Fig. 6.1
Herpes viral simplex infection. Clinically, it presents as multiple grouped vesicles with an erythematous base
Originally, HSV shedding was thought to be infrequent given the low rate of symptomatic recurrences. Numerous studies are refuting this concept; daily samples study with PCR amplification have documented asymptomatic shedding in the genital tract of 80–90 % of HSV type 2 seropositive individuals around 10 % of days [17]. The same occurs with infected patients with HSV type 1 [18]. The viral shedding are typically brief and can be simultaneous or subsequent to reactivations and continues to occur over time [19]. These silent reactivations with frequent shedding episodes have a tremendous public health impact. Even more, studies indicate that most of HSV transmission occurs during these asymptomatic viral shedding episodes. Although the risk is higher with active lesions as the viral load is greater.
Histopathology
An early intact herpes simplex vesicle usually involves the entire epidermis with ballooning and reticular degeneration (Fig. 6.2a). Ballooning degeneration presents in the form of intense cytoplasmic eosinophilia with loss of intercellular cohesion leading to acantholysis. On the other hand, reticular degeneration typically shows hydropic swelling and rupture of the epidermal cells. Classically, infected keratinocytes demonstrate multinucleation with molding blue-gray nuclei with margination of the chromatin (“ground glass”) (Fig. 6.2b). Cowdry A inclusions are eosinophilic intranuclear inclusions of 3–8 μm in diameter that are surrounded by a halo. With time, the cells undergo karyorrhexis and lysis, that clinically translate in erosions and ulcerations. At this stage, many times it is difficult to identify the characteristic features of HSV infection in the epidermis. Occasionally, sebaceous gland and follicular involvement can still harbor the diagnostic viral cytopathic changes (Fig. 6.3a, b). Herpetic syringitis, viral cytopathic changes in eccrine epithelium with necrosis and syringosquamous metaplasia, has been reported especially in immunocompromised patients [20]. The nerves are also affected by the infection in the form of neuronal atrophy and Schwann cell hypertrophy. Biopsy of nodular lesions demonstrates pseudoepitheliomatous hyperplasia of the epidermis and a dense inflammatory dermal process simulating a lymphoma. Cytological evaluation of the scraping of the base and edges of a fresh ulcer, or freshly opened vesicle, usually will show the multinucleated cells with viral cytopathic effects, characteristic of HSV infection. Moistening the ulcer with a saline-soaked sponge and then scraping it with a wooden spatula may improve the diagnostic yield from ulcerative lesions. Morphologic changes seen with HSV infection are not reliable in separating from secondary infection or in distinguishing HSV type 1 from type 2 infection. Furthermore, herpes zoster can involve the vulva and may have similar cytological findings.
Fig. 6.2
(a, b) Herpes viral simplex infection. Intraepidermal vesicle with keratinocytic ballooning and reticular degeneration (a). Infected keratinocytes demonstrate multinucleation with molding and blue-gray nuclei with margination of the chromatin (b)
Fig. 6.3
(a, b) Herpes simplex viral infection. Follicular involvement is noted (a) and confirmed by viral cytopathic changes in high power (b)
In difficult cases, HSV-specific fluorescein-conjugated antiserum may be placed on smears of ulcers or vesicles to identify HSV antigens. Immunoperoxidase techniques, utilizing HSV-specific antibodies, may be of value if the histopathological findings are nonspecific and can be employed on paraffin-embedded tissue to separate the different types of the herpes virus family.
Isolation of HSV types 1 or 2 can be achieved by the inoculation of tissue culture monolayers, such as WI-38 human embryonic lung fibroblasts or monkey kidney cells. Culture sensitivity increases if the sample lesion is a blister than an ulcer, from 10 % to 33 %. Both types of HSV produce characteristic cytopathic changes on these cell lines, which are confirmed by direct immunofluorescence employing monoclonal antibodies to HSV. Virus isolation can be achieved within 4 days. Rapid viral culture over 24 h, followed by a search for HSV antigen using immunoperoxidase technique, can give a prompt result (Fig. 6.4a, b). In situ hybridization and polymerase chain reaction (PCR) techniques, employing HSV-specific primers, are another approach to the positive identification of HSV infections. In considering diagnostic techniques for genital herpes infection, culture and direct fluorescent antibody staining of infected cells are labor-intensive techniques that require skilled laboratories and are impacted by collection technique, transport conditions, and subjective interpretation [21, 22]. On the other hand, PCR assays are more sensitive and are less time consuming that result in a fast turnaround time [22].
Fig. 6.4
(a, b) Herpes simplex viral infection. The infected keratinocytes (a) react with immunohistochemical antibody against the virus (b)
Differential Diagnosis
The identification of viral cytopathic changes confirms the diagnosis. However, the reactive changes associated with the infection and unusual clinical presentation can be misleading. Verrucous herpes infection, seen in HIV-infected patients, raises the differential diagnosis of squamous cell carcinoma. The infiltrative irregular nests of severely atypical keratinocytes associated with desmoplastic stromal reaction as well as the presence of atypical mitoses support the interpretation of squamous cell carcinoma. Furthermore, no viral cytopathic changes will be noted. A dense lymphoid infiltrate can be associated with herpes viral infection. This may raise the consideration of a T-cell process like lymphomatoid papulosis due to the presence of CD30-positive reactive lymphocytes. The detection of T-cell clonality in the lymphoid infiltrate secondary to the viral infection can be even more confusing. The clinical history of a vesicular eruption and acute inflammation associated with CD4- and CD8-positive T-cell lymphocytes will support the diagnosis of herpes infection over lymphoproliferative disorders. Serial sections can be a useful tool to detect the viral changes. Lastly, reparative changes near an ulcer can produce multinucleated cells; the absence of “ground-glass” changes in the nuclei or the presence of Cowdry A cytopathic effect supports secondary reactive changes over viral infection. If in doubt, immunohistochemical stain can be of help.
Summary
Clinical Presentation
Three stages: primary infection (initial manifestations in women without preexisting antibodies), latency (time after initial infection before reactivation periods), and recurrent episodes (reactivation of HSV after latency).
Primary infection: 3–7 days after exposure, with a prodrome of general malaise.
Classic clinical presentation consists of vesicles on an erythematous base arranged in clusters that evolve to pustules and/or erosions.
