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Over the past decade, there have been many improvements in the field of metagenomics, including sequencing technologies, advances in bioinformatics and the development of reference databases, but a one-size-fits-all sequencing and bioinformatics pipeline does not yet seem achievable. In this study, we address the bioinformatics part of the analysis by combining three methods into a three-step workflow that increases the sensitivity and specificity of clinical metagenomics and improves pathogen detection. The individual tools are combined into a user-friendly workflow suitable for analysing short paired-end (PE) and long reads from metagenomics datasets—MetaAll. To demonstrate the applicability of the developed workflow, four complicated clinical cases with different disease presentations and multiple samples collected from different biological sites as well as the CAMI Clinical pathogen detection challenge dataset were used. MetaAll was able to identify putative pathogens in all but one case. In this case, however, traditional microbiological diagnostics were also unsuccessful. In addition, co-infection with Haemophilus influenzae and Human rhinovirus C54 was detected in case 1 and co-infection with SARS-Cov-2 and Influenza A virus (FluA) subtype H3N2 was detected in case 3. In case 2, in which conventional diagnostics could not find a pathogen, mNGS pointed to Klebsiella pneumoniae as the suspected pathogen. Finally, this study demonstrated the importance of combining read classification, contig validation and targeted reference mapping for more reliable detection of infectious agents in clinical metagenome samples. [ABSTRACT FROM AUTHOR]

Objectives This study aimed to explore the prevalence of macrolide resistance and the underlying resistance mechanisms in Haemophilus influenzae (n  = 2556) and Haemophilus parainfluenzae (n  = 510) collected between 2018 and 2021 from Bellvitge University Hospital, Spain. Methods Antimicrobial susceptibility was tested by microdilution. Whole-genome sequencing was performed using Illumina MiSeq and Oxford Nanopore technologies, and sequences were examined for macrolide resistance determinants and mobile genetic structures. Results Macrolide resistance was detected in 67 H. influenzae (2.6%) and 52 (10.2%) H. parainfluenzae strains and associated with resistance to other antimicrobials (co-trimoxazole, chloramphenicol, tetracycline). Differences in macrolide resistance existed between the two species. Acquired resistance genes were more prevalent in H. parainfluenzae (35/52; 67.3%) than in H. influenzae (12/67; 17.9%). Gene mutations and amino acid substitutions were more common in H. influenzae (57/67; 85%) than in H. parainfluenzae (16/52; 30.8%). Substitutions in L22 and in 23S rRNA were only detected in H. influenzae (34.3% and 29.0%, respectively), while substitutions in L4 and AcrAB/AcrR were observed in both species. The MEGA element was identified in 35 (67.3%) H. parainfluenzae strains, five located in an integrative and conjugative element (ICE); by contrast, 11 (16.4%) H. influenzae strains contained the MEGA element (all in an ICE). A new ICE HpaHUB8 was described in H. parainfluenzae. Conclusions Macrolide resistance was higher in H. parainfluenzae than in H. influenzae , with differences in the underlying mechanisms. H. parainfluenzae exhibits co-resistance to other antimicrobials, often leading to an extensively drug-resistant phenotype. This highlights the importance of conducting antimicrobial resistance surveillance. [ABSTRACT FROM AUTHOR]

Background Despite vaccination, influenza and otitis media (OM) remain leading causes of illness. We previously found that the human respiratory commensal Haemophilus haemolyticus prevents bacterial infection in vitro and that the related murine commensal Muribacter muris delays OM development in mice. The observation that M muris pretreatment reduced lung influenza titer and inflammation suggests that these bacteria could be exploited for protection against influenza/OM. Methods Safety and efficacy of intranasal H haemolyticus at 5 × 107 colony-forming units (CFU) was tested in female BALB/cARC mice using an influenza model and influenza-driven nontypeable Haemophilus influenzae (NTHi) OM model. Weight, symptoms, viral/bacterial levels, and immune responses were measured. Results Intranasal delivery of H haemolyticus was safe and reduced severity of influenza, with quicker recovery, reduced inflammation, and lower lung influenza virus titers (up to 8-fold decrease vs placebo; P ≤.01). Haemophilus haemolyticus reduced NTHi colonization density (day 5 median NTHi CFU/mL = 1.79 × 103 in treatment group vs 4.04 × 104 in placebo, P =.041; day 7 median NTHi CFU/mL = 28.18 vs 1.03 × 104; P =.028) and prevented OM (17% OM in treatment group, 83% in placebo group; P =.015). Conclusions Haemophilus haemolyticus has potential as a live biotherapeutic for prevention or early treatment of influenza and influenza-driven NTHi OM. Additional studies will deem whether these findings translate to humans and other respiratory infections. [ABSTRACT FROM AUTHOR]

Biofilm formation has been suggested to be associated with phenotype changes compared with the planktonic form. We screened 1092 Haemophilus influenzae isolates for their genetic relationships and then selected 29 isolates from different genotypes and phenotypes and tested their ability to form biofilm. Our data showed a higher capacity of nontypable isolates, particularly isolates from respiratory and genital infections to form biofilm, compared with typable isolates. This ability to form biofilm was also correlated with reduced deposition of the complement component C3b on biofilm-involved bacteria. These data suggest that the biofilm formation contributes to the virulence of nontypable H. influenzae. [ABSTRACT FROM AUTHOR]

Transmission rates among children with conjunctivitis were low and antibiotic use was not associated with reduced transmission. Policies recommending exclusion from daycare and school for conjunctivitis should be scrutinized as they may not reduce transmission and may increase unnecessary antibiotic use. [ABSTRACT FROM AUTHOR]

Objectives Haemophilus parainfluenzae is an opportunistic pathogen causing respiratory tract infection and sexually transmitted diseases. The emergence of multidrug resistance in this species is particularly worrisome, especially since the recent description of CTX-M-15 ESBL-producing isolates in Spain. The aim of this study was to characterize a CTX-M-15-producing H. parainfluenzae clinical isolate, HP01, obtained from a urethral swab. Methods MICs were determined with gradient strips for this isolate. Hydrolysis assays were performed with the β LACTA test. Genomic DNA from HP01 was subjected to Illumina and Oxford Nanopore sequencing to investigate the genetic environment of bla CTX-M-15. Phylogenetic analysis was performed with available H. parainfluenzae genomes from the NCBI database, including CTX-M-15 producers. Results HP01, an XDR isolate, was resistant to penicillin, third-generation cephalosporins, fluoroquinolones, macrolides, cyclines and co-trimoxazole and susceptible only to carbapenems and rifampicin. HP01 carried bla TEM-1, bla CTX-M-15, tet (M), catS and mef (E) /mel and harboured amino acid substitutions in PBP3, PBP5, GyrA, ParC and FolA implicated in resistance. Genomic analysis revealed that bla CTX-M-15 was carried by a Tn 3 -like transposon inserted into a novel integrative and conjugative element (ICE), ICE Hpa SLS, present on the chromosome and belonging to the ICE Hin1056 family described in Haemophilus influenzae. The tet (M)-MEGA element was also detected on the chromosome. No plasmid was found. The phylogenetic analysis showed that four H. parainfluenzae producing CTX-M-15 clustered in the same clade. Conclusions Here we report the description of an XDR H. parainfluenzae producing bla CTX-M-15 isolated from a urethral swab. The bla CTX-M-15 gene was inserted into an ICE structure similar to those recently described in CTX-M-15 producers in Spain. The emergence of XDR H. parainfluenzae producing bla CTX-M-15 is a matter of great concern. Careful surveillance is required to prevent its spread. [ABSTRACT FROM AUTHOR]

Objectives Lefamulin (Xenleta™), a pleuromutilin antibiotic, was approved for the oral and IV treatment of community-acquired bacterial pneumonia (CABP) in adults in 2019/2020. This study evaluated the in vitro activity of lefamulin and comparators against 19 584 unique bacterial isolates collected from patients with community-acquired respiratory tract infections and hospitalized patients with pneumonia within the global SENTRY Antimicrobial Surveillance Program during 2015–21. Methods Isolates were susceptibility tested by the CLSI broth microdilution method, and resistance mechanisms were investigated in isolates with elevated lefamulin MICs. Results Lefamulin exhibited potent antibacterial activity against the most common and typical CABP pathogens tested, including Streptococcus pneumoniae [MIC50/90, 0.06/0.25 mg/L; 99.9% susceptible (S)], Staphylococcus aureus (MIC50/90, 0.06/0.12 mg/L; 99.6% S), Haemophilus influenzae (MIC50/90, 0.5/2 mg/L; 99.1% S) and Moraxella catarrhalis (MIC50/90, 0.06/0.12 mg/L; 100.0% S). Potent activity was also observed against the less common pneumonia pathogens: β-haemolytic (MIC50/90 of 0.03/0.06 mg/L) and viridans group Streptococcus spp. (MIC50/90 of 0.06/0.25 mg/L) and Haemophilus parainfluenzae (MIC50/90 of 1/4 mg/L). Lefamulin's activity was not adversely affected by resistance to macrolides, penicillin, tetracyclines, fluoroquinolones and other resistance phenotypes. Non-susceptibility/resistance to lefamulin was rare and primarily determined by ribosomal protection through vga (A) variants in S. aureus, overexpression of AcrAB-TolC efflux pump in H. influenzae or modifications in L3, L4 and 23SrRNA in Streptococcus spp. Conclusions Based on the coverage of the most important CABP pathogens and lacking cross-resistance, lefamulin may represent a valuable empirical treatment option for ambulatory and hospitalized patients with CABP, particularly in settings with high prevalence of resistance. [ABSTRACT FROM AUTHOR]

