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Abstract Background Research in children is essential for them to benefit from the outcomes of research but involvement must be weighed against potential harms. In many countries and circumstances, medical research legally requires parental consent until the age of 18 years, with poorly defined recommendations for assent prior to this. However, there is little research exploring how these decisions are made by families and the ethical implications of this. Aim To explore key ethical debates in decision-making for participation of children and adolescents in a human papillomavirus (HPV) vaccine trial. Methods Semi-structured interviews were undertaken with Tanzanian girls (aged 9–16 years) who had participated in an HPV vaccine trial (n = 13), their parents or guardians (n = 12), and girls together with their parents (in paired parent-child interviews) (n = 6). The interviews were analysed using thematic analysis. Interview data came from a qualitative acceptability study undertaken as part of the Dose Reduction Immunobridging and Safety Study of Two Human Papillomavirus (HPV) Vaccines in Tanzanian Girls (DoRIS) trial. Results Girls and parents desired collaborative decision-making, with parents ultimately making the decision to consent. However, girls wanted a larger part in decision-making. Decisions to consent involved many people, including extended social networks, the trial team, media outlets and healthcare professionals and this resulted in conflicts to be negotiated. Deciding where to place trust was central in participants and parents considering decisions to consent and overcoming rumours about trial involvement. Conclusions Existing models of decision-making help to understand dynamics between parents, adolescents and researchers but neglect the important wider social impacts and the fundamental nature of trust. Children’s roles in discussions can be evaluated using the principles of consent: autonomy, freedom and information. Concepts such as relational autonomy help to explain mechanisms families use to negotiate complex consent decisions. Whilst interviewees supported the maintenance of legal parental consent, researchers must design consent processes centring the child to ensure that whole family decision-making processes are supported.

Introduction: Wastewater and environmental surveillance (WES) has provided valuable quantitation and sequencing data for polio, SARS-CoV-2 and Mpox to support public health decision-making. We aimed to determine if WES for vaccine-preventable diseases (VPDs) is possible and if it may complement clinical surveillance for hepatitis A, E, measles, rubella and influenza viruses. Methods: Digital PCR (dPCR) assays using disease-specific primers were incorporated into single and multiplex reactions that were optimized using positive controls (viral culture, RNA plasmids or known clinical samples). Limits of quantification for each pathogen were determined using quantitative (qPCR) and dPCR on serial dilutions of positive controls. Retained concentrates from wastewater samples collected from 48 national wastewater treatment plants or district sampling networks in nine South African provinces for SARS-CoV-2 wastewater surveillance were subjected to batched RNA extraction and dPCR testing for VPDs. Results of wastewater testing and national notifiable medical conditions surveillance data were compared by epidemiological week and district of collection. Influenza wastewater results were compared with reference to the influenza season (during, before and after) as determined by clinical sentinel site surveillance data. Results: Limits of quantification in genome copies/mL were as follows hepatitis A: 260; hepatitis E: 400; measles: 590; rubella: 460; influenza A&B: 1000. Amongst retrospective wastewater samples the proportion testing positive were; hepatitis A: 425/2329 (18%); hepatitis E: 280/2329 (12%), measles: 47/2422 (1.9%); rubella 39/2422 (1.6%), influenza A: 113/2422 (4.7%); influenza B: 72/2422 (3%). When compared with clinical results, wastewater results were positive when clinical cases were found in the same district and epidemiological week amongst 26%, 8%, 3% and 1% of time-district pairs for hepatitis A, E, measles and rubella respectively. However, the virus was detected in wastewater when no clinical cases were reported that week amongst 4%, 25%, 3% and 4% of time-district pairs for hepatitis A, E, measles and rubella respectively. Regarding influenza A, the majority of wastewater samples tested positive during the season (44/80 positive samples, 55%) than before or after, whilst the majority of influenza B detections in 2023 were after the season (28/36 positive samples, 77%) Discussion: In our comparison of clinical and wastewater data for hepatitis A, E, measles and rubella, WES found evidence of circulating virus in wastewater on multiple occasions in districts when clinical surveillance failed to find cases. Clinical surveillance may be limited on account of test unavailability, absent health seeking, and cases being asymptomatic or minimally symptomatic. Public health action following wastewater detection in the absence of clinical cases should be determined by contextual factors (availability and coverage of vaccination, consequences of ongoing transmission, disease burden) and specific aims of disease control programmes. Conclusion: Wastewater detection of vaccine preventable diseases is possible and may complement VPD surveillance and control programmes

Introduction: Wastewater surveillance for infectious disease has demonstrated its potential to support clinical surveillance of infectious disease through the COVID-19 and Mpox pandemics and the polio environmental surveillance. The National Institute for Communicable Diseases (NICD) provides disease surveillance, specialised diagnostics, outbreak response, public health research and capacity building for communicable diseases. Through grant funding, the NICD developed a national and district wastewater surveillance network to support infectious disease surveillance in South Africa. We describe lessons learned sample collection, laboratory processing and data analysis and reporting as the surveillance network has become established. Methods: A surveillance network comprising 48 wastewater treatment plant (WWTP) and district sampling sites, with a minimum of once weekly sampling, was set up across 9 provinces in South Africa. Samples are collected according to standard procedures, maintained at 40C and transported to the NICD laboratory, In the laboratory, samples undergo concentration, storage of residual concentrate, RNA extraction, PCR detection and quantification. Positive samples are sequenced. All data are recorded into RedCap. Processing data including turn-around-times, sequencing quality data and results are reported weekly. Clinical case data as (determined by case definitions from national notifiable medical conditions surveillance) from the National Health Laboratory Service (NHLS) and NICD laboratory testing (measles and rubella) are downloaded weekly and epidemiological curves are generated. Results: To date, 3350 samples have been collected from June 2020 until April 2024, with 25-58 samples processed weekly. Turn-around times for quantification and sequencing were 5-7 days, and 3-4 weeks. Challenges included delays in new site activation due to co-ordination across multiple layers of authority. Dedicated fridges were required in some sites to maintain sample integrity. Failure or delay in submitting samples occurred because of challenges with communication, co-ordination for the supply of sampling consumables to collection sites, and delays with processes for procurement of specialised courier services. In the laboratory, delays were experienced with shipment of filtration apparatus and PCR reagents. In addition, contamination in sequencing laboratories intermittently disrupted sequencing as deep cleaning was conducted. Analysis and reporting challenges were encountered in the introduction of RedCap data management system. The programme has also not been able to identify and attract senior bioinformatics staff. Discussion: A stable network of sentinel sites with regular sampling, testing and reporting has been established. Challenges have provided learning and development opportunities. Research efforts are now focusing on exploring and interpreting the relationship between the clinical and wastewater based epidemiology of the pathogens, and development of models to support public health decision making. Conclusion: Wastewater sentinel site surveillance networks, testing and analytic infrastructure are necessary components of pandemic preparedness. We have demonstrated successful creation of this network that will provide ongoing evidence to inform data to policy and practice pathways.

Background/Introduction: Limited studies report on the implementation of antimicrobial stewardship programmes (ASPs) in resource-limited neonatal inpatient settings. Undertaking qualitative research to better understand the contextual drivers for implementation of ASPs is increasingly recognized as means to enable sustainable stewardship programs. Methods & Materials: As part of a sequential three-phase, mixed-methods, multidisciplinary, prospective neonatal stewardship interventional study in 14 South African hospitals (7 public, 7 private), we conducted a pre-intervention barriers and enablers survey (BES) to help understand the context for implementing a multidisciplinary approach to neonatal AMS. The survey included 82 potential barriers to neonatal antibiotic stewardship with a five-point rating scale and an open question on enablers. We used a purposive sampling method including neonatal unit multidisciplinary professionals and members of the hospital leadership team at each hospital. Surveys were completed anonymously via an electronic link provided and entered directly into a RedCap database system. Results: Responses received from 100 health professionals and leaders were equally distributed between public and private sector hospitals. There was a higher proportion of nurse respondents, and a lower proportion of clinicians (neonatologists/paediatricians) respondents from hospitals with smaller neonatal units (

Introduction: The epidemiology of Group C and Group G Streptococcal bloodstream infections (GCS and GGS respectively) in England has not previously been investigated in detail. Considering longitudinal increases in these pathogens, we assessed trends and health inequalities of GCS and GGS bacteraemia in England to support and prioritise development of appropriate healthcare and public health interventions. Methods: GCS and GGS bacteraemia data between 01/01/2015 and 31/12/2022 for England were extracted from UKHSA's national laboratory reporting database and reference laboratory. Data were augmented through linkage to Office for National Statistics and Hospital Episode Statistics to capture socioeconomic deprivation score (index of multiple-deprivation, IMD) based on residential location, and ethnicity. Infection rates by ethnicity and IMD were direct age-standardised. Results: Between 2015 and 2022, 11,275 GCS (range 932-1693 per-year) and 10,068 GGS (range 1070-1411) were reported in England. For GCS, the median age was 77y and 54.7% (n=6164) of cases were female, and for GGS, the median age was 79y with 52.3% female (n=5265).Annual rates of GCS increased 71% (1.7 to 2.9/100,000 population) and GGS 12% (2.0 to 2.2) between 2015 and 2022, both peaking in 2019 (3.0 and 2.5 respectively). North East England had the highest annual rates of GCS (up to 9.0/100,000 per-year); up to 2.4 times higher than the London region after age-adjustment (3.7 vs 9.0 per 100,000 in 2019, and 2.9 vs 7.0 per 100,000 in 2022).There was a disproportionate burden of GCS in patients in the most-deprived IMD-quintile of the population in England (23.7% of total; n=2677), with the least-deprived IMD-quintile comprising the smallest contribution to the total (16.5%; n=1864). Age-adjusted annual rates in the most-deprived areas were double those in the least-deprived (4.3 vs 2.3/100,000 in 2022). For GGS there was a disproportionate burden in the 40%-60% most-deprived IMD-quintile (21.4% of total; n=2152); after age-standardisation, rates were highest in the most-deprived and lowest in the least-deprived IMD-quintiles (3.4 vs 1.9/100,000 in 2022).In 2022, 95.1% of GCS (n=1,516) and 94.9% of GGS (n=1,144) cases respectively were from patients of ‘White’ ethnicity with ‘Black’, ‘Asian’, ‘Mixed’, and ‘Other’ ethnicities comprising

Introduction: Ticks transmit a greater variety of pathogenic agents than any other blood-feeding arthropods, posing a profound impact on the economy of livestock farming and human health concerns. Cambodia already has an enormously high burden of infectious diseases; however, little is known about potential hotspots of tick-borne pathogens. The diversity and distribution of tick species remain poorly characterized. The medical, veterinary, and socio-economic burdens of tick-borne diseases remain also largely unknown. In the present study, we aim to assess the diversity of tick species and associated pathogens circulating in a rural community of Cambodia and to evaluate the transmission risk of these pathogens in people living in the area where ticks have been collected. Methods: The study was conducted in rural communities of Kampong Thom province located in the central part of Cambodia. Ticks were collected from animals and the vegetation surrounding human households. All collected ticks were screened for pathogens considered major threats to human health including severe fever with thrombocytopenia syndrome virus (SFTSV), tick-borne encephalitis virus (TBEV), and Crimean-Congo hemorrhagic fever virus (CCHFV), using PCR assays. Human blood samples collected from 200 residents of tick-affected households underwent ELISA assay for detection of IgG antibodies against these pathogens. Results: From 82 households, 1,433 ticks were collected, primarily from dogs (97.3%). Among them, 91% were adult, dominated by Rhipicephalus sanguineus (97.3%) followed by Rhipicephalus microplus (2.4%) and Rhipicephalus haemaphysaloides (0.3%). None of the targeted viruses were detected. Of the 200 human samples, 56% tested positive for anti-TBEV IgG, 6.7% for anti-SFTSV IgG, 2.5% for anti-CCHFV IgG, 10.5% for anti-scrub typhus IgG, 5% for anti-Borrelia, and 0.6% for anti-Coxiella IgG. Discussion: Given the absence of highly pathogenic pathogens in our findings, further analysis utilizing metagenomics approaches is necessary to identify and characterize other tick-borne pathogens potentially circulating in Cambodia. The presence of antibodies against tick-borne pathogens in humans, particularly anti-TBEV IgG antibodies, might be overestimated in the present study due to cross-reactions with other flaviviruses. Thus, further confirmatory tests are anticipated for samples positive for anti-SFTSV, TBEV, and CCHFV IgG antibodies using seroneutralization assay. Conclusion: Our study provided preliminary data on ticks and the potential risks of tick-borne pathogens circulating in the rural community of Cambodia. Large-scale studies implementing one health approach must be considered to better assess the threats of tick-borne diseases at the intersection of animal health, human health, and the environment in Cambodia.

