Efficient Detection of Pathogenic Leptospires Using 16S Ribosomal RNA

Pathogenic Leptospira species cause a prevalent yet neglected zoonotic disease with mild to life-threatening complications in a variety of susceptible animals and humans. Diagnosis of leptospirosis, which primarily relies on antiquated serotyping methods, is particularly challenging due to presentation of non-specific symptoms shared by other febrile illnesses, often leading to misdiagnosis. Initiation of antimicrobial therapy during early infection to prevent more serious complications of disseminated infection is often not performed because of a lack of efficient diagnostic tests. Here we report that specific regions of leptospiral 16S ribosomal RNA molecules constitute a novel and efficient diagnostic target for PCR-based detection of pathogenic Leptospira serovars. Our diagnostic test using spiked human blood was at least 100-fold more sensitive than corresponding leptospiral DNA-based quantitative PCR assays, targeting the same 16S nucleotide sequence in the RNA and DNA molecules. The sensitivity and specificity of our RNA assay against laboratory-confirmed human leptospirosis clinical samples were 64% and 100%, respectively, which was superior then an established parallel DNA detection assay. Remarkably, we discovered that 16S transcripts remain appreciably stable ex vivo, including untreated and stored human blood samples, further highlighting their use for clinical detection of L. interrogans. Together, these studies underscore a novel utility of RNA targets, specifically 16S rRNA, for development of PCR-based modalities for diagnosis of human leptospirosis, and also may serve as paradigm for detection of additional bacterial pathogens for which early diagnosis is warranted.

致病性钩端螺旋体属可引发一种流行却被忽视的人畜共患病，会在多种易感动物与人类身上引发从轻症到危及生命的各类并发症。钩端螺旋体病的诊断主要依赖陈旧的血清分型方法，且由于其临床表现与其他发热性疾病存在大量非特异性症状，诊断极具挑战性，常导致误诊。由于缺乏高效的诊断检测手段，感染早期为预防播散性感染引发的更严重并发症而启动抗菌治疗的情况往往难以实现。本研究发现，钩端螺旋体16S核糖体RNA（16S ribosomal RNA）的特定区域可作为新型高效的诊断靶点，用于基于聚合酶链反应（PCR）的致病性钩端螺旋体血清型检测。我们针对加标人类血液样本开展的诊断检测，其灵敏度至少是靶向RNA与DNA分子中同一16S核苷酸序列的相应钩端螺旋体DNA定量PCR检测的100倍。针对经实验室确认的人类钩端螺旋体病临床样本，该RNA检测方法的灵敏度与特异性分别为64%与100%，优于现有成熟的平行DNA检测方法。值得注意的是，我们发现16S转录本在离体状态下仍保持显著稳定性，包括未经处理与已储存的人类血液样本，这进一步凸显了其用于临床检测问号钩端螺旋体（Leptospira interrogans）的应用价值。综上，本研究证实了RNA靶点（尤其是16S rRNA）在开发基于PCR的人类钩端螺旋体病诊断方法中的新型应用价值，同时也可为亟需早期诊断的其他细菌性病原体的检测提供参考范式。