Intensity of resistance in Kinshasa Province before and after LLIN mass distribution using CDC bottle bioassay
Over the four periods of testing, a total of 15,200 An. gambiae s.l. were used for resistance intensity bioassays in Kinshasa Province. Resistance to permethrin and deltamethrin was found in all sites (Fig. 3). In June 2016, before LLIN distribution mean results for Kinshasa (mean of Kingasani, Kinkole, Kimpoko, Bu) showed that permethrin resistance intensity was high and the mean mortality rate was 43% at the 5 × concentration and 73% at the 10 × concentration. After the mass distribution of LLINs in December 2016 (mean results for February, June and October tests) the mean mortality rate in Kinshasa was 32% for 5 × and 60% with permethrin at the 10 × concentration. The mean resistance intensity to deltamethrin was also high before LLIN distribution (75% at 5 × and 94% mortality at 10 × concentration) but decreased after LLIN distribution to a mean of 95% and 99% mortality at 5 and 10X concentrations, respectively. In general, levels of resistance were lower for deltamethrin, compared to permethrin. However, there was considerable variation in the results by site (Table 1). The hypothesis was that resistance intensity to permethrin and deltamethrin would increase in Kinshasa following LLIN distribution, compared to the control site of Kasangulu. In the control site of Kasangulu, mortality in permethrin intensity tests decreased significantly in 2017 (indicating an increase in resistance). Resistance intensity was greater in Kinkole for permethrin (OR 11.49, p-value < 0.001) and deltamethrin (OR 22.00, p-value < 0.001) compared to Kasangulu post-LLIN distribution and also in Kimpoko for deltamethrin (OR 2.57, p-value < 0.001). However, in Kingasani the opposite trend was recorded with a significantly lower resistance intensity following LLIN distribution for permethrin (OR 0.11, p-value < 0.001) and deltamethrin (OR 0.21, p-value < 0.001) compared to the control site, while in Bu there was no significant change post-distribution for both insecticides.
Table 1
Odds ratios and p-values for permethrin and deltamethrin susceptibility bioassays conducted in and around Kinshasa, DRC, taking into account dose, site, evaluation period, and the change in the distribution site relative to the control site.
| Permethrin | | Deltamethrin |
| Odds Ratio | p-value | | Odds Ratio | p-value |
Dose | | | | | |
1x | reference | | | reference | |
2x | 3.44 | < 0.001 | | 2.49 | < 0.001 |
5x | 13.61 | < 0.001 | | 10.48 | < 0.001 |
10x | 79.97 | < 0.001 | | undefined | < 0.001 |
Site | | | | | |
Kasangulu | reference | | | reference | |
Bu | 0.23 | 0.003 | | 2.27 | 0.010 |
Kimpoko | 0.47 | 0.045 | | 0.42 | 0.008 |
Kingasani | 1.62 | 0.153 | | 5.75 | < 0.001 |
Kinkole | 0.02 | < 0.001 | | 0.07 | < 0.001 |
Evaluation period | | | | | |
Pre-distribution | reference | | | reference | |
Post-distribution | 0.49 | < 0.001 | | 1.09 | 0.568 |
Change in distribution site relative to the control site (Kasangulu) | |
Bu | 0.70 | 0.099 | | 0.66 | 0.072 |
Kimpoko | 0.95 | 0.791 | | 2.57 | < 0.001 |
Kingasani | 0.11 | < 0.001 | | 0.21 | < 0.001 |
Kinkole | 11.49 | < 0.001 | | 22.00 | < 0.001 |
Intensity of permethrin and deltamethrin resistance in six sites in DRC in 2017 using CDC bottle bioassay
Nationwide bioassay testing showed that permethrin resistance was present in all 6 sites, with less than 10% mortality at the diagnostic dose. Mortality rates increased slightly with increased concentration, but high intensity permethrin resistance was present in all sites, with considerably less than 98% mortality at 10x the diagnostic concentration of permethrin (Fig. 4).
An. gambiae s.l. populations were less intensely resistant to deltamethrin, although all populations tested were resistant at 1x and 2x the diagnostic dose. The resistance intensity was low (> 98% mortality at 5x dose) in Kabondo and Inongo, moderate in Kalemie and Katana (> 98% mortality at 10x dose) and high (< 98% mortality at 10x dose) in Mikalayi and Kapolowe (Fig. 5).