Introduction: Cholera is a public health threat in Africa, with an estimated 68,480 cases and 895 deaths reported across seven of 14 Member States in the Eastern region in 2023. Within the region, cholera outbreak has been protracted in the horn of Africa, with countries responding to the outbreak amid prolonged harsh drought, floods and complex humanitarian emergencies. In recent months, an upsurge in cases and deaths has been observed in the region, with five countries reporting active outbreaks this year. We describe the trend of cholera cases in Comoros, Ethiopia, Kenya, Somalia and Tanzania for 2007-2024. Methods: Descriptive trend analysis of cholera case data for Comoros, Ethiopia, Kenya, Somalia and Tanzania, from 2007-2024, was performed. Data were obtained from the Event Management System of the Africa Centres for Disease Control and Prevention (2023-2024) and the Global Infectious Disease and Epidemiology Network (GIDEON) online resource for data (2007-2022). Water, Sanitation and Hygiene (WASH) data was sourced from WHO/UNICEF Joint Monitoring Program for WASH. The term 'cholera case' includes both confirmed and suspected. Results: From 2007 to 5 April 2024, a total of 578,449 cholera cases and 8,514 deaths [case fatality rate (CFR): 1.5%] were reported from Comoros, Ethiopia, Kenya, Somalia and Tanzania. Somalia accounted for 55.2% (315,972) of the cases and 54.6% (4,700) of the corresponding deaths. The average CFR per year was 1.4%, ranging from 0.3% in 2014 to 2.4% in 2016. During the review period, notable geographical patterns that were triggered by climate change were observed. Somalia experienced annual cholera outbreaks beginning November and December and receding in May, with largest occurrences in 2011 (77,636 cases) and 2017 (75,414 cases). Widespread outbreaks occurred in Ethiopia from 2007-2010, with a high peak in 2009 (31,509 cases) and recently in 2023 (29,869 cases). For this year, cases in Kenya appear to be on the decline while Tanzania shows an increasing pattern. Comoros reported its first cholera outbreak since 2007 in February 2024 through a cross-border event with Tanzania. Ethiopia is among countries in Africa with the largest population practicing open defecation in 2022. Discussion: The trend analysis underscores the persistent nature of cholera outbreak in the Eastern region. Climate change impacts the dynamics of the outbreak by limiting access to safe water and sanitation, and triggering increased cross-border movements. The average CFR per year surpassed the recommended

Introduction: Africa CDC is a continental autonomous health agency of the African Union with the mandate to support public health initiatives of Member States and strengthen the capacity of their public health institutions to detect, prevent, control and respond quickly and effectively to disease threats. Africa reports the heaviest burden of public health emergencies globally. In the past 22 years, over 100 public health events were reported annually, predominantly infectious (92%) and zoonotic diseases (30%) coupled with humanitarian crises (5%). The incidence of zoonotic diseases continues to increase significantly among countries with similar geographical and epidemiological profiles, leading to cross-border spill overs of outbreaks. Africa CDC developed the continental strategic framework for strengthening cross-border surveillance coordination, collaboration, and information sharing in Africa to address cross-border health threats in the continent. Methods: Africa CDC developed this strategic framework through a participatory and consultative process in collaboration with representatives of regional economic communities (RECs), regional and country stakeholders from African Union Member States, and experts in port health surveillance. Technical input from international partners including the US CDC, IOM, WHO, and UKHSA was also obtained through technical working group sessions. In October 2022, a continental consultative workshop on cross border surveillance and coordination with a representation from 50 AU member states and four regional economic communities (RECs) was held in Kigali, Rwanda. From the workshop, a technical working group with MS representatives from the 5 AU regions, RECs, Africa CDC and technical partners was established to guide the framework development. Among the key performance indicators include number of member states supported to develop Cross-border coordination mechanisms. A detailed implementation roadmap coupled with a robust monitoring and evaluation matrix anchors this framework. Results: The framework fosters partnerships and collaborations that support countries in implementing cross-border surveillance (CBS) initiatives and key interventions, including establishing border health committees, developing SOPs and guidelines, joint action plans, and holding regional trainings, especially during outbreak responses. To date, the framework has supported various notable outbreak responses through the development process, like Ebola Virus Disease (EVD) 2022-2023 in Uganda and Marburg Virus Disease (MVD) in 2023 in Equatorial Guinea and Tanzania. Discussion: This strategic framework is intended for use by National Public Health Institutes, Ministries in charge of human, animal and environmental health, trade, immigration, foreign affairs and other relevant Ministries, departments, organizations, agencies, and sectors involved in border health activities, academia and other stakeholders, including non-state actors to build CBS capacity with MS and to support early detection, prevent and timely response to the public health threats in the continent. Conclusion: This CBS framework is aligned with the Africa CDC Strategic Plan (2023-2027)’s strategic priorities and its ambitious vision of a New Public Health Order for Africa.

Background: Arboviruses represent a serious concern in tropical/subtropical countries. In Cameroon, port cities and cities surrounded by forests constitute the main hot-spot of dengue and yellow fever. Due to the absence of effective treatments and vaccines against most arboviruses diseases, vector control remains the most effective measure to prevent further resurgence. To date, little is known about the bionomics of their main vectors Aedes aegypti and Aedes albopictus in such environments. This study aim to assess the bioecology, distribution and susceptibility profiles to insecticides of these vectors in three cities of Cameroon. Methods: Entomological surveys were undertaken from September 2021 to October 2022 in Bertoua (savanna area), Kribi (city port) and Sangmelima (forested area). Immature stages of Aedes spp. were collected by deeping and Stegomyia indexes were estimated. After emergence and morphological identification, F0 and F1 of Aedes aegypti and Aedes albopictus progeny will be used to assess the current phenotypic resistance status to the commonly used insecticides using WHO bioassays and mortality rates were assess according to the different insecticides tested. Results: A total 475 breeding sites in Sangmelima (41.47%), Kribi (33.47%) and Bertoua (25.05%) were identified. Tires and plastic containers were most abundant. A total of 2364 mosquitoes belonging to 4 genera and 9 species were collected. Aedes species recorded (1864) included Ae. albopictus (72%), Ae. aegypti (25.91%), and Aedes spp. (2.09%). These main vectors were present in all the study sites. Ae. albopictus was predominant in Sangmelima and Kribi while Ae. aegypti was mostly found in Bertoua. According to house indexes, transmission of dengue and yellow fever were high in Kribi and Bertoua. Ae. aegypti across study sites were found to be resistant to permethrin, deltamethrin and DDT, while Ae. albopictus was resistant to bendiocarb in Kribi and Bertoua. However, all these species were susceptible to malathion. Discussion: The study shows that Ae. albopictus and Ae. aegypti larvae live in a wide variety of breeding sites (Ngo et al., 2020), those species are largely distributed across the country(Tedjou et al., 2019). Ae. albopictus and Aedes aegypti were sensitive to organophosphates (malathion). These results are similar to previous studies (Kamgang et al., 2017). The tolerance and resistance observed could be due to the use of insecticides such as sprays and reminders, which are becoming more and more common(Nchoutpouen et al., 2019). Conclusion: The implementation of surveillance and control programmes for arbovirus vectors is urgent required in view of the increase in cases of arboviroses. To control arboviruses effectively, a targeted approach is needed to limit the spread of these vectors in our environment. This work is a contribution to vector control using ecological and susceptibility parameters with the aim of preventing a possible arbovirus epidemic in Cameroon.

Introduction: In Italy, despite the existence of an epidemiological surveillance system for Arboviruses, managed in a One Health perspective, the implementation of genomic characterisation remains fragmented and no systematic genomic surveillance initiative integrating human and animal data has been established. The purpose of this study is to investigate the contribution of Whole Genome Sequencing (WGS) in the integrated genomic characterization of West Nile virus in target avian reservoirs, mosquito vectors and infected humans in Romagna (northeastern Italy). Methods: The study was conducted in Romagna, northeastern Italy, between May and October 2023 involving patients with encephalitis/meningitis and healthy blood donors who tested positive for WNV during screening programmes. Mosquitoes were sampled with attractive traps, and resident and migratory birds were collected as of active or passive surveillance. Samples were screened with WNV-specific PCR (Polymerase Chain Reaction) assays, and positive samples were subjected to WGS by tagmentation and hybrid-capture-mediated target enrichment (Viral Surveillance Panel, Illumina). Assemblies and consensus sequences were obtained with Bowtie2 v.2.4.1 and BCFtools. Maximum likelihood trees for viral phylogenetic inference were constructed with IQ-TREE 2.0.3 with automatic selection of the best nucleotide substitution model, 1000 ultrafast bootstrap replicates and midpoint rooted. Results: Eleven WNV lineage 2 sequences were obtained (4 meningitis cases, 3 blood donors, 3 Culex pipiens and 1 Pica pica). Genome sequencing yielded 98.8-100% coverage. Coding sequences maintained high nucleotide and deduced aminoacid conservation, with an average identity of 99.82% and 99.92%. Mutations of virulent clade 2d were identified, affecting infection rates in avian hosts and mosquito vectors and neuroinvasiveness in humans. Obtained sequences clustered within the Central-Southern European clade with robust bootstrap support, forming a distinct cluster with other viruses isolated in Italy from 2021 to 2023. Discussion: Our study, albeit with preliminary and limited results, provides an insight into integrated genomic characterisation of WNV strains circulating in the study area, where WNV is endemic and which is considered a high-transmission-risk hotspot due to its geoclimatic characteristics and its proximity with one of the main stop-overs and breeding ground for migratory birds in Central and Southern Europe (Delta of Po river). Continued genomic surveillance, throughout future seasons, will support the tracking of arboviruses genetic diversity, evolution, emergence of new variants or introduction of genetically diverse strains. Conclusion: Our approach suggests the importance of adopting an interdisciplinary multistakeholder strategy to integrate the steps from clinical diagnosis to entomological and ornithological monitoring, up to epidemiological and genomic surveillance to create an operational process involving human and animal health suitable for the genomic characterisation of both autochthonous and imported Arboviruses. Engaging both human and animal health sectors, will facilitate a comprehensive characterisation of endemic and emerging Arboviruses to mitigate their impact on public health.

Introduction: Yellow fever (YF) remains a major global health threat, complicated by climate change, vector expansion, and insufficient health system infrastructure in endemic regions. Recent shortages of licensed vaccines during regional outbreaks underscore the need for next-generation YF vaccines that retain safety and effectiveness standards while enhancing manufacturing efficiency. Grown in serum-free Vero cells, vYF is a live-attenuated YF vaccine developed to ensure a sustainable and robust global supply. Methods: In two ongoing Phase II randomized, observer-blind, active-controlled, non-inferiority multicenter clinical trials of vYF in 18-60-year-old adults in the US (vs. YF-VAX – VYF02 study) or in Europe and Asia (vs. Stamaril – VYF03 study), the neutralizing antibody (NAb) responses are being measured by a YF microneutralization assay on day 29 (D29), month 6 (M6), and then annually from year 1 (Y1) until Y5. Seroprotection is defined as NAb titers ≥ 10 (1/dil). Interim analyses conducted one-month post-vaccination demonstrated that the vYF immune response was non-inferior to YF-VAX or Stamaril. Here, we report the immunogenicity up to Y1.Adults were randomized 2:1 to receive one dose of vYF or YF active control: 568 in the US (VYF02) and 690 in Europe and Asia (VYF03). Results: Twenty-eight days after a vYF dose administration, over 99% of YF-naive participants in the US, and over 98% in the EU and over 95% in Asia were seroprotected.YF-naive recipients (FAS) exhibited D29 post-vaccination geometric mean titers (GMT) well above the seroprotective threshold: 2654, 1/dil, in the US; 2508, 1/dil, in EU; 1374, 1/dil, in Asia. As expected, GMTs waned after D29 but more than 97% of participants in the US, over 98% in Europe and more than 94% in Asia maintained titers above the threshold of protection at Y1, with GMTs of 401, 1/dil, in the US; 469, 1/dil, in EU; and 205, 1/dil, in Asia. Discussion: Overall, 94% or more of YF-naive adults in US, EU and Asia were seroprotected 28 days after receiving vYF and remained seroprotected at Y1. Seroprotection rates and GMT values following administration of the control vaccines (YF-VAX or Stamaril) were in similar ranges at all evaluated timepoints. Despite lower NAb titers observed in Asia from D29, GMT values remain well above the threshold considered for protection in all regions. Conclusion: Immunogenicity of vYF is non-inferior to YF-VAX and Stamaril, inducing seroprotective NAb responses in 18 to 60-year-old vaccinees to 1-year post-vaccination. Reduced NAb titers in Asia relative to the US and Europe did not diminish rates of seroprotection. These results are encouraging for addressing unmet public health needs during YF outbreaks.

Background: Multi-drug-resistant microorganisms are emerging at a rapid pace, and the burden of infectious disease on our healthcare system is still enormous. Point prevalence surveys of antimicrobials in admitted patients often exclude emergency units. However, emergency units in LMIC often double up as wards due to delays in transfer. Further, they reflect on antimicrobial practices in the referral unit and hence, antimicrobial stewardship is of utmost importance in the emergency units. With this background, we undertook a point prevalence survey in the emergency unit of a tertiary care centre which sees an annual footfall averaging 47,000 per year. Methods: This inpatient PPS was conducted during May 2023 in triage areas of the emergency unit of a tertiary care hospital. We collected data on all patients in the emergency unit triage areas between 8 am and 5 pm. All patients referred from any hospital, clinic and nursing home in the triage area till 11:59 pm of the previous day of the survey were assessed for antibiotic use, either ongoing or pre-administered during the stay in the referring unit. The survey team was trained to fill in and capture the data. Predesigned forms were used to collect data about patient demographics, diagnosis, antimicrobial indications and supportive investigation reports.Compliance with the Institute Antimicrobial Policy was also undertaken. Results: Of the 145 admitted patients in the triage areas of the emergency unit, 94 were included in the survey, 34% were referred from public and 36% from private hospitals, of which 42.5% had discharge summaries.Around 68 % had a co-morbid illness and 65% of patients had the risk factor for multidrug-resistant pathogens Among these, 82% were prescribed antimicrobials, of which 32.9% were ongoing at the time of ER visit.26.5% of the patients were on two antimicrobials.Of the total antimicrobials prescribed, 55.3% were prescribed for community-acquired infections requiring hospitalization. In 49%, no evidence of infectious aetiology was found, while 36 % of patients had sepsis. Drugs from the ‘Watch’ category were prescribed most commonly. Over 60 % of the antimicrobial treatment given was in concordance between the treating team and the surveyor with respect to the purpose of antibiotic. Importantly, nearly 2/3rd of the prescriptions were in accordance with the antimicrobial policy for infection management.The problems identified were in the antibiotic choices for pneumonia, meningoencephalitis, MRSA and acute pancreatitis, utilization of diagnostics and de-escalation. Conclusion: Our survey demonstrated a high use of antimicrobials in patients admitted in triage areas of emergency unit with a considerable proportion of drugs from the ‘Watch” category. Also, a large number of patients have a very high risk for development of infections caused by MDR pathogens. The survey was efficient in identifying points for intervention for antimicrobial stewardship in the emergency unit.

