Selection of pfdhfr/pfdhps alleles and declining artesunate/sulphadoxine-pyrimethamine efficacy against Plasmodium falciparum eight years after deployment in eastern Sudan.

BACKGROUND: Artesunate/sulphadoxine-pyrimethamine (AS/SP) has been the first-line treatment for falciparum malaria in Sudan since 2004. The impact of this combination on anti-malarial resistance-associated molecular markers has not been investigated. In this study, an evaluation of the efficacy and prevalence of drug resistance alleles (pfcrt, pfmdr1, pfdhfr and pfdhps) eight years after the adoption of AS/SP in eastern Sudan is reported. METHODS: A 28-day follow-up efficacy trial of AS/SP was conducted in eastern Sudan during the 2012 transmission season. Blood smears were collected from patients on days 0, 1, 2, 3, 7, 14, 21 and 28. Blood spots on filter paper were obtained pre-treatment and on the day the patient was parasite positive by microscopy. Genotyping of alleles was performed by qPCR (pfcrt 72-76 and pfmdr1 copy number) and direct sequencing of pfmdr1, pfdhfr and pfdhps. RESULTS: Sixty-three patients out of 68 (93%) completed the 28-day follow-up, adequate clinical, and parasitological response occurred in 90.5% and 85.3% of the patients in the per-protocol and intent-to-treat analyses, respectively. PCR corrected per-protocol efficacy was 93.7%. The enrolment prevalence of pfcrt-CVMNK was 30.2% and pfmdr1-N86 was 40.3%. The pfmdr1 haplotype NFD occurred in 32.8% of pre-treatment samples and was significantly higher than previous reports (Fisher's exact p = 0.0001). The pfdhfr-51I/108N combination occurred in all sequenced isolates and 59R was observed in a single individual. pfdhps substitutions 436A, 437G, 540E, 581G and 613S were observed at 7.8, 77.3, 76.9%, 33.8% and 0.0%, respectively. Treatment failures were associated with the pfdhps haplotype SGEGA at these five codons (OR 7.3; 95% CI 0.65 - 368; p = 0.048). CONCLUSION: The decrease of CQR associated genotypes reflects the formal policy of complete removal of CQ in Sudan. However, the frequency of markers associated with SP failure is increasing in this study area and may be contributing to the treatment efficacy falling below 90%. Further monitoring of AS/SP efficacy and of post-treatment selection of pfdhfr and pfdhps alleles in vivo is required to inform future treatment guidelines

Shigella MreB promotes polar IcsA positioning for actin tail formation.

Pathogenic Shigella bacteria are a paradigm to address key issues of cell and infection biology. Polar localisation of the Shigella autotransporter protein IcsA is essential for actin tail formation, which is necessary for the bacterium to travel from cell-to-cell; yet how proteins are targeted to the bacterial cell pole is poorly understood. The bacterial actin homologue MreB has been extensively studied in broth culture using model organisms including Escherichia coli, Bacillus subtilis and Caulobacter crescentus, but has never been visualised in rod-shaped pathogenic bacteria during infection of host cells. Here, using single-cell analysis of intracellular Shigella, we discover that MreB accumulates at the cell pole of bacteria forming actin tails, where it colocalises with IcsA. Pharmacological inhibition of host cell actin polymerisation and genetic deletion of IcsA is used to show, respectively, that localisation of MreB to the cell poles precedes actin tail formation and polar localisation of IcsA. Finally, by exploiting the MreB inhibitors A22 and MP265, we demonstrate that MreB polymerisation can support actin tail formation. We conclude that Shigella MreB promotes polar IcsA positioning for actin tail formation, and suggest that understanding the bacterial cytoskeleton during host-pathogen interactions can inspire development of new therapeutic regimes for infection control.This article has an associated First Person interview with the first author of the paper

The obligate intracellular bacterium Orientia tsutsugamushi differentiates into a developmentally distinct extracellular state

Orientia tsutsugamushi (Ot) is an obligate intracellular bacterium in the family Rickettsiaceae that causes scrub typhus, a severe mite-borne human disease. Its mechanism of cell exit is unusual amongst Rickettsiaceae, as Ot buds off the surface of infected cells enveloped in plasma membrane. Here, we show that Ot bacteria that have budded out of host cells are in a distinct developmental stage compared with intracellular bacteria. We refer to these two stages as intracellular and extracellular bacteria (IB and EB, respectively). These two forms differ in physical properties: IB is both round and elongated, and EB is round. Additionally, IB has higher levels of peptidoglycan and is physically robust compared with EB. The two bacterial forms differentially express proteins involved in bacterial physiology and host-pathogen interactions, specifically those involved in bacterial dormancy and stress response, and outer membrane autotransporter proteins ScaA and ScaC. Whilst both populations are infectious, entry of IB Ot is sensitive to inhibitors of both clathrin-mediated endocytosis and macropinocytosis, whereas entry of EB Ot is only sensitive to a macropinocytosis inhibitor. Our identification and detailed characterization of two developmental forms of Ot significantly advances our understanding of the intracellular lifecycle of an important human pathogen

Seasonal malaria chemoprevention combined with community case management of malaria in children under 10 years of age, over 5 months, in south-east Senegal: A cluster-randomised trial.

