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In 2011 mecC, a new mecA gene homologue, was described in a bovine isolate in the UK. Since then, mecC-positive methicillin-resistant Staphylococcus aureus (mecC-MRSA) has also been found in wild animals. An especially high prevalence of mecC-MRSA has been reported among hedgehogs in Sweden (64%) and Denmark (61%). Based on these findings we aimed to survey the hedgehog population for mecC-MRSA in Hungary.\ud \ud Altogether 200 hedgehogs were screened for Staphylococcus aureus using a culture-based method. The antibiotic susceptibility of the isolates to nine drugs was determined, their genetic relatedness was established by PFGE and spa-typing, and virulence genes were identified by PCR. Whole genome sequencing was performed for the single mecC-MRSA isolate found.\ud \ud Of the 200 animals, 13 were carriers of S. aureus (6.5%). Among these, one isolate was mecA positive and one was mecC positive. The isolates were susceptible to non-beta-lactam antibiotics. Toxin genes were not found, but the majority carried genes responsible for adhesion and biofilm production. The mecC-MRSA isolate was a single-locus variant of ST130, had a new spa type (t19701) and belonged to SCCmec type XI. It carried a recently described, novel exfoliative toxin (etE).\ud \ud This is the first report of mecC-MRSA in Hungary and the first survey of staphylococcus carriage among wild animals in the country. The mecC prevalence was much lower than in Northern European countries and rather similar to other countries in our region. MecC-MRSA could potentially emerge as a novel human pathogen, especially where close contact occurs between humans and animals.

Intestinal parasitic infections affect more than 2 billion people throughout the world with disproportionately high prevalence rates in Low- and Middle-Income Countries (LMICs) (Herricks et al., 2017). Multicellular and highly complex parasites such as Ascaris, hook worm, Trichuris, Enterobius and Schistosoma, as well as unicellular organisms including Entamoeba, Giardia, Toxoplasma, Cyclospora, and Cryptosporidium are among major pathogens that contribute to the global intestinal parasitic disease burden.\ud \ud Parasites can cause persistent infection due to their ability to resist immune-mediated expulsion by modulating the host's immune response (McSorley and Maizels, 2012; Wammes et al., 2014; Chabé et al., 2017; Burrows et al., 2019; Ryan et al., 2020). There is a complex interaction between parasites and human microbiota which can triangulate with host's immune homeostasis and host responses to bystander antigens, vaccines or other unrelated diseases, both infectious and non-communicable diseases (McSorley and Maizels, 2012; Wammes et al., 2014). Recently, the world has grappled with an unprecedented pandemic due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that causes coronavirus disease 2019 (COVID-19) (WHO, 2020). The pathogenesis of severe disease in COVID-19 has been linked to the phenomenon of immune hyperactivation (Sinha et al., 2020; Tay et al., 2020). Here, we propose that the interplay between intestinal parasites and microbiome may have a potential direct or indirect effects on the pathogenesis of SARS-CoV-2 infection, in particular in the context of LMICs.

Background In cerebral malaria, the retina can be used to understand disease pathogenesis. The mechanisms linking sequestration, brain swelling, and death remain poorly understood. We hypothesized that retinal vascular leakage would be associated with brain swelling. Methods We used retinal angiography to study blood-retinal barrier integrity. We analyzed retinal leakage, histopathology, brain magnatic resonance imaging (MRI), and associations with death and neurological disability in prospective cohorts of Malawian children with cerebral malaria. Results Three types of retinal leakage were seen: large focal leak (LFL), punctate leak (PL), and vessel leak. The LFL and PL were associated with death (odds ratio [OR] = 13.20, 95% confidence interval [CI] = 5.21–33.78 and OR = 8.58, 95% CI = 2.56–29.08, respectively) and brain swelling (P Conclusions Blood-retina barrier breakdown occurs in 3 patterns in cerebral malaria. Associations between LFL, brain swelling, and death suggest that the rapid accumulation of cerebral hemorrhages, with accompanying fluid egress, may cause fatal brain swelling. Vessel leak, from barrier dysfunction, and nonperfusion were not associated with severe brain swelling but with neurological deficits, suggesting hypoxic injury in survivors.

