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Larvae of the greater wax moth, Galleria mellonella, are a convenient in vivo model for assessing the activity and toxicity of antimicrobial agents and for studying the immune response to pathogens and provide results similar to those from mammals. G. mellonella larvae are now widely used in academia and industry and their use can assist in the identification and evaluation of novel antimicrobial agents. Galleria larvae are inexpensive to purchase and house, easy to inoculate, generate results within 24–48 h and their use is not restricted by legal or ethical considerations. This review will highlight how Galleria larvae can be used to assess the efficacy of novel antimicrobial therapies (photodynamic therapy, phage therapy, metal-based drugs, triazole-amino acid hybrids) and for determining the in vivo toxicity of compounds (e.g., food preservatives, ionic liquids) and/or solvents (polysorbate 80). In addition, the disease development processes are associated with a variety of pathogens (e.g., Staphylococcus aureus, Listeria monocytogenes, Aspergillus fumigatus, Madurella mycotomatis) in mammals are also present in Galleria larvae thus providing a simple in vivo model for characterising disease progression. The use of Galleria larvae offers many advantages and can lead to an acceleration in the development of novel antimicrobials and may be a prerequisite to mammalian testing.

The immune system of insects and the innate immune response of mammals share many similarities and, as a result, insects may be used to assess the virulence of fungal pathogens and give results similar to those from mammals. Larvae of the greater wax moth Galleria mellonella are widely used in this capacity and also for assessing the toxicity and in vivo efficacy of antifungal drugs. G. mellonella larvae are easy to use, inexpensive to purchase and house, and have none of the legal/ethical restrictions that are associated with use of mammals. Larvae may be inoculated by intra-hemocoel injection or by force-feeding. Larvae can be used to assess the in vivo toxicity of antifungal drugs using a variety of cellular, proteomic, and molecular techniques. Larvae have also been used to identify the optimum combinations of antifungal drugs for use in the treatment of recalcitrant fungal infections in mammals. The introduction of foreign material into the hemocoel of larvae can induce an immune priming effect which may operate independently with the activity of the antifungal drug. Procedures to identify this effect and limit its action are required.

Background Galleria mellonella larvae were infected with conidia of Aspergillus fumigatus and the cellular and humoral immune responses of larvae to the pathogen were characterized as invasive aspergillosis developed. Results At 2 h post-infection there was an increase in hemocyte density to 7.43 ± 0.50 × 10 6 /ml from 0.98 ± 0.08 × 10 6 /ml at 0 h. Hemocytes from larvae immune primed for 6 h with heat killed A. fumigatus conidia displayed superior anti-fungal activity. Examination of the spread of the fungus by Cryo-imaging and fluorescent microscopy revealed dissemination of the fungus through the larvae by 6 h and the formation of distinct nodules in tissue. By 24 h a range of nodules were visible at the site of infection and at sites distant from that indicating invasion of tissue. Proteomic analysis of larvae infected with viable conidia for 6 h demonstrated an increase in the abundance of gustatory receptor candidate 25 (37 fold), gloverin-like protein (14 fold), cecropin-A (11 fold). At 24 h post-infection gustatory receptor candidate 25 (126 fold), moricin-like peptide D (33 fold) and muscle protein 20-like protein (12 fold) were increased in abundance. Proteins decreased in abundance included fibrohexamerin (13 fold) and dimeric dihydrodiol dehydrogenase (8 fold). Conclusion The results presented here indicate that G. mellonella larvae may be a convenient model for studying the stages in the development of invasive aspergillosis and may offer an insight into this process in mammals.

Although effective antiretroviral therapy(ART) increases CD4+ T-cell count, responses to ART vary considerably and only a minority of patients normalise their CD4+/CD8+ ratio. Although retention of naïve CD4+ T-cells is thought to predict better immune responses, relationships between CD4+ and CD8+ T-cell subsets and CD4+/CD8+ ratio have not been well described. A cross-sectional study in a cohort of ambulatory HIV+ patients. We used flow cytometry on fresh blood to determine expanded CD4+ and CD8+ T-cell subsets; CD45RO+CD62L+(central memory), CD45RO+CD62L-(effector memory) and CD45RO-CD62L+(naïve) alongside routine T-cell subsets(absolute, percentage CD4+ and CD8+ counts), HIVRNA and collected demographic and treatment data. Relationship between CD4+/CD8+ T-cell ratio and expanded T-cell subsets was determined using linear regression analysis. Results are median[IQR] and regression coefficients unless stated. We recruited 190 subjects, age 42(36-48) years, 65% male, 65.3% Caucasian, 91% on ART(52.6% on protease inhibitors), 78.4% with HIVRNA<40cps/ml and median ART duration 6.8(2.6-10.2) years. Nadir and current CD4+ counts were 200(112-309) and 465(335-607) cells/mm3 respectively. Median CD4+/CD8+ ratio was 0.6(0.4-1.0), with 26.3% of subjects achieving CD4+/CD8+ ratio>1. Of the expanded CD4+ T-cell subsets, 27.3(18.0-38.3)% were naïve, 36.8(29.0-40.0)% central memory and 27.4(20.0-38.5)% effector memory. Of the CD8+ T-cells subsets, 16.5(10.2-25.5)% were naïve, 19.9(12.7-26.6)% central memory and 41.0(31.8-52.5)% effector memory. In the multivariable adjusted analysis, total cumulative-ART exposure(+0.15,p = 0.007), higher nadir CD4+ count(+0.011,p<0.001) and higher %CD8+ naive T-cells(+0.0085,p<0.001) were associated with higher CD4+/CD8+ ratio, higher absolute CD8+ T-cell(-0.0044,p<0.001) and higher %CD4+ effector memory T-cells(-0.004,p = 0.0036) were associated with lower CD4+/CD8+ ratio. Those with CD4+/CD8+ ratio>1 had significantly higher median %CD8+ naive T-cells; 25.4(14.0-36.0)% versus 14.4(9.4-21.6)%, p<0.0001, but significantly lower absolute CD8+ count; 464(384.5-567) versus 765(603-1084) cells/mm3, p<0.001. Study suggests important role for naïve CD8+ T-cell populations in normalisation of the immune response to HIV-infection. How these findings relate to persistent immune activation on ART requires further study.

