Identification of bacterial pathogens in sudden unexpected death in infancy and childhood using 16S rRNA gene sequencing

Background Sudden unexpected death in infancy (SUDI) is the most common cause of post-neonatal death in the developed world. Following an extensive investigation, the cause of ~40% of deaths remains unknown. It is hypothesized that a proportion of deaths are due to an infection that remains undetected due to limitations in routine techniques. This study aimed to apply 16S rRNA gene sequencing to post-mortem (PM) tissues collected from cases of SUDI, as well as those from the childhood equivalent (collectively known as sudden unexpected death in infancy and childhood or SUDIC), to investigate whether this molecular approach could help identify potential infection-causing bacteria to enhance the diagnosis of infection. Methods In this study, 16S rRNA gene sequencing was applied to de-identified frozen post-mortem (PM) tissues from the diagnostic archive of Great Ormond Street Hospital. The cases were grouped depending on the cause of death: (i) explained non-infectious, (ii) infectious, and (iii) unknown. Results and conclusions In the cases of known bacterial infection, the likely causative pathogen was identified in 3/5 cases using bacterial culture at PM compared to 5/5 cases using 16S rRNA gene sequencing. Where a bacterial infection was identified at routine investigation, the same organism was identified by 16S rRNA gene sequencing. Using these findings, we defined criteria based on sequencing reads and alpha diversity to identify PM tissues with likely infection. Using these criteria, 4/20 (20%) cases of unexplained SUDIC were identified which may be due to bacterial infection that was previously undetected. This study demonstrates the potential feasibility and effectiveness of 16S rRNA gene sequencing in PM tissue investigation to improve the diagnosis of infection, potentially reducing the number of unexplained deaths and improving the understanding of the mechanisms involved

Condensed-Phase Photochemistry in the Absence of Radiation Chemistry

We report post-irradiation photochemistry studies of condensed ammonia using photons of energies below condensed ammonia’s ionization threshold of ~ 9 eV. Hydrazine (N2H4), diazene (also known as diimide and diimine) (N2H2), triazane (N3H5), and one or more isomers of N3H3 are detected as photochemistry products during temperature-programmed desorption. Product yields increase monotonically with (1) photon fluence and (2) film thickness. In the studies reported herein, the energies of photons responsible for product formation are constrained to less than 7.4 eV. Previous post-irradiation photochemistry studies of condensed ammonia employed photons sufficiently energetic to ionize condensed ammonia and initiate radiation chemistry. Such studies typically involve ion-molecule reactions and electron-induced reactions in addition to photochemistry. Although photochemistry is cited as a dominant mechanism for the synthesis of prebiotic molecules in interstellar ices, to the best of our knowledge, ours is one of the first astrochemically-relevant studies that has found unambiguous evidence for condensed-phase chemical synthesis induced by photons in the absence of ionization

Testing the effects of mass drug administration of azithromycin on mortality and other outcomes among 1–11-month-old infants in Mali (LAKANA) : study protocol for a cluster-randomized, placebo-controlled, double-blinded, parallel-group, three-arm clinical trial

Background: Mass drug administration (MDA) of azithromycin (AZI) has been shown to reduce under-5 mortality in some but not all sub-Saharan African settings. A large-scale cluster-randomized trial conducted in Malawi, Niger, and Tanzania suggested that the effect differs by country, may be stronger in infants, and may be concentrated within the first 3 months after treatment. Another study found no effect when azithromycin was given concomitantly with seasonal malaria chemoprevention (SMC). Given the observed heterogeneity and possible effect modification by other co-interventions, further trials are needed to determine the efficacy in additional settings and to determine the most effective treatment regimen. Methods: LAKANA stands for Large-scale Assessment of the Key health-promoting Activities of two New mass drug administration regimens with Azithromycin. The LAKANA trial is designed to address the mortality and health impacts of 4 or 2 annual rounds of azithromycin MDA delivered to 1–11-month-old (29–364 days) infants, in a high-mortality and malaria holoendemic Malian setting where there is a national SMC program. Participating villages (clusters) are randomly allocated in a ratio of 3:2:4 to three groups: placebo (control):4-dose AZI:2-dose AZI. The primary outcome measured is mortality. Antimicrobial resistance (AMR) will be monitored closely before, during, and after the intervention and both among those receiving and those not receiving MDA with the study drugs. Other outcomes, from a subset of villages, comprise efficacy outcomes related to morbidity, growth and nutritional status, outcomes related to the mechanism of azithromycin activity through measures of malaria parasitemia and inflammation, safety outcomes (AMR, adverse and serious adverse events), and outcomes related to the implementation of the intervention documenting feasibility, acceptability, and economic aspects. The enrolment commenced in October 2020 and is planned to be completed by the end of 2022. The expected date of study completion is December 2024. Discussion: If LAKANA provides evidence in support of a positive mortality benefit resulting from azithromycin MDA, it will significantly contribute to the options for successfully promoting child survival in Mali, and elsewhere in sub-Saharan Africa. Trial registration: ClinicalTrials.gov NCT04424511. Registered on 11 June 2020.publishedVersionPeer reviewe

Investigating a potential infectious cause of death in Sudden Unexpected Death in Infancy and Childhood using 16S rRNA gene sequencing

