Molecular surveillance leads to the first detection of Anopheles stephensi in Kenya

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Molecular surveillance leads to the first detection of Anopheles stephensi in Kenya

https://doi.org/10.21203/rs.3.rs-2498485/v1

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Anopheles stephensi is an invasive malaria vector that is endemic to south Asia and the Arabian Peninsula. It was recently reported in the Horn of Africa countries including Djibouti (2012), Ethiopia, Sudan (2019), Somalia (2019) and most recently Nigeria (2020). This mosquito is a competent vector for both Plasmodium falciparum and P. vivax. It is characterized by a high degree of behavioral plasticity and the ability to reproduce in various types of breeding sites including containers and therefore has the potential to propagate malaria transmission in rapidly urbanizing settings with poor drainage and disposal of waste containers. The World Health Organization (WHO) has called on all countries to scale up surveillance efforts to detect and report invasion by this vector and institute appropriate and effective control mechanisms. In Kenya, the Division for National Malaria Program (DNMP) and its partners have been conducting entomological surveillance in all coastal and northern counties that are suspected to be at risk of An. stephensi invasion as well as in all counties at risk of malaria. These efforts were supported by molecular surveillance of all unidentified Anopheles mosquitoes from other studies conducted by the Kenya Medical Research Institute (KEMRI) to try and identify An. stephensi. In this article, we report the first detection of An. stephensi in two sub counties of Marsabit County, Kenya in December 2022. We used Polymerase Chain Reaction (PCR) as the primary method of identification and confirmed results using morphological keys and sequencing of the ITS2 region. With the detection of this vector in Kenya, there is an urgent need for intensified surveillance to determine its occurrence and distribution and develop tailored approaches towards control to prevent further spread.

Health sciences/Diseases

Health sciences/Diseases/Infectious diseases/Malaria

Anopheles stephensi is a major vector of malaria in south Asia, Middle East and southern China where it is endemic and is known to transmit both Plasmodium falciparum and as P. vivax. This vector differs from other malaria vectors due to its ability to develop in man-made containers, preferably in clean water, but also in contaminated water. These traits have enabled An. stephensi to colonize urban settings where it can sustain transmission of malaria. Additionally, it is an efficient vector in rural areas. Anopheles stephensi was first reported in the Horn of Africa in Djibouti in 2012 [1]. Since then, it has now been reported in multiple urban and rural settings in Ethiopia, Sudan and Somalia [2–4] and could be responsible for sustaining malaria transmission in Ethiopia with the potential to increase P. falciparum incidence by 50% according to recent mathematical modelling of the potential impact of An. stephensi [5, 6] as has been observed in Djibouti [7].

There is a risk of An. stephensi spreading further south and west from its original foci of detection in the Horn of Africa region, as has been observed in Nigeria (2020) [3]. Importantly, this vector has the potential to establish or increase transmission in urban settings where the malaria burden is generally lower compared to rural settings, particularly where there are poorly planned drainage and waste disposal systems, creating conducive larval habitats [8]. The adult mosquito behaviour is poorly understood, especially as it colonizes new areas in Africa. WHO recently called for heightened surveillance and the development of response strategies to limit the spread of the vector[3]. This document highlights five key areas of focus; increased collaboration across sectors and borders, strengthening surveillance, improving information exchange, developing national guidelines, and prioritizing research to evaluate tools against this vector.

In Kenya, the Division for National Malaria Program (DNMP) at the Ministry of Health and its partners have been on high alert and instituted surveillance efforts following WHO’s initiative[3]. Surveillance efforts have been focused along the Kenyan coast and the northern counties bordering Sudan and Ethiopia. The current surveillance efforts are aimed at the collection of both larval and adult mosquito samples. Samples collected are identified using morphological keys and further by PCR at the reference laboratories located at the Kenya Medical Research Institute (KEMRI). Here we report the process leading to the first detection and identification of An. stephensi in Kenya.

Surveillance sites

Mosquitoes were collected in 14 Counties in December 2022 as part of a routine surveillance exercise conducted by the DNMP and its partners. The following counties where DNMP supported vector surveillance in December 2022 are categorized as malaria endemic (Kilifi, Taita Taveta), highland epidemic prone (Elgeyo Marakwet, West Pokot, Kisii, Nandi), low risk (Garissa, Makueni, Kajiado, Kirinyaga and Laikipia), or seasonal (Marsabit, Baringo and Turkana) (Fig. 1). For the purpose of this work, we shall only describe and present results for Marsabit County, where samples were collected, identified and confirmed to be An. stephensi. Marsabit County is in the northern part of Kenya and borders Ethiopia to the North, Turkana County to the west, Samburu County to the South and Wajir and Isiolo Counties to the East. The county lies at between 300m and 900m above sea level. The major economic activities are livestock rearing and cross-border trade. Sampling was conducted in Laisamis and Saku sub-counties of Marsabit County and focused on urban and rural settings along the northern transport corridor connecting Kenya and Ethiopia (Table 1). The main economic activities of the rural population is nomadic pastoralism due to the mostly semi-arid terrain while urban trade centers are set up the northern transport corridor and was the focus of the sampling efforts.

