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Background The use of in vivo and ex vivo diagnostic tools for delayed immune-mediated adverse drug reactions is currently ill defined. Objective To determine whether the combination of skin testing and/or IFN-γ enzyme-linked immunoSpot assay (ELISpot) can aid diagnosis of these allergy phenotypes. Methods Patients with antibiotic-associated severe delayed immune-mediated adverse drug reaction hypersensitivity, including Stevens-Johnson syndrome and toxic epidermal necrolysis, drug reaction with eosinophilia and systemic symptoms (DRESS), acute generalized exanthematous pustulosis, generalized bullous fixed drug eruption, and severe maculopapular exanthema, were prospectively recruited. In vivo testing was completed to the implicated drug(s), and ex vivo testing was performed with the patient's PBMCs stimulated with the relevant antibiotic concentrations for IFN-γ release ELISpot measurement. Results Eighty-one patients met the inclusion criteria, with DRESS (42; 51.9%) accounting for most cases. Among the 63 (78%) who had an ELISpot assay performed, 34 (54%) were positive to at least 1 implicated antibiotic (median spot-forming units/million cells, 99.5; interquartile range, 68-187), with glycopeptide being a strong predictor of positivity (adjusted odds ratio, 6.11; 95% CI, 1.74-21.42). In combination (in vivo and ex vivo), 51 (63%) of those tested were positive to an implicated antibiotic. For DRESS and severe maculopapular exanthema associated with penicillins and cephalosporins, this combination confirmed the culprit agent in 11 of the 12 cases and in 6 of 7 for DRESS associated with glycopeptides. Conclusions This study demonstrates that using in vivo in combination with ex vivo testing can enhance the diagnostic approach in these severe phenotypes by assisting with the identification of possible culprit antibiotics.

Background Penicillin allergies are associated with inferior patient and antimicrobial stewardship outcomes. We implemented a whole-of-hospital program to assess the efficacy of inpatient delabeling for low-risk penicillin allergies in hospitalized inpatients. Methods Patients ≥ 18 years of age with a low-risk penicillin allergy were offered a single-dose oral penicillin challenge or direct label removal based on history (direct delabeling). The primary endpoint was the proportion of patients delabeled. Key secondary endpoints were antibiotic utilization pre- (index admission) and post-delabeling (index admission and 90 days). Results Between 21 January 2019 and 31 August 2019, we assessed 1791 patients reporting 2315 antibiotic allergies, 1225 with a penicillin allergy. Three hundred fifty-five patients were delabeled: 161 by direct delabeling and 194 via oral penicillin challenge. Ninety-seven percent (194/200) of patients were negative upon oral penicillin challenge. In the delabeled patients, we observed an increase in narrow-spectrum penicillin usage (adjusted odds ratio [OR], 10.51 [95% confidence interval {CI}, 5.39–20.48]), improved appropriate antibiotic prescribing (adjusted OR, 2.13 [95% CI, 1.45–3.13]), and a reduction in restricted antibiotic usage (adjusted OR, 0.38 [95% CI, .27–.54]). In the propensity score analysis, there was an increase in narrow-spectrum penicillins (OR, 10.89 [95% CI, 5.09–23.31]) and β-lactam/β-lactamase inhibitors (OR, 6.68 [95% CI, 3.94–11.35]) and a reduction in restricted antibiotic use (OR, 0.52 [95% CI, .36–.74]) and inappropriate prescriptions (relative risk ratio, 0.43 [95% CI, .26–.72]) in the delabeled group compared with the group who retained their allergy label. Conclusions This health services program using a combination of direct delabeling and oral penicillin challenge resulted in significant impacts on the use of preferred antibiotics and appropriate prescribing.

Importance Penicillin allergy is a significant public health issue for patients, antimicrobial stewardship programs, and health services. Validated clinical decision rules are urgently needed to identify low-risk penicillin allergies that potentially do not require penicillin skin testing by a specialist. Objective To develop and validate a penicillin allergy clinical decision rule that enables point-of-care risk assessment of patient-reported penicillin allergies. Design, Setting, and Participants In this diagnostic study, a multicenter prospective antibiotic allergy–tested cohort of 622 patients from 2 tertiary care sites in Melbourne, Australia (Austin Health and Peter MacCallum Cancer Centre) was used for derivation and internal validation of a penicillin allergy decision rule. Backward stepwise logistic regression was used to derive the model, including clinical variables predictive of a positive penicillin allergy test result. Internal validation of the final model used bootstrapped samples and the model scoring derived from the coefficients. External validation was performed in retrospective penicillin allergy–tested cohorts consisting of 945 patients from Sydney and Perth, Australia, and Nashville, Tennessee. Patients who reported a penicillin allergy underwent penicillin allergy testing using skin prick, intradermal, or patch testing and/or oral challenge (direct or after skin testing). Data were collected from June 26, 2008, to June 3, 2019, and analyzed from January 9 to 12, 2019. Main Outcomes and Measures The primary outcome for the model was any positive result of penicillin allergy testing performed during outpatient or inpatient assessment. Results From an internal derivation and validation cohort of 622 patients (367 female [59.0%]; median age, 60 [interquartile range{IQR}, 48-71] years) and an external validation cohort of 945 patients (662 female [70.1%]; median age, 55 [IQR, 38-68] years), the 4 features associated with a positive penicillin alle

