Validation of an HIV whole genome sequencing method for HIV drug resistance testing in an Australian clinical microbiology laboratory.

Detection of HIV drug resistance (HIVDR) is vital to successful anti-retroviral therapy (ART). HIVDR testing to determine drug-resistance mutations is routinely performed in Australia to guide ART choice in newly diagnosed people living with HIV or in cases of treatment failure. In 2022, our clinical microbiology laboratory sought to validate a next-generation sequencing (NGS)-based HIVDR assay to replace the previous Sanger-sequencing (SS)-based ViroSeq. NGS solutions for HIVDR offer higher throughput, lower costs and higher sensitivity for variant detection. We sought to validate the previously described low-cost probe-based NGS method (veSEQ-HIV) for whole-genome recovery and HIVDR-testing in a diagnostic setting. veSEQ-HIV displayed 100% and 98% accuracy in major and minor mutation detection, respectively, and 100% accuracy of subtyping (provided > 1000 mapped reads were obtained). Pairwise comparison exhibited low inter-and intrarun variability across the whole-genome (Jaccard index [J] = 0.993; J = 0.972) and the Pol gene (J = 0.999; J = 0.999), respectively. veSEQ-HIV met all our pre-set criteria based on WHO recommendations and successfully replaced ViroSeq in our laboratory. Scaling-down veSEQ-HIV to a limited batch size and sequencing on Illumina iSeq. 100, allowed easy implementation of the assay into the workflow of a small sequencing laboratory with minimal staff and equipment and the ability to meet clinically relevant test turn-around times. As HIVDR-testing moves from SS- to NGS-based methods and new ART drugs come to market (particularly those with targets outside the Pol region), whole-genome recovery using veSEQ-HIV provides a robust, cost-effective and "future-proof" NGS method for HIVDR-testing.

HIV: the changing paradigm.

The past four decades have seen enormous progress in the diagnosis and management of human immunodeficiency virus (HIV) infection. There have been significant advances spanning the approval of the first antiretroviral agents, the advent of combination antiretroviral therapy to single tablet regimens with minimal toxicity. Although these remarkable developments have on the surface led to the 'end of AIDS', there are still key populations being left behind. This clinical update will describe the diagnosis and management of HIV, and the changing paradigms that have seen HIV transform from a life-limiting condition to a manageable chronic disease over a few decades.

Episodic fevers and vasodilatory shock mimicking urosepsis in a patient with HIV-associated multicentric Castleman's Disease: a case report.

BACKGROUND: Multicentric Castleman's disease (MCD) is a pre-malignancy that presents with lymphadenopathy and features of systemic inflammation. Human immunodeficiency virus (HIV)-associated MCD is associated with human herpesvirus-8 (HHV-8) infection. If untreated MCD has a relapsing and remitting course that is eventually fatal. CASE PRESENTATION: A 67-year-old man had six hospital admissions over 20 months characterised by fever, urinary frequency and CRP >100 mg/L. The final admission was complicated by hypotension requiring intensive care unit admission and ionotropic support. His history included HIV and Hepatitis B virus (HBV) co-infection on suppressive therapy. Each presentation was managed as presumed urosepsis with use of empirical antibiotics, however numerous blood and urine cultures failed to identify a pathogen. A bone-marrow aspirate and trephine found no evidence of haematological malignancy. A positron emission tomography scan found active lymph nodes, one of which was biopsied and found to contain the plasma-cell variant of Castleman's disease. Ultimately the cause for the recurrent presentations was attributed to progressive MCD. The patient received rituximab monotherapy and has had no further related admissions. CONCLUSIONS: MCD should be considered in patients with chronic HIV infection presenting with recurrent sepsis-like episodes and/or vasodilatory shock, particularly if no pathogen is identified or lymphadenopathy is evident.

Poor efficacy of oral tacrolimus in the treatment of severe generalized atopic eczema in adults: a small retrospective case series.

We report a small, but novel case series of four adults with severe generalized atopic eczema (AE) not responsive to several other immunomodulatory therapies, who were treated with oral tacrolimus (5 mg twice-daily). Three of the four patients failed therapy with systemic tacrolimus, despite two of these showing an initial clinical response; the fourth patient remains on tacrolimus monotherapy with good control of skin disease. Although oral tacrolimus was well-tolerated in this small group of adults, the clinical efficacy in this series for severe AE was poor. Tacrolimus may yet have a role in less severe disease, but larger prospective studies are required to qualify its place as a treatment option in AE.

Lack of standardisation in interpretation and reporting of autoantibody assays: a survey analysis of Australasian laboratories with focus on line immunoassays.

