Glurp Genes Research Articles

Plasmodium falciparum genetic diversity; implications for malaria control in Ethiopia: Systematic review and meta-analysis.

In malaria endemic regions, Plasmodium falciparum infection is characterized by variable genetic diversity at different settings. The parasite's various forms are found at varied frequency in different geographic areas. Understanding malaria parasite diversity and transmission is vital to evaluate control interventions. The aim of this study was under taken to determine the status of P. falciparum genetic diversity and MOI in different regions of Ethiopia. Relevant publications were identified from electronic databases such as; PubMed, EMBASE, Google scholar and Google. Besides, an online search was done using the above databases for all articles published in English on genetic diversity of P. falciparum in Ethiopia. STATA software was used for data analysis. The pooled estimates were calculated using random effect model. The summary estimates were presented using forest plots and tables. A total of 11 studies were included in the systematic review. However, only 8, 10 and 2 studies were included for Pfmsp-1, Pfmsp-2 and glurp gene meta-analysis, respectively. However, the meta-analysis result showed that the pooled prevalence of Pfmsp-1, msp-2 and glurp gene were 84% for both msp-1/2% and 51%, respectively. The pooled prevalence of msp-1 gene was higher in Amhara followed by Oromia region and lower in SNNPR while, for msp-2 gene the pooled prevalence was higher in Benshangul gumez region. Among the allelic family of msp-1 and msp-2 genes, MAD20 (34%) and FC27 (44%) were the most predominant respectively. Based on the review, there is evidence of the presence of high genetic diversity of P. falciparum parasites in Ethiopia, suggesting that malaria transmission remain high and that strengthened control efforts are needed. The approaches and methods used for investigation of diversified parasites have similarity between studies and should use advanced molecular techniques, like microsatellite, to assess the genetic diversity of P. falciparum for better results.

Therapeutic efficacy and tolerability of artemether–lumefantrine for uncomplicated Plasmodium falciparum malaria in Niger, 2020

BackgroundMonitoring therapeutic efficacy is important to ensure the efficacy of artemisinin-based combination therapy (ACT) for malaria. The current first-line treatment for uncomplicated malaria recommended by the National Malaria Control Program in Niger is artemether–lumefantrine (AL). In 2020, an in vivo study was carried out to evaluate clinical and parasitological responses to AL as well as the molecular resistance to the drug in three sentinel sites: Agadez, Tessaoua and Gaya, in Niger.MethodsA multi-center, single-arm trial was conducted according to the 28-day World Health Organization (WHO) 2009 therapeutic efficacy study protocol. Children between 6 months and 15 years with confirmed uncomplicated Plasmodium falciparum infection and 1000–200,000 asexual parasites/μL of blood were enrolled and followed up for 28 days. Uncorrected and PCR-corrected efficacy results at day 28 were calculated, and molecular correction was performed by genotyping the msp1, msp2, and glurp genes. The pfk13, pfdhfr, pfdhps, pfcrt and pfmdr genes were analyzed by PCR and Sanger sequencing. The Kaplan–Meier curve assessed parasite clearance.ResultsA total of 255 patients were enrolled in the study. The adequate clinical and parasitological response after PCR correction was 98.9% (95% CI 96.4–101.0%), 92.2% (85.0–98.5%) and 97.1% (93.1–101.0%) in Gaya, Tessaoua and Agadez, respectively. No adverse events were observed. Ten mutations (SNP) were found, including 7 synonyms (K248K, G690G, E691E, E612E, C469C, G496G, P718P) and 3 non-synonyms (N594K, R255K, V714S). Two mutations emerged: N594K and V714S. The R255K mutation detected in Southeast Asia was also detected. The pfdhpsK540E and pfdhfrI164L mutations associated with high levels of resistance are absent. There is a reversal of chloroquine resistance.ConclusionThe study findings indicate that AL is effective and well tolerated for the treatment of uncomplicated malaria in three sites in Niger. The emergence of a pfk13 mutation requires additional testing such as the Ring Stage Assay and CRISPR/Cas9 to confirm the role of these emerging mutations.Trial registration NCT05070520, October 7, 2021.

