The expansion of an invasive malaria vector: Anopheles stephensi emergence in Arba Minch town in the southern Rift Valley of Ethiopia.

Urban areas in malaria-endemic countries in East Africa are experiencing a significant increase in malaria cases, with the establishment of an "exotic" urban malaria vector, Anopheles stephensi, increasing the risk of urban malaria. To this end, the present study aimed to investigate the emergence of this species in Arba Minch, Ethiopia. Following the detection of An. stephensi in other parts of Ethiopia, 76 artificial containers (55 discarded tyres, 18 concrete water storage, and three plastic containers) were sampled in 21 locations in Arba Minch town, for immature Anopheles mosquito stages, using the standard dipping technique. Larvae were reared into adults which were morphologically identified at the species level 2-3 days after emergence. Morphological identification results were confirmed by species-specific polymerase chain reaction. Of the examined containers, 67 (88%) had at least one Anopheles larva. Thirty-two of the adults emerged were morphologically identified as An. stephensi, with 26 (81%) confirmed by molecular analysis. This is the first study to report An. stephensi from Arba Minch, one of South Ethiopia's largest towns, highlighting the need for increased vigilance. The planned and ongoing study in and around Arba Minch will contribute to understanding the bionomics and role of An. stephensi in malaria parasite transmission, helping develop a strategy to address the impending risk of urban malaria in Ethiopia.

Prevalence of malaria and associated risk factors among household members in South Ethiopia: a multi-site cross-sectional study.

BACKGROUND: Despite continuous prevention and control strategies in place, malaria remains a major public health problem in sub-Saharan Africa including Ethiopia. Moreover, prevalence of malaria differs in different geographical settings and epidemiological data were inadequate to assure disease status in the study area. This study was aimed to determine the prevalence of malaria and associated risk factors in selected rural kebeles in South Ethiopia. METHODS: A community-based cross-sectional study was conducted between February to June 2019 in eight malaria-endemic kebeles situated in four zones in South Ethiopia. Mult-stage sampling techniques were employed to select the study zones, districts, kebeles and households. Blood sample were collected from 1674 participants in 345 households by finger prick and smears were examined by microscopy. Sociodemographic data as well as risk factors for Plasmodium infection were collected using questionnaires. Bivariate and multivariate logistic regressions were used to analyse the data. RESULTS: The overall prevalence of malaria in the study localities was 4.5% (76/1674). The prevalence was varied among the study localities with high prevalence in Bashilo (14.6%; 33/226) followed by Mehal Korga (12.1%; 26/214). Plasmodium falciparum was the dominant parasite accounted for 65.8% (50/76), while Plasmodium vivax accounted 18.4% (14/76). Co-infection of P. falciparum and P. vivax was 15.8% (12/76). Among the three age groups prevalence was 7.8% (27/346) in age less than 5 years and 7.5% (40/531) in 5-14 years. The age groups > 14years were less likely infected with Plasmodium parasite (AOR = 0.14, 95% CI 0.02-0.82) than under five children. Non-febrile individuals 1638 (97.8%) were more likely to had Plasmodium infection (AOR = 28.4, 95% CI 011.4-70.6) than febrile 36 (2.2%). Individuals living proximity to mosquito breeding sites have higher Plasmodium infection (AOR = 6.17, 95% CI 2.66-14.3) than those at distant of breeding sites. CONCLUSIONS: Malaria remains a public health problem in the study localities. Thus, malaria prevention and control strategies targeting children, non-febrile cases and individuals living proximity to breeding sites are crucial to reduce malaria related morbidity and mortality.

Anopheles arabiensis continues to be the primary vector of Plasmodium falciparum after decades of malaria control in southwestern Ethiopia.

