A novel nested polymerase chain reaction assay targeting Plasmodium mitochondrial DNA in field‐collected Anopheles mosquitoes

Abstract

Sensitive techniques for the detection of Plasmodium (Aconoidasida: Plasmodiidae) sporozoites in field-collected malaria vectors are essential for the correct assessment of risk for malaria transmission. A real-time polymerase chain reaction (RT-PCR) protocol targeting Plasmodium mtDNA proved to be much more sensitive in detecting sporozoites in mosquitoes than the widely used enzyme-linked immunosorbent assay targeting Plasmodium circumsporozoite protein (CSP-ELISA). However, because of the relatively high costs associated with equipment and reagents, RT-PCRs are mostly used to assess the outcomes of experimental infections in the frame of research experiments, rather than in routine monitoring of mosquito infection in the field. The present authors developed a novel mtDNA-based nested PCR protocol, modified from a loop-mediated isothermal amplification (LAMP) assay for Plasmodium recognition in human blood samples, and compared its performance with that of routinely used CSP-ELISAs in field-collected Anopheles coluzzii (Diptera: Culicidae) samples. The nested PCR showed 1.4-fold higher sensitivity than the CSP-ELISA. However, nested PCR results obtained in two laboratories and in different replicates within the same laboratory were not 100% consistent, probably because the copy number of amplifiable Plasmodium mtDNA was close in some specimens to the threshold of nested PCR sensitivity. This implies that Plasmodium-positive specimens should be confirmed by a second nested PCR to avoid false positives. Overall, the results emphasize the need to use molecular approaches to obtain accurate estimates of the actual level of Plasmodium circulation within malaria vector populations.

Malaria surveillance from both ends: concurrent detection of Plasmodium falciparum in saliva and excreta harvested from Anopheles mosquitoes

“…Rapid diagnostic tests in dipstick format have also been developed [13, 14], with results comparable to those obtained by ELISA [15]. A variety of polymerase chain reaction (PCR) methods are available, with high sensitivity and versatility [16–18]. However, although certainly useful, these techniques have limitations.…”

Behavioural plasticity of Anopheles coluzzii and Anopheles arabiensis undermines LLIN community protective effect in a Sudanese-savannah village in Burkina Faso

“…gambiae s.l. in Goden (1.8<SR<12.1[43])and in the same geographical area before LLIN implementation (0.5<HBR<26.3[64,65]; 3<SR<10[64][65][66] in 6 villages in a radius of 40 km from Ouagadougou) and comparable to SR in Goden in 2011 (6.9%,[24]) and 2012 (6.6%,[25]). Notably, even though SRs were assessed by different approaches in different studies, results by recently developed rDNA-based TaqMan assays [33,67] do not significantly differ to those obtained by traditionally used CSP-ELISA[67].Data also shed some light on other entomological factors, which could have reduced LLINeffectiveness after 5-year implementation (i.e.…”

Behavioural plasticity of Anopheles coluzzii and Anopheles arabiensis undermines LLIN community protective effect in a Sudanese-savannah village in Burkina Faso

“…gambiae (s.l.) in Goden (1.8 < SR < 12.1 [43]) and in the same geographical area before LLIN implementation (0.5 < HBR < 26.3 [64,65]; 3 < SR < 10 [64-66] in 6 villages in a radius of 40 km from Ouagadougou) and comparable to SR in Goden in 2011 (6.9% [24]) and 2012 (6.6% [25]). Notably, even though SRs were assessed by different approaches in different studies, results by recently developed rDNA-based TaqMan assays [33,67] do not significantly differ to those obtained by traditionally used CSP-ELISA [67].…”

“…Similar infective rates were also confirmed in a subsequent entomological survey conducted in the same village in 2012 (SR = 6.6% in An. coluzzii [25]). These observations suggest that, despite LLINs having significantly reduced human/vector contact in holo-endemic areas such as Burkina Faso, they have not apparently led to a substantial reduction of mosquito infection rates.…”