Microscopic examination using negative staining for rapid diagnosis of syphilis

Main Article Content

Yeva Rosana
Ida Effendi
Wresti Indriatmi
Andi Yasmon


Syphilis is a global health problem, especially in developing countries including Indonesia. Treponema pallidum, the etiologic agent of syphilis, cannot be cultured in vitro. Syphilis has several clinical manifestations, making laboratory testing a very important aspect of diagnosis. Microscopic examination may support the diagnosis but is rarely used in Indonesia. The aim of this study was to evaluate negative staining using the light microscope to detect T. pallidum in syphilitic lesions.

A cross-sectional study was conducted involving 27 subjects who came to several dermato-venereology clinics in Jakarta. Exudates were collected from genital ulcers, condylomata lata, and dry mucocutaneous rash on palms and soles of syphilis patients. Negative staining using one drop of Indian ink was used to examine for treponemas under the light microscope at 10x100 magnification.

Microscopic examination using negative staining showed a few clusters of small and spiral shaped bacteria. Of the 39 specimens from 27 subjects, microscopic examinations were successfully done on 10 specimens. Observations could only be conducted on 5 specimens, 3 (60.0%) of which showed the morphology of spirochetes. This examination is the easiest method for detecting the bacteria. Moreover, the bacteria that were isolated from painless genital ulcers could be observed more clearly than those from erythematous maculopapular lesions.

Treponema pallidum was successfully detected by microscopic examination in all moist lesions, but was difficult to detect in dry lesions. Negative staining under the light microscope appears to be simple, affordable, and available in most microbiology laboratories in Indonesia.

Article Details

How to Cite
Rosana, Y., Effendi, I. ., Indriatmi, W. ., & Yasmon, A. . (2022). Microscopic examination using negative staining for rapid diagnosis of syphilis. Universa Medicina, 41(1), 64–70. https://doi.org/10.18051/UnivMed.2022.v41.64-70
Original Articles


World Health Organization. Global incidence and prevalence of selected curable sexually transmitted infections-2008. Switzerland: WHO Library Cataloguing-in-publication data; 2012.

Tramont EC. Treponema pallidum (syphilis). In: Bennett JE, Dolin R, Blaser MJ, eds. Mandell, Douglas, and Bennett’s principles and practice of infectious diseases. Philadelphia: Elsevier Saunders; 2015. pp.3035-55.

Kementerian Kesehatan Republik Indonesia. Pedoman Tata Laksana Sifilis Untuk Pengendalian Sifilis Di Layanan Kesehatan Dasar; 2017.

Kementerian Kesehatan Republik Indonesia. Pedoman Nasional Penanganan Infeksi Menular Seksual; 2016.

Edmondson DG, Hu B, Norris SJ. Long-term in vitro culture of the syphilis spirochete Treponema pallidum subsp. pallidum. mBio 2018;9:e01153-18. https://doi.org/10.1128/mBio.01153-18.

Sharma S, Chaudhary J, Hans C. VDRL v/s TPHA for diagnosis of syphilis among HIV sero-reactive patients in a tertiary care hospital. Int J Curr Microbiol App Sci 2014;3:726-30.

Shah D, Marfatia YS. Serological tests for syphilis. Indian J Sex Transm Dis AIDS 2019; 40: 186–91. doi: 10.4103/ijstd.IJSTD_86_19.

Theel ES, Katz SS, Pillay A. Molecular and direct detection tests for Treponema pallidum subspecies pallidum: a review of the literature, 1964–2017. CID 2020;71(Suppl 1):S1-S12. DOI: 10.1093/cid/ciaa176.

Luo Y, Xie Y and Xiao Y. Laboratory diagnostic tools for syphilis: current status and future prospects. Front Cell Infect Microbiol 2021;10:574806. doi: 10.3389/fcimb.2020.574806.

Workowski KA, Bolan GA. Sexually transmitted diseases treatment guidelines, 2015. MMWR Recomm Rep 2015;64;1-140.

Pierce EF, Katz KA. Darkfield microscopy for point-of-care syphilis diagnosis. Med Lab Obs 2011;43:30-1.

Hajian-Tilaki K. Sample size estimation in diagnostic test studies of biomedical informatics. J Biomed Informatics 2014;48:193–204. http://dx.doi.org/10.1016/j.jbi.2014.02.013.

Department of Health and Human Services Centers for Disease Control and Prevention. Sexually transmitted disease: summary of CDC treatment guidelines 2015 US; 2015.

Byrne PO, MacPherson P. Syphilis. BMJ 2019; 366:l4746. doi: 10.1136/bmj.l4746.

Çakmak SK, Tamer E, Karadað AS, Waugh M. Syphilis: a great imitator. Clin Dermatol 2019; 37:182-91. doi: 10.1016/j.clindermatol.2019. 01.007.

Nyatsanza F, Tipple C. Syphilis: presentations in general medicine. Clin Med (Lond) 2016;16:184-8. 10.7861/clinmedicine.16-2-184.

Klausner JD. The great imitator revealed: syphilis. Top Antivir Med 2019;27:71-4.

Cruz AR, Pillay A, Zuluaga AV, et al. Secondary syphilis in Cali, Colombia: new concepts in disease pathogenesis. PLoS Negl Trop Dis 2010;4:e690.

Tuddenham S, Ghanem KG. Emerging trends and persistent challenges in the management of adult syphilis. BMC Infect Dis 2015;15:351. doi: 10.1186/s12879-015-1028-3.

Sato NS. Laboratorial diagnosis of syphilis. In: Sato NS (editor). Syphilis - recognition, description and diagnosis. Croatia: INTECH; 2011.pp.87-108.

Arando M, Naval CF, Foix MM, et al. Early syphilis: risk factors and clinical manifestations focusing on HIV-positive patients. BMC Infect Dis 2019;19:727. https://doi.org/10.1186/s12879-019-4269-8.