Table 1

Characteristics of the habitats from which larvae were collected
County	Subcounty	Locality Village	Habitat Type	Larvae presence	Latitude	Longitude	Elevation
Marsabit	Saku	Mountain	Tank	Yes	2.329833	37.996728	1319.14
Marsabit	Saku	Nagayo	Tyre	Yes	2.337666	37.990045	1353.7
Marsabit	Saku	Larare	Stream bed	No	2.227091	37.938076	1057.33
Marsabit	Laisamis	Malgis	River bed	Yes	1.832333	37.86083	453.76
Marsabit	Laisamis	Laisamis	Animal drinking points	Yes	1.587676	37.80819	541.84

Sampling: Mosquito sampling was conducted for both adult and larval samples. Adults were collected using CDC light traps while larvae were collected by dipping. CDC light traps were set overnight from 1800 to 1900hrs and collected the next morning between 0700-0800hrs. Additionally, the teams dipped for larvae in animal watering pens, containers, tyres, and other water bodies in the area (Fig. 2). Anopheles larvae were collected and placed in whirlpacks for transportation to the entomology laboratory at KEMRI in Kisumu for additional assays. The mosquitoes were reared in the infection room, with a triple door and curtains at the entrance and sealed windows to prevent any escapees. Surviving larvae were reared to adults for morphological identification using standard conditions (25 ± 2°C; 80% ± 4% relative humidity with a 12 h: 12 h light/dark cycle). Larvae were fed on Tetramin baby fish food and brewer’s yeast daily and adults maintained on 10% sugar solution.

Molecular characterization

DNA from 55 either whole or legs and wings of the mosquito samples was isolated using the ethanol precipitation method [9]. Amplification was carried out using an endpoint PCR assay previously described in Singh and others alongside a positive control using 0.15ul of the DNA template [10]. Thereafter, 15 µl of each of the PCR products was run on 2% agarose gel alongside 3 µl of a 100bp DNA ladder for size comparison. The products were visualized in the gel documentation system for an expected amplicon size of approximately 438bp. This was used as the primary method of identification given the relative inexperience of the laboratory teams in morphological identification of An. stephensi.

Morphological identification

Emerging adults were taxonomically identified using the keys described by Coetzee [11] to detect the distinct banding on the maxillary palps, pale scales on the scutum, and the 3 dark spots on wing vein 1A.

Sequencing

A sample of four adult specimens identified as An. stephensi by morphology were randomly selected and shipped to the US Centers for Disease Control and Prevention (CDC), Atlanta, USA where DNA from a single mosquito leg was extracted using the Extracta DNA Prep for PCR ®kit (Quantabio Beverly, MA) and Sanger sequencing was conducted using ITS2 primers[12]. Thereafter, Basic Local Alignment Search Tool (BLAST) homology searches of sequences was done using the default parameters on the sequences to confirm what species they matched to. Additionally, the phylogeny of ITS2 sequences from An. stephensi isolates from Kenya and other countries was performed using the construct/Test Maximum likelihood Tree approach on MEGA software. We used the default phylogeny reconstruction parameters apart from the number of bootstrap replications which was set to 100.

Anopheles larvae were collected from the sources illustrated in Table 1 in 4 of the 5 villages the team visited. A total of 59 larvae were collected. Eleven died in transit and were immediately prepared for PCR identification using the An. stephensi protocol, 7 of which were confirmed as An. stephensi (Table 2). The remaining larvae were pooled by sub-county for rearing.

Table 2

Numbers of mosquitoes sampled as larvae and either used for PCR or reared to adults then used for PCR. Unamplified samples are those that failed to amplify with the An. stephensi, An. gambiae and An. funestus PCR protocols
		Larvae used for PCR			Adults used for PCR
Sub-County	Total larvae collected	Sample size	An. stephensi	Unamplified	Sample size	An. stephensi	Unamplified
Saku	42	9	6	3	33	8	25
Laisamis	17	2	1	1	15	8	7
Total	59	11	7	4	48*	16	32
* Four adults were shipped to CDC for sequencing.