BACKGROUND: Investigating the species distribution and their role in malaria transmission is important as it varies from place to place and is highly needed to design interventions appropriate to the site. The current study aimed to investigate the Anopheles mosquito species distribution and their infection rate in southwestern Ethiopia. METHODS: The study was conducted in 14 malaria-endemic kebeles (the smallest administrative unit), which were situated in eight different malaria-endemic districts and four zones in southwestern Ethiopia. Ten per cent of households in each village were visited to collect adult mosquitoes using Centers for Disease Control and Prevention (CDC) light traps. The larval and pupal collection was done from breeding sites within the villages, and reared to adults. Female mosquitoes were morphologically identified. The head and thorax of adult Anopheles mosquitoes were tested for circumsporozoite proteins (CSPs) using ELISA. At the same time, legs, wings, and abdomen were used to identify sibling species using PCR targeting the rDNA intergenic spacers region for species typing of the Anopheles funestus group and the internal transcribed spacer 2 region genes for Anopheles gambiae complex. RESULTS: A total of 1445 Anopheles mosquitoes comprising eight species were collected. Of 813 An. gambiae complex tested by PCR, 785 (97%) were Anopheles arabiensis, and the remaining 28 (3%) were not amplified. There were 133 An. funestus group captured and tested to identify the species, of which 117 (88%) were positive for Anopheles parensis, and 15 (11%) were not amplified. A single specimen (1%) showed a band with a different base pair length from the known An. funestus group species. Sequencing revealed this was Anopheles sergentii. Among 1399 Anopheles tested for CSPs by ELISA, 5 (0.4%) An. arabiensis were positive for Plasmodium falciparum and a single (0.07%) was positive for Plasmodium vivax. CONCLUSIONS: Anopheles arabiensis continues to play the principal role in malaria transmission despite implementing indoor-based interventions for decades. Sequencing results suggest that An. sergentii was amplified by the An. funestus group primer, producing PCR amplicon size of different length. Therefore, relying solely on amplifying a specific gene of interest in grouping species could be misleading, as different species may share the same gene.

High human blood meal index of mosquitoes in Arba Minch town, southwest Ethiopia: an implication for urban mosquito-borne disease transmission.

Unplanned human population shifts in urban areas are expected to increase the prevalence of vector-borne diseases. This study aimed to investigate mosquito species composition, blood meal sources, and malaria vectors in an urban area. Indoor-resting adult mosquitoes were collected using Prokopack and host-seeking mosquitoes using Centers for Disease Control and Prevention light traps in Arba Minch town. Larval collection from artificial containers was done in those houses selected for adult mosquito collection. Anopheles adults collected and emerged from larvae were identified morphologically using a taxonomic key. ELISA was used to identify blood meal sources in freshly fed Anopheles and Culex mosquitoes, and CSP of Anopheles mosquitoes. A total of 16,756 female mosquitoes were collected. Of these, 93% (15,571) were Culex, 6% (1016) were Anopheles, and 1% (169) were Aedes mosquitoes. Out of the 130 adult mosquitoes that were raised from larvae collected from the containers, 20% were An. rhodesiensis, while the remaining 80% were Aedes mosquitoes. Out of 823 mosquitoes tested for blood meal origins, 86.3% (710/823) tested positive for human blood, 2.2% (18/823) tested positive for bovine blood, and 11.5% (95/823) were negative for human and bovine antibodies. Anopheles gambiae complex had a human blood meal index (HBI) of 50% (90/180; CI 42.3-57.5%) and a bovine blood meal index (BBI) of only 0.5% (95% CI 0.01-3.1%). Culex HBI was 96.7% (620/641), and its BBI index was 2.4% (15/641). While it was low (0.8%) in Culex, the proportion of An. gambiae complex with unidentified blood meal sources was 49.5% (95 CI% 41.9-56.9%). Among the 1016 Anopheles mosquitoes tested, a single An. gambiae complex (0.1%; 1/1016) was positive for P. vivax CSP. The high HBI indicates frequent contact between humans and vectors. To reduce human exposure, personal protection tools should be implemented.

Maize pollen diet enhances malaria mosquito longevity and infectivity to Plasmodium parasites in Ethiopia.

