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.

Genetic Variations 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 Plasmodium falciparum malaria. However, false negative results of RDT caused by genetic variations 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 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 repeat type isolates were less conserved compared to the PfHRP3 repeat type. A total of eleven and eight different PfHRP2 and PfHRP3 amino acid repeat types were identified, respectively. Type 1, 4 and 7 repeats were shared by PfHRP2 and PfHRP3 isolates. 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 isolate at parasitemia levels >250 parasites/µl. Using the Baker model, 100 % of the isolates in group A and 73.7% of the isolates in group B were predicted to be detected by PfHRP2 RDT at parasitemia 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.

HPREP: a comprehensive database for human proteome repeats

Amino acid repeats are found to play important roles in both structures and functions of the proteins. These are commonly found in all kingdoms of life, especially in eukaryotes and a larger fraction of human proteins composed of repeats. Further, the abnormal expansions of shorter repeats cause various diseases to humans. Therefore, the analysis of repeats of the entire human proteome along with functional, mutational and disease information would help to better understand their roles in proteins. To fulfill this need, we developed a web database HPREP (http://bioinfo.bdu.ac.in/hprep) for human proteome repeats using Perl and HTML programming. We identified different categories of well-characterized repeats and domain repeats that are present in the human proteome of UniProtKB/Swiss-Prot by using in-house Perl programming and novel repeats by using the repeat detection T-REKS tool as well as XSTREAM web server. Further, these proteins are annotated with functional, mutational and disease information and grouped according to specific repeat types. The developed database enables the users to search by specific repeat type in order to understand their involvement in proteins. Thus, the HPREP database is expected to be a useful resource to gain better insight regarding the different repeats in human proteome and their biological roles.

Detection of simple sequence repeats in the chloroplast genome of Tetraphis pellucida Hedw.

Simple sequence repeats (SSRs) consist of short repeat motifs of 1-6 nucleotides and are found in DNA sequences.The present study was conducted to detect SSRs in chloroplast genome of Tetraphis pellucida (Accession number: NC_024291), downloaded from the National Center for Biotechnology Information (NCBI). The sequence was mined with the help of MISA, a Perl script, to detect SSRs. The length of SSRs defined as ≥12 for mono, di, tri and tetranucleotide, ≥15 for pentanucleotide and ≥18 for hexanucleotide repeats. In total, 41 perfect microsatellites were identified in 127.489 kb sequence mined. An average length of 13.56 bp was calculated for mined SSRs with a density of 1 SSR/3.04 kb. Depending on the repeat units, the length of SSRs ranged from 12 to 20 nt. Dinucleotides (14, 34.15%) were the most frequent repeat type, followed by tetranucleotides (10, 24.39%), trinucleotides (7, 17.07%), mononucleotides (6, 14.63%) and pentanucleotide (4, 9.76%) repeats. Hexanucleotide repeats were completely absent in chloroplast genome of Tetraphis pellucida. The mined SSRs can be used to develop molecular markers and genetic diversity studies in Tetraphis species.

Computational Mining of Microsatellites in the Chloroplast Genome of Ptilidium pulcherrimum, a Liverwort

Microsatellites also known as simple sequence repeats (SSRs) are found in DNA sequences. These repeats consist of short motifs of 1-6 bp and play important role in population genetics, phylogenetics and also in the development of molecular markers. In this study chloroplastic SSRs (cpSSRs) in the chloroplast genome of Ptilidium pulcherrimum, downloaded from the National Center for Biotechnology Information (NCBI), were detected. The chloroplast genome sequence of P. pulcherrimum was mined with the help of a Perl script named MISA. A total of 23 perfect cpSSRs were detected in 119.007 kb sequence mined showing density of 1 SSR/5.17 kb. Depending on the repeat units, the length of SSRs found to be 12 bp for mono and tri, 12 to “22 bp for di, 12 to 16 bp for tetra nucleotide repeats. Penta and hexanucleotide repeats were completely absent in chloroplast genome of P. pulcherrimum. Dinucleotide repeats were the most frequent repeat type (47.83%) followed by tri (21.74%) and tetranucleotide (21.74%) repeats. Out of 23 SSRs detected, PCR primers were successfully designed for 22 (95.65%) cpSSRs. DOI: http://dx.doi.org/10.3126/ije.v3i3.11063 International Journal of Environment Vol.3(3) 2014: 50-58