Gene knock-in as a tool to phenotype clinically relevant varient alleles for studies on malaria pathobiology: proof of concept using the plasmodium knowlesi normocyte binding protein Xa gene

The zoonotic parasite, Plasmodium knowlesi, is one of five human malaria species. P. knowlesi is geographically restricted to locations of the long-tailed and pig-tailed macaque, indigenous across South-East Asia (SEA). Initial research showed that P. knowlesi was present in the human population in Malaysian Borneo, with subsequent studies confirming P. knowlesi throughout SEA. P. knowlesi cases were shown to be both severe (10%) and lethal (2%), with hyperparasitaemia correlating with severe malaria. Recent work has identified a polymorphism in the essential P. knowlesi normocyte binding protein Xa (Pknbpxa) gene that associates with high parasitaemia. The aims of this study were firstly to enhance and standardise the isolation of parasite DNA from a Biobank of frozen clinical isolates. Alongside this we aimed to take P. knowlesi whole genome sequence data and identify further mutations in the Pknbpxa gene linked to severe disease and express these in an in vitro P. knowlesi experimental line. Detailed here is the optimisation of the Whatman-Plasmodipur method to purify P. knowlesi DNA from a Biobank of frozen clinical samples. This resulted in 13/22 samples returning greater than 70% P. knowlesi DNA and within this, 8 samples were suitable for genome sequencing. Genome sequence data representing cluster type 2 was used to inform the synthesis and construction of a Pknbpxa synthetic gene representing clinical alleles. Single-crossover homologous recombination was used to replace the native Pknbpxa with this synthetic copy, containing polymorphisms associated with high parasitaemia. This was subsequently transfected into the P. knowlesi A.1-H.1 clone via nucleofection, resulting in an experimental line expressing clinically relevant mutations within the essential PkNBPXa invasion gene. The construction of this translational approach enables functional examination for mutation involvement in parasite erythrocyte invasion and contribution to severe disease

Normal left ventricular function does not protect against propafenone-induced incessant ventricular tachycardia

Propafenone is a class Ic anti-arrhythmic agent with mild B-blocking properties which has recently become available in South Africa. We have used the drug in 3 patients with sustained m.onomorphic ventricular tachycardia not due to ischaemic heart disease. All had norm.al left ventricular function; 1 had Wegener's granulom.atosis and 2 had arrhythmogenic right ventricular dysplasia. In the latter 2, propafenone provoked incessant monomorphic ventricular tachycardia which persisted for m.ore than 24 hours despite repeated efforts at term.ination. The morphology was similar to the patients' spontaneous ventricular tachycardia, but the rate was slower and the QRS complexes broader, consistent with propafenone's marked ability to slow intraventricular conduction. It is postulated that incessant tachycardia results from. perpetuation of re-entry due to marked conduction slowing produced by the drug. Previous reports have suggested that this is most likely to occur in patients with poor left ventricular function, but our experience indicates that those with normal left ventricular function are also at risk, particularly if the substrate for reentry is present. Propafenone, like all other powerful antiarrhythmic agents, may provoke life-threatening arrhythmias and should be used with great caution after due consideration of the indications, even in patients with norm.al left ventricular function

Surgery for the Wolff-Parkinson-White syndrome - the Groote Schuur Hospital experience

