Molecular Confirmation of Anopheles stephensi Mosquitoes in the Al Hudaydah Governorate, Yemen, 2021 and 2022.

We detected malaria vector Anopheles stephensi mosquitoes in the Al Hudaydah governorate in Yemen by using DNA sequencing. We report 2 cytochrome c oxidase subunit I haplotypes, 1 previously found in Ethiopia, Somalia, Djibouti, and Yemen. These findings provide insight into invasive An. stephensi mosquitoes in Yemen and their connection to East Africa.

Population genomic analyses reveal population structure and major hubs of invasive Anopheles stephensi in the Horn of Africa.

Anopheles stephensi invasion in the Horn of Africa (HoA) poses a substantial risk of increased malaria disease burden in the region. An understanding of the history of introduction(s), establishment(s) and potential A. stephensi sources in the HoA is needed to predict future expansions and establish where they may be effectively controlled. To this end, we take a landscape genomic approach to assess A. stephensi origins and spread throughout the HoA, information essential for vector control. Specifically, we assayed 2070 genome-wide single nucleotide polymorphisms across 214 samples spanning 13 populations of A. stephensi from Ethiopia and Somaliland collected in 2018 and 2020, respectively. Principal component and genetic ancestry analyses revealed clustering that followed an isolation-by-distance pattern, with genetic divergence among the Ethiopian samples significantly correlating with geographical distance. Additionally, genetic relatedness was observed between the northeastern and east central Ethiopian A. stephensi populations and the Somaliland A. stephensi populations. These results reveal population differentiation and genetic connectivity within HoA A. stephensi populations. Furthermore, based on genetic network analysis, we uncovered that Dire Dawa, the site of a spring 2022 malaria outbreak, was one of the major hubs from which sequential founder events occurred in the rest of the eastern Ethiopian region. These findings can be useful for the selection of sites for heightened control to prevent future malaria outbreaks. Finally, we did not detect significant genotype-environmental associations, potentially due to the recency of their colonization and/or other anthropogenic factors leading to the initial spread and establishment of A. stephensi. Our study highlights how coupling genomic data at landscape levels can shed light into even ongoing invasions.

Public health impact of the spread of Anopheles stephensi in the WHO Eastern Mediterranean Region countries in Horn of Africa and Yemen: need for integrated vector surveillance and control.

BACKGROUND: Anopheles stephensi is an efficient vector of both Plasmodium falciparum and Plasmodium vivax in South Asia and the Middle East. The spread of An. stephensi to countries within the Horn of Africa threatens progress in malaria control in this region as well as the rest of sub-Saharan Africa. METHODS: The available malaria data and the timeline for the detection of An. stephensi was reviewed to analyse the role of An. stephensi in malaria transmission in Horn of Africa of the Eastern Mediterranean Region (EMR) in Djibouti, Somalia, Sudan and Yemen. RESULTS: Malaria incidence in Horn of Africa of EMR and Yemen, increased from 41.6 in 2015 to 61.5 cases per 1000 in 2020. The four countries from this region, Djibouti, Somalia, Sudan and Yemen had reported the detection of An. stephensi as of 2021. In Djibouti City, following its detection in 2012, the estimated incidence increased from 2.5 cases per 1000 in 2013 to 97.6 cases per 1000 in 2020. However, its contribution to malaria transmission in other major cities and in other countries, is unclear because of other factors, quality of the urban malaria data, human mobility, uncertainty about the actual arrival time of An. stephensi and poor entomological surveillance. CONCLUSIONS: While An. stephensi may explain a resurgence of malaria in Djibouti, further investigations are needed to understand its interpretation trends in urban malaria across the greater region. More investment for multisectoral approach and integrated surveillance and control should target all vectors particularly malaria and dengue vectors to guide interventions in urban areas.

Variation of the response to metal ions and nonsense-mediated mRNA decay across different Saccharomyces cerevisiae genetic backgrounds.

