Infection of Leishmania donovani in Phlebotomus orientalis Sand Flies at Different Microhabitats of a Kala-Azar Endemic Village in Eastern Sudan.

A study was carried out to compare the infection rates of Leishmania donovani in Phlebotomus orientalis sandflies at different microhabitats of a VL endemic village in Gedarif state, Sudan. DNA extracts of 1078 P. orientalis sand fly females sampled by CDC light traps from indoor, outdoor, peri-domestic, and sylvatic sites, in three transmission seasons, March-June 2016-18, in Helat-Belo village, were subjected to independent PCR amplifications targeting Leishmania kDNA and the cpb gene followed by ITS1 region sequencing. Leishmania kDNA was detected in 1.4% of the 1078 P. orientalis females captured in the area. Two of these specimens showed a characteristic 741 bp band of L. donovani after cpb gene amplification. The DNA sequence of the ITS1 region of the parasites matched the ITS1 L. donovani genotype F. There were no signficant differences between rates of infection of L. donovani in P. orientalis captured at different sites. Blood meals found in infected flies origninated from human (5 specimens), cattle (4 specimens) and donkey (2 specimens). The finding of fresh cow and donkey blood in the infected flies suggests the possible role of these animals in the zoopotentiation and/or zooprophylaxis against VL. The study provides important information for VL transmission models and control programs in East Africa.

Understanding the risk perception of visceral leishmaniasis exposure and the acceptability of sandfly protection measures among migrant workers in the lowlands of Northwest Ethiopia: a health belief model perspective.

BACKGROUND: Visceral leishmaniasis (VL) is the leading cause of health concerns among Ethiopian migrant workers. Understanding risk perception and health-protective behavior are significant challenges in the prevention and eradication of the disease. As a result, studies are required to assess these important epidemiological factors, which will provide guidance on how to assist migrant workers in taking preventive measures against VL. METHOD: We conducted qualitative research among migrant workers on seasonal agricultural farms in Northwest Ethiopia between June and November 2019 to assess their perception of the risk of contracting VL and their willingness to use protective measures against the disease. Seventeen focus group discussions and 16 key informant interviews were conducted to study migrant workers' risk perception in relation to sandfly bite exposure and use of sandfly control measures. For analysis, all interviews were recorded, transcribed, and translated. ATLASti was used to perform qualitative content analysis on the data. RESULT: Migrant workers are fearful of VL because of previous exposure and the disease's prevalence in the area. They believe, however, that VL is a minor illness that is easily treated. While Insecticide Treated Nets (ITNs) are widely accepted as a protective measure, there are still reservations about using them due to the seasonality of the transmission, difficulties in hanging them on farm areas, and a preference for alternative traditional practices. Regardless of perceived self-efficacy, the central cues were the message delivered by the health workers and an increase in sandfly bite irritation. Based on the findings, three levels of intervention modalities are suggested: 1) increasing pre-arrival awareness through outdoor media (posters, stickers, billboards), 2) encouraging proper use of protective measures upon arrival at farm camps, and 3) informing departing workers on disease recognition and best practices for health-seeking continuous use of protective measures at home. CONCLUSION: This finding suggests that VL prevention interventions should focus on individuals' perceptions in order to promote consistent use of protective measures. The findings are highly useful in planning effective interventions against VL.

Outdoor Residual Insecticide Spraying (ODRS), a New Approach for the Control of the Exophilic Vectors of Human Visceral Leishmaniasis: Phlebotomus orientalis in East Africa.