Microsatellites reveal high polymorphism and high potential for use in anti-malarial efficacy studies in areas with different transmission intensities in mainland Tanzania

BackgroundTanzania is currently implementing therapeutic efficacy studies (TES) in areas of varying malaria transmission intensities as per the World Health Organization (WHO) recommendations. In TES, distinguishing reinfection from recrudescence is critical for the determination of anti-malarial efficacy. Recently, the WHO recommended genotyping polymorphic coding genes, merozoite surface proteins 1 and 2 (msp1 and msp2), and replacing the glutamate-rich protein (glurp) gene with one of the highly polymorphic microsatellites in Plasmodium falciparum to adjust the efficacy of antimalarials in TES. This study assessed the polymorphisms of six neutral microsatellite markers and their potential use in TES, which is routinely performed in Tanzania.MethodsPlasmodium falciparum samples were obtained from four TES sentinel sites, Kibaha (Pwani), Mkuzi (Tanga), Mlimba (Morogoro) and Ujiji (Kigoma), between April and September 2016. Parasite genomic DNA was extracted from dried blood spots on filter papers using commercial kits. Genotyping was done using six microsatellites (Poly-α, PfPK2, TA1, C3M69, C2M34 and M2490) by capillary method, and the data were analysed to determine the extent of their polymorphisms and genetic diversity at the four sites.ResultsOverall, 83 (88.3%) of the 94 samples were successfully genotyped (with positive results for ≥ 50.0% of the markers), and > 50.0% of the samples (range = 47.6–59.1%) were polyclonal, with a mean multiplicity of infection (MOI) ranging from 1.68 to 1.88 among the four sites. There was high genetic diversity but limited variability among the four sites based on mean allelic richness (RS = 7.48, range = 7.27–8.03, for an adjusted minimum sample size of 18 per site) and mean expected heterozygosity (He = 0.83, range = 0.80–0.85). Cluster analysis of haplotypes using STRUCTURE, principal component analysis, and pairwise genetic differentiation (FST) did not reveal population structure or clustering of parasites according to geographic origin. Of the six markers, Poly-α was the most polymorphic, followed by C2M34, TA1 and C3M69, while M2490 was the least polymorphic.ConclusionMicrosatellite genotyping revealed high polyclonality and genetic diversity but no significant population structure. Poly-α, C2M34, TA1 and C3M69 were the most polymorphic markers, and Poly-α alone or with any of the other three markers could be adopted for use in TES in Tanzania.

Genetic diversity of Plasmodium falciparum among asymptomatic pregnant women on intermittent preventive treatment with sulfadoxine-pyrimethamine in Nigeria.

This study investigated the genetic diversity of Plasmodium falciparum among asymptomatic pregnant women on intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp-Sp) in Osogbo, southwest Nigeria. Blood sample was obtained from consenting pregnant women attending antenatal clinics. Microscopy and Polymerase chain reaction (PCR) were employed to diagnose and analyse genetic diversity. Of the 301 samples, 53 (18%) and 83 (28%) were positive for P. falciparum by microscopy and PCR, respectively. Using the merozoite surface protein (msp)-1, msp-2, and glutamate-rich protein (glurp) genes of P. falciparum as polymorphic markers, the msp-1 gene showed nine alleles with R033 (66.7%) being predominant, followed by K1 (45.5%) and MAD20 (33.3%). The msp-2 gene had 16 alleles (eight each for FC27 and 3D7). The 3D7 alleles (82.1%) was significantly more than FC27 alleles (48.2%) (p = 0.0093). Nine alleles were detected with glurp gene, presenting with the highest monoclonal and the lowest polyclonal infection. The multiplicity of infection (MOI) of 1.5, 1.8, and 1.2 were obtained for msp-1, msp-2 and glurp genes. In light of the high P. falciparum genetic diversity among pregnant women on IPT-Sp in this study, additional strategies for preventing and controlling malaria in pregnancy might be required.

High prevalence of persistent residual parasitemia on days 3 and 14 after artemether-lumefantrine or pyronaridine-artesunate treatment of uncomplicated Plasmodium falciparum malaria in Nigeria.

Microscopic evaluation of parasite clearance is the gold standard in antimalarial drug efficacy trials. However, the presence of sub-microscopic residual parasitemia after artemisinin-based combination therapy (ACT) needs to be investigated. One hundred and twenty (AL: n = 60, PA: n = 60) days 3 and 14 dried blood spots, negative by microscopy were analysed for residual parasitemia using nested PCR. Isolates with residual parasitemia on days 3 and 14 were further genotyped with their corresponding day-0 isolates using merozoite surface proteins msp-1, msp-2, and glurp genes for allelic similarity. Persistent PCR-determined sub-microscopic residual parasitemia at day 3 post ACT treatment was 83.3 (AL) and 88.3% (PA), respectively (ρ = 0.600), while 63.6 and 36.4% (ρ = 0.066) isolates were parasitemic at day 14 for AL and PA, respectively. Microscopy-confirmed gametocytemia persisted from days 0 to 7 and from days 0 to 21 for AL and PA. When the alleles of day 3 versus day 0 were compared according to base pair sizes, 59% of parasites shared identical alleles for glurp, 36% each for 3D7 and FC27, while K1 was 77%, RO33 64%, and MAD20 23%, respectively. Similarly, day 14 versus day 0 was 36% (glurp), 64% (3D7), and 32% (FC27), while 73% (K1), 77% (RO33), and 41% (MAD20), respectively. The occurrence of residual parasitemia on days 3 and 14 following AL or PA treatment may be attributable to the presence of either viable asexual, gametocytes, or dead parasite DNAs, which requires further investigation.

