Evaluation of nanopore sequencing as a diagnostic tool for the rapid identification of Mycoplasma bovis from individual and pooled respiratory tract samples. Evaluation of nanopore sequencing as a diagnostic tool for Mycoplasma bovis

Rapid identification of Mycoplasma bovis infections in cattle is a key factor to guide antimicrobial therapy and biosecurity measures. Recently, nanopore sequencing became an affordable diagnostic tool for both clinically relevant viruses and bacteria, but the diagnostic accuracy for M. bovis identification is undocumented. Therefore, in this study nanopore sequencing was compared to rapid identification of M. bovis with MALDI-TOF MS (RIMM), and triplex real-time PCR in a Bayesian latent class model (BLCM) for M. bovis in bronchoalveolar lavage fluid (BALf) obtained from calves. In practice, pooling of samples is often used to save money, but the influence on diagnostic accuracy has not been described for M. bovis. Therefore, a convenience sample of 17 pooled samples containing 5 individual BALf samples per farm was analyzed as well. The results of the pooled samples were compared to the individual samples, to determine sensitivity (Se) and specificity (Sp). The BLCM showed a good Se (77.3%; 95% Credible Interval: 57.8%-92.8%) and high Sp (97.4%; 91.5%-99.7%) for nanopore sequencing compared to RIMM (Se: 93.0%; 76.8%-99.5%, Sp: 91.3; 82.5%-97.0%) and real-time PCR (Se: 94.6%; 89.7%-97.7%, Sp: 86.0%; 76.1-93.6%). Se and Sp of pooled analysis for M. bovis were 85.7% (95% confidence interval: 59.8-111.6%) and 90.0% (71.4-108.6%%) for nanopore sequencing and 100% (100%-100%) and 88.9% (68.4-109.4%) for RIMM, respectively. In conclusion, nanopore sequencing is a rapid, reliable tool for the identification of M. bovis. To reduce costs and increase the chance of M. bovis identification, pooling of 5 samples for nanopore sequencing and RIMM is possible.

快速鉴定牛支原体（Mycoplasma bovis）感染，是指导抗菌治疗与生物安全防控措施的核心环节。近年来，纳米孔测序（nanopore sequencing）已成为兼具临床相关性的病毒与细菌的低成本诊断工具，但目前尚未见其用于牛支原体鉴定的诊断准确性相关报道。为此，本研究以犊牛支气管肺泡灌洗液（BALf）中的牛支原体为检测对象，采用贝叶斯隐类别模型（BLCM），将纳米孔测序分别与基于基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）的牛支原体快速鉴定（RIMM）及三重实时荧光定量PCR进行对比分析。实际操作中，为节约成本常采用样本混检策略，但目前尚无其对牛支原体诊断准确性影响的相关研究报道。为此，本研究同时分析了17份混检样本，每份混样包含来自同一养殖场的5份个体支气管肺泡灌洗液样本。将混检样本的检测结果与个体样本结果进行比对，以计算诊断灵敏度（Se）与特异度（Sp）。相较于RIMM（灵敏度：93.0%；95%置信区间：76.8%~99.5%，特异度：91.3%；82.5%~97.0%）与三重实时荧光定量PCR（灵敏度：94.6%；95%置信区间：89.7%~97.7%，特异度：86.0%；76.1%~93.6%），贝叶斯隐类别模型下纳米孔测序展现出良好的灵敏度（77.3%；95%可信区间：57.8%~92.8%）与较高的特异度（97.4%；91.5%~99.7%）。纳米孔测序用于牛支原体混检分析的灵敏度与特异度分别为85.7%（95%置信区间：59.8%~111.6%）与90.0%（71.4%~108.6%）；RIMM的对应值则为100%（100%~100%）与88.9%（68.4%~109.4%）。综上，纳米孔测序是一种快速可靠的牛支原体鉴定工具。为降低检测成本并提升牛支原体的检出率，采用每份混样包含5份个体样本的混检策略应用于纳米孔测序与RIMM检测均具备可行性。