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The population structure of Haemophilus influenzae serotype a (Hia) was examined by interrogation of the H. influenzae MLST website. There were 196 entries of Hia with 55 sequence types (STs) identified (as of 3 September 2020). BURST analysis clustered related STs into four complexes with ST-23, ST-4, ST-21, and ST-62 identified as their ancestral STs. The majority of Hia entries (73.4%) and STs (65.5%) were identified as clonal division I (ST-23 and ST-4 complexes). Only 43 (21.9%) entries and 14 STs (25.5%) were identified as clonal division II (ST-62 and ST-21 complexes). Current data suggest that most invasive Hia belonged to clonal division I and the ST-23 complex, while most clonal division II Hia were respiratory isolates, with the exception of ST-62 which was common among invasive Hia in the US southwest. Comparison of the capsule bexABCD genes from clonal divisions I and II strains showed sequence diversity with variations following the pattern of clonal divisions. Evidence from the literature and the current study suggests that the recent emergence of invasive Hia might be related to capsule replacement subsequent to the implementation of the Hib conjugate vaccine and possibly exacerbated by other conjugate vaccines that may have altered the microbial flora of the human respiratory tract.

This study examined the phylogenetic structure of serotype a Haemophilus influenzae (Hia) isolates recovered from patients in Canada. Hia isolates from 490 separate patients and an American Type Culture Collection (ATCC) strain were analyzed by multilocus sequence typing (MLST), with 18 different sequence types (STs) identified. Most (85.7%) Hia patient isolates were typed as ST-23 and another 12.7% belonged to 14 different STs with 6, 5, or 4 MLST gene loci related to ST-23 (ST-23 complex). Core genome single-nucleotide variation phylogeny (SNVPhyl) on whole genome sequence (WGS) data of 121 Hia patient isolates representing all identified STs and the ATCC strain revealed 2 phylogenetic populations, with all the ST-23 complex isolates within 1 population. The other phylogenetic population contained only the ATCC strain and 3 patient isolates. Concatenated hitABC sequences retrieved from WGS data and analyzed by MEGA (Molecular Evolutionary Genetic Analysis) alignment confirmed the phylogeny obtained by SNVPhyl. The sodC gene was found only in isolates in the minor phylogenetic population. The 2 phylogenetic populations of the Canadian Hia isolates are similar to the 2 clonal divisions described for serotype b H. influenzae . Combining MLST, core SNVPhyl, and hitABC gene sequence alignment showed that most (99.4%) Canadian Hia patient isolates belonged to 1 major phylogenetic population.

Haemophilus influenzae is a well-established human pathogen capable of causing a range of respiratory and invasive diseases. Since the 1970s, it has been observed that a nontypeable cryptic genospecies of H. influenzae , most often biotype IV, has been associated with the genitourinary tracts of females and with invasive neonatal infections. This distinct genospecies has been provisionally named " Haemophilus quentini " Here, we report seven cases of invasive H. quentini disease in patients from Ontario, Canada, over a 2-year period. Significantly, while most reports of invasive disease with H. quentini to date have been in neonates, we observed five cases in adults (three in women of childbearing age and two in seniors) as well as two in neonates. Identification of H. quentini is challenging and was not possible for frontline laboratories, requiring work at the reference laboratory level. We describe in detail the biochemical results, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-Tof MS) results, and PCR results with several targets, including the 16S rRNA gene and multilocus sequence typing (MLST) genes, for the seven Ontario H. quentini isolates and several controls. Our data, combined with those of other publications, support the fact that H. quentini is distinct from H. influenzae and Haemophilus haemolyticus This organism is recognized as a pathogen of neonates, but we hypothesize that it may be underrecognized as an important pathogen in adults as well, particularly pregnant women. By sharing the detailed descriptions of these isolates, we hope to enable other laboratories to better identify H. quentini so that the true prevalence of this organism and disease can be explored., (© Crown copyright 2019.)