In patients early post–autologous stem cell transplant, seroprotection rates were high for Hemophilus influenzae type B and tetanus toxoid (70%–90%) but lower for Streptococcus pneumoniae (30%–50%) including after revaccination. There were high rates of seropositivity (67%–86%) to measles, mumps, and rubella and varicella zoster virus. Durability of protection requires assessment. [ABSTRACT FROM AUTHOR]

Background Rapidly instituted antimicrobial therapy is important in severe infections, and reduced time to the antimicrobial susceptibility testing (AST) report is thus of importance. Disc diffusion (DD) is a cheap, rapidly adaptable, flexible and comprehensive method for phenotypic AST. Previous studies have shown that early reading of inhibition zones for non-fastidious species is possible. Objectives To evaluate zone reading after short incubation of DD in Haemophilus influenzae (n  = 73) and Streptococcus pneumoniae (n  = 112). Methods The readability was evaluated and susceptibility interpretation (SIR) was performed, using the EUCAST 18 ± 2 h incubation breakpoint table (version 12.0), after 6 and 8 h of incubation. Categorical agreement (CA) and error rates were calculated using standard DD and broth microdilution as reference. Results The proportion of readable zones in H. influenzae was 19% (6 h) and 89% (8 h). The CA was 98% after 8 h. The corresponding readability in S. pneumoniae was 63%/98% and CA was 95%/97% after 6 and 8 h, respectively. Early reading of the screening discs (benzylpenicillin 1 unit in H. influenzae and oxacillin 1 µg in S. pneumoniae) correctly identified 18/22 of the H. influenzae isolates and all the readable S. pneumoniae isolates with reduced β-lactam susceptibility. For non-β-lactam agents, very major errors were most common for quinolones in S. pneumoniae. Introduction of areas of technical uncertainty (ATUs) reduced the error rate to ≤1.1%. Conclusions We conclude that shortened incubation is feasible for H. influenzae and S. pneumoniae. To reduce the risk of false categorization a buffer zone (i.e. ATU) near the breakpoints must be used. [ABSTRACT FROM AUTHOR]

Pasteurella multocida , an encapsulated gram-negative bacterium, is a significant veterinary pathogen. The P. multocida is classified into 5 serogroups (A, B, D, E, and F) based on the bacterial capsular polysaccharide (CPS), which is important for virulence. Serogroups B and E are the primary causative agents of bovine hemorrhagic septicemia that is associated with significant yearly losses of livestock worldwide, primarily in low- and middle-income countries. The P. multocida disease is currently managed by whole-cell vaccination, albeit with limited efficacy. CPS is an attractive antigen target for an improved vaccine: CPS-based vaccines have proven highly effective against human bacterial diseases and could provide longer-term protection against P. multocida. The recently elucidated CPS repeat units of serogroups B and E both comprise a N-acetyl-β-D-mannosaminuronic acid/N-acetyl-β-D-glucosamine disaccharide backbone with β-D-fructofuranose (Fru f) side chain, but differ in their glycosidic linkages, and a glycine (Gly) side chain in serogroup B. Interestingly, the Haemophilus influenzae types e and d CPS have the same backbone residues. Here, comparative modeling of P. multocida serogroups B and E and H. influenzae types e and d CPS identifies a significant impact of small structural differences on both the chain conformation and the exposed potential antibody-binding epitopes (Ep). Further, Fru f and/or Gly side chains shield the immunogenic amino-sugar CPS backbone—a possible common strategy for immune evasion in both P. multocida and H. influenzae. As the lack of common epitopes suggests limited potential for cross-reactivity, a bivalent CPS-based vaccine may be necessary to provide adequate protection against P. multocida types B and E. [ABSTRACT FROM AUTHOR]

Background This retrospective hospital-based surveillance aimed to assess the epidemiology, causative pathogens trend, and serotypes distribution of pneumococcal meningitis among children aged under 5 years with bacterial meningitis in Southern Vietnam after the introduction of pentavalent vaccine in the Expanded Program on Immunization (EPI). Methods From 2012 to 2021, cerebrospinal fluid samples were collected from children aged under 5 years with suspected bacterial meningitis at Children's Hospitals 1 and 2 in Ho Chi Minh City. Probable bacterial meningitis (PBM) cases were identified using biochemistry and cytology. Real-time polymerase chain reaction was used to confirm cases of confirmed bacterial meningitis (CBM) caused by Streptococcus pneumoniae , Haemophilus influenzae , or Neisseria meningitidis. Streptococcus pneumoniae serotyping was performed. Results Of the 2560 PBM cases, 158 (6.2%) were laboratory-confirmed. The CBM proportion decreased during the 10-year study and was associated with age, seasonality, and permanent residence. Streptococcus pneumoniae was the most common pathogen causing bacterial meningitis (86.1%), followed by H influenzae (7.6%) and N meningitidis (6.3%). The case-fatality rate was 8.2% (95% confidence interval, 4.2%–12.2%). Pneumococcal serotypes 6A/B, 19F, 14, and 23F were the most prevalent, and the proportion of pneumococcal meningitis cases caused by the 10-valent pneumococcal conjugate vaccine (PCV) serotypes decreased from 96.2% to 57.1% during the PCV eras. Conclusions Streptococcus pneumoniae is the most frequent causative agent of bacterial meningitis in children aged under 5 years in Southern Vietnam over the last decade. Policymakers may need to consider introducing PCVs into the EPI to effectively prevent and control bacterial meningitis. [ABSTRACT FROM AUTHOR]

Background Cefepime and aztreonam are highly efficacious against H. influenzae , and resistant strains are rare. In this study, we isolated cefepime- and aztreonam-nonsusceptible H. influenzae strains and addressed the molecular basis of their resistance to cefepime and aztreonam. Methods Two hundred and 28 specimens containing H. influenzae were screened, of which 32 isolates were enrolled and applied to antimicrobial susceptibility testing and whole-genome sequencing. Genetic variations that were detected in all nonsusceptible isolates with statistical significance by Fisher's exact tests were identified as cefepime or aztreonam nonsusceptibility related. Functional complementation assays were conducted to assess the in vitro effects of proteins with sequence substitutions on drug susceptibility. Results Three H. influenzae isolates were nonsusceptible to cefepime, one of which was also nonsusceptible to aztreonam. Genes encoding TEM, SHV and CTX-M extended-spectrum β-lactamases were not detected in the cefepime- and aztreonam-nonsusceptible isolates. Five genetic variations in four genes and 10 genetic variations in five genes were associated with cefepime and aztreonam nonsusceptibility, respectively. Phylogenetic analyses revealed that changes in FtsI were correlated strongly with the MIC of cefepime and moderately with aztreonam. FtsI Thr532Ser-Tyr557His cosubstitution linked to cefepime nonsusceptibility and Asn305Lys-Ser385Asn-Glu416Asp cosubstitution to aztreonam nonsusceptibility. Functional complementation assays revealed that these cosubstitutions increased MICs of cefepime and aztreonam in susceptible H. influenzae isolates, respectively. Conclusions Genetic variations relevant to resistant phenotypes of cefepime and aztreonam nonsusceptibility in H. influenzae were identified. Moreover, the effects of FtsI cosubstitutions on increasing MICs of cefepime and aztreonam in H. influenzae were demonstrated. [ABSTRACT FROM AUTHOR]

Background Nontypeable Haemophilus influenzae (NTHi) is the most common cause of invasive H. influenzae disease in the United States (US). We evaluated the epidemiology of invasive NTHi disease in the US, including among pregnant women, infants, and people with human immunodeficiency virus (PWH). Methods We used data from population- and laboratory-based surveillance for invasive H. influenzae disease conducted in 10 sites to estimate national incidence of NTHi, and to describe epidemiology in women of childbearing age, infants aged ≤30 days (neonates), and PWH living in the surveillance catchment areas. H. influenzae isolates were sent to the Centers for Disease Control and Prevention for species confirmation, serotyping, and whole genome sequencing of select isolates. Results During 2008–⁠2019, average annual NTHi incidence in the US was 1.3/100 000 population overall, 5.8/100 000 among children aged <1 year, and 10.2/100 000 among adults aged ≥80 years. Among 225 reported neonates with NTHi, 92% had a positive culture within the first week of life and 72% were preterm. NTHi risk was 23 times higher among preterm compared to term neonates, and 5.6 times higher in pregnant/postpartum compared to nonpregnant women. More than half of pregnant women with invasive NTHi had loss of pregnancy postinfection. Incidence among PWH aged ≥13 years was 9.5 cases per 100 000, compared to 1.1 cases per 100 000 for non-PWH (rate ratio, 8.3 [95% confidence interval, 7.1–9.7]; P <.0001). Conclusions NTHi causes substantial invasive disease, especially among older adults, pregnant/postpartum women, and neonates. Enhanced surveillance and evaluation of targeted interventions to prevent perinatal NTHi infections may be warranted. [ABSTRACT FROM AUTHOR]