Although larval diet quality may affect adult mosquito fitness, its impact on parasite development is scarce. Plant pollen from Zea mays, Typha latifolia, and Prosopis juliflora was ultraviolet-sterilized and examined for effects on larval development, pupation rate, adult mosquito longevity, survival and infectivity. The control larvae were fed Tetramin fish food as a comparator food. Four treatment and two control groups were used for each pollen diet, and each experimental tray had 25 larvae. Female An. arabiensis were starved overnight and exposed to infectious blood using a membrane-feeding system. The Kaplan-Meier curves and log-rank test were used for analysis. The Z. mays pollen diet increased malaria mosquito survival and pupation rate (91.3%) and adult emergence (85%). Zea mays and Tetramin fish food had comparable adulthood development times. Adults who emerged from larvae fed Z. mays pollen had the longest average wing length (3.72 mm) and were more permissive to P. vivax (45%) and P. falciparum (27.5%). They also survived longer after feeding on infectious blood and had the highest number of P. vivax oocysts. Zea mays pollen improved larval development, adult mosquito longevity, survival and infectivity to Plasmodium. Our findings suggest that malaria transmission in Z. mays growing villages should be monitored.

Malaria misdiagnosis in the routine health system in Arba Minch area district in southwest Ethiopia: an implication for malaria control and elimination.

BACKGROUND: Plasmodium falciparum and Plasmodium vivax are coendemic in Ethiopia, with different proportion in different settings. Microscopy is the diagnostic tool in Ethiopian health centres. Accurate species-specific diagnosis is vital for appropriate treatment of cases to interrupt its transmission. Therefore, this study assessed the status of species-specific misdiagnosis by microscope compared with polymerase chain reaction (PCR). METHODS: A health facility based cross-sectional study was conducted from November 2019 to January 2020 in Kolla Shelle Health centre, Arba Minch Zuria district. The study population were suspected malaria cases, who visited the health centre for a diagnosis and treatment. Consecutive microscopy positive cases as well as a sample of microscopically negative cases were included for molecular analysis by polymerase chain reaction (PCR). RESULTS: 254 microscopically negative and 193 microscopically positive malaria suspects were included. Of the 193 malaria positive cases, 46.1% [95% confidence interval (CI) 38.9-53.4] (89/193) were P. falciparum infection, 52.3% (95% CI 45.0-59.5) (101/193) were P. vivax infection, and 1.6% (3/193) had mixed infection of P. falciparum and P. vivax. Of the microscopically positive cases of P. falciparum, 3.4% (3/89) were P. vivax and 11.2% (10/89) were mixed infections with P. falciparum and P. vivax and a single case was negative molecularly. Similarly, of the microscopically positive P. vivax cases, 5.9% (6/101) were P. falciparum and 1% (1/101) was mixed infection. Single case was negative by molecular technique. Of the 254 microscopically negative cases, 0.8% were tested positive for P. falciparum and 2% for P. vivax by PCR. Considering molecular technique as a reference, the sensitivity of microscopy for detecting P. falciparum was 89.2% and for P. vivax, it was 91.2%. The specificity of microscopy for detecting P. falciparum was 96.1% and for P. vivax, it was 97.7%. However, the sensitivity of microscopy in detecting mixed infection of P. falciparum and P. vivax was low (8.3%). CONCLUSION: There were cases left untreated or inappropriately treated due to the species misidentification. Therefore, to minimize this problem, the gaps in the microscopic-based malaria diagnosis should be identified. It is recommended to regularly monitor the competency of malaria microscopists in the study area to improve species identification and diagnosis accuracy.

Infectivity of symptomatic Plasmodium vivax cases to different generations of wild-caught and laboratory-adapted Anopheles arabiensis using a membrane feeding assay, Ethiopia.

