Untargeted Metabolomics Reveals the Potential Antidepressant Activity of a Novel Adenosine Receptor Antagonist.

Depression is the most common mental illness, affecting approximately 4.4% of the global population. Despite many available treatments, some patients exhibit treatment-resistant depression. Thus, the need to develop new and alternative treatments cannot be overstated. Adenosine receptor antagonists have emerged as a promising new class of antidepressants. The current study investigates a novel dual A1/A2A adenosine receptor antagonist, namely 2-(3,4-dihydroxybenzylidene)-4-methoxy-2,3-dihydro-1H-inden-1-one (1a), for antidepressant capabilities by determining its metabolic profiles and comparing them to those of two reference compounds (imipramine and KW-6002). The metabolic profiles were obtained by treating male Sprague-Dawley rats with 1a and the reference compounds and subjecting them to the forced swim test. Serum and brain material was consequently collected from the animals following euthanasia, after which the metabolites were extracted and analyzed through untargeted metabolomics using both 1H-NMR and GC-TOFMS. The current study provides insight into compound 1a's metabolic profile. The metabolic profile of 1a was similar to those of the reference compounds. They potentially exhibit their antidepressive capabilities via downstream effects on amino acid and lipid metabolism.

A Three-Dimensional Bioabsorbable Tissue Marker for Volume Replacement and Radiation Planning: A Multicenter Study of Surgical and Patient-Reported Outcomes for 818 Patients with Breast Cancer.

BACKGROUND: Accurate identification of the tumor bed after breast-conserving surgery (BCS) ensures appropriate radiation to the tumor bed while minimizing normal tissue exposure. The BioZorb® three-dimensional (3D) bioabsorbable tissue marker provides a reliable target for radiation therapy (RT) planning and follow-up evaluation while serving as a scaffold to maintain breast contour. METHODS: After informed consent, 818 patients (826 breasts) implanted with the BioZorb® at 14 U.S. sites were enrolled in a national registry. All the patients were prospectively followed with the BioZorb® implant after BCS. The data collected at 3, 6, 12, and 24 months included all demographics, treatment parameters, and provider/patient-assessed cosmesis. RESULTS: The median follow-up period was 18.2 months (range, 0.2-53.4 months). The 30-day breast infection rate was 0.5 % of the patients (n = 4), and re-excision was performed for 8.1 % of the patients (n = 66), whereas 2.6 % of the patients (n = 21) underwent mastectomy. Two patients (0.2 %) had local recurrence. The patient-reported cosmetic outcomes at 6, 12, and 24 months were rated as good-to-excellent by 92.4 %, 90.6 %, and 87.3 % of the patients, respectively and similarly by the surgeons. The radiation oncologists reported planning of target volume (PTV) reduction for 46.2 % of the patients receiving radiation boost, with PTV reduction most commonly estimated at 30 %. CONCLUSIONS: This report describes the first large multicenter study of 818 patients implanted with the BioZorb® tissue marker during BCS. Radiation oncologists found that the device yielded reduced PTVs and that both the patients and the surgeons reported good-to-excellent long-term cosmetic outcomes, with low adverse effects. The BioZorb® 3D tissue marker is a safe adjunct to BCS and may add benefits for both surgeons and radiation oncologists.

Impact of a Novel Bioabsorbable Implant on Radiation Treatment Planning for Breast Cancer.

