Epidemiology of childhood invasive pneumococcal disease in Australia: a prospective cohort study.

BACKGROUND: The widespread use of pneumococcal conjugate vaccines (PCV) has changed the epidemiology of invasive pneumococcal disease (IPD) in children globally. METHODS: Multicentre prospective audit of IPD episodes from five paediatric hospitals in Australia over 5.5 years between 2016 and June 2021. Children (<18 years) with Streptococcus pneumoniae isolated from a sterile site were included. RESULTS: There were 377 IPD episodes in 375 children: 338 (90%) had received ≥3 PCV doses; 42 (11%) had IPD risk factors. The most common presentations were complicated pneumonia (254, 67%), bacteraemia (65, 17%) and meningitis (29, 8%). Five (1%) children died.Serotype information was available for 230 (61%) episodes; 140 (61%) were 13vPCV vaccine serotypes (VTs). The majority (85%) of episodes of complicated pneumonia were due to a VT; predominantly 3, 19A, 19F. Children with risk factors were more likely to present with bacteraemia ± sepsis (42% vs 12%) and to have a non-vaccine serotype (NVT) (74% vs 32%). Resistance to ceftriaxone (meningitis cut-off) occurred in 17% of 23B isolates (n=12) and accounted for 22% (5/23) of meningitis cases. CONCLUSIONS: Complicated pneumonia is the most common IPD presentation. NVTs account for the majority of bacteraemia and meningitis episodes. High rates of ceftriaxone resistance for NVT 23B support the addition of vancomycin for empiric treatment of suspected meningitis.

"Hurts less, lasts longer"; a qualitative study on experiences of young people receiving high-dose subcutaneous injections of benzathine penicillin G to prevent rheumatic heart disease in New Zealand.

BACKGROUND: Four-weekly intramuscular (IM) benzathine penicillin G (BPG) injections to prevent acute rheumatic fever (ARF) progression have remained unchanged since 1955. A Phase-I trial in healthy volunteers demonstrated the safety and tolerability of high-dose subcutaneous infusions of BPG which resulted in a much longer effective penicillin exposure, and fewer injections. Here we describe the experiences of young people living with ARF participating in a Phase-II trial of SubCutaneous Injections of BPG (SCIP). METHODOLOGY: Participants (n = 20) attended a clinic in Wellington, New Zealand (NZ). After a physical examination, participants received 2% lignocaine followed by 13.8mL to 20.7mL of BPG (Bicillin-LA®; determined by weight), into the abdominal subcutaneous tissue. A Kaupapa Maori consistent methodology was used to explore experiences of SCIP, through semi-structured interviews and observations taken during/after the injection, and on days 28 and 70. All interviews were recorded, transcribed verbatim, and thematically analysed. PRINCIPAL FINDINGS: Low levels of pain were reported on needle insertion, during and following the injection. Some participants experienced discomfort and bruising on days one and two post dose; however, the pain was reported to be less severe than their usual IM BPG. Participants were 'relieved' to only need injections quarterly and the majority (95%) reported a preference for SCIP over IM BPG. CONCLUSIONS: Participants preferred SCIP over their usual regimen, reporting less pain and a preference for the longer time gap between treatments. Recommending SCIP as standard of care for most patients needing long-term prophylaxis has the potential to transform secondary prophylaxis of ARF/RHD in NZ and globally.

Impact of the COVID-19 related border restrictions on influenza and other common respiratory viral infections in New Zealand.

BACKGROUND: New Zealand's (NZ) complete absence of community transmission of influenza and respiratory syncytial virus (RSV) after May 2020, likely due to COVID-19 elimination measures, provided a rare opportunity to assess the impact of border restrictions on common respiratory viral infections over the ensuing 2 years. METHODS: We collected the data from multiple surveillance systems, including hospital-based severe acute respiratory infection surveillance, SHIVERS-II, -III and -IV community cohorts for acute respiratory infection (ARI) surveillance, HealthStat sentinel general practice (GP) based influenza-like illness surveillance and SHIVERS-V sentinel GP-based ARI surveillance, SHIVERS-V traveller ARI surveillance and laboratory-based surveillance. We described the data on influenza, RSV and other respiratory viral infections in NZ before, during and after various stages of the COVID related border restrictions. RESULTS: We observed that border closure to most people, and mandatory government-managed isolation and quarantine on arrival for those allowed to enter, appeared to be effective in keeping influenza and RSV infections out of the NZ community. Border restrictions did not affect community transmission of other respiratory viruses such as rhinovirus and parainfluenza virus type-1. Partial border relaxations through quarantine-free travel with Australia and other countries were quickly followed by importation of RSV in 2021 and influenza in 2022. CONCLUSION: Our findings inform future pandemic preparedness and strategies to model and manage the impact of influenza and other respiratory viral threats.

