355 INTRODUCTION Syphilis is a complex, sexually transmitted disease with diverse clinical manifestations. According to World Health Organization data in 2005 and 2008, there was an increase in the prevalence of syphilis in Southeast Asian countries, from 1.37% to 6.1% in women and 1.25% to 6.3% in men (1). The prevalence of syphilis among direct and indirect female sex workers (FSWs) in Indonesia was reported as 7.5% and 3.1%, respectively. Moreover, an increasing prevalence was found among brothel-based FSWs from 7.8% to 14.5% in 2005–2007 (2). A problem in diagnosing syphilis is the incapability of Treponema pallidum to be cultured outside the human body; this impairs the ability to detect and identify T. pallidum and makes it difficult to determine the antibiotic sensitivity pattern (1,3–5). The most commonly used diagnostic test for syphilis is a nontreponemal serological examination followed by a treponemal confirmation test (5–7). To minimize false positive and negative results in serological examinations, such as rapid plasma reagin (RPR) and T. pallidum hemagglutination (TPHA) vs. clinical symptoms, molecular methods such as polymerase chain reaction (PCR) having suitable sensitivity Original Article Detection of A2058G and A2059G on the 23S rRNA Gene by Multiplex Nested PCR to Identify Treponema pallidum Resistance to Azithromycin in Indonesia Yeva Rosana 1 * , Andi Yasmon 1 , Wresti Indriatmi 2 , Ida Effendi 3,4 , Raden Lia Kusumawati 5 , Rasmia Rowawi 6 , Sunarjati Sudigdoadi 7 , Gita Widya Pradini 7 , Anak Agung Gde Putra Wiraguna 8 , Ni Made Dwi Puspawati 8 , Maryam Kusumawaty 9 , and Muhammad Nasrum Massi 10 1 Department of Microbiology and 2 Department of Dermato-Venereology, Medical Faculty Universitas Indonesia – Ciptomangunkusumo Hospital, Jakarta; 3 Clinical Microbiologist Program, Faculty of Medicine, Universitas Indonesia, Jakarta; 4 Department of Microbiology, Faculty of Medicine, Trisakti University, Jakarta; 5 Department of Microbiology, Medical Faculty University of Sumatera Utara, Medan; 6 Department of Dermato-Venereology, Hasan Sadikin Hospital, and 7 Microbiology Division, Department of Biomedical Science, Faculty of Medicine, Universitas Padjadjaran, Bandung; 8 Department of Dermato-Venereology, Medical Faculty University of Udayana-Sanglah Hospital, Bali; and 9 Department of Dermato-Venereology and 10 Department of Microbiology, Medical Faculty University of Hasanuddin, Makassar, Indonesia ABSTRACT: Azithromycin is an antibiotic used to treat syphilis, especially in the context of penicillin allergy. Although resistance to azithromycin has been widely reported to be associated with one- and/ or two-point mutations on the 23S rRNA gene, it has yet to be described in Indonesia. Specimens were collected from 220 patients diagnosed with secondary syphilis. A multiplex nested polymerase chain reaction (PCR) testing system using the 23S rRNA target gene of Treponema pallidum was designed using three primer pairs. The first step involved the use of PCR primer pairs to detect T. pallidum. In the second step, two PCR primer pairs were constructed to identify azithromycin-resistant T. pallidum based on A2058G and A2059G point mutations. T. pallidum detected in samples from Jakarta or Bandung were not resistant to azithromycin. However, azithromycin-resistant T. pallidum were found in samples from Makassar, Medan, and Bali. Specimens from heterosexual males and patients with HIV accounted for the majority of azithromycin resistance noted in the study. This study demonstrated that the azithromycin-resistant T. pallidum detected in Indonesia appear to be a novel variant of resistance, containing both the A2058G and A2059G mutations found in Medan and Makassar. Received October 30, 2021. Accepted December 9, 2021. J-STAGE Advance Publication December 28, 2021. DOI: 10.7883/yoken.JJID.2021.738 *Corresponding author: Mailing address: Department of Microbiology, Medical Faculty Universitas Indonesia – Ciptomangunkusumo Hospital, Jl. Pegangsaan Timur no.16, Jakarta 10320, Indonesia. Tel: +62 3160492, Fax: +62 3100810, E-mail: yeva.rosana@ui.ac.id Jpn. J. Infect. Dis., 75, 355-360, 2022