BACKGROUND: Techniques for accurately delineating the tumor bed after breast-conserving surgery (BCS) can be challenging. As a result, the accuracy, and efficiency of radiation treatment (RT) planning can be negatively impacted. Surgically placed clips or the post-surgical seroma are commonly used to determine target volume; however, these methods can lead to a high degree of uncertainty and variability. A novel 3-dimensional bioabsorbable marker was used during BCS and assessed for its impact on RT planning. METHODS: One hundred and ten implants were sutured to the margins of the tumor bed excision site in 108 patients undergoing BCS. Routine CT imaging of the breast tissue was performed for RT planning, and the marker was assessed for visibility and utility in target delineation. RT regimens, target volumes and associated treatment costs were analyzed. RESULTS: In all patients, the marker was easily visible and in 95.7 % of cases, it proved useful for RT planning. 36.8 % of patients received conventional whole breast irradiation plus boost, 56.6 % received hypo-fractionation plus boost, and 6.6 % received accelerated partial breast irradiation. A shift toward increased use of hypo-fractionated regimens was noted over the three year period of this study. There were no device-related complications or cancer recurrences in this group of patients. CONCLUSIONS: This study demonstrated the use of a novel 3-dimensional marker as a safe and effective method for delineating the tumor bed with a significant utility for RT planning. With routine use of the device, an increased use of hypofractionation with a resultant 25 % cost savings was noted.

Dirty Money: A Matter of Bacterial Survival, Adherence, and Toxicity.

In this study we report the underlying reasons to why bacteria are present on banknotes and coins. Despite the use of credit cards, mobile phone apps, near-field-communication systems, and cryptocurrencies such as bitcoins which are replacing the use of hard currencies, cash exchanges still make up a significant means of exchange for a wide range of purchases. The literature is awash with data that highlights that both coins and banknotes are frequently identified as fomites for a wide range of microorganisms. However, most of these publications fail to provide any insight into the extent to which bacteria adhere and persist on money. We treated the various currencies used in this study as microcosms, and the bacterial loading from human hands as the corresponding microbiome. We show that the substrate from which banknotes are produced have a significant influence on both the survival and adherence of bacteria to banknotes. Smooth, polymer surfaces provide a poor means of adherence and survival, while coarser and more fibrous surfaces provide strong bacterial adherence and an environment to survive on. Coins were found to be strongly inhibitory to bacteria with a relatively rapid decline in survival on almost all coin surfaces tested. The inhibitory influence of coins was demonstrated through the use of antimicrobial disks made from coins. Despite the toxic effects of coins on many bacteria, bacteria do have the ability to adapt to the presence of coins in their environment which goes some way to explain the persistent presence of low levels of bacteria on coins in circulation.

The modA10 phasevarion of nontypeable Haemophilus influenzae R2866 regulates multiple virulence-associated traits.

Non-typeable Haemophilus influenzae (NTHi) is a human restricted commensal and pathogen that elicits inflammation by adhering to and invading airway epithelia cells: transcytosis across these cells can result in systemic infection. NTHi strain R2866 was isolated from the blood of a normal 30-month old infant with meningitis, and is unusual for NTHi in that it is able to cause systemic infection. Strain R2866 is able to replicate in normal human serum due to expression of lgtC which mimics human blood group p(k). R2866 contains a phase-variable DNA methyltransferase, modA10 which switches ON and OFF randomly and reversibly due to polymerase slippage over a long tetrameric repeat tract located in its open reading frame. Random gain or loss of repeats during replication can results in expressed (ON), or not expressed (OFF) states, the latter due to a frameshift or transcriptional termination at a premature stop codon. We sought to determine if the unusual virulence of R2866 was modified by modA10 phase-variation. A modA10 knockout mutant was found to have increased adherence to, and invasion of, human ear and airway monolayers in culture, and increased invasion and transcytosis of polarized human bronchial epithelial cells. Intriguingly, the rate of bacteremia was lower in the infant rat model of infection than a wild-type R2866 strain, but the fatality rate was greater. Transcriptional analysis comparing the modA10 knockout to the R2866 wild-type parent strain showed increased expression of genes in the modA10 knockout whose products mediate cellular adherence. We conclude that loss of ModA10 function in strain R2866 enhances colonization and invasion by increasing expression of genes that allow for increased adherence, which can contribute to the increased virulence of this strain.

A biphasic epigenetic switch controls immunoevasion, virulence and niche adaptation in non-typeable Haemophilus influenzae.

