- Email: [email protected]
Reflectance confocal microscopy for the in vivo detection of Treponema pallidum in skin lesions of secondary syphilis
Reflectance confocal microscopy for the in vivo detection of Treponema pallidum in skin lesions of secondary syphilis Marina Venturini, MD,a Raffaella Sala, MD,a Diego Semenza, MD,a Amerigo Santoro, MD,b Fabio Facchetti, MD,b and Piergiacomo Calzavara-Pinton, MDa Brescia, Italy Background: Secondary syphilis is a diagnostic challenge that relies on microscopic and laboratory tests. Objective: We sought to assess the usefulness of in vivo reflectance confocal microscopy (RCM) to detect Treponema pallidum in lesions suggestive of secondary syphilis. Methods: Macular and papular skin lesions from 3 patients clinically suggestive of secondary syphilis were imaged by RCM and confirmed by skin punch biopsy. Results: In all lesions RCM demonstrated elongated small bright particles with a spiral shape intermingled with the keratinocytes. These features corresponded with immunohistochemical findings that revealed several spirochetes infiltrating the epidermis. Limitations: Unlike immunohistochemistry, RCM did not visualize T pallidum in the dermis and vascular walls because of limited imaging depth. The specificity and sensitivity of this technique need to be assessed. Conclusion: RCM may be an effective diagnostic tool for in vivo real-time imaging of T pallidum in skin lesions of secondary syphilis, and seems to correlate well with immunohistochemistry. ( J Am Acad Dermatol 2009;60:639-42.)
T
he diagnosis of secondary syphilis is challenging because of the wide range of clinical forms it assumes and, therefore, physicians must always rely on microscopic and laboratory tests to verify their diagnoses. Dark-field microscopy is an effective real-time aid in most cases, mainly for moist lesions.1 However, it has some drawbacks. First, it requires trained specialists2 and second, when it is used for nonmoist lesions, the stratum corneum must be removed by repeated adhesive tape stripping. Serology and immunohistochemistry are diagnostic mainstays as well, but require several days before the results are available, and their use is limited by the From the Departments of Dermatologya and Pathology,b University of Brescia, Spedali Civili Hospital. Funding sources: None. Conflicts of interest: None declared. Reprint requests: Piergiacomo Calzavara-Pinton, MD, Department of Dermatology, Spedali Civili Hospital, P.le Spedali Civili 1, 25123 Brescia, Italy. E-mail: [email protected]. 0190-9622/$36.00 ª 2008 by the American Academy of Dermatology, Inc. doi:10.1016/j.jaad.2008.11.901
invasiveness of taking blood and tissue samples. In addition, serology will show a negative response in the first days of an infection, and this seronegative window may be even longer in cases of HIV infection. False-negative results in screening with nontreponemal tests, such as the VDRL test or rapid plasma reagin assays, may occur as a result of the prozone phenomenon, which is a false-negative reaction to a serologic test that occurs when very high antibody titers in the serum prevent the formation of the antibody-antigen lattice needed to visualize a flocculation reaction.3-5 The prozone phenomenon occurs in 0.3% to 2% of all cases of syphilis6 and may be more common in diseases associated with B-cell dysregulation and excess antibody, such as in patients with HIV7,8 and transplantation.9 In addition, the use of immunohistochemistry is limited because it is only available in a few specialized laboratories.2 Therefore, a new affordable real-time diagnostic tool for secondary syphilis is needed. Reflectance confocal microscopy (RCM) is a novel noninvasive technique for in vivo, real-time, en face imaging of epidermis and papillary dermis,10 with a resolution power close to that available with 639
640 Venturini et al
J AM ACAD DERMATOL APRIL 2009
Fig 1. Generalized erythematous macules and keratotic papules.
conventional histology. RCM has been previously reported to be useful in the in vivo assessment of skin tumors and various inflammatory skin diseases including contact dermatitis, psoriasis, and discoid lupus erythematosus.11-19 Furthermore, it has been proven effective in the imaging of pathogens in onychomycosis and scabies.20,21 In this article we describe the in vivo real-time disclosure of Treponema pallidum by RCM in 3 cases of secondary syphilis.
