Evidence of intensification of pyrethroid resistance in the major malaria vectors in Kinshasa, Democratic Republic of Congo.

Assessing patterns and evolution of insecticide resistance in malaria vectors is a prerequisite to design suitable control strategies. Here, we characterised resistance profile in Anopheles gambiae and Anopheles funestus in Kinshasa and assess the level of aggravation by comparing to previous 2015 estimates. Both species collected in July 2021 were highly resistant to pyrethroids at 1×, 5× and 10× concentrations (mortality < 90%) and remain fully susceptible to bendiocarb and pirimiphos methyl. Compared to 2015, Partial recovery of susceptibility was observed in A. gambiae after PBO synergist assays for both permethrin and α-cypermethrin and total recovery of susceptibility was observed for deltamethrin in 2021. In addition, the efficacy of most bednets decreased significantly in 2021. Genotyping of resistance markers revealed a near fixation of the L1014-Kdr mutation (98.3%) in A. gambiae in 2021. The frequency of the 119F-GSTe2 resistant significantly increased between 2015 and 2021 (19.6% vs 33.3%; P = 0.02) in A. funestus. Transcriptomic analysis also revealed a significant increased expression (P < 0.001) of key cytochrome P450s in A. funestus notably CYP6P9a. The escalation of pyrethroid resistance observed in Anopheles populations from Kinshasa coupled with increased frequency/expression level of resistance genes highlights an urgent need to implement tools to improve malaria vector control.

Behavior of Adult Aedes aegypti and Aedes albopictus in Kinshasa, DRC, and the Implications for Control.

Yellow fever and chikungunya outbreaks-and a few dengue cases-have been reported in the Democratic Republic of the Congo (DRC) in recent years. However, little is known about the ecology and behavior of the adult disease vector species, Aedes aegypti and Aedes albopictus, in DRC. Preliminary studies showed important differences in Aedes behavior in DRC and Latin-American sites. Therefore, this study aimed to assess the host-seeking and resting behaviors of female Ae. aegypti and Ae. albopictus, and their densities in four communes of Kinshasa (Kalamu, Lingwala, Mont Ngafula and Ndjili). Two cross-sectional surveys were carried out, one in the dry season (July 2019) and one in the rainy season (February 2020). We used three different adult vector collection methods: BG-Sentinel 2, BG-GAT, and prokopack. Both Aedes species were clearly exophagic, exophilic, and sought breeding sites outdoors. The adult house index for Ae. aegypti exceeded 55% in all communes except Lingwala, where it was only 27%. The Adult Breteau Index (ABI) for Ae. aegypti was 190.77 mosquitoes per 100 houses inspected in the rainy season and 6.03 in the dry season. For Ae. albopictus, the ABI was 11.79 and 3.52 in the rainy and dry seasons, respectively. Aedes aegypti showed unimodal host-seeking activity between 6 h and 21 h. The exophagic and exophilic behaviors of both species point to the need to target adult mosquitoes outdoors when implementing vector control.

Comparative study of the effect of solvents on the efficacy of neonicotinoid insecticides against malaria vector populations across Africa.

BACKGROUND: New insecticides with a novel mode of action such as neonicotinoids have recently been recommended for public health by WHO. Resistance monitoring of such novel insecticides requires a robust protocol to monitor the development of resistance in natural populations. In this study, we comparatively used three different solvents to assess the susceptibility of malaria vectors to neonicotinoids across Africa. METHODS: Mosquitoes were collected from May to July 2021 from three agricultural settings in Cameroon (Njombe-Penja, Nkolondom, and Mangoum), the Democratic Republic of Congo (Ndjili-Brasserie), Ghana (Obuasi), and Uganda (Mayuge). Using the CDC bottle test, we compared the effect of three different solvents (ethanol, acetone, MERO) on the efficacy of neonicotinoids against Anopheles gambiae s.l. In addition, TaqMan assays were used to genotype key pyrethroid-resistant markers in An. gambiae and odds ratio based on Fisher exact test were used to evaluate potential cross-resistance between pyrethroids and clothianidin. RESULTS: Lower mortality was observed when using absolute ethanol or acetone alone as solvent for clothianidin (11.4‒51.9% mortality in Nkolondom, 31.7‒48.2% in Mangoum, 34.6‒56.1% in Mayuge, 39.4‒45.6% in Obuasi, 83.7‒89.3% in Congo and 71.1‒95.9% in Njombe pendja) compared to acetone + MERO for which 100% mortality were observed for all the populations. Similar observations were done for imidacloprid and acetamiprid. Synergist assays (PBO, DEM and DEF) with clothianidin revealed a significant increase of mortality suggesting that metabolic resistance mechanisms are contributing to the reduced susceptibility. A negative association was observed between the L1014F-kdr mutation and clothianidin resistance with a greater frequency of homozygote resistant mosquitoes among the dead than among survivors (OR = 0.5; P = 0.02). However, the I114T-GSTe2 was in contrast significantly associated with a greater ability to survive clothianidin with a higher frequency of homozygote resistant among survivors than other genotypes (OR = 2.10; P = 0.013). CONCLUSIONS: This study revealed a contrasted susceptibility pattern depending on the solvents with ethanol/acetone resulting to lower mortality, thus possibly overestimating resistance, whereas the MERO consistently showed a greater efficacy of neonicotinoids but it could prevent to detect early resistance development. Therefore, we recommend monitoring the susceptibility using both acetone alone and acetone + MERO (4 µg/ml for clothianidin) to capture the accurate resistance profile of the mosquito populations.