Non-typeable Haemophilus influenzae contains an N(6)-adenine DNA-methyltransferase (ModA) that is subject to phase-variable expression (random ON/OFF switching). Five modA alleles, modA2, modA4, modA5, modA9 and modA10, account for over two-thirds of clinical otitis media isolates surveyed. Here, we use single molecule, real-time (SMRT) methylome analysis to identify the DNA-recognition motifs for all five of these modA alleles. Phase variation of these alleles regulates multiple proteins including vaccine candidates, and key virulence phenotypes such as antibiotic resistance (modA2, modA5, modA10), biofilm formation (modA2) and immunoevasion (modA4). Analyses of a modA2 strain in the chinchilla model of otitis media show a clear selection for ON switching of modA2 in the middle ear. Our results indicate that a biphasic epigenetic switch can control bacterial virulence, immunoevasion and niche adaptation in an animal model system.

Bacteremia and malaria in Tanzanian children hospitalized for acute febrile illness.

We recorded the reason for presentation to a rural hospital in an area endemic for malaria in 909 children between January 2006 and March 2009. Blood smears were examined for Plasmodium falciparum parasites, and blood spots dried on filter paper were prepared for 464 children. A PCR assay utilizing the stored blood spots was developed for Streptococcus pneumoniae (lytA) and Haemophilus influenzae (pal). Malaria was present in 299 children whose blood was tested by polymerase chain reaction (PCR); 19 had lytA and 15 had pal. The overall prevalence of lytA was 25 of the 464 children, while that of pal was 18 children. Fever was present in 369 children of whom 19 had lytA DNA while 11 had pal DNA detected. Of the 95 afebrile children, six had lytA and seven pal. We conclude that there are no clinical features that distinguish malaria alone from bacteremia alone or the presence of both infections.

Open versus minimally invasive sacroiliac joint fusion: a multi-center comparison of perioperative measures and clinical outcomes.

BACKGROUND: Sacroiliac (SI) joint pain is an under diagnosed source of low back pain due in part to lack of visible pathology on radiographs and symptoms mimicking other back-related disorders. Open SI joint fusion has been performed since the 1920s. This technique has fallen out of favor with the introduction of minimally invasive options. To date there has been no direct comparison between open and MIS SI joint fusion. METHODS: We conducted a multi-center, retrospective comparative cohort study of patients who underwent SI joint fusion using either an open surgical (OS) technique using a combination of screws and cages or a minimally invasive surgical (MIS) technique with a series of titanium plasma spray (TPS) coated triangular implants. Operative measures including surgical operating time, length of hospitalization and estimated blood loss (EBL) were collected along with demographics and medical history, surgical complications, and 12- and 24-month pain scores. Improvements in pain were compared after matching for age and gender and controlling for a history of lumbar spine fusion using repeated measures analysis of variance. RESULTS: Data were available for 263 patients treated by 7 surgeons; 149 patients treated with OS and 114 treated with MIS SI joint fusion. Compared to OS patients, MIS patients were on average 10 years older (mean age 57 vs. 46) and 69% of all patients were female. MIS operative measures of EBL, operating time and length of hospitalization were significantly lower than open surgery (p < 0.001). Pain relief, measured as change from baseline to 12 months in VAS pain rating, was 3.5 points lower in the MIS vs. OS group (-6.2 vs. -2.7 points, p < 0.001). When matched for age, gender and a history of prior lumbar spinal fusion, postoperative pain scores were on average 3.0 points (95% CI 2.1 - 4.0) lower in MIS vs. OS (rANOVA p < 0.001). CONCLUSIONS: In this multi-center comparative study, patients who underwent either OS or MIS SI joint fusion showed postoperative improvements in pain score. Compared to OS patients, patients who underwent MIS SI joint fusion had significantly greater pain relief and more favorable perioperative surgical measures.

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.

"Affect of anaerobiosis on the antibiotic susceptibility of H. influenzae".