Surgical division of accessory atrioventricular (AV) connections has been performetl on 9 patients with the WolffParkinson- White (WPW) syndrome at Groote Schuur Hospital. All patients had symptomatic paroxysmal tachycardia. The indication for surgery in 5 patients was poor control on antiarrhythmic drugs. Surgery was performed on a 15-year-old boy to prevent lifelong dependence on drugs, although his atrial fibrillation (ventricular rate> 300/min) was controllable with sotalol 1 280 mg daily. The remaining 3 patients required cilrdiac surgery for other indications and therefore their accessory pathways (APs) were divided concurrently. The AP was localised by pre-operative endocardial mapping and intra-operative epicardial mapping. There were 4 posteroseptal, 3 left free-wall and 2 right free-wall pathways. An endocardial approach was used to divide the pathways. All 5 free-wall APs were successfully divided without complications or recurrence. However, 1 patient with paroxysmal atrial fibrillation and severe unstable angina·due to coronary artery disease died unexpectedly 10 days after 4-vessel coronary bypass grafting and division of a posteroseptal AP. Postoperative complications occurred in a further 2 patients with posteroseptal APs. One patient developed complete heart block and is now asymptomatic with a DDD pacemaker, while the other had recurrence of retrograde bypass conduction postoperatively, but is now successfully controlled on sotalol. Therefore 7 of the 8 survivors are free of recurrence of tachycardia on no anti-arrhythmic drugs after a mean follow-up of 14,3 months. New insights into the surgical technique, particularly for division of posteroseptal pathways, can be expected to improve the outlook

Deuterated Ammonia in Galactic Protostellar Cores

We report on a survey of \nh2d towards protostellar cores in low-mass star formation and quiescent regions in the Galaxy. Twenty-three out of thirty-two observed sources have significant (\gsim 5\sigma) \nh2d emission. Ion-molecule chemistry, which preferentially enhances deuterium in molecules above its cosmological value of \scnot{1.6}{-5} sufficiently explains these abundances. NH2D/NH3 ratios towards Class 0 sources yields information about the ``fossil remnants'' from the era prior to the onset of core collapse and star formation. We compare our observations with predictions of gas-phase chemical networks.Comment: 16 Pages, 7 Figures, Accepted to Ap.J., to appear in the June 20, 2001 editio

Plasmodium knowlesi Genome Sequences from Clinical Isolates Reveal Extensive Genomic Dimorphism.

Plasmodium knowlesi is a newly described zoonosis that causes malaria in the human population that can be severe and fatal. The study of P. knowlesi parasites from human clinical isolates is relatively new and, in order to obtain maximum information from patient sample collections, we explored the possibility of generating P. knowlesi genome sequences from archived clinical isolates. Our patient sample collection consisted of frozen whole blood samples that contained excessive human DNA contamination and, in that form, were not suitable for parasite genome sequencing. We developed a method to reduce the amount of human DNA in the thawed blood samples in preparation for high throughput parasite genome sequencing using Illumina HiSeq and MiSeq sequencing platforms. Seven of fifteen samples processed had sufficiently pure P. knowlesi DNA for whole genome sequencing. The reads were mapped to the P. knowlesi H strain reference genome and an average mapping of 90% was obtained. Genes with low coverage were removed leaving 4623 genes for subsequent analyses. Previously we identified a DNA sequence dimorphism on a small fragment of the P. knowlesi normocyte binding protein xa gene on chromosome 14. We used the genome data to assemble full-length Pknbpxa sequences and discovered that the dimorphism extended along the gene. An in-house algorithm was developed to detect SNP sites co-associating with the dimorphism. More than half of the P. knowlesi genome was dimorphic, involving genes on all chromosomes and suggesting that two distinct types of P. knowlesi infect the human population in Sarawak, Malaysian Borneo. We use P. knowlesi clinical samples to demonstrate that Plasmodium DNA from archived patient samples can produce high quality genome data. We show that analyses, of even small numbers of difficult clinical malaria isolates, can generate comprehensive genomic information that will improve our understanding of malaria parasite diversity and pathobiology

Pneumococcal carriage in sub-Saharan Africa--a systematic review.

