Blood meal profile and positivity rate with malaria parasites among different malaria vectors in Sudan.

BACKGROUND: Malaria is a life-threatening public health problem globally with particularly heavy burden in the sub-Saharan Africa including Sudan. The understanding of feeding preference of malaria vectors on different hosts is a major challenge for hindering the transmission cycle of malaria. In this study, blood meals taken by blood-fed Anopheles mosquitoes collected from the field in malaria endemic areas of Sudan were analysed for source of blood meal and malaria parasite presence. METHODS: Anopheles mosquitoes were collected from different regions in Sudan: Khartoum state, Sennar state, Northern state, and El Gedarif state between September 2020 and February 2021. Anopheles mosquitoes were collected using the standard pyrethrum spray catch and back-pack aspirator. Mosquito samples were sorted and morphologically identified to species level using international identification keys. Morphologically identified mosquito species were also confirmed using PCR. Genomic DNA was extracted from mosquitoes for molecular identification of blood meal source and parasite detection. The presence of Plasmodium species DNA in each mosquito sample was investigated using semi-nested PCR. Frequency of each blood meal source, Anopheles mosquito vector, and malaria parasite detected was calculated. Positivity rate of each fed female Anopheles mosquito was calculated for each species. RESULTS: A total of 2132 Anopheles mosquitoes were collected. 571 (26.8%) were males and 1561 (73.2%) were females classified based on their abdominal status into 1048 (67.1%) gravid, 274 (17.6%) fed, and 239 (15.3%) unfed females. Among the blood fed Anopheles mosquitoes, 263 (96.0%) were morphologically identified and confirmed using PCR to Anopheles arabiensis, 9 (3.3%) to Anopheles stephensi, and 2 (0.7%) to Anopheles rufipes. Of 274 blood-fed An. arabiensis, 68 (25.9%) fed on mixed blood meals from human and cattle, 8 (3.0%) fed on cattle and goat, and 13 (4.8%) fed on human, cattle and goat. For single blood meal sources, 70 (26.6%) fed on human, 95 (36.1%) fed on cattle, 8 (3.0%) fed on goat, and 1 (0.4%) fed on dog. While An. rufipes and An. stephensi fed on dog (2; 0.75%) and cattle (9; 3.3%), respectively. Plasmodium parasite detection in the blood meals showed that 25/274 (9.1%) An. arabiensis meals were positive for Plasmodium vivax and 19/274 (6.9%) An. arabiensis meals were positive for Plasmodium falciparum. The rate of positivity of An. arabiensis with any Plasmodium species was 16.7%. However, the positivity rate with P. falciparum only was 7.2%, while P. vivax was 9.5%. Both An. rufipes and An. stephensi were having positivity rates of 0.0% each. CONCLUSIONS: This study which was mainly on blood-fed Anopheles mosquitoes showed a diversity in the type of diet from human, cattle, and goat. Anopheles mosquitoes especially An. arabiensis in Sudan, are opportunistic blood feeders and can feed broadly on both human and cattle. The application of blood meal identification is not only important in malaria vector epidemiological surveillance but also is very useful in areas where arthropods exhibit zoophilic feeding behaviour for mammals.

Assessment of genetic diversity of Plasmodium falciparum circumsporozoite protein in Sudan: the RTS,S leading malaria vaccine candidate.

BACKGROUND: The currently used malaria vaccine, RTS,S, is designed based on the Plasmodium falciparum circumsporozoite protein (PfCSP). The pfcsp gene, besides having different polymorphic patterns, can vary between P. falciparum isolates due to geographical origin and host immune response. Such aspects are essential when considering the deployment of the RTS,S vaccine in a certain region. Therefore, this study assessed the genetic diversity of P. falciparum in Sudan based on the pfcsp gene by investigating the diversity at the N-terminal, central repeat, and the C-terminal regions. METHODS: A cross-sectional molecular study was conducted; P. falciparum isolates were collected from different health centres in Khartoum State between January and December 2019. During the study period, a total of 261 febrile patients were recruited. Malaria diagnosis was made by expert microscopists using Giemsa-stained thick and thin blood films. DNA samples were examined by the semi-nested polymerase chain reaction (PCR). Single clonal infection of the confirmed P. falciparum cases, were used to amplify the pfcsp gene. The amplified amplicons of pfcsp have been sequenced using the Sanger dideoxy method. The obtained sequences of pfcsp nucleotide diversity parameters including the numbers of haplotypes (Hap), haplotypes diversity (Hapd), the average number of nucleotide differences between two sequences (p), and the numbers of segregating sites (S) were obtained. The haplotype networks were constructed using the online tcsBU software. Natural selection theory was also tested on pfcsp using Fuand Li's D, Fuand Li's F statistics, and Tajima's D test using DnaSP. RESULTS: In comparison with the different pfcsp reference strains, the Sudanese isolates showed high similarity with other African isolates. The results of the N-terminal region showed the presence of 2 different haplotypes with a Hapd of 0.425 ± 0.00727. The presence of the unique insertion of NNNGDNGREGKDEDKRDGNN was reported. The KLKQP motif was conserved in all the studied isolates. At the central repeat region, 11 haplotypes were seen with a Hapd of 0.779 ± 0.00097. The analysis of the genetic diversity in the C-terminal region showed the presence of 10 haplotypes with a Hapd of 0.457 ± 0.073. Several non-synonymous amino acids changes were also seen at the Th2R and the Th3R T-cell epitope regions including T317K, E317K, Q318E, K321N, I322K, T322K, R322K, K324Q, I327L, G352N, S354P, R355K, N356D, Q357E, and E361A. CONCLUSIONS: In this study, the results indicated a high conservation at the pfcsp gene. This may further contribute in understanding the genetic polymorphisms of P. falciparum prior to the deployment of the RTS,S vaccine in Sudan.

