Macrophage and neutrophil death programs differentially confer resistance to tuberculosis.

Apoptosis can potently defend against intracellular pathogens by directly killing microbes and eliminating their replicative niche. However, the reported ability of Mycobacterium tuberculosis to restrict apoptotic pathways in macrophages in vitro has led to apoptosis being dismissed as a host-protective process in tuberculosis despite a lack of in vivo evidence. Here we define crucial in vivo functions of the death receptor-mediated and BCL-2-regulated apoptosis pathways in mediating protection against tuberculosis by eliminating distinct populations of infected macrophages and neutrophils and priming T cell responses. We further show that apoptotic pathways can be targeted therapeutically with clinical-stage compounds that antagonize inhibitor of apoptosis (IAP) proteins to promote clearance of M. tuberculosis in mice. These findings reveal that any inhibition of apoptosis by M. tuberculosis is incomplete in vivo, advancing our understanding of host-protective responses to tuberculosis (TB) and revealing host pathways that may be targetable for treatment of disease.

Combined immunodeficiency and impaired PI3K signaling in a patient with biallelic LCP2 variants.

BACKGROUND: Inborn errors affecting components of the T-cell receptor signaling cascade cause combined immunodeficiency with various degrees of severity. Recently, homozygous variants in LCP2 were reported to cause pediatric onset of severe combined immunodeficiency with neutrophil, platelet, and T- and B-cell defects. OBJECTIVE: We sought to unravel the genetic cause of combined immunodeficiency and early-onset immune dysregulation in a 26-year-old man who presented with specific antibody deficiency, autoimmunity, and inflammatory bowel disease since early childhood. METHODS: The patient was subjected to whole-exome sequencing of genomic DNA and examination of blood neutrophils, platelets, and T and B cells. Expression levels of the Src homology domain 2-containing leukocyte protein of 76 kDa (SLP76) and tonic and ligand-induced PI3K signaling were evaluated by flow-cytometric detection of phosphorylated ribosomal protein S6 in B and T cells. RESULTS: Compound heterozygous missense variants were identified in LCP2, affecting the proline-rich repeat domain of SLP76 (p.P190R and p.R204W). The patient's total B- and T-cell numbers were within the normal range, as was platelet function. However, neutrophil function, numbers of unswitched and class-switched memory B cells, and serum IgA were decreased. Moreover, intracellular SLP76 protein levels were reduced in the patient's B cells, CD4+ and CD8+ T cells, and natural killer cells. Tonic and ligand-induced levels of phosphorylated ribosomal protein S6 and ligand-induced phosphorylated PLCγ1 were decreased in the patient's B cells and CD4+ and CD8+ T cells. CONCLUSIONS: Biallelic variants in LCP2 impair neutrophil function and T-cell and B-cell antigen-receptor signaling and can cause combined immunodeficiency with early-onset immune dysregulation, even in the absence of platelet defects.

Utility of repeat extractable nuclear antigen antibody testing: a retrospective audit.

OBJECTIVES: Autoantibodies to ENA are frequently ordered during the workup of suspected autoimmune connective tissue diseases. There are no current guidelines for repeat test ordering. The objective of this study was to assess the utility of repeat ENA testing after an initial negative result. METHODS: A retrospective study was conducted in a single, multicentre tertiary health network in Melbourne, Australia. Results of all ENA tests were extracted from the hospital laboratory information system. For patients who had a change in ENA result from negative to positive, clinical information was obtained from the hospital records regarding new diagnosis of an ANA-associated rheumatic disease (AARD). RESULTS: A total of 23 438 ENA tests were performed in 19 603 patients from 29 July 2013 to 28 September 2020. In total, 20 918 (89.2%) were negative with 215 (0.9%) being equivocal. Of the 2305 positive tests, the most common ENA auto-antibody specificity detected was anti-Ro52 (1185, 51.4%). A total of 2636 of 19 603 patients (13.4%) had more than one ENA test performed during the study period. Of these, most (2523, 95.7%) had stable ENA results with no change compared with the first test. Only 53 patients (2.2%) had an ENA result that changed from negative to positive. Excluding patients with pre-existing rheumatic conditions and those under 18, there were five new AARDs found in the remaining 34 patients. CONCLUSION: Repeat ENA test results rarely change or result in a new diagnosis of an AARD, with repeated testing only warranted if there is a change in clinical manifestations.

