背景:由溃疡分枝杆菌(MU)引起的布鲁里溃疡(BU)是一种破坏性的坏死性皮肤病。PCR,世卫组织建议确认BU,需要一个设备齐全的实验室,因此,经常在远程设置中延迟BU患者的及时诊断和治疗。环介导等温扩增(LAMP)是一种基于PCR的DNA等温扩增方案,已被建议用于在低资源环境中诊断BU。
■这是一项探索性诊断测试评估研究,具有嵌入式定性子研究。它的目标是双重的:第一,与更复杂的常规DNA提取方法(CM)相比,评估基于简单的快速注射器的DNA提取方法(SM),然后是针对IS2404的LAMP测定法,用于检测MU,使用市售的口袋加热器(pw)或加热块(hb)进行孵育。第二,通过在加纳农村流行地区的社区卫生中心探索BU的诊断工作流程,作为潜在目标设置的示例,使用与研究人员和医护人员(HCWs)的访谈。针对BU诊断的目标产品配置文件(TPP)的要求和目标设置,讨论了诊断测试评估结果。
结果:使用SM进行DNA提取,然后进行IS2404PCR(IS2404PCRSM)的方案能够在提交诊断的83份BU临床标本中的73份中鉴定出MUDNA。敏感性,特异性,IS2404PCRSM的阳性预测值(PPV)和阴性预测值(NPV)分别为90.12%,100%,分别为100%和65.21%,与参考标准IS2404PCR结合分枝杆菌DNA的标准提取方案相比。对64个SMDNA提取物的LAMP测定的评估显示出灵敏度,特异性,PPV和NPV为83.6%,100%,100%和50%,分别,使用口袋加热器(pwLAMPSM)或加热块(hbLAMPSM)孵育反应,与相同的参考标准相比。发现pwLAMPSM的检测极限是IS2404靶标的30个拷贝。访谈结果探讨了BU诊断和治疗的障碍,包括对疾病的看法,成本,和运输的可用性。参与者确认PoC的诊断,除了根据临床标准进行筛查外,将有利于防止后续行动的延误和损失。
结论:pwLAMP的高诊断和分析准确性,由我们结合基于注射器的DNA提取方法进行评估,支持在没有可靠电力供应的情况下,在社区或初级卫生保健水平上快速检测可疑BU样本中的MU的潜在用途。进一步的优化需求包括裂解缓冲液,直接在PoC和/或其他站点进行评估,评估员工培训要求和质量控制。
BACKGROUND: Buruli ulcer (BU) caused by Mycobacterium ulcerans (MU) is a devastating necrotic skin disease. PCR, recommended for confirmation of BU by WHO, requires an adequately equipped laboratory, therefore often delaying timely diagnosis and treatment of BU patients in remote settings. Loop-mediated isothermal amplification (LAMP) is a PCR-based protocol for isothermal amplification of DNA that has been suggested for diagnosis of BU in low-resource settings.
UNASSIGNED: This is an exploratory diagnostic test evaluation study, with an embedded qualitative sub-study. Its aims are two-fold: First, to evaluate a simple rapid syringe-based DNA extraction method (SM) in comparison with a more elaborate conventional DNA extraction method (CM), followed by a LAMP assay targeting IS2404 for the detection of MU, either using a commercially available pocket warmer (pw) or a heat block (hb) for incubation. Second, to complement this by exploring the diagnostic workflow for BU at a community-based health centre in an endemic area in rural Ghana as an example of a potential target setting, using interviews with researchers and health care workers (HCWs). Diagnostic test evaluation results are discussed in relation to the requirements of a target product profile (TPP) for BU diagnosis and the target setting.
RESULTS: A protocol using SM for DNA extraction followed by IS2404 PCR (IS2404 PCRSM) was able to identify MU DNA in 73 out of 83 BU clinical specimens submitted for diagnosis. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of IS2404 PCRSM were 90.12%, 100%, 100% and 65.21% respectively, as compared to the reference standard IS2404 PCR in combination with a standard extraction protocol for mycobacterial DNA. Evaluation of the LAMP assay on 64 SM DNA extracts showed a sensitivity, specificity, PPV and NPV of 83.6%, 100%, 100% and 50%, respectively, using either pocket warmer (pwLAMPSM) or heat block (hbLAMPSM) for incubation of the reaction, as compared to the same reference standard. The limit of detection of pwLAMPSM was found to be 30 copies of the IS2404 target. Interview findings explored barriers to BU diagnosis and treatment, including perceptions of the disease, costs, and availability of transport. Participants confirmed that a diagnosis at the PoC, in addition to screening based on clinical criteria, would be advantageous in order to prevent delays and loss to follow-up.
CONCLUSIONS: The high diagnostic and analytic accuracy of the pwLAMP, evaluated by us in combination with a syringe-based DNA extraction method, supports its potential use for the rapid detection of MU in suspected BU samples at the community or primary health care level without reliable electricity supply. Further optimization needs include a lysis buffer, evaluation directly at the PoC and/or other sites, assessing staff training requirements and quality control.