Background: We report the first human case of an invasive novel Arsenophonus species. The genus Arsenophonus of the order Enterobacterales, comprises of insect symbiotic intracellular bacteria with a wide host spectrum including triatomine bugs, bees, ticks, lice and parasitic wasps. Case Description: The patient, a 70-year-old homeless man from Johannesburg, South Africa was admitted in February 2020 with symptomatic iron-deficiency anaemia. His physical examination revealed pedal oedema, pallor and bibasal lung crackles. He had poor dentition and body lice. He had evidence of systemic infection, with leukocytosis and a raised C-reactive protein. He was diagnosed with iron deficiency anaemia secondary to confirmed duodenitis, had chronic kidney disease and monoclonal gammopathy of unknown significance. He was treated empirically with amoxicillin-clavulanate for four days. He received a further four days of ceftriaxone after admission blood cultures flagged positive with an unknown Gram-negative bacillus. The bacterium was cultured from blood cultures on two separate occasions. The patient responded favourably to antibiotics and was discharged from the hospital.The Vitek®2 instrument misidentified the bacterium as Aeromonas salmonicida with 94% probability. The isolate was unidentifiable on the Vitek® MALDI mass spectrometer (MS) but was identified as Arsenophonus nasoniae with a low score of 1.62 on the Bruker MS.Whole genome sequencing was undertaken by extracting genomic DNA and sequencing it on the Illumina MiSeq platform. The genome of the new bacterium is 3158693 bp in size with a G+C content of 36.95 mol%. It has a genome-based average nucleotide identity of 92% with A. nasoniae DMS15247. We propose this bacterium to be a new species within the genus Arsenophonus, family Morganellaceae, order Enterobacterales, phylum Proteobacteria. The bacterium will be deposited in two different strain collections. Discussion: There are only two previous reports of possible human infection with A. nasoniae, one from a skin eschar and the other from a blood culture. The Arsenophonus species infecting our patient possibly originated from his body lice. Arsenophonus species are insect symbiotic bacteria and human infection is a rare phenomenon. Our patient had several immunocompromising conditions that may have predisposed him to this unusual infection. Infections with insect symbiotic bacteria are emerging in humans and should be considered as potential pathogens in patients with a clear history of insect infestation. Conclusion: We present the first description of human invasive infection with a new species of Arsenophonus genus that resolved on broad spectrum antibiotics. Rare infections with insect symbiotic bacteria are difficult to identify on routine microbiological testing but should be considered in patients with insect infestations.

Introduction: The Integrated Disease Surveillance and Response system relies mainly on aggregated, paper-based, error-prone routine data from health facilities. Lack of timeliness, inadequate data management and analysis, and weak laboratory capabilities result in low capacity for early detection and rapid response to disease outbreaks.The objective was to evaluate a novel surveillance strategy, based on a clinical e-tool for patient management implemented in primary care health facilities in Tanzania, enhanced by sentinel sites and mobile clinically targeted microbiological investigations. Methods: An electronic clinical decision support algorithm (CDSA) was provided to healthcare workers to guide them during consultations with children aged 1 day to 14 years. A real-time syndromic surveillance dashboard (medAL-outbreak) displaying the recorded clinical data was developed and adapted after feedback from end-users at the health facility and district levels. This syndromic surveillance was supplemented by microbiological investigations using two approaches: 1) A static approach, in 6 sentinel facilities, for children with specific pre-established conditions: severe respiratory disease (nasal swab sample), severe gastrointestinal disease (stool sample), and unspecific or prolonged fever (blood sample); 2) A mobile approach, where a surveillance team intervened in health facilities when alerts were triggered by medAL-outbreak. Priority pathogens of public health relevance were tested for at the two local reference laboratories. Results: From April 2023 to March 2024 (the study is ongoing until September 2024), we included 31,525 acutely ill children across 60 healthcare facilities in 5 districts from 2 regions (Mbeya and Morogoro). MedAL-outbreak allowed monitoring of 22 syndromes, including severe cases, pneumonia, malaria, severe/non-severe acute diarrhea, meningitis, anemia, malnutrition, and sexually transmitted infections. Malaria prevalence was low in Mbeya CC, Mbeya DC, and Ifakara TC (1%, 1%, and 3% of febrile children, respectively), while much higher in Ulanga and Mlimba (45% and 14%, respectively), with some seasonality (42% from April to November, and 53% from December to March in Ulanga). Pneumonia was more prevalent in Mbeya DC, accounting for 22% of all cases (compared with 8% in Mbeya CC and 11% in the 3 Morogoro districts), with no clear seasonal pattern. Acute watery diarrhea was also more frequent in Mbeya CC and DC, occurring in 19% of the cases, against 9% in the other 3 districts. Microbiological surveillance is ongoing until September 2024; results will be presented during the congress. Discussion: Utilization of a CDSA in health facilities enabled reliable real-time syndromic surveillance. Conclusion: Adding static and mobile targeted microbiological surveillance, triggered by alerts based on clinical and spatio-temporal abnormalities, could potentially allow Tanzania to advance to a smart resource-sparing surveillance system with early detection of outbreaks.

Introduction: Pathogenic RNA viruses pose a significant threat to global health due to their high rates of morbidity, mortality, and substantial economic impact. The rapid development of antiviral drugs, therapeutics, and vaccines during an outbreak requires adequate virus samples for efficacy evaluation. However, obtaining viruses or clinical samples during outbreaks is challenging. Reverse genetics offers a powerful solution to this problem by enabling the rapid and efficient production of viruses. This technique enhances containment and response strategies, thereby accelerating the development of preventive measures. Materials & Methods: We established a reverse genetics system for RNA viruses, focusing on vesicular stomatitis virus (VSV), a prototypical negative-sense RNA virus. Using a rapid single-tube method, we assembled the full-length cDNA clone of the VSV antigenome into a single plasmid. This involved amplifying the antigenome as five overlapping fragments, excluding the G protein genes, and integrating regulatory elements (T7 promoter, ribozyme, and T7 terminator) into a plasmid vector (pLinker). To ensure virus rescue, we cloned minigenome plasmids encoding essential accessory proteins (N, P, and L) with regulatory elements. Co-transfection of plasmids encoding VSV antisense genome, minigenome, and T7 polymerase into BHK-21 cells facilitated virus rescue. Virus titration was performed by infecting Vero-E6 cells with the collected supernatant. Results: To construct functional VSV full-length genome and minigenome plasmids, we integrated regulatory elements like leader, trailer, and ribozyme sequences (

Background: In 2016, the World Health Organization recommended that a fractional dose of yellow fever vaccine could be used in persons 2 years of age or older in response to an emergency situation that resulted in a global shortage of available yellow fever vaccine. However, this recommendation did not extend to the youngest age group licensed for yellow fever vaccine as no published data were available on the use or safety of fractional dose yellow fever vaccination among children aged 9–23 months. We conducted a single-blind randomized controlled trial comparing the immunogenicity and safety of fractional one-fifth and one-half doses of Bio-Manguinhos 17DD yellow fever vaccine with full dose in children 9-23 months old in Uganda. In this paper, we present the interim analysis on safety. Methods: Children aged 9–23 months presenting for routine well-child services were recruited for inclusion at one of three study sites. We collected data on all adverse event(s) following immunization (AEFI) during active AEFI surveillance for 28 days post-vaccination using multiple data collection tools including a home diary card with an objective measurement of fever. An independent team from the Uganda national AEFI Committee investigated and classified serious AEFI (SAE) according to Brighton Collaboration Criteria. Results: Among 1053 enrolled children, 672 (64%) were reported to have a non-serious AEFI (NSAE) and 18 (2%) were reported to have a SAE. The most commonly reported AEFI were diarrhoea, fever and rash, each reported by 355 (34%), 338 (33%) and 188 (18%) participants, respectively. Among 18 participants with SAE, eight were reported to have seizures and five were hospitalised for seizures or other causes (respiratory symptoms, gastrointestinal illness, malaria). Four SAEs (deaths) occurred >28 days after vaccination. There were no reported cases of pre-specified or vaccine-related SAEs. We observed no significant difference in frequency or severity of adverse event among the study groups. Conclusions: Using comprehensive active surveillance monitoring, we did not identify any unexpected safety concerns among children aged

Background: First-line pathogen detection and antimicrobial resistance (AMR) profiling, using culture, requires viable organisms. Metagenomic next generation sequencing (mNGS) can identify and characterize pathogens directly from clinical material without culture. This proof-of-concept study aimed to compare bacterial identification and AMR profiling using mNGS and culture. Methods: To date, 24/48 samples comprising of ascitic fluid, cerebrospinal fluid (CSF), urine, and pleural fluid were tested on routine culture and mNGS (using the Oxford Nanopore Technology (ONT) MinION platform). Results were analysed using EPI2ME wf-metagenomics, following host-depletion using Bowtie2, to characterise pathogens and AMR profiles. Pathogen identification was expressed as relative abundance (RA), and sequencing results were compared to those obtained by routine culture. Results: Culture identified a pathogen in 9/24 (38%) specimens while mNGS identified a potential pathogen in 12/24 (50%). mNGS was able to identify a potential pathogen in 6/15 (40%) culture negative specimens. There was congruence in microbes identified using culture and mNGS in 5/9 (56%) culture positive specimens; however, the species matching culture results was rarely also dominant in terms of abundance (3/9, 30%), complicating causality assignment. Co-detections were also common with mNGS, with ≥5 species detected in the majority of the samples. Further, 9/24 (38%) specimens were interpreted as negative using mNGS due to the exclusion of unknown reads which did not match to any taxonomic group. Phenotypic resistance was seen in 7/24 (29%) samples, with correlation between culture and mNGS in 3/7 (42%) samples. mNGS detected AMR genes in 5/17 (29%) samples without phenotypic resistance. Discussion: mNGS was able to resolve 40% of the culture negative samples, majority of which were urine. The culture negative results may have been due to the fastidious nature of the organisms or prior antibiotic administration. A substantial portion of the reads among the samples could not be classified; this may be attributed to poor sequence quality, resulting from poor DNA, or sequencing library, quality, and low DNA input. Further analysis is needed to decern whether unknown reads could be classified with alternate methods or are true negatives. Co-detections were common in polymicrobial sample types, such as urine, albeit majority were normal flora. There was a low correlation between AMR profiles identified using mNGS and phenotypic drug susceptibility testing, indicating that mNGS alone may not be a suitable approach for describing clinically actionable AMR results. Conclusion: In our proof-of-concept study, mNGS was able to identify a potential pathogen in 50% of the samples, including 40% which were unresolved using culture; however, congruence for pathogen identification with culture was moderate. For future research, a larger sample size should be considered as well as additional measures in conjunction with RA to determine the pathogen of interest and assessing the clinical significance of results.

Introduction: Thousands of human infective viruses spread worldwide, yet protective measures exist for only a fraction, with internationally accepted vaccines available for an even smaller subset. Treatment options are limited, often restricted to symptom management, as specific antiviral agents remain elusive for most viruses, including the Sindbis virus. Originating in Africa, Sindbis virus, a member of the Alphavirus genus, is primarily transmitted by arthropod vectors, notably mosquitoes, leading to the manifestation of Sindbis fever characterized by symptoms including rash, headache, joint pain, and fatigue, highlighting its significance as a vector-borne human pathogen. While historically associated with major epidemics in northern European countries such as Ockelbo and Pogosta disease, and Karelian fever, the virus's potential for global spread necessitates vigilance and preparedness for the emergence of outbreaks in new regions. In this context, the exploration of repurposed therapeutics, such as derivatives of glycopeptide antibiotics, holds promise for combating Sindbis virus and other viral pathogens, given their observed antiviral activity against a spectrum of viruses including influenza, Dengue virus, West-Nile virus and Human Immundeficiency virus. Methods: We investigated the antiviral efficacy of 26 synthetic antibiotics and derivatives against Sindbis virus in vitro, using Vero E6 cells. Initial compounds included teicoplanin, ristocetin, and vancomycin and their derivatives. Cytotoxicity assessment preceded evaluation of antiviral potential via microscopic analysis, MTT assay, and quantitative PCR analysis to measure changes in viral copy numbers. Results: Four compounds exhibited potent inhibitory effects, with two demonstrating moderate antiviral activity. Among them, a ristocetine derivative (ERJ-552) displayed exceptional potency against Sindbis virus. Further investigations into its mechanism of action were conducted using a time addition assay to determine its antiviral effects during the infection cycle. Discussion: Our findings underscore the potential of repurposed antibiotics and their derivatives in combating Sindbis virus. The identification of ERJ-552 as a potent antiviral compound highlights avenues for further research into novel therapeutics against alphaviruses and other viral pathogens. The study elucidates the importance of exploring existing drugs for new applications in viral therapy. Conclusion: In conclusion, our study demonstrates the antiviral efficacy of synthetic antibiotics and derivatives against Sindbis virus, with ERJ-552 emerging as a particularly promising candidate. These findings contribute to the ongoing efforts to develop effective treatments against emerging viral pathogens, emphasizing the significance of repurposing existing drugs in the fight against infectious diseases.

Background/Introduction: The autogenous vaccine in livestock is an alternative application to reduce loss and less antibiotics usage in swine production. However, the results of autogenous vaccine in terms of an impact on other environment pathogens were quiet limit. In this study we assessed the antimicrobial susceptibility of E. coli isolated from pig farm waste (feces and wastewater) in Thailand. Methods & Materials: Feces and wastewater samples were collected 4 times from nursery to finishing phase (at the age of 6, 10, 20 and 26 weeks). Two herds were divided to get commercial or autogenous vaccine against APP disease. Bacterial identification was conducted by culture method and antimicrobial susceptibility test were performed by VITEK-2. Results: Among overall isolations of E. coli (99 isolates), high resistance rates (>60%) to ampicillin, cefalexin, cefalotin, cefpodoxime, cefovecin, ceftiofur, gentamicin, enrofloxacin, marbofloxacin, pradofloxacin, doxycycline, tetracycline were documented. Low resistance rates were found in amoxicillin-clavulanic acid, neomycin and nitrofurantoin while their susceptibility rates to imipenem and amikacin were 100%. Multidrug-resistance was more than 90%. The group of E. coli at the age of 6 weeks (pre-vaccination phase) appeared to have more percentage of resistant to antimicrobial drugs than the group from 10 - 26 weeks (after vaccination). The E. coli from age: 20 weeks had significant relationship with ampicillin and doxycycline resistance though the age: 26 weeks had significant relationship with pradofloxacin resistance. Furthermore, the types of APP vaccines usage had significant relationship with enrofloxacin and marbofloxacin resistance that was higher resistance rates in the group of APP commercial vaccine usage than the group of autogenous vaccine usage. Discussion: The results demonstrate that autogenous vaccine might be used as a tool to reduce E. coli resistant in the environment, particularly in pig farm waste. Conclusion: Therefore, the impact of autogenous vaccine on other environment pathogens still was restricted and more studies should be done in a greater number of farms and genera of bacteria.

