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Background. The most prominent variant surface antigens (VSAs) of Plasmodium falciparum are the var gene-encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family, which serves as a parasite-sequestering ligand to endothelial cells. In this study we have examined the antibody reactivity of autologous plasma from symptomatic and asymptomatic malaria infected children against the infected erythrocytes’ surface antigens using flow cytometry. Methods. Ethidium-bromide-labelled erythrocytic mature forms of P. falciparum parasites obtained from symptomatic and asymptomatic children were sequentially incubated with autologous plasma and fluorescein isothiocyanate-conjugated (FITC) antihuman IgG. Plasma antibody reactivity was detected by flow cytometry. Results. Asymptomatic children had more prevalence of trophozoites in peripheral blood (66%) compared to symptomatic children (16%), p=0.002. The mean percentage of infected RBCs reacting with autologous sera was 89.78 among symptomatic children compared to 79.62 among asymptomatic children (p=0.09). Moreover, the mean fluorescence intensity (MFI) in the asymptomatic was significantly higher compared to symptomatic children (p value = 0.040). Conclusion. Variant surface antigens on Plasmodium falciparum infected RBCs from symptomatic malaria children tend to be better recognized by IgG antibodies. This may suggest a role of some IgG antibodies in severity of malaria. [ABSTRACT FROM AUTHOR]

Numerous studies have indicated a strong association between amplification of the multidrug resistance-1 gene and in vivo and in vitro mefloquine resistance of Plasmodium falciparum. Although falciparum infection usually is not treated with mefloquine, incorrect diagnosis, high frequency of undetected mixed infections, or relapses of P. vivax infection triggered by P. falciparum infections expose non-P. falciparum parasites to mefloquine. To assess the consequences of such unintentional treatments on P. vivax, we studied variations in number of Pvmdr-1 (PlasmoDB accession no. PVX_080100, NCBI reference sequence NC_009915.1) copies worldwide in 607 samples collected in areas with different histories of mefloquine use from residents and from travelers returning to France. Number of Pvmdr-1 copies correlated with drug use history. Treatment against P. falciparum exerts substantial collateral pressure against sympatric P. vivax, jeopardizing future use of mefloquine against P. vivax. A drug policy is needed that takes into consideration all co-endemic species of malaria parasites. [ABSTRACT FROM AUTHOR]

Background The Erythrocyte Binding Antigen (EBA) 175 has been considered as one of the most important Plasmodium falciparum (P. falciparum) merozoite ligands that mediate invasion of the erythrocytes through their sialated receptor: Glycophorin A (GPA). The effect of the EBA 175 dimorphic alleles (F and C) on the severity of the disease is not yet fully understood. Therefore this study was designed to assess the distribution of the divergent dimorphic alleles of P. falciparum EBA-175 (F and C) in three different geographical areas in Sudan and the possible association of this dimorphism with the severity of the disease. Methods A sum of 339 field isolates of P. falciparum obtained from patients in three different geographical areas in Sudan were screened for the dimorphic alleles (F, C) of the EBA-175 using nested PCR. Results The percentage of F, C, and mixed F/C alleles were; 41%, 51%, and 8% respectively. F and C alleles showed significantly different distributions in the various geographic areas (p = 0.00). There was no significant association between malaria clinical manifestation and P. falciparum EBA-175 F and C alleles frequencies Conclusions This study showed a significant differential distribution of F and C alleles in different geographical malaria endemic areas. No significant association was observed between F and C alleles and different malaria phenotypes. [ABSTRACT FROM AUTHOR]