Early transcriptional responses to human enteric fever challenge.

Enteric fever, caused by oral infection with typhoidal Salmonella serovars, presents as a non-specific febrile illness preceded by an incubation period of 5 days or more. The enteric fever human challenge model provides a unique opportunity to investigate the innate immune response during this incubation period, and how this response is altered by vaccination with the Vi polysaccharide or conjugate vaccine. We find that on the same day as ingestion of typhoidal Salmonella, there is already evidence of an immune response, with 199 genes upregulated in the peripheral blood transcriptome 12 hours post-challenge (false discovery rate <0.05). Gene sets relating to neutrophils, monocytes, and innate immunity were over-represented (false discovery rate <0.05). Estimating cell proportions from gene expression data suggested a possible increase in activated monocytes 12 hours post-challenge (P = 0.036, paired Wilcoxon signed-rank test). Furthermore, plasma TNF-α rose following exposure (P = 0.011, paired Wilcoxon signed-rank test). There were no significant differences in gene expression (false discovery rate <0.05) in the 12 hours response between those who did and did not subsequently develop clinical or blood culture confirmed enteric fever or between vaccination groups. Together, these results demonstrate early perturbation of the peripheral blood transcriptome after enteric fever challenge and provide initial insight into early mechanisms of protection.

Inborn Errors of Immunity in Children With Invasive Pneumococcal Disease: A Multicenter Prospective Study.

BACKGROUND: In settings with universal conjugate pneumococcal vaccination, invasive pneumococcal disease (IPD) can be a marker of an underlying inborn error of immunity. The aim of this study was to determine the prevalence and characterize the types of immunodeficiencies in children presenting with IPD. METHODS: Multicenter prospective audit following the introduction of routinely recommended immunological screening in children presenting with IPD. The minimum immunological evaluation comprised a full blood examination and film, serum immunoglobulins (IgG, IgA and IgM), complement levels and function. Included participants were children in whom Streptococcus pneumoniae was isolated from a normally sterile site (cerebrospinal fluid, pleura, peritoneum and synovium). If isolated from blood, features of sepsis needed to be present. Children with predisposing factors for IPD (nephrotic syndrome, anatomical defect or malignancy) were excluded. RESULTS: Overall, there were 379 episodes of IPD of which 313 (83%) were eligible for inclusion and 143/313 (46%) had an immunologic evaluation. Of these, 17/143 (12%) were diagnosed with a clinically significant abnormality: hypogammaglobulinemia (n = 4), IgA deficiency (n = 3), common variable immunodeficiency (n = 2), asplenia (n = 2), specific antibody deficiency (n = 2), incontinentia pigmenti with immunologic dysfunction (n = 1), alternative complement deficiency (n = 1), complement factor H deficiency (n = 1) and congenital disorder of glycosylation (n = 1). The number needed to investigate to identify 1 child presenting with IPD with an immunologic abnormality was 7 for children under 2 years and 9 for those 2 years old and over. CONCLUSIONS: This study supports the routine immune evaluation of children presenting with IPD of any age, with consideration of referral to a pediatric immunologist.

Time to first dose of antibiotics in febrile neonates.

