- Email: [email protected]
Aminolevulinic acid (ALA)-assisted photodynamic diagnosis of subclinical and latent HPV infection of external genital region
Photodiagnosis and Photodynamic Therapy (2008) 5, 251—255
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/pdpdt
Aminolevulinic acid (ALA)-assisted photodynamic diagnosis of subclinical and latent HPV infection of external genital region Hong-Wei Wang MD a,∗, Xiu-Li Wang a, Ling-Lin Zhang a, Ming-Xia Guo a, Zheng Huang b a
Shanghai Skin Diseases and STD Hospital, Shanghai 200050, China University of Colorado Denver, Aurora 80045, USA Available online 14 January 2009
b
KEYWORDS 5-Aminolevulinic acid (ALA); Fluorescence; Photodynamic diagnosis (PDD); Condylomata acuminata; Human papillomavirus (HPV)
Summary The relatively high recurrence rate of genital warts can be attributed to the unsuccessful elimination of viruses in areas of subclinical and latent infection. Therefore, the identification and treatment of the subclinical and latent infection is a key to reduce the recurrence. The goal of this study is to investigate the usefulness of 5-aminolevulinic acid (ALA)-assisted in situ fluorescence diagnosis of subclinical lesion and latent HPV infection. A total of 30 patients with histologically confirmed genital warts (condylomata acuminata) were subjected to topical application of ALA, acetic acid test, histopathologic examination and HPV DNA subtyping. Topical application of ALA was performed by applying 20% ALA cream to the lesion plus 2-cm margin for 2 h followed by fluorescence examination. Correlations between histopathologic examination, aceto-whitening test, HPV DNA subtyping and fluorescence were examined. All warty lesions and subclinical lesions (n = 25) showed red fluorescence and harbored HPV DNA (HPV6 or 11). Latent HPV infections at 0.5—2 cm away from the warty lesion also showed red fluorescence. Nonspecific fluorescence was associated with mucosa, inflammatory infiltration and erosive lesion. ALA-assisted photodynamic diagnosis could be employed for the detection of the lesion and subclinical lesion of genital warts. It is also useful in detecting latent HPV infection. © 2008 Elsevier B.V. All rights reserved.
Introduction Condylomata acuminata are benign anogenital warts caused by human papillomavirus (HPV), mainly type 6 and 11.
∗
Corresponding author. E-mail address: [email protected] (H.-W. Wang).
Manifestations of this HPV-associated disease range from clinically visible warty lesions to non-symptomatic subclinical lesion and latent infection. Although a reasonable expectation of treatment outcome is to achieve cure or long lasting remission, recurrence rates including new lesions at previously treated or new remote sites are estimated at 20—30% [1]. The relatively high recurrence might be caused by unsuccessful clearance of HPV-infected lesion and elimination of the virus in areas of subclinical and latent infection. Therefore, the identification and treatment
1572-1000/$ — see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.pdpdt.2008.11.004
252 of the subclinical lesion and latent infection might be a key to reduce the recurrence. Aceto-whitening test has been used to visualize subclinical lesion. But this simple test suffers poor specificity [2]. Early study suggests that 5-aminolevulinic acid (ALA)-based fluorescence detection might provide an alternative for visualization of subclinical lesions [3]. Since the discovery of endogenous fluorescent protoporphyrin IX (PpIX) induced by exogenous supply of ALA, skin premalignant and malignant lesions have become a favorite target of ALA-based photodynamic diagnosis (PDD) and therapy (PDT). Previous studies demonstrate that condylomata acuminata lesions of skin and mucosal surfaces can selectively accumulate PpIX through the heme metabolic pathway [4—7]. Our previous studies demonstrate that ALA-based PDT is an effective means for treatment of urethral condylomata acuminata and is associated with low incidence of side effects (e.g. ulcer and scarring) and recurrence [8]. Pharmacokinetics study indicates that ALA-induced PpIX fluorescence are dominantly distributed in the epidermis and accumulated in the rete Malpighii and koilocytes due to their high proliferation [9]. In this pilot study, we investigated the potential of ALA-assisted fluorescence examination for detecting the subclinical and latent HPV infection of genital condylomata acuminata.
Materials and methods Subjects From June 2006 to June 2007, 30 patients (24 male, 6 female) were clinically diagnosed as genital wart at the outpatient clinic of the Shanghai Skin Diseases and STD Hospital. The average age of those patients was 36.8 years old (23—54 years old). The average length of history was 5.03 months (1—24 months). Lesions were located at the external urethral orifice, balanus, coronary sulcus, medial prepuce and penis shaft in male patients and the labia majora, labia minora and around vagina opening in female patients. Condylomata acuminata was confirmed in all patients by biopsy and histopathological examination. Lesions were not accompanied by gonococcal or nongonococcal urethritis or cervicitis, autoimmune diseases, human immunodeficiency virus (HIV) infection or diabetics. None of the patients received systemic corticosteroid therapy or laser therapy or local chemotherapy within a month. All patients received and signed the consent form following approval by the Hospital Research Committee.
