Molecular surveillance reveals the presence of pfhrp2 and pfhrp3 gene deletions in Plasmodium falciparum parasite populations in Uganda, 2017–2019

Abstract Background Histidine-rich protein-2 (HRP2)-based rapid diagnostic tests (RDTs) are the only RDTs recommended for malaria diagnosis in Uganda. However, the emergence of Plasmodium falciparum histidine rich protein 2 and 3 (pfhrp2 and pfhrp3) gene deletions threatens their usefulness as malaria diagnostic and surveillance tools. The pfhrp2 and pfhrp3 gene deletions surveillance was conducted in P. falciparum parasite populations in Uganda. Methods Three-hundred (n = 300) P. falciparum isolates collected from cross-sectional malaria surveys in symptomatic individuals in 48 districts of eastern and western Uganda were analysed for the presence of pfhrp2 and pfhrp3 genes. Presence of parasite DNA was confirmed by PCR amplification of the 18s rRNA gene, msp1 and msp2 single copy genes. Presence or absence of deletions was confirmed by amplification of exon1 and exon2 of pfhrp2 and pfhrp3 using gene specific PCR. Results Overall, pfhrp2 and pfhrp3 gene deletions were detected in 29/300 (9.7%, 95% CI 6.6–13.6%) parasite isolates. The pfhrp2 gene was deleted in 10/300 (3.3%, 95% CI 1.6–6.0%) isolates, pfhrp3 in 9/300 (3.0%, 95% CI 1.4–5.6%) while both pfhrp2 and pfhrp3 were deleted in 10/300 (3.3%, 95% CI 1.6–6.0%) parasite isolates. Proportion of pfhrp2/3 deletions was higher in the eastern 14.7% (95% CI 9.7–20.0%) compared to the western region 3.1% (95% CI 0.8–7.7%), p = 0.001. Geographical location was associated with gene deletions aOR 6.25 (2.02–23.55), p = 0.003. Conclusions This is the first large-scale survey reporting the presence of pfhrp2/3 gene deletions in P. falciparum isolates in Uganda. Roll out of RDTs for malaria diagnosis should take into consideration the existence of pfhrp2/3 gene deletions particularly in areas where they were detected. Periodic pfhrp2/3 surveys are recommended to inform future decisions for deployment of alternative RDTs

Genetic surveillance and molecular epidemiology of human malaria parasites

This study investigates P. falciparum adaption to human activities to control malaria. Patient data analysis, genetic screening and molecular phylogeny were used to understand the drug resistance and diagnostic test-evasion profiles of P. falciparum imported to NSW and collected in Thailand. Artemisinin resistance (ART Rx) markers were found, including from PNG (resistance not previously reported). A geographically informative barcoding protocol was developed to identify evolutionary origins. Findings support the hypothesis that genetic background is crucial to the (ART Rx) phenotype. Putative P. vivax drug resistance markers (Pvmdr, Pvcrt-o, Pvdfr, and Pvdhps) and P. falciparum ART Rx markers (Pfkelch13) were analysed from samples collected in four provinces of South Thailand. Numerous P. vivax chloroquine and antifolate Rx-associated mutations were observed. The C580Y coding mutation was detected in 60-100% of P. falciparum samples originating from Ranong and Yala - the first reports of ART Rx genotypes south of the Rx perimeter in Southeast Asia. This study determined the pfhrp2/pfhrp3 deletion status of imported P. falciparum (genes underlying the protein detected by HPR2-based Rapid Diagnostic Tests). The study detected gene-deleted parasites from several African countries without reported data, with >10% from Sudan, South Sudan, and Nigeria, posing a risk of false-negative HRP2-RDT results. Microsatellite analysis was used to investigate genetic diversity and relatedness. Country cohorts were compared to Eritrean parasites, which have undergone selection for gene-deleted lineages. Selection signatures were not observed. The surprising diversity (including Rx genotypes) from populations lacking genetic data redefined ongoing surveillance targets. We conclude that malaria surveillance in Australia is a valuable global data gathering tool and necessary to pre-empt treatment failure and modify diagnostic testing algorithms in light of the genetic changes detected