Prevalence of malaria and dengue co-infections among febrile patients during dengue transmission season in Kassala, eastern Sudan.

BACKGROUND: Malaria and dengue are common mosquito-borne diseases around the world that cause high mortality and morbidity. The number of cases of both diseases is currently rising in Sudan and is associated with climate and environmental changes. Limited information is available on malaria and dengue co-infections and the severity of the two diseases among febrile patients in eastern Sudan. Thus, this study aimed to estimate the prevalence of malaria and dengue co-infections among febrile patients in Kassala, eastern Sudan. METHODOLOGY/PRINCIPAL FINDINGS: A cross-sectional hospital-based study was conducted among febrile patients from September to December 2019. A total of 395 patients were enrolled after consenting to participate in the study. Demographic and clinical data were collected by structured questionnaires. Blood samples were provided to diagnose malaria infections using microscopy and polymerase chain reaction (PCR) and for serology diagnosis of dengue using enzyme-linked immune sorbent assay (ELISA) IgM. Multiple logistic regression analysis was used to assess the association between demographic information, clinical symptoms and malaria and dengue co-infections. Out of 395 febrile patients examined 158 (40%) were malaria positive and 67 (17%) were dengue positive. The prevalence of malaria and dengue co-infections was 6.6% (26/395). Results of multiple logistic regression indicated that elder patients (41-60 years) had less rate of co-infections (OR = 0.3, 95% CI 0.11 to 0.81, p-value = 0.018), while patients of co-infections were eight times more likely to have fatigue, and two times more likely to suffer from joint and muscle pain and this difference was statistically significant with (OR = 8.3, 95% CI: 1.89 to 37.22, p-value = 0.005) and (OR = 2.4, 95% CI 1.10 to 5.39, p-value = 0.027), respectively. CONCLUSIONS/SIGNIFICANCE: This study confirmed the existence of malaria and dengue co-infections among febrile patients in Kassala, eastern Sudan for the first time. The severity of clinical symptoms of patients with malaria and dengue co-infections was observed, and the co-infections were found prevalent among young people.

The Phylodynamic and Spread of the Invasive Asian Malaria Vectors, Anopheles stephensi, in Sudan.

Anopheles stephensi is an invasive Asian malaria vector that initially emerged in Africa in 2012 and was reported in Sudan in 2019. We investigated the distribution and population structure of An. stephensi throughout Sudan by using sequencing and molecular tools. We confirmed the presence of An. stephensi in eight border-states, identifying both natural and human-made breeding sites. Our analysis revealed the presence of 20 haplotypes with different distributions per state. This study revealed a countrywide spread of An. stephensi in Sudan, with confirmed presence in borders states with Chad, Egypt, Eritrea, Ethiopia, Libya, Republic of Central Africa, and South Sudan. Detection of An. stephensi at points of entry with these countries, particularly Chad, Libya, and South Sudan, indicates the rapid previously undetected spread of this invasive vector. Our phylogenetic and haplotype analysis suggested local establishment and evolutionary adaptation of the vector to different ecological and environmental conditions in Sudan. Urgent engagement of the global community is essential to control and prevent further spread into Africa.

Seroprevalence and associated risk factors of Dengue fever in Kassala state, eastern Sudan.

Dengue is a rapidly growing public health threat in Kassala state, eastern Sudan. The objective of this study was to determine the seroprevalence, entomological transmission indices, and socioeconomic risk factors associated with dengue in this region. A cross-sectional community-based study was conducted in four dengue-endemic sites; Khatmia, West Gash, Thoriba, and Shokriya between March 2016 to March 2017. Enzyme-linked immunosorbent assay (ELISA) of immunoglobulin G (IgG) was used to determine the prevalence of dengue virus among the study participants. An entomological survey was conducted using pyrethrum spray catch and dipping for the collection of adults and aquatic stages of Aedes aegypti, respectively. Ribonucleic acid was extracted from the buffy coat of participants as well as from adult female Ae. aegypti to assess the possible circulation of dengue virus using Reverse Transcription Polymerase Chain Reaction (RT-PCR). Multiple logistic regression model was used to estimate the association between potential risk factors and dengue seropositivity. A total of 409 persons were recruited to the study: 45.5% were in the 20-39 years' age category; 57.9% were living in houses with 6-10 persons; and 29.1% had at most secondary school education. In the majority (65.8%) of the households, the socioeconomic status was low (P<0.001). Long-lasting insecticide-treated bed nets were used in 56.5% of the households. Over three-quarters (77.8%) claimed not to have experienced febrile illness in the last three months. Routine entomological survey across Kassala state identified a total of 3,304 larvae and 390 pupae Ae. aegypti, respectively. The overall house index was 32.8% and Breteau Index was 35.96% (146/406). The overall pupal demographic index was 13.31%, and the pupal children index was 97.26%. Antibodies against IgG were detected from 66 (42.04%) out of a total of 157 sera. Twenty-two positive sera (75.9%) were collected from Khatmia. A total of 329 adults Ae. aegypti were identified but only one (0.3%) was positive for DENV in Khatmia. Finally, four independent risk factors were identified to derive dengue circulation in Kassala: elder age (> 60 years) (OR 6.31, CI 1.09-36.36); type of bathroom (OR 3.52, CI 1.35-9.20); using water-based air conditioner (OR 6.90, CI 1.78-26.85) and previous infection of any household member with dengue (OR 28.73, CI 3.31-249.63). Our findings suggest that Kassala state is facing an increasing occurrence of dengue and emphasizes the need for developing appropriate interventions to address the identified risk factors, and place control programs into actions. Establishment of routine dengue epidemiological and entomological surveillance, and climate warning systems will contribute to early warning and timely detection and response to emerging outbreaks.