Genetic Diversity of Merozoite Surface Protein-1 and -2 Genes in Plasmodium falciparum Isolates among Asymptomatic Population in Boset and Badewacho Districts, Southern Ethiopia.

Background: The genetic variation of Plasmodium falciparum has been studied to assess local malaria transmission genetic profile using evidence-based intervention measures. However, there are no known previous reports of P. falciparum polymorphism in Badewacho and Boset districts, Southern Ethiopia. The purpose of this study was to determine the genetic diversity of the merozoite surface protein-1 and -2 (msp-1 and msp-2) allelic families in P. falciparum isolates from an asymptomatic populations. Methods: This study was conducted from finger-prick blood samples spotted on 3 mm Whatman filter paper collected during a community-based cross-sectional study. Nested polymerase chain reaction amplification was used to type the allelic variants of msp-1 and msp-2. Results: From 669 asymptomatic study participants, a total of 50 samples positive for P. falciparum were included for molecular analysis. Of 50 positive samples, 43 P. falciparum isolates were successfully amplified for the msp-1 and msp-2 allelic families. A total of twelve different allele sizes (75-250 bp) were identified within the three allelic families of msp-1, whereas ten different allele sizes (250-500 bp) were detected within the two allelic families of msp-2. MAD20 had a higher allelic proportion, 65% among allelic families of msp-1, whereas the 3D7 allelic family 90.7% was higher in msp-2. A slightly higher frequency of polyclonal infection 53.5% was found in msp-2 allelic family, whereas a low proportion polyclonal infection 46.5% was found in msp-1 allelic family. The overall mean multiplicity of infection (MOI) for msp-1 and msp-2 was identical (MOI = 1.56). Correspondingly, the expected heterozygosity (He) value for msp-1 (He = 0.23) and msp-2 (He = 0.22) was almost similar. Conclusions: The findings of this study revealed low genetic diversity of the msp-1 and msp-2 allelic families in P. falciparum isolates. However, continued monitoring status of the local genetic diversity profile in the P. falciparum population is required to support current malaria control and elimination strategies.

High prevalence of Pfcrt 76T and Pfmdr1 N86 genotypes in malaria infected patients attending health facilities in East Shewa zone, Oromia Regional State, Ethiopia.

BACKGROUND: Plasmodium falciparum resistance to series of anti-malarial drugs is a major challenge in efforts to control and/or eliminate malaria globally. In 1998, following the widespread of chloroquine (CQ) resistant P. falciparum, Ethiopia switched from CQ to sulfadoxine-pyrimethamine (SP) and subsequently in 2004 from SP to artemether-lumefantrine (AL) for the treatment of uncomplicated falciparum malaria. Data on the prevalence of CQ resistance markers after more than two decades of its removal is important to map the selection pressure behind the targets codons of interest. The present study was conducted to determine the prevalence of mutations in Pfcrt K76T and Pfmdr1 N86Y codons among malaria-infected patients from Adama, Olenchiti and Metehara sites of East Shewa zone, Oromia Regional State, Ethiopia. METHODS: Finger-prick whole blood samples were collected on 3MM Whatman ® filter papers from a total of 121 microscopically confirmed P. falciparum infected patients. Extraction of parasite DNA was done by Chelex-100 method from dried blood spot (DBS). Genomic DNA template was used to amplify Pfcrt K76T and Pfmdr1 N86Y codons by nested PCR. Nested PCR products were subjected to Artherobacter protophormiae-I (APoI) restriction enzyme digestion to determine mutations at codons 76 and 86 of Pfcrt and Pfmdr1 genes, respectively. RESULTS: Of 83 P. falciparum isolates successfully genotyped for Pfcrt K76T, 91.6% carried the mutant genotypes (76T). The prevalence of Pfcrt 76T was 95.7%, 92.5% and 84.5% in Adama, Metehara and Olenchiti, respectively. The prevalence of Pfcrt 76T mutations in three of the study sites showed no statistical significance difference (χ2 = 1.895; P = 0.388). On the other hand, of the 80 P. falciparum samples successfully amplified for Pfmdr1, all carried the wild-type genotypes (Pfmdr1 N86). CONCLUSION: Although CQ officially has been ceased for the treatment of falciparum malaria for more than two decades in Ethiopia, greater proportions of P. falciparum clinical isolates circulating in the study areas carry the mutant 76T genotypes indicating the presence of indirect CQ pressure in the country. However, the return of Pfmdr1 N86 wild-type allele may be favoured by the use of AL for the treatment of uncomplicated falciparum malaria.

Genetic variation of Plasmodium falciparum histidine-rich protein 2 and 3 in Assosa zone, Ethiopia: its impact on the performance of malaria rapid diagnostic tests.