AIM: To assess whether febrile neonates from the community received their first dose of intravenous antibiotics within 1 h from time of arrival, as per the regional paediatric sepsis pathway, at a tertiary combined adult/child emergency department in New Zealand. METHOD: Retrospective data were collected from January 2018 to December 2019 with 28 patients included. RESULTS: Mean time to first antibiotic dose for all neonates and those with serious bacterial infection was 3 h 20 min and 2 h 53 min respectively. No case used the paediatric sepsis pathway. A pathogen was identified in 19/28 (67%) neonates and 16/28 (57%) had clinical signs of shock. CONCLUSION: This study adds to Australasian data on community neonatal sepsis. Antibiotic administration was delayed for neonates with serious bacterial infection, clinical signs of shock and raised lactate. The reasons for delay are examined, with a number of potential areas for improvement identified.

"I Don't Have to be Sick to Still Be Worthy": The Barriers Experienced by Adolescent and Young Adult Cancer Survivors in New Zealand.

Purpose: To identify the spectrum and nature of survivorship barriers experienced by New Zealand's adolescent and young adult (AYA) cancer survivor population. In addition, we explore associations between survivorship barriers and sociodemographic characteristics, cancer type, and day-to-day happiness following the end of treatment. Methodology: Participants were recruited for the online survey from AYA cancer service patient databases. Eligibility criteria included: aged 12-24 years at diagnosis, diagnosed between 2010 and 2019, and completed treatment at least one year prior. The analysis focused on 11 barriers (domains, issues, or concerns) which respondents may have faced during survivorship. Results: Two hundred and eighteen AYA survivors participated in the study. The mean number of impactful survivorship barriers was 2.5 (standard deviation 1.7), with 13 respondents (6.0%) reporting no barriers of concern and 31 (14.2%) reporting 5 or more. A higher number of impactful barriers was associated with lower day-to-day happiness (r = -0.34, p ≤ 0.001). The most commonly identified impactful survivorship barriers were mental health (50.0% of respondents), physical health (43.1%), thinking and memory (33.0%), education and work (27.1%), social life (26.1%), and fertility (22.5%). Subgroup analysis identified significant differences according to gender, age at diagnosis, tumor group, ethnicity, and time since diagnosis. Poor access to health care and unmet needs were common themes. Positive impacts, particularly with regards to family relationships, were also identified. Conclusion: These results will inform initiatives to improve AYA survivorship care in New Zealand. Gaps in service delivery and funding will need to be overcome by utilizing innovative strategies and broad sector engagement.

Genetic Susceptibility to Enteric Fever in Experimentally Challenged Human Volunteers.

Infections with Salmonella enterica serovars Typhi and Paratyphi A cause an estimated 14 million cases of enteric fever annually. Here, the controlled nature of challenge studies is exploited to identify genetic variants associated with enteric fever susceptibility. Human challenge participants were genotyped by Illumina OmniExpress-24 BeadChip array (n = 176) and/or transcriptionally profiled by RNA sequencing (n = 174). While the study was underpowered to detect any single nucleotide polymorphisms (SNPs) significant at the whole-genome level, two SNPs within CAPN14 and MIATNB were identified with P < 10-5 for association with development of symptoms or bacteremia following oral S. Typhi or S. Paratyphi A challenge. Imputation of classical human leukocyte antigen (HLA) types from genomic and transcriptomic data identified HLA-B*27:05, previously associated with nontyphoidal Salmonella-induced reactive arthritis, as the HLA type most strongly associated with enteric fever susceptibility (P = 0.011). Gene sets relating to the unfolded protein response/heat shock and endoplasmic reticulum-associated protein degradation were overrepresented in HLA-B*27:05+ participants following challenge. Furthermore, intracellular replication of S. Typhi is higher in C1R cells transfected with HLA-B*27:05 (P = 0.02). These data suggest that activation of the unfolded protein response by HLA-B*27:05 misfolding may create an intracellular environment conducive to S. Typhi replication, increasing susceptibility to enteric fever.

The role of Kingella kingae in pre-school aged children with bone and joint infections.