Autoantibody assays are reported in a variety of formats. Results only slightly above established cut-offs provide lower likelihood ratios; therefore, their clinical significance may be more uncertain, which is not readily communicated with dichotomous qualitative reporting. Line immunoassays (LIA) are a common method for detecting antibodies to extractable nuclear antigens (ENA) and myositis-associated antibodies. However, recommended positive cut-offs are contentious. We distributed a survey via e-mail to participants in the Royal College of Pathologists of Australasia Quality Assurance Program (RCPAQAP) Immunology modules and to a dedicated immunology mailing list in Australasia. Questions explored general viewpoints surrounding autoantibody reporting, as well as current laboratory practices, with particular focus on interpretation and reporting of the most commonly used ENA LIA manufactured by Euroimmun. There were 31 responders, representative of at least 17 unique laboratories across Australia (8 public, 5 private) and New Zealand (4 laboratories). Responses suggest that autoantibody reporting is not standardised; there was variation in general viewpoints and reporting practices, particularly regarding the interpretation of and positive cut-offs used for the Euroimmun ENA LIA, which were contrary to the manufacturer's guidelines in a majority of the responses. Interpretative qualitative reporting based on results from other investigations and the clinical history was a common theme. There is large variation in the reporting of autoantibody assays within Australasia, especially by LIA. A majority of respondents report the most commonly used ENA LIA contrary to manufacturer's guidelines; alternative positive cut-offs are commonly utilised. LIA reports should indicate the level of positivity to enhance their relevance in the clinical decision-making process.

Subtype-specific differences in transmission cluster dynamics of HIV-1 B and CRF01_AE in New South Wales, Australia.

INTRODUCTION: The human immunodeficiency virus 1 (HIV-1) pandemic is characterized by numerous distinct sub-epidemics (clusters) that continually fuel local transmission. The aims of this study were to identify active growing clusters, to understand which factors most influence the transmission dynamics, how these vary between different subtypes and how this information might contribute to effective public health responses. METHODS: We used HIV-1 genomic sequence data linked to demographic factors that accounted for approximately 70% of all new HIV-1 notifications in New South Wales (NSW). We assessed differences in transmission cluster dynamics between subtype B and circulating recombinant form 01_AE (CRF01_AE). Separate phylogenetic trees were estimated using 2919 subtype B and 473 CRF01_AE sequences sampled between 2004 and 2018 in combination with global sequence data and NSW-specific clades were classified as clusters, pairs or singletons. Significant differences in demographics between subtypes were assessed with Chi-Square statistics. RESULTS: We identified 104 subtype B and 11 CRF01_AE growing clusters containing a maximum of 29 and 11 sequences for subtype B and CRF01_AE respectively. We observed a > 2-fold increase in the number of NSW-specific CRF01_AE clades over time. Subtype B clusters were associated with individuals reporting men who have sex with men (MSM) as their transmission risk factor, being born in Australia, and being diagnosed during the early stage of infection (p < 0.01). CRF01_AE infections clusters were associated with infections among individuals diagnosed during the early stage of infection (p < 0.05) and CRF01_AE singletons were more likely to be from infections among individuals reporting heterosexual transmission (p < 0.05). We found six subtype B clusters with an above-average growth rate (>1.5 sequences / 6-months) and which consisted of a majority of infections among MSM. We also found four active growing CRF01_AE clusters containing only infections among MSM. Finally, we found 47 subtype B and seven CRF01_AE clusters that contained a large gap in time (>1 year) between infections and may be indicative of intermediate transmissions via undiagnosed individuals. CONCLUSIONS: The large number of active and growing clusters among MSM are the driving force of the ongoing epidemic in NSW for subtype B and CRF01_AE.

Regulation of axial and head patterning during planarian regeneration by a commensal bacterium.

Some animals, such as planaria, can regenerate complex anatomical structures in a process regulated by genetic and biophysical factors, but additional external inputs into regeneration remain to be uncovered. Microbial communities inhabiting metazoan organisms are important for metabolic, immune, and disease processes, but their instructive influence over host structures remains largely unexplored. Here, we show that Aquitalea sp. FJL05, an endogenous commensal bacterium of Dugesia japonica planarians, and one of the small molecules it produces, indole, can influence axial and head patterning during regeneration, leading to regeneration of permanently two-headed animals. Testing the impact of indole on planaria tissues via RNA sequencing, we find that indole alters the regenerative outcomes in planarians through changes in expression to patterning genes, including a downregulation of Wnt pathway genes. These data provide a unique example of the product of a commensal bacterium modulating transcription of patterning genes to affect the host's anatomical structure during regeneration.

Increased HIV Subtype Diversity Reflecting Demographic Changes in the HIV Epidemic in New South Wales, Australia.