Materials Drug grade ALA (Aila® - Aminolevulinic Acid Hydrochloride for Topical Powder® ) was provided by Shanghai FudanZhangjiang Bio-Pharmaceutical Co., Ltd. (Shanghai, China) and used for preparing ALA cream. A handhold UV LED device (410 nm) was used as an excitation light source. A Kodak digital camera equipped with a UV filter was used to take fluorescence pictures.
H.-W. Wang et al.
Aceto-whitening test One day prior to photodynamic diagnosis procedure, the conventional aceto-whitening test was performed by applying 5% acetic acid solution to the warty lesion plus 2-cm margin for 5 min. Greyish white color with well-defined margin was considered as positive. The area showing white color but without visible clinical signs was classified as subclinical lesion.
Photodynamic diagnosis procedure Lesion and surrounding skin and mucosa were first cleared with 0.9% NaCl solution. A freshly prepared 20% ALA cream was then applied to the lesion plus 2-cm margin. The whole area was occluded with cling film and covered with thick gauze for light protection. After 2 h incubation, lesion was examined with the LED light source to visualize the presence of fluorescent PpIX. Bright red fluorescence (also known as ‘‘bright brick-red fluorescence’’) with welldefined margin was considered as positive.
Biopsy procedure Photographs were taken prior to and post-applying acetic acid and ALA. Biopsies were taken from four different locations: lesion, subclinical lesion (no visible lesion but aceto-whitening test positive), 0.5—1 cm away from the lesion (no visible lesion and aceto-whitening test negative but showing red fluorescence), and 1—2 cm away from the lesions (no visible lesion and aceto-whitening test negative but showing red fluorescence). Biopsies were divided into two portions. One was used for standard H&E staining and another used for DNA extraction and HPV DNA subtyping.
Laboratory analysis HPV DNA typing of 36 specimens was performed using a commercial HPV genotyping assay kit. The cytometric bead array (CBA) kit (Tellgen Life Science Corporation, Shanghai, China) could detect a total of 26 HPV DNA subtypes: including high-risk types — 16, 18, 31, 33, 35, 39, 45, 52, 58, 68, 51/59, 56/66, 26/55/61 and 73/83 and low-risk types — 6, 11, 40, 42, 44, 53, and 54. Briefly, the genomic DNA was extracted from each biopsy specimen and used as a template. Fragments of 26 HPV DNA subtypes were amplified by multiplex PCR. The resulting amplicons were then directly hybridized with microbeads array. The hybrid productions with matching sequences were labeled with strepavidin-PE and quantified by the Luminex 100 analyzer. Each subtype assay contained one positive control and one negative control.
Results Condylomata acuminata lesions in all 30 patients showed aceto-whitening test positive. In addition, there were 25 cases (83.3%) clearly showing white color spots with well-defined margin outside the lesion—–indicating possible subclinical lesions (Fig. 1b and e). Histological examina-
Photodynamic diagnosis of HPV infection
253
Figure 1 Representative images of aceto-whitening test and fluorescence examination. Case 1: (a) gross view of a male patient with 5-month history of condylomata acuminate. Yellow arrow indicates the lesion (HPV+). (b) Aceto-whitening test. White arrow indicates the subclinical lesion (HPV+). (c) Fluorescence examination. Green arrow indicates latent infection at 1 cm (HPV+) and blue arrow indicates latent infection at 2 cm (HPV+). Case 2: (d) gross view of a male patient with 2-month history of condylomata acuminata. Yellow arrow indicates the lesion (HPV+). (e) Aceto-whitening test. White arrow indicates the subclinical lesion (HPV+). (f) Fluorescence examination. Green arrow indicates latent infection at 0.5 cm (HPV+) and blue arrow indicates latent infection at 1.5 cm (HPV+). Case 3: (g) gross view of a male patient with 2-month history of condylomata acuminata (HPV+), accompanied with balanitis. (h) Fluorescence examination showing non-specific red fluorescence (HPV− in 0.5—2 cm zone). Case 4: (i) gross view of a female patient with 5-month history of condylomata acuminata (HPV+), accompanied with monilial vulvitis. (j) Fluorescence examination showing non-specific red fluorescence (HPV+ in 0.5—1 cm zone and HPV− in 1—2 cm zone).