Letter to the editor

Letter to the editor

Background: The community-associated methicillin-resistant S. aureus (CA-MRSA) ST93 clone is becoming dominant in Australia and is clinically highly virulent. In addition, sepsis and skin infection models demonstrate that ST93 CA-MRSA is the most virulent global clone of S. aureus tested to date. While the determinants of virulence have been studied in other clones of CA-MRSA, the basis for hypervirulence in ST93 CA-MRSA has not been defined. Results: Here, using a geographically and temporally dispersed collection of ST93 isolates we demonstrate that the ST93 population hyperexpresses key CA-MRSA exotoxins, in particular -hemolysin, in comparison to other global clones. Gene deletion and complementation studies, and virulence comparisons in a murine skin infection model, showed unequivocally that increased expression of -hemolysin is the key staphylococcal virulence determinant for this clone. Genome sequencing and comparative genomics of strains with divergent exotoxin profiles demonstrated that, like other S. aureus clones, the quorum sensing agr system is the master regulator of toxin expression and virulence in ST93 CA-MRSA. However, we also identified a previously uncharacterized AraC/XylS family regulator (AryK) that potentiates toxin expression and virulence in S. aureus. Conclusions: These data demonstrate that hyperexpression of -hemolysin mediates enhanced virulence in ST93 CA-MRSA, and additional control of exotoxin production, in particular -hemolysin, mediated by regulatory systems other than agr have the potential to fine-tune virulence in CA-MRSA.

In Australia the PVL - positive ST93-IV [2B], colloquially known as “Queensland CA-MRSA” has become the dominant CA-MRSA clone. First described in the early 2000s, ST93-IV [2B] is associated with skin and severe invasive infections including necrotizing pneumonia. A singleton by multilocus sequence typing (MLST) eBURST analysis ST93 is distinct from other S aureus clones. To determine if the increased prevalence of ST93-IV [2B] is due to the widespread transmission of a single strain of ST93-IV [2B] the genetic relatedness of 58 S. aureus ST93 isolated throughout Australia over an extended period were studied in detail using a variety of molecular methods including pulsed-field gel electrophoresis, spa typing, MLST, microarray DNA, SCCmec typing and dru typing. Identification of the phage harbouring the lukS-PV/lukF-PV Panton Valentine leucocidin genes, detection of allelic variations in lukS-PV/lukF-PV, and quantification of LukF-PV expression was also performed. Although ST93-IV [2B] is known to have an apparent enhanced clinical virulence, the isolates harboured few known virulence determinants. All PVL-positive isolates carried the PVL-encoding phage ΦSa2USA and the lukS-PV/lukF-PV genes had the same R variant SNP profile. The isolates produced similar expression levels of LukF-PV. Although multiple rearrangements of the spa sequence have occurred, the core genome in ST93 is very stable. The emergence of ST93-MRSA is due to independent acquisitions of different dru-defined type IV and type V SCCmec elements in several spa-defined ST93-MSSA backgrounds. Rearrangement of the spa sequence in ST93-MRSA has subsequently occurred in some of these strains. Although multiple ST93-MRSA strains were characterised, little genetic diversity was identified for most isolates, with PVL-positive ST93-IVa [2B]-t202-dt10 predominant across Australia. Whether ST93-IVa [2B] t202-dt10 arose from one PVL-positive ST93-MSSA-t202, or by independent acquisitions of SCCmec-IVa [2B]-dt10

