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BACKGROUND: The study objective was to reveal reservoirs potentially leading to Staphylococcus aureus infections in haemodialysis clinic clients in the tropical north of the Australian Northern Territory (NT). This client population are primarily Aboriginal Australians who have a greater burden of ill health than other Australians. Reservoir identification will enhance infection control in this client group, including informing potential S. aureus decolonisation strategies. METHODS AND FINDINGS: The study participants were 83 clients of four haemodialysis clinics in the Darwin region of the NT, and 46 clinical staff and researchers who had contact with the clinic clients. The study design was longitudinal, encompassing swabbing of anatomical sites at two month intervals to yield carriage isolates, and also progressive collection of infection isolates. Swab sampling was performed for all participants, and infection isolates collected for dialysis clients only. Analysis was based on the comparison of 139 carriage isolates and 27 infection isolates using whole genome sequencing. Genome comparisons were based on of 20,651 genome-wide orthologous SNPs, presence/absence of the mecA and pvl genes, and inferred multilocus sequence type and clonal complex. Pairs of genomes meeting the definition of "not discriminated" were classed as defining potential transmission events. The primary outcome was instances of potential transmission between a carriage site other than a skin lesion and an infection site, in the same individual. Three such instances were identified. Two involved ST762 (CC1) PVL- MRSA, and one instance ST121 PVL+ MSSA. Three additional instances were identified where the carriage strains were derived from skin lesions. Also identified were six instances of potential transmission of a carriage strains between participants, including transmission of strains between dialysis clients and staff/researchers, and one potential transmission of a clinical strain between

Group A Streptococcus (GAS) skin infections are caused by a diverse array of strain types and are highly prevalent in disadvantaged populations. The role of strain-specific immunity in preventing GAS infections is poorly understood, representing a critical knowledge gap in vaccine development. A recent GAS murine challenge study showed evidence that sterilising strain-specific and enduring immunity required two skin infections by the same GAS strain within three weeks. This mechanism of developing enduring immunity may be a significant impediment to the accumulation of immunity in populations. We used an agent-based mathematical model of GAS transmission to investigate the epidemiological consequences of enduring strain-specific immunity developing only after two infections with the same strain within a specified interval. Accounting for uncertainty when correlating murine timeframes to humans, we varied this maximum inter-infection interval from 3 to 420 weeks to assess its impact on prevalence and strain diversity, and considered additional scenarios where no maximum inter-infection interval was specified. Model outputs were compared with longitudinal GAS surveillance observations from northern Australia, a region with endemic infection. We also assessed the likely impact of a targeted strain-specific multivalent vaccine in this context. Our model produced patterns of transmission consistent with observations when the maximum inter-infection interval for developing enduring immunity was 19 weeks. Our vaccine analysis suggests that the leading multivalent GAS vaccine may have limited impact on the prevalence of GAS in populations in northern Australia if strain-specific immunity requires repeated episodes of infection. Our results suggest that observed GAS epidemiology from disease endemic settings is consistent with enduring strain-specific immunity being dependent on repeated infections with the same strain, and provide additional motivation for relevant human st

The prevalence of rheumatic heart disease (RHD) in the Aboriginal population of the Australian Northern Territory is high, and Streptococcus pyogenes skin infections likely contribute to this. A promising candidate S. pyogenes "30mer" vaccine is composed of 30 pharyngitis associated type-specific antigens from the S. pyogenes M protein. Cross opsonisation experiments suggest that 30mer vaccine protection may extend to non-cognate emm types. A new "emm cluster" scheme for classifying M protein is based on the full-length coding sequence, and correlates with functional and immunological properties, and anatomical tropism. Twenty-seven years of research in the Northern Territory has yielded 1810 S. pyogenes isolates with clinical and emm type data. The primary aim was to analyse these data with reference to the emm cluster scheme and cross opsonisation information, to inform estimation of 30mer vaccine efficacy in the Northern Territory. The isolates encompass 101 emm types. Variants of cluster A-C were enriched in throat isolates, and variants of emm cluster D enriched in skin isolates. Throat isolates were enriched for 30mer vaccine cognate emm types in comparison with skin isolates of which only 25% were vaccine emm types. While cross opsonisation data indicates potential for enhancing 30mer vaccine coverage, more than one third of skin isolates were within 38 emm types untested for cross opsonisation. Emm cluster D variants, in particular emm cluster D4, were not only all non-cognate with the vaccine, but were abundant and diverse, and less likely to be cross-opsonisation positive than other emm clusters. Long term persistence of many emm types in the study area was revealed. It was concluded that the 30mer vaccine efficacy in the Northern Territory will likely require both cross protection, and additional measures to elicit immunity against variants of emm cluster D.

