Study Sites
Flies were collected from four sites undergoing MDA with ivermectin and from a non-MDA site (Batouri health district) in Eastern Cameroon. The MDA sites included two Community Directed Treatment with Ivermectin (CDTI) Project sites in the South West (SW1 and SW2) as well as sites in Eastern (Messamena health district) and North West Cameroon (Nwa health district). The CDTI SW1 site operates within the Mungo and Meme drainage basins and SW2 operates in the Manyu drainage basin [26]. Each of these sites has a different ivermectin MDA treatment profile. SW1 (Kumba health district) and SW2 (Mamfe health district) situated in areas of L. loa mild endemicity, had been under CDTI for more than a decade (12–14 years) by the time of the study [27]. Whereas the Eastern and North West project sites situated in areas of high L. loa endemicity, had respectively, been under CDTI for 10 and 9 years prior to the study [28, 29]. A CDTI-naïve site (Batouri health district) in the East region was also surveyed. This site is known to be L. loa hyper-endemic from a previous study [30].
The climate in the southwest and northwest is tropical with two seasons, one wet season of about 9 months, lasting from March to November and a short dry season from mid-November to mid-March. The mean annual rainfall in these areas varies from 2,500 to 4,000 millimeters. Ambient temperature ranges from 20˚C to 40˚C depending on the seasons. The climate of the east region is a Type A wet equatorial climate [31], with an annual rainfall of 1500–2000 millimeters and average temperatures of about 24˚C with four seasons (a long dry season from December to May, a light wet season from May to June, a short dry season from July to October, and a heavy wet season from October to November)[29].
Figure 1: Study sites with fly collection points
Study design
To evaluate the performance and suitability of the LAMP assay as a surveillance tool, the study comprised two phases: one involving the use of experimentally-infected flies to determine sensitivity and a field phase using wild-caught insects.
Collection and laboratory maintenance of experimentally fed Chrysops flies
Chrysops flies were allowed to take blood from consenting microfilaraemic volunteers, caught by the human landing method using 50 mL Falcon tubes (Corning, USA). Each tube was prepared to provide suitable conditions for the survival and transport of a single fly as described by Wanji et al. [32]. For the experimental infections, two batches of 18 flies were each fed on either a microfilaraemic volunteer with a low (< 10 mf/ml) microfilarial load (Lot 1), or with high (> 30000 mf/ml of blood) microfilarial load (Lot 2). Once back in the laboratory, flies were maintained for up to 14 days to monitor larval development (time for the microfilariae to develop to L3 stage) in the insectarium. Within this period, flies were fed daily with sterile 15% sucrose solution. The temperature of the insectarium was maintained between 23–28 °C and the relative humidity between 79% − 80% as described by Tendongfor et al. [33]. Two flies from each lot were frozen at -20 ̊C on day 0 (< 7 hours post infection), 1, 4, 6, 7, 10, 11, 12 and 14 post infection. At the end of the experiment, the flies were separated into the head, thorax and abdomen, and DNA was extracted from each body part and subjected to the RF4 LAMP assay for detection of infection.
Field collection of wild Chrysops flies
Insect collections were performed, essentially as described [34], between 7 a.m. and 6 p.m. from the month of August to October 2016 for a period of 5days/community. Five trained collectors, dressed in thick, long-sleeved and long-legged clothing to avoid being bitten by the flies, were stationed near a wood fire. Blood-seeking female flies attracted by the smoke were caught when attempting to take a blood meal at the different study sites using sweep nets. The number of flies caught per hour was recorded. At the end of each session, wild-caught flies were then randomly separated into three groups. The first group served as a control group, as flies from this group were dissected to check for parity and a total of 138 nulliparous flies were retained to be further evaluated using the RF4 LAMP assay. This was in order to ascertain the specificity of the assay in the detection of L. loa parasite, so as to cancel any issues of confounding factors arising from the flies. The remaining two groups were evaluated using either conventional dissection and microscopy or stored in 80% alcohol for DNA extraction followed by LAMP to detect L. loa infection.