When measuring human to mosquito transmission of Plasmodium spp., laboratory-adapted (colony) mosquitoes can be utilized. To connect transmission studies to the local epidemiology, it can be important to comprehend the relationship between infectivity in laboratory-adapted (colony) and wild-caught (wild) mosquitoes of the same species. Microscopically confirmed Plasmodium vivax cases were recruited from health facilities in Arba Minch town, and a nested polymerase chain reaction (nPCR) was used for subsequent confirmation. We performed paired membrane-feeding assays using colony An. arabiensis and three generations of wild origin An. arabiensis. Anopheles arabiensis aged 3-6 days were fed after being starved for 8-14 h. Microscopically, the oocyst development was evaluated at day 7 after feeding. Circumsporozoite proteins (CSPs) assay was carried out by enzyme-linked immunosorbent assay (ELISA). In 19 paired feeding experiments, the feeding efficiency was more than doubled in colony (median: 62.5%; interquartile range, IQR: 35-78%) than in wild mosquitoes (median: 28.5%; IQR: 17.5-40%; P < 0.001). Among the 19 P. vivax gametocyte-positive blood samples, 63.2% (n = 12) were infective to wild An. arabiensis and 73.7% (n = 14) were infective to colony An. arabiensis. The median infection rate was twice as high (26%) in the colony than in the wild (13%) An. arabiensis, although the difference was marginally insignificant (P = 0.06). Although the observed difference was not statistically significant (P = 0.19), the median number of oocysts per midgut was more than twice as high (17.8/midgut) in colony than in wild (7.2/midgut) An. arabiensis. The median feeding efficiency was 26.5% (IQR: 18-37%) in F1, 29.3% (IQR: 28-40%) in F2 and 31.2% (IQR: 30-37%) in F3 generations of wild An. arabiensis. Also, no significant difference was observed in oocyst infection rate and load between generations of wild An. arabiensis. CSP rate of P. vivax was 3.1% (3/97; 95% CI: 0.6-8.8%) in wild and 3.6% (3/84; 95% CI: 0.7-10.1%) in colony An. arabiensis. The results of the present study revealed that oocyst infection and load/midgut, and CSP rate were roughly comparable, indicating that colony mosquitoes can be employed for infectivity studies, while larger sample sizes may be necessary in future studies.

Anopheles mosquito diversity, entomological indicators of malaria transmission and challenges of morphological identification in southwestern Ethiopia.

BACKGROUND: A number of Anopheles species play either a primary or secondary role in malaria transmission. This necessitates understanding the species composition, bionomics, and behaviors of malaria mosquitoes in a particular geographic area, which is relevant to design and implement tailored intervention tools. This study aimed to assess the species composition, sporozoite infection rate, and blood meal origins of malaria mosquitoes in two malaria-endemic villages of Boreda district in Gamo Zone, southwest Ethiopia. METHODS: Thirty houses, 20 for Center for Disease Control and Prevention (CDC) light traps and 10 for Pyrethrum Spray Catches (PSC) were randomly selected for bimonthly mosquito collection from October 2019 to February 2020. An enzyme-linked immunosorbent assay (ELISA) was carried out to detect the blood meal origins and circumsporozoite proteins (CSPs). The entomological inoculation rate (EIR) was calculated by multiplying the sporozoite and human biting rates from PSCs. Anopheles gambiae complex and An. funestus group samples were further identified to species by the polymerase chain reaction (PCR). Anopheles species with some morphological similarity with An. gambiae complex or An. funestus group were tested using the primers of the two species complexes. RESULTS: A total of 14 Anopheles species were documented, of which An. demeilloni was found to be the dominant species. An. arabiensis was found to be positive for P. falciparum CSP with the overall CSP rate of 0.53% (1/190: 95% CI 0.01-2.9). The overall estimated P. falciparum EIR of An. arabiensis from PSC was 1.5 infectious bites/person/5 months. Of the 145 freshly fed Anopheles mosquitoes tested for blood meal sources, 57.9% (84/145) had bovine blood meal, 15.2% (22/145) had human blood meal origin alone, and 16.5% (24/145) had a mixed blood meal origin of human and bovine. Anopheles demeilloni were more likely to feed on blood meals of bovine origin (102/126 = 80.9%), while An. arabiensis were more likely to have blood meals of human origin. Eleven samples (2.6%; 11/420) were morphologically categorized as An. demeilloni, but it has been identified as An. leesoni (the only An. funestus group identified in the area) by PCR, though it requires additional verification by sequencing, because different species genes may have amplified for these species specific primers. Similarly, a small number of An. arabiensis were morphologically identified as An. salbaii, An. maculipalpis and An. fuscivenosus. CONCLUSIONS AND RECOMMENDATIONS: In spite of the wide variety of Anopheles mosquito species, An. arabiensis dominates indoor malaria transmission, necessitating additional interventions targeting this species. In addition, increasing entomological knowledge may make morphological identification less difficult.

