Bacterial ‘colonisation’ of the nasopharynx by potential bacterial pathogens in early childhood is a normal, important series of events. They are part of a dynamic process in which the microbiota of the upper airways becomes established and evolves in early childhood. The potential pathogens ‘colonising’ the upper airways interact with resident, apparently non-pathogenic, bacteria; other potential pathogens including those from their own species; viruses and the hosts immune response. Environmental factors such as family dynamics, child care, antibiotic usage, smoking and indeed vaccines all have an impact. Understanding the significant of potential beneficial interactions as well as those factors that are detrimental to the host will be important in disease prevention in the future.
Streptococcus pneumoniae; Haemophilus influenzae; Microbial populations; Bacterial interference; Bacterial competition; commensals
Health benefits and costs of combined reduced-antigen-content tetanus, diphtheria, and pertussis (Tdap) immunization among adults ≥65 years have not been evaluated. In February 2012, the Advisory Committee on Immunization Practices (ACIP) recommended expanding Tdap vaccination (one single dose) to include adults ≥65 years not previously vaccinated with Tdap. Our study estimated the health and economic outcomes of one-time replacement of the decennial tetanus and diphtheria (Td) booster with Tdap in the 10% of individuals aged 65 years assumed eligible each year compared with a baseline scenario of continued Td vaccination.
We constructed a model evaluating the cost-effectiveness of vaccinating a cohort of adults aged 65 with Tdap, by calculating pertussis cases averted due to direct vaccine effects only. Results are presented from societal and payer perspectives for a range of pertussis incidences (25–200 cases per 100,000), due to the uncertainty in estimating true annual incidence. Cases averted were accrued throughout the patient 's lifetime, and a probability tree used to estimate the clinical outcomes and costs (US$ 2010) for each case. Quality-adjusted life-years (QALYs) lost to acute disease were calculated by multiplying cases of mild/moderate/severe pertussis by the associated health-state disutility; QALY losses due to death and long-term sequelae were also considered. Incremental costs and QALYs were summed over the cohort to derive incremental cost-effectiveness ratios. Scenario analyses evaluated the effect of alternative plausible parameter estimates on results.
At incidence levels of 25, 100, 200 cases/100,000, vaccinating adults aged 65 years costs an additional $336,000, $63,000 and $17,000/QALY gained, respectively. Vaccination has a cost-effectiveness ratio less than $50,000/QALY if pertussis incidence is >116 cases/100,000 from societal and payer perspectives. Results were robust to scenario analyses.
Tdap immunization of adults aged 65 years according to current ACIP recommendations is a cost-effective health-care intervention at plausible incidence assumptions.
Background. Response rates and immunologic memory following measles vaccination are reduced in human immunodeficiency virus (HIV)–infected children in the absence of highly active antiretroviral therapy (HAART).
Methods. HIV-infected children 2 to <19 years old receiving HAART and with HIV loads <30 000 copies/mL, CD4% ≥15, and ≥1 prior measles-mumps-rubella vaccination (MMR) were given another MMR. Measles antibody concentrations before and 8, 32, and 80 weeks postvaccination were determined by plaque reduction neutralization (PRN). A subset was given another MMR 4–5 years later, and PRN antibody was measured before and 7 and 28 days later.
Results. At entry, 52% of 193 subjects were seroprotected (PRN ≥120 mIU/mL). Seroprotection increased to 89% 8 weeks postvaccination, and remained at 80% 80 weeks postvaccination. Of 65 subjects revaccinated 4–5 years later, 85% demonstrated memory based on seroprotection before or 7 days after vaccination. HIV load ≤400 copies/mL at initial study vaccination was associated with higher seroprotection rates, greater antibody concentrations, and memory. Grade 3 fever or fatigue occurred in 2% of subjects.
Conclusions. Measles revaccination induced high rates of seroprotection and memory in children receiving HAART. Both endpoints were associated with HIV viral load suppression.
Clinical Trials Registration: NCT00013871 (www.clinicaltrials.gov).