DNA recovery from used malaria RDT to detect Plasmodium species and to assess Plasmodium falciparum genetic diversity: a pilot study in Madagascar

BackgroundRapid diagnostic tests (RDT) are widely used for malaria diagnosis in Madagascar, where Plasmodium falciparum is the predominant species. Molecular diagnosis is essential for malaria surveillance, but requires additional blood samples for DNA extraction. Used RDTs is an attractive alternative that can be used as a source of DNA. Plasmodium falciparum genetic diversity and multiplicity of infection, usually determined by the genotyping of polymorphic regions of merozoite surface proteins 1 and 2 genes (msp1, msp2), and the repeated region RII of the glutamate-rich protein gene (glurp) have been associated with malaria transmission levels and subsequently with the impact of the deployed control strategies.Thus, the study aims to use RDT as DNA source to detect Plasmodium species, to characterize Plasmodium falciparum genetic diversity and determine the multiplicity of infection.MethodsA pilot study was conducted in two sites with different epidemiological patterns: Ankazomborona (low transmission area) and Matanga (high transmission area). On May 2018, used RDT (SD BIOLINE Malaria Ag P.f/Pan, 05FK63) were collected as DNA source. Plasmodium DNA was extracted by simple elution with nuclease free water. Nested-PCR were performed to confirm Plasmodium species and to analyse P. falciparum msp1, msp2 and glurp genes polymorphisms.ResultsAmongst the 170 obtained samples (N = 74 from Ankazomborona and N = 96 from Matanga), Plasmodium positivity rate was 23.5% (40/170) [95% CI 17.5–30.8%] by nested-PCR with 92.2% (37/40) positive to P. falciparum, 5% (2/40) to Plasmodium vivax and 2.5% (1/40) to P. falciparum/P. vivax mixed infection. Results showed high polymorphisms in P. falciparum msp1, msp2 and glurp genes. Multiple infection rate was 28.6% [95% CI 12.2–52.3%]. The mean of MOI was 1.79 ± 0.74.ConclusionThis pilot study highlighted that malaria diagnosis and molecular analysis are possible by using used malaria RDT. A large-scale study needs to be conducted to assess more comprehensively malaria parasites transmission levels and provide new data for guiding the implementation of local strategies for malaria control and elimination.Trial registration Retrospectively registered

Genetic diversity and allelic frequency of antigenic markers in Plasmodium falciparum isolates from Nnewi district in Nigeria.

The genetic diversity of Plasmodium falciparum poses a threat to the development and implementation of malaria control strategies. Thus, there is a need for continuous surveillance of its genetic diversity, especially amongst the parasite's reservoir's asymptomatic population. Three cohorts comprising children under ten years old, pregnant women and other adults were recruited into this study. Blood sample was collected from all consenting individuals and screened by the polymerase chain reaction (PCR) method. The genetic diversity of P. falciparum was determined by genotyping the merozoite surface protein-1 (msp-1), merozoite surface protein-2 (msp-2) and glutamate-rich protein (glurp). The size of alleles was visualized on the agarose gel. The multiplicity of infection (MOI) and expected heterozygosity (He) were determined. The majority of the patients showed polyclonal infections, while the multiplicity of infection with msp-2 and glurp of isolates from pregnant women were 2.5 and 1.8, respectively. Children and adults were 2.3 and 1.1; 2.4 and 1.3, respectively. The estimated number of genotypes was 10 msp-1 (4 KI; 4 MAD; 2 RO33), 27 msp-2 (14 FC27; 13 IC/3D7) and 8 glurp. K1 (36/100) was more frequent than the MAD20 (22.33/100) allele, which was, in turn, more frequent than the RO33 (13.59/100). The samples with the 3D7 allele (53.40/100) of msp-2 occurred more frequently than the FC27 type (45.63/100). Polymorphism in the glurp gene occurred most frequently (72.82/100). The study samples exhibited a high degree of genetic polymorphism in msp-2 allele typing with multiple clones, reflecting the complexity of parasite populations.