OBJECTIVE: We conducted a comprehensive, systematic review of the global childhood population prevalence of impetigo and the broader condition pyoderma. METHODS: PubMed was systematically searched for impetigo or pyoderma studies published between January 1 1970 and September 30 2014. Two independent reviewers extracted data from each relevant article on the prevalence of impetigo. FINDINGS: Sixty-six articles relating to 89 studies met our inclusion criteria. Based on population surveillance, 82 studies included data on 145,028 children assessed for pyoderma or impetigo. Median childhood prevalence was 12·3% (IQR 4·2-19·4%). Fifty-eight (65%) studies were from low or low-middle income countries, where median childhood prevalences were 8·4% (IQR 4·2-16·1%) and 14·5% (IQR 8·3-20·9%), respectively. However, the highest burden was seen in underprivileged children from marginalised communities of high-income countries; median prevalence 19·4%, (IQR 3·9-43·3%). CONCLUSION: Based on data from studies published since 2000 from low and low-middle income countries, we estimate the global population of children suffering from impetigo at any one time to be in excess of 162 million, predominantly in tropical, resource-poor contexts. Impetigo is an under-recognised disease and in conjunction with scabies, comprises a major childhood dermatological condition with potential lifelong consequences if untreated.

With the increasing availability of high quality digital cameras that are easily operated by the non-professional photographer, the utility of using digital images to assess endpoints in clinical research of skin lesions has growing acceptance. However, rigorous protocols and description of experiences for digital image collection and assessment are not readily available, particularly for research conducted in remote settings. We describe the development and evaluation of a protocol for digital image collection by the non-professional photographer in a remote setting research trial, together with a novel methodology for assessment of clinical outcomes by an expert panel blinded to treatment allocation.

BACKGROUND: Crusted scabies, or hyperinfestation with Sarcoptes scabiei, occurs in people with an inadequate immune response to the mite. In recent decades, data have emerged suggesting that treatment of crusted scabies with oral ivermectin combined with topical agents leads to lower mortality, but there are no generally accepted tools for describing disease severity. Here, we describe a clinical grading scale for crusted scabies and its utility in real world practice. METHODOLOGY/PRINCIPAL FINDINGS: In 2002, Royal Darwin Hospital (RDH), a hospital in tropical Australia developed and began using a clinical grading scale to guide the treatment of crusted scabies. We conducted a retrospective observational study including all episodes of admission to RDH for crusted scabies during the period October 2002-December 2010 inclusive. Patients who were managed according to the grading scale were compared with those in whom the scale was not used at the time of admission but was calculated retrospectively. There were 49 admissions in 30 patients during the study period, of which 49 (100%) were in Indigenous Australians, 29 (59%) were male and the median age was 44.1 years. According to the grading scale, 8 (16%) episodes were mild, 24 (49%) were moderate, and 17 (35%) were severe. Readmission within the study period was significantly more likely with increasing disease severity, with an odds ratio (95% CI) of 12.8 (1.3-130) for severe disease compared with mild. The patients managed according to the grading scale (29 episodes) did not differ from those who were not (20 episodes), but they received fewer doses of ivermectin and had a shorter length of stay (11 vs. 16 days, p = 0.02). Despite this the outcomes were no different, with no deaths in either group and a similar readmission rate. CONCLUSIONS/SIGNIFICANCE: Our grading scale is a useful tool for the assessment and management of crusted scabies.