Pediatric Staphylococcus aureus Bacteremia: Clinical Spectrum and Predictors of Poor Outcome.

BACKGROUND: Staphylococcus aureus is a common cause of bacteremia, yet the epidemiology and predictors of poor outcome remain inadequately defined in childhood. METHODS: ISAIAH (Invasive Staphylococcus aureus Infections and Hospitalizations in children) is a prospective, cross-sectional study of S. aureus bacteremia (SAB) in children hospitalized in Australia and New Zealand over 24 months (2017-2018). RESULTS: Overall, 552 SABs were identified (incidence 4.4/100 000/year). Indigenous children, those from lower socioeconomic areas and neonates were overrepresented. Although 90-day mortality was infrequent, one-third experienced the composite of: length of stay >30 days (26%), intensive care unit admission (20%), relapse (4%), or death (3%). Predictors of mortality included prematurity (adjusted odds ratio [aOR],16.8; 95% confidence interval [CI], 1.6-296.9), multifocal infection (aOR, 22.6; CI, 1.4-498.5), necrotizing pneumonia (aOR, 38.9; CI, 1.7-1754.6), multiorgan dysfunction (aOR, 26.5; CI, 4.1-268.8), and empiric vancomycin (aOR, 15.7; CI, 1.6-434.4); while infectious diseases (ID) consultation (aOR, 0.07; CI .004-.9) was protective. Neither MRSA nor vancomycin trough targets impacted survival; however, empiric vancomycin was associated with nephrotoxicity (OR, 3.1; 95% CI 1.3-8.1). CONCLUSIONS: High SAB incidence was demonstrated and for the first time in a pediatric setting, necrotizing pneumonia and multifocal infection were predictors of mortality, while ID consultation was protective. The need to reevaluate pediatric vancomycin trough targets and limit unnecessary empiric vancomycin exposure to reduce poor outcomes and nephrotoxicity is highlighted. One in 3 children experienced considerable SAB morbidity; therefore, pediatric inclusion in future SAB comparator trials is paramount to improve outcomes.

Congenital asplenia study: clinical and laboratory characterisation of adults with congenital asplenia.

Congenital asplenia is a rare disorder commonly associated with other visceral and cardiac congenital anomalies. Isolated congenital asplenia is even less common than syndromic forms. The risk of severe bacterial infections associated with asplenia is the most concerning clinical implication and carries a significant mortality risk. Prophylactic measures against the clinical syndrome known as overwhelming postsplenectomy infections (OPSI) include vaccination, prophylactic and emergency antibiotics and health education including fever management and travel advice. This case series describes fourteen adults with congenital asplenia and polysplenia syndrome, most of whom were diagnosed incidentally as adults, and outlines the nature of their diagnosis, clinical phenotype, family history and key pathology findings.

Underutilised human immunodeficiency virus testing in the setting of invasive pneumococcal disease.

People living with human immunodeficiency virus (HIV) are at increased risk of invasive pneumococcal disease (IPD). We assessed whether patients with invasive Streptococcus pneumoniae, in blood or cerebrospinal fluid, underwent HIV serology testing over a 5-year period. We found that only 39 inpatients out of 156 (25%) with IPD were tested for HIV and thus conclude that such testing is not being undertaken according to some guidelines in patients with IPD. Education and implementation strategies are required to increase testing.

Prevalence and distribution of functional splenic tissue after splenectomy.