Visceral Leishmaniasis (VL) due to Leishmania donovani is a neglected protozoan parasitic disease in humans, which is usually fatal if untreated. Phlebotomus orientalis, the predominant VL vector in East Africa, is a highly exophilic/exophagic species that poses a major challenge to current Integrated Vector Management (IVM). Here we report results of pilot studies conducted in rural villages in Gedarif state, Sudan, to evaluate outdoor residual spraying of 20mg active ingredient (a.i.) /m2 deltamethrin insecticide applied to the characteristic household compound boundary reed fence and to the outside of household buildings (Outdoor Residual Insecticide Spraying, ODRS), and as an alternative, spraying restricted to the boundary fence only (Restricted Outdoor Residual Insecticide Spraying, RODRS). Four to six clusters of 20 households were assigned to insecticide treatments or control in three experiments. Changes in sand fly numbers were monitored over 2,033 trap-nights over 43-76 days follow-up in four sentinel houses per cluster relative to unsprayed control clusters. Sand fly numbers were monitored by sticky traps placed on the ground on the inside ("outdoor") and the outside ("peridomestic") of the boundary fence, and by CDC light traps suspended outdoors in the household compound. The effects of ODRS on sand fly numbers inside sleeping huts were monitored by insecticide knockdown. After a single application, ODRS reduced P. orientalis abundance by 83%-99% in outdoor and peridomestic trap locations. ODRS also reduced numbers of P. orientalis found resting inside sleeping huts. RODRS reduced outdoor and peridomestic P. orientalis by 60%-88%. By direct comparison, RODRS was 58%-100% as effective as ODRS depending on the trapping method. These impacts were immediate on intervention and persisted during follow-up, representing a large fraction of the P. orientalis activity season. Relative costs of ODRS and RODRS delivery were $5.76 and $3.48 per household, respectively. The study demonstrates the feasibility and high entomological efficacy of ODRS and RODRS, and the expected low costs relative to current IVM practises. These methods represent novel sand fly vector control tools against predominantly exophilic/exophagic sand fly vectors, aimed to lower VL burdens in Sudan, with potential application in other endemic regions in East Africa.

Impact of Education on Knowledge and Practice of Kala Azar Preventive Measures among Seasonal and Migrant Agricultural Workers in Northwest Ethiopia.

Kala azar occurs among seasonal and migrant agricultural workers in northwest Ethiopia and accounts for almost 60% of the disease burden in the country. We conducted a quantitative study on the level of knowledge and practice of this vulnerable group in relation to kala azar transmission and acceptability of its vector control tools. A total of 403 workers were randomly selected from eight farms using a purposive sampling technique. Knowledge and practice scores were calculated based on 12 and 9 core questions, respectively. Binary logistic regression was used to identify factors associated with knowledge and practice. A large gap in knowledge of the disease and the vector was evident with 61.8%, 24.6%, and 13.6% of the workers having poor, moderate, and good levels of knowledge scores, respectively. Similarly, 95% of the seasonal workers reported poor level of use of protective measures against the bite of the sand fly vector. Good level of knowledge about kala azar and its sand fly vector was statistically associated with formal education (adjusted odds ratio [AOR] = 2.11; 95% CI = 1.17, 3.80; P < 0.05) and previous exposure to health education (AOR = 4.72; 95% CI = 1.99, 11.16; P < 0.001). Despite poor current knowledge and practice, a large proportion of the study participants showed interest in using vector control tools if made available, with 78% of the seasonal and migrant workers expressing some willingness to pay for different measures that can protect against sand fly bites. Therefore, we strongly recommend that comprehensive health education and vector control programs should be provided to these workers.

Human antibody reaction against recombinant salivary proteins of Phlebotomus orientalis in Eastern Africa.

BACKGROUND: Phlebotomus orientalis is a vector of Leishmania donovani, the causative agent of life threatening visceral leishmaniasis spread in Eastern Africa. During blood-feeding, sand fly females salivate into the skin of the host. Sand fly saliva contains a large variety of proteins, some of which elicit specific antibody responses in the bitten hosts. To evaluate the exposure to sand fly bites in human populations from disease endemic areas, we tested the antibody reactions of volunteers' sera against recombinant P. orientalis salivary antigens. METHODOLOGY/PRINCIPAL FINDINGS: Recombinant proteins derived from sequence data on P. orientalis secreted salivary proteins, were produced using either bacterial (five proteins) or mammalian (four proteins) expression systems and tested as antigens applicable for detection of anti-P. orientalis IgG in human sera. Using these recombinant proteins, human sera from Sudan and Ethiopia, countries endemic for visceral leishmaniasis, were screened by ELISA and immunoblotting to identify the potential markers of exposure to P. orientalis bites. Two recombinant proteins; mAG5 and mYEL1, were identified as the most promising antigens showing high correlation coefficients as well as good specificity in comparison to the whole sand fly salivary gland homogenate. Combination of both proteins led to a further increase of correlation coefficients as well as both positive and negative predictive values of P. orientalis exposure. CONCLUSIONS/SIGNIFICANCE: This is the first report of screening human sera for anti-P. orientalis antibodies using recombinant salivary proteins. The recombinant salivary proteins mYEL1 and mAG5 proved to be valid antigens for screening human sera from both Sudan and Ethiopia for exposure to P. orientalis bites. The utilization of equal amounts of these two proteins significantly increased the capability to detect anti-P. orientalis antibody responses.