The long-term effects of SARS-CoV-2 infection and optimal follow-up approach are not well-recognised. Here we describe the implementation of a post-COVID clinic in an Irish tertiary centre after the first wave of the pandemic. This study describes the characteristics of our patient cohort and the operations and outcomes of the clinic, exploring some of the risk factors for developing post-COVID syndrome and the appropriateness of the triage system employed. All SARS-CoV-2 positive patients from March 10th to June 14th 2020 were telephone-triaged as red, amber or green based on ongoing symptoms with clinic appointments scheduled accordingly. All clinic visits were face-to-face with the infectious diseases medical team and a proforma for each patient was completed. Data were collected retrospectively by reviewing the proformas and the electronic medical record (EMR). 311 patients attended the clinic. Median time from illness to clinic appointment was 95 days (IQR 77-105.5). 204 patients (66%) were female, 192 (62%) were hospital staff, and the median age was 43 years (IQR 31-53). 138 patients (44%) had required hospital admission. At their first clinic visit 219 patients (70%) had ongoing symptoms. A further appointment was made for 62 patients (20%). 34 patients (11%) were discussed at an MDT meeting, and 55 (18%) were referred onward to a specialist service. 85% of those triaged green, 73% of those triaged amber, and 39% of those triaged red did not receive further follow up after one clinic visit. Patients were more likely to require follow up with reported dyspnoea (OR 5.6; 95% CI 2.8-11.3; p <0.001), cough (OR 3.0; 95% CI 1.1-8.4, p = 0.04), and palpitations (OR 3.6; 95% CI 1.0-12.3; p = 0.04). Female sex was associated with increased odds of a higher triage category (OR 1.8; 95% CI 1.08 to 3.20; p = 0.02), as was requiring admission to hospital (OR 4.0; 95% CI 2.34 to 6.90; p < 0.001). The long-term effects of COVID-19 are significant with 70% of our cohort experiencing persistent symptoms. Persistent dyspnoea, cough and palpitations were associated with increased need for follow up. This study also suggests that a traffic light telephone-triage service followed by a face-to-face medical-led clinic could be an effective way of identifying patients who require further management.

This study assessed the development of disseminated candidiasis within Galleria mellonella larvae and characterized the proteomic responses of Candida albicans to larval hemolymph. Infection of larvae with an inoculum of 1 × 106 yeast cells reduced larval viability 24 (53.33 ± 3.33%), 48 (33.33 ± 3.33%) and 72 (6.66 ± 3.33%) h post infection. C. albicans infection quickly disseminated from the site of inoculation and the presence of yeast and hyphal forms were found in nodules extracted from infected larvae at 6 and 24 h. A range of proteins secreted during infection of G. mellonella by C. albicans were detected in larval hemolymph and these were enriched for biological processes such as interaction with host and pathogenesis. The candicidal activity of hemolymph after immediate incubation of yeast cells resulted in a decrease in yeast cell viability (0.23 ± 0.03 × 106 yeast cells/mL), p < 0.05) as compared to control (0.99 ± 0.01 × 106 yeast cells/mL). C. albicans responded to incubation in hemolymph ex vivo by the induction of an oxidative stress response, a decrease in proteins associated with protein synthesis and an increase in glycolytic proteins. The results presented here indicate that C. albicans can overcome the fungicidal activity of hemolymph by altering protein synthesis and cellular respiration, and commence invasion and dissemination throughout the host.