BACKGROUND: Haemophilus influenzae is a human-restricted facultative anaerobe which resides mostly in the oropharynx. The majority of isolates recovered from the throat are unencapsulated commensals (NTHi), but depending on host susceptibility they cause bronchitis, otitis media and on occasion bacteremia and meningitis. Because of the variable oxygen availability in the various niche permitting bacterium replication, the organism must thrive in well oxygenated surfaces, such as pharyngeal epithelium to anoxic environments like the bottom of a Biofilm and in airway mucus. Other reports indicate that H. influenzae use aerobic respiration, anaerobic respiration and fermentation to generate ATP. To gain insight in to the activity of several classes of antibiotics against five well-characterized unencapsulated H. influenzae in room air, in 5% CO2 and under strict anaerobiosis. We also tested for the role of oxidative killing by all cidal antibiotics. RESULTS: In comparison to room air, testing in 5% CO2 had minimal effects on the susceptibility to aminoglycosides, cephalosporins, tetracycline and chloramphenicol: the MIC of rifampin and ciprofloxacin increased eight fold with certain strains in 5% CO2. All antibiotics, except trimethoprim were cidal under both growth conditions. Aminoglycosides remained bactericidal in a strict anaerobic environment, while a reliable MBC was obtained with trimethoprim only under anaerobic conditions. Kinetic analysis of the cidal action of spectinomycin and tetracycline indicated slower killing anaerobically. An oxidative mechanism for aerobic killing could not be demonstrated. CONCLUSIONS: We conclude that ß-lactams, cephalosporins, macrolides, tetracycline's, aminoglycosides, chloramphenicol, rifampin and ciprofloxacin are bactericidal against five well-characterizes H. influenzae in an aerobic and anaerobic environment. The activity of trimethoprim was increased in anaerobic conditions.

Mycotic aneurysm of the abdominal aorta caused by Haemophilus influenzae type f.

Prior to the advent of the Haemophilus influenzae type b vaccine, invasive infections due to H. influenzae type f were rarely described. However, the epidemiology of H. influenzae is changing. While the incidence of invasive infections due to H. influenzae is declining in children, such infections are becoming more common in adults, particularly in the elderly. Here, we report an unusual case of infective aortic aneurysm caused by H. influenzae type f that underlines the emerging clinical relevance and pathogenic capability of this organism.

Adhesin genes and serum resistance in Haemophilus influenzae type f isolates.

The incidence of invasive infections due to Haemophilus influenzae has decreased significantly in developed countries with high rates of vaccination against H. influenzae serotype b (Hib). This vaccine provides no protection against H. influenzae serotype f (Hif), typically associated with invasive infections in adults with chronic disease and/or immunodeficiency, and rarely in otherwise healthy adults and children. The specific properties of Hif associated with virulence remain largely uncharacterized. A panel of 26 Hif strains consisting of both invasive disease-associated and mucosal surface non-invasive disease-associated isolates was surveyed by DNA fingerprinting, biotyping and PCR detection of hmw1, hmw2, hsf, the hif fimbrial locus and the lipo-oligosaccharide (LOS) biosynthetic island, and assessment of ß-lactamase expression and determination of resistance to the bactericidal activity of normal adult human serum. Repetitive sequence-based PCR fingerprinting differentiated the 26 strains into three clusters, with the majority of isolates (22/26, 84.6 %) clustered into a single indistinguishable group. Most isolates (24/26, 92.3 %) were of biotype I and two isolates produced ß-lactamase with detection of a conjugative plasmid, and the isolates displayed a range of resistances to the bactericidal activity of human serum. All 26 isolates carried the adhesin hsf, 21 carried a partial hif fimbrial operon and 4 had the adhesin genes hmw1/2. A LOS biosynthetic island was detected in 20 isolates consisting of the genes lic2BC. It was concluded that Hif has many recognized virulence properties and comprises a relatively homogeneous group independent of the anatomical source from which it was isolated.

Characterization of extended co-culture of non-typeable Haemophilus influenzae with primary human respiratory tissues.