In the post- Haemophilus influenzae serotype b (Hib) vaccine era, invasive H. influenzae serotype a (Hia) disease emerged in Canadian First Nation, Inuit, and Alaskan Indigenous populations. Previous studies by our group found a high incidence of invasive Hia disease in northwestern Ontario. We retrospectively reviewed 24 cases (4 pediatric and 20 adult) of invasive H. influenzae disease hospitalized at the northwestern Ontario regional hospital between August 2011 and June 2018. The objectives were to further document the changing epidemiology of invasive H. influenzae disease in the region and to discuss potential control measures. Twenty-two H. influenzae isolates were serotyped and characterized using molecular-biological methods. Of the serotyped cases, there were 2 Hib, 9 Hia, and 11 non-typeable (NTHi). All Hia isolates belonged to the most common sequence types (ST) found in Canada (ST-23 and ST-929); 8 out of 9 were pan susceptible to antibiotics. One (11%) of 9 Hia and 5 (45%) of 11 NTHi cases were fatal. Our data on the consistent presence of serious invasive H. influenzae disease, with 41% prevalence of Hia (9 out of 22 serotyped isolates) and 50% prevalence of NTHi strains (11 out of 22), emphasize the importance of continued surveillance of H. influenzae in the post-Hib vaccine era and are critical information to inform potential vaccine development.

During the last two decades, Haemophilus influenzae serotype a (Hia) emerged as an important cause of invasive disease in Canadian First Nations and Inuit, and Alaskan Native populations, with the highest rates reported in young children. Immunocompetent adults, in contrast to children, do not typically develop invasive Hia disease. To clarify factors responsible for an increased burden of invasive Hia disease in certain population groups we studied serum bactericidal activity (SBA) against Hia and quantified IgG and IgM specific to Hia capsular polysaccharide in healthy adult members of two First Nations communities: 1) with reported cases of invasive Hia disease (Northern Ontario, NO), and 2) without reported cases (Southern Ontario, SO), in comparison to non-First Nations living in proximity to the NO First Nations community, and non-First Nations elderly non-frail Canadians from across the country (total of 110 First Nations and 76 non-First Nations). To elucidate the specificity of bactericidal antibodies, sera were absorbed with various Hia antigens. Naturally acquired SBA against Hia was detected at higher rates in First Nations (NO, 80%; SO, 96%) than non-First Nations elderly Canadians (64%); the SBA titres in First Nations were higher than in non-First Nations elderly Canadians (P<0.001) and NO non-First Nations adults (P>0.05). Among First Nations, SBA was mediated predominantly by IgM, and by both antibodies specific to Hia capsular polysaccharide and lipooligosaccharide., Conclusions: The SBA against Hia is frequently present in sera of First Nations adults regardless of the burden of Hia disease observed in their community; it may represent part of the natural antibody repertoire, which is potentially formed in this population under the influence of certain epigenetic factors. Although the nature of these antibodies deserves further studies to understand their origin, the data suggest that they may represent important protective mechanism against invasive Hia disease., Competing Interests: The authors have declared that no competing interests exist.

Introduction: North American indigenous populations experience a high burden of invasive bacterial infections. Because Streptococcus pneumoniae and Haemophilus influenzae have multiple antigenic variants, the existing vaccines cannot prevent all cases. This study addresses the current epidemiology of invasive H. influenzae and pneumococcal disease (IPD) in a region of Northwestern Ontario, Canada with a relatively high (82%) indigenous population., Methods: Data were retrieved from a retrospective chart review at a hospital servicing a population of 29000 (82% indigenous), during January 2010-July 2015., Results: Ten cases of invasive H. influenzae disease and 37 cases of IPD were identified. The incidence of both in the study population (6.3 and 23.1/100000/year, respectively) exceeded national rates (1.6 and 9.0/100000/year). H. influenzae serotype a (Hia) was the most common (50%), followed by non-typeable H. influenzae (20%). In adults, 77% of IPD cases were caused by serotypes included in the 23-valent pneumococcal polysaccharide vaccine. All paediatric IPD cases were caused by serotypes not included in the 13-valent pneumococcal conjugate vaccine. The case-fatality rate was 10% for invasive H. influenzae and 2.7% for IPD. Most cases exhibited substantial co-morbidity., Conclusions: In Northwestern Ontario, the incidence of invasive Hia disease exceeds that of H. influenzae type b (Hib) in the pre-Hib vaccine era. This provides strong support for the development of a new Hia vaccine. Improved pneumococcal vaccination of high-risk adults in the region is warranted., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: More than two decades after the implementation of the Hib conjugate vaccine in North America, Haemophilus influenzae serotype a (Hia) has emerged as a significant cause of invasive disease in Indigenous communities. However, little is known about the global presence of this pathogen., Methods: We interrogated the H. influenzae Multi-Locus Sequence Typing (MLST) website (https://pubmlst.org/hinfluenzae/) by selecting for serotype a records. We also updated our previous literature review on this subject matter., Results: Hia has been reported from at least 35 countries on six major continents. However, most Hia diseases were associated with Indigenous communities. Clonal analysis identified two clonal populations with one typified as ST-23 responsible for most invasive disease in North America and being the predominant clone described on the H. influenzae MLST website. Incidence of invasive Hia disease in Indigenous communities in North America are similar to the rates of Hib disease reported prior to the Hib conjugate vaccine era. Hia causes severe clinical diseases, such as meningitis, septicaemia, pneumonia, and septic arthritis with case-fatality rates between 5.6% and 33% depending on the age of the patient and the genetic makeup of the Hia strain., Conclusion: Although invasive Hia disease can be found globally, the current epidemiological data suggest that this infection predominantly affects Indigenous communities in North America. The clinical disease of Hia and the clonal nature of the bacteria resemble that of Hib. The high incidence of invasive Hia disease in Indigenous communities, along with potential fatality and severe sequelae causing long-term disability in survivors, may support the development of a new Hia conjugate vaccine for protection against this infection similar in design to the one introduced in the 1990s to control invasive Hib disease., (Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.)

