Targeted Amplicon Deep Sequencing Research Articles

Genotyping methods to distinguish Plasmodium falciparum recrudescence from new infection for the assessment of antimalarial drug efficacy: an observational, single-centre, comparison study

Distinguishing Plasmodium falciparum recrudescence from new infections is crucial for the assessment of antimalarial drug efficacy against Pfalciparum. We aimed to compare the efficacy of different genotyping methods to assess their effect on drug efficacy estimates, particularly in patients from high-transmission settings with polyclonal infections. In this head-to-head comparison study, we compared five different genotyping methods currently used: fast capillary electrophoresis (F-CE) using msp1, msp2, and glurp; high-resolution capillary electrophoresis (H-CE) using msp1, msp2, and glurp; H-CE using microsatellites; targeted amplicon deep sequencing (TADS) using single nucleotide polymorphism (SNP)-rich markers; and high-resolution melting (HRM) analysis using msp1 and msp2. We assessed their sensitivity in detecting minority clones in polyclonal infections, their reproducibility, and the genetic diversity of the markers used. Our study used four well characterised Pfalciparum laboratory strains mixed in varying ratios, and 20paired samples collected from an in-vivo clinical trial. The experiments were performed at the Swiss Tropical and Public Health Institute in Basel, Switzerland between May 5, 2020, and Aug 23,2021. H-CE using msp1 and msp2 and TADS revealed the highest sensitivity in detecting minority clones (up to ratios of 1:100 for H-CE and 50:1:1:1 for TADS in the FCB1:HB3 and 3D7:K1:HB3:FCB1 laboratory strain mixtures, respectively), highest reproducibility (intra-assay: 99% and 91% for H-CE and TADS, respectively; inter-assay: 98% and 92% for H-CE and TADS, respectively), and highest genetic diversity in the used markers (up to 36and32unique genotypes in 20paired samples for H-CE using msp2 and TADS using cpmp, respectively). Microsatellites assessed by H-CE had a lower genetic diversity compared with msp1, msp2, and glurp assessed by H-CE and the SNP-rich markers assessed by TADS, with a maximum of 13unique genotypes, and some genotypes having allelic frequencies larger than 30%.Markers used by TADS gave the most consistent results in distinguishing recrudescence from new infection across all methods (in 18of 20pairs of samples vs 15of 20pairs for H-CE). WHO currently recommends replacing glurp with microsatellites. However, in this study, the replacement of glurp with microsatellites did not change the genotyping outcome, probably due to the lower genetic diversity of microsatellites. More studies with large sample sizes are required to identify the most suitablemicrosatellites that could replace glurp. Our study indicates that TADS should be considered the gold standardfor genotyping to distinguish recrudescence from new infection, and that it should be used to validate other methods. Swiss Tropical and Public Health Institute.

Targeted amplicon deep sequencing of ama1 and mdr1 to track within-host P. falciparum diversity throughout treatment in a clinical drug trial

Introduction Antimalarial therapeutic efficacy studies are routinely conducted in malaria-endemic countries to assess the effectiveness of antimalarial treatment strategies. Targeted amplicon sequencing (AmpSeq) uniquely identifies and quantifies genetically distinct parasites within an infection. In this study, AmpSeq of Plasmodium falciparum apical membrane antigen 1 (ama1), and multidrug resistance gene 1 (mdr1), were used to characterise the complexity of infection (COI) and drug-resistance genotypes, respectively. Methods P. falciparum-positive samples were obtained from a triple artemisinin combination therapy clinical trial conducted in 30 children under 13 years of age between 2018 and 2019 in Kilifi, Kenya. Nine of the 30 participants presented with recurrent parasitemia from day 26 (624h) onwards. The ama1 and mdr1 genes were amplified and sequenced, while msp1, msp2 and glurp data were obtained from the original clinical study. Results The COI was comparable between ama1 and msp1, msp2 and glurp; overall, ama1 detected more microhaplotypes. Based on ama1, a stable number of microhaplotypes were detected throughout treatment until day 3. Additionally, a recrudescent infection was identified with an ama1 microhaplotype initially observed at 30h and later in an unscheduled follow-up visit. Using the relative frequencies of ama1 microhaplotypes and parasitemia, we identified a fast (<1h) and slow (>5h) clearing microhaplotype. As expected, only two mdr1 microhaplotypes (NF and NY) were identified based on the combination of amino acid polymorphisms at codons 86 and 184. Conclusions This study highlights AmpSeq as a tool for highly-resolution tracking of parasite microhaplotypes throughout treatment and can detect variation in microhaplotype clearance estimates. AmpSeq can also identify slow-clearing microhaplotypes, a potential early sign of selection during treatment. Consequently, AmpSeq has the capability of improving the discriminatory power to distinguish recrudescences from reinfections accurately.