METHODS We enrolled patients who had been sent to our university outpatient clinic for sexually transmitted diseases because they had skin lesions and anamnestic findings that raised suspicion that secondary syphilis could have been the cause. Routine blood chemistries, hematology, urinalysis, and T-cell counts were performed by standard methods. In addition, both enzyme-linked immunosorbent assay and Western blot techniques for the detection of HIV antibodies, and a panel of serology tests for syphilis including rapid plasma reagin test, T pallidum particle agglutination assay, and the fluorescent treponemal antibody absorption (IgG and IgM) test, were applied. Skin lesions were investigated with the dark-field examination of scrapings and in vivo RCM, and with the histologic and immunohistochemical examination of biopsy samples. The RCM images were taken with a Vivascope 1500 (Lucid Technologies, Henrietta, NY). This system uses a 330 water immersion objective lens of numeric aperture 0.9 and 830-nm laser source with a power less than 35 mW/cm2 at tissue level. The device provides a lateral resolution of 1.0 m, an axial resolution of 3.0 m, and an imaging depth of 200 to 250 m in vivo.10 In addition, VivaScan 7.0 software (Lucid Technologies) saves still images as bmp files. The system captures 9 video frames per second as a 24-bit uncompressed avi file, with a digital resolution
of 1000 3 1000 pixels and 256 levels of grey. The system can register movies with a time length up to 2 minutes. The size of 10 spirochetes was measured with the software. Results were given as mean 6 SD. RCM images were taken from a lesion of the arm in all patients and the same lesion was biopsied to compare the histologic and confocal findings. Biopsy samples were examined with standard histology after staining with hematoxylin-eosin, and with immunohistochemistry after heating for 15 minutes in EDTA buffer and staining with anti-T pallidum antibodies (Biocare Medical, Concord, CA) at a 1:100 dilution. Image analysis of spirochetes was performed with the software Cell^F 2.5 (Olympus Soft Imaging Solutions GmbH, Hamburg, Germany).
RESULTS Three young patients, two homosexual men and one woman, were seen for skin eruptions clinically suggestive of secondary syphilis. Two of the patients said they had noted an ulcerative nodule on the genitalia in the recent past. These lesions had disappeared spontaneously without any systemic antibiotic therapy. Dermatologic examination showed the skin lesions to be macular in one patient (Fig 1) and papular in two. All patients had lesions on the trunk and two showed in addition involvement of limbs, palms, and soles. Slight fever, malaise, and weight loss were reported by all patients and generalized lymphadenopathy was detected in two. Serology for syphilis was positive in two patients. All patients were HIV positive with a remarkable decrease in CD31 and CD41 T-cell counts. Pertinent clinical and laboratory findings are reported in detail in Table I. No other significant abnormalities of blood chemistries or hematology were detected. RCM examination disclosed small elongated bright particles with a spiral shape intermingled with the keratinocytes of the stratum granulosum, stratum spinosum, and basal layers (Figs 2 and 3). Their sizes ranged from 4.3 to 15.7 m (mean 6 SD: 9.4 6 3.4 m). Particles were grouped in clusters in the stratum granulosum and isolated in the stratum spinosum and basal layers. No particles were seen in the stratum corneum. RCM investigations of the papillary dermis did not show particles. In realtime video recording up to 1 minute in length, the bright helical-shaped particles appeared to be motionless in the epidermis. The lesions were subsequently biopsied. Histologic examination showed dilatation and thickening of blood vessels with a proliferation of endothelial cells and a papillary and reticular perivascular
Venturini et al 641
J AM ACAD DERMATOL VOLUME 60, NUMBER 4
Table I. Clinical and laboratory findings of the 3 cases Patient No.
