Evaluation of a multiplex-qPCR for paediatric pleural empyema-An observational study in hospitalised children.

Pleural empyema is a serious complication of pneumonia in children. Negative bacterial cultures commonly impede optimal antibiotic therapy. To improve bacterial identification, we developed a molecular assay and evaluated its performance compared with bacterial culture. Our multiplex-quantitative PCR to detect Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus and Haemophilus influenzae was assessed using bacterial genomic DNA and laboratory-prepared samples (n = 267). To evaluate clinical performance, we conducted the Molecular Assessment of Thoracic Empyema (MATE) observational study, enrolling children hospitalised with empyema. Pleural fluids were tested by bacterial culture and multiplex-qPCR, and performance determined using a study gold standard. We determined clinical sensitivity and time-to-organism-identification to assess the potential of the multiplex-qPCR to reduce the duration of empiric untargeted antibiotic therapy. Using spiked samples, the multiplex-qPCR demonstrated 213/215 (99.1%) sensitivity and 52/52 (100%) specificity for all organisms. During May 2019-March 2023, 100 children were enrolled in the MATE study; median age was 3.9 years (IQR 2-5.6). A bacterial pathogen was identified in 90/100 (90%) specimens by multiplex-qPCR, and 24/100 (24%) by bacterial culture (P <0.001). Multiplex-qPCR identified a bacterial cause in 68/76 (90%) culture-negative specimens. S. pneumoniae was the most common pathogen, identified in 67/100 (67%) specimens. We estimate our multiplex-qPCR would have reduced the duration of untargeted antibiotic therapy in 61% of cases by a median 20 days (IQR 17.5-23, range 1-55). Multiplex-qPCR significantly increased pathogen detection compared with culture and may allow for reducing the duration of untargeted antibiotic therapy.

Detection of group a streptococcal pharyngitis by quantitative PCR.

BACKGROUND: Group A streptococcus (GAS) is the most common bacterial cause of sore throat. School-age children bear the highest burden of GAS pharyngitis. Accurate diagnosis is difficult: the majority of sore throats are viral in origin, culture-based identification of GAS requires 24-48 hours, and up to 15% of children are asymptomatic throat carriers of GAS. The aim of this study was to develop a quantitative polymerase chain reaction (qPCR) assay for detecting GAS pharyngitis and assess its suitability for clinical diagnosis. METHODS: Pharyngeal swabs were collected from children aged 3-18 years (n = 91) and adults (n = 36) located in the Melbourne area who presented with sore throat. Six candidate PCR assays were screened using a panel of reference isolates, and two of these assays, targeting speB and spy1258, were developed into qPCR assays. The qPCR assays were compared to standard culture-based methods for their ability to detect GAS pharyngitis. GAS isolates from culture positive swabs underwent emm-typing. Clinical data were used to calculate McIsaac scores as an indicator of disease severity. RESULTS: Twenty-four of the 127 samples (18.9%) were culture-positive for GAS, and all were in children (26%). The speB qPCR had 100% sensitivity and 100% specificity compared with gold-standard culture, whereas the spy1258 qPCR had 87% sensitivity and 100% specificity. Nine different emm types were found, of which emm 89, 3, and 28 were most common. Bacterial load as measured by qPCR correlated with culture load. There were no associations between symptom severity as indicated by McIsaac scores and GAS bacterial load. CONCLUSIONS: The speB qPCR displayed high sensitivity and specificity and may be a useful tool for GAS pharyngitis diagnosis and research.

Prospective Surveillance of Pediatric Invasive Group A Streptococcus Infection.