BACKGROUND: Pneumococcal epidemiology varies geographically and few data are available from the African continent. We assess pneumococcal carriage from studies conducted in sub-Saharan Africa (sSA) before and after the pneumococcal conjugate vaccine (PCV) era. METHODS: A search for pneumococcal carriage studies published before 2012 was conducted to describe carriage in sSA. The review also describes pneumococcal serotypes and assesses the impact of vaccination on carriage in this region. RESULTS: Fifty-seven studies were included in this review with the majority (40.3%) from South Africa. There was considerable variability in the prevalence of carriage between studies (I-squared statistic = 99%). Carriage was higher in children and decreased with increasing age, 63.2% (95% CI: 55.6-70.8) in children less than 5 years, 42.6% (95% CI: 29.9-55.4) in children 5-15 years and 28.0% (95% CI: 19.0-37.0) in adults older than 15 years. There was no difference in the prevalence of carriage between males and females in 9/11 studies. Serotypes 19F, 6B, 6A, 14 and 23F were the five most common isolates. A meta-analysis of four randomized trials of PCV vaccination in children aged 9-24 months showed that carriage of vaccine type (VT) serotypes decreased with PCV vaccination; however, overall carriage remained the same because of a concomitant increase in non-vaccine type (NVT) serotypes. CONCLUSION: Pneumococcal carriage is generally high in the African continent, particularly in young children. The five most common serotypes in sSA are among the top seven serotypes that cause invasive pneumococcal disease in children globally. These serotypes are covered by the two PCVs recommended for routine childhood immunization by the WHO. The distribution of serotypes found in the nasopharynx is altered by PCV vaccination

Disease progression in Plasmodium knowlesi malaria is linked to variation in invasion gene family members.

Emerging pathogens undermine initiatives to control the global health impact of infectious diseases. Zoonotic malaria is no exception. Plasmodium knowlesi, a malaria parasite of Southeast Asian macaques, has entered the human population. P. knowlesi, like Plasmodium falciparum, can reach high parasitaemia in human infections, and the World Health Organization guidelines for severe malaria list hyperparasitaemia among the measures of severe malaria in both infections. Not all patients with P. knowlesi infections develop hyperparasitaemia, and it is important to determine why. Between isolate variability in erythrocyte invasion, efficiency seems key. Here we investigate the idea that particular alleles of two P. knowlesi erythrocyte invasion genes, P. knowlesi normocyte binding protein Pknbpxa and Pknbpxb, influence parasitaemia and human disease progression. Pknbpxa and Pknbpxb reference DNA sequences were generated from five geographically and temporally distinct P. knowlesi patient isolates. Polymorphic regions of each gene (approximately 800 bp) were identified by haplotyping 147 patient isolates at each locus. Parasitaemia in the study cohort was associated with markers of disease severity including liver and renal dysfunction, haemoglobin, platelets and lactate, (r = ≥ 0.34, p =  <0.0001 for all). Seventy-five and 51 Pknbpxa and Pknbpxb haplotypes were resolved in 138 (94%) and 134 (92%) patient isolates respectively. The haplotypes formed twelve Pknbpxa and two Pknbpxb allelic groups. Patients infected with parasites with particular Pknbpxa and Pknbpxb alleles within the groups had significantly higher parasitaemia and other markers of disease severity. Our study strongly suggests that P. knowlesi invasion gene variants contribute to parasite virulence. We focused on two invasion genes, and we anticipate that additional virulent loci will be identified in pathogen genome-wide studies. The multiple sustained entries of this diverse pathogen into the human population must give cause for concern to malaria elimination strategists in the Southeast Asian region

Observations of Massive Star Forming Regions with Water Masers: Mid-Infrared Imaging

We present here a mid-infrared imaging survey of 26 sites of water maser emission. Observations were obtained at the InfraRed Telescope Facility 3-m telescope with the University of Florida mid-infrared imager/spectrometer OSCIR, and the JPL mid-infrared camera MIRLIN. The main purpose of the survey was to explore the relationship between water masers and the massive star formation process. It is generally believed that water masers predominantly trace outflows and embedded massive stellar objects, but may also exist in circumstellar disks around young stars. We investigate each of these possibilities in light of our mid-infrared imaging. We find that mid-infrared emission seems to be more closely associated with water and OH maser emission than cm radio continuum emission from UC HII regions. We also find from the sample of sources in our survey that, like groups of methanol masers, both water and OH masers have a proclivity for grouping into linear or elongated distributions. We conclude that the vast majority of linearly distributed masers are not tracing circumstellar disks, but outflows and shocks instead.Comment: 49 pages; 23 figures; To appear in February 2005 ApJS; To download a version with better quality figures, go to http://www.ctio.noao.edu/~debuizer

Is heavy eccentric calf training superior to wait-and-see, sham rehabilitation, traditional physiotherapy and other exercise interventions for pain and function in mid-portion Achilles tendinopathy?