The 48 remaining larvae were used to rear adult samples. Of the first 11 samples that emerged, 9 adults were identified by morphology (Fig. 3). The team correctly identified 7 of the 9 samples as An. stephensi which were later confirmed by PCR (Table 2), the other two were identified as An. gambiae by morphology but confirmed to be An. stephensi by PCR. Four of these samples were shipped to the US Centers for Disease Control and Prevention (CDC) for sequencing. Thirty-six samples did not amplify using An. stephensi, An. gambiae or An. funestus PCR protocols and are the subject of further investigation. Morphological identification was not conducted on these samples prior to DNA extraction; therefore, they will be sequenced to determine species at a later date. Light traps did not collect any adult mosquitoes.

Of the 59 mosquito samples tested by PCR, 23 were confirmed to be An. stephensi. The experiment was repeated with different concentrations of DNA but 0.15ul gave the clearest distinction.

Sequencing

The sequence for 3 of the 4 samples matched and were confirmed as An. stephensi. One sample failed to amplify possibly due to DNA degradation (Table 3).

Table 3

Results for sequencing analysis of the 4 samples from Kenya
Origin Sample ID	CDC Sample ID	Morphology ID	Confirmed Species	Accession Number of closest match	%Identity match
KE83GY	2023KEN0001	Suspected An. stephensi	An. stephensi	FJ526599.1	99.40%
KE83QF	2023KEN0002	Suspected An. stephensi	An. stephensi	MW732931.1	100%
KE83FZ	2023KEN0003	Suspected An. stephensi	An. stephensi	MW732931.1	100%
KE831Q	2023KEN0004	Suspected An. stephensi	Did not amplify

BLAST searches using default parameters for isolates 2 and 3 matched to An. stephensi sequences with 100% identity to hap 10 5.8S ribosomal RNA gene, ITS2 while isolate 1 was at 99.4% identity to the same gene but 100% identity to An. stephensi isolate 141 steph 5.8S ribosomal RNA gene, ITS2.

Phylogenetic analysis of sequenced isolates with 31 other isolates of ITS2 for An. stephensi available on GenBank showed that our isolates closely matched the Iraq, India, Yemen and Nigerian isolates (Fig. 4).

In this article, we report the first collection and detection of An. stephensi from Marsabit County in northern Kenya. From the samples collected, multiple methods were used to conduct the identification, including morphological keys, standard PCR, and Sanger sequencing. Molecular methods were instrumental in confirming the presence of An. stephensi in Marsabit county, Kenya. Additional surveys conducted at the same time in Turkana County in the northwestern parts of the country, bordering Marsabit County did not yield a similar finding; there have been no recent surveys on the northeastern part of the country. The An. stephensi mosquitoes were collected as larvae but no adults were found in the light traps set at the same locations indicating the need for studies to characterize vector bionomics and behavior to understand its contribution/potential contribution to malaria transmission and to design appropriate tools for conducting surveillance for adult An. stephensi.

From our surveillance efforts, we collected 59 larvae but did not trap any adults in the same area using CDC light traps. Reports from other sites have documented the difficulty in trapping adult mosquitoes [5]. The bionomics and behaviour of this vector in its recent invasive geographic foci are poorly understood, with the only detailed descriptions available to this point being from Asia [3, 13, 14]. However, reports have indicated that crepuscular biting and resting outside houses could translate to reduced efficacy of core vector control interventions, Insecticide treated nets (ITNs) and indoor residual spray (IRS) indicating the importance of accurate parameterization [6]. Additionally, the potential for any insecticide-based control methods to be effective will depend on the insecticide resistance status of the An. stephensi mosquitoes and therefore necessitating insecticide resistance surveys [6, 15]. Based on the phylogenetic analysis, the Kenyan An. stephensi isolates matched closely with isolates from India, Iraq, Yemen, Iran and Nigeria but more distant to the isolates from Ethiopia which could indicate multiple clades of An. stephensi invading different parts of Africa. There is a need for additional population genetics studies using whole genome sequencing approaches to describe these clades, coupled with intensive surveillance to describe their bionomics and behaviour. Our findings also suggest potential introduction routes, the An. stephensi were found along the main highway that connects Kenya to Ethiopia, highlighting the need for increased surveillance along major transportation routes and targeting areas such as truck stops and resting sites, weighbridges, and borders. That said, it is important to note that phylogenetic analyses of the ITS2 isolates from Kenya did not match those from Ethiopia which may mean multiple introductions of different clades simultaneously in different parts of Africa. Further, it will be important to track parasites causing malaria cases around the areas where An. stephensi has been introduced given that it is an efficient vector of both P. falciparum and P. vivax.