OBJECTIVES: The Pre-school Osteoarticular Infection (POI) study aimed to describe the burden of disease, epidemiology, microbiology and treatment of acute osteoarticular infections (OAI) and the role of Kingella kingae in these infections. METHODS: Information about children 3-60 months of age who were hospitalized with an OAI to 11 different hospitals across Australia and New Zealand between January 2012 and December 2016 was collected retrospectively. RESULTS: A total of 907 cases (73%) were included. Blood cultures grew a likely pathogen in only 18% (140/781). The peak age of presentation was 12 to 24 months (466/907, 51%) and Kingella kingae was the most frequently detected microorganism in this age group (60/466, 13%). In the majority of cases, no microorganism was detected (517/907, 57%). Addition of PCR to culture increased detection rates of K. kingae. However, PCR was performed infrequently (63/907, 7%). CONCLUSIONS: This large multi-national study highlights the need for more widespread use of molecular diagnostic techniques for accurate microbiological diagnosis of OAI in pre-school aged children. The data from this study supports the hypothesis that a substantial proportion of pre-school aged children with OAI and no organism identified may in fact have undiagnosed K. kingae infection. Improved detection of Kingella cases is likely to reduce the average length of antimicrobial treatment.

Impact of the COVID-19 nonpharmaceutical interventions on influenza and other respiratory viral infections in New Zealand.

Stringent nonpharmaceutical interventions (NPIs) such as lockdowns and border closures are not currently recommended for pandemic influenza control. New Zealand used these NPIs to eliminate coronavirus disease 2019 during its first wave. Using multiple surveillance systems, we observed a parallel and unprecedented reduction of influenza and other respiratory viral infections in 2020. This finding supports the use of these NPIs for controlling pandemic influenza and other severe respiratory viral threats.

Impact of the COVID-19 nonpharmaceutical interventions on influenza and other respiratory viral infections in New Zealand.

Stringent nonpharmaceutical interventions (NPIs) such as lockdowns and border closures are not currently recommended for pandemic influenza control. New Zealand used these NPIs to eliminate coronavirus disease 2019 during its first wave. Using multiple surveillance systems, we observed a parallel and unprecedented reduction of influenza and other respiratory viral infections in 2020. This finding supports the use of these NPIs for controlling pandemic influenza and other severe respiratory viral threats.

Homologous and heterologous re-challenge with Salmonella Typhi and Salmonella Paratyphi A in a randomised controlled human infection model.

Enteric fever is a systemic infection caused by Salmonella Typhi or Paratyphi A. In many endemic areas, these serovars co-circulate and can cause multiple infection-episodes in childhood. Prior exposure is thought to confer partial, but incomplete, protection against subsequent attacks of enteric fever. Empirical data to support this hypothesis are limited, and there are few studies describing the occurrence of heterologous-protection between these closely related serovars. We performed a challenge-re-challenge study using a controlled human infection model (CHIM) to investigate the extent of infection-derived immunity to Salmonella Typhi or Paratyphi A infection. We recruited healthy volunteers into two groups: naïve volunteers with no prior exposure to Salmonella Typhi/Paratyphi A and volunteers previously-exposed to Salmonella Typhi or Paratyphi A in earlier CHIM studies. Within each group, participants were randomised 1:1 to oral challenge with either Salmonella Typhi (104 CFU) or Paratyphi A (103 CFU). The primary objective was to compare the attack rate between naïve and previously challenged individuals, defined as the proportion of participants per group meeting the diagnostic criteria of temperature of ≥38°C persisting for ≥12 hours and/or S. Typhi/Paratyphi bacteraemia up to day 14 post challenge. The attack-rate in participants who underwent homologous re-challenge with Salmonella Typhi was reduced compared with challenged naïve controls, although this reduction was not statistically significant (12/27[44%] vs. 12/19[63%]; Relative risk 0.70; 95% CI 0.41-1.21; p = 0.24). Homologous re-challenge with Salmonella Paratyphi A also resulted in a lower attack-rate than was seen in challenged naïve controls (3/12[25%] vs. 10/18[56%]; RR0.45; 95% CI 0.16-1.30; p = 0.14). Evidence of protection was supported by a post hoc analysis in which previous exposure was associated with an approximately 36% and 57% reduced risk of typhoid or paratyphoid disease respectively on re-challenge. Individuals who did not develop enteric fever on primary exposure were significantly more likely to be protected on re-challenge, compared with individuals who developed disease on primary exposure. Heterologous re-challenge with Salmonella Typhi or Salmonella Paratyphi A was not associated with a reduced attack rate following challenge. Within the context of the model, prior exposure was not associated with reduced disease severity, altered microbiological profile or boosting of humoral immune responses. We conclude that prior Salmonella Typhi and Paratyphi A exposure may confer partial but incomplete protection against subsequent infection, but with a comparable clinical and microbiological phenotype. There is no demonstrable cross-protection between these serovars, consistent with the co-circulation of Salmonella Typhi and Paratyphi A. Collectively, these data are consistent with surveillance and modelling studies that indicate multiple infections can occur in high transmission settings, supporting the need for vaccines to reduce the burden of disease in childhood and achieve disease control. Trial registration NCT02192008; clinicaltrials.gov.