Resistance screening and trend analysis of imported <i>falciparum</i> malaria in NSW, Australia (2010 to 2016)

<div><p>Background</p><p>The World Health Organization currently recommends artemisinin (along with a partner drug) as the global frontline treatment for <i>Plasmodium falciparum</i> malaria. Artemisinin resistant <i>P</i>. <i>falciparum</i> are now found throughout the greater Mekong subregion of South East Asia. Several polymorphisms in the parasite’s kelch gene have been demonstrated to confer artemisinin resistance. While genotypes within the greater Mekong subregion are thoroughly examined in the literature, <i>P</i>. <i>falciparum</i> populations within several areas that do not (yet) have endemic resistance are underrepresented.</p><p>Results</p><p>This investigation characterised the <i>Pf</i>kelch13 propeller domains from 153 blood samples of 140 imported cases of <i>P</i>. <i>falciparum</i> malaria in New South Wales from 2010 to 2016. A low level of propeller domain diversity was observed, including the C580<u><b>Y</b></u> coding mutation most strongly associated with artemisinin resistance in South East Asia. The resistance genotype was found in a sample originating in Papua New Guinea, where this mutation, or artemisinin treatment failure, have not been previously reported. Sequencing a panel of geographically informative polymorphisms within the organellar genomes identified the C580<u><b>Y</b></u> parasite as having Oceanic origins. Patient data analysis revealed that New South Wales, Australia, <i>P</i>. <i>falciparum</i> malaria cases often originated from regions with limited drug resistance screening.</p><p>Conclusions</p><p>The C580<u><b>Y</b></u> finding from outside of the greater Mekong subregion supports the consensus to upscale molecular surveillance of artemisinin resistance outside of South East Asia. The genetic screening results identify a risk of importing resistant <i>falciparum</i> malaria to Australia, supporting an ongoing surveillance protocol to pre-empt treatment failure and contribute to global data gathering.</p></div

Mutations found in <i>Pfkelch13</i> propeller domain.

<p>SNPs found within <i>Pfkelch13</i> propeller consensus sequences (n = 153) as compared to reference 3D7 <i>P</i>. <i>falciparum</i> sequence.</p

Geographically informative barcode of <i>P</i>. <i>falciparum</i> organellar genome SNPs.

<p>The C580<u><b>Y</b></u> Papua New Guinea (BDB5) sample sequence results for the 23 predictive geotyping SNP <i>loci</i> are compared to the geographically informative haplotype barcodes previously defined (predictive accuracy 92.1%) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197369#pone.0197369.ref017" target="_blank">17</a>]. SAM (South America); WAF: West Africa; EAF: East Africa; SEA: South East Asia, OCE: Oceania. Haplotype 10 represents the 3D7 reference <i>P</i>. <i>falciparum</i>. *SNP <i>loci</i> api2772 additionally occurs at nucleotide position api31461due to an inverted repeat within the apicoplast genome.</p

Limitations of rapid diagnostic tests in malaria surveys in areas with varied transmission intensity in Uganda 2017-2019: Implications for selection and use of HRP2 RDTs.

BackgroundPlasmodium falciparum histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs) are exclusively recommended for malaria diagnosis in Uganda; however, their functionality can be affected by parasite-related factors that have not been investigated in field settings.MethodsUsing a cross-sectional design, we analysed 219 RDT-/microscopy+ and 140 RDT+/microscopy+ dried blood spots obtained from symptomatic children aged 2-10 years from 48 districts in Uganda between 2017 and 2019. We aimed to investigate parasite-related factors contributing to false RDT results by molecular characterization of parasite isolates. ArcGIS software was used to map the geographical distribution of parasites. Statistical analysis was performed using chi-square or Fisher's exact tests, with P ≤ 0.05 indicating significance. Odds ratios (ORs) were used to assess associations, while logistic regression was performed to explore possible factors associated with false RDT results.ResultsThe presence of parasite DNA was confirmed in 92.5% (332/359) of the blood samples. The levels of agreement between the HRP2 RDT and PCR assay results in the (RDT+/microscopy+) and (RDT-/microscopy+) sample subsets were 97.8% (137/140) and 10.9% (24/219), respectively. Factors associated with false-negative RDT results in the (RDT-/microscopy+) samples were parasite density (ConclusionThis is the first evaluation and report of the contributions of pfhrp2/3 gene deletion, non-P. falciparum species, and low-density infections to false-negative RDT results under field conditions in Uganda. In view of these findings, the use of HRP2 RDTs should be reconsidered; possibly, switching to combination RDTs that target alternative antigens, particularly in affected areas, may be beneficial. Future evaluations should consider larger and more representative surveys covering other regions of Uganda