With rapid, often unplanned urbanization in Africa, many of the urban centers have poor refuse disposal and drainage systems which are potential larval habitats of An. stephensi [1, 8]. Coupled with climate change, which creates suitable climatic conditions for mosquito breeding, there is great potential for the spread and establishment of An. stephensi in African cities. At the point of introduction of An. stephensi into Djibouti [1], the country was at pre-elimination, with under 3000 malaria cases reported in 2013, one year after this mosquito was first reported. Six years later in 2019, Djibouti reported 49,402 malaria cases [16]. Modelling of the potential impact of An. stephensi establishment in Ethiopia predicts a surge in P. falciparum cases by 50% overall if no additional interventions are put in place with areas of lowest transmission (~ 0.1%) being impacted the most [6]. There is a need for similar models to be conducted in all the areas that are newly invaded such as Kenya, to predict the spread and potential impact of the vector and to understand the potential impact of additional interventions.

Anopheles stephensi breeding habitats are similar to Aedes aegypti habitats, with a poorer understanding of adult resting and biting behaviour [17, 18]. Evidence of outdoor, crepuscular feeding by this species suggests it may be less affected by ITNs or IRS. Furthermore, An. stephensi in Ethiopia was reported to be highly resistant to pyrethroids, carbamates and organophosphates [13]. These traits indicate that alternative vector control and non-vector control measures may be needed to address the threat of this invasive mosquito. As the national malaria control program develops a vector control strategy, integrated vector management (IVM) approaches offer advantages due to of the potential benefit of targeting additional vectors based on WHO guidance [19, 20] but more importantly, because of how little of this vector’s behaviour is understood, especially as it colonizes new areas. Deploying an integrated approach provides opportunities to target the two vectors for surveillance and control operations using similar interventions thus optimizing resource allocation and use in resource limited settings where this vector is currently being reported. Other potential vector control tools, including those currently under evaluation, include spatial repellents [21], attractive targeted sugar baits [22], endectocides [23], and genetically modified mosquitoes [24]. Given the outdoor, early evening biting behaviors, resistance to multiple insecticides, and the threat to malaria control efforts, these alternative vector control approaches may be necessary to sustain the gains made against malaria over the last two decades.

We confirm the presence of An. stephensi in northern Kenya. This finding is critical for the DNMP as it points to the urgent need to re-examine and expand the vector surveillance and control efforts to include An stephensi which is likely to sustain and possibly increase transmission in northern Kenya and spread further southwards to highly populated urban areas and existing malaria-endemic counties further compounding the problem of malaria control in the country. This report emphasizes the need for heightened and tailored surveillance to understand the scope of the spread of this invasive vector and to advise on targeted control using all the existing interventions, including those currently under trial given the emergency.

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ITNs

Insecticide Treated Nets

IRS

Indoor Residual Spraying

Anopheles

KEMRI

Kenya Medical Research Institute

CDC

Centers for Disease Control and Prevention

DNMP

Division for National Malaria Program

IVM

Integrated Vector Management

WHO

World Health Organization

BLAST

Basic Local Alignment Search Tool

ITS2

Internal Transcribed Spacer 2

Acknowledgement

We acknowledge the support of Paul Omondi Osodo, Stephen Okeyo, George Marube, James Opalla who conducted the survey leading to the discovery of An. stephensi in Marsabit. Ian Mabiria and Blantine Akinyi who supported the molecular identification process, and Richard Amito who supported the rearing of the collected larvae. James Kiarie of DNMP and of KEMRI who helped with the data computation and development of the maps.

Authors’ contribution

EOO, EO, LN, EJ, JDO, DM, LK, JEG, MS, DW, JM, Marta Maia, CC, AO, CM, Lenson Kariuki: Conceptualization of the study, data analysis, drafting of the manuscript

SM, BA, BO, Margaret Muchoki, DO, CR: Study design, sample collection, sample processing, data collection, data analysis, drafting the manuscript

All authors read and approved this manuscript

Competing interests

The authors declare that they have no competing interests

Funding

This work was supported by funding from NIH_NIAID through the Liverpool School of Tropical Medicine (LSTM) and Bill and Melinda Gates Foundation Grant #INV-024969 to KEMRI and GF NFM III to the DNMP.

Data Availability

The An. stephensi sequences have now been uploaded on GenBank with accession numbers: OQ275144 (https://www.ncbi.nlm.nih.gov/nuccore/OQ275144), OQ275145 (https://www.ncbi.nlm.nih.gov/nuccore/OQ275145) and OQ275146 (https://www.ncbi.nlm.nih.gov/nuccore/OQ275146).

Consent for publication

This manuscript is published with the permission of the Director-General of the Kenya Medical Research Institute.

Participation of human subjects

Humans were not involved in this study

Disclaimer

The findings and conclusions expressed herein are those of the author(s) and do not necessarily represent the official position of USAID, PMI, or the Centers for Disease Control and Prevention (CDC).

No competing interests reported.

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Molecular surveillance leads to the first detection of Anopheles stephensi in Kenya

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