Cattle feeding tendency of Anopheles mosquitoes and their infection rates in Aradum village, North Wollo, Ethiopia: an implication for animal-based malaria control strategies.

BACKGROUND: Surveillance of indoor and outdoor resting malaria vector populations is crucial to monitor possible changes in vector resting and feeding behaviours. This study was conducted to assess the resting behaviour, blood meal sources and circumsporozoite (CSP) rates of Anopheles mosquito in Aradum village, Northern Ethiopia. METHODS: Mosquito collection was conducted from September 2019 to February 2020 using clay pots (indoor and outdoor), pit shelter and pyrethrum spray catches (PSC). The species of Anopheles gambiae complex and Anopheles funestus group were identified using polymerase chain reaction (PCR). Enzyme-linked immunosorbent assay (ELISA) was done to determine CSP and blood meal sources of malaria vectors. RESULTS: A total of 775 female Anopheles mosquitoes were collected using the clay pot, PSC and pit shelter. Seven Anopheles mosquito species were identified morphologically, of which Anopheles demeilloni (593; 76.5%) was the dominant species followed by An. funestus group (73; 9.4%). Seventy-three An. funestus group screened by PCR, 91.8% (67/73) were identified as Anopheles leesoni and only 2.7% (2/73) were found to be Anopheles parensis. The molecular speciation of 71 An. gambiae complex confirmed 91.5% (65/71) of Anopheles arabiensis. The majority of Anopheles mosquitoes were collected from outdoor pit shelter (42.2%) followed by outdoor clay pots. The majority of the blood meal of An. demeilloni (57.5%; 161/280), An. funestus sensu lato 10 (43.5%) and An. gambiae (33.3%; 14/42) originated from bovine. None of the 364 Anopheles mosquitoes tested for Plasmodium falciparum and Plasmodium vivax sporozoite infections were positive. CONCLUSION: Since the Anopheles mosquitoes in the area prefer to bite cattle, it may be best to target them with an animal-based intervention. Clay pots could be an alternative tool for outdoor monitoring of malaria vectors in areas where pit shelter construction is not possible.

Outdoor and early hour human biting activities of malaria mosquitoes and the suitability of clay pot for outdoor resting mosquito collection in malaria endemic villages of southern Rift Valley, Ethiopia.

Background: Sampling adult Anopheles mosquitoes is important for assessing vector density, estimating the sporozoite infection rate, and quantifying the impact of vector control interventions. The objective of this study was to assess the Anopheles mosquito species composition, and their outdoor and indoor biting activities, and to evaluate the suitability of clay pots for indoor and outdoor resting mosquito collections. Methods: Two malaria-endemic villages in the Gamo zone were purposely selected. Forty clay pots were deployed for outdoor resting mosquitoes sampling and another forty for indoor resting sampling. Twenty pit shelters were constructed for outdoor resting mosquito collection. The human landing catch (HLC) technique was employed to collect indoor and outdoor host-seeking mosquitoes in two households in each village. Morphological identification of the Anopheles mosquito was done using an identification key. Enzyme-linked immunosorbent assay technique was used for blood meal origin and circumsporozoite proteins (CSP) test. Speciation of An. gambiae complex was done using polymerase chain reaction. A Chi-square test was used to compare the effectiveness of clay pot and pit shelters for outdoor resting sampling. Results: A total of 904 female Anopheles mosquitoes comprising An. gambiae complex, An. pharoensis, An. tenebrosus, An. dencalicus and An. demelloni were sampled. The majority (64%) of them were sampled by the HLC technique. There was a slight difference between the outdoor clay pot (19%) and pit shelter (17%) collection. No Anopheles mosquitoes were collected indoor using clay pots. All mosquitoes were tested for CSPs, but none of them were found to be positive. Anopheles mosquitoes were tending to bite humans outdoor than indoors, and their peak biting hours was 10-11 pm. The human blood meal index of Anopheles mosquitoes was 0.07 from pit shelters and it was 0.04 from clay pots. The bovine blood meal index was 0.45 for mosquitoes from both pit shelters and clay pot collections. Conclusion: Anopheles arabiensis was the predominant species and it was tending to bite cattle more than humans. Clay pot could be suitable for outdoor resting mosquito collection, but not for indoor resting species.