Age, y/sex Skin lesions Skin distribution Slight fever, malaise, and weight loss Generalized lymphoadenopathy Duration of lesions, wk Previous ulcerated nodule on genitalia RPR TPPA FTA-ABS (IgG and IgM) HIV serology T-cell counts (CD31/CD41/CD81), cells/mm3 Dark-field examination RCM investigation Immunohistochemistry
1
2
3
34/M Macules Generalized Yes Yes 3 Yes 1:8 1:320 Positive Positive 530/51/142 Negative Positive Positive
36/M Papules Generalized Yes Yes 4 Yes Negative Negative Negative Positive 480/73/223 Positive Positive Positive
33/F Papules Trunk Yes No 2 No 1:4 1:320 Positive Positive 602/100/410 Positive Positive Positive
F, Female; FTA-ABS, fluorescent treponemal antibody absorption; M, male; RCM, reflectance confocal microscopy; RPR, rapid plasma reagin; TPPA, Treponema pallidum particle agglutination assay.
Fig 3. Reflectance confocal microscopy image shows bright particles with helical shape (arrows) corresponding to spirochetes in stratum spinosum (scale bar: 50 m). Fig 2. Group of spirochetes in stratum granulosum. Reflectance confocal microscopy image shows clusters of small elongated bright particles (white circle) corresponding to spirochetes in superficial epidermis (scale bar: 50 m).
dermal infiltrate containing numerous plasma cells. Immunohistochemical investigation revealed several spirochetes infiltrating the epidermis, dermis, and walls of blood vessels (Fig 4). The spirochetes were helical in shape and, using electronic image analysis to measure the length of 10 micro-organisms, we found them to range from 1.9 to 12.5 m (mean 6 SD: 6.2 6 3.2 m). On the basis of the clinical, serologic, and histopathological data, a final diagnosis of secondary syphilis was made in the 3 patients. Lumbar puncture was performed in all patients and cerebrospinal fluid evaluation excluded neurosyphilis (VDRL test # 1:32; leukocyte count # 5/mm3).
A single intramuscular injection of 2.4 million units of benzathine benzylpenicillin was administered to the patients, and their lesions disappeared within a few days. At a follow-up visit 30 days later, the serologic tests for syphilis of patient 2 were positive (rapid plasma reagin 1:2; T pallidum particle agglutination assay 1:320; fluorescent treponemal antibody absorption positive).
DISCUSSION In this investigation, RCM proved to be a noninvasive, real-time tool for the in vivo imaging of T pallidum in skin lesions of secondary syphilis. We emphasize that RCM disclosed spirochetes in all 3 patients whereas dark-field examination was negative in the patient with the macular eruption and serology was negative in another when first tested.7,22 Spirochetes were visualized in the superficial layers of epidermis as small, bright, elongated, and spiral-shaped particles. Their brightness is likely
642 Venturini et al
J AM ACAD DERMATOL APRIL 2009
Fig 4. Immunohistochemistry for Treponema pallidum: numerous spirochetes in epidermis and dermis (scale bar: 50 m). (Original magnification: 3400.)
to be caused by the highly reflective protein content of their cellular structures (outer membrane, peptide-glycan-cytoplasmic membrane complex, and endoflagella).23 In the horizontal sections of RCM we found the spirochetes to be mostly grouped in clusters in the stratum granulosum and sparse and isolated in the stratum spinosum and basal layers. The morphology, grouping, and location of these particles correlated well with those of the spirochetes seen with immunohistochemistry. Although the mean length of spirochetes was less with immunostaining in comparison with RCM (6.2 6 3.2 vs 9.4 6 3.4 m, respectively), this difference may be reasonably related to retraction as a result of tissue fixation. The absence of motility of T pallidum in real-time RCM observation suggests that spirochetes are restricted in their tiny movements by the adhesion systems of keratinocytes. However, to be able to observe slower movements we would need an RCM with a longer capturing time. The main limitation of RCM is the reduced depth of light penetration that, unlike immunohistochemistry, does not allow visualization of T pallidum in the dermis and vascular walls, where it is often located. In conclusion, these findings suggest that RCM could be an effective diagnostic tool for in vivo real-time imaging of T pallidum in skin lesions. The specificity and sensitivity of this technique remains to be clarified. However, like immunohistochemistry, RCM was positive in all patients whereas dark-field microscopy and serology were each negative in one patient. REFERENCES 1. Sanchez MR. Syphilis. In: Freedberg IM, Eisen AZ, Wolff K, Austen KF, Goldsmith LA, Kats SI, editors. Fitzpatrick’s Dermatology in General Medicine. New York: Publishing Division; 2003. pp. 2163-88. 2. Buffet M, Grange PA, Gerhardt P, Carlotti A, Calvez V, Bianchi A, et al. Diagnosing Treponema pallidum in secondary syphilis by PCR and immunohistochemistry. J Invest Dermatol 2007; 127:2345-50.
