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Background Chloroquine (CQ) was the cornerstone of anti-malarial treatment in Africa for almost 50 years, but has been widely withdrawn due to the emergence and spread of resistance. Recent reports have suggested that CQ-susceptibility may return following the cessation of CQ usage. Here, we monitor CQ sensitivity and determine the prevalence of genetic polymorphisms in the CQ resistance transporter gene ( pfcrt ) of Plasmodium falciparum isolates recently imported from Africa to China. Methods Blood samples were collected from falciparum malaria patients returning to China from various countries in Africa. Isolates were tested for their sensitivity to CQ using the SYBR Green I test ex vivo, and for a subset of samples, in vitro following culture adaptation. Mutations at positions 72–76 and codon 220 of the pfcrt gene were analyzed by sequencing and confirmed by PCR-RFLP. Correlations between drug sensitivity and pfcrt polymorphisms were investigated. Results Of 32 culture adapted isolates assayed, 17 (53.1%), 6 (18.8%) and 9 (28.1%) were classified as sensitive, moderately resistant, and highly resistant, respectively. In vitro CQ susceptibility was related to point mutations in the pfcrt gene, the results indicating a strong association between pfcrt genotype and drug sensitivity. A total of 292 isolates were typed at the pfcrt locus, and the prevalence of the wild type (CQ sensitive) haplotype CVMNK in isolates from East, South, North, West and Central Africa were 91.4%, 80.0%, 73.3%, 53.3% and 51.7%, respectively. The only mutant haplotype observed was CVIET, and this was almost always linked to an additional mutation at A220S. Conclusions Our results suggest that a reduction in drug pressure following withdrawal of CQ as a first-line drug may lead to a resurgence in CQ sensitive parasites. The prevalence of wild-type pfcrt CQ sensitive parasites from East, South and North Africa was higher than from the West and Central areas, but this varied greatly between countries. Further surveillance is required to assess whether the prevalence of CQ resistant parasites will continue to decrease in the absence of widespread CQ usage.

BACKGROUND: Loop-mediated isothermal amplification (LAMP) is a high performance method for detecting DNA and holds promise for use in the molecular detection of infectious pathogens, including Plasmodium spp. However, in most malaria-endemic areas, which are often resource-limited, current LAMP methods are not feasible for diagnosis due to difficulties in accurately interpreting results with problems of sensitive visualization of amplified products, and the risk of contamination resulting from the high quantity of amplified DNA produced. In this study, we establish a novel visualized LAMP method in a closed-tube system, and validate it for the diagnosis of malaria under simulated field conditions. METHODS: A visualized LAMP method was established by the addition of a microcrystalline wax-dye capsule containing the highly sensitive DNA fluorescence dye SYBR Green I to a normal LAMP reaction prior to the initiation of the reaction. A total of 89 blood samples were collected on filter paper and processed using a simple boiling method for DNA extraction, and then tested by the visualized LAMP method for Plasmodium vivax infection. RESULTS: The wax capsule remained intact during isothermal amplification, and released the DNA dye to the reaction mixture only when the temperature was raised to the melting point following amplification. Soon after cooling down, the solidified wax sealed the reaction mix at the bottom of the tube, thus minimizing the risk of aerosol contamination. Compared to microscopy, the sensitivity and specificity of LAMP were 98.3% (95% confidence interval (CI): 91.1-99.7%) and 100% (95% CI: 88.3-100%), and were in close agreement with a nested polymerase chain reaction method. CONCLUSIONS: This novel, cheap and quick visualized LAMP method is feasible for malaria diagnosis in resource-limited field settings.

Variation in the multiplication rate of blood stage malaria parasites is often positively correlated with the severity of the disease they cause. The rodent malaria parasite Plasmodium yoelii yoelii has strains with marked differences in multiplication rate and pathogenicity in the blood. We have used genetic analysis by linkage group selection (LGS) to identify genes that determine differences in multiplication rate. Genetic crosses were generated between genetically unrelated, fast- (17XYM) and slowly multiplying (33XC) clones of P. y. yoelii. The uncloned progenies of these crosses were placed under multiplication rate selection in blood infections in mice. The selected progenies were screened for reduction in intensity of quantitative genetic markers of the slowly multiplying parent. A small number of strongly selected markers formed a linkage group on P. y. yoelii chromosome 13. Of these, that most strongly selected marked the gene encoding the P. yoelii erythrocyte binding ligand (pyebl), which has been independently identified by Otsuki and colleagues [Otsuki H, et al. (2009) Proc Natl Acad Sci USA 106:10.1073/pnas.0811313106] as a major determinant of virulence in these parasites. In an analysis of a previous genetic cross in P. y. yoelii, pyebl alleles of fast- and slowly multiplying parents segregated with the fast and slow multiplication rate phenotype in the cloned recombinant progeny, implying the involvement of the pyebl locus in determining the multiplication rate. Our genome-wide LGS analysis also indicated effects of at least 1 other locus on multiplication rate, as did the findings of Otsuki and colleagues on virulence in P. y. yoelii.