Evaluation of conventional and four real-time PCR methods for the detection of Leishmania on field-collected samples in Ethiopia.

In most low-resource settings, microscopy still is the standard method for diagnosis of cutaneous leishmaniasis, despite its limited sensitivity. In Ethiopia, the more sensitive molecular methods are not yet routinely used. This study compared five PCR methods with microscopy on two sample types collected from patients with a suspected lesion to advise on optimal diagnosis of Leishmania aethiopica. Between May and July 2018, skin scrapings (SS) and blood exudate from the lesion spotted on filter paper (dry blood spot, DBS) were collected for PCR from 111 patients of four zones in Southern Ethiopia. DNA and RNA were simultaneously extracted from both sample types. DNA was evaluated by a conventional PCR targeting ITS-1 and three probe-based real-time PCRs: one targeting the SSU 18S rRNA and two targeting the kDNA minicircle sequence (the 'Mary kDNA PCR' and a newly designed 'LC kDNA PCR' for improved L. aethiopica detection). RNAs were tested with a SYBR Green-based RT-PCR targeting spliced leader (SL) RNA. Giemsa-stained SS smears were examined by microscopy. Of the 111 SS, 100 were positive with at least two methods. Sensitivity of microscopy, ITS PCR, SSU PCR, Mary kDNA PCR, LC kDNA PCR and SL RNA PCR were respectively 52%, 22%, 64%, 99%, 100% and 94%. Microscopy-based parasite load correlated well with real-time PCR Ct-values. Despite suboptimal sample storage for RNA detection, the SL RNA PCR resulted in congruent results with low Ct-values. DBS collected from the same lesion showed lower PCR positivity rates compared to SS. The kDNA PCRs showed excellent performance for diagnosis of L. aethiopica on SS. Lower-cost SL RNA detection can be a complementary high-throughput tool. DBS can be used for PCR in case microscopy is negative, the SS sample can be sent to the referral health facility where kDNA PCR method is available.

Feeding behavior and activity of Phlebotomus pedifer and potential reservoir hosts of Leishmania aethiopica in southwestern Ethiopia.

BACKGROUND: Cutaneous leishmaniasis (CL) is a major public health concern in Ethiopia. However, knowledge about the complex zoonotic transmission cycle is limited, hampering implementation of control strategies. We explored the feeding behavior and activity of the vector (Phlebotomus pedifer) and studied the role of livestock in CL transmission in southwestern Ethiopia. METHODS: Blood meal origins of engorged sand flies were determined by sequencing host DNA. A host choice experiment was performed to assess the feeding preference of P. pedifer when humans and hyraxes are equally accessible. Ear and nose biopsies from livestock were screened for the presence of Leishmania parasites. Sand flies were captured indoor and outdoor with human landing catches and CDC light traps to determine at which time and where P. pedifer is mostly active. PRINCIPAL FINDINGS: A total of 180 P. pedifer sand flies were found to bite hosts of 12 genera. Humans were the predominant blood meal source indoors (65.9%, p < 0.001), while no significant differences were determined outdoors and in caves. In caves, hyraxes were represented in blood meals equally as humans (45.5% and 42.4%, respectively), but the host choice experiment revealed that sand flies have a significant preference for feeding on hyraxes (p = 0.009). Only a single goat nose biopsy from 412 animal samples was found with Leishmania RNA. We found that P. pedifer is predominantly endophagic (p = 0.003), but occurs both indoors and outdoors. A substantial number of sand flies was active in the early evening, which increased over time reaching its maximum around midnight. CONCLUSION: In contrast to earlier suggestions of exclusive zoonotic Leishmania transmission, we propose that there is also human-to-human transmission of CL in southwestern Ethiopia. Livestock does not play a role in CL transmission and combined indoor and outdoor vector control measures at night are required for efficient vector control.