Extensive Homoplasy but No Evidence of Convergent Evolution of Repeat Numbers at MIRU Loci in Modern Mycobacterium tuberculosis Lineages.

More human deaths have been attributable to Mycobacterium tuberculosis than any other pathogen, and the epidemic is sustained by ongoing transmission. Various typing schemes have been developed to identify strain-specific differences and track transmission dynamics in affected communities, with recent introduction of whole genome sequencing providing the most accurate assessment. Mycobacterial interspersed repetitive unit (MIRU) typing is a family of variable number tandem repeat schemes that have been widely used to study the molecular epidemiology of M. tuberculosis. MIRU typing was used in most well-resourced settings to perform routine molecular epidemiology. Instances of MIRU homoplasy have been observed in comparison with sequence-based phylogenies, limiting its discriminatory value. A fundamental question is whether the observed homoplasy arises purely through stochastic processes, or whether there is evidence of natural selection. We compared repeat numbers at 24 MIRU loci with a whole genome sequence-based phylogeny of 245 isolates representing three modern M. tuberculosis lineages. This analysis demonstrated extensive homoplasy of repeat numbers, but did not detect any evidence of natural selection of repeat numbers, at least since the ancestral branching of the three modern lineages of M. tuberculosis. In addition, we observed good sensitivity but poor specificity and positive predictive values of MIRU-24 to detect clusters of recent transmission, as defined by whole-genome single nucleotide polymorphism analysis. These findings provide mechanistic insight, and support a transition away from VNTR-based typing toward sequence-based typing schemes for both research and public health purposes.

Culture-proven Bloodstream Infections at a Specialist Pediatric Hospital.

BACKGROUND: Bloodstream infection (BSI) is one of the leading causes of morbidity and mortality in children. This study was done to assess the local epidemiology and outcome of BSIs managed at a large specialist pediatric hospital with a focus on community-onset BSI. METHODS: We retrospectively reviewed laboratory-confirmed BSI in children (0-18 years) at The Children's Hospital at Westmead over a 3-year period (2014-2016). Laboratory data and patient medical records were used to determine BSI rates, blood culture contamination rates, patient demographics, isolate profile, antimicrobial resistance and mortality rate in this cohort. RESULTS: In total, 47,368 blood cultures were collected; 1027 (2.2%) grew probable contaminants and 991 (2.1%) grew clinically significant isolates. Clinically significant bacteremia accounted for 4.8 per 1000 admissions, with 391 children managed for 465 culture-proven BSI episodes. One hundred thirty-one (28.2%) episodes were community-onset community-associated, and 334 (71.8%) were either community-onset healthcare-associated (HCA) (187; 40.2 %) or hospital-onset (147; 31.6%). Of the significant isolates, 243 (52.3%) were Gram-positive bacteria, 198 (42.6%) were Gram-negative bacteria, 6 (1.3%) were polymicrobial infections and 18 (3.9%) were yeast. Staphylococcus aureus (115; 24.7%) and Escherichia coli (54; 11.6%) were the most common organisms identified. Osteoarticular infection (44; 33.6%) and urosepsis (23; 17.6%) were the most frequent sites of infection associated with non-HCA BSI. Mortality at 30 days was reported in 15 (3.3%) children, all whom had preexisting comorbidities. CONCLUSIONS: The majority of BSI episodes managed in our hospital were either community-onset HCA or hospital-onset infections. This highlights the considerable importance of infection control and central venous catheter device care initiatives. Among community-associated BSI, S. aureus in association with osteoarticular infection was predominant.

Drug-Resistant tuberculosis - primary transmission and management.

