main benefit, seen in twelve patients, was the reduction of outbreak frequencies (Figure 30B).

Figure 30: Patients on prophylactic medication. A. How many take aciclovir, valaciclovir, or famciclovir by virus type; B. In what way was the treatment beneficial

65 Results

Prior to commencing prophylactic medication, patients had a mean of 14 episodes per year (range 3 to 30), which reduced to a mean of 5 episodes per year (range 0 to 24) when on prophylactic medication (Figure 31). Looking at this by virus type, pre-prophylactic medication, the HSV or VZV group had the highest mean of 17 outbreaks per year, followed by the VZV group with a mean of 15, and the HSV group with a mean of 12; on prophylactic medication, the VZV and HSV or VZV groups had a significant reduction to a mean of zero and 1 episode respectively, but the HSV had only a small reduction to a mean of 10 episodes (Figure 31). Six out of 16 patients were outbreak free on prophylactic medication; three of these patients were in the VZV and three in the HSV or VZV group, while none of the HSV patients was free from outbreaks (Figure 32). Only two of the 21 herpes patients received the shingles vaccine Zostavax, but no benefit was reported (Figure 33).

Figure 31: Frequency of outbreaks pre- and on prophylactic medication, by virus type

66 Results

Figure 32: A. Number of patients with outbreaks on prophylactic medication. B. Number of patients with outbreaks on prophylactic medication by virus type.

Figure 33: How many patients have had Zostavax vaccination?

67 Discussion

5 Discussion

5.1 Clinical Features of Patients

5.1.1 Characteristics of Patients with Warts

5.1.1.1 HPV Type Analysis Specific types of HPVs correlate with histological and clinical characteristics of warts (Bruggink et al. 2012). In 2017 the most frequently detected HPV types in cutaneous warts are in general HPV 2, 27 and 57 from the α-HPV genus; HPV 4 and 65 from the γ-HPV genus; and HPV-1 from the µ-HPV genus (Hagiwara et al. 2005; Iftner et al. 2003; Rübben et al. 1997; Porro et al. 2003).

In this study, α-HPV 2, 27, 28 and 57 were detected. The most prevalent HPV type was 27 (36%), followed by 57 (29%), 28 (21%) and 2 (7%). These results differ slightly from larger HPV prevalence studies in which HPV 1, 2, 27 and 57 were found most frequently although the prevalence of each of these 4 types has varied in these studies (Bruggink et al. 2012; Iftner et al. 2003; Rübben et al. 1997; Porro et al. 2003). There may be different explanations for this: Firstly, in this study HPV types were only analysed for β- and α-genus and as HPV 1 belongs to the µ-HPV types we would not have been able to detect it. In addition, there are observations that HPV 1 is more prevalent in young patients and is of short duration (Rübben et al. 1997; Bruggink et al. 2012; Jabłonska et al. 1997), whereas the patients in this study had a mean age of 46 years and a mean duration of warts of 18 years. However, it would have been interesting to analyse the HPV DNA also for HPV types from the µ- and γ-genus. HPV 28, together with HPV 3 and 10, are associated with plane warts and are preferentially located on the face (Jabłonska et al. 1997). The single HPV 28 type in this study was from a plane wart on the patient’s face. The other two HPV 28 types where detected in warts with double HPV types and therefore may have not been responsible for the development of the warts. Lastly the sample size of 20 wart swabs from 14 patients, compared to sample sizes of over 200 in other HPV prevalence studies, is very small. In a larger sample size the prevalence of HPV 28 may have been less.

In this study, α-HPV DNA was detected in 70% of the wart swabs, which differs to recent studies in where α-HPV DNA was detected in over 90% of swabs of cutaneous warts (Bruggink et al. 2012; Iftner et al. 2003; Koning et al. 2010). This study used a modified degenerate PCR technique by Harwood et al (1999) which has been shown to detect a broad spectrum of mucosal, cutaneous and EV HPV types to a high degree of sensitivity. The reduced sensitivity in this study may again be explained by the possibility that in wart swabs defined negative for α-HPV DNA, other HPV types were present that were not analysed such

68 Discussion as HPV types from γ- or µ-genus. All wart swabs were positive for β-HPV DNA so the warts from the patients in which α-HPV DNA could not be detected, could be caused by HPV types from the β-genus. However, a quantitative analysis would be needed to confirm this, as β- HPVs are highly prevalent but non-pathogenic in the general population (Boxman et al. 1997; Astori et al. 1998; Antonsson et al. 2000).

In this study, the presence of multiple α-HPV types was detected in 10% of all swabs. One hypothesis here is that only one HPV type is the driving force for the development of the wart and that the additional viruses are passengers (Schmitt et al. 2011). In this study, non- invasive swabs of the warts were used so passenger HPV types present on the skin could have been additionally detected by the sensitive modified degenerate PCR. To confirm this, we would have needed to identify the HPV type with the highest viral load but unfortunately, we did not quantify the viral loads of the HPV types detected. In this study two cases were found in which two different HPV types were sequenced in one wart swab (one case of HPV 28 and 57, and in one case 28 and 27). HPV 27 and 57 are frequently found in common warts, while HPV 28 is associated with plane warts of the face. As both swabs were taken from common warts, it can be hypothesised that HPV 27 or HPV 57 was responsible for the development of the patient´s wart and that HPV 28 was only a passenger on the skin. In addition, it is also not clear whether the existence of multiple HPV types within the swab represents co-infection of single cells or the presence of different cells infected with single HPV types (Harwood et al. 1999). In this study, we did not localise the genomes of these HPVs within the wart and therefore we cannot state whether or not the two different HPV types in these cases infected the same nucleus.

Although cutaneous viral warts are common in the general population (see section 1.2.2), this patient cohort had an unusually long history of persistence (mean duration of 19 years), often numerous (50% of patients had well over 20 warts) and severe warts (Figure 34). Therefore, we investigated whether these patients had an underlying immunodeficiency and studied a possible role of EV-HPV types (β-HPVs) in the development of the patient´s severe persistent warts (see section 1.4.1.1). EV patients, like the patients in this study, suffer from life-long persistent cutaneous warts (Orth 2008). EV-HPVs are highly prevalent but non- pathogenic in the general population (Astori et al. 1998; Antonsson et al. 2000; Boxman et al. 1997). However, they have been found to be activated not only in patients diagnosed with EV but also in patients with (Favre et al. 1998; Weissenborn et al. 1999), in patients on after renal transplantation (Bens et al. 1998; Jong-Tieben et al. 1995; Berkhout et al. 1995; Shamanin et al. 1994) and in patients with immune deficiencies such as CVID (Vu et al. 2007) and HIV (Rogers et al. 2009). In this study β-HPV DNA was identified in all patients. In one patient, only a single β-HPV was detected, however, all other patients revealed multiple types, ranging from 2 to 8 with a mean of 5 different types. These

69 Discussion observations are in line with other results. For example, the median number of infecting β- HPV types in a large cross-sectional study of 845 immunocompetent and 560 immunosuppressed individuals from six countries, ranged from 3 to 6, where the prevalence of β-HPV DNA reached 91 % in the immunocompetent and 98 % in the immunosuppressed population (Koning et al. 2009).

Figure 34: Examples of patients with severe warts

To determine which of the HPV type in our patients was responsible for the development of the patient’s warts, the viral load would have been a good indicator, as it is regarded to reflect viral replication (Weissenborn et al. 2005). Unfortunately, we did not measure the viral load.

In this study, the most prevalent beta HPV types were 93 (64%), 8 (50%), 24 (43%), 23 (36%) and 76 (28%) and 92 (28%). In the literature, depending on which typing technique was used, there is a very large variation in the frequency and HPV types detected. Also, over the last decade, the sensitivity and specificity of the detection techniques have improved considerably, enabling the identification of many more HPV types (Weissenborn et al. 2010).

5.1.2 Characteristics of Patients with Herpes

Patients in this study experienced a mean of 9 herpes episodes per year, ranging from 2 to 30 episodes, which is unusually high compared to the literature (see section 1.2.1.1 and 1.2.1.2). In addition, the age of this patient cohort differed to the expected age of re- occurrence. HSV reactivation appears to be declining after the age 35 (Steiner et al. 2007;

70 Discussion

Esmann 2001) while the incidence of herpes zoster increases with age (Steiner et al. 2007). The mean age of the HSV and VZV patients in this study was 38 and 52 years respectively. The diagnosis of most patients was not virologically confirmed so that some VZV may have been HSV patients. Still, the unusual high frequency and age of recurrences in this cohort aroused suspicion of an underlying immune deficiency. Therefore, an immunology workup was performed, the results of which will be discussed later.

In this study 71% of patients reported trigger factors which would cause their herpetic infection to reactivate. Physical or emotional stress was stated as a trigger factor most frequently in 66% of these patients followed by other infections and menstruation in 27% of patients (see Table 10). This finding is in line with the literature where emotional or physical stress, common cold, fever, menstruation, immunosuppressant or chemotherapy, corticosteroid administration and exposure to heat, cold, or sunlight are reported as typical trigger factors (Arduino and Porter, 2008; Fatahzaden and Schwarz, 2008). In addition, patients in this study were asked whether they related an event to the set off of their herpes infections and 48% experienced one. Set off events as such are not reported in the literature, however the ones stated from the patients in this study (see Table 9) can be summarised into physical or emotional stress and other infections which are typical reported trigger factors of clinical outbreaks.

Prodromal symptoms at the site of reactivation occur in 46% to 60% of herpes labialis patients (Arduino und Porter 2008) and in about 80% of patients with acute VZV infection (Wittek et al. 2010). The number of patients with a prodrome in this study was slightly higher with 70% in the HSV and 100% in the VZV cohort. This may be related to the higher frequency and severity of herpes infection in our patients as those who experience prodromal symptoms appear to have larger lesions (Spruance et al. 1977).

In this study, genital herpes was experienced most frequently in 52% patients closely followed by herpes orolabialis in 48% of patients and facial herpes in 38% of patients (see Table 8). The high frequency of genital herpes arose the suspicion that these patients might have actually suffered from HSV-2 rather than HSV-1 infection, as HSV-2 is predominantly associated with genital disease (Arduino und Porter 2008). However, crossover infection via sexual practice is possible as more than 25% of genital herpes infections are attributable to HSV-1 (Fatahzadeh und Schwartz 2007b; Siegel 2002). Also, in some of the patients in this study with genital herpes the virus was confirmed by PCR as HSV-1, supporting the findings from the literature. Also 53% of the patients in this study experienced reactivation at 2 or 3 different locations. The patients with genital herpes were within this group suffering in addition with orolabial and facial herpes. These patients may have suffered from both, HSV-1 and HSV-2infections.

71 Discussion

5.2 Immunology Features of Patients

Host defence against viruses relies on intact functioning of the innate and cellular immune responses. In this patient cohort, concerns for immune defects were raised because HPV or herpes infections were severe or recalcitrant, and therefore an immune workup was performed.

Analysis of immunoglobulins showed values well within the range of healthy control (HCR) for the no-PID warts patients and the herpes patients, while CVID patients revealed low or no IgA and IgM. The latter was expected as the diagnostic criteria for CVID requires serum IgG and IgA with or without IgM levels to be at least two standard deviations less than the mean for the patient’s age (Gathmann 2016). CVID patient’s IgG levels were not analysed as they were within the normal range, confounded by the fact that all CVID patients received immunoglobulin replacement therapy.

Analysis of B cells showed that the majority of CVID patients in this study were below the lower limit of the HCR for absolute CD19 count (Figure 13B). According to the literature, for most CVID patients, the absolute B cell count tends to be normal or are reduced (Berron- Ruiz et al. 2014). Low B cell numbers in CVID patients are associated with progressive disease and reduced survival (Cunningham-Rundles und Bodian 1999). Also regarding the percentage of CD19+ B cells (Figure 15B), the majority of CVID patients revealed results below the HCR, including three patients with B cell count less than 1% of the total peripheral blood lymphocyte population; this suggests a defect at the early stage of B cell differentiation (Gaspar und Conley 2000) in these three patients.

Phenotypic analysis of peripheral B cell subsets revealed that all CVID patients, apart from one, had reduced numbers of switched memory cells (CD27+IgM-IgD-) (Figure 20B). Moreover, IgM memory cells (CD27+IgM+IgD+) were at the lower end of the HCR (Figure 19B). Neutralising antibodies provide an effective line of antiviral defence (Burton 2002). A decreased humural compartment with lower serum immunoglobulin levels and less effective antibody response, may therefore predispose patients to viral infections. A significantly reduced switched memory B cell compartment is a hallmark of CVID (Agematsu et al. 2002; Berron-Ruiz et al. 2014; Ko et al. 2005; Warnatz 2002), indicating an impaired germinal centre function. Reduced switched memory B cell counts are correlated with complications in CVID such as bronchiectasis, , splenomegaly and autoimmunity (Alachkar et al. 2006; Sanchez-Ramon et al. 2008). Also, patients with lower numbers of IgM+CD27+ memory B cells have been shown to have a higher risk for developing chronic lung disease (Ahn und Cunningham-Rundles 2009). These complications however, were not the focus of this study.

72 Discussion

Apart from the characteristic B cell defect, numerous T cell abnormalities in CVID patients have been reported, including reduced T cells counts, reduced proliferative responses to mitogens, reduced cytokine production, and a paucity of regulatory T cells (Tregs) (Giovannetti et al. 2007; Bateman et al. 2012; Cunningham-Rundles und Bodian 1999). As T cells are considered the major defence against viruses, it is surprising that, compared to bacterial infections, patients with CVID respond relatively well to viruses. A possible explanation for this is that, when considering the ESID clinical criteria for CVID, there is no evidence of a profound T cell deficiency in CVID patients (Azizi et al, 2016). According to the ESID clinical criteria, profound T cell deficiency is defined as: CD4+ cells/μL at 2-6 years <300, 6-12 years <250, >12 years <200; percentage of naive CD4+ T cells at 2-6 years <25%, 6-16 years <20%, >16 years <10%; and absence of T cell proliferation.

CVID patients who present with two out of three of the above criteria are considered to have a late onset combined immunodeficiency (LOCID) (Gathmann 2016). In our cohort, none of the CVID patients had a profound T cell defect. The percentage of CD4 naive cells were low with a mean of 18%, absolute CD4 and absolute CD8 count results were close but above the lower HCR limit and proliferation response was normal (Figure 11B, 12B, 17B). However, our patients still suffered from severe and recalcitrant warts. Warts are uncommon in CVID, but a few cases with disseminated or unremitting warts are reported (Lin et al. 2009; Lynn et al. 2004; Reid et al. 1976; Uluhan et al. 1998). Like the patients in our cohort, in addition to low immunoglobulin levels, all published patients presented with T lymphopenia and/or impaired T cell proliferation (Table 18). This decreased cell-mediated immunity is likely to contribute to susceptibility to warts in CVID patients. However, the small number of patients reported, makes it difficult to determine which T cell subsets are significantly affected. It would have been interesting to compare the T cell counts, phenotypes and function of this cohort to matched CVID patients without warts, but this could be the subject of a future study.

73 Discussion

Table 18: Laboratory comparison of CVID patients with warts reported in the literature

Lin et al Lynn et al Reid et al Uluhan et al This CVID cohort

Lymphocyte count Normal Reduced Reduced Normal Low* Severly Absolute CD4 count Reduced n/a Normal Low* reduced Absolute CD8 Count Normal Reduced n/a Normal Low* Absolute CD19 Count Normal Reduced Normal Normal Low* Absolute CD16+CD56 Normal Reduced n/a Normal Low* Count Serverly Serverly % of CD4 Naive n/a n/a n/a reduced reduced Serverly Lymphocyte proliferation Normal n/a Reduced Normal** reduced Immunoglobulins Reduced Reduced Reduced Reduced Reduced * Close to the lower limit of the HCR ** Reduced in 3 patients n/a not applicable (value was not measured)

In addition, the absolute count and the percentage of CD16+ CD56+ NK cells were low in the CVID cohort. NK cells play an important role in the innate immune response against viral infections through their rapid cytotoxic activity and their IFN-ɣ production (Lodoen und Lanier 2006). Although, NK cells in CVID patients have been observed to be reduced in absolute numbers and percentages (Aspalter et al. 2000; Berron-Ruiz et al. 2014; Kutukculer et al. 2015) this appears not to have any clinical consequence as CVID patients are not as susceptible to viral infections compared to patients with isolated NK cell deficiencies (Orange 2013). In the literature, a compensatory mechanism for the low numbers is suggested as studies on in-vitro cytotoxicity of PBMC from CVID patients to the K562 cell line (presumed NK activity) have been normal (Eckert et al. 1994; Kutukculer et al. 2015), and another paper demonstrated normal IFN-ɣ by CVID NK cells (Aspalter et al. 2000). In this study, NK cell function was not tested.

Interestingly, regarding the absolute count and percentage of CD16+ CD56+ NK cells, most of the no-PID wart patients and the herpes cohort had values close to the lower limit of the HCR (Figure 16B). For the no-PID wart cohort this was the only cell subset that revealed low values. The low NK cell numbers in these patients may have a relevant influence on the severity and persistence of their infections as research has shown that NK cell-deficient individuals are exceptionally susceptible to infection with viruses, particularly to those of the herpesvirus and papillomavirus families (Orange 2012).

A few mutations in human genes have been identified to affect NK cell biology in the context of naturally occurring disease. For example, GATA2 mutations lead to decreased or absent circulating monocytes, B cells, dentritic cells and NK cells (Bigley et al. 2011; Vinh et al. 2010). Patients present with infectious phenotypes characteristic of NK deficiency,

74 Discussion particularly with HPV infections, causing recalcitrant warts followed by herpes family viruses’ infections including HSV, VZV and EBV (Vinh et al. 2010; Spinner et al. 2014).

Another PID that has been associated with an NK cell abnormality is that of autosomal recessive hyper-IgE syndrome caused by mutations or deletions in the gene encoding DOCK8 resulting in a combined immunodeficiency (Engelhardt et al. 2009; Zhang et al. 2009), affecting B cells, NK cells, and various T cell subsets (Aydin et al. 2015). These patients’ NK cells can bind to their target cell, but do not accumulate actin filaments at the lytic immunological synapse, which is required for effective cytotoxicity (Mizesko et al. 2013). This NK cell impairment may contribute to the increased susceptibility of these patients to cutaneous viral infections including HPV, HSV, MCV and VZV. The DOCK8-deficient patient in this study presented with severe warts (Figure 35) but his absolute count and percentage of NK cells were within the HCR, although his cytotoxicity function was not investigated.

