Four human Plasmodium species quantification using droplet digital PCR

Srisutham, Suttipat; Saralamba, Naowarat; Malleret, Benoît; Rénia, Laurent; Dondorp, Arjen M.; Imwong, Mallika

doi:10.1371/journal.pone.0175771

Cited by 63 publications

(58 citation statements)

References 42 publications

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“…DRC samples were first subjected to a pan- Plasmodium real-time PCR assay targeting the 18S rRNA gene. (35) Positive samples were then subjected to four species-specific, semi-quantitative 18S rRNA real-time PCR assays for P. falciparum,(36) P. ovale,(37) P. malariae,(36) and P. vivax,(38) respectively. Parasite densities for all DRC and Uganda samples were determined using quantitative real-time PCR (qPCR) targeting the single-copy P. falciparum lactate dehydrogenase ( pfldh ) gene as previously described.…”

Section: Methodsmentioning

confidence: 99%

A novel CRISPR-based malaria diagnostic capable ofPlasmodiumdetection, speciation, and drug-resistance genotyping

Hennelly

et al. 2020

Preprint

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One-sentence summary: Novel malaria SHERLOCK assays enabled robust detection, 16 speciation, and genotyping of Plasmodium spp. in diverse samples collected in Africa Abstract word count: 228 20 Manuscript word count: 5291 21 # Corresponding author information: Jonathan B. Parr, MD, MPH; 130 Mason Farm 22Rd., Chapel HillABSTRACT 24 25 CRISPR-based diagnostics are a new class of highly sensitive and specific assays with 26 multiple applications in infectious disease diagnosis. SHERLOCK, Sensitivity Enzymatic Reporter UnLOCKing, is one such CRISPR-based diagnostic that 28 combines recombinase polymerase pre-amplification, CRISPR-RNA base-pairing, and 29LwCas13a activity for nucleic acid detection. We developed SHERLOCK assays for 30 malaria capable of detecting all Plasmodium species known to cause malaria in humans 31 and species-specific detection of P. vivax and P. falciparum, the species responsible for 32 the majority of malaria cases worldwide. We validated these assays against parasite 33 genomic DNA and achieved analytical sensitivities ranging from 2.5-18.8 parasites per 34 reaction. We further tested these assays using a diverse panel of 123 clinical samples 35 from the Democratic Republic of the Congo, Uganda, and Thailand and pools of 36 Anopheles mosquitoes from Thailand. When compared to real-time PCR, the P. 37 falciparum assay achieved 94% sensitivity and 94% specificity in clinical samples. In 38 addition, we developed a SHERLOCK assay capable of detecting the dihydropteroate 39 synthetase (dhps) single nucleotide variant A581G associated with P. falciparum 40 sulfadoxine-pyrimethamine resistance. Compared to amplicon-based deep sequencing, 41 the dhps SHERLOCK assay achieved 73% sensitivity and 100% specificity when 42 applied to a panel of 43 clinical samples, with false-negative calls only at lower parasite 43 densities. These novel SHERLOCK assays have potential to spawn a new generation of 44 molecular diagnostics for malaria and demonstrate the versatility of CRISPR-based 45 diagnostic approaches. 46 47 48 Newly developed technologies that utilize Clustered, Regularly-Interspaced Palindromic 49 Repeat (CRISPR) systems have the potential to revolutionize infectious disease 50 diagnostic testing.(1) SHERLOCK, or Specific High-Sensitivity Enzymatic Reporter 51UnLOCKing, is a CRISPR-based diagnostic assay that has now been used to detect 52 dengue and Zika viruses with excellent sensitivity and specificity.(2) Its simple workflow 53 and robust performance characteristics have enabled multiplexed detection and 54 genotyping of Zika and dengue viruses, with increasingly streamlined protocols that 55 facilitate use at the point-of-care.(3, 4) SHERLOCK's potential is perhaps greatest in 56 low-resource settings where improved, reliable diagnostics are urgently needed for 57 multiple pathogens and for malaria, in particular. 58 59 Timely and accurate diagnosis is an important component of malaria control and 60 elimination efforts. The current generation of rapid diagnostic tests (RDTs) that detect 61 ...

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Section: Methodsmentioning

confidence: 99%

A novel CRISPR-based malaria diagnostic capable ofPlasmodiumdetection, speciation, and drug-resistance genotyping

Hennelly

et al. 2020

Preprint

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“…The specificity of primers was assessed on samples from symptomatic P. malariae patients (Table S2) compared to the reference nested-PCR method targeting the 18S rRNA [60]. Specificity of all primer products were checked for amplification of unspecific products to access true positive results.…”

Section: Sensitivity and Specificitymentioning

confidence: 99%

Polymorphic markers for identification of parasite population in Plasmodium malariae