Background: Mid-portion Achilles tendinopathy (AT) is prevalent amongst athletic and non-athletic populations with pain, stiffness and impaired function typically reported. While different management options exist, loading protocols remain the best available intervention and have been shown to be effective in the management of AT. Trials investigating loading in AT have used a variety of different protocols, and recent narrative reviews suggest that no protocol is superior to another when comparing outcomes in pain and function. However, there has been no systematic review or meta-analysis completed to determine this. Furthermore, the narrative review did not consider wait-and-see or sham interventions, thus a systematic review and met-analysis which includes wait-and see or sham interventions is warranted. Methods: A systematic review and meta-analyses will be conducted as per the PRISMA guidelines. The databases PUBMED, CINAHL (Ovid) and CINAHL (EBSCO) will be searched for articles published from inception to 31 December 2017. Our search focuses on studies examining the improvement of pain and function when completing a loading program for mid-portion AT. Only randomised/ quasi-randomised trials will be included while case reports and case series will be excluded. The primary outcome assessing pain and function will be the Victorian Institute Sports Assessment - Achilles (VISA-A). Two reviewers will screen articles, extract data and assess the risk of bias independently with a third reviewer resolving any disagreements between the two reviewers. A meta-analysis will then be performed on the data (if appropriate) to determine if the traditional heavy load calf training protocol described by Alfredson is superior to wait-and-see, sham intervention, traditional physiotherapy, and other forms of exercise rehabilitation. Discussion: This systematic review and meta-analysis will allow us to investigate if there are difference in pain and function when comparing wait-and-see, sham interventions, traditional physiotherapy and different exercise interventions to the traditional heavy eccentric calf training protocol for mid-portion Achilles tendon pain. Systematic review registration: PROSPERO registration number CRD42018084493

Use of complementary and alternative medicine by those with a chronic disease and the general population - results of a national population based survey

<p>Abstract</p> <p>Background</p> <p>The use of complementary and alternative medicine (CAM) is becoming more common, but population-based descriptions of its patterns of use are lacking. This study aimed to determine the prevalence of CAM use in the general population and for those with asthma, diabetes, epilepsy and migraine.</p> <p>Methods</p> <p>Data from cycles 1.1, 2.1 and 3.1 of the Canadian Community Health Survey (CCHS) were used for the study. The CCHS is a national cross-sectional survey administered to 400,055 Canadians aged ≥12 between 2001-2005. Self-reported information about professionally diagnosed health conditions was elicited. CCHS surveys use a multistage stratified cluster design to randomly select a representative sample of Canadian household residents. Descriptive data on the utilization of CAM services was calculated and logistic regression was used to determine what sociodemographic factors predict CAM use.</p> <p>Results</p> <p>Weighted estimates show that 12.4% (95% Confidence Interval (CI): 12.2-12.5) of Canadians visited a CAM practitioner in the year they were surveyed; this rate was significantly higher for those with asthma 15.1% (95% CI: 14.5-15.7) and migraine 19.0% (95% CI: 18.4-19.6), and significantly lower for those with diabetes 8.0% (95% CI: 7.4-8.6) while the rate in those with epilepsy (10.3%, 95% CI: 8.4-12.2) was not significantly different from the general population.</p> <p>Conclusion</p> <p>A large proportion of Canadians use CAM services. Physicians should be aware that their patients may be accessing other services and should be prepared to ask and answer questions about the risks and benefits of CAM services in conjunction with standard medical care.</p