Changes over time in HIV-1 subtype diversity within a population reflect changes in factors influencing the development of local epidemics. Here we report on the genetic diversity of 2364 reverse transcriptase sequences from people living with HIV-1 in New South Wales (NSW) notified between 2004 and 2018. These data represent >70% of all new HIV-1 notifications in the state over this period. Phylogenetic analysis was performed to identify subtype-specific transmission clusters. Subtype B and non-B infections differed across all demographics analysed (p < 0.001). We found a strong positive association for infections among females, individuals not born in Australia or reporting heterosexual transmission being of non-B origin. Further, we found an overall increase in non-B infections among men who have sex with men from 50 to 79% in the last 10 years. However, we also found differences between non-B subtypes; heterosexual transmission was positively associated with subtype C only. In addition, the majority of subtype B infections were associated with clusters, while the majority of non-B infections were singletons. However, we found seven non-B clusters (≥5 sequences) indicative of local ongoing transmission. In conclusion, we present how the HIV-1 epidemic has changed over time in NSW, becoming more heterogeneous with distinct subtype-specific demographic associations.

Limited Sustained Local Transmission of HIV-1 CRF01_AE in New South Wales, Australia.

Australia's response to the human immunodeficiency virus type 1 (HIV-1) pandemic led to effective control of HIV transmission and one of the world's lowest HIV incidence rates-0.14%. Although there has been a recent decline in new HIV diagnoses in New South Wales (NSW), the most populous state in Australia, there has been a concomitant increase with non-B subtype infections, particularly for the HIV-1 circulating recombinant form CRF01_AE. This aforementioned CRF01_AE sampled in NSW, were combined with those sampled globally to identify NSW-specific viral clades. The population growth of these clades was assessed in two-year period intervals from 2009 to 2017. Overall, 109 NSW-specific clades were identified, most comprising pairs of sequences; however, five large clades comprising ≥10 sequences were also found. Forty-four clades grew over time with one or two sequences added to each in different two-year periods. Importantly, while 10 of these clades have seemingly discontinued, the remaining 34 were still active in 2016/2017. Seven such clades each comprised ≥10 sequences, and are representative of individual sub-epidemics in NSW. Thus, although the majority of new CRF01_AE infections were associated with small clades that rarely establish ongoing chains of local transmission, individual sub-epidemics are present and should be closely monitored.

T-cell-mediated drug hypersensitivity: immune mechanisms and their clinical relevance.

T-cell-mediated drug hypersensitivity represents a significant proportion of immune mediated drug hypersensitivity reactions. In the recent years, there has been an increase in understanding the immune mechanisms behind T-cell-mediated drug hypersensitivity. According to hapten mechanism, drug specific T-cell response is stimulated by drug-protein conjugate presented on major histocompatibility complex (MHC) as it is presented as a new antigenic determinant. On the other hand, p-i concept suggests that a drug can stimulate T cells via noncovalent direct interaction with T-cell receptor and/or peptide-MHC. The drug binding site is quite variable and this leads to several different mechanisms within p-i concept. Altered peptide repertoire can be regarded as an 'atypical' subset of p-i concept since the mode of the drug binding and the binding site are essentially identical to p-i concept. However, the intracellular binding of abacavir to HLA-B(*)57:01 additionally results in alteration in peptide repertoire. Furthermore the T-cell response to altered peptide repertoire model is only shown for abacavir and HLA-B(*)57:01 and therefore it may not be generalised to other drug hypersensitivity. Danger hypothesis has been postulated to play an important role in drug hypersensitivity by providing signal 2 but its experimental data is lacking at this point in time. Furthermore, the recently described allo-immune response suggests that danger signal may be unnecessary. Finally, in view of these new understanding, the classification and the definition of type B adverse drug reaction should be revised.

Limited reporting of major harms in studies of initial combination antiretroviral therapy: a systematic review.

OBJECTIVES: Risk-benefit assessment of combination antiretroviral therapy (cART) requires consideration of all potential serious harms. Studies of initial cART may permit identification of associations between particular regimens and uncommon harms, but only if comprehensively reported in the public domain. DESIGN: Study-based, systematic review of published initial cART studies (in adult patients) for completeness of serious harms reporting. METHODS: Electronic databases, abstracts, and regulatory/sponsor reports were searched (1 January 1996 - 31 December 2012). Reporting of pre-specified harms - deaths, new/recurrent AIDS events, serious non-AIDS events (2010 INSIGHT classification) and serious adverse events (SAEs) - were assessed as the proportion of studies providing data (reporting frequency). Pharmaceutical sponsors were approached for unreported data. RESULTS: 103 studies (86% randomized, 54% industry-sponsored) were included. Deaths, AIDS events, serious non-AIDS events and SAEs were reported for 85 (83%), 55 (53%), 26 (25%) and 43 (42%) studies, respectively. Deaths were better reported for academic than industry-sponsored studies (91 vs. 75%; P = 0.03); the converse applied for SAEs (26 vs. 55%; P = 0.002). SAEs were better reported for randomized than cohort studies (46 vs. 14%; P = 0.03), and for phase 3 than phase 2 or 4 studies (58 vs. 32 and 29%, respectively; P = 0.02). SAE reporting increased over time [ρ = 0.704, P = 0.002 (Spearman)]. Unreported data acquired for 34 (61%) of 56 industry-sponsored studies improved ascertainment in these studies to between 82 and 100% (P < 0.001). CONCLUSION: Public domain reporting of serious harms for initial cART studies is limited. Insufficient data exist to determine if particular ART drugs/regimens are associated with most serious harms.