Abstract Genes encoded within organelle genomes often evolve at rates different from those in the nuclear genome. Here, we analyzed the relative rates of nucleotide substitution in the mitochondrial, apicoplast, and nuclear genomes in four different lineages of Plasmodium species (malaria parasites) infecting mammals. The rates of substitution in the three genomes exhibit substantial variation among lineages, with the relative rates of nuclear and mitochondrial DNA being particularly divergent between the Laverania (including Plasmodium falciparum) and Vivax lineages (including Plasmodium vivax). Consideration of synonymous and nonsynonymous substitution rates suggests that their variation is largely due to changes in mutation rates, with constraints on amino acid replacements remaining more similar among lineages. Mitochondrial DNA mutation rate variations among lineages may reflect differences in the long-term average lengths of the sexual and asexual stages of the life cycle. These rate variations have far-reaching implications for the use of molecular clocks to date Plasmodium evolution.

Plasmodium rhoptry neck protein 2 (RON2), which is released from the neck portion of the merozoite rhoptries and interacts with the microneme protein Apical Membrane Antigen 1 (AMA1), plays a crucial role in erythrocyte invasion. In this study, we sequenced the Plasmodium vivax RON2 gene from 19 P. vivax isolates collected in central China in order to establish whether this protein is under positive diversifying selection, which may occur as a result of protective host immune pressure†. In comparison with the P. vivax Sal-1 reference line, we found 10 amino acid substitutions dispersed throughout the open reading frame as well as indels caused by polymorphism in a repeat unit (21–23 repeats of (Q/E/K/N/H)(G/D)G(H/L/Y/P)G) in the second tandem repeat region located at amino acid positions 541–650. A McDonald-Kreitman test with RON2 sequences from the primate malaria parasite Plasmodium knowlesi, detected significant departure from neutrality in the PvRON2 3′ region (nucleotide positions 2668–6609). These results suggest that the PvRON2 gene has evolved under positive diversifying selection.

Variation in the multiplication rate of blood stage malaria parasites is often positively correlated with the severity of the disease they cause. The rodent malaria parasite Plasmodium yoelii yoelii has strains with marked differences in multiplication rate and pathogenicity in the blood. We have used genetic analysis by linkage group selection (LGS) to identify genes that determine differences in multiplication rate. Genetic crosses were generated between genetically unrelated, fast- (17XYM) and slowly multiplying (33XC) clones of P. y. yoelii. The uncloned progenies of these crosses were placed under multiplication rate selection in blood infections in mice. The selected progenies were screened for reduction in intensity of quantitative genetic markers of the slowly multiplying parent. A small number of strongly selected markers formed a linkage group on P. y. yoelii chromosome 13. Of these, that most strongly selected marked the gene encoding the P. yoelii erythrocyte binding ligand (pyebl), which has been independently identified by Otsuki and colleagues [Otsuki H, et al. (2009) Proc Natl Acad Sci USA 106:10.1073/pnas.0811313106] as a major determinant of virulence in these parasites. In an analysis of a previous genetic cross in P. y. yoelii, pyebl alleles of fast- and slowly multiplying parents segregated with the fast and slow multiplication rate phenotype in the cloned recombinant progeny, implying the involvement of the pyebl locus in determining the multiplication rate. Our genome-wide LGS analysis also indicated effects of at least 1 other locus on multiplication rate, as did the findings of Otsuki and colleagues on virulence in P. y. yoelii.