Background: Previously we studied the antibiotic susceptibility of invasive Haemophilus influenzae collected in Canada from 1990 to 2006 and characterized isolates by serotype, MLST and ftsI gene sequencing for significant PBP3 mutations., Objectives: To provide an update based on isolates collected from 2007 to 2014., Methods: A total of 882 case isolates were characterized by serotype using slide agglutination and PCR. MLST was carried out to determine ST. Isolates were tested for β-lactamase production, presence of significant PBP3 mutations and antibiotic susceptibility by disc diffusion against 14 antibiotics. MIC values of three antibiotics were determined for 316 isolates using microbroth dilution., Results: Non-typeable H. influenzae accounted for 54.6% of the isolates and 45.4% were serotypeable, predominantly type a (23.1%), type b (8.3%) and type f (10.8%). The overall rate of ampicillin resistance due to β-lactamase production was 16.4% and increased from 13.5% in 2007-10 to 19% in 2011-14. Significant PBP3 mutations were identified in 129 isolates (14.6%) with 23 (2.6%) also producing β-lactamase. MLST identified related STs (ST-136, ST-14 and ST-367) associated exclusively with genetically β-lactamase-negative, ampicillin-resistant isolates and confirmed previously reported associations between significant PBP3 mutations and ST., Conclusions: A significant increase in β-lactamase-producing isolates was observed from 2007 to 2014; the rate of significant PBP3 mutations has increased since previously reported and 52.5% of non-typeable H. influenzae now show resistance markers. Resistance to trimethoprim/sulfamethoxazole was common and no resistance to fluoroquinolones or third-generation cephalosporins was found., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: With invasive Haemophilus influenzae serotype b (Hib) disease controlled by vaccination with conjugate Hib vaccines, there is concern that invasive disease due to non-serotype b strains may emerge., Objective: This study characterized invasive H. influenzae (Hi) isolates from Nunavut, Canada, in the post-Hib vaccine era., Methods: Invasive H. influenzae isolates were identified by conventional methods at local hospitals; and further characterized at the provincial and federal public health laboratories, including detection of serotype antigens and genes, multi-locus sequence typing and antibiotic susceptibility., Results: Of the 89 invasive H. influenzae cases identified from 2000 to 2012, 71 case isolates were available for study. There were 43 serotype a (Hia), 12 Hib, 2 Hic, 1 Hid, 1 Hie, 2 Hif and 10 were non-typeable (NT). All 43 Hia were biotype II, sequence type (ST)-23. Three related STs were found among the Hib isolates: ST-95 (n=9), ST-635 (n=2) and ST-44 (n=1). Both Hif belonged to ST-124 and the 2 Hic were typed as ST-9. The remaining Hid (ST-1288) and Hie (ST-18) belonged to 2 separate clones. Of the 10 NT strains, 3 were typed as ST-23 and the remaining 7 isolates each belonged to a unique ST. Eight Hib and 1 NT-Hi were found to be resistant to ampicillin due to β-lactamase production. No resistance to other antibiotics was detected., Conclusion: During the period of 2000-2012, Hia was the predominant serotype causing invasive disease in Nunavut. This presents a public health concern due to an emerging clone of Hia as a cause of invasive H. influenzae disease and the lack of published guidelines for the prophylaxis of contacts. The clonal nature of Hia could be the result of spread within an isolated population, and/or unique characteristics of this strain to cause invasive disease. Further study of Hia in other populations may provide important information on this emerging pathogen. No antibiotic resistance was detected among Hia isolates; a small proportion of Hib and NT-Hi isolates demonstrated resistance to ampicillin due to β-lactamase production.