Introduction: Dengue, Zika, and Chikungunya are endemic arboviruses in Brazil, presenting significant healthcare challenges due to their similar symptoms and the difficulties in laboratory diagnosis, which lead to an underestimation of the true number of infections. This study aims to elucidate the emergence and reemergence of these arboviruses in new regions. Methods: A cross-sectional study was conducted at two emergency care units in Salvador, Brazil, from April 2023 to March 2024. Patients with fever in the last seven days and associated symptoms such as skin rash, retroocular pain, myalgia, and arthralgia were included. Respiratory symptoms were exclusion criteria. Data were collected using REDCap (v.9.3.1 © 2023), and blood samples were taken. RNA extraction was performed using the QIAamp MinElute Virus Spin Kit (QIAGEN), and RT-qPCR was conducted with the ZDC kit (IBMP). Serological analysis was carried out using Euroimmun (Lübeck, Germany) kits to detect Dengue and Chikungunya antibodies. Results: A total of 351 participants were enrolled, with 327 providing samples. The majority were women (218; 62.1%) of mixed race (156; 44.8%), with a median age of 29 years. RT-qPCR identified 25 samples positive for DENV1, two for DENV2, and eight for Chikungunya. Other serotypes were not detected. Serological analysis showed 64 (25.9%) samples positive for NS1 Dengue antigen, and 124 (68.9%) positive for Dengue IgM. For Chikungunya, six (6.6%) samples were positive for IgM, and one (1%) was positive for Zika IgM. IgG seroprevalence was 70.1% for Dengue (n = 124), 27.6% for Chikungunya (n = 58), and 56.6% for Zika (n = 164). Discussion: Positive qPCR samples were forwarded for sequencing. A metagenomic approach will be applied to samples that were negative in all tests (PCR, IgM, and NS1). Conclusion: The study identified a high prevalence of arboviruses, underscoring their extensive circulation in the region. Genomic analysis of the viruses is essential to better understand their dynamics and introduction in the area.

Background: Healthcare-associated bacterial and fungal infections pose a significant public health concern in Southern Africa, with high rates of antimicrobial resistance. Compared to bacterial infections, the epidemiology of fungal infections remains poorly described in most Southern African countries, including Zambia. Colonisation of the skin of critically-ill inpatients by Candida species and multidrug-resistant bacteria often precedes invasive infections. This study aimed to investigate the prevalence of Candida and bacterial colonisation in critically ill patients in Zambia. Methods: In a Point-prevalence survey (PPS) for Candida and bacterial skin colonisation conducted on October 2 and 3, 2023, skin swab samples (composite axilla/groin) were collected from critically-ill patients who were admitted for at least 48 hours to two tertiary hospitals in Zambia. The flocked swabs were immediately placed in Amies transport medium and processed at a local laboratory. Candida and bacteria were isolated in culture on Candida Plus chromogenic agar and a combination of blood and MacConkey agar, respectively. Species identification was performed using MALDI-TOF (Bruker) at the National Institute for Communicable Diseases in South Africa. Antifungal susceptibility testing for Candida parapsilosis and C. glabrata isolates was determined using Sensititre YeastOne (Thermo Fisher Scientific) and Etest (bioMerieux) adhering to Clinical and Laboratory Standards Institute guidelines. Results: Of the 139 patients swabbed, 73% (101/139) were admitted to a neonatal intensive care unit (ICU), 14% (19/139) to a kangaroo mother and child care unit, 5% (7/139) to an adult ICU and 4% (5/139) to a paediatric ICU. Candida colonisation was detected in 17% (23/139) of the samples, with Candida albicans being the most predominant (65%, 15/23), followed by C. glabrata (17%, 4/23), C. parapsilosis (17%, 4/23), and a rare species, C. allociferrii (4%, 1/23). One non-Candida rare yeast species, Kluyveromyces marxianus, was also isolated. C. parapsilosis and C. glabrata isolates were both pan-susceptible. Notably, 94% (131/139) of patients showed colonisation by one or more bacterial species, with K. pneumoniae being the most prevalent (11%, 37/349), followed by E. faecium (8%, 28/349), A. baumannii (3%, 10/349), and Enterobacter species (3%, 9/349). Discussion: The results show a significant difference in the rates of fungal and bacterial colonization. Given the prevalence of multidrug-resistant organisms, the dominance of bacterial colonization in these settings presents serious challenges for infection control. In high-risk intensive care unit settings where horizontal transmission can result in severe outbreaks, this study emphasises the need for continuous surveillance and effective infection prevention strategies. Conclusion: The survey highlights the high prevalence bacterial colonization over fungal colonization among critically-ill patients in ICU patients. The data support the necessity of heightened preventative measures and ongoing surveillance to mitigate these pathogens' impact. Future national surveys are planned to offer insights for developing targeted interventions in hospital environments.

Background: Genetically altered viruses or variants have the potential to increase their virulence, pathogenicity, transmission, and ability to evade both natural and vaccine-induced immune responses leading to diarrhoeal disease in under five-year-old children who have received all recommended doses of Rotavirus vaccines, also known as breakthrough infections. Studies characterising Rotavirus strains responsible for breakthrough infections are rare in resource-limited countries like Zambia where Rotavirus-associated diarrhoeal disease is endemic. This study characterised Rotavirus strains detected in fully vaccinated under five-year-old children residing in Zambia using next generation sequencing. Methods: This was a case study nested under an open label randomised controlled Rotavirus vaccine clinical trial that evaluated safety and immune boosting effects of a third dose of Rotarix compared to a two-dose schedule. The Rotaclone kit was used to screen for RV in stool. Sanger sequencing was used to genotype VP7 and VP4 in Rotvirus positive stool samples. Whole genome sequencing was done on the Illumina Miseq platform. Genome assembly was done using Geneious software and multiple sequence alignment using Muscle in MEGA version 6. Results: A total of 76 diarrhoeal stool specimens were collected and screened for Rotavirus of which 4/76 (5.2%) were positive. Sanger sequencing confirmed the genotypes in three of the four cases as G1P[4], G12P[4] and G12P[8]. Whole genome analysis revealed that the RVA/Human-wt/ZMB/CIDRZ-RV2088/2020/G1P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2 and RVA/Human-wt/ZMB/CIDRZ-RV2106/ 2020/G12P[4]-I1-R2-C2-M2-A2-N1-T2-E1-H2 strains were mostly DS-1-like with mono and multiple reassortant respectively, whilst the RVA/Human-wt/ZMB/CIDRZ-RV2150/2020/G12P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 was a typical Wa-like strain. Comparison of the VP7 antigenic epitope of strains causing breakthrough infections and Rotarix vaccine strains revealed several amino acid differences, e.g., G96P and M217E. Comparison of P[4] strains with VP4 of the Rotarix vaccine strain demonstrated two amino acids differences (P114Q and V115T) were P114Q was an immune escape mutation. Discussion: Differences observed in amino acids in antigenic epitopes suggested its role in the immune evasion of neutralising antibodies elicited by the vaccine. Findings from this study have the potential to inform national Rotavirus vaccination strategies and design highly efficacious universal Rotavirus vaccines. Furthermore, there might be need to monitor strains that have escaped vaccine-induced immunity to prevent diarrhoeal diseases in children under five years of age. Conclusion: This study confirmed the importance of post-market surveillance of vaccines, in identifying and characterising Rotavirus strains responsible for breakthrough infections to monitor viral evolution and inform next generation of vaccines. A strain with 4 gene reassortment was reported, which is implicated in breakthrough infections. A reassortment of vaccine strain with DS-1-like wild-type strain was also reported. Furthermore, differences observed in amino acids in the VP7 and VP4 antigenic epitopes at immune escape positions might have played a role in the evasion of antibodies elicited by the vaccine.

Introduction: The prevalence of non-tuberculous mycobacteria (NTM) infection is increasing worldwide, presenting challenges for clinicians and microbiologists alike. This emerging infection is becoming more common in developed countries. While the clinical presentation of Mycobacterium tuberculosis complex (MTBC) and NTM may resemble each other, their treatment regimens differ significantly. The aim of this study is to comprehensively investigate and characterize the clinical and microbiological aspects of NTM in presenting presumption TB cases attending tertiary care center in central India. Methods: A total of 970 suspected tuberculosis (TB) cases, comprising 512 pulmonary (52.7%) and 458 extrapulmonary (47.3%) cases, were included in this study. Data encompassing clinical, laboratory, and radiological findings were recorded. NTM diseases were verified using the criteria set forth by the American Thoracic Society/Infectious Diseases Society of America. All samples underwent Ziehl- and Neelsen staining, NALC-NaOH decontamination method for cultivation in the MGIT-960 system (Becton Dickinson Diagnostic Instrument Systems). Final identification and differentiation between Mycobacterium tuberculosis complex (MTBC) and various NTM species were confirmed through the GenoType Mycobacterium CM/AS assay. Results: Among the 970 samples tested, 37 cases (3.8%) tested positive for acid-fast bacilli (AFB) via Ziehl-Neelsen (ZN) staining, and 160 cases (16.5%) showed positive mycobacterial cultures. Of these, 20 out of 160 cases (12.5%) were confirmed as non-tuberculous mycobacteria (NTM) using the GenoType Mycobacterium CM assay. Among the NTM isolates, 75% (15 cases) originated from pulmonary sources. In pulmonary NTM cases, the most commonly observed species were M. fortuitum, M. abscessus, and M. senegalens. Discussion: The study highlights a significant presence of NTM in presumed TB cases, emphasizing the necessity for differential diagnosis to ensure appropriate treatment. The findings underline the importance of incorporating advanced diagnostic tools, such as the GenoType Mycobacterium CM/AS assay, into routine practice. This will facilitate timely and accurate identification of NTM, improving patient outcomes. Conclusion: The predominant isolation of and M. fortuitum and M. abscessus as the most prevalent NTM, particularly among males in the 30-50 age group from pulmonary sources, has been observed in tertiary care centers in central India.

Introduction: Despite significant improvements in laboratory diagnostic methods, direct parasitological methods remain the gold standard for diagnosing parasitic diseases of the gastrointestinal tract. The purpose of our work was to conduct a comparative analysis of the effectiveness of methods for direct diagnosis of parasitoses of the gastrointestinal tract, most often used in clinical practice in Russia. Methods: The study was carried out in the laboratory of the clinic of infectious and parasitic diseases of the Rostov Research Institute of Microbiology and Parasitology in 2021-2023. The following methods were used: native and stained smears, Kato-Katz thick smear, flotation method with NaNO₃, ether-formalin sedimentation (EFS). The material was also studied by sedimentation method using an intestinal parasite concentrator containing ethyl acetate.Under experimental conditions, 180 fecal samples were studied. The model was Ascaris lumbricoides eggs due to the possibility of obtaining a large amount of material (240,000 eggs from 1 female roundworm). Previously, the feces from patients of Clinic were examined for all types of helminths and protozoan cysts. Using a laboratory pipette, a suspension of Ascaris lumbricoides eggs in the amount of 500 ± 10 eggs was added to each sample (15 grams of feces,), and the material was thoroughly mixed. Each method was carried out in 30 replicates. Results: Analysis of the results showed that the sensitivity of the stained and native smear method was 10% (out of 30 samples, eggs were found in only 3), in an amount of 0.1 egg in 1 field of view. The sensitivity of the flotation method with NaNO₃ was 46.7% - eggs were found in 14 samples, 1 egg in 1 field of view. The sensitivity of the Kato-Katz thick smear was 60%, eggs were found only in 18 samples out of 30, 2 eggs in 1 field of view. The sensitivity of the EFS method was 100% eggs were detected in all samples in the amount of 4 eggs in 1 field of view. When using a concentrator for intestinal parasites, eggs were detected in 100% of samples, but in the amount of 3.5 eggs in 1 field of view. Discussion: Currently, Russian laboratories use sedimentation methods in various modifications, flotation methods (with NaCl and NaNO₃) to detect helminth eggs and cyst of pathogenic protozoa. Also the Kato-Katz thick smear kits are used, as well as the study of stained and native smears. The use of direct microscopic methods is attractive due to its low cost, but the main problem of these methods is the need for highly qualified laboratory diagnostic specialists. Conclusion: Analysis of the data obtained led to the conclusion that the most effective method for detecting A. lumbricoides eggs in feces is the ether-formalin sedimentation method.

Introduction: Antimicrobial resistance represents a growing global concern, with methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci representing major contributors. Linezolid (LNZ) is one of the few available treatments for such infections, yet resistance has already been documented. It is therefore essential to accurately detect resistant strains to provide adequate treatment to patients and prevent nosocomial dissemination. This study evaluates the performance of four commercial methods employed by laboratories for determining Gram-positive cocci susceptibility to LNZ. Methods: A panel of 99 stains from our routine and National Reference Centre (NRC) collections were studied, including 26 S. aureus (15 LNZ-resistant), 26 S. epidermidis (14 LNZ-resistant), 27 E. faecium (23 LNZ-resistant) and 20 E. faecalis (6 LNZ-resistant). Susceptibility to LNZ was determined by home-made broth microdilution method (BMD), considered as gold standard, and by four commercial methods namely disk diffusion (LNZ10, Bio-Rad, USA), gradient strip diffusion (E-TEST®, bioMérieux, France), broth microdilution assay (UMIC, Biocentric, France) and automated antimicrobial susceptibilty testing (AST; VITEK 2, bioMérieux). For the diffusion methods and UMIC, two incubation times were evaluated (18h and 48h). EUCAST guidelines and breakpoints were used. Categorical agreement (CA), major errors (ME's) and very major errors (VME's) were defined as recommended by the FDA: commercially available antibiotics methods should have a CA ≥ 90%, and a percentage of ME's ≤ 3% and of VME's ≤ 1.5%. Results: All commercial methods exhibited similar performance, with accurate classification of LNZ- susceptible strains. However, all failed in detecting LNZ-resistance, except for S. epidermidis. The overall percentage of VME among S. aureus was 66.7%. Among E. faecium, it was 78.3% and 82.6% when using Vitek2 or another method, respectively. Among E. faecalis, it was 21.4% and 14.3% when using UMIC or another method, respectively. The percentage of VME's decreased with additional incubation times. The use of E-test exhibited the lowest percentage of VME's. However, none of the methods achieved the expected 90% CA. Discussion: Even though discrepancies in LNZ AST methods have been reported, no practical guidelines are available for routine laboratories. According to this study, the following algorithm is proposed: if the strain is classified as LNZ-resistant, it should be considered as such and a PCR could be performed to reveal LNZ resistance genes, if the strain is classified as susceptible and if the patient must be treated with LNZ, an E-test read at 48h is recommended. Given the potential for false-susceptible results, particularly with S. aureus and E. faecium, it is crucial to monitor patients undergoing LNZ treatment. Should further analysis be required, the strains can be submitted to the NRC. Conclusion: There is currently no optimal commercial method for the detection of LNZ-resistance. We therefore propose an algorithm that should assist laboratories in a routine practice context.