BACKGROUND: Rapid diagnostic tests (RDT) are commonly used for the diagnosis of malaria caused by Plasmodium falciparum. However, false negative results of RDT caused by genetic variation of P. falciparum histidine-rich protein 2 and 3 genes (pfhrp2/3) threaten existing malaria case management and control efforts. The main objective of this study was to investigate the genetic variations of the pfhrp2/3 genes. METHODS: A cross-sectional study was conducted from malaria symptomatic individuals in 2018 in Assosa zone, Ethiopia. Finger-prick blood samples were collected for RDT and microscopic examination of thick and thin blood films. Dried blood spots (DBS) were used for genomic parasite DNA extraction and molecular detection. Amplification of parasite DNA was made by quantitative PCR. DNA amplicons of pfhrp2/3 were purified and sequenced. RESULTS: The PfHRP2 amino acid repeat type isolates were less conserved compared to the PfHRP3 repeat type. Eleven and eight previously characterized PfHRP2 and PfHRP3 amino acid repeat types were identified, respectively. Type 1, 4 and 7 repeats were shared by PfHRP2 and PfHRP3 proteins. Type 2 repeats were found only in PfHRP2, while types 16 and 17 were found only in PfHRP3 with a high frequency in all isolates. 18 novel repeat types were found in PfHRP2 and 13 novel repeat types were found in PfHRP3 in single or multiple copies per isolate. The positivity rate for PfHRP2 RDT was high, 82.9% in PfHRP2 and 84.3% in PfHRP3 sequence isolates at parasitaemia levels > 250 parasites/µl. Using the Baker model, 100% of the isolates in group A (If product of types 2 × type 7 repeats ≥ 100) and 73.7% of the isolates in group B (If product of types 2 × type 7 repeats 50-99) were predicted to be detected by PfHRP2 RDT at parasitaemia level > 250 parasite/µl. CONCLUSION: The findings of this study indicate the presence of different PfHRP2 and PfHRP3 amino acid repeat including novel repeats in P. falciparum from Ethiopia. These results indicate that there is a need to closely monitor the performance of PfHRP2 RDT associated with the genetic variation of the pfhrp2 and pfhrp3 gene in P. falciparum isolates at the country-wide level.

Detection of high prevalence of Plasmodium falciparum histidine-rich protein 2/3 gene deletions in Assosa zone, Ethiopia: implication for malaria diagnosis.

BACKGROUND: Rapid diagnostic tests (RDTs) targeting histidine rich protein 2(HRP2) are widely used for diagnosis of Plasmodium falciparum infections. Besides PfHRP2, the PfHRP3 antigen contributes to the detection of P. falciparum infections in PfHRP2 RDTs. However, the performance HRP2-based RDT is affected by pfhrp2/3 gene deletions resulting in false-negative test results. The objective of this study was to determine the presence and prevalence of pfhrp2/3 gene deletions including the respective flanking regions among symptomatic patients in Assosa zone, Northwest Ethiopia. METHODS: A health-facility based cross-sectional study was conducted in febrile patients seeking a malaria diagnosis in 2018. Blood samples were collected by finger-prick for microscopic examination of blood smears, malaria RDT, and molecular analysis using dried blood spots (DBS) prepared on Whatman filter paper. A total of 218 P. falciparum positive samples confirmed by quantitative PCR were included for molecular assay of pfhrp2/3 target gene. RESULTS: Of 218 P. falciparum positive samples, exon 2 deletions were observed in 17.9% of pfhrp2 gene and in 9.2% of pfhrp3 gene. A high proportion of deletions in short segments of pfhrp2 exon1-2 (50%) was also detected while the deletions of the pfhrp3 exon1-2 gene were 4.1%. The deletions were extended to the downstream and upstream of the flanking regions in pfhrp2/3 gene (above 30%). Of eighty-six PfHRP2 RDT negative samples, thirty-six lacked pfhrp2 exon 2. Five PfHRP2 RDT negative samples had double deletions in pfhrp2 exon 2 and pfhrp3 exon2. Of these double deletions, only two of the samples with a parasite density above 2000 parasite/µl were positive by the microscopy. Three samples with intact pfhrp3 exon2 in the pfhrp2 exon2 deleted parasite isolates were found to be positive by PfHRP2 RDT and microscopy with a parasite density above 10,000/µl. CONCLUSION: This study confirms the presence of deletions of pfhrp2/3 gene including the flanking regions. Pfhrp2/3 gene deletions results in false-negative results undoubtedly affect the current malaria control and elimination effort in the country. However, further countrywide investigations are required to determine the magnitude of pfhrp2/3 gene deletions and its consequences on routine malaria diagnosis.

Evaluation of PfHRP2 and PfLDH Malaria Rapid Diagnostic Test Performance in Assosa Zone, Ethiopia.

In malaria-endemic countries, rapid diagnostic tests (RDTs) targeting Plasmodium falciparum histidine-rich protein 2 (PfHRP2) and lactate dehydrogenase (PfLDH) have been widely used. However, little is known regarding the diagnostic performances of these RDTs in the Assosa zone of northwest Ethiopia. The objective of this study was to determine the diagnostic performances of PfHRP2 and PfLDH RDTs using microscopy and quantitative PCR (qPCR) as a reference test. A health facility-based cross-sectional study design was conducted from malaria-suspected study participants at selected health centers from November to December 2018. Finger-prick blood samples were collected for microscopy, RDTs, and qPCR method. The prevalence of P. falciparum was 26.4%, 30.3%, and 24.1% as determined by microscopy, PfHRP2 RDT, and PfLDH RDT, respectively. Compared with microscopy, the sensitivity and specificity of the PfHRP2 RDT were 96% and 93%, respectively, and those of the PfLDH RDT were 89% and 99%, respectively. Compared with qPCR, the specificity of the PfHRP2 RDT (93%) and PfLDH RDT (98%) was high, but the sensitivity of the PfHRP2 RDT (77%) and PfLDH RDT (70%) was relatively low. These malaria RDTs and reference microscopy methods showed reasonable agreement with a kappa value above 0.85 and provided accurate diagnosis of P. falciparum malaria. Thus, the current malaria RDT in the Ministry of Health program can be used in the Assosa zone of Ethiopia. However, continuous monitoring of the performance of PfHRP2 RDT is important to support control and elimination of malaria in Ethiopia.