Background In 2019, a high-level quinolone-resistant Haemophilus haemolyticus strain (levofloxacin MIC = 16 mg/L) was isolated from a paediatric patient. In this study, we aimed to determine whether the quinolone resistance of H. haemolyticus could be transferred to Haemophilus influenzae and to identify the mechanism underlying the high-level quinolone resistance of H. haemolyticus. Methods A horizontal gene transfer assay to H. influenzae was performed using genomic DNA or PCR-amplified quinolone-targeting genes from the high-level quinolone-resistant H. haemolyticus 2019-19 strain. The amino acids responsible for conferring quinolone resistance were identified through site-directed mutagenesis. Results By adding the genomic DNA of H. haemolyticus 2019-19, resistant colonies were obtained on agar plates containing quinolones. Notably, H. influenzae grown on levofloxacin agar showed the same level of resistance as H. haemolyticus. Sequencing analysis showed that gyrA , parC and parE of H. influenzae were replaced by those of H. haemolyticus , suggesting that horizontal transfer occurred between the two strains. When the quinolone-targeting gene fragments were added sequentially, the addition of parE , as well as gyrA and parC , contributed to high-level resistance. In particular, amino acid substitutions at both the 439th and 502nd residues of ParE were associated with high-level resistance. Conclusions These findings indicate that quinolone resistance can be transferred between species and that amino acid substitutions at the 439th and 502nd residues of ParE, in addition to amino acid substitutions in both GyrA and ParC, contribute to high-level quinolone resistance. [ABSTRACT FROM AUTHOR]

Background: The Biofire® FilmArray® Pneumonia Panel (PN Panel) provides a more rapid and sensitive method of respiratory pathogen detection than standard culture. However, it is often unclear how to apply the results clinically, especially in the case of discordant culture results. We evaluated the concordance of bacterial organism and resistance gene identification between the PN Panel and standard culture methods in hospitalized patients with a clinical diagnosis of pneumonia. Methods: This single-center retrospective observational study of 274 inpatients assessed the positive predictive value (PPV) and described the prevalence of individual bacterial organism and resistance marker targets on the PN Panel. Results: The overall PPV of the PN Panel in identifying bacteria was 70.1%, with individual organism PPV ranging from 50.0% to 90.9%. For resistance gene identification, the PN Panel's PPV ranged from 46.2% for CTX-M to 68.4% for mecA/C and the staphylococcal cassette chromosome mec element right extremity junction (MREJ), although resistance was uncommon. Staphylococcus aureus was the most common bacterial pathogen detected by the PN Panel (38.7%), followed by Pseudomonas aeruginosa (22.3%), and Haemophilus influenzae (12.0%). Conclusions: The PN Panel detected more bacteria and resistance gene targets than standard culture methods. To optimize the use of this technology for both patient care and antimicrobial stewardship, results should be coupled with clinical assessment and clinician education. [ABSTRACT FROM AUTHOR]

Background Streptococcus pneumoniae (Spn), Haemophilus influenzae (Hflu), and Moraxella catarrhalis (Mcat) nasopharyngeal colonization precedes disease pathogenesis and varies among settings and countries. We sought to assess colonization prevalence, density, Spn serotypes, and antibiotic resistance in children in the first 6 months of life in pediatric primary care settings. Methods Prospective cohort study in Rochester, NY during 2018–2020. Nasopharyngeal swabs were collected from 101 children at age 1, 2, and 3 weeks, then 1, 2, 4, 6, 9, 12, 15, 18, and 24 months. Spn serotypes were determined by Quellung. Oxacillin resistance for Spn and β-lactamase production by Hflu and Mcat was tested. All children received PCV13 vaccine according to U.S. recommended schedule. Results Spn, Hflu , and Mcat colonization was detected in only 5% of infants before age 2 months old. Cumulative prevalence was 34% for Spn , 10% for Hflu , and 53% for Mcat in children ≤6 months of age. Nasopharyngeal bacterial density of Spn, Hflu , and Mcat (x = 2.71 log) in children ≤6 months of age was lower than at 7–24 months of age (x = 3.15 log, p < 0.0001). Predominant serotypes detected ≤6 months of age were 23B (16.7%), 22F (12.9%), 15B/C (11%), and 16F (9.2%). In total, 14.8% of Spn isolates were oxacillin resistant and 66.7% of Hflu isolates were β-lactamase producing. Conclusion Spn, Hflu , and Mcat nasopharyngeal colonization was uncommon and of low density among children ≤6 months old, especially among children <2 months of age. Non-PCV13 serotypes predominated and a different serotype distribution was observed in ≤6-month olds compared to 7- to 24-month olds. [ABSTRACT FROM AUTHOR]

Background Pneumococcal conjugate vaccines (PCVs) effectively reduce infection and asymptomatic carriage of Streptococcus pneumoniae vaccine serotypes. In 2016, Belgium replaced its infant PCV13 program by a 4-year period of PCV10. Concomitantly, S. pneumoniae serotype carriage was monitored together with the carriage of other nasopharyngeal pathogens in children attending day-care centers. Methods From 2016 to 2019, a total of 3459 nasopharyngeal swabs were obtained from children aged 6–30 months. Culture and qPCR were used for the identification of S. pneumoniae , Haemophilus influenzae, Moraxella catarrhalis , and Staphylococcus aureus and for serotyping and antimicrobial susceptibility assessment of S. pneumoniae strains. Results S. pneumoniae colonization was frequent and stable over the study years. H. influenzae and M. catarrhalis were more frequently carried (P  < .001) than S. pneumoniae , by, respectively, 92.3% and 91.0% of children. Prevalence of all PCV13 serotypes together increased significantly over time from 5.8% to 19.6% (P  < .001) and was attributable to the increasing prevalence of serotype 19A. Coincidently, non-vaccine serotype 6C increased (P  < .001) and the overall pneumococcal non-susceptibility to tetracycline and erythromycin. Non-susceptibility to cotrimoxazole decreased (P  < .001). Conclusions The switch to a PCV program no longer covering serotypes 19A, 6A, and 3 was associated with a sustained increase of serotypes 19A and 6C in healthy children, similarly as in invasive pneumococcal disease. This resulted in a re-introduction of the 13-valent conjugate vaccine during the summer of 2019. [ABSTRACT FROM AUTHOR]

Background: Quinolone-resistant bacteria are known to emerge via the accumulation of mutations in a stepwise manner. Recent studies reported the emergence of quinolone low-susceptible Haemophilus influenzae ST422 isolates harbouring two relevant mutations, although ST422 isolates harbouring one mutation were never identified.Objectives: To investigate if GyrA and ParC from quinolone low-susceptible isolates can be transferred horizontally and simultaneously to susceptible isolates.Methods: Genomic DNA was extracted from an H. influenzae isolate harbouring amino acid substitutions in both gyrA and parC and mixed with clinical isolates. The emergence of resistant isolates was compared, and WGS analysis was performed.Results: By adding the genomic DNA harbouring both mutated gyrA and parC, resistant bacteria exhibiting recombination at gyrA only or both gyrA and parC loci were obtained on nalidixic acid and pipemidic acid plates, and the frequency was found to increase with the amount of DNA. Recombination events in gyrA only and in both gyrA and parC occurred with at least 1 and 1-100 ng of DNA, respectively. The genome sequence of a representative strain showed recombination events throughout the genome. The MIC of quinolone for the resulting strains was found to be similar to that of the donor. Although the recombination efficacy was different among the various strains, all strains used in this study obtained multiple genes simultaneously.Conclusions: These findings indicate that H. influenzae can simultaneously obtain more than two mutated genes. This mechanism of horizontal transfer could be an alternative pathway for attaining quinolone resistance. [ABSTRACT FROM AUTHOR]

Background: The role of nasopharyngeal bacteria in respiratory syncytial virus (RSV) disease has been underestimated. We measured the frequency and burden of respiratory bacteria in the upper respiratory tract of infants with RSV infection over 7 respiratory seasons, and their impact on clinical outcomes.Methods: Children <2 years old with mild (outpatients, n=115) or severe (inpatients, n=566) RSV infection, and matched healthy controls (n=161) were enrolled. Nasopharyngeal samples were obtained for RSV, Streptococcus pneumoniae, Staphylococcus aureus, Moraxella catarrhalis, and Haemophilus influenzae detection and quantitation by PCR. Multivariable models were constructed to identify variables predictive of severe disease.Results: S. pneumoniae, H. influenzae, and M. catarrhalis, but not S. aureus, were detected more frequently in RSV-infected children (84%) than healthy controls (46%; P<.001). Detection of S. pneumoniae and/or H. influenzae was associated with fever, more frequent antibiotic treatment, worse radiologic findings, and higher neutrophil counts (P<.01). In adjusted analyses, S. pneumoniae/H. influenzae codetection was independentlyassociated with greater odds of hospitalization, higher disease severity scores, need for supplemental oxygen, and longer hospitalization.Conclusions: Nasopharyngeal codetection of S. pneumoniae and H. influenzae in infants with RSV infection is associated with increased disease severity. [ABSTRACT FROM AUTHOR]

Background: Haemophilus influenzae serotype a (Hia) can cause severe invasive disease, especially in young children. In 2018, 4 invasive Hia cases occurred in an Alaska community. We used whole-genome sequencing (WGS) to evaluate the relationship of the bacteria from this community and other Alaska patients with invasive Hia.Methods: All carriage (n = 15) and invasive (n = 4) Hia isolates from the outbreak community, together with 15 nonoutbreak Alaska invasive Hia surveillance isolates from 2018, were tested for antimicrobial susceptibility and characterized using WGS.Results: Phylogenetic analysis of both invasive and carriage Hia isolates revealed 2 major clades that differed by an average of 300 core single-nucleotide polymorphisms (SNPs). All isolates from the outbreak community were clustered in 1 subclade, within a larger clade containing 3 nonoutbreak invasive Hia isolates. Comparative genomics did not reveal any genetic mutations that distinguished carriage from invasive isolates. Three (20%) community isolates were rifampin resistant and had a previously unreported mutation in the rpoB gene.Conclusions: In the outbreak community, Hia isolates from carriers were indistinguishable from the invasive Hia isolates. Overall, invasive Hia isolates from Alaska in 2018 were genetically similar. The rifampin resistance mutation is concerning as rifampin is the first-line medication for Hia prophylaxis. [ABSTRACT FROM AUTHOR]

