BACKGROUND & OBJECTIVES Submicroscopic malaria infections with low parasite density serve as a silent reservoir for maintaining residual transmission in the population. These infections should be identified and targeted to be eliminated for sustained malaria control. The conventional methods of diagnosis such as light microscopy and rapid diagnostic kits often fail to detect low density infections. Therefore, the more sensitive molecular techniques should be employed to detect low density infections. The objectives of the study was to explore the prevalence of sub-microscopic infections in low transmission areas of Punjab using highly sensitive molecular tool. METHODS A total of 1114 finger prick blood samples were collected through active surveillance and tested for malaria diagnosis using light microscopy, RDT and PCR. Nested PCR amplification was performed using a pair of Plasmodium genus-specific primers from the 18S rRNA small subunit gene (18S rRNA). The amplified PCR products were analysed using a 2% agarose gel, stained with ethidium bromide and observed under transilluminator. RESULTS Test positive rate (TPR) by microscopy, RDT and PCR was 4.4, 3.95 and 5.75%, respectively. Microscopy and RDT failed to detect mixed infections whereas 0.26% cases were found to be mixed infection in PCR. Compared to LM and RDT, PCR has detected 1.3% additional positive cases. However, of the total positive cases detected by PCR, 23.4% infections were found to be submicroscopic, which could not be detected by conventional methods of diagnosis. INTERPRETATION & CONCLUSIONS The molecular study revealed the existence of submicroscopic malaria cases in the study population which would have remained undetected by conventional methods of diagnosis. This is particularly important because Punjab state is in malaria elimination phase and targeted to achieve elimination in 2021. However, such undetected parasite positive cases may pose bigger problem any time due to continued transmission. Therefore, application of more sensitive diagnostic tools like PCR and LAMP with conventional methods may be much more useful in case detection particularly in low transmission settings for malaria elimination.

中文翻译：

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旁遮普省低传播状态下亚显微疟疾的流行：消除疟疾的潜在威胁。

背景和目标 具有低寄生虫密度的亚显微疟疾感染可作为维持人群中残留传播的无声水库。应确定这些感染并有针对性地消除这些感染，以实现持续的疟疾控制。传统的诊断方法，如光学显微镜和快速诊断试剂盒，通常无法检测到低密度感染。因此，应采用更灵敏的分子技术来检测低密度感染。该研究的目的是使用高度敏感的分子工具探索旁遮普省低传播地区亚微观感染的流行情况。方法通过主动监测收集1114份手指采血样本，采用光学显微镜、RDT和PCR检测疟疾诊断。使用一对来自 18S rRNA 小亚基基因 (18S rRNA) 的疟原虫属特异性引物进行嵌套 PCR 扩增。使用 2% 琼脂糖凝胶分析扩增的 PCR 产物，用溴化乙锭染色并在透射仪下观察。结果显微镜、RDT和PCR检测阳性率（TPR）分别为4.4%、3.95%和5.75%。显微镜和 RDT 未能检测到混合感染，而 PCR 发现 0.26% 的病例为混合感染。与 LM 和 RDT 相比，PCR 检测到的阳性病例增加了 1.3%。然而，在PCR检测到的全部阳性病例中，发现23.4%的感染是亚微观的，这是常规诊断方法无法检测到的。解释 ＆结论 分子研究揭示了研究人群中存在亚显微疟疾病例，而传统的诊断方法无法检测到这些病例。这一点尤为重要，因为旁遮普邦正处于疟疾消除阶段，目标是在 2021 年实现消除疟疾。但是，由于持续传播，这种未被发现的寄生虫阳性病例随时可能构成更大的问题。因此，在病例检测中应用更敏感的诊断工具（如 PCR 和 LAMP）可能更有用，特别是在消除疟疾的低传播环境中。由于持续传播，这种未被发现的寄生虫阳性病例随时可能造成更大的问题。因此，在病例检测中应用更敏感的诊断工具（如 PCR 和 LAMP）可能更有用，特别是在消除疟疾的低传播环境中。由于持续传播，这种未被发现的寄生虫阳性病例随时可能造成更大的问题。因此，在病例检测中应用更敏感的诊断工具（如 PCR 和 LAMP）可能更有用，特别是在消除疟疾的低传播环境中。