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BackgroundUnregulated antimicrobial use is common in both hospital and community settings of low- and middle-income countries (LMICs). However, discrete data regarding the use/misuse of antimicrobials at pharmacies in LMICs are limited. This study was conducted to understand the knowledge, attitude, and practice of pharmacy employees on antimicrobial dispensing in Nepal.MethodsWe conducted a cross-sectional survey using a structured questionnaire on 801 pharmacy employees working in community and hospital pharmacies located in Lalitpur metropolitan city (LMC) of Kathmandu, Nepal between April 2017 and March 2019.ResultsNot all respondents replied to all questions asked. A majority (92%, 738/801) of respondents agreed that the demand for non-prescription antimicrobials was common. Sixty nine percent (437/635) of participants responded that they would ask of prescription before dispensing. Suspected respiratory tract infection was the most common reason demanding for non-prescription antimicrobials, identified by 68% (535/792) of respondents. Azithromycin was the most commonly prescribed and sold antimicrobials, as reported by 46%, (361/787) and 48% (377/779) of participants respectively. A majority (87%; 696/800) of respondents agreed on antimicrobial resistance (AMR) to be a global public health threat; 54% (429/796) perceived antimicrobial misuse to be an important driver of AMR, while only 39% (315/801) replied that judicious dispensing of antimicrobials can help curb AMR.ConclusionOur study revealed that the unfounded dispensing and use of antimicrobials is prevalent among pharmacies in Kathmandu, Nepal. This overreliance on antimicrobials, notably azithromycin, may escalated the burden of AMR. We identified several drivers of inappropriate antimicrobial dispensing practice in pharmacies, which will aid public health authorities in addressing these issues. Further studies considering the role of other stakeholders, such as physicians, veterinarians, general public, and policy makers are required to obtain a more holistic perspectives on the practices of antimicrobial use so to curb the extant AMR crisis.

Antibiotic resistance is increasing at an alarming rate, and three related mycobacteria are the source of widespread infections in humans. According to the World Health Organization,Mycobacterium leprae, which causes leprosy, is still endemic in tropical countries; Mycobacterium tuberculosis is the second largest infectious disease killer worldwide after COVID-19;Mycobacteroides abscessus, a group of non-tuberculous mycobacteria, causes lung infections and other health-care-associated infections in humans. Due to the rise in resistance to common antibacterial drugs, it is critical to develop alternatives to traditional treatment procedures. Furthermore, an understanding of the biochemical mechanisms underlying pathogenic evolution is important for the treatment and management of these diseased conditions.In this study, metabolic models have been developed for two bacterial pathogens,M. leprae, andM. abscessus, and a new computational tool has been used to identify potential drug targets, which are referred to as bottleneck reactions. The genes, reactions, and pathways in each of these organisms have been highlighted; the potential drug targets can be further explored as broad-spectrum antibacterials and the unique drug targets to each pathogen are significant for precision medicine initiatives.The models and associating datasets are available in GigaScience and the following repositories:M. abscessusBiomodels:https://www.ebi.ac.uk/biomodels/MODEL2203300002https://www.patmedb.org/Bacteria/MabscessusM. lepraeBiomodels:https://www.ebi.ac.uk/biomodels/MODEL2203300001https://www.patmedb.org/Bacteria/Mleprae