Regulation of mRNA steady-state levels is important in controlling gene expression particularly in response to environmental stimuli. This allows cells to rapidly respond to environment changes. The highly conserved nonsense-mediated mRNA decay (NMD) pathway was initially identified as a pathway that degrades aberrant mRNAs. NMD is now recognized as a pathway with additional functions including precisely regulating the expression of select natural mRNAs. Majority of these natural mRNAs encode fully functional proteins. Regulation of natural mRNAs by NMD is activated by NMD targeting features and environmental cues. Here, we show that Saccharomyces cerevisiae strains from three genetic backgrounds respond differentially to NMD depending on the environmental stimuli. We found that wild type and NMD mutant W303a, BY4741, and RM11-1a yeast strains respond similarly to copper in the environment but respond differentially to toxic cadmium. Furthermore, the PCA1 alleles encoding different mRNAs from W303a and RM11-1a strains are regulated similarly by NMD in response to the bio-metal copper but differentially in response to toxic cadmium.

Investigations of socioeconomic factors associated with follow-up compliance with malaria treatment in Haiti.

OBJECTIVE: To identify factors affecting compliance with follow-up during treatment in confirmed malaria patients at two health centers in Haiti. METHODS: A prospective observational study of malaria patients undergoing treatment over a six-week period. Patients' return visits (follow-up visits) to the health centers for consultation in accordance with the physicians' requests were recorded and used to determine compliance. Socioeconomic data were obtained from patient enrollment questionnaires and through post-treatment interviews. The management practices and procedures at the health centers to retain patients were also reviewed. Descriptive statistics and Spearman's rank correlation were used to identify significant factors, which were used as variables in a logistic regression model. RESULTS: Sixty-eight percent of the malaria patients completed follow-up, with higher compliance being recorded in the larger, more established health center of Leogane (67%) than Cite Soleil (33%). The patient socioeconomic profiles differed between the two health center locations by level of education, religious diversity, household size, and percentage of married individuals. Crude logistic regression analyses identified health center location (OR = 0.179 [95% CI 0.064, 0.504]) and household size (OR = 1.374 [95% CI 1.056, 1.787]) to be associated with compliance. The adjusted model only identified health center location (OR = 0.226 [95% CI 0.056, 0.918]) as significantly associated with compliance. CONCLUSION: Although patients' household size may be important according to the crude logistic regression analysis, in the adjusted analysis the site location of the health center where patients receive treatment was identified as the only important factor associated with follow-up compliance in malaria patients during treatment in Haiti. This information might be helpful to improve treatment outcomes and contribute to the monitoring of antimalarial resistance in Haiti.

Insecticide resistance in Anopheles stephensi in Somali Region, eastern Ethiopia.

BACKGROUND: The movement of malaria vectors into new areas is a growing concern in the efforts to control malaria. The recent report of Anopheles stephensi in eastern Ethiopia has raised the necessity to understand the insecticide resistance status of the vector in the region to better inform vector-based interventions. The aim of this study was to evaluate insecticide resistance in An. stephensi in eastern Ethiopia using two approaches: (1) World Health Organization (WHO) bioassay tests in An. stephensi; and (2) genetic analysis of insecticide resistance genes in An. stephensi in eastern Ethiopia. METHODS: Mosquito larvae and pupae were collected from Kebri Dehar. Insecticide susceptibility of An. stephensi was tested with malathion 5%, bendiocarb 0.1%, propoxur 0.1%, deltamethrin 0.05%, permethrin 0.75%, pirimiphos-methyl 0.25% and DDT 4%, according to WHO standard protocols. In this study, the knockdown resistance locus (kdr) in the voltage gated sodium channel (vgsc) and ace1R locus in the acetylcholinesterase gene (ace-1) were analysed in An. stephensi. RESULTS: All An. stephensi samples were resistant to carbamates, with mortality rates of 23% and 21% for bendiocarb and propoxur, respectively. Adult An. stephensi was also resistant to pyrethroid insecticides with mortality rates 67% for deltamethrin and 53% for permethrin. Resistance to DDT and malathion was detected in An. stephensi with mortality rates of 32% as well as An. stephensi was resistance to pirimiphos-methyl with mortality rates 14%. Analysis of the insecticide resistance loci revealed the absence of kdr L1014F and L1014S mutations and the ace1R G119S mutation. CONCLUSION: Overall, these findings support that An. stephensi is resistant to several classes of insecticides, most notably pyrethroids. However, the absence of the kdr L1014 gene may suggest non-target site resistance mechanisms. Continuous insecticide resistance monitoring should be carried out in the region to confirm the documented resistance and exploring mechanisms conferring resistance in An. stephensi in Ethiopia.