Here we report a single nucleotide polymorphism (SNP) based genotyping method for Klebsiella pneumoniae utilising high-resolution melting (HRM) analysis of fragments within the multilocus sequence typing (MLST) loci. The approach is termed mini-MLST or Minim typing and it has previously been applied to Streptococcus pyogenes, Staphylococcus aureus and Enterococcus faecium. Six SNPs were derived from concatenated MLST sequences on the basis of maximisation of the Simpsons Index of Diversity (D). DNA fragments incorporating these SNPs and predicted to be suitable for HRM analysis were designed. Using the assumption that HRM alleles are defined by G+C content, Minim typing using six fragments was predicted to provide a D = 0.979 against known STs. The method was tested against 202 K. pneumoniae using a blinded approach in which the MLST analyses were performed after the HRM analyses. The HRM-based alleles were indeed in accordance with G+C content, and the Minim typing identified known STs and flagged new STs. The tonB MLST locus was determined to be very diverse, and the two Minim fragments located herein contribute greatly to the resolving power. However these fragments are refractory to amplification in a minority of isolates. Therefore, we assessed the performance of two additional formats: one using only the four fragments located outside the tonB gene (D = 0.929), and the other using HRM data from these four fragments in conjunction with sequencing of the tonB MLST fragment (D = 0.995). The HRM assays were developed on the Rotorgene 6000, and the method was shown to also be robust on the LightCycler 480, allowing a 384-well high through-put format. The assay provides rapid, robust and low-cost typing with fully portable results that can directly be related to current MLST data. Minim typing in combination with molecular screening for antibiotic resistance markers can be a powerful surveillance tool kit.

The impact of Panton-Valentine leukocidin (PVL) on the severity of complicated skin and skin structure infections (cSSSI) caused by Staphylococcus aureus is controversial. We evaluated potential associations between clinical outcome and PVL presence in both methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) isolates from patients enrolled in two large, multinational phase three clinical trials assessing ceftaroline fosamil for the treatment of cSSSI (the CANVAS 1 and 2 programs). Isolates from all microbiologically evaluable patients with monomicrobial MRSA or MSSA infections (n = 473) were genotyped by PCR for pvl and underwent pulsed-field gel electrophoresis (PFGE). Genes encoding pvl were present in 266/473 (56.2%) isolates. Infections caused by pvl-positive S. aureus were associated with younger patient age, North American acquisition, and presence of major abscesses (P<0.001 for each). Cure rates of patients infected with pvl-positive and pvl-negative S. aureus were similar overall (93.6% versus 92.8%; P = 0.72), and within MRSA-infected (94.5% vs. 93.1%; P = 0.67) and MSSA-infected patients (92.2% vs. 92.7%; P = 1.00). This finding persisted after adjustment for multiple patient characteristics. Outcomes were also similar when USA300 PVL+ and non-USA300 PVL+ infections were compared. The results of this contemporary, international study suggest that pvl presence was not the primary determinant of outcome in patients with cSSSI due to either MRSA or MSSA.

INTRODUCTION: We aimed to design a real-time reverse-transcriptase-PCR (rRT-PCR), high-resolution melting (HRM) assay to detect the H275Y mutation that confers oseltamivir resistance in influenza A/H1N1 2009 viruses. FINDINGS: A novel strategy of amplifying a single base pair, the relevant SNP at position 823 of the neuraminidase gene, was chosen to maintain specificity of the assay. Wildtype and mutant virus were differentiated when using known reference samples of cell-cultured virus. However, when dilutions of these reference samples were assayed, amplification of non-specific primer-dimer was evident and affected the overall melting temperature (T(m)) of the amplified products. Due to primer-dimer appearance at >30 cycles we found that if the cycle threshold (C(T)) for a dilution was >30, the HRM assay did not consistently discriminate mutant from wildtype. Where the C(T) was <30 we noted an inverse relationship between C(T) and T(m) and fitted quadratic curves allowed the discrimination of wildtype, mutant and 30∶70 mutant∶wildtype virus mixtures. We compared the C(T) values for a TaqMan H1N1 09 detection assay with those for the HRM assay using 59 clinical samples and demonstrated that samples with a TaqMan detection assay C(T)>32.98 would have an H275Y assay C(T)>30. Analysis of the TaqMan C(T) values for 609 consecutive clinical samples predicted that 207 (34%) of the samples would result in an HRM assay C(T)>30 and therefore not be amenable to the HRM assay. CONCLUSIONS: The use of single base pair PCR and HRM can be useful for specifically interrogating SNPs. When applied to H1N1 09, the constraints this placed on primer design resulted in amplification of primer-dimer products. The impact primer-dimer had on HRM curves was adjusted for by plotting T(m) against C(T). Although less sensitive than TaqMan assays, the HRM assay can rapidly, and at low cost, screen samples with moderate viral concentrations.