Intensity of permethrin, deltamethrin and alpha-cypermethrin resistance in eleven sites in 2018 in DRC using the WHO tube test
In 2018, nationwide WHO insecticide susceptibility and resistance intensity tests were completed with An. gambiae s.l. populations in 11 sites. The data is presented in Figs. 6, 7, and 8 for permethrin, deltamethrin and alpha-cypermethrin, respectively. In Kabondo, testing with alpha-cypermethrin 5 × and 10 × was not completed as mortality was > 20% in the control and the field team was unable to find sufficient larvae for repeat tests. Resistance to permethrin (< 90% mortality) was observed in all sites at the diagnostic dose (1×), except Katana, where there was possible resistance (90–98% mortality). Resistance intensity was low in Katana, Inongo, and Kapolowe; moderate (> 98% mortality at 5x dose) in Karawa, Kimpese, Mikalayi, and Pawa; and high (< 98% mortality at 10x dose) in Kingasani, Lodja, and Kalemie (Fig. 6).
Resistance to deltamethrin was recorded in all sites, except Katana (possible resistance),Lodja and Inongo (susceptible). The intensity of resistance was low in Kapolowe and Kabondo, moderate in Mikalayi, and high in Kingasani, Karawa, Kimpese, Kalemie, and Pawa (Fig. 6).
Resistance to alpha-cypermethrin was also observed in all sites. The intensity was low in Kalemie and Kapolowe, high in Katana, Mikalayi, and Lodja, and moderate in the remaining five sites (Fig. 7).
Species identification within the An. gambiae s.l. complex and frequency of VGSC-1014F and 1014S resistance alleles
Kinshasa resistance intensity study post-LLIN distribution (2017)
A total of 217 An. gambiae s.l. were tested for species identification and of those samples, 189 were analyzedforVGSC-1014F and 1014S frequency. The primary species found was An. gambiae s.s. (91%). An. coluzzii were only identified in Kimpoko (9%) and Kinkole (32%). Hybrid An. gambiae s.s./An. coluzzii were only found in Kinkole (2/44) (Table 2). One percent of samples did not amplify.
Table 2
Species identification within the complex An. gambiae s.l.from study sites in the province of Kinshasa
| An. gambiae speciesn(%) | |
Site | An. gambiaes.s. | An. coluzzii | Hybrid | Total |
Bu | 32 (100%) | 0 | 0 | 32 (100%) |
Kasangulu | 46 (100%) | 0 | 0 | 46 (100%) |
Kimpoko | 30 (91%) | 3 (9%) | 0 | 33 (100%) |
Kingasani | 62 (100%) | 0 | 0 | 62 (100%) |
Kinkole | 28 (64%) | 14 (32%) | 2 (5%) | 44 (100%) |
Total | 198 (91%) | 17 (8%) | 2 (1%) | 217 (100%) |
Overall, both An. gambiae s.s. and An. coluzzii carried VGSC-1014F and 1014S resistance alleles, albeit at different frequencies. The mean frequency of resistance alleles determined across all sites for An. gambiae s.s. was 83% for homozygote VGSC-1014F, 3% for homozygote 1014S and 14% for heterozygous 1014F/1014S (Table 3). All VGSC-1014F frequencies were over 70% for An. gambiae s.s. and the highest was observed in Kingasani and Kinkole (both 91%). The trend was different for An. coluzzii, with 94% (16/17) being homozygous for the VGSC-1014S allele.