Non-typeable Haemophilus influenzae (NTHi) is a major human pathogen for which there is no globally licensed vaccine. NTHi has a strict growth requirement for iron and encodes several systems to scavenge elemental iron and heme from the host. An effective NTHi vaccine would target conserved, essential surface factors, such as those involved in iron acquisition. Haemoglobin–haptoglobin binding proteins (Hgps) are iron-uptake proteins localized on the outer-membrane of NTHi. If the Hgps are to be included as components of a rationally designed subunit vaccine against NTHi, it is important to understand their prevalence and diversity. Following analysis of all available Hgp sequences, we propose a standardized grouping method for Hgps, and demonstrate increased diversity of these proteins than previously determined. This analysis demonstrated that genes encoding variants HgpB and HgpC are present in all strains examined, and almost 40% of strains had a duplicate, nonidentical hgpB gene. Hgps are also phase-variably expressed; the encoding genes contain a CCAA(n) simple DNA sequence repeat tract, resulting in biphasic ON–OFF switching of expression. Examination of the ON–OFF state of hgpB and hgpC genes in a collection of invasive NTHi isolates demonstrated that 58% of isolates had at least one of hgpB or hgpC expressed (ON). Varying expression of a diverse repertoire of hgp genes would provide strains a method of evading an immune response while maintaining the ability to acquire iron via heme. Structural analysis of Hgps also revealed high sequence variability at the sites predicted to be surface exposed, demonstrating a further mechanism to evade the immune system—through varying the surface, immune-exposed regions of the membrane anchored protein. This information will direct and inform the choice of candidates to include in a vaccine against NTHi. [ABSTRACT FROM AUTHOR]

Background Incidence of invasive disease due to Haemophilus influenzae serotype a (Hia) increased an average of 13% annually from 2002 through 2015. We describe clinical characteristics and adverse clinical outcomes of US invasive Hia cases detected through multistate surveillance during 2011–2015. Methods Medical record data were abstracted for cases reported in 8 jurisdictions conducting active population- and laboratory-based surveillance for invasive Hia disease across the United States. Isolates from sterile sites were serotyped using real-time polymerase chain reaction. Adverse clinical outcomes were defined as any possible complication of meningitis, bacteremic pneumonia, or bacteremia (including hearing loss and developmental delay, but excluding death) and were assessed at hospital discharge and one-year post-disease onset. Results During 2011–2015, 190 Hia cases were reported to the 8 participating sites; 169 (88.9%) had data abstracted. Many patients were aged <5 years (42.6%). Meningitis was the most common clinical presentation among those aged <1 year (71.4%); bacteremic pneumonia was the most common presentation among persons aged ≥50 years (78.7%). Overall, 95.9% of patients were hospitalized. Among those hospitalized, 47.5% were admitted to an intensive care unit and 6.2% died during hospitalization. At hospital discharge and one-year post-disease onset, adverse outcomes were identified in 17.7% and 17.8% of patients overall and in 43.9% and 48.5% of patients with meningitis (primarily children). Conclusions Hia infection can cause severe disease that requires hospitalization and may also cause short- and long-term adverse clinical outcomes, especially among children. Novel vaccines could prevent morbidity and mortality. [ABSTRACT FROM AUTHOR]

Background Community-acquired pneumonia (CAP) is one of the most common infectious diseases and is a significant cause of mortality and morbidity globally. A microbial cause was not determined in a sizable percentage of patients with CAP; there are increasing data to suggest regional differences in bacterial aetiology. We devised a multiplex real-time PCR assay for detecting four microorganisms (Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae and Burkholderia pseudomallei) of relevance to CAP infections in Asia. Methods Analytical validation was accomplished using bacterial isolates (n=10–33 of each target organism for analytical sensitivity and n=117 for analytical sensitivity) and clinical validation using 58 culture-positive respiratory tract specimens. Results The qPCR assay exhibited 100% analytical sensitivity and analytical specificity, and 100% clinical sensitivity and 94–100% clinical specificity. The limit of detection and efficiency for the multiplex PCR assay were 3–33 CFU/mL and 93–110%, respectively. The results showed that the PCR-based method had higher sensitivity than traditional culture-based methods. The assay also demonstrated an ability to semiquantify bacterial loads. Conclusion We have devised a reliable laboratory-developed multiplex qPCR assay, with a turnaround time of within one working day, for detection of four clinically important CAP-associated microorganisms in Asia. The availability of a test with improved diagnostic capabilities potentially leads to an informed choice of antibiotic usage and appropriate management of the patient to achieve a better treatment outcome and financial savings. [ABSTRACT FROM AUTHOR]

Background Since the introduction of Haemophilus influenzae serotype b (Hib) conjugate vaccines in the United States, invasive H. influenzae disease epidemiology has changed, and racial disparities have not been recently described. Methods Active population- and laboratory-based surveillance for H. influenzae was conducted through Active Bacterial Core surveillance at 10 US sites. Data from 2008–2017 were used to estimate projected nationwide annual incidence, as cases per 100 000. Results During 2008–2017, Active Bacterial Core surveillance identified 7379 H. influenzae cases. Of 6705 patients (90.9%) with reported race, 76.2% were White, 18.6% were Black, 2.8% were Asian/Pacific Islander, and 2.4% were American Indian or Alaska Native (AI/AN). The nationwide annual incidence was 1.8 cases/100 000. By race, incidence was highest among AI/AN populations (3.1) and lowest among Asian/Pacific Islander populations (0.8). Nontypeable H. influenzae caused the largest incidence within all races (1.3), with no striking disparities identified. Among AI/AN children aged <5 years, incidence of H. influenzae serotype a (Hia) was 16.7 times higher and Hib incidence was 22.4 times higher than among White children. Although Hia incidence was lower among White and Black populations than among AI/AN populations, Hia incidence increased 13.6% annually among White children and 40.4% annually among Black children aged <5 years. Conclusions While nontypeable H. influenzae causes the largest H. influenzae burden overall, AI/AN populations experience disproportionately high rates of Hia and Hib, with the greatest disparity among AI/AN children aged <5 years. Prevention tools are needed to reduce disparities affecting AI/AN children and address increasing Hia incidence in other communities. [ABSTRACT FROM AUTHOR]

Background: Based on guidelines from the Infectious Diseases Society of America and the American Society for Microbiology, many pediatric hospitals are implementing weight-based collection guidelines for blood cultures. To simplify the process of culture collection, there has been interest in validating the use of adult blood culture bottles with low volumes, which may allow for a "one size fits all" bottle. Method: This study examined 9 clinically relevant organisms (Staphylococcus aureus, Streptococcus pneumonia, Streptococcus agalactiae, Enterococcus faecium, Escherichia coli, Haemophilus influenzae, Pseudomonas aeruginosa, Candida glabrata, and Candida albicans) utilizing the BD BacTec system at various inoculation volumes and dilutions to assess performance, based on time to positivity, of adult blood culture bottles compared with pediatric blood culture bottles. Results: There was a lack of detection of H. influenzae using adult blood culture bottles inoculated at low volumes, whereas pediatric bottles detected H. influenzae regardless of dilution–volume combinations tested. Conclusions: Exclusive use of adult blood culture bottles may not detect H. influenzae bacteremia in the setting of low-volume inoculum. [ABSTRACT FROM AUTHOR]

Background Nongonococcal urethritis (NGU) is a common syndrome with no known etiology in ≤50% of cases. We estimated associations between urethral bacteria and NGU in men who have sex with men (MSM) and men who have sex with women (MSW). Methods Urine was collected from NGU cases (129 MSM, 121 MSW) and controls (70 MSM, 114 MSW) attending a Seattle STD clinic. Cases had ≥5 polymorphonuclear leukocytes on Gram stain plus symptoms or discharge; controls had <5 PMNs, no symptoms, no discharge. NGU was considered idiopathic when Neisseria gonorrhoeae , Chlamydia trachomatis , Mycoplasma genitalium , Trichomonas vaginalis , adenovirus, and herpes simplex virus were absent. The urethral microbiota was characterized using 16S rRNA gene sequencing. Compositional lasso analysis was conducted to identify associations between bacterial taxa and NGU and to select bacteria for targeted qPCR. Results Among NGU cases, 45.2% were idiopathic. Based on compositional lasso analysis, we selected Haemophilus influenzae (HI) and Mycoplasma penetrans (MP) for targeted qPCR. Compared with 182 men without NGU, the 249 men with NGU were more likely to have HI (14% vs 2%) and MP (21% vs 1%) (both P ≤.001). In stratified analyses, detection of HI was associated with NGU among MSM (12% vs 3%, P =.036) and MSW (17% vs 1%, P <.001), but MP was associated with NGU only among MSM (13% vs 1%, P =.004). Associations were stronger in men with idiopathic NGU. Conclusions HI and MP are potential causes of male urethritis. MP was more often detected among MSM than MSW with urethritis. [ABSTRACT FROM AUTHOR]

