Deletion patterns, genetic variability and protein structure of pfhrp2 and pfhrp3: implications for malaria rapid diagnostic test in Amhara region, Ethiopia

Background: Although rapid diagnostic tests (RDTs) play a key role in malaria-control strategies, their efficacy has been threatened by deletion and genetic variability of the genes pfhrp2/3. This study aims to characterize the deletion, genetic patterns and diversity of these genes and their implication for malaria RDT effectiveness, as well as their genetic evolution in the Amhara region of Ethiopia. Methods: The study included 354 isolates from symptomatic patients from the Amhara region of Ethiopia who tested positive by microscopy. Exon 1?2 and exon 2 of genes pfhrp2 and -3 were amplified, and exon 2 was sequenced to analyse the genetic diversity, phylogenetic relationship and epitope availability. Results: The deletion frequency in exon 1?2 and exon 2 was 22 and 4.6% for pfhrp2, and 68 and 18% for pfhrp3, respectively. Double deletion frequency for pfhrp2 and pfhrp3 was 1.4%. High genetic diversity, lack of clustering by phylogenetic analysis and evidence of positive selection suggested a diversifying selection for both genes. The amino-acid sequences, classified into different haplotypes, varied widely in terms of frequency of repeats, with novel amino-acid changes. Aminoacidic repetition type 2 and type 7 were the most frequent in all the sequences. The most frequent epitopes among protein sequences were those recognized by MAbs 3A4 and C1-13. Conclusion: Deletions and high amino acidic variation in pfhrp2 and pfhrp3 suggest their possible impact on RDT use in the Amhara region, and the high genetic diversity of these genes could be associated with a diversifying selection in Ethiopia. Surveillance of these genes is, therefore, essential to ensure the effectiveness of public health interventions in this region.Instituto de Salud Carlos II

Prevalence and Genetic Diversity of <i>Giardia duodenalis</i> and <i>Cryptosporidium</i> spp. among School Children in a Rural Area of the Amhara Region, North-West Ethiopia

<div><p>Backgroud</p><p><i>Giardia duodenalis</i> and <i>Cryptosporidium</i> spp. are enteric protozoan causing gastrointestinal illness in humans and animals. Giardiasis and cryptosporidiosis are not formally considered as neglected tropical diseases, but belong to the group of poverty-related infectious diseases that impair the development and socio-economic potential of infected individuals in developing countries.</p><p>Methods</p><p>We report here the prevalence and genetic diversity of <i>G</i>. <i>duodenalis</i> and <i>Cryptosporidium</i> spp. in children attending rural primary schools in the Bahir Dar district of the Amhara Region, Ethiopia. Stool samples were collected from 393 children and analysed by molecular methods. <i>G</i>. <i>duodenalis</i> was detected by real-time PCR, and the assemblages and sub-assemblages were determined by multilocus sequence-based genotyping of the glutamate dehydrogenase and β-giardin genes of the parasite. Detection and identification of <i>Cryptosporidium</i> species was carried out by sequencing of a partial fragment of the small-subunit ribosomal RNA gene.</p><p>Principal Findings</p><p>The PCR-based prevalences of <i>G</i>. <i>duodenalis</i> and <i>Cryptosporidium</i> spp. were 55.0% (216/393) and 4.6% (18/393), respectively. A total of 78 <i>G</i>. <i>duodenalis</i> isolates were successfully characterized, revealing the presence of sub-assemblages AII (10.3%), BIII (28.2%), and BIV (32.0%). Discordant typing results AII/AIII and BIII/BIV were identified in 7.7% and 15.4% of the isolates, respectively. An additional five (6.4%) isolates were assigned to assemblage B. No mixed infections of assemblages A+B were found. Extensive genetic variation at the nucleotide level was observed within assemblage B (but no within assemblage A), resulting in the identification of a large number of sub-types. <i>Cryptosporidium</i> diversity was demonstrated by the occurrence of <i>C</i>. <i>hominis</i>, <i>C</i>. <i>parvum</i>, and <i>C</i>. <i>viatorum</i> in the population under study.</p><p>Conclusions</p><p>Our data suggest an epidemiological scenario with an elevated transmission intensity of a wide range of <i>G</i>. <i>duodenalis</i> genetic variants. Importantly, the elevated degree of genetic diversity observed within assemblage B is consistent with the occurrence of intra-assemblage recombination in <i>G</i>. <i>duodenalis</i>.</p></div