Our herpes cohort, in addition to low NK cell numbers, revealed values close to the lower limit of HCR in the percentage of CD4+ CD45RA+ CD4+ naive T cells and in percentage of CD27+ IgD+ IgM+ memory B cells (Figure 17B and 19B). To date there are no publications on phenotypic analysis of peripheral B or T cell subsets in patients with recurrent herpes infections. Once CD4+ naive T cells are activated, they undergo clonal expansion and differentiate into effector T cells including different T helper (TH) cell subsets and CD4+ T cells with cytotoxic function. TH cells play a key role in antiviral response by promoting B cell antibody production and enhancing CD8+ T cells response, producing cytokines and chemokines, and through direct cytotoxic effects on virus-infected cells (Kaech et al. 2002; Swain et al. 2012). Low numbers of CD4+ naive T cells may lead to a limited antiviral response in this herpes cohort making them more susceptible to recurrent infections. The IgM+IgD+CD27+ B lymphocytes represent memory B cells that have undergone somatic hypermutation of Ig V genes but not Ig isotype switching (Tangye und Good 2007). Although their immunological functions are still poorly understood, recently authors have shown that stimulation with IFN-γ, but also with immunomodulatory neutrophils, caused these IgM memory B cells to differentiate and secrete IgM or class switch to IgG2. This highlights their potentially special role in the early inflammatory response (Seifert et al. 2015). In addition, there is a correlation between impaired immunity to encapsulated bacteria and reduced numbers of IgM memory B cells, suggesting their function in controlling responses against encapsulated bacteria such as Streptococcus pneumoniae infections (Kruetzmann et al. 2003; Weller et al. 2004). Hence, reduced number of IgM memory B cells in this cohort may potentiate the recurrent herpes infections.

Overall, the low percentage of NK cells in the no-PID wart cohort and the low percentage of CD4+naive, IgM memory and NK cells in the herpes cohort may contribute to the increased

75 Discussion susceptibility of these patients to cutaneous viral infections. However, once the CVID patients were excluded, laboratory evaluation of the wart- and herpes cohort did not raise a suspicion of an underlying PID. In addition to the above PIDs, associated with an NK abnormality, other PIDs with a susceptibility for HSV or HPV infections have been described, including EV, SCID, deficiency in the TLR3 pathway, and WHIM syndrome. Therefore, to exclude a causative mutation in the patient´s immune system, DNA of this cohort was collected and patients who consented were included in a BRIDGE study. BRIDGE studies are Next Generation Sequencing (NGS) projects with two aims: 1. to discover sequence variants underlying hitherto unresolved inherited Rare Diseases and; 2. to evaluate the sensitivity and specificity of exome sequencing or other NGS approaches to identify already known high penetrance Rare Disease-causing variants (Cambridge Biomedical Research Centre). However, none of the patient did achieve a genetic diagnosis.

5.3 Dermatology Quality of Life Index (DLQI)

5.3.1 Patients with warts

It is well established that skin disease has a detrimental effect on the patient’s quality of Life (QoL) (Finlay und KHAN 1994; Jobanputra und Bachmann 2000; Jowett und Ryan 1985) and that it especially causes distress when visible areas such as the hands or the face are affected (Moloney et al. 2005). Cutaneous warts for example are viewed as socially challenging, when located on visible sites. They are common, often benign and most warts clear spontaneously within months or years due to natural immunity. However, they may become recalcitrant and depending on their location they can be painful (Zachariae et al. 2012). Cutaneous warts can be difficult to treat, especially in immunosuppressed individuals, and it has been reported that this has an impact on these patient’s QoL. For example, Zacharia et al (2012) reported that 12.3% of kidney transplanted patients with warts in their study had a DLQI above 6 (on a scale of 0-30), indicating a moderate effect on patient´s life (Table 13). A review article of the DLQI by Lewis and Finlay (2004) provided the means and ranges of all DLQI scores reported in the literature in the last 10 years. Here the mean and the range of the DLQI of 24 wart patients from three studies were 4.7 and 3.8 to 6.7 respectively. Similarly, in another study the mean DLQI score in outpatients with warts was 4.4 (Zachariae et al. 2000).

The DLQI scores in our wart cohort, was lower than in the literature, with a mean of 3. The reason for the lower score in our study might be explained by the fact that most of our patients were not primarily visiting the immunology clinic because of their warts. Nine of the patients with warts had CVID, suffering from recurrent chronic infections, chronic lung disease, gastrointestinal disease, or autoimmune disorders. Their persistent severe warts

76 Discussion might have felt less burdensome compared to their other health problems. This hypothesis is strengthened when looking at the scores from CVID and no-PID patients separately. Here the mean score in the no-PID group was 4, similar to the scores reported in the literature, while the mean score of the CVID patients was only 2. However, the warts still had an impact on the CVID patient’s QoL: This is seen when comparing their score to a population of healthy controls which has been reported to be 0.4 (Zachariae et al. 2000). Alternatively, there is no significant difference between the QoL scores of the CVID patients, the no-PID patients and of the patients reported in the literature. According to the banding system to aid the interpretation of DLQI scores (see Table 13) all assessed patients fall into Band 1 that includes scores 2 to 5 and indicates that the warts have a small effect of patient’s QoL. Overall, these are surprisingly low values when considering that 50% of the patients in this cohort had over 20 warts and 83% had lived with them for over 5 years. Only two patients, the DOCK8-deficient and one CVID patient, in this study had a DLQI score greater than 10. This score falls into Band 3, indicating a very large effect on the patient’s life (Hongbo et al. 2005). The severity of the warts in these two patients differed to the rest of the cohort, as they had well over 20 warts, mostly over their hands and feet (Figure 35). Another patient whose warts were not as extensive as in the latter two patients but more severe compared to the rest of the cohort, revealed a score of 7, which is Band 2, indicating a moderate effect on the patient’s life. The results of these three patients indicate a correlation between the severity of warts and a higher DLQI score.

Figure 35: Warts from the DOCK 8 deficient and one CVID patient

77 Discussion

5.3.2 Herpes Patients

The DLQI scores of the herpes no outbreak patients had a mean of 2, similar to the wart cohort. However, in a week of an outbreak, the scores in the herpes cohort were considerably higher with a mean of 15 (Band 3) indicating a large effect on patient’s lives. Unfortunately, according to the review article of the DLQI by Lewis and Finlay (2004), DLQI scores of herpes patients were not reported in the literature. Only one study assessing the effect of skin disease on QoL after renal transplant concluded that a history of at least four cold sores in the past year had an important impact on the QoL measure but the actual DLQI score was not stated (Moloney et al. 2005). In healthy hosts, most HSV-1 lesions are mild and self-limiting. Nevertheless, frequent outbreaks are associated with significant irritation, discomfort and pain, inconvenience, cosmetic and loss of self-esteem (Esmann 2001; Spruance et al. 2006). In this study, patients also experienced pain, inconvenience, and loss of self-esteem (see Table 9). In addition, patients had difficulty functioning normally, were not able to get out of bed, experienced overall tiredness, and reported lack of stamina. This suggests that patients not only had local symptoms but that the virus possible affected them systemically. Dividing the cohort into HSV and VZV patients revealed a very high DLQI score of 22 in VZV cohort compared to a score of 13 in the HSV group. According to the banding system, a score of 22 indicates an extremely large effect on a patient’s life (see Table 13). Unfortunately, there are no DLQI scores for VZV patients reported in the literature. Two studies however assessed the impact of acute zoster pain on health-related QoL. These analyses showed that moderate to severe pain was common during the acute rash phase and that the overall pain burden was significantly correlated with poor physical and social functioning, and increased emotional distress (Katz et al. 2004; Schmader et al. 2007). Regrettably, our study did not assess the acute pain intensity as it would have been interesting to see whether greater pain burden was associated with a higher DLQI score.

Only 10 of the 21 herpes patients returned a second DLQI, completed in a week of a herpes virus infection outbreak. In a week of no outbreak, the one-questionnaire herpes group (Q 1/1) had a mean score of 1 and the two-questionnaire herpes group (Q 1/2) a mean score of 3 (Figure 4). This demonstrates a slight difference between the groups, as a score of 1 suggests ‘no effect at all on patient’s life’ and a score of 3 suggests a ‘small effect on patient’s life’ (Hongbo et al. 2005). One reason for not returning a second questionnaire is that in most of these patients the virus was controlled with antiviral medication so that they did not suffer from outbreaks anymore. This may also explain the difference between the two groups. The group that did not suffer outbreaks with antiviral medication may have felt in control of their health so that their skin condition did not affect their life anymore, while patients who still experienced outbreaks were subconsciously worried about, it even in 78 Discussion weeks without symptoms. Analysing the various aspects of impairment covered in the DLQI, outbreak Q (1/1) and Q (1/2) groups scored equally high in the question related to physical symptoms (Question 1). This is surprising as it suggest that patients do not suffer physical symptoms during outbreak free periods. One explanation could be that patients experienced prodromal symptoms such as tenderness, pain, burning sensation or paraesthesia at the site of previous reactivation, at the time of completing the questionnaire. Another explanation is that there is a psychosomatic component to their symptoms. In all other questions, Q (1/2) patients reported a larger influence on their QoL with the highest effect on work or study (Question 7) and their emotional wellbeing (Question 2). The fact that emotional wellbeing was one of their highest scoring questions strengthens the argument that patients who still experience outbreaks were still subconsciously worried about their skin condition even in times without visible symptoms. The highest score in Question 7 can be accounted for by some patients who worked with vulnerable people in the health care system. They reported not being allowed to go work to during flare up times or having to do alternative tasks while having frequent recurrences. The effect of recurrent herpes labialis on patients working life was also demonstrated in two large surveys in France and the USA in which 21% of 437 patients and 45% of 476 patients respectively had to stop working due to their episode (Dreno et al. 2012).

The DOCK8-deficient patient felt that the treatment had the most effect on his QoL with a score of 3 (out of 3), a question which showed low scores in the other groups. This patient had very extensive severe warts (see Figure 35) and was daily soaking his feet in ointment before applying local treatment and wrapping up his feet to maximise the benefit. In addition, he was suffering from a dry mouth and lips, one of the side effects of the acitretin tablets he was taking. His skin condition had a major effect on his working life (score 2/3); he was unemployed and reported that he would regularly be invited to interviews but would not get the job once employers saw that his hands were covered with warts.

Overall, this study showed that all warts and herpes patients experienced physical symptoms and unpleasant feelings of embarrassment or felt that their skin condition had an impact on their self-esteem. Feelings of embarrassment or low self-esteem can lead to reduced self- confidence; this was stated by several patients when asked why they had difficulties due to their skin condition. Similar findings were reported by Ciconte et al (2003) who assessed the morbidity associated with having viral cutaneous warts and showed that out of 85 patients, 52% had moderate to extreme discomfort, 81% felt embarrassed, 70% feared negative appraisal by others due to their warts, and 39% stated an effect on social and leisure activities. In our study, the social and leisure activities question scored highly in the herpes outbreak but not in the warts cohort. Similarly, Schmader et al (2007), using the Zoster Impact Questionnaire in 160 patients, reported that leisure activities were particularly

79 Discussion affected after herpes zoster rash onset; Katz et al (2004) showed a significant association of acute zoster pain with poorer social functioning; and Dreno et al (2012) showed about two- thirds of patients reported an impact on their physical and ⁄ or emotional well-being and the majority of patients were disturbed in their social life.

5.4 Treatment of Patients

5.4.1 Wart Patient’s Treatment

Although cutaneous viral warts are ubiquitous, no single therapy has been established to achieve complete remission in every patient and high quality evidence based studies for the optimal treatment are limited (see section 1.2.2). In this study, wart patients tried cryotherapy, salicylic acid, imiquimod, systemic acitretin, cautery, and CO2 laser therapy to clear their warts.

In the general population, cutaneous warts are common in children and young adults with a lower prevalence in adults. Hence, the majority of treatment modalities are assessed in young individuals. Other factors such as time of presence of the wart before commencing treatment and number of warts may play a role in the success rate. For example, Bruggink et al (2010), investigating the effectiveness of cryotherapy and salicylic acid, found that cure rates were considerably lower in participants over twelve years and older and among those who had suffered warts for more than six months. Similarly, Harwood et al (2005) noted that best response of imiquimod was seen in patients that had a relatively short duration of warts prior to treatment. In some studies, patients with more than five warts were excluded and duration of warts are inconsistently documented. In our study patients’ mean age was 46 years, they had suffered from warts for between 3 to 40 years and 78 percent of participants had more than 5 warts. These facts should be kept in mind when comparing the treatment outcome of our patients with the literature.

5.4.1.1 Cryotherapy Cryotherapy was the most frequently used treatment in this study (12 patients). However, only in one patient this modality was curative and only two patients stated a temporary benefit in reducing the number and size of warts for a short period of time. This is similar to findings in the literature, where cure rates have a large range from 0% to 69% (Sterling et al. 2014) with aggressive cryotherapy reported to be more effective than gentle freezing (Kwok et al. 2011).

Treatment regimens in this study were not uniform and ranged between weekly and monthly treatment, suggesting that some patients carried out home cryotherapy while others went to see their dermatologist. The patients with curative or temporary benefits were treated at the

80 Discussion dermatologist so it is assumed that they received aggressive cryotherapy. As warts may naturally regress over time among immunocompetent people, and a recent meta-analysis found no benefit of cryotherapy over placebo (Kwok et al. 2012), it is difficult to determine whether the cure of the one patient in this study can be directly attributed to the therapy.

5.4.1.2 Salicylic Acid Salicylic acid was the second most used treatment in this study; seven CVID and four no-PID patients applied salicylic acid daily or three times a week for several months, but none of the 11 patients gained any benefit. These findings differ to the literature where numerous studies found salicylic acid to be significantly more effective than placebo (Kwok et al. 2011; Gibbs et al. 2002).

In this study patients were asked retrospectively about their treatment so they may not have remembered their exact treatment regime, especially if they used salicylic acid many years ago, and had tried in addition several other treatment modalities. This made it difficult to estimate their compliance so we do not know whether the treatment may have been successful with a strict treatment regime. Qualitative research for example has revealed poor compliance for salicylic acid because patients did not believe in its efficiency, found application time consuming and complained about irritation of the surrounding skin (Thomas et al. 2006). Also, we do not know which salicylic acid product patients used and this may impact its effectiveness as products differ in strength from less than 7% in over-the counter lotions to up to 70% salicylic acid in physician-prescribed preparations (Bacelieri und Johnson 2005). However, data comparing different preparations are lacking.

5.4.1.3 Imiquimod In this study, imiquimod was the most successful treatment, and was used by three CVID and seven no-PID patients showing a nearly complete resolution in three no-PID patients (30%) and a temporary benefit in one no-PID patient. This is similar to the literature in which numerous small open-label studies demonstrated different success rates, but overall encouraging results in both immunocompetent and immunosuppressed individuals (see section 1.5.1.2).

Imiquimod is so far approved for treating anogenital warts, and basal cell carcinoma. The recommended regime for anogenital warts is to apply 5% imiquimod cream three times weekly at night for a maximum of 16 weeks, with similar regimes in the other two conditions (Rote Liste® Service GmbH 2017). While in our study patients applied imiquimod 3 times a week, all studies referenced here administered imiquimod daily. A more frequent application might have increased the cure rate in our study but might have also enhanced healing time as our patients applied imiquimod for 6 to 24 months compared to 3 weeks to 6 months in the referenced studies. While local skin reactions and systemic adverse effects

81 Discussion

(such as headache, fever, myalgia and nausea) are reported in clinical trials applying imiquimod for anogenital warts, no systemic and only mild and well-tolerated local side effects (such as pruritus, , burning and pain) were experienced during daily application in the referenced studies (Kim et al. 2013; Harwood et al. 2004; Grussendorf‐Conen und Jacobs 2002; Micali et al. 2003). This indicates that 5% imiquimod cream, when used for cutaneous warts compared to when used for anogenital warts, is better tolerated even when applied more than 3 times a week and that daily application may enhance success rates and healing time. However, larger studies and RCT are needed to confirm this hypothesis and to determine the appropriate dosing regimen for the treatment of non-genital warts.

In one of our patients who was treated successfully, dramatic improvement was only seen after starting the combination of 20g of acitretin daily and imiquimod. An explanation for this could be that the volume of this patient’s warts was reduced with acitretin, an oral retinoid that disrupts epidermal proliferation and differentiation (Lipke 2006; Sterling et al. 2014; Sterling et al. 2001). This may have then optimised imiquimod penetration. This theory is supported by other studies that de-bulked warts in different ways to enhance drug penetration: Grussendorf-Conen and Jacobs (2002) cut back patient’s warts with a scalpel every 2 weeks; Kim et al (2013) (2013) used duke tape occlusion therapy; and Micali et al (2003) applied 50% salicylic acid under occlusive dressing for 5 days prior to imiquimod therapy.

5.4.1.4 Systemic Acitretin Systemic acitretin was tried by 7 patients in this study, but was only successful in one patient in combination with imiquimod (as described in the previous section). Another patient took 25mg of acitretin daily for 6 months and saw a reduction in size and softening of the warts, but they recurred when the treatment ended. Recurrence is expected, as retinoids such as acitretin, disrupt epidermal proliferation and differentiation but cannot eradicate HPV (Choi et al. 2006).

There are only a limited number of small trials or case reports assessing the effectiveness of retinoids in the treatment of warts: In a small study of 23 organ transplant patients, there was a 45% reduction in the number of warts after 3 months topical retinoid treatment but 3 months post-treatment there was only a 29% reduction (Euvrard et al. 1992), indicating recurrence. A more recent study assessed the benefit of low dose oral isotretinoin (0.5mg/kg/day) for 2 months in the treatment of recalcitrant facial plane warts in 26 patients, of which 19 patients showed a complete regression but 23% of these 19 had recurrences 4 months post-treatment (Al-Hamamy et al. 2012).