Sequence-based identification of Anopheles species in eastern Ethiopia.

BACKGROUND: The recent finding of a typically non-African Anopheles species in eastern Ethiopia emphasizes the need for detailed species identification and characterization for effective malaria vector surveillance. Molecular approaches increase the accuracy and interoperability of vector surveillance data. To develop effective molecular assays for Anopheles identification, it is important to evaluate different genetic loci for the ability to characterize species and population level variation. Here the utility of the internal transcribed spacer 2 (ITS2) and cytochrome oxidase I (COI) loci for detection of Anopheles species from understudied regions of eastern Ethiopia was investigated. METHODS: Adult mosquitoes were collected from the Harewe locality (east) and Meki (east central) Ethiopia. PCR and Sanger sequencing were performed for portions of the ITS2 and COI loci. Both NCBI's Basic Local Alignment Search tool (BLAST) and phylogenetic analysis using a maximum-likelihood approach were performed to identify species of Anopheles specimens. RESULTS: Two species from the east Ethiopian collection, Anopheles arabiensis and Anopheles pretoriensis were identified. Analyses of ITS2 locus resulted in delineation of both species. In contrast, analysis of COI locus could not be used to delineate An. arabiensis from other taxa in Anopheles gambiae complex, but could distinguish An. pretoriensis sequences from sister taxa. CONCLUSION: The lack of clarity from COI sequence analysis highlights potential challenges of species identification within species complexes. These results provide supporting data for the development of molecular assays for delineation of Anopheles in east Ethiopia.

Massive single visit cervical pre-cancer and cancer screening in eastern Democratic Republic of Congo.

BACKGROUND: In the Democratic Republic of Congo (DRC), practical and affordable strategies for cervical cancer screening are needed to detect and treat pre-cancerous and cancerous lesions in a timely fashion. This study presents the results of mass cervical cancer screenings in eastern DRC using a "screen and treat" approach. METHODS: In two mass cervical cancer screening campaigns, patients underwent a combination of visual inspection of the cervix with acetic acid, visual inspection of the cervix with Lugol iodine solution, and colposcopy with or without loop electrosurgical excision procedure. Cervical biopsy samples were taken for histology analysis. Marital status, age, history of abnormal bleeding, and number of pregnancies were recorded for each patient and association analyses were performed. RESULTS: Of the 644 women who received cervical pre-cancer and cancer screening, 48 had suspicious pre-cancer and cancer lesions that were biopsied (7.45%). On histology analysis cervical intraepithelial neoplasia (CIN) was identified in 15 (2.33%), squamous cell carcinoma (SCC) was identified in 6 (0.93%) and non-neoplastic cervicitis was identified in 11 (1.71%). Abnormal bleeding was significantly associated with CIN/SCC but no significant association was observed for prior pregnancy, patients' home region, or age. CONCLUSION: Forty-eight women with suspicious pre-cancerous or cancerous lesions were successfully identified using the "screen and treat" approach in eastern DRC, suggesting that this approach is feasible for reducing cervical cancer morbidity and mortality. However, community awareness would be necessary, providers would have to be properly trained, referral and follow up mechanisms would have to be put in place, and equipment / supplies would have to be secured if the "screen and treat" approach is to be successful on a wider scale. There is ongoing need for HPV vaccination in DRC as a primary prevention strategy against cervical cancer.

Malaria elimination in Haiti by the year 2020: an achievable goal?

