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.

Port-site infection due to nontuberculous mycobacteria following laparoscopic surgery.

Nontuberculous mycobacteria (NTM) are commonly found in soil and water and can cause nosocomial infections by contaminating equipment and disinfectants solution used in hospitals. NTM port-site infection after laparoscopic surgery is increasingly observed, but its clinical features, management, and prevention have not been reviewed adequately. We performed a comprehensive literature review of reports that described the clinical manifestation and management of NTM port-site infections following laparoscopic surgery. The perceived increase in NTM port-site infections is likely multifactorial, influenced by greater awareness, better diagnostics, changes in medical practice, increased prevalence of immunosuppression, and potential pathogen spread. Widespread resistance to common disinfectants is a major concern. Patients with NTM port-site infections typically present 1-3 months after the laparoscopic intervention with chronic local and minimal systemic symptoms. Surgical excision plays an important role in localized or refractory cases. Medical treatment should be guided by species identification and in vitro drug-susceptibility testing (DST) of the infecting NTM strain, with a combination of second-line antituberculosis agents, given for a prolonged duration. NTM port site infection is best prevented by meticulous skin preparation and infection control, using only sterilized supplies for laparoscopic surgery.

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.

Rapid direct identification of positive paediatric blood cultures by MALDI-TOF MS technology and its clinical impact in the paediatric hospital setting.

OBJECTIVE: Rapid diagnostic tools are imperative for timely clinical decision making, particularly in bacteraemic patients. This study evaluated the performance of a fast, inexpensive novel in house method for processing positive blood cultures for immediate identification of microorganisms by matrix-assisted laser desorption ionization-time of flight mass spectrometry (Vitek MS bioMérieux). We prospectively analyzed the clinical impact of such method on the management of pediatric patients. RESULT: In total, 360 positive blood cultures were included. Among 318 mono-microbial cultures, in-house method achieved correct identification in 270 (85%) cultures to the species level, whilst 43 (13.5%) gave no identification, and 7 (2.2%) gave discordant identifications. Identification of Gram-negative organisms was accurate to both species and genus level in 99% of isolates, and for Gram positives accuracy was 84% to genus and 81% to species level overall, with accuracy of 100% for Staphylococcus aureus and Enterococcus to the species level. Assessment of the potential impact of direct identification in sixty sequential cases revealed a clear clinical benefit in 35.5% of cases. Benefits included timely antibiotic rationalization, change of medical intervention, and early confirmation of contamination. This study demonstrates a highly accurate in-house method with considerable potential clinical benefits for paediatric care.

Educational intervention does not reduce non-invasive urine contamination rates in children presenting to the emergency department.

AIM: Urinary tract infection is common in children with high contamination rates with non-invasive urine sampling (NIU). Our aims were to evaluate an educational tool for decreasing contamination rates and find factors associated with contamination. METHODS: This was a prospective cohort interventional study with a review of microbiology data and medical records of all NIU specimens collected at a large tertiary children's emergency department (ED) over a 1-year period. The intervention was the provision of a urine collection kit and educational pamphlet and education of staff. NIU contamination was calculated for 6 months pre-intervention and 6 months post-intervention. The association of factors with NIU contamination was evaluated for all cohorts (age, gender, presence of diarrhoea, season, time of day, time to incubation and activity of the ED). RESULTS: A total of 2104 NIU samples were included (median age 3 years, 52% females). There was no difference between periods in contamination rates (29.2% and 31.2%, respectively, P = 0.322). Collectively, high monthly activity of the department, age and female gender were associated with contamination. The highest contamination rates were among children aged 0-3 months and 12 years and older (38.1 and 48.9%, respectively). CONCLUSIONS: The urine collection kit and educational tool did not decrease NIU contamination rates in our ED. Contamination rates were correlated with the monthly activity of our department and female gender and were noticeably high among infants and adolescents. Given the high prevalence of urinary tract infection among these age groups, measures should be taken to reassess indications and methods for urine collection.

Overview of paediatric tuberculosis cases treated in the Sydney Children's Hospitals Network, Australia.

BACKGROUND: Sydney has a large and highly mobile immigrant community. The pattern of paediatric tuberculosis (TB) disease in this highly cosmopolitan city is not well documented. METHODS: We reviewed data on all children notified with TB in New South Wales (NSW), Australia, from January 2014 to December 2015, complemented by an expanded dataset for children managed within the Sydney Children's Hospitals Network (SCHN). RESULTS: Over the 2-year study period, 921 TB cases were identified in NSW, including 26 (2.8%) children younger than 15 years of age. Of 23 children and adolescents treated for TB in the SCHN, 21 (91.3%) had a history of recent immigration from, or travel to, a country with high TB incidence, and 7 (30.4%) reported contact with an infectious TB case in Australia. Fourteen (60.9%) children had microbiologically confirmed TB; of these, 5 (21.7%) had acid-fast bacilli on microscopy, 8 (34.8%) were positive by polymerase chain reaction and 11 (47.8%) were positive by culture. All Mycobacterium tuberculosis isolates were susceptible to first-line drugs. Ten (43.5%) cases were not vaccinated with bacille Calmette-Guérin (BCG), including all cases with severe disease: 2 with disseminated (miliary) TB and 3 with tuberculous meningitis. CONCLUSION: Our findings emphasise the need for improved TB prevention and surveillance in children at high risk of exposure, particularly young children travelling to areas of high TB incidence.

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.

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.

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.