82 Discussion

In our study three patients stopped acitretin treatment due to side effects. To my knowledge there are no clinical trials specifically assessing the side effects of systemic retinoids in the treatment of cutaneous warts. Al-Hamamy et al (2012) listed but did not comment on the side effects of his 26 patients which included dry lips and skin in most cases. Unfortunately, we did not ask our patients about their side effects but in the experience of the clinicians in our clinic, a lot of patients stop treatment because of dry lips and skin.

Overall systemic retinoids appear to have a beneficial effect as an alternative management in the treatment of extensive cutaneous warts, especially when used as part of a combination treatment modality. However, larger trials are needed to confirm its benefit, to investigate the side effects, and to find a dosing regimen which maximises the treatment response, minimises relapse rates, and has tolerable side effects.

5.4.1.5 Cautery Four patients in this study had surgical removal of warts by cautery; all report a successful removal but recurrence. Cautery is widely practiced but no RCT has been published (Lipke 2006; Sterling et al. 2001). Successful removal is reported in 65 to 85% of patients and recurrence rates may be as high as 30% (Sterling et al. 2001; Mulhem und Pinelis 2011).

5.4.1.6 CO2 Laser CO2 Laser treatment was tried in two patients of this study. On the efficacy of CO2 laser therapy, no RCTs have been published (Lipke 2006) but in two case series a cure rate of 64% to 71% at 12 months is reported (Sloan et al. 1998; Street und Roenigk 1990). In a more recent study, promising results have been found in immunosuppressed patients with recalcitrant warts (Läuchli et al. 2003). Unfortunately, in our study CO2 laser treatment was without success.

5.4.1.7 Vaccine Two patients in our cohort had a HPV vaccine (Gardasil) but no improvement of their warts was observed. To date there are no published clinical trials investigating the quadrivalent HPV vaccine as a treatment modality for recalcitrant warts (Cyrus et al. 2015), but case reports have shown promising results (Landis et al. 2012; Kreuter et al. 2010; Silling et al. 2014; Venugopal und Murrell 2010), indicating that further research may be worthwhile in assessing vaccines as a treatment modality for extensive, persistent warts.

5.4.2 Herpes Patient’s Treatment

Out of the 21 herpes patients, 17 (81%) were treated with prophylactic antiviral medication while the remaining four patients (19%) only took antiviral medication during an acute episode.

83 Discussion

5.4.2.1 Symptomatic Therapy Taking acute antiviral medication is appropriate for patients with mild and infrequent outbreaks preceded by prodromal symptoms (Esmann 2001). The patients in this study who used acute medication, all had prodromal symptoms and experienced infrequent outbreaks with a mean of four episodes per year compared to 14 episodes in prophylactic medication patients.

Two of the patients on acute medication, one with HSV and one with VZV, were treated with aciclovir and the other two with valaciclovir, initiated when prodromal symptoms were felt. Several trials assessing the effect of oral aciclovir or valaciclovir in non-genital HSV and VZV infections have shown modest treatment benefits. This included shortened lesion healing time and duration of lesion pain, and reduced viral shedding (Beutner et al. 1995; Chosidow et al. 2003; Laiskonis et al. 2002; Spruance et al. 1990; Raborn et al. 1987; Tyring et al. 1995; Wood et al. 1996). Two patients of our study (one on aciclovir and one on valaciclovir) reported treatment success including reduced severity, smaller lesions and shorter duration. Viral titers were not measured in this study. The other valaciclovir patient had just commenced treatment, so no judgements could be made while the other aciclovir patient reported no benefit. Maximal clinical benefit from acute antiviral treatment is gained when medication is initiated early, since most viral replication occurs in the first 24 hours of lesion onset (Spruance et al. 2006). This requires patient’s compliance and ability to recognise early clinical signs to start therapy as soon as prodromal symptoms are noticed (Esmann 2001). The lack of any benefit of aciclovir treatment in one patient may be due to a delayed start of medication. Therefore, patient education about its timely use may ameliorate patients’ symptoms during an outbreak. In case of aciclovir failure, changing to another cyclic guanosine analogue with better bioavailability such as valaciclovir and famciclovir could be considered.

5.4.2.2 Prophylactic Therapy For patients with severe or frequent herpes infection, prophylactic suppression with oral antiviral medication may be warranted (Esmann 2001). In this study, 17 patients were on antiviral prophylactic medication of which eight patients took aciclovir, eight valaciclovir and one patient took famciclovir. In the aciclovir group most patients took a daily dose of 800mg, in the valaciclovir group most patients took 1g a day and the patient taking famciclovir had a daily dose of 500mg. The optimal dose for prophylactic therapy still remains to be established but Woo et al (2007), reviewing the literature for evidence of efficacy of antiviral agents in the prophylaxis of recurrent HSV infections, recommended an oral dose of 400- 800mg aciclovir 3 times a day, an oral dose of 500-1000mg valaciclovir and famciclovir twice a day in immunocompromised patients. The doses taken by patients in our study were lower,

84 Discussion but none of these patients were diagnosed with having a primary or secondary immunodeficiency.

Before commencing treatment, patients in the aciclovir group had a mean of eight outbreaks per year compared to 20 in the valaciclovir group indicating that patients with a higher number of outbreaks were treated with valaciclovir. However, it is not clear why this is the case, as in our study both drugs achieved an approximate reduction of outbreaks between 70% and 80% and there are no clinical trials directly comparing any of these antiviral agents. The disadvantage of oral aciclovir is its poor bioavailability of only 10-20% and its short plasma life time, making frequent dosing necessary (Brown et al. 2002), while valaciclovir, the prodrug of aciclovir, has a 3- to 5-times greater bioavailability (Brown et al. 2002; Arduino und Porter 2006; Tyring et al. 2002), allowing a less frequent oral dosing which may improve patient’s compliance (Fatahzadeh und Schwartz 2007a; Spruance et al. 2006). It would have been interesting to know whether patients or the therapist had chosen the more convenient dosing regimen of valaciclovir and whether patients had tried aciclovir unsuccessfully before and their therapy was changed to valaciclovir.

In this study, the patients were asked about the treatment effect they had experienced since commencing prophylactic medication. Some patients reported to have smaller lesions, a shorter duration of the outbreak and less severe symptoms. An explanation for these treatment effects could be that antiviral agents target viral DNA replication at both the epithelial and neuronal sites. Hence, virions reaching the skin along axonal pathways, initiate epithelial replication with reduced viral load, which, in turn, may reduce the severity of the outbreak (Miller et al. 2004).

The benefit reported most frequently in twelve out of 17 patients was a reduction in outbreak numbers, with six patients stating to be outbreak free. This has also been shown in small trials studying the effect of prophylactic aciclovir (Rooney et al. 1993; Thomas et al. 1985) and valaciclovir in non-genital HSV infections (Baker und Eisen 2003; Miller et al. 2004). In these trials, compared to a placebo, daily oral antiviral medication reduced the number of recurrent infections and prolonged the median time to the first clinical documented recurrence.

To reduce the number of recurrent infections has not been confirmed for famciclovir. Although it has only been assessed as a prophylactic treatment for non-genital herpes in one trial, where a limited reduction in lesion healing time was observed, but the drug was unsuccessful in preventing the development of new lesions (Spruance et al. 1999) (see section 1.5.1.2). Interestingly, in our study famciclovir also failed to prevent recurrences, however only one patient was treated with this antiviral agent.

85 Discussion

Analysing frequencies of outbreaks in patients on prophylactic medication more precisely revealed that, with a yearly mean of 14 episodes pre-treatment and only 5 episodes during treatment, the herpes cohort had an average reduction in outbreaks of 68%. This is higher than in the literature where rates between 46% and 60% are reported for HSV patients (Baker und Eisen 2003; Miller et al. 2004). This cohort and also the referenced studies only have a small number of patients, thus larger studies are needed to confirm these results.

The cohort in our study includes HSV-1 but also VZV patients. VZV patients were not included in the referenced trials and to my knowledge there are no studies on prophylactic antiviral medication in patients with recurrent episodes of zoster. The reason for this could be that recurrent herpes zoster infections in immunocompetent patients are uncommon with an estimate range from only 1% to 5% (Gebo et al. 2005; Yawn et al. 2011) making it difficult to reach patients numbers for trials. Also in our study, only five patients had a diagnosis of recurrent VZV. This diagnosed was not virologically confirmed meaning that these patients could be mislabelled HSV patients.

Overall larger studies are needed to confirm the benefit of prophylactic antiviral medication in the treatment of recurrent HSV infection. Especially studies using famciclovir and head-to- head studies comparing the efficacy of different available cyclic guanosine analogues are missing.

5.4.2.3 Vaccines Patients in our study were asked whether they had received the herpes zoster vaccine (Zostavax) and whether they had gained any benefit from it. Zostavax is approved in the EU for the prevention of herpes zoster (HZ) and (PHN) in adults aged ≥50 years (Schmader et al. 2012; Oxman et al. 2005). This vaccine has shown a reduction in the incidence of herpes zoster of 70% in adults aged 50 to 55, of 64% in adults aged 60 to 69, and of 38% in adults 70 or above (Schmader et al. 2012; Oxman et al. 2005). In our study two zoster patients had received Zostavax but no benefit was reported. The referenced trials excluded patients with recurrent zoster infections so no direct comparison can be made and to my knowledge there is no study investigating the efficacy of the herpes zoster vaccine in patients with recurrent VZV infections.

86 Summary and Conclusion

6 Summary and Conclusion

Viral skin infections are frequently seen in children and adolescents due to their developing immune system, while they are rarely seen in healthy adults. This study investigated 39 adults with severe viral skin infections, including 18 with persistent warts and 21 with recurrent herpes infections. Investigations included, HPV typing and blood value analysis to describe the clinical and immunological features and define any underlying PID. The DLQI was used to understand whether and how viral skin infections impact patients`QoL, and a clinical questionnaire to explore the treatment modalities and responses was deployed. In the wart cohort, nine patients were diagnosed with CVID, one with DOCK8- and one with IL7Ra- deficiency. Warts had effected the patients for a mean of 19 years with 50% having more than 20 warts, with the DOCK8- and the IL17Ra-deficiency patient having the highest number and severity. Patients tried six therapies with cryotherapy used most frequently and imiquimod the most successful. Treatment regimens were not uniform and none was convincingly successful. In the HPV DNA type analysis of wart samples, α-HPV was identified in 70% of samples, with 27 (36%), 57 (29%), and 28 (21%) the most common types. β-HPV was identified in all patients with 93 (64%), 8 (50%), and 24 (43%), being the most common types. In the herpes cohort, ten patients were clinically diagnosed with HSV, seven with VZV, four with no definite diagnosis, and none with an underlying PID. Genital herpes was experienced most frequently (52% of patients), followed by herpes orolabialis (48% of patients) and facial herpes (38% of patients). 81 % of the herpes patients were treated with prophylactic antiviral medication while the remaining took symptomatic medication. All patients reported having smaller lesions, a shorter outbreak duration and less severe symptoms since starting medication. Taking prophylactic aciclovir or valaciclovir achieved a significant reduction of outbreaks (70% and 80%). The DLQI scores indicated a large impact on patient’s QoL in the herpes outbreak cohort, and a small impact on patient’s QoL in the wart- and herpes no-outbreak cohort. All patients experienced physical and emotional impacts. Blood value analysis of the CVID patients with warts indicated a reduced or lacking IgA and IgM, low absolute CD4, CD8, CD19, and NK cell counts, a low percentage of CD19+ B cells, NK cells, and IgM memory cells; and in the majority of patients a reduced percentage of switched memory cells and CD4+ naive T cells. Both, the no-PID wart patients and the herpes cohort showed low percentages of NK cells while the herpes cohort, in addition, had a low percentage of CD4+ naive T cells, and a decreased IgM memory. When removing the CVID patients from analysis, laboratory evaluation of the wart and herpes cohort did not raise a suspicion of an underlying PID. However, the low percentage of CD4+naive, IgM memory and NK cells might have contributed to the increased susceptibility of these patients to cutaneous viral infections.

87 Zusammenfassung

7 Zusammenfassung

Diese Studie hat 39 erwachsene Patienten mit schwerwiegenden viralen Hautinfektionen untersucht. Darunter waren 18 mit persistierenden Warzen und 21 mit häufig wiederkehrenden Herpesinfektionen. Die Untersuchungen beinhalteten HPV-Typisierungen, die Analyse spezifischer Blutwerte, einen Dermatologischen Lebens Qualität Index (DLQI) und einen klinischen Fragebogen. In der Warzen-Kohorte befanden sich neun Patienten mit CVID, bei einem Patienten wurde eine DOCK8-Defizienz und bei einem weiteren ein IL7Ra- Mangel diagnostiziert. Die Patienten litten im Durchschnitt 19 Jahre unter Warzen. Die Hälfte aller Betroffenen hatte mehr als 20 Warzen, wobei die Patienten mit dem DOCK8- und IL17Ra-Mangel die größte Anzahl und den höchsten Schweregrad aufwiesen. Sechs Behandlungsarten wurden durchgeführt, die häufigste war die Kryotherapie und die erfolgreichste war Imiquimod. In der HPV-Typisierung wurde α-HPV in 70% der Proben identifiziert. Die häufigsten waren Typ 27 (36%), Typ 57 (29%), und Typ 28 (21%). Bei allen Patienten wurde β-HPV gefunden. Hierbei waren Typ 93 (64%), Typ 8 (50%), und Typ 24 (43%) die häufigsten. In der Herpes-Kohorte wurde bei zehn Patienten jeweils klinisch HSV und bei sieben VZV diagnostiziert. Vier Patienten hatten keine klare Diagnose. Kein Patient wies eine PID auf. Genitaler Herpes wurde am häufigsten beschrieben (52% der Patienten), gefolgt von Mund- und Lippenherpes (48%) und fazialem Herpes (38%). 81 % der Patienten wurden prophylaktisch mit antiviraler Medikation behandelt, während die restlichen nur bei Symptomen antivirale Medikamente einnahmen. Alle Patienten gaben an, nach Behandlungsbeginn, kleinere Läsionen, kürzere Ausbrüche und weniger schwere Symptome zu haben. Prophylaktisches Aciclovir oder Valaciclovir zeigte eine signifikante Reduktion der Ausbrüche (70% und 80%). Das Ergebnis des DLQI zeigte, dass Infektionen bei Herpes- Patienten während eines Ausbruches einen großen Einfluss auf die Lebensqualität hatten. Auch in einem ausbruchsfreien Intervall wurde ein messbarer Einfluss nachgewiesen. Hingegen hatten die HPV-Infektionen generell nur einen kleinen Einfluss auf die Lebensqualität der Warzen-Patienten. Jedoch beschrieben alle Patienten körperliche und emotionale Beeinträchtigungen. Die Analyse der Blutwerte der Warzen-Patienten mit CVID zeigte: reduziertes oder fehlendes IgA und IgM, niedrige Zahlen absoluter CD4, CD8, CD19 und NK-Zellen; einen niedrigen Prozentsatz der CD19+ B-, NK-, und IgM-Memory-Zellen; und in den meisten Patienten einen reduzierten Prozentsatz der Switched-Memory- und CD4-Naive-Zellen. Die Warzen-Patienten ohne PID wiesen einen niedrigen Prozentsatz von NK-Zellen auf. Auch die Herpes-Kohorte hatte eine niedrigen Prozentsatz von NK-Zellen und darüber hinaus einen niedrigen Prozentsatz von CD4-Naive T-Zellen und IgM-Memory- Zellen. Bei der Laboranalyse der Warzen- und Herpes- Patienten (CVID Patienten ausgeschlossen) wurde kein Verdacht auf eine zugrundeliegende PID erhoben.

88 References

8 References

Abendroth, A.; Arvin, A. M. (2001): Immune evasion as a pathogenic mechanism of varicella zoster virus. In: Seminars in immunology 13 (1), S. 27–39.

Agematsu, Kazunaga; Futatani, Takeshi; Hokibara, Sho; Kobayashi, Norimoto; Takamoto, Masaya; Tsukada, Satoshi et al. (2002): Absence of memory B cells in patients with common variable immunodeficiency. In: Clinical immunology (Orlando, Fla.) 103 (1), S. 34–42.

Ahn, Sam; Cunningham-Rundles, Charlotte (2009): Role of B cells in common variable immune deficiency. In: Expert review of clinical immunology 5 (5), S. 557–564.

Akira, Shizuo; Uematsu, Satoshi; Takeuchi, Osamu (2006): Pathogen recognition and innate immunity. In: Cell 124 (4), S. 783–801.

Alachkar, Hana; Taubenheim, Nadine; Haeney, Mansel R.; Durandy, Anne; Arkwright, Peter D. (2006): Memory switched B cell percentage and not serum immunoglobulin concentration is associated with clinical complications in children and adults with specific antibody deficiency and common variable immunodeficiency. In: Clinical immunology (Orlando, Fla.) 120 (3), S. 310–318.

Alexopoulou, Lena; Holt, Agnieszka Czopik; Medzhitov, Ruslan; Flavell, Richard A. (2001): Recognition of double-stranded RNA and activation of NF-κB by Toll-like receptor 3. In: Nature 413 (6857), S. 732–738.

Al-Hamamy, Hayder R.; Salman, Husam Ali; Abdulsattar, Nawar A. (2012): Treatment of plane warts with a low-dose oral isotretinoin. In: ISRN dermatology 2012, S. 163929.

Alster, Tina S.; Nanni, Christopher A. (1999): Famciclovir prophylaxis of herpes simplex virus reactivation after laser skin resurfacing. In: Dermatologic 25 (3), S. 242–246.

Amir, Jacob (2001): Clinical aspects and antiviral therapy in primary herpetic gingivostomatitis. In: Paediatric drugs 3 (8), S. 593–597.

Antonsson, A.; Karanfilovska, S.; Lindqvist, P. G.; Hansson, B. G. (2003): General Acquisition of Human Papillomavirus Infections of Skin Occurs in Early Infancy. In: Journal of Clinical Microbiology 41 (6), S. 2509–2514.

Antonsson, Annika (2012): Review: antibodies to cutaneous human papillomaviruses. In: Journal of medical virology 84 (5), S. 814–822.

