Low HIV drug resistance prevalence among recently diagnosed HIV-positive men who have sex with men in a setting of high PrEP use.

INTRODUCTION: New South Wales (NSW) has one of the world's highest uptake rates of HIV pre-exposure prophylaxis (PrEP). This uptake has been credited with sharp declines in HIV transmission, particularly among Australian-born gay and bisexual men. Concerns have been raised around the potential for the emergence of tenofovir (TFV) and XTC (lamivudine/emtricitabine) resistance in settings of high PrEP use. Such an emergence could also increase treatment failure and associated clinical outcomes among people living with HIV (PLHIV). Despite low levels of nucleoside reverse-transcriptase inhibitor (NRTI) resistance relating to PrEP use in clinical settings, there are few published studies describing the prevalence of NRTI resistance among people newly diagnosed with HIV in a setting of high PrEP use. METHODS: Using HIV antiretroviral drug resistance data linked to NSW HIV notifications records of people diagnosed from 1 January 2015 to 31 December 2021 and with HIV attributed to male-to-male sex, we described trends in TFV and XTC resistance. Resistance was identified using the Stanford HIV Drug Resistance genotypic resistance interpretation system. To focus on transmitted drug resistance, resistance prevalence estimates were generated using sequences taken less than 3 months post-HIV diagnosis. These estimates were stratified by timing of sequencing relative to the date of diagnosis, year of sequencing, birthplace, likely place of HIV acquisition, and stage of HIV at diagnosis. RESULTS: Among 1119 diagnoses linked to HIV genomes sequenced less than 3 months following diagnosis, overall XTC resistance prevalence was 1.3%. Between 2015 and 2021, XTC resistance fluctuated between 0.5% to 2.9% and was 1.0% in 2021. No TFV resistance was found over the study period in any of the sequences analysed. Higher XTC resistance prevalence was observed among people with newly acquired HIV (evidence of HIV acquisition in the 12 months prior to diagnosis; 2.9%, p = 0.008). CONCLUSIONS: In this Australian setting, TFV and XTC resistance prevalence in new HIV diagnoses remained low. Our findings offer further evidence for the safe scale-up of PrEP in high-income settings, without jeopardizing the treatment of those living with HIV.

Trends in HIV testing and HIV stage at diagnosis among people newly diagnosed with HIV.

OBJECTIVE: To identify groups more likely to be referred for HIV testing because of symptomatic presentation rather than as part of asymptomatic screening. DESIGN: A retrospective analysis of Australian National HIV Registry (NHR) surveillance data including sociodemographic and clinical data, as well as reasons for HIV test. METHODS: Using notification records from 2017 to 2022, we summarised reasons for testing leading to an HIV diagnosis. Reasons for testing were combined with clinical status at diagnosis to derive HIV testing categories: testing while symptomatic; asymptomatic HIV screening; seroconversion; and other test reason. We stratified these categories by stage of HIV at diagnosis with late-stage HIV defined as a CD4 + cell count <350 cells/µl at time of diagnosis. RESULTS: Among 4134 HIV notifications with at least one reason for testing recorded, STI screening was the predominant reason for test referral (38%), followed by HIV indicative symptoms (31%), and risk behaviour (13%). By testing category, people aged 50 years or older (24%), people with HIV attributed to heterosexual sex (21%), people born in sub-Saharan Africa (19%), and women (17%) had lower levels of asymptomatic screening. More late-stage HIV diagnoses resulted from testing while symptomatic (58%) compared with asymptomatic screening (25%). CONCLUSIONS: Older people and heterosexuals may not access HIV focused healthcare where HIV screening is routinely offered. Instead, HIV testing opportunities may arise in other settings. By normalising HIV testing and offering low-cost HIV screening in a range of settings, it may be possible to facilitate earlier HIV diagnoses, better health outcomes, and reduced onward transmission.

Acinetobacter baumannii GC2 Sublineage Carrying the aac(6')-Im Amikacin, Netilmicin, and Tobramycin Resistance Gene Cassette.