Characterization of the early inflammatory infiltrate at the feeding site of infected sand flies in mice protected from vector-transmitted Leishmania major by exposure to uninfected bites.

BACKGROUND: Mice exposed to sand fly saliva are protected against vector-transmitted Leishmania major. Although protection has been related to IFN-γ producing T cells, the early inflammatory response orchestrating this outcome has not been defined. METHODOLOGY/PRINCIPAL FINDINGS: Mice exposed to uninfected P. duboscqi bites and naïve mice were challenged with L. major-infected flies to characterize their early immune response at the bite site. Mostly, chemokine and cytokine transcript expression post-infected bites was amplified in exposed compared to naïve mice. In exposed mice, induced chemokines were mostly involved in leukocyte recruitment and T cell and NK cell activation; IL-4 was expressed at 6 h followed by IFN-γ and iNOS2 as well as IL-5 and IL-10 expression. In naïve animals, the transcript expression following Leishmania-infected sand fly bites was suppressed. Expression profiles translated to an earlier and significantly larger recruitment of leukocytes including neutrophils, macrophages, Gr+ monocytes, NK cells and CD4+ T cells to the bite site of exposed compared to naïve mice post-infected bites. Additionally, up to 48 hours post-infected bites the number of IFN-γ-producing CD4+ T cells and NK cells arriving at the bite site was significantly higher in exposed compared to naïve mice. Thereafter, NK cells become cytolytic and persist at the bite site up to a week post-bite. CONCLUSION/SIGNIFICANCE: The quiet environment induced by a Leishmania-infected sand fly bite in naïve mice was significantly altered in animals previously exposed to saliva of uninfected flies. We propose that the enhanced recruitment of Gr+ monocytes, NK cells and CD4 Th1 cells observed at the bite site of exposed mice creates an inhospitable environment that counters the establishment of L. major infection.

The mating competence of geographically diverse Leishmania major strains in their natural and unnatural sand fly vectors.

Invertebrate stages of Leishmania are capable of genetic exchange during their extracellular growth and development in the sand fly vector. Here we explore two variables: the ability of diverse L. major strains from across its natural range to undergo mating in pairwise tests; and the timing of the appearance of hybrids and their developmental stage associations within both natural (Phlebotomus duboscqi) and unnatural (Lutzomyia longipalpis) sand fly vectors. Following co-infection of flies with parental lines bearing independent drug markers, doubly-drug resistant hybrid progeny were selected, from which 96 clonal lines were analyzed for DNA content and genotyped for parent alleles at 4-6 unlinked nuclear loci as well as the maxicircle DNA. As seen previously, the majority of hybrids showed '2n' DNA contents, but with a significant number of '3n' and one '4n' offspring. In the natural vector, 97% of the nuclear loci showed both parental alleles; however, 3% (4/150) showed only one parental allele. In the unnatural vector, the frequency of uniparental inheritance rose to 10% (27/275). We attribute this to loss of heterozygosity after mating, most likely arising from aneuploidy which is both common and temporally variable in Leishmania. As seen previously, only uniparental inheritance of maxicircle kDNA was observed. Hybrids were recovered at similar efficiencies in all pairwise crosses tested, suggesting that L. major lacks detectable 'mating types' that limit free genetic exchange. In the natural vector, comparisons of the timing of hybrid formation with the presence of developmental stages suggest nectomonads as the most likely sexually competent stage, with hybrids emerging well before the first appearance of metacyclic promastigotes. These studies provide an important perspective on the prevalence of genetic exchange in natural populations of L. major and a guide for experimental studies to understand the biology of mating.

Evidence for genetic differentiation at the microgeographic scale in Phlebotomus papatasi populations from Sudan.