Women suffering from HIV present with unusual clinical presentations.
Histologic Features
Epidermis with ballooning and reticular degeneration.
Epidermal blister +/− ulceration.
Viral cytopathic changes:
Common: cells with multinucleated, molding nuclei with margination of chromatin (ground glass)
Less common: Cowdry A cytopathic effect (eosinophilic nuclear inclusion with clear halo)
Involvement of folliculosebaceous units and eccrine gland occurs.
Florid mixed inflammatory reaction.
Differential Diagnosis
Squamous cell carcinoma
CD30-positive lymphoproliferative disorder
Reparative changes
Takeaway Essentials
Clinical Relevant Pearls
There is a shift in the epidemiologic landscape of genital herpes infection.
Increase risk associated with increased number of sexual partners, lower education, and poverty.
Infected individuals experience frequent, asymptomatic brief shedding episodes.
Prior infection likely reduces the burden of disease when infected by a second HSV type.
Atypical presentation can be seen in HIV-infected patients with genital herpes.
When considering cultures, selection of a blister increases the sensitivity.
Pathology Interpretation Pearls
When the lesion is old and only an ulcer is present, adnexal structures like folliculosebaceous units and eccrine glands may demonstrate the characteristic viral cytopathic changes.
Sometimes multiple levels are needed to detect the viral cytopathic changes.
Immunohistochemical Findings
Immunohistochemical stains in paraffin-embedded tissue separate the different types of herpes virus implicated in the infection.
Varicella Zoster
Varicella zoster virus (VZV) is a ubiquitous, neurotropic alpha herpes virus. Primary infection results in varicella (chickenpox). This is followed by a latent infection of neurons of cranial nerve, dorsal root, and autonomic ganglia along the neuraxis. Twenty percent of immunocompetent hosts and half of immunocompromised infected people will undergo reactivation of the virus [23].
Clinical Features
Clinical reactivation is frequently seen in women around the fifth decade. An episode of herpes zoster usually starts with a prodrome of pain, pruritus, or tingling in a dermatomal distribution. In a short period of time, clusters of papules appear and change into vesicles with an erythematous base (Fig. 6.5). In the vulva, the vesicles have a short life span ending in erosions. Postherpetic neuralgia can be a debilitating sequelae of this infection [24]. Anogenital VZV infection in the pediatric population can be confused with child sexual abuse [25].
Fig. 6.5
Varicella zoster. Cluster of papules appear and change into vesicles with an erythematous base
Histopathology
The histologic features of VZV infection overlap with those seen in HSV infection (Fig. 6.6a, b). VZV infection is less inflammatory than HSV but with a more prominent vasculitic component (Fig. 6.7a, b). When the vasculitis is severe, necrotizing lesions elicit. Confirmation of the infection can be reached using immunohistochemistry, direct florescence antibodies, or PCR test [26].
Fig. 6.6
(a, b) Varicella zoster. Its histologic features overlap with those of herpes viral simplex infection (a). An immunohistochemical stain can separate these viruses (b)
Fig. 6.7
(a, b) Varicella zoster. This viral infection is less inflammatory (a) than herpes simplex virus infection, but with a more prominent vasculitic component (b)
Differential Diagnosis
The identification of viral cytopathic changes confirms the diagnosis. Due to the overlap in histologic features between VZV and HSV, please refer to HSV section for discussion on differential diagnosis.
Summary
Clinical Presentation
Dermatomal distribution
Prodrome of burning or tingling
Papules and vesicles rapidly turning into erosions
Histologic Features
Similar to HSV
Differential Diagnosis
Squamous cell carcinoma
CD30-positive lymphoproliferative disorder
Reparative epithelial changes
Takeaway Essentials
Clinical Relevant Pearls
The distribution of the rash is a strong clue to reach the diagnosis.
Atypical forms can be seen in immunosuppressed patients.
Postherpetic neuralgia should be considered in women with unexplained dysesthesia.
Anogenital Varicella zoster in the pediatric population can be confused with sexual abuse.
Pathology Interpretation Pearls
Histology of VZV is similar to HSV infection.
Immunohistochemical Findings
Immunohistochemical stains are simple, reliable, and time-efficient diagnostic tools to separate HSV from VZV infection.
Cytomegalovirus
Cytomegalovirus (CMV) is part of the subgroup of beta herpesviruses. As other members of the Herpesviridae family, they have a primary infection, latent phase, and reinfection. CMV is one of the most fatal infection in immunocompromised individuals [27].
Clinical Features
Female genital CMV is a rare event. Routes of transmission are sexual, close contact, blood/tissue exposure, and perinatal. Clinical presentation exhibits a wide range of nonspecific cutaneous manifestations including generalized maculopapular rash, ulcers, hyperpigmented nodules, vesicles, and petechias, among others [27]. Single or multiple ulcers can be seen in the vulva, perineal, and perianal region [28]. In immunosuppressed patients, polymicrobial anogenital ulcers are the most frequent cutaneous presentation of CMV [29]. The detection of CMV in the lesion can be pathogenic, but in some cases, it is believed to be an epiphenomenon with colonization of endothelial cells during CMV viremia [30]. The latter has been argued in polymicrobial lesions in HIV-positive patients, especially in the presence of HSV and VZV virus infection. Intraneural location in the vulva and perianal skin is considered to be potential latency sites for this virus as seen in shingles [28].
Histopathology
The pathognomonic change in CMV consists of vascular dilatation with endothelial cells exhibiting large, eosinophilic intranuclear inclusions of 10 μm in size surrounded by a clear halo (owl’s eye inclusions) (Fig. 6.8a, b). These inclusions can be seen not only in endothelial cells but also in keratinocytes, eccrine epithelium, macrophages, and fibroblasts [30]. Although the owl’s eye intranuclear inclusions are the more characteristic viral cytopathic change; it is rare, with the most frequent form of viral change presenting as irregular-shaped cytomegaly and bubbly cytoplasm. Furthermore, the morphology of the viral inclusion varies through the different stages of the infection with an initial phase of nuclear enlargement, followed by cytoplasmic enlargement and viral inclusions in the cytoplasm and nucleus, and ends with nuclear inclusion fragmentation and disappearance of cytoplasmic inclusions [31]. Leukocytoclastic vasculitis, pauci-inflammatory thrombogenic vasculopathy [32], and eccrine squamous syringometaplasia [33] are reactive phenomenon associated with CMV infection.