BACKGROUND: Individuals splenectomised for trauma have lower infection rates than those splenectomised for other conditions. Residual functional splenic tissue (FST) after splenectomy may provide ongoing immunological protection. AIMS: To quantify the prevalence and volume of residual FST post-splenectomy using standard testing. METHODS: Splenectomised adults were recruited from the Spleen Australia clinical registry. Eligible individuals had been splenectomised at least 1 year prior to their visit and resided in Victoria. Splenic function was identified by evaluating Howell-Jolly bodies and IgM memory B cells. A 99m-Technetium-labelled, heat-denatured erythrocyte scintigraphic scan was performed if splenic function was detected. RESULTS: Initially, 75 splenectomised individuals (all cause) were recruited, with a median of 58 years of age and who were splenectomised a median of 14 years previously. The most common indications for splenectomy were trauma (30.7%) and haematological disease (28.0%). Scintigraphy identified FST in nine individuals (12.0%). Eight had been splenectomised for trauma. In this cohort, 34.8% of individuals splenectomised for trauma had residual FST. To explore our findings further, 45 additional individuals were recruited, predominately individuals splenectomised for trauma. Twenty-five individuals completed assessments by December 2018. An additional 11 individuals had FST, of whom 9 had been splenectomised for trauma. Overall, we identified 20 individuals with residual FST. Volumes ranged from 2.2 to 216.0 cc. We saw individuals with accessory spleens and splenotic nodules and an individual with both. Seventeen individuals had been splenectomised for trauma. CONCLUSIONS: Residual FST is commonly seen in individuals splenectomised for trauma. It can present in varying distributions and of varying volume. The clinical significance is unclear.

Human immunodeficiency virus-infected young people in Australia: data from the Australian HIV Observational Database.

BACKGROUND: Individuals aged 13-24 years undergo vast physical, cognitive, social and psychological changes. Australian data regarding clinical outcomes of those diagnosed with HIV in this age are sparse. AIM: We aimed to describe demographic factors, virologic and clinical outcomes of individuals aged 13-24 years diagnosed with human immunodeficiency virus (HIV). METHODS: Patients diagnosed with HIV after 1997 in the Australian HIV Observational Database were divided into young adults, diagnosed at age <25 years (n = 223), and older adults (n = 1957). Demographic and clinical factors were compared between groups. RESULTS: Young adults had a median age at diagnosis of 22 years (inter quartile range (IQR) 20-24) and median age at treatment initiation of 24 years (IQR 22-26). They were more likely to be female than the older cohort (21.1 vs 10.8%; P < 0.001). Men who have sex with men was the most common exposure category in both groups. CD4 count at diagnosis was significantly higher in younger than older adults (median 460 vs 400 cells/mm3 , P = 0.006), whereas HIV viral load at diagnosis was lower (35 400 vs 61 659 copies/mL, P = 0.011). The rate of loss to follow up (LTFU) was higher in young adults (8.0 vs 4.3 per 100PY, P < 0.001). Young adults were more likely to have a treatment interruption compared to older adults (5.3 vs 4.0 per 100PY, P = 0.039). Rates of treatment switch, time to treatment change, and CD4 and viral load responses to treatment were similar between groups. CONCLUSIONS: Young adults were diagnosed with HIV at higher CD4 counts and lower viral loads than their older counterparts. LTFU and treatment interruption were more common highlighting the need for extra efforts directed towards retention in care and education regarding the risks of treatment interruptions.

Eliminating hepatitis B by antagonizing cellular inhibitors of apoptosis.

