Tenfold difference in DNA recovery rate: systematic comparison of whole blood vs. dried blood spot sample collection for malaria molecular surveillance

BACKGROUND: Molecular and genomic surveillance is becoming increasingly used to track malaria control and elimination efforts. Blood samples can be collected as whole blood and stored at - 20 degrees C until DNA extraction, or as dried blood spots (DBS), circumventing the need for a cold chain. Despite the wide use of either method, systematic comparisons of how the method of blood sample preservation affects the limit of detection (LOD) of molecular diagnosis and the proportion of DNA recovered for downstream applications are lacking. METHODS: Extractions based on spin columns, magnetic beads, Tween-Chelex, and direct PCR without prior extraction were compared for whole blood and dried blood spots (DBS) using dilution series of Plasmodium falciparum culture samples. Extracted DNA was quantified by qPCR and droplet digital PCR (ddPCR). RESULTS: DNA recovery was 5- to 10-fold higher for whole blood compared to DBS, resulting in a 2- to 3-fold lower LOD for both extraction methods compared to DBS. For whole blood, a magnetic bead-based method resulted in a DNA recovery rate of 88-98% when extracting from whole blood compared to 17-33% for a spin-column based method. For extractions from DBS, the magnetic bead-based method resulted in 8-20% DNA recovery, while the spin-column based method resulted in only 2% DNA recovery. The Tween-Chelex method was superior to other methods with 15-21% DNA recovery, and even more sensitive than extractions from whole blood samples. The direct PCR method was found to have the lowest LOD overall for both, whole blood and DBS. CONCLUSIONS: Pronounced differences in LOD and DNA yield need to be considered when comparing prevalence estimates based on molecular methods and when selecting sampling protocols for other molecular surveillance applications

Left bundle branch block causes relative but not absolute septal underperfusion during exercise

Aims Left bundle branch block (LBBB) often causes septal perfusion defects in radionuclide myocardial perfusion imaging using exercise (Ex) but rarely using vasodilator stress. We studied whether this is due to an underlying structural disease inherent to spontaneous LBBB or whether it is also found in temporary LBBB induced by right ventricular pacing (PM) indicating a functional rather than a structural alteration. Methods and results Regional myocardial blood flow (MBF) at rest and at Ex was measured with(15)O-H(2)O and PET in 10 age-matched healthy volunteers (controls), 10 LBBB patients and 10 PM patients with right ventricular pacing off and on (PM off and PM on). Although at Ex septal MBF tended to be higher in LBBB than in controls (3.04 +/- 1.18 vs. 2.27 +/- 0.72 mL/min/g; P= ns), the ratio septal/lateral MBF was 19% lower in LBBB than in controls (P < 0.05). Similarly, switching PM on at Ex decreased the ratio septal/lateral MBF by 17% (P < 0.005). Conclusion The apparent septal perfusion defect in LBBB is mainly due to a relative lateral hyperperfusion rather than to an absolute septal flow decrease. This pattern seems to be reversibly inducible by right ventricular pacing, suggesting a functional rather than a structural alteratio

Significant geographical differences in prevalence of mutations associated with Plasmodium falciparum and Plasmodium vivax drug resistance in two regions from Papua New Guinea