Increases in colonization with serotypes of Streptococcus pneumoniae not contained within the 7-valent pneumococcal conjugate vaccine (PCV) have been reported among children following introduction. Serotype 6C has emerged as prevalent in nasopharyngeal colonization and acute otitis media (AOM), though it is uncommonly recovered from children with invasive pneumococcal disease. Vaccine serotypes within PCV7 have been replaced by nonvaccine serotypes without significant changes in the overall carriage rate. We hypothesize 1) that serotypes vary in their ability to evade host defenses and establish AOM following colonization and 2) the observed reduction in pneumococcal otitis results from a reduced disease potential by some ‘replacement serotypes’. We compared the capacity of S. pneumoniae serotypes 6C and 19A to produce experimental otitis media (EOM) in a chinchilla model. The proportion of chinchillas that developed culture positive EOM and density of middle ear infection was evaluated. EOM was found in 28/82 (34%) ears challenged with 6C compared to 13/18(72.2%) with 19A [p=0.0003]. When disease due to 6C did occur, it was characterized by lowdensity infection. Our findings demonstrate that challenge with serotype 6C results in EOM less frequently than 19A. These data support the need for greater knowledge regarding differences among serotypes to produce AOM.
Streptococcus pneumoniae; complement; virulence
Streptococcus pneumoniae strains expressing serotype 11E commonly occur among disease isolates, rarely occur among carriage isolates, and are clonally unrelated. Thus, 11E strains seem to have emerged after dissemination of serotype 11A progenitors to deeper tissues outside the nasopharynx.
Background. Streptococcus pneumoniae is a commensal colonizer of the human nasopharynx (NP) that causes disease after evasion of host defenses and dissemination. Pneumococcal strains expressing the newly identified serotype 11E arise from antigenically similar 11A progenitors by genetic inactivation of the O-acetyltransferase gene wcjE. Each 11E strain contains a distinct mutation to wcjE, suggesting that 11E strains are not transmitted among hosts despite their recovery from multiple patients with pneumococcal disease. We investigated whether the presumed lack of transmission of serotype 11E is consistent with its inability to survive in the NP.
Methods. More than 400 pneumococcal carriage, middle ear, conjunctiva, and blood isolates, serotyped as 11A by Quellung reaction, were reexamined for reactivity to 11A- and 11E-specific antibodies. We confirmed serotyping of isolates with sequencing of wcjE alleles.
Results. Serotype 11E strains were statistically more likely to occur among blood (4 of 15), conjunctiva (1 of 14), or middle ear (2 of 21) isolates than among carriage isolates (2 of 355). All 11E isolates contained unique mutations that putatively decrease wcjE expression.
Conclusions. The lack of a circulating 11E clone and the increased occurrence of 11E strains among disease isolates supports the idea that serotype 11E independently arises during infection after initial colonization with a serotype 11A progenitor. Factors encountered in the NP likely contribute to relative rarity of 11E among carriage isolates, whereas selective pressures in deeper tissues possibly promote 11E emergence. These findings illustrate a novel model of microevolution that transpires during the span of a single encounter with serotype 11A, highlighting the adaptability of bacterial pathogens within hosts.
We sought to measure trends in Streptococcus pneumoniae (SP) carriage and antibiotic resistance in young children in Massachusetts communities after widespread adoption of heptavalent pneumococcal conjugate vaccine (PCV7) and before the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13).
We conducted a cross-sectional study including collection of questionnaire data and nasopharyngeal specimens among children <7 years in primary care practices from 8 Massachusetts communities during the winter season of 2008–9 and compared with to similar studies performed in 2001, 2003–4, and 2006–7. Antimicrobial susceptibility testing and serotyping were performed on pneumococcal isolates, and risk factors for colonization in recent seasons (2006–07 and 2008–09) were evaluated.
We collected nasopharyngeal specimens from 1,011 children, 290 (29%) of whom were colonized with pneumococcus. Non-PCV7 serotypes accounted for 98% of pneumococcal isolates, most commonly 19A (14%), 6C (11%), and 15B/C (11%). In 2008–09, newly-targeted PCV13 serotypes accounted for 20% of carriage isolates and 41% of penicillin non-susceptible S. pneumoniae (PNSP). In multivariate models, younger age, child care, young siblings, and upper respiratory illness remained predictors of pneumococcal carriage, despite near-complete serotype replacement. Only young age and child care were significantly associated with PNSP carriage.