Background A considerable portion of the HIV pandemic is composed of people under antiretroviral therapy, many of whom get a late diagnosis. Patients starting antiretroviral therapy (ART) at a very advanced stage of HIV disease attain a low recovery of CD4 T cells. Factors associated with poor recovery are incompletely described. This study aimed at finding variables associated with CD4 T cell recovery in late-presenting HIV patients. Methods We studied a cohort of HIV+ patients initiating ART with very low basal CD4 T cell counts. We defined immune recovery as the net increase in circulating CD4 T cell counts after one year on ART. We analyzed diverse routine laboratory determinations at different times using Least Absolute Shrinkage and Selection Operator (LASSO), adaptive LASSO and Conditional Inference Random Forest. Results CD4/CD8 ratio, % CD4 T cells and CD8 T cell counts at different times were the main recovery correlates, validated by all approaches. Unexpectedly, basal hematocrit was a consistent predictor. Additionally, week 24 creatinine had a high lasso coefficient, and alkaline phosphatase had a high conditional inference random forest coefficients, although neither was verified by other tests. Conclusions CD4 T cell proportions are associated with CD4 T cell recovery, independently of cell counts. Inflammation-related variables could also affect reconstitution. These accessible variables may reflect underlying mechanisms and could improve the follow up of patients starting ART with an advanced HIV infection.

Funder: Medimmune; Id: http://dx.doi.org/10.13039/501100004628, Funder: Infinitus (China) Ltd., Wound biofilms represent a particularly challenging problem in modern medicine. They are increasingly antibiotic resistant and can prevent the healing of chronic wounds. However, current treatment and diagnostic options are hampered by the complexity of the biofilm environment. In this review, we present new chemical avenues in biofilm sensors and new materials to treat wound biofilms, offering promise for better detection, chemical specificity, and biocompatibility. We briefly discuss existing methods for biofilm detection and focus on novel, sensor‐based approaches that show promise for early, accurate detection of biofilm formation on wound sites and that can be translated to point‐of‐care settings. We then discuss technologies inspired by new materials for efficient biofilm eradication. We focus on ultrasound‐induced microbubbles and nanomaterials that can both penetrate the biofilm and simultaneously carry active antimicrobials and discuss the benefits of those approaches in comparison to conventional methods.

Background Typhoid fever remains a significant cause of morbidity and mortality in Asia and Africa. The emergence of azithromycin resistance in South Asia is concerning, as azithromycin is one of the last effective oral drugs for treating typhoid. Objectives To describe the molecular mechanism and phylogenetics of azithromycin-resistant (AzithR) Salmonella Typhi isolates from Patan Hospital, Kathmandu, Nepal. Methods Whole-genome sequences of three AzithR S. Typhi isolates (MIC >256 mg/L) were analysed and compared with a global collection to investigate the azithromycin resistance mechanism and phylogenetic structure. Clinical information is reported for one of the three patients infected with AzithR S. Typhi. Results The three AzithR isolates belonged to the H58 lineage and were genetically identical; they were distantly related to contemporaneous S. Typhi from Nepal and AzithR S. Typhi recently described in Bangladesh. Azithromycin resistance was mediated by a non-synonymous mutation in the acrB gene (R717L). The three AzithR isolates showed reduced susceptibility to ciprofloxacin (double mutation in the gyrA: S83F and D87G), and were susceptible to ampicillin, chloramphenicol and co-trimoxazole. Clinical information from one patient suggested non-response to azithromycin treatment. Conclusions This is the first molecular description of AzithR S. Typhi in Nepal. These organisms showed no phylogenetic link to AzithR S. Typhi in Bangladesh. Our data suggest that increasing use of azithromycin may pose a strong selective pressure driving the emergence of AzithR S. Typhi in South Asia. Further investigations are needed to evaluate treatment responses to azithromycin, predict evolutionary trajectories, and track the transmission of these organisms.

Arming oncolytic adenoviruses with therapeutic transgenes is a well-established strategy for multimodal tumour attack. However, this strategy sometimes leads to unexpected attenuated viral replication and a loss of oncolytic effects, preventing these viruses from reaching the clinic. Previous work has shown that altering codon usage in viral genes can hamper viral fitness. Here, we have analysed how transgene codon usage impacts viral replication and oncolytic activity. We observe that, although transgenes with optimized codons show high expression levels at the first round of infection, they impair viral fitness and are therefore not expressed in a sustained manner. Conversely, transgenes encoded by suboptimal codons do not compromise viral replication and are thus stably expressed over time, allowing a greater oncolytic activity both in vitro and in vivo. Altogether, our work shows that fine-tuning codon usage leads to a concerted optimization of transgene expression and viral replication paving the way for the rational design of more efficacious oncolytic therapies.