tion of biopsies take at these areas showed acanthosis or epidermal thickening, papillomatous hyperplasia, elongated rete ridges, and vacuolar or ballooning degeneration with basophilic inclusions in the upper layer of epidermis. These histopathologic presentations were similar to characteristics of condylomata acuminate. Tissue specimens collected at 0.5—2 cm away from the lesion and subclinical showed no obvious histological changes. ALA cream was applied to the skin lesion plus 2 cm margin. After 2 h incubation in the dark, lesion and surrounding skin were examined using the UV LED light source. Atten-
tions were given to lesion, subclinical lesion, and 0.5—2 cm zone. All condylomata acuminata lesions showed intense red fluorescence. Among them, 21 cases (70%) showed distinct red fluorescence spots with well-defined margin (Fig. 1c and f). However, 9 cases (30%) showed diffused red fluorescence without well-defined margin (Fig. 1h and j). Such non-specific red fluorescence were mainly seen at (i) the adjacent mucosal area, (ii) areas showing obvious inflammatory signs (e.g. congestion, inflammatory papules, etc.) that were often accompanied with balanoposthitis or monilial vulvitis, or (iii) areas showing denuded epidermis accom-
254 Table 1
H.-W. Wang et al. Summary of aceto-whitening test, fluorescence examination and DNA subtyping. N
Aceto-whitening test
Fluorescence examination
DNA subtyping HPV6
HPV11
Lesion Subclinical lesion 0.5—1 cm
30 25 14
30 25 0
30 25 14
16 14 9
14 11 5
2 cm Fluorescence + Fluorescence −
4 26
0 0
4 0
2 2
2 1
panied with oozing. At the meantime, all subclinical lesions also showed the red fluorescence (Fig. 1c and f). In addition, the characteristic red fluorescence was also observed in 14 out of 30 cases (46.7%) at the 0.5—1 cm zone and 4 out of 30 cases (13.3%) at the 1—2 cm zone (Table 1). HPV DNA typing was performed using a commercial genotyping assay kit. Results showed that 16 out of 30 cases were HPV6 positive and other 14 cases were HPV11 positive. High risk HPV16, 18 and 45 were not detected in any specimens. Among 25 cases of subclinical lesions, 14 cases were HPV6 positive and 11 cases were HPV11 positive. At the 0.5—1 cm zone, among the 14 fluorescence positive cases, 9 cases were HPV6 positive and 5 cases were HPV11 positive. At the 1—2 cm zone, among the 4 fluorescence positive cases, 2 cases were HPV6 positive and 2 cases were HPV11 positive, whereas among the 26 fluorescence negative cases, HPV DNA were detected in 3 cases (2 cases of HPV6, 1 case of HPV11) (Table 1). The DNA subtyping of different locations of the same patient was consistence.
Discussion Human papillomavirus is one of the most common sexually transmitted infections (STI) in adolescents. The incidence of this infection has been on the rise in recent years. It has been estimated that the incidence of symptomatic condylomata acuminata approaches 0.5—1% in sexually active or young adults in the United States alone although the Chinese data is unavailable. Noticeably, the average age of this particular group of patients seen at our clinic is relatively older (i.e. 36.8 years). Recent studies also have shown that the incidence of subclinical HPV infection far exceeds the incidence of symptomatic infection [10,11]. Subclinical and latent infection might play roles in high recurrence and their non-invasive detection poses a great challenge. This pilot study investigated the usefulness of ALA-assisted photodynamic diagnosis in detecting subclinical lesion of condylomata acuminata. Many studies show that condylomata acuminata can selectively accumulate PpIX after topical application of ALA suggesting that ALA/PpIX mediated photoactivation is a potential diagnostic and therapeutic tool [3,4,7,9]. Our pharmacokinetics study indicates that ALA-induced PpIX fluorescence are dominantly distributed in the epidermis and accumulated in the rete Malpighii and koilocytes. The rete Malpighii is the initial infection and replication site and koilocytes are involved in virus particle assembly [12]. The
intensity of transient cellular PpIX fluorescence is affected by ALA incubation time. After 2 h incubation of 20% ALA solution, there is a significant increase in PpIX fluorescence intensity. The intensity reaches its peak at 3 h [9]. Ross et al. report that the fluorescence intensity ratio between lesion and surrounding normal tissues is determined by the incubation time. After 2 h incubation, there is a significant increase in the ratio. After reaching its peak at 3 h it begins to decrease [9], which implies that a prolonged incubation will increase the PpIX accumulation in normal tissue and therefore generate strong non-specific fluorescence. Therefore, we chose 2 h incubation for fluorescence examination in this study. Due to the long time interval between acetowhitening test and ALA application (>24 h), we believe there is no interference between those two tests. After 2 h incubation in the dark, all condylomas lesions showed intense red fluorescence (see Fig. 1). All subclinical lesions also showed the red fluorescence and the identical HPV subtype as detected at the lesion site (i.e. HPV6 or 11). These findings suggest that ALA-induced PpIX fluorescence might be a useful tool for the non-invasive detection of subclinical HPV lesion. Early diagnosis and treatment of subclinical HPV lesion and latent infection might reduce recurrence. During our practice, ALA and light application will also cover those areas. In this study, nine cases (30%) showed diffused red fluorescence without well-defined margin (see Fig. 1h and j). Such non-specific red fluorescence presented as an intense fluorescence background around the lesion and mainly found at the mucosal area or associated with inflammatory or denuded epidermis. This suggests that the damage of skin barrier can promote ALA absorption and PpIX production. Therefore, PpIX fluorescence examination has limited diagnostic value for areas involving female genital mucosa, urethral mucosa and overt inflammatory reactions. Aceto-whitening test is simple and fast. Its mechanism might be attributed to the molecular composition change when cells are affected by viral infection and undergo malignant growth [13]. But its clinical value in detecting subclinical and latent infection is still need to be determined. In this study, at the 0.5—1 cm zone, there were 14 out of 30 cases (46.7%) showed the red fluorescence and HPV positive but aceto-whitening test negative. Again, at the 1—2 cm zone, there was 4 out of 30 cases (13.3%) also showed the red fluorescence and HPV positive but aceto-whitening test negative (see Table 1). These results suggest that ALA-assisted fluorescence examination is superior to aceto-whitening test in finding latent injection sites. Furthermore, this also con-
Photodynamic diagnosis of HPV infection firms an early suggestion that ‘‘normal skin’’ 1 cm away from the condylomas lesion edge can indeed harbor HPV [14]. Our findings are also consistence with previous report and support that latent HPV infection can also exist at 2 cm away from the lesion site and beyond [15]. Although ALA-assisted fluorescence examination shows great potential in identifying latent infection sites, we also noticed that there were three cases showing HPV positive at the 2 cm away from the lesion site without fluorescence. This is possibly due to insufficient ALA absorption and/or inadequate sensitivity of this modality for detecting latent infection. Optimization of ALA formulation and utilization of ALA esters derivatives might further improve specificity and sensitivity [16,17]. In conclusion, although ALA/PpIX-assisted fluorescence examination cannot replace aceto-whitening test and use as a quick routine diagnostic tool because of its cost, multiplestep procedure and requirement of dark environment, it is superior to aceto-whitening test and histological examination in detecting subclinical lesion and latent infection. Its clinical value should be recognized. It might be a useful modality in localization of HPV infection site and determination of treatment zone during laser therapy and surgery.
