MALDI-TOF MS: optimization for future uses in entomological surveillance and identification of mosquitoes from New Caledonia

Abstract

Background: Mosquito vectors cause a significant human public health burden through the transmission of pathogens. Due to the expansion of international travel and trade, the dispersal of these mosquito vectors and the pathogens they carry is on the rise. Entomological surveillance is therefore required which relies on accurate mosquito species identification. This study aimed to optimize the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for mosquito identification. Methods: Aedes aegypti of the Bora-Bora strain and 11 field-sampled mosquito species were used in this study. Analyses were performed to study the impact of the trapping duration on mosquito identification with MALDI-TOF MS. The best preservation methods to use for short, medium and long-term preservation before MALDI-TOF MS analysis were also assessed. In addition, the number of specimens per species required for MALDI-TOF MS database creation was determined. The first MALDI-TOF database of New Caledonian mosquitoes was assembled and the optimal threshold for mosquito species identification according to the sensitivity and specificity of this technique was determined. Results: This study showed that the identification scores decreased as the trapping duration increased. High identification scores were obtained for mosquitoes preserved on silica gel and cotton at room temperature and those frozen at − 20 °C, even after two months of preservation. In addition, the results showed that the scores increased according to the number of main spectrum patterns (MSPs) used until they reached a plateau at 5 MSPs for Ae. aegypti. Mosquitoes (n = 67) belonging to 11 species were used to create the MALDI-TOF reference database. During blind test analysis, 96% of mosquitoes tested (n = 224) were correctly identified. Finally, based on MALDI-TOF MS sensitivity and specificity, the threshold value of 1.8 was retained for a secure identification score. Conclusions: MALDI-TOF MS allows accurate species identification with high sensitivity and specificity and is a promising tool in public health for mosquito vector surveillance.

New assessment of Anopheles vector species identification using MALDI-TOF MS

“…Indeed, with consideration of the highest scoring spectrum, the legs required the using of four replicates of protein extract whereas for the head, only one replicate provided almost equivalent results as using four replicates, at the LSV threshold of 1.7 and 1.8. These results are concordant with a recent study on mosquitoes [ 25 ] that observed that using the legs, the LSVs were improved when three spots of each sample were deposited onto the target plate, compared to the using of only one spot. Therefore, the using of the head will represent a concrete improvement of the routine use of MALDI-TOF for Anopheles identification, as it will allow to gain rapidity of analysis by decreasing the number of deposits.…”

“…As previously mentioned [16], the fragility of the legs, which are breakable and can be lost during collection, transportation, storage or processing, may lead to partial or total loss of the protein content. Indeed, a study showed that legs were prone to degradation during the trapping, with modification of protein profiles and a decrease of identification log(scores) as the trapping duration increased, even after 24 h of trapping [25]. Similarly, disparities have been observed in this study, between spectra from laboratory-reared specimens and from field-caught specimens for every anatomic parts.…”

“…Previous studies concluded that legs provided sufficient protein material to give reproducible and specific mass spectra [ 10 – 14 ]. However, a recent study reported that the using of less than four legs could compromise the MALDI-TOF MS identification of mosquito species, showing that at least four legs are required to get sufficient protein material [ 25 ]. In addition, one of the previous studies observed that the quality of legs spectra from field-caught Anopheles was lower than that from colony specimens, with a decreased intensity [ 14 ].…”

“…Similarly, a study reported that specimens both from the same Anopheles species and colony were split in different groups of a dendrogram [ 15 ]. Therefore, it is supposed that variability of mass spectra can also result from the method of storage or other experimental conditions such as trapping method or trapping duration [ 25 ], quality of protein extraction and homogenization [ 34 ]. In addition, even if we did not know precisely the age of the colonies, as there was no clear clustering of the spectra from colony specimens, it probably not has impacted the results.…”

“…Indeed, some specimens were dry frozen preserved and analysed several months or years later, whereas other specimens were stored at ambient temperature and analysed in a few weeks. However, as these various storage conditions have been shown to preserve the quality of spectra, the results were most likely only partially affected [ 8 , 25 ]. Another limitation is that only one field-caught species was tested in the panels, which was the dominant species An.…”