Mycetoma is a neglected chronic and granulomatous infection primarily associated with the fungal pathogen Madurella mycetomatis. Characteristic of this infection is the formation of grains. However, the processes leading to grain formation are not known. In this study, we employed a proteomic approach to characterise M. mycetomatis grain formation in Galleria mellonella larvae and map the processes leading to grain formation over time. For this, at 1 day, 3 days and 7 days post-inoculation, proteins from grains and hemolymph were extracted and analysed by label-free mass spectrometry. A total of 87, 51 and 48 M. mycetomatis proteins and 713, 997, 18 G. mellonella proteins were found in grains on day 1, 3 and 7 post-inoculation respectively. M. mycetomatis proteins were mainly involved in cellular metabolic processes and numerous enzymes were encountered. G. mellonella proteins were primarily involved in the nodulation process. The proteins identified were linked to nodulation and grain formation and four steps of grain formation were identified. The results of this proteomic approach could in the future be used to design novel strategies to interfere with mycetoma grain formation and to combat this difficult to treat infection.

Aspergillus fumigatus is a serious cause of disease in immune-deficient patients and in those with pulmonary malfunction (e.g., cystic fibrosis (CF), asthma). Atorvastatin is a member of the statin drug family, which are the main therapeutic agents used to decrease high serum cholesterol levels by inhibiting (HMG-CoA) reductase enzyme. The aim of the work presented here was to analyse the antifungal activity of atorvastatin and assess its effect on the virulence of A. fumigatus. Atorvastatin demonstrated strong antifungal activity and reduced the growth and viability of A. fumigatus. Exposure of A. fumigatus to atorvastatin led to a reduction in ergosterol content and increased membrane permeability, as evidenced by the release of protein, amino acids and gliotoxin. Proteomic analysis revealed an increased abundance of proteins associated with an oxidative stress response, such as the glutathione s-transferase family protein (+8.43-fold), heat shock protein Hsp30/Hsp42 (+2.02-fold) and 5-demethoxyubiquinone hydroxylase, mitochondrial (+1.73-fold), as well as secondary metabolites such as isocyanide synthase A icsA (+8.52-fold) and non-ribosomal peptide synthetase fmpE (+3.06-fold). The results presented here indicate that atorvastatin has strong antifungal properties and may have potential application in the treatment of A. fumigatus infections alone or in combination with existing antifungal agents.

The larvae of the insect Galleria mellonella , have recently been established as a non-mammalian infection model for the Mycobacterium tuberculosis complex (MTBC). To gain further insight into the potential of this model, we applied proteomic (label-free quantification) and transcriptomic (gene expression) approaches to characterise the innate immune response of G. mellonella to infection with Mycobacterium bovis BCG lux over a 168 h time course. Proteomic analysis of the haemolymph from infected larvae revealed distinct changes in the proteome at all time points (4, 48, 168 h). Reverse transcriptase quantitative PCR confirmed induction of five genes ( gloverin , cecropin , IMPI , hemolin , and Hdd11 ), which encoded proteins found to be differentially abundant from the proteomic analysis. However, the trend between gene expression and protein abundance were largely inconsistent (20%). Overall, the data are in agreement with previous phenotypic observations such as haemocyte internalization of mycobacterial bacilli (hemolin/β-actin), formation of granuloma-like structures (Hdd11), and melanization (phenoloxidase activating enzyme 3 and serpins). Furthermore, similarities in immune expression in G. mellonella , mouse, zebrafish and in vitro cell-line models of tuberculosis infection were also identified for the mechanism of phagocytosis (β-actin). Cecropins (antimicrobial peptides), which share the same α-helical motif as a highly potent peptide expressed in humans (h-CAP-18), were induced in G. mellonella in response to infection, giving insight into a potential starting point for novel antimycobacterial agents. We believe that these novel insights into the innate immune response further contribute to the validation of this cost-effective and ethically acceptable insect model to study members of the MTBC.

Background. Current or recent use of abacavir for treating human immunodeficiency virus type 1 (HIV-1) infection has been associated with increased rates of myocardial infarction (MI). Given the role of platelet aggregation in thrombus formation in MI and the reversible nature of the abacavir association, we hypothesized that patients treated with abacavir would have increased platelet reactivity. Methods. In a prospective study in adult HIV-infected patients, we determined associations between antiretro virais (ARVs), and in particular the nucleoside reverse transcriptase inhibitor abacavir, and platelet reactivity by measuring time-dependent platelet aggregation in response to agonists: adenosine diphosphate (ADP), thrombin receptor-activating peptide (TRAP), collagen, and epinephrine. Results. Of 120 subjects, 40 were ARV-naive and 80 ARV-treated, 40 of whom were receiving abacavir. No consistent differences in platelet reactivity were observed between the ARV-naive and ARV-treated groups. In contrast, within the ARV-treated group, abacavir-treated subjects had consistently higher percentages of platelet aggregation upon exposure to ADP, collagen, and epinephrine (P = .037, P = .022, and P = .032, respectively) and had platelets that were more sensitive to aggregation upon exposure to TRAP (P = .025). Conclusions. The consistent increases in platelet reactivity observed in response to a range of agonists provides a plausible underlying mechanism to explain the reversible increased rates of MI observed in abacavir-treated patients.