Department of Medical Microbiology & Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia.
Bacteriology Unit, Infectious Disease Research Center (IDRC), Institute for Medical Research, Complex National Institutes of Health, Section U13 Setia Alam, Shah Alam 43200, Selangor, Malaysia.
Int J Environ Res Public Health. 2020 Feb 18;17(4):1307. doi: 10.3390/ijerph17041307.
: Isolation of by culture represents a definitive growth and confirmation of the disease, yet it is hampered with its nature of slow growth. With slight modification of culture method, the study aims to isolate and characterize spp. from patients with acute febrile illness. : A total of 109 blood samples were collected from patients with acute febrile illness that presented at the Emergency Department of Hospital Universiti Sains Malaysia, Malaysia. Clinical samples were subjected to IgM Rapid test, microscopic agglutination test (MAT), isolation by culture method, and direct real-time PCR test. For leptospiral isolation, the samples (whole blood and deposit from spun plasma) were cultured into modified Ellinghausen McCullough Johnson Harris (EMJH) media with and without 5'-fluorouracil (5-FU). In every culture positive sample, partial 16S rRNA gene sequencing was performed for molecular identification of the isolates. Phylogenetic analysis was carried out to determine the genetic relatedness among the isolates. An inhibition of 5-FU study was performed on serovar Canicola with different concentrations to compare the growth detection of the tested with or without 5-FU within 7 days of incubation. : Leptospirosis was diagnosed in 14.7% of patients with acute febrile illness. Two spp. (n = 2/109, 1.85%) were successfully isolated from whole blood and deposit from spun plasma samples. B004 and B208 samples were positive at day 11 and day 7, respectively, in EMJH media without addition of 5-FU. Sample B004 was identified as Leptospira interrogans and B208 as . Phylogenetic analysis confirmed that both of them were within pathogenic group and they were not related. The 5-FU inhibition study revealed that additional of 5-FU at final concentration of 200 µg/mL to EMJH media demonstrated an inhibitory effect on the growth of the tested strain : Isolation of spp. using EMJH media without addition of 5'-fluorouracil resulted in a better outcome. Two pathogenic isolates were successfully cultivated from patients with acute febrile illness that were genetically not related.
从患者急性发热性疾病中分离和鉴定 spp. ：采用略微改良的培养方法。共采集马来西亚大学科学医院急诊科就诊的 109 例急性发热性疾病患者的 109 份血样。对临床标本进行 IgM 快速检测、显微镜凝集试验（MAT）、培养方法分离和直接实时 PCR 检测。为了进行钩端螺旋体分离，将样本（全血和离心血浆沉淀）培养于改良 Ellinghausen McCullough Johnson Harris（EMJH）培养基中，有或没有 5'-氟尿嘧啶（5-FU）。对每一个培养阳性的样本，进行部分 16S rRNA 基因测序，以对分离株进行分子鉴定。进行系统发育分析以确定分离株之间的遗传相关性。对不同浓度的 Canicola 血清型进行 5-FU 抑制研究，以比较在 7 天孵育期内有无 5-FU 存在时，测试的 生长检测情况。在急性发热性疾病患者中，钩端螺旋体病的诊断率为 14.7%。从全血和离心血浆沉淀样本中成功分离出 2 株 spp.（n=109，1.85%）。B004 和 B208 样本分别在无添加 5-FU 的 EMJH 培养基中于第 11 天和第 7 天呈阳性。B004 样本鉴定为问号钩端螺旋体，B208 样本鉴定为。系统发育分析证实，两者均属于致病性群，彼此没有关联。5-FU 抑制研究表明，在 EMJH 培养基中最终浓度为 200μg/ml 的 5-FU 添加物对测试菌株的生长具有抑制作用。从急性发热性疾病患者中分离 spp.，不添加 5'-氟尿嘧啶，效果更好。从急性发热性疾病患者中成功培养出 2 株具有遗传关系的致病性 分离株。