255
[5]
[6]
[7]
[8]
[9]
[10] [11]
Acknowledgement
[12]
Authors are grateful to the Laser Research Institute of Munich University for providing the fluorescence imaging devices.
[13]
References [14] [1] Wilson J. Treatment of genital warts—–what’s the evidence? Int J STD AIDS 2002;13:216—22. [2] Wikstrom A, Hedblad MA, Johansson B, et al. The acetic acid test in evaluation of subclinical genital papillomavirus infection: a comparative study on penoscopy, histopathology, virology and scanning electron microscopy findings. Sex Transm Inf 1992;68:90—9. [3] Schneede P, Munch P, Wagner S, et al. Fluorescence urethroscopy following instillation of 5-aminolevulinic acid: a new procedure for detecting clinical and subclinical HPV lesions of the urethra. J Eur Acad Dermatol Venereol 2000;15:121—5. [4] Fehr MK, Chapman CF, Krasieva T, et al. Selective photosensitizer distribution in vulvar condyloma acuminatum after topical
[15]
[16] [17]
application of 5-aminolevulinic acid. Am J Obstet Gynecol 1996;174:951—7. Fehr MK, Hornung R, Degen A, et al. Photodynamic therapy of vulvar and vaginal condyloma and intraepithelial neoplasia using topically applied 5-aminolevulinic acid. Lasers Surg Med 2002;30:273—9. Frank GB, Bos JD. Photodynamic therapy for condylomata acuminata with local application of 5-aminolevulinic acid. Sex Transm Inf 1996;72:70—1. Ross EV, Romero R, Kollias N, Crum C, Anderson RR. Selectivity of protoporphyrin IX fluorescence for condylomata after topical application of 5-aminolaevulinic acid: implications for photodynamic treatment. Br J Dermatol 1997;137: 736—42. Wang XL, Wang HW, Wang HS, et al. Topical 5-aminolevulinic acid-photodynamic therapy for the treatment of urethral condylomata acuminata. Br J Dermatol 2004;151:880—5. Wang XL, Wang HW, Huang Z, et al. Study of protoporphyrin IX (PpIX) pharmacokinetics after topical application of ALA in urethral condylomata acuminata. Photochem Photobiol 2007;83:1—5. Anorlu RI. What is the significance of the HPV epidemic? Can J Urol 2008;15:3860—5. Dempsey AF, Koutsky LA. National burden of genital warts: a first step in defining the problem. Sex Transm Dis 2008;35:361—2. Chow LT, Broker TR. Mechanisms and regulation of papillomavirus DNA replication. In: Saveria Campo M, editor. Papillomavirus research: from natural history to vaccines and beyond. Caister Academic Press; 2006. p. 193—206. Wu TT, Qu JY. Assessment of the relative contribution of cellular components to the acetowhitening effect in cell cultures and suspensions using elastic light-scattering spectroscopy. Appl Opt 2007;46:4834—42. Ferenczy A, Behelak Y, Haber G, Wright Jr TC, Richart RM. Treating vaginal and external anogenital condylomas with electrosurgery vs CO2 laser ablation. J Gynecol Surg 1995;11:41—50. Stefanaki IM, Georgiou S, Themelis GC, Vazgiouraki EM, Tosca AD. In vivo fluorescence kinetics and photodynamic therapy in condylomata acuminata. Br J Dermatol 2003;149: 972—6. Krammer B, Plaetzer K. ALA and its clinical impact, from bench to bedside. Photochem Photobiol Sci 2008;7:283—9. Lesar A, Padgett M, O’Dwyer M, Ferguson J, Moseley H. Fluorescence induced by aminolevulinic acid and methyl aminolevulinate on normal skin. Photodiagn Photodyn Ther 2007;4:224—9.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/pdpdt
Aminolevulinic acid (ALA)-assisted photodynamic diagnosis of subclinical and latent HPV infection of external genital region Hong-Wei Wang MD a,∗, Xiu-Li Wang a, Ling-Lin Zhang a, Ming-Xia Guo a, Zheng Huang b a
Shanghai Skin Diseases and STD Hospital, Shanghai 200050, China University of Colorado Denver, Aurora 80045, USA Available online 14 January 2009
b
KEYWORDS 5-Aminolevulinic acid (ALA); Fluorescence; Photodynamic diagnosis (PDD); Condylomata acuminata; Human papillomavirus (HPV)