BACKGROUND: Cutaneous Leishmaniasis (CL) is endemic in Sudan. It is caused by Leishmania major parasites and transmitted by Phlebotomus papatasi sandflies. Recently, uncommon clinical manifestations of CL have been reported. Moreover, L. donovani parasites that cause Visceral Leishmaniasis (VL) have been isolated from CL lesions of some patients who contracted the disease in Khartoum State, Central Sudan with no history of travelling to VL endemic sites on south-eastern Sudan. Because different clinical manifestations and the parasite behaviour could be related to genetic differentiation, or even sub-structuring within sandfly vector populations, a population genetic study was conducted on P. papatasi populations collected from different localities in Khartoum State known for their uncommon CL cases and characterized by contrasting environmental conditions. METHODS: A set of seven microsatellite loci was used to investigate the population structure of P. papatasi samples collected from different localities in Khartoum State, Central Sudan. Populations from Kassala State, Eastern Sudan and Egypt were also included in the analyses as outgroups. The level of genetic diversity and genetic differentiation among natural populations of P. papatasi was determined using FST statistics and Bayesian assignments. RESULTS: Genetic analyses revealed significant genetic differentiation (FST) between the Sudanese and the Egyptian populations. Within the Sudanese P. papatasi populations, one population from Gerif West, Khartoum State, exhibited significant genetic differentiation from all other populations including those collected as near as 22 km. CONCLUSION: The significant genetic differentiation of Gerif West P. papatasi population from other Sudanese populations may have important implication for the epidemiology of leishmaniasis in Khartoum State and needs to be further investigated. Primarily, it could be linked to the unique location of Gerif West which is confined by the River Nile and its tributaries that may act as a natural barrier for gene flow between this site and the other rural sites. The observed high migration rates and lack of genetic differentiation among the other P. papatasi populations could be attributed to the continuous human and cattle movement between these localities.

The role of Palmyra palm trees (Borassus flabellifer) and sand fly distribution in northeastern India.

Visceral leishmaniasis (VL), known as Kala-azar in India, is a parasite transmitted by the bite of the sand fly vector Phlebotomus argentipes. Published information on the species indicates it is a poor flyer, mainly hopping and gliding. This study describes the vector as more arboreal than previously documented. Data collected indicate the ability of P. argentipes and Sergentomyia spp to attain vertical heights in Palmyra palm trees Borassus flabellifer up to 18.4 m above ground level. To determine if sand flies were either climbing the tree trunk to rest in the canopy or flying, sticky traps were set around the tree trunk and checked for captures overnight. CDC traps set in the palm tree canopy resulted in the capture of 5,067 sand flies, 3,990 of which were P. argentipes. Traps were set during daylight hours to determine if sand flies remained and rested in the canopy. A total of 128 sand flies were trapped over 29 trap days in the palm trees. With the CDC traps, 130 P. argentipes and no Sergentomyia spp were captured. The converse was true for the sticky traps set around tree trunks 3 m below the CDC traps. Of the 105 sand flies collected, only one was P. argentipes and 104 were Sergentomyia spp. As reported elsewhere, this indicates Sergentomyia spp tend to climb and hop, wheareas P. argentipes are capable of longer and more sustained flight. Data presented herein suggest that P. argentipes is more exophylic and exophagic than previously reported. These findings have implications for sand fly control.

A possible role for Phlebotomus (Anaphlebotomus) rodhaini (Parrot, 1930) in transmission of Leishmania donovani.

BACKGROUND: Visceral leishmaniasis (VL, kala azar), caused by Leishmania donovani is a major health problem in Sudan and other East African countries. In this region the only proven vectors of L. donovani are Phlebotomus orientalis in eastern Sudan, Ethiopia and Upper Nile areas of Southern Sudan and Phlebotomus martini in Ethiopia, Kenya and Southern Sudan. In this report, we present the first evidence that Phlebotomus rodhaini may also play a role in maintaining transmission of L. donovani between animal reservoir hosts in eastern Sudan. The study was conducted in a zoonotic focus of visceral leishmaniasis in Dinder National Park, eastern Sudan, where previous work showed high infection rates of L. donovani in P. orientalis. Sand flies, captured by CDC traps were dissected and examined for infection with Leishmania parasites. Parasite isolates were subjected to L. donovani specific PCR. Field experiments were also carried out to compare efficiency of rodent baited and un-baited CDC traps in collection of P. rodhaini and determine its man-biting rate. RESULTS: Three female P. rodhaini were found infected with Leishmania parasites in an astonishingly small number of flies captured in three separate field trips. Two of these isolates were typed by molecular methods as L. donovani, while the third isolate was inoculated into a hamster that was subsequently lost. Although P. rodhaini is generally considered a rare species, results obtained in this study indicate that it can readily be captured by rodent-baited traps. Results of human landing collection showed that it rarely bites humans in the area. CONCLUSION: It is concluded that P. rodhaini is a possible vector of L. donovani between animal reservoir hosts but is not responsible for infecting humans. It is suggested that the role of P. rodhaini in transmission of L. donovani in other zoonotic foci of visceral leishmaniasis in Africa should be re-examined.