Fig. 6.8
(a, b) Cytomegalovirus. The dermal abscess (a) shows around the vessels the characteristic large, eosinophilic intranuclear inclusions surrounded by a clear halo (owl’s eye inclusions) (b)
Although light microscopy is a sensitive tool to detect CMV, immunohistochemistry (Fig. 6.9a, b), in situ hybridization, and molecular investigations can also be used to confirm the diagnosis in challenging cases.
Fig. 6.9
(a, b) Cytomegalovirus. The infected cells (a) react with immunohistochemical antibody against the virus (b)
Differential Diagnosis
The presence of the intranuclear inclusion in CMV is quite specific and definitive for a diagnosis. However, these inclusions are not present in all biopsies, and the differential diagnosis would include an anogenital ulcer due to a variety of etiologies including syphilis and other sexually transmitted diseases. For relevant information in the differential diagnosis, refer to Table 6.1.
Table 6.1
Sexually transmitted diseases
Chancroid | Lymphogranuloma venereum | Granuloma inguinale (donovanosis) | Syphilis | |
|---|---|---|---|---|
Etiology | Haemophilus ducreyi (Gram-negative anaerobic coccobacillus) | Chlamydia trachomatis (obligatory intracellular Gram-negative coccoid or rod, serotype L1, L2, and L3) | Klebsiella granulomatis (obligatory intracellular Gram-negative rod) | Treponema pallidum (spirochete) |
Location | Fourchette and labia minora | No specific location | Vulvar labia | Fourchette, labia majora |
Incubation | 1–14 days | 3–42 days | 2 weeks to 6 months after exposure | 3 weeks |
Clinical presentation | Chancroid: tender non-indurated ulcer with irregular undermined edges and purulent base | Three phases: (1) ulcerated skin lesion, (2) adenitis, and (3) fibrosis (genitoanorectal syndrome with perirectal abscesses, fistulas, and stenosis) | Ulcerated painless papules that merged to form a velvety beefy-erythematous granulation tissue surrounded by rolled edges | Primary: painless indurated ulceration with clean base |
Usually multiple | Primary lesion: inconspicuous ulcerated nontender papule healing without scar | Late manifestation: esthiomene | Secondary: maculopapular rash, pustular syphilis, lues maligna, and condyloma lata | |
Late manifestation: esthiomene | If not treated is mutilating | Tertiary: nodules or ulcerated masses | ||
Adenopathy | Inguinal adenopathy with flocculent nodes and drainage by fistula | Painful enlarged inguinal and/or femoral adenitis with bubo formation and rupture with drainage through sinus tract | Usually absent | Mildly enlarged |
Histopathology | Lesion with three zones (1) ulcer, (2) granulation tissue and thrombosed vessels, and (3) chronic inflammation | Primary cutaneous lesion: nonspecific findings | Ulceration and granulation tissue. Donovan bodies: histiocytes with rod-shaped oval organisms with the form of a safety pin decorated by Warthin–Starry or Giemsa stain | Primary: ulcer with pseudoepitheliomatous hyperplasia |
Organisms: Gram-negative arranged as school of fish in the superficial aspect of lesion | Lymph nodes: serpiginous or stellate abscesses with granulomatous reaction | Endarteritis obliterans | ||
Secondary: psoriasiform pattern and lichenoid pattern with plasma cells component Condylomata lata: epidermal acanthosis with lymphoplasmacytic infiltrate and neutrophils in the surface | ||||
Pustular syphilis: folliculocentric neutrophilic inflammation | ||||
Lues maligna: thrombotic endarteritis | ||||
Tertiary: granulomatous inflammation and endarteritis obliterans | ||||
Diagnostic tests | Cultures with selective agar medium or smears (better to show coccobacilli than histopathology) | Cultures with yolk sac of eggs, complement fixation antibodies, and PCR | Detection of Donovan bodies from smear of ulcer, culture, and PCR | Immunohistochemical stains, serologic tests (RPR, VDRRL, and PTHA), and molecular tests |
Immunofluorescent test and PCR |
Summary
Clinical Presentation
Single or multiple ulcers can be seen in the vulva, perineal, and perianal region.
Histologic Features
CMV inclusions (“owl’s eye inclusions): large, eosinophilic intranuclear inclusions of 10 μm in size surrounded by a clear halo.
Inclusions can be seen in endothelial cells but also in keratinocytes, eccrine epithelium, macrophages, and fibroblasts.
Frequently the viral inclusions present as irregular-shaped cytomegaly and bubbly cytoplasm.
Differential Diagnosis
Ulceration of other causes such as sexually transmitted diseases like syphilis.
Takeaway Essentials
Clinical Relevant Pearls
Protean clinical presentation, anogenital region frequently present in the form of ulcers.
The dermis is believed to be an inhospitable location for CMV.
In immunosuppressed patient, the lesions are usually polymicrobial (HSV, VZV, and CMV).
Pathology Interpretation Pearls
The “owl eye inclusion” however, it is not always present.
Morphology of the inclusion will vary depending on the phase of the viral cycle.
Serial sections may help to detect the viral cytopathic changes.
Immunohistochemical Findings
Immunohistochemical stains are a useful tool to detect this virus in densely inflamed cases.
Molluscum Contagiosum
Molluscum contagiosum (MC) is a moderately contagious viral disease that, in adults, is often related to intimate and/or sexual contact. This poxvirus infection can present at any age, but it is frequently seen in children, sexually active adolescents, and immunocompromised individuals [34].
Clinical Features
Lesions of MC are usually asymptomatic; however, perianal lesions can become pruritic and undergo secondary infection. The lesions present as small, smooth papules, 3–6 mm in diameter with a central punctum or umbilication (Fig. 6.10). The most frequent affected anatomic locations include the labia majora, labia minora, and mons pubis [35, 36]. They are usually multiple and separated lesions, an unusual form of presentation is a plaque, which is made up of 50–100 individual clustered lesions. The incubation period varies between 14 and 50 days. The characteristic clinical presentation usually does not require biopsy. Cytological identification of the typical intracytoplasmic inclusion bodies (molluscum bodies or Henderson–Patterson bodies) within scrapings from the interior of molluscum papule is sufficient to confirm the diagnosis. Molluscum dermatitis (eczematous reaction to the viral infection) and Gianotti–Crosti-like reactions (erythematous papules or papulovesicles) can be seen in these patients [37]. Classically, lesions of molluscum contagiosum commonly regress within 6–12 months.