Haiti and the Dominican Republic, which share the island of Hispaniola, are the last locations in the Caribbean where malaria still persists. Malaria is an important public health concern in Haiti with 17,094 reported cases in 2014. Further, on January 12, 2010, a record earthquake devastated densely populated areas in Haiti including many healthcare and laboratory facilities. Weakened infrastructure provided fertile reservoirs for uncontrolled transmission of infectious pathogens. This situation results in unique challenges for malaria epidemiology and elimination efforts. To help Haiti achieve its malaria elimination goals by year 2020, the Laboratoire National de Santé Publique and Henry Ford Health System, in close collaboration with the Direction d'Épidémiologie, de Laboratoire et de Recherches and the Programme National de Contrôle de la Malaria, hosted a scientific meeting on "Elimination Strategies for Malaria in Haiti" on January 29-30, 2015 at the National Laboratory in Port-au-Prince, Haiti. The meeting brought together laboratory personnel, researchers, clinicians, academics, public health professionals, and other stakeholders to discuss main stakes and perspectives on malaria elimination. Several themes and recommendations emerged during discussions at this meeting. First, more information and research on malaria transmission in Haiti are needed including information from active surveillance of cases and vectors. Second, many healthcare personnel need additional training and critical resources on how to properly identify malaria cases so as to improve accurate and timely case reporting. Third, it is necessary to continue studies genotyping strains of Plasmodium falciparum in different sites with active transmission to evaluate for drug resistance and impacts on health. Fourth, elimination strategies outlined in this report will continue to incorporate use of primaquine in addition to chloroquine and active surveillance of cases. Elimination of malaria in Haiti will require collaborative multidisciplinary approaches, sound strategic planning, and strong ownership of strategies by the Haiti Ministère de la Santé Publique et de la Population.

A decade of invasive Anopheles stephensi sequence-based identification: toward a global standard.

Anopheles stephensi is an invasive malaria vector in Africa that has been implicated in malaria outbreaks in the Horn of Africa. In 10 years, it has been detected as far east as Djibouti and as far west as Ghana. Early detections were mostly incidental, but now active surveillance in Africa has been updated to include An. stephensi. Morphological identification of An. stephensi from native vectors can be challenging, thus, sequence-based assays have been used to confirm identification during initial detections. Methods of sequence-based identification of An. stephensi have varied across initial detections to date. Here, we summarize initial detections, make suggestions that could provide a standardized approach, and discuss how sequences can inform additional genomic studies beyond species identification.

Breeding habitats, bionomics and phylogenetic analysis of Aedes aegypti and first detection of Culiseta longiareolata, and Ae. hirsutus in Somali Region, eastern Ethiopia.

INTRODUCTION: Arboviral diseases, such as dengue, chikungunya, yellow fever, and Zika, are caused by viruses that are transmitted to humans through mosquito bites. However, the status of arbovirus vectors in eastern Ethiopia is unknown. The aim of this study was to investigate distribution, breeding habitat, bionomics and phylogenetic relationship of Aedes aegypti mosquito species in Somali Regional State, Eastern Ethiopia. METHODS: Entomological surveys were conducted in four sites including Jigjiga, Degehabur, Kebridehar and Godey in 2018 (October to December) to study the distribution of Ae. aegypti and with a follow-up collection in 2020 (July-December). In addition, an investigation into the seasonality and bionomics of Ae. aegypti was conducted in 2021 (January-April) in Kebridehar town. Adult mosquitoes were collected from indoor and outdoor locations using CDC light traps (LTs), pyrethrum spray collection (PSCs), and aspirators. Larvae and pupae were also collected from a total of 169 water-holding containers using a dipper between October and November 2020 (rainy season) in Kebridehar town. The species identification of wild caught and reared adults was conducted using a taxonomic key. In addition, species identification using mitochondrial and nuclear genes maximum likelihood-based phylogenetic analysis was performed. RESULTS: In the 2018 collection, Ae. aegypti was found in all study sites (Jigjiga, Degahabour, Kebridehar and Godey). In the 2020-2021 collection, a total of 470 (Female = 341, Male = 129) wild caught adult Ae. aegypti mosquitoes were collected, mostly during the rainy season with the highest frequency in November (n = 177) while the lowest abundance was in the dry season (n = 14) for both February and March. The majority of Ae. aegypt were caught using PSC (n = 365) followed by CDC LT (n = 102) and least were collected by aspirator from an animal shelter (n = 3). Aedes aegypti larval density was highest in tires (0.97 larvae per dip) followed by cemented cisterns (0.73 larvae per dip) and the Relative Breeding Index (RBI) was 0.87 and Container Index (CI) was 0.56. Genetic analysis of ITS2 and COI revealed one and 18 haplotypes, respectively and phylogenetic analysis confirmed species identification. The 2022 collection revealed no Ae. aegpti, but two previously uncharacterized species to that region. Phylogenetic analysis of these two species revealed their identities as Ae. hirsutus and Culiseta longiareolata. CONCLUSION: Data from our study indicate that, Ae. aegypti is present both during the wet and dry seasons due to the availability of breeding habitats, including water containers like cemented cisterns, tires, barrels, and plastic containers. This study emphasizes the necessity of establishing a national entomological surveillance program for Aedes in Somali region.