Serotype replacement post-PCV7 is essentially complete and has been sustained in young children, with the relatively virulent 19A being the most common serotype. Predictors of carriage remained similar despite serotype replacement. PCV13 may reduce 19A and decrease antibiotic-resistant strains, but monitoring for new serotype replacement is warranted.
Streptococcus pneumoniae; pneumococcal conjugate vaccine; antibiotic resistance; serotype; colonization
Nontypeable Haemophilus influenzae (NTHi) causes otitis media, sinusitis, and likely lower respiratory tract infections in children. Colonization, strain diversity, transmission, and antimicrobial susceptibility have implications for both children and their caregivers.
For 13 months, we conducted a cross-sectional study of NTHi colonization. 273 infants and children aged 2 to 26 months old and their primary caregivers had upper respiratory tract cultures performed. NTHi isolates were characterized by multilocus sequence typing (MLST) and antibiotic resistance was examined.
Of the 273 infants, 44 (16.1%) were colonized with NTHi. Prevalence of NTHi varied from 14% in infants less than 6 months of age to 32% in infants 19-26 months of age (p=0.003). NTHi colonized infants were more likely to attend daycare (30% vs. 11%), have a recent respiratory infection (68% vs. 38%), recent antibiotic use (27% vs. 9%), and caregiver reported asthma (11% vs. 1%) compared with other infants (p<0.001). Of the 44 infants colonized with NTHi, we identified 33 different MLSTs. Nine (20.5%) of the 44 infant-primary caregiver dyads were colonized with NTHi and 7/9 shared identical NTHi strains. We also found beta-lactamase negative NTHi with minimum inhibitory concentrations >2 μg/mL for amoxicillin and beta-lactamase positive NTHi with minimum inhibitory concentrations >2 μg/mL for amoxicillin clavulanate.
We found substantial diversity by MLST analysis among NTHi isolates from this community. Infant-primary caregiver dyads usually carried the same strain of NTHi, suggesting that infant-primary caregiver transmission is occurring.
Nontypeable Haemophilus influenzae; multilocus sequence typing; prevalence; diversity; transmission
A second generation 13 valent pneumococcal conjugate vaccine (PCV13) was licensed and recommended for universal immunization of children through age five years in 2010. Its introduction is intended to address the residual burden of pneumococcal diseases that persists a decade after the introduction of PCV7.
Immunization with PCV7 has resulted in a substantial decline in pneumococcal diseases caused by vaccine serotypes in both vaccinated and unvaccinated persons in the US. However an increase in disease due to non vaccine serotypes, including empyema; the emergence of multidrug, including ceftriaxone, resistant serotype 19A strains; and the need for broader serotype coverage to address the global disease burden provides a rationale for a second generation conjugate vaccine that includes serotypes 1, 3, 5, 6A, 7F and 19A.
This article reviews the lessons learned from a decade of experience with PCV7, the increasing problem of disease due to non-vaccine serotypes, and the likelihood of PCV13 to impact the residual disease burden. We contrast the potential differences in prevention of invasive pneumococcal disease (IPD) compared to nonbacteremic pneumonia and acute otitis media. We conclude with the current recommendations for PCV13 providing a rationale for immunization through age 5 years to create both direct and indirect protection in the population.
Pneumococcal disease; Conjugate vaccine; Nonvaccine serotypes; AAP recommendations; Catch up regimen
The acute phase response is an evolutionarily conserved reaction in which physiological stress triggers the liver to remodel the blood proteome. Although thought to be involved in immune defense, the net biological effect of the acute phase response remains unknown. As the acute phase response is stimulated by diverse cytokines that activate either NF-κB or STAT3, we hypothesized that it could be eliminated by hepatocyte-specific interruption of both transcription factors. Here, we report that the elimination in mice of both NF-κB p65 (RelA) and STAT3, but neither alone, abrogated all acute phase responses measured. The failure to respond was consistent across multiple different infectious, inflammatory, and noxious stimuli, including pneumococcal pneumonia. When the effects of infection were analyzed in detail, pneumococcal pneumonia was found to alter the expression of over a thousand transcripts in the liver. This outcome was inhibited by the combined loss of RelA and STAT3. Moreover, this interruption of the acute phase response increased mortality and exacerbated bacterial dissemination during pneumonia, possibly as a result of acute humoral enhancement of macrophage opsonophagocytosis, which was impaired in the mutant mice. Thus, we conclude that RelA and STAT3 are essential for stress-induced transcriptional remodeling in the liver and the subsequent activation of the acute phase response, whose functional role includes compartmentalization of local infection.