Haemophilus influenzae, particularly H influenzae serotype b (Hib), is an important pathogen that causes serious diseases like meningitis and septicaemia. Since the introduction of Hib conjugate vaccines in the 1990s, the epidemiology of invasive H influenzae disease has changed substantially, with most infections now caused by non-Hib strains. We discuss the importance of H influenzae serotype a (Hia) as a cause of serious morbidity and mortality and its global epidemiology, clinical presentation, microbiology, immunology, prevention, and control. Much like Hib, the capsule of Hia is an important virulence factor contributing to the development of invasive disease. Molecular typing of Hia has identified distinct clonal groups, with some linked to severe disease and high case-fatality rates. Similarities between Hia and Hib capsules, their clinical presentation, and immunology of infection suggest that a bivalent Hia-Hib capsular polysaccharide-protein conjugate vaccine could offer protection against these two important serotypes of H influenzae., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Haemophilus influenzae serotype a (Hia) is an important pathogen since the introduction of vaccines for control of disease due to serotype b strains. Using a sodC-based polymerase chain reaction, Hia can be divided into 2 phylogenetic divisions, each with their own unique multilocus sequence types. Most Canadian Hia belongs to clonal division I and the ST-23 clonal complex. The recently described hypervirulent clone of ST-4 was found in a single Canadian isolate. Therefore, surveillance of invasive H. influenzae disease should include serotyping to detect Hia and multilocus sequence typing to detect hypervirulent clones.

A real-time polymerase chain reaction assay that uses degenerate primers and a dual-labelled probe was developed to detect the bexA gene of Haemophilus influenzae, including those belonging to non-b serotypes as well as clonal division II strains. This assay is sensitive and specific, detecting 20 copies of the gene, but negative with a variety of bacteria associated with meningitis and bacteremia or septicemia.

Seven epidemiologically unrelated cases of invasive Haemophilus influenzae type a (Hia) disease were identified in First Nations communities of Northwestern Ontario, Canada, in 2004-2008. In all cases, Hia was isolated from blood. The clinical presentation in most of the cases was moderately severe and all patients responded to antibiotic therapy. Laboratory analysis of Hia isolates from Northwestern Ontario indicated striking similarities in their phenotypic and genotypic characteristics. The findings are discussed in the context of current epidemiology of invasive Hia disease. Our data along with some published studies by others suggest an increased susceptibility to this infection among North American indigenous populations.

With the introduction of the Haemophilus influenzae serotype b (Hib) vaccine, invasive Hib disease has decreased substantially, but nontypeable H. influenzae (NT Hi) disease appears to be increasing. In order to understand the origin of NT Hi strains and their relationship with serotypeable strains, we analysed 125 NT Hi isolates collected from individual patients with either invasive disease (70 isolates) or respiratory tract infections (55 isolates). Serotype-specific and capsular transport genes were absent by PCR analysis, confirming their nonencapsulated status, which also suggested the NT Hi isolates were not encapsulated strains that shed their capsules. Multilocus sequence typing confirmed the NT Hi isolates did not have the same genetic background as serotypeable strains, including Hib. Despite the genetic heterogeneity found, two major genetic clusters were identified, both containing invasive and respiratory isolates. Fourteen invasive isolates and nine respiratory isolates produced beta-lactamase and were ampicillin resistant. More invasive (26.8%) than respiratory isolates (10.9%) showed decreased susceptibility towards ampicillin by a mechanism unrelated to beta-lactamase production. Besides a change in the capsule status of invasive Hi strains, the burden of invasive Hi disease, which used to be mainly a childhood disease, has now shifted to involve both adults and infants.

A high incidence of invasive non-type b Haemophilus influenzae disease was found in Northwestern Ontario, Canada; H. influenzae type a was the most prevalent serotype (42%). Clinical and demographic analyses indicate that aboriginal children aged <5 years and adults with predisposing medical conditions are the most affected by invasive H. influenzae disease in the post-H. influenzae vaccine era.

Introduction of Haemophilus influenzae type b (Hib) conjugate vaccines in late 1980s-early 1990s made a dramatic effect on the incidence of invasive Hib disease among children in many industrialized countries with routine Hib immunization programs. However, cases of vaccine failure and an increased susceptibility to invasive Hib as well as non-type b H. influenzae disease have been consistently reported among individuals with various congenital and acquired immunodeficiencies. Remarkably, in the 21st century, diseases due to non-type b strains of H. influenzae are becoming relatively more frequent than before. Despite the overall successful immunization against Hib, some indigenous populations, i.e. Australian Aboriginal and North American Indian children still experience increased rates of invasive H. influenzae disease. In order to monitor the evolving nature of invasive H. influenzae disease, carefully documented surveillance data is required to capture the true magnitude of the problem. Developing new vaccines against non-type b H. influenzae is a potential solution to protect some vulnerable populations against the invasive disease.