Antonsson, Annika; Forslund, Ola; Ekberg, Henrik; Sterner, Gunnar; Hansson, Bengt Göran (2000): The ubiquity and impressive genomic diversity of human skin papillomaviruses suggest a commensalic nature of these viruses. In: Journal of virology 74 (24), S. 11636– 11641.

IX References

Arduino, P. G.; Porter, S. R. (2006): Oral and perioral herpes simplex virus type 1 (HSV-1) infection: review of its management. In: Oral diseases 12 (3), S. 254–270.

Arduino, Paolo G.; Porter, Stephen R. (2008): Herpes Simplex Virus Type 1 infection: overview on relevant clinico‐pathological features. In: Journal of oral pathology & medicine 37 (2), S. 107–121.

Arvin, Ann (2005): Aging, immunity, and the varicella–zoster virus. In: New England Journal of Medicine 352 (22), S. 2266–2267.

Arvin, Ann M. (2008): Humoral and cellular immunity to varicella-zoster virus: an overview. In: The Journal of infectious diseases 197 Suppl 2, S. 60.

Aspalter, R. M.; Sewell, W. A.C.; Dolman, K.; Farrant, J.; Webster, A. D.B. (2000): Deficiency in circulating natural killer (NK) cell subsets in common variable immunodeficiency and X‐linked agammaglobulinaemia. In: Clinical & Experimental Immunology 121 (3), S. 506– 514.

Astori, Giuseppe; Lavergne, Donna; Benton, Claire; Höckmayr, Birgit; Egawa, Kiyofumi; Garbe, Claus; Villiers, Ethel-Michele de (1998): Human papillomaviruses are commonly found in normal skin of immunocompetent hosts. In: Journal of Investigative Dermatology 110 (5), S. 752–755.

Ault, Kevin A. (2007): Human papillomavirus vaccines and the potential for cross-protection between related HPV types. In: Gynecologic Oncology 107 (2), S. S31-S33.

Aumakhan, Bulbulgul; Gaydos, Charlotte A.; Quinn, Thomas C.; Beyrer, Chris; Benning, Lorie; Minkoff, Howard et al. (2010): Clinical reactivations of herpes simplex virus type 2 infection and human immunodeficiency virus disease progression markers. In: PloS one 5 (4), S. e9973.

Aydin, Susanne E.; Kilic, Sara Sebnem; Aytekin, Caner; Kumar, Ashish; Porras, Oscar; Kainulainen, Leena et al. (2015): DOCK8 deficiency: clinical and immunological phenotype and treatment options - a review of 136 patients. In: Journal of clinical immunology 35 (2), S. 189–198.

Bacelieri, Rocky; Johnson, Sandra Marchese (2005): Cutaneous warts: an evidence-based approach to therapy. In: Am Fam Physician 72 (4), S. 647–652.

Baker, David; Eisen, Drore (2003): Valacyclovir for prevention of recurrent herpes labialis: 2 double-blind, placebo-controlled studies. In: Cutis 71 (3), S. 239–242.

Balabanian, Karl; Lagane, Bernard; Pablos, Jose Luis; Laurent, Lysiane; Planchenault, Thierry; Verola, Olivier et al. (2005): WHIM syndromes with different genetic anomalies are

X References accounted for by impaired CXCR4 desensitization to CXCL12. In: Blood 105 (6), S. 2449– 2457.

Ballow, Mark (2002): Primary immunodeficiency disorders. Antibody deficiency. In: Journal of Allergy and Clinical Immunology 109 (4), S. 581–591.

Basra, M. K. A.; Fenech, R.; Gatt, R. M.; Salek, M. S.; Finlay, A. Y. (2008): The Dermatology Life Quality Index 1994-2007: a comprehensive review of validation data and clinical results. In: The British journal of dermatology 159 (5), S. 997–1035.

Bateman, E. A. L.; Ayers, L.; Sadler, R.; Lucas, M.; Roberts, C.; Woods, A. et al. (2012): T cell phenotypes in patients with common variable immunodeficiency disorders: associations with clinical phenotypes in comparison with other groups with recurrent infections. In: Clinical and experimental immunology 170 (2), S. 202–211.

Bens, G.; Wieland, U.; Hofmann, A.; Pfister, H. (1998): Detection of new human papillomavirus sequences in skin lesions of a renal transplant recipient and characterization of one complete genome related to epidermodysplasia verruciformis-associated types. In: Journal of general virology 79 (4), S. 779–787.

Berkhout, R. J.; Tieben, Linda M.; Smits, Henk L.; Bavinck, J. N.; Vermeer, Bert J.; Ter Schegget, J. (1995): Nested PCR approach for detection and typing of epidermodysplasia verruciformis-associated human papillomavirus types in cutaneous from renal transplant recipients. In: Journal of Clinical Microbiology 33 (3), S. 690–695.

Bernard, Hans-Ulrich; Burk, Robert D.; Chen, Zigui; van Doorslaer, Koenraad; Zur Hausen, Harald; Villiers, Ethel-Michele de (2010): Classification of papillomaviruses (PVs) based on 189 PV types and proposal of taxonomic amendments. In: Virology 401 (1), S. 70–79.

Berron-Ruiz, L.; Lopez-Herrera, G.; Vargas-Hernandez, A.; Mogica-Martinez, D.; Garcia- Latorre, E.; Blancas-Galicia, L. et al. (2014): Lymphocytes and B-cell abnormalities in patients with common variable immunodeficiency (CVID). In: Allergologia et immunopathologia 42 (1), S. 35–43.

Beutner, Karl R.; Friedman, David J.; Forszpaniak, Christine; Andersen, Paul L.; Wood, Martin J. (1995): Valaciclovir compared with acyclovir for improved therapy for herpes zoster in immunocompetent adults. In: Antimicrobial Agents and Chemotheraphy 39 (7), S. 1546– 1553.

Bigley, Venetia; Haniffa, Muzlifah; Doulatov, Sergei; Wang, Xiao-Nong; Dickinson, Rachel; McGovern, Naomi et al. (2011): The human syndrome of dendritic cell, monocyte, B and NK lymphoid deficiency. In: The Journal of experimental medicine 208 (2), S. 227–234.

XI References

Biron, C. A. (1998): Role of early cytokines, including alpha and beta interferons (IFN - alpha/beta), in innate and adaptive immune responses to viral infections. In: Seminars in immunology 10 (5), S. 383–390.

Biron, Christine A.; Nguyen, Khuong B.; Pien, Gary C.; Cousens, Leslie P.; Salazar-Mather, Thais P. (1999): Natural killer cells in antiviral defense: function and regulation by innate cytokines. In: Annual review of immunology 17 (1), S. 189–220.

Bisaccia, Emil; Scarborough, Dwight (2003): Herpes simplex virus prophylaxis with famciclovir in patients undergoing aesthetic facial CO2 laser resurfacing. In: Cutis 72 (4), S. 327–328.

Boxman, Ingeborg; Berkhout, Ron J. M.; Mulder, Linda H. C.; Wolkers, Monika C.; Bavinck, Jan N. Bouwes; Vermeer, Bert Jan; ter Schegget, Jan (1997): Detection of human papillomavirus DNA in plucked hairs from renal transplant recipients and healthy volunteers. In: Journal of Investigative Dermatology 108 (5), S. 712–715.

Brady, Rebecca C.; Bernstein, David I. (2004): Treatment of herpes simplex virus infections. In: Antiviral research 61 (2), S. 73–81.

Brown, Tricia J.; McCrary, Monica; Tyring, Stephen K. (2002): Antiviral agents. Nonantiviral drugs. In: Journal of the American Academy of Dermatology 47 (4), S. 581–599.

Bruggink, Sjoerd C.; de Koning, Maurits N C; Gussekloo, Jacobijn; Egberts, Paulette F.; ter Schegget, Jan; Feltkamp, Mariet C. W. et al. (2012): Cutaneous wart-associated HPV types: prevalence and relation with patient characteristics. In: Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology 55 (3), S. 250–255.

Bruggink, Sjoerd C.; Eekhof, Just A. H.; Egberts, Paulette F.; van Blijswijk, Sophie C E; Assendelft, Willem J. J.; Gussekloo, Jacobijn (2013): Natural course of cutaneous warts among primary schoolchildren: a prospective cohort study. In: Annals of family medicine 11 (5), S. 437–441.

Bruggink, Sjoerd C.; Gussekloo, Jacobijn; Berger, Marjolein Y.; Zaaijer, Krista; Assendelft, Willem J. J.; de Waal, Margot W M et al. (2010): Cryotherapy with liquid nitrogen versus topical salicylic acid application for cutaneous warts in primary care: randomized controlled trial. In: CMAJ : Canadian Medical Association journal = journal de l'Association medicale canadienne 182 (15), S. 1624–1630.

Burton, Dennis R. (2002): Antibodies, viruses and vaccines. In: Nature reviews. Immunology 2 (9), S. 706–713.

XII References

Cambridge Biomedical Research Centre: What is the BRIDGE Project. National Institute for Health Research. Online verfügbar unter https://bridgestudy.medschl.cam.ac.uk/whatis.shtml, zuletzt geprüft am 18.05.2017.

Casrouge, Armanda; Zhang, Shen-Ying; Eidenschenk, Céline; Jouanguy, Emmanuelle; Puel, Anne; Yang, Kun et al. (2006): Herpes simplex virus encephalitis in human UNC-93B deficiency. In: Science 314 (5797), S. 308–312.

Chapel, Helen; Lucas, Mary; Lee, Martin; Bjorkander, Janne; Webster, David; Grimbacher, Bodo et al. (2008): Common variable immunodeficiency disorders: division into distinct clinical phenotypes. In: Blood 112 (2), S. 277–286.

Choi, Yun‐Lim; Lee, Kwang‐Jun; Kim, Won‐Serk; Lee, Dong‐Youn; Lee, Joo‐Heung; Lee, Eil‐Soo; Yang, Jun‐Mo (2006): Treatment of extensive and recalcitrant viral warts with acitretin. In: International journal of dermatology 45 (4), S. 480–482.

Chosidow, O.; Drouault, Y.; Garraffo, R.; Veyssier, P. (2003): Valaciclovir as a single dose during prodrome of herpes facialis: a pilot randomized double‐blind clinical trial. In: British Journal of Dermatology 148 (1), S. 142–146.

Chow, Ken Y. C.; Brotin, Émilie; Khalifa, Youcef Ben; Carthagena, Laetitia; Teissier, Sébastien; Danckaert, Anne et al. (2010): A pivotal role for CXCL12 signaling in HPV- mediated transformation of keratinocytes: clues to understanding HPV-pathogenesis in WHIM syndrome. In: Cell host & microbe 8 (6), S. 523–533.

Ciconte, Antoinette; Campbell, Jan; Tabrizi, Sepehr; Garland, Suzanne; Marks, Robin (2003): Warts are not merely blemishes on the skin: A study on the morbidity associated with having viral cutaneous warts. In: Australasian journal of dermatology 44 (3), S. 169–173.

Clercq, E. (2004): Discovery and development of BVDU (brivudin) as a therapeutic for the treatment of herpes zoster. In: Biochemical pharmacology 68 (12), S. 2301–2315.

Clifton, Missy M.; Johnson, Sandra Marchese; Roberson, Paula K.; Kincannon, Jay; Horn, Thomas D. (2003): Immunotherapy for Recalcitrant Warts in Children Using Intralesional Mumps or Candida Antigens. In: Pediatr Dermatol 20 (3), S. 268–271.

Cockayne, Sarah; Hewitt, Catherine; Hicks, Kate; Jayakody, Shalmini; Kang'ombe, Arthur Ricky; Stamuli, Eugena et al. (2011): Cryotherapy versus salicylic acid for the treatment of plantar warts (verrucae): a randomised controlled trial. In: BMJ (Clinical research ed.) 342, S. d3271.

Cohen, Kenneth R.; Salbu, Rebecca L.; Frank, Jerry; Israel, Igor (2013): Presentation and Management of Herpes Zoster (Shingles) in the Geriatric Population. In: and Therapeutics 38 (4), S. 217.

XIII References

Coleman, Nicholas; Birley, Humphrey D. L.; Renton, Adrian M.; Hanna, Nashat F.; Ryait, Balvinder K.; Byrne, Micheline et al. (1994): Immunological events in regressing genital warts. In: American journal of clinical pathology 102 (6), S. 768–774.

Croen, Kenneth D. (1993): Evidence for antiviral effect of nitric oxide. Inhibition of herpes simplex virus type 1 replication. In: Journal of Clinical Investigation 91 (6), S. 2446.

Cullen, S. P.; Martin, S. J. (2008): Mechanisms of granule-dependent killing. In: Cell Death & Differentiation 15 (2), S. 251–262.

Cunningham, A. L.; Diefenbach, R. J.; Miranda-Saksena, M.; Bosnjak, L.; Kim, M. (2006): The Cycle of Human Herpes Simplex Virus Infection: Virus Transport and Immune Control. In: Journal of Infectious Diseases 194, S. 11–18.

Cunningham-Rundles, C.; Bodian, C. (1999): Common variable immunodeficiency: clinical and immunological features of 248 patients. In: Clinical immunology (Orlando, Fla.) 92 (1), S. 34–48.

Cyrus, Nika; Blechman, Adam B.; Leboeuf, Matthew; Belyaeva, Elizaveta A.; Koning, Maurits N. C. de; Quint, Koen D.; Stern, John J. (2015): Effect of Quadrivalent Human Papillomavirus Vaccination on Oral Squamous Cell . In: JAMA dermatology 151 (12), S. 1359– 1363.

Decman, Vilma; Freeman, Michael L.; Kinchington, Paul R.; Hendricks, Robert L. (2005): Immune control of HSV-1 latency. In: Viral immunology 18 (3), S. 466–473.

Degreef, Hugo (1994): Famciclovir, a new oral antiherpes drug: results of the first controlled clinical study demonstrating its efficacy and safety in the treatment of uncomplicated herpes zoster in immunocompetent patients. In: International journal of antimicrobial agents 4 (4), S. 241–246.

Delves, Peter J.; Martin, Seamus J.; Burton, Dennis R.; Roitt, Ivan M. (2014): Roitt's essential immunology. 12. Aufl.: John Wiley & Sons.

Diaz, George A.; Gulino, A. Virginia (2005): WHIM syndrome: a defect in CXCR4 signaling. In: Current allergy and asthma reports 5 (5), S. 350–355.

Dimmock, N. J. (1995): Update on the neutralisation of animal viruses. In: Rev. Med. Virol. 5 (3), S. 165–179.

Dochez, Carine; Bogers, Johannes J.; Verhelst, Rita; Rees, Helen (2014): HPV vaccines to prevent and genital warts: an update. In: Vaccine 32 (14), S. 1595–1601.

Dreno, B.; Malkin, J. E.; Saiag, P.; Batuji-Garin, S. (2012): Patients' profile, burden of disease and quality of life in recurrent herpes labialis: a survey conducted in USA and France. In:

XIV References

Journal of the European Academy of Dermatology and Venereology : JEADV 26 (11), S. 1458–1460.

Dworkin, Robert H.; Johnson, Robert W.; Breuer, Judith; Gnann, John W.; Levin, Myron J.; Backonja, Miroslav et al. (2007): Recommendations for the management of herpes zoster. In: Clinical infectious diseases 44 (Supplement 1), S. S1-S26.

Eckert, K.; Schmitt, M.; Garbin, F.; Wahn, U.; Maurer, H. R. (1994): Thymosin α1 effects, in vitro, on lymphokine-activated killer cells from patients with primary immunodeficiencies: preliminary results. In: International journal of immunopharmacology 16 (12), S. 1019–1025.

Engelhardt, Karin R.; McGhee, Sean; Winkler, Sabine; Sassi, Atfa; Woellner, Cristina; Lopez- Herrera, Gabriela et al. (2009): Large deletions and point mutations involving the dedicator of cytokinesis 8 (DOCK8) in the autosomal-recessive form of hyper-IgE syndrome. In: The Journal of allergy and clinical immunology 124 (6), S. 1289.

Erickson, Britt K.; Landers, Emily E.; Huh, Warner K. (2014): Update on vaccination clinical trials for HPV-related disease. In: Clinical therapeutics 36 (1), S. 8–16.

Esmann, J. (2001): The many challenges of facial herpes simplex virus infection. In: Journal of Antimicrobial Chemotherapy 47 (suppl 1), S. 17–27.

Euvrard, S.; Verschoore, M.; Touraine, J. L.; Dureau, G.; Cochat, P.; Czernielewski, J.; THIVOLET, J. (1992): Topical retinoids for warts and keratoses in transplant recipients. In: The Lancet 340 (8810), S. 48–49.

Farina, Cinthia; Krumbholz, Markus; Giese, Thomas; Hartmann, Gunther; Aloisi, Francesca; Meinl, Edgar (2005): Preferential expression and function of Toll-like receptor 3 in human astrocytes. In: Journal of neuroimmunology 159 (1-2), S. 12–19.

Fatahzadeh, M.; Schwartz, R. A. (2007a): Human herpes simplex labialis. In: Clinical and experimental dermatology 32 (6), S. 625–630.

Fatahzadeh, Mahnaz; Schwartz, Robert A. (2007b): Human herpes simplex virus infections: epidemiology, pathogenesis, symptomatology, diagnosis, and management. In: Journal of the American Academy of Dermatology 57 (5), S. 737–763.

Favre, M.; Orth, G.; Majewski, S.; Baloul, S.; Pura, A.; Jablonska, S. (1998): Psoriasis: A possible reservoir for human papillomavirus type 5, the virus associated with skin of epidermodysplasia verruciformis. In: The Journal of investigative dermatology 110 (4), S. 311–317.

Feduchi, Elena; Carrasco, Luis (1991): Mechanism of inhibition of HSV-1 replication by tumor necrosis factor and interferon γ. In: Virology 180 (2), S. 822–825.

XV References

Finlay, A. Y.; KHAN, G. K. (1994): Dermatology Life Quality Index (DLQI)-a simple practical measure for routine clinical use. In: Clin Exp Dermatol 19 (3), S. 210–216.

Fischer, A. (2000): Severe combined immunodeficiencies (SCID). In: Clin Exp Immunol 122 (2), S. 143–149.