Background: Plasmodium vivax is the most prevalent human malaria parasite, causing serious public health problems in malaria-endemic countries. Until recently the Duffy-negative blood group phenotype was considered to confer resistance to vivax malaria for most African ethnicities. We and others have reported that P. vivax strains in African countries from Madagascar to Mauritania display capacity to cause clinical vivax malaria in Duffy-negative people. New insights must now explain Duffy-independent P. vivax invasion of human erythrocytes. Methods/Principal Findings: Through recent whole genome sequencing we obtained ≥70× coverage of the P. vivax genome from five field-isolates, resulting in ≥93% of the Sal I reference sequenced at coverage greater than 20×. Combined with sequences from one additional Malagasy field isolate and from five monkey-adapted strains, we describe here identification of DNA sequence rearrangements in the P. vivax genome, including discovery of a duplication of the P. vivax Duffy binding protein (PvDBP) gene. A survey of Malagasy patients infected with P. vivax showed that the PvDBP duplication was present in numerous locations in Madagascar and found in over 50% of infected patients evaluated. Extended geographic surveys showed that the PvDBP duplication was detected frequently in vivax patients living in East Africa and in some residents of non-African P. vivax-endemic countries. Additionally, the PvDBP duplication was observed in travelers seeking treatment of vivax malaria upon returning home. PvDBP duplication prevalence was highest in west-central Madagascar sites where the highest frequencies of P. vivax-infected, Duffy-negative people were reported. Conclusions/Significance: The highly conserved nature of the sequence involved in the PvDBP duplication suggests that it has occurred in a recent evolutionary time frame. These data suggest that PvDBP, a merozoite surface protein involved in red cell adhesion is rapidly evolving, possibly in response to constraints imposed by erythrocyte Duffy negativity in some human populations. [ABSTRACT FROM AUTHOR]

Background: In areas where visceral leishmaniasis (VL) and malaria are co-endemic, co-infections are common. Clinical implications range from potential diagnostic delay to increased disease-related morbidity, as compared to VL patients. Nevertheless, public awareness of the disease remains limited. In VL-endemic areas with unstable and seasonal malaria, vulnerability to the disease persists through all age-groups, suggesting that in these populations, malaria may easily co-occur with VL, with potentially severe clinical effects. Methods: A retrospective case-control study was performed using medical records of VL patients admitted to Tabarakallah and Gedarif Teaching Hospitals (Gedarif State) and Al`Azaza kala-azar Clinic (Sennar State), Sudan (2005-2010). Patients positively diagnosed with VL and malaria were identified as cases, and VL patients without microscopy-detectable malaria as controls. Associations between patient characteristics and the occurrence of the co-infection were investigated using logistic regression analysis. Confirmation of epidemiological outcomes was obtained with an independently collected dataset, composed by Médecins Sans Frontières (MSF) at Um-el-Kher and Kassab Hospitals, Gedarif State (1998). Results: The prevalence of malaria co-infection among VL surveyed patients ranged from 3.8 to 60.8%, with a median of 26.2%. Co-infected patients presented at hospital with deteriorated clinical pictures. Emaciation (Odds Ratio (OR): 2.46; 95% Confidence Interval (95% CI): 1.72-3.50), jaundice (OR: 2.52; 95% CI: 1.04-6.09) and moderate anemia (OR: 1.58; 95% CI: 1.10-2.28) were found to be positively associated with the co-infection, while severity of splenomegaly (OR: 0.53; 95% CI: 0.35-0.81) and, to a less extent, hepatomegaly (OR: 0.52; 95% CI: 0.27-1.01) appeared to be reduced by concomitant VL and malaria. The in-hospital case-fatality rates did not significantly differ between co- and mono-infected patients (OR: 1.13; 95% CI: 0.59-2.17). Conversely, a significantly increased mortality rate (OR: 4.38; 95% CI: 1.83-10.48) was observed by MSF amongst co-infected patients enrolled at Um-el-Kher and Kassab Hospitals, who also suffered an enhanced risk of severe anemia (OR: 3.44; 95% CI: 1.68-7.02) compared to VL monoinfections. Conclusions: In endemic VL areas with unstable seasonal malaria, like eastern Sudan, VL patients are highly exposed to the risk of developing concomitant malaria. Prompt diagnosis and effective treatment of malaria are essential to ensure that its co-infection does not result into poor prognoses. [ABSTRACT FROM AUTHOR]