Simultaneous targeted amplicon deep sequencing and library preparation for a time and cost-effective universal parasite diagnostic sequencing approach.

We recently described a targeted amplicon deep sequencing (TADS) strategy that utilizes a nested PCR targeting the 18S rDNA gene of blood-borne parasites. The assay facilitates selective digestion of host DNA by targeting enzyme restriction sites present in vertebrates but absent in parasites. This enriching of parasite-derived amplicon drastically reduces the proportion of host-derived reads during sequencing and results in the sensitive detection of several clinically important blood parasites including Plasmodium spp., Babesia spp., kinetoplastids, and filarial nematodes. Despite these promising results, high costs and the laborious nature of metagenomics sequencing are prohibitive to the routine use of this assay in most laboratories. We describe and evaluate a new metagenomic approach that utilizes a set of primers modified from our original assay that incorporates Illumina barcodes and adapters during the PCR steps. This modification makes amplicons immediately compatible with sequencing on the Illumina MiSeq platform, removing the need for a separate library preparation, which is expensive and time-consuming. We compared this modified assay to our previous nested TADS assay in terms of preparation speed, limit of detection (LOD), and cost. Our modifications reduced assay turnaround times from 7 to 5days. The cost decreased from approximately $40 per sample to $11 per sample. The modified assay displayed comparable performance in the detection and differentiation of human-infecting Plasmodium spp., Babesia spp., kinetoplastids, and filarial nematodes in clinical samples. The LOD of this modified approach was determined for malaria parasites and remained similar to that previously reported for our earlier assay (0.58 Plasmodium falciparum parasites/µL of blood). These modifications markedly reduced costs and turnaround times, making the assay more amenable to routine diagnostic applications.

Targeted amplicon deep sequencing of ama1 and mdr1 to track within-host P. falciparum diversity throughout treatment in a clinical drug trial

Introduction Antimalarial therapeutic efficacy studies are routinely conducted in malaria-endemic countries to assess the effectiveness of antimalarial treatment strategies. Targeted amplicon sequencing (AmpSeq) uniquely identifies and quantifies genetically distinct parasites within an infection. In this study, AmpSeq of Plasmodium falciparum apical membrane antigen 1 (ama1), and multidrug resistance gene 1 (mdr1), were used to characterise the complexity of infection (COI) and drug-resistance genotypes, respectively. Methods P. falciparum-positive samples were obtained from a triple artemisinin combination therapy clinical trial conducted in 30 children under 13 years of age between 2018 and 2019 in Kilifi, Kenya. Nine of the 30 participants presented with recurrent parasitemia from day 26 (624h) onwards. The ama1 and mdr1 genes were amplified and sequenced, while msp1, msp2 and glurp data were obtained from the original clinical study. Results The COI was comparable between ama1 and msp1, msp2 and glurp; overall, ama1 detected more microhaplotypes. Based on ama1, a stable number of microhaplotypes were detected throughout treatment until day 3. Additionally, a recrudescent infection was identified with an ama1 microhaplotype initially observed at 30h and later in an unscheduled follow-up visit. Using the relative frequencies of ama1 microhaplotypes and parasitemia, we identified a fast (<1h) and slow (>5h) clearing microhaplotype. As expected, only two mdr1 microhaplotypes (NF and NY) were identified based on the combination of amino acid polymorphisms at codons 86 and 184. Conclusions This study highlights AmpSeq as a tool for highly-resolution tracking of parasite microhaplotypes throughout treatment and can detect variation in microhaplotype clearance estimates. AmpSeq can also identify slow-clearing microhaplotypes, a potential early sign of selection during treatment. Consequently, AmpSeq has the capability of improving the discriminatory power to distinguish recrudescences from reinfections accurately.

Non-human primate malaria in travellers.

Emerging zoonotic malaria caused by Plasmodium knowlesi, P. cynomolgi and P. inui occurred in humans amid a decline of malaria prevalence in Thailand during the past decade. Diagnosis of non-human primate malaria in ecotourists is challenging. Molecular diagnosis seems to be mandatory such as species-specific PCR and targeted amplicon deep sequencing.

Bovine Piroplasma Populations in the Philippines Characterized Using Targeted Amplicon Deep Sequencing.