Background. Hypervirulent Klebsiella pneumoniae causes severe disseminated infections, typically with hepatic and central nervous system involvement including endophthalmitis. Case Presentation. We report a fatal case of an undocumented Chinese migrant in her 60s who presented to the emergency department with abdominal pain, lethargy, and headache over the preceding two weeks. She had a new diagnosis of diabetes mellitus on admission. Computed tomography scan of the thorax, abdomen, and pelvis showed bilateral pneumonia with liver abscesses. The patient was treated with empirical broad-spectrum antibiotics before K. pneumoniae was isolated from cerebrospinal fluid and blood cultures. The isolate was further characterised as a ST23 (ST: sequence type), serotype K1 hypervirulent strain using Nanopore sequencing. Despite admission to the intensive care unit, the patient died within 48 hrs of admission. Conclusions. This case demonstrates the need for increased awareness of hypervirulent K. pneumoniae, even in countries where it occurs infrequently. Novel, rapid, sequencing technologies can support diagnosis in unusual presentations.

New malaria control tools and tailoring interventions to local contexts are needed to reduce the malaria burden and meet global goals. The housing modification, screening plus a targeted house-based insecticide delivery system called the In2Care® Eave Tubes, has been shown to reduce clinical malaria in a large cluster randomised controlled trial. However, the widescale suitability of this approach is unknown. We aimed to predict household suitability and define the most appropriate locations for ground-truthing where Screening + Eave Tubes (SET) could be implemented across Côte d'Ivoire. We classified DHS sampled households into suitable for SET based on the walls and roof materials. We fitted a Bayesian beta-binomial logistic model using the integrated nested Laplace approximation (INLA) to predict suitability of SET and to define priority locations for ground-truthing and to calculate the potential population coverage and costs. Based on currently available data on house type and malaria infection rate, 31% of the total population and 17.5% of the population in areas of high malaria transmission live in areas suitable for SET. The estimated cost of implementing SET in suitable high malaria transmission areas would be $46m ($13m -$108m). Ground-truthing and more studies should be conducted to evaluate the efficacy and feasibility of SET in these settings. The study provides an example of implementing strategies to reflect local socio-economic and epidemiological factors, and move beyond blanket, one-size-fits-all strategies.

The interaction between immune cells and virus-infected targets involves multiple plasma membrane (PM) proteins. A systematic study of PM protein modulation by vaccinia virus (VACV), the paradigm of host regulation, has the potential to reveal not only novel viral immune evasion mechanisms, but also novel factors critical in host immunity. Here, >1000 PM proteins were quantified throughout VACV infection, revealing selective downregulation of known T and NK cell ligands including HLA-C, downregulation of cytokine receptors including IFNAR2, IL-6ST and IL-10RB, and rapid inhibition of expression of certain protocadherins and ephrins, candidate activating immune ligands. Downregulation of most PM proteins occurred via a proteasome-independent mechanism. Upregulated proteins included a decoy receptor for TRAIL. Twenty VACV-encoded PM proteins were identified, of which five were not recognised previously as such. Collectively, this dataset constitutes a valuable resource for future studies on antiviral immunity, host-pathogen interaction, poxvirus biology, vector-based vaccine design and oncolytic therapy.

Expansion microscopy is a sample preparation technique that enables the optical imaging of biological specimens at super-resolution owing to their physical magnification, which is achieved through water-absorbing polymers. The technique uses readily available chemicals and does not require sophisticated equipment, thus offering super-resolution to laboratories that are not microscopy-specialised. Here we present a protocol combining sample expansion with light sheet microscopy to generate high-contrast, high-resolution 3D reconstructions of whole virus-infected cells. The results are superior to those achievable with comparable imaging modalities and reveal details of the infection cycle that are not discernible before expansion. An image resolution of approximately 95 nm could be achieved in samples labelled in 3 colours. We resolve that the viral nucleoprotein is accumulated at the membrane of vesicular structures within the cell cytoplasm and how these vesicles are positioned relative to cellular structures. We provide detailed guidance and a video protocol for the optimal application of the method and demonstrate its potential to study virus-host cell interactions.