Infection parameters in the sand fly vector that predict transmission of Leishmania major.

To identify parameters of Leishmania infection within a population of infected sand flies that reliably predict subsequent transmission to the mammalian host, we sampled groups of infected flies and compared infection intensity and degree of metacyclogenesis with the frequency of transmission. The percentage of parasites within the midgut that were metacyclic promastigotes had the highest correlation with the frequency of transmission. Meta-analysis of multiple transmission experiments allowed us to establish a percent-metacyclic "cutoff" value that predicted transmission competence. Sand fly infections initiated with variable doses of parasites resulted in correspondingly altered percentages of metacyclic promastigotes, resulting in altered transmission frequency and disease severity. Lastly, alteration of sand fly oviposition status and environmental conditions at the time of transmission also influenced transmission frequency. These observations have implications for transmission of Leishmania by the sand fly vector in both the laboratory and in nature, including how the number of organisms acquired by the sand fly from an infection reservoir may influence the clinical outcome of infection following transmission by bite.

Ecology and control of the sand fly vectors of Leishmania donovani in East Africa, with special emphasis on Phlebotomus orientalis.

A literature review is provided on the state of knowledge of the ecology and control of the sand fly vectors of Leishmania donovani in East Africa, with a special emphasis on Phlebotomus orientalis. Visceral leishmaniasis caused by L. donovani is a major health problem in several areas in East Africa. Studies conducted in the past 70 years identified P. orientalis Parrot and P. martini Parrot as the principal vectors of L. donovani in Sudan, Ethiopia and Kenya and P. celiae Minter as the secondary vector of the parasite in one focus in Ethiopia. Findings on sand fly fauna and other circumstantial evidence indicate that P. martini is also responsible for transmission of L. donovani in VL endemic foci of Somalia and Uganda. Several studies showed that P. orientalis occupy distinct habitat characterized by black cotton soil and Acacia seyal-Balanites aegyptiaca vegetation, whereas P. martini and P. celiae are associated with termite mounds. Little knowledge exists on effective control measures of sand fly vectors of L. donovani in East Africa. However, recent evidence showed that use of insecticide impregnated bednets and insect repellents may reduce exposure to the bites of P. orientalis.

Role of the domestic dog as a reservoir host of Leishmania donovani in eastern Sudan.

BACKGROUND: The study aims to determine the role of domestic dogs in transmission of visceral leishmaniasis in eastern Sudan. A cross-sectional survey was conducted in 10 villages along the River Rahad in eastern Sudan to elucidate the role of domestic dogs (Canis familiaris, Linnaeus, 1758) as a reservoir host of Leishmania donovani. In this study, 87 dogs were screened for infection by Leishmania donovani. Blood and lymph node samples were taken from 87 and 33 dogs respectively and subsequently screened by the Polymerase Chain Reaction (PCR) and Direct Agglutination Test (DAT) test. Additional lymph node smears were processed for microscopy and parasite culture. Host preference of the visceral leishmaniasis (VL) vector in the area, Phlebotomus orientalis, and other sandflies for the Nile rat (Arvicanthis niloticus, E. Geoffrey, 1803), the genet (Genetta genetta, Linnaeus, 1758), the mongoose (Herpeistes ichneumon, Linnaeus, 1758), and the domestic dog were determined by counting numbers of sand flies attracted to CDC traps that were baited by these animals. RESULTS: DAT on blood samples detected anti-Leishmania antibodies in 6 samples (6.9%). Two out of 87 (2.3%) blood samples tested were PCR positive, giving an amplification product of 560 bp. The two positive samples by PCR were also positive by DAT. However, none of the 33 lymph nodes aspirates were Leishmania positive when screened by microscopy, culture and genus-specific PCR. The dog-baited trap significantly attracted the highest number of P. orientalis and sand fly species (P < 0.001). This was followed by the Egyptian mongoose baited trap and less frequently by the genet baited trap. CONCLUSION: It is concluded that the results obtained from host attraction studies indicate that dog is more attractive for P. orientalis than Egyptian mongoose, common genet and Nile rat.