BACKGROUND: Seasonal malaria chemoprevention (SMC) is recommended in the Sahel region of Africa for children under 5 years of age, for up to 4 months of the year. It may be appropriate to include older children, and to provide protection for more than 4 months. We evaluated the effectiveness of SMC using sulfadoxine-pyrimethamine plus amodiaquine given over 5 months to children under 10 years of age in Saraya district in south-east Senegal in 2011. METHODS AND FINDINGS: Twenty-four villages, including 2,301 children aged 3-59 months and 2,245 aged 5-9 years, were randomised to receive SMC with community case management (CCM) (SMC villages) or CCM alone (control villages). In all villages, community health workers (CHWs) were trained to treat malaria cases with artemisinin combination therapy after testing with a rapid diagnostic test (RDT). In SMC villages, CHWs administered SMC to children aged 3 months to 9 years once a month for 5 months. The study was conducted from 27 July to 31 December 2011. The primary outcome was malaria (fever or history of fever with a positive RDT). The prevalence of anaemia and parasitaemia was measured in a survey at the end of the transmission season. Molecular markers associated with resistance to SMC drugs were analysed in samples from incident malaria cases and from children with parasitaemia in the survey. SMC was well tolerated with no serious adverse reactions. There were 1,472 RDT-confirmed malaria cases in the control villages and 270 in the SMC villages. Among children under 5 years of age, the rate difference was 110.8/1,000/month (95% CI 64.7, 156.8; p < 0.001) and among children 5-9 years of age, 101.3/1,000/month (95% CI 66.7, 136.0; p < 0.001). The mean haemoglobin concentration at the end of the transmission season was higher in SMC than control villages, by 6.5 g/l (95% CI 2.0, 11; p = 0.007) among children under 5 years of age, and by 5.2 g/l (95% CI 0.4, 9.9; p = 0.035) among children 5-9 years of age. The prevalence of parasitaemia was 18% in children under 5 years of age and 25% in children 5-9 years of age in the control villages, and 5.7% and 5.8%, respectively, in these 2 age groups in the SMC villages, with prevalence differences of 12.5% (95% CI 6.8%, 18.2%; p < 0.001) in children under 5 years of age and 19.3% (95% CI 8.3%, 30.2%; p < 0.001) in children 5-9 years of age. The pfdhps-540E mutation associated with clinical resistance to sulfadoxine-pyrimethamine was found in 0.8% of samples from malaria cases but not in the final survey. Twelve children died in the control group and 14 in the SMC group, a rate difference of 0.096/1,000 child-months (95% CI 0.99, 1.18; p = 0.895). Limitations of this study include that we were not able to obtain blood smears for microscopy for all suspected malaria cases, such that we had to rely on RDTs for confirmation, which may have included false positives. CONCLUSIONS: In this study SMC for children under 10 years of age given over 5 months was feasible, well tolerated, and effective in preventing malaria episodes, and reduced the prevalence of parasitaemia and anaemia. SMC with CCM achieved high coverage and ensured children with malaria were promptly treated with artemether-lumefantrine. TRIAL REGISTRATION: www.clinicaltrials.gov NCT01449045

Optimising lysis of L929 host cells infected with <i>O</i>. <i>tsutsugamushi</i>.

<p>Panel A. Confocal fluorescence microscopy images showing the effect on host and bacterial cells of different lysis methods. Blue = nuclei (DAPI), red = host cells (Evans blue) and green = bacteria (Alexafluor 488-labelled antibody). Scale bar = 40 μm. Panel B. The effect of different host cell lysis methods on subsequent growth of bacteria. Panel C. The effect of purification temperature on subsequent growth of bacteria. Panel D. The yield of bacteria after different stages of purification. Panel E. the pelleting of bacteria after centrifugation at different g-forces. All samples were spun for 3 mins. Panel F. The effect of different centrifugation speeds on subsequent bacterial growth.</p