Post-acute COVID-19 condition (PACC): a perspective on collaborative Australian research imperatives and primary health models of care.

Post-acute COVID-19 condition (PACC) - also known as long COVID - is a serious and growing problem in primary health. This letter describes the work of the Victorian Post-Acute COVID-19 Study (VPACS) group, which comprises clinician researchers, basic scientists and consumers. Two key priorities for PACC research in Australia are identified and discussed: (1) the establishment of COVID-19 patient registries and data linkage; and (2) the consolidation of clinical guidelines. Collaboration between consumers, researchers, clinicians and institutions must be the foundation of PACC management in Australia. Ongoing research should focus on large, multicentre controlled studies, the protective effect of vaccination, differential impacts from variants, pathobiological underpinnings, disease mechanisms to avoid severe and enduring impacts on the Australian economy. The lived experience of people with PACC is also essential to enable the design and implementation of effective models of care. VPACS brings a diverse group of people together to work on a shared vision of holistic and high-quality care, and collectively maximise their impact on outcomes for patients and the broader community.

Actinomyces spp. bloodstream and deep vein thrombus infections in people who inject drugs.

INTRODUCTION: Actinomyces spp. cause several well-described syndromes including cervicofacial and pelvic infections. Actinomyces spp. infection as an opportunistic infection among people who inject drugs has rarely been described with few case reports published. METHODS AND RESULTS: Here we describe four people who inject drugs admitted with Actinomyces spp. infections, all with an overlapping syndrome and who presented a challenge to both diagnose and to manage. DISCUSSION: This case series highlights the potential to overlook Actinomyces spp. infection in people who inject drugs and aims to increase clinician awareness of diagnosis, empirical and directed treatment, and potential complications of this infection.

Mycobacterium abscessus complex: Natural history and treatment outcomes at a tertiary adult cystic fibrosis center.

Background: Mycobacterium abscessus complex (MAbsC) is a significant management dilemma when taking care of patients with cystic fibrosis (CF). Methods: We undertook a retrospective cohort analysis of all CF patients in whom MAbsC was isolated from 2005 to 2014. The natural history of MAbsC was determined and clinical factors examined in an attempt to predict transient compared to persistent colonization. Results: No correlation was found between recurrent MAbsC isolation and clinical factors such as body mass index, respiratory function, or age. Over two-thirds of our cohort cleared MAbsC colonization with no intervention and no consistent effect on lung function was identified. Four CF patients were initiated on treatment with only one successful outcome. Conclusion: This analysis demonstrates there are no clear predictors of those CF patients who will become persistently colonized with MAbsC and that a significant proportion will spontaneously clear carriage. As treatment success rate is poor, more work is urgently required in improving patient outcomes.

Benign acute childhood myositis following human parainfluenza virus type-1 infection.

Benign acute childhood myositis (BACM) is a post-respiratory tract infection condition of school-age children. Presentation is typically with acute onset calf pain and tenderness and refusal to walk or altered gait during the convalescent period of an influenza A or B infection. We describe a unique cluster of children with BACM following infection with human parainfluenza 1 virus, with no evidence of influenza A or B infection. BACM is a commonly missed diagnosis of altered gait in children presenting to the emergency department. This is the first report to describe a cluster of human parainfluenza virus type-1 associated BACM. We discuss the presentation, clinical examination and investigation results of the children identified. Furthermore, we review the current research surrounding BACM, overview the clinical presentation to healthcare professionals, and present an interesting case of a child presenting for the second time with BACM.

Characterization of tetraspanins CD9, CD53, CD63, and CD81 in monocytes and macrophages in HIV-1 infection.

