A Pediatric Investigators Collaborative Network on Infections in Children (PICNIC) multi-centre Canadian descriptive analysis of Haemophilus influenzae bacteremia in children: Emerging serotypes

Background There has been dramatic reduction in Haemophilus influenzae serotype b (Hib) since introduction of Hib vaccines, but children still experience serious invasive Haemophilus influenzae (Hi) disease caused by various serotype and non-typeable bacteria. The object of this study was to describe the serotype distribution and clinical spectrum of Hi bacteremia in children admitted to Canadian hospitals. Methods All children with Hi bacteremia admitted 2013 through 2017 to 10 centres across Canada were included. Demographic, clinical, treatment and outcome data were collected. Results Haemophilus influenzae bacteremia occurred in 118 children of median age 12 months (inter-quartile range: 7–48 months). Forty-three (36%) isolates were non-typeable (NTHi) and 8 were not typed. Of the 67 typeable (THi), Hia (H. influenzae serotype a) (n=36, 54%), Hif (serotype f) (n=19, 26%) and Hib (serotype b) (n=9, 13%) dominated. The THi was more likely than NTHi bacteremia to present as meningitis (p<0.001), particularly serotype a (p=0.04) and less likely to present as pneumonia (p<0.001). Complicated disease (defined as intensive care unit admission, need for surgery, long-term sequelae or death) occurred in 31 (26%) cases and were more likely to have meningitis (p<0.001) than were those with uncomplicated disease. Conclusion In the era of efficacious conjugate Hib vaccines, NTHi, Hia and Hif have emerged as the leading causes of invasive Hi in Canadian children, with Hia being most likely to result in meningitis and complicated disease. A vaccine for all NTHi and THi would be ideal, but knowledge of the current disease burden from circulating strains will inform prioritization of vaccine targets.


Introduction
Haemophilus influenzae serotype b (Hib) was overwhelmingly the leading cause of invasive Hi disease until the introduction of the Hib vaccines into the routine childhood immunization schedule in the United States (US) and Canada in the late 1980s (1).This was followed by the emergence of Hi serotype a, particularly in Indigenous children in Canada and in Alaska (2)(3)(4).Recent publications from the US reported an increase in the incidence of invasive Hi disease in children, possibly due to an increase in the incidence of cases due to non-typeable Hi (NTHi) (5,6).We sought to describe the serotype distribution and clinical spectrum of Hi bacteremia in Canadian children across several provinces and to determine factors associated with complicated disease.

Study population and design
Ten centres within the Pediatric Investigators Collaborative Network on Infections in Canada (PICNIC) retrospectively enrolled all hospitalized children younger than 18 years of age with blood culture isolates of Hi from January 1, 2013, through December 31, 2017.For nine centres (London, Ontario; Hamilton, Ontario; Ottawa, Ontario; Kingston, Ontario; Winnipeg, Manitoba; Edmonton, Alberta; Vancouver, British Columbia; Halifax, Nova Scotia and Montréal, Québec), this was a sub-study arising from a retrospective cohort of Gram-negative bacteremia, while the tenth centre (Sioux Lookout, Ontario) was added for this sub-study due to their known high incidence of Hi infections.Ethics approval was obtained at each participating centre and the need for parental consent was waived.

Study definitions
The focus of infection was classified as meningitis, pneumonia, epiglottitis, skin and soft tissue infection, osteoarticular infection, other or none (isolated bacteremia with no focus).Multifocal disease was defined as bacteremia with two or more foci.
Disease was defined as complicated if any of the following occurred related to Hi disease: intensive care unit (ICU) admission; organ failure; surgical interventions including amputations for purpura fulminans or drainage of purulent collections (arthrocentesis did not qualify unless performed more than once); complications relating to disease focus including motor deficits, seizures, hydrocephalus, visual or hearing deficits, or necrotizing skin or lung infections; and death.

Serotyping
Serotyping of isolates was completed using monovalent antisera at reference laboratories.Strains were classified by capsular type (a to f) or as non-typeable.When serotyping was not available, the isolates were recorded as not typed.