Molecular assays and capillary electrophoresis sequencing have been used to identify parasites in livestock. The low sample capacity, which increases labor and processing time, is one drawback. Targeted amplicon sequencing (Ampliseq) uses the fast and large sample capacity platform to identify parasites in the target host, overcoming this limitation. DNA was extracted from 162 whole blood samples collected from cattle in three provinces in the Philippines. Using Illumina's Miseq platform, the V4 hypervariable region of the piroplasma 18S rRNA gene was amplified and sequenced. The AMPtk pipeline was used to obtain distinct amplicon sequence variants (ASVs) and the NCBI BLAST non-redundant database was used to assign taxonomy. In total, 95 (58.64%) samples were positive for piroplasma. Using the AMPTk pipeline, 2179 ASVs were obtained. A total of 79 distinct ASVs were obtained after clustering and filtering, which belonged to genera Babesia (n = 58), Theileria (n = 17), Hepatozoon (n = 2), and Sarcocystis (n = 2). The ASV top hits were composed of 10 species: Babesia bovis, B. bigemina, Theileria orientalis, Babesia sp., Hepatozoon canis, Sarcocystis cruzi, T. annulata, T. equi, T. mutans, and Theileria sp. Thung Song. The results generated in this study demonstrated the applicability of Ampliseq in detecting piroplasmid parasites infecting cattle in the Philippines.

Efficient detection of somatic UBA1 variants and clinical scoring system predicting patients with variants in VEXAS syndrome.

To efficiently detect somatic UBA1 variants and establish a clinical scoring system predicting patients with pathogenic variants in VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome. Eighty-nine Japanese patients with clinically suspected VEXAS syndrome were recruited [81 males and 8 females; median age of onset 69.3 years (interquartile range 62.1-77.6)]. Peptide nucleic acid-clamping PCR (PNA-PCR), regular PCR targeting exon 3 clustering UBA1 variants and subsequent Sanger sequencing were conducted for variant screening. Partitioning digital PCR or targeted amplicon deep sequencing was also performed to evaluate the variant allele frequency (VAF). We developed our clinical scoring system to predict UBA1 variant-positive and -negative patients and assessed the diagnostic value of our system using receiver operating characteristics (ROC) curve analysis. Forty patients (44.9%) with reported pathogenic UBA1 variants were identified, including a case having a variant with VAF of 1.7%, using a highly sensitive method. Our clinical scoring system considering age >50 years, cutaneous lesions, lung involvement, chondritis and macrocytic anaemia efficiently predicted patients with UBA1 variants (the area under the curve for the scoring total was 0.908). Genetic screening with the combination of regular PCR and PNA-PCR detected somatic UBA1 variants with high sensitivity and specificity. Our scoring system could efficiently predict patients with UBA1 variants.

Polymorphism analysis of drug resistance markers in Plasmodium falciparum isolates from Benin

Like most countries in sub-Saharan African countries, Benin continues to bear a heavy malaria burden. In 2014, the National Malaria Control Programme (NMCP) changed its treatment policy, and recommended the use of artemisinin-based combination therapy (ACT) as first-line treatment for uncomplicated Plasmodium falciparum cases. The study presented here was conducted to investigate the impact of current antimalarial drug resistance on the country. Molecular surveillance targeting the Pfcrt, Pfmdr1, Pfkelch13, dhfr, and dhps genes was carried out on samples from patients positive for P. falciparum malaria by microscopy, LAMP and PCR diagnostic test. Molecular analysis was performed using targeted amplicon deep sequencing (TADS). In addition, the frequency of parasites with dual deletion of the histidine-rich protein 2 and 3 genes (pfhrp2 and pfhrp3), known to be responsible of the performance of HRP-based malaria rapid diagnostic tests (HRP-RDT), was estimated. Fifty-three falciparum samples collected at the Saint Jean de Dieu hospital in Tanguiéta, Benin, were tested. No Pfkelch13 validated or candidate artemisinin partial resistant variants were identified. A marked prevalence of Asn51Ile (N51I), Cys59Arg (C59R), and Ser108Asn (S108N) mutant alleles was found in the dhfr gene, representing the most frequent genotype (64%). Five-point mutations were detected in dhps, Ile431Val (I431V), Ser436Ala (S436A), Ala437Gly (A437G), Ala581Gly (A581G), Ala613Ser (A613S) of which the third was the most common (92%). No mutation was identified in dhps Lys540Glu (K540E). The quintuple mutant genotype resulting from the combination of the dhfr triple mutant (51I/59R/108N) with the dhps double mutant 436A/437G was detected at a frequency of 30%. Low levels of mutations in Pfcrt and no mutation at codon 86 in the Pfmdr1 DNA fragment were observed, whereas a high level of Tyr184Phe (Y184F) polymorphism in the Pfmdr1 gene was found. These results could be indicative, over a decade after the implementation of ACT therapy, of the return of chloroquine-sensitive but artemether-lumefantrine resistant falciparum genotypes in Benin. There was no evidence of HRP2 and HRP3 deletions. Data from the present study support the need for routine monitoring of molecular markers of antimalarial drug resistance as part of surveillance activities aimed to make informed treatment policy decisions at the national level.

