A comparison between manual count, flow cytometry and qPCR as a means of determining Babesia rossi parasitaemia in naturally infected dogs

Light microscopic manual count is the current gold standard for parasite quantification. The ability to determine parasite density in whole blood is crucial to understanding disease pathogenesis and finding a suitable automated method of B. rossi parasite quantification would facilitate higher throughput and provide results that are more objective. This study investigated both peripheral capillary and central venous whole blood to estimate the correlations between light microscopy, flow cytometry and quantitative real-time PCR (qPCR). Furthermore, the use of SYBR Green I as a nuclear marker for the detection and quantification of B. rossi by flow cytometric analysis was explored. Peripheral capillary and central venous blood were sampled from forty naturally B. rossi-infected dogs and ten healthy control dogs. Samples were analysed by reverse line blot hybridization assay to confirm a mono-B. rossi infection. Capillary blood parasite density was quantified using light microscopic manual counting and venous blood parasitaemia quantified by manual counts, flow cytometry and qPCR. Flow cytometry, using SYBR Green I staining, showed promise in quantifying B. rossi nucleic acid in venous blood. A significant correlation was found between the venous manual counts and adjusted flow cytometric results, as well as qPCR. A significant correlation was also observed between the capillary manual counts compared to venous manual counts, adjusted flow cytometric results, and qPCR. The study results suggest that qPCR is of value as an alternative to the gold standard manual count for quantifying B. rossi parasitaemia in canine whole blood and that flow cytometry may be useful with further refinement of issues such as background fluorescence and the influence of reticulocytes. The data presented in Tables A-C represent the raw field data used during data analysis.光学显微镜手动计数（light microscopic manual count）是当前寄生虫定量检测的金标准。全血寄生虫密度的测定对于阐明疾病发病机制至关重要；而建立针对犬巴贝斯虫（B. rossi）的自动化定量检测方法，将有助于提升检测通量并获得更客观的检测结果。本研究同时采集外周毛细血管血与中心静脉全血，以探究光学显微镜手动计数、流式细胞术（flow cytometry）与实时定量聚合酶链反应（quantitative real-time PCR，qPCR）之间的相关性。此外，本研究还探索了SYBR Green I作为核标记物，通过流式细胞术分析检测并定量犬巴贝斯虫的应用潜力。研究共纳入40头自然感染犬巴贝斯虫的犬只与10头健康对照犬，采集其毛细血管血与静脉血样本。所有样本均通过反向线点杂交试验（reverse line blot hybridization assay）确认仅为犬巴贝斯虫单一感染。毛细血管血的寄生虫密度采用光学显微镜手动计数法定量，静脉血的寄生虫血症则分别通过手动计数、流式细胞术与qPCR进行定量。采用SYBR Green I染色的流式细胞术在静脉血犬巴贝斯虫核酸定量中展现出应用潜力。研究发现，静脉血手动计数结果与校正后的流式细胞术检测结果、qPCR结果均存在显著相关性；毛细血管血手动计数结果与静脉血手动计数结果、校正后的流式细胞术检测结果以及qPCR结果同样存在显著相关性。本研究结果表明，qPCR可作为替代金标准手动计数法的备选方案，用于犬全血中犬巴贝斯虫寄生虫血症的定量检测；而流式细胞术在进一步优化背景荧光、网织红细胞影响等问题后，也具备应用价值。表A至C中呈现的数据为本次数据分析所用的原始现场数据。