Detection of Nonsynonymous Single Variants in Human HLA-DRB1 Exon 2 Associated with Renal Transplant Rejection.

Background: HLA-DRB1 is the most polymorphic gene in the human leukocyte antigen (HLA) class II, and exon 2 is critical because it encodes antigen-binding sites. This study aimed to detect functional or marker genetic variants of HLA-DRB1 exon 2 in renal transplant recipients (acceptance and rejection) using Sanger sequencing. Methods: This hospital-based case-control study collected samples from two hospitals over seven months. The 60 participants were equally divided into three groups: rejection, acceptance, and control. The target regions were amplified and sequenced by PCR and Sanger sequencing. Several bioinformatics tools have been used to assess the impact of non-synonymous single-nucleotide variants (nsSNVs) on protein function and structure. The sequences data that support the findings of this study with accession numbers (OQ747803-OQ747862) are available in National Center for Biotechnology Information (GenBank database). Results: Seven SNVs were identified, two of which were novel (chr6(GRCh38.p12): 32584356C>A (K41N) and 32584113C>A (R122R)). Three of the seven SNVs were non-synonymous and found in the rejection group (chr6(GRCh38.p12): 32584356C>A (K41N), 32584304A>G (Y59H), and 32584152T>A (R109S)). The nsSNVs had varying effects on protein function, structure, and physicochemical parameters and could play a role in renal transplant rejection. The chr6(GRCh38.p12):32584152T>A variant showed the greatest impact. This is because of its conserved nature, main domain location, and pathogenic effects on protein structure, function, and stability. Finally, no significant markers were identified in the acceptance samples. Conclusion: Pathogenic variants can affect intramolecular/intermolecular interactions of amino acid residues, protein function/structure, and disease risk. HLA typing based on functional SNVs could be a comprehensive, accurate, and low-cost method for covering all HLA genes while shedding light on previously unknown causes in many graft rejection cases.

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.

Assessment of Plasmodium falciparum drug resistance molecular markers from the Blue Nile State, Southeast Sudan.

BACKGROUND: Plasmodium falciparum malaria is a public health problem worldwide. Malaria treatment policy has faced periodic changes due to emergence of drug resistant parasites. In Sudan chloroquine has been replaced by artesunate and sulfadoxine/pyrimethamine (AS/SP) in 2005 and to artemether-lumefantrine (AL) in 2017, due to the development of drug resistance. Different molecular markers have been used to monitor the status of drug resistant P. falciparum. This study aimed to determine the frequency of malaria drug resistance molecular markers in Southeast Sudan. METHODS: The samples of this study were day zero dried blood spot samples collected from efficacy studies in the Blue Nile State from November 2015 to January 2016. A total of 130 samples were amplified and sequenced using illumina Miseq platform. The molecular markers included were Pfcrt, Pfmdr1, Pfdhfr, Pfdhps, Pfk13, exonuclease and artemisinin resistant (ART-R) genetic background (Pfmdr2, ferroredoxine, Pfcrt and Pfarps10). RESULTS: Resistance markers for chloroquine were detected in 25.8% of the samples as mutant haplotype Pfcrt 72-76 CVIET and 21.7% Pfmdr1 86Y. Pfdhfr mutations were detected in codons 51, 59 and 108. The ICNI double-mutant haplotype was the most prevalent (69%). Pfdhps mutations were detected in codons 436, 437, 540, 581 and 613. The SGEGA triple-mutant haplotype was the most prevalent (43%). In Pfdhfr/Pfdhps combined mutation, quintuple mutation ICNI/SGEGA is the most frequent one (29%). Six of the seven treatment failure samples had quintuple mutation and the seventh was quadruple. This was significantly higher from the adequately responsive group (P < 0.01). Pfk13 novel mutations were found in 7 (8.8%) samples, which were not linked to artemisinin resistance. Mutations in ART-R genetic background genes ranged from zero to 7%. Exonuclease mutation was not detected. CONCLUSION: In this study, moderate resistance to chloroquine and high resistance to SP was observed. Novel mutations of Pfk13 gene not linked to treatment failure were described. There was no resistance to piperaquine the partner drug of dihydroartemisinin/piperaquine (DHA-PPQ).

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.

Low risk of recurrence following artesunate-Sulphadoxine-pyrimethamine plus primaquine for uncomplicated Plasmodium falciparum and Plasmodium vivax infections in the Republic of the Sudan.