Ex vivo RSA and pfkelch13 targeted-amplicon deep sequencing reveal parasites susceptibility to artemisinin in Senegal, 2017

BackgroundMalaria control is highly dependent on the effectiveness of artemisinin-based combination therapy (ACT), the current frontline malaria curative treatment. Unfortunately, the emergence and spread of parasites resistant to artemisinin (ART) derivatives in Southeast Asia and South America, and more recently in Rwanda and Uganda (East Africa), compromise their long-term use in sub-Saharan Africa, where most malaria deaths occur.MethodsHere, ex vivo susceptibility to dihydroartemisinin (DHA) was evaluated from 38 Plasmodium falciparum isolates collected in 2017 in Thiès (Senegal) expressed in the Ring-stage Survival Assay (RSA). Both major and minor variants were explored in the three conserved-encoding domains of the pfkelch13 gene, the main determinant of ART resistance using a targeted-amplicon deep sequencing (TADS) approach.ResultsAll samples tested in the ex vivo RSA were found to be susceptible to DHA (parasite survival rate < 1%). The non-synonymous mutations K189T and K248R in pfkelch13 were observed each in one isolate, as major (99%) or minor (5%) variants, respectively.ConclusionThe results suggest that ART is still fully effective in the Thiès region of Senegal in 2017. Investigations combining ex vivo RSA and TADS are a useful approach for monitoring ART resistance in Africa.

Evolution of Plasmodium falciparum antimalarial drug resistance markers post-adoption of artemisinin-based combination therapies in Yaounde, Cameroon

ObjectivesThis study aimed to investigate the evolution of Plasmodium falciparum antimalarial drug resistance markers by comparing the pre- and post-adoption of artemisinin-based combination therapies (ACTs) in Yaounde, Cameroon. MethodsThe molecular characterization of known antimalarial drug resistance markers (Pfcrt, Pfmdr1, Pfdhfr, Pfdhps, and Pfk13) in P. falciparum-positive samples collected in 2014 and 2019-2020 was achieved using nested polymerase chain reaction, followed by targeted amplicon deep sequencing on the Illumina MiSeq platform. Data derived were compared with those published during the pre-ACT adoption period from 2004 to 2006. ResultsA high prevalence of Pfmdr1 184F, Pfdhfr 51I/59R/108N, and Pfdhps 437G mutant alleles was observed during the post-ACT adoption period. The Pfcrt 76T and Pfmdr1 86Y mutant alleles significantly declined between 2004 and 2020 (P <0.0001). Conversely, the resistance markers to antifolates, Pfdhfr 51I/59R/108N and Pfdhps 437G, significantly increased during the same study period (P <0.0001). We identified nine mutations in the propeller domains of Pfk13; although they were all present in single parasite isolates, none of them are known to confer artemisinin resistance. ConclusionThis study documented a near-complete reversion to sensitive parasites for markers conferring resistance to the 4-aminoquinolines and arylamino alcohols in Yaounde. In contrast, the Pfdhfr mutations associated with pyrimethamine resistance are moving toward saturation.

Targeted amplicon deep sequencing of ama1 and mdr1 to track within-host P. falciparum diversity throughout treatment in a clinical drug trial.