The aminoglycoside antibiotics amikacin, gentamicin, and tobramycin are important therapeutic options for Acinetobacter iinfections. Several genes that confer resistance to one or more of these antibiotics are prevalent in the globally distributed resistant clones of Acinetobacter baumannii, but the aac(6')-Im (aacA16) gene (amikacin, netilmicin, and tobramycin resistance), first reported in isolates from South Korea, has rarely been reported since. In this study, GC2 isolates (1999 to 2002) from Brisbane, Australia, carrying aac(6')-Im and belonging to the ST2:ST423:KL6:OCL1 type were identified and sequenced. The aac(6')-Im gene and surrounds have been incorporated into one end of the IS26-bounded AbGRI2 antibiotic resistance island and are accompanied by a characteristic 70.3-kbp deletion of adjacent chromosome. The compete genome of the 1999 isolate F46 (RBH46) includes only two copies of ISAba1 (in AbGRI1-3 and upstream of ampC) but later isolates, which differ from one another by <10 single nucleotide differences (SND), carry two to seven additional shared copies. Several complete GC2 genomes with aac(6')-Im in an AbGRI2 island (2004 to 2017; several countries) found in GenBank and two additional Australian A. baumannii isolates (2006) carry different gene sets, KL2, KL9, KL40, or KL52, at the capsule locus. These genomes include ISAba1 copies in a different set of shared locations. The distribution of SND between F46 and AYP-A2, a 2013 ST2:ST208:KL2:OCL1 isolate from Victoria, Australia, revealed that a 640-kbp segment that includes KL2 and the AbGRI1 resistance island replaces the corresponding region in F46. Over 1,000 A. baumannii draft genomes also include aac(6')-Im, indicating that it is currently globally disseminated and significantly underreported. IMPORTANCE Aminoglycosides are important therapeutic options for treatment of Acinetobacter infections. Here, we show that a little-known aminoglycoside resistance gene, aac(6')-Im (aacA16), that confers amikacin, netilmicin, and tobramycin resistance has been circulating undetected for many years in a sublineage of A. baumannii global clone 2 (GC2), generally with a second aminoglycoside resistance gene, aacC1, which confers resistance to gentamicin. These two genes are commonly found together in GC2 complete and draft genomes and globally distributed. One isolate appears to be ancestral, as its genome contains few ISAba1 copies, providing insight into the original source of this insertion sequence (IS), which is abundant in most GC2 isolates. Tracking ISAba1 spread can provide a simple means to track the development and ongoing evolution as well as the dissemination of specific lineages and detect the formation of many sublineages. The complete ancestral genome will provide an essential base point for tracking this process.

A population-level application of a method for estimating the timing of HIV acquisition among migrants to Australia.

INTRODUCTION: Australia has set the goal for the virtual elimination of HIV transmission by the end of 2022, yet accurate information is lacking on the level of HIV transmission occurring among residents. We developed a method for estimating the timing of HIV acquisition among migrants, relative to their arrival in Australia. We then applied this method to surveillance data from the Australian National HIV Registry with the aim of ascertaining the level of HIV transmission among migrants to Australia occurring before and after migration, and to inform appropriate local public health interventions. METHODS: We developed an algorithm incorporating CD4+ T-cell decline back-projection and enhanced variables (clinical presentation, past HIV testing history and clinician estimate of the place of HIV acquisition) and compared it to a standard algorithm which uses CD4+ T-cell back-projection only. We applied both algorithms to all new HIV diagnoses among migrants to estimate whether HIV infection occurred before or after arrival in Australia. RESULTS: Between 1 January 2016 and 31 December 2020, 1909 migrants were newly diagnosed with HIV in Australia, 85% were men, and the median age was 33 years. Using the enhanced algorithm, 932 (49%) were estimated to have acquired HIV after arrival in Australia, 629 (33%) before arrival (from overseas), 250 (13%) close to arrival and 98 (5%) were unable to be classified. Using the standard algorithm, 622 (33%) were estimated to have acquired HIV in Australia, 472 (25%) before arrival, 321 (17%) close to arrival and 494 (26%) were unable to be classified. CONCLUSIONS: Using our algorithm, close to half of migrants diagnosed with HIV were estimated to have acquired HIV after arrival in Australia, highlighting the need for tailored culturally appropriate testing and prevention programmes to limit HIV transmission and achieve elimination targets. Our method reduced the proportion of HIV cases unable to be classified and can be adopted in other countries with similar HIV surveillance protocols, to inform epidemiology and elimination efforts.