Frazer, Ian H. (2009): Interaction of human papillomaviruses with the host immune system: a well evolved relationship. In: Virology 384 (2), S. 410–414.

Freeman, Alexandra F.; Holland, Steven M. (2008): The hyper-IgE syndromes. In: Immunology and allergy clinics of North America 28 (2), S. 277.

Freeman, Alexandra F.; Holland, Steven M. (2010): Clinical manifestations of hyper IgE syndromes. In: Disease markers 29 (3-4), S. 123–130.

Gaspar, H. B.; Conley, M. E. (2000): Immunodeficiency Review: Early B cell defects. In: Clinical & Experimental Immunology 119 (3), S. 383–389.

Gaspar, H. Bobby; Harwood, Catherine; Leigh, Irene; Thrasher, Adrian J. (2004): Severe cutaneous papillomavirus disease after haematopoietic stem-cell transplantation in patients with severe combined immunodeficiency. In: British journal of haematology 127 (2), S. 232– 233.

Gathmann, B.; Goldacker, S.; Klima, M.; Belohradsky, B. H.; Notheis, G.; Ehl, S. et al. (2013): The German national registry for primary immunodeficiencies (PID). In: Clinical and experimental immunology 173 (2), S. 372–380.

Gathmann, B.; Grimbacher, B.; Beaute, J.; Dudoit, Y.; Mahlaoui, N.; Fischer, A. et al. (2009): The European internet-based patient and research database for primary immunodeficiencies: results 2006-2008. In: Clinical and experimental immunology 157 Suppl 1, S. 3–11.

Gathmann, Benjamin (2016): ESID Registry - Working definitions for clinical diagnosis of PID, S. 11, zuletzt geprüft am 22.05.2017.

Gathmann, Benjamin; Mahlaoui, Nizar; Gerard, Laurence; Oksenhendler, Eric; Warnatz, Klaus; Schulze, Ilka et al. (2014): Clinical picture and treatment of 2212 patients with common variable immunodeficiency. In: The Journal of allergy and clinical immunology 134 (1), S. 116–126.

Gebo, Kelly A.; Kalyani, Rita; Moore, Richard D.; Polydefkis, Michael J. (2005): The Incidence of, Risk Factors for, and Sequelae of Herpes Zoster Among HIV Patients in the Highly Active Antiretroviral Therapy Era. In: JAIDS Journal of Acquired Immune Deficiency Syndromes 40 (2), S. 169–174.

XVI References

Gelmetti, Carlo; Cerri, Danilo; Schiuma, Aldo Antonio; Menni, Silvano (1987): Treatment of extensive warts with etretinate: a clinical trial in 20 children. In: Pediatric dermatology 4 (3), S. 254–258.

Gershon, Anne A.; Mervish, Nancy; LaRussa, Philip; Steinberg, Sharon; Lo, Shaw Hwa; Hodes, David et al. (1997): Varicella-zoster virus infection in children with underlying human immunodeficiency virus infection. In: Journal of Infectious Diseases 176 (6), S. 1496–1500.

Gibbs, Sam; Altman, Douglas G.; Harvey, Ian; Sterling, Jane; Stark, Rosemary (2002): PapersLocal treatments for cutaneous warts: systematic reviewCommentary: Systematic reviewers face challenges from varied study designs. In: Bmj 325 (7362), S. 461.

Gilbert, Stanley C. (2007): Suppressive therapy versus episodic therapy with oral valacyclovir for recurrent herpes labialis: efficacy and tolerability in an open-label, crossover study. In: Journal of drugs in dermatology: JDD 6 (4), S. 400–405.

Gill, Kanwar S.; Wood, Martin J. (1996): The clinical pharmacokinetics of famciclovir. In: Clinical pharmacokinetics 31 (1), S. 1–8.

Giovannetti, A.; Pierdominici, M.; Mazzetta, F.; Marziali, M.; Renzi, C.; Mileo, A. M. et al. (2007): Unravelling the Complexity of T Cell Abnormalities in Common Variable Immunodeficiency. In: The Journal of Immunology 178 (6), S. 3932–3943.

Gonçalves, Maria Alice Guimarães; Donadi, Eduardo Antonio (2004): Immune cellular response to HPV: current concepts. In: Brazilian Journal of Infectious Diseases 8 (1), S. 1–9.

Gorlin, Robert J.; Gelb, Bruce; Diaz, George A.; Lofsness, Karen G.; Pittelkow, Mark R.; Fenyk, John R. (2000): WHIM syndrome, an autosomal dominant disorder: clinical, hematological, and molecular studies. In: American journal of medical genetics 91 (5), S. 368–376.

Gross, G.; Schöfer, H.; Wassilew, S.; Friese, K.; Timm, A.; Guthoff, R. et al. (2003): Herpes zoster of the German Dermatology Society (DDG). In: Journal of Clinical Virology 26 (3), S. 277–289.

Grussendorf‐Conen, E‐I; Jacobs, S. (2002): Efficacy of imiquimod 5% cream in the treatment of recalcitrant warts in children. In: Pediatric dermatology 19 (3), S. 263–266.

Gulino, A. Virginia (2003): WHIM syndrome: a genetic disorder of leukocyte trafficking. In: Current opinion in allergy and clinical immunology 3 (6), S. 443–450.

Guo, Yiqi; Audry, Magali; Ciancanelli, Michael; Alsina, Laia; Azevedo, Joana; Herman, Melina et al. (2011): Herpes simplex virus encephalitis in a patient with complete TLR3 deficiency: TLR3 is otherwise redundant in protective immunity. In: The Journal of experimental medicine 208 (10), S. 2083–2098.

XVII References

Gupta, Rachna; Warren, Terri; Wald, Anna (2008): Genital herpes. In: The Lancet 370 (9605), S. 2127–2137.

Hagiwara, Keisuke; Uezato, Hiroshi; Arakaki, Hajime; Nonaka, Shigeo; Nonaka, Kimiko; Nonaka, Hideo et al. (2005): A genotype distribution of human papillomaviruses detected by polymerase chain reaction and direct sequencing analysis in a large sample of common warts in Japan. In: Journal of medical virology 77 (1), S. 107–112.

Hammarström, L.; Vorechovsky, I.; Webster, D. (2000): Selective IgA deficiency (SIgAD) and common variable immunodeficiency (CVID). In: Clinical & Experimental Immunology 120 (2), S. 225–231.

Handisurya, Alessandra; Schellenbacher, Christina; Kirnbauer, Reinhard (2009): Diseases caused by human papillomaviruses (HPV). In: JDDG: Journal der Deutschen Dermatologischen Gesellschaft 7 (5), S. 453–466.

Harpaz, R.; Ortega-Sanchez, I. R.; Seward, J. F. (2008): Advisory Committee on Immunization Practices Centers for Disease C, Prevention. Prevention of herpes zoster: recommendations of the advisory committee on immunization practices (ACIP). In: MMWR Recomm Rep 57 (1), S. e30.

Harwood, C. A.; Perrett, C. M.; Brown, V. L.; Leigh, I. M.; McGregor, J. M.; Proby, C. M. (2005): Imiquimod cream 5% for recalcitrant cutaneous warts in immunosuppressed individuals. In: The British journal of dermatology 152 (1), S. 122–129.

Harwood, C. A.; Surentheran, T.; Sasieni, P.; Proby, C. M.; Bordea, C.; Im Leigh et al. (2004): Increased risk of associated with the presence of epidermodysplasia verruciformis human papillomavirus types in normal skin. In: British Journal of Dermatology 150 (5), S. 949–957.

Harwood, Catherine A.; Spink, Patricia J.; Surentheran, T.; Leigh, Irene M.; Villiers, Ethel- Michele de; McGregor, Jane M. et al. (1999): Degenerate and nested PCR: a highly sensitive and specific method for detection of human papillomavirus infection in cutaneous warts. In: Journal of Clinical Microbiology 37 (11), S. 3545–3555.

Hay, Roderick J.; Johns, Nicole E.; Williams, Hywel C.; Bolliger, Ian W.; Dellavalle, Robert P.; Margolis, David J. et al. (2014): The global burden of skin disease in 2010: an analysis of the prevalence and impact of skin conditions. In: The Journal of investigative dermatology 134 (6), S. 1527–1534.

Hay, Roderick James; Fuller, Lucinda Claire (2011): The assessment of dermatological needs in resource-poor regions. In: International journal of dermatology 50 (5), S. 552–557.

XVIII References

Heininger, Ulrich; Seward, Jane F. (2006): Varicella. In: The Lancet 368 (9544), S. 1365– 1376.

Hengge, U. R.; Esser, S.; Schultewolter, T.; Behrendt, C.; Meyer, T.; Stockfleth, E.; Goos, M. (2000): Self‐administered topical 5% imiquimod for the treatment of common warts and molluscum contagiosum. In: British Journal of Dermatology 143 (5), S. 1026–1031.

Holland, Steven M.; DeLeo, Frank R.; Elloumi, Houda Z.; Hsu, Amy P.; Uzel, Gulbu; Brodsky, Nina et al. (2007): STAT3 mutations in the hyper-IgE syndrome. In: New England Journal of Medicine 357 (16), S. 1608–1619.

Hongbo, Yan; Thomas, Charles L.; Harrison, Michael A.; Salek, M. Sam; Finlay, Andrew Y. (2005): Translating the science of quality of life into practice: What do dermatology life quality index scores mean? In: The Journal of investigative dermatology 125 (4), S. 659–664.

Hsu, Amy P.; McReynolds, Lisa J.; Holland, Steven M. (2015): GATA2 deficiency. In: Current opinion in allergy and clinical immunology 15 (1), S. 104–109.

Huber, Michaell A. (2003): Herpes simplex type-1 virus infection. In: Quintessence international 34 (6).

Huff, J. C.; Bean, B.; Balfour Jr, H. H.; Laskin, O. L.; Connor, J. D.; Corey, Lawrence et al. (1988): Therapy of herpes zoster with oral acyclovir. In: The American journal of medicine 85 (2A), S. 84–89.

Iftner, Angelika; Klug, Stefanie J.; Garbe, Claus; Blum, Andreas; Stancu, Alice; Wilczynski, Sharon P.; Iftner, Thomas (2003): The prevalence of human papillomavirus genotypes in nonmelanoma skin cancers of nonimmunosuppressed individuals identifies high-risk genital types as possible risk factors. In: Cancer research 63 (21), S. 7515–7519.

Iwasaki, Akiko; Medzhitov, Ruslan (2004): Toll-like receptor control of the adaptive immune responses. In: Nature immunology 5 (10), S. 987–995.

Jabłonska, Stefania; Majewski, Slawomir; Obalek, Slawomir; Orth, Gérard (1997): Cutaneous warts. In: Clinics in dermatology 15 (3), S. 309–319.

Janeway, Charles A.; Medzhitov, Ruslan (2002): Innate immune recognition. In: Annual review of immunology 20, S. 197–216.

Jobanputra, Raj; Bachmann, Max (2000): The effect of skin diseases on quality of life in patients from different social and ethnic groups in Cape Town, South Africa. In: Int J Dermatol 39 (11), S. 826–831.

Jong-Tieben, Linda M. de; Berkhout, Ron J. M.; Vermeer, Bert J.; van der Woude, Fokko J.; ter Schegget, Jan; Smits, Henk L.; Bouwes Bavinck, Jan N. (1995): High Frequency of Detection of Epidermodysplasia Verruciformis–Associated Human Papillomavirus DNA in XIX References

Biopsies from Malignant and Premalignant Skin Lesions from Renal Transplant Recipients. In: Journal of Investigative Dermatology 105 (3), S. 367–371.

Jowett, Sandra; Ryan, Terence (1985): Skin disease and handicap: an analysis of the impact of skin conditions. In: Social science & medicine 20 (4), S. 425–429.

Kaech, Susan M.; Wherry, E. John; Ahmed, Raft (2002): Effector and memory T-cell differentiation: implications for vaccine development. In: Nature reviews. Immunology 2 (4), S. 251–262.

Kamili, Qurat Ul Ain; Seeborg, Filiz O.; Saxena, Kapil; Nicholas, Sarah K.; Banerjee, Pinaki P.; Angelo, Laura S. et al. (2014): Severe cutaneous human papillomavirus infection associated with natural killer cell deficiency following stem cell transplantation for severe combined immunodeficiency. In: The Journal of allergy and clinical immunology 134 (6), S. 1451–1453.

Katz, Jennifer; Cooper, Edith M.; Walther, Robert R.; Sweeney, Eugene W.; Dworkin, Robert H. (2004): Acute pain in herpes zoster and its impact on health-related quality of life. In: Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 39 (3), S. 342–348.

Khanna, Kamal M.; Lepisto, Andrew J.; Decman, Vilma; Hendricks, Robert L. (2004): Immune control of herpes simplex virus during latency. In: Current opinion in immunology 16 (4), S. 463–469.

Kim, Sun Yae; Jung, Sung Kyu; Lee, Sang Geun; Yi, Sang Min; Kim, Jae Hwan; Kim, Il- Hwan (2013): New alternative combination therapy for recalcitrant common warts: the efficacy of imiquimod 5% cream and duct tape combination therapy. In: Annals of dermatology 25 (2), S. 261–263.

Klasse, P. J.; Sattentau, Q. J. (2002): Occupancy and mechanism in antibody-mediated neutralization of animal viruses. In: Journal of general virology 83 (9), S. 2091–2108.

Klenner, Lars; Hafezi, Wali; Clausen, Björn E.; Lorentzen, Eva U.; Luger, Thomas A.; Beissert, Stefan et al. (2015): Cutaneous RANK–RANKL Signaling Upregulates CD8- Mediated Antiviral Immunity during Herpes simplex Virus Infection by Preventing Virus- Induced Langerhans Cell Apoptosis. In: Journal of Investigative Dermatology 135 (11), S. 2676–2687.

Ko, Jimmy; Radigan, Lin; Cunningham-Rundles, Charlotte (2005): Immune competence and switched memory B cells in common variable immunodeficiency. In: Clinical immunology (Orlando, Fla.) 116 (1), S. 37–41.

XX References

Kodukula, Padma; Liu, Ting; van Rooijen, Nico; Jager, Martine J.; Hendricks, Robert L. (1999): Macrophage control of herpes simplex virus type 1 replication in the peripheral nervous system. In: The Journal of Immunology 162 (5), S. 2895–2905.

Koning, Maurits; Quint, Wim; Struijk, Linda; Kleter, Bernhard; Wanningen, Patrick; van Doorn, Leen-Jan et al. (2006): Evaluation of a novel highly sensitive, broad-spectrum PCR- reverse hybridization assay for detection and identification of beta-papillomavirus DNA. In: Journal of Clinical Microbiology 44 (5), S. 1792–1800.

Koning, Maurits; Schegget, Jan; Eekhof, Just A. H.; Kamp, Marga; Kleter, Bernhard; Gussekloo, Jacobijn et al. (2010): Evaluation of a novel broad-spectrum PCR-multiplex genotyping assay for identification of cutaneous wart-associated human papillomavirus types. In: Journal of Clinical Microbiology 48 (5), S. 1706–1711.

Koning, Maurits; Weissenborn, Sonke Jan; Abeni, Damiano; Bouwes Bavinck, Jan Nico; Euvrard, Sylvie; Green, Adele C. et al. (2009): Prevalence and associated factors of betapapillomavirus infections in individuals without cutaneous . In: The Journal of general virology 90 (Pt 7), S. 1611–1621.

Kreuter, Alexander; Waterboer, Tim; Wieland, Ulrike (2010): Regression of cutaneous warts in a patient with WILD syndrome following recombinant quadrivalent human papillomavirus vaccination. In: Archives of dermatology 146 (10), S. 1196–1197.

Kruetzmann, Stephanie; Rosado, M. Manuela; Weber, Holger; Germing, Ulrich; Tournilhac, Olivier; Peter, Hans-Hartmut et al. (2003): Human immunoglobulin M memory B cells controlling Streptococcus pneumoniae infections are generated in the spleen. In: The Journal of experimental medicine 197 (7), S. 939–945.

Ku, C.-C.; Padilla, J. A.; Grose, C.; Butcher, E. C.; Arvin, A. M. (2002): Tropism of Varicella- Zoster Virus for Human Tonsillar CD4+ T Lymphocytes That Express Activation, Memory, and Skin Homing Markers. In: Journal of virology 76 (22), S. 11425–11433.

Ku, Chia-Chi; Besser, Jaya; Abendroth, Allison; Grose, Charles; Arvin, Ann M. (2005): Varicella-Zoster virus pathogenesis and immunobiology: new concepts emerging from investigations with the SCIDhu mouse model. In: Journal of virology 79 (5), S. 2651–2658.

Ku, Chia-Chi; Zerboni, Leigh; Ito, Hideki; Graham, Brad S.; Wallace, Mark; Arvin, Ann M. (2004): Varicella-zoster virus transfer to skin by T Cells and modulation of viral replication by epidermal cell interferon-alpha. In: The Journal of experimental medicine 200 (7), S. 917– 925.

Kubeyinje, Ep (2009): Evaluation of the efficacy and safety of 0.05% tretinoin cream in the treatment of plane warts in Arab children. In: Journal of Dermatological Treatment 7 (1), S. 21–22. XXI References

Kusne, S.; Schwartz, M.; Breinig, M. K.; Dummer, J. S.; Lee, R. E.; Selby, R. et al. (1991): Herpes Simplex Virus Hepatitis after Solid Organ Transplantation in Adults. In: Journal of Infectious Diseases 163 (5), S. 1001–1007.

Kutukculer, Necil; Azarsiz, Elif; Karaca, Neslihan Edeer; Ulusoy, Ezgi; Koturoglu, Guldane; Aksu, Guzide (2015): A Clinical and Laboratory Approach to the Evaluation of Innate Immunity in Pediatric CVID Patients. In: Frontiers in immunology 6, S. 145.

Kwok, C. S.; Holland, R.; Gibbs, S. (2011): Efficacy of topical treatments for cutaneous warts: a meta-analysis and pooled analysis of randomized controlled trials. In: The British journal of dermatology 165 (2), S. 233–246.

Kwok, Chun Shing; Gibbs, Sam; Bennett, Cathy; Holland, Richard; Abbott, Rachel (2012): Topical treatments for cutaneous warts. In: The Cochrane database of systematic reviews (9), S. CD001781.