MLST; conjugate vaccination; Streptococcus pneumoniae; nasopharyngeal carriage
Pneumococcal type 1 pilus proteins have been proposed as potential vaccine candidates. Following conjugate pneumococcal vaccination, the prevalence of the pneumococcal type 1 pilus declined dramatically, a decline associated with the elimination of vaccine-type (VT) strains. Here we show that between 2004 and 2007, there has been a significant increase in pilus prevalence, now exceeding rates from the pre-conjugate vaccine era. This increase is primarily due to non-VT strains. These emerging piliated non-VT strains are mostly novel clones, with some exceptions. The rise in pilus type 1 frequency across multiple distinct genetic backgrounds suggests that the pilus may confer an intrinsic advantage.
S. pneumoniae pilus; PCV7; vaccine- and non-vaccine-types
Invasive pneumococcal disease (IPD) has been reduced in the US following conjugate vaccination (PCV7) targeting seven pneumococcal serotypes in 2000. However, increases in IPD due to other serotypes have been observed, in particular 19A. How much this “serotype replacement” will erode the benefits of vaccination and over what timescale is unknown. We used a population genetic approach to test first whether the selective impact of vaccination could be detected in a longitudinal carriage sample, and secondly how long it persisted for following introduction of vaccine in 2000. To detect the selective impact of the vaccine we compared the serotype diversity of samples from pneumococcal carriage in Massachusetts children collected in 2001, 2004 and 2007 with others collected in the pre-vaccine era in Massachusetts, the UK and Finland. The 2004 sample was significantly (p >0.0001) more diverse than pre-vaccine samples, indicating the selective pressure of vaccination. The 2007 sample showed no significant difference in diversity from the pre-vaccine period, and exhibited similar population structure, but with different serotypes. In 2007 the carriage frequency of 19A was similar to that of the most common serotype in pre-vaccine samples. We suggest that serotype replacement involving 19A may be complete in Massachusetts due to similarities in population structure to pre-vaccine samples. These results suggest that the replacement phenomenon occurs rapidly with high vaccine coverage, and may allay concerns about future increases in disease due to 19A. For other serotypes, the future course of replacement disease remains to be determined.
Streptococcus pneumoniae; Infectious disease epidemiology; Nasopharyngeal carriage; Population genetics
Non-vaccine Streptococcus pneumoniae serotypes are increasingly associated with disease. We evaluated isolates of the same sequence type (ST199) but different serotype (15B/C, 19A) for growth in vitro, and pathogenic potential in a chinchilla otitis media model. We also developed a qPCR assay to quantitatively assess each isolate, circumventing the need for selectable markers. In vitro studies showed faster growth of serotype 19A over 15B/C. Both were equally capable of colonization and middle ear infection in this model. Serotype 19A is included in new conjugate vaccine formulations while serotype 15B/C is not. Non-capsular vaccine targets will be important in disease prevention efforts.
Streptococcus pneumoniae; conjugate vaccine; qPCR assay
The goals were to assess serial changes in Streptococcus pneumoniae serotypes and antibiotic resistance in young children and to evaluate whether risk factors for carriage have been altered by heptavalent pneumococcal conjugate vaccine (PCV7).
Nasopharyngeal specimens and questionnaire/medical record data were obtained from children 3 months to <7 years of age in primary care practices in 16 Massachusetts communities during the winter seasons of 2000–2001 and 2003–2004 and in 8 communities in 2006–2007. Antimicrobial susceptibility testing and serotyping were performed with S pneumoniae isolates.