Median real-time PCR cycle threshold (Ct) values from <i>Giardia duodenali</i>s positive samples by age group in children aged 6–15 years in the Amhara Region, Ethiopia.

<p>Statistical significance (P < 0.01) is indicated by asterisks.</p

Diversity and frequency of single-nucleotide polymorphisms displayed by <i>Giardia duodenalis</i> sub-assemblage BIV isolates at the glutamate dehydrogenase locus (partial sequence between positions 80 to 467) identified in the present study.

<p>Novel sub-types were shown underlined. Transversion mutations were highlighted in bold. Point mutations inducing amino-acid substitutions were marked as superscript indicating the amino-acid change.</p

Prevalence and assemblage frequencies of <i>Giardia duodenalis</i> in children attending rural schools in the Bahir Dar district, Amhara Region, Ethiopia, 2013.

<p>Prevalence and assemblage frequencies of <i>Giardia duodenalis</i> in children attending rural schools in the Bahir Dar district, Amhara Region, Ethiopia, 2013.</p

Evolutionary relationships among assemblages of <i>G</i>. <i>duodenalis</i> at the <i>GDH</i> locus inferred by a Neighbor-Joining analysis of the nucleotide sequence covering a 403-bp region (positions 80 to 467 of GenBank accession number L40508) of the gene.

<p>The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 iterations) is indicated next to the branches. Bootstrap values lower than 50% were not displayed. The evolutionary distances were computed using the Kimura 2-parameter method. The rate variation among sites was modelled with a gamma distribution (shape parameter = 2). Filled circles and squares represent BIII and BIV sequences, respectively, from this study. Open squares indicate BIV sequences previously reported in human isolates from Ethiopia [see ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159992#pone.0159992.ref022" target="_blank">22</a>]. <i>Spironucleus vortens</i> was used as outgroup taxa.</p

Diversity and frequency of single-nucleotide polymorphisms displayed by <i>Giardia duodenalis</i> assemblage B isolates at the beta giardin locus (partial sequence between positions 108 to 573) identified in the present study.

<p>Novel sub-types were shown underlined. Point mutations inducing amino-acid substitutions were marked as superscript indicating the amino-acid change.</p

Evolutionary relationships among assemblages of <i>G</i>. <i>duodenalis</i> at the <i>BG</i> locus inferred by a Neighbor-Joining analysis of the nucleotide sequence covering a 487-bp region (positions 108 to 573 of GenBank accession number AY072727) of the gene.

<p>The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 iterations) is indicated next to the branches. Bootstrap values lower than 50% were not displayed. The evolutionary distances were computed using the Kimura 2-parameter method. The rate variation among sites was modelled with a gamma distribution (shape parameter = 2). Filled circles represent B sequences from this study, whereas open circles indicate B sequences previously reported in human isolates from Ethiopia (see ref. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159992#pone.0159992.ref022" target="_blank">22</a>]).No outgroup taxa was used as beta-giardin is a <i>Giardia</i>-specific structural protein.</p

Sub-assemblage frequencies of <i>Giardia duodenalis</i> in children attending rural schools in the Bahir Dar district, Amhara Region, Ethiopia, 2013.

<p>Sub-assemblage frequencies of <i>Giardia duodenalis</i> in children attending rural schools in the Bahir Dar district, Amhara Region, Ethiopia, 2013.</p