A regional One Health approach to the risk of invasion by Anopheles stephensi in Mauritius.

BACKGROUND: Anopheles stephensi is an invasive malaria vector in Africa that threatens to put an additional 126 million people at risk of malaria if it continues to spread. The island nation of Mauritius is highly connected to Asia and Africa and is at risk of introduction due to this connectivity. For early detection of An. stephensi, the Vector Biology and Control Division under the Ministry of Health in Mauritius, leveraged a well-established Aedes program, as An. stephensi is known to share Aedes habitats. These efforts triggered multisectoral coordination and cascading benefits of integrated vector and One Health approaches. METHODS: Beginning June 2021, entomological surveys were conducted at points of entry (seaport, airport) and on ships transporting livestock in collaboration with the Civil Aviation Department, the Mauritian Port Authority and National Veterinary Services. A total of 18, 39, 723 mosquito larval surveys were respectively conducted in the airport, seaport, and other localities in Mauritius while two, 20, and 26 adult mosquito surveys were respectively conducted in the airport, seaport, and twenty-six animal assembly points. Alongside adult mosquito surveys, surveillance of vectors of veterinary importance (e.g.- Culicoides spp.) was also carried out in collaboration with National Parks and Conservation Service and land owners. RESULTS: A total of 8,428 adult mosquitoes were collected and 1,844 larval habitats were positive for mosquitoes. All collected mosquitoes were morphologically identified and 151 Anopheles and 339 Aedes mosquitoes were also molecularly characterized. Mosquito species detected were Aedes albopictus, Anopheles arabiensis, An. coustani, An. merus, Culex quinquefasciatus, Cx. thalassius and Lutzia tigripes. Anopheles stephensi was not detected. The One Health approach was shared with the French Agricultural Research Centre for International Development (CIRAD), strengthening collaboration between Mauritius and Réunion Island on vector surveillance at entry points and insecticide resistance monitoring. The Indian Ocean Commission (IOC) was also alerted to the risk of An. stephensi, leading to regional efforts supporting trainings and development of a response strategy to An. stephensi bringing together stakeholders from Comoros, Madagascar, Mauritius, Réunion Island and Seychelles. CONCLUSIONS: Mauritius is a model system showing how existing public health entomology capabilities can be used to enhance vector surveillance and control and create multisectoral networks to respond to any emerging public and veterinary health vector-borne disease threat.

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.

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.

Building the vector in: construction practices and the invasion and persistence of Anopheles stephensi in Jigjiga, Ethiopia.

Anopheles stephensi is a major vector of malaria in Asia and the Arabian Peninsula, and its recent invasion into Africa poses a major threat to malaria control and elimination efforts on the continent. The mosquito is well adapted to urban environments, and its presence in Africa could potentially lead to an increase in malaria transmission in cities. Most of the knowledge about An stephensi ecology in Africa has been generated from studies conducted during the rainy season, when vectors are most abundant. Here, we provide evidence from the peak of the dry season in the city of Jigjiga in Ethiopia, and report An stephensi immature stages infesting predominantly in water reservoirs made to support construction operations (ie, in construction sites or associated with brick-manufacturing businesses). Political and economic changes in Ethiopia (particularly the Somali Region) have fuelled an unprecedented construction boom since 2018 that, in our opinion, has been instrumental in the establishment, persistence, and propagation of An stephensi via the year-round availability of perennial larval habitats associated with construction. We argue that larval source management during the dry season might provide a unique opportunity for focused control of An stephensi in Jigjiga and similar areas.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

First detection of Anopheles stephensi Liston, 1901 (Diptera: culicidae) in Ethiopia using molecular and morphological approaches.

Malaria is a major public health concern in Ethiopia. With the increase in malaria cases in the Somali Region of Ethiopia, understanding the distribution and identifying the species of malaria vectors is vital to public health. Here we report the first detection of Anopheles stephensi in Ethiopia, a malaria vector typically found in the Middle East, the Indian subcontinent, and China, but recently found in Djibouti. An entomological investigation was conducted during November to December 2016 in Kebri Dehar town of the Ethiopian Somali Regional State as ancillary work for Anopheles spp. surveillance. Mosquito larvae were collected from water reservoirs. Larvae were reared in the laboratory to the adult stage and identified morphologically. PCR and sequencing of cytochrome oxidase 1 (COI) and internal transcribed spacer 2 (ITS2) loci were performed. Basic Local Alignment Search Tool (BLAST) was used to compare sample sequences to sequences in the NCBI nucleotide database for species identification. To further analyze the relationship between the specimen we collected in Kebri Dehar and other Anopheles samples available in Genbank, phylogenetic analysis was performed using a maximum likelihood approach. Molecular and morphological results confirm specimens were An. stephensi. The closest high scoring hit was for all specimens was for the An. stephensi sequence. Independent phylogenetic analyses of COI and ITS2 sequences revealed in both cases strong bootstrap (100) support for our sequence forming a clade with other An. stephensi sequences to the exclusion of any other species of Anopheles. In conclusion, Anopheles stephensi is present in Kebri Dehar town in Ethiopia. These findings highlight the need for additional research to examine the role of An. stephensi in malaria transmission in Ethiopia.

Glucose-6-Phosphate Dehydrogenase Deficiency Genetic Variants in Malaria Patients in Southwestern Ethiopia.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked erythrocyte enzyme disorder with relevance to malaria treatment policy. Treatment with the antimalarial primaquine can result in hemolytic anemia in G6PD-deficient patients. With increased interest in primaquine use, it is important to identify G6PD variants in Ethiopia to inform malaria treatment policy. In the present study, mutations in the G6PD gene are identified in a sample of patients with malaria in Jimma town in southwest Ethiopia. Plasmodium species of infection were confirmed using polymerase chain reaction (PCR) and gel electrophoresis. PCR and Sanger sequencing were performed to observe a portion of the G6PD gene where the common G6PD mutations (A376G, G202A, and C563T) are found. Molecular analysis revealed that most of the samples were single Plasmodium vivax infections (83.7%). For G6PD genotyping, A376G was detected in 23.26% of individuals, whereas G202A and C563T were absent. Three other uncommon mutations were identified: rs782669677 (535G→A), rs370658483, (485 + 37 G→T), and a new mutation at chrX:154535443(C→T). Bioinformatic analysis of these mutations' potential functional impact suggests minimal effect on protein function. The discovery of both common and uncommon G6PD mutations contributes to the discussion on G6PD deficiency and appropriate primaquine treatment in Ethiopia.