兽研1. 引言
猫血支原体病，通常称为猫血原体病，是一种重要的跨界媒介传播传染病，其临床表现从轻度或隐性携带状态到危及生命的溶血性贫血和全身性疾病不等，呈全球性分布，影响家猫和野猫[1,2,3]。该病主要由柔膜菌纲中三种嗜血性、细胞壁缺陷、多形性细菌引起，这些细菌专性寄生于宿主红细胞表面：猫血支原体（Mhf）、‘暂定种猫血小支原体’（CMhm）和‘暂定种苏黎世血支原体’（CMt）[4,5,6]。在这三种中，Mhf毒力最强，常引起严重的溶血性贫血，伴有广泛的血管内和/或血管外溶血[7]。相比之下，CMhm和CMt传统上被认为毒力较弱，在免疫功能正常的猫中通常引起亚临床感染或仅轻度血液学异常[4,5,8]。然而，近期报道记录了以CMhm为唯一检出病原体的严重急性贫血病例，表明该菌种偶尔也可引起严重疾病。此外，混合性血支原体感染和并发逆转录病毒感染可显著加重临床表现并增加疾病严重程度[4,5,8,9]。
猫血支原体的全球分布和流行病学动态高度异质，在不同地理区域差异显著。近期一项系统综述和荟萃分析估计，家猫中血支原体的全球合并患病率为19.8%，其中CMhm为主要菌种，其次为Mhf和CMt[10]。传播主要通过吸血节肢动物发生，尤其是猫栉首蚤（Ctenocephalides felis）和血红扇头蜱（Rhipicephalus sanguineus），二者被认为是重要的潜在媒介[4,5,6,11,12]。除媒介传播外，通过攻击性互动尤其是咬伤导致的水平传播已被广泛记录。因此，未去势的户外公猫以及生活在高密度收容环境中的猫感染率 consistently 较高[4,6,13]。最近，分子监测研究揭示了非典型血支原体菌种在伴侣动物和猫宿主中的存在，包括牛血支原体病的主要病原体温氏支原体（Mycoplasma wenyonii）。这些发现提示可能存在跨物种传播或此前未识别的宿主关联[4,6]。总体而言，这些观察结果强调了在“同一健康”框架内开展全面分子监测的重要性，以增进对宿主适应、病原传播和新发感染风险的认识[6]。
在东南亚，尤其是泰国，猫血原体病是一种地方性传染病，影响多种猫群[5,8,14]。分子监测研究报告的患病率从住院猫的11.2%到栖息于公共区域和寺庙环境的半家养猫和野猫的38.05%不等[6,8,15]。在已报道的泰国分离株中，CMhm为主要菌种，Mhf较少见，CMt仅偶尔检出[14]。鉴于感染率高且CMhm在泰国占主导地位，准确及时的诊断对于有效疾病管理仍然至关重要。尽管具有临床和兽医重要性，猫血原体病的早期诊断面临重大技术挑战[4,5]。吉姆萨染色血涂片的光学显微镜检查在资源有限的兽医环境中仍被广泛使用。然而，该方法敏感性有限（约51.47%），可能无法检测低水平菌血症，且易受观察者偏倚和染色伪影影响[4,5,16]。分子诊断方法，特别是靶向16S rRNA基因的常规PCR和多位点序列分析（MLSA），因其高分析敏感性和特异性而被广泛视为参考标准[4,5,8,17,18]。然而，基于常规PCR的方法需要专门的实验室基础设施、相对较高的运行成本和较长的周转时间，限制了其在快速现场筛查和疫情应对中的适用性。
为克服集中式PCR检测和传统显微镜筛查的局限性，已开发出多种先进的即时检验（POCT）技术，以提供快速可靠的分子诊断。在这些技术中，近期在伴侣动物和 livestock 环境中开展的验证研究表明，RNase杂交辅助扩增（RHAM）技术具有前景良好的诊断性能。RHAM是一种等温分子检测方法，将环介导等温扩增（LAMP）与RNase HII介导的检测系统整合于全自动封闭卡盒平台中。该设计提供与qPCR相当的分析灵敏度和特异性，可在35分钟内生成结果，并最大限度降低气溶胶介导交叉污染的风险[19,20,21]。尽管RHAM已成功验证用于检测伴侣动物中其他血源性病原体和传染性逆转录病毒，但支持其在临床现场条件下对猫血支原体诊断性能的证据仍然有限。因此，需要进一步评估以确定其是否适合猫血支原体病的常规诊断。
因此，本研究旨在通过与靶向16S rRNA基因的传统PCR直接比较，评估商业化Pluslife®猫血支原体（FHM）核酸检测卡的诊断性能，包括其灵敏度、特异性和精密度。研究使用从泰国曼谷一所兽医教学医院就诊猫采集的血液样本，还评估了这种快速即时分子检测在支持猫血支原体病及时诊断和临床决策方面的临床实用性。本研究进一步旨在描述泰国猫血支原体的检测频率和分子流行病学特征。
2. 材料与方法
2.1. 伦理批准与知情同意
研究方案在2025年9月开始样本采集前，已获得泰国马哈那空理工大学兽医学院动物研究伦理委员会批准（批准号ACUC-MUT-2025/011）以及泰国农业大学机构动物护理与使用委员会批准（批准号ACKU66-VET-002）。所有猫主人在入组前均签署了参与研究和发表的书面知情同意书。
2.2. 研究期间与地点
2025年9月至2026年5月期间，从兽医教学医院就诊的病猫采集血液样本，并送至泰国曼谷农业大学兽医学院诊断寄生虫学实验室进行后续分析。
2.3. 样本采集与研究方案设计及样本量论证
共有114只不同年龄和品种的猫入组研究。所有入组猫均表现出一项或多项可能与血支原体感染相符的临床症状，包括发热、黏膜苍白、贫血、黄疸和体重减轻。最低所需样本量采用Buderer公式针对诊断准确性研究进行估算。假设预期灵敏度和特异性均为90%、置信水平为95%，并根据历史监测数据假设泰国临床患病猫中血支原体预期患病率约为45%，计算得出最低所需样本量为107只猫。因此，最终入组114只猫超过了最低所需样本量，被认为足以评估RHAM检测相对于参考方法的诊断性能和一致性。
全血样本采集于EDTA管中，收到后分为两份等分试样，以确保两种诊断检测均使用来自同一标本的材料进行。为尽量减少潜在的识别和观察者偏倚，每份标本由未参与任何诊断检测的独立技术人员分配匿名追踪码。第一份等分试样立即由对传统PCR结果和猫详细病历不知情的操作人员使用Pluslife® FHM核酸检测卡进行检测。第二份等分试样储存于−20 °C，直至由对Pluslife®检测结果不知情的独立实验室人员进行批量DNA提取和传统PCR分析。两种诊断方法的结果在所有检测完成后才揭盲并制表。由于寄生虫学诊断单位的常规诊断送检流程，包括全血细胞计数、血细胞压积值在内的详细血液学数据，以及流行病学和生活方式信息（如体外寄生虫预防史和室内/室外状态），并非对所有入组猫均可系统获取。
