Validation of Loop Mediated Isothermal Amplification for the detection of Loa loa infection in Chrysops spp in experimental and natural field conditions

Abstract

Background: The Mass Drug Administration of ivermectin for onchocerciasis control has contributed to a significant drop in Loa loa microfilaria loads in humans that led to reduction of the levels of infection in Chrysops vectors. Accurate parasite detection is essential for assessing loiasis transmission; as such a potential alternative or an indirect strategy to address the problem co-endemic loiasis presents to the LF and onchocerciasis elimination programme and also, further reflecting the true magnitude of the loiasis problem as excess human mortality has been reported to be associated with the disease. Although microscopy is the gold standard for detecting the infection, its sensitivity is compromised when the intensity of infection is low. The Loop-mediated isothermal amplification (LAMP) of parasite DNA is an alternative method for detecting infection which offers operational simplicity, rapidity and versatility of visual readout options. This study aimed at validating the Loa loa LAMP assay for the detection of infected Chrysops spp. under experimental and natural field conditions. Methods: Two sets of 18 flies were fed on volunteers with either a low (<10mf/ml) or high (>30,000mf/ml) microfilarial load. The fed flies were maintained under laboratory conditions for 14 days and then analysed with LAMP for the detection of L. loa infection. In addition, a total of 9,270 flies were collected from the North West, East, and South West regions of Cameroon using sweep nets and subjected to microscopy (7,841 flies) and LAMP (1,291 flies plus 138 nulliparous flies) analyses. Results: LAMP assay successfully detected parasites in Chrysops fed on both low and high microfilariaemic volunteers. Field validation and surveillance studies phase revealed LAMP-based infection rates ranging from 0.5 % to 31.6 %, with the lowest levels in the South West 2 and highest infection rates in South West 1. LAMP assay detected significantly higher infection rates than microscopy in 4 of 5 study sites. Conclusion: This study demonstrated the potential of LAMP as a simple surveillance tool, and is more sensitive than microscopy for the detection of experimental and natural L. loa infections in Chrysops vectors.