Summary The relatively high recurrence rate of genital warts can be attributed to the unsuccessful elimination of viruses in areas of subclinical and latent infection. Therefore, the identification and treatment of the subclinical and latent infection is a key to reduce the recurrence. The goal of this study is to investigate the usefulness of 5-aminolevulinic acid (ALA)-assisted in situ fluorescence diagnosis of subclinical lesion and latent HPV infection. A total of 30 patients with histologically confirmed genital warts (condylomata acuminata) were subjected to topical application of ALA, acetic acid test, histopathologic examination and HPV DNA subtyping. Topical application of ALA was performed by applying 20% ALA cream to the lesion plus 2-cm margin for 2 h followed by fluorescence examination. Correlations between histopathologic examination, aceto-whitening test, HPV DNA subtyping and fluorescence were examined. All warty lesions and subclinical lesions (n = 25) showed red fluorescence and harbored HPV DNA (HPV6 or 11). Latent HPV infections at 0.5—2 cm away from the warty lesion also showed red fluorescence. Nonspecific fluorescence was associated with mucosa, inflammatory infiltration and erosive lesion. ALA-assisted photodynamic diagnosis could be employed for the detection of the lesion and subclinical lesion of genital warts. It is also useful in detecting latent HPV infection. © 2008 Elsevier B.V. All rights reserved.
Introduction Condylomata acuminata are benign anogenital warts caused by human papillomavirus (HPV), mainly type 6 and 11.
∗
Corresponding author. E-mail address: [email protected] (H.-W. Wang).
Manifestations of this HPV-associated disease range from clinically visible warty lesions to non-symptomatic subclinical lesion and latent infection. Although a reasonable expectation of treatment outcome is to achieve cure or long lasting remission, recurrence rates including new lesions at previously treated or new remote sites are estimated at 20—30% [1]. The relatively high recurrence might be caused by unsuccessful clearance of HPV-infected lesion and elimination of the virus in areas of subclinical and latent infection. Therefore, the identification and treatment
1572-1000/$ — see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.pdpdt.2008.11.004
252 of the subclinical lesion and latent infection might be a key to reduce the recurrence. Aceto-whitening test has been used to visualize subclinical lesion. But this simple test suffers poor specificity [2]. Early study suggests that 5-aminolevulinic acid (ALA)-based fluorescence detection might provide an alternative for visualization of subclinical lesions [3]. Since the discovery of endogenous fluorescent protoporphyrin IX (PpIX) induced by exogenous supply of ALA, skin premalignant and malignant lesions have become a favorite target of ALA-based photodynamic diagnosis (PDD) and therapy (PDT). Previous studies demonstrate that condylomata acuminata lesions of skin and mucosal surfaces can selectively accumulate PpIX through the heme metabolic pathway [4—7]. Our previous studies demonstrate that ALA-based PDT is an effective means for treatment of urethral condylomata acuminata and is associated with low incidence of side effects (e.g. ulcer and scarring) and recurrence [8]. Pharmacokinetics study indicates that ALA-induced PpIX fluorescence are dominantly distributed in the epidermis and accumulated in the rete Malpighii and koilocytes due to their high proliferation [9]. In this pilot study, we investigated the potential of ALA-assisted fluorescence examination for detecting the subclinical and latent HPV infection of genital condylomata acuminata.
Materials and methods Subjects From June 2006 to June 2007, 30 patients (24 male, 6 female) were clinically diagnosed as genital wart at the outpatient clinic of the Shanghai Skin Diseases and STD Hospital. The average age of those patients was 36.8 years old (23—54 years old). The average length of history was 5.03 months (1—24 months). Lesions were located at the external urethral orifice, balanus, coronary sulcus, medial prepuce and penis shaft in male patients and the labia majora, labia minora and around vagina opening in female patients. Condylomata acuminata was confirmed in all patients by biopsy and histopathological examination. Lesions were not accompanied by gonococcal or nongonococcal urethritis or cervicitis, autoimmune diseases, human immunodeficiency virus (HIV) infection or diabetics. None of the patients received systemic corticosteroid therapy or laser therapy or local chemotherapy within a month. All patients received and signed the consent form following approval by the Hospital Research Committee.