Introduction: Ferrets are widely recognized as valuable small animal models for studying respiratory viruses, including influenza and SARS-CoV, due to their susceptibility to these viruses and the similarity of their respiratory tract to that of humans. Despite their importance in virology, there is a notable lack of established cellular assays to detect cellular responses in ferrets. This limitation hinders our detailed understanding of immune mechanisms and the development of targeted therapies. In this study, we established and optimized various cellular assays for ferrets and developed monoclonal antibodies against ferret IL-2. Methods: To validate the qRT-PCR assay, we synthesized plasmid constructs encoding 13 ferret cytokine genes, in viro transcribed RNA transcripts of IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12b, IL-17, IFN-α, IFN-β, IFN-γ, and TNF-α. By using this assay, we then measured cytokine expression from PBMCs of four ferrets, stimulated with or without ConA and PMA+Iono. For intracellular cytokine staining and T-cell quantification, influenza virus-infected ferret tissue sections were stained with cross-reactive antibodies. Recombinant ferret cytokines IL-2, IFN-α, and IFN-β were cloned and produced. Mice immunized with these proteins provided spleen cells to generate hybridomas. Selected clones facilitated the development of ELISA, immunofluorescent staining, epitope prediction, and western blot assays. Results: Our study validates a qRT-PCR assay enabling the detection of 13 diverse ferret cytokines. Notably, PMA+Iono stimulation elicited significantly higher levels of IFN-γ compared to ConA. Utilizing cDNA from PMA+Iono-stimulated PBMCs, we successfully detected a range of cytokines including IL-2, IL-4, IL-6, IL-10, IFN-α, IFN-β, and TNF-α using gene-specific primers. Furthermore, a PCR-based assay was established, facilitating amplification of TNF-α, IFN-α, IFN-B, TNF-α, IL-2, and IL-4 from ferret PBMCs, albeit excluding IL-6 and IL-10. In influenza-infected ferret lymph nodes, we observed significant levels of TNF-α, IFN-γ, and infiltrating T cell populations (CD4+ and CD8+). Immunization with recombinant ferret cytokines, specifically IL-2, IFN-α, and IFN-β, elicited a robust immune response. Furthermore, from spleen cells, we generated multiple monoclonal antibodies targeting IL-2. These antibodies demonstrated binding affinity to both linear and conformational epitopes, facilitating assays including western blotting, ELISA, and immunofluorescent staining. Discussion: We developed robust cellular assays for detecting ferret cytokines, useful for measuring immune responses during viral infections and vaccine efficacy in ferrets. Our study validated a reliable qRT-PCR assay for 13 ferret cytokines, revealing differential responses and highlighting key immune players in ferret immunology. The successful use of recombinant cytokine immunization and monoclonal antibody development presents promising tools for advancing future ferret immunological research. Conclusion: We developed robust cellular assays for detecting ferret cytokines, enabling precise measurement of ferret immune responses during viral infections, and assessing vaccine efficacy in ferrets.

Introduction: There is limited knowledge on dengue transmission in the Democratic Republic of Congo (DRC). However, recent publications highlight an increasing dengue prevalence among individuals presenting with febrile symptoms in Kinshasa, the capital of DRC. The extent of dengue circulation in the country is unknown, which poses questions around the burden of dengue infection and the potential impact of new interventions. The objective of our analysis was to assess the age-stratified dengue seroprevalence and estimate the force of infection (FOI, per-capita risk of dengue infection for a susceptible subject) in Kinshasa and Matadi. Methods: By leveraging samples collected from individuals aged 0 to 94 years in a SARS-CoV-2 serosurvey, we assessed the age-stratified dengue seroprevalence using a combination of two Enzyme-Linked Immunosorbent Assay (ELISA) tests, respectively for anti-dengue serotype 1-4 IgG and NS1 IgG and a Plaque Reduction Neutralization Test (PRNT) for all 4 dengue serotypes. We employed a Bayesian framework to estimate the FOI for each location and reconstruct the results obtained across each test. Results: 3580 plasma samples were tested by IgG ELISA serotype 1-4 in DRC and a subset of 202 samples was also tested by IgG ELISA NS1 of which 70 were also tested by PRNT. Our findings reveal endemic dengue circulation in DRC, with an average FOI of 2.9% (95% CrI: 2.2-3.9) in Kinshasa and 1.4% (95% CrI: 0.9-2.0) in Matadi and an overall population seroprevalence of 41% (95% CrI: 34-48) in Kinshasa and 23% (95% CrI: 17, 32) in Matadi respectively. Conclusion: These results confirm endemic dengue transmission in different regions of DRC and highlight the dengue transmission differences between the two cities. These findings underscore the importance of establishing dengue surveillance in the country, to monitor prevalence and changes in transmission in a changing climate.

Introduction: Arboviruses such as Dengue (DENV) and Chikungunya (CHIKV) pose a significant global public health challenge. Dengue has been endemic in Brazil for decades, causing annual outbreaks. CHIKV was introduced in Brazil in 2014 and has since established endemic transmission, resulting in successive outbreaks. Both infections can cause acute febrile, systemic, and debilitating illnesses, with considerable morbidity and substantial impacts on public health. In some cases, infections can progress to severe forms, such as severe dengue and chronic polyarthritis/arthralgia associated with CHIKV.The indigenous population in Brazil consists of approximately 1.7 million individuals from 303 ethnic groups. These populations are considered socially vulnerable and have limited access to healthcare services. Data regarding arboviral infections in these populations are scarce. Objetive: To evaluate the seroprevalence of Dengue and Chikungunya viruses in indigenous populations in Brazil. Methods: A cross-sectional study was conducted, enrolling individuals of all genders and ages residing in indigenous territories in Bahia, Brazil. Surveys were conducted using REDCap v9.3.1 (2023), and serological testing was performed on serum samples to detect IgM and IgG anti-DENV and anti-CHIKV specific markers using the ELISA (Enzyme-Linked Immunosorbent Assay) method. Results: A total of 2,058 individuals from 33 villages and 10 ethnic groups were recruited for this study. Of the participants, 60.2% were female, with a median age of 33 years. Additionally, 48.5% were single, 46.1% had incomplete primary education, and the majority, 67.8%, had a family income below the minimum wage. The seroprevalence of DENV infection was 61.6% for IgG and 4.3% for IgM, while for CHIKV, it was 14.2% for IgG and 9% for IgM. Villages near urban centers had a higher prevalence of DENV and CHIKV antibodies compared to remote villages. Discussion: The studied indigenous population exhibits significant socioeconomic vulnerability, characterized by low levels of education and insufficient family income. Conclusions: high rates of both acute and past exposure to DENV and CHIKV were identified. These findings highlight the challenges of diagnosing arboviral infections in these populations and the underreporting of cases.

Introduction: Syphilis is a sexually transmitted infection caused by the spirochaete Treponema pallidum subsp. pallidum. Early infectious syphilis (EIS) is characterised by mucocutaneous lesions. Neurosyphilis is a serious complication of treponemal infection. While penicillin is standard antibiotic treatment for syphilis, doxycycline and azithromycin can be prescribed for patients allergic to β-lactams. Furthermore, doxycycline can be used as postexposure prophylaxis against syphilis. Dark ground microscopy (DGM) of samples from mucocutaneous lesions identifies motile treponemes confirming EIS. However, commercial controls for DGM are no longer available. Therefore, we introduced realtime-PCR (RT-PCR) for detection of T.pallidum (Tp) DNA in lesions. Since diagnosis of neurosyphilis is based on detection of serological markers in CSF, we also assessed the role of RT-PCR. Furthermore, detection of Tp-DNA in mucocutaneous lesions enabled identification of antibiotic resistance to doxycycline and azithromycin. Methods: Fifty-two samples were collected over a year at St James's Hospital. RT-PCR allowed detection of Tp-DNA in swabs collected from suspected syphilitic mucocutaneous lesions. Cerebrospinal fluid (CSF) samples collected from 23 patients with neurosyphilis were tested. Sequencing of Tp 16SrRNA and 23SrRNA identified antibiotic resistance. Results: Tp-DNA was detected in swabs of lesions from 25 patients with clinical and serological evidence of EIS. Tp-DNA was not detected in swabs from 27 patients without serological evidence of recent syphilis but with genital ulcers caused by Mpox, HSV-1/2 and LGV. TpDNA was not detected in CSF samples from a cohort of 23 patients with neurosyphilis. Resistance determinants to azithromycin were identified in all 25 patients with EIS. No molecular evidence of resistance to doxycycline was found. Discussion: The use of RT-PCR testing in a clinical context is immensely valuable. Treponemal PCR testing holds much value allowing for the rapid and accurate diagnosis of EIS. Serological testing is the current mainstay form of diagnosis, although challenging to perform and interpret at various stages of the laboratory process. The identification of highly infectious individuals can allow for treatment prioritization, whilst concurrently facilitating the development of guideline recommendations for high-risk population groups.The limitations of this study included a gender disparity among patients. Most of the participants in this study were men, and the samples were collected at the Genito-Urinary Medicine (GUM) clinics. This does highlight the disproportionate burden of syphilis infection particularly in high-risk communities. Conclusion: Detection of Tp-DNA in swabs from mucocutaneous lesions showed excellent sensitivity and specificity for identification of EIS, but performed poorly in diagnosis of neurosyphilis. Doxycycline retained activity against T. pallidum but azithromycin should not be used for the treatment of syphilis.

Introduction: Dirofilariasis is a relatively new medical problem caused by changes in natural, climatic and social conditions. Methods: In 2023, venous blood of 233 dogs was collected from shelters for homeless animals in the city of Rostov-on-Don and the Rostov region. Testing the blood of dogs for dirofilariasis was carried out using two Methods: the morphological (Knott's method with 3% acetic acid solution) and real-time polymerase chain reaction (RT-PCR). RT-PCR was performed using primers developed by us for Dirofilaria repens and Dirofilaria immitis in accordance with improved isolation protocols.The preparation of the PCR mixture for the reaction was carried out using a set of reagents for RT-PCR in the presence of EVAGreen (SINTOL) with highly specific primer pairs. For D. repens: (F2690DR) 5’-АAGTGTTGATGGTCAACCTGAA-3’ and (R2812DR) 5’- GTAGAACGCATATTCTGAGT-3’. For D. immitis: (F2690DI) 5’- GAGTGTAGAGGGTCAGCCTGAG-3’ and (R2812DI) 5’- GTAGAACGTATATTCTGAAC- 3’. DNA amplification modes were selected empirically. Results: As a result of research using larvoscopy, dirofilaria larvae were found in 53 samples with an intensity of invasion from one to one hundred larvae in 0.2 ml of blood, the extent of invasion was 22.75%. The PCR method detected dirofilaria in 60 samples. The prevalence of dogs was 25.75%, of which D. repens – 21.67%, D. immitis – 41.67%, mixed invasion was 36.67%. The results obtained were analyzed in a four-field contingency table. As the analysis showed, the sensitivity of the PCR method was 90.57%, and the microscopy method was 81.36%. The specificity of the first method was 93.33%, the second – 97.13%. Discussion: Despite the increase in the quality and quantity of deworming activities for dogs, which are the most epidemiologically significant source of dirofilaria infection, in recent decades there has been an increase in the prevalence of these nematodes in definitive hosts in Europe. The expansion of the range of dirofilariasis has been facilitated by animal migration and climate change, which affects mosquito vectors. The main factor in the unfavorable epidemiological situation is the significant number of stray dogs infected with dirofilaria that have not undergone veterinary examination. Direct diagnostic methods remain the «gold standard», but require highly qualified specialists. The polymerase chain reaction (PCR) method is also a direct diagnostic method that can detect the DNA of parasitic pathogens in various biological substrates. Conclusion: The results of our study indicate a continuing high risk of dirofilariasis in the population of a number of regions of southern Russia. The main cause of epizootological problems remains the high intensity of invasion of animals not covered by veterinary examination.The developed test system allows you to determine the presence of two species of dirofilaria in real time and in a short time. This system can improve the work of veterinary laboratories.