Background & objectives: Vector-borne pathogen surveillance programmes typically rely on the collection of large numbers of potential vectors followed by screening protocols focused on detecting pathogens in the arthropods. These processes are laborious, time consuming, expensive, and require screening of large numbers of samples. To streamline the surveillance process, increase sample throughput, and improve cost-effectiveness, a method to detect dengue virus and malaria parasites (Plasmodium falciparum) by leveraging the sugar-feeding behaviour of mosquitoes and their habit of expectorating infectious agents in their saliva during feeding was investigated in this study. Methods: Dengue virus 2 (DENV-2) infected female Aedes aegypti mosquitoes and P. falciparum infected female Anopheles stephensi mosquitoes were allowed to feed on honey coated Flinders Technical Associates —FTA® cards dyed with blue food colouring. The feeding resulted in deposition of saliva containing either DENV-2 particles or P. falciparum sporozoites onto the FTA card. Nucleic acid was extracted from each card and the appropriate real-time PCR (qPCR) assay was run to detect the pathogen of interest. Results: As little as one plaque forming unit (PFU) of DENV-2 and as few as 60 P. falciparum parasites deposited on FTA cards from infected mosquitoes were detected via qPCR. Hence, their use to collect mosquito saliva for pathogen detection is a relevant technique for vector surveillance. Interpretation & conclusion: This study provides laboratory confirmation that FTA cards can be used to capture and stabilize expectorated DENV-2 particles and P. falciparum sporozoites from infectious, sugar-feeding mosquitoes in very low numbers. Thus, the FTA card-based mosquito saliva capture method offers promise to overcome current limitations and revolutionize traditional mosquito-based pathogen surveillance programmes. Field testing and further method development are required to optimize this strategy.

12 Páginas, 5 Figuras, Facultative parthenogenesis (FP) is asexual reproduction in plant and animal species that would otherwise reproduce sexually. This process in vertebrates typically results from automictic development (likely terminal fusion) and is phylogenetically widespread. In squamate reptiles and chondrichthyan fishes, FP has been reported to occur in nature and can result in the production of reproductively viable offspring; suggesting that it is of ecological and evolutionary significance. However, terminal fusion automixis is believed to result in near genome-wide reductions in heterozygosity; thus, FP seems likely to affect key phenotypic characters, yet this remains almost completely unstudied. Snake venom is a complex phenotypic character primarily used to subjugate prey and is thus tightly linked to individual fitness. Surprisingly, the composition and function of venom produced by a parthenogenetic pitviper exhibits a high degree of similarity to that of its mother and conspecifics from the same population. Therefore, the apparent loss of allelic diversity caused by FP appears unlikely to have a significant impact on the prey-capturing ability of this snake. Accordingly, the pitviper offspring produced by FP retained complex phenotypic characteristics associated with fitness. This result reinforces the potential ecological and evolutionary importance of FP and questions our understanding of the inheritance of venom-associated genes., NRC was supported by a Sir Henry Dale Fellowship (200517/Z/16/Z) jointly funded by the Wellcome Trust and the Royal Society. None of the authors of this manuscript have a financial interest related to this work

BACKGROUND: The antiretroviral nevirapine is associated with hypersensitivity reactions in 6%-10% of patients, including hepatotoxicity, maculopapular exanthema, Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). OBJECTIVES: To undertake a genome-wide association study (GWAS) to identify genetic predisposing factors for the different clinical phenotypes associated with nevirapine hypersensitivity. METHODS: A GWAS was undertaken in a discovery cohort of 151 nevirapine-hypersensitive and 182 tolerant, HIV-infected Malawian adults. Replication of signals was determined in a cohort of 116 cases and 68 controls obtained from Malawi, Uganda and Mozambique. Interaction with ERAP genes was determined in patients positive for HLA-C*04:01 In silico docking studies were also performed for HLA-C*04:01 RESULTS: Fifteen SNPs demonstrated nominal significance (P

Despite decades of research on cerebral malaria (CM) there is still a paucity of knowledge about what actual causes CM and why certain people develop it. Although sequestration of P. falciparum infected red blood cells has been linked to pathology, it is still not clear if this is directly or solely responsible for this clinical syndrome. Recent data have suggested that a combination of parasite variant types, mainly defined by the variant surface antigen, P. falciparum erythrocyte membrane protein 1 (PfEMP1), its receptors, coagulation and host endothelial cell activation (or inflammation) are equally important. This makes CM a multi-factorial disease and a challenge to unravel its causes to decrease its detrimental impact.