Sudden unexpected death in infancy (SUDI) is the most common cause of post-neonatal infant death in the developed world. Following thorough investigation approximately 40% of cases remain unexplained. It has been hypothesised that a subset of these currently unexplained infant deaths are due to infection that could not be diagnosed at post-mortem (PM) examination due to limitations in current diagnostic techniques. The aim of this study was to optimise the 16S rRNA sequencing technique for PM tissue samples from both SUDI cases and their childhood equivalent (collectively referred to as sudden unexpected death in infancy and childhood, or SUDIC) to investigate whether this technique can reliably identify infected tissues and assist in the PM diagnosis of infection. This thesis investigated several aspects of the PM process in order to determine the use of the 16S rRNA gene sequencing technique in routine clinical diagnosis of infection. The process of PM bacterial translocation was investigated using the 16S rRNA gene sequencing technique in two animal models over a 14-day study period in order to determine the effects this process may have on bacteria identified within internal organs in a PM setting. This investigation found no evidence for significant PM bacterial translocation in the mouse model. In the piglet model, low-levels of bacteria were detected in some tissues, but there was no evidence for significant translocation from the gastrointestinal tract or nasal cavity. With this insight into the PM process, a 16S rRNA gene sequencing method was successfully optimised for use in both formalin-fixed paraffin-embedded (FFPE) and frozen tissues from archived cases of SUDI. These investigations included examination of the effects of different sequencing protocols and tissue processing. The optimised 16S rRNA gene sequencing method was then performed on PM tissues from SUDIC cases. These cases were assigned to one of three groups depending on the final cause of death (CoD) as recorded on the autopsy report: (i) infectious, (ii) explained non-infectious, and (iii) unexplained. Results showed that the technique could successfully distinguish between explained non-infectious and infectious cases of SUDIC. The method correctly identified the causative organism in all cases of infectious SUDIC due to a bacterial infection where the pathogen had been identified at PM investigation. In four cases of infectious SUDIC where the causative organism was not identified at PM investigation using current routine techniques, a dominant bacterial pathogen was identified in this study using the sequencing technique. Using control groups a threshold was defined to identify infected PM tissue samples. When applied to the unexplained cohort four cases of SUDIC were identified where overwhelming bacterial colonisation of PM tissues was observed. Considering clinical history and other PM findings, it is possible that these deaths had a potentially unidentified infectious cause. The results of this thesis support the further evaluation and use of 16S rRNA gene sequencing in PM tissues from cases of SUDIC. The technique has the potential to identify infectious agents which are undetected using current techniques. Further work should increase the study cohort and perform this investigation prospectively as a part of routine clinical diagnosis of SUDIC

FcMBL magnetic bead-based MALDI-TOF MS rapidly identifies paediatric blood stream infections from positive blood cultures

Rapid identification of potentially life-threatening blood stream infections (BSI) improves clinical outcomes, yet conventional blood culture (BC) identification methods require ~24-72 hours of liquid culture, plus 24-48 hours to generate single colonies on solid media suitable for identification by mass spectrometry (MS). Newer rapid centrifugation techniques, such as the Bruker MBT-Sepsityper® IVD, replace culturing on solid media and expedite the diagnosis of BCs but frequently demonstrate reduced sensitivity for identifying clinically significant Gram-positive bacterial or fungal infections. This study introduces a protocol that utilises the broad-range binding properties of an engineered version of mannose-binding lectin linked to the Fc portion of immunoglobulin (FcMBL) to capture and enrich pathogens combined with matrix-assisted laser desorption-ionisation time-of-flight (MALDI-TOF) MS for enhanced infection identification in BCs. The FcMBL method identified 94.1% (64 of 68) of clinical BCs processed, with a high sensitivity for both Gram-negative and Gram-positive bacteria (94.7 and 93.2%, respectively). The FcMBL method identified more patient positive BCs than the Sepsityper® (25 of 25 vs 17 of 25), notably with 100% (3/3) sensitivity for clinical candidemia, compared to only 33% (1/3) for the Sepsityper®. Additionally, during inoculation experiments, the FcMBL method demonstrated a greater sensitivity, identifying 100% (24/24) of candida to genus level and 9/24 (37.5%) top species level compared to 70.8% (17/24) to genus and 6/24 to species (25%) using the Sepsityper®. This study demonstrates that capture and enrichment of samples using magnetic FcMBL-conjugated beads is superior to rapid centrifugation methods for identification of BCs by MALDI-TOF MS. Deploying the FcMBL method therefore offers potential clinical benefits in sensitivity and reduced turnaround times for BC diagnosis compared to the standard Sepsityper® kit, especially for fungal diagnosis

Comparison of the FcMBL and Sepsityper^® protocols.

Time to identification is ~7 minutes for the full Sepsityper® workflow compared to ~9 minutes for the FcMBL protocol.</p

Bruker Biotyper log score comparison of the Sepsityper^<i>®</i> and FcMBL methods for the detection of pathogens in positive clinical blood culture samples.

Bold Log scores indicate the higher score of the two methods.</p

Heatmap of spectral similarities with dendrogram clustering analysis for FcMBL candida mass spectra (2,500–7,400m/z).

Heatmap of spectral similarities with dendrogram clustering analysis for FcMBL candida mass spectra (2,500–7,400m/z).</p

MALDI-TOF MS identification of paediatric samples using FcMBL beads.

Seven patients had both an anaerobic and aerobic bottle processed if available, accounting for the higher number of FcMBL positive samples seen for some species.</p

Extreme enrichment of <i>E</i>. <i>coli</i> from spiked blood samples compared to centrifuged samples.

E. coli specific peaks are found in all concentrations using the FcMBL method. The limit of detection for the centrifuged samples was found to be at 106 CFU/ml.</p