Tetraspanins are a family of membrane-organizing proteins that mediate diverse functions. Little is known of their expression or function in myeloid cells. Here, expression of CD9, CD53, CD63, and CD81, tetraspanins that have been implicated in HIV-1 pathogenesis, were characterized in normal monocyte subsets, in MDM, and in HIV-1-infected donors. We show that tetraspanins are expressed differentially by monocyte subsets, with higher CD9 and CD63 and lower CD53 and CD81 levels on CD14++CD16- monocytes compared with CD14++CD16+ and CD14+CD16++ subsets. Maturation of monocytes resulted in increased CD9 expression and apparent relocation of CD63 and CD53 from surface to intracellular membranes. Expression was modulated by cytokines, and CD9 was a marker of anti-inflammatory and CD53 a marker of proinflammatory MDM. Tetraspanin expression on monocyte subsets from HIV-1-infected donors receiving antiretroviral therapy was unchanged compared with that in uninfected donors. However, CD53 expression was inversely correlated with viral load in HIV-1-infected donors not on therapy. This study is the first to comprehensively characterize tetraspanin expression on monocyte subsets and macrophages in health and during HIV-1 infection. It demonstrates regulation of tetraspanin expression by cytokines, and CD53 expression as a novel correlate of a proinflammatory phenotype. This paper characterizes tetraspanins in myeloid cells and shows that tetraspanins are expressed differentially in monocyte subsets and are modified in inflammatory conditions.

HIV-1 inhibits phagocytosis and inflammatory cytokine responses of human monocyte-derived macrophages to P. falciparum infected erythrocytes.

HIV-1 infection increases the risk and severity of malaria by poorly defined mechanisms. We investigated the effect of HIV-1(Ba-L) infection of monocyte-derived macrophages (MDM) on phagocytosis of opsonised P. falciparum infected erythrocytes (IE) and subsequent proinflammatory cytokine secretion. Compared to mock-infected MDM, HIV-1 infection significantly inhibited phagocytosis of IE (median (IQR) (10 (0-28) versus (34 (27-108); IE internalised/100 MDM; p = 0.001) and decreased secretion of IL-6 (1,116 (352-3,387) versus 1,552 (889-6,331); pg/mL; p = 0.0078) and IL-1ß (16 (7-21) versus 33 (27-65); pg/mL; p = 0.0078). Thus inadequate phagocytosis and cytokine production may contribute to impaired control of malaria in HIV-1 infected individuals.

Differential expression of CD163 on monocyte subsets in healthy and HIV-1 infected individuals.

CD163, a haptoglobin-hemoglobin (Hp-Hb) scavenger receptor, expressed by monocytes and macrophages, is important in resolution of inflammation. Age-related non-AIDS co-morbidities in HIV-infected individuals, particularly dementia and cardiovascular disease, result in part from effects of HIV-1 infection on monocyte and macrophage biology. CD163 co-expression on CD14+CD16++ monocytes has been proposed as a useful biomarker for HIV-1 disease progression and the presence of HIV associated dementia. Here we investigated CD163 expression on monocyte subsets ex vivo, on cultured macrophages, and soluble in plasma, in the setting of HIV-1 infection. Whole blood immunophenotyping revealed CD163 expression on CD14++CD16- monocytes but not on CD14+CD16++ monocytes (P = 0.004), supported by CD163 mRNA levels. Incubation with M-CSF induced CD163 protein expression on CD14+CD16++ monocytes to the same extent as CD14++CD16- monocytes. CD163 expression on CD14++CD16+ monocytes from HIV-infected subjects was significantly higher than from uninfected individuals, with a trend towards increased expression on CD14++CD16- monocytes (P = 0.019 and 0.069 respectively), which is accounted for by HIV-1 therapy including protease inhibitors. Shedding of CD163 was shown to predominantly occur from the CD14++CD16- subset after Ficoll isolation and LPS stimulation. Soluble CD163 concentration in plasma from HIV-1 infected donors was similar to HIV-1 uninfected donors. Monocyte CD163 expression in HIV-1 infected patients showed a complicated relationship with classical measures of disease progression. Our findings clarify technical issues regarding CD163 expression on monocyte subsets and further elucidates its role in HIV-associated inflammation by demonstrating that CD163 is readily lost from CD14++CD16- monocytes and induced in pro-inflammatory CD14+CD16++ monocytes by M-CSF. Our data show that all monocyte subsets are potentially capable of differentiating into CD163-expressing anti-inflammatory macrophages given appropriate stimuli. Levels of CD163 expression on monocytes may be a potential biomarker reflecting efforts by the immune system to resolve immune activation and inflammation in HIV-infected individuals.