Antimalarial therapeutic efficacy studies are routinely conducted in malaria-endemic countries to assess the effectiveness of antimalarial treatment strategies. Targeted amplicon sequencing (AmpSeq) uniquely identifies and quantifies genetically distinct parasites within an infection. In this study, AmpSeq of Plasmodium falciparum apical membrane antigen 1 ( ama1), and multidrug resistance gene 1 ( mdr1), were used to characterise the complexity of infection (COI) and drug-resistance genotypes, respectively. P. falciparum-positive samples were obtained from a triple artemisinin combination therapy clinical trial conducted in 30 children under 13 years of age between 2018 and 2019 in Kilifi, Kenya. Nine of the 30 participants presented with recurrent parasitemia from day 26 (624h) onwards. The ama1 and mdr1 genes were amplified and sequenced, while msp1, msp2 and glurp data were obtained from the original clinical study. The COI was comparable between ama1 and msp1, msp2 and glurp; overall, ama1 detected more microhaplotypes. Based on ama1, a stable number of microhaplotypes were detected throughout treatment until day 3. Additionally, a recrudescent infection was identified with an ama1 microhaplotype initially observed at 30h and later in an unscheduled follow-up visit. Using the relative frequencies of ama1 microhaplotypes and parasitemia, we identified a fast (<1h) and slow (>5h) clearing microhaplotype. As expected, only two mdr1 microhaplotypes (NF and NY) were identified based on the combination of amino acid polymorphisms at codons 86 and 184. This study highlights AmpSeq as a tool for highly-resolution tracking of parasite microhaplotypes throughout treatment and can detect variation in microhaplotype clearance estimates. AmpSeq can also identify slow-clearing microhaplotypes, a potential early sign of selection during treatment. Consequently, AmpSeq has the capability of improving the discriminatory power to distinguish recrudescences from reinfections accurately.

Comparative analysis of targeted next-generation sequencing for Plasmodium falciparum drug resistance markers

Well-defined molecular resistance markers are available for a range of antimalarial drugs, and molecular surveillance is increasingly important for monitoring antimalarial drug resistance. Different genotyping platforms are available, but these have not been compared in detail. We compared Targeted Amplicon Deep sequencing (TADs) using Ion Torrent PGM with Illumina MiSeq for the typing of antimalarial drug resistance genes. We developed and validated protocols to type the molecular resistance markers pfcrt, pfdhfr, pfdhps, pfmdr1, pfkelch, and pfcytochrome b, in Plasmodium falciparum for the Ion Torrent PGM and Illumina MiSeq sequencing platforms. With P. falciparum 3D7 and K1 as reference strains, whole blood samples (N = 20) and blood spots from Rapid Diagnostic Test (RDT) samples (N = 5) from patients with uncomplicated falciparum malaria from Ubon Ratchathani were assessed on both platforms and compared for coverage (average reads per amplicon), sequencing accuracy, variant accuracy, false positive rate, false negative rate, and alternative allele detection, with conventional Sanger sequencing as the reference method for SNP calling. Both whole blood and RDT samples could be successfully sequenced using the Ion Torrent PGM and Illumina MiSeq platforms. Coverage of reads per amplicon was higher with Illumina MiSeq (28,886 reads) than with Ion Torrent PGM (1754 reads). In laboratory generated artificial mixed infections, the two platforms could detect the minor allele down to 1% density at 500X coverage. SNPs calls from both platforms were in complete agreement with conventional Sanger sequencing. The methods can be multiplexed with up to 96 samples per run, which reduces cost by 86% compared to conventional Sanger sequencing. Both platforms, using the developed TAD protocols, provide an accurate method for molecular surveillance of drug resistance markers in P. falciparum, but Illumina MiSeq provides higher coverage than Ion Torrent PGM.

Targeted Amplicon deep sequencing of ama1 and mdr1 to track within-host P. falciparum diversity throughout treatment in a clinical drug trial

Antimalarial therapeutic efficacy studies are routinely conducted in malaria-endemic countries to assess the effectiveness of antimalarial treatment strategies. Targeted amplicon deep sequencing (TADS) uniquely identifies and quantifies genetically distinct parasites within an infection. In this study, TADS Plasmodium falciparum apical membrane antigen 1 (ama1), and multidrug resistance gene 1 (mdr1), were used to characterize the complexity of infection (COI) and drug-resistance genotypes, respectively. P. falciparum positive samples were obtained from a triple artemisinin combination therapy clinical trial conducted in 30 children under 13 years of age between 2018 and 2019 in Kilifi, Kenya. Of the 30 participants, 9 presented with recurrent parasitemia from day 26 (624h) onwards. The ama1 and mdr1 genes were amplified and sequenced, while msp1, msp2 and glurp data were obtained from the original clinical study. The COI was comparable between ama1 and msp1, msp2 and glurp, however, overall ama1 detected more haplotypes. Based on ama1, a stable number of haplotypes were detected throughout treatment up until day 3. Additionally, a recrudescent infection was identified with an ama1 haplotype initially observed at 30h and later in an unscheduled follow-up visit. Using the relative frequencies of ama1 haplotypes and parasitaemia, we identified a fast (&lt;1h) and slow (&gt;5h) clearing haplotype. As expected, only two mdr1 haplotypes (NF and NY) were identified based on the combination of amino acid polymorphisms at codons 86 and 184. This study highlights TADS as a sensitive tool for tracking parasite haplotypes throughout treatment and can detect variation in haplotype clearance estimates. TADS can also identify slow clearing haplotypes, a potential early sign of selection during treatment. Consequently, TADS has the capability of improving the discriminatory power to accurately distinguish recrudescences from reinfections.