Background The Plasmodium genus of malaria parasites encodes several families of antigen-encoding genes. These genes tend to be hyper-variable, highly recombinogenic and variantly expressed. The best-characterized family is the var genes, exclusively found in the Laveranian subgenus of malaria parasites infecting humans and great apes. Var genes encode major virulence factors involved in immune evasion and the maintenance of chronic infections. In the human parasite P. falciparum, var gene recombination and diversification appear to be promoted by G-quadruplex (G4) DNA motifs, which are strongly associated with var genes in P. falciparum. Here, we investigated how this association might have evolved across Plasmodium species – both Laverania and also more distantly related species which lack vars but encode other, more ancient variant gene families. Results The association between var genes and G4-forming motifs was conserved across Laverania, spanning ~ 1 million years of evolutionary time, with suggestive evidence for evolution of the association occurring within this subgenus. In rodent malaria species, G4-forming motifs were somewhat associated with pir genes, but this was not conserved in the Laverania, nor did we find a strong association of these motifs with any gene family in a second outgroup of avian malaria parasites. Secondly, we compared two different G4 prediction algorithms in their performance on extremely A/T-rich Plasmodium genomes, and also compared these predictions with experimental data from G4-seq, a DNA sequencing method for identifying G4-forming motifs. We found a surprising lack of concordance between the two algorithms and also between the algorithms and G4-seq data. Conclusions G4-forming motifs are uniquely strongly associated with Plasmodium var genes, suggesting a particular role for G4s in recombination and diversification of these genes. Secondly, in the A/T-rich genomes of Plasmodium species, the choice of prediction algorithm may be particularly influential when studying G4s in these important protozoan pathogens.

Background: Norovirus, also known as the winter vomiting bug, is the predominant cause of non-bacterial gastroenteritis worldwide. Disease control is predicated on a robust innate immune response during the early stages of infection. Double-stranded RNA intermediates generated during viral genome replication are recognised by host innate immune sensors in the cytoplasm, activating the strongly antiviral interferon gene programme. Ifit proteins (interferon induced proteins with tetratricopeptide repeats), which are highly expressed during the interferon response, have been shown to directly inhibit viral protein synthesis as well as regulate innate immune signalling pathways. Ifit1 is well-characterised to inhibit viral translation by sequestration of eukaryotic initiation factors or by directly binding to the 5' terminus of foreign RNA, particularly those with non-self cap structures. However, noroviruses have a viral protein, VPg, covalently linked to the 5' end of the genomic RNA, which acts as a cap substitute to recruit the translation initiation machinery. Methods: : Ifit1 knockout RAW264.7 murine macrophage-like cells were generated using CRISPR-Cas9 gene editing. These cells were analysed for their ability to support murine norovirus infection, determined by virus yield, and respond to different immune stimuli, assayed by quantitative PCR. The effect of Ifit proteins on norovirus translation was also tested in vitro . Results: : Here, we show that VPg-dependent translation is completely refractory to Ifit1-mediated translation inhibition in vitro and Ifit1 cannot bind the 5' end of VPg-linked RNA. Nevertheless, knockout of Ifit1 promoted viral replication in murine norovirus infected cells. We then demonstrate that Ifit1 promoted interferon-beta expression following transfection of synthetic double-stranded RNA but had little effect on toll-like receptor 3 and 4 signalling. Conclusions: : Ifit1 is an antiviral factor during norovirus infection but cannot directly inhibit viral translation. Instead, Ifit1 stimulates the antiviral state following cytoplasmic RNA sensing, contributing to restriction of norovirus replication.

