Development of a TaqMan Allelic Discrimination Assay for detection of Single Nucleotides Polymorphisms associated with anti-malarial drug resistance

<p>Abstract</p> <p>Background</p> <p>Anti-malarial drug resistance poses a threat to current global efforts towards control and elimination of malaria. Several methods are used in monitoring anti-malarial drug resistance. Molecular markers such as single nucleotide polymorphism (SNP) for example are increasingly being used to identify genetic mutations related to anti-malarial drug resistance. Several methods are currently being used in analysis of SNP associated with anti-malarial drug resistance and although each one of these methods has unique strengths and shortcoming, there is still need to improve and/or develop new methods that will close the gap found in the current methods.</p> <p>Methods</p> <p>TaqMan Allelic Discrimination assays for detection of SNPs associated with anti-malarial drug resistance were designed for analysis on Applied Biosystems PCR platform. These assays were designed by submitting SNP sequences associated with anti-malarial drug resistance to Applied Biosystems website. Eleven SNPs associated with resistance to anti-malarial drugs were selected and tested. The performance of each SNP assay was tested by creating plasmid DNAs carrying codons of interests and analysing them for analysis. To test the sensitivity and specificity of each SNP assay, 12 clinical samples were sequenced at codons of interest and used in the analysis. Plasmid DNAs were used to establish the Limit of Detection (LoD) for each assay.</p> <p>Results</p> <p>Data from genetic profiles of the <it>Plasmodium falciparum </it>laboratory strains and sequence data from 12 clinical samples was used as the reference method with which the performance of the SNP assays were compared to. The sensitivity and specificity of each SNP assay was establish at 100%. LoD for each assay was established at 2 GE, equivalent to less than 1 parasite/μL. SNP assays performed well in detecting mixed infection and analysis of clinical samples.</p> <p>Conclusion</p> <p>TaqMan Allelic Discrimination assay provides a good alternative tool in detection of SNPs associated with anti-malarial drug.</p

Effect of malaria transmission reduction by insecticide-treated bed nets (ITNs) on the genetic diversity of Plasmodium falciparum merozoite surface protein (MSP-1) and circumsporozoite (CSP) in western Kenya

Background Although several studies have investigated the impact of reduced malaria transmission due to insecticide-treated bed nets (ITNs) on the patterns of morbidity and mortality, there is limited information on their effect on parasite diversity. Methods Sequencing was used to investigate the effect of ITNs on polymorphisms in two genes encoding leading Plasmodium falciparum vaccine candidate antigens, the 19 kilodalton blood stage merozoite surface protein-1 (MSP-119kDa) and the Th2R and Th3R T-cell epitopes of the pre-erythrocytic stage circumsporozoite protein (CSP) in a large community-based ITN trial site in western Kenya. The number and frequency of haplotypes as well as nucleotide and haplotype diversity were compared among parasites obtained from children <5 years old prior to the introduction of ITNs (1996) and after 5 years of high coverage ITN use (2001). Results A total of 12 MSP-119kDa haplotypes were detected in 1996 and 2001. The Q-KSNG-L and E-KSNG-L haplotypes corresponding to the FVO and FUP strains of P. falciparum were the most prevalent (range 32–37%), with an overall haplotype diversity of > 0.7. No MSP-119kDa 3D7 sequence-types were detected in 1996 and the frequency was less than 4% in 2001. The CSP Th2R and Th3R domains were highly polymorphic with a total of 26 and 14 haplotypes, respectively detected in 1996 and 34 and 13 haplotypes in 2001, with an overall haplotype diversity of > 0.9 and 0.75 respectively. The frequency of the most predominant Th2R and Th3R haplotypes was 14 and 36%, respectively. The frequency of Th2R and Th3R haplotypes corresponding to the 3D7 parasite strain was less than 4% at both time points. There was no significant difference in nucleotide and haplotype diversity in parasite isolates collected at both time points. Conclusion High diversity in these two genes has been maintained overtime despite marked reductions in malaria transmission due to ITNs use. The frequency of 3D7 sequence-types was very low in this area. These findings provide information that could be useful in the design of future malaria vaccines for deployment in endemic areas with high ITN coverage and in interpretation of efficacy data for malaria vaccines based on 3D7 parasite strains

Trends in the density (Number /km²) of vervet monkey, guinea fowl, giraffe, wildebeest, impala, buffalo, warthog and waterbuck for the entire Nakuru Wildlife Conservancy during 1996–2015.