2.4. RNase杂交辅助扩增（RHAM）检测
用于检测CMhm、Mhf和CMt的RHAM检测包括三个主要组成部分：Pluslife一体化核酸检测设备、核酸释放管和Pluslife®猫血支原体（FHM）核酸检测卡（广州Pluslife生物技术有限公司，中国广州）。所有检测程序均按照制造商说明进行。
检测前，将设备预热并连接至电脑上的Pluslife Pet应用程序（版本1.7）。预热后，使用一次性巴斯德吸管从EDTA抗凝标本中吸取50 µL全血转移至核酸释放管中。随后将管置于65 °C孵育5 min以促进核酸释放。将所得裂解液转移至FHM核酸检测卡的指定样本入口。将检测卡保持直立位置15 s，然后按下卡盖上的空气按钮。随后短暂振荡检测卡并将其插入一体化核酸检测仪。通过在Pluslife Pet应用程序中选择“开始”功能启动检测，约30 min后显示检测结果。
2.5. 常规聚合酶链反应（PCR）
使用NucleoSpin® Blood试剂盒（Macherey-Nagel GmbH & Co.，Düren，德国）按照制造商说明书从EDTA抗凝全血中提取基因组DNA。DNA以100 µL终体积洗脱，保存于−20 °C直至后续PCR分析。
通过靶向16S rRNA基因的常规PCR检测嗜血支原体DNA。使用Criado-Fornelio等[22]描述的引物对：正向引物5′-ATACGGCCCATATTCCTACG-3′和反向引物5′-TGCTCCACCACTTGTTCA-3′。每份PCR反应在20 µL终体积中进行，包含1× PCR缓冲液、1.5 mM MgCl2、0.2 mM dNTPs、各0.1 µM引物、1.0 U Taq DNA聚合酶（Invitrogen™，Thermo Fisher Scientific，Carlsbad，CA，USA）和2 µL DNA模板。
为监测检测性能和潜在污染，每次PCR运行均包含阳性对照和无模板对照。经序列确认的Candidatus Mycoplasma haemominutum DNA作为阳性对照，无核酸酶水作为无模板对照。PCR产物通过琼脂糖凝胶电泳分析，方法如先前所述[23,24,25,26,27]。预期扩增子大小约为580至620 bp，取决于嗜血支原体种类。
2.6. 分析
嗜血支原体感染检出频率按PCR阳性猫占所有受检猫的比例计算，以百分比表示。以常规PCR为参考方法评估Pluslife® FHM核酸检测卡的诊断性能。根据与传统PCR结果的一致性，将Pluslife® FHM检测结果分类为真阳性、真阴性、假阳性或假阴性。Pluslife® FHM检测相对于常规PCR的敏感性、特异性、准确性和精确度（阳性预测值）使用以下标准公式计算[28,29]：诊断敏感性=真阳性数真阳性数+假阴性数×100 特异性=真阴性数真阴性数+假阳性数×100 准确性=真阳性数+(真阴性数)真阳性数+真阴性数+假阳性数+(假阴性数)×100 精确度=真阳性数真阳性数+假阳性数×100
2.7. DNA测序
为探究诊断结果不一致的原因并确认嗜血支原体种类，对所有PCR阳性样本的扩增子进行DNA测序，包括四份结果不一致的样本（Pluslife® FHM核酸检测卡阴性但常规PCR阳性）。16S rRNA基因常规PCR后，从凝胶中切取阳性产物并使用PCR纯化凝胶提取试剂盒（NucleoSpin® Gel and PCR Clean-up，MACHEREY-NAGEL GmbH & Co，Düren，德国）纯化。随后，将纯化产物送交Sanger DNA测序（Macrogen，Beotkkot-ro，Geumcheon-gu，Seoul，Republic of Korea）。 for分子鉴定，将一致性部分16S rRNA序列使用基础局部比对搜索工具（BLAST）与美国国家生物技术信息中心（NCBI）数据库中的参考序列进行比较。物种鉴定主要基于最高序列同一性百分比和相应E值，同时记录最接近匹配的参考登录号。测序分析包括四份假阴性样本（66、68、105和107），以确定它们是否代表已识别的猫嗜血支原体种类，包括CMhm、Mhf和CMt，或其他非目标或跨物种。
3. 结果
3.1. 研究人群的人口学特征
研究人群（包括114只猫）的人口学特征总结于表1。雄性猫占人群的大多数（n = 76，66.67%），而雌性猫占其余38只动物（33.33%）。最大年龄组为1–5岁猫（n = 47，41.23%），其次为6–10岁猫（n = 41，35.96%）、11岁以上猫（n = 17，14.91%）和1岁以下幼猫（n = 9，7.89%）。家养短毛猫（DSH）为主要品种，占87只动物（76.32%）。其余猫为纯种猫；苏格兰折耳猫（n = 15，13.16%）占多数；其他纯种猫见表1。
3.2. Pluslife® FHM核酸检测卡与常规PCR相比的诊断性能
采用常规PCR作为参考标准，对Pluslife® FHM核酸检测卡的诊断性能进行了评估。所有114份临床标本的单项检测结果见补充表S1，交叉制表结果及计算的诊断性能指标汇总于表2。
在分析的114份样本中，两种方法一致阳性（真阳性）55份，一致阴性（真阴性）53份。共发现6份不一致结果：4份假阴性样本（编号66、68、105和107），Pluslife® FHM检测为阴性但常规PCR为阳性；2份假阳性样本（编号24和26），Pluslife® FHM检测为阳性但常规PCR为阴性。
具体而言，样本编号24和26在Pluslife® FHM检测中产生CMhm阳性信号，但常规PCR及琼脂糖凝胶电泳后未出现可检测的扩增条带。由于常规PCR作为参考标准，这两份样本在诊断性能评估中被归类为假阳性。此外，由于缺乏PCR扩增产物，无法进行Sanger测序以解决不一致结果。
相对于常规PCR，Pluslife® FHM检测的敏感性为93.22%，特异性为96.36%。总体准确率为94.74%，精密度（阳性预测值）为96.49%。
3.3. 不一致分离株的分子特征与序列分析