Table 3
Kdr L1014F and L1014S(n(proportion))resistance alleles from the study sites in the province of Kinshasa
Anopheles species | Site | Homozygous kdr-west (L1014F/L1014F) | Homozygous kdr-east (L1014S/L1014S) | Heterozygous kdr-west/kdr-east (L1014F/L1014S) |
Anopheles gambiae s.s. | Kasangulu | 28 (0.70) | 3 (0.08) | 9 (0.23) |
Kingasani | 52 (0.91) | 1 (0.02) | 4 (0.07) |
Kinkole | 21 (0.91) | 1 (0.04) | 1 (0.04) |
Kimpoko | 18 (0.86) | 0 (0.00) | 3 (0.14) |
Bu | 21 (0.78) | 0 (0.00) | 6 (0.22) |
Total An. gambiaes.s. | All sites | 140 (0.83) | 5 (0.03) | 23 (0.14) |
An. coluzzii | Kinkole | 0 (0.00) | 14 (1.00) | 0 (0.00) |
Kimpoko | 1 (0.33) | 2 (0.67) | 0 (0.00) |
TotalAn. coluzzii | All sites | 1 (0.06) | 16 (0.94) | 0 (0.00) |
Anopheles gambiae s.s./An. coluzzii | Kinkole | 0 (0.00) | 0 (0.00) | 2 (1.00) |
An. gambiae s.l. Overall | 141 (0.75) | 21 (0.11) | 25 (0.13) |
Resistance intensity survey covering 11 sites nationwide (2018)
A total of 998/1100 (91%) An. gambiae s.l. were successfully amplified for species identification and 862/1100 (78%) forL1014F. Overall, An. gambiae s.s. (98.5%) was the primary species in all 11 sites. An. coluzzii were only found in Kingasani (1%) and Mikalayi (1%) and 1% of hybrid An. gambiae s.s./An. coluzzii were found in Kingasani (Table 4). TheL1014F frequency for An. gambiae s.l. recorded over eleven sites in 2018 varied between 0.85 (Kingasani) and 1.0 (Pawa and Karawa). The mean L1014F wasclose to fixation at 0.98 (Table 5). Evidence for significant deviations from Hardy-Weinberg equilibrium were observed for L1014F in Kingasani, Kalemie, Kabondo and Katana (Table 5).
Table 4
Species identification within the complex An. gambiae s.l.over eleven study sites in 2018
| An. gambiae speciesn(%) | | |
Site | An. gambiaes.s. | An. coluzzii | Hybrid | Total | Did not amplify |
Lodja | 65 (65%) | 0 | 0 | 100 | 35 (35%) |
Kapolowe | 88 (88%) | 0 | 0 | 100 | 12 (12%) |
Kingasani | 82 (82%) | 4 (4%) | 3 (3%) | 100 | 11 (11%) |
Mikalayi | 53 (53%) | 8 (8%) | 0 | 100 | 39 (39%) |
Kalemie | 96 (96%) | 0 | 0 | 100 | 4 (4%) |
Kimpese | 99 (99%) | 0 | 0 | 100 | 1 (1%) |
Pawa | 100 (100%) | 0 | 0 | 100 | 0 |
Karawa | 100 (100%) | 0 | 0 | 100 | 0 |
Inongo | 100 (100%) | 0 | 0 | 100 | 0 |
Kabondo | 100 (100%) | 0 | 0 | 100 | 0 |
Katana | 100 (100%) | 0 | 0 | 100 | 0 |
Overall | 983 (89%) | 12 (1%) | 3 (1%) | 1100 | 102 (9%) |
Table 5
Kdr L1014F resistance alleles over eleven study sites in 2018
Site | Number tested | RR | RS | SS | Did not amplify | Frequency 1014F | χ2 | p-value |
Lodja | 100 | 98 | 1 | 0 | 1 | 0.99 | 0.0026 | 0.96 |
Kapolowe | 100 | 98 | 1 | 0 | 1 | 0.99 | 0.0026 | 0.96 |
Kingasani | 100 | 64 | 0 | 11 | 25 | 0.85 | 61 | < 0.000 |
Mikalayi | 39 | 31 | 0 | 4 | 4 | 0.89 | - | - |
Kalemie | 100 | 90 | 0 | 3 | 7 | 0.97 | 93 | < 0.000 |
Kimpese | 100 | 96 | 1 | 0 | 3 | 0.99 | 0.0026 | 0.96 |
Pawa | 100 | 86 | 0 | 0 | 14 | 1 | - | - |
Karawa | 100 | 95 | 0 | 0 | 5 | 1 | - | - |
Inongo | 100 | 95 | 3 | 0 | 2 | 0.98 | 0.024 | 0.88 |
Kabondo | 100 | 50 | 0 | 1 | 49 | 0.98 | 51 | < 0.000 |
Katana | 100 | 80 | 0 | 3 | 17 | 0.96 | 83 | < 0.000 |
Overall | 1,039 | 883 | 6 | 22 | 128 | 0.97 | | |
RR means homozygote resistant; RS means heterozygote resistant and SS means homozygote susceptible |