Targeted Amplicon Deep Sequencing for Monitoring Antimalarial Resistance Markers in Western Kenya.

ABSTRACTMolecular surveillance of Plasmodium falciparum parasites is important to track emerging and new mutations and trends in established mutations and should serve as an early warning system for antimalarial resistance. Dried blood spots were obtained from a Plasmodium falciparum malaria survey in school children conducted across eight counties in western Kenya in 2019. Real-time PCR identified 500 P. falciparum-positive samples that were amplified at five drug resistance loci for targeted amplicon deep sequencing (TADS). The absence of important kelch 13 mutations was similar to previous findings in Kenya pre-2019, and low-frequency mutations were observed in codons 569 and 578. The chloroquine resistance transporter gene codons 76 and 145 were wild type, indicating that the parasites were chloroquine and piperaquine sensitive, respectively. The multidrug resistance gene 1 haplotypes based on codons 86, 184, and 199 were predominantly present in mixed infections with haplotypes NYT and NFT, driven by the absence of chloroquine pressure and the use of lumefantrine, respectively. The sulfadoxine-pyrimethamine resistance profile was a “superresistant” combination of triple mutations in both Pfdhfr (51I 59R 108N) and Pfdhps (436H 437G 540E), rendering sulfadoxine-pyrimethamine ineffective. TADS highlighted the low-frequency variants, allowing the early identification of new mutations, Pfmdr1 codon 199S and Pfdhfr codon 85I and emerging 164L mutations. The added value of TADS is its accuracy in identifying mixed-genotype infections and for high-throughput monitoring of antimalarial resistance markers.

Evaluation of a parasite-density based pooled targeted amplicon deep sequencing (TADS) method for molecular surveillance of Plasmodium falciparum drug resistance genes in Haiti.

Sequencing large numbers of individual samples is often needed for countrywide antimalarial drug resistance surveillance. Pooling DNA from several individual samples is an alternative cost and time saving approach for providing allele frequency (AF) estimates at a population level. Using 100 individual patient DNA samples of dried blood spots from a 2017 nationwide drug resistance surveillance study in Haiti, we compared codon coverage of drug resistance-conferring mutations in four Plasmodium falciparum genes (crt, dhps, dhfr, and mdr1), for the same deep sequenced samples run individually and pooled. Samples with similar real-time PCR cycle threshold (Ct) values (+/- 1.0 Ct value) were combined with ten samples per pool. The sequencing success for samples in pools were higher at a lower parasite density than the individual samples sequence method. The median codon coverage for drug resistance-associated mutations in all four genes were greater than 3-fold higher in the pooled samples than in individual samples. The overall codon coverage distribution for pooled samples was wider than the individual samples. The sample pools with < 40 parasites/μL blood showed more discordance in AF calls for dhfr and mdr1 between the individual and pooled samples. This discordance in AF estimation may be due to low amounts of parasite DNA, which could lead to variable PCR amplification efficiencies. Grouping samples with an estimated ≥ 40 parasites/μL blood prior to pooling and deep sequencing yielded the expected population level AF. Pooling DNA samples based on estimates of > 40 parasites/μL prior to deep sequencing can be used for rapid genotyping of a large number of samples for these four genes and possibly other drug resistant markers in population-based studies. As Haiti is a low malaria transmission country with very few mixed infections and continued chloroquine sensitivity, the pooled sequencing approach can be used for routine national molecular surveillance of resistant parasites.

Whole genome sequencing of low input circulating cell-free DNA obtained from normal human subjects.

