Hib Vaccines: Their Impact on Haemophilus influenzae Type b Disease.

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.

Remembering and Enhancing the Impact of Women in Infectious Diseases.

Following the establishment of the Infectious Diseases Society of America (IDSA), women played a minor role as IDSA leaders, awards recipients, and presenters at the national meeting. Since the formation of the IDSA Women's Committee in 1992, women have played an increasing role in all of these domains of the Society. Two subsequent IDSA task forces have emphasized the importance of women, and other unrepresented minorities, to the success of the core missions of the Society. Ongoing efforts to maintain the presence of women and their unique talents, experiences, and understandings in the Society will sustain the strengths of IDSA.

Into Darkness and Silence: What Caused Helen Keller's Deafblindness?

In 1882, at 19 months of age, Helen Keller developed a febrile illness that left her both deaf and blind. Historical biographies attribute the illness to rubella, scarlet fever, encephalitis, or meningitis. This analysis of her illness suggests she likely had bacterial meningitis, caused by Neisseria meningitidis or possibly Haemophilus influenzae.

Capsule Typing of Haemophilus influenzae by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

Encapsulated Haemophilus influenzae strains belong to type-specific genetic lineages. Reliable capsule typing requires PCR, but a more efficient method would be useful. We evaluated capsule typing by using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Isolates of all capsule types (a-f and nontypeable; n = 258) and isogenic capsule transformants (types a-d) were investigated. Principal component and biomarker analyses of mass spectra showed clustering, and mass peaks correlated with capsule type-specific genetic lineages. We used 31 selected isolates to construct a capsule typing database. Validation with the remaining isolates (n = 227) showed 100% sensitivity and 92.2% specificity for encapsulated strains (a-f; n = 61). Blinded validation of a supplemented database (n = 50) using clinical isolates (n = 126) showed 100% sensitivity and 100% specificity for encapsulated strains (b, e, and f; n = 28). MALDI-TOF mass spectrometry is an accurate method for capsule typing of H. influenzae.

Life Is Not Fair.

A pediatrian's elegy to a dear friend recounts in this narrative medicine essay how the obstacles from cancer did not impede her from living fully, allowing the friends to share life's pleasures for many years.

Haemophilus influenzae Type b Invasive Disease in Amish Children, Missouri, USA, 2014.

During 5 months in 2014, three Amish children in Missouri, USA, were diagnosed with invasive Haemophilus influenzae type b infection. Two were rural neighbors infected with a genetically similar rare strain, sequence type 45. One child had recently traveled, raising the possibility of maintenance of this strain among unvaccinated carriers in Amish communities.

Comparative Genomic Analysis of Haemophilus haemolyticus and Nontypeable Haemophilus influenzae and a New Testing Scheme for Their Discrimination.

Haemophilus haemolyticus has been recently discovered to have the potential to cause invasive disease. It is closely related to nontypeable Haemophilus influenzae (NT H. influenzae). NT H. influenzae and H. haemolyticus are often misidentified because none of the existing tests targeting the known phenotypes of H. haemolyticus are able to specifically identify H. haemolyticus Through comparative genomic analysis of H. haemolyticus and NT H. influenzae, we identified genes unique to H. haemolyticus that can be used as targets for the identification of H. haemolyticus A real-time PCR targeting purT (encoding phosphoribosylglycinamide formyltransferase 2 in the purine synthesis pathway) was developed and evaluated. The lower limit of detection was 40 genomes/PCR; the sensitivity and specificity in detecting H. haemolyticus were 98.9% and 97%, respectively. To improve the discrimination of H. haemolyticus and NT H. influenzae, a testing scheme combining two targets (H. haemolyticus purT and H. influenzae hpd, encoding protein D lipoprotein) was also evaluated and showed 96.7% sensitivity and 98.2% specificity for the identification of H. haemolyticus and 92.8% sensitivity and 100% specificity for the identification of H. influenzae, respectively. The dual-target testing scheme can be used for the diagnosis and surveillance of infection and disease caused by H. haemolyticus and NT H. influenzae.