3. Jurado RL, Campbell J, Martin PD. Prozone phenomenon in secondary syphilis: has its time arrived? Arch Intern Med 1993; 153:2496-8. 4. Smith G, Holman RP. The prozone phenomenon with syphilis and HIV-1 co-infection. South Med J 2004;97:379-82. 5. Geisler WM. The prozone phenomenon in syphilis testing. South Med J 2004;97:327-8. 6. el-Zaatari MM, Martens MG, Anderson GD. Incidence of the prozone phenomenon in syphilis serology. Obstet Gynecol 1994;84:609-12. 7. Hicks CB, Benson PM, Lupton GP, Tramont EC. Seronegative secondary syphilis in a patient infected with the human immunodeficiency virus (HIV) with Kaposi sarcoma: a diagnostic dilemma. Ann Intern Med 1987;107:492-5. 8. Johnson PD, Graves SR, Stewart L, Warren R, Dwyer B, Lucas CR. Specific syphilis serological tests may become negative in HIV infection. AIDS 1991;5:419-23. 9. Taniguchi S, Osato K, Hamada T. The prozone phenomenon in secondary syphilis. Acta Derm Venereol 1995;75:153-4. 10. Rajadhyaksha M, Gonza´lez S, Zavislan JM, Anderson RR, Webb RH. In vivo confocal scanning laser microscopy of human skin II: advances in instrumentation and comparison with histology. J Invest Dermatol 1999;113:293-303. 11. Pellacani G, Cesinaro AM, Seidenari S. In vivo assessment of melanocytic nests in nevi and melanomas by reflectance confocal microscopy. Mod Pathol 2005;18:469-74. 12. Gerger A, Koller S, Weger W, Richtig E, Kerl H, Samonigg H, et al. Sensitivity and specificity of confocal laser-scanning microscopy for in vivo diagnosis of malignant skin tumors. Cancer 2006;107:193-200. 13. Ulrich M, Maltusch A, Ro¨wert-Huber J, Gonza´lez S, Sterry W, Stockfleth E, et al. Actinic keratoses: non-invasive diagnosis for field cancerization. Br J Dermatol 2007;156(Suppl):S13-7. 14. Nori S, Rius-Dı´az F, Cuevas J, Goldgeier M, Jaen P, Torres A, et al. Sensitivity and specificity of reflectance-mode confocal microscopy for in vivo diagnosis of basal cell carcinoma: a multicenter study. J Am Acad Dermatol 2004;51:923-30. 15. Horn M, Gerger A, Koller S, Weger W, Langsenlehner U, Krippl P, et al. The use of confocal laser-scanning microscopy in microsurgery for invasive squamous cell carcinoma. Br J Dermatol 2007;156:81-4. 16. Agero AL, Gill M, Ardigo M, Myskowski P, Halpern AC, Gonza´lez S. In vivo reflectance confocal microscopy of mycosis fungoides: a preliminary study. J Am Acad Dermatol 2007;57:435-41. 17. Swindells K, Burnett N, Rius-Diaz F, Gonza´lez E, Mihm MC, Gonza´lez S. Reflectance confocal microscopy may differentiate acute allergic and irritant contact dermatitis in vivo. J Am Acad Dermatol 2004;50:220-8. 18. Gonza´lez S, Rajadhyaksha M, Rubinstein G, Anderson RR. Characterization of psoriasis in vivo by reflectance confocal microscopy. J Med 1999;30:337-56. 19. Ardigo` M, Maliszewski I, Cota C, Scope A, Sacerdoti G, Gonzalez S, et al. Preliminary evaluation of in vivo reflectance confocal microscopy features of discoid lupus erythematosus. Br J Dermatol 2007;156:1196-203. 20. Hongcharu W, Dwyer P, Gonzalez S, Anderson RR. Confirmation of onychomycosis by in vivo confocal microscopy. J Am Acad Dermatol 2000;42:214-6. 21. Longo C, Bassoli S, Monari P, Seidenari S, Pellacani G. Reflectance-mode confocal microscopy for the in vivo detection of Sarcoptes scabiei. Arch Dermatol 2005;141:1336. 22. Baughn RE, Musher DM. Secondary syphilitic lesions. Clin Microbiol Rev 2005;18:205-16. 23. Holt SC. Anatomy and chemistry of spirochetes. Microbiol Rev 1978;42:114-60.