Mathema

Nakeesathit

Pagornrat

et al. 2020

Preprint

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Background Molecular genotyping in Plasmodium serves many aims including providing tools for studying parasite population genetics and distinguishing recrudescence from reinfection. Microsatellite typing, insertion-deletion (INDEL) and single nucleotide polymorphisms is used for genotyping, but only limited information is available for Plasmodium malariae, an important human malaria species. This study aimed to provide a set of genetic markers to facilitate the study of P. malariae population genetics. Methods Markers for microsatellite genotyping and pmmsp1 gene polymorphisms were developed and validated in symptomatic P. malariae field isolates from Myanmar (N=37). Fragment analysis was used to determine allele sizes at each locus to calculate multiplicity of infections (MOI), linkage disequilibrium, genetic richness index, heterozygosity and construct dendrograms. Nucleotide diversity (π), number of haplotypes, and genetic diversity (Hd) were assessed and a phylogenetic tree was constructed. Genome-wide microsatellite maps with annotated regions of newly identified markers were constructed. Results Six microsatellite markers were developed and tested in 37 P. malariae isolates which showed sufficient heterozygosity (0.530-0.922), genetic richness index (0.050-0.250) and absence of linkage disequilibrium (IAS = 0.03, p-value > 0.05)(N=37). In addition, a tandem repeat (VNTR)-based pmmsp1 INDEL polymorphisms marker was developed and assessed in 27 P. malariae isolates showing a nucleotide diversity of 0.0976, haplotype gene diversity of 0.698 and identified 14 unique variants. The size of VNTR consensus repeat unit adopted as allele was 27 base pairs. The markers Pm12_426 andpmmsp1 showed greatest diversity with heterozygosity scores of 0.920 and 0.835, respectively. Using six microsatellites markers, the likelihood that any two parasite strains would have the same microsatellite genotypes was 8.46 × 10-4 and was further reduced to 1.66 × 10-4 when pmmsp1 polymorphisms were included. Conclusions Six novel microsatellites genotyping markers and a set of pmmsp1 VNTR-based INDEL polymorphisms markers for P. malariae were developed and validated. Each marker could be independently or in combination employed to access genotyping of the parasite. The newly developed markers may serve as a useful tool for investigating parasite diversity, population genetics, molecular epidemiology and for distinguishing recrudescence from reinfection in drug efficacy studies.

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“…Parasite DNA was extracted using QIAamp DNA mini kit (Qiagen, Germany). All samples were confirmed as P. malariae using 18 small-subunit ribosomal RNA (18S rRNA)-based PCR [60] following the Standards for the Reporting of Diagnostic Accuracy (STARD) [61][62][63]. Ethical approval for the study was obtained from the ethical review board of the Faculty of Tropical Medicine, Mahidol University.…”

Section: Study Site Dna Sampling and Reference Sequencesmentioning

confidence: 99%

“…The specificity of primers was assessed on samples from symptomatic P. malariae patients (Additional file 1: Table S2) compared to the reference nested-PCR method targeting the 18S rRNA [60]. Specificity of all primer products were checked for amplification of unspecific products to access true positive results.…”

Section: Sensitivity and Specificitymentioning

confidence: 99%

Polymorphic markers for identification of parasite population in Plasmodium malariae

Mathema

Nakeesathit

Pagornrat

et al. 2020

Malar J

Self Cite

View full text Add to dashboard Cite

Background: Molecular genotyping in Plasmodium serves many aims including providing tools for studying parasite population genetics and distinguishing recrudescence from reinfection. Microsatellite typing, insertion-deletion (INDEL) and single nucleotide polymorphisms is used for genotyping, but only limited information is available for Plasmodium malariae, an important human malaria species. This study aimed to provide a set of genetic markers to facilitate the study of P. malariae population genetics. Methods: Markers for microsatellite genotyping and pmmsp1 gene polymorphisms were developed and validated in symptomatic P. malariae field isolates from Myanmar (N = 37). Fragment analysis was used to determine allele sizes at each locus to calculate multiplicity of infections (MOI), linkage disequilibrium, heterozygosity and construct dendrograms. Nucleotide diversity (π), number of haplotypes, and genetic diversity (H d) were assessed and a phylogenetic tree was constructed. Genome-wide microsatellite maps with annotated regions of newly identified markers were constructed. Results: Six microsatellite markers were developed and tested in 37 P. malariae isolates which showed sufficient heterozygosity (0.530-0.922), and absence of linkage disequilibrium (I A S =0.03, p value > 0.05) (N = 37). In addition, a tandem repeat (VNTR)-based pmmsp1 INDEL polymorphisms marker was developed and assessed in 27 P. malariae isolates showing a nucleotide diversity of 0.0976, haplotype gene diversity of 0.698 and identified 14 unique variants. The size of VNTR consensus repeat unit adopted as allele was 27 base pairs. The markers Pm12_426 and pmmsp1 showed greatest diversity with heterozygosity scores of 0.920 and 0.835, respectively. Using six microsatellites markers, the likelihood that any two parasite strains would have the same microsatellite genotypes was 8.46 × 10 −4 and was further reduced to 1.66 × 10 −4 when pmmsp1 polymorphisms were included. Conclusions: Six novel microsatellites genotyping markers and a set of pmmsp1 VNTR-based INDEL polymorphisms markers for P. malariae were developed and validated. Each marker could be independently or in combination employed to access genotyping of the parasite. The newly developed markers may serve as a useful tool for investigating parasite diversity, population genetics, molecular epidemiology and for distinguishing recrudescence from reinfection in drug efficacy studies.

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Four human Plasmodium species quantification using droplet digital PCR

Cited by 63 publications

References 42 publications

A novel CRISPR-based malaria diagnostic capable ofPlasmodiumdetection, speciation, and drug-resistance genotyping

A novel CRISPR-based malaria diagnostic capable ofPlasmodiumdetection, speciation, and drug-resistance genotyping

Polymorphic markers for identification of parasite population in Plasmodium malariae

Polymorphic markers for identification of parasite population in Plasmodium malariae

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