Comparative Profile of Heme Acquisition Genes in Disease-Causing and Colonizing Nontypeable Haemophilus influenzae and Haemophilus haemolyticus.

Nontypeable Haemophilus influenzae (NTHI) are Gram-negative bacteria that colonize the human pharynx and can cause respiratory tract infections, such as acute otitis media (AOM). Since NTHI require iron from their hosts for aerobic growth, the heme acquisition genes may play a significant role in avoiding host nutritional immunity and determining virulence. Therefore, we employed a hybridization-based technique to compare the prevalence of five heme acquisition genes (hxuA, hxuB, hxuC, hemR, and hup) between 514 middle ear strains from children with AOM and 235 throat strains from healthy children. We also investigated their prevalences in 148 Haemophilus haemolyticus strains, a closely related species that colonizes the human pharynx and is considered to be nonpathogenic. Four out of five genes (hxuA, hxuB, hxuC, and hemR) were significantly more prevalent in the middle ear strains (96%, 100%, 100%, and 97%, respectively) than in throat strains (80%, 92%, 93%, and 85%, respectively) of NTHI, suggesting that strains possessing these genes have a virulence advantage over those lacking them. All five genes were dramatically more prevalent in NTHI strains than in H. haemolyticus, with 91% versus 9% hxuA, 98% versus 11% hxuB, 98% versus 11% hxuC, 93% versus 20% hemR, and 97% versus 34% hup, supporting their potential role in virulence and highlighting their possibility to serve as biomarkers to distinguish H. influenzae from H. haemolyticus. In summary, this study demonstrates that heme acquisition genes are more prevalent in disease-causing NTHI strains isolated from the middle ear than in colonizing NTHI strains and H. haemolyticus isolated from the pharynx.

Phase variation and host immunity against high molecular weight (HMW) adhesins shape population dynamics of nontypeable Haemophilus influenzae within human hosts.

Nontypeable Haemophilus influenzae (NTHi) is a bacterium that resides within the human pharynx. Because NTHi is human-restricted, its long-term survival is dependent upon its ability to successfully colonize new hosts. Adherence to host epithelium, mediated by bacterial adhesins, is one of the first steps in NTHi colonization. NTHi express several adhesins, including the high molecular weight (HMW) adhesins that mediate attachment to the respiratory epithelium where they interact with the host immune system to elicit a strong humoral response. hmwA, which encodes the HMW adhesin, undergoes phase variation mediated by 7-base pair tandem repeats located within its promoter region. Repeat number affects both hmwA transcription and HMW-adhesin production such that as the number of repeats increases, adhesin production decreases. Cells expressing large amounts of HMW adhesins may be critical for the establishment and maintenance of NTHi colonization, but they might also incur greater fitness costs when faced with an adhesin-specific antibody-mediated immune response. We hypothesized that the occurrence of large deletion events within the hmwA repeat region allows NTHi cells to maintain adherence in the presence of antibody-mediated immunity. To study this, we developed a mathematical model, incorporating hmwA phase variation and antibody-mediated immunity, to explore the trade-off between bacterial adherence and immune evasion. The model predicts that antibody levels and avidity, catastrophic loss rates, and population carrying capacity all significantly affected numbers of adherent NTHi cells within a host. These results suggest that the occurrence of large, yet rare, deletion events allows for stable maintenance of a small population of adherent cells in spite of HMW adhesin specific antibody-mediated immunity. These adherent subpopulations may be important for sustaining colonization and/or maintaining transmission.

Postoperative spine and VEPTR infections in children: a case-control study.