T
he diagnosis of secondary syphilis is challenging because of the wide range of clinical forms it assumes and, therefore, physicians must always rely on microscopic and laboratory tests to verify their diagnoses. Dark-field microscopy is an effective real-time aid in most cases, mainly for moist lesions.1 However, it has some drawbacks. First, it requires trained specialists2 and second, when it is used for nonmoist lesions, the stratum corneum must be removed by repeated adhesive tape stripping. Serology and immunohistochemistry are diagnostic mainstays as well, but require several days before the results are available, and their use is limited by the From the Departments of Dermatologya and Pathology,b University of Brescia, Spedali Civili Hospital. Funding sources: None. Conflicts of interest: None declared. Reprint requests: Piergiacomo Calzavara-Pinton, MD, Department of Dermatology, Spedali Civili Hospital, P.le Spedali Civili 1, 25123 Brescia, Italy. E-mail: [email protected]. 0190-9622/$36.00 ª 2008 by the American Academy of Dermatology, Inc. doi:10.1016/j.jaad.2008.11.901
invasiveness of taking blood and tissue samples. In addition, serology will show a negative response in the first days of an infection, and this seronegative window may be even longer in cases of HIV infection. False-negative results in screening with nontreponemal tests, such as the VDRL test or rapid plasma reagin assays, may occur as a result of the prozone phenomenon, which is a false-negative reaction to a serologic test that occurs when very high antibody titers in the serum prevent the formation of the antibody-antigen lattice needed to visualize a flocculation reaction.3-5 The prozone phenomenon occurs in 0.3% to 2% of all cases of syphilis6 and may be more common in diseases associated with B-cell dysregulation and excess antibody, such as in patients with HIV7,8 and transplantation.9 In addition, the use of immunohistochemistry is limited because it is only available in a few specialized laboratories.2 Therefore, a new affordable real-time diagnostic tool for secondary syphilis is needed. Reflectance confocal microscopy (RCM) is a novel noninvasive technique for in vivo, real-time, en face imaging of epidermis and papillary dermis,10 with a resolution power close to that available with 639
640 Venturini et al
J AM ACAD DERMATOL APRIL 2009
Fig 1. Generalized erythematous macules and keratotic papules.
conventional histology. RCM has been previously reported to be useful in the in vivo assessment of skin tumors and various inflammatory skin diseases including contact dermatitis, psoriasis, and discoid lupus erythematosus.11-19 Furthermore, it has been proven effective in the imaging of pathogens in onychomycosis and scabies.20,21 In this article we describe the in vivo real-time disclosure of Treponema pallidum by RCM in 3 cases of secondary syphilis.
