Abstract
The filarial parasite Loa loa, the causative agent of loiasis, is endemic in Central and Western Africa infecting 3–13 million people. L. loa has been associated with fatal encephalopathic reactions in high Loa-infected individuals receiving ivermectin during mass drug administration programs for the control of onchocerciasis and lymphatic filariasis. In endemic areas, the only diagnostic method routinely used is the microscopic examination of mid-day blood samples by thick blood film. Improved methods for detection of L. loa are needed in endemic regions with limited resources, where delayed diagnosis results in high mortality. We have investigated the use of a loop-mediated isothermal amplification (LAMP) assay to facilitate rapid, inexpensive, molecular diagnosis of loiasis. Primers for LAMP were designed from a species-specific repetitive DNA sequence from L. loa retrieved from GenBank. Genomic DNA of a L. loa adult worm was used to optimize the LAMP conditions using a thermocycler or a conventional heating block. Amplification of DNA in the LAMP mixture was visually inspected for turbidity as well as addition of fluorescent dye. LAMP specificity was evaluated using DNA from other parasites; sensitivity was evaluated using DNA from L. loa 10-fold serially diluted. Simulated human blood samples spiked with DNA from L. loa were also tested for sensitivity. Upon addition of fluorescent dye, all positive reactions turned green while the negative controls remained orange under ambient light. After electrophoresis on agarose gels, a ladder of multiple bands of different sizes could be observed in positive samples. The detection limit of the assay was found to be as little as 0.5 ag of L. loa genomic DNA when using a heating block. We have designed, for the first time, a highly sensitive LAMP assay for the detection of L. loa which is potentially adaptable for field diagnosis and disease surveillance in loiasis-endemic areas.
Highlights
Loiasis is a neglected tropical disease caused by the nematode Loa loa, an endemic filarial parasite transmitted by Tabanid flies of the genus Chrysops in the forest and savannah regions of Central and Western Africa infecting between 3 and 13 million people
A 839 bp species-specific repetitive DNA sequence from L. loa [10] was selected as a target for a loop-mediated isothermal amplification (LAMP)-based method to detect purified L. loa DNA
To determine the specificity of L. loa LAMP assay, reactions were conducted for amplification of L. loa genomic DNA including heterogeneous DNA from other parasites
Summary
Loiasis is a neglected tropical disease caused by the nematode Loa loa, an endemic filarial parasite transmitted by Tabanid flies of the genus Chrysops in the forest and savannah regions of Central and Western Africa infecting between 3 and 13 million people. L. loa has come increasingly to light as it has been associated with severe adverse reactions (including fatal encephalopathies) in high Loa-infected individuals receiving ivermectin during mass drug administration programs for the control of onchocerciasis and lymphatic filariasis. This had a negative impact on control of these two diseases in several areas of co-endemicity with loiasis [2,3]. A greatest risk for Loa-related post-ivermectin encephalopathy has been reported to appear with microfilaremia above 8,000 organism/mL [4,5] In this context, it is necessary the identification of individuals with high-level L. loa microfilaremia in order to prevent the interruption of mass drug administration programs in potentially loiasis endemic areas where ivermectin treatment was initially planned
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