The DOTS strategy assisted global tuberculosis (TB) control, but was unable to prevent the emergence and spread of drug-resistant strains. Genomic evidence confirms the transmission of drug-resistant Mycobacterium tuberculosis strains in many different settings, indicative of epidemic spread. These findings emphasise the need for enhanced infection control measures in health care and congregate settings. Young children in TB endemic areas are particularly vulnerable. Although advances in TB drug and vaccine development are urgently needed, improved access to currently available preventive therapy and treatment for drug resistant TB could reduce the disease burden and adverse outcomes experienced by children. We review new insights into the transmission dynamics of drug resistant TB, the estimated disease burden in children and optimal management strategies to consider.

Nosocomial Transmission from an Adolescent with Sputum Smear-Negative Pulmonary Tuberculosis.

We describe a case of sputum smear-negative pulmonary tuberculosis in an adolescent boy, where a delay in diagnosis and institution of appropriate infection control measures resulted in transmission of infection to at least 3 and possibly as many as 6 healthcare workers. Lapses in the use of standard precautions for infection control were also identified.

Low prevalence of Kingella kingae carriage in children aged 6-48 months in Sydney, Australia.

AIM: A prospective observational study was conducted to estimate the prevalence of oropharyngeal carriage of Kingella kingae in healthy Australian pre-school children. METHODS: Screening for carriage of K. kingae as well as Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, Staphylococcus aureus, Haemophilus influenzae, and K. kingae was undertaken using a single bacterial throat swab taken from well children aged 6 months to 4 years. Standard laboratory procedures were used for culture and identification of organisms. RESULTS: One hundred children were enrolled between October and December 2014 at the Children's Hospital at Westmead. Median age was 24.0 months (range 6.1-48.8 months); 52 children were male and 36 attended day-care facilities. Forty-one children had siblings aged less than 5 years and 67 children had siblings of any age. K. kingae oropharyngeal carriage was not detected in any of the children. Rates of carriage of other organisms were: 30% S. aureus, 21% H. influenzae, 2% S. pneumoniae and 2% S. pyogenes. Thirty-eight children were colonised with Kingella denitrificans. CONCLUSIONS: Our results suggest that prevalence of K. kingae carriage in pre-school children in Sydney is very low and support local and national guidelines that recommend flucloxacillin as empiric first-line therapy for children with osteoarticular infections. Studies conducted over the winter months and in other Australian centres could help answer outstanding questions regarding differences in carriage rates of K. kingae in children.

Paediatric Staphylococcus aureus bacteraemia: A single-centre retrospective cohort.

AIM: We aimed to describe the clinical epidemiology of Staphylococcus aureus bacteraemia (SAB) at a large, tertiary/quaternary children's hospital in Australia. METHODS: We performed a retrospective chart review of SAB cases at the Children's Hospital at Westmead (CHW) over 5 years; 2006-2011. We compared frequency, clinical profile and outcomes of SAB with published data from CHW; 1994-1998. We compared health-care associated with community-associated (HCA-SAB and CA-SAB; defined epidemiologically) and methicillin-resistant with methicillin susceptible S. aureus (MRSA and MSSA). RESULTS: We identified 174 episodes of paediatric SAB with an average annual admission rate of 1.3/1000 which has not increased compared with a decade earlier. Half of the cases (49%) were CA-SAB; 18% were MRSA. The proportion of CA-MRSA bacteraemia (22%) has increased. The proportion of SAB associated with central venous access devices (CVADs; 40%) has increased. CA-SAB cases were more likely to present with a tissue focus of disease (e.g. osteo-articular, pneumonia) and often required surgery. HCA-SAB less frequently required surgery, a minority is MRSA, and vascular device intervention (removal, sterilisation) is common. Six cases (4%) of infective endocarditis (IE) were identified; three with a history of congenital heart disease, two with CVADs in situ. There were no deaths in this cohort. CONCLUSIONS: Over an 18-year period, the proportion of SAB due to CA-MRSA and SAB associated with CVADs has increased. Categorisation of SAB as HCA and CA reveals two broad phenotypes of paediatric SAB. SAB in children is infrequently associated with IE. The health-care burden of paediatric SAB is considerable', but mortality is low.