Nineteen beta-lactamase-negative ampicillin-resistant (BLNAR) and 2 beta-lactamase-positive amoxicillin-clavulanic acid-resistant Canadian Haemophilus influenzae strains were characterized. All 21 isolates were found to have the N526K mutations in their ftsI genes, and their ampicillin MIC(50) values were 4-8 times that of beta-lactamase-negative ampicillin-susceptible strains. The difficulty in detection of BLNAR strains was discussed.

One hundred and twenty-two isolates of Haemophilus influenzae causing invasive disease were collected in Manitoba, Canada, from 2000 to 2006 and examined for serotype, biotype, sequence type (ST) by multilocus sequence typing and antibiotic susceptibility. Nonserotypeable (NST) isolates accounted for over half of the isolates collected (69 isolates, 56.6%). There were 36 serotype a, five serotype b, two serotype c, one serotype d, four serotype e and five serotype f isolates collected. The 69 NST isolates were found to be very diverse, with isolates representing six biotypes and 45 STs. The serotypeable isolates were more clonal, with each of the serotypes showing little diversity in their biotypes and STs. Of the 122 isolates, 17% were resistant to ampicillin due to beta-lactamase production, 10.7% were resistant to trimethoprim-sulfamethoxazole, 1.6% were resistant to clarithromycin, 2.5% were resistant to amoxicillin-clavulanic acid and none was resistant to ciprofloxacin or moxifloxacin. Antibiotic resistance was more common in the NST strains, with 37.7% showing resistance to at least one antibiotic compared to 15% in the serotypeable strains. The results of this study suggest a shift in the epidemiology of invasive H. influenzae infections in the post-Hib vaccine era, and surveillance should include all serotypeable and NST isolates.

Four epidemiologically unrelated Haemophilus influenzae serotype a (Hia) strains from patients in Quebec, Canada, were characterized and found to represent 3 distinct groups. One isolate, found to be biotype I and sequence type (ST)-62 by multilocus sequence typing, was shown to possess the copper- and zinc-containing superoxide dismutase gene, sodC, and was suspected to belong to clonal division II. The other 3 isolates were classified as clonal division I based on the absence of the sodC gene. Among the 3 sodC-negative Hia strains, 2 were biotype II and had related STs (ST-23 and ST-403) and highly similar DNA fingerprints, similar to a group of previously described Hia isolates causing invasive disease in Manitoba, Canada. The remaining sodC-negative strain belonged to biotype I and ST-4 and shared no common allele with ST-23, ST-403, or ST-62. This isolate also possessed the IS1016-bexA partial deletion, which is often associated with increased virulence. Despite the small number of isolates used in this study, our finding of 3 distinct groups shows the existence of a potential genetic diversity not previously described for Hia. Whether this genetic diversity is related to the severity and epidemiology of Hia disease requires further studies.

In addition to the proportional increase in cases of non-type b Haemophilus influenzae disease in the post-H. influenzae type b vaccine era, the incidence of invasive H. influenzae disease was found to be approaching the rates of H. influenzae type b disease that were documented in the prevaccine period. Fifty-six percent of invasive disease now occurs in individuals aged >10 years.

The bexA genes of 36 Haemophilus influenzae isolates were sequenced to reveal their nucleotide sequence diversity, which divided them into two groups, similar to clonal divisions I and II. This sequence diversity may lead to false-negative PCR results for H. influenzae infections if bexA is the chosen gene target.

Fifty-two Haemophilus influenzae isolates from patients with invasive disease in the province of Manitoba, Canada, were examined for serotype, biotype, genotype, and antibiotic susceptibility. Half of the 52 isolates were found to be serotype a, and 38.5% (20 isolates) were found to be nonserotypeable (NST). There were only three serotype b strains and one each for serotypes c, d, and f. All 26 serotype a isolates belonged to biotype II and demonstrated identical or highly similar DNA fingerprints by pulsed-field gel electrophoresis. An analysis of these isolates by multilocus sequence typing showed that they belong to the clonal complex ST-23. While 69% (18 of 26) of the serotype a cases were found in males, only 9 (45%) of the 20 patients with NST isolates were males. Twenty (77%) of the 26 serotype a isolates were from patients who were