Epidemiology of Haemophilus influenzae in the Republic of Ireland, 2010-2018.

The purpose of this study was to investigate H. influenzae epidemiology in the Republic of Ireland. We performed serotyping, multi-locus sequence typing (MLST) and susceptibility testing on H. influenzae isolates received by the Irish Meningitis and Sepsis Reference Laboratory from 2010 to 2018. Three hundred sixty-seven invasive and 41 non-invasive infection (NII) isolates were received. Invasive isolates were mostly recovered from paediatric (21%) and elderly (42%) populations. Invasive disease was more prevalent in females of childbearing age (72%) compared with males the same age (28%). Non-typeable H. influenzae (NTHi) predominated among invasive (83%) and NII (95%). Invasive Hib disease isolates were infrequent (4%, n = 15). Among invasive disease, Hif was the commonest encapsulated serotype (10%, n = 37), and the only encapsulated serotype detected in NII (5%, 2/41). The first PCR-confirmed serotypes d and a in Ireland were characterised among invasive disease in 2017 and 2018, respectively. MLST revealed a diverse NTHi population, while encapsulated serotypes were clonal. Sequence type (ST) 103 (n = 14) occurred exclusively in invasive NTHi disease. Ampicillin resistance (AmpR) was 18% among invasive isolates and 22% in NII. ß-Lactamase production was the main source of ampicillin resistance in invasive and NII isolates. We detected ß-lactamase negative ampicillin resistance (BLNAR) among invasive isolates. We report an NTHi fluoroquinolone-resistant clone: ST1524 among invasive (n = 2) and NII isolates (n = 2). The Hib vaccine has positively impacted on Hib disease in Ireland, given the low frequency of Hib. The dominance of NTHi, emergence of serotypes a and d and BLNAR suggest a changing H. influenzae epidemiology in Ireland.

Resolution of a Protracted Serogroup B Meningococcal Outbreak with Whole-Genome Sequencing Shows Interspecies Genetic Transfer.

A carriage study was undertaken (n = 112) to ascertain the prevalence of Neisseria spp. following the eighth case of invasive meningococcal disease in young children (5 to 46 months) and members of a large extended indigenous ethnic minority Traveller family (n = 123), typically associated with high-occupancy living conditions. Nested multilocus sequence typing (MLST) was employed for case specimen extracts. Isolates were genome sequenced and then were assembled de novo and deposited into the Bacterial Isolate Genome Sequencing Database (BIGSdb). This facilitated an expanded MLST approach utilizing large numbers of loci for isolate characterization and discrimination. A rare sequence type, ST-6697, predominated in disease specimens and isolates that were carried (n = 8/14), persisting for at least 44 months, likely driven by the high population density of houses (n = 67/112) and trailers (n = 45/112). Carriage for Neisseria meningitidis (P < 0.05) and Neisseria lactamica (P < 0.002) (2-sided Fisher's exact test) was more likely in the smaller, more densely populated trailers. Meningococcal carriage was highest in 24- to 39-year-olds (45%, n = 9/20). Evidence of horizontal gene transfer (HGT) was observed in four individuals cocolonized by Neisseria lactamica and Neisseria meningitidis One HGT event resulted in the acquisition of 26 consecutive N. lactamica alleles. This study demonstrates how housing density can drive meningococcal transmission and carriage, which likely facilitated the persistence of ST-6697 and prolonged the outbreak. Whole-genome MLST effectively distinguished between highly similar outbreak strain isolates, including those isolated from person-to-person transmission, and also highlighted how a few HGT events can distort the true phylogenetic relationship between highly similar clonal isolates.

Diversity of meningococci associated with invasive meningococcal disease in the Republic of Ireland over a 19 year period, 1996-2015.

This study examined the capsular phenotype and genotype of invasive meningococcal disease (IMD)-associated Neisseria meningitidis recovered in the Republic of Ireland (RoI) between 1996 and 2015. This time period encompasses both pre- (when IMD was hyperendemic in the RoI) and post- meningococcal serogroup C conjugate (MCC) vaccine introduction. In total, 1327 isolates representing over one-third of all laboratory-confirmed cases of IMD diagnosed each epidemiological year (EY), were characterised. Serogroups B (menB) and C (menC) predominated throughout, although their relative abundance changed; with an initial increase in the proportion of menC in the late 1990s followed by their dramatic reduction post-MCC vaccine implementation and a concomitant dominance of menB, despite an overall decline in IMD incidence. While the increase in menC was associated with expansion of specific clonal-complexes (cc), cc11 and cc8; the dominance of menB was not. There was considerable variation in menB-associated cc with declines in cc41/44 and cc32, and increases in cc269 and cc461, contributing to a significant increase in the clonal diversity of menB isolates over the study. This increase in diversity was also displayed among the serosubtyping data, with significant declines in proportions of menB isolates expressing p1.4 and p1.15 antigens. These data highlight the changing diversity of IMD-associated meningococci since 1996 in the RoI and emphasise the need for on-going surveillance particularly in view of the recent introduction of a menB vaccine.

Development of a diagnostic real-time polymerase chain reaction assay for the detection of invasive Haemophilus influenzae in clinical samples.

Since the introduction of the Haemophilus influenzae serotype b vaccine, invasive H. influenzae disease has become dominated by nontypeable (NT) strains. Several widely used molecular diagnostic methods have been shown to lack sensitivity or specificity in the detection of some of these strains. Novel real-time assays targeting the fucK, licA, and ompP2 genes were developed and evaluated. The fucK assay detected all strains of H. influenzae tested (n = 116) and had an analytical sensitivity of 10 genome copies/polymerase chain reaction (PCR). This assay detected both serotype b and NT H. influenzae in 12 previously positive specimens (culture and/or bexA PCR) and also detected H. influenzae in a further 5 of 883 culture-negative blood and cerebrospinal fluid (CSF) samples. The fucK assay has excellent potential as a diagnostic test for detection of typeable and nontypeable strains of invasive H. influenzae in clinical samples of blood and CSF.