Fig. 6.10
Molluscum contagiosum. Clinically, the lesions consist of small, smooth papules, 3–6 mm in diameter with a central punctum or umbilication
Histopathology
MC presents as a cup-shaped keratinocytic endophytic growth containing the characteristic intracytoplasmic viral inclusions, which impart an eosinophilic appearance to the keratinocytic cytoplasm (molluscum body or Henderson–Patterson body) (Fig. 6.11a, b). The viral particles increase in size with progression toward the surface of the invagination causing the peripheral displacement of the nuclei. In older lesions, the cytoplasmic bodies take on a more basophilic appearance preceding lysis of the cell. The central dimple of the lesion is seen histologically if the lesion is carefully bisected. In the dermis, there is often an inflammatory infiltrate, which sometimes can be very prominent and simulate a lymphoma or a pseudolymphoma. Most lesions regress spontaneously; however, untreated lesions may persist for years, during which time they may be spread by close contact. Recently, a fluorescence resonance energy transfer-based real-time PCR is available for a sensitive and specific diagnosis of this infection [38].
Fig. 6.11
(a, b) Molluscum contagiosum. Cup-shaped keratinocytic endophytic growth (a) containing the characteristic intracytoplasmic viral inclusions (Henderson–Patterson body) (b)
Differential Diagnosis
The clinical and histologic appearance of MC is pathognomonic. The differential diagnosis in large lesions may include condyloma acuminatum. The presence of Henderson–Patterson bodies and the absence of koilocytes support the interpretation of MC.
Summary
Clinical Presentation
Affect labia majora, labia minora, and mons pubis.
The incubation period varies between 14 and 50 days.
The lesions present as small, smooth papules, 3–6 mm in diameter with a central punctum or umbilication.
Histologic Features
Cup-shaped endophytic epidermal growth
Henderson–Patterson bodies
Differential Diagnosis
Condyloma accuminata
Takeaway Essentials
Clinical Relevant Pearls
Classic clinical presentation would not need a biopsy to confirm this infection.
Pathology Interpretation Pearls
Exuberant lymphocytic reaction may mimic a lymphoproliferative disease.
Serially sections through the block may be necessary to identify the virus.
Human Papillomavirus Infection (Condyloma Cuminata: Genital Warts)
Human papilloma virus (HPV) is a family of non-enveloped, circular, double-stranded DNA viruses. HPV is responsible for benign and malignant processes that affect the vulva, including condyloma acuminata and squamous cell carcinomas. Genital human papillomavirus is a common sexually transmitted infection (STI) with a transmission rate of approximately 65 % [39]. There are more than 40 types of HPV that can infect the genital areas of males and females, and they can infect the mouth and throat as well.
Condyloma acuminata (genital warts) are sexually transmitted benign neoplasms that may involve the vulva, vagina, cervix, urethra, anal canal, and perianal skin. Recurrent anogenital warts is a frequent cause of medical vulvitis with a reported 360,000 occurrences in 2008 in the United States [39]. However, in recent years, the appearance of a quadrivalent (HPV genotypes 6, 11, 16, and 18) or bivalent (HPV types 6 and 11) vaccine is changing the epidemiologic landscape of genital warts. Future studies will reveal the impact of this vaccination in the patterns of origin and distribution of this infection. The prevalence of HPV infection varies greatly, depending on the analyzed population; in most studies, clinically evident vulvar involvement is less common than cervical HPV infection.
Molecular biology studies utilizing in situ hybridization have identified HPV-6 as the most prevalent HPV type in genital condylomata acuminata. HPV-11 is present in 25 % of genital warts [40]. These are low-risk HPV types in opposition to the high-risk (HPV16/16) types seen in vulva intraepithelial lesions and squamous cell carcinoma (see Chaps. 9 and 10).
Clinical Features
Clinically, condylomas present as flesh color or hyperpigmented, confluent papillary, verrucous, filiform, flat, or papular lesions of the skin and mucous membrane of the vulva that may extend to perineum and perianal region (Fig. 6.12a, b). Affects 1 % of sexually active adults with the highest incidence in the second and third decade [39]. The incubation period can range from 3 weeks to 8 months with a clinically apparent lesion within 2–3 months after infection [41]. Most lesions are asymptomatic unless secondarily infected. Small lesions are best appreciated with the application of 3–5 % acetic acid for 3–5 min, followed by colposcopic examination. Lesions that are only detected by colposcopic examination after performing HPV testing on the tissue in question are considered subclinical. Autoinoculation explains the high recurrence rates of condyloma, approximately 20–50 % [42]. Frequently, large proportion of genital warts may spontaneously regress within 1–2 years. Genotype-specific immunity may develop and protect against reinfection in the patient [43]. In immunocompromised patients, these lesions are frequently overgrown, large masses with a prologue history of recurrences [44, 45]. These large irregular and heavily keratinized lesions are frequently clinically confused with squamous cell carcinoma.
Fig. 6.12
(a, b) Condyloma acuminata. Clinically, condylomas present as flesh color or hyperpigmented multiple (a), confluent papillary, verrucous, filiform, flat (b), or papular lesions
Condyloma acuminata are commonly associated with vaginitis, pregnancy, diabetes mellitus, oral contraceptive use, poor perineal hygiene, immunosuppression, and sexual activity with multiple partners. Approximately 30–50 % of women with vulvar condyloma acuminatum have associated cervical HPV infection. The presence of vulvar condyloma acuminatum in children is usually related to sexual abuse. A potential pitfall is the presence of HPV2 verruca vulgaris in young girls; their clinical presentation is similar to the nongenital counterpart with more hyperkeratosis and papillomatosis than condylomas accuminatum [46]. When in doubt, DNA studies may help to distinguish these two clinical scenarios. To take into consideration is the fact that HPV-DNA has been detected on the fingers of children suffering from genital wart; therefore, it has been proposed that sexual contact is not always the route of transmission [47]. Other potential explanation for the presence of condylomas in children is the transmission from the mother to the infant during birth with a period of latency until clinical visible condylomas. Thorough clinical and social history and molecular tests are essential tools in the investigation of cases of potential sexual abuse.