Haemophilus influenzae serotype b (Hib) is an important cause of serious, invasive infections, particularly in young children. Since 1985, a series of vaccines composed of the type b capsular polysaccharide polyribosylribitol phosphate (PRP), followed by PRP conjugated to various proteins, have been licensed for use in the United States and worldwide. The conjugated vaccines offer increased immunogenicity and prolonged durability of immune protection compared to the plain PRP vaccine and increasingly are combined with other childhood vaccines for decreased cost and increased ease of vaccination. Hib vaccines have a very favorable safety profile, have been found to be either cost-saving or cost-effective by many public health agencies, and, in most countries, are initiated during early infancy as part of routine childhood immunization programs. As a result of widespread use of the vaccines, the incidence of Hib infections, and their associated morbidity and mortality, has fallen dramatically across the globe. Yet, many children remain unimmunized or underimmunized against Hib, particularly in limited-resource countries. Future efforts to further reduce the disease burden of Hib infections remain a high priority. [ABSTRACT FROM AUTHOR]

Background: The World Health Organization (WHO) coordinates the Global Invasive Bacterial Vaccine-Preventable Diseases (IB-VPD) Surveillance Network to support vaccine introduction decisions and use. The network was established to strengthen surveillance and laboratory confirmation of meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis.Methods: Sentinel hospitals report cases of children <5 years of age hospitalized for suspected meningitis. Laboratories report confirmatory testing results and strain characterization tested by polymerase chain reaction. In 2019, the network included 123 laboratories that follow validated, standardized testing and reporting strategies.Results: From 2014 through 2019, >137 000 suspected meningitis cases were reported by 58 participating countries, with 44.6% (n = 61 386) reported from countries in the WHO African Region. More than half (56.6%, n = 77 873) were among children <1 year of age, and 4.0% (n = 4010) died among those with reported disease outcome. Among suspected meningitis cases, 8.6% (n = 11 798) were classified as probable bacterial meningitis. One of 3 bacterial pathogens was identified in 30.3% (n = 3576) of these cases, namely S. pneumoniae (n = 2177 [60.9%]), H. influenzae (n = 633 [17.7%]), and N. meningitidis (n = 766 [21.4%]). Among confirmed bacterial meningitis cases with outcome reported, 11.0% died; case fatality ratio varied by pathogen (S. pneumoniae, 12.2%; H. influenzae, 6.1%; N. meningitidis, 11.0%). Among the 277 children who died with confirmed bacterial meningitis, 189 (68.2%) had confirmed S. pneumoniae. The proportion of pneumococcal cases with pneumococcal conjugate vaccine (PCV) serotypes decreased as the number of countries implementing PCV increased, from 77.8% (n = 273) to 47.5% (n = 248). Of 397 H. influenzae specimens serotyped, 49.1% (n = 195) were type b. Predominant N. meningitidis serogroups varied by region.Conclusions: This multitier, global surveillance network has supported countries in detecting and serotyping the 3 principal invasive bacterial pathogens that cause pediatric meningitis. Streptococcus pneumoniae was the most common bacterial pathogen detected globally despite the growing number of countries that have nationally introduced PCV. The large proportions of deaths due to S. pneumoniae reflect the high proportion of meningitis cases caused by this pathogen. This global network demonstrated a strong correlation between PCV introduction status and reduction in the proportion of pneumococcal meningitis infections caused by vaccine serotypes. Maintaining case-based, active surveillance with laboratory confirmation for prioritized vaccine-preventable diseases remains a critical component of the global agenda in public health.The World Health Organization (WHO)-coordinated Invasive Bacterial Vaccine-Preventable Disease (IB-VPD) Surveillance Network reported data from 2014 to 2019, contributing to the estimates of the disease burden and serotypes of pediatric meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis. [ABSTRACT FROM AUTHOR]

Background: Despite the availability of vaccines, invasive bacterial diseases remain a public health concern and cause childhood morbidity and mortality. We investigated the characteristics of etiological agents causing bacterial meningitis in children <5 years in the years pre- (2010-2012) and post- (2014-2019) 10-valent pneumococcal conjugate vaccine (PCV10) introduction in Zambia.Methods: Streptococcus pneumoniae (Spn), Haemophilus influenzae (Hi), and Neisseria meningitidis (Nm) from cerebrospinal fluid (CSF) were identified by microbiological culture and/or real-time polymerase chain reaction.Results: During the surveillance period, a total of 3811 children were admitted with suspected meningitis, 16% (598 of 3811) of which were probable cases. Bacterial meningitis was confirmed in 37% (221 of 598) of the probable cases. Spn pneumoniae, Hi, and Nm accounted for 67% (148 of 221), 14% (31 of 221), and 19% (42 of 221) of confirmed cases, respectively. Thirty-six percent of pneumococcal meningitis was caused by 10-valent pneumococcal conjugate vaccine (PCV10) serotypes, 16% 13-valent pneumococcal conjugate vaccine and 39% by nonvaccine serotype (NVS). There was an association between the introduction of PCV10 vaccination and a decrease in both Spn meningitis and the proportion of PVC10 serotypes in the postvaccination period. Antimicrobial susceptibility of 47 Spn isolates revealed 34% (16 of 47) penicillin resistance. The 31 serotyped Hi accounted for 74% type b (Hib) and 10% type a (Hia). All 42 serogrouped Nm belonged to serogroup W.Conclusions: There was a decline in pneumococcal meningitis and proportion of PCV10 serotypes in the postvaccination period. However, the serotype replacement with non-PCV10 serotypes and penicillin resistance warrant continued surveillance to inform policy. [ABSTRACT FROM AUTHOR]

Background: As part of the global Invasive Bacterial Vaccine-Preventable Diseases Surveillance Network, 12 African countries referred cerebrospinal fluid (CSF) samples to South Africa's regional reference laboratory. We evaluated the utility of real-time polymerase chain reaction (PCR) in detecting and serotyping/grouping Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumoniae (HNS).Methods: From 2008 to 2017, CSF samples collected from children <5 years old with suspected meningitis underwent routine microbiology testing in-country, and 11 680 samples were submitted for HNS PCR at the regional reference laboratory. Unconditional logistic regression, with adjustment for geographic location, was performed to identify factors associated with PCR positivity.Results: The overall HNS PCR positivity rate for all countries was 10% (1195 of 11 626 samples). In samples with both PCR and culture results, HNS PCR positivity was 11% (744 of 6747 samples), and HNS culture positivity was 3% (207 of 6747). Molecular serotype/serogroup was assigned in 75% of PCR-positive specimens (762 of 1016). Compared with PCR-negative CSF samples, PCR-positive samples were more often turbid (adjusted odds ratio, 6.80; 95% confidence interval, 5.67-8.17) and xanthochromic (1.72; 1.29-2.28), had elevated white blood cell counts (6.13; 4.71-7.99) and high protein concentrations (5.80; 4.34-7.75), and were more often HNS culture positive (32.70; 23.18-46.12).Conclusion: PCR increased detection of vaccine-preventable bacterial meningitis in countries where confirmation of suspected meningitis cases is impeded by limited culture capacity. [ABSTRACT FROM AUTHOR]

Background: Streptococcus pneumoniae, or pneumococcus, is a leading cause of morbidity and mortality in children worldwide. Pneumococcal conjugate vaccines (PCV) reduce carriage in the nasopharynx, preventing disease. We conducted a pneumococcal carriage study to estimate the prevalence of pneumococcal colonization, identify risk factors for colonization, and describe antimicrobial susceptibility patterns among pneumococci colonizing young children in Port-au-Prince, Haiti, before introduction of 13-valent PCV (PCV13).Methods: We conducted a cross-sectional study of children aged 6-24 months at an immunization clinic in Port-au-Prince between September 2015 and January 2016. Consenting parents were interviewed about factors associated with pneumococcal carriage; nasopharyngeal swabs were collected from each child and cultured for pneumococcus after broth enrichment. Pneumococcal isolates were serotyped and underwent antimicrobial susceptibility testing. We compared frequency of demographic, clinical, and environmental factors among pneumococcus-colonized children (carriers) to those who were not colonized (noncarriers) using unadjusted bivariate analysis and multivariate logistic regression.Results: Pneumococcus was isolated from 308 of the 685 (45.0%) children enrolled. Overall, 157 isolates (50.8%) were PCV13 vaccine-type serotypes; most common were 6A (13.3%), 19F (12.6%), 6B (9.7%), and 23F (6.1%). Vaccine-type isolates were significantly more likely to be nonsusceptible to ≥1 antimicrobial (63.1% vs 45.4%, P = .002). On bivariate analysis, carriers were significantly more likely than noncarriers to live in a household without electricity or running water, to share a bedroom with ≥3 people, to have a mother or father who did not complete secondary education, and to have respiratory symptoms in the 24 hours before enrollment (P < .05 for all comparisons). On multivariable analysis, completion of the pentavalent vaccination series (targeting diphtheria, pertussis, tetanus, hepatitis B, and Haemophilus influenzae type b) remained significantly more common among noncarriers.Conclusions: Nearly a quarter of healthy children surveyed in Haiti were colonized with vaccine-type pneumococcal serotypes. This baseline carriage study will enable estimation of vaccine impact following nationwide introduction of PCV13. [ABSTRACT FROM AUTHOR]

