Department of International Health, University of Tampere Medical School, Tampere, Finland.
Malar J. 2010 Oct 6;9:269. doi: 10.1186/1475-2875-9-269.
New diagnostic tools for malaria are required owing to the changing epidemiology of malaria, particularly among pregnant women in sub-Saharan Africa. Real-time PCR assays targeting Plasmodium falciparum lactate dehydrogenase (pfldh) gene may facilitate the identification of a high proportion of pregnant women with a P. falciparum parasitaemia below the threshold of microscopy. These molecular methods will enable further studies on the effects of these submicroscopic infections on maternal health and birth outcomes.
The pfldh real-time PCR assay and conventional microscopy were compared for the detection of P. falciparum from dried blood spots and blood smears collected from the peripheral blood of 475 Malawian women at delivery. A cycle threshold (Ct) of the real-time PCR was determined optimizing the sensitivity and specificity of the pfldh PCR assay compared to microscopy. A real-time PCR species-specific assay was applied to identify the contribution to malaria infections of three Plasmodium species (P. falciparum P. ovale and P. malariae) in 44 discordant smear and pfldh PCR assay results.
Of the 475 women, P. falciparum was detected in 11 (2.3%) by microscopy and in 51 (10.7%) by real-time PCR; compared to microscopy, the sensitivity of real-time PCR was 90.9% and the specificity 91.2%. If a Ct value of 38 was used as a cut-off, specificity improved to 94.6% with no change in sensitivity. The real-time PCR species-specific assay detected P. falciparum alone in all but four samples: two samples were mixed infections with P. falciparum and P. malariae, one was a pure P. malariae infection and one was a pfldh PCR assay-positive/species-specific assay-negative sample. Of three P. malariae infections detected by microscopy, only one was confirmed by the species-specific assay.
Although microscopy remains the most appropriate method for clinical malaria diagnosis in field settings, molecular diagnostics such as real-time PCR offer a more reliable means to detect malaria parasites, particularly at low levels. Determination of the possible contribution of these submicroscopic infections to poor birth outcomes and maternal health is critical. For future studies to investigate these effects, this pfldh real-time PCR assay offers a reliable detection method.
由于疟疾的流行病学变化，特别是在撒哈拉以南非洲的孕妇中，需要新的诊断工具。针对恶性疟原虫乳酸脱氢酶（pfldh）基因的实时 PCR 检测可能有助于鉴定出大量疟原虫寄生血症低于显微镜检测阈值的孕妇。这些分子方法将使我们能够进一步研究这些亚微观感染对产妇健康和出生结局的影响。
比较了 pfldh 实时 PCR 检测和常规显微镜检查，以检测来自 475 名马拉维妇女分娩时外周血的干血斑和血涂片中的恶性疟原虫。通过优化 pfldh PCR 检测的灵敏度和特异性，确定了实时 PCR 的循环阈值（Ct）。应用实时 PCR 种特异性检测方法，鉴定了三种疟原虫（恶性疟原虫、卵形疟原虫和间日疟原虫）在 44 份与涂片和 pfldh PCR 检测结果不一致的样本中的疟原虫感染情况。
在 475 名妇女中，显微镜检查发现 11 名（2.3%）妇女感染恶性疟原虫，51 名（10.7%）妇女感染实时 PCR；与显微镜检查相比，实时 PCR 的灵敏度为 90.9%，特异性为 91.2%。如果将 Ct 值 38 作为截断值，特异性提高到 94.6%，而灵敏度不变。实时 PCR 种特异性检测方法仅在 4 个样本中检测到恶性疟原虫，其中 2 个样本为恶性疟原虫和间日疟原虫混合感染，1 个样本为间日疟原虫感染，1 个样本为 pfldh PCR 检测阳性/种特异性检测阴性样本。在显微镜检查发现的 3 例间日疟原虫感染中，只有 1 例被种特异性检测方法证实。
尽管显微镜检查仍然是现场环境中临床疟疾诊断的最适当方法，但实时 PCR 等分子诊断方法为检测疟原虫提供了更可靠的手段，特别是在低水平时。确定这些亚微观感染对不良出生结局和产妇健康的可能贡献至关重要。为了未来的研究来调查这些影响，这种 pfldh 实时 PCR 检测方法提供了一种可靠的检测方法。