Genomic miscellany and allelic frequencies of Plasmodium falciparum msp-1, msp-2 and glurp in parasite isolates

IntroductionThe genomic miscellany of malaria parasites can help inform the intensity of malaria transmission and identify potential deficiencies in malaria control programs. This study was aimed at investigating the genomic miscellany, allele frequencies, and MOI of P. falciparum infection.MethodsA total of 85 P. falciparum confirmed isolates out of 100 were included in this study that were collected from P. falciparum patients aged 4 months to 60 years in nine districts of Khyber Pakhtunkhwa Province. Parasite DNA was extracted from 200µL whole blood samples using the Qiagen DNA extraction kit following the manufacturer’s instructions. The polymorphic regions of msp-1, msp-2 and glurp loci were genotyped using nested PCR followed by gel electrophoresis for amplified fragments identification and subsequent data analysis.ResultsOut of 85 P. falciparum infections detected, 30 were msp-1 and 32 were msp-2 alleles specific. Successful amplification occurred in 88.23% (75/85) isolates for msp-1, 78.9% (67/85) for msp-2 and 70% (60/85) for glurp gene. In msp-1, the K1 allelic family was predominantly prevalent as 66.66% (50/75), followed by RO33 and MAD20. The frequency of samples with single infection having only K1, MAD20 and RO33 were 21.34% (16/75), 8% (6/75), and 10.67% (8/75), respectively. In msp-2, both the FC27 and 3D7 allelic families revealed almost the same frequencies as 70.14% (47/67) and 67.16% (45/67), respectively. Nine glurp RII region alleles were identified in 60 isolates. The overall mean multiplicity of infection for msp genes was 1.6 with 1.8 for msp-1 and 1.4 for msp-2, while for glurp the MOI was 1.03. There was no significant association between multiplicity of infection and age groups (Spearman’s rank coefficient = 0.050; P = 0.6) while MOI and parasite density correlated for only msp-2 allelic marker.ConclusionsThe study showed high genetic diversity and allelic frequency with multiple clones of msp-1, msp-2 and glurp in P. falciparum isolates in Khyber Pakhtunkhwa, Pakistan. In the present study the genotype data may provide valuable information essential for monitoring the impact of malaria eradication efforts in this region.

High genetic diversity in Plasmodium falciparum isolates among Chinese migrant workers returnee from Africa.

Malaria is one of the most important parasitic diseases that causes a serious public health problem. The genetic diversity of malaria parasites may affect malaria transmission and malaria control strategies. In China, imported malaria was significantly increased in recent years, among which numerous migrant workers were infected with Plasmodium falciparum from Africa. However, little was known about genetic diversity of these populations in China. In this study, we evaluated genetic polymorphism and allele frequencies of msp1, msp2, and glurp genes in P. falciparum among Chinese migrant workers returnee from Africa between 2013 and 2017. Of the 381 P. falciparum isolates, 89.0% for msp1 gene, 71.7% for msp2 gene, and 78.0% for glurp gene were successfully genotyped. In msp1, 29 different alleles were observed, among which the K1 allelic family (71.7%) was predominant. In msp2, 21 different alleles were detected, of which the 3D7 allelic family (91.2%) was more frequent than FC27 allelic family (72.5%). For glurp, 12 individual alleles were detected in the samples. Taken together, the findings showed a high genetic diversity of these isolates, which provided the baseline data for African P. falciparum population imported to China.

Evaluation of pfmdr-1 Polymorphisms and Parasites’ Population Diversity in Children with Acute Uncomplicated Malaria 5 Years Post-Adoption of Artemisinin-Based Combination Therapies

Mutatons on pfmdr1 gene have been implicated in drug resistance to chloroquine and the partner drugs in artemisinin-based combination therapies (ACTs), hence the need to evaluate the impact of ACTs five years after its adoption in Nigeria on pfmdr1 polymorphisms and parasite diversity. Parasite genomic DNA was isolated from children below 5 years in Ibadan in 2010. Nested PCR followed by restriction fragment length polymorphism (RFLP) detected pfmdr1 Y86, F184 and Y1246 mutant alleles were present in 27%, 56% and 48% of the isolates respectively, while nested PCR evaluated polymorphic regions of MSP-1, MSP-2 and GLURP genes and monoclonal infections were observed in 81.6%, 51.6% and 5.6% with multiplicity of infection being 1.8, 2.0 and 2.4 respectively. This study showed a relative decline in the prevalence of Y86, F184 and Y1246 mutant alleles, but no significant change in the parasite population diversity of P.falciparum in children in Ibadan, Nigeria.

Genetic Diversity of Plasmodium falciparum Glutamate Rich Protein in Patients Attending the Merauke Hospital in Papua Province, Indonesia