Monitoring the presence of trypanosomes' DNA - Including Trypanosoma brucei gambiense DNA - From the midguts of riverine Glossina trapped in the south east outskirts of Kinshasa City (Democratic Republic of Congo).

Even if the number of Human African Trypanosomiasis (HAT) cases from Kinshasa province in DRC is going towards elimination for the last decade, cases still occur in the periphery of the city. The diagnosis of 21 cases in the south periphery of Kinshasa, between 2015 and 2017 gives evidence of the existence of an active focus in this area. Here, we present the results of a punctual entomological survey that was realized in july 2014 in the outskirts of the southeast of Kinshasa. Using pyramidal traps, we caught tsetse flies during 2 days, dissecting the fresh ones for further molecular analysis. The average Apparent Density of flies per Trap and per Day was three with a maximum of 5.6 flies in Nganda PIO. Polymerase chain reaction analysis of the midguts provided evidence of a high prevalence (57.2%) of infected flies. Ninety three percent of the trypanosomes that were identified belonged to the Nanomonas species, but Trypanozoon trypanosomes were also present in 24% of the infected flies, including mixed infections with Nanomonas, including 3 flies carrying Trypanosoma brucei gambiense, the human pathogen of trypanosomiasis. These results show that at the time of the field's study there was an active reservoir of trypanosomes, closed to pigsties, knowing that pig is a potential animal reservoir. It also demonstrates that xenomonitoring using the entomological approach can be an efficient tool for monitoring sleeping sickness. Finally, results are discussed in the frame of WHO's HAT elimination project. Regarding Kinshasa, it points out the need of regular epidemiologic surveys.

Nationwide insecticide resistance status and biting behaviour of malaria vector species in the Democratic Republic of Congo.

BACKGROUND: Globally, the Democratic Republic of Congo (DRC) accounted for 9% of malaria cases and 10% of malaria deaths in 2015. As part of control efforts, more than 40 million long-lasting insecticidal nets (LLINs) were distributed between 2008 and 2013, resulting in 70% of households owning one or more LLINs in 2014. To optimize vector control efforts, it is critical to monitor vector behaviour and insecticide resistance trends. Entomological data was collected from eight sentinel sites throughout DRC between 2013 and 2016 in Kingasani, Mikalayi, Lodja, Kabondo, Katana, Kapolowe, Tshikaji and Kalemie. Mosquito species present, relative densities and biting times were monitored using human landing catches (HLC) conducted in eight houses, three times per year. HLC was conducted monthly in Lodja and Kapolowe during 2016 to assess seasonal dynamics. Laboratory data included resistance mechanism frequency and sporozoite rates. Insecticide susceptibility testing was conducted with commonly used insecticides including deltamethrin and permethrin. Synergist bioassays were conducted with PBO to determine the role of oxidases in permethrin resistance. RESULTS: In Lodja, monthly Anopheles gambiae s.l. biting rates were consistently high at > 10 bites/person/night indoors and outdoors. In Kapolowe, An. gambiae s.l. dominated during the rainy season, and Anopheles funestus s.l. during the dry season. In all sites, An. gambiae and An. funestus biting occurred mostly late at night. In Kapolowe, significant biting of both species started around 19:00, typically before householders use nets. Sporozoite rates were high, with a mean of 4.3% (95% CI 3.4-5.2) for An. gambiae and 3.3% (95% CI 1.3-5.3) for An. funestus. Anopheles gambiae were resistant to permethrin in six out of seven sites in 2016. In three sites, susceptibility to deltamethrin was observed despite high frequency permethrin resistance, indicating the presence of pyrethroid-specific resistance mechanisms. Pre-exposure to PBO increased absolute permethrin-associated mortality by 24%, indicating that resistance was partly due to metabolic mechanisms. The kdr-1014F mutation in An. gambiae was present at high frequency (> 70%) in three sites (Kabondo, Kingasani and Tshikaji), and lower frequency (< 20%) in two sites (Lodja and Kapolowe). CONCLUSION: The finding of widespread resistance to permethrin in DRC is concerning and alternative insecticides should be evaluated.

Characterization of sleeping sickness transmission sites in rural and periurban areas of Kinshasa (République Démocratique du Congo).

To characterize the potential transmission sites of sleeping sickness in Kinshasa, two entomologic surveys were carried out during the dry and the rainy seasons in rural and periurban areas of Kinshasa in 2005. About 610 pyramidal traps were set up, and 897 Glossina fuscipes quanzensis were captured. Environmental and biologic factors were reported, and relationships between these factors were evaluated using logistic regression and multiple correspondence analysis. The biologic factors (the presence of tsetse flies, human blood meals, and teneral flies) were progressively accumulated at each capture site to permit the characterization of the sleeping sickness transmission risk. The dry season was found to be a more favorable period for the disease transmission than the rainy season. Moreover, the landscapes characterized by the presence of argillaceous soils, raised ground cover with forest residues and rivers, were identified as types of environments with greater risk of sleeping sickness transmission. Pig breeding appeared as an important factor increasing the disease transmission. If vector control is continuously performed along rivers segments at high risk, the transmission of sleeping sickness in rural and periurban areas of Kinshasa will considerably decrease.

Human African trypanosomiasis transmission, Kinshasa, Democratic Republic of Congo.

To investigate the epidemiology of human African trypanosomiasis (sleeping sickness) in Kinshasa, Democratic Republic of Congo, 2 entomologic surveys were conducted in 2005. Trypanosoma brucei gambiense and human-blood meals were found in tsetse fly midguts, which suggested active disease transmission. Vector control should be used to improve human African trypanosomiasis control efforts.