Materials Drug grade ALA (Aila® - Aminolevulinic Acid Hydrochloride for Topical Powder® ) was provided by Shanghai FudanZhangjiang Bio-Pharmaceutical Co., Ltd. (Shanghai, China) and used for preparing ALA cream. A handhold UV LED device (410 nm) was used as an excitation light source. A Kodak digital camera equipped with a UV filter was used to take fluorescence pictures.
H.-W. Wang et al.
Aceto-whitening test One day prior to photodynamic diagnosis procedure, the conventional aceto-whitening test was performed by applying 5% acetic acid solution to the warty lesion plus 2-cm margin for 5 min. Greyish white color with well-defined margin was considered as positive. The area showing white color but without visible clinical signs was classified as subclinical lesion.
Photodynamic diagnosis procedure Lesion and surrounding skin and mucosa were first cleared with 0.9% NaCl solution. A freshly prepared 20% ALA cream was then applied to the lesion plus 2-cm margin. The whole area was occluded with cling film and covered with thick gauze for light protection. After 2 h incubation, lesion was examined with the LED light source to visualize the presence of fluorescent PpIX. Bright red fluorescence (also known as ‘‘bright brick-red fluorescence’’) with welldefined margin was considered as positive.
Biopsy procedure Photographs were taken prior to and post-applying acetic acid and ALA. Biopsies were taken from four different locations: lesion, subclinical lesion (no visible lesion but aceto-whitening test positive), 0.5—1 cm away from the lesion (no visible lesion and aceto-whitening test negative but showing red fluorescence), and 1—2 cm away from the lesions (no visible lesion and aceto-whitening test negative but showing red fluorescence). Biopsies were divided into two portions. One was used for standard H&E staining and another used for DNA extraction and HPV DNA subtyping.
Laboratory analysis HPV DNA typing of 36 specimens was performed using a commercial HPV genotyping assay kit. The cytometric bead array (CBA) kit (Tellgen Life Science Corporation, Shanghai, China) could detect a total of 26 HPV DNA subtypes: including high-risk types — 16, 18, 31, 33, 35, 39, 45, 52, 58, 68, 51/59, 56/66, 26/55/61 and 73/83 and low-risk types — 6, 11, 40, 42, 44, 53, and 54. Briefly, the genomic DNA was extracted from each biopsy specimen and used as a template. Fragments of 26 HPV DNA subtypes were amplified by multiplex PCR. The resulting amplicons were then directly hybridized with microbeads array. The hybrid productions with matching sequences were labeled with strepavidin-PE and quantified by the Luminex 100 analyzer. Each subtype assay contained one positive control and one negative control.
Results Condylomata acuminata lesions in all 30 patients showed aceto-whitening test positive. In addition, there were 25 cases (83.3%) clearly showing white color spots with well-defined margin outside the lesion—–indicating possible subclinical lesions (Fig. 1b and e). Histological examina-
Photodynamic diagnosis of HPV infection
253
Figure 1 Representative images of aceto-whitening test and fluorescence examination. Case 1: (a) gross view of a male patient with 5-month history of condylomata acuminate. Yellow arrow indicates the lesion (HPV+). (b) Aceto-whitening test. White arrow indicates the subclinical lesion (HPV+). (c) Fluorescence examination. Green arrow indicates latent infection at 1 cm (HPV+) and blue arrow indicates latent infection at 2 cm (HPV+). Case 2: (d) gross view of a male patient with 2-month history of condylomata acuminata. Yellow arrow indicates the lesion (HPV+). (e) Aceto-whitening test. White arrow indicates the subclinical lesion (HPV+). (f) Fluorescence examination. Green arrow indicates latent infection at 0.5 cm (HPV+) and blue arrow indicates latent infection at 1.5 cm (HPV+). Case 3: (g) gross view of a male patient with 2-month history of condylomata acuminata (HPV+), accompanied with balanitis. (h) Fluorescence examination showing non-specific red fluorescence (HPV− in 0.5—2 cm zone). Case 4: (i) gross view of a female patient with 5-month history of condylomata acuminata (HPV+), accompanied with monilial vulvitis. (j) Fluorescence examination showing non-specific red fluorescence (HPV+ in 0.5—1 cm zone and HPV− in 1—2 cm zone).