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 has spread rapidly across North America, and CA-MRSA is also increasing in Australia. However, the dominant Australian CA-MRSA strain, ST93-IV [2B] appears distantly related to USA300 despite strikingly similar clinical and epidemiological profiles. Here, we compared the virulence of a recent Australian ST93 isolate (JKD6159) to other MRSA, including USA300, and found that JKD6159 was the most virulent in a mouse skin infection model. We fully sequenced the genome of JKD6159 and confirmed that JKD6159 is a distinct clone with 7616 single nucleotide polymorphisms (SNPs) distinguishing this strain from all other S. aureus genomes. Despite its high virulence there were surprisingly few virulence determinants. However, genes encoding α-hemolysin, Panton-Valentine leukocidin (PVL) and α-type phenol soluble modulins were present. Genome comparisons revealed 32 additional CDS in JKD6159 but none appeared to encode new virulence factors, suggesting that this clone's enhanced pathogenicity could lie within subtler genome changes, such as SNPs within regulatory genes. To investigate the role of accessory genome elements in CA-MRSA epidemiology, we next sequenced three additional Australian non-ST93 CA-MRSA strains and compared them with JKD6159, 19 completed S. aureus genomes and 59 additional S. aureus genomes for which unassembled genome sequence data was publicly available (82 genomes in total). These comparisons showed that despite its distinctive genotype, JKD6159 and other CA-MRSA clones (including USA300) share a conserved repertoire of three notable accessory elements (SSCmecIV, PVL prophage, and pMW2). This study demonstrates that the genetically distinct ST93 CA-MRSA from Australia is highly virulent. Our comparisons of geographically and genetically diverse CA-MRSA genomes suggest that apparent convergent evolution in CA-MRSA may be better explained by the rapid dissemination of a hig

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 has spread rapidly across North America, and CA-MRSA is also increasing in Australia. However, the dominant Australian CA-MRSA strain, ST93-IV [2B] appears distantly related to USA300 despite strikingly similar clinical and epidemiological profiles. Here, we compared the virulence of a recent Australian ST93 isolate (JKD6159) to other MRSA, including USA300, and found that JKD6159 was the most virulent in a mouse skin infection model. We fully sequenced the genome of JKD6159 and confirmed that JKD6159 is a distinct clone with 7616 single nucleotide polymorphisms (SNPs) distinguishing this strain from all other S. aureus genomes. Despite its high virulence there were surprisingly few virulence determinants. However, genes encoding alpha-hemolysin, Panton-Valentine leukocidin (PVL) and alpha-type phenol soluble modulins were present. Genome comparisons revealed 32 additional CDS in JKD6159 but none appeared to encode new virulence factors, suggesting that this clone's enhanced pathogenicity could lie within subtler genome changes, such as SNPs within regulatory genes. To investigate the role of accessory genome elements in CA-MRSA epidemiology, we next sequenced three additional Australian non-ST93 CA-MRSA strains and compared them with JKD6159, 19 completed S. aureus genomes and 59 additional S. aureus genomes for which unassembled genome sequence data was publicly available (82 genomes in total). These comparisons showed that despite its distinctive genotype, JKD6159 and other CA-MRSA clones (including USA300) share a conserved repertoire of three notable accessory elements (SSCmecIV, PVL prophage, and pMW2). This study demonstrates that the genetically distinct ST93 CA-MRSA from Australia is highly virulent. Our comparisons of geographically and genetically diverse CA-MRSA genomes suggest that apparent convergent evolution in CA-MRSA may be better explained by the rapid dissemination

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 has spread rapidly across North America, and CA-MRSA is also increasing in Australia. However, the dominant Australian CA-MRSA strain, ST93-IV [2B] appears distantly related to USA300 despite strikingly similar clinical and epidemiological profiles. Here, we compared the virulence of a recent Australian ST93 isolate (JKD6159) to other MRSA, including USA300, and found that JKD6159 was the most virulent in a mouse skin infection model. We fully sequenced the genome of JKD6159 and confirmed that JKD6159 is a distinct clone with 7616 single nucleotide polymorphisms (SNPs) distinguishing this strain from all other S. aureus genomes. Despite its high virulence there were surprisingly few virulence determinants. However, genes encoding alpha-hemolysin, Panton-Valentine leukocidin (PVL) and alpha-type phenol soluble modulins were present. Genome comparisons revealed 32 additional CDS in JKD6159 but none appeared to encode new virulence factors, suggesting that this clone's enhanced pathogenicity could lie within subtler genome changes, such as SNPs within regulatory genes. To investigate the role of accessory genome elements in CA-MRSA epidemiology, we next sequenced three additional Australian non-ST93 CA-MRSA strains and compared them with JKD6159, 19 completed S. aureus genomes and 59 additional S. aureus genomes for which unassembled genome sequence data was publicly available (82 genomes in total). These comparisons showed that despite its distinctive genotype, JKD6159 and other CA-MRSA clones (including USA300) share a conserved repertoire of three notable accessory elements (SSCmecIV, PVL prophage, and pMW2). This study demonstrates that the genetically distinct ST93 CA-MRSA from Australia is highly virulent. Our comparisons of geographically and genetically diverse CA-MRSA genomes suggest that apparent convergent evolution in CA-MRSA may be better explained by the rapid dissemination