臺灣淋病雙球菌分子流行病學、抗藥性分析及抗藥性機轉研究

Abstract

淋病，是由淋病雙球菌引起的感染症，其臨床表現的差異性甚大，從無症狀感染、瘙癢、排尿有刺痛或燒灼感、到尿道有膿性分泌物，皆有可能；如果不及時治療，將引起嚴重的並發症，例如附睾炎和盆腔炎。從感染部位取得檢體，經由培養方式或核酸擴增試驗，證實淋病雙球菌的存在，是主要的診斷的方法。由於缺乏有效的疫苗，抗生素治療仍然是控制淋病感染的主要方法。不幸的是，由於日益嚴重的抗藥性，使得抗生素的療效逐漸喪失。面對這一艱鉅的挑戰，許多國際治療指引建議以合併ceftriaxone及azithromycin的雙重處方為首選的治療藥物，期望能夠延緩抗藥性菌種的產生。各國學者亦著手許多淋病的相關研究，比如分子流行病學的監測及抗藥性機轉的探討。本研究的目的即在描述台灣淋病雙球菌的分子流行病學，同時觀察抗藥性變化的趨勢及探討抗藥性的相關機轉，以期提供臨床醫療人員適當的診治建議。 我們回顧9家台灣醫療機構於2001-2018年的病歷記錄及微生物學實驗室記錄，收集整理淋病患者的資料及其淋病雙球菌菌株。通過瓊脂稀釋法決定各菌株對抗生素的最小抑制濃度，並根據美國臨床暨實驗室標準協會和歐洲抗菌藥敏試驗委員會的標準，來判定各菌株對抗生素的藥敏性。我們使用多基因座序列分型和淋病雙球菌多抗原序列分型確定各菌株的遺傳相關性。對azithromycin高抗藥性(最小抑菌濃度 256 mg/L)的菌株，再進行脈衝場凝膠電泳分析，以決定彼此間的相關性。以聚合酶連鎖反應的方式擴增23S rRNA，mtrR和penA基因的序列，並將基因序列上傳下達到NG-STAR網站上，以決定突變的存在與否。 從2001年至2013年，我們在台大醫院共收集了279株淋病雙球菌菌株，這些菌株對ceftriaxone和cefotaxime沒有抗藥性，對cefixime、spectinomycin、cefpodoxime、ciprofloxacin、penicillin的抗藥性則分別為0.4％、0.4％、13.3％、91.6％和87.6％。對azithromycin產生抗藥性的比例為14.6％(歐洲抗菌藥敏試驗委員會標準)，高於各國以往的監測研究。以多基因座序列分型後，三個主要基因型別為ST1901 (14.2％)，ST7365 (9.3％)，ST1927 (8.0％)。與其他基因型別相比，ST1901菌株對ceftriaxone和azithromycin的最小抑菌濃度相對較高。男女性別的差異對cephalosprin的藥敏性結果沒有影響。基於上述發現，我們再從台灣其他醫療機構收集2014-2018年的372株淋病雙球菌菌株，以了解azithromycin在台灣的抗藥性狀況。我們鑑定出10株azithromycin高抗藥性的淋病雙球菌菌株，基因型別分別為MSLT 12039/10899和NG-MAST 1866/16497。它們都具有非鑲嵌(non-mosaic)的penA基因和相同的azithromycin抗藥性決定因子，包括具有4個A2059G突變的23S rRNA，mtrR基因啟動子區域的-35A缺失和mtrR編碼區域的G45D突變。對10株azithromycin高抗藥性的菌株以脈衝場凝膠電泳分析，可鑑定出4種不同的基因型別(cluster)，顯示有azithromycin高抗藥性的淋病雙球菌菌株在台灣的某特定族群間傳染感染。在台灣，ceftriaxone仍是治療淋病的有效選擇。但是，由於發現了azithromycin高抗藥性的淋病雙球菌菌株在台灣傳播，因此我們應監測並仔細重新評估ceftriaxone及azithromycin雙重處方的療效。此外，我們發現A2059G突變的23S rRNA和鑲嵌penA基因，所以我們也要密切關注淋病雙球菌菌株可能通過自發突變和DNA轉化產生對ceftriaxone和azithromcyin的抗藥性。

