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.

Association of interleukin-17A rs2275913 polymorphism with rheumatoid arthritis susceptibility in Sudanese population.

OBJECTIVES: Rheumatoid arthritis is a chronic inflammatory autoimmune disease. This study aimed to determine the association of interleukin-17A-197G/A polymorphism with rheumatoid arthritis in Sudanese patients. METHODS: A case-control study was conducted between March and December 2018. Clinical and demographic data of the study participants were collected and analyzed. Polymerase chain reaction restriction fragment length polymorphism molecular technique was done to investigate interleukin-17A-197G/A polymorphisms. All statistical tests were considered statistically significant when p < 0.05. RESULTS: The study population included 266 participants aged between 1 and 85 years, with an average of 40 years, classified into 85 (31.2%) cases (mean age 48.5 ± 11.3 years), and 181 (68.8%) controls (mean age 35.3 ± 15.9 years). The interleukin-17A homozygote AA genotype was more frequent among the control group compared to the case group; 95 (52.5%) and 7 (8.2%), respectively. The homozygote GG and the heterozygote AG genotypes were proportionally not different among the cases and control groups; 13 (54.2%) and 11 (45.8%), and 65 (46.4%) and 75 (53.6%), respectively. According to the distribution of interleukin-17A genotypes, a statistically significant difference was observed among cases with the interleukin-17A AA and AG genotypes, p values 0.001 and 0.004, respectively. For the association interleukin-17A genotypes and family history a negatively significant association was reported (95% confidence interval, -0.219, p value = 0.001). There was also a negatively significant association of interleukin-17A genotypes and anti-cyclic citrullinated peptide (95% confidence interval, -0.141, p value = 0.002). CONCLUSION: This study is the first study in Sudan established the association between interleukin-17A-197G/A (rs2275913) polymorphisms and susceptibly to rheumatoid arthritis. These findings appeal for further research in Sudan to investigate the exact role of IL-17A in immunopathology and disease severity among Sudanese rheumatoid arthritis.

Genetic polymorphism of the N-terminal region in circumsporozoite surface protein of Plasmodium falciparum field isolates from Sudan.

BACKGROUND: Malaria caused by Plasmodium falciparum parasite is still known to be one of the most significant public health problems in sub-Saharan Africa. Genetic diversity of the Sudanese P. falciparum based on the diversity in the circumsporozoite surface protein (PfCSP) has not been previously studied. Therefore, this study aimed to investigate the genetic diversity of the N-terminal region of the pfcsp gene. METHODS: A cross-sectional molecular study was conducted; 50 blood samples have been analysed from different regions in Sudan. Patients were recruited from the health facilities of Khartoum, New Halfa, Red Sea, White Nile, Al Qadarif, Gezira, River Nile, and Ad Damazin during malaria transmission seasons between June to October and December to February 2017-2018. Microscopic and nested PCR was performed for detection of P. falciparum. Merozoite surface protein-1 was performed to differentiate single and multiple clonal infections. The N-terminal of the pfcsp gene has been sequenced using PCR-Sanger dideoxy method and analysed to sequences polymorphism including the numbers of haplotypes (H), segregating sites (S), haplotypes diversity (Hd) and the average number of nucleotide differences between two sequences (Pi) were obtained using the software DnaSP v5.10. As well as neutrality testing, Tajima's D test, Fu and Li's D and F statistics. RESULTS: PCR amplification resulted in 1200 bp of the pfcsp gene. Only 21 PCR products were successfully sequenced while 29 were presenting multiple clonal P. falciparum parasite were not sequenced. The analysis of the N-terminal region of the PfCSP amino acids sequence compared to the reference strains showed five different haplotypes. H1 consisted of 3D7, NF54, HB3 and 13 isolates of the Sudanese pfcsp. H2 comprised of 7G8, Dd2, MAD20, RO33, Wellcome strain, and 5 isolates of the Sudanese pfcsp. H3, H4, and H5 were found in 3 distinct isolates. Hd was 0.594 ± 0.065, and S was 12. The most common polymorphic site was A98G; other sites were D82Y, N83H, N83M, K85L, L86F, R87L, R87F, and A98S. Fu and Li's D* test value was - 2.70818, Fu and Li's F* test value was - 2.83907, indicating a role of negative balancing selection in the pfcsp N-terminal region. Analysis with the global pfcsp N-terminal regions showed the presence of 13 haplotypes. Haplotypes frequencies were 79.4%, 17.0%, 1.6% and 1.0% for H1, H2, H3 and H4, respectively. Remaining haplotypes frequency was 0.1% for each. Hd was 0.340 ± 0.017 with a Pi of 0.00485, S was 18 sites, and Pi was 0.00030. Amino acid polymorphisms identified in the N-terminal region of global pfcsp were present at eight positions (D82Y, N83H/M, K85L/T/N, L86F, R87L/F, A98G/V/S, D99G, and G100D). CONCLUSIONS: Sudanese pfcsp N-terminal region was well-conserved with only a few polymorphic sites. Geographical distribution of genetic diversity showed high similarity to the African isolates, and this will help and contribute in the deployment of RTS,S, a PfCSP-based vaccine, in Sudan.