对所有PCR阳性样本（表S1）的部分16S rRNA基因扩增产物进行了双向Sanger测序及BLASTn分析，包括4份不一致（假阴性）样本，以探究观察到的诊断差异。如表3所示，BLASTn分析将全部4份假阴性样本鉴定为目标猫血支原体种CMhm。这4株分离株与参考CMhm菌株显示高度序列相似性，核苷酸一致性范围为95.38%至99.49%，E值为0.0。
3.4. 嗜血支原体的检测频率与物种分布
对2025年9月至2026年5月期间在泰国农业大学兽医教学医院就诊的114只临床患病猫，比较了常规PCR与Pluslife® FHM检测对嗜血支原体的检测频率和物种分布（表4）。
常规PCR鉴定CMhm为最常检出的单一物种感染（n = 52/114，45.61%），其次为Mhf（n = 6/114，5.26%）和CMt（n = 1/114，0.88%）。相比之下，Pluslife® FHM检测在48只猫中鉴定出单一物种CMhm感染（48/114，42.11%），5只猫为Mhf感染（5/114，4.39%），1只猫为CMt感染（1/114，0.88%）。值得注意的是，Pluslife® FHM检测在3只DSH猫中检出了嗜血支原体物种共感染（CMhm和Mhf），而常规PCR未检出任何共感染。
4. 讨论
快速准确的POCT平台在兽医诊断中日益重要，尤其是在临床和现场环境中管理传染病方面[19,20,21]。在此背景下，我们以靶向16S rRNA基因的常规PCR为参照，评估了Pluslife® FHM核酸检测卡对泰国某兽医教学医院自然感染猫的诊断性能。相对于常规PCR，Pluslife® FHM检测的敏感性为93.22%，特异性为96.36%，总体准确率为94.74%，精密度（阳性预测值）为96.49%，表明在研究人群中具有强大的诊断性能。这些发现与先前关于等温核酸扩增方法在兽医诊断中的研究基本一致。环介导等温扩增（LAMP）和重组酶聚合酶扩增（RPA）检测对多种兽医病原体显示出良好的诊断性能和快速周转时间，包括猫媒介传播病原体、猫冠状病毒、犬细小病毒和牛血支原体[30,31,32,33,34]。更直接地说，针对伴侣动物病原体开发的基于RHAM的检测方法，包括猫白血病病毒（FeLV）、猫免疫缺陷病毒（FIV）、犬埃立克体及多种人类病原体，已证明其诊断性能接近实验室分子方法，而其封闭卡盒设计可能降低扩增后携带污染的风险[19,20,21,35,36,37]。
在本研究中，住院猫中经常检测到嗜血支原体感染，其中CMhm是传统PCR（45.61%）和Pluslife® FHM检测（42.11%）均检测到的主要菌种。本研究中观察到的CMhm优势与最近的一项全球荟萃分析一致，该分析确定CMhm是全球范围内最常检测到的猫嗜血支原体菌种[10]。我们的发现也与之前在泰国进行的分子流行病学研究一致。例如，Kamyingkird等人[15]报告CMhm是居住在曼谷寺庙的流浪猫中的主要嗜血支原体菌种，个体感染率为24.53%，且在所有嗜血支原体阳性采样点均有检出。同样，Bui等人[8]通过多位点序列分析证实，CMhm仍然是泰国家猫中最常检测到的嗜血支原体菌种。本研究中大多数感染猫为雄性和DSH猫，反映了研究人群的人口学构成。先前的研究表明，行为因素，包括领地争斗和户外游荡，会增加接触猫栉首蚤等外寄生虫的机会，从而促进嗜血支原体菌种的传播[10,15]。
对Pluslife® FHM检测与传统PCR之间不一致结果的调查为诊断不一致的潜在来源提供了重要见解。对四个传统PCR阳性/Pluslife阴性的样本进行序列分析，为检测到的微生物的身份和遗传特征提供了额外信息。BLASTn分析显示，所有四个不一致样本（编号66、68、105和107）获得的序列与CMhm参考序列最密切匹配，序列一致性范围为95.38%至99.49%。这些假阴性结果的一个可能解释是细菌载量低于Pluslife® FHM检测的分析检测限。尽管本研究中未对细菌载量进行定量，但已有报道称，针对其他动物病原体开发的基于RHAM的检测方法在低目标浓度下检测能力降低[19,20,21]。其他等温扩增平台，包括LAMP和RPA，也报道了在低目标浓度下的类似局限性，特别是与qPCR或巢式PCR等高灵敏度实验室分子方法相比[30,38,39,40]。CMhm内部的遗传多样性是不一致结果的另一种可能解释。多位点序列分析已鉴定出几个遗传上不同的CMhm亚群（A、B和C）[8]。如果检测靶向区域内发生多态性，这种遗传多样性可能潜在地影响引物或探针的结合；然而，本研究无法直接评估这种可能性。两个明显的假阳性结果（样本编号24和26），即Pluslife® FHM检测阳性但传统PCR阴性，也值得考虑。一个可能的解释是这些样本含有可被RHAM检测检测到但低于传统凝胶PCR检测限的目标DNA浓度。然而，由于没有可用于测序的传统PCR扩增产物，这些样本的真实感染状态无法独立确定，也不能排除非特异性检测信号或其他技术因素。
此外，这两种检测方法在检测单一感染和混合感染方面的差异需要仔细解读。Pluslife® FHM检测鉴定出三只CMhm–Mhf混合感染猫，而传统PCR将其归类为单一菌种感染。一个可能的解释是当存在多个目标模板时扩增效率存在差异。在传统PCR中，目标丰度或扩增效率的差异可能降低对丰度较低的共感染微生物的检测，尽管本研究未直接研究这一机制。或者，Pluslife® FHM平台的检测设计可能有助于同时检测多个目标，从而可能改善混合感染的鉴定。先前验证研究已报道了基于RHAM的分子检测方法的类似优势，尽管需要额外的分析研究来确定这一机制是否有助于改善本研究中观察到的猫嗜血支原体菌种混合感染的检测[19,20,21]。
当将这些诊断发现外推至更广泛的家猫群体（包括无症状猫和收容所猫）时，应考虑感染流行率以及菌血症幅度和时间动态的差异。在无症状携带者中，血支原体菌血症可能以低水平存在并随时间波动，可能接近或低于分子检测方法的检测限[4,7]。因此，Pluslife® FHM检测在无症状群体中的诊断敏感性可能不同于本研究中临床患病猫所观察到的93.22%，尤其是当细菌载量较低时。此外，由于预测值取决于感染流行率，假设敏感性和特异性相似，在低流行率筛查人群中使用该检测通常预期会降低阳性预测值（PPV）并提高阴性预测值（NPV）。相反，在血支原体感染流行率较高的收容所人群中，PPV预期会升高，可能支持使用快速检测进行分诊并为适当的感染控制或管理决策提供依据。
