In the context of an increase in malaria cases in the first 8 months of 2023 compared to the same period of 2022 in three communes (Khanh Thuong, Khanh Thanh, and Son Thai) of Khanh Vinh district, Khanh Hoa province, the Institute of Malariology, Parasitology and Entomology Quy Nhon has enhanced its support in containment of the disease.

This includes monitoring malaria vectors in areas with increasing cases with the following objectives: Assess the malaria situation in Khanh Vinh district, Khanh Hoa province; Determine the composition and density of Anopheles mosquitoes at the surveyed sites; Evaluate the insecticide resistance status of malaria vectors; and Design the research: descriptive cross-sectional study and bioassays.

Anopheles mosquito collection methods

Adult mosquito collection will be conducted according to WHO Guidelines (WHO, 2013) and Technical handbook for malaria prevention of National Institute of Malariology, Parasitology and Entomology (NIMPE, 2011). Three different sampling methods will be applied concurrently at each selected site:

- Cow-baited tent (CBT): CBT will be conducted overnight from 6 p.m. to 6 a.m for 4 nights. An adult cow was loosely tied to a stake in the ground at the centre of a large tent (Coleman 13 × 15 ft. screened canopy tent). Notably, tent is raised off the ground (20 cm), so that mosquitoes attracted to the cow-bait are collected using manual aspirator.

- Human lading catch (HLC): HLC were conducted for 4 nights by investigators who collected mosquitoes landing o­n their exposed legs with a mouth aspirator and a flashlight. Collectors worked in 6-h shifts, switching out with other collectors at midnight. o­ne participant collected mosquitoes for six hours (18 pm to 24 pm) and switching out with other collectors to mosquito collection for six hours next time (0 am to 6 am). Collections were performed for 50 minutes every hour with a 10-minute break. During collection investigators sat o­n a 40 cm high stool and were protected from the elements by plastic-sheeting roofs. Mosquitoes were caught from the exposed legs and collected in paper cups covered with netting. Specimens collected will be labelled with information (location, method and time of collection) outside the paper cup (1 hour apart).

- CDC light trap: Traps will be hung indoor and outdoor overnight from 18:00 to 06:00h for 4 nights.

- All mosquitoes will be morphologically identified using identification keys (NIMPE 2008). After that, those species identified as malaria vectors (An. dirus, An. aconitus, An. minimus, An. maculatus) will be kept alive separated by species, in separate cages with humid conditions, until obtain sufficient numbers, when possible, for insecticide susceptibility bioassays.

- WHO susceptibility bioassays: The procedure used for adult bioassays followed the standard WHO protocol

Insecticide susceptibility bioassays (tube tests) will be performed o­n collected mosquitoes following WHO standard protocol (WHO 2018). Adult females will be exposed to lambda cyhalothrin 0,05%, and alpha-cypermethrin 30 mg/m2 or other insecticides. Mosquitoes exposed to untreated papers will be used as control.

Ideally, 120-150 adult female mosquitoes of each species are required to conduct a single set of WHO insecticide susceptibility tests; of these, 100 will be exposed to the insecticide that is being tested (four or five replicates each, with 20-25 mosquitoes). The remaining 50 mosquitoes will be served as "controls" (i.e. two replicates each of 20-25 mosquitoes); additional batches of mosquitoes will be required for each insecticide.Mosquito will be exposed for 60 minutes to estimate the knocked-down time (KDT).

The observed mortality = (Total number of dead mosquitoes/Total tested) x 100, will be recorded 24 hours after exposure. Tests will be replicated if there will be a sufficient number of collected specimens. According to WHO criteria, a mosquito population is considered resistant when the 24-hour mortality was < 90%, resistance is suspected when the mortality was between 90 and 97%, and the population is susceptible when the mortality was >= 98%.

Notes: Bioassay tests will be performed when there is a sufficient amount of mosquito to test for each insecticide. If the number of Anopheles mosquitoes is not enough, to obtain adult mosquitoes, larvae collected in the field will be reared and/or mosquitoes will be bred (F1 progeny) in the laboratory from wild-caught female until obtain sufficient number of mosquitoes for the insecticide susceptibility bioassay. For Aedes mosquitoes, testing of susceptibility to insecticides will be conducted in Laboratory of Entomology Department.

Results

Surveyed sites

The vector survey was conducted in the communes of Khanh Thanh, Son Thai, and Khanh Thuong of Khanh Vinh district, Khanh Hoa province. These communes situated to the west of Khanh Vinh district, Khanh Hoa province. According to the 2019 malaria epidemiological stratification results from the Ministry of Health, Khanh Thanh, Son Thai, and Khanh Thuong were all in malaria highly-endemic zone.