High frequency of the erythroid silent Duffy antigen genotype and lack of Plasmodium vivax infections in Haiti.

BACKGROUND: Malaria is a significant public health concern in Haiti where approximately 30,000 cases are reported annually with CDC estimates as high as 200,000. Malaria infections in Haiti are caused almost exclusively by Plasmodium falciparum, while a small number of Plasmodium malariae and an even smaller number of putative Plasmodium vivax infections have been reported. The lack of confirmed P. vivax infections in Haiti could be due to the genetic background of native Haitians. Having descended from West African populations, many Haitians could be Duffy negative due to a single nucleotide polymorphism from thymine to cytosine in the GATA box of the promoter region of the Duffy antigen receptor for chemokines (DARC) gene. This mutation, encoded by the FYES allele, eliminates the expression of the Duffy antigen on erythrocytes, which reduces invasion by P. vivax. This study investigated the frequency of the FYES allele and P. vivax infections in malaria patients with the goal of uncovering factors for the lack of P. vivax infections reported in Haiti. METHODS: DNA was extracted from dried blood spots collected from malaria patients at four clinic locations in Haiti. The samples were analysed by polymerase chain reaction (PCR) for the presence of the P. vivax small subunit ribosomal RNA gene. PCR, sequencing, and restriction enzyme digestion were used to detect the presence of the FYES allele. Matched samples were examined for both presence of P. vivax and the FYES allele. RESULTS: No cases of P. vivax were detected in any of the samples (0/136). Of all samples tested for the FYES allele, 99.4% had the FYES allele (163/164). Of the matched samples, 99% had the FYES allele (98/99). CONCLUSIONS: In this preliminary study, no cases of P. vivax were confirmed by PCR and 99% of the malaria patients tested carried the FYES allele. The high frequency of the FYES allele that silences erythroid expression of the Duffy antigen offers a biologically plausible explanation for the lack of P. vivax infections observed. These results provide insights on the host susceptibility for P. vivax infections that has never before been investigated in Haiti.

Evaluation of dihydrofolate reductase and dihydropteroate synthetase genotypes that confer resistance to sulphadoxine-pyrimethamine in Plasmodium falciparum in Haiti.

BACKGROUND: Malaria caused by Plasmodium falciparum infects roughly 30,000 individuals in Haiti each year. Haiti has used chloroquine (CQ) as a first-line treatment for malaria for many years and as a result there are concerns that malaria parasites may develop resistance to CQ over time. Therefore it is important to prepare for alternative malaria treatment options should CQ resistance develop. In many other malaria-endemic regions, antifolates, particularly pyrimethamine (PYR) and sulphadoxine (SDX) treatment combination (SP), have been used as an alternative when CQ resistance has developed. This study evaluated mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes that confer PYR and SDX resistance, respectively, in P. falciparum to provide baseline data in Haiti. This study is the first comprehensive study to examine PYR and SDX resistance genotypes in P. falciparum in Haiti. METHODS: DNA was extracted from dried blood spots and genotyped for PYR and SDX resistance mutations in P. falciparum using PCR and DNA sequencing methods. Sixty-one samples were genotyped for PYR resistance in codons 51, 59, 108 and 164 of the dhfr gene and 58 samples were genotyped for SDX resistance codons 436, 437, 540 of the dhps gene in P. falciparum. RESULTS: Thirty-three percent (20/61) of the samples carried a mutation at codon 108 (S108N) of the dhfr gene. No mutations in dhfr at codons 51, 59, 164 were observed in any of the samples. In addition, no mutations were observed in dhps at the three codons (436, 437, 540) examined. No significant difference was observed between samples collected in urban vs rural sites (Welch's T-test p-value = 0.53 and permutations p-value = 0.59). CONCLUSION: This study has shown the presence of the S108N mutation in P. falciparum that confers low-level PYR resistance in Haiti. However, the absence of SDX resistance mutations suggests that SP resistance may not be present in Haiti. These results have important implications for ongoing discussions on alternative malaria treatment options in Haiti.