尽管RHAM平台具有快速周转时间（约30分钟）和封闭卡盒格式，但在分散的兽医环境中实施该平台可能面临若干实际挑战，尤其是在资源有限的环境中。首先，仪器购置和经常性耗材成本可能限制较小兽医诊所的采用，尤其是当检测费用由客户直接承担时。虽然传统PCR在批量处理样本时可能实现较低的试剂成本，但有意义的经济学比较还应考虑实验室基础设施、人员、样本运输和周转时间。其次，尽管封闭卡盒格式降低了对专业分子实验室基础设施的要求，但分析前程序——包括准确的微量移液、样本裂解和卡盒加载——仍需要适当的人员培训，以尽量减少操作错误、污染和无效检测结果。第三，遵守制造商推荐的运输和储存条件对于维持试剂稳定性至关重要，尤其是在高温和高湿度可能使产品分销和储存复杂化的热带环境中。应对这些挑战需要因地制宜的定价和分销策略、可靠的供应和储存系统，以及充分的人员培训和质量控制程序。
在解释这些发现时，应承认本研究存在若干局限性。首先，研究人群完全由从单一兽医教学医院招募的临床患病猫组成，这可能限制研究结果对无症状猫以及其他临床或地理环境人群的普适性。其次，有限的样本量阻碍了定量实时PCR（qPCR）分析，因此无法评估假阴性样本中的细菌载量。 consequently，无法直接评估低细菌载量导致所观察到的诊断不一致的可能性。第三，由于标本是通过常规诊断送检流程获得的，并非所有猫都系统性地具备详细的血液学和临床数据——包括贫血严重程度和再生状态、血细胞比容、其他血细胞指标以及发热状态。这些数据的缺失阻碍了评估物种特异性感染（尤其是Mhf和CMhm）与贫血严重程度或其他临床表现之间潜在关联的能力。最后，并非所有入组猫都系统性地记录了流行病学和行为学变量，包括外寄生虫暴露或感染、跳蚤控制史以及室内/室外生活方式。因此，这些流行病学数据的缺失限制了对可能促进传播的潜在风险因素或行为参数的评估。未来需要开展涉及更大规模、更多样化的临床患病猫和无症状猫群体的多中心研究，结合定量细菌载量评估以及临床和流行病学数据的标准化收集，以进一步验证Pluslife® FHM检测的诊断性能并阐明其在不同家猫群体中的诊断效用。
5. 结论
Pluslife® FHM核酸检测卡对猫嗜血支原体感染的检测表现出较强的诊断性能，与传统PCR相比，灵敏度为93.22%，特异性为96.36%，总体准确率为94.74%，精密度（阳性预测值）为96.49%。该检测约30分钟即可提供结果，实现了快速、物种特异性的分子检测，同时降低了对专业分子实验室基础设施的要求。尽管在114份样本中观察到6份结果不一致，但由于缺乏细菌载量定量以及对所有不一致样本的独立确认，其潜在原因未能完全阐明。尽管如此，在本研究评估的条件下，Pluslife® FHM检测与传统PCR相比被证明是一种可靠的诊断工具。其快速周转时间和简化的工作流程支持其作为即时分子诊断辅助工具的潜在用途，用于猫血支原体病的及时诊断和临床管理，尤其是在难以获得集中化分子实验室的兽医环境中。
查看英文原文▾
1. Introduction
Feline hemotropic mycoplasmosis, commonly referred to as feline hemoplasmosis, is a significant transboundary vector-borne infectious disease whose signs range from mild or silent carrier stages to life-threatening hemolytic anemia and systemic illness in domestic and wild felines worldwide [1,2,3]. The disease is primarily caused by three hemotropic, cell wall-deficient, pleomorphic bacterial species within the class Mollicutes that obligately parasitize the epicellular surfaces of host erythrocytes: Mycoplasma haemofelis (Mhf), ‘Candidatus Mycoplasma haemominutum’ (CMhm), and ‘Candidatus Mycoplasma turicensis’ (CMt) [4,5,6]. Among these three species, Mhf is the most virulent and frequently causes severe hemolytic anemia accompanied by extensive intravascular and/or extravascular hemolysis [7]. In contrast, CMhm and CMt have traditionally been regarded as less virulent species, typically causing subclinical infections or only mild hematological abnormalities in immunocompetent cats [4,5,8]. Nevertheless, recent reports have documented cases of severe acute anemia associated with CMhm as the sole detected pathogen, indicating that this species may occasionally cause severe disease. Furthermore, mixed hemotropic Mycoplasma infections and concurrent retroviral infections can substantially worsen clinical manifestations and increase disease severity [4,5,8,9].