Drug resistance remains a major obstacle to malaria treatment and control. It can arise and spread rapidly, and vary substantially even at sub-national level. National malaria programmes require cost-effective and timely ways of characterizing drug-resistance at multiple sites within their countries.; An improved multiplexed post-PCR ligase detection reaction-fluorescent microsphere assay (LDR-FMA) was used to simultaneously determine the presence of mutations in chloroquine resistance transporter (crt), multidrug resistance 1 (mdr1), dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes in Plasmodium falciparum (n = 727) and Plasmodium vivax (n = 574) isolates collected in 2006 from cross-sectional community population surveys in two geographically distinct regions (Madang and East Sepik) of Papua New Guinea (PNG) where strong regional differences in in vivo aminoquinoline and antifolate therapeutic efficacy had previously been observed. Data were compared to those of a follow-up survey conducted in 2010.; Despite some very low parasite densities, the assay successfully amplified all P. falciparum and P. vivax loci in 77 and 69 % of samples, respectively. In 2006, prevalences of pfdhfr (59R-108 N) double mutation/wild type pfdhps haplotype, pfcrt SVMNT haplotype (72S-76T double mutation), and 86Y pfmdr1 mutation all exceeded 90 %. For P. vivax, 65 % carried at least two pvdhfr mutations, 97 % the 647P pvdhps mutation and 54 % the 976F pvmdr1 mutation. Prevalence of mutant haplotypes was higher in Madang than East Sepik for pfcrt SVMNT (97.4 vs 83.3 %, p = 0.001), pfdhfr (59R-108 N) (100 vs 90.6 %, p = 0.001), pvdhfr haplotypes (75.8 vs 47.6 %, p = 0.001) and pvmdr1 976F (71.2 vs 26.2 %, p &lt; 0.001). Data from a subsequent Madang survey in 2010 showed that the prevalence of pfdhps mutations increased significantly from &lt;5 % to &gt;30 % (p &lt; 0.001) as did the prevalence of pvdhfr mutant haplotypes (from 75.8 to 97.4 %, p = 0.012).; This LDR-FMA multiplex platform shows feasibility for low-cost, high-throughput, rapid characterization of a broad range of drug-resistance markers in low parasitaemia infections. Significant geographical differences in mutation prevalence correlate with previous genotyping surveys and in vivo trials and may reflect variable drug pressure and differences in health-care access in these two PNG populations

Plasmodium vivax and Plasmodium falciparum infection dynamics: re-infections, recrudescences and relapses

Background: In malaria endemic populations, complex patterns of Plasmodium vivax and Plasmodium falciparum blood-stage infection dynamics may be observed. Genotyping samples from longitudinal cohort studies for merozoite surface protein (msp) variants increases the information available in the data, allowing multiple infecting parasite clones in a single individual to be identified. msp genotyped samples from two longitudinal cohorts in Papua New Guinea (PNG) and Thailand were analysed using a statistical model where the times of acquisition and clearance of each clone in every individual were estimated using a process of data augmentation. Results: For the populations analysed, the duration of blood-stage P. falciparum infection was estimated as 36 (95% Credible Interval (CrI): 29, 44) days in PNG, and 135 (95% CrI 94, 191) days in Thailand. Experiments on simulated data indicated that it was not possible to accurately estimate the duration of blood-stage P. vivax infections due to the lack of identifiability between a single blood-stage infection and multiple, sequential blood-stage infections caused by relapses. Despite this limitation, the method and data point towards short duration of blood-stage P. vivax infection with a lower bound of 24 days in PNG, and 29 days in Thailand. On an individual level, P. vivax recurrences cannot be definitively classified into re-infections, recrudescences or relapses, but a probabilistic relapse phenotype can be assigned to each P. vivax sample, allowing investigation of the association between epidemiological covariates and the incidence of relapses. Conclusion: The statistical model developed here provides a useful new tool for in-depth analysis of malaria data from longitudinal cohort studies, and future application to data sets with multi-locus genotyping will allow more detailed investigation of infection dynamics

Tissue Sampling Methods and Standards for Vertebrate Genomics

The recent rise in speed and efficiency of new sequencing technologies have facilitated high-throughput sequencing, assembly and analyses of genomes, advancing ongoing efforts to analyze genetic sequences across major vertebrate groups. Standardized procedures in acquiring high quality DNA and RNA and establishing cell lines from target species will facilitate these initiatives. We provide a legal and methodological guide according to four standards of acquiring and storing tissue for the Genome 10K Project and similar initiatives as follows: four-star (banked tissue/cell cultures, RNA from multiple types of tissue for transcriptomes, and sufficient flash-frozen tissue for 1 mg of DNA, all from a single individual);three-star (RNA as above and frozen tissue for 1 mg of DNA); two-star (frozen tissue for at least 700 μg of DNA); and one-star (ethanol-preserved tissue for 700 μg of DNA or less of mixed quality). At a minimum, all tissues collected for the Genome 10K and other genomic projects should consider each species’ natural history and follow institutional and legal requirements. Associated documentation should detail as much information as possible about provenance to ensure representative sampling and subsequent sequencing. Hopefully, the procedures outlined here will not only encourage success in the Genome 10K Project but also inspire the adaptation of standards by other genomic projects, including those involving other biota