Background: Malaria infection and disease exhibit microgeographic heterogeneity which if predictable could have implications for designing small-area intervention. Here, the space-time clustering of Plasmodium falciparum infections using data from repeat cross-sectional surveys in Gezira State, a low transmission area in northern Sudan, is investigated. Methods: Data from cross-sectional surveys undertaken in January each year from 1999-2009 in 88 villages in the Gezira state were assembled. During each survey, about a 100 children between the ages two to ten years were sampled to examine the presence of P. falciparum parasites. In 2009, all the villages were mapped using global positioning systems. Cluster level data were analysed for spatial-only and space-time clustering using the Bernoulli model and the significance of clusters were tested using the Kulldorff scan statistic. Results: Over the study period, 96,022 malaria slide examinations were undertaken and the P. falciparum prevalence was 8.6% in 1999 and by 2009 this had reduced to 1.6%. The cluster analysis showed the presence of one significant spatial-only cluster in each survey year and one significant space-time cluster over the whole study period. The primary spatial-only clusters in 10/11 years were either contained within or overlapped with the primary space-time cluster. Conclusion: The results of the study confirm the generally low malaria transmission in the state of Gezira and the presence of spatial and space-time clusters concentrated around a specific area in the south of the state. Improved surveillance data that allows for the analysis of seasonality, age and other risk factors need to be collected to design effective small area interventions as Gezira state targets malaria elimination. [ABSTRACT FROM AUTHOR]

Background: This study describes the laboratory evaluation of a novel diagnostic platform for malaria. The Magneto Optical Test (MOT) is based on the bio-physical detection of haemozoin in clinical samples. Having an assay time of around one minute, it offers the potential of high throughput screening. Methods: Blood samples of confirmed malaria patients from different regions of Africa, patients with other diseases and healthy non-endemic controls were used in the present study. The samples were analysed with two reference tests, i.e. an histidine rich protein-2 based rapid diagnostic test (RDT) and a conventional Pan-Plasmodium PCR, and the MOT as index test. Data were entered in 2 × 2 tables and analysed for sensitivity and specificity. The agreement between microscopy, RDT and PCR and the MOT assay was determined by calculating Kappa values with a 95% confidence interval. Results: The observed sensitivity/specificity of the MOT test in comparison with clinical description, RDT or PCR ranged from 77.2 - 78.8% (sensitivity) and from 72.5 - 74.6% (specificity). In general, the agreement between MOT and the other assays is around 0.5 indicating a moderate agreement between the reference and the index test. However, when RDT and PCR are compared to each other, an almost perfect agreement can be observed (k = 0.97) with a sensitivity and specificity of >95%. Conclusions: Although MOT sensitivity and specificity are currently not yet at a competing level compared to other diagnostic test, such as PCR and RDTs, it has a potential to rapidly screen patients for malaria in endemic as well as non-endemic countries. [ABSTRACT FROM AUTHOR]

Background In view of widespread drug resistance of malaria parasites, laboratory techniques are becoming more important to assess treatment efficacy.Availability of a fast, sensitive, reliable and simple test to detect parasite survival during and after drug treatment could help clinicians to monitor and -if needed-adjust treatment regimen(s). The use of rapid diagnostic tests (RDTs) as an alternative to monitor treatment efficacy was evaluated in a prospective assessor-blind comparative study. Methods From September 2003 to March 2004, 51 P.falciparum infected patients attending one of the participating health centers in Wad Medani-Central Sudan were treated with standard dose of chloroquine (CQ) and subsequently followed up for 28 days according to WHO in vivo protocol 2002. Parallel to microscopic examination, RDTs (i.e. Paracheck® Pf which detects histidine-rich protein II or OptiMAL® test detecting lactate dehydrogenase (PLDH) enzyme) were used during follow up to determine the presence of P. falciparum infection. Results The total number of blood samples investigated was 370 (270 from the enrolled P. falciparum malaria cases during the follow up days and 100 from the blood bank donors as a control).Microscopy showed that 118 cases were positive and 252 were negative. The Paracheck test (HRPII) showed that 149 individuals were positive and 221 negative, where as the optiMAL (pLDH) detected that 114 were positive and 256 were negative. The findings indicated that there were 31 false positives demonstrated by the Paracheck test and no false negatives with this test. In contrast, the optiMAL test resulted in only 4 false negative samples and no false positive ones. The results of Paracheck test showed that its sensitivity was 88.9%, its specificity was 91.2%, and its reliability was 80.1%. While the optiMAL test had in this study a sensitivity of 98.2%, a specificity of 98.9%and reliability of 97%. The study revealed that the CQ resistance frequencies were 43.7%, 47.9% and 41.7% (P-value = 0.001) by using microscopy, Paracheck test and optiMAL tests respectively. Conclusions The OptiMal test is the most sensitive and specific RDT for the diagnosis of malaria, and for monitoring antimalarial drugs efficacy. While the Paracheck test is less sensitive to monitor therapeutic efficacy, but it is sensitive for the diagnosis of acute P. falciparum infection with a parasitaemia of > 100 parasites/μl of blood. [ABSTRACT FROM AUTHOR]