Urolitíase em cães: avaliação quantitativa da composição mineral de 156 urólitos Canine urolithiasis: quantitative evaluation of mineral composition of 156 uroliths

O estudo teve como objetivo avaliar os casos de urolitíase canina em que a composição mineral dos urólitos foi analisada quantitativamente. Foi avaliada quantitativamente a composição mineral de 156 urólitos obtidos de cães (nefrólitos, ureterólitos, urocistólitos e uretrólitos). Desse total, 79,5% (n=124) eram simples, 18% (n=28) eram compostos e apenas 2,5% (n=4) eram mistos. A estruvita foi o tipo mineral mais frequente nos urólitos simples (47,6%; n=59), em todos os mistos (100%; n=4) e nas camadas núcleo e pedra de urólitos compostos (32,1 e 75%, respectivamente). O oxalato de cálcio foi o segundo mineral mais frequente dos urólitos simples (37,9%, n=47). Ao contrário do que é preconizado para os urólitos simples, as recomendações para o tratamento de urólitos compostos são mais complexas, tais como protocolos de tratamento de dissolução diferentes (se composto por minerais distintos e passíveis de dissolução como urato e estruvita). Além disso, a dissolução pode não ser viável, caso ocorra presença de material insolúvel envolvendo o urólito ou se este representar mais de 20% da camada. Vinte e dois urólitos compostos (78,7%) apresentaram uma camada externa não passível de dissolução (oxalato de cálcio ou fosfato de cálcio); dois (7,1%) apresentaram camadas externas passíveis de dissolução (estruvita ou urato), porém camadas mais internas não solúveis, o que permitiria apenas a dissolução parcial do urólito. Assim, o conhecimento da composição de todas as camadas que compõem o urólito é essencial para o entendimento da formação do cálculo e consequentemente para a indicação do tratamento adequado, assim como para prevenção de recidivas.<br>The aim of this study was to evaluate dogs with urolithiasis in which mineral composition of calculi was quantitatively analyzed. Quantitative mineral composition was performed in 156 canine uroliths. Simple uroliths represented 79.5% (n=124) of the cases, 18% were compound (n=28) and only 2.5% (n=4) of the calculi were mixed. Struvite was the most frequent mineral type of simple uroliths (47.6%; n=59) as well as in all mixed (100%; n=4) and in the core and stone uroliths (32.1% and 75%, respectively). Calcium oxalate was the second more frequent mineral composition of simple uroliths (37.9%; n=47). Unlike simple uroliths, recommendation for the treatment of compound uroliths is more complex, and diet protocols for calculi dissolution may be different when the calculus is composed by different minerals that are possible to be dissolved (e.g. urate and stuvite). Besides, dissolution may not be feasible if it occurs in presence of insoluble material involving urolith or if it represents more than 20% of the layer. Twenty two compound uroliths (78.7%) presented an external layer that was not possible to be dissolved (calcium oxalate or calcium phosphate); two calculi (7.1%) had superficial layers dissolvable (struvite or urate), but inner layers were not soluble, which allowed only partial dissolution of urolith. Knowledge of all urolith layers mineral composition is essential for the understanding of calculus formation and for the adequate treatment indication as well as for the procedures to prevent recurrence