tion of biopsies take at these areas showed acanthosis or epidermal thickening, papillomatous hyperplasia, elongated rete ridges, and vacuolar or ballooning degeneration with basophilic inclusions in the upper layer of epidermis. These histopathologic presentations were similar to characteristics of condylomata acuminate. Tissue specimens collected at 0.5—2 cm away from the lesion and subclinical showed no obvious histological changes. ALA cream was applied to the skin lesion plus 2 cm margin. After 2 h incubation in the dark, lesion and surrounding skin were examined using the UV LED light source. Atten-
tions were given to lesion, subclinical lesion, and 0.5—2 cm zone. All condylomata acuminata lesions showed intense red fluorescence. Among them, 21 cases (70%) showed distinct red fluorescence spots with well-defined margin (Fig. 1c and f). However, 9 cases (30%) showed diffused red fluorescence without well-defined margin (Fig. 1h and j). Such non-specific red fluorescence were mainly seen at (i) the adjacent mucosal area, (ii) areas showing obvious inflammatory signs (e.g. congestion, inflammatory papules, etc.) that were often accompanied with balanoposthitis or monilial vulvitis, or (iii) areas showing denuded epidermis accom-
254 Table 1
H.-W. Wang et al. Summary of aceto-whitening test, fluorescence examination and DNA subtyping. N
Aceto-whitening test
Fluorescence examination
DNA subtyping HPV6
HPV11
Lesion Subclinical lesion 0.5—1 cm
30 25 14
30 25 0
30 25 14
16 14 9
14 11 5
2 cm Fluorescence + Fluorescence −
4 26
0 0
4 0
2 2
2 1
panied with oozing. At the meantime, all subclinical lesions also showed the red fluorescence (Fig. 1c and f). In addition, the characteristic red fluorescence was also observed in 14 out of 30 cases (46.7%) at the 0.5—1 cm zone and 4 out of 30 cases (13.3%) at the 1—2 cm zone (Table 1). HPV DNA typing was performed using a commercial genotyping assay kit. Results showed that 16 out of 30 cases were HPV6 positive and other 14 cases were HPV11 positive. High risk HPV16, 18 and 45 were not detected in any specimens. Among 25 cases of subclinical lesions, 14 cases were HPV6 positive and 11 cases were HPV11 positive. At the 0.5—1 cm zone, among the 14 fluorescence positive cases, 9 cases were HPV6 positive and 5 cases were HPV11 positive. At the 1—2 cm zone, among the 4 fluorescence positive cases, 2 cases were HPV6 positive and 2 cases were HPV11 positive, whereas among the 26 fluorescence negative cases, HPV DNA were detected in 3 cases (2 cases of HPV6, 1 case of HPV11) (Table 1). The DNA subtyping of different locations of the same patient was consistence.
Discussion Human papillomavirus is one of the most common sexually transmitted infections (STI) in adolescents. The incidence of this infection has been on the rise in recent years. It has been estimated that the incidence of symptomatic condylomata acuminata approaches 0.5—1% in sexually active or young adults in the United States alone although the Chinese data is unavailable. Noticeably, the average age of this particular group of patients seen at our clinic is relatively older (i.e. 36.8 years). Recent studies also have shown that the incidence of subclinical HPV infection far exceeds the incidence of symptomatic infection [10,11]. Subclinical and latent infection might play roles in high recurrence and their non-invasive detection poses a great challenge. This pilot study investigated the usefulness of ALA-assisted photodynamic diagnosis in detecting subclinical lesion of condylomata acuminata. Many studies show that condylomata acuminata can selectively accumulate PpIX after topical application of ALA suggesting that ALA/PpIX mediated photoactivation is a potential diagnostic and therapeutic tool [3,4,7,9]. Our pharmacokinetics study indicates that ALA-induced PpIX fluorescence are dominantly distributed in the epidermis and accumulated in the rete Malpighii and koilocytes. The rete Malpighii is the initial infection and replication site and koilocytes are involved in virus particle assembly [12]. The
intensity of transient cellular PpIX fluorescence is affected by ALA incubation time. After 2 h incubation of 20% ALA solution, there is a significant increase in PpIX fluorescence intensity. The intensity reaches its peak at 3 h [9]. Ross et al. report that the fluorescence intensity ratio between lesion and surrounding normal tissues is determined by the incubation time. After 2 h incubation, there is a significant increase in the ratio. After reaching its peak at 3 h it begins to decrease [9], which implies that a prolonged incubation will increase the PpIX accumulation in normal tissue and therefore generate strong non-specific fluorescence. Therefore, we chose 2 h incubation for fluorescence examination in this study. Due to the long time interval between acetowhitening test and ALA application (>24 h), we believe there is no interference between those two tests. After 2 h incubation in the dark, all condylomas lesions showed intense red fluorescence (see Fig. 1). All subclinical lesions also showed the red fluorescence and the identical HPV subtype as detected at the lesion site (i.e. HPV6 or 11). These findings suggest that ALA-induced PpIX fluorescence might be a useful tool for the non-invasive detection of subclinical HPV lesion. Early diagnosis and treatment of subclinical HPV lesion and latent infection might reduce recurrence. During our practice, ALA and light application will also cover those areas. In this study, nine cases (30%) showed diffused red fluorescence without well-defined margin (see Fig. 1h and j). Such non-specific red fluorescence presented as an intense fluorescence background around the lesion and mainly found at the mucosal area or associated with inflammatory or denuded epidermis. This suggests that the damage of skin barrier can promote ALA absorption and PpIX production. Therefore, PpIX fluorescence examination has limited diagnostic value for areas involving female genital mucosa, urethral mucosa and overt inflammatory reactions. Aceto-whitening test is simple and fast. Its mechanism might be attributed to the molecular composition change when cells are affected by viral infection and undergo malignant growth [13]. But its clinical value in detecting subclinical and latent infection is still need to be determined. In this study, at the 0.5—1 cm zone, there were 14 out of 30 cases (46.7%) showed the red fluorescence and HPV positive but aceto-whitening test negative. Again, at the 1—2 cm zone, there was 4 out of 30 cases (13.3%) also showed the red fluorescence and HPV positive but aceto-whitening test negative (see Table 1). These results suggest that ALA-assisted fluorescence examination is superior to aceto-whitening test in finding latent injection sites. Furthermore, this also con-
Photodynamic diagnosis of HPV infection firms an early suggestion that ‘‘normal skin’’ 1 cm away from the condylomas lesion edge can indeed harbor HPV [14]. Our findings are also consistence with previous report and support that latent HPV infection can also exist at 2 cm away from the lesion site and beyond [15]. Although ALA-assisted fluorescence examination shows great potential in identifying latent infection sites, we also noticed that there were three cases showing HPV positive at the 2 cm away from the lesion site without fluorescence. This is possibly due to insufficient ALA absorption and/or inadequate sensitivity of this modality for detecting latent infection. Optimization of ALA formulation and utilization of ALA esters derivatives might further improve specificity and sensitivity [16,17]. In conclusion, although ALA/PpIX-assisted fluorescence examination cannot replace aceto-whitening test and use as a quick routine diagnostic tool because of its cost, multiplestep procedure and requirement of dark environment, it is superior to aceto-whitening test and histological examination in detecting subclinical lesion and latent infection. Its clinical value should be recognized. It might be a useful modality in localization of HPV infection site and determination of treatment zone during laser therapy and surgery.