We have shown that cellular inhibitor of apoptosis proteins (cIAPs) impair clearance of hepatitis B virus (HBV) infection by preventing TNF-mediated killing/death of infected cells. A key question, with profound therapeutic implications, is whether this finding can be translated to the development of drugs that promote elimination of infected cells. Drug inhibitors of cIAPs were developed as cancer therapeutics to promote TNF-mediated tumor killing. These drugs are also known as Smac mimetics, because they mimic the action of the endogenous protein Smac/Diablo that antagonizes cIAP function. Here, we show using an immunocompetent mouse model of chronic HBV infection that birinapant and other Smac mimetics are able to rapidly reduce serum HBV DNA and serum HBV surface antigen, and they promote the elimination of hepatocytes containing HBV core antigen. The efficacy of Smac mimetics in treating HBV infection is dependent on their chemistry, host CD4(+) T cells, and TNF. Birinapant enhances the ability of entecavir, an antiviral nucleoside analog, to reduce viral DNA production in HBV-infected animals. These results indicate that birinapant and other Smac mimetics may have efficacy in treating HBV infection and perhaps, other intracellular infections.

Cellular inhibitor of apoptosis proteins prevent clearance of hepatitis B virus.

Hepatitis B virus (HBV) infection can result in a spectrum of outcomes from immune-mediated control to disease progression, cirrhosis, and liver cancer. The host molecular pathways that influence and contribute to these outcomes need to be defined. Using an immunocompetent mouse model of chronic HBV infection, we identified some of the host cellular and molecular factors that impact on infection outcomes. Here, we show that cellular inhibitor of apoptosis proteins (cIAPs) attenuate TNF signaling during hepatitis B infection, and they restrict the death of infected hepatocytes, thus allowing viral persistence. Animals with a liver-specific cIAP1 and total cIAP2 deficiency efficiently control HBV infection compared with WT mice. This phenotype was partly recapitulated in mice that were deficient in cIAP2 alone. These results indicate that antagonizing the function of cIAPs may promote the clearance of HBV infection.

Vitamin D deficiency impacts on expression of toll-like receptor-2 and cytokine profile: a pilot study.

BACKGROUND: Vitamin D is believed to play an important role outside the endocrine system in the regulation of the immune system, and in cellular proliferation and differentiation. The aim of the study was to investigate the impact of vitamin D levels on innate immunity. METHODS: Participants for this prospective, longitudinal study were recruited amongst otherwise healthy staff of a large hospital in Victoria, Australia. Those fulfilling the inclusion criteria, including a vitamin D level of <50 nmol/L, were supplemented. Using flow cytometry, expression of the innate immune receptors TLR2, TLR4 and CD86 was measured on peripheral blood mononuclear cells (PBMCs) collected prior to vitamin D treatment and then at 1 and 3 months. Additonally, PBMCs at each timepoint were stimulated with specific TLR ligands and resultant supernatants were assayed for the cytokines TNFα, IL-6, IFN-α and IP-10. RESULTS: In participants whose vitamin D level was >100 nmol/L post supplementation (n=11), TLR2 expression on PBMCs increased significantly, with no change noted in TLR4 or CD86 expression. Stimulation of vitamin D deficient samples with TLR ligands produced a number of proinflammatory cytokines, which were significantly reduced upon vitamin D normalisation. In patients whose levels returned to a deficient level at 3 months despite ongoing low-level supplementation, an increase in the pro-inflamamtory state returned. This suggests that vitamin D may play an important role in ensuring an appropriate baseline pro-inflammatory state. CONCLUSIONS: This ex-vivo pilot study adds clinical evidence supporting a possibly important role for vitamin D in innate immunity. If confirmed, this unique clinical study has potentially significant implications for the treatment of a variety of inflammatory conditions, where achieving optimal vitamin D levels may help reduce inflammation.

Frequency and Clinical Utility of Antibodies to Extractable Nuclear Antigen in the Setting of a Negative Antinuclear Antibody Test.