<p>Trends in the density (Number /km²) of vervet monkey, guinea fowl, giraffe, wildebeest, impala, buffalo, warthog and waterbuck for the entire Nakuru Wildlife Conservancy during 1996–2015.</p

Temporal trends in the aggregate biomass of the 32 herbivore species, 4 primate species and 8 carnivore species counted in the Nakuru Wildlife Conservancy from 1996 to 2015.

<p>The species and their unit weights [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169730#pone.0169730.ref039" target="_blank">39</a>] are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169730#pone.0169730.s008" target="_blank">S2 Table</a> in the supplementary materials. The vertical needles are the biomass estimates, the thick solid line is the trend line and the band around the line is the 95% confidence band fitted by penalized cubic basis spline regression.</p

Trends in the density (Number /km²) of steinbuck, bushbuck, hartebeest, Greater kudu, klipspringer, mongoose, oribi, porcupine, cheetah, leopard, ostrich and Sykes monkey for the entire Nakuru Wildlife Conservancy during 1996–2015.

<p>Trends in the density (Number /km²) of steinbuck, bushbuck, hartebeest, Greater kudu, klipspringer, mongoose, oribi, porcupine, cheetah, leopard, ostrich and Sykes monkey for the entire Nakuru Wildlife Conservancy during 1996–2015.</p

Trends in the density (Number /km²) of hyena, baboon, Grant’s gazelle, Grevy’s zebra, lion, spring hare, Burchell’s zebra, bushpig, white rhino, rock hyrax, topi and oryx for the entire Nakuru Wildlife Conservancy during 1996–2015.

<p>Trends in the density (Number /km²) of hyena, baboon, Grant’s gazelle, Grevy’s zebra, lion, spring hare, Burchell’s zebra, bushpig, white rhino, rock hyrax, topi and oryx for the entire Nakuru Wildlife Conservancy during 1996–2015.</p

Trends in the density (Number /km²) of dik dik, eland, African hare, jackal, duiker, hippopotamus, Thomson’s gazelle, aardvark, serval cat, colobus monkey, bat-eared fox and reedbuck for the entire Nakuru Wildlife Conservancy during 1996–2015.

<p>Trends in the density (Number /km²) of dik dik, eland, African hare, jackal, duiker, hippopotamus, Thomson’s gazelle, aardvark, serval cat, colobus monkey, bat-eared fox and reedbuck for the entire Nakuru Wildlife Conservancy during 1996–2015.</p

The estimated variances of the disturbance terms, the variances of the irregular component, damping factor and period of the cycles in annual, wet and dry season rainfall components.

<p>The estimated variances of the disturbance terms, the variances of the irregular component, damping factor and period of the cycles in annual, wet and dry season rainfall components.</p

a) The distribution of the total monthly rainfall across months in Nakuru Wildlife conservancy averaged over 1967 and 2011 and the standardized deviates for the b) annual, c) wet season and d) dry season rainfall components.

<p>The vertical needles in panels <b>b</b>-<b>d</b> are the observed standardized deviates, the thick solid lines are the 4-year moving averages for the annual and wet season rainfall components and the 3-year moving average for the dry season rainfall component. The dashed horizontal lines are the percentiles of the standardized deviates for each rainfall component. The two dashed curves in the panel for the wet season rainfall are the secondary cycles with 3.5 and 2.2-year periods.</p

Significance analysis of components (based on the final state).

<p>Significance analysis of components (based on the final state).</p