Histopathology
Histologically, condylomas present as dome-shaped lesions with areas of acanthosis, papillomatosis, and hyperkeratosis. There is also a prominent granular layer with large and irregular keratohyaline granules and a few dyskeratotic keratinocytes. In some cases, the so-called koilocytes are seen in the superficial epidermis (Figs. 6.13a–c and 6.14a–d). These cells are characterized by the presence of cytoplasmic halos and enlarged irregular nuclei with slight hyperchromasia. Binucleated and multinucleated squamous cells often are found. The typical regular maturation upward from the basal layer is present, and the mitotic activity is restricted to the basal layer. A superficial chronic inflammatory infiltrate often is present in the dermis.
Fig. 6.13
(a–c) Condyloma acuminata. Verrucous filiform lesions (a) histologically present as a papillomatous acanthosis (b) with classic koilocytes (c)
Fig. 6.14
(a–d) Condyloma acuminata. Clinically flat lesions (a) present as dome shaped (b) with koilocytes (c). Chromogenic in situ hybridization for HPV6/11 demonstrates strong nuclear staining of infected keratinocytes (d)
It is well recognized that treatment of genital warts with podophyllin leads to the presence of apoptosis, nuclear enlargement, and hyperchromasia. It is the clinical history and absence of atypia in the basal layer that can clarify the nature of these changes. Lately, the changes associated with podophyllin treatment were described in condylomas of young women in the absence of such treatment under the name of pseudobowenoid changes [48]. Here again, it is the absence of basal layer atypia that is a clue to separate these changes from high-grade vulva intraepithelial lesions.
On tissue specimen, tests available for detection of HPV include PCR, in situ hybridization, and ISH. PCR is considered the most effective tests for HPV-DNA detection, but limitations include the absence of architecture preservation due to DNA extraction and the need of high expertise and rigorous laboratory conditions to avoid contaminations. ISH has the advantage of easy-implemented technique with preservation of morphology; however, it lacks in sensitivity. A solution to this limitation is the tyramide-based signal amplification kit, based on HPV chromogenic in situ (CISH) technology. This technology would help to distinguish episomal from integrated HPV [49].
Differential Diagnosis
The main differential diagnosis for condyloma acuminata is with high-grade vulva intraepithelial lesion. Condylomata are typically verrucous or papillary and have normal mitoses located in the basal layer, koilocytosis, parabasal hyperplasia, accentuated intracellular bridges, dyskeratosis, hypergranulosis, and compact hyperkeratosis. In contrast, high-grade vulva intraepithelial lesions present as flat macules with enlarged keratinocytes and prominent nuclei and nucleoli. However, in many cases, high-grade vulva intraepithelial lesions may develop in the setting of a condyloma. In those cases, the lesions at low power demonstrate the typical appearance of condylomas, but they usually show abnormal cytology with numerous atypical mitotic figures in the upper third of the epithelium.
Bowenoid papulosis that histologically presents with identical histologic features to squamous cell carcinoma in situ needs to be distinguished from condyloma acuminatum. Clinically, bowenoid papulosis usually presents with multiple small papules and histologically shows multiple mitotic figures and cellular atypia.
Small lesions of lichen simplex chronicus may resemble regressing or early flat condylomata acuminatum. In those cases, the presence of koilocytes may help in the differential diagnosis. If this cannot be resolved by histopathological examination, molecular biologic methods such as a polymerase chain reaction (PCR) or in situ hybridization to detect the presence of HPV may help to make a more definitive diagnosis.
Vulvar vestibular papillomatosis also enters in the differential diagnosis of condylomata. In the former, the epithelium lacks hyperkeratosis and other typical microscopic features of condyloma, and the lesions are confined to the vulva vestibule [50] (see Chap. 12). Fibroepithelial polyps may have the shape of large condylomas; however, the epithelium also lacks evidence of HPV infection in the form of koilocytes. Seborrheic keratosis in this area can also create a diagnostic conundrum. The absence of koilocytes and thick keratohyaline granules are clues for seborrheic keratosis. Condylomata lata may resemble condylomata acuminata clinically; however, on biopsy, the presence of prominent inflammatory infiltrates composed mainly of plasma cells and the presence of spirochetes distinguishes these lesions. Warty surface of acquired lymphangiectasis can also mimic a genital wart; the presence of thin-walled lymphatics vessels in the superficial stroma confirms the diagnosis of lymphatic ectasias. Lastly, in systemic amyloidosis, vulva deposits can be associated with squamous proliferations like condylomas [51, 52]. Congo red stain can confirm the nature of the deposit for a final diagnosis of amyloidosis.
In large condylomas, the main differential diagnosis includes verrucous carcinoma. The latter has minimal cytologic atypia, no koilocytes, and a characteristic pushing broad-based rete ridges. HPV is commonly negative.
Summary
Clinical Presentation
HPV-6 is the most prevalent HPV type in condylomata acuminata
Affect the vulva, vagina, cervix, urethra, anal canal, and perianal skin.
Present as multiple flesh color or hyperpigmented, confluent papillary, verrucous, filiform, flat, or papular lesions of the skin and mucous membrane.
Immunosuppressed women have large hyperkeratotic lesions.
Histologic Features
Dome-shaped lesions with areas of acanthosis, papillomatosis, and hyperkeratosis
Koilocytes and prominent keratohyaline granules
Presence of keratinocytic maturation upward with absence of basal layer atypia and superficial mitoses
Differential Diagnosis
High-grade vulva intraepithelial lesion
Bowenoid papulosis
Small lesions of lichen simplex chronicus
Vulvar vestibular papillomatosis
Fibroepithelial polyp
Condylomata lata
Warty surface of acquired lymphan-giectasis
Verrucous carcinoma
Takeaway Essentials
Clinical Relevant Pearls
The different HPV subtypes can cause a wide spectrum of diseases in humans with varied morbidity and mortality.
Most lesions are asymptomatic unless secondarily infected.
Lesions that are only detected by colposcopic examination after performing HPV testing on the tissue in question are considered subclinical.
In immunosuppressed women, condyloma accuminatum is frequently confused with squamous cell carcinoma due to the large size and hyperkeratosis.
Pathology Interpretation Pearls
Changes associated with podophyllin treatment were described in condylomas of young women in the absence of such treatment under the name of pseudobowenoid changes.