Whole Genome Sequencing Demonstrates Limited Transmission within Identified Mycobacterium tuberculosis Clusters in New South Wales, Australia.

Australia has a low tuberculosis incidence rate with most cases occurring among recent immigrants. Given suboptimal cluster resolution achieved with 24-locus mycobacterium interspersed repetitive unit (MIRU-24) genotyping, the added value of whole genome sequencing was explored. MIRU-24 profiles of all Mycobacterium tuberculosis culture-confirmed tuberculosis cases diagnosed between 2009 and 2013 in New South Wales (NSW), Australia, were examined and clusters identified. The relatedness of cases within the largest MIRU-24 clusters was assessed using whole genome sequencing and phylogenetic analyses. Of 1841 culture-confirmed TB cases, 91.9% (1692/1841) had complete demographic and genotyping data. East-African Indian (474; 28.0%) and Beijing (470; 27.8%) lineage strains predominated. The overall rate of MIRU-24 clustering was 20.1% (340/1692) and was highest among Beijing lineage strains (35.7%; 168/470). One Beijing and three East-African Indian (EAI) clonal complexes were responsible for the majority of observed clusters. Whole genome sequencing of the 4 largest clusters (30 isolates) demonstrated diverse single nucleotide polymorphisms (SNPs) within identified clusters. All sequenced EAI strains and 70% of Beijing lineage strains clustered by MIRU-24 typing demonstrated distinct SNP profiles. The superior resolution provided by whole genome sequencing demonstrated limited M. tuberculosis transmission within NSW, even within identified MIRU-24 clusters. Routine whole genome sequencing could provide valuable public health guidance in low burden settings.

Identifying Likely Transmission Pathways within a 10-Year Community Outbreak of Tuberculosis by High-Depth Whole Genome Sequencing.

BACKGROUND: Improved tuberculosis control and the need to contain the spread of drug-resistant strains provide a strong rationale for exploring tuberculosis transmission dynamics at the population level. Whole-genome sequencing provides optimal strain resolution, facilitating detailed mapping of potential transmission pathways. METHODS: We sequenced 22 isolates from a Mycobacterium tuberculosis cluster in New South Wales, Australia, identified during routine 24-locus mycobacterial interspersed repetitive unit typing. Following high-depth paired-end sequencing using the Illumina HiSeq 2000 platform, two independent pipelines were employed for analysis, both employing read mapping onto reference genomes as well as de novo assembly, to control biases in variant detection. In addition to single-nucleotide polymorphisms, the analyses also sought to identify insertions, deletions and structural variants. RESULTS: Isolates were highly similar, with a distance of 13 variants between the most distant members of the cluster. The most sensitive analysis classified the 22 isolates into 18 groups. Four of the isolates did not appear to share a recent common ancestor with the largest clade; another four isolates had an uncertain ancestral relationship with the largest clade. CONCLUSION: Whole genome sequencing, with analysis of single-nucleotide polymorphisms, insertions, deletions, structural variants and subpopulations, enabled the highest possible level of discrimination between cluster members, clarifying likely transmission pathways and exposing the complexity of strain origin. The analysis provides a basis for targeted public health intervention and enhanced classification of future isolates linked to the cluster.

Added value of whole-genome sequencing for management of highly drug-resistant TB.

OBJECTIVES: Phenotypic drug susceptibility testing (DST) for Mycobacterium tuberculosis takes several weeks to complete and second-line DST is often poorly reproducible, potentially leading to compromised clinical decisions. Following a fatal case of XDR TB, we investigated the potential benefit of using whole-genome sequencing to generate an in silico drug susceptibility profile. METHODS: The clinical course of the patient was reviewed, assessing the times at which phenotypic DST data became available and changes made to the therapeutic regimen. Whole-genome sequencing was performed on the earliest available isolate and variants associated with drug resistance were identified. RESULTS: The final DST report, including second-line drugs, was issued 10 weeks after patient presentation and 8 weeks after initial growth of M. tuberculosis. In the interim, the patient may have received a compromised regimen that had the potential to select for further drug resistance. The in silico susceptibility profile, extrapolated from evolving evidence in the literature, provided comparable or superior data to the DST results for second-line drugs and could be generated in a much shorter timeframe. CONCLUSIONS: We propose routine whole-genome sequencing of all MDR M. tuberculosis isolates in adequately resourced settings. This will improve individual patient care, monitor for transmission events and advance our understanding of resistance-associated mutations.