Dissection of wild Chrysops flies
Wild Chrysops were transported in a cold box to the field laboratory and dissected immediately after collection. After a slight knock down using a needle tip, flies were then dissected in physiological saline (0.9% NaCl) under a dissecting microscope. The head, thorax and abdomen of each fly was separated and placed on slides containing a drop of dissecting medium. The abdomen was teased gently to pull out the ovarioles and spread out to determine the presence (parous) or absence (nulliparous) of follicular relic on the pedicel as described by Duke [35]. Parous flies were further dissected for the presence or absence of L. loa larvae. Larvae were classified into sausage (L1), larval stage 2 (L2) and larval stage 3 or infective larvae (L3) following the methods of Duke, 1958 [36] and Orihel, 1975 [37]. The infection rates were generated as described by Duke [38] and Noireau et al. [39].
Purification of DNA from Chrysops flies
DNA was extracted using the Zymo Research Genomic DNA Tissue™ MiniPrep Kit, following manufacturers’ protocol. Briefly, Chrysops spp were crushed individually with the help of sterile micro pestles in eppendorf tubes containing 95 µL of water, 95 µL 2 X digestion buffer and 10 µL Proteinase K Solution. The mixtures were incubated at 55 °C in a water bath for 1–3 hours to denature the nucleases. Seven hundred microliters of genomic lysis buffer was added and the samples were mixed thoroughly using a vortex machine followed by a centrifugation step at 10000 g for 1 minute to remove insoluble debris. The individual supernatants were carefully transferred to Zymo-spin columns in different collection tubes and then centrifuged at 10,000 g for one minute. The columns were removed and inserted into 2 mL Collection Tubes, and 200 µL of DNA Pre-wash buffer was added to the different spin columns in the new collection tubes followed by centrifugation at 10,000 g for one minute. Four hundred microliters of genomic-DNA wash buffer was added to the spin columns and centrifuged at 10,000 g for one minute. The spin columns were later transferred into clean 1.5 mL eppendorf tubes. DNA in the spin columns were reconstituted in 200 µL of elution buffer, incubated for two to five minutes at room temperature. A centrifugation step was performed at maximum speed for thirty seconds and DNA finally stored at -20 °C until use.
LAMP Assay to detect L. loa
The L. loa LAMP primers (Poole et al., 2015), targeting the RF4 family repeat, used for the colorimetric assay were synthesized and HPLC purified by Integrated DNA Technologies (Iowa, USA). The primers used have the following sequence (5’-3’): F3-TCTTTCYTTTTATCGAGTCGTT, FIP-CGACGTCTTCACAAGGTAAGCC-GTTTAGCCTTGAGTTAGGATC, BIP-AGGACACAGAGTAAAATTTACCGCT-CGATTTYCTACTCGTTATTCTTCAA, B3-AACAGCYTTTGACTCACG, LF-TTAATTAAAGTTCTGCT, and LB-TACAGAGTTGATCAGTAGG.
LAMP reactions contained 1.6 µM each of primers FIP and BIP, 0.2 µM each of F3 and B3, 0.4 µM each of LF and LB, 12.5 µl of WarmStart Colorimetric LAMP 2X Master Mix (New England Biolabs Inc., USA) with 2 µl of template DNA, or molecular biology grade H20 for non-template controls (NTCs), in a total volume of 25 µl. Reactions were incubated at 61 °C for up to 40 minutes in a GeneAmp®, PCR System 9700 Thermal Cycler (Applied Biosystems, USA). A detailed method for reaction setup can be found in Additional file 1. Samples were considered positive for L. loa DNA if an obvious colour change from pink to yellow was observed, while negative samples remained pink (Fig. 2). Non template controls were included in each LAMP reaction. These controls never amplified.
Due to the high sensitivity of LAMP, precautions were taken to prevent cross-contamination in every experiment. DNA contamination and carry-over of amplified products was prevented by using filter tips at all times, cleaning all work surfaces with a 10% bleach solution before and after each session of work, performing each step of the analysis in separate work areas and minimizing manipulation of the reaction tubes. All tubes are tightly closed and never opened after amplification to avoid contaminating the work area.