Impact of aerial spraying of pyrethrin insecticide on Culex pipiens and Culex tarsalis (Diptera: Culicidae) abundance and West Nile virus infection rates in an urban/suburban area of Sacramento County, California.

In response to an epidemic amplification of West Nile virus (family Flaviviridae, genus Flavivirus, WNV), the Sacramento and Yolo Mosquito and Vector Control District (SYMVCD) sprayed ultralow-volume (ULV) formulations of pyrethrin insecticide (Evergreen EC 60-6: 6% pyrethrin insecticide, 60% piperonylbutoxide; MGK, Minneapolis, MN, applied as 0.003 kg/ha [0.0025 lb/acre] ) over 218 km2 in north Sacramento and 243.5 km2 in south Sacramento on three consecutive evenings in August 2005. We evaluated the impact of this intervention in north Sacramento on the abundance and WNV infection rates of Culex pipiens L. and Culex tarsalis Coquillett. Mortality rates of caged Cx. tarsalis sentinels ranged from 0% under dense canopy to 100% in open fields. A comparison of weekly geometric mean mosquito abundance in CO2-baited traps in sprayed and unsprayed areas before and after treatment indicated a 75.0 and 48.7% reduction in the abundance of Cx. pipiens and Cx. tarsalis, respectively. This reduction was statistically significant for Cx. pipiens, the primary vector of WNV, with highest abundance in this urban area, but not for Cx. tarsalis, which is more associated with rural areas. The infection rates of WNV in Cx. pipiens and Cx. tarsalis collected from the spray zone were 8.2 and 4.3 per 1,000 female mosquitoes in the 2 wk before and the 2 wk after applications of insecticide, respectively. In comparison, WNV infection rates in Cx. pipiens and Cx. tarsalis collected at same time interval in the unsprayed zone were 2.0 and 8.7 per 1,000, respectively. Based on the reduction in vector abundance and its effects on number of infective bites received by human population, we concluded that the aerial application ofpyrethrin insecticide reduced the transmission intensity of WNV and decreased the risk of human infection.

Risk mapping of visceral leishmaniasis: the role of local variation in rainfall and altitude on the presence and incidence of kala-azar in eastern Sudan.

Visceral leishmaniasis (VL) is a vector-borne disease highly influenced by environmental factors. A model was developed for mapping the distribution and incidence of VL in Gedaref State, eastern Sudan, in relation to different environmental factors. Geographical information systems (GIS) were used to extract and map regression results for environmental variables of 190 villages in Gedaref State, including rainfall, vegetation status, soil type, altitude, distance from river, topography, wetness indexes, and average rainfall estimates. VL incidence in each village was calculated from hospital records. By use of logistic and linear multivariate regression analyses, models were developed to determine which environmental factors explain variability in VL presence and incidence. We found that average rainfall and the altitude were the best predictors of VL incidence. The resulting models were mapped by GIS software predicting both VL presence or absence and incidence at any locality in Gedaref State. The results are discussed in relation to VL control.

A marked seasonality of malaria transmission in two rural sites in eastern Sudan.

The ecology of Anopheles arabiensis and its relationship to malaria transmission was investigated in two villages in eastern Sudan. Seasonal malaria case incidence was compared with the number of vectors detected and with climatic variables. Following the end of the short rainy season in October the number of A. arabiensis detected dropped gradually until February when neither outdoor human bait trapping nor indoor spray catches revealed any mosquitoes. Vectors re-appeared in June as humidity rose with the onset of rain. Despite the apparent absence of the vector at the height of the long, hot dry season between February and May, sporadic asymptomatic malaria infections were detected in the two villages. The low endemicity of malaria in the area was reflected by the relatively low total September-December parasite and sporozoite rates (15 and 1.4%, respectively) measured in the villages. The entomological inoculation rate (EIR) was estimated to be around two to three infective bites per person per year, although heterogeneity in the transmission indices of malaria between the two villages was observed. The implications of these patterns of anopheline population dynamics for the epidemiology and control of malaria in eastern Sudan are considered.