Immunohistochemical Findings
Immunohistochemical stains and CISH are tools to detect and subtype this virus in paraffin-embedded tissue.
Epstein–Barr Virus Infection
Nearly four decades ago, the first documented case of Epstein–Barr virus (EBV)-associated genital ulcers was published in the literature. It is believed that some of the cases described earlier under the name of “Lipschutz syndrome” in reality represent patients affected by EBV genital infection.
Clinical Features
Clinical presentation can be seen in the form of a single large (>1 cm in diameter) or multiple small ulcers. Characteristic ulcers are deep and necrotic with irregular borders and covered by a yellow adherent membrane. The mean age of these patients is 14.5 years (range 2–51 years old) [53]. Pain is the main complaint and usually proceeds to a prodrome of fatigue, fever, and headache. Symptoms similar to mononucleosis like tonsillitis and adenopathy can also be seen in these patients [54]. Detection of IgM in the absence of antibody to EBV nuclear antigen and later on in the disease identification of IgG antibody against EBV viral capsid confirms a primary EBV infection. PCR using a sample of the ulcer can also be helpful for the diagnosis.
Histopathology
Sections of the ulcer demonstrate nonspecific features like granulation tissue with acute and chronic inflammation and debris. Vessels with reactive endothelial swelling and thrombosis can be seen as secondary changes to the ulcer (Fig. 6.15a, b).
Fig. 6.15
(a, b) Epstein–Barr virus infection. Histology demonstrates ulcer with nonspecific features like acute and chronic inflammation, debris (a), and vascular thrombosis (b)
Differential Diagnosis
The two most frequent etiologies of genital ulcers are herpes viral infection and syphilis. Primary syphilis is painless in contrary to EBV ulcers. Serologies and immunohistochemical stains against the spirochete can be useful diagnostic tools. Multinucleated infected keratinocytes with molding nuclei exhibiting margination of the chromatin support the diagnosis of genital herpes infection. Aphthous ulcer can be excluded based on the serologic findings. Localized papulonecrotic tuberculid is also a consideration. The wedge-shaped dermal necrosis associated with leukocytoclastic vasculitis and granulomas will separate this hypersensitivity reaction to mycobacteria from EBV infection. Long-lasting history of the lesions and a distant focus of tuberculosis will confirm the diagnosis.
Summary
Clinical Presentation
Single large ulcer (>1 cm in diameter) or multiple small ulcers.
Ulcers are deep and necrotic with irregular borders and covered by a yellow adherent membrane.
Prodrome of general malaise.
Mononucleosis-like presentation can also be seen.
Histologic Features
Sections of the ulcer demonstrate nonspecific features like granulation tissue with acute and chronic inflammation and debris.
Vessels with reactive endothelial swelling and thrombosis are also present.
Differential Diagnosis
Syphilis
Herpes virus infection
Aphthous ulcer
Tuberculosis
Takeaway Essentials
Clinical Relevant Pearls
Detection of IgM in the absence of antibody to EBV nuclear antigen and later on in the disease identification of IgG antibody against EBV viral capsid confirms a primary EBV infection.
Pathology Interpretation Pearls
Vessels with marked reactive endothelialitis should not be confused with vascular changes seen in syphilis. When in doubt, immunohistochemical stain can be a rapid tool to reach the correct diagnosis.
Bacterial Infections
Bacterial vaginal and cervical infections often cause vulvar symptoms, partly by the production of an irritant discharge.
Bacterial Vaginosis
Bacterial vaginosis results from an overgrowth of commensal bacterial (Gardnerella vaginalis) and anaerobic organism (e.g., Mobiluncus or Bacteroides) producing an increased volume of vaginal secretions [55, 56]. The characteristic unpleasant fishy odor is the product of bacterial amines released after exposure to an alkaline substance such as a potassium hydroxide or semen.
Clinical Features
Although bacterial vaginosis lacks symptomatic manifestations (e.g., burning, itchiness, or erythema) and vulvitis is uncommon, it carries an increased risk of preterm labor in pregnant women.
Histopathology
Rarely, a biopsy is performed. The diagnosis is made with the presence of profuse milky vaginal discharge, with fishy odor when exposed to 10–20 % potassium hydroxide (positive whiff test) [57], and the presence of clue cells (squamous epithelial cells covered by coccobacilli producing a ground-glass appearance of the cytoplasm and obscuring the cell borders) on vaginal smear (Fig. 6.16).
Fig. 6.16
Bacterial vaginosis. Clue cells: squamous epithelial cells covered by coccobacilli
Summary
Clinical Presentation
Vaginal discharge can cause vulvitis by irritation.
Histologic Features
Clue cells (squamous epithelial cells covered by coccobacilli producing a ground-glass appearance of the cytoplasm and obscuring the cell borders) on vaginal smear
Takeaway Essentials
Clinical Relevant Pearls
Rarely biopsy due to the presence of milky vaginal discharge
Pathology Interpretation Pearls
Unspecific inflammatory changes
Staphylococcal Infections
Like any hair-bearing skin, the labia or mons pubis may be the site of a superficial staphylococcal folliculitis and deep folliculitis producing furunculosis (boils). Occlusion, intertrigo, and depilation of pubic hair (particularly by shaving), all predispose to clinical infection. Other organisms producing similar changes include Pseudomonas, Malassezia furfur, and dermatophyte fungi.
Clinical Features
The warmth and moisture of the vulva provide the ideal environment for this infection. Clinical inspection reveals a variety of manifestations, from folliculitis presenting as perifollicular erythema with suppuration and follicular destruction to bullous impetigo showing yellow crust covering residual superficial erosions immediately after superficial fragile blisters rupture. Deep-seated abscess may arise upon infection of Bartholin gland, Skene ducts, Gartner ducts, and urethra diverticula. Although cultures identified in most of these settings are S. aureus, other bacteria like E. coli, N. gonorrhea, K. pneumonia, and Bacteroides have also been detected [58–61]. In recurrent abscesses, the possibility of a methicillin-resistant S. aureus infection should be suspected [62–64].