Ghrelin in gastrointestinal disease.

Enteroendocrine cells of the gastric fundus are the predominant source of ghrelin production, although ghrelin gene transcripts and ghrelin-producing cells have been identified throughout the gastrointestinal tract. Various infectious, inflammatory and malignant disorders of the gastrointestinal system have been shown to alter ghrelin production and secretion and consequently to affect endocrine ghrelin levels and activity. Animal studies have demonstrated that ghrelin and synthetic ghrelin mimetics can reduce the severity of gastric and colonic inflammation and human clinical trials are underway to determine the efficacy of ghrelin in improving motility disorders. This review summarises the impact of gastrointestinal disease on ghrelin synthesis and secretion and the potential use of ghrelin and its mimetics for the treatment of these diseases.

Extremely prolonged HIV seroconversion associated with an MHC haplotype carrying disease susceptibility genes for antibody deficiency disorders.

Severe immunodeficiency during primary human immunodeficiency virus (HIV) infection is unusual. Here, we characterized viral and immunological parameters in a subject presenting with Pneumocystis jirovecii pneumonia in the setting of prolonged primary HIV illness and delayed seroconversion. HIV antibody was only detected by enzyme-linked immunosorbent assay 12 months after presentation, and Western blot profiles remain indeterminate. Isolated virus was of R5 phenotype, exhibited poor viral fitness, but was otherwise unremarkable. Analysis of HIV antibody isotypes showed failure to mount a detectable HIV IgG response over nearly 2 years of infection, in particular IgG(1)- and IgG(3)-specific responses, despite normal responses to common infections and vaccines. Genetic analysis demonstrated homozygosity for part of an MHC haplotype containing susceptibility genes for common variable immunodeficiency (CVID) syndrome and other antibody deficiency disorders. Thus, a primary disorder of specific antibody production may explain exceptionally slow antibody development in an otherwise severe seroconversion illness. This highlights the need for multiparameter testing, in particular use of a fourth generation HIV test, for confirming HIV infection and underscores the importance of host factors in HIV pathogenesis.

A novel flow cytometric phagocytosis assay of malaria-infected erythrocytes.

Monocytes play a crucial role in controlling malaria infection. To facilitate our research into the development of antibody-mediated immunity against pregnancy-associated malaria we have established several novel malaria-specific flow cytometric phagocytosis assays based on ethidium bromide staining of DNA present in blood stage trophozoites. The first assay quantifies the ability of sera to opsonise trophozoites and promotes phagocytosis by the monocytic cell line THP1. This measures the levels of functional antibodies to the chosen strain of parasite. The second assay is a whole blood phagocytosis assay which measures the phagocytic ability of patient monocytes ex vivo. The third assay employs simultaneous labelling of trophozoites with ethidium bromide and erythrocytes with fluorescein isothiocyanate to compare phagocytosis of both non-infected and parasitised erythrocytes to assess possible bystander effects on uninfected erythrocytes. These assays have the advantage over other malaria phagocytosis assays in that they are rapid, simple and specific to malaria-infected cells and avoid potential bias associated with manual counting.

The CD16+ monocyte subset is more permissive to infection and preferentially harbors HIV-1 in vivo.

HIV-1 persists in peripheral blood monocytes in individuals receiving highly active antiretroviral therapy (HAART) with viral suppression, despite these cells being poorly susceptible to infection in vitro. Because very few monocytes harbor HIV-1 in vivo, we considered whether a subset of monocytes might be more permissive to infection. We show that a minor CD16+ monocyte subset preferentially harbors HIV-1 in infected individuals on HAART when compared with the majority of monocytes (CD14highCD16-). We confirmed this by in vitro experiments showing that CD16+ monocytes were more susceptible to CCR5-using strains of HIV-1, a finding that is associated with higher CCR5 expression on these cells. CD16+ monocytes were also more permissive to infection with a vesicular stomatitis virus G protein-pseudotyped reporter strain of HIV-1 than the majority of monocytes, suggesting that they are better able to support HIV-1 replication after entry. Consistent with this observation, high molecular mass complexes of apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G) were observed in CD16+ monocytes that were similar to those observed in highly permissive T cells. In contrast, CD14highCD16- monocytes contained low molecular mass active APOBEC3G, suggesting this is a mechanism of resistance to HIV-1 infection in these cells. Collectively, these data show that CD16+ monocytes are preferentially susceptible to HIV-1 entry, more permissive for replication, and constitute a continuing source of viral persistence during HAART.