Efficacy of initial antiretroviral therapy for HIV-1 infection in adults: a systematic review and meta-analysis of 114 studies with up to 144 weeks' follow-up.

BACKGROUND: A comprehensive assessment of initial HIV-1 treatment success may inform study design and treatment guidelines. METHODS: Group-based, systematic review and meta-analysis of initial antiretroviral therapy studies, in adults, of ≥ 48 weeks duration, reported through December 31, 2012. Size-weighted, intention-to-treat efficacy was calculated. Parameters of study design/eligibility, participant and treatment characteristics were abstracted. Multivariable, random effects, linear regression models with backwards, stepwise selection were then used to identify variables associated with efficacy. OUTCOME MEASURES: Antiviral efficacy (undetectable plasma viral load) and premature cessation of therapy. RESULTS: 114 studies were included (216 treatment groups; 40,124 participants; mean CD4 count 248 cells/µL [SD 81]; mean HIV-1 plasma viral load log10 4.9 [SD 0.2]). Mean efficacy across all groups was 60% (SD 16) over a mean 82 weeks' follow-up (SD 38). Efficacy declined over time: 66% (SD 16) at 48 weeks, 60% (SD 16) at 96 weeks, 52% (SD 18) at 144 weeks. The most common reason for treatment cessation was participant decision (11%, SD 6.6). Efficacy was higher with 'Preferred' than 'Alternative' regimens (as defined by 2013 United States antiretroviral guidelines): 75% vs. 65%, respectively, difference 10%; 95%CI 7.6 to 15.4; p<0.001. In 98 groups (45%) reporting efficacy stratified by pre-treatment viral load (< or ≥100,000 copies/mL), efficacy was greater for the lower stratum (70% vs. 62%, respectively, difference 8.4%; 95%CI 6.0 to 10.9; p<0.001). This difference persisted within 'Preferred' regimens. Greatest efficacy was associated with use of tenofovir-emtricitabine (vs. other nucleoside analogue backbones) and integrase strand transfer inhibitors (vs. other third drug classes). CONCLUSION: Initial antiretroviral treatments for HIV-1 to date appear to have suboptimal long-term efficacy, but are more effective when commenced at plasma viral loads <100,000 copies/mL. Rising viral load should be considered an indication for starting treatment. Integrase inhibitors offer a treatment advantage (vs. other third drug classes).

Skeletal muscle toxicity associated with raltegravir-based combination antiretroviral therapy in HIV-infected adults.

OBJECTIVE/DESIGN: Raltegravir is uncommonly associated with rhabdomyolysis and grade 3-4 creatine kinase (CK) elevation. In this cross-sectional study, we compared the prevalence of skeletal muscle toxicity in HIV-infected adults receiving raltegravir with that of a control group. METHODS: Adults receiving combination antiretroviral therapy were recruited consecutively. Assessments included physical examination, an exercise questionnaire, and blood testing for CK, troponin T, and raltegravir trough levels. The primary endpoint was the prevalence of skeletal muscle toxicity, defined as a composite of any of the following: (1) isolated CK elevation; (2) myalgia; (3) proximal myopathy on examination; or (4) rhabdomyolysis. RESULTS: A total of 318 participants (159 raltegravir, 159 control) were evaluated; 98% were male, 89% white, with median age 51 years, and 91% had HIV-1 RNA <50 copies per milliliter. Mean raltegravir exposure was 28 months. Skeletal muscle toxicity was present in 37% of the raltegravir vs. 19% of the control group (P < 0.001). By component, there were significant respective differences in myalgia (19% vs. 3%, P < 0.001) and proximal myopathy (4% vs. 0%, P = 0.030) but not CK elevation (14% vs. 16%, P = 0.639). No patient had rhabdomyolysis. In multivariate analysis, raltegravir therapy (P < 0.001) and strenuous exercise (P = 0.002) were independently associated with overall muscle toxicity. No component of muscle toxicity was associated with duration of raltegravir or the raltegravir level. CONCLUSIONS: Raltegravir-based therapy is associated with a higher prevalence of symptomatic skeletal muscle toxicity, which does not seem to be concentration or time dependent, nor associated with elevated CK. Proximal myopathy may be an uncommon but significant side effect of raltegravir exposure.