Cell‐free DNA circulates in plasma at low levels as a normal by‐product of cellular apoptosis. Multiple clinical pathologies, as well as environmental stressors can lead to increased circulating cell‐free DNA (ccfDNA) levels. Plasma DNA studies frequently employ targeted amplicon deep sequencing platforms due to limited concentrations (ng/ml) of ccfDNA in the blood. Here, we report whole genome sequencing (WGS) and read distribution across chromosomes of ccfDNA extracted from two human plasma samples from normal, healthy subjects, representative of limited clinical samples at <1 ml. Amplification was sufficiently robust with ~90% of the reference genome (GRCh38.p2) exhibiting 10X coverage. Chromosome read coverage was uniform and directly proportional to the number of reads for each chromosome across both samples. Almost 99% of the identified genomic sequence variants were known annotated dbSNP variants in the hg38 reference genome. A high prevalence of C>T and T>C mutations was present along with a strong concordance of variants shared between the germline genome databases; gnomAD (81.1%) and the 1000 Genome Project (93.6%). This study demonstrates isolation and amplification procedures from low input ccfDNA samples that can detect sequence variants across the whole genome from amplified human plasma ccfDNA that can translate to multiple clinical research disciplines.

Targeted deep amplicon sequencing of antimalarial resistance markers in Plasmodium falciparum isolates from Cameroon

BackgroundRecent studies showed the first emergence of the R561H artemisinin-associated resistance marker in Africa, which highlights the importance of continued molecular surveillance to assess the selection and spread of this and other drug resistance markers in the region. MethodIn this study, we used targeted amplicon deep sequencing of 116 isolates collected in two areas of Cameroon to genotype the major drug resistance genes, k13, crt, mdr1, dhfr, and dhps, and the cytochrome b gene (cytb) in Plasmodium falciparum. ResultsNo confirmed or associated artemisinin resistance markers were observed in Pfk13. In comparison, both major and minor alleles associated with drug resistance were found in Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps. Notably, a high frequency of other nonsynonymous mutations was observed across all the genes, except for Pfcytb, suggesting continued selection pressure. ConclusionsThe results from this study supported the continued use of artemisinin-based combination therapy and administration of sulfadoxine-pyrimethamine for intermittent preventive therapy in pregnant women, and for seasonal chemoprevention in these study sites in Cameroon.

Intrahost cytomegalovirus population genetics following antibody pretreatment in a monkey model of congenital transmission.

Human cytomegalovirus (HCMV) infection is the leading non-genetic cause of congenital birth defects worldwide. While several studies have addressed the genetic composition of viral populations in newborns diagnosed with HCMV, little is known regarding mother-to-child viral transmission dynamics and how therapeutic interventions may impact within-host viral populations. Here, we investigate how preexisting CMV-specific antibodies shape the maternal viral population and intrauterine virus transmission. Specifically, we characterize the genetic composition of CMV populations in a monkey model of congenital CMV infection to examine the effects of passively-infused hyperimmune globulin (HIG) on viral population genetics in both maternal and fetal compartments. In this study, 11 seronegative, pregnant monkeys were challenged with rhesus CMV (RhCMV), including a group pretreated with a standard potency HIG preparation (n = 3), a group pretreated with a high-neutralizing potency HIG preparation (n = 3), and an untreated control group (n = 5). Targeted amplicon deep sequencing of RhCMV glycoprotein B and L genes revealed that one of the three strains present in the viral inoculum (UCD52) dominated maternal and fetal viral populations. We identified minor haplotypes of this strain and characterized their dynamics. Many of the identified haplotypes were consistently detected at multiple timepoints within sampled maternal tissues, as well as across tissue compartments, indicating haplotype persistence over time and transmission between maternal compartments. However, haplotype numbers and diversity levels were not appreciably different between control, standard-potency, and high-potency pretreatment groups. We found that while the presence of maternal antibodies reduced viral load and congenital infection, it had no apparent impact on intrahost viral genetic diversity at the investigated loci. Interestingly, some minor haplotypes present in fetal and maternal-fetal interface tissues were also identified as minor haplotypes in corresponding maternal tissues, providing evidence for a loose RhCMV mother-to-fetus transmission bottleneck even in the presence of preexisting antibodies.

Evaluation of an ensemble-based distance statistic for clustering MLST datasets using epidemiologically defined clusters of cyclosporiasis.