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.

An outbreak of multiply antibiotic-resistant ST49:ST128:KL11:OCL8 Acinetobacter baumannii isolates at a Sydney hospital.

OBJECTIVES: To understand the acquisition of resistance genes by a non-GC1, non-GC2 Acinetobacter baumannii strain responsible for a 4 year outbreak at a Sydney hospital. METHODS: Representative isolates were screened for resistance to antibiotics. Three were subjected to WGS using Illumina HiSeq. One genome was completed with MinION long reads. Resistance regions were compared with known sequences using bioinformatics. RESULTS: Isolates were resistant to third-generation cephalosporins, gentamicin and tobramycin, sulfamethoxazole and erythromycin. Sequenced isolates were ST49 (Institut Pasteur scheme) and ST128 (Oxford scheme) and carried KL11 at the capsule locus and OCL8 at the lipooligosaccharide outer core locus. The complete genome of isolate J9 revealed that the resistance genes were all in plasmids; pRAY* contained aadB, and a large plasmid, pJ9-3, contained sul2 and floR genes and a dif module containing the mph(E)-msr(E) macrolide resistance genes. Transposon Tn6168, consisting of a second copy of the chromosomal ampC gene region flanked by ISAba1s, confers resistance to third-generation cephalosporins. Tn6168 is located inside the mph(E)-msr(E) dif module. pJ9-3 includes a set of four dif modules and the orientation of the pdif sites, XerC-XerD or XerD-XerC, alternates. A large transposon, Tn6175, containing tniCABDE transposition genes and genes annotated as being involved in heavy metal metabolism, uptake or export was found in the comM gene. Other ST49:ST128:KL11:OCL8 genomes found in the GenBank WGS database carried Tn6175 but neither of the plasmids carrying the resistance genes. CONCLUSIONS: An early carbapenem-susceptible A. baumannii outbreak recorded in Australia was caused by an unusual clone that had acquired plasmids carrying antibiotic resistance genes.

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.

Emergence, molecular mechanisms and global spread of carbapenem-resistant Acinetobacter baumannii.

Acinetobacter baumannii is a nosocomial pathogen that has emerged as a global threat because of high levels of resistance to many antibiotics, particularly those considered to be last-resort antibiotics, such as carbapenems. Although alterations in the efflux pump and outer membrane proteins can cause carbapenem resistance, the main mechanism is the acquisition of carbapenem-hydrolyzing oxacillinase-encoding genes. Of these, oxa23 is by far the most widespread in most countries, while oxa24 and oxa58 appear to be dominant in specific regions. Historically, much of the global spread of carbapenem resistance has been due to the dissemination of two major clones, known as global clones 1 and 2, although new lineages are now common in some parts of the world. The analysis of all publicly available genome sequences performed here indicates that ST2, ST1, ST79 and ST25 account for over 71 % of all genomes sequenced to date, with ST2 by far the most dominant type and oxa23 the most widespread carbapenem resistance determinant globally, regardless of clonal type. Whilst this highlights the global spread of ST1 and ST2, and the dominance of oxa23 in both clones, it could also be a result of preferential selection of carbapenem-resistant strains, which mainly belong to the two major clones. Furthermore, ~70 % of the sequenced strains have been isolated from five countries, namely the USA, PR China, Australia, Thailand and Pakistan, with only a limited number from other countries. These genomes are a vital resource, but it is currently difficult to draw an accurate global picture of this important superbug, highlighting the need for more comprehensive genome sequence data and genomic analysis.

Complete Genome Sequence of WM99c, an Antibiotic-Resistant Acinetobacter baumannii Global Clone 2 (GC2) Strain Representing an Australian GC2 Lineage.

The extensively antibiotic-resistant Acinetobacter baumannii isolate WM99c recovered in Sydney, Australia, in 1999 is an early representative of a distinct lineage of global clone 2 (GC2) seen on the east coast of Australia. We present the complete 4.121-Mbp genome sequence (chromosome plus 2 plasmids), generated via long-read sequencing (PacBio).