BACKGROUND: First-line schizontocidal treatment for uncomplicated malaria in the Republic of the Sudan is artesunate (total dose 12 mg/kg) plus Sulphadoxine/pyrimethamine (25/1.25 mg/kg) (AS/SP). Patients with Plasmodium vivax are also treated with 14 days primaquine (total dose 3.5 mg/kg) (PQ). The aim of this study was to assess the efficacy of the national policy. METHODS: Patients above 1 year, with microscopy-confirmed, Plasmodium falciparum and/or P. vivax malaria were treated with AS/SP. Patients with P. falciparum were randomized to no primaquine (Pf-noPQ) or a single 0.25 mg/kg dose of PQ (Pf-PQ1). Patients with P. vivax received 14 days unsupervised 3.5 mg/kg PQ (Pv-PQ14) on day 2 or at the end of follow up (Pv-noPQ). Primary endpoint was the risk of recurrent parasitaemia at day 42. G6PD activity was measured by spectrophotometry and the Accessbio Biosensor™. RESULTS: 231 patients with P. falciparum (74.8%), 77 (24.9%) with P. vivax and 1 (0.3%) patient with mixed infection were enrolled. The PCR corrected cumulative risk of recurrent parasitaemia on day 42 was 3.8% (95% CI 1.2-11.2%) in the Pf-noPQ arm compared to 0.9% (95% CI 0.1-6.0%) in the Pf-PQ1 arm; (HR = 0.25 [95% CI 0.03-2.38], p = 0.189). The corresponding risks of recurrence were 13.4% (95% CI 5.2-31.9%) in the Pv-noPQ arm and 5.3% (95% CI 1.3-19.4%) in the Pv-PQ14 arm (HR 0.36 [95% CI 0.1-2.0], p = 0.212). Two (0.9%) patients had G6PD enzyme activity below 10%, 19 (8.9%) patients below 60% of the adjusted male median. Correlation between spectrophotometry and Biosensor™ was low (rs = 0.330, p < 0.001). CONCLUSION: AS/SP remains effective for the treatment of P. falciparum and P. vivax. The addition of PQ reduced the risk of recurrent P. falciparum and P. vivax by day 42, although this did not reach statistical significance. The version of the Biosensor™ assessed is not suitable for routine use. Trial registration https://clinicaltrials.gov/ct2/show/NCT02592408.

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.

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.

A snapshot of Plasmodium falciparum malaria drug resistance markers in Sudan: a pilot study.

OBJECTIVES: Malaria infection is still known to be a worldwide public health problem, especially in tropical and sub-tropical African countries like Sudan. A pilot study conducted to describe the trend of P. falciparum drug resistance markers in 2017-2018 in comparison to CQ and AS/SP eras in Sudan. The Pfcrt, Pfmdr-1, Pfdhfr, and Pfdhps genes were investigated. Data deposited by the worldwide antimalarial resistance network was consulted, and the molecular markers previously reported from Sudan were analyzed. RESULTS: Drug molecular markers analysis was successfully done on 20 P. falciparum isolates. The Pfcrt K76 showed high frequency; 16 (80%). For the Pfmdr-1, 9 (45%) isolates were carrying the N86 allele, and 11 (55%) were 86Y allele. While the Y184F of the Pfmdr-1 showed a higher frequency of 184F compared to Y184; 16 (80%) and 4 (20%), respectively. In the Pfdhfr, 51I allele showed higher frequency compared to N51; 18 (90%) and 2 (10%), respectively. For S108N, 18 (90%) were 108 N and 2 (10%) were S108. In the Pfdhps, all isolates were carrying the mutant alleles; 437G and 540E. The frequency distribution of the Pfcrt, Pfmdr-1, Pfdhfr, Pfdhps was significantly different across the whole years in Sudan.

Frequency distribution of IL-17A G197A (rs2275913) and IL-17F A7488G (rs763780) polymorphisms among healthy Sudanese population.

OBJECTIVES: IL-17A G197A and IL-17F A7488G polymorphisms has been identified to be associated with the susceptibility to many diseases. This study aimed to investigate the frequency distribution of IL-17A G197A and IL-17F A7488G polymorphisms among healthy Sudanese population. A descriptive cross-sectional hospital-based molecular study conducted in different sites throughout Sudan. Two ml blood samples were collected from 717 healthy participants. Demographic data and the medical history of the participants were collected. RESULTS: Of the 717 participants, 355 (49.5%) were males and 362 (50.5%) were females, their mean age was 30.2 ± 17.2 and 32.2 ± 16.5, respectively. For IL-17A, the most frequent genotype detected among males and females was IL-17A heterozygote allele (AG); 215 (60.6%) and 194 (53.6%), respectively. Whereas, for IL-17F, the most frequent allele among males and females was the homozygote allele (AA); 298 (83.9%) for males and 322 (89.0%) for females. HWE for genotype distributions of IL-17A was showing statistical insignificance for IL-17A among males and females, P value 0.614. While HWE for IL-17F reached the equilibrium level, P value 0.048. The most frequent age group was those aged between 21 to 40 years; 281 (39.2%). Arab constituted the major ethnicity of the study participants; 418 (58.3%), P value 0.034.