Introduction: Acinetobacter baumannii (Ab) is one the major threat, causing nosocomial infections and getting resistant to almost all the available antibiotics. Biofilm formation is one the strategy opted by Ab to overcome the limited sensitivity against various small molecules, AMPs, antibiotics etc. Hence, deciphering the role of biofilm in AMR, we must study the composition of Ab biofilm. Method: We employed various methodologies to elucidate the precise composition of Acinetobacter baumannii biofilm. These included topological analyses (AFM, SEM, Confocal microscopy), biochemical assays (Phenol-sulfuric assay (PSA), BCA, and gel electrophoresis for eDNA), chemical composition studies (XPS, FTIR, Zeta Potential), and molecular investigations (Proteomics and Metabolomics). Results: After growing A. baumannii biofilms for three days on 2xYT agar, AFM, SEM, and confocal microscopy showed increased biofilm density, with cells completely encapsulated. PSA and BSA assays indicated higher carbohydrate and protein levels in the biofilm matrix. Agarose gel electrophoresis revealed the presence of eDNA.Chemical composition analysis using XPS, FTIR, and zeta potential showed consistent percentages of carbon, oxygen, and nitrogen over three days, but an increase in phosphorus. FTIR data indicated peaks for water, DNA, RNA, proteins, carbohydrates, phosphorylated proteins, phospholipids, and Amide I and II. Zeta potential measurements confirmed the net charge of the biofilm. Metabolomic studies shows upregulated fatty acid biosynthesis, cell structure synthesis and carbohydrate biosynthesis pathways. Discussion: This study represents the first comprehensive analysis of the biofilm composition of Acinetobacter baumannii. Biofilm formation by bacteria enhances their stability and survivability under adverse conditions, such as antimicrobial pressure and nutrient-depleted environments. Biofilms serve as a source of nutrition, shelter, and protection against a variety of antimicrobial agents. Our investigation reveals the detailed composition of the extracellular matrix (ECM) of A. baumannii biofilms, which includes phosphorylated proteins, phospholipids, extracellular DNA (eDNA), water, DNA, RNA, proteins, and carbohydrates. Topological analysis indicates that the bacteria are completely encased within the matrix, providing robust structural support. Zeta potential measurements indicate that the A. baumannii biofilm carries a net charge, which aids in neutralizing various antimicrobial peptides (AMPs), small molecules, immune responses, and antibiotics. Conclusion: This study comprehensively characterize the biofilm composition of A. baumannii. It provides deep insight into the nature of pathogenic material, potentially generating a complex cascade of immune escape events. From this study, we can conclude that the biofilm composition of Ab is so complex and yet to explore because there are so many components of biofilm which help bacteria in escaping antibiotics and host mediated immune responses. Net charge of biofilm, physical shelter formation by biofilm over bacteria, various modified proteins, lipids and carbohydrates may putatively enables bacteria to survive under antibiotic stress and escape host immune response.

Introduction: Haemophilus influenzae (Hi) is a significant cause of invasive infections, including meningitis and sepsis. The emergence of resistant strains has prompted the World Health Organization to include Hi on its priority list for research and development of new antibiotics. The objective of this study was to describe the epidemiology of invasive Hi strains in Belgium, with a particular focus on beta-lactam resistance. Methods: A retrospective analysis of invasive Hi strains referred to the NRC from 2018 to 2023 was conducted. All national laboratories are required to submit invasive Hi strains to the NRC. There, the strains are identified using MALDI-TOF MS and antimicrobial susceptibility testing is performed following EUCAST guidelines. For isolates showing reduced susceptibility to beta-lactams, the ftsI gene is sequenced. Selected isolates (e.g. in the case of suspicion of a cluster) undergo whole-genome sequencing. Results: A total of 804 strains were included, with the lowest incidence rate per 100.000 population observed in 2020 and 2021 (0.75/100.000 inhabitants). The male-to-female ratio was 1.13, with a male predominance observed in individuals under the age of 5. Over 75% of strains were non-typeable Hi (NTHi). The resistance rates for ampicillin and amoxicillin-clavulanic acid were 18.7% and 4.6%, respectively. Mutations in the ftsl gene associated with decreased susceptibility to beta-lactams varied from 11.5% to 17.7%. In 2023, mutations associated with high levels of beta-lactam resistance (group III/III-like) were detected in six NTHi strains (3.3%), five of which exhibited identical ftsI mutations (D350N, S357N, M377I, S385T, R517H, T532S). Four of the five isolates belonged to the MLST clonal complex 3 and exhibited a similar phenotypic profile namely biotype II, absence of beta-lactamase, resistance to cefuroxime and trimethoprim-sulfamethoxazole. Three of the four isolates were recovered at the beginning of the year from patients with pneumonia residing in Limburg. A whole genome single nucleotide polymorphism (SNP) analysis demonstrated they had less than four SNPs of difference. Discussion: This study provides a comprehensive overview of the epidemiology of invasive Hi infections in Belgium. As with other respiratory pathogens, the COVID-19 pandemic has had a significant impact on the epidemiology of invasive Hi infections. The distribution of sex and age was consistent with global trends, as was the prevalence of NTHi. The prevalence of beta-lactam resistance was comparable to that observed in other European countries. It is noteworthy that, for the first time in Belgium, six strains of Hi have emerged, displaying mutations associated with high-level beta-lactam resistance. Furthermore, WGS analysis has confirmed the existence of a cluster of three of these strains. Conclusion: Vigilant surveillance of circulating Hi strains and research are fundamental to addressing evolving challenges and guiding future interventions, including the adaptation of treatment guidelines.

Introduction: Vector surveillance is essential to control several infectious vector-borne diseases. Aedes Mosquitoes are the main vectors of arboviruses diseases affecting humans such as Dengue, Chikungunya, yellow fever and Zika represent a serious concern in tropical/subtropical countries and are considered emerging or re-emerging. Due to the absence of effective treatments and vaccines against most arboviruses diseases, vector control remains the most effective measure to prevent further resurgence. It is important to measure variation in their population to predict the environmental conditions promoting their development. In this line, a large-scale surveillance was undertaken in a peri-urban area of Yaounde, Cameroon to study the spatio-temporal distribution of Aedes population. Methods: The study was conducted in Nkolbisson, a peri-urban district of Yaounde over one year. A total of 100 ovitraps were placed in 14 randomly selected stations across the study site, they were fixed to supports such as banana tree trunks, stakes and tree trunks (height: 1m) around dwellings. Those ovitraps containing filter paper soaked in spring water to collect Aedes eggs. Seven days later, ovitraps were checked and ovitrap-related indices were estimated. The filter paper was replaced each time the sites were visited. Immature stages were counted, kept and raised until the adult stage and morphologically identified after emergence using the identification keys. Results: A total of 51,499 mosquitoes belonging to 3 genus namely Aedes, Culex and Eretmapodites and 14 species were collected. Aedes genus was predominant (n=51,146; 99.31%). Morphological identification of the collected species mostly reveals the presence of Aedes albopictus (88.14%), followed by Aedes simpsoni (5.97%) and Aedes aegypti (5.12%) and we see that the main vector in this site was Aedes albopictus and there was no significant difference in the adult mosquito's numbers Aedes simpsoni and Aedes aegypti (P˃0.05). The highest values for Aedes egg density and positive ovitraps percentage were recorded in Mai 2023 and April 2023 respectively. High densities recorded in ovitraps through the study site highlights a potentially high-risk area for dengue epidemics. Discussion: The eggs abundance and density per traps indicates a high level of infestation at the study site. The widespread distribution of Ae. albopictus highlights its adaptability and competitive advantage over Ae. aegypti and native species. The presence of other mosquito species alongside Aedes points to competition and potential co-transmission of diseases. This finding highlights the utility of ovitraps for broader vector surveillance. Conclusion: The implementation of surveillance and control programs for arboviruses vectors is urgent required in view of the increase in cases of arboviruses. Ovitraps can therefore be an effective tool for arboviruses surveillance. These data provide important entomological information that could be useful in designing an effective integrated program to control Aedes vectors in the city of Yaounde.

Introduction: The increasing incidence of Neisseria gonorrhoeae (NG) has become a global health threat due to drug resistance and potentially invasive disease. Few studies in Brazil have analyzed the presence of resistance genes and its correlation with invasive clinical presentations. To assess the genotypic virulence profile in both invasive and non-invasive N. gonorrhoeae strains from patients with gonococcal disease at Hospital das Clínicas of the Faculdade de Medicina of the University of São Paulo, Brazil (HC-FMUSP). Methods: To analyze genotypic profiles of 25 NG clinical isolates from 25 hospitalized patients at Hospital das Clínicas da FMUSP from January 2017 to March 2020. The isolates were submitted to whole genome sequencing, typed by MLST and resistome analysis was performed. Results: Of the 25 isolates, 10 (40%) were from sterile sites (blood, biopsy and articular fluid) and classified as invasive. The phylogenetic tree analyses showed that invasive specimens were grouped in 5 STs (8143, 1588, 16704, 14939, 12894) and noninvasive specimens clustered in 11 STs, with 3 STs in common between the groups. The resistance genes blaTEM-1B (7% vs. 50%, p = 0.023) and the tetM (13% vs. 80%, p = 0.002) were more frequently found in isolates from patients with invasive disease and they were always accompanied by the mobile genetic elements pJD4 and pEP5289, respectively. Those elements were also associated with toxins and resistance elements described previously as possible virulence factors in vitro studies. Conclusion: Our results observed that invasive strains of NG are genotypically clustered and frequently harbor two plasmids that had never been associated with invasive disease before. These results evidence the need for more NG surveillance projects in Brazil.

Background: Severe acute malnutrition (SAM) is strongly associated with mortality in young children, and infections are thought to be a major driver of that mortality. The World Health Organization (WHO) therefore recommends that all children hospitalized with SAM receive empiric intravenous broad-spectrum antibiotics. Most data on the burden of infections in children with SAM are outdated given recent dramatic changes in antimicrobial resistance (AMR) and HIV epidemiology. The aim of this study was to give a contemporary description of the prevalence of treatable infections and AMR in children hospitalized with SAM. Methods: This prospective cohort study enrolled children 6-59 months old hospitalized with SAM at a tertiary referral hospital in Malawi. The prevalence of various infections was estimated with a combination of routine testing and clinical diagnoses, plus study-specific testing that included: blood and urine cultures; gastric aspirate Xpert MTB/RIF Ultra; and for participants with concern for central nervous system infection, cell counts, microscopy, culture, and Xpert MTB/RIF Ultra on cerebrospinal fluid. Results: From February 2023 to January 2024, 212 participants were enrolled. Mean age was 21 months, 48% were female, and 12% were HIV-infected. In-hospital mortality rate was 11%. The composite outcome of serious bacterial infection (SBI: bacteremia, meningitis, or pneumonia) was found in 24% of participants, and SBI was strongly associated with mortality (odds ratio, OR: 3.4; 95% confidence interval, CI: 1.4-8.3). Tuberculosis disease (TB) was diagnosed in 9% and was also associated with mortality (OR: 3.4, 95% CI: 1.1-10.4). Blood cultures detected likely pathogens for 10% of participants. Urine for culture was obtained on 136 participants and 20% had urinary tract infection (UTI). For the bacteria isolated from bacteremia and UTI cases, resistance to first-line antibiotics was 88% for ampicillin and 41% for gentamicin, and 38% for second-line antibiotics (third-generation cephalosporins). Discussion: This contemporary evaluation of infections in children hospitalized with SAM at a single site in Malawi reconfirms high rates of SBI, UTI, and TB that have been demonstrated in older studies of this vulnerable population in other settings. Given the persistently high mortality of children hospitalized with SAM and the demonstrated association of mortality with SBI and TB, more research is urgently needed to improve early diagnosis and optimized treatment of these deadly infections. High rates of resistance to first- and second-line antibiotics raises the question of whether alternative empiric antibiotics should be recommended in SAM. Conclusion: These results support the WHO recommendation of treating children hospitalized with SAM with empiric broad spectrum antibiotics. To improve antibiotic stewardship, better tools are needed to identify those children at high risk for severe infection (including TB). In addition, reconsideration of the recommended empiric antibiotic regimens may be required to reflect increases in the prevalence of AMR.

Introduction: In many areas of Africa, recent investigations underscore the great influence of ticks on the health of both animals and humans across the continent. Identification of ticks and tick-borne diseases is crucial for epidemiological research and assessing the risks associated with disease transmission among humans and animals. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) has emerged as an alternative method for tick species identification, based on leg protein spectra. This study aimed to characterize tick species collected from domestic and wild animals in Malawi, employing morphological, MALDI-TOF MS and molecular tools. Methods: Ticks were systematically harvested from various locations across Malawi, covering diverse sources such as wild animals in protected areas, as well as ticks found on domestic animals and in the surrounding environment. The collected ticks were grouped by animal host, stored in labelled vials and preserved in 95% ethanol.Ticks were morphologically identified at genus level using dichotomous keys. Subsequently, ticks were individually dissected in three subsets of samples: four legs for MALDI-TOF identification, four legs for biomolecular identification amplifying and sequencing the 16S gene, and the remaining idiosome was preserved for further analysis. Ticks were subjected to MALDI-TOF MS analysis to evaluate the intraspecies reproducibility and interspecies specificity of MS profiles obtained from the different species. Results: In this study we present preliminary results from MALDI-TOF MS analysis. In total, 995 ticks were collected from Malawi. Morphologically, the ticks belonged to five different genera Amblyomma spp., Hyalomma spp., Boophilus spp., Rhipicephalus spp., Haemaphysalis spp.To date, 171 African ticks have been processed, resulting in the identification of 13 species through 16S analysis. Moreover, within the processed ticks, 17 new spectra have been incorporated into the MALDI-TOF library, belonging to 5 different tick species (Amblyomma tholloni, Amblyomma variegatum, Rhipicephalus sanguineus, Rhipicephalus/Boophilus microplus, Rhipicephalus maculatus). These spectra were obtained from samples collected from African bush elephants (Loxodonta africana), Zebras (Equus quagga), Impalas (Aepyceros melampus), Bell's hinge-back tortoises (Kinixys belliana), domestic dogs, Malawi Zebu cattle, and Black rhinos (Diceros bicornis). In addition, after incorporating these 17 new spectra into the library, further specimens were tested. Among these, 20 specimens produced high-quality scores (>1.70), facilitating their identification. The library is continually expanded to include new species. Discussion: Our efforts focus on creating a comprehensive database for identifying tick species in Malawi and potentially across other Sub-Saharan African countries. This database will enable thorough risk analysis for vector-borne diseases at the human-animal interface. Conclusion: In conclusion, accurate identification of tick species and their associated pathogens is crucial for improving vector control measures aimed at surveillance and prevention of tick-borne disease outbreaks. MALDI-TOF MS emerges as a promising tool for arthropod identification, offering advantages like simple specimen processing and low consumable costs.