Background Resource-limited tropical countries are home to numerous infectious pathogens of both human and zoonotic origin. A capability for early detection to allow rapid outbreak containment and prevent spread to non-endemic regions is severely impaired by inadequate diagnostic laboratory capacity, the absence of a “cold chain” and the lack of highly trained personnel. Building up detection capacity in these countries by direct replication of the systems existing in developed countries is not a feasible approach and instead requires “leapfrogging” to the deployment of the newest diagnostic systems that do not have the infrastructure requirements of systems used in developed countries. Methods A laboratory for molecular diagnostics of infectious agents was established in Bo, Sierra Leone with a hybrid solar/diesel/battery system to ensure stable power supply and a satellite modem to enable efficient communication. An array of room temperature stabilization and refrigeration technologies for reliable transport and storage of reagents and biological samples were also tested to ensure sustainable laboratory supplies for diagnostic assays. Results The laboratory demonstrated its operational proficiency by conducting an investigation of a suspected avian influenza outbreak at a commercial poultry farm at Bo using broad range resequencing microarrays and real time RT-PCR. The results of the investigation excluded influenza viruses as a possible cause of the outbreak and indicated a link between the outbreak and the presence of Klebsiella pneumoniae. Conclusions This study demonstrated that by application of a carefully selected set of technologies and sufficient personnel training, it is feasible to deploy and effectively use a broad-range infectious pathogen detection technology in a severely resource-limited setting.

Background Persistent diarrhoea in children is a common problem in low and middle income countries. To help target appropriate treatment for specific pathogens in the absence of diagnostic tests, we systematically reviewed pathogens most commonly associated with persistent diarrhoea in children. Methods We sought all descriptive studies of pathogens in the stool of children with diarrhoea of over 14 days duration in low and middle income countries with a comprehensive search of the MEDLINE, EMBASE, LILACS and WEB OF SCIENCE databases. We described the study designs and populations, assessed the quality of the laboratory tests, and extracted and summarised data on pathogens. For Escherichia coli, we calculated high and low prevalence estimates of all enteropathic types combined. Results across studies were compared for geographical patterns. Results Nineteen studies were included. Some used episodes of diarrhoea as the unit of analysis, others used children. The quality of reporting of laboratory procedures varied, and pathogens (particularly E. coli types) were classified in different ways. As there were no apparent regional differences in pathogen prevalence, we aggregated data between studies to give a guide to overall prevalence. Enteropathic E. coli types were commonly found in children with persistent diarrhoea (up to 63%). Various other organisms, including viruses, bacteria and parasites, were detected but across all studies their prevalence was under 10%. However, these pathogens were also found in similar frequencies in children without diarrhoea. Conclusion A number of pathogens are commonly associated with persistent diarrhoea in children, but in children without diarrhoea the pathogens are found with similar frequencies. New research with carefully selected controls and standardised laboratory investigations across countries will help map causes and help explore effective options for presumptive treatment.