The ATP-binding cassette transporter ABCB6 was recently discovered to encode the Langereis (Lan) blood group antigen. Lan null individuals are asymptomatic, and the function of ABCB6 in mature erythrocytes is not understood. Here, we assessed ABCB6 as a host factor for Plasmodium falciparum malaria parasites during erythrocyte invasion. We show that Lan null erythrocytes are highly resistant to invasion by P. falciparum, in a strain-transcendent manner. Although both Lan null and Jr(a-) erythrocytes harbor excess porphyrin, only Lan null erythrocytes exhibit a P. falciparum invasion defect. Further, the zoonotic parasite P. knowlesi invades Lan null and control cells with similar efficiency, suggesting that ABCB6 may mediate P. falciparum invasion through species-specific molecular interactions. Using tandem mass tag-based proteomics, we find that the only consistent difference in membrane proteins between Lan null and control cells is absence of ABCB6. Our results demonstrate that a newly identified naturally occurring blood group variant is associated with resistance to Plasmodium falciparum. Elizabeth Egan and colleagues demonstrate that host ATP binding cassette transporter ABCB6, which encodes the Langereis blood group antigen, promotes erythrocyte invasion by the malaria parasite Plasmodium falciparum. This study suggests that asymptomatic Langereis null individuals may be better protected from malaria.

Background: The infection of a participant with norovirus during the adaptive study of interleukin-2 dose on regulatory T cells in type 1 diabetes (DILT1D) allowed a detailed insight into the cellular and cytokine immune responses to this prevalent gastrointestinal pathogen. Methods: Serial blood, serum and peripheral blood mononuclear cell (PBMC) samples were collected pre-, and post-development of the infection. To differentiate between the immune response to norovirus and to control for the administration of a single dose of aldesleukin (recombinant interleukin-2, rIL-2) alone, samples from five non-infected participants administered similar doses were analysed in parallel. Results: Norovirus infection was self-limited and resolved within 24 hours, with the subsequent development of anti-norovirus antibodies. Serum pro- and anti-inflammatory cytokine levels, including IL-10, peaked during the symptomatic period of infection, coincident with increased frequencies of monocytes and neutrophils. At the same time, the frequency of regulatory CD4+ T cell (Treg), effector T cell (Teff) CD4+ and CD8+ subsets were dynamically reduced, rebounding to baseline levels or above at the next sampling point 24 hours later. NK cells and NKT cells transiently increased CD69 expression and classical monocytes expressed increased levels of CD40, HLA-DR and SIGLEC-1, biomarkers of an interferon response. We also observed activation and mobilisation of Teffs, where increased frequencies of CD69+ and Ki-67+ effector memory Teffs were followed by the emergence of memory CD8+ Teff expressing the mucosal tissue homing markers CD103 and β7 integrin. Treg responses were coincident with the innate cell, Teff and cytokine response. Key Treg molecules FOXP3, CTLA-4, and CD25 were upregulated following infection, alongside an increase in frequency of Tregs with the capacity to home to tissues. Conclusions: The results illustrate the innate, adaptive and counter-regulatory immune responses to norovirus infection. Low-dose IL-2 administration induces many of the Treg responses observed during infection.