Clinical experience of intramuscular immunoglobulin for measles prophylaxis in children: Is it practical?

Measles continues to be a public health concern world-wide. Vulnerable individuals including those in which vaccinations is contraindicated, may be reliant on normal human immunoglobulin (NHIG) prophylaxis in an aim to prevent disease. This paper will summarise and discuss a tertiary paediatric hospital's clinical experience and the practicalities of administering intramuscular (IM) NHIG to paediatric patients as per the current measles prophylaxis guidelines in Australia. Following potential exposure within the emergency department, 17 paediatric patients (0-15 years) were recommended IM NHIG for prophylaxis. The dose of NHIG ranged from 0.6 to 15 mL and required multiple (2-8) injections. Two patients required sedation for staff to safely administer the injections. Staff involved with these cases reported administering multiple injections to paediatric patients to be a traumatising experience. They also expressed views that the injection of large volumes via the IM route was an impractical method of administration. Based on this experience, we recommend intravenous immunoglobulin be considered when large volumes of NHIG are recommended intramuscularly.

Diagnostic host gene signature for distinguishing enteric fever from other febrile diseases.

Misdiagnosis of enteric fever is a major global health problem, resulting in patient mismanagement, antimicrobial misuse and inaccurate disease burden estimates. Applying a machine learning algorithm to host gene expression profiles, we identified a diagnostic signature, which could distinguish culture-confirmed enteric fever cases from other febrile illnesses (area under receiver operating characteristic curve > 95%). Applying this signature to a culture-negative suspected enteric fever cohort in Nepal identified a further 12.6% as likely true cases. Our analysis highlights the power of data-driven approaches to identify host response patterns for the diagnosis of febrile illnesses. Expression signatures were validated using qPCR, highlighting their utility as PCR-based diagnostics for use in endemic settings.

Publisher Correction: Clonal analysis of Salmonella-specific effector T cells reveals serovar-specific and cross-reactive T cell responses.

In the version of this article initially published, the first affiliation lacked 'MRC'; the correct name of the institution is 'MRC Weatherall Institute of Molecular Medicine'. Two designations (SP110Y and ST110H) were incorrect in the legend to Fig. 6f,h,i. The correct text is as follows: for panel f, "...loaded with either the CdtB(105-125)SP110Y (DRB4*SP110Y) or the CdtB(105-125)ST110H (DRB4*ST110H) peptide variants..."; for panel h, "...decorated by the DRB4*SP110Y tetramer (lower-right quadrant), the DRB4*ST110H (upper-left quadrant)..."; and for panel i, "...stained ex vivo with DRB4*SP110Y, DRB4*ST110H...". In Fig. 8e, the final six residues (LTEAFF) of the sequence in the far right column of the third row of the table were missing; the correct sequence is 'CASSYRRTPPLTEAFF'. In the legend to Fig. 8d, a designation (HLyE) was incorrect; the correct text is as follows: "(HlyE?)." Portions of the Acknowledgements section were incorrect; the correct text is as follows: "This work was supported by the UK Medical Research Council (MRC) (MR/K021222/1) (G.N., M.A.G., A.S., V.C., A.J.P.),...the Oxford Biomedical Research Centre (A.J.P., V.C.),...and core funding from the Singapore Immunology Network (SIgN) (E.W.N.) and the SIgN immunomonitoring platform (E.W.N.)." Finally, a parenthetical element was phrased incorrectly in the final paragraph of the Methods subsection "T cell cloning and live fluorescence barcoding"; the correct phrasing is as follows: "...(which in all cases included HlyE, CdtB, Ty21a, Quailes, NVGH308, and LT2 strains and in volunteers T5 and T6 included PhoN)...". Also, in Figs. 3c and 4a, the right outlines of the plots were not visible; in the legend to Fig. 3, panel letter 'f' was not bold; and in Fig. 8f, 'ND' should be aligned directly beneath DRB4 in the key and 'ND' should be removed from the diagram at right, and the legend should be revised accordingly as follows: "...colors indicate the HLA class II restriction (gray indicates clones for which restriction was not determined (ND)). Clonotypes are grouped on the basis of pathogen selectivity (continuous line), protein specificity (dashed line) and epitope specificity; for ten HlyE-specific clones (pixilated squares), the epitope specificity was not determined...". The errors have been corrected in the HTML and PDF versions of the article.