Data collection
Demographic, clinical, microbiological, treatment, outcome and follow-up data were extracted from medical records and entered into REDCap (Research Electronic Data Capture) tools hosted at the University of Alberta by each participating centre (7).

Statistical analysis
Descriptive analysis was conducted.Chi-square or Fisher's exact test was used to compare categorical variables and nonparametric tests were used to compare continuous variables.Univariate analysis was used to explore potential factors associated with Hia disease and complicated disease course.Variables with a univariate p value of ≤0.2 and potential confounding factors (e.g.age and sex) were considered for inclusion in multivariable logistic regression model aimed at determining independent risk factors for complicated disease.The IBM SPSS version 28 was used for statistical analysis.
Multifocal disease occurred in seven other children (five due to Hia and two due to Hif), with five cases having meningitis as one of the foci.

The median duration of antibiotic therapy in
Complicated disease was more common with THi than with NTHi (n=24/67, 36% vs. n=6/43, 14%; p=0.015).The fatal cases were both caused by Hia and occurred in an infant with meningitis and endocarditis despite no congenital heart disease and in a four-year-old with meningitis complicated by subdural empyema.One infant with Hib meningitis required bilateral limb amputations due to purpura fulminans but survived.The composite outcome of mortality or sequelae at discharge was significantly associated with Hia as compared to non-Hia disease (n=9/36, 25% vs. n=7/74, 9%; p<0.001) (see Appendix).
In the univariate analysis (after adjusting for multiple comparisons), Hi cases with meningitis (p<0.001) were more likely to have a complicated clinical course whereas those with isolated bacteremia were less likely (p<0.001)(Table 2).

Discussion
This multicentre study from 10 sites across Canada provides insight into the current serotype distribution of Hi bacteremia in Canadian children.Prior to the era of Hib vaccine, invasive Hi disease was very rare in children over four years of age, while approximately one quarter of our cases were older.Invasive and non-invasive Hi disease from 2013 to 2019 has been reviewed from epidemiological data from a single Canadian site with a predominant Indigenous population (Sioux Lookout) (8).At this centre, invasive Hi disease was identified in 10 children under 4 years, in two aged 5-15 years and in eight aged 16 years and older (8), suggesting a bimodal distribution which differs from the pre-Hib vaccine era where younger children were primarily affected.Our data highlight the pediatric centres in Winnipeg, Montréal, London and Edmonton as the highest contributors to THi, and may reflect that these centres are referral centres for large communities of Indigenous children as well as communities with reduced Hib vaccine uptake.The Hib cases still accounted for almost 10% of cases in the current study.As expected, Hib occurred predominantly in unimmunized or under-immunized children.14, [10][11][12][13][14][15][16][17][18][19][20][21][22][23] 26,  13, [10][11][12][13][14][15][16][17][18][19][20] 0 Serotypes a and f were the most common, accounting for 55% and 25% of THi, respectively, consistent with recent literature (4,6).While Hia meningitis was significantly associated with a complicated course, it otherwise mirrored the Hib experience in terms of age and clinical syndromes.Children with Hif were often older with pneumonia as the focus of infection.
The less virulent NTHi accounted for just over one third of all Hi bacteremic events, where fortunately the incidence of complicated disease was only 4%.The NTHi cases presented as isolated bacteremia or pneumonia and rarely as central nervous system disease, in keeping with recent US data (5,6).Comorbid medical conditions were a risk factor for developing NTHi disease.In a retrospective surveillance study of NTHi invasive disease in children and adults in the Netherlands, comorbid conditions in children, including immunocompromise, malignancy, neurological disease and other conditions, were identified in a large proportion of NTHi cases (n=327/396, 83%) over a seven-year span (9).
It is exciting that Phase I trials of a Hia vaccine will begin in Canada in 2023 (10) (personal communication, M. Ulanova, Canadian Immunization Research Network, 2022).A vaccine has been shown to be cost-effective in the Canadian territory of Nunavut, given the high incidence of Hia among Indigenous children and their high risk of disease (11).Post-marketing studies of the multivalent pneumococcal vaccines that employ protein D from NTHi as the carrier protein suggest that they prevent some otitis media due to NHTi diseases, so it is plausible that these vaccines may also prevent Hi bacteremia (12).