These surveyed communes are commonly characterized by a mixture of natural forests and cultivated forests. Currently, the cultivated forest area (mainly acacia tree cultivation) is o­n the rise, and the local habitat in these communes typically feature streams and almost year-round flowing water in major streams that lead to villages and farmhouses. Thanks to the regular water source, households in these areas cultivate fruit trees such as pomelo, durian, and oranges, as well as short-term crops like cassava, rice, corn, bananas, and engage in forestry activities by planting acacia trees o­n the hilly slopes.

The local residents frequently work and live in various farm houses located 3-15 km away from the town centers of different communes. Access to these farm house areas can be relatively challenging, often involving crossing streams like in Khanh Thanh and Son Thai commune, and traversing high mountainous terrain as in Khanh Thuong commune. During rainy days, even walking becomes difficult due to the swift-flowing streams, causing people to stay overnight and sometimes for several days in the farmhouses. Some local farmhouses are small but accommodate multiple household members who stay overnight to work o­n the fields and clear vegetation to prepare for acacia tree cultivation. Additionally, the current period is the peak acacia tree harvesting season, especially in Khanh Thuong commune, which has led to the formation of small, rudimentary cluster camps with makeshift canvas roofs and no walls. People living in these temporary camp clusters include both local residents from Khanh Thuong commune and others from neighboring communes who gather for exploiting and peeling acacia.

Many households live at the edge of the forest, and occasionally, the primary malaria vector An. dirus can be found in this area. Furthermore, some households reside in the farm areas year-round and engage in livestock and poultry farming o­n the farms, raising cows, pigs, and chickens. Particularly, the current summer season has led many families to bring their young children to the farms to facilitate farm work and childcare.

Habitats and cluster camps in Khanh Vinh district

With these favorable conditions, malaria-transmitting mosquitoes can breed and thrive year-round. Additionally, the economic activities and habits of the local population also favor the spread of malaria within the community, especially in farm areas and residential areas near forests.

Malaria situation in Khanh Vinh district

As of July 25, 2023, the entire Khanh Vinh district has recorded 57 malaria cases in 10 out of 14 communes and townships. The majority of these cases are concentrated in Khanh Thuong commune with 36 cases, accounting for 63.2% of the total malaria cases in the whole district. The communes with low malaria cases include Son Thai (5 cases), Khanh Trung (4 cases), Khanh Thanh (2 cases), Giang Ly (2 cases), Cau Ba (2 cases), Khanh Hiep (2 cases), Khanh Vinh town (1 case), Khanh Nam (2 cases), and Khanh Binh (1 case) (Figure 1).

Figure 1. Distribution of malaria cases from January to July 2023 in Khanh Vinh district

Table 1. Distribution of malaria cases by month in Khanh Thuong commune, 2018-2023

(Source: Statistics at Khanh Thuong commune health station)

In Khanh Thuong commune, during the period from 2018 to 2022, malaria cases were recorded in low numbers and were sporadically distributed throughout the year, particularly in 2021-2022 o­nly o­ne cases was reported. However, from January 1, 2023, to July 25, 2023, a dramatic increase of malaria patients was seen in Khanh Thuong commune with 36 confirmed cases. Remarkably, during the same period in 2022, there were no malaria cases reported.

In Khanh Thanh commune, from 2019 to 2022, no malaria cases were reported. However, from the beginning of the year to July 25, 2023, the commune has recorded 2 confirmed malaria cases. For Son Thai commune, there have been 3 confirmed malaria cases reported from the beginning of the year to July 25, 2023.

Malaria vector control in Khanh Vinh district

In 2022, the Khanh Vinh District Health Center implemented impregnation of bed-nets with insecticides in the communes. The results showed that 9,995 double bed nets were treated with a protected population of 19,989. The population coverage rate achieved 99.95% compared to the planned target. Specifically, in Khanh Thuong commune, 675 bed nets were treated with insecticides, covering a protected population of 1,350, reaching 100% of the planned target. However, from the beginning of 2023 until now, malaria vector control measures (as shown in Table 2) have not been implemented.

Table 2. Results of malaria vector control in Khanh Vinh district in 2022

The results of bed-nets and hammocks distribution for the communes in Khanh Vinh district in 2023 are presented in Table 3 as follows: Khanh Vinh district has distributed 21,800 bed-nets and 1,750 hammocks, providing protection for a total population of 45,350. Specifically, Khanh Thuong commune received 1,400 double bed-nets and 110 hammocks - hammock nets, with a protected population of 2,910. From the beginning of 2023 until now, Khanh Vinh district has not implemented any vector control activities.

Table 3. Results of bed-nets and hammocks allocation for the communes of Khanh Vinh district in 2022.

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