Malaria remains an important health problem in Indonesia with the highest transmission in Papua Province, an easternpart of this country. The genetic diversity of malaria parasites is the main problem in understanding several aspects ofmalaria infections and the dynamics of their transmission, which also play a role in the development of a vaccine.Plasmodium falciparum is the deadliest of the human malaria parasites. Plasmodium falciparum glutamate-rich protein(Pfglurp) is one of the many erythrocytic stages antigens currently under development for a vaccine. The Pfglurp gene hasbeen extensively used as a marker to investigate the genetic diversity, Multiplicity of Infection (MOI), the level of malariatransmission, immunity against malaria, as well as a discriminatory instrument to distinguish new from recrudescentinfections of the field parasite population. Thus, this genotyping study aimed to find out the genetic population ofP.falciparum at the Merauke District, Province of Papua, Indonesia. DNA samples were isolated from Dried Blood Spots(DBS) obtained from P.falciparum infected patients in the Regional Public Hospital of Merauke, Province of Papua, Indonesiaduring May 2019-July 2019. The isolated DNAs were then amplified for nested Polymerase Chain Reaction (PCR) prior toPfglurp genotyping. The glurp gene was identified in all 51 DBS samples of P.falciparum-infected patients, and 18 variants ofallele were found. Among them, 45.10% were found to bear multigenotype infections. The size of the dominant allele(12.5%) was 701-750 bp. The MOI was 1.58. The genetic population of P.falciparum in Merauke Hospital has contained ahigher percentage of multigenotypes compared with monogenotypes indicating the high transmission of malaria in thestudied area.

Plasmodium falciparum Genetic Diversity in Panamá Based on glurp, msp-1 and msp-2 Genes: Implications for Malaria Elimination in Mesoamerica.

Panamá, together with all the nations in Mesoamerica, has committed to eliminate malaria from the region by 2020. As these countries approach malaria elimination and local transmission decreases, an active molecular surveillance to identify genotypes circulating along the border areas is particularly needed to accurately infer infection origin, drug resistance and disease propagation patterns in the region. This study evaluated the genetic diversity and allele frequencies of msp-1, msp-2 and glurp genes using different molecular analyses (nested PCR, PCR-restriction fragment length polymorphism (RFLP) and sequencing) from 106 autochthonous and imported P. falciparum isolates collected from different endemic areas in Panamá between 2003 and 2019. We also explored if P. falciparum genotypes assessed with these molecular markers were associated with relevant malaria epidemiological parameters using a multiple correspondence analysis. A strong association of certain local haplotypes with their geographic distribution in endemic areas, but also with parasite load and presence of gametocytes, was evidenced. Few multiclonal infections and low genetic diversity among locally transmitted P. falciparum samples were detected, consequent with the low transmission intensity of this parasite in Panamá, a pattern likely to be extended across Mesoamerica. In addition, several imported cases were genetically dissimilar to local infections and representative of more diverse extra-continental lineages.

Contribution of P. falciparum parasites with Pfhrp 2 gene deletions to false negative PfHRP 2 based malaria RDT results in Ghana: A nationwide study of symptomatic malaria patients

IntroductionFalse-negative malaria rapid diagnostic test (RDT) results amongst symptomatic malaria patients are detrimental as they could lead to ineffective malaria case management. This study determined the nationwide contribution of parasites with Pfhrp2 and Pfhrp 3 gene deletions to false negative malaria RDT results in Ghana.MethodsThis was a cross sectional study where whole blood (~2 ml) was collected from patients presenting with malaria symptoms at 100 health facilities in all the regions in Ghana from May to August 2018. An aliquot of the blood was used to prepare thin and thick blood smears, filter paper blood spots (DBS) and spot a PfHRP 2 RDT kit. The remaining blood was separated into plasma and blood cells and stored at -20°C. Plasmodium parasite density and species identity was estimated from the blood smears. Plasmodium falciparum specific 18S rRNA PCR, merozoite surface protein (msp 1) and glutamate rich protein (glurp) gene PCR were used to identify P. falciparum positive samples, which were subjected to Pfhrp 2/3 exon1-2 and exon2 genotyping.ResultsOf the 2,860 microscopically P. falciparum positive patients analyzed, 134 (4.69%) had false negative P. falciparum specific RDT results. Samples for PCR analysis was available for 127 of the false negative patients, and the analysis identified 116 (91.3%) as positive for P. falciparum. Only 58.1% (79/116) of the false negative RDT samples tested positive by msp 1 and glurp PCR. Genotyping of exon 1–2 and exon 2 of the Pfhrp 2 gene identified 12.9% (10/79) and 39.5% (31/79) of samples respectively to have deletions. Genotyping exon 1–2 and exon 2 of the Pfhrp 3 gene identified 15.2% (12/79) and 40.5% (32/79) of samples respectively to have deletions. Only 5% (4/79) of the false negative samples had deletions in both exon 1–2 and exon 2 of the Pfhrp 2 gene. Out of the 49 samples that tested positive for aldolase by luminex, 32.6% (16/49) and) had deletions in Pfhrp 2 exon 2 and 2% (1/49) had deletions in both exon 2 and exon 1–2 of the Pfhrp 2 gene.ConclusionsThe low prevalence of false negative RDT test results provides assurance that PfHRP 2 based malaria RDT kits remain effective in diagnosing symptomatic malaria patients across all the Regions of Ghana. Although there was a low prevalence of parasites with deletions in exon 2 and exon 1–2 of the Pfhrp 2 gene the prevalence of parasites with deletions in Pfhrp 2 exon 2 was about a third of the false negative RDT results. The need to ensure rapid, accurate and reliable malaria diagnosis requires continuous surveillance of parasites with Pfhrp 2 gene deletions.