RCH51, a multiply antibiotic-resistant Acinetobacter baumannii ST103IP isolate, carries resistance genes in three plasmids, including a novel potentially conjugative plasmid carrying oxa235 in transposon Tn6252.

Objectives: To determine the identity and context of genes conferring antibiotic resistance in a sporadic multiply antibiotic-resistant Acinetobacter baumannii recovered at Royal Children's Hospital, Brisbane. Methods: The antibiotic resistance phenotype for 23 antibiotics was determined using disc diffusion or MIC determination. The whole-genome sequence of RCH51 was determined using the Illumina HiSeq platform. Antibiotic resistance determinants were identified using ResFinder. Plasmids were recovered by transformation. Results: Isolate RCH51 belongs to the uncommon STs ST103 IP (7-3-2-1-7-1-4) and ST514 OX (1-52-29-28-18-114-7). It was found to be resistant to sulfamethoxazole, tetracycline, gentamicin, tobramycin and kanamycin and also exhibited reduced susceptibility to imipenem (MIC 2 mg/L) and meropenem (MIC 6 mg/L). RCH51 carries the oxa235 , sul2 , floR , aadB and tet39 resistance genes, all located on plasmids. The largest of the three plasmids, pRCH51-3, is 52 789 bp and carries oxa235 in the ISAba1-bounded transposon Tn 6252 , as well as sul2 and floR . pRCH51-3 represents a new A. baumannii plasmid family that is potentially conjugative as it contains several genes predicted to encode transfer functions. However, conjugation of pRCH51-3 was not detected. The aadB and tet39 resistance genes were each found in small plasmids identical to the known plasmids pRAY*-v1 and pRCH52-1, respectively. Conclusions: The resistance gene complement of RCH51 was found in three plasmids. pRCH51-3, which carries the oxa235 , sul2 and floR resistance genes, represents a new, potentially conjugative A. baumannii plasmid type.

Loss and gain of aminoglycoside resistance in global clone 2 Acinetobacter baumannii in Australia via modification of genomic resistance islands and acquisition of plasmids.

OBJECTIVES: The objective of this study was to examine the evolution of carbapenem-resistant global clone 2 (GC2) Acinetobacter baumannii in Australia focusing on the complement of aminoglycoside resistance genes and their location in resistance islands and plasmids. METHODS: Sixty-two carbapenem-resistant GC2 A. baumannii isolates with various aminoglycoside resistance profiles and resistance gene content that were recovered over the period 1999-2010 from hospitals on the east coast of Australia were examined. PCR was used to link relevant contigs retrieved from whole genomes sequenced using Illumina HiSeq and assembled de novo using Velvet. Resistance phenotypes were extended to include additional antibiotics using a disc diffusion assay. RESULTS: Sixty-one isolates were ST208 (formerly ST92; Oxford scheme) and one was ST425. All isolates included the oxa23 carbapenem resistance gene in Tn2006 located in the same position in AbGRI1-2, along with the ISAba1-sul2-CR2Δ-tetA(B)-tetA(R)-CR2-strB-strA configuration. All isolates harboured either AbGRI2-1 carrying the aacC1 (gentamicin resistance) cassette or a variant derived from it via loss of some of the island content. When aacC1 was lost, aminoglycoside resistance was sometimes regained via acquisition of aadB (gentamicin, kanamycin and tobramycin resistance) in pRAY*-v1 or TnaphA6 (amikacin, kanamycin and neomycin resistance) in a repAci6 plasmid. A small cryptic plasmid or a deletion variant of this plasmid was always present and a large cryptic plasmid was also variably present. CONCLUSIONS: The extensively antibiotic-resistant GC2 isolates from Sydney, Brisbane and Canberra appear to have arisen from a single import that was introduced into Australia in, or prior to, 1999 that then evolved and spread.

Structure and context of Acinetobacter transposons carrying the oxa23 carbapenemase gene.