METHODS We enrolled patients who had been sent to our university outpatient clinic for sexually transmitted diseases because they had skin lesions and anamnestic findings that raised suspicion that secondary syphilis could have been the cause. Routine blood chemistries, hematology, urinalysis, and T-cell counts were performed by standard methods. In addition, both enzyme-linked immunosorbent assay and Western blot techniques for the detection of HIV antibodies, and a panel of serology tests for syphilis including rapid plasma reagin test, T pallidum particle agglutination assay, and the fluorescent treponemal antibody absorption (IgG and IgM) test, were applied. Skin lesions were investigated with the dark-field examination of scrapings and in vivo RCM, and with the histologic and immunohistochemical examination of biopsy samples. The RCM images were taken with a Vivascope 1500 (Lucid Technologies, Henrietta, NY). This system uses a 330 water immersion objective lens of numeric aperture 0.9 and 830-nm laser source with a power less than 35 mW/cm2 at tissue level. The device provides a lateral resolution of 1.0 m, an axial resolution of 3.0 m, and an imaging depth of 200 to 250 m in vivo.10 In addition, VivaScan 7.0 software (Lucid Technologies) saves still images as bmp files. The system captures 9 video frames per second as a 24-bit uncompressed avi file, with a digital resolution
of 1000 3 1000 pixels and 256 levels of grey. The system can register movies with a time length up to 2 minutes. The size of 10 spirochetes was measured with the software. Results were given as mean 6 SD. RCM images were taken from a lesion of the arm in all patients and the same lesion was biopsied to compare the histologic and confocal findings. Biopsy samples were examined with standard histology after staining with hematoxylin-eosin, and with immunohistochemistry after heating for 15 minutes in EDTA buffer and staining with anti-T pallidum antibodies (Biocare Medical, Concord, CA) at a 1:100 dilution. Image analysis of spirochetes was performed with the software Cell^F 2.5 (Olympus Soft Imaging Solutions GmbH, Hamburg, Germany).
RESULTS Three young patients, two homosexual men and one woman, were seen for skin eruptions clinically suggestive of secondary syphilis. Two of the patients said they had noted an ulcerative nodule on the genitalia in the recent past. These lesions had disappeared spontaneously without any systemic antibiotic therapy. Dermatologic examination showed the skin lesions to be macular in one patient (Fig 1) and papular in two. All patients had lesions on the trunk and two showed in addition involvement of limbs, palms, and soles. Slight fever, malaise, and weight loss were reported by all patients and generalized lymphadenopathy was detected in two. Serology for syphilis was positive in two patients. All patients were HIV positive with a remarkable decrease in CD31 and CD41 T-cell counts. Pertinent clinical and laboratory findings are reported in detail in Table I. No other significant abnormalities of blood chemistries or hematology were detected. RCM examination disclosed small elongated bright particles with a spiral shape intermingled with the keratinocytes of the stratum granulosum, stratum spinosum, and basal layers (Figs 2 and 3). Their sizes ranged from 4.3 to 15.7 m (mean 6 SD: 9.4 6 3.4 m). Particles were grouped in clusters in the stratum granulosum and isolated in the stratum spinosum and basal layers. No particles were seen in the stratum corneum. RCM investigations of the papillary dermis did not show particles. In realtime video recording up to 1 minute in length, the bright helical-shaped particles appeared to be motionless in the epidermis. The lesions were subsequently biopsied. Histologic examination showed dilatation and thickening of blood vessels with a proliferation of endothelial cells and a papillary and reticular perivascular
Venturini et al 641
J AM ACAD DERMATOL VOLUME 60, NUMBER 4
Table I. Clinical and laboratory findings of the 3 cases Patient No.
Age, y/sex Skin lesions Skin distribution Slight fever, malaise, and weight loss Generalized lymphoadenopathy Duration of lesions, wk Previous ulcerated nodule on genitalia RPR TPPA FTA-ABS (IgG and IgM) HIV serology T-cell counts (CD31/CD41/CD81), cells/mm3 Dark-field examination RCM investigation Immunohistochemistry
1
2
3
34/M Macules Generalized Yes Yes 3 Yes 1:8 1:320 Positive Positive 530/51/142 Negative Positive Positive
36/M Papules Generalized Yes Yes 4 Yes Negative Negative Negative Positive 480/73/223 Positive Positive Positive
33/F Papules Trunk Yes No 2 No 1:4 1:320 Positive Positive 602/100/410 Positive Positive Positive
F, Female; FTA-ABS, fluorescent treponemal antibody absorption; M, male; RCM, reflectance confocal microscopy; RPR, rapid plasma reagin; TPPA, Treponema pallidum particle agglutination assay.