Chromosomal-level assembly of the Asian Seabass genome using long sequence reads and multi-layered scaffolding

We report here the ~670 Mb genome assembly of the Asian seabass (Lates calcarifer), a tropical marine teleost. We used long-read sequencing augmented by transcriptomics, optical and genetic mapping along with shared synteny from closely related fish species to derive a chromosome-level assembly with a contig N50 size over 1 Mb and scaffold N50 size over 25 Mb that span ~90% of the genome. The population structure of L. calcarifer species complex was analyzed by re-sequencing 61 individuals representing various regions across the species' native range. SNP analyses identified high levels of genetic diversity and confirmed earlier indications of a population stratification comprising three clades with signs of admixture apparent in the South-East Asian population. The quality of the Asian seabass genome assembly far exceeds that of any other fish species, and will serve as a new standard for fish genomics

Persistence of Plasmodium falciparum parasitemia after artemisinin combination therapy: evidence from a randomized trial in Uganda

Artemisinin resistance is rapidly spreading in Southeast Asia. The efficacy of artemisinin-combination therapy (ACT) continues to be excellent across Africa. We performed parasite transcriptional profiling and genotyping on samples from an antimalarial treatment trial in Uganda. We used qRT-PCR and genotyping to characterize residual circulating parasite populations after treatment with either ACT or ACT-primaquine. Transcripts suggestive of circulating ring stage parasites were present after treatment at a prevalence of >25% until at least 14 days post initiation of treatment. Greater than 98% of all ring stage parasites were cleared within the first 3 days, but subsequently persisted at low concentrations until day 14 after treatment. Genotyping demonstrated a significant decrease in multiplicity of infection within the first 2 days in both ACT and ACT-primaquine arms. However, multiple clone infections persisted until day 14 post treatment. Our data suggest the presence of genetically diverse persisting parasite populations after ACT treatment. Although we did not demonstrate clinical treatment failures after ACT and the viability and transmissibility of persisting ring stage parasites remain to be shown, these findings are of relevance for the interpretation of parasite clearance transmission dynamics and for monitoring drug effects in Plasmodium falciparum parasites

Extensive population genetic structure in the giraffe

<p>Abstract</p> <p>Background</p> <p>A central question in the evolutionary diversification of large, widespread, mobile mammals is how substantial differentiation can arise, particularly in the absence of topographic or habitat barriers to dispersal. All extant giraffes (<it>Giraffa camelopardalis</it>) are currently considered to represent a single species classified into multiple subspecies. However, geographic variation in traits such as pelage pattern is clearly evident across the range in sub-Saharan Africa and abrupt transition zones between different pelage types are typically not associated with extrinsic barriers to gene flow, suggesting reproductive isolation.</p> <p>Results</p> <p>By analyzing mitochondrial DNA sequences and nuclear microsatellite loci, we show that there are at least six genealogically distinct lineages of giraffe in Africa, with little evidence of interbreeding between them. Some of these lineages appear to be maintained in the absence of contemporary barriers to gene flow, possibly by differences in reproductive timing or pelage-based assortative mating, suggesting that populations usually recognized as subspecies have a long history of reproductive isolation. Further, five of the six putative lineages also contain genetically discrete populations, yielding at least 11 genetically distinct populations.</p> <p>Conclusion</p> <p>Such extreme genetic subdivision within a large vertebrate with high dispersal capabilities is unprecedented and exceeds that of any other large African mammal. Our results have significant implications for giraffe conservation, and imply separate <it>in situ </it>and <it>ex situ </it>management, not only of pelage morphs, but also of local populations.</p