Laffort, Caroline; Le Deist, Françoise; Favre, Michel; Caillat-Zucman, Sophie; Radford- Weiss, Isabelle; Fraitag, Sylvie et al. (2004): Severe cutaneous papillomavirus disease after haemopoietic stem-cell transplantation in patients with severe combined immune deficiency caused by common γc cytokine receptor subunit or JAK-3 deficiency. In: The Lancet 363 (9426), S. 2051–2054.

Laiskonis, A.; Thune, T.; Neldam, S.; Hiltunen-Back, E. (2002): Valacyclovir in the treatment of facial herpes simplex virus infection. In: Journal of Infectious Diseases 186 (Supplement 1), S. S66-S70.

Lambe, Teresa; Crawford, Greg; Johnson, Andy L.; Crockford, Tanya L.; Bouriez-Jones, Tiphaine; Smyth, Aisling M. et al. (2011): DOCK8 is essential for T-cell survival and the maintenance of CD8+ T-cell memory. In: European journal of immunology 41 (12), S. 3423– 3435.

Landis, Megan N.; Lookingbill, Donald P.; Sluzevich, Jason C. (2012): Recalcitrant plantar warts treated with recombinant quadrivalent human papillomavirus vaccine. In: Journal of the American Academy of Dermatology 67 (2), S. 4.

Lanzavecchia, Antonio; Sallusto, Federica (2001): Regulation of T Cell Immunity by Dendritic Cells. In: Cell 106 (3), S. 263–266.

Läuchli, Severin; Kempf, Werner; Dragieva, Galya; Burg, Gü nter; Hafner, Jü rg (2003): CO2 laser treatment of warts in immunosuppressed patients. In: Dermatology 206 (2), S. 148–152.

Lazarczyk, Maciej; Cassonnet, Patricia; Pons, Christian; Jacob, Yves; Favre, Michel (2009): The EVER proteins as a natural barrier against papillomaviruses: a new insight into the

XXII References pathogenesis of human papillomavirus infections. In: Microbiology and molecular biology reviews : MMBR 73 (2), S. 348–370.

Lazarczyk, Maciej; Dalard, Cecile; Hayder, Myriam; Dupre, Loic; Pignolet, Beatrice; Majewski, Slawomir et al. (2012): EVER proteins, key elements of the natural anti-human papillomavirus barrier, are regulated upon T-cell activation. In: PloS one 7 (6), S. e39995.

Lazarczyk, Maciej; Favre, Michel (2008): Role of Zn2+ ions in host-virus interactions. In: Journal of virology 82 (23), S. 11486–11494.

Lazarczyk, Maciej; Pons, Christian; Mendoza, Jose-Andres; Cassonnet, Patricia; Jacob, Yves; Favre, Michel (2008): Regulation of cellular zinc balance as a potential mechanism of EVER-mediated protection against pathogenesis by cutaneous oncogenic human papillomaviruses. In: The Journal of experimental medicine 205 (1), S. 35–42.

Le Bon, Agnes; Tough, David F. (2002): Links between innate and adaptive immunity via type I interferon. In: Current opinion in immunology 14 (4), S. 432–436.

Lee, Seung-Hwan; Biron, Christine A. (2010): Here today--not gone tomorrow: roles for activating receptors in sustaining NK cells during viral infections. In: European journal of immunology 40 (4), S. 923–932.

Leiding, Jennifer W.; Holland, Steven M. (2012): Warts and all: human papillomavirus in primary immunodeficiencies. In: The Journal of allergy and clinical immunology 130 (5), S. 1030–1048.

Leung, Lawrence (2011): Recalcitrant nongenital warts. In: Australian family physician 40 (1/2), S. 40, zuletzt geprüft am 19.05.2017.

Lewis, Victoria; Finlay, Andrew Y. (2004): 10 years experience of the Dermatology Life Quality Index (DLQI). In: The journal of investigative dermatology. Symposium proceedings 9 (2), S. 169–180.

Lim, Kim-Chew; Hosoya, Tomonori; Brandt, William; Ku, Chia-Jui; Hosoya-Ohmura, Sakie; Camper, Sally A. et al. (2012): Conditional Gata2 inactivation results in HSC loss and lymphatic mispatterning. In: The Journal of clinical investigation 122 (10), S. 3705–3717.

Lin, Joann H.; Wang, Kevin Y.; Kraft, Sheryl; Roberts, Robert L. (2009): Resolution of warts in association with subcutaneous immunoglobulin in immune deficiency. In: Pediatric dermatology 26 (2), S. 155–158.

Lindelöf, B.; Sigurgeirsson, B.; Gäbel, H.; Stern, R. S. (2000): Incidence of skin cancer in 5356 patients following organ transplantation. In: British Journal of Dermatology 143 (3), S. 513–519.

XXIII References

Lipke, M. M. (2006): An Armamentarium of Wart Treatments. In: Clinical Medicine & Research 4 (4), S. 273–293.

Lodoen, Melissa B.; Lanier, Lewis L. (2006): Natural killer cells as an initial defense against pathogens. In: Current opinion in immunology 18 (4), S. 391–398.

Lynch, Magnus D.; Cliffe, Jane; Morris-Jones, Rachael (2014): Management of cutaneous viral warts. In: BMJ (Clinical research ed.) 348, S. g3339.

Lynn, Jerry; Knight, Adina K.; Kamoun, Malek; Levinson, Arnold I. (2004): A 55-year-old man with hypogammaglobulinemia, lymphopenia, and unrelenting cutaneous warts. In: The Journal of allergy and clinical immunology 114 (2), S. 409–414.

Mace, Emily M.; Hsu, Amy P.; Monaco-Shawver, Linda; Makedonas, George; Rosen, Joshua B.; Dropulic, Lesia et al. (2013): Mutations in GATA2 cause human NK cell deficiency with specific loss of the CD56(bright) subset. In: Blood 121 (14), S. 2669–2677.

Matsumoto, Misako; Funami, Kenji; Tatematsu, Megumi; Azuma, Masahiro; Seya, Tsukasa (2014): Assessment of the Toll-like receptor 3 pathway in endosomal signaling. In: Methods in enzymology 535, S. 149–165.

Matsumoto, Misako; Oshiumi, Hiroyuki; Seya, Tsukasa (2011): Antiviral responses induced by the TLR3 pathway. In: Reviews in medical virology 21 (2), S. 67–77.

McKendrick, M. W.; J I McGill, J. I.; J E White, J. E.; Wood, M. J. (1986): Oral acyclovir in acute herpes zoster. In: BMJ (Clinical research ed.) 293, S. 1529–1532.

Micali, G.; Dall'Oglio, F.; Nasca, M. R. (2003): An open label evaluation of the efficacy of imiquimod 5% cream in the treatment of recalcitrant subungual and periungual cutaneous warts. In: The Journal of dermatological treatment 14 (4), S. 233–236.

Miklos, Zorka; Danis, Vic A.; Adams, Susan; Lloyd, Andrew R.; Adrian, Deborah L.; Cunningham, Anthony L. (1998): In vivo production of cytokines and β (CC) chemokines in human recurrent herpes simplex lesions—do herpes simplex virus-infected keratinocytes contribute to their production? In: Journal of Infectious Diseases 177 (4), S. 827–838.

Miller, Craig S.; Cunningham, Larry L.; Lindroth, John E.; Avdiushko, Sergei A. (2004): The efficacy of valacyclovir in preventing recurrent herpes simplex virus infections associated with dental procedures. In: The Journal of the American Dental Association 135 (9), S. 1311– 1318.

Minegishi, Yoshiyuki; Saito, Masako; Tsuchiya, Shigeru; Tsuge, Ikuya; Takada, Hidetoshi; Hara, Toshiro et al. (2007): Dominant-negative mutations in the DNA-binding domain of STAT3 cause hyper-IgE syndrome. In: Nature 448 (7157), S. 1058–1062.

XXIV References

Mizesko, Melissa C.; Banerjee, Pinaki P.; Monaco-Shawver, Linda; Mace, Emily M.; Bernal, William E.; Sawalle-Belohradsky, Julie et al. (2013): Defective actin accumulation impairs human natural killer cell function in patients with dedicator of cytokinesis 8 deficiency. In: The Journal of allergy and clinical immunology 131 (3), S. 840–848.

Moloney, F. J.; Keane, S.; O'Kelly, P.; Conlon, P. J.; Murphy, G. M. (2005): The impact of skin disease following renal transplantation on quality of life. In: The British journal of dermatology 153 (3), S. 574–578.

Mulhem, Elie; Pinelis, Susanna (2011): Treatment of nongenital cutaneous warts. In: American family physician 84 (3).

Nandakumar, Subhadra; Woolard, Stacie N.; Yuan, Dorothy; Rouse, Barry T.; Kumaraguru, Uday (2008): Natural killer cells as novel helpers in anti-herpes simplex virus immune response. In: Journal of virology 82 (21), S. 10820–10831.

Newton, Robert (2000): Molecular mechanisms of glucocorticoid action: what is important? In: Thorax 55 (7), S. 603–613.

Nishimura, Masuhiro; Naito, Shinsaku (2005): Tissue-specific mRNA expression profiles of human toll-like receptors and related genes. In: Biological and Pharmaceutical Bulletin 28 (5), S. 886–892.

Ono, Fumitake; Yasumoto, Shinichiro; Furumura, Minao; Hamada, Takahiro; Ishii, Norito; Gyotoku, Takashi et al. (2012): Comparison between famciclovir and valacyclovir for acute pain in adult Japanese immunocompetent patients with herpes zoster. In: The Journal of dermatology 39 (11), S. 902–908.

Orange, Jordan S. (2012): Unraveling human natural killer cell deficiency. In: The Journal of clinical investigation 122 (3), S. 798–801.

Orange, Jordan S. (2013): Natural killer cell deficiency. In: The Journal of allergy and clinical immunology 132 (3), S. 515.

Orth, G. (2008): Host defenses against human papillomaviruses: lessons from epidermodysplasia verruciformis. In: Immunology, Phenotype First: How Mutations Have Established New Principles and Pathways in Immunology: Springer, S. 59–83.

Oxman, Michael N.; Levin, Myron J.; Johnson, Gary R.; Schmader, K. E.; Straus, S. E.; Gelb, L. D. et al. (2005): A vaccine to prevent herpes zoster and postherpetic neuralgia in older adults. In: New England Journal of Medicine 352 (22), S. 2271–2284.

Patel, Tejas; Morrison, L. Katie; Rady, Peter; Tyring, Stephen (2010): Epidermodysplasia verruciformis and susceptibility to HPV. In: Disease markers 29 (3-4), S. 199–206.

XXV References

Perez de Diego, Rebeca; Sancho-Shimizu, Vanessa; Lorenzo, Lazaro; Puel, Anne; Plancoulaine, Sabine; Picard, Capucine et al. (2010): Human TRAF3 adaptor molecule deficiency leads to impaired Toll-like receptor 3 response and susceptibility to herpes simplex encephalitis. In: Immunity 33 (3), S. 400–411.

Pisegna, Simona; Pirozzi, Gianluca; Piccoli, Mario; Frati, Luigi; Santoni, Angela; Palmieri, Gabriella (2004): p38 MAPK activation controls the TLR3-mediated up-regulation of cytotoxicity and cytokine production in human NK cells. In: Blood 104 (13), S. 4157–4164.

Plotkin, Stanley A. (2008): Vaccines: correlates of vaccine-induced immunity. In: Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 47 (3), S. 401–409.

Porro, A. M.; Alchorne, M. M. A.; Mota, G. R.; Michalany, N.; Pignatari, A. C.C.; Souza, I. E. (2003): Detection and typing of human papillomavirus in cutaneous warts of patients infected with human immunodeficiency virus type 1. In: British Journal of Dermatology 149 (6), S. 1192–1199.

Préhaud, Christophe; Mégret, Françoise; Lafage, Mireille; Lafon, Monique (2005): Virus infection switches TLR-3-positive human neurons to become strong producers of beta interferon. In: Journal of virology 79 (20), S. 12893–12904.

Raborn, G. Wayne; McGaw, W. T.; Grace, M.; Tyrrell, L. David; Samuels, Sheri M. (1987): Oral acyclovir and herpes labialis: a randomized, double-blind, placebo-controlled study. In: The Journal of the American Dental Association 115 (1), S. 38–42.

Rahimi, Hanieh; Mara, Tom; Costella, John; Speechley, Mark; Bohay, Richard (2012): Effectiveness of antiviral agents for the prevention of recurrent herpes labialis: a systematic review and meta-analysis. In: Oral surgery, oral medicine, oral pathology and oral radiology 113 (5), S. 618–627.

Ramoz, Nicolas; Rueda, Luis-Alfredo; Bouadjar, Bakar; Montoya, Luz-Stella; Orth, Gerard; Favre, Michel (2002): Mutations in two adjacent novel genes are associated with epidermodysplasia verruciformis. In: Nature genetics 32 (4), S. 579–581.

Reading, Patrick C.; Whitney, Paul G.; Barr, Daniel P.; Smyth, Mark J.; Brooks, Andrew G. (2006): NK cells contribute to the early clearance of HSV-1 from the lung but cannot control replication in the central nervous system following intranasal infection. In: European journal of immunology 36 (4), S. 897–905.

Reid, T.M.S.; Fraser, N. G.; Kernohan, I. R. (1976): Generalized warts and immune deficiency. In: Br J Dermatol 95 (5), S. 559–564.

XXVI References

Rodrigues, Neil P.; Janzen, Viktor; Forkert, Randolf; Dombkowski, David M.; Boyd, Ashleigh S.; Orkin, Stuart H. et al. (2005): Haploinsufficiency of GATA-2 perturbs adult hematopoietic stem-cell homeostasis. In: Blood 106 (2), S. 477–484.

Rogers, Heather D.; Macgregor, Jennifer L.; Nord, Kristin M.; Tyring, Stephen; Rady, Peter; Engler, Danielle E.; Grossman, Marc E. (2009): Acquired epidermodysplasia verruciformis. In: Journal of the American Academy of Dermatology 60 (2), S. 315–320.

Rooney, James F.; Straus, Stephen E.; Mannix, Margaret L.; Wohlenberg, Charles R.; Alling, David W.; Dumois, Juan A.; Notkins, Abner Louis (1993): Oral acyclovir to suppress frequently recurrent herpes labialis: a double-blind, placebo-controlled trial. In: Annals of internal medicine 118 (4), S. 268–272.

Rote Liste® Service GmbH (2017): Aldara® 5% Creme. Online verfügbar unter https://online.rote-liste.de/suche/praep/16273/Aldara%C2%AE%205%25%20Creme, zuletzt aktualisiert am 22.05.2017.

Rübben, A. (2011): Klinischer Algorithmus zur Therapie von kutanen, extragenitalen HPV- induzierten Warzen. In: Der Hautarzt 62 (1), S. 6–16.

Rübben, A.; Kalka, Katrin; Spelten, Bettina; Grußendorf-Conen, Elke-Ingrid (1997): Clinical features and age distribution of patients with HPV 2/27/57-induced common warts. In: Archives of Dermatological Research 289 (6), S. 337–340.

Sabatini, Linda; Yungbluth, Peggy (2006): Herpes simplex virus infections and current methods for laboratory detection. In: Clinical Microbiology Newsletter 28 (24), S. 185–192.

Sanchez-Ramon, Silvia; Radigan, Lin; Yu, Joyce E.; Bard, Susan; Cunningham-Rundles, Charlotte (2008): Memory B cells in common variable immunodeficiency: clinical associations and sex differences. In: Clinical immunology (Orlando, Fla.) 128 (3), S. 314–321.

Schmader, Kenneth E.; Dworkin, Robert H. (2008): Natural history and treatment of herpes zoster. In: The journal of pain : official journal of the American Pain Society 9 (1 Suppl 1), S. 9.

Schmader, Kenneth E.; Levin, Myron J.; Gnann, John W., JR; McNeil, Shelly A.; Vesikari, Timo; Betts, Robert F. et al. (2012): Efficacy, safety, and tolerability of herpes zoster vaccine in persons aged 50-59 years. In: Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 54 (7), S. 922–928.

Schmader, Kenneth E.; Sloane, Richard; Pieper, Carl; Coplan, Paul M.; Nikas, Alexander; Saddier, Patricia et al. (2007): The impact of acute herpes zoster pain and discomfort on functional status and quality of life in older adults. In: The Clinical journal of pain 23 (6), S. 490–496.

XXVII References

Schmitt, Markus; Koning, Maurits N. C. de; Eekhof, Just A. H.; Quint, Wim G. V.; Pawlita, Michael (2011): Evaluation of a novel multiplex human papillomavirus (HPV) genotyping assay for HPV types in skin warts. In: Journal of Clinical Microbiology 49 (9), S. 3262–3267.

Schofield, Julia; Grindlay, Douglas; Williams, Hywel (2009): Skin conditions in the UK: a health care needs assessment: Centre of Evidence Based Dermatology, University of Nottingham.

Schroder, Martina; Bowie, Andrew G. (2005): TLR3 in antiviral immunity: key player or bystander? In: Trends in immunology 26 (9), S. 462–468.

Sciubba, J. J. (2002): Herpes simplex and aphthous ulcerations: presentation, diagnosis and management--an update. In: General dentistry 51 (6), S. 510–516.

Seifert, Marc; Przekopowitz, Martina; Taudien, Sarah; Lollies, Anna; Ronge, Viola; Drees, Britta et al. (2015): Functional capacities of human IgM memory B cells in early inflammatory responses and secondary germinal center reactions. In: Proceedings of the National Academy of Sciences 112 (6), S. E546-E555.

Sen, Nandini; Che, Xibing; Rajamani, Jaya; Zerboni, Leigh; Sung, Phillip; Ptacek, Jason; Arvin, Ann M. (2012): Signal transducer and activator of transcription 3 (STAT3) and survivin induction by varicella-zoster virus promote replication and skin pathogenesis. In: Proceedings of the National Academy of Sciences of the United States of America 109 (2), S. 600–605.