The direct agglutination test (DAT) based on freeze-dried (FD)Leishmania donovaniantigen was evaluated for the serodiagnosis of kala-azar in a rural setting in eastern Sudan. The performance of the FD-DAT was compared with standard liquid antigen (LQ) by testing serum samples and blood samples collected on filter paper of microscopically and PCR-confirmed VL patients, apparently healthy endemic controls and patients with other relevant infectious diseases for the region. In the present study, the FD-DAT had a sensitivity of 96.8% and a specificity of 96.2%. The LQ-DAT had a sensitivity of 91.0% and a specificity of 96.6%. A high degree of agreement (97.3%;r-value 0.94) was observed between the FD-DAT and the LQ-DAT, as well as between the FD-DAT performed on serum samples and corresponding blood samples collected on filter paper (agreement 97.8%;r-value 0.79). The FD-DAT is very suitable as diagnostic test for kala-azar in remote rural conditions as it is sensitive, specific and stable. The antigen is affordable, reproducible and available, which contributes to the sustainability of the DAT as a diagnostic test for VL. [ABSTRACT FROM AUTHOR]

An abstract of the article "Informed decision making before changing to RDT: a comparison of microscopy, Rapid Diagnostic Test and molecular techniques for the Diagnosis and Identification of Malaria Parasites in Kassala Eastern Sudan" that was presented at a conference titled "From Parasite to Prevention: Advances in the understanding of malaria" held in Edinburgh, Great Britain on October 20-22, 2010, is presented.

Background: Due to the increasing resistance of Plasmodium falciparum to chloroquine (CQ) in Sudan, a shift from CQ to artesunate combined with sulfadoxine/pyrimethamine as a first-line treatment for uncomplicated falciparum malaria was adopted in 2004. This study aimed to determine the frequency distribution of K76T and N86Y mutations in P. falciparum chloroquine resistance transporter ( pfcrt ) and P. falciparum multidrug resistance 1 ( pfmdr1 ) genes as key markers of resistance to CQ among P. falciparum isolates from patients in Nyala district of South Darfur state, west of Sudan., Methods: A descriptive, cross-sectional study was conducted among 75 P. falciparum isolates from Sudanese patients diagnosed with falciparum malaria mono-infection. Parasite DNA was extracted from dried blood spots and amplified using a nested polymerase chain reaction (PCR). Then, restriction fragment length polymorphism (RFLP) was used to detect the genetic polymorphisms in codons 76 of pfcrt and 86 of pfmdr1 . PCR-RFLP products were analyzed using 1.5% gel electrophoresis to identify the genetic polymorphisms in the studied codons. The wild-type (pfcrt K76 and pfmdr1 N86), mutant ( pfcrt 76T and pfmdr1 86Y) and mixed-type ( pfcrt K76T and pfmdr1 N86Y) alleles were expressed as frequencies and proportions., Results: The wild-type pfcrt K76 allele was observed among 34.7% of isolates and the mutant 76T allele among 20% of isolates, while the mixed-type K76T allele was observed among 45.3% of isolates. On the other hand, 54.7% of isolates harbored the wild-type pfmdr1 N86 allele and 5.3% of isolates had the mutant 86Y allele, while the mixed-type N86Y allele was observed among 40% of isolates., Conclusion: The key molecular markers associated with CQ resistance ( pfcrt 76T and pfmdr1 86Y) are still circulating in high frequency among P. falciparum isolates in South Darfur state, about twelve years after the official withdrawal of the drug as a treatment for uncomplicated falciparum malaria., Competing Interests: The authors report no conflicts of interest in this work., (© 2024 Magboul et al.)

