Experimental Evolution of Resistance to Artemisinin Combination Therapy Results in Amplification of the mdr1 Gene in a Rodent Malaria Parasite

Background: Lacking suitable alternatives, the control of malaria increasingly depends upon Artemisinin Combination Treatments (ACT): resistance to these drugs would therefore be disastrous. For ACTs, the biology of resistance to the individual components has been investigated, but experimentally induced resistance to component drugs in combination has not been generated. Methodology/Principal Findings: We have used the rodent malaria parasite Plasmodium chabaudi to select in vivo resistance to the artesunate (ATN) + mefloquine (MF) version of ACT, through prolonged exposure of parasites to both drugs over many generations. The selection procedure was carried out over twenty-seven consecutive sub-inoculations under increasing ATN + MF doses, after which a genetically stable resistant parasite, AS-ATNMF1, was cloned. AS-ATNMF1 showed increased resistance to ATN + MF treatment and to artesunate or mefloquine administered separately. Investigation of candidate genes revealed an mdr1 duplication in the resistant parasites and increased levels of mdr1 transcripts and protein. There were no point mutations in the atpase6 or ubp1genes. Conclusion: Resistance to ACTs may evolve even when the two drugs within the combination are taken simultaneously and amplification of the mdr1 gene may contribute to this phenotype. However, we propose that other gene(s), as ye

Global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in 2015:a systematic review and modelling study

Background: We have previously estimated that respiratory syncytial virus (RSV) was associated with 22% of all episodes of (severe) acute lower respiratory infection (ALRI) resulting in 55 000 to 199 000 deaths in children younger than 5 years in 2005. In the past 5 years, major research activity on RSV has yielded substantial new data from developing countries. With a considerably expanded dataset from a large international collaboration, we aimed to estimate the global incidence, hospital admission rate, and mortality from RSV-ALRI episodes in young children in 2015. Methods: We estimated the incidence and hospital admission rate of RSV-associated ALRI (RSV-ALRI) in children younger than 5 years stratified by age and World Bank income regions from a systematic review of studies published between Jan 1, 1995, and Dec 31, 2016, and unpublished data from 76 high quality population-based studies. We estimated the RSV-ALRI incidence for 132 developing countries using a risk factor-based model and 2015 population estimates. We estimated the in-hospital RSV-ALRI mortality by combining in-hospital case fatality ratios with hospital admission estimates from hospital-based (published and unpublished) studies. We also estimated overall RSV-ALRI mortality by identifying studies reporting monthly data for ALRI mortality in the community and RSV activity. Findings: We estimated that globally in 2015, 33·1 million (uncertainty range [UR] 21·6–50·3) episodes of RSV-ALRI, resulted in about 3·2 million (2·7–3·8) hospital admissions, and 59 600 (48 000–74 500) in-hospital deaths in children younger than 5 years. In children younger than 6 months, 1·4 million (UR 1·2–1·7) hospital admissions, and 27 300 (UR 20 700–36 200) in-hospital deaths were due to RSV-ALRI. We also estimated that the overall RSV-ALRI mortality could be as high as 118 200 (UR 94 600–149 400). Incidence and mortality varied substantially from year to year in any given population. Interpretation: Globally, RSV is a common cause of childhood ALRI and a major cause of hospital admissions in young children, resulting in a substantial burden on health-care services. About 45% of hospital admissions and in-hospital deaths due to RSV-ALRI occur in children younger than 6 months. An effective maternal RSV vaccine or monoclonal antibody could have a substantial effect on disease burden in this age group

Drug resistance lineage of <i>Plasmodium chabaudi</i> AS.

<p>Note: the Figure does not depict all drug-resistant clones within the AS lineage. Only those relevant to the present work are represented. Each drug used to select resistant clones is noted inside each arrow and the increasing colour tonality of the arrow represents an approximation of the increase in drug doses during the evolution of resistance. The length of each arrow depicts the approximate relative time for generating the resistance phenotype. The generation of pyrimethamine resistance was a result of a single-step selection, whilst for all other drugs the evolution of resistance resulted from prolonged exposures to small increments in drug concentrations over many generations. The genotypic differences between a particular parasite and its progenitor are noted in brackets. Asterisks depict the clones used in this work.</p

<i>In vivo</i> evolution of resistance to artesunate + mefloquine.

<p>Artesunate (ATN) and mefloquine (MF) were given together to <i>P. chabaudi</i>-infected mice over many generations. After twenty seven sub-inoculations under increasing ATN + MF exposure, the drug-resistant population was cloned. One of these clones, denoted AS-ATNMF1, was selected from subsequent studies.</p

Typical Western blot result depicting MDR1 expression in the artesunate + mefloquine resistant clone AS-ATNMF1.

<p>Hybridization was carried out with α anti Pgh1 antibodies and α anti tubulin antibodies. AS-3CQ and AS-ATN were used as one-<i>mdr1</i> copy controls and AS-15MF was used as the two-<i>mdr1</i> copies control.</p

Drug test results.

<p>Each line represents the evolution of parasitaemia in each parasite clone from day 4 post-inoculum (p.i) in the absence of treatment (a), under artesunate + meloquine treatment (b), under mefloquine treatment (c) or under artesunate treatment (d). Each data point is a mean of % parasitaemias ± SE resulting from reads of groups of two and five mice in the untreated and treated groups, respectively.</p