Microscopic Features
Histologically, areas of suppuration affecting the epidermis and epithelium of hair follicles are identified. The hair follicles are surrounded and infiltrated by neutrophils associated with edema (Fig. 6.17a–c). In advance stages of the infection, destruction of the follicle leads to the formation of granulomas and presence of naked hair shafts frequently surrounded by multinucleated giant cells. Bullous impetigo presents as a subcorneal bullae filled with neutrophils associated with a superficial perivascular lymphocytic infiltrate admixed with neutrophils. Brown and Brenn stain can be used to highlight the presence of bacteria in the bullae. The exfoliative toxin produced by S. aureus can induce flaccid blisters or pustules in the genitalia and thighs. In those cases, a subcorneal blister is formed with numerous neutrophils in the stratum corneum with areas of acantholysis (see Chap. 4). The latter is accompanied by a dermal sparse perivascular mixed inflammatory cell infiltrate and absence of identifiable bacteria. This is in contrast to bullous impetigo in which the dermal inflammatory component is heavier and bacteria colonies can be seen.
Fig. 6.17
(a–c) Staphylococcal infection. (a) Follicle with acute inflammation and (b, c) presence of Gram-positive cocci
Differential Diagnosis
Neutrophilic folliculitis can be seen associated with a wide range of infections, from bacteria to virus to fungus. Herpes virus can affect the hair follicle and sebaceous glands; the presence of multinucleated keratinocytes with molding nuclei and margination of the chromatin will support a viral etiology. If in doubt, immunohistochemical stains can help to detect the virus. Dermatophytes can also produce a neutrophilic suppurative folliculitis; periodic acid–Schiff (PAS) and Gomori methenamine silver (GMS) stains would highlight the fungal forms. A subcorneal collection of neutrophils raises the differential diagnosis of infections (e.g., candida) and blistering disorders (e.g., IgA pemphigus and pemphigus foliaceus). The presence of budding yeast and pseudohyphae will point toward the diagnosis of candida, while immunofluorescent tests will be able to identify the blistering disorder.
Summary
Clinical Presentation
Affect predominantly hair-bearing areas: labia majora and pubis
Suppurative folliculitis
Furunculosis (boils)
Flaccid blisters or pustules
Histologic Features
Similar to superficial or deep folliculitis elsewhere in the body.
Subcorneal blister with numerous neutrophils.
Brown and Brenn stain will highlight the bacterial forms in the folliculitis or bullae.
Differential Diagnosis
Folliculitis caused by dermatophytes
Herpetic folliculitis
IgA pemphigus
Pemphigus foliaceus
Takeaway Essentials
Clinical Relevant Pearls
Location of the abscess may hint to the diagnosis (e.g., abscesses located at 4:00 and 8:00 o’clock positions in the vulva should raise concern of Bartholin gland abscess).
Diagnosis is made by cultures, especially in recurrent infection in search of methicillin-resistant species.
The epidermolytic toxin by staphylococci is responsible of bullous impetigo and staphylococcal scalded skin syndrome.
Pathology Interpretation Pearls
In bullous impetigo, a Brown–Brenn stain would highlight the presence of bacteria producing the toxin in situ, while in staphylococcal scalded skin syndrome, the flaccid bullae is formed by a toxin that originated at a distant site.
Serial histologic section may be necessary to identify the residual component of the infected glands (e.g., Bartholin gland abscess) or follicle.
Histochemical stains to identify bacteria and fungus should be considered as part of the study of these cases (e.g., PAS, GMS, and B&B).
A skin biopsy for immunofluorescent tests should be consider to workup patient with flaccid bullae.
Streptococcal Infections
Streptococcal infections can cause several alterations in the genital area, ranging from superficial cutaneous infection to necrotizing fasciitis, which causes a rapidly progressive necrosis of the skin and subcutaneous tissue. Superficial bacterial infections are common in children between 3 and 5 years and have a predominant perianal distribution that can extend to the vulva and vagina. These lesions commonly yield group A or C hemolytic streptococci. In adults, streptococcal infections are uncommon and can occur alone or in conjunction with another skin disorder. Sometimes they are associated with diseases such as lichen sclerosus or other dermatoses.
The most severe presentation of this bacterial infection is necrotizing fasciitis caused by group A streptococci, which has increased in frequency over the past decade. However, polymicrobial infection with anaerobic and aerobic bacteria like Streptococcus , Staphylococcus aureus, Escherichia coli, Bacteroides, and Clostridium spp. is very common. This rare and distinct form of rapidly progressive necrosis of subcutaneous tissue and fascia can be fatal if not recognized early and if aggressive therapeutic intervention is not used. Vulvar lesions can extend to the perineum and abdominal wall. It usually occurs in diabetics, particularly when complicated by obesity, hypertension, and peripheral vascular disease, or in immunocompromised host [65, 66]. The infection’s origin can be a surgical incision (e.g., episiotomy) or a local abscess of skin or Bartholin gland. Mortality is up to 40 % if the disease is not recognized and debridement is delay or group A streptococcus is involved.
Clinical Features
Streptococcal infection clinical features are heterogeneous. Streptococcal vulvitis and perianal dermatitis produce tender, well-demarcated red skin with or without scale. The vagina shows erythematous mucosa and purulent discharge. Occasionally, anal fistulas with mucoid discharge and vulvar fissures develop. Lesions show excoriations and lichenification secondary to scratching and rubbing.
The patient suffering from necrotizing fasciitis presents with a hot, very tender, erythematous, swollen areas resistant to antibiotic treatment. Very rapidly, the skin changes from red to dusky-blue with bulla formation and necrosis. The soft tissue frequently feels indurated and wooden to palpation. Patients usually show signs or toxicity including fever, chills, malaise, shock, and tachycardia.
Microscopic Features
The characteristic features of necrotizing fasciitis are the presence of extensive necrosis extending from the epidermis to the subcutaneous tissue and sometimes to the muscle. Numerous polymorphonuclear leukocytes and mononuclear cells infiltrate the tissue and secondary vasculitis with trombi is present as large number bacterial colonies in the upper dermis [65]. Necrotizing fasciitis has been postulated as a form of septic vasculitis.
Differential Diagnosis
The differential diagnosis would include pyoderma gangrenosum and necrotizing Sweet syndrome. Pyodermal gangrenosum will be characterized by a folliculocentric neutrophilic dermal infiltrate with central necrotizing suppurative inflammation and peripheral lymphocytic vascular reaction. These histologic features, the absence of myonecrosis, and the clinical history of worsening after surgical procedures support the diagnosis of pyodermal gangrenosum. Necrotizing Sweet syndrome needs to be considered in patients with risk factors for this syndrome presenting with clinicopathologic features mimicking necrotizing fasciitis. Although histologic overlap preclude separation of these two diseases, multiple negative cultures, absence of significant myonecrosis, and improvement after steroid treatment will support the diagnosis of necrotizing Sweet syndrome [67].