Genetic Diversity of Plasmodium falciparum Isolated from Symptomatic and Asymptomatic Individuals in Parts of Kaduna Metropolis, Kaduna State, Nigeria

Malaria due to Plasmodium falciparum is still a major public health problem and cause of high morbidity and mortality in Nigeria despite many efforts and interventions programmes put in place to control malaria, with transmission occurring throughout the year. Characterization of P. falciparum isolates from different geographical locations of Nigeria could provide the much needed information on the genetic composition of P. falciparum natural populations with regard to three polymorphic genes: Merozoite Surface Protein 1 (MSP 1), Merozoite Surface Protein (MSP 2) and Glutamate-Rich Protein (GLURP) genes, the findings of this study will assist in adopting more strategies and intervention programmes aim at controlling P. falciparum infection in Nigeria. This study was conducted to determine the genetic diversity and elucidate possible genomic variation between isolates of P. falciparum from symptomatic and asymptomatic malaria cases in Kaduna metropolis. Plasmodium falciparum genomic DNA was extracted from 42 positive blood samples collected onto whatmani¯s filter paper using the phenol-chloroform DNA extraction method. Genotyping of P. falciparum was based on the amplification of MSP 1, MSP 2 and glurp genes. Analysis of PCR products of P. falciparum isolates shows the presence of 33 alleles spread across MSP 1, MSP 2 and glurp genes at a frequency of 5, 24 and 4 respectively. Detection of the three genes; (MSP1); MAD20 and KI, (MSP2); FC27 and 3D7/ICI and (glurp) gene and the occurrence of single and multiple genotypic infection recorded showed the diverse genetic composition of P. falciparum population, an indication of the endemicity of malaria. This information is essential for effective malaria control programme.

Genetic diversity of Plasmodium falciparum and genetic profile in children affected by uncomplicated malaria in Cameroon

BackgroundMalaria is a major public health problem in Cameroon. The study of the genetic diversity within parasite population is essential for understanding the mechanism underlying malaria pathology and to determine parasite clones profile in an infection, for proper malaria control strategies. The objective of this study was to perform a molecular characterization of highly polymorphic genetic markers of Plasmodium falciparum, and to determine allelic distribution with their influencing factors valuable to investigate malaria transmission dynamics in Cameroon.MethodsA total of 350 P. falciparum clinical isolates were characterized by genotyping block 2 of msp-1, block 3 of msp-2, and region II of glurp gene using nested PCR and DNA sequencing between 2012 and 2013.ResultsA total of 5 different genotypes with fragment sizes ranging from 597 to 817 bp were recorded for GLURP. Overall, 16 MSP-1 genotypes, including K1, MAD20 and RO33 were identified, ranging from 153 to 335 bp. A peculiarity about this study is the RO33 monomorphic pattern revealed among the Pfmsp-1 allelic type. Again, this study identified 27 different Pfmsp-2 genotypes, ranging from 140 to 568 bp in size, including 15 belonging to the 3D7-type and 12 to the FC27 allelic families. The analysis of the MSP-1 and MSP-2 peptides indicates that the region of the alignment corresponding K1 polymorphism had the highest similarity in the MSP1and MSP2 clade followed by MAD20 with 93% to 100% homology. Therefore, population structure of P. falciparum isolates is identical to that of other areas in Africa, suggesting that vaccine developed with K1 and MAD20 of Pfmsp1 allelic variant could be protective for Africa children but these findings requires further genetic and immunological investigations. The multiplicity of infection (MOI) was significantly higher (P < 0.05) for Pfmsp-2 loci (3.82), as compare with Pfmsp-1 (2.51) and heterozygotes ranged from 0.55 for Pfmsp-1 to 0.96 for Pfmsp-2.ConclusionHigh genetic diversity and allelic frequencies in P. falciparum isolates indicate a persisting high level of transmission. This study advocate for an intensification of the malaria control strategies in Cameroon.Trial registration This study was approved by Cameroon National Ethics Committee. It is a randomized controlled trial retrospectively registered in NIH U.S. National Library of Medicine, ClinicalTrials.gov on the 28/11/2016 at https://clinicaltrials.gov/ct2/show/NCT02974348 with the registration number NCT02974348

PO 8607 DETECTION OFPLASMODIUM FALCIPARUMHISTIDINE-RICH PROTEIN 2/3(PFHRP-2/PFHRP-3) GENES DELETION AND AMINO ACID NUCLEOTIDE SEQUENCE VARIABILITY IN NIGERIA