The global distribution and epidemiological dynamics of feline hemotropic mycoplasmas are highly heterogeneous and vary considerably across geographic regions. A recent systematic review and meta-analysis estimated a global pooled hemotropic Mycoplasma prevalence of 19.8% in domestic cats, with CMhm identified as the predominant species, followed by Mhf and CMt [10]. Transmission is believed to occur primarily through hematophagous arthropods, particularly the cat flea (Ctenocephalides felis) and the brown dog tick (Rhipicephalus sanguineus), which are considered important potential vectors [4,5,6,11,12]. In addition to vector-borne transmission, horizontal spread through aggressive interactions, especially bite wounds, has been widely documented. Consequently, intact outdoor male cats and cats living in high-density shelter environments consistently exhibit higher infection rates [4,6,13]. More recently, molecular surveillance studies have revealed the presence of atypical hemotropic Mycoplasma species, including Mycoplasma wenyonii, the primary causative agent of bovine hemotrophic mycoplasmosis, in companion animals and feline hosts. These findings suggest possible cross-species transmission or previously unrecognized host associations [4,6]. Collectively, these observations underscore the importance of comprehensive molecular surveillance within a One Health framework to improve understanding of host adaptation, pathogen transmission, and the emergence of novel infection risks [6].
In Southeast Asia, particularly in Thailand, feline hemoplasmosis is an endemic infectious disease affecting diverse feline populations [5,8,14]. Molecular surveillance studies have reported prevalence rates ranging from 11.2% in hospitalized cats to 38.05% in semi-domesticated and feral cats inhabiting public areas and temple environments [6,8,15]. Among reported Thai isolates, CMhm is the predominant species, whereas Mhf occurs less frequently and CMt is detected only sporadically [14]. Given the high prevalence of infection and the predominance of CMhm in Thailand, accurate and timely diagnosis remains essential for effective disease management. Despite its clinical and veterinary importance, early diagnosis of feline hemoplasmosis faces substantial technical challenges [4,5]. Light microscopic examination of Giemsa-stained blood smears remains widely used in resource-limited veterinary settings. However, this method has limited sensitivity (approximately 51.47%), may fail to detect low-level bacteremia, and is susceptible to observer bias and staining artifacts [4,5,16]. Molecular diagnostic methods, particularly conventional PCR targeting the 16S rRNA gene and multi-locus sequence analysis (MLSA), are widely regarded as reference standards because of their high analytical sensitivity and specificity [4,5,8,17,18]. Nevertheless, conventional PCR-based methods require specialized laboratory infrastructure, relatively high operation costs, and longer turnaround times, limiting their applicability for rapid field-based screening and outbreak response.
To overcome the limitations of both centralized PCR assays and conventional microscopic screening, several advanced point-of-care testing (POCT) technologies have been developed to provide rapid and reliable molecular diagnosis. Among these technologies, recent validation studies conducted in companion animal and livestock settings have demonstrated the promising diagnostic performance of RNase hybridization-assisted amplification (RHAM) technology. RHAM is an isothermal molecular assay that integrates loop-mediated isothermal amplification (LAMP) with an RNase HII-mediated detection system within a fully automated closed-cartridge platform. This design provides qPCR-comparable analytical sensitivity and specificity, generates results in less than 35 min, and minimizes the risk of aerosol-mediated cross-contamination [19,20,21]. Although RHAM has been successfully validated for detecting other blood-borne pathogens and infectious retroviruses in companion animals, evidence supporting its diagnostic performance for feline hemotropic mycoplasmas under clinical field conditions remains limited. Consequently, further evaluation is required to determine its suitability for routine diagnosis of feline hemoplasmosis.
Therefore, this study aimed to evaluate the diagnostic performance of the commercial Pluslife® Feline Hemotropic Mycoplasma (FHM) nucleic acid test card, including its sensitivity, specificity, and precision, by direct comparison with conventional PCR targeting the 16S rRNA gene. Using blood samples collected from cats presented to a veterinary teaching hospital in Bangkok, Thailand, the study also assessed the clinical utility of this rapid point-of-care molecular assay for supporting timely diagnosis and clinical decision-making in feline hemoplasmosis. The study further aimed to characterize the detection frequency and molecular epidemiology of feline hemotropic mycoplasmas in Thailand.
2. Materials and Methods
2.1. Ethical Approval and Informed Consent
The study protocol was approved by the Animal Research Ethics Committee, Faculty of Veterinary Medicine, Mahanakorn University of Technology, Thailand (approval no. ACUC-MUT-2025/011), and the Institutional Animal Care and Use Committee of Kasetsart University, Thailand (approval no. ACKU66-VET-002), before sample collection commenced in September 2025. Written informed consent for study participation and publication was obtained from all cat owners before enrollment.
2.2. Study Period and Location
Between September 2025 and May 2026, blood samples were collected from sick cats presented at the Veterinary Teaching Hospital and sent to the Diagnostic Parasitology Laboratory, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand, for subsequent analysis.
2.3. Sample Collection and Study Protocol Design, and Sample Size Justification
A total of 114 cats of various ages and breeds were enrolled in the study. All enrolled cats exhibited one or more clinical signs potentially compatible with hemotropic Mycoplasma infection, including fever, pale mucous membranes, anemia, jaundice, and weight loss. The minimum required sample size was estimated using Buderer’s formula for diagnostic accuracy studies. Assuming an anticipated sensitivity and specificity of 90%, a 95% confidence level, and an expected hemoplasma prevalence of approximately 45% among clinically ill cats in Thailand based on historical surveillance, the minimum required sample size was calculated to be 107 cats. The final enrollment of 114 cats therefore exceeded the minimum required sample size and was considered adequate for evaluating the diagnostic performance and agreement of the RHAM assay relative to the reference method.
