Alexander C. Outhred

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.

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.

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.

Anncaliia algerae Microsporidial Myositis

Immunosuppression is a risk factor for serious infection in humans.

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.

Nontuberculous Mycobacteria in Children: A Focus on Bloodstream Infections

Background: Nontuberculous mycobacteria (NTM) are ubiquitous organisms with variable disease-causing potential. Bloodstream infections caused by NTM in children are poorly described. Methods: We describe a retrospective case series of children with culture-confirmed mycobacterial disease managed at the Children’s Hospital at Westmead between July 2005 and June 2015. Results: Sixty-five patients had 149 positive NTM cultures; 55 (83.0%) episodes in 54 patients were considered clinically significant. Of the 54 children who met criteria for NTM disease, 25 (46.3%) had lymphadenitis, 13 (24.1%) lung disease, 8 (14.8%) had soft tissue infection or osteomyelitis and 8 (14.8%) had bacteremia. All children with bacteremia had a central venous catheter; those with pulmonary infection had underlying lung disease and all children with soft tissue infection or osteomyelitis had a history of recent penetrating injury. Disease caused by Mycobacterium avium–intracellulare complex was most common, accounting for 19 (76.0%) and 7 (53.8%) lymph node and lung infections, respectively. The most frequently isolated rapid growing mycobacteria were Mycobacterium fortuitum (8; 15%) and Mycobacterium abscessus (6; 11%), with M. fortuitum accounting for the majority (6; 75%) of bloodstream infections. Six (75%) patients with bacteremia had their intravenous catheter removed and all had a favorable outcome. A single disease relapse was reported in 1 of 2 patients with a retained catheter. Conclusion: Lymphadenitis was the most common NTM disease manifestation and not associated with comorbidity. NTM bacteremia was always associated with a central line and catheter removal with cure. We were unable to assess the added value of various antibiotic regimens.

Paediatric Staphylococcus aureus bacteraemia: A single-centre retrospective cohort

We aimed to describe the clinical epidemiology of Staphylococcus aureus bacteraemia (SAB) at a large, tertiary/quaternary childrens hospital in Australia.

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

A prospective observational study was conducted to estimate the prevalence of oropharyngeal carriage of Kingella kingae in healthy Australian pre‐school children.

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.