BACKGROUND: Surgical site infection (SSI) after pediatric scoliosis surgery is a major cause of morbidity. We compared the odds ratios of various potential risk factors for infection among patients who developed a deep SSI following spinal deformity surgery and those who remained infection free. METHODS: This was a case-control study, not a matched study. More noninfection cases (50) than infection cases (20) were selected because more were available. Twenty children with a deep SSI after scoliosis surgery were compared with 50 similar children who did not develop a deep SSI. Fourteen perioperative factors were examined in both the groups. RESULTS: Of the 20 patients who had a deep SSI, 14 had neuromuscular scoliosis. In the infected group, 6 patients had undergone vertical expandable prosthetic titanium rib placement, 2 had undergone growing rod insertion, and 12 had undergone posterior spinal fusion. Eighteen patients developed a SSI within 1 year of the operation and 2 patients presented with a SSI >1 year after surgery. Sixteen patients had positive cultures. Majority were skin flora: coagulase-negative Staphylococcus (8) and Propionibacterium acnes (4). Both patients with tracheostomies had Enterococcus faecalis infections. When comparing the 20 patients with deep SSI to the 50 controls, increased preoperative Cobb angle (P=0.011), increased postoperative Cobb angle (P=0.0043), nonambulatory status (P=0.0002), and increased length of stay (P=0.015) were associated with significantly increased odds of infection. CONCLUSIONS: Our study shows that patients with neuromuscular scoliosis are at higher risk of developing a deep SSI after spinal deformity surgery. Skin flora is a common cause of deep SSI. We have now instituted a standard skin preparation protocol to include alcohol and chlorhexidine washes the night before and the morning of surgery. We have altered our prophylactic antibiotic regimen to cover skin flora in all patients and gastrointestinal flora in patients with a tracheostomy. We have counseled the families of nonambulatory children with large neuromuscular curves regarding the significantly increased odds of postoperative deep SSI. LEVEL OF EVIDENCE: Level III.

Design and validation of a supragenome array for determination of the genomic content of Haemophilus influenzae isolates.

BACKGROUND: Haemophilus influenzae colonizes the human nasopharynx as a commensal, and is etiologically associated with numerous opportunistic infections of the airway; it is also less commonly associated with invasive disease. Clinical isolates of H. influenzae display extensive genomic diversity and plasticity. The development of strategies to successfully prevent, diagnose and treat H. influenzae infections depends on tools to ascertain the gene content of individual isolates. RESULTS: We describe and validate a Haemophilus influenzae supragenome hybridization (SGH) array that can be used to characterize the full genic complement of any strain within the species, as well as strains from several highly related species. The array contains 31,307 probes that collectively cover essentially all alleles of the 2890 gene clusters identified from the whole genome sequencing of 24 clinical H. influenzae strains. The finite supragenome model predicts that these data include greater than 85% of all non-rare genes (where rare genes are defined as those present in less than 10% of sequenced strains). The veracity of the array was tested by comparing the whole genome sequences of eight strains with their hybridization data obtained using the supragenome array. The array predictions were correct and reproducible for ~ 98% of the gene content of all of the sequenced strains. This technology was then applied to an investigation of the gene content of 193 geographically and clinically diverse H. influenzae clinical strains. These strains came from multiple locations from five different continents and Papua New Guinea and include isolates from: the middle ears of persons with otitis media and otorrhea; lung aspirates and sputum samples from pneumonia and COPD patients, blood specimens from patients with sepsis; cerebrospinal fluid from patients with meningitis, as well as from pharyngeal specimens from healthy persons. CONCLUSIONS: These analyses provided the most comprehensive and detailed genomic/phylogenetic look at this species to date, and identified a subset of highly divergent strains that form a separate lineage within the species. This array provides a cost-effective and high-throughput tool to determine the gene content of any H. influenzae isolate or lineage. Furthermore, the method for probe selection can be applied to any species, given a group of available whole genome sequences.

Urease operon and urease activity in commensal and disease-causing nontypeable Haemophilus influenzae.

The ure operon was significantly more prevalent in Haemophilus influenzae isolates causing otitis media and chronic obstructive pulmonary disease (COPD)-associated bronchitis than in those from throats of healthy individuals (97% versus 78.1%, P < 0.001). Strains lacking the ure operon are over 8 times more likely to be from the throat than either otitis media or COPD isolates.

Molecular basis of increased serum resistance among pulmonary isolates of non-typeable Haemophilus influenzae.