Background: Routine molecular surveillance for imported drug-resistant malaria parasites to the USA and European Union is an important public health activity. The obtained molecular data are used to help keep chemoprophylaxis and treatment guidelines up to date for persons traveling to malaria endemic countries. Recent advances in next-generation sequencing (NGS) technologies provide a new and effective way of tracking malaria drug-resistant parasites., Methods: As part of a technology transfer arrangement between the CDC Malaria Branch and the Istituto Superiore di Sanità (ISS), Rome, Italy, the recently described Malaria Resistance Surveillance (MaRS) protocol was used to genotype 148 Plasmodium falciparum isolates from Eritrea for kelch 13 (k13) and cytochrome b (cytb) genes, molecular markers associated with resistance to artemisinin (ART) and atovaquone/proguanil (AP), respectively., Results: Spanning the full-length k13 gene, seven non-synonymous single nucleotide polymorphisms (SNPs) were found (K189N, K189T, E208K, D281V, E401Q, R622I and T535M), of which none have been associated with artemisinin resistance. No mutations were found in cytochrome b., Conclusion: All patients successfully genotyped carried parasites susceptible to ART and AP treatment. Future studies between CDC Malaria Branch and ISS are planned to expand the MaRS system, including data sharing, in an effort to maintain up to date treatment guidelines for travelers to malaria endemic countries.