Morphological identification and genetic characterization of Anopheles stephensi in Somaliland.

Malaria control in Somaliland depends on the effective identification of potential malaria vectors, particularly those that may be invasive. The malaria vector Anopheles stephensi has been detected in multiple countries in the Horn of Africa (HOA), but data on its geographic distribution and population genetic diversity are incomplete. We implemented a vector surveillance program and performed molecular analysis of Anopheles in three urban areas in Somaliland. Our study confirmed the presence of both the invasive An. stephensi and the long-established HOA malaria vector Anopheles arabiensis. Further analysis of An. stephensi genetic diversity revealed three cytochrome oxidase I (COI) haplotypes, all of which have been observed in other countries in East Africa and one also observed in South Asia. We also detected the knockdown resistance (kdr) L1014F mutation, which is associated with pyrethroid resistance; this finding supports the need for further assessment of the potential for insecticide resistance. The detection of multiple haplotypes previously observed in other regions of East Africa indicates that An. stephensi is an established population in Somaliland and likely shares its origin with other newly identified An. stephensi populations in East Africa. The detection of genetic diversity in An. stephensi in Somaliland provides a basis for future studies on the history of the species in the region and its dispersal throughout East Africa.

Wolbachia 16S rRNA haplotypes detected in wild Anopheles stephensi in eastern Ethiopia.

BACKGROUND: About two out of three Ethiopians are at risk of malaria, a disease caused by the parasites Plasmodium falciparum and Plasmodium vivax. Anopheles stephensi, an invasive vector typically found in South Asia and the Middle East, was recently found to be distributed across eastern and central Ethiopia and is capable of transmitting both P. falciparum and P. vivax. The detection of this vector in the Horn of Africa (HOA) coupled with widespread insecticide resistance requires that new methods of vector control be investigated in order to control the spread of malaria. Wolbachia, a naturally occurring endosymbiotic bacterium of mosquitoes, has been identified as a potential vector control tool that can be explored for the control of malaria transmission. Wolbachia could be used to control the mosquito population through suppression or potentially decrease malaria transmission through population replacement. However, the presence of Wolbachia in wild An. stephensi in eastern Ethiopia is unknown. This study aimed to identify the presence and diversity of Wolbachia in An. stephensi across eastern Ethiopia. METHODS: DNA was extracted from An. stephensi collected from eastern Ethiopia in 2018 and screened for Wolbachia using a 16S targeted PCR assay, as well as multilocus strain typing (MLST) PCR assays. Haplotype and phylogenetic analysis of the sequenced 16S amplicons were conducted to compare with Wolbachia from countries across Africa and Asia. RESULTS: Twenty out of the 184 mosquitoes screened were positive for Wolbachia, with multiple haplotypes detected. In addition, phylogenetic analysis revealed two superclades, representing Wolbachia supergroups A and B (bootstrap values of 81 and 72, respectively) with no significant grouping of geographic location or species. A subclade with a bootstrap value of 89 separates the Ethiopian haplotype 2 from other sequences in that superclade. CONCLUSIONS: These findings provide the first evidence of natural Wolbachia populations in wild An. stephensi in the HOA. They also identify the need for further research to confirm the endosymbiotic relationship between Wolbachia and An. stephensi and to investigate its utility for malaria control in the HOA.

Analysis of the Knockdown Resistance Locus (kdr) in Anopheles stephensi, An. arabiensis, and Culex pipiens s.l. for Insight Into the Evolution of Target-site Pyrethroid Resistance in Eastern Ethiopia.