Seward, Jane F.; Zhang, John X.; Maupin, Teresa J.; Mascola, Laurene; Jumaan, Aisha O. (2004): Contagiousness of varicella in vaccinated cases: a household contact study. In: JAMA 292 (6), S. 704–708.

Shafran, Stephen D.; Tyring, Stephen K.; Ashton, Richard; Decroix, Jacque; Forszpaniak, Chistine; Wade, Alan et al. (2004): Once, twice, or three times daily famciclovir compared with aciclovir for the oral treatment of herpes zoster in immunocompetent adults. A randomized, multicenter, double-blind clinical trial. In: Journal of Clinical Virology 29 (4), S. 248–253.

Shamanin, Vladimir; Glover, Mary; Rausch, Christine; Proby, Charlotte; Leigh, Irene M.; Zur Hausen, Harald; Villiers, Ethel-Michele de (1994): Specific types of human papillomavirus found in benign proliferations and carcinomas of the skin in immunosuppressed patients. In: Cancer research 54 (17), S. 4610–4613.

Sheng, Kuo-Ching; Day, Stephanie; Wright, Mark D.; Stojanovska, Lily; Apostolopoulos, Vasso (2013): Enhanced Dendritic Cell-Mediated Antigen-Specific CD4+ T Cell Responses: IFN-Gamma Aids TLR Stimulation. In: Journal of drug delivery 2013, S. 516749.

XXVIII References

Siegel, Michael A. (2002): Diagnosis and management of recurrent herpes simplex infections. In: The Journal of the American Dental Association 133 (9), S. 1245–1249.

Siegrist, Claire-Anne (2008): Vaccine immunology. In: Vaccines 5, S. 1725.

Silling, Steffi; Wieland, Ulrike; Werner, Marko; Pfister, Herbert; Potthoff, Anja; Kreuter, Alexander (2014): Resolution of novel human papillomavirus-induced warts after HPV vaccination. In: Emerging infectious diseases 20 (1), S. 142–145.

Simmons, Anthony (2002): Clinical manifestations and treatment considerations of herpes simplex virus infection. In: Journal of Infectious Diseases 186 (Supplement 1), S. S71-S77.

Simon, A. Katharina; Hollander, Georg A.; McMichael, Andrew (2015): Evolution of the immune system in humans from infancy to old age. In: Proceedings. Biological sciences 282 (1821), S. 20143085.

Sladden, Michael J.; Johnston, Graham A. (2004): Common skin infections in children. In: BMJ (Clinical research ed.) 329 (7457), S. 95–99.

Sloan, Kevin; Haberman, Herbert; Lynde, Charles W. (1998): Carbon dioxide laser-treatment of resistant verrucae vulgaris: retrospective analysis. In: Journal of cutaneous medicine and surgery 2 (3), S. 142–145.

Spinner, Michael A.; Sanchez, Lauren A.; Hsu, Amy P.; Shaw, Pamela A.; Zerbe, Christa S.; Calvo, Katherine R. et al. (2014): GATA2 deficiency: a protean disorder of hematopoiesis, lymphatics, and immunity. In: Blood 123 (6), S. 809–821.

Spruance, S. L.; Nett, R.; Marbury, T.; Wolff, R.; Johnson, J. (2002): Acyclovir Cream for Treatment of Herpes Simplex Labialis: Results of Two Randomized, Double-Blind, Vehicle- Controlled, Multicenter Clinial Trials. In: Antimicrobial Agents and Chemotheraphy 46 (7), S. 2238–2243.

Spruance, S. L.; Stewart, J. C. B.; Rowe, N. H.; McKeough, M.B., Wenerstrom, G.; Freeman, D. J. (1990): Treatment of Recurrent Herpes Simplex Labialis with Oral Acyclovir. In: The Journal of infectious diseases 161, S. 185–190.

Spruance, Spotswood L.; Bodsworth, Neil; Resnick, Harvey; Conant, Marcus; Oeuvray, Claude; Gao, Joseph; Hamed, Kamal (2006): Single-dose, patient-initiated famciclovir: a randomized, double-blind, placebo-controlled trial for episodic treatment of herpes labialis. In: Journal of the American Academy of Dermatology 55 (1), S. 47–53.

Spruance, Spotswood L.; Jones, Terry M.; Blatter, Mark M.; Vargas-Cortes, Mauricio; Barber, Judy; Hill, Joanne et al. (2003): High-dose, short-duration, early valacyclovir therapy for episodic treatment of cold sores: results of two randomized, placebo-controlled, multicenter studies. In: Antimicrobial Agents and Chemotheraphy 47 (3), S. 1072–1080.

XXIX References

Spruance, Spotswood L.; Overall Jr, James C.; Kern, Earl R.; Krueger, Gerald G.; Pliam, Victoria; Miller, William (1977): The natural history of recurrent herpes simplex labialis: implications for antiviral therapy. In: New England Journal of Medicine 297 (2), S. 69–75.

Spruance, Spotswood L.; Rowe, Nathaniel H.; Raborn, G. Wayne; Thibodeau, Edward A.; D’Ambrosio, Joseph A.; Bernstein, David I. (1999): Peroral Famciclovir in the Treatment of Experimental Ultraviolet Radiation–Induced Herpes Simplex Labialis: A Double-Blind, Dose- Ranging,Placebo-Controlled, Multicenter Trial. In: The Journal of infectious diseases 179, S. 303–310.

Sri, Jennifer C.; Dubina, Meghan I.; Kao, Grace F.; Rady, Peter L.; Tyring, Stephen K.; Gaspari, Anthony A. (2012): Generalized verrucosis: a review of the associated diseases, evaluation, and treatments. In: Journal of the American Academy of Dermatology 66 (2), S. 292–311.

Stanley, M. A. (2009): Immune responses to human papilloma viruses.

Stanley, Margaret; Gissmann, Lutz; Nardelli-Haefliger, Denise (2008): Immunobiology of human papillomavirus infection and vaccination - implications for second generation vaccines. In: Vaccine 26 Suppl 10, S. 7.

Stanley, Margaret; Pinto, Ligia A.; Trimble, Connie (2012): Human papillomavirus vaccines-- immune responses. In: Vaccine 30 Suppl 5, S. 7.

Stanley, Margaret A. (2001): Immunobiology of papillomavirus infections. In: Journal of reproductive immunology 52 (1), S. 45–59.

Steiner, Israel; Kennedy, Peter G. E.; Pachner, Andrew R. (2007): The neurotropic herpes viruses. Herpes simplex and varicella-zoster. In: The Lancet Neurology 6 (11), S. 1015– 1028.

Steinman, Ralph M.; Banchereau, Jacques (2007): Taking dendritic cells into medicine. In: Nature 449 (7161), S. 419–426.

Sterling, J. C.; Gibbs, S.; Haque Hussain, S. S.; Mohd Mustapa, M. F.; Handfield-Jones, S. E. (2014): British Association of Dermatologists' guidelines for the management of cutaneous warts 2014. In: The British journal of dermatology 171 (4), S. 696–712.

Sterling, J. C.; Handfield‐Jones, S.; Hudson, P. M. (2001): Guidelines for the management of cutaneous warts. In: British Journal of Dermatology 144 (1), S. 4–11.

Stock, Christiane; Guillén-Grima, Francisco; Mendoza, Juan Hermoso de; Marin-Fernandez, Blanca; Aguinaga-Ontoso, Ines; Krämer, Alexander (2001): Risk factors of herpes simplex type 1 (HSV-1) infection and lifestyle factors associated with HSV-1 manifestations. In: European journal of epidemiology 17 (9), S. 885–890.

XXX References

Street, Marcy L.; Roenigk, Randall K. (1990): Recalcitrant periungual verrucae: the role of carbon dioxide laser vaporization. In: Journal of the American Academy of Dermatology 23 (1), S. 115–120.

Swain, Susan L.; McKinstry, K. Kai; Strutt, Tara M. (2012): Expanding roles for CD4(+) T cells in immunity to viruses. In: Nature reviews. Immunology 12 (2), S. 136–148.

Tangye, S. G.; Good, K. L. (2007): Human IgM+CD27+ B Cells. Memory B Cells or "Memory" B Cells? In: The Journal of Immunology 179 (1), S. 13–19.

Tassone, Laura; Moratto, Daniele; Vermi, William; Francesco, Maria de; Notarangelo, Lucia D.; Porta, Fulvio et al. (2010): Defect of plasmacytoid dendritic cells in warts, hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome patients. In: Blood 116 (23), S. 4870–4873.

Theofilopoulos, Argyrios N.; Baccala, Roberto; Beutler, Bruce; Kono, Dwight H. (2005): Type I interferons (alpha/beta) in immunity and autoimmunity. In: Annual review of immunology 23, S. 307–336.

Thomas, K.; Keogh-Brown, M.; Chalmers, J.; Fordham, R.; Holland, R.; Armstrong, S. et al. (2006): Effectiveness and cost-effectiveness of salicylic acid and cryotherapy for cutaneous warts. An economic decision model. In: Health Technol Assess 10 (25).

Thomas, R. MeyrickH; Dodd, H. J.; YEO, JANE M.; Kirby, J. D.T. (1985): Oral acyclovir in the suppression of recurrent non‐genital herpes simplex virus infection. In: British Journal of Dermatology 113 (6), S. 731–735.

Town, T.; Jeng, D.; Alexopoulou, L.; Tan, J.; Flavell, R. A. (2006): Microglia Recognize Double-Stranded RNA via TLR3. In: The Journal of Immunology 176 (6), S. 3804–3812.

Trinchieri, GIORGIO; Santoli, DANIELA (1978): Anti-viral activity induced by culturing lymphocytes with tumor-derived or virus-transformed cells. Enhancement of human natural killer cell activity by interferon and antagonistic inhibition of susceptibility of target cells to lysis. In: Journal of Experimental Medicine 147 (5), S. 1314–1333.

Tseng, Hung Fu; Chi, Margaret; Smith, Ning; Marcy, Stephen M.; Sy, Lina S.; Jacobsen, Steven J. (2012): Herpes zoster vaccine and the incidence of recurrent herpes zoster in an immunocompetent elderly population. In: The Journal of infectious diseases 206 (2), S. 190– 196.

Tyring, S. K. (2000): Antiviral Therapy for Herpes Zoster. Randomized, Controlled Clinical Trial of Valacyclovir and Famciclovir Therapy in Immunocompetent Patients 50 Years and Older. In: Archives of Family Medicine 9 (9), S. 863–869.

XXXI References

Tyring, Stephen; Barbarash, Rick A.; Nahlik, James E.; Cunningham, Anthony; Marley, John; Heng, Madalene et al. (1995): Famciclovir for the treatment of acute herpes zoster: effects on acute disease and postherpetic neuralgia: a randomized, double-blind, placebo-controlled trial. In: Annals of internal medicine 123 (2), S. 89–96.

Tyring, Stephen K. (1998): Advances in the treatment of herpesvirus infection: the role of famciclovir. In: Clinical therapeutics 20 (4), S. 661–670.

Tyring, Stephen K. (2007): Management of herpes zoster and postherpetic neuralgia. In: Journal of the American Academy of Dermatology 57 (6 Suppl), S. 42.

Tyring, Stephen K.; Baker, David; Snowden, Wendy (2002): Valacyclovir for herpes simplex virus infection: long-term safety and sustained efficacy after 20 years’ experience with acyclovir. In: Journal of Infectious Diseases 186 (Supplement 1), S. S40-S46.

Uluhan, A.; Sager, D.; Jasin, H. E. (1998): Juvenile rheumatoid arthritis and common variable hypogammaglobulinemia. In: The Journal of rheumatology 25 (6), S. 1205–1210. van der Burg, Mirjam; Gennery, Andy R. (2011): Educational paper. The expanding clinical and immunological spectrum of severe combined immunodeficiency. In: European journal of pediatrics 170 (5), S. 561–571.

Venugopal, Supriya S.; Murrell, Dedee F. (2010): Recalcitrant cutaneous warts treated with recombinant quadrivalent human papillomavirus vaccine (types 6, 11, 16, and 18) in a developmentally delayed, 31-year-old white man. In: Archives of dermatology 146 (5), S. 475–477.

Vicente, Carmen; Conchillo, Ana; Garcia-Sanchez, Maria A.; Odero, Maria D. (2012): The role of the GATA2 transcription factor in normal and malignant hematopoiesis. In: Critical reviews in oncology/hematology 82 (1), S. 1–17.

Villiers, Ethel-Michele de; Fauquet, Claude; Broker, Thomas R.; Bernard, Hans-Ulrich; Zur Hausen, Harald (2004): Classification of papillomaviruses. In: Virology 324 (1), S. 17–27.

Vinh, Donald C.; Patel, Smita Y.; Uzel, Gulbu; Anderson, Victoria L.; Freeman, Alexandra F.; Olivier, Kenneth N. et al. (2010): Autosomal dominant and sporadic monocytopenia with susceptibility to mycobacteria, fungi, papillomaviruses, and myelodysplasia. In: Blood 115 (8), S. 1519–1529.

Vu, Jenny; Wallace, Genevieve R.; Singh, Rajendra; Diwan, Hafeez; Prieto, Victor; Rady, Peter et al. (2007): Common variable immunodeficiency syndrome associated with epidermodysplasia verruciformis. In: American journal of clinical dermatology 8 (5), S. 307– 310.

XXXII References

Wagstaff, Antona J.; Perry, Caroline M. (2007): Topical Imiquimod. In: Drugs 67 (15), S. 2187–2210.

Wall, Simeon H.; Ramey, Stephen J.; Wall, Forrest (1999): Famciclovir as antiviral prophylaxis in laser resurfacing procedures. In: Plastic and reconstructive surgery 104 (4), S. 1103–1108.

Warnatz, K. (2002): Severe deficiency of switched memory B cells (CD27+IgM-IgD-) in subgroups of patients with common variable immunodeficiency. A new approach to classify a heterogeneous disease. In: Blood 99 (5), S. 1544–1551.

Weissenborn, S. J.; Hopfl, R.; Weber, F.; Smola, H.; Pfister, H. J.; Fuchs, P. G. (1999): High prevalence of a variety of epidermodysplasia verruciformis-associated human papillomaviruses in psoriatic skin of patients treated or not treated with PUVA. In: The Journal of investigative dermatology 113 (1), S. 122–126.

Weissenborn, Soenke Jan; Nindl, Ingo; Purdie, Karin; Harwood, Catherine; Proby, Charlotte; Breuer, Judy et al. (2005): Human papillomavirus-DNA loads in actinic keratoses exceed those in non- skin cancers. In: The Journal of investigative dermatology 125 (1), S. 93–97.

Weissenborn, Sonke J.; Wieland, Ulrike; Junk, Monika; Pfister, Herbert (2010): Quantification of beta-human papillomavirus DNA by real-time PCR. In: Nature protocols 5 (1), S. 1–13.

Weller, Sandra; Braun, Moritz C.; Tan, Bruce K.; Rosenwald, Andreas; Cordier, Corinne; Conley, Mary Ellen et al. (2004): Human blood IgM “memory” B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire. In: Blood 104 (12), S. 3647–3654.

Wesch, D.; Beetz, S.; Oberg, H.-H.; Marget, M.; Krengel, K.; Kabelitz, D. (2006): Direct Costimulatory Effect of TLR3 Ligand Poly(I. C) on Human T Lymphocytes. In: The Journal of Immunology 176 (3), S. 1348–1354.

Whitley, Richard J.; Roizman, Bernard (2001): Herpes simplex virus infections. In: The Lancet 357 (9267), S. 1513–1518.

Whitley, Richard J.; Volpi, Antonio; McKendrick, Mike; van Wijck, Albert; Oaklander, Anne Louise (2010): Management of herpes zoster and post-herpetic neuralgia now and in the future. In: Journal of Clinical Virology 48, S. S20-S28.

Wittek, Miriam; Doerr, Hans Wilhelm; Allwinn, Regina (2010): Varizellen und Herpes zoster. Teil 1: Virologie, Epidemiologie, Klinik, Labordiagnostik. In: Medizinische Klinik (Munich, Germany : 1983) 105 (5), S. 334–338.

XXXIII References

Woo, Sook-Bin; Challacombe, Stephen J. (2007): Management of recurrent oral herpes simplex infections. In: Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics 103 Suppl, S. 18.

Wood, M. J.; Kay, R.; Dworkin, R. H.; Soong, S-J; Whitley, R. J. (1996): Oral acyclovir therapy accelerates pain resolution in patients with herpes zoster: a meta-analysis of placebo-controlled trials. In: Clinical infectious diseases 22 (2), S. 341–347.

Woodworth, Craig D. (2002): HPV innate immunity. In: Frontiers in bioscience: a journal and virtual library 7, S. 71.

Yawn, Barbara P.; Gilden, Don (2013): The global epidemiology of herpes zoster. In: Neurology 81 (10), S. 928–930.

Yawn, Barbara P.; Saddier, Patricia; Wollan, Peter C.; Sauver, Jennifer L. St; Kurland, Marge J.; Sy, Lina S. (Hg.) (2007): A population-based study of the incidence and complication rates of herpes zoster before zoster vaccine introduction: Elsevier (82).

Yawn, Barbara P.; Wollan, Peter C.; Kurland, Marge J.; Sauver, Jennifer L. St; Saddier, Patricia (Hg.) (2011): Herpes zoster recurrences more frequent than previously reported: Elsevier (86).

Yong, Patrick F. K.; Freeman, Alexandra F.; Engelhardt, Karin R.; Holland, Steven; Puck, Jennifer M.; Grimbacher, Bodo (2012): An update on the hyper-IgE syndromes. In: Arthritis research & therapy 14 (6), S. 228.

Zachariae, Claus; Sand, Carsten; Hansen, Jesper Melchior; Sorensen, Soren Schwartz; Koch, Karen; Villumsen, John; Axelsen, Mads (2012): Warts in a cohort of Danish kidney transplanted patients: impact on quality of life. In: Acta dermato-venereologica 92 (6), S. 615–618.

Zachariae, Robert; Zachariae, C. O.C.; Ibsen, Hans; Mortensen, J. Touborg; Wulf, H. Christian (2000): Dermatology life quality index: data from Danish inpatients and outpatients. In: ACTA DERMATOVENEREOLOGICA-STOCKHOLM- 80 (4), S. 272–276.