Summary
Clinical Presentation
Two main clinical forms: one as a limited superficial infection and the other as a fulminant life-threatening deep infection.
In children, it presents as tender well-demarcated erythematous macules of the vulva with perianal involvement.
Necrotizing fasciitis shows rapid progression from red tender swollen areas to dusky red-blue bullae leading to extensive necrosis up to the muscle.
Histologic Features
Necrotizing fasciitis: extensive tissue necrosis affecting dermis up to the fascia and muscle with obliterative endarteritis and thrombosis
Differential Diagnosis
Pyoderma gangrenosum
Necrotizing Sweet syndrome
Takeaway Essentials
Clinical Relevant Pearls
Necrotizing fasciitis of the external genitalia is an emergency due to the high rate of lethality if not recognized early, and aggressive therapeutic intervention is pursued.
Superficial streptococcal infections in children should be considered in the differential diagnosis of sexual abuse.
Superficial streptococcal infection can coexist with other inflammatory disorders of the vulva like lichen sclerosus.
Pathology Interpretation Pearls
Cultures are useful to identify the infectious agent.
Syphilis
Syphilis is a worldwide highly contagious sexually transmitted disease that develops in four stages: primary, secondary, latent, and tertiary [68, 69]. The spirochete Treponema pallidum is the etiologic factor, and like other sexually transmitted diseases, coinfection with human immunodeficiency virus (HIV) is well recognized. Congenital and acquired syphilis present with cutaneous lesions including vulvar manifestation [70].
Clinical Features
Primary lesions (chancre) present as a painless, indurated, shallow, clean appearing ulcers with raised edges. The chancre usually presents within 3 weeks after initial contact at fourchette or labia majora; the range, however, is 7–90 days [69]. Spirochetes, Gram-negative slender and spiral-shaped bacteria, can be detected in the lesion. If secondarily infected, the chancre may become soft and painful and show an ulcerated surface. Most of the time, chancres are solitary lesions, but they can be multiple especially in immunocompromised women. Chancres may occur on inconspicuous surfaces, such as the vulva, perineum, cervix, anal mucosa, or oropharynx. In approximately 50 % of women and 30 % of men, the primary lesion cannot be identified. Lymphadenopathy presents 3–4 days after the chancre. The nodes are nontender, freely moveable, and rubbery. The exudate from the chancre is rich in spirochetes that can be detected using dark-field microscopy allowing a prompt diagnosis. Left untreated, the chancre will heal within 2–6 weeks and typically does not leave a scar; but the occurrence of vascular invasion will facilitate the progression to the secondary stage [69].
The secondary stage of the disease will become evident within 6 weeks to 6 months. At this point, the patient may present with a skin rash that often involves the trunk, genital area, flexor aspect of limbs, as well as the palms of the hands and soles of the feet. The cutaneous clinical phenotype is heterogeneous including maculopapular, annular, psoriasiform, pustular, lichenoid, and ulcerative lesions [71]. On occasion, the secondary lesions are papular, especially about the vulva, presenting as elevated flat, fleshy, moist plaques up to 3 cm in diameter (Fig. 6.18). These are known as condyloma lata and clinically may mimic condylomata acuminata. Condyloma lata are among the most contagious lesions in syphilis. Such lesions also may occur on other mucocutaneous areas. After 3–8 weeks, they disappear spontaneously. Pregnant women may infect the fetus via transplacental passage of the spirochete. In secondary stage, diagnostic tools include the serologic tests that detect the presence of antibodies to cardiolipin by rapid plasma reagin (RPR) and venereal disease research laboratory (VDRL) assay [72, 73]. Interactions between HIV and T. pallidum can accelerate the disease with atypical clinical presentation (e.g., present with lues maligna and severe ulcerations and pseudolymphomatous lesion) [74, 75], change laboratory presentation, as well as increase the risk for complications [76, 77]. In women infected with HIV or immunosuppressed, a false-negative RPR results or the prozone effect can be encountered [78]. This effect results from extremely high antibody titers in the infected women that the visualization of the reaction is compromised. Other diagnostic methods include microhemagglutination assay for antibodies to T. pallidum (MHA-TP) or detection of antibodies to surface proteins of T. pallidum by T. pallidum hemadsorption test (PTHA).
Fig. 6.18
Secondary syphilis. Clinically it presents as flat, fleshy, moist papules, and plaques. These lesions are known as Condyloma lata
Latency is the period of time between healing of the clinical lesions and appearance of late manifestations and may last for years. The tertiary gumma of syphilis, an exuberant systemic immune response to a low border spirochete, is rarely seen on the vulva.
Microscopic Findings
If syphilis was not considered in the clinical differential, diagnosis may be quite difficult from histologic findings alone. A biopsy is necessary when the chancre has an atypical clinical presentation or it is located in an unusual anatomic site. Microscopically, the diagnosis of syphilis may be difficult, especially in the genital area, because the presence of plasma cells, which is the hallmark for the diagnosis of syphilis in other areas of the body, is not very reliable in genital areas. Plasma cells are usually present in many inflammatory processes. Other features commonly present in primary lesions of syphilis are areas of ulceration covered by cellular debris and mixed inflammatory infiltrates including neutrophils and plasma cells. Acanthosis of the epidermis is common at the edges of the ulcer, and blood vessels show prominent endothelial swelling and hyperplasia leading to endarteritis obliterans. If the diagnosis of primary syphilis is suspected clinically, immunoperoxidase stains for the identification of T. pallidum should be performed. Serologic tests to confirm the diagnosis are essential. The Warthin–Starry stain or other silver impregnation techniques were used in the past to demonstrate the spirochetes; nowadays, however, more sensitive immunohistochemical stains have replaced this capricious method [79]. Immunohistochemical stains while extremely useful, they are hampered by false positivity for other spirochetal infections and borreliosis [72]. In primary syphilis, spirochetes are usually identified at the dermoepidermal junction and within and around superficial dermal blood vessels.
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