The malaria vector, Anopheles stephensi, which is typically restricted to South Asia and the Middle East, was recently detected in the Horn of Africa. Addressing the spread of this vector could involve integrated vector control that considers the status of insecticide resistance of multiple vector species in the region. Previous reports indicate that the knockdown resistance mutations (kdr) in the voltage-gated sodium channel (vgsc) are absent in both pyrethroid-resistant and pyrethroid-sensitive An. stephensi in eastern Ethiopia; however, similar information about other vector species in the same areas is limited. In this study, kdr and the neighboring intron were analyzed in An. stephensi, An. arabiensis, and Culex pipiens s.l. collected between 2016 and 2017 to determine the evolutionary history of kdr in eastern Ethiopia. A sequence analysis revealed that all of Cx. pipiens s.l. (N = 42) and 71.6% of the An. arabiensis (N = 67) carried kdr L1014F, which is known to confer target-site pyrethroid resistance. Intronic variation was only observed in An. stephensi (six segregating sites, three haplotypes), which was previously shown to have no kdr mutations. In addition, no evidence of non-neutral evolutionary processes was detected at the An. stephensi kdr intron, thereby further supporting the target-site mechanism not being a major resistance mechanism in this An. stephensi population. Overall, these results show key differences in the evolution of target-site pyrethroid/dichlorodiphenyltrichloroethane resistance mutations in populations of vector species from the same region. Variations in insecticide resistance mechanism profiles between eastern Ethiopian mosquito vectors may lead to different responses to insecticides used in integrated vector control.

Detection and population genetic analysis of kdr L1014F variant in eastern Ethiopian Anopheles stephensi.

Anopheles stephensi is a malaria vector that has been recently introduced into East Africa, where it threatens to increase malaria disease burden. The use of insecticides, especially pyrethroids, is still one of the primary malaria vector control strategies worldwide. The knockdown resistance (kdr) mutation in the IIS6 transmembrane segment of the voltage-gated sodium channel (vgsc) is one of the main molecular mechanisms of pyrethroid resistance in Anopheles. Extensive pyrethroid resistance in An. stephensi has been previously reported in Ethiopia. Thus, it is important to determine whether or not the kdr mutation is present in An. stephensi populations in Ethiopia to inform vector control strategies. In the present study, the kdr locus was analyzed in An. stephensi collected from ten urban sites (Awash Sebat Kilo, Bati, Dire Dawa, Degehabur, Erer Gota, Godey, Gewane, Jigjiga, Semera, and Kebridehar) situated in Somali, Afar, and Amhara regions, and Dire Dawa Administrative City, to evaluate the frequency and evolution of kdr mutations and the association of the mutation with permethrin resistance phenotypes. Permethrin is one of the pyrethroid insecticides used for vector control in eastern Ethiopia. DNA extractions were performed on adult mosquitoes from CDC light trap collections and those raised from larval and pupal collections. PCR and targeted sequencing were used to analyze the IIS6 transmembrane segment of the vgsc gene. Of 159 An. stephensi specimens analyzed from the population survey, nine (5.7%) carried the kdr mutation (L1014F). An. stephensi with kdr mutations were only observed from Bati, Degehabur, Dire Dawa, Gewane, and Semera. We further selected randomly twenty resistant and twenty susceptible An. stephensi mosquitoes from Dire Dawa post-exposure to permethrin and investigated the role of kdr in pyrethroid resistance by comparing the vgsc gene in the two populations. We found no kdr mutations in the permethrin-resistant mosquitoes. Population genetic analysis of the sequences, including neighboring introns, revealed limited evidence of non-neutral evolution (e.g., selection) at this locus. The low kdr mutation frequency detected and the lack of kdr mutation in the permethrin-resistant mosquitoes suggest the existence of other molecular mechanisms of pyrethroid resistance in eastern Ethiopian An. stephensi.

Genetic diversity of Anopheles stephensi in Ethiopia provides insight into patterns of spread.