Limitations
A significant limitation of our study is that it did not capture ethnicity/race data as this variable is not reliably recorded in health records in Canada.This is especially important given the established high rates of Hia in Indigenous populations (4).Our study may underestimate the burden and spectrum of Hi disease given that it was limited to bacteremic cases; blood cultures are not always drawn prior to administration of antibiotics and can be falsely negative if an inadequate volume is obtained.As neurodevelopmental and long-term follow-up data were not uniformly available, we reported sequelae documented at or before discharge.

Conclusion
Our study provides detailed clinical comparisons of THi and NTHi, highlighting serotype-specific clinical patterns and outcomes.Although there has been dramatic reduction in Hib since introduction of Hib vaccines, children still experience serious invasive Hi disease caused by both NHTi and by non-b serotypes, especially Hia.Preventive strategies are needed to reduce the morbidity associated with this disease.

Figure 1 :
Figure 1: Haemophilus influenzae serotype distribution by year in paediatric population, Canada

Figure 2 :
Figure 2: Serotype-specific distribution of Haemophilus influenzae invasive disease in ten centres across Canada a

Table 1 :
Demographic, clinical, microbiological and outcome-related patterns across serotypes in paediatric population, Canada (continued) Pairwise comparison of medians across serotype distribution was not significantly different after adjusting by Bonferroni correction for multiple tests c Unless listed as multifocal disease (more than two systems) or same system disease in distant sites, the focus listed was the sole focus identified during the admission.A sinusitis case was classified as pneumonia given that both are respiratory foci.Skin and soft tissue infections included unspecified cellulitis, facial cellulitis (n=3) and cellulitis with fasciitis (n=1; caused by Hia).The latter case with associated suppurative myositis d Infective endocarditis refers to endovascular infection (n=1) or endocarditis (n=2); the latter occurred in absence of preceding cardiac disease.Two cases caused by Hia and Hif respectively were multifocal with CNS involvement.The endovascular infection occurred in an adolescent (without immunodeficiency) who presented with Lemierre syndrome with thrombosis of both internal jugular veins with associated Hif meningitis (patient also grew Streptococcus anginosus in blood) 13%)Abbreviations: CNS, central nervous system; Hia, Haemophilus influenzae serotype a; Hib, Haemophilus influenzae serotype b; Hic, Haemophilus influenzae serotype c; Hie, Haemophilus influenzae serotype e; Hif, Haemophilus influenzae serotype f; ICU, intensive care unit; IE, infective endocarditis; IQR, interquartile range; NTHi, non-typeable Haemophilus influenzae; OI, osteoarticular infections; SSTI, skin and soft tissue infection; THi, typeable Haemophilus influenzae a Values shown as totals (%) for categorical variables and medians with IQRs for continuous variables b f Courses were considered complicated if there was ICU requirement or if disease-specific complications (e.g.ventriculitis, necrotizing pneumonia, need for infectious foci-related surgery, longer than usual course of antimicrobial therapy for a given focus.Complications in THi were mainly related to CNS infections.For NTHi, two of six children with complicated courses had CNS complications (ventriculitis and seizures).Other complications in NTHi were due to pneumonia (three needing chest tube, one of whom was ventilated) and another developing shock requiring ICU admission.
Centre-specific contributions are shown in Figure 2. Centres in Winnipeg, Montréal and London contributed most Hi cases, with disease predominantly caused by THi.Yearly case frequency ranged from 15-34 cases annually with the peak frequency occurring in 2016.Non-typeable H. influenzae followed by serotype a then serotype f disease were the leading causes of Hi disease in 2013, 2014 and 2017.Serotype a followed by NTHi and then serotype f were the leading causes in 2015 and 2016.

Table 2 :
Univariate and multivariate analysis of factors associated with complicated Haemophilus influenzae disease