Fig 3. Reflectance confocal microscopy image shows bright particles with helical shape (arrows) corresponding to spirochetes in stratum spinosum (scale bar: 50 m). Fig 2. Group of spirochetes in stratum granulosum. Reflectance confocal microscopy image shows clusters of small elongated bright particles (white circle) corresponding to spirochetes in superficial epidermis (scale bar: 50 m).
dermal infiltrate containing numerous plasma cells. Immunohistochemical investigation revealed several spirochetes infiltrating the epidermis, dermis, and walls of blood vessels (Fig 4). The spirochetes were helical in shape and, using electronic image analysis to measure the length of 10 micro-organisms, we found them to range from 1.9 to 12.5 m (mean 6 SD: 6.2 6 3.2 m). On the basis of the clinical, serologic, and histopathological data, a final diagnosis of secondary syphilis was made in the 3 patients. Lumbar puncture was performed in all patients and cerebrospinal fluid evaluation excluded neurosyphilis (VDRL test # 1:32; leukocyte count # 5/mm3).
A single intramuscular injection of 2.4 million units of benzathine benzylpenicillin was administered to the patients, and their lesions disappeared within a few days. At a follow-up visit 30 days later, the serologic tests for syphilis of patient 2 were positive (rapid plasma reagin 1:2; T pallidum particle agglutination assay 1:320; fluorescent treponemal antibody absorption positive).
DISCUSSION In this investigation, RCM proved to be a noninvasive, real-time tool for the in vivo imaging of T pallidum in skin lesions of secondary syphilis. We emphasize that RCM disclosed spirochetes in all 3 patients whereas dark-field examination was negative in the patient with the macular eruption and serology was negative in another when first tested.7,22 Spirochetes were visualized in the superficial layers of epidermis as small, bright, elongated, and spiral-shaped particles. Their brightness is likely
642 Venturini et al
J AM ACAD DERMATOL APRIL 2009
Fig 4. Immunohistochemistry for Treponema pallidum: numerous spirochetes in epidermis and dermis (scale bar: 50 m). (Original magnification: 3400.)
to be caused by the highly reflective protein content of their cellular structures (outer membrane, peptide-glycan-cytoplasmic membrane complex, and endoflagella).23 In the horizontal sections of RCM we found the spirochetes to be mostly grouped in clusters in the stratum granulosum and sparse and isolated in the stratum spinosum and basal layers. The morphology, grouping, and location of these particles correlated well with those of the spirochetes seen with immunohistochemistry. Although the mean length of spirochetes was less with immunostaining in comparison with RCM (6.2 6 3.2 vs 9.4 6 3.4 m, respectively), this difference may be reasonably related to retraction as a result of tissue fixation. The absence of motility of T pallidum in real-time RCM observation suggests that spirochetes are restricted in their tiny movements by the adhesion systems of keratinocytes. However, to be able to observe slower movements we would need an RCM with a longer capturing time. The main limitation of RCM is the reduced depth of light penetration that, unlike immunohistochemistry, does not allow visualization of T pallidum in the dermis and vascular walls, where it is often located. In conclusion, these findings suggest that RCM could be an effective diagnostic tool for in vivo real-time imaging of T pallidum in skin lesions. The specificity and sensitivity of this technique remains to be clarified. However, like immunohistochemistry, RCM was positive in all patients whereas dark-field microscopy and serology were each negative in one patient. REFERENCES 1. Sanchez MR. Syphilis. In: Freedberg IM, Eisen AZ, Wolff K, Austen KF, Goldsmith LA, Kats SI, editors. Fitzpatrick’s Dermatology in General Medicine. New York: Publishing Division; 2003. pp. 2163-88. 2. Buffet M, Grange PA, Gerhardt P, Carlotti A, Calvez V, Bianchi A, et al. Diagnosing Treponema pallidum in secondary syphilis by PCR and immunohistochemistry. J Invest Dermatol 2007; 127:2345-50.