Non-typeable Haemophilus influenzae (NTHi), a common commensal of the human pharynx, is also an opportunistic pathogen if it becomes established in the lower respiratory tract (LRT). In comparison to colonizing isolates from the upper airway, LRT isolates, especially those associated with exacerbations of chronic obstructive pulmonary disease, have increased resistance to the complement- and antibody-dependent, bactericidal effect of serum. To define the molecular basis of this resistance, mutants constructed in a serum resistant strain using the mariner transposon were screened for loss of survival in normal human serum. The loci required for serum resistance contribute to the structure of the exposed surface of the bacterial outer membrane. These included loci involved in biosynthesis of the oligosaccharide component of lipooligosaccharide (LOS), and vacJ, which functions with an ABC transporter encoded by yrb genes in retrograde trafficking of phospholipids from the outer to inner leaflet of the cell envelope. Mutations in vacJ and yrb genes reduced the stability of the outer membrane and were associated with increased cell surface hyrophobicity and phospholipid content. Loss of serum resistance in vacJ and yrb mutants correlated with increased binding of natural immunoglobulin M in serum as well as anti-oligosaccharide mAbs. Expression of vacJ and the yrb genes was positively correlated with serum resistance among clinical isolates. Our findings suggest that NTHi adapts to inflammation encountered during infection of the LRT by modulation of its outer leaflet through increased expression of vacJ and yrb genes to minimize recognition by bactericidal anti-oligosaccharide antibodies.

Use of bexB to detect the capsule locus in Haemophilus influenzae.

Haemophilus influenzae strains are classified as typeable or nontypeable H. influenzae (NTHI) based upon the presence or absence of capsule. In addition to serotyping, which is subject to false-positive results, typeable strains can be identified through the detection of the capsular export gene bexA and one of six capsule-specific genes, but this method is resource intensive, especially in characterizing large numbers of strains. To address these challenges, we developed a bexB-based method to differentiate true NTHI strains from typeable strains. We validated a PCR-based method to detect bexB in 10 strains whose capsule status was well defined. Among 40 strains that were previously serotype positive in clinical microbiology laboratories, 5 lacked bexA, bexB, and capsule type-specific genes by PCR analysis and thus likely represent false-positive serotyping results. Among 94 additional otitis media, commensal, and serotype b-negative invasive strains, 85 were bexA and bexB negative and 9 contained either a complete or partial capsule locus, i.e., 8 were bexA and bexB positive and 1 was bexA negative but bexB positive. Finally, we adapted the method for use in a high-throughput DNA hybridization-based microarray method, which showed 98.75 and 97.5% concordance to the PCR methods for bexA and bexB, respectively. In addition, bexB showed 84% or greater nucleotide identity among strains containing the capsule locus. In this study, we demonstrate that bexB is a reliable proxy for the capsule locus and that its detection provides a simple and reliable method for differentiating strains that lack the entire capsule locus from those containing a partial or complete capsule locus.

Prevalence of Haemophilus influenzae type b genetic islands among clinical and commensal H. influenzae and H. haemolyticus isolates.

Five genetic islands (HiGI) found in Haemophilus influenzae type b strain Eagan were used as hybridization probes on type b, Haemophilus haemolyticus, and nontypeable H. influenzae (NTHi) isolates. HiGI2 and HiGI7 were significantly more prevalent in NTHi isolates from children with otitis media than in those from the throats of healthy children.

Prevalence of the sodC gene in nontypeable Haemophilus influenzae and Haemophilus haemolyticus by microarray-based hybridization.

The sodC gene has been reported to be a useful marker for differentiating nontypeable (NT) Haemophilus influenzae from Haemophilus haemolyticus in respiratory-tract samples, but discrepancies exist as to the prevalence of sodC in NT H. influenzae. Therefore, we used a microarray-based, "library-on-a-slide" method to differentiate the species and found that 21 of 169 (12.4%) NT H. influenzae strains and all 110 (100%) H. haemolyticus strains possessed the sodC gene. Multilocus sequence analysis confirmed that the 21 NT H. influenzae strains were H. influenzae and not H. haemolyticus. An inactive sodC gene has been reported in encapsulated H. influenzae strains belonging to phylogenetic division II. Capsule-specific Southern hybridization and PCR and a lack of copper/zinc-cofactored superoxide dismutase (CuZnSOD) expression indicated that 6 of the 21 sodC-containing NT H. influenzae strains in our study were likely capsule-deficient mutants belonging to phylogenetic division II. DNA sequence comparisons of the 21 H. influenzae sodC genes with sodC from H. haemolyticus or encapsulated H. influenzae demonstrated that the sodC genes of the six H. influenzae capsule-deficient mutants were, on average, 99% identical to sodC from encapsulated H. influenzae but only 85% identical to sodC from H. haemolyticus. The sodC genes from 2/15 NT H. influenzae strains were similarly more closely related to sodC from encapsulated strains, while sodC genes from 13 NT H. influenzae strains were almost 95% identical to sodC genes from H. haemolyticus, suggesting the possibility of interspecies recombination in these strains. In summary, this study demonstrates that sodC is not completely absent (9.2%) in true NT H. influenzae strains.