BackgroundPrompt diagnosis and appropriate treatment remain the hallmark needed to reduce malaria-related mortality in areas of high transmission. Rapid diagnostic tests (RDTs) that target the Pfhrp-2 gene, are essential in resource-limited settings where microscopy is not available. However, Pfhrp-2 gene deletion is implicated in limiting RDT sensitivity. Studies evaluating Pfhrp-2 and Pfhrp-3 deletion and the amino acid sequence diversity has not been investigated in Nigeria. We therefore hypothesised that malaria parasites in Nigeria are lacking Pfhrp-2/Pfhrp-3 genes with variable amino acid repeats sequences.MethodsThe study was part of a prospective cohort study evaluating RDTs performance. We pooled 66 samples comprising false negatives (n=31) and true positives (n=35) to elucidate Pfhrp-2/Pfhrp-3 gene deletion, RDT cross-reactivity with Pfhrp-3 antigen and amino acid sequence diversity. The 18SrRNA, msp 1, msp2 and glurp genes were amplified to establish activePlasmodium falciparuminfection and the exon-2 regions of Pfhrp-2 and Pfhrp-3 genes were amplified to determine the presence or absence of Pfhrp-2 and Pfhrp-3 genes. Isolates with conserved Pfhrp-2/Pfhrp-3 were sequenced.ResultsAll 66 samples were positive for 18SrRNA, msp1, msp2 and glurp, indicating activeP. falciparuminfection. However, 16.7% and 6.0% of the samples were lacking Pfhrp-2 and Pfhrp-3 genes. Of the false negative samples, 25.8% and 12.9% has Pfhrp-2 and Pfhrp-3 deletions. Three Pfhrp-3 conserved antigens cross reacted to give RDT positive results. An extensive diversity in the amino acid sequence was observed.ConclusionPlasmodium falciparumparasites in Nigeria lack Pfhrp-2 and Pfhrp-3 genes. However, the proportion of deletions is low compared to reports from other malaria-endemic regions. In addition, a high amino acid tandem repeat was observed. A combination of pLDH and Pfhrp-2 based RDTs is recommended for accurate malaria diagnosis.

Characterization of Plasmodium falciparum structure in Nigeria with malaria SNPs barcode

BackgroundPlasmodium falciparum malaria remains a major health challenge in Nigeria despite the global decline of its incidence and mortality rates. Although significant progress has been made in preventing the transmission of P. falciparum and controlling the spread of the infection, there is much to be done in the area of proper monitoring, surveillance of the parasite, investigating the population dynamics and drug resistance profiling of the parasite as these are important to its eventual eradication. Polymorphic loci of msp1, msp2 and/or glurp genes or microsatellites have been traditionally used to characterize P. falciparum population structure in various parts of Nigeria. The lack of standardization in the interpretation of results, as well as the inability of these methods to distinguish closely related parasites, remains a limitation of these techniques. Conversely, the recently developed 24 single nucleotide polymorphism (SNP)-based molecular barcode assay has the possibility of differentiating between closely related parasites and offer additional information in determining the population diversity of P. falciparum within and between parasite populations. This study is therefore aimed at defining the population diversity of P. falciparum in and between two localities in Nigeria using the SNPs barcode technique.MethodsThe 24-SNP high-resolution melt (HRM) barcode assay and msp2 genotyping was used to investigate both intra and inter population diversity of the parasite population in two urban cities of Nigeria.ResultsBased on SNP barcode analysis, polygenomic malaria infections were observed in 17.9% and 13.5% of population from Enugu and Ibadan, respectively, while msp2 analyses showed 21% and 19.4% polygenomic infections in Enugu and Ibadan, respectively. Low levels of genetic diversity (π) of 0.328 and 0.318 were observed in Enugu and Ibadan parasite populations, respectively, while the FST value of 0.02 (p = 0.055) was obtained when the genetic divergence of both populations was considered.ConclusionsThe 24-SNP barcode assay was effective in analysing P. falciparum population diversity. This study also showed that P. falciparum populations in Enugu and Ibadan had a degree of intra-population diversity, but very low divergence between the population. A low degree of polygenomic infections were also observed in the two parasite populations unlike previous years. This maybe as a result of the effect of artemisinin-based combination therapy (ACT), long-lasting insecticide-treated nets (LLITNs) and intermittent preventive treatments in the study populations.