Non-typeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that comprise part of the normal flora of the human upper airway, but are also responsible for a number of mucosal infections such as otitis media and bronchitis. These infections often recur and can become chronic. To characterize the effect of long-term co-culture of NTHi with human tissues, we infected primary respiratory epithelial cells grown at the air-liquid interface with three NTHi strains over a range of 1-10 days. Scanning and transmission electron microscopy of tissues confirmed that intact NTHi were persisting paracellularly, while organisms observed in intracellular vacuoles appeared degraded. Furthermore, the apical surface and tight junctions of the infected tissues were undisturbed, with high transepithelial electrical resistances, while the basal cell layer displayed more junctional disorganization and wider intercellular spaces than the uninfected control tissues. Although the tissues elaborated the cytokine profile reported for NTHi-caused otitis media in vivo, there was little change in the dynamics of cytokine secretion over the time points tested. Finally, we report that NTHi strains released outer membrane vesicles (OMVs) during extended co-culture with the tissues, and show that these OMVs directly interact with host cell membranes.

Infant rat infection modifies phenotypic properties of an invasive nontypeable Haemophilus influenzae.

Enhancing the virulence trait of a specific bacterium in an animal model is often performed prior to the use of the strain for ex vivo human studies, such as reactivity with complement and antibody, or with phagocytic cells. For example, in Streptococcus pneumoniae mouse passage is used to enhance capsule production. While investigating an unusual serum-resistant unencapsulated Haemophilus influenzae (R2866), we found that animal passage yielded an isolate (R3392) which had decreased resistance to human serum, but increased virulence in Chang conjunctival cell monolayers, but with less invasion and transcytosis of polar H292 cells. We examined 90 colonies recovered from three infant rats for phase variants of LPS biosynthetic genes. In 88 colonies lgtC was OFF due to tetrameric repeat mediated slipped-strand mispairing at the time of DNA replication, while there was no variation in lic1A, lic2A, lic3A, lexA and oaf A. With lgtC OFF the LPS lacks Galα1-4ßGal, an epitope mimicking the human p(k) blood group, and molecular mimicry is lost. Selection for strain susceptible to NHS in the infant rat was not antibody mediated. We conclude that the passage of pathogens virulent in humans and animals may select for phenotypes only relevant for the animal species used.

Biology and data-intensive scientific discovery in the beginning of the 21st century.

The life sciences are poised at the beginning of a paradigm-changing evolution in the way scientific questions are answered. Data-Intensive Science (DIS) promise to provide new ways of approaching scientific challenges and answering questions. This article is a summary of the life sciences issues and challenges as discussed in the DIS workshop in Seattle, September 19-20, 2010.

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.

Dirty money: an investigation into the hygiene status of some of the world's currencies as obtained from food outlets.

A total of 1280 banknotes were obtained from food outlets in 10 different countries (Australia, Burkina Faso, China, Ireland, the Netherlands, New Zealand, Nigeria, Mexico, the United Kingdom, and the United States), and their bacterial content was enumerated. The presence of bacteria on banknotes was found to be influenced by the material of the notes, and there was a strong correlation between the number of bacteria per square centimeter and a series of indicators of economic prosperity of the various countries. The strongest correlation was found with the "index of economic freedom," indicating that the lower the index value, the higher the typical bacterial content on the banknotes in circulation. Other factors that appear to influence the number of bacteria on banknotes were the age of the banknotes and the material used to produce the notes (polymer-based vs. cotton-based). The banknotes were also screened for the presence of a range of pathogens. It was found that pathogens could only be isolated after enrichment and their mere presence does not appear to be alarming. In light of our international findings, it is recommended that current guidelines as they apply in most countries with regard to the concurrent hygienic handling of foods and money should be universally adopted. This includes that, in some instances, the handling of food and money have to be physically separated by employing separate individuals to carry out one task each; whereas in other instances, it could be advantageous to handle food only with a gloved hand and money with the other hand. If neither of these precautions can be effectively implemented, it is highly recommended that food service personnel practice proper hand washing procedures after handling money and before handling food.

Determination of capsulation status in Haemophilus influenzae by multiplex polymerase chain reaction.