Theoxa23gene encoding the OXA-23 carbapenemase (and several minor variants of it) is widespread inAcinetobacter baumanniiclinical isolates and compromises treatment with carbapenem antibiotics. The gene is derived from the chromosome ofAcinetobacter radioresistenswhere it is an intrinsic gene, here designatedoxaAr InA. baumanniiand otherAcinetobacterspecies,oxa23is usually preceded by an IS, ISAba1, which supplies the strong promoter required for the gene to confer clinically relevant levels of resistance. TheoxaArgene appears to have been mobilized twice creating Tn2008and Tn2008B, both of which consist of a single ISAba1 and anA. radioresistens-derived fragment. Tn2006and Tn2009are clearly derived from Tn2008Band are each made up of Tn2008Bwith an additional segment of unknown origin and an additional ISAba1, creating a compound transposon. Tn2006, Tn2008and possibly Tn2008Bare globally disseminated, while Tn2009has as yet only been found in China. Of the four ISAba1-associated transposons, Tn2006has been most frequently observed worldwide and Tn2006in Tn6022, known as AbaR4, appears to contribute significantly to the dissemination ofoxa23 Moreover, AbaR4, Tn2006, Tn2008and Tn2009have each been found in conjugative plasmids, further facilitating their spread.

Evolution of AbGRI2-0, the Progenitor of the AbGRI2 Resistance Island in Global Clone 2 of Acinetobacter baumannii.

A320, isolated in the Netherlands in 1982 and also known as RUH134, is the earliest available multiply antibiotic-resistant (MAR) Acinetobacter baumannii isolate belonging to global clone 2 (GC2) and is the reference strain for this clone. The draft genome sequence of A320 was used to investigate the original location and configuration of the IS26-bounded AbGRI2 resistance island found in current GC2 isolates. PCR mapping and sequencing were used to order contigs composing the resistance islands. A320 contains two IS26-bounded resistance islands, AbGRI2-0a and AbGRI2-0b, of 7.8 kb and 25.4 kb, respectively. Together they contain blaTEM, aacC1, aadA1, sul1, catA1, and aphA1b genes, which confer resistance to antibiotics used clinically in the 1970s, as well as an incomplete mercury resistance module. Tracking the continuity of the chromosome and the target site duplications revealed that the two resistance islands were originally together as AbGRI2-0, an island of 32.4 kb, and were subsequently separated via an IS26-mediated intramolecular inversion that reversed the orientation of 1.54 Mb of the chromosome and duplicated an IS26. A320 contains an ancestral form of AbGRI2, and the original insertion site of the AbGRI2 island was identified. Many of the AbGRI2 versions present in the completed GC2 genomes can be derived from it via the variant AbGRI2-1. IS26-mediated inversions have also played a part in forming AbGRI2-0, and, upon reversal, large regions of AbGRI2-0 are identical to parts of AbaR0, the ancestral version of the AbaR islands present in GC1 isolates. This indicates a common source.

Variation in the OC locus of Acinetobacter baumannii genomes predicts extensive structural diversity in the lipooligosaccharide.

Lipooligosaccharide (LOS) is a complex surface structure that is linked to many pathogenic properties of Acinetobacter baumannii. In A. baumannii, the genes responsible for the synthesis of the outer core (OC) component of the LOS are located between ilvE and aspS. The content of the OC locus is usually variable within a species, and examination of 6 complete and 227 draft A. baumannii genome sequences available in GenBank non-redundant and Whole Genome Shotgun databases revealed nine distinct new types, OCL4-OCL12, in addition to the three known ones. The twelve gene clusters fell into two distinct groups, designated Group A and Group B, based on similarities in the genes present. OCL6 (Group B) was unique in that it included genes for the synthesis of L-Rhamnosep. Genetic exchange of the different configurations between strains has occurred as some OC forms were found in several different sequence types (STs). OCL1 (Group A) was the most widely distributed being present in 18 STs, and OCL6 was found in 16 STs. Variation within clones was also observed, with more than one OC locus type found in the two globally disseminated clones, GC1 and GC2, that include the majority of multiply antibiotic resistant isolates. OCL1 was the most abundant gene cluster in both GC1 and GC2 genomes but GC1 isolates also carried OCL2, OCL3 or OCL5, and OCL3 was also present in GC2. As replacement of the OC locus in the major global clones indicates the presence of sub-lineages, a PCR typing scheme was developed to rapidly distinguish Group A and Group B types, and to distinguish the specific forms found in GC1 and GC2 isolates.