Whole blood samples were collected into EDTA tubes and divided into two aliquots upon receipt to ensure that both diagnostic assays were performed using material derived from the same specimen. To minimize potential identification and observer bias, each specimen was assigned an anonymized tracking code by an independent technician who was not involved in performing either diagnostic assay. The first aliquot was tested immediately using the Pluslife® FHM nucleic acid test card by an operator blinded to the conventional PCR results and the cat’s detailed medical records. The second aliquot was stored at −20 °C until batched DNA extraction and conventional PCR analysis by independent laboratory personnel who were blinded to the Pluslife® assay results. Results from the two diagnostic methods were unblinded and tabulated only after all assays had been completed. Because of the routine diagnostic submission workflow of the parasitology diagnostic unit, detailed hematological data, including complete blood count, packed cell volume values, and epidemiological and lifestyle information, such as ectoparasite prevention history and indoor/outdoor status, were not systematically available for all enrolled cats.
2.4. RNase Hybridization-Assisted Amplification (RHAM) Assay
The RHAM assay for detecting CMhm, Mhf, and CMt comprises three main components: the Pluslife Integrated Nucleic Acid Testing Device, a nucleic acid releaser tube, and the Pluslife® Feline Hemotropic Mycoplasma (FHM) nucleic acid test card (Guangzhou Pluslife Biotech, Guangzhou, China). All testing procedures were performed according to the manufacturer’s instructions.
Before testing, the device was preheated and connected to the Pluslife Pet application (version 1.7) on a computer. After preheating, 50 µL of whole blood was transferred from the EDTA specimen to the nucleic acid releaser tube using a disposable Pasteur pipette. The tube was then incubated at 65 °C for 5 min to facilitate nucleic acid release. The resulting lysate was transferred to the designated sample inlet of the FHM nucleic acid test card. The test card was maintained in an upright position for 15 s, after which the air button on the cartridge cap was pressed. The card was then shaken briefly and inserted into the Integrated Nucleic Acid Testing Device. The assay was initiated by selecting the “Start” function in the Pluslife Pet application, and test results were displayed after approximately 30 min.
2.5. Conventional Polymerase Chain Reaction (PCR)
Genomic DNA was extracted from EDTA-anticoagulated whole blood using the NucleoSpin® Blood kit (Macherey-Nagel GmbH & Co., Düren, Germany) according to the manufacturer’s instructions. DNA was eluted in a final volume of 100 µL and stored at −20 °C until subsequent PCR analysis.
Hemotropic Mycoplasma DNA was detected by conventional PCR targeting the 16S rRNA gene. The primer pair described by Criado-Fornelio et al. [22] was used: forward primer 5′-ATACGGCCCATATTCCTACG-3′ and reverse primer 5′-TGCTCCACCACTTGTTCA-3′. Each PCR was performed in a final volume of 20 µL containing 1× PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.1 µM of each primer, 1.0 U of Taq DNA polymerase (Invitrogen™, Thermo Fisher Scientific, Carlsbad, CA, USA), and 2 µL of DNA template.
To monitor assay performance and potential contamination, positive and no-template controls were included in each PCR run. Sequence-confirmed Candidatus Mycoplasma haemominutum DNA served as the positive control, whereas nuclease-free water served as the no-template control. PCR products were analyzed by agarose gel electrophoresis as previously described [23,24,25,26,27]. The expected amplicon size was approximately 580 to 620 bp, depending on the hemoplasma species.
2.6. Analysis
The detection frequency of hemotropic Mycoplasma infection was calculated as the proportion of PCR-positive cats among all cats tested and expressed as a percentage. The diagnostic performance of the Pluslife® FHM nucleic acid test card was evaluated using conventional PCR as the reference method. Pluslife® FHM assay results were classified as true positives, true negatives, false positives, or false negatives according to their agreement with the conventional PCR results. Sensitivity, specificity, accuracy, and precision (positive predictive value) of the Pluslife® FHM assay relative to conventional PCR were calculated using the following standard formulas [28,29]: Diagnostic sensitivity=No. of true positiveNo. of true positive+No. of false negative×100 Specificity=No. of true negativeNo. of true negative+No. of false positive×100 Accuracy=No. of true positive+(No. of true negative)No. of true positive+No. of true negative+No. of false positive+(No. of false negative)×100 Precision=No. of true positiveNo. of true positive+No. of false positive×100
2.7. DNA Sequencing
To investigate the causes of discordant diagnostic results and confirm hemotropic Mycoplasma species, all amplicons of PCR-positive samples, including four discordant samples (negative by the Pluslife® FHM nucleic acid test card but positive by conventional PCR) were subjected to DNA sequencing. After 16S rRNA gene conventional PCR, the positive products were cut from the gel and purified using a PCR clean-up gel extraction kit (NucleoSpin® Gel and PCR Clean-up, MACHEREY-NAGEL GmbH & Co, Düren, Germany). Subsequently, the purified product was submitted for Sanger DNA sequencing (Macrogen, Beotkkot-ro, Geumcheon-gu, Seoul, Republic of Korea). For molecular identification, the consensus partial 16S rRNA sequences were compared with reference sequences in the National Center for Biotechnology Information (NCBI) database using the Basic Local Alignment Search Tool (BLAST). Species identification was based primarily on the highest percentage sequence identity and corresponding E-values, while the closest matching reference accession numbers were recorded. The sequencing analysis included four false-negative samples (66, 68, 105, and 107) to determine whether they represented recognized feline hemotropic Mycoplasma species, including CMhm, Mhf, and CMt, or other non-target or cross-species.
3. Results
3.1. Demographic Characteristics of the Study Population
The demographic characteristics of the study population, comprising 114 cats, are summarized in Table 1. Male cats were the majority of the population (n = 76, 66.67%), whereas female cats accounted for the remaining 38 animals (33.33%). The largest age group comprised cats aged 1–5 years (n = 47, 41.23%), followed by those aged 6–10 years (n = 41, 35.96%), cats older than 11 years (n = 17, 14.91%), and kittens younger than 1 year (n = 9, 7.89%). Domestic shorthair (DSH) cats were the predominant breed, accounting for 87 animals (76.32%). The remaining cats were purebred; Scottish Fold (n = 15, 13.16%) was the majority; the other purebreds are shown in Table 1.