First detection of Echinococcus granulosus sensu stricto (G1) in dogs in central Sudan.

Eighty-four stray dogs shot as a part of a governmental rabies control program in two neighboring towns of central Sudan were examined for the presence of Echinococcus spp. and other intestinal helminths. Echinococcus worms were identified to species level by PCR and gene sequencing. For comparative reasons, rectal content of the necropsied dogs was examined for helminth eggs and subjected to copro-PCR for Echinococcus. At necropsy, 51.2% (43/84) of the dogs harbored Echinococcus canadensis (G6/7) worms with worm burdens ranging from 22,000 to 80,000. Dipylidiun caninum was found in 53.6% of the dogs. At coproscopy, taeniid eggs were found in 37 of the 43 dogs which were positive for Echinococcus at necropsy, but none in the 41 necropsy-negative dogs. In addition, 58% of the rectal samples contained eggs of Toxocara spp., 34.5% eggs of Trichuris spp. (34.5%), and 26% eggs of Ancylostoma caninum. Copro-PCR gave positive results for E. canadensis with 97.5% (39/40) of nonhibiting samples from the necropsy positive dogs; the one remaining dog tested positive for E. granulosus sensu stricto (G1), whose partial cox1 and nad1 sequences showed a 100% identity with various reference sequences of the G1 genotype. 100% of 38 non-inhibited samples taken from the necropsy-negative dogs were also negative in copro-PCR. This is the first study which combines prevalence and genetic identification of Echinococcus spp. in dogs of Sudan. Together with a recent report from cattle, it confirms the autochthonous presence, at low level, of E. granulosus sensu stricto in Central Sudan.

A molecular survey on cystic echinococcosis in Sinnar area, Blue Nile state (Sudan).

BACKGROUND: Cystic echinococcosis (CE) is a zoonosis caused by the cestodes of the Echinococcus species. Its life cycle involves dogs and other canids as definitive hosts for the intestinal tapeworm, as well as domestic and wild ungulates as intermediate hosts for the tissue-invading metacestode (larval) stage. The disease has a special impact on disadvantaged pastoralist communities and is listed now among the three top priority neglected tropical disease (NTD). Therefore, CE is a neglected disease even in high endemicity regions. This study aimed at investigation of the prevalence of CE in different animals slaughtered for food consumption in Sinnar area, Blue Nile states in Sudan. METHODS: A survey of CE in livestock was conducted from April 2009 to March 2011 in Sinnar area, Blue Nile state in Sudan. Location, parasitological status and fertility conditions were determined. In addition, 120 hydatid cysts (30 from camels, 62 from cattle and 28 from sheep) were examined by polymerase chain reaction (PCR) and mitochondrial gene sequencing for the genetic allocation of Echinococcus strains or species RESULTS: The prevalence of CE was 29.7% (30/101) in camels, 2.7% (62/2310) in cattle and 0.6% (26/4378) in sheep. It was shown that infection rates increased with age in camels, cattle and sheep. In camels, 67% (20/30) of the infected animals were aged between 2 - 5 years whereas 58% (36/62) of the infected cattle were > 5 years. In sheep, the prevalence rate was distributed equally between animals ranging 2 - 5 years and > 5 years. Even though multiple cysts were found in some animals, the average number of cysts per animal was close to 1 in all examined species. Lungs were found to be the predilection sites for the parasite in both camels and cattle, while most of the cysts found in sheep were located in the liver. About 63.4% of cysts encountered in camels were considered as large (5 - 7 cm), whereas those in cattle and sheep were medium (2 - 4 cm) and small (< 2 cm) respectively. The highest fertility rate was found in camel cysts with 85.4% (35/41) followed by cattle (50.0%, 32/64) and sheep (39.0%, 11/28). All examined cysts belonged to Echinococcus canadensis G6, which was confirmed to be the overwhelmingly predominant species in that area. CONCLUSION: The epidemiological situation in Sinnar area, Blue Nile state is characterized by intense transmission of Echinococcus canadensis G6, thereby closely resembling the situation in most other regions of Sudan.