High resolution melting (HRM) analysis is gaining prominence as a method for discriminating DNA sequence variants. Its advantage is that it is performed in a real-time PCR device, and the PCR amplification and HRM analysis are closed tube, and effectively single step. We have developed an HRM-based method for Staphylococcus aureus genotyping. Eight single nucleotide polymorphisms (SNPs) were derived from the S. aureus multi-locus sequence typing (MLST) database on the basis of maximized Simpson's Index of Diversity. Only G↔A, G↔T, C↔A, C↔T SNPs were considered for inclusion, to facilitate allele discrimination by HRM. In silico experiments revealed that DNA fragments incorporating the SNPs give much higher resolving power than randomly selected fragments. It was shown that the predicted optimum fragment size for HRM analysis was 200 bp, and that other SNPs within the fragments contribute to the resolving power. Six DNA fragments ranging from 83 bp to 219 bp, incorporating the resolution optimized SNPs were designed. HRM analysis of these fragments using 94 diverse S. aureus isolates of known sequence type or clonal complex (CC) revealed that sequence variants are resolved largely in accordance with G+C content. A combination of experimental results and in silico prediction indicates that HRM analysis resolves S. aureus into 268 "melt types" (MelTs), and provides a Simpson's Index of Diversity of 0.978 with respect to MLST. There is a high concordance between HRM analysis and the MLST defined CCs. We have generated a Microsoft Excel key which facilitates data interpretation and translation between MelT and MLST data. The potential of this approach for genotyping other bacterial pathogens was investigated using a computerized approach to estimate the densities of SNPs with unlinked allelic states. The MLST databases for all species tested contained abundant unlinked SNPs, thus suggesting that high resolving power is not dependent upon large numbers of SNPs.

We have developed a single nucleotide polymorphism (SNP) nucleated high-resolution melting (HRM) technique to genotype Enterococcus faecium. Eight SNPs were derived from the E. faecium multilocus sequence typing (MLST) database and amplified fragments containing these SNPs were interrogated by HRM. We tested the HRM genotyping scheme on 85 E. faecium bloodstream isolates and compared the results with MLST, pulsed-field gel electrophoresis (PFGE) and an allele specific real-time PCR (AS kinetic PCR) SNP typing method. In silico analysis based on predicted HRM curves according to the G+C content of each fragment for all 567 sequence types (STs) in the MLST database together with empiric data from the 85 isolates demonstrated that HRM analysis resolves E. faecium into 231 "melting types" (MelTs) and provides a Simpson's Index of Diversity (D) of 0.991 with respect to MLST. This is a significant improvement on the AS kinetic PCR SNP typing scheme that resolves 61 SNP types with D of 0.95. The MelTs were concordant with the known ST of the isolates. For the 85 isolates, there were 13 PFGE patterns, 17 STs, 14 MelTs and eight SNP types. There was excellent concordance between PFGE, MLST and MelTs with Adjusted Rand Indices of PFGE to MelT 0.936 and ST to MelT 0.973. In conclusion, this HRM based method appears rapid and reproducible. The results are concordant with MLST and the MLST based population structure.