The histidine-rich protein 2 of Plasmodium falciparum is the most common malaria antigen targeted by rapid diagnostic tests for the specific diagnosis of P. falciparum. Recently, pfhrp2 gene deletions have been documented in P. falciparum isolates from South America and some multiple endemic countries in Africa and Asia. Parasites with such gene deletions can produce false negative diagnostic results using HRP2-based rapid diagnostic kits. In the present work, the prevalence of P. falciparum parasites lacking pfhrp2, pfhrp3, which produces a second P. falciparum antigen that is recognized by PfHRP2 -based rapid diagnostic tests, and their flanking genes was evaluated in 135 P. falciparum isolates from Gash Barka region and in 9 isolates from Debub region, in Eritrea. In the analyzed samples, 56% (81/144) of isolates were pfhrp2/pfhrp3 positive, while 9.7% (14/144) showed deletion of exon 2 of pfhrp2 gene and 43% (62/144) of isolates lacked the pfhrp3 gene. These results suggest that the pfhrp2 and pfhrp3 deletion phenomenon is present in a considerable proportion in the study areas, thus making the HRP2/3 based rapid diagnostic tests not completely reliable for malaria diagnosis in Eritrea., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The introduction of artemisinin-based combination therapy has led to extraordinary results in malaria control, however the recent emergence of partial resistance to artemisinin therapy in Southeast Asia jeopardizes these successes. This study aimed at investigating resistance to the antimalarial drugs by evaluating the polymorphisms in the PfK13, Pfcrt and Pfmdr1 genes in Plasmodium falciparum isolates obtained from patients in Eritrea., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In Sudan, Plasmodium vivax accounts for approximately 5-10% of malaria cases. This study was carried out to determine the genetic diversity of P. vivax population from Sudan by analyzing the polymorphism of P. vivax csp (pvcsp) and pvmsp-3α genes. Blood samples (n=76) were taken from suspected malaria cases from 2012-2013 in three health centers of Eastern and Central Sudan. Parasite detection was performed by microscopy and molecular techniques, and genotyping of both genes was performed by PCR-RFLP followed by DNA sequence for only pvcsp gene (n=30). Based on microscopy analysis, 76 (%100) patients were infected with P. vivax, whereas nested-PCR results showed that 86.8% (n=66), 3.9% (n=3), and 3.9% (n=3) of tested samples had P. vivax as well as Plasmodium falciparum mono- and mixed infections, respectively. Four out of 76 samples had no results in molecular diagnosis. All sequenced samples were found to be of VK210 (100%) genotype with six distinct amino acid haplotypes, and 210A (66.7%) was the most prevalent haplotype. The Sudanese isolates displayed variations in the peptide repeat motifs (PRMs) ranging from 17 to 19 with GDRADGQPA (PRM1), GDRAAGQPA (PRM2) and DDRAAGQPA (PRM3). Also, 54 polymorphic sites with 56 mutations were found in repeat and post-repeat regions of the pvcsp and the overall nucleotide diversity (π) was 0.02149±0.00539. A negative value of dN-dS (-0.0344) was found that suggested a significant purifying selection of Sudanese pvcsp, (Z test, P<0.05). Regarding pvmsp-3α, three types were detected: types A (94.6%, 52/55), type C (3.6%, 2/55), and type B (1.8%, 1/55). No multiclonal infections were detected, and RFLP analysis identified 13 (Hha I, A1-A11, B1, and C1) and 16 (Alu I, A1-A14, B1, and C1) distinct allelic forms. In conclusion, genetic investigation among Sudanese P. vivax isolates indicated that this antigen showed limited antigenic diversity., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Plasmodium vivax is the most geographically widespread species, and its burden has been increasingly documented in Eastern and Central Sudan. P. vivax becomes the crucial challenge during elimination programs; thus an effective treatment is necessary to prevent the development and the spread of resistant parasites. Therefore, the main objective of the present study was to provide data on the prevalence of molecular markers in two genes (pvdhfr and pvdhps) associated with SP resistance after nine years of AS+SP deployment among P. vivax parasites from Eastern and Central Sudan using PCR-RFLP. During 2012-2013, a number of 66 blood spots were obtained on filter paper. The samples were collected before treatment from febrile patients who were microscopically positive for P. vivax, from three states in Eastern and Central Sudan (Gezira, Gedarif, and Kassala). Mutations were detected in three codons of pvdhfr (I13L, S58R, and S117N) and none in pvdhps. The majority of P. vivax parasites had double mutations (58R/117N, 58%) in dhfr gene, while all parasites were wild type in dhps gene. In addition, limited distinct haplotypes (n=4) were detected. In conclusion, the prevalence of mutations associated with SP resistance is low in Eastern and Central Sudan. Such information is necessary for guiding malaria control measures in the frame of Roll Back Malaria strategies for the elimination of malaria in the world., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Objective: To assess the in vitro response of Plasmodium falciparum malaria to chloroquine (CQ), sulfadoxine/pyrimethamine (SDX/PYR), Quinine (QU) and Mefloquine (MQ) and monitoring their resistance., Methods: In 1999 to 2000, an in vitro study was carried out in Wad Medani district in Sudan. The standard protocol of the WHO in vitro micro-test Mark II was used for the selection of the subjects, the collection of blood samples, the culture techniques, the examination of the post-culture blood slides and the interpretation of the results., Results: In vitro micro-test Mark II were performed on 62 Plasmodium falciparum isolates. Of these isolates, 42 produced successful schizonts growth. The data obtained showed that 29 of 42 isolates (69%) were CQ resistant with an effective concentrations (EC); EC50 = 399.621 nM, EC90 = 2754.145 nM and EC99 = 13284.967 nM to inhibit the schizont maturation, the values of SDX/PYR showed a flat regression line as an indication of in vitro reduced response with an EC50 = 0.262 nM, EC90 = 147.390 nM and EC99 = 25722.296 nM, and the response to the QU indicated only one of the 42 isolates (2.4%) was resistant with an EC50 = 150.085 nM, EC90 = 822.825 nM and EC99 = 3293.667 nM, while all the 42 isolates were sensitive to MQ with an EC50 = 190.763 nM, EC90 = 615.125 nM and EC99 = 1597.504 nM., Conclusion: The results of this study revealed a high degree of in vitro resistance to CQ and reduced in vitro response to SDX/PYR and QU, while MQ was fully sensitive. The effective concentrations to inhibit 50, 90 and 99% of the parasite maturation were determined for antimalarial drugs efficacy monitoring surveillance in Sudan.