Genetic diversity of the msp-1, msp-2, and glurp genes of Plasmodium falciparum isolates in Northwest Ethiopia

BackgroundDetermination of the genetic diversity of malaria parasites can inform the intensity of transmission and identify potential deficiencies in malaria control programmes. This study was conducted to characterize the genetic diversity and allele frequencies of Plasmodium falciparum in Northwest Ethiopia along the Eritrea and Sudan border.MethodsA total of 90 isolates from patients presenting to the local health centre with uncomplicated P. falciparum were collected from October 2014 to January 2015. DNA was extracted and the polymorphic regions of the msp-1, msp-2 and glurp loci were genotyped by nested polymerase chain reactions followed by gel electrophoresis for fragment analysis.ResultsAllelic variation in msp-1, msp-2 and glurp were identified in 90 blood samples. A total of 34 msp alleles (12 for msp-1 and 22 for msp-2) were detected. For msp-1 97.8% (88/90), msp-2 82.2% (74/90) and glurp 46.7% (42/90) were detected. In msp-1, MAD20 was the predominant allelic family detected in 47.7% (42/88) of the isolates followed by RO33 and K1. For msp-2, the frequency of FC27 and IC/3D7 were 77% (57/74) and 76% (56/74), respectively. Nine glurp RII region genotypes were identified. Seventy percent of isolates had multiple genotypes and the overall mean multiplicity of infection was 2.6 (95% CI 2.25–2.97). The heterozygosity index was 0.82, 0.62 and 0.20 for msp-1, msp-2 and glurp, respectively. There was no significant association between multiplicity of infection and age or parasite density.ConclusionsThere was a high degree of genetic diversity with multiple clones in P. falciparum isolates from Northwest Ethiopia suggesting that there is a need for improved malaria control efforts in this region.

Extensive diversity in the allelic frequency of Plasmodium falciparum merozoite surface proteins and glutamate-rich protein in rural and urban settings of southwestern Nigeria

BackgroundNigeria carries a high burden of malaria which makes continuous surveillance for current information on genetic diversity imperative. In this study, the merozoite surface proteins (msp-1, msp-2) and glutamate-rich protein (glurp) of Plasmodium falciparum collected from two communities representing rural and urban settings in Ibadan, southwestern Nigeria were analysed.MethodsA total of 511 febrile children, aged 3–59 months, whose parents/guardians provided informed consent, were recruited into the study. Capillary blood was obtained for malaria rapid diagnostic test, thick blood smears for parasite count and blood spots on filter paper for molecular analysis.ResultsThree-hundred and nine samples were successfully genotyped for msp-1, msp-2 and glurp genes. The allelic distribution of the three genes was not significantly different in the rural and urban communities. R033 and 3D7 were the most prevalent alleles in both rural and urban communities for msp-1 and msp-2, respectively. Eleven of glurp RII region genotypes, coded I–XII, with sizes ranging from 500 to 1100 base pairs were detected in the rural setting. Genotype XI (1000–1050 bp) had the highest prevalence of 41.5 and 38.5% in rural and urban settings, respectively. Overall, 82.1 and 70.0% of samples had multiclonal infection with msp-1 gene resulting in a mean multiplicity of infection (MOI) of 2.8 and 2.6 for rural and urban samples, respectively. Msp-1 and msp-2 genes displayed higher levels of diversity and higher MOI rates than the glurp gene.ConclusionSignificant genetic diversity was observed between rural and urban parasite populations in Ibadan, southwestern Nigeria. The results of this study show that malaria transmission intensity in these regions is still high. No significant difference was observed between rural and urban settings, except for a completely different msp-1 allele, compared to previous reports, thereby confirming the changing face of malaria transmission in these communities. This study provides important baseline information required for monitoring the impact of malaria elimination efforts in this region and data points useful in revising current protocols.

Genetic diversity and antibody responses against Plasmodium falciparum vaccine candidate genes from Chhattisgarh, Central India: Implication for vaccine development.

The genetic diversity in Plasmodium falciparum antigens is a major hurdle in developing an effective malaria vaccine. Protective efficacy of the vaccine is dependent on the polymorphic alleles of the vaccine candidate antigens. Therefore, we investigated the genetic diversity of the potential vaccine candidate antigens i.e. msp-1, msp-2, glurp, csp and pfs25 from field isolates of P.falciparum and determined the natural immune response against the synthetic peptide of these antigens. Genotyping was performed using Sanger method and size of alleles, multiplicity of infection, heterogeneity and recombination rate were analyzed. Asexual stage antigens were highly polymorphic with 55 and 50 unique alleles in msp-1 and msp-2 genes, respectively. The MOI for msp-1 and msp-2 were 1.67 and 1.28 respectively. A total 59 genotype was found in glurp gene with 8 types of amino acid repeats in the conserved part of RII repeat region. The number of NANP repeats from 40 to 44 was found among 55% samples in csp gene while pfs25 was found almost conserved with only two amino acid substitution site. The level of genetic diversity in the present study population was very similar to that from Asian countries. A higher IgG response was found in the B-cell epitopes of msp-1 and csp antigens and higher level of antibodies against csp B-cell epitope and glurp antigen were recorded with increasing age groups. Significantly, higher positive responses were observed in the csp antigen among the samples with ≥42 NANP repeats. The present finding showed extensive diversity in the asexual stage antigens.