Skin-based samples (leather, skin, and parchment) in archaeological, historic and museum settings are among the most challenging materials to radiocarbon (14C) date in terms of removing exogenous carbon sources—comparable to bone collagen in many respects but with much less empirical study to guide pretreatment approaches. In the case of leather, the 14C content of materials used in manufacturing the leather can vary greatly. The presence of leather manufacturing chemicals before pretreatment and their absence afterward is difficult to demonstrate, and the accuracy of dates depends upon isolating the original animal proteins and removing exogenous carbon. Parchments differ in production technique from leather but include similar unknowns. It is not clear that lessons learned in the treatment of one are always salient for treating the other. We measured the 14C content of variously pretreated leather, parchment, skin samples, and extracts, producing apparent ages that varied by hundreds or occasionally thousands of years depending upon sample pretreatment. Fourier Transform Infrared Spectroscopy (FTIR) and C:N ratios provided insight into the chemical composition of carbon reservoirs contributing to age differences. The results of these analyses demonstrated that XAD column chromatography resulted in the most accurate 14C dates for leather and samples of unknown tannage, and FTIR allowed for the detection of contamination that might have otherwise been overlooked.

Hundreds of scarlet macaw (Ara macao cyanoptera) skeletons have been recovered from archaeological contexts in the southwestern United States and northwestern Mexico (SW/NW). The location of these skeletons, >1,000 km outside their Neotropical endemic range, has suggested a far-reaching pre-Hispanic acquisition network. Clear evidence for scarlet macaw breeding within this network is only known from the settlement of Paquimé in NW dating between 1250 and 1450 CE. Although some scholars have speculated on the probable existence of earlier breeding centers in the SW/NW region, there has been no supporting evidence. In this study, we performed an ancient DNA analysis of scarlet macaws recovered from archaeological sites in Chaco Canyon and the contemporaneous Mimbres area of New Mexico. All samples were directly radiocarbon dated between 900 and 1200 CE. We reconstructed complete or near-complete mitochondrial genome sequences of 14 scarlet macaws from five different sites. We observed remarkably low genetic diversity in this sample, consistent with breeding of a small founder population translocated outside their natural range. Phylogeographic comparisons of our ancient DNA mitogenomes with mitochondrial sequences from macaws collected during the last 200 years from their endemic Neotropical range identified genetic affinity between the ancient macaws and a single rare haplogroup (Haplo6) observed only among wild macaws in Mexico and northern Guatemala. Our results suggest that people at an undiscovered pre-Hispanic settlement dating between 900 and 1200 CE managed a macaw breeding colony outside their endemic range and distributed these symbolically important birds through the SW.

The influence of climate change on civil conflict and societal instability in the premodern world is a subject of much debate, in part because of the limited temporal or disciplinary scope of case studies. We present a transdisciplinary case study that combines archeological, historical, and paleoclimate datasets to explore the dynamic, shifting relationships among climate change, civil conflict, and political collapse at Mayapan, the largest Postclassic Maya capital of the Yucatán Peninsula in the thirteenth and fourteenth centuries CE. Multiple data sources indicate that civil conflict increased significantly and generalized linear modeling correlates strife in the city with drought conditions between 1400 and 1450 cal. CE. We argue that prolonged drought escalated rival factional tensions, but subsequent adaptations reveal regional-scale resiliency, ensuring that Maya political and economic structures endured until European contact in the early sixteenth century CE. The influence of climate on premodern civil conflict and societal instability is debated. Here, the authors combine archeological, historical, and paleoclimatic datasets to show that drought between 1400-1450 cal. CE escalated civil conflict at Mayapan, the largest Postclassic Maya capital of the Yucatán Peninsula.

The Younger Dryas impact hypothesis posits that a cosmic impact across much of the Northern Hemisphere deposited the Younger Dryas boundary (YDB) layer, containing peak abundances in a variable assemblage of proxies, including magnetic and glassy impact-related spherules, high-temperature minerals and melt glass, nanodiamonds, carbon spherules, aciniform carbon, platinum, and osmium. Bayesian chronological modeling was applied to 354 dates from 23 stratigraphic sections in 12 countries on four continents to establish a modeled YDB age range for this event of 12,835–12,735 Cal B.P. at 95% probability. This range overlaps that of a peak in extraterrestrial platinum in the Greenland Ice Sheet and of the earliest age of the Younger Dryas climate episode in six proxy records, suggesting a causal connection between the YDB impact event and the Younger Dryas. Two statistical tests indicate that both modeled and unmodeled ages in the 30 records are consistent with synchronous deposition of the YDB layer within the limits of dating uncertainty (∼100 y). The widespread distribution of the YDB layer suggests that it may serve as a datum layer.