3.2. Diagnostic Performance of the Pluslife® FHM Nucleic Acid Test Card Compared with Conventional PCR
The diagnostic performance of the Pluslife® FHM nucleic acid test card was evaluated using conventional PCR as the reference standard. Individual test results for all 114 clinical specimens are provided in Supplementary Table S1, while the cross-tabulation results and calculated diagnostic performance metrics are summarized in Table 2.
Of the 114 samples analyzed, 55 were concordantly positive (true positive) and 53 were concordantly negative (true negative) by both methods. Six discordant results were identified: four false-negative samples (Nos. 66, 68, 105, and 107), which were negative by the Pluslife® FHM assay but positive by conventional PCR; and two false-positive samples (Nos. 24 and 26), which were positive by the Pluslife® FHM assay but negative by conventional PCR.
Specifically, samples No. 24 and 26 generated positive CMhm signals with the Pluslife® FHM assay but yielded no detectable amplicon bands following conventional PCR and agarose gel electrophoresis. As conventional PCR served as the reference standard, these two samples were classified as false positives for diagnostic performance evaluations. Furthermore, the absence of PCR amplicons prevented Sanger sequencing from being performed to resolve the discordant results.
Relative to conventional PCR, the Pluslife® FHM assay demonstrated a sensitivity of 93.22% and a specificity of 96.36%. Overall accuracy was 94.74%, and precision (positive predictive value) was 96.49%.
3.3. Molecular Characterization and Sequence Analysis of Discrepant Isolates
Bidirectional Sanger sequencing followed by BLASTn analysis was performed on partial 16S rRNA gene amplicons from all PCR-positive samples (Table S1), including the four discordant (false-negative) samples, to investigate the observed diagnostic discrepancies. As shown in Table 3, BLASTn analysis identified all four false-negative samples as the target feline hemoplasma species CMhm. These four isolates showed high sequence similarity to reference CMhm strains, with nucleotide identities ranging from 95.38% to 99.49% and E-values of 0.0.
3.4. Detection Frequency and Species Distribution of Hemotropic Mycoplasma
The detection frequencies and species distributions of hemotropic Mycoplasma were compared between conventional PCR and the Pluslife® FHM assay in 114 clinically ill cats presented to Kasetsart University Veterinary Teaching Hospital between September 2025 and May 2026 (Table 4).
Conventional PCR identified CMhm as the most frequently detected single-species infection (n = 52/114, 45.61%), followed by Mhf (n = 6/114, 5.26%) and CMt (n = 1/114, 0.88%). By comparison, the Pluslife® FHM assay identified single-species CMhm infection in 48 cats (48/114, 42.11%), Mhf infection in 5 cats (5/114, 4.39%), and CMt infection in 1 cat (1/114, 0.88%). Notably, the Pluslife® FHM assay detected co-infections of hemotropic Mycoplasma species (CMhm and Mhf) in three DSH cats, whereas no co-infections were detected by conventional PCR.
4. Discussion
Rapid and accurate POCT platforms have become increasingly important in veterinary diagnostics, particularly for management of infectious diseases in clinical and field settings [19,20,21]. In this context, we evaluated the diagnostic performance of the Pluslife® FHM nucleic acid test card relative to conventional PCR targeting the 16S rRNA gene in naturally infected cats presented to a veterinary teaching hospital in Thailand. Relative to conventional PCR, the Pluslife® FHM assay demonstrated a sensitivity of 93.22%, specificity of 96.36%, overall accuracy of 94.74%, and precision (positive predictive value) of 96.49%, indicating strong diagnostic performance in the study population. These findings are broadly consistent with previous studies of isothermal nucleic acid amplification methods in veterinary diagnostics. Loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) assays have shown favorable diagnostic performance and rapid turnaround times for several veterinary pathogens, including feline vector-borne agents, feline coronavirus, canine parvovirus, and bovine hemoplasmas [30,31,32,33,34]. More directly, RHAM-based assays developed for companion animal pathogens, including feline leukemia virus (FeLV), feline immunodeficiency virus (FIV), Ehrlichia canis, and several human pathogens, have demonstrated diagnostic performance approaching that of laboratory-based molecular methods, while their closed-cartridge design may reduce the risk of post-amplification carry-over contamination [19,20,21,35,36,37].
Hemotropic Mycoplasma infections were frequently detected among hospitalized cats in this study, with CMhm representing the predominant species detected by both conventional PCR (45.61%) and the Pluslife® FHM assay (42.11%). The predominance of CMhm observed in the present study is consistent with recent global meta-analysis that identified CMhm as the most frequently detected feline hemoplasma species worldwide [10]. Our findings are also consistent with previous molecular epidemiological studies conducted in Thailand. For example, Kamyingkird et al. [15] reported CMhm as the predominant hemotropic Mycoplasma species among stray cats living in Bangkok monasteries, with an individual prevalence of 24.53% and detection at all hemoplasma-positive sampling sites. Similarly, Bui et al. [8] confirmed through multi-locus sequence analysis that CMhm remained the most frequently detected hemotropic Mycoplasma species among domestic cats in Thailand. Most infected cats in the present study were male and DSH cats, reflecting the demographic composition of the study population. Previous studies have suggested that behavioral factors, including territorial fighting and outdoor roaming, increase exposure to ectoparasites such as Ctenocephalides felis, thereby facilitating the transmission of hemotropic Mycoplasma species [10,15].