Discrimination of live and early apoptotic mononuclear cells by the fluorescent SYTO 16 vital dye.

Accurate detection of apoptotic cells is important for the determination of cell viability. The aim of this study was to compare the sensitivity of the cell permeant SYTO 16 fluorescent dye for detecting early apoptotic mononuclear cells (MNCs) in normal donor blood with other apoptosis assays [i.e. Annexin-V, light scatter/7-amino-actinomycin-D (7-AAD) and chloromethyl-X-rosamine (CMXRos)] and to identify critical parameters for optimal SYTO 16 staining. Apoptosis was induced in normal human leukocytes from adult peripheral blood or cord blood, or the Jurkat T-lymphocytic cell line and assessed by fluorescence microscopy and flow cytometry. Dual labelling showed that SYTO 16 detected more apoptotic MNCs compared to Annexin-V. SYTO 16 staining intensity was consistent with the light scatter profiles expected of live, apoptotic and necrotic MNCs and was more objective than light scatter/7-AAD. CMXRos staining required considerable care and may not be a robust marker of apoptotic primary MNCs. For SYTO 16 flow cytometric analysis, the optimal conditions for staining 1x10(6) leukocytes were 4 nM SYTO 16 in the presence of 30 muM verapamil for 25-45 min at 37 degrees C in media containing calcium/magnesium supplemented with protein. A P-glycoprotein inhibitor, such as verapamil, and calcium/magnesium are essential for optimal loading of SYTO 16 into live MNCs and discrimination of apoptotic MNCs in normal blood samples. SYTO 16 is a sensitive, simple, inexpensive 'live cell' method for the discrimination of live, apoptotic and necrotic normal blood MNCs and is more sensitive for detecting apoptosis in these cells than Annexin-V or light scatter/7-AAD.

Poststorage leuko-depleted plasma inhibits T-cell proliferation and Th1 response in vitro: characterization of TGFbeta-1 as an important immunomodulatory component in stored blood.

BACKGROUND: Poststorage, leuko-depleted blood transfusions have been associated with increased postoperative infections and improved allograft survival compared with prestorage leukocyte-depleted blood transfusion. Although the mechanism of this phenomenon remains to be fully elucidated, it is clear that the immunomodulatory effect is mediated by leukocytes/platelets or their products. METHOD: The aim of this study was to investigate the in vitro effects of pre- and poststorage leuko-depleted plasma (LDP) and buffy coat LDP on T-cell proliferation and cytokine synthesis using multiparameter flow cytometry. RESULTS: In cell cultures exposed to prestorage LDP and buffy coat LDP there were no significant changes compared with fresh blood. In cell cultures exposed to poststorage LDP, T-cells showed reduced expression of CD69, CD25 (IL-2Ralpha), CD122 (IL-2Rbeta) and CD132 (IL-2Rtau) and production of TNF-alpha and IL-2 but there was no significant alteration for IFN-tau or IL-4. Changes in cytokine/cytokine receptor synthesis and T-cell proliferation were shown to be directly proportional to poststorage LDP concentration. Some of these changes were characteristic of TGFbeta-1. Addition of TGFbeta-1 neutralising antibody to poststorage LDP, negated the immunosuppressive effect on PHA-stimulated PBMC cultures. CONCLUSIONS: The decrease in T-cell proliferation and Th1 cytokines TNF-alpha and IL-2, may be one basis of altered immunoregulation resulting in increased rates of certain types of infections and increased graft tolerance reported in patients receiving poststorage LD blood transfusions. TGFbeta-1 is a major immunomodulatory component of poststorage LD blood transfusions.