Differentiation of naïve CD4+ T cells into functionally distinct T helper (TH) subsets is crucial for the orchestration of immune responses. Because of extensive heterogeneity and multiple overlapping transcriptional programs in differentiating T cell populations, this process has remained a challenge for systematic dissection in vivo. By using single-cell transcriptomics and computational analysis with a temporal mixtures of Gaussian processes model, termed GPfates, we reconstructed the developmental trajectories of TH1 and TFH (T follicular helper) cells during blood-stage Plasmodium infection in mice. By tracking clonality using endogenous T cell receptor sequences, we first demonstrated that TH1/TFH bifurcation had occurred at both population and single-clone levels. Next, we identified genes whose expression was associated with TH1 or TFH fates and demonstrated a T cell–intrinsic role for Galectin-1 in supporting TH1 differentiation. We also revealed the close molecular relationship between TH1 and interleukin-10–producing Tr1 cells in this infection. TH1 and TFH fates emerged from a highly proliferative precursor that up-regulated aerobic glycolysis and accelerated cell cycling as cytokine expression began. Dynamic gene expression of chemokine receptors around bifurcation predicted roles for cell-cell interaction in driving TH1/TFH fates. In particular, we found that precursor TH cells were coached toward a TH1 but not a TFH fate by inflammatory monocytes. Thus, by integrating genomic and computational approaches, our study has provided two unique resources: a database, www.PlasmoTH.org, which facilitates discovery of novel factors controlling TH1/TFH fate commitment, and, more generally, GPfates, a modeling framework for characterizing cell differentiation toward multiple fates.

Human cytomegalovirus (HCMV) is a human herpesvirus which causes little or no disease in the immunocompetent. However, in immunocompromised individuals, neonates, or patients on immune suppressive therapies, HCMV can cause significant morbidity and mortality in some patient groups. As with all herpesviruses, HCMV has two life cycle phases: a productive phase, where new virions are produced and a latent phase where there is a restricted gene transcription profile and no new virion production. Currently available antivirals target the productive phase of HCMV infection and, although these have greatly decreased the severity of HCMV-induced disease in immunocompromised or immunosuppressed individuals, they often have associated toxicities, routinely result in selection of drug resistant viral mutants, and, importantly, they do not target cells latently infected with virus. Thus, there is a real need to derive novel antiviral therapies which, not least, are also able to target latent infection. In this paper, we describe recent work which has begun to analyse changes in the cell associated with latent infection and the possibility that these latency-associated changes in cell phenotype could be targeted by novel chemo- or immunotherapeutic strategies in order to diminish, or even clear, latent infection at least in some specific clinical settings.

HPV infection in the genital tract is common in young sexually active individuals, the majority of whom clear the infection without overt clinical disease. However most of those who develop benign lesions eventually mount an effective cell mediated immune (CMI) response and the lesions regress. Failure to develop effective CMI to clear or control infection results in persistent infection and, in the case of the oncogenic HPVs, an increased probability of progression to CIN3 and invasive carcinoma. The prolonged duration of infection associated with HPV seems to be associated with effective evasion of innate immunity thus delaying the activation of adaptive immunity. Natural infections in animals show that neutralising antibody to the virus coat protein L1 is protective suggesting that this would be an effective prophylactic vaccine strategy. The current prophylactic HPV VLP vaccines are delivered i.m. circumventing the intra-epithelial immune evasion strategies. These vaccines generate high levels of antibody and both serological and B cell memory as evidenced by persistence of antibody and robust recall responses. However there is no immune correlate - no antibody level that correlates with protection. Recent data on how HPV infects basal epithelial cells and how antibody can prevent this provides a mechanistic explanation for the effectiveness of HPV VLP vaccines.

Following recent large scale-up of malaria control interventions in Ethiopia, this study aimed to compare ownership and use of long-lasting insecticidal nets (LLIN), and the change in malaria prevalence using two population-based household surveys in three regions of the country. Each survey used multistage cluster random sampling with 25 households per cluster. Household net ownership tripled from 19.6% in 2006 to 68.4% in 2007, with mean LLIN per household increasing from 0.3 to 1.2. Net use overall more than doubled from 15.3% to 34.5%, but in households owning LLIN, use declined from 71.7% to 48.3%. Parasitemia declined from 4.1% to 0.4%. Large scale-up of net ownership over a short period of time was possible. However, a large increase in net ownership was not necessarily mirrored directly by increased net use. Better targeting of nets to malaria-risk areas and sustained behavioural change communication are needed to increase and maintain net use.