Investigation of discordant results between the Pluslife® FHM assay and conventional PCR provided important insights into potential sources of diagnostic disagreement. Sequence analysis of the four conventional PCR-positive/Pluslife-negative samples provided additional information on the identity and genetic characteristics of the detected organisms. BLASTn analysis showed that sequences obtained from all four discordant samples (Nos. 66, 68, 105 and 107) most closely matched CMhm reference sequences, with sequence identities ranging from 95.38% to 99.49%. One possible explanation for these false-negative results is a bacterial burden below the analytical detection limit of the Pluslife® FHM assay. Although bacterial loads were not quantified in the present study, reduced detection at low target concentrations has been reported for RHAM-based assays developed for other animal pathogens [19,20,21]. Similar limitations at low target concentrations have been reported for other isothermal amplification platforms, including LAMP and RPA, particularly when compared with highly sensitive laboratory-based molecular methods such as qPCR or nested PCR [30,38,39,40]. Genetic diversity within CMhm represents another possible explanation for the discordant results. Multilocus sequence analyses have identified several genetically distinct CMhm subgroups (A, B, and C) [8]. Such genetic diversity could potentially affect primer or probe binding if polymorphisms occur within assay-targeted regions; however, this possibility could not be directly evaluated in the present study. The two apparent false-positive results (samples Nos. 24 and 26), which were positive by the Pluslife® FHM assay but negative by conventional PCR, also warrant consideration. One possible explanation is that these samples contained target DNA at concentrations detectable by the RHAM assay but below the detection limit of conventional gel-based PCR. However, because no conventional PCR amplicons were available for sequencing, the true infection status of these samples could not be independently resolved, and nonspecific assay signals or other technical factors cannot be excluded.
Furthermore, discrepancies between these two assays in detecting single and mixed infections warrant careful interpretation. The Pluslife® FHM assay identified three cats with CMhm–Mhf mixed infection that were classified as single-species infections by conventional PCR. One possible explanation is differential amplification efficiency when multiple target templates are present. In conventional PCR, differences in target abundance or amplification efficiency may reduce the detection of less abundant co-infecting organisms, although this mechanism was not directly investigated in the present study. Alternatively, the assay design of the Pluslife® FHM platform may facilitate the simultaneous detection of multiple targets, potentially improving the identification of mixed infections. Similar advantages have been reported for RHAM-based molecular assays in previous validation studies, although additional analytical investigations are required to determine whether this mechanism contributes to the improved detection of mixed infections of feline hemotropic Mycoplasma species observed in the present study [19,20,21].
When these diagnostic findings are extrapolated to broader feline populations, including asymptomatic and shelter-housed cats, differences in infection prevalence and the magnitude and temporal dynamics of bacteremia should be considered. In asymptomatic carriers, hemoplasma bacteremia may occur at low levels and fluctuate over time, potentially approaching or falling below the detection limits of molecular assays [4,7]. Accordingly, the diagnostic sensitivity of the Pluslife® FHM assay in asymptomatic populations may differ from the 93.22% observed in the clinically ill cats evaluated in the present study, particularly when bacterial loads are low. Furthermore, because predictive values depend on infection prevalence, the use of the assay in lower-prevalence screening populations would generally be expected to decrease the positive predictive value (PPV) and increase the negative predictive value (NPV), assuming similar sensitivity and specificity. Conversely, in shelter populations with a higher prevalence of hemoplasma infection, PPV would be expected to increase, potentially supporting the use of rapid testing for triage and informing appropriate infection-control or management decisions.
Despite its rapid turnaround time (~30 min) and closed-cartridge format, implementation of the RHAM platform in decentralized veterinary settings may face several practical challenges, particularly in resource-limited settings. First, instrument acquisition and recurring consumable costs may limit adoption by smaller veterinary practices, particularly when test costs are borne directly by clients. Although conventional PCR may achieve lower reagent costs when samples are processed in batches, meaningful economic comparisons should also account for laboratory infrastructure, personnel, sample transport, and turnaround time. Second, although the closed-cartridge format reduces the requirement for specialized molecular laboratory infrastructure, pre-analytical procedures—including accurate micropipetting, sample lysis, and cartridge loading—still require appropriate staff training to minimize handling errors, contamination, and invalid test results. Third, adherence to manufacturer-recommended transport and storage conditions is essential for maintaining reagent stability, particularly in tropical environments where high temperature and humidity may complicate product distribution and storage. Addressing these challenges will require context-appropriate pricing and distribution strategies, reliable supply and storage systems, and adequate staff training and quality-control procedures.
Several limitations of the present study should be acknowledged when interpreting these findings. First, the study population consisted exclusively of clinically affected cats recruited from a single veterinary teaching hospital, which may limit the generalizability of the findings to asymptomatic cats and populations from other clinical or geographic settings. Second, limited sample volumes prevented quantitative real-time PCR (qPCR) analysis and therefore precluded assessment of bacterial loads in false-negative samples. Consequently, the possibility that a low bacterial burden contributed to the observed diagnostic discordance could not be directly evaluated. Third, detailed hematological and clinical data—including anemia severity and regenerative status, packed cell volume, other blood cell indices, and febrile status—were not systematically available for all cats because specimens were obtained through a routine diagnostic submission workflow. The absence of these data prevented assessment of potential associations between species-specific infection, particularly Mhf and CMhm, and anemia severity or other clinical manifestations. Finally, epidemiological and behavioral variables, including ectoparasite exposure or infestation, flea-control history, and indoor/outdoor lifestyle, were not systematically recorded for all enrolled cats. Consequently, the absence of these epidemiological data limited the assessment of potential risk factors or behavioral parameters that may facilitate transmission. Future multicenter studies involving larger and more diverse populations of clinically ill and asymptomatic cats, together with quantitative bacterial-load assessment and standardized collection of clinical and epidemiological data, are warranted to further validate the diagnostic performance of the Pluslife® FHM assay and clarify its diagnostic utility across different feline populations.
5. Conclusions
The Pluslife® FHM nucleic acid test card demonstrated strong diagnostic performance for the detection of feline hemotropic Mycoplasma infections, showing a sensitivity of 93.22%, specificity of 96.36%, overall accuracy of 94.74%, and precision (positive predictive value) of 96.49% relative to conventional PCR. Providing results in approximately 30 min, the assay provides rapid, species-specific molecular detection while reducing the requirement for specialized molecular laboratory infrastructure. Although six discordant results were observed among the 114 samples, the underlying causes could not be fully resolved due to the lack of bacterial load quantification and independent confirmation for all discordant specimens. Nevertheless, under the conditions evaluated in this study, the Pluslife® FHM assay proved to be a reliable diagnostic tool when compared with conventional PCR. Its rapid turnaround time and simplified workflow support its potential use as a point-of-care molecular diagnostic aid for timely diagnosis and clinical management of feline hemoplasmosis, particularly in veterinary settings with limited access to centralized molecular laboratories.