OBJECTIVE: Simultaneous antibody testing during screening for autoimmune conditions is discouraged. The incidence of positive extractable nuclear antigen (ENA) in the setting of a negative antinuclear antibody (ANA) has been reported as low. Our objective was to characterize the frequency of diagnosis of new ANA-associated rheumatic disease (AARD) in the setting of a negative ANA with a positive ENA. METHODS: This was a 7-year retrospective study from a multicenter tertiary health network in Australia. Clinical information was sought on patients over 18 years old who had a negative ANA but positive ENA test result. Results were extracted from hospital computer systems. RESULTS: From March 19, 2011, to July 23, 2018, ENA testing was ordered simultaneously with an ANA test on 4,248 occasions in 3,484 patients. ANA was positive in 2,520 patients (59.3%) and ENA was positive in 1,980 patients (46.6%). Among positive ANA patients, ENA was positive in 1,563 patients (62.0%). Among 1,728 negative ANA tests, ENA was positive in 417 (24.1%) (P < 0.001). A total of 328 patients with discordant ANA/ENA results had data available for further analysis, of whom 279 had no pre-established rheumatologic condition. A new AARD was diagnosed in 17 of 279 patients, yielding a positive predictive value of 6.09% (95% confidence interval 3.59-9.58). CONCLUSION: Despite the higher-than-expected incidence of positive ENA in the setting of a negative ANA, the yield of newly diagnosed rheumatic diseases was low. Our findings support the stepwise addition of ENA requests when an ANA test result is positive and clinical suspicion of an AARD is high.

Inborn Errors of Immunity in Children With Invasive Pneumococcal Disease: A Multicenter Prospective Study.

BACKGROUND: In settings with universal conjugate pneumococcal vaccination, invasive pneumococcal disease (IPD) can be a marker of an underlying inborn error of immunity. The aim of this study was to determine the prevalence and characterize the types of immunodeficiencies in children presenting with IPD. METHODS: Multicenter prospective audit following the introduction of routinely recommended immunological screening in children presenting with IPD. The minimum immunological evaluation comprised a full blood examination and film, serum immunoglobulins (IgG, IgA and IgM), complement levels and function. Included participants were children in whom Streptococcus pneumoniae was isolated from a normally sterile site (cerebrospinal fluid, pleura, peritoneum and synovium). If isolated from blood, features of sepsis needed to be present. Children with predisposing factors for IPD (nephrotic syndrome, anatomical defect or malignancy) were excluded. RESULTS: Overall, there were 379 episodes of IPD of which 313 (83%) were eligible for inclusion and 143/313 (46%) had an immunologic evaluation. Of these, 17/143 (12%) were diagnosed with a clinically significant abnormality: hypogammaglobulinemia (n = 4), IgA deficiency (n = 3), common variable immunodeficiency (n = 2), asplenia (n = 2), specific antibody deficiency (n = 2), incontinentia pigmenti with immunologic dysfunction (n = 1), alternative complement deficiency (n = 1), complement factor H deficiency (n = 1) and congenital disorder of glycosylation (n = 1). The number needed to investigate to identify 1 child presenting with IPD with an immunologic abnormality was 7 for children under 2 years and 9 for those 2 years old and over. CONCLUSIONS: This study supports the routine immune evaluation of children presenting with IPD of any age, with consideration of referral to a pediatric immunologist.

Missed Opportunities? A Retrospective Study Into Adults Hospitalized With Invasive Infection From Airway Pathogens.

Background: Invasive disease caused by airway pathogens, including Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, and Moraxella catarrhalis, has high morbidity and mortality worldwide, with immunodeficiency being a known association with recurrent disease. The study aimed to describe the frequency of known immunodeficiency and predisposing factors in adult patients presenting with invasive infections and determine the frequency of screening for and detection of immunodeficiency. Methods: A retrospective analysis was conducted at a large tertiary Australian health service, comprising multiple centers. Patients aged 18 years or older, in whom the above pathogens were isolated from sterile sites, were included as identified through a microbiology database, between 2015 and 2020. Using electronic medical records, patient demographics, medical history, outcomes of admission, and pathology results were captured and reviewed to address the aims. Results: In 252 patients, S pneumoniae was the most common culprit, isolated in 73% (185/252), compared to 14.3% (36/252) and 11.5% (29/252) of infections caused by H influenzae and N meningitidis, respectively. Known diagnoses of secondary immunodeficiency were common (31% of patients). Of those presenting with invasive pneumococcal disease, 78% had at least 1 predisposing condition, though only 9 patients (6%) had previously received pneumococcal vaccination. Despite poor screening for immunodeficiency, 12 new diagnoses were made. While the commonest immunodeficiency was secondary, due to hematological and solid organ malignancies, 3 new primary immunodeficiency diagnoses were made. Conclusions: Immunodeficiency is common in this patient population. Screening should be undertaken to ensure timely diagnosis and treatment of the underlying condition to avoid future morbidity and mortality.

