Assessing Aedes mosquito larval indicators, dengue virus infection rates, and risk factors in Khyber Pakhtunkhwa: Insights for improved vector control strategies

Abstract

Background: Effective dengue management hinges on targeting key vector breeding sites and understanding transmission risks. Despite recurring outbreaks in Pakistan's Khyber Pakhtunkhwa (KP) Province since 2013, comprehensive entomological and virological data remain scarce. This study identified key larval-based indicators (habitats, Stegomyia indices), mosquito species composition, and dengue virus (DENV) infection rates in Aedes mosquitoes, evaluating their contributions to outbreak risk.

Methodology/principal findings: From July to December 2021, a cross-sectional larval survey of Aedes aegypti and Ae. albopictus was conducted across epidemiologically high-risk KP districts, inspecting water-holding containers located indoors, outdoors, and on rooftops. Additionally, adult mosquitoes were collected using aspirators and nets, with weekly dengue case data sourced from Peshawar's Directorate of Health Services. A subsample of 200 adult mosquito pools (20 per district) underwent RT-PCR to determine minimum infection rates (MIR). Larval indices revealed a House Index (HI) of 19.4%, a Container Index (CI) of 20.4%, and a Breteau Index (BI) of 89%. Aedes aegypti was the dominant species, accounting for 62% of larvae and 67.8% of adult mosquitoes. Peshawar (BI = 89.3), Nowshera (BI = 71.4), and Mardan (BI = 57) reported the highest Breteau indices and corresponding dengue case counts: 2,584 (48.8%), 404 (7.6%), and 327 (6.2%), respectively. The peak larval positivity was recorded in October (29.3%) and September (24.7%), aligning with dengue patient hospitalization rates of 52.8% and 46.8%, respectively. Common breeding sites included indoor flowerpots (25.4%), outdoor rubber tyres (16%), and roof tap water (23.7%). Container type and location significantly (P < 0.00) predicted larval abundance. Regression analysis revealed significant associations between dengue incidence, population density, and stegomyia indices. Of 38 positive pools (19%), DENV-2 and DENV-3 predominated (47.4% each), with peak MIRs recorded in Peshawar (30), Mardan (25), and Haripur (25).

Conclusions/significance: High larval indices and dual-serotype circulation in adaptable Aedes vectors signal substantial outbreak risk in KP. These findings underscore the need for targeted vector strategies, focusing on containers with the highest breeding potentials and epidemiological significance, particularly in high-transmission areas. Further molecular and entomological investigations are critical to corroborate these findings and inform more effective interventions.

Fig 1. Map of Khyber Pakhtunkhwa (KP), Pakistan, showing the sampled districts for Aedes mosquito collection.

The elevation gradient (meters) is represented, with lower elevations in blue and higher elevations in red. The mosquito icons indicate the districts where sampling was conducted. The inset map in the upper left highlights KP within Pakistan, while the bottom right inset zooms in on Rustam, a Union Council in the Mardan District. Data sources: DIVA-GIS (https://diva-gis.org/).

Fig 2. Summary of larval collection from multiple breeding sites/containers across KP, where the two letter subscripts represents: BT: Bucket, CB: Can & bottle, CD: Car wash dicks, CM: Cement basin, CS: Canals, DC: Disposable container, DT: Discarded Rubber tyres, EP: Earthen pot, FP: Flower pot, PB: Plastic bowls, RT: Rubber tyres, RW: Rooftop water, TW: Tube wells, WD: Water dispenser, WS: Water storing drums, WT: Water tanks.

The y-axis represents the log10-transformed counts of larvae per container type. The black points indicate data points, while the colored points show mean values with 95% confidence intervals based on bootstrapped standard errors.

Fig 3. The box plot representing cumulative distribution of Ae. aegypti and Ae. albopictus larvae across different container placement (outdoors, indoors, and roof tops), irrespective of container type.

The P values (at 5% level of significance) were computed using the Chi Square test statistic for independence. Notched Boxplots showing the significant differences in larval indices between A. aegypti and A. albopictus. The p-values represent the results of statistical tests comparing the two species, indicating inter-species differences rather than associations with external factors such as climatic conditions or container types. The boxes represent the interquartile range (IQR), the horizontal line inside each box represents the median, and the whiskers extend to the minimum and maximum values within 1.5 times the IQR. Outliers are shown as individual points. The notches represent approximate 95% confidence intervals for the medians, and the appearance of the lower quantile line being higher than the edges of the box is due to the notch design, not an error. The boxes represent the interquartile range (IQR), the horizontal line inside each box indicates the median, and the whiskers extend to the minimum and maximum values within 1.5 times the IQR.

Fig 4. Stegomyia indices across various districts in the province.

Fig 5. Maps showing the distribution of Ae. aegypti (panel a, left) and Ae. albopictus (panel b, right) (adults and larvae) across Khyber Pakhtunkhwa Province. Panel (c) illustrates dengue incidence per 100,000 population in the province.

The shapefile for these maps was sourced from DIVA-GIS (https://diva-gis.org/).