Since the introduction of Haemophilus influenzae type b conjugate vaccines, there have been concerns regarding the emergence of invasive non-type b strains. Serotyping of H. influenzae with commercially available reagents is subjective. Definitive characterization of the capsulation status can be performed by polymerase chain reaction (PCR) amplification of capsular genes. However, PCR amplification of the conserved export locus in the 2 known phylogenic lines of type b strains and detection of serotype conferring genes in each of the 6 serotypes require multiple assays. To rapidly screen multiple isolates, we devised a multiplex method using 15 primers, which produced a serotype-specific, distinct pattern of amplicons with reference-encapsulated H. influenzae. We applied this technique to a panel of 35 clinical isolates that had been serotyped as type a, c, d, e, or f by slide agglutination; 15 strains lacked capsular genes. Conversely, of 69 invasive isolates that were not serotypeable, all but 11 contained capsule genes. We conclude that this technique will be useful in screening recently isolated H. influenzae for capsulation status.

Health-related quality of life in children with hepatitis C acquired in the first year of life.

AIMS: The first aim of this study was to determine the health-related quality of life (HRQoL) of children with chronic hepatitis C virus (HCV) infection and compare HRQoL as reported by parents. The second aim was to ascertain parents' perceptions and concerns about current and future life for their child with HCV, and compare these findings with those reported by adolescents. METHODS: The study group comprised children attending a tertiary pediatric HCV-clinic in Melbourne, Australia, who acquired HCV prior to 12 months of age by vertical transmission or blood transfusion. Two validated (parent- and self-reported) questionnaires of HRQoL were completed (CHQ-PF 50 and CHQ-CF 50). Scores for children with HCV were compared with normative data (representative sample of 3119 age-matched Victorian children). A study-designed questionnaire relating to the impact of the diagnosis of HCV on parent and child perceptions of current and future health was administered. RESULTS: In total, 83% (19/23) questionnaires were returned. Physical and psychosocial summary scores were significantly lower in HCV than non-HCV children (45.3 vs 49.6 and 44.0 vs 50.1, respectively). Nine out of 11 scale scores were significantly lower in children with HCV, most notably the General health (49.9 vs 77.1; P < 0.001) and Parent impact-emotional (45.6 vs 80.3; P < 0.001) scales. Children reported reduced physical functioning (82.8% vs 94.4%) but were otherwise less concerned than their parents about their future health. CONCLUSIONS: Despite being "asymptomatic" on routine medical history, children with early acquired HCV have significantly poorer health status than community controls. These findings suggest the need for services currently available for adult HCV patients to support families and children with HCV.

Analysis of genetic relatedness of Haemophilus influenzae isolates by multilocus sequence typing.

The gram-negative bacterium Haemophilus influenzae is a human-restricted commensal of the nasopharynx that can also be associated with disease. The majority of H. influenzae respiratory isolates lack the genes for capsule production and are nontypeable (NTHI). Whereas encapsulated strains are known to belong to serotype-specific phylogenetic groups, the structure of the NTHI population has not been previously described. A total of 656 H. influenzae strains, including 322 NTHI strains, have been typed by multilocus sequence typing and found to have 359 sequence types (ST). We performed maximum-parsimony analysis of the 359 sequences and calculated the majority-rule consensus of 4,545 resulting equally most parsimonious trees. Eleven clades were identified, consisting of six or more ST on a branch that was present in 100% of trees. Two additional clades were defined by branches present in 91% and 82% of trees, respectively. Of these 13 clades, 8 consisted predominantly of NTHI strains, three were serotype specific, and 2 contained distinct NTHI-specific and serotype-specific clusters of strains. Sixty percent of NTHI strains have ST within one of the 13 clades, and eBURST analysis identified an additional phylogenetic group that contained 20% of NTHI strains. There was concordant clustering of certain metabolic reactions and putative virulence loci but not of disease source or geographic origin. We conclude that well-defined phylogenetic groups of NTHI strains exist and that these groups differ in genetic content. These observations will provide a framework for further study of the effect of genetic diversity on the interaction of NTHI with the host.