实时 PCR 检测法和快速诊断检测法在孟加拉国用于疑似临床疟疾患者的诊断。
Real-time PCR assay and rapid diagnostic tests for the diagnosis of clinically suspected malaria patients in Bangladesh.
机构信息
Parasitology Laboratory, ICDDR.B. GPO Box 128, Dhaka-1000, Bangladesh.
出版信息
Malar J. 2011 Jun 26;10:175. doi: 10.1186/1475-2875-10-175.
BACKGROUND
More than 95% of total malaria cases in Bangladesh are reported from the 13 high endemic districts. Plasmodium falciparum and Plasmodium vivax are the two most abundant malaria parasites in the country. To improve the detection and management of malaria patients, the National Malaria Control Programme (NMCP) has been using rapid diagnostic test (RDT) in the endemic areas. A study was conducted to establish a SYBR Green-based modified real-time PCR assay as a gold standard to evaluate the performance of four commercially-available malaria RDTs, along with the classical gold standard- microscopy.
METHODS
Blood samples were collected from 338 febrile patients referred for the diagnosis of malaria by the attending physician at MatirangaUpazila Health Complex (UHC) from May 2009 to August 2010. Paracheck RDT and microscopy were performed at the UHC. The blood samples were preserved in EDTA tubes. A SYBR Green-based real-time PCR assay was performed and evaluated. The performances of the remaining three RDTs (Falcivax, Onsite Pf and Onsite Pf/Pv) were also evaluated against microscopy and real-time PCR using the stored blood samples.
RESULT
In total, 338 febrile patients were enrolled in the study. Malaria parasites were detected in 189 (55.9%) and 188 (55.6%) patients by microscopy and real-time PCR respectively. Among the RDTs, the highest sensitivity for the detection of P. falciparum (including mixed infection) was obtained by Paracheck [98.8%, 95% confidence interval (CI) 95.8-99.9] and Falcivax (97.6%, 95% CI 94.1-99.4) compared to microscopy and real-time PCR respectively. Paracheck and Onsite Pf/Pv gave the highest specificity (98.8%, 95% CI 95.7-99.9) compared to microscopy and Onsite Pf/Pv (98.8, 95% CI 95.8-99.9) compared to real-time PCR respectively for the detection of P. falciparum. On the other hand Falcivax and Onsite Pf/Pv had equal sensitivity (90.5%, 95% CI 69.6-98.8) and almost 100% specificity compared to microscopy for the detection of P. vivax. However, compared to real-time PCR assay RDTs and microscopy gave low sensitivity (76.9%, 95% CI 56.4-91) in detecting of P. vivax although a very high specificity was obtained (99-100%).
CONCLUSION
The results of this study suggest that the SYBR Green-based real-time PCR assay could be used as an alternative gold standard method in a reference setting. Commercially-available RDTs used in the study are quite sensitive and specific in detecting P. falciparum, although their sensitivity in detecting P. vivax was not satisfactory compared to the real-time PCR assay.
背景
孟加拉国超过 95%的疟疾病例报告来自 13 个高度流行地区。在该国，疟原虫恶性疟原虫和间日疟原虫是两种最丰富的疟疾病原体。为了提高疟疾患者的检测和管理水平，国家疟疾控制规划（NMCP）一直在流行地区使用快速诊断检测（RDT）。本研究旨在建立一种基于 SYBR Green 的改良实时 PCR 检测方法作为金标准，以评估四种市售疟疾 RDT 的性能，同时评估传统金标准——显微镜检查。
方法
2009 年 5 月至 2010 年 8 月，从 MatirangaUpazila 卫生综合大楼（UHC）就诊的 338 例发热患者中采集血样，用于疟疾诊断。在 UHC 进行 Paracheck RDT 和显微镜检查。将血液样本保存在 EDTA 管中。进行基于 SYBR Green 的实时 PCR 检测，并进行评估。还使用储存的血液样本，根据显微镜检查和实时 PCR 评估其余三种 RDT（Falcivax、Onsite Pf 和 Onsite Pf/Pv）的性能。
结果
共纳入 338 例发热患者。显微镜检查和实时 PCR 分别检测到 189 例（55.9%）和 188 例（55.6%）疟疾病例。在 RDT 中，Paracheck [98.8%，95%置信区间（CI）95.8-99.9]和 Falcivax（97.6%，95% CI 94.1-99.4）对恶性疟原虫（包括混合感染）的检测灵敏度最高，与显微镜检查和实时 PCR 相比。Paracheck 和 Onsite Pf/Pv 的检测特异性最高（98.8%，95% CI 95.7-99.9），与显微镜检查相比，Paracheck 和 Onsite Pf/Pv 的检测特异性最高（98.8%，95% CI 95.7-99.9），与实时 PCR 相比，Onsite Pf/Pv 的检测特异性最高（98.8%，95% CI 95.8-99.9）。另一方面，Falcivax 和 Onsite Pf/Pv 的检测灵敏度相当（90.5%，95% CI 69.6-98.8），与显微镜检查相比，检测间日疟原虫的特异性几乎为 100%。然而，与实时 PCR 检测相比，RDT 和显微镜检查对间日疟原虫的检测灵敏度较低（76.9%，95% CI 56.4-91），尽管获得了非常高的特异性（99-100%）。
结论
本研究结果表明，基于 SYBR Green 的实时 PCR 检测方法可作为参考环境中的替代金标准方法。本研究中使用的市售 RDT 在检测恶性疟原虫方面非常敏感和特异，尽管与实时 PCR 检测相比，其检测间日疟原虫的灵敏度并不令人满意。
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