Whole genome sequencing and molecular epidemiology of paediatric Staphylococcus aureus bacteraemia.

OBJECTIVES: The role Staphylococcus aureus antimicrobial resistance genes and toxins play in disease severity, management and outcome in childhood is an emerging field requiring further exploration. METHODS: A prospective multisite study of Australian and New Zealand children hospitalised with S. aureus bacteraemia (SAB) occurred over 24 months (2017-2018). Whole genome sequencing (WGS) data were paired with clinical information from the ISAIAH cohort. RESULTS: 353 SAB isolates were sequenced; 85% methicillin-susceptible S. aureus ([MSSA], 301/353) and 15% methicillin-resistant S. aureus ([MRSA], 52/353). There were 92 sequence types (STs), most commonly ST5 (18%) and ST30 (8%), grouped into 23 clonal complexes (CCs), most frequently CC5 (21%) and CC30 (12%). MSSA comprised the majority of healthcare-associated SAB (87%, 109/125), with principal clones CC15 (48%, 11/21) and CC8 (33%, 7/21). Panton-Valentine leukocidin (PVL)-positive SAB occurred in 22% (76/353); predominantly MSSA (59%, 45/76), community-onset (92%, 70/76) infections. For community-onset SAB, the only microbiological independent predictor of poor outcomes was PVL positivity (aOR 2.6 [CI 1.0-6.2]). CONCLUSION: From this WGS paediatric SAB data, we demonstrate the previously under-recognized role MSSA has in harbouring genetic virulence and causing healthcare-associated infections. PVL positivity was the only molecular independent predictor of poor outcomes in children. These findings underscore the need for further research to define the potential implications PVL-producing strains may have on approaches to S. aureus clinical management.

Curtobacterium flaccumfaciens septic arthritis following puncture with a Coxspur Hawthorn thorn.

Curtobacterium species are recognized plant pathogens. We report the first well-documented case of Curtobacterium human infection, a child with septic arthritis following puncture with a Coxspur Hawthorn plant thorn. The organism isolated from synovial tissue and the plant thorn was identified as Curtobacterium flaccumfaciens by 16S rRNA gene sequence analysis.

Bartonella henselae masquerading as possible gamma-delta T-cell lymphoma in a paediatric patient with 22q11.2 deletion syndrome.

A 14-year-old boy with 22q11.2 deletion syndrome and a right ventricular to pulmonary artery xenograft conduit presented to an Australian tertiary children's hospital with prolonged fevers, weight loss, splenomegaly and a high proportion of gamma-delta T cells in peripheral blood and bone marrow, concerning for possible gamma-delta T-cell lymphoma. However, investigations did not reveal evidence of lymphoma or autoimmune disease. After 5 months of intermittent fever episodes and ongoing symptoms, he was found to have an extremely high Bartonella henselae titre (8192) on serological testing, with the organism also detected on blood PCR. After 6 months of oral azithromycin and rifampicin, with complete resolution of his symptoms 3 months into treatment, his blood PCR was negative and gamma-delta T cells in peripheral blood were decreasing. The B. henselae titre remained unchanged for some time, but decreased to 2048 around 1 year after treatment was started.