BACKGROUND: The recent detection of the South Asian malaria vector Anopheles stephensi in the Horn of Africa (HOA) raises concerns about the impact of this mosquito on malaria transmission in the region. Analysis of An. stephensi genetic diversity and population structure can provide insight into the history of the mosquito in the HOA to improve predictions of future spread. We investigated the genetic diversity of An. stephensi in eastern Ethiopia, where detection suggests a range expansion into this region, in order to understand the history of this invasive population. METHODS: We sequenced the cytochrome oxidase subunit I (COI) and cytochrome B gene (CytB) in 187 An. stephensi collected from 10 sites in Ethiopia in 2018. Population genetic, phylogenetic, and minimum spanning network analyses were conducted for Ethiopian sequences. Molecular identification of blood meal sources was also performed using universal vertebrate CytB sequencing. RESULTS: Six An. stephensi COI-CytB haplotypes were observed, with the highest number of haplotypes in the northeastern sites (Semera, Bati, and Gewana towns) relative to the southeastern sites (Kebridehar, Godey, and Degehabur) in eastern Ethiopia. We observed population differentiation, with the highest differentiation between the northeastern sites compared to central sites (Erer Gota, Dire Dawa, and Awash Sebat Kilo) and the southeastern sites. Phylogenetic and network analysis revealed that the HOA An. stephensi are more genetically similar to An. stephensi from southern Asia than from the Arabian Peninsula. Finally, molecular blood meal analysis revealed evidence of feeding on cows, goats, dogs, and humans, as well as evidence of multiple (mixed) blood meals. CONCLUSION: We show that An. stephensi is genetically diverse in Ethiopia and with evidence of geographical structure. Variation in the level of diversity supports the hypothesis for a more recent introduction of An. stephensi into southeastern Ethiopia relative to the northeastern region. We also find evidence that supports the hypothesis that HOA An. stephensi populations originate from South Asia rather than the Arabian Peninsula. The evidence of both zoophagic and anthropophagic feeding support the need for additional investigation into the potential for livestock movement to play a role in vector spread in this region.

Geographical distribution of Anopheles stephensi in eastern Ethiopia.

BACKGROUND: The recent detection of the South Asian malaria vector Anopheles stephensi in Ethiopia and other regions in the Horn of Africa has raised concerns about its potential impact on malaria transmission. We report here the findings of a survey for this species in eastern Ethiopia using both morphological and molecular methods for species identification. METHODS: Adult and larval/pupal collections were conducted at ten sites in eastern Ethiopia and Anopheles specimens were identified using standard morphological keys and genetic analysis. RESULTS: In total, 2231 morphologically identified An. stephensi were collected. A molecular approach incorporating both PCR endpoint assay and sequencing of portions of the internal transcribed spacer 2 (ITS2) and cytochrome c oxidase subunit 1 (cox1) loci confirmed the identity of the An. stephensi in most cases (119/124 of the morphologically identified An. stephensi confirmed molecularly). Additionally, we observed Aedes aegypti larvae and pupae at many of the An. stephensi larval habitats. CONCLUSIONS: Our findings show that An. stephensi is widely distributed in eastern Ethiopia and highlight the need for further surveillance in the southern, western and northern parts of the country and throughout the Horn of Africa.

Geographic distributions and origins of human head lice (Pediculus humanus capitis) based on mitochondrial data.

Human head lice (Pediculus humanus capitis) are subdivided into 3 deeply divergent mitochondrial clades (Clades A, B, and C), each having unique geographical distributions. Determining the evolutionary history and geographic distribution of these mitochondrial clades can elucidate the evolutionary history of the lice as well as their human hosts. Previous data suggest that lice belonging to mitochondrial Clade B may have originated in North America or Asia; however, geographic sampling and sample sizes have been limited. With newly collected lice, we calculate the relative frequency, geographic distribution, and genetic diversity of louse mitochondrial clades to determine the geographic origin of lice belonging to Clade B. In agreement with previous studies, genetic diversity data support a North American origin of Clade B lice. It is likely that lice belonging to this mitochondrial clade recently migrated to other geographic localities, e.g., Europe and Australia, and, if not already present, may disperse further to occupy all geographic regions.