A miniPCR-duplex Lateral Flow Dipstick Platform in Combination with the Microfluidic Device for Rapid and Visual Diagnosis of Lymphatic Filariae Infection

Abstract

Background: Lymphatic filariasis is a major neglected tropical disease that is a leading cause of permanent and long-term disability worldwide. Significant progress made by the Global Programme to Eliminate Lymphatic Filariasis (GPELF) has led to a substantial decrease in the levels of infection. Accordingly, access is now needed to a cost-efficient, non-laborious diagnostic assay(s) capable of detecting low levels of microfilariae and without the need for infrastructure and expensive equipment.Methods: We developed a diagnostic assay that combines a miniPCR with a duplex lateral flow dipstick (DLFD) assay. The design of the PCR primers was guided by sequence alignments of the HhaI and SspI genes of Brugia malayi and Wuchereria bancrofti, respectively. The limits of detection, accuracy, and cross-reactivity of the assay were evaluated, after which the assay was deployed to assess infection using blood samples obtained from Myanmar study participants living along the Thailand-Myanmar border, a region in Tak province endemic W. bancrofti. In addition, blood samples were provided by from Thais in Narathiwat province residing in an area endemic for brugian filariasis. We also combined a previously reported semi-automated microfluidic device with the miniPCR-DLFD to facilitate rapid detection and species identification of microfilariae in human blood.Results: The miniPCR-DLFD assay exhibited a detection limit of two microfilariae per mililiter blood sample, and cross-amplification was not observed with from other parasites. For field validation, microfilariae and DNA of W. bancrofti were detected from two (0.6%) and five (1.5%) out of 328 (100%) blood samples, respectively. For the Narathiwat samples, microfilariae and DNA of B. malayi were detected from one (0.46%) and two (0.9%) out of 216 (100%) blood samples, respectively. A rapid and visual of species identification was accurately obtained in all microfilariae entrapped by our previously developed microfluidic device.Conclusion: A miniPCR-DLFD platform alone or coupled with the microfluidic device provided rapid and visual diagnosis of lymphatic filariasis. The microfluidic device, the miniPCR, and the DLFD are all portable. Coupled with a portable microcentrifuge for DNA extraction, this is a promising platform for the diagnosis of lymphatic filariasis in point-of-collection settings with a modest cost (~USD $ 5) per sample.