Exserohilum infections in Australian Queensland children.

BACKGROUND: Exserohilum species are environmental moulds that can cause skin infection and sinusitis in both normal and immunosuppressed children. This study reviews paediatric cases of Exserohilum infection in Queensland, Australia, to identify the spectrum of disease and its clinical course. METHODS: All culture-positive samples of Exserohilum species in children <18 years of age were identified from the Queensland Health Laboratory database (April 2003-April 2018). Clinical information was recorded from medical records. RESULTS: Eleven children were identified, and all had isolated Exserohilum rostratum. The mean age was 7.4 years (range 2.3-17.8) and 64% female. Four immunocompetent children (36%) had a skin infection (2/4), chronic sinusitis (1/4) or otitis externa (1/4). Seven children (64%) had an underlying oncological diagnosis with E. rostratum causing local skin infection (2/7), invasive rhinosinus disease (3/7) or disseminated infection (2/7). All oncological patients were empirically started on liposomal amphotericin B with addition, or switch, to posaconazole or voriconazole. CONCLUSION: Exserohilum rostratum infection of the skin has a favourable course, whereas rhinosinus infection can be rapidly invasive in the immunocompromised child requiring prompt surgical intervention and antifungal therapy. Susceptibility data support empiric use of liposomal amphotericin and/or posaconazole.

Clonal analysis of Salmonella-specific effector T cells reveals serovar-specific and cross-reactive T cell responses.

To tackle the complexity of cross-reactive and pathogen-specific T cell responses against related Salmonella serovars, we used mass cytometry, unbiased single-cell cloning, live fluorescence barcoding, and T cell-receptor sequencing to reconstruct the Salmonella-specific repertoire of circulating effector CD4+ T cells, isolated from volunteers challenged with Salmonella enterica serovar Typhi (S. Typhi) or Salmonella Paratyphi A (S. Paratyphi). We describe the expansion of cross-reactive responses against distantly related Salmonella serovars and of clonotypes recognizing immunodominant antigens uniquely expressed by S. Typhi or S. Paratyphi A. In addition, single-amino acid variations in two immunodominant proteins, CdtB and PhoN, lead to the accumulation of T cells that do not cross-react against the different serovars, thus demonstrating how minor sequence variations in a complex microorganism shape the pathogen-specific T cell repertoire. Our results identify immune-dominant, serovar-specific, and cross-reactive T cell antigens, which should aid in the design of T cell-vaccination strategies against Salmonella.

MAIT cell clonal expansion and TCR repertoire shaping in human volunteers challenged with Salmonella Paratyphi A.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that can detect bacteria-derived metabolites presented on MR1. Here we show, using a controlled infection of humans with live Salmonella enterica serovar Paratyphi A, that MAIT cells are activated during infection, an effect maintained even after antibiotic treatment. At the peak of infection MAIT cell T-cell receptor (TCR)ß clonotypes that are over-represented prior to infection transiently contract. Select MAIT cell TCRß clonotypes that expand after infection have stronger TCR-dependent activation than do contracted clonotypes. Our results demonstrate that host exposure to antigen may drive clonal expansion of MAIT cells with increased functional avidity, suggesting a role for specific vaccination strategies to increase the frequency and potency of MAIT cells to optimize effector function.