Introduction: Russia is a high burden country for multidrug-resistant tuberculosis (MDR-TB) but some of its regions such as, Siberia and Far East, have especially high prevalence rate of primary MDR-TB (35.4% and 31.8%, respectively, in 2021). Mycobacterium tuberculosis Beijing genotype is the most prevalent here and its B0/W148 subtype is strongly MDR and transmissible. In addition, epidemic strains of the Euro-American lineage also circulate. We aimed to perform molecular characterization of MDR M. tuberculosis isolates in the Omsk province in West Siberia, to identify the most epidemically relevant strain clusters. Methods: A total of 617 M. tuberculosis isolates were recovered from newly diagnosed patients with TB. The Beijing genotype and its main sublineages and Russian epidemic and endemic clusters (B0/W148 and Central Asian/Russian), and ancient Beijing sublineage were detected by PCR assays. Non-Beijing isolates were spoligotyped and compared to SITVIT2 database. This research was supported by Russian Science Foundation (grant 24-44-00004). Results: Of the 617 M. tuberculosis strains, 250 (40.5%) were MDR, of which 83 (33.2%) were pre-extensively drug-resistant (pre-XDR). Resistances only to fluoroquinolones (FQs) and only to injectable drugs were detected in 17.6% (44) and in 15.2% (38) of MDR strains, respectively. Resistance to FQs and the injectable drugs together was found in 15.6% (39) of MDR strains. The majority of MDR strains belonged to the Beijing family (86.8%; 217/250). Main Beijing subtypes were Central Asian/Russian (34.4%; 86) and B0/W148 (39.2%; 98). Twenty-nine (11.6%) MDR strains of the ancient Beijing sublineage belonged to the 1071-32 cluster (24) and 14717-15 cluster (5), respectively. The 33 non-Beijing strains represented LAM (12), Ural (12), T/L4-unclassified (7), S (1) and ‘unknown’ family (1). The most common LAM spoligotypes were SIT254 (4) and SIT803 (4). All Ural genotype isolates had the SIT262 spoligotype. Beijing B0/W148 strains were more frequently pre-XDR than Central Asian/Russian strains: 43.9% vs. 26.7%, p=0.0165. The resistance to the injectable drugs and FQs together was more common among B0/W148 vs. Central Asian/Russian subtypes (24.5% vs. 9.3%, p=0.0039). Discussion: The genotypes identified in this study are known to be endemic for Russia and former Soviet Union countries: “Russian successful Beijing B0/W148 strain”, LAM SIT254 that is widespread across all Russia, and Ural SIT262 that is epidemic MDR strain in Eastern Europe. On the other hand, some MDR subtypes are endemic for Siberia and Asian Russia, as a whole, such as hypervirulent Beijing 14717-15-cluster; low-virulent Beijing 1071-32 although the latter was already detected in southern and eastern Europe. Conclusion: Beijing genotype strains are highly prevalent (86.8%) in МDR M. tuberculosis population in West Siberia, Russia. Among Beijing B0/W148 strains, 43.9% were pre-XDR. Thus, increasing proportion of primary MDR-TB in Siberia, Russia, is due to active transmission of Beijing strains, mostly MDR B0/W148 strains.

Background: Frequency of bloodstream infections (BSI) caused by Escherichia coli have increased steadily in recent years. The extra intestinal pathogenic Escherichia coli (ExPEC) are implicated in causing UTI, pneumonia, intra-abdominal infection and meningitis often leading to secondary bacteraemia. Outcome of E.coli bacteraemia is dependent on the virulence of pathogen and host specific factors. Role of ExPEC virulence genes (VGs) in the pathogenesis and outcome is not much explored. E.coli possesses moreVGs (adhesins, protectins, capsules, toxins, iron acquisition) compared to other Enterobacterales. There are very few studies from India reporting the role of VGs of ExPEC in the development of sepsis. Present study was proposed to look for the overall frequency and microbiological susceptibility of E.coli in BSIs, associated risk factors, possession of VGs and clinical outcome. Methods: Clinically suspected BSI growing E.coli from blood cultures (1st Feb 2022- 31st Jan 2023) were subjected for susceptibility test by Vitek2 and conventional PCR using primers for - adhesins (fimH, papA eaeA), siderophores (feoB, iutA), capsule (kpsMTII), and invasion (ibeA).Demographics, risk factors for survival and outcome were summarized and analysed by Chi-square test, Kaplan–Meier curve & Cox regression. Possession of VGs were correlated with clinical outcome and a P value 75 %) was shown to ciprofloxacin, amoxiclav and all cephalosporins. Susceptibility to colistin was 100% followed by amikacin (>80%) and carbapenems (70-80 %). The thirty-day mortality was 23% (23/100) and 30-days survival analysis was statistically significant. Based on Cox-Regression Analysis, CVC (HR = 6.9, p = 0.001), solid tumor (HR = 8.8, p = 0.003) and, urinary catheterization (HR=6.8, p=0.013) were significantly contributing to the mortality. Among the VGs, feoB was possessed by 83% of the isolates followed by kpsMTIII (73%), fimH (70%), iutA (59%), and others. The most frequent combination observed was feoB +fimH (68%). However, correlation between 30-day mortality & possession of feoB +fimH combination. was statistically insignificant (p= 0.12). Discussion: The finding of 23% thirty-day mortality in our study was comparable to a similar study from Europe (2021) where the probability of death was 19.5% and. there was a strong positive correlation with the virulence genes. In our study, although majority had single or combination genes but the correlation was not significant which could be due to small sample size. Conclusion: All isolates possessed differentVGs encoding multiple virulence factors and > 50% mortality were associated with feoB +fimH combination. This knowledge may be useful for detecting virulent strains and predicting clinical outcome in E.coli BSI.

Introduction: Toscana virus (TOSV, Phlebovirus genus) is a neurotropic sandfly-transmitted virus whose distribution encompasses various countries within the Mediterranean basin. TOSV infection remains a non-notifiable disease in Europe with no official surveillance plans in place despite being considered a significant public health concern in Southern Mediterranean countries. In order to mitigate the impact of this virus on human health, it is crucial to gain a deeper understanding beyond the anecdotal and fragmented evidence that currently exists. The aim of this study is to develop an amplicon-based whole genome sequencing method suitable for the genomic characterization of TOSV. Methods: Primers were designed on TOSV lineage A S, M and L segments reference sequences (GenBank: GCA_031497085.1) using Primal Scheme web tool to generate 400 bp tiled amplicons. To guarantee coverage and sensitivity, a multi-sequence alignment of GenBank-available complete sequences was used to identify mismatches and introduce degenerations in relevant ambiguous sites. Two-pool multiplex amplicon-based library preparation was carried out with Illumina Microbial Amplicon Prep (iMAP, Illumina) and custom designed primers. Two viral isolates on Vero E6 cells and three clinical samples of patients presenting with meningitis were sequenced. De novo assembly was performed with BaseSpace DRAGEN Targeted Microbial (version 1.3.1). Results: A set of 45 oligonucleotide primer pairs (26 for segment L, 13 for segment M and 6 for segment S) that amplify overlapping segments spanning TOSV genome was generated. The primers sets were subsequently compared to an alignment of 8 sequences for segment L, 16 for segment M and 32 for segment S to introduce degenerations. Genome sequencing yielded a mean 98.2% coverage for high-viral-titre propagates on Vero E6 cell culture. Two human samples (1 urine and 1 cerebrospinal fluid) with Ct (Cycle threshold) values of 32 and 33 yielded to sequences with coverage of 91.3% and 80.3%. Another urine sample with Ct = 35 yielded to 47% of mean coverage. Discussion: Our study, albeit with preliminary and limited but very promising results for propagates and samples with relatively high Ct values, proposes a new whole genome amplicon-based sequencing method for genomic characterization of TOSV one of the most neglected and least monitored arboviruses. Alongside an increasingly necessary systematic epidemiological monitoring of TOSV infections, WGS may be instrumental to unravel its genomic makeup and to gain insights into its biology and transmission dynamics. Conclusion: Taking advantage and making the most of the sequencing infrastructures, bioinformatics pipelines, and data-sharing platforms, established during the COVID-19 pandemic, it will make possible to establish an integrated surveillance led by sentinel territorial network to targeting TOSV and other endemic and emerging Arboviruses. This knowledge can inform the development of new diagnostics, therapeutics, and preventive measures, particularly in TOSV high risk regions.

Introduction: The recent emergence and re-emergence of infectious diseases across Africa have emphasised the critical need for vigorous pathogen genomic surveillance systems on the continent. Effective pathogen genomic surveillance relies heavily on training and capacity development in genomic sequencing and bioinformatics, as rapid public health responses to disease outbreaks depend on the continuous enhancement of these skills. Methods: Over the past four years, we have developed and conducted hybrid training in pathogen genomic surveillance and bioinformatics, for different personas in various national public health institutions across Africa. To date, we have trained over 290 participants from 36 African countries, utilising trainers and facilitators from across the continent to address various competency levels.We have also implemented comprehensive resources to support our training initiatives, including a user-friendly helpdesk ticketing system for ongoing assistance, a robust trainer database for easy access to expert knowledge, and user intuitive websites that host training materials. These tools work jointly to ensure that training and related resources are widely accessible, while also providing participants with support and engagement opportunities long after receiving training. Results: Feedback on our training initiatives, from 75% of participants via post-training evaluation surveys, have underscored the necessity for ongoing efforts in training and capacity building in pathogen genomic surveillance and bioinformatics across the continent. The qualitative feedback emphasised the necessity of further developing improved course materials, sustaining support for participants, and enhancing engagement between trainers and participants. Discussion: Based on our post-training feedback, we are currently consolidating a standardised pathogen genomics surveillance training curriculum to strengthen training and capacity development in Africa. The curriculum is designed to serve as a comprehensive resource for trainers, encompassing content that ranges from foundational courses in generic, wet-lab, and bioinformatics topics to advanced pathogen-specific courses that include tailored genomic surveillance workflows.A future step involves the implementation of more training initiatives across public health institutes in Africa, utilising the standardised pathogen genomics surveillance training curriculum to reinforce training and capacity development. Conclusion: The critical necessity for effective pathogen genomic surveillance systems in Africa has motivated our training and capacity development initiatives in pathogen genomic surveillance and bioinformatics across the continent over the past four years. By maintaining a focus on these training initiatives in the future, Africa's capacity to respond to infectious disease threats can be enhanced, ultimately contributing to the safeguarding of public health throughout the region.

Introduction: Respiratory infections, affecting mainly young children and the elderly, pose significant global health threats. While protection from related strains of viruses like influenza is well-documented, cross-protection among different viruses remains less understood. The disappearance of influenza during the COVID-19 pandemic prompted investigation into viral interference and non-pharmaceutical interventions' role. Studies suggest interactions between different viruses and host responses influence infection rates. In a large retrospective review, we assessed the impact of initial respiratory infections on subsequent viral infections, studying prevalent viruses including Influenza, Adenovirus, EV/RV, Parainfluenza, RSV, and coronaviruses. This aimed to elucidate the dynamics of viral interference independent of pandemic-related interventions. Methods: We analysed all patients tested for positive respiratory viruses between January 2016– December 2019 just prior to the pandemic in the National University Hospital laboratory, Singapore. Nasal, throat, and/ or nasopharyngeal swabs were collected by treating clinicians and tested by either multiplex PCR or immunofluorescence. Survival plots were created to study the time for a second viral infection following the initial respiratory viral detection. Results: RSV (HR: 0.63, 95%CI: 0.43–0.92; p

Backgrounds: HIV-associated neurocognitive disorders (HAND) are the common complications in the central nervous system (CNS), mainly causing HIV dementia and leading to serious social economic load. HIV-1 gp120, the envelope protein of HIV-1, has been confirmed to be an important virulence factor of HAND, and the expression levels of pyroptosis-related genes in the brains of HIV-1 gp120 transgenic mice are significantly increased. Several studies have confirmed the broad neuroprotective potential of Kynurenic acid(KYNA) as a derivative of tryptophan metabolism. Our previous study found that tryptophan-fed mice had significantly increased KYNA levels in the brain and ameliorated HIV-1 gp120-induced neurological damage, but the mechanism is still unclear. In this study, we initially investigated the protective effect of KYNA against gp120-induced neuronal pyroptosis. Methods and materials: Sy5y-SH cells were used as the neuronal model in this study and grouped into control, LPS(2.5µg/L)+ATP(5mM), HIV-1 gp120(5nM), KYNA(25µM), HIV-1 gp120+KYNA(5µM), and HIV-1 gp120+KYNA(25µM) groups. The cells in the HIV-1 gp120 group were treated with gp120 (5nM) for 24h, and the cells in the ATP group were pretreated with ATP for 6h and LPS for 1h. After the treatment, the cells were lysed with RIPA, and the protein suspension was collected. Immunofluorescence was used to detect the expression level of ASC in each group, and Western blot was used to detect the expression of pyroptosis-related proteins in each group. Results: Immunofluorescence assay showed that KYNA treated did not affect ASC expression. HIV-1 gp120 and LPS+ATP significantly enhanced ASC expression compared control and KYNA-treated groups (P

Introduction: ProMED-mail Service in Russian (ProMED-RUS) informs readers about the most important events related to infectious diseases in the world with especial focus to countries of the former Soviet Union and other countries of the WHO European Region (WHO/EURO). There are several countries in WHO/EURO, which territories are endemic for Crimean-Congo hemorrhagic fever (CCHF). Objective: To estimate the incidence of CCHF in WHO/EURO region within 2005-2023 based on ProMED-RUS data. Methods and materials: We reviewed 345 ProMED-RUS CCHF reports published between 2005 and 2023. Comments from moderators were also taken into account. Results: According to ProMED-RUS, total of 2047 cases of CCHF were recorded during the observation period, including 60 deaths (CFR 2.8%) in the Southern and North Caucasus Federal Districts of Russia. During 2005-2023 cases of CCHF were registered in the Astrakhan, Volgograd, Stavropol, Rostov regions, the republics of Dagestan, Ingushetia, Kabardino-Balkaria, Kalmykia. The expansion of the epidemically active territory of the CCHF natural focus continues in Russia. Thus, in 2023, for the first time, 1 case of CCHF was registered in the Botlikh and Novolaksky districts of the Republic of Dagestan. In total, 26 cases of CCHF were identified in Russia in 2023 (25 endemic cases, 1 case was imported to Moscow from Georgia (lethal). The number of people infected with CCHF in RF in 2023 is 2.3 times less than in 2022 (59 cases), and 71.6% lower than the long-term average. In 2013-2022 this figure averaged 88.8 cases per year.There are 10-20 cases of CCHF registered in Kazakhstan annually, more than 297 in total. In 2023, 19 cases of CCHF were registered in the Republic of Kazakhstan: 6 cases in the each of Kyzylorda, Turkestan and Zhambyl regions, 1 case in the Aktobe region.Cases of CCHF were also recorded in Georgia (12 cases in 2023), Tajikistan (10 cases, 3 - fatal in 2007- 2009), Uzbekistan (7 cases in 2014-2022, 4 - fatal), Kyrgyzstan (imported from Kazakhstan in 2019). Case of CCHF was registered in Greece (2008). Since 2016 CCHF cases also started to record in Spain (8 cases, 3 - fatal). Annually CCHF cases (from 1 to 6) record in Bulgaria. In France (Corsica) in 2023 cases of CCHF were recorded in cattle (imported from Senegal). Discussion: The expansion of the epidemically active territory of the CCHF has been observed in recent years in the WHO/EURO, which probably connected with global warning, fitness of ticks and CCHV virus, intensification of people, and cattle migration. Conclusion: ProMED-RUS reports on CCHF in the countries of the former USSR and other countries of the WHO European Region contain relevant and useful information and allow to monitor the epidemiological situation of CCHF.