BACKGROUND: Invasive group A Streptococcus (GAS) disease has an incidence in high-income countries of 3 to 5 per 100000 per annum and a case-fatality ratio of 10% to 15%. Although these rates are comparable to those of invasive meningococcal disease in Australia before vaccine introduction, invasive GAS disease currently requires reporting in only 2 jurisdictions. METHODS: Data were collected prospectively through active surveillance at the Royal Children's Hospital, Melbourne (October 2014 to September 2016). Isolation of GAS from a sterile site was required for inclusion. Comprehensive demographic and clinical data were collected, and emm typing was performed on all isolates. Disease was considered severe if the patient required inotropic support or mechanical ventilation. RESULTS: We recruited 28 patients. The median age of the patients was 3.5 years (range, 4 days to 11 years). Ten (36%) patients had severe disease. Fifteen (54%) children had presented to a medical practitioner for review in the 48 hours before their eventual admission, including 7 of the 10 patients with severe GAS infection. Complications 6 months after discharge persisted in 21% of the patients. emm1 was the most common emm type (29%). CONCLUSION: We found considerable short- and longer-term morbidity associated with pediatric invasive GAS disease in our study. Disease manifestations were frequently severe, and more than one-third of the patients required cardiorespiratory support. More than one-half of the patients attended a medical practitioner for assessment but were discharged in the 48-hour period before admission, which suggests that there might have been a window for earlier diagnosis. Our methodology was easy to implement as a surveillance system.

Nontuberculous Mycobacterial Disease in Children - Epidemiology, Diagnosis & Management at a Tertiary Center.

BACKGROUND: There are limited data on the epidemiology, diagnosis and optimal management of nontuberculous mycobacterial (NTM) disease in children. METHODS: Retrospective cohort study of NTM cases over a 10-year-period at a tertiary referral hospital in Australia. RESULTS: A total of 140 children with NTM disease, including 107 with lymphadenitis and 25 with skin and soft tissue infections (SSTIs), were identified. The estimated incidence of NTM disease was 0.6-1.6 cases / 100,000 children / year; no increasing trend was observed over the study period. Temporal analyses revealed a seasonal incidence cycle around 12 months, with peaks in late winter/spring and troughs in autumn. Mycobacterium-avium-complex accounted for most cases (77.8%), followed by Mycobacterium ulcerans (14.4%) and Mycobacterium marinum (3.3%). Polymerase chain reaction testing had higher sensitivity than culture and microscopy for acid-fast bacilli (92.0%, 67.2% and 35.7%, respectively). The majority of lymphadenitis cases underwent surgical excision (97.2%); multiple recurrences in this group were less common in cases treated with clarithromycin and rifampicin compared with clarithromycin alone or no anti-mycobacterial drugs (0% versus 7.1%; OR:0.73). SSTI recurrences were also less common in cases treated with two anti-mycobacterial drugs compared with one or none (10.5% versus 33.3%; OR:0.23). CONCLUSIONS: There was seasonal variation in the incidence of NTM disease, analogous to recently published observations in tuberculosis, which have been linked to seasonal variation in vitamin D. Our finding that anti-mycobacterial combination therapy was associated with a reduced risk of recurrences in patients with NTM lymphadenitis or SSTI requires further confirmation in prospective trials.

The age-related risk of co-existing meningitis in children with urinary tract infection.

OBJECTIVE: The primary aim of this study was to determine age-stratified rates of co-existing bacterial meningitis in children with urinary tract infection (UTI). The secondary aims of this study were to determine the causative pathogens of UTI, and the clinical features and outcome of children with co-existing meningitis. METHODS: Analysis of data collected over a nine-year period at a tertiary pediatric hospital in Australia. STUDY POPULATION: children below 16 years of age with culture-confirmed UTI and a paired CSF sample. RESULTS: A total of 748 episodes in 735 cases were included in the final analysis. The commonest pathogens causing UTI were Escherichia coli (67.4%), Enterococcus faecalis (8.4%), Klebsiella oxytoca (3.5%) and Klebsiella pneumoniae (3.5%). Only two (1.2%; 95% CI: 0.15-4.36%) of 163 neonates (between 0 and 28 days of age) with UTI had co-existing meningitis. Both presented with pyrexia, irritability and lethargy, and recovered uneventfully with antibiotic treatment. There were no cases of co-existing meningitis among 499 infants (between 29 days and 12 months of age) with UTI (95% CI: 0.00-0.74%), or any of the 86 children aged 12 months or over (95% CI: 0.00-4.20%). CONCLUSIONS: These findings indicate that clinicians should have a low threshold to perform a lumbar puncture in neonates with UTI, as the risk of co-existing meningitis is not insignificant in this age group. In contrast, beyond the neonatal period, the risk is small and a more selective approach is warranted.