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[5]
[6]
[7]
[8]
[9]
[10] [11]
Acknowledgement
[12]
Authors are grateful to the Laser Research Institute of Munich University for providing the fluorescence imaging devices.
[13]
References [14] [1] Wilson J. Treatment of genital warts—–what’s the evidence? Int J STD AIDS 2002;13:216—22. [2] Wikstrom A, Hedblad MA, Johansson B, et al. The acetic acid test in evaluation of subclinical genital papillomavirus infection: a comparative study on penoscopy, histopathology, virology and scanning electron microscopy findings. Sex Transm Inf 1992;68:90—9. [3] Schneede P, Munch P, Wagner S, et al. Fluorescence urethroscopy following instillation of 5-aminolevulinic acid: a new procedure for detecting clinical and subclinical HPV lesions of the urethra. J Eur Acad Dermatol Venereol 2000;15:121—5. [4] Fehr MK, Chapman CF, Krasieva T, et al. Selective photosensitizer distribution in vulvar condyloma acuminatum after topical
[15]
[16] [17]
application of 5-aminolevulinic acid. Am J Obstet Gynecol 1996;174:951—7. Fehr MK, Hornung R, Degen A, et al. Photodynamic therapy of vulvar and vaginal condyloma and intraepithelial neoplasia using topically applied 5-aminolevulinic acid. Lasers Surg Med 2002;30:273—9. Frank GB, Bos JD. Photodynamic therapy for condylomata acuminata with local application of 5-aminolevulinic acid. Sex Transm Inf 1996;72:70—1. Ross EV, Romero R, Kollias N, Crum C, Anderson RR. Selectivity of protoporphyrin IX fluorescence for condylomata after topical application of 5-aminolaevulinic acid: implications for photodynamic treatment. Br J Dermatol 1997;137: 736—42. Wang XL, Wang HW, Wang HS, et al. Topical 5-aminolevulinic acid-photodynamic therapy for the treatment of urethral condylomata acuminata. Br J Dermatol 2004;151:880—5. Wang XL, Wang HW, Huang Z, et al. Study of protoporphyrin IX (PpIX) pharmacokinetics after topical application of ALA in urethral condylomata acuminata. Photochem Photobiol 2007;83:1—5. Anorlu RI. What is the significance of the HPV epidemic? Can J Urol 2008;15:3860—5. Dempsey AF, Koutsky LA. National burden of genital warts: a first step in defining the problem. Sex Transm Dis 2008;35:361—2. Chow LT, Broker TR. Mechanisms and regulation of papillomavirus DNA replication. In: Saveria Campo M, editor. Papillomavirus research: from natural history to vaccines and beyond. Caister Academic Press; 2006. p. 193—206. Wu TT, Qu JY. Assessment of the relative contribution of cellular components to the acetowhitening effect in cell cultures and suspensions using elastic light-scattering spectroscopy. Appl Opt 2007;46:4834—42. Ferenczy A, Behelak Y, Haber G, Wright Jr TC, Richart RM. Treating vaginal and external anogenital condylomas with electrosurgery vs CO2 laser ablation. J Gynecol Surg 1995;11:41—50. Stefanaki IM, Georgiou S, Themelis GC, Vazgiouraki EM, Tosca AD. In vivo fluorescence kinetics and photodynamic therapy in condylomata acuminata. Br J Dermatol 2003;149: 972—6. Krammer B, Plaetzer K. ALA and its clinical impact, from bench to bedside. Photochem Photobiol Sci 2008;7:283—9. Lesar A, Padgett M, O’Dwyer M, Ferguson J, Moseley H. Fluorescence induced by aminolevulinic acid and methyl aminolevulinate on normal skin. Photodiagn Photodyn Ther 2007;4:224—9.