Zarember, K. A.; Godowski, P. J. (2002): Tissue Expression of Human Toll-Like Receptors and Differential Regulation of Toll-Like Receptor mRNAs in Leukocytes in Response to Microbes, Their Products, and Cytokines. In: The Journal of Immunology 168 (2), S. 554– 561.

Zerboni, Leigh; Sen, Nandini; Oliver, Stefan L.; Arvin, Ann M. (2014): Molecular mechanisms of varicella zoster virus pathogenesis. In: Nature Reviews Microbiology 12 (3), S. 197–210.

XXXIV References

Zhang, Qian; Davis, Jeremiah C.; Lamborn, Ian T.; Freeman, Alexandra F.; Jing, Huie; Favreau, Amanda J. et al. (2009): Combined immunodeficiency associated with DOCK8 mutations. In: The New England journal of medicine 361 (21), S. 2046–2055.

Zhang, Shen-Ying; Jouanguy, Emmanuelle; Sancho-Shimizu, Vanessa; Bernuth, Horst von; Yang, Kun; Abel, Laurent et al. (2007a): Human Toll-like receptor-dependent induction of interferons in protective immunity to viruses. In: Immunological reviews 220, S. 225–236.

Zhang, Shen-Ying; Jouanguy, Emmanuelle; Ugolini, Sophie; Smahi, Asma; Elain, Gaelle; Romero, Pedro et al. (2007b): TLR3 deficiency in patients with herpes simplex encephalitis. In: Science (New York, N.Y.) 317 (5844), S. 1522–1527.

XXXV Appendix

9 Appendix

9.1 Blood Values Spreadsheet

XXXVI Appendix

XXXVII Appendix

XXXVIII Appendix

XXXIX Appendix

9.2 Questionnaire Dermatology Quality of Life Index (DLQI)

DERMATOLOGY QUALITY OF LIFE INDEX

DLQI Hospital No: Date: Name: Score: Address: Diagnosis:

The aim of this questionnaire is to measure how much your skin problem has affected your life OVER THE LAST WEEK. Please tick one box for each question.

1. Over the last week, how itchy, sore, Very much  painful or stinging has your skin A lot  been? A little  Not at all 

2. Over the last week, how embarrassed Very much  or self conscious have you been because A lot  of your skin? A little  Not at all 

3. Over the last week, how much has your Very much  skin interfered with you going A lot  shopping or looking after your home or A little  garden? Not at all  Not relevant 

4. Over the last week, how much has your Very much  skin influenced the clothes A lot  you wear? A little  Not at all  Not relevant 

5. Over the last week, how much has your Very much  skin affected any social or A lot  leisure activities? A little  Not at all  Not relevant 

XL Appendix

6. Over the last week, how much has your Very much  skin made it difficult for A lot  you to do any sport? A little  Not at all  Not relevant 

7. Over the last week, has your skin prevented Yes  you from working or studying? No           Not relevant 

If "No", over the last week how much has A lot  your skin been a problem at A little  work or studying? Not at all 

8. Over the last week, how much has your Very much  skin created problems with your A lot  partner or any of your close friends A little  or relatives? Not at all           Not relevant 

9. Over the last week, how much has your Very much  skin caused any sexual A lot  difficulties? A little  Not at all           Not relevant 

10. Over the last week, how much of a Very much  problem has the treatment for your A lot  skin been, for example by making A little  your home messy, or by taking up time? Not at all           Not relevant  Please check you have answered EVERY question. Thank you.

AY Finlay, GK Khan, April 1992 www.dermatology.org.uk, this must not be copied without the permission of the authors.

XLI Appendix

9.3 Questionnaire Herpes Patients

Department of Clinical Immunology IMMUNOLOGY CLINIC (Incorporating The Immunodeficiency Clinic)

Head of Department Specialist Dr Magdalena Dziadzio Royal Free Hospital Prof Hans Stauss Registrars Dr. Jaroslava Orosova Pond Street Dr Mari Campbell Clinical Lead Clinical Svenja Abel London NW3 2QG Dr Siobhan Burns Psychologist Sarita Workman Switchboard: 020 7794 0500 Medical Student Andrew Symes www.royalfree.nhs.uk Dr R Chee Sisters Irene Wahlberg Prof. B Grimbacher Chanell Pritchard Dr David Lowe Staff Nurse Tel: 0207 830 2141 Dr Suranjith Seneviratne Secretary Fax: 0207 830 2224 Clinic and Appointment Enquiries on Tuesdays, Wednesdays, and Thursdays Tel: 020 7794 0500 ext 22525

Questionnaire for patients with recurrent herpes infections

We are currently taking a closer look at patients with recurrent herpes infection. This questionnaire will give us a better overall picture of this condition and shall be used as a foundation for further, more in-depth research. We would be more than grateful if you supported our research by completing this questionnaire. It will take about 10 minutes to answer.

NAME: ______DATE: ______

1. When did you start to get herpes infections?

Age of onset: or Year of onset:

2. Do you know which herpes virus is causing your problems? ☐ Herpes simplex virus Type 1 (HSV 1), ☐ Herpes simplex virus Type 2 (HSV2), ☐ Varizella Zoster Virus (VZV), ☐ Other :______☐ I don’t know, ☐No clear diagnosis

3. Was there a trigger for your herpes infections to start? ☐ YES: ______☐ NO

4. Where do you have herpes infections? (please tick all that apply)

Orolabial herpes: ☐ lips, ☐ mouth, ☐ gums, ☐ tongue Facial herpes: ☐ chin, ☐ nose, ☐ ear, ☐ cheek, ☐ eye (Herpes Keratitis) Genital herpes: ☐ buttocks, ☐thigh, ☐perianal, ☐labia, ☐penis, ☐internal genital

XLII Appendix

☐ (finger/thumb) ☐ other: ______Please in addition mark in Picture A where you have herpes infections.

Picture A

5. Are your herpes infections always in the same location or do the locations vary? ☐ always in the same spots, ☐ locations vary

6. How often did you get outbreaks before you started treatment?

times a month OR times a year OR every years

7. What treatment do you have? ☐ I only take medication during an acute episode please go on to Question 8 A).

☐ I take medication prophylactically all the time please go on to Question 8 B).

8. Please give more precise information about your medication A) (acute therapy only) Medication How many mg or g per For how long? day/week? ☐ Aciclovir ☐ Valaciclovir ☐ Famciclovir

XLIII Appendix

☐ other:

B) (prophylactic medication only) Medication How many mg or g per How many mg or g per day/week? day/week during flare up? ☐ Aciclovir ☐ Valaciclovir

☐ Famciclovir ☐ other:

9. Does the medication help? ☐ YES ☐ NO If yes how? : ☐ symptoms are less severe, ☐ lesion is smaller, ☐ duration of outbreak is shorter, ☐ frequency of outbreaks are less: times per month OR times per year (please state number of outbreaks) ☐ other: ______

9. If you are on prophylactic medication, do you still get breakthroughs? ☐ YES ☐ NO

If yes how often? times a month OR times a year OR every years

10. Have you had a shingles vaccine (Zostavax)? This is a vaccination against Varicella Zoster virus, the same virus that causes chickenpox. ☐ YES ☐ NO If yes, when did you have the vaccine? ______and did it improve your symptoms? ☐ YES ☐ NO If yes how? : ☐ symptoms are less severe, ☐ lesion is smaller, ☐ duration of outbreak is shorter, ☐ frequency of outbreaks are lower: times per month OR times per year (please state number of outbreaks) ☐ other: ______

11. Is there a trigger now for getting flare ups?

XLIV Appendix

☐ YES ☐ NO If yes, which? : ☐other infections, ☐physical or emotional stress, ☐ sun exposure, ☐ menstruation, ☐ other: ______

12. Do you have warning symptoms (tingling, pain, burning sensation, or itching at the site of reactivation) before you have a flare up? ☐ YES ☐ NO If yes, please list those: ______

13. Is there anything you have difficulty doing because of herpes infections? ☐ YES ☐ NO If yes, what is difficult and why? ______

14. Have you had any of the following herpes virus complication? ☐ , ☐ , ☐ Herpes encephalitis, ☐urinary retention, ☐ herpes meningitis, ☐ other ______☐ NONE of them

15. Does anyone in your family suffer from recurrent herpes infections (6 or more annual outbreaks)? ☐ YES ☐ NO If yes, who is it? ☐ mother, ☐ father, ☐ sister, ☐ brother, ☐ son, ☐ daughter, ☐ other: ______

16. Are you on any immunosuppressive medication (e.g. Steroids, Chemotherapy, Antibodies or drugs that suppress your immune system) ? ☐ YES ☐ NO If yes, what is the name of the medication? ______

17. Have you been diagnosed with a primary immunodeficiency (PID)? ☐ YES ☐ NO If yes, what is your diagnosis? ______

XLV Appendix

18. Do you suffer from other recurrent infections (infections you needed medication from your doctor for)? ☐YES ☐NO If yes: a) Site of infection: ☐ chest, ☐ear, nose or throat, ☐sinuses, ☐ skin, ☐ gastrointestinal, ☐other:______b) Organism: ☐ bacterial, ☐ fungal, ☐ viral, ☐ parasitic, ☐ I don’t know

19. Have you had antibiotic treatment in the last 12 months? ☐ YES, ☐ NO

If yes, why? ______how many times? : and for how long ?: days OR months.

20. Medical History Please tick if you have been diagnosed with any of the following: ☐ Autoimmune neutropenia, ☐Autoimmune haemolytic anaemia, ☐Autoimmune thrombocytopenia, ☐Gastritis, ☐Colitis, ☐Autoimmune hepatitis, ☐Cholangitis, ☐Biliary cirrhosis, ☐, ☐Bronchiectasis, ☐GLILD, ☐Alopecia, ☐, ☐, ☐Psoariasis, ☐Thyroid disease, ☐Diabetes, ☐Uveitis, ☐Keratitis, ☐Rheumatoid Arthritis, ☐Seronegative Arthritis, ☐Systemic erythematous, ☐Raynaud’s disease, ☐ NONE of them

21. Do you suffer from Allergies/Atopy? ☐YES ☐NO If yes, from which Allergy? ☐Eczema, ☐ Allergic rhinitis, ☐ allergic Asthma, ☐food allergy, ☐ Urticaria, ☐other:______

22. Have you ever been diagnosed with cancer? ☐ YES, ☐ NO If yes, with which cancer?______when were you diagnosed?

(year) and how were/are you treated?______

XLVI Appendix

23. If I have any further questions, may I contact you? ☐ YES ☐ NO If yes, please give preferred Phone number:______AND/OR Email:______

24. Would you like to be informed about the result of my research project? ☐ YES ☐ NO If yes, please give your email address: ______

Thank you very much for completing this questionnaire and supporting our research on your condition!

XLVII Appendix

9.4 Questionnaire Wart Patients

Department of Clinical Immunology IMMUNOLOGY CLINIC (Incorporating The Immunodeficiency Clinic)

Head of Department Specialist Dr Magdalena Dziadzio Royal Free Hospital Prof Hans Stauss Registrars Dr. Jaroslava Orosova Pond Street Dr Mari Campbell Clinical Lead Clinical Svenja Abel London NW3 2QG Dr Siobhan Burns Psychologist Sarita Workman Switchboard: 020 7794 0500 Medical Student Andrew Symes www.royalfree.nhs.uk Dr R Chee Sisters Irene Wahlberg Prof. B Grimbacher Chanell Pritchard Dr David Lowe Staff Nurse Tel: 0207 830 2141 Dr Suranjith Seneviratne Secretary Fax: 0207 830 2224 Clinic and Appointment Enquiries on Tuesdays, Wednesdays, and Thursdays Tel: 020 7794 0500 ext 22525

Questionnaire for patients with persistent skin warts

We are currently taking a closer look at patients with persistent skin warts. This questionnaire will give us a better overall picture of this condition and shall be used as a foundation for further, more in-depth research. We would be more than grateful if you supported our research by completing this questionnaire. It will take about 10 minutes to answer.

NAME: ______DATE: ______

1. For how long have you had warts? ☐ < 6 month, ☐ 6- 24months, ☐ > 2 years, ☐ > 5 years

2. Where do you have warts? a) Please mark in Picture A where you have warts.

Picture A

XLVIII Appendix b) If you have warts on your hands or feet please in addition mark Picture B and C to be more precise.

Picture C

Picture B

2. How many warts in total do you have? ☐ < 5, ☐ 5-20, ☐ > 20

3. Is there anything you have difficulty doing because of the warts? ☐ YES ☐ NO If yes, what is difficult and why?______

4. Does anyone in your family suffer from persistent skin warts? ☐ YES ☐ NO If yes, who is it? ☐ mother, ☐ father, ☐ sister, ☐ brother, ☐ son, ☐ daughter, ☐ other: ______

5. What treatment have you tried? a) Topical (local)

XLIX Appendix

Treatment How often? For how long? Did it help? ☐ 5-Fluorouracil (Acktikerall) ☐ Cautery

☐ Cryotherapy

☐ Curettage

☐ CO2 Laser

☐ DCP

☐ Imiquimod (Aldara) Treatment How often? For how long? Did it help? ☐ Salicylic acid

☐ Imiquimod + Salicylic acid ☐ Retinoids (Acitretin) ☐ 0ther, please list in spaces below

b) Have you had any systemic (oral) treatment? ☐ YES ☐ NO If yes, Which drug? How often? For how long? Did it help?

L Appendix c) Have you had a HPV vaccine? This is a vaccination against Human Papillomavirus, the virus that causes warts. ☐ YES ☐ NO If yes, which vaccine? : ☐Gardasil, ☐ other:______☐ I don’t know When did you have the vaccine? ______and did it improve your warts?______

6. Have you ever had a bone marrow/ haematopoietic stem cell transplant? ☐ YES ☐ NO

If yes, when?

7. Are you on any immunosuppressive medication (e.g. Steroids, Chemotherapy, Antibodies or drugs that suppress your immune system) ? ☐ YES ☐ NO If yes, what is the name of the medication? ______

8. Have you been diagnosed with a primary immunodeficiency (PID)? ☐ YES ☐ NO If yes, what is your diagnosis? ______

9. Do you suffer from recurrent infections (infections you needed medication from your doctor for)? ☐YES ☐NO If yes: a) Site of infection: ☐ chest, ☐ear, nose or throat, ☐sinuses, ☐ skin, ☐ gastrointestinal,

☐other:______b) Organism: ☐ bacterial, ☐ fungal, ☐ viral, ☐ parasitic, ☐ I don’t know

LI Appendix

10. Are you on prophylactic antibiotic treatment? ☐ YES, ☐ NO If yes, go to question 12.

11. Have you had antibiotic treatment in the last 12 months? ☐ YES, ☐ NO

If yes, why? ______how many times? and how long for? days.

12. Medical History Please tick if you have been diagnosed with any of the following: ☐ Autoimmune neutropenia, ☐Autoimmune haemolytic anaemia, ☐Autoimmune thrombocytopenia, ☐Gastritis, ☐Colitis, ☐Autoimmune hepatitis, ☐Cholangitis, ☐Biliary cirrhosis, ☐Vasculitis, ☐Bronchiectasis, ☐GLILD, ☐Alopecia, ☐Vitiligo,

☐Folliculitis, ☐Psoariasis, ☐Thyroid disease, ☐Diabetes, ☐Uveitis, ☐Keratitis, ☐Rheumatoid Arthritis, ☐Seronegative Arthritis, ☐Systemic Lupus erythematous, ☐Raynaud’s disease, ☐ NONE of them

13. Do you suffer from Allergies/ Atopy? ☐YES ☐NO If yes, from which Allergy? ☐Eczema, ☐ allergic rhinitis, ☐allergic Asthma, ☐Food allergy, ☐Urticaria, ☐other:______

14. Have you ever been diagnosed with cancer? ☐ YES, ☐ NO If yes, with which cancer?______when have you been diagnosed? and how were/are you treated?______

15. If I have any further questions, may I contact you? ☐ YES ☐ NO

LII Appendix

If yes, please give preferred Phone number: ______Email:______

16. Would you like to be informed about the result of my research project? ☐ YES ☐ NO If yes, please give your email address: ______

17. Have we taken photos for documentation? ☐ YES ☐ NO If we have taken photos, do you agree with us using these in a research paper (anonymously) to illustrate the results of our research? ☐ YES ☐ NO

18. Have we taken scrapings of your warts to see which types of the virus exactly are causing your warts? ☐ YES ☐ NO If yes, when (date)? ______and which site? ______

Thank you very much for completing this questionnaire and supporting our research on your condition!

LIII Appendix

9.5 Eidesstattliche Versicherung

Abel, Svenja, geboren am 28.08.1983 in Emmendingen

Ich erkläre hiermit an Eides statt, dass ich die vorliegende Dissertation mit dem Thema

„The Characteristics of Viral Skin Infections in the Immunocompromised Host“

selbständig verfasst, mich außer der angegebenen keiner weiteren Hilfsmittel bedient und alle Erkenntnisse, die aus dem Schrifttum ganz oder annähernd übernommen sind, als solche kenntlich gemacht und nach ihrer Herkunft unter Bezeichnung der Fundstelle einzeln nachgewiesen habe.

Ich erkläre des Weiteren, dass die hier vorgelegte Dissertation nicht in gleicher oder in ähnlicher Form bei einer anderen Stelle zur Erlangung eines akademischen Grades eingereicht wurde.

Lahr, den 14. Dezember 2018

LIV Acknowledgements

10 Acknowledgements

A special “Thank you” goes to Prof Bodo Grimbacher who gave me the opportunity to conduct a clinical based dissertation in London. He made sure that my project at the Royal Free Hospital would succeed and gave me the support from Freiburg I needed.

A special “Thank you” also goes to Dr Siobhan Burns, my supervisor at the Royal Free Hospital, who came up with the idea of my project and was very supportive whenever I needed her input.

In addition, I would like to thank Dr. Karin Purdie who performed the HPV typing, Fernando Moreira who made sure I did not miss any patients in clinic, and the rest of the team in the Immunology and Dermatology Department of the Royal Free Hospital London who were very supportive and without their help my project would not have succeeded.

Lastly, I would like to thank my parents for giving me the opportunity to study a year longer to be able to concentrate on my dissertation.

LV