Anncaliia algerae microsporidial myositis.

The insect microsporidian Anncaliia algerae was first described in 2004 as a cause of fatal myositis in an immunosuppressed person from Pennsylvania, USA. Two cases were subsequently reported, and we detail 2 additional cases, including the only nonfatal case. We reviewed all 5 case histories with respect to clinical characteristics, diagnosis, and management and summarized organism life cycle and epidemiology. Before infection, all case-patients were using immunosuppressive medications for rheumatoid arthritis or solid-organ transplantation. Four of the 5 case-patients were from Australia. All diagnoses were confirmed by skeletal muscle biopsy; however, peripheral nerves and other tissues may be infected. The surviving patient received albendazole and had a reduction of immunosuppressive medications and measures to prevent complications. Although insects are the natural hosts for A. algerae, human contact with water contaminated by spores may be a mode of transmission. A. algerae has emerged as a cause of myositis, particularly in coastal Australia.

A loop-mediated isothermal amplification (LAMP) assay for Strongyloides stercoralis in stool that uses a visual detection method with SYTO-82 fluorescent dye.

An assay to detect Strongyloides stercoralis in stool specimens was developed using the loop-mediated isothermal amplification (LAMP) method. Primers were based on the 28S ribosomal subunit gene. The reaction conditions were optimized and SYTO-82 fluorescent dye was used to allow real-time and visual detection of the product. The product identity was confirmed with restriction enzyme digestion, cloning, and sequence analysis. The assay was specific when tested against DNA from bacteria, fungi and parasites, and 30 normal stool samples. Analytical sensitivity was to < 10 copies of target sequence in a plasmid and up to a 10(-2) dilution of DNA extracted from a Strongyloides ratti larva spiked into stool. Sensitivity was increased when further dilutions were made in water, indicative of reduced reaction inhibition. Twenty-seven of 28 stool samples microscopy and polymerase chain reaction positive for S. stercoralis were positive with the LAMP method. On the basis of these findings, the assay warrants further clinical validation.

Viral arthritides.

Viral infections may manifest as acute or chronic arthritis. Joint involvement arises from either direct infection of the joint, through an immunological response directed towards the virus or autoimmunity. Epidemiological clues to the diagnosis include geographic location and exposure to vector-borne, blood-borne or sexually transmitted viruses. Although not always possible, it is important to diagnose the pathogenic virus, usually by serology, nucleic acid tests or rarely, viral culture. In general, viral arthritides are self-limiting and treatment is targeted at symptomatic relief. This article focuses on the causes, clinical features, diagnosis and treatment of viral arthritides.

Nocardia infections of the face and neck.

Involvement of the soft tissues of the face and neck by Nocardia spp. is uncommon. We review the epidemiology, clinical features, diagnosis, and management of such infections in the setting of primary cutaneous nocardiosis and disseminated disease. Although immune compromise is an important risk factor for these infections, they also occur in healthy individuals. Infection may arise through direct inoculation following injury or by hematogenous spread from a primary site, usually the lung. The rare variant of lymphocutaneous disease-cervicofacial nocardiosis-typically affects children, but can occur in adults. The diagnosis is made by conventional microscopy and culture, but radiological imaging is useful to delineate disease extent, and molecular methods are increasingly assisting the diagnosis by providing rapid detection and identification of the pathogen. Sulfonamides remain the preferred treatment for many cases and are an important component of the therapeutic armamentarium. Other therapeutic options include minocycline, the carbapenems, and linezolid.