Neisseria gonorrhoeae (N. gonorrhoeae) infection is a huge burden to human health and can be asymptomatic or present multiple symptoms such as mucopurulent discharge, pruritus, and dysuria. If left untreated, it can cause severe complications, such as epididymitis and pelvic inflammatory disease. Positive results of discharge culture and nucleic acid amplification test (NAAT) are the commonly used methods for diagnosis. Antimicrobial treatment remains the principal method to control N. gonorrhoeae infection. Unfortunately, we have limited options for gonorrhea treatment due to the emergence of antimicrobial resistance. Facing this tough challenge, multifaceted approaches have been taken, such as the recommendation of a dual antimicrobial regimen with ceftriaxone (CRO) and azithromycin (AZM), intensive molecular surveillance, and researches on relevant antimicrobial resistance. The aims of this research are to explore the molecular epidemiology, annual antimicrobial susceptibility trends, and the genetic resistance determinants of N. gonorrhoeae. Patients with N. gonorrhoeae infection were identified through the review of microbiology laboratory records from nine medical institutions in Taiwan during 2001-2018. Minimal inhibitory concentrations (MICs) of the isolates to antimicrobial agents were determined by the agar dilution method. The MIC interpretive breakpoints used to determine susceptibility, intermediate susceptibility, and resistance to the agents tested were in accordance with the guidelines recommended by the Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST). Genetic relatedness of the isolates was determined using multilocus sequence typing (MLST) and N. gonorrhoeae multi-antigen sequence typing (NG-MAST). Pulsed-field gel electrophoresis (PFGE) analysis was performed for high-level AZM-resistant (AZM-HLR) isolates. To recognize the presence of mutations, domain V gene sequences of the 23S rRNA, mtrR, and penA genes were amplified and the DNA sequencing data was uploaded to a publicly accessible database on the NG-STAR website. A total of 279 non-duplicate isolates of N. gonorrhoeae were recovered from 279 patients who were treated at NTUH from 2001 to 2013. None of the isolates were resistant to CRO and cefotaxime, and the resistance rates for spectinomycin, cefixime, cefpodoxime, ciprofloxacin, and penicillin were 0.4%, 0.4%, 13.3%, 91.6%, and 87.6%, respectively. The rate of isolates resistant to AZM was 14.6% (EUCAST criteria), which is higher than in previous surveillance studies. The three main sequence types (STs) included ST1901 (14.2%), ST7365 (9.3%), ST1927 (8.0%). ST1901 isolates had relatively higher MIC values for CRO and AZM than those for the other STs. No difference in susceptibility for cephalosporins existed between genders. In addition to the previously mentioned 226 isolates of N. gonorrhoeae recovered from NTUH during 2001-2013, a further 372 N. gonorrhoeae isolates were collected during 2014-2018 from other hospitals in Taiwan. Ten isolates were identified as 10 AZM-HLR isolates. These isolates were sequenced as MSLT 12039/10899 and NG-MAST 1866/16497, respectively. All of them had non-mosaic penA alleles and the same AZM resistance determinants, including 4 mutated 23S rRNA copies with the A2059G mutation, -35A deletion in the promoter region of the mtrR gene, and G45D mutation in the mtrR coding region. PFGE analysis of the 10 AZM-HLR isolates resulted in dendrograms defined four clusters. CRO is an effective drug of choice for managing gonorrhea, but the efficacy of dual therapy, namely CRO plus AZM, should be closely monitored and carefully re-evaluated as the clonal spread of AZM-HLR isolates was found through the sexual network in Taiwan. The A2059G mutated 23S rRNA and mosaic penA alleles were depicted in our study so that we should pay close attention to the possible emergence of N. gonorrhoeae isolates with resistance to both CRO and AZM through spontaneous mutation and DNA transformation.