Evaluation of the Clinical and Microbiological Response to Salmonella Paratyphi A Infection in the First Paratyphoid Human Challenge Model.

Background: To expedite the evaluation of vaccines against paratyphoid fever, we aimed to develop the first human challenge model of Salmonella enterica serovar Paratyphi A infection. Methods: Two groups of 20 participants underwent oral challenge with S. Paratyphi A following sodium bicarbonate pretreatment at 1 of 2 dose levels (group 1: 1-5 × 103 colony-forming units [CFU] and group 2: 0.5-1 × 103 CFU). Participants were monitored in an outpatient setting with daily clinical review and collection of blood and stool cultures. Antibiotic treatment was started when prespecified diagnostic criteria were met (temperature ≥38°C for ≥12 hours and/or bacteremia) or at day 14 postchallenge. Results: The primary study objective was achieved following challenge with 1-5 × 103 CFU (group 1), which resulted in an attack rate of 12 of 20 (60%). Compared with typhoid challenge, paratyphoid was notable for high rates of subclinical bacteremia (at this dose, 11/20 [55%]). Despite limited symptoms, bacteremia persisted for up to 96 hours after antibiotic treatment (median duration of bacteremia, 53 hours [interquartile range, 24-85 hours]). Shedding of S. Paratyphi A in stool typically preceded onset of bacteremia. Conclusions: Challenge with S. Paratyphi A at a dose of 1-5 × 103 CFU was well tolerated and associated with an acceptable safety profile. The frequency and persistence of bacteremia in the absence of clinical symptoms was notable, and markedly different from that seen in previous typhoid challenge studies. We conclude that the paratyphoid challenge model is suitable for the assessment of vaccine efficacy using endpoints that include bacteremia and/or symptomatology. Clinical Trials Registration: NCT02100397.

Development and Evaluation of a Blood Culture PCR Assay for Rapid Detection of Salmonella Paratyphi A in Clinical Samples.

BACKGROUND: Enteric fever remains an important cause of morbidity in many low-income countries and Salmonella Paratyphi A has emerged as the aetiological agent in an increasing proportion of cases. Lack of adequate diagnostics hinders early diagnosis and prompt treatment of both typhoid and paratyphoid but development of assays to identify paratyphoid has been particularly neglected. Here we describe the development of a rapid and sensitive blood culture PCR method for detection of Salmonella Paratyphi A from blood, potentially allowing for appropriate diagnosis and antimicrobial treatment to be initiated on the same day. METHODS: Venous blood samples from volunteers experimentally challenged orally with Salmonella Paratyphi A, who subsequently developed paratyphoid, were taken on the day of diagnosis; 10 ml for quantitative blood culture and automated blood culture, and 5 ml for blood culture PCR. In the latter assay, bacteria were grown in tryptone soy broth containing 2.4% ox bile and micrococcal nuclease for 5 hours (37°C) before bacterial DNA was isolated for PCR detection targeting the fliC-a gene of Salmonella Paratyphi A. RESULTS: An optimized broth containing 2.4% ox bile and micrococcal nuclease, as well as a PCR test was developed for a blood culture PCR assay of Salmonella Paratyphi A. The volunteers diagnosed with paratyphoid had a median bacterial burden of 1 (range 0.1-6.9) CFU/ml blood. All the blood culture PCR positive cases where a positive bacterial growth was shown by quantitative blood culture had a bacterial burden of ≥ 0.3 CFU/ ml blood. The blood culture PCR assay identified an equal number of positive cases as automated blood culture at higher bacterial loads (≥0.3 CFU/ml blood), but utilized only half the volume of specimens. CONCLUSIONS: The blood culture PCR method for detection of Salmonella Paratyphi A can be completed within 9 hours and offers the potential for same-day diagnosis of enteric fever. Using 5 ml blood, it exhibited a lower limit of detection equal to 0.3 CFU/ml blood, and it performed at least as well as automated blood culture at higher bacterial loads (≥0.3 CFU/ml blood) of clinical specimens despite using half the volume of blood. The findings warrant its further study in endemic populations with a potential use as a novel diagnostic which fills the present gap of paratyphoid diagnostics.