Background: Reduction in detection of asymptomatic carriage of Haemophilus influenzae type b (Hib) can be used to assess vaccine impact. In Nepal, routine vaccination against Hib in children at 6, 10, and 14 weeks of age was introduced in 2009. Before vaccine introduction, Hib carriage was estimated at 5.0% among children aged <13 years in Nepal, with higher rates among children under 5. Large-scale evaluation of Hib carriage in children has not been investigated since the introduction of the pentavalent diphtheria-tetanus-pertussis/Hib/hepatitis B (DTP-Hib-HepB) vaccine in Nepal.Methods: A total of 666 oropharyngeal swabs were collected between August and December 2018 from healthy children between 6 months and 5 years of age attending the vaccination clinic at Patan Hospital, Kathmandu, Nepal. Of these 666 swabs, 528 (79.3%) were tested for Hib by culture. Demographic and vaccination data were collected.Results: Among 528 swabs tested for Hib, 100% came from fully vaccinated children. No swabs were positive for Hib (95% confidence interval, .0-.7). The absence of Hib in 2018 suggests vaccine-induced protection against Hib carriage 9 years after vaccine introduction.Conclusions: Following 3 doses of pentavalent DTP-Hib-HepB vaccine, Hib carriage in children under the age of 5 years in Nepal is no longer common. Ongoing high coverage with Hib vaccine in early childhood is expected to maintain protection against Hib disease in Nepal. [ABSTRACT FROM AUTHOR]

Background Haemophilus influenzae serotype a (Hia) can cause invasive disease similar to serotype b; no Hia vaccine is available. We describe the epidemiology of invasive Hia disease in the United States overall and specifically in Alaska during 2008–2017. Methods Active population- and laboratory-based surveillance for invasive Hia disease was conducted through Active Bacterial Core surveillance sites and from Alaska statewide invasive bacterial disease surveillance. Sterile-site isolates were serotyped via slide agglutination or real-time polymerase chain reaction. Incidences in cases per 100 000 were calculated. Results From 2008 to 2017, an estimated average of 306 invasive Hia disease cases occurred annually in the United States (estimated annual incidence: 0.10); incidence increased by an average of 11.1% annually. Overall, 42.7% of cases were in children aged <5 years (incidence: 0.64), with highest incidence among children aged <1 year (1.60). Case fatality was 7.8% overall and was highest among adults aged ≥65 years (15.1%). Among children aged <5 years, the incidence was 17 times higher among American Indian and Alaska Native (AI/AN) children (8.29) than among children of all other races combined (0.49). In Alaska, incidences among all ages (0.68) and among children aged <1 year (24.73) were nearly 6 and 14 times higher, respectively, than corresponding US incidences. Case fatality in Alaska was 10.2%, and the vast majority (93.9%) of cases occurred among AI/AN. Conclusions Incidence of invasive Hia disease has increased since 2008, with the highest burden among AI/AN children. These data can inform prevention strategies, including Hia vaccine development. [ABSTRACT FROM AUTHOR]

Background Between May and July 2018, 4 Haemophilus influenzae serotype a (Hia) infections occurred in a remote Alaska community. We performed a public health response to prevent further illness and understand Hia carriage. Methods We collected oropharyngeal samples community-wide to evaluate baseline carriage. Risk factors were evaluated by interview. We offered prophylactic rifampin to individuals in contact with invasive Hia patients (contacts) and to all children aged <10 years. Oropharyngeal samples were collected again 8 weeks after rifampin distribution. Samples were tested using real-time polymerase chain reaction and culture. Results At baseline, 4 of 27 (14.8%) contacts and 7 of 364 (1.9%) noncontacts (P <.01) carried Hia. Contacts aged <10 years were more likely to carry Hia at any timepoint (11/18 [61%]) compared to contacts aged ≥10 years (3/34 [8.8%]), noncontacts aged <10 years (2/139 [1.4%]), and noncontacts ≥10 years (6/276 [2.2%]) (P <.001 for all). Hia carriers were clustered in 9 households (7% of total households). At the household level, carriage was associated with households with ≥1 contact (prevalence ratio [PR], 5.6 [95% confidence interval {CI}, 1.3–21.6]), crowding (PR, 7.7 [95% CI, 1.1–199.5]), and ≥3 tobacco users (PR, 5.0 [95% CI, 1.2–19.6]). Elevated carriage prevalence persisted in contacts compared to noncontacts 8 weeks after rifampin distribution (6/25 [24%] contacts, 2/114 [1.8%] noncontacts; P <.001). Conclusions Hia carriage prevalence was significantly higher among contacts than noncontacts. Rifampin prophylaxis did not result in a reduction of Hia carriage prevalence in this community. [ABSTRACT FROM AUTHOR]

Objectives: To characterize the mechanisms of antimicrobial resistance and the prevalence of the polysaccharide capsule among urogenital and respiratory Haemophilus parainfluenzae isolates.Methods: Antimicrobial susceptibility was tested by microdilution. Fifty-five MDR strains were subjected to WGS and were phylogenetically compared with all the available H. parainfluenzae genomes from the NCBI database. The identification of the capsular bexA gene was performed by PCR in 266 non-MDR strains.Results: In 31 of the 42 ampicillin-resistant strains, blaTEM-1 located within Tn3 was identified. β-Lactamase-negative cefuroxime-resistant strains (n = 12) presented PBP3 substitutions. The catS gene (n = 14), the tet(M)-MEGA element (n = 18) and FolA substitutions (I95L and F154V/S) (n = 41) were associated with resistance to chloramphenicol, tetracycline plus macrolides, and co-trimoxazole, respectively. Thirty-seven isolates had a Tn10 harbouring tet(B)/(C)/(D)/(R) genes with (n = 15) or without (n = 22) catA2. Putative transposons (Tn7076-Tn7079), including aminoglycoside and co-trimoxazole resistance genes, were identified in 10 strains (18.2%). These transposons were integrated into three new integrative and conjugative elements (ICEs), which also included the resistance-associated transposons Tn3 and Tn10. The capsular operon was found only in the urogenital isolates (18/154, 11.7%), but no phylogenetic clustering was observed. The capsular operons identified were similar to those of Haemophilus influenzae serotype c and Haemophilus sputorum type 2.Conclusions: The identification of ICEs with up to three resistance-associated transposons suggests that these transferable elements play an important role in the acquisition of multidrug resistance in H. parainfluenzae. Moreover, the presence of polysaccharide capsules in some of these urogenital isolates is a cause for concern. [ABSTRACT FROM AUTHOR]

Background Long regarded as the second most common cause of community-acquired pneumonia (CAP), Haemophilus influenzae has recently been identified with almost equal frequency as pneumococcus in patients hospitalized for CAP. The literature lacks a detailed description of the presentation, clinical features, laboratory and radiologic findings, and outcomes in Haemophilus pneumonia. Methods During 2 prospective studies of patients hospitalized for CAP, we identified 33 patients with Haemophilus pneumonia. In order to provide context, we compared clinical findings in these patients with findings in 36 patients with pneumococcal pneumonia identified during the same period. We included and analyzed separately data from patients with viral coinfection. Patients with coinfection by other bacteria were excluded. Results Haemophilus pneumonia occurred in older adults who had underlying chronic lung disease, cardiac conditions, and alcohol use disorder, the same population at risk for pneumococcal pneumonia. However, in contrast to pneumococcal pneumonia, patients with Haemophilus pneumonia had less severe infection as shown by absence of septic shock on admission, less confusion, fewer cases of leukopenia or extreme leukocytosis, and no deaths at 30 days. Viral coinfection greatly increased the severity of Haemophilus , but not pneumococcal pneumonia. Conclusions We present the first thorough description of Haemophilus pneumonia, show that it is less severe than pneumococcal pneumonia, and document that viral coinfection greatly increases its severity. These distinctions are lost when the label CAP is liberally applied to all patients who come to the hospital from the community for pneumonia. [ABSTRACT FROM AUTHOR]

Background: Haemophilus influenzae can cause invasive infections, in which cefotaxime is among the first-line antibiotics for treatment. The prevalence of cefotaxime-resistant H. influenzae in Europe is reported to be on a low level. Nevertheless, systematic studies with a large set of invasive isolates are scarce.Objectives: To provide prevalence data for cefotaxime resistance in invasive H. influenzae isolates in Germany 2016-19 and investigate the epidemiological relevance of PBP3 mutations known to elevate the cefotaxime MIC.Methods: Cefotaxime susceptibility of invasive H. influenzae isolates, collected in the national laboratory surveillance programme, was examined by gradient agar diffusion (GAD) testing. Cefotaxime resistance was determined according to EUCAST guidelines (resistance breakpoint MIC >0.125 mg/L). Therefore, the MIC for all resistant isolates was verified by broth microdilution method (BMD). WGS was performed to investigate the genetic relationship of cefotaxime-resistant isolates and to analyse alterations in the PBP3. An analysis of the geographic distribution of the resistant isolates was performed.Results: From 2016 to 2019, the German National Reference Laboratory for Meningococci and H. influenzae received 2432 invasive H. influenzae isolates from blood and CSF. According to GAD results, 27 strains were resistant to cefotaxime. BMD confirmed the resistance in 22 of these isolates, which equals a prevalence of cefotaxime resistance of 0.90% in invasive H. influenzae in Germany. Among cefotaxime-resistant isolates cgMLST revealed three clusters. PBP3 analysis showed previously described mutations in our strains. In comparison with cefotaxime-susceptible strains, the alterations L389F and Y557H were significantly associated with cefotaxime resistance, but were not present in all resistant strains. Geographic analysis showed that the disease cases with cefotaxime-resistant H. influenzae were evenly spread throughout the population in Germany.Conclusions: Cefotaxime is still well suited for the treatment of invasive H. influenzae infections. Rarely occurring cefotaxime resistance is caused by sporadic mutations. The role of PBP3 mutations needs further investigation. [ABSTRACT FROM AUTHOR]