Introduction\ud \ud The causes of coma and death in cerebral malaria remain unknown. Malarial retinopathy has been identified as an important clinical sign in the diagnosis and prognosis of cerebral malaria. As part of a larger autopsy study to determine causes of death in children with coma presenting to hospital in Blantyre, Malawi, who were fully evaluated clinically prior to death, we examined the histopathology of eyes of patients who died and underwent autopsy. \ud \ud Methodology/Principal Findings\ud \ud Children with coma were admitted to the pediatric research ward, classified according to clinical definitions as having cerebral malaria or another cause of coma, evaluated and treated. The eyes were examined by direct and indirect ophthalmoscopy. If a child died and permission was given, a standardized autopsy was carried out. The patient was then assigned an actual cause of death according to the autopsy findings. The eyes were examined pathologically for hemorrhages, cystoid macular edema, parasite sequestration and thrombi. They were stained immunohistochemically for fibrin and CD61 to identify the components of thrombi, β-amyloid precursor protein to detect axonal damage, for fibrinogen to identify vascular leakage and for glial fibrillary acidic protein to detect gliosis. Sixty-four eyes from 64 patients were examined: 35 with cerebral malaria and 29 with comas of other causes. Cerebral malaria was distinguished by sequestration of parasitized erythrocytes, the presence and severity of retinal hemorrhages, the presence of cystoid macular edema, the occurrence and number of fibrin-platelet thrombi, the presence and amount of axonal damage and vascular leakage. \ud \ud Conclusions/Significance\ud \ud We found significant differences in retinal histopathology between patients who died of cerebral malaria and those with other diagnoses. These histopathological findings offer insights into the etiology of malarial retinopathy and provide a pathological basis for recently described retinal capillary non-perfusion in children with malarial retinopathy. Because of the similarities between the retina and the brain it also suggests mechanisms that may contribute to coma and death in cerebral malaria.

Introduction \ud Streptococcus pneumoniae (or pneumococcus) is a common commensal (coloniser) of the human upper respiratory tract. Colonisation is likely a prerequisite for respiratory tract infections and invasive pneumococcal disease. Colonisation also has a significant role in the horizontal spread of this pathogen within communities, but paradoxically could also lead to boosting of the host’s immune system. We use the unique experimental human pneumococcal challenge (EHPC) model to study pneumococcal transmission and colonisation in healthy adults. This novel study design allows us to investigate bacteriological and immune factors associated with colonisation and to examine the density and duration of colonisation episodes.\ud Project Aims\ud 1) Investigation of the transmission dynamics of Streptococcus pneumoniae. Can the hands can be a vector for transmission of S. pneumoniae into the nasopharynx, leading to colonisation? Does concurrent asymptomatic viral infection affect transmission? \ud 2) Investigation of the propensity of two pneumococcal serotypes to cause experimental pneumococcal colonisation, to improve the generalisability of the model and to investigate if immunological responses to serotype 6B are similar to other serotypes. We also wanted to investigate if colonisation is an asymptomatic process in healthy adults? How do the host’s polysaccharide specific antibody responses affect colonisation?\ud Main findings \ud Using our unique controlled human pneumococcal challenge model, we have demonstrated the viability of transmission of pneumococcus from the hand into the nasopharynx, leading to colonisation. We were unable to investigate the relationship between colonisation acquisition and concurrent viral infection due to the absence of viral infection in our participants. The data presented in this thesis showed that the experimental human pneumococcal carriage model can successfully investigate transmission dynamics of pneumococcus. We also demonstrated the varying propensity of two pneumococcal serotypes, 23F and 15B to experimentally colonise the nasopharynx of healthy adults. Nasopharyngeal colonisation was shown not to cause nasal symptoms; however, the data suggested that colonisation may cause a cough in healthy adults. No relationship was found between the level of serum IgG to 15B capsular polysaccharide at screening and colonisation outcome after intranasal inoculation. Nasopharyngeal colonisation with 15B was however, found to boost polysaccharide specific immunity; colonisation positive participants had a significant increase in serum IgG levels to 15B capsular PS.\ud Implications \ud Data presented in this thesis suggest that good hand hygiene practices, already known to reduce enteric bacterial and viral disease, may also prevent the spread of pneumococcus which is thought to be spread primarily through aerosolisation. Results support epidemiological studies which have shown the varying propensity of different pneumococcal serotypes to cause colonisation. We can build upon this work by investigating serotypes in vitro and in vivo to understand bacterial factors that impact the pneumococcus’ ability to colonise the nasopharynx in humans. The EHPC model will be useful in further studies to better understand the dynamics and drivers of pneumococcal transmission, bacterial factors which support successful colonisation and host responses to pneumococcal exposure and colonisation.