3. Jurado RL, Campbell J, Martin PD. Prozone phenomenon in secondary syphilis: has its time arrived? Arch Intern Med 1993; 153:2496-8. 4. Smith G, Holman RP. The prozone phenomenon with syphilis and HIV-1 co-infection. South Med J 2004;97:379-82. 5. Geisler WM. The prozone phenomenon in syphilis testing. South Med J 2004;97:327-8. 6. el-Zaatari MM, Martens MG, Anderson GD. Incidence of the prozone phenomenon in syphilis serology. Obstet Gynecol 1994;84:609-12. 7. Hicks CB, Benson PM, Lupton GP, Tramont EC. Seronegative secondary syphilis in a patient infected with the human immunodeficiency virus (HIV) with Kaposi sarcoma: a diagnostic dilemma. Ann Intern Med 1987;107:492-5. 8. Johnson PD, Graves SR, Stewart L, Warren R, Dwyer B, Lucas CR. Specific syphilis serological tests may become negative in HIV infection. AIDS 1991;5:419-23. 9. Taniguchi S, Osato K, Hamada T. The prozone phenomenon in secondary syphilis. Acta Derm Venereol 1995;75:153-4. 10. Rajadhyaksha M, Gonza´lez S, Zavislan JM, Anderson RR, Webb RH. In vivo confocal scanning laser microscopy of human skin II: advances in instrumentation and comparison with histology. J Invest Dermatol 1999;113:293-303. 11. Pellacani G, Cesinaro AM, Seidenari S. In vivo assessment of melanocytic nests in nevi and melanomas by reflectance confocal microscopy. Mod Pathol 2005;18:469-74. 12. Gerger A, Koller S, Weger W, Richtig E, Kerl H, Samonigg H, et al. Sensitivity and specificity of confocal laser-scanning microscopy for in vivo diagnosis of malignant skin tumors. Cancer 2006;107:193-200. 13. Ulrich M, Maltusch A, Ro¨wert-Huber J, Gonza´lez S, Sterry W, Stockfleth E, et al. Actinic keratoses: non-invasive diagnosis for field cancerization. Br J Dermatol 2007;156(Suppl):S13-7. 14. Nori S, Rius-Dı´az F, Cuevas J, Goldgeier M, Jaen P, Torres A, et al. Sensitivity and specificity of reflectance-mode confocal microscopy for in vivo diagnosis of basal cell carcinoma: a multicenter study. J Am Acad Dermatol 2004;51:923-30. 15. Horn M, Gerger A, Koller S, Weger W, Langsenlehner U, Krippl P, et al. The use of confocal laser-scanning microscopy in microsurgery for invasive squamous cell carcinoma. Br J Dermatol 2007;156:81-4. 16. Agero AL, Gill M, Ardigo M, Myskowski P, Halpern AC, Gonza´lez S. In vivo reflectance confocal microscopy of mycosis fungoides: a preliminary study. J Am Acad Dermatol 2007;57:435-41. 17. Swindells K, Burnett N, Rius-Diaz F, Gonza´lez E, Mihm MC, Gonza´lez S. Reflectance confocal microscopy may differentiate acute allergic and irritant contact dermatitis in vivo. J Am Acad Dermatol 2004;50:220-8. 18. Gonza´lez S, Rajadhyaksha M, Rubinstein G, Anderson RR. Characterization of psoriasis in vivo by reflectance confocal microscopy. J Med 1999;30:337-56. 19. Ardigo` M, Maliszewski I, Cota C, Scope A, Sacerdoti G, Gonzalez S, et al. Preliminary evaluation of in vivo reflectance confocal microscopy features of discoid lupus erythematosus. Br J Dermatol 2007;156:1196-203. 20. Hongcharu W, Dwyer P, Gonzalez S, Anderson RR. Confirmation of onychomycosis by in vivo confocal microscopy. J Am Acad Dermatol 2000;42:214-6. 21. Longo C, Bassoli S, Monari P, Seidenari S, Pellacani G. Reflectance-mode confocal microscopy for the in vivo detection of Sarcoptes scabiei. Arch Dermatol 2005;141:1336. 22. Baughn RE, Musher DM. Secondary syphilitic lesions. Clin Microbiol Rev 2005;18:205-16. 23. Holt SC. Anatomy and chemistry of spirochetes. Microbiol Rev 1978;42:114-60.