Background: Haemophilus influenzae bacteria can cause asymptomatic carriage and invasive disease. Haemophilus influenzae serotype a (Hia) is an emerging cause of invasive disease in Alaska, with greatest burden occurring among rural Alaska Native (AN) children. The first case of invasive Hia (iHia) in Alaska was reported in 2002; however, it is unclear how long the pathogen has been in Alaska.Methods: We quantified immunoglobulin G antibodies against Hia (anti-Hia) in 839 banked serum samples from Alaska residents, comparing antibody concentrations in samples drawn in the decades before (1980s and 1990s) and after (2000s) the emergence of iHia. We also assessed serum antibody concentration by age group, region of residence, and race.Results: The anti-Hia was >0.1 µg/mL in 88.1% (348 of 395) and 91.0% (404 of 444) of samples from the decades prior and after the emergence of Hia, respectively (P = .17). No significant differences in antibody levels were detected between people from rural and urban regions (1.55 vs 2.08 µg/mL, P = .91 for age ≥5) or between AN and non-AN people (2.50 vs 2.60 µg/mL, P = .26).Conclusions: Our results are consistent with widespread Hia exposure in Alaska predating the first iHia case. No difference in Hia antibody prevalence was detected between populations with differing levels of invasive disease. [ABSTRACT FROM AUTHOR]

Background: Otitis media (OM) is a common and potentially serious disease of childhood. Although OM is multifactorial on origin, bacterial infection is a unifying component. Many studies have established a critical role for innate immunity in bacterial clearance and OM resolution. A key component of innate immunity is the recruitment of immune and inflammatory cells, including macrophages.Methods: To explore the role of macrophages in OM, we evaluated the expression of genes related to macrophage function during a complete episode of acute OM in the mouse caused by middle ear (ME) inoculation with Haemophilus influenzae. We also combined CCR2 deficiency with chlodronate liposome toxicity to deplete macrophages during OM.Results: Macrophage genes were robustly regulated during OM. Moreover, macrophage depletion enhanced and prolonged the infiltration of neutrophils into the infected ME and increased the persistence of bacterial infection.Conclusions: The results illustrate the critical role played by macrophages in OM resolution. [ABSTRACT FROM AUTHOR]

Homologous recombination is a key pathway found in nearly all bacterial taxa. The recombination complex not only allows bacteria to repair DNA double-strand breaks but also promotes adaption through the exchange of DNA between cells. In Proteobacteria, this process is mediated by the RecBCD complex, which relies on the recognition of a DNA motif named Chi to initiate recombination. The Chi motif has been characterized in Escherichia coli and analogous sequences have been found in several other species from diverse families, suggesting that this mode of action is widespread across bacteria. However, the sequences of Chi-like motifs are known for only five bacterial species: E. coli, Haemophilus influenzae, Bacillus subtilis, Lactococcus lactis, and Staphylococcus aureus. In this study, we detected putative Chi motifs in a large dataset of Proteobacteria and identified four additional motifs sharing high sequence similarity and similar properties to the Chi motif of E. coli in 85 species of Proteobacteria. Most Chi motifs were detected in Enterobacteriaceae and this motif appears well conserved in this family. However, we did not detect Chi motifs for the majority of Proteobacteria, suggesting that different motifs are used in these species. Altogether these results substantially expand our knowledge on the evolution of Chi motifs and on the recombination process in bacteria. [ABSTRACT FROM AUTHOR]

Background Following widespread use of the Haemophilus influenzae serotype b (Hib) vaccine, H. influenzae serotype a (Hia) has emerged as an important pathogen in children in some regions. We describe the clinical features and molecular epidemiology of invasive Hia disease in children in Utah over an 11-year period. Methods We identified cases of invasive Hia disease, defined as detection of Hia from a normally sterile site, in children aged <18 years from Utah between 2007 and 2017. Medical records were reviewed to determine demographic characteristics and clinical outcomes. Available Hia isolates were genotyped using multilocus sequence typing, and phylogenetic division was determined using sodC polymerase chain reaction. Presence of the putative virulence-associated IS 1016-bexA duplication-deletion was evaluated. Results We identified 51 children with invasive Hia. The average annual incidence was 1.7 cases per 100 000 children aged <5 years; 4.8 cases per 100 000 children aged <1 year. The median age was 11.3 months. The most common clinical presentation was meningitis (53%), followed by pneumonia (14%) and septic arthritis (14%). Twenty-two children (43%) required admission to an intensive care unit; 1 died. Sequence type (ST) 62, phylogenetic division II isolates caused 75% (21/28) of disease. No isolates contained the virulence-associated IS 1016 - bexA duplication-deletion. Conclusions Hia is a significant cause of severe invasive bacterial infection in Utah. The majority of infections were caused by ST62 isolates, a phylogenetic division II Hia type that lacks the IS 1016 - bexA duplication-deletion. Hia ST62 has not been commonly reported elsewhere, suggesting a unique molecular epidemiology in our population. [ABSTRACT FROM AUTHOR]

Objectives: Implementation of EUCAST susceptibility testing in an Australian hospital laboratory demonstrated higher rates of aminopenicillin and amoxicillin/clavulanate resistance in Haemophilus influenzae than previously recognized. This study aimed to better define the variability in the detection of β-lactam resistance based on EUCAST and CLSI disc diffusion (DD) methodology, by comparison with the recommended reference method, broth microdilution (BMD), and by concordance with genomic analysis.Methods: A total of 100 random H. influenzae isolates were assessed for ampicillin and amoxicillin/clavulanate susceptibility by EUCAST and CLSI DD and BMD. WGS was used to analyse the ftsI gene of a subset of isolates with β-lactam resistance, other than that due to isolated β-lactamase production.Results: Of the 100 isolates, 32 were categorized as either β-lactamase negative, ampicillin resistant (BLNAR) (n = 18) or β-lactamase positive, amoxicillin/clavulanate resistant (BLPACR) (n = 14) by EUCAST DD. All 18 EUCAST BLNAR isolates were genotypically confirmed by WGS. Five of 18 BLNAR isolates were concordant by CLSI DD, 12 by EUCAST BMD and 4 by CLSI BMD. Nine of 14 EUCAST BLPACR isolates were confirmed by WGS; the remaining 5 were 1 mm below the EUCAST DD breakpoint. Only one isolate was detected as BLPACR by CLSI DD. Group III mutations associated with high-level ampicillin resistance were identified in 10/32 isolates.Conclusions: The EUCAST DD susceptibility method is more reliable than either CLSI or BMD for the detection of genotypically defined BLNAR resistance. However, accurate categorization of amoxicillin/clavulanate resistance remains problematic. Continuous and reproducible surveillance of resistance is needed; for this to be possible, robust susceptibility methods are required. [ABSTRACT FROM AUTHOR]

Background: The carbapenems imipenem and meropenem play an important role in the empirical anti-infective treatment of critically ill patients. Carbapenem resistance in Haemophilus influenzae (Hi) has rarely been reported.Objectives: We provide prevalence data for resistance to carbapenems from laboratory surveillance of invasive Hi infections in Germany in 2016.Methods: Phenotypic susceptibility testing against ampicillin, amoxicillin/clavulanate, cefotaxime and imipenem was carried out on 474 isolates from blood and CSF. The isolates were collected as part of the national laboratory surveillance programme. Imipenem-resistant strains were further tested for meropenem susceptibility. Molecular analysis was done by ftsI sequencing to detect mutations in PBP3, by acrR sequencing to detect alterations in the regulatory protein of the AcrAB-TolC efflux pump and by MLST.Results: No resistance to meropenem was detected. Cefotaxime resistance was rare (n = 3; 0.6%). Imipenem resistance was found in 64 strains (13.5%) using gradient agar diffusion and was confirmed in 26 isolates by broth microdilution (5.5%). Imipenem resistance occurred predominantly in Hi that were β-lactamase negative but ampicillin resistant and in those that were β-lactamase positive but nevertheless amoxicillin/clavulanate resistant. This finding suggested a β-lactamase-independent mechanism. Accordingly, sequence analysis of PBP3 identified previously described mutations. MLST of the imipenem-resistant strains, which were all non-typeable Hi, revealed a high diversity.Conclusions: We conclude that imipenem, but not meropenem, resistance is frequent in Hi. It is likely to be supported by PBP3 mutations. [ABSTRACT FROM AUTHOR]

Aims: To study the association between number and positions of mutations with MICs of fluoroquinolone non‐susceptible Haemophilus influenzae. Methods and Results: More than 40% of 48 H. influenzae isolated from nursing home residents were not susceptible to fluoroquinolone. Amino acid changes in the quinolone resistance determining regions, and correlation with MICs and inhibition zone diameters were analysed. All isolates with reduced susceptibility to fluoroquinolones (MIC ≥0·125 µg ml−1) had at least one mutation in gyrA at position 84 and were resistant to nalidixic acid. Compared to isolates with reduced susceptibility, resistant isolates were associated with mutations in gyrA at positions 88 and 134, and in parC at position 88 (P < 0·001). Inhibition zone diameter for nalidixic acid disk ≥23 mm may detect susceptible isolates. Conclusions: Reduced susceptibility to fluoroquinolones was associated with mutations at position 84 in gyrA. A further increase in fluoroquinolone MIC was associated with mutations in gyrA at positions 88 and 134, and parC at position 88. Significance and Impact of the Study: Due to limited resistant H. influenzae strains, prior studies on association between positions of mutations and fluoroquinolone MICs were inconclusive. The comparison of mutations between isolates with susceptibility, reduced susceptibility and high resistance supported the importance of the present study. [ABSTRACT FROM AUTHOR]