Introduction: Misinformation and vaccine hesitancy pose a significant challenge to uptake and adherence to public health interventions, especially during public health emergencies. Social listening involves actively monitoring and analysing online and offline conversations, offering insights into public sentiments, concerns, and perceptions of public health interventions. Nigeria Health Watch conducted an online social listening analysis on the introduction of the Human Papillomavirus (HPV) vaccine in Nigeria between January and July 2023. The aim was to identify and promptly address misinformation about the HPV vaccine to reduce hesitance and improve vaccine uptake. Methods: This study employed a retrospective social listening methodology to identify and analyse discussions related to HPV vaccine introduction in Nigeria between the months of January and July 2023. We searched a variety of online spaces, such as news, blogs, and social media platforms (Twitter, Facebook, Instagram, and YouTube), for relevant discussions using keywords like ‘Human papillomavirus’, ‘Human papillomavirus vaccine’, ‘HPV vaccine’, and ‘cervical cancer’. Predefined prioritisation indicators were used to collate and analyse mentions thematically. Eligibility for inclusion was based on relevance to HPV vaccine introduction in Nigeria. The analysis was based on metrics such as the mentions' volume, reach, and engagement. Results: Twitter had the highest volume of conversations with 294 mentions, followed by news media/blogs (241), YouTube (4), Facebook (3) and Instagram (3). The cumulative reach across all platforms was 4,580,586, showing a strong online presence of HPV-related discussions. Twitter demonstrated the most significant level of engagement with 13,784 interactions, trailed by Facebook with 626 engagements. Discussion: Data from this study indicates a considerable amount of online discussion about HPV, particularly on Twitter and News Media/Blogs platforms. There is a consistent upward trend in HPV vaccine -related discussions across the social media platforms. Twitter emerged as the primary platform for these conversations, with a substantial increase in volume over the reporting period. Research has shown that social media platforms provide valuable insights and knowledge that can positively influence the perception of the public on HPV vaccine. Conclusion: There has been a consistent upward trend in the volume of HPV vaccine recommendations-related conversations across various platforms. Among these, twitter and other social media platforms exhibited the highest levels of engagement and reach, indicating the public's keen interest in obtaining information from these sources. Therefore, we recommend the development of clear and concise messaging that directly addresses common HPV vaccine misconceptions and dissemination on these platforms.

Introduction: The emergence of the pandemic severe acute respiratory syndrome coronavirus (SARS-CoV)-2, following significant outbreaks of SARS-CoV-1 and MERS-CoV, serves as a stark reminder of the potential for future outbreaks. This underscores the urgent need for the development of rapid and effective therapeutic and vaccine strategies to mitigate such threats. In this study, our objective is to characterize the spike protein of CoVs and explore vaccine strategies that can target multiple virus variants, aiming to bolster our defences against potential future outbreaks. Methods: In vitro studies were conducted to investigate the interactions between the SARS-CoV-2 spike protein and its receptors, along with assessing the mechanisms of viral entry using pseudotyped CoVs. Using a group of 74 experimentally proven neutralizing monoclonal antibodies, this method made it possible to find escape variants and map out the epitopes of SARS-CoV-2 variants. Furthermore, a self-assembled nanoparticle decorated with the SARS-CoV-2 spike protein, including escape mutants, as well as SARS-CoV-1 and MERS-CoV, was developed and subjected to various biochemical analyses for characterization. Subsequently, mice were immunized with these nanoparticles to evaluate their immunological responses. Results: In this study, we fully characterized the spike proteins of SARS-CoV-1, SARS-CoV-2, and MERS-CoV. This led to the creation of pseudotyped CoVs that bind specifically to their receptors, making entry into cells easier. In addition, our study includes mapping 74 experimentally proven neutralizing monoclonal antibodies (mAbs) binding epitopes for all major variants of SARS-CoV-2, such as Delta and Omicron, and structural annotation of these variants. Most of these neutralizing epitopes (87.84%) are found in the receptor-binding domain (RBD), where they overlap with other epitopes. A smaller percentage (12.16%) is found in the N-terminal domain (NTD), but it works less well than RBD antibodies. Moreover, we observed mutations in the antibody-binding epitope residues of 69 out of 74 mAbs, with the most potent RBD epitopes displaying higher mutation rates (4-10 amino acids) than less effective antibodies, indicating potential co-evolution with immune pressure. Subsequently, we engineered a self-assembled nanoparticle decorated with a SARS-CoV-2 spike and variants, demonstrating significantly enhanced immune responses against SARS-CoV-2 and its variants. Similarly, SARS-CoV-1 and MERS-CoV spike decorated nanoparticles elicit potent neutralizing antibodies against both viruses. Discussion: Our research underscores the invaluable utility of pseudotyped viruses in unraveling the intricacies of viral entry mechanisms. Furthermore, the multimeric presentation of self-assembled protein nanoparticles has shown to be very effective at producing strong antibodies that can neutralize SARS-CoV-2 antibody escape variants and also SARS-CoV-1, and MERS-CoV. Conclusion: In conclusion, the development of self-assembled protein nanoparticle vaccines represents a promising approach in our ongoing battle against coronaviruses. These innovative vaccines hold significant potential to serve as effective countermeasures, offering a versatile and robust strategy for combating future outbreaks.

Introduction: Vaccine-preventable disease outbreak response and vaccination rates are negatively impacted by rumors and false information. Erroneous information persists even after it has been corrected, negatively influencing health decisions and exacerbating health disparities while disproportionately impacting underprivileged communities. To overcome these challenges, community networks are essential - they can effectively advocate for public health initiatives while delivering accurate and culturally relevant information, which have a significant impact on community perception. By strengthening and leveraging these networks, we can be sure that accurate health information is distributed in a targeted and efficient manner to a variety of demographic groups, strengthening community resistance to false information. Thus, a fellowship program was piloted in Niger State, Nigeria to improve community networks’ capacities and reinforce their involvement in public health advocacy. Methods: The health misinformation management fellowship was designed using a bottom-up approach, starting with a needs assessment. It was conducted between August 2023 to January 2024. Following online applications, applicants went through a competitive selection process, and approximately 25 fellows representing 10 community network categories of religious, community, traditional, and media groups were selected. Fellows were initially trained in person over 3 days, with subsequent remote mentoring and support provided over subsequent months during which fellows conducted community listening and sensitization activities. The training approach was andragogic, with regular check-ins instituted for reporting and peer learning over the course of the fellowship. Results: The findings from the fellowship pre and post-test showed a significant 10% increase in knowledge and skill across the key knowledge areas of health misinformation, identification of health misinformation, and misinformation management. Fellows identified and addressed 15 rumours and misinformation within their communities and successfully engaged about 7,979 individuals, with 47% reached online and 53% offline; 33% of the offline participants were men, 60% were women, and 7% were children. Discussion: The Program demonstrated how community networks can be strengthened to identify and health misinformation. Comparable efforts, such as those by community health workers in South Asia and Sub-Saharan Africa, successfully improved health outcomes by addressing local misinformation and increasing immunization rates. While the participants of this fellowship were not healthcare workers, they proved to be effective as they leveraged their networks and the trust of their communities to reach a broader, more diverse audience, both online and offline. Conclusion: Non-health community actors are effective in the management of health misinformation when trained, as they are able to leverage the trust of their communities to reach a broader audience. Future research should focus on quantifying the long-term impacts of such interventions on vaccine uptake and public health outcomes, as well as exploring the scalability of this model to other regions and health challenges.

Introduction: Candida spp. are a common cause of healthcare-associated infections globally and Candida bloodstream infections (BSI) are associated with ∼30% mortality. Candida auris, a multidrug-resistant yeast which can be challenging to detect using traditional laboratory methods, was first reported in Botswana in 2022. This study aimed to analyze surveillance data to characterize patterns of Candida BSI and colonization among hospitalized patients in Botswana. Methods: We reviewed microbiology surveillance records over a 16-month period (October 2022 – January 2024) from a 530-bed tertiary referral hospital in Botswana. Blood cultures were collected per routine work-up for sepsis, incubated in an automated system (BacT/ALERT, bioMérieux), and identified using a combination of manual and automated methods (e.g. VITEK 2). Colonization samples (skin and perirectal swabs) were collected during twice-monthly point prevalence surveys in the hospital's intensive care unit (ICU) and neonatal intensive care unit (NICU), transported in ESwab media (COPAN Diagnostics), and cultured with chromogenic media (CHROMagar, Candida Plus). Identification of preserved presumptive Candida spp. isolates from both BSIs and colonization samples was confirmed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) at the National Institute for Communicable Diseases, South Africa. Results: A total of 70 Candida BSI were detected during the surveillance period, making it the 6th leading cause of BSI (995 all-cause BSIs detected from a total of 6895 blood cultures). Of 43 preserved BSI isolates which underwent confirmation, C. albicans was most commonly identified species (20%), followed by C. auris (17%), C. krusei (15%), C. parapsilosis (10%) and C. glabrata (10%) (Figure 1). Among 190 samples collected from ICU, Candida colonization was identified from 181 (95%) patient samples, most commonly C. albicans (35% of 181), followed by C. auris (32% of 181). Among 116 samples collected from ICU, Candida colonization was identified from 103 (89%) patient samples, most commonly C. albicans (56% of 103), followed by C. parapsilosis (17% of 103). The relative frequency of Candida spp. causing BSI [data not shown] roughly aligned with the species identified through concurrent colonization screening, with the exception of C. krusei, which can be attributed to a neonatal C. krusei outbreak linked to contaminated medication (Figure 2). Discussion: Candida spp. accounted for a significant portion of BSIs at this hospital and were responsible for at least one hospital outbreak during this time period. The repeated detection of C. auris among hospitalized patients, including neonates, over several months demonstrates the potential for sustained transmission. Conclusion: Routine colonization screening may serve as an early signal for disease transmission and help in the development of targeted containment strategies by infection prevention/control teams. Further analysis with whole genome sequencing could be used to produce a phylogenetic comparison with other clinical isolates in the southern African region.

Introduction: Antimicrobial resistance (AMR) poses a significant global threat. In 2019, AMR contributed to an estimated 4.95 million deaths with 1.27 million deaths attributed to AMR. It is influenced by factors including antibiotic usage and supply chain patterns, patient-provider dynamics, and community attitudes. Understanding patient views regarding the appropriate use of antibiotics for bacterial infections and inappropriately for viral infections, is crucial for addressing inappropriate antibiotic use and combating AMR. Africa experiences high death rates from AMR but there are little data or understanding of patient views especially in primary healthcare (PHC). This scoping review focused on PHC settings in low- and middle-income countries (LMICs) like South Africa. It aimed to identify key themes surrounding patient and community members views regarding antimicrobial use in PHC in LMICs. Methods: The scoping review followed the Joanna Briggs Institute (JBI) methodology and PRISMA-ScR guidelines and included qualitative studies, close-ended questionnaires, and descriptive observational designs. The Covidence web-based platform was used for literature screening and data extraction including population characteristics, methods, tools and/or questionnaires used, and key findings and the Critical Appraisal Skills Programme (CASP) qualitative checklist was used for qualitative papers. Results: The review identified several key themes related to: 1) patient factors including patient beliefs, expectations, knowledge and understanding, and perceptions of antibiotics, resistance and illness severity; satisfaction with and trust in healthcare providers and shared decision making and empowerment; community attitudes, perceptions and beliefs, and sociocultural factors; 2) provider factors including patient-provider relationship, healthcare utilisation and antibiotic prescribing provision and practices and healthcare delivery in PHC; 3) healthcare system factors including resource constraints and healthcare delivery, diagnostic tools and testing and technological advances and access to care, and 4) uptake, implementation, effectiveness and impact of educational interventions. Patients demonstrated varied levels of knowledge regarding infections and antibiotic resistance, influenced by factors such as migration, cultural background, and health literacy. Motivations for seeking antibiotics included cultural beliefs, family influences, and concerns about illness severity. Discussion: Understanding patient and healthcare provider perspectives and understanding are crucial for designing effective interventions to combat AMR. This study emphasizes the need for enhancing antibiotic stewardship programs, addressing information needs, and considering population diversity and health literacy in educational interventions. Challenges related to resource scarcity and power dynamics within the healthcare system must also be considered, Conclusion: Despite the growing awareness that a substantial portion of antibiotic consumption occurs within PHC settings, patients' roles in the use of antibiotics have often been overlooked. This review emphasizes the importance of understanding patient perspectives in LMICs regarding antibiotic use in PHC settings. The focus should be on implementing targeted interventions for both patients and providers to promote judicious antibiotic use and mitigate the global threat of AMR.