Prevalence of genetic differences in phosphorylcholine expression between nontypeable Haemophilus influenzae and Haemophilus haemolyticus.

BACKGROUND: Although non-typeable (NT) Haemophilus influenzae and Haemophilus haemolyticus are closely related human commensals, H. haemolyticus is non-pathogenic while NT H. influenzae is an important cause of respiratory tract infections. Phase-variable phosphorylcholine (ChoP) modification of lipooligosaccharide (LOS) is a NT H. influenzae virulence factor that, paradoxically, may also promote complement activation by binding C-reactive protein (CRP). CRP is known to bind more to ChoP positioned distally than proximally in LOS, and the position of ChoP within LOS is dictated by specific licD alleles (designated here as licDI, licDIII, and licDIV) that are present in a lic1 locus. The lic1 locus contains the licA-licD genes, and ChoP-host interactions may also be influenced by a second lic1 locus that allows for dual ChoP substitutions in the same strain, or by the number of licA gene tetranucleotide repeats (5'-CAAT-3') that reflect phase-variation mutation rates. RESULTS: Using dot-blot hybridization, 92% of 88 NT H. influenzae and 42.6% of 109 H. haemolyticus strains possessed a lic1 locus. Eight percent of NT H. influenzae and none of the H. haemolyticus strains possessed dual copies of lic1. The licDIII and licDIV gene alleles were distributed similarly (18-22%) among the NT H. influenzae and H. haemolyticus strains while licDI alleles were present in 45.5% of NT H. influenzae but in less than 1% of H. haemolyticus strains (P < .0001). NT H. influenzae had an average of 26.8 tetranucleotide repeats in licA compared to 14.8 repeats in H. haemolyticus (P < .05). In addition, NT H. influenzae strains that possessed a licDIII allele had increased numbers of repeats compared to NT H. influenzae with other licD alleles (P < .05). CONCLUSIONS: These data demonstrate that genetic similarities and differences of ChoP expression exist between NT H. influenzae and H. haemolyticus and strengthen the hypothesis that, at the population level, these differences may, in part, provide an advantage in the virulence of NT H. influenzae.

VIOLIN: vaccine investigation and online information network.

Vaccines are among the most efficacious and cost-effective tools for reducing morbidity and mortality caused by infectious diseases. The vaccine investigation and online information network (VIOLIN) is a web-based central resource, allowing easy curation, comparison and analysis of vaccine-related research data across various human pathogens (e.g. Haemophilus influenzae, human immunodeficiency virus (HIV) and Plasmodium falciparum) of medical importance and across humans, other natural hosts and laboratory animals. Vaccine-related peer-reviewed literature data have been downloaded into the database from PubMed and are searchable through various literature search programs. Vaccine data are also annotated, edited and submitted to the database through a web-based interactive system that integrates efficient computational literature mining and accurate manual curation. Curated information includes general microbial pathogenesis and host protective immunity, vaccine preparation and characteristics, stimulated host responses after vaccination and protection efficacy after challenge. Vaccine-related pathogen and host genes are also annotated and available for searching through customized BLAST programs. All VIOLIN data are available for download in an eXtensible Markup Language (XML)-based data exchange format. VIOLIN is expected to become a centralized source of vaccine information and to provide investigators in basic and clinical sciences with curated data and bioinformatics tools for vaccine research and development. VIOLIN is publicly available at http://www.violinet.org.