Background There are limited data on the etiology of respiratory infections in human immunodeficiency virus (HIV)–infected patients in resource-limited settings. Methods We performed quantitative multiplex real-time polymerase chain reaction (PCR) for Pneumocystis jirovecii and common bacterial and viral respiratory pathogens on sputum samples (spontaneous or induced) from a prospective cohort study of HIV-infected inpatients with World Health Organization danger signs and cough. Mycobacterial culture was done on 2 sputum samples, blood cultures, and relevant extrapulmonary samples. Results We enrolled 284 participants from 2 secondary-level hospitals in Cape Town, South Africa: median CD4 count was 97 cells/μL, 64% were women, and 38% were on antiretroviral therapy. One hundred forty-eight had culture-positive tuberculosis, 100 had community-acquired pneumonia (CAP), 26 had P. jirovecii pneumonia (PJP), and 64 had other diagnoses. Probable bacterial infection (>105 copies/mL) was detected in 133 participants; the prevalence was highest in those with CAP (52%). Haemophilus influenzae and Streptococcus pneumoniae were the commonest bacterial pathogens detected; atypical bacteria were uncommon. Viruses were detected in 203 participants; the prevalence was highest in those with PJP (85%). Human metapneumovirus was the commonest virus detected. Multiple coinfections were commonly detected. Conclusions Sputum multiplex PCR could become a useful diagnostic tool for bacterial respiratory infections in HIV-infected inpatients, but its value is limited as quantitative cutoffs have only been established for a few bacterial pathogens and validation has not been done in this patient population. We found a high prevalence of respiratory viruses, but it is unclear whether these viruses were causing infection as there are no accepted quantitative PCR cutoffs for diagnosing respiratory viral infections. [ABSTRACT FROM AUTHOR]

Objectives: To determine antibiotic susceptibility of Streptococcus pneumoniae and Haemophilus influenzae isolates from community-acquired respiratory tract infections (CA-RTIs) collected in 2015–17 from Kuwait, Lebanon and Saudi Arabia. Methods: MICs were determined by CLSI broth microdilution and susceptibility was assessed using CLSI, EUCAST (dose-specific) and pharmacokinetic/pharmacodynamic (PK/PD) breakpoints. Results: A total of 139 S. pneumoniae isolates were collected from four centres in Kuwait, Lebanon and Saudi Arabia in 2015–17 and 55 H. influenzae isolates were collected and analysed from Saudi Arabia over the same time period. Pneumococci from all three countries were commonly non-susceptible to penicillin based on CLSI oral or low-dose IV penicillin using EUCAST breakpoints (39% in Kuwait to 57.1% in Lebanon) but by CLSI IV and EUCAST high-dose breakpoints most isolates were susceptible (∼90% in Kuwait and Saudi Arabia, and 100% in Lebanon). Isolates from Lebanon were highly susceptible to most other antibiotics (>90%) except cefaclor, oral cefuroxime and cefpodoxime (EUCAST breakpoints only). Overall, susceptibility was significantly lower in Kuwait and Saudi Arabia than Lebanon. Although all H. influenzae isolates (Saudi Arabia only) were β-lactamase negative, 3.6% and 12.7% were ampicillin resistant by CLSI and EUCAST breakpoints, respectively. Otherwise susceptibility was high in H. influenzae. The application of different EUCAST breakpoints for low and higher doses for some of the antibiotics (amoxicillin, amoxicillin/clavulanic acid, ampicillin, penicillin, ceftriaxone, clarithromycin, erythromycin, levofloxacin and trimethoprim/sulfamethoxazole) allowed, for the first time in a SOAR study, the effect of raising the dosage on susceptibility to be quantified. Conclusions: Relatively low antibiotic susceptibility was observed in S. pneumoniae from Kuwait and Saudi Arabia in contrast to Lebanon, where rates of susceptibility were generally higher. Isolates of H. influenzae from Saudi Arabia were susceptible to most antibiotics. These factors are important in decision making for empirical therapy of CA-RTIs. [ABSTRACT FROM AUTHOR]

An editor is presented on the Invasive Haemophilus influenzae Type a Disease. The article discusses that use of conjugate Hib vaccines starting in the 1990s resulted in a dramatic decline in Hib morbidity and mortality; and the relative importance of infections caused by nontypeable H. influenzae (NTHi) and non– type b encapsulated strains has increased.

An editorial is presented on the Invasive Haemophilus influenzae Serotype a Disease in the H. influenzae Serotype b Conjugate Vaccine Era. The article discusses that Encapsulated strains of the gram-negative pathogen Haemophilus influenzae are categorized into 6 serotypes on the basis of antigenically distinct polysaccharide capsules; and it is also important to consider that Indigenous populations experience environmental factors.

Background: Previous simulation studies of the case-control study design using incidence density sampling, which required individual matching for time, showed biased estimates of association from conditional logistic regression (CLR) analysis; however, the reason for this is unknown. Separately, in the analysis of case-control studies using the exclusive sampling design, it has been shown that unconditional logistic regression (ULR) with adjustment for an individually matched binary factor can give unbiased estimates. The validity of this analytic approach in incidence density sampling needs evaluation.Methods: In extensive simulations using incidence density sampling, we evaluated various analytic methods: CLR with and without a bias-reduction method, ULR with adjustment for time in quintiles (and residual time within quintiles) and ULR with adjustment for matched sets and bias reduction. We re-analysed a case-control study of Haemophilus influenzae type B vaccine using these methods.Results: We found that the bias in the CLR analysis from previous studies was due to sparse data bias. It can be controlled by the bias-reduction method for CLR or by increasing the number of cases and/or controls. ULR with adjustment for time in quintiles usually gave results highly comparable to CLR, despite breaking the matches. Further adjustment for residual time trends was needed in the case of time-varying effects. ULR with adjustment for matched sets tended to perform poorly despite bias reduction.Conclusions: Studies using incidence density sampling may be analysed by either ULR with adjustment for time or CLR, possibly with bias reduction. [ABSTRACT FROM AUTHOR]

Background Meningococcal serogroup A conjugate vaccine (MACV) was introduced in Chad during 2011–2012. Meningitis surveillance has been conducted nationwide since 2003, with case-based surveillance (CBS) in select districts from 2012. In 2016, the MenAfriNet consortium supported Chad to implement CBS in 4 additional districts and real-time polymerase chain reaction (rt-PCR) at the national reference laboratory (NRL) to improve pathogen detection. We describe analysis of bacterial meningitis cases during 3 periods: pre-MACV (2010–2012), pre-MenAfriNet (2013–2015), and post-MenAfriNet (2016–2018). Methods National surveillance targeted meningitis cases caused by Neisseria meningitidis , Haemophilus influenzae , and Streptococcus pneumoniae. Cerebrospinal fluid specimens, inoculated trans-isolate media, and/or isolates from suspected meningitis cases were tested via culture, latex, and/or rt-PCR; confirmed bacterial meningitis was defined by a positive result on any test. We calculated proportion of suspected cases with a specimen received by period, and proportion of specimens with a bacterial meningitis pathogen identified, by period, pathogen, and test. Results The NRL received specimens for 6.8% (876/12813), 46.4% (316/681), and 79.1% (787/995) of suspected meningitis cases in 2010–2012, 2013–2015, and 2016–2018, respectively, with a bacterial meningitis pathogen detected in 33.6% (294/876), 27.8% (88/316), and 33.2% (261/787) of tested specimens. The number of N. meningitidis serogroup A (NmA) among confirmed bacterial meningitis cases decreased from 254 (86.4%) during 2010–2012 to 2 (2.3%) during 2013–2015, with zero NmA cases detected after 2014. In contrast, proportional and absolute increases were seen between 2010–2012, 2013–2015, and 2016–2018 in cases caused by S. pneumoniae (5.1% [15/294], 65.9% [58/88], and 52.1% [136/261]), NmX (0.7% [2/294], 1.1% [1/88], and 22.2% [58/261]), and Hib (0.3% [1/294], 11.4% [10/88], and 14.9% [39/261]). Of specimens received at the NRL, proportions tested during the 3 periods were 47.7% (418), 53.2% (168), and 9.0% (71) by latex; 81.4% (713), 98.4% (311), and 93.9% (739) by culture; and 0.0% (0), 0.0% (0), and 90.5% (712) by rt-PCR, respectively. During the post-MenAfriNet period (2016–2018), 86.1% (678) of confirmed cases were tested by both culture and rt-PCR, with 12.5% (85) and 32.4% (220) positive by culture and rt-PCR, respectively. Conclusions CBS implementation was associated with increased specimen referral. Increased detection of non-NmA cases could reflect changes in incidence or increased sensitivity of case detection with rt-PCR. Continued surveillance with the use of rt-PCR to monitor changing epidemiology could inform the development of effective vaccination strategies. [ABSTRACT FROM AUTHOR]