Outbreaks of cyclosporiasis, a food-borne illness caused by the coccidian parasite Cyclospora cayetanensis have increased in the USA in recent years, with approximately 2300 laboratory-confirmed cases reported in 2018. Genotyping tools are needed to inform epidemiological investigations, yet genotyping Cyclospora has proven challenging due to its sexual reproductive cycle which produces complex infections characterized by high genetic heterogeneity. We used targeted amplicon deep sequencing and a recently described ensemble-based distance statistic that accommodates heterogeneous (mixed) genotypes and specimens with partial genotyping data, to genotype and cluster 648 C. cayetanensis samples submitted to CDC in 2018. The performance of the ensemble was assessed by comparing ensemble-identified genetic clusters to analogous clusters identified independently based on common food exposures. Using these epidemiologic clusters as a gold standard, the ensemble facilitated genetic clustering with 93.8% sensitivity and 99.7% specificity. Hence, we anticipate that this procedure will greatly complement epidemiologic investigations of cyclosporiasis.

Abstract GMM-048: CTDNA RESPONSE TO THE PARP INHIBITOR RUCAPARIB PREDICTS PROGRESSION-FREE SURVIVAL AND BEST OVERALL RESPONSE ON THE ARIEL2 TRIAL

Abstract BACKGROUND: High grade serous ovarian carcinoma (HGSOC) is characterized by ubiquitous TP53 mutation and has the highest genomic complexity of all epithelial neoplasms. Sensitivity to PARP inhibitor therapy is strongly associated with homologous recombination deficiency (HRD). Genomic biomarkers of HRD such as genome-wide loss of heterozygosity (LOH) scores predict HRD and response to rucaparib. We hypothesized that functional testing of response during PARP inhibitor treatment using changes in circulating tumour DNA (ctDNA) could improve prediction of patient outcome. We tested whether the change in ctDNA TP53 mutant allele fraction (MAF) after one cycle of rucaparib treatment was predictive of progression free survival (PFS) and response in patients from the phase 2 ARIEL2 trial in women with platinum-sensitive recurrent high grade ovarian cancer (NCT01891344). METHODS: We analyzed serial plasma samples (n = 636) from 142 HGSOC patients during screening, on day 1 of each treatment cycle, and at the end of rucaparib treatment. Targeted amplicon deep sequencing (TADS) of TP53 was performed on DNA extracted from plasma (median depth 6916×). Somatic TP53 mutation and loss of heterozygosity score (LOH) were available from archival and biopsy specimens. Statistical analyses were pre-specified and ctDNA analysis was carried out blinded to visit and response data. TP53 MAF changes after one cycle of treatment were compared with PFS and best overall response assessed by RECIST v1.1 and GCIG CA-125 criteria. Optimal cut points for ctDNA response were determined using a cross-validation analysis. In cases with &amp;gt;1 TP53 mutation, response assessment was performed using the mutation with highest MAF. RESULTS: We detected TP53 mutations in plasma from 134 patients; all cases were concordant between tumour and plasma except for one patient (present in plasma but not tumour). In 18 patients (13%), 2 or more TP53 mutations were detected in ctDNA. The median TP53 MAF prior to cycle 1 was 2.6% (IQR 0.3–8.6). Reduction of &amp;gt;70% of TP53 MAF in ctDNA between cycle 1 and 2 was significantly predictive of improved PFS (n = 97; HR = 0.53, 95% CI 0.34-0.85, p = 0.008, median 273 vs 158 days, sensitivity 76%, specificity 62%) and best overall response (n = 97; OR = 7.04, 95% CI 2.69–21.06, p &amp;lt; 0.001). Combining ctDNA and LOH scores did not improve prediction of response. CONCLUSIONS Response measured by &amp;gt;70% fall in TP53 ctDNA between pre-cycle 1 and pre-cycle 2 of rucaparib therapy was significantly associated with best overall response and improved PFS. Similar findings were observed in a retrospective study of recurrent HGSOC treated with standard of care chemotherapy. The pathological or genomic factors causing multiple TP53 mutations in ctDNA are unknown. The association between early fall in ctDNA and validated RECIST and CA-125 response markers provides strong evidence that ctDNA may have utility for detecting early response to targeted therapy. Further analyses in randomized studies should be performed to confirm that ctDNA response has strong predictive value. Citation Format: Anna Piskorz, David Robertson, Kevin K. Lin, James Morris, Elaina Mann, Amit Oza, Robert L. Coleman, David M. O'Malley, Michael Friedlander, Janiel M. Cragun, Ling Ma, Heidi Giordano, Iain A. McNeish, Elizabeth Swisher, James Wason, James D. Brenton. CTDNA RESPONSE TO THE PARP INHIBITOR RUCAPARIB PREDICTS PROGRESSION-FREE SURVIVAL AND BEST OVERALL RESPONSE ON THE ARIEL2 TRIAL [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-048.