We collected 678, 988, and 972 specimens during the sampling periods in 2000–2001, 2003–2004, and 2006–2007, respectively. Carriage of non-PCV7 serotypes increased from 15% to 19% and 29% (P < .001), with vaccine serotypes decreasing to 3% of carried serotypes in 2006–2007. The relative contribution of several non-PCV7 serotypes, including 19A, 35B, and 23A, increased across sampling periods. By 2007, commonly carried serotypes included 19A (16%), 6A (12%), 15B/C (11%), 35B (9%), and 11A (8%), and high-prevalence serotypes seemed to have greater proportions of penicillin nonsusceptibility. In multivariate models, common predictors of pneumococcal carriage, such as child care attendance, upper respiratory tract infection, and the presence of young siblings, persisted.
The virtual disappearance of vaccine serotypes in S pneumoniae carriage has occurred in young children, with rapid replacement with penicillin-nonsusceptible nonvaccine serotypes, particularly 19A and 35B. Except for the age group at highest risk, previous predictors of carriage, such as child care attendance and the presence of young siblings, have not been changed by the vaccine.
Streptococcus pneumoniae; pneumococcal conjugate vaccine; antibiotic resistance; serotype; colonization
Sialic acid has been shown to be a major virulence determinant in the pathogenesis of otitis media caused by the bacterium Haemophilus influenzae. This study aimed to characterise the expression of genes required for the metabolism of sialic acid and to investigate the role of these genes in virulence.
Using qRT-PCR, we observed decreased transcriptional activity of genes within a cluster that are required for uptake and catabolism of 5-acetyl neuraminic acid (Neu5Ac), when bacteria were cultured in the presence of the sugar. We show that these uptake and catabolic genes, including a sialic acid regulatory gene (siaR), are highly conserved in the H. influenzae natural population. Mutant strains were constructed for seven of the nine genes and their influence upon LPS sialylation and resistance of the bacteria to the killing effect of normal human serum were assessed. Mutations in the Neu5Ac uptake (TRAP transporter) genes decreased virulence in the chinchilla model of otitis media, but the attenuation was strain dependent. In contrast, mutations in catabolism genes and genes regulating sialic acid metabolism (siaR and crp) did not attenuate virulence.
The commensal and pathogenic behaviour of H. influenzae involves LPS sialylation that can be influenced by a complex regulatory interplay of sialometabolism genes.
Hepatitis B virus (HBV) is an important cause of comorbidity in human immunodeficiency virus (HIV)–infected individuals. The immunogenicity of HBV vaccination in children receiving highly active antiretroviral therapy (HAART) was investigated.
HIV-infected children receiving HAART who had low to moderate HIV loads and who had previously received ≥3 doses of HBV vaccine were given an HBV vaccine booster. Concentrations of antibody to hepatitis B surface antigen (anti-HBs) were determined before vaccination and at weeks 8, 48, and 96. A subset of subjects was administered a subsequent dose, and anti-HBs was measured before and 1 and 4 weeks later.
At entry, 24% of 204 subjects were seropositive. Vaccine response occurred in 46% on the basis of seropositivity 8 weeks after vaccination and in 37% on the basis of a ≥4-fold rise in antibody concentration. Of 69 subjects given another vaccination 4–5 years later, immunologic memory was exhibited by 45% on the basis of seropositivity 1 week after vaccination and by 29% on the basis of a ≥4-fold rise in antibody concentration at 1 week. Predictors of response and memory included higher nadir and current CD4 cell percentage, higher CD19 cell percentage, and undetectable HIV load.
HIV-infected children frequently lack protective levels of anti-HBs after previous HBV vaccination, and a significant proportion of them do not respond to booster vaccination or demonstrate memory despite receiving HAART, leaving this population insufficiently protected from infection with HBV.
Because pneumococcal serotype 6C was previously not distinguished from serotype 6A, the impact of the 7-valent pneumococcal conjugate vaccine (PCV7) on the carriage of serotype 6C is unknown.
The nasopharyngeal (NP) prevalence of the 6C serotype was determined using 1326 pneumococcal isolates collected from 7 cohorts of Massachusetts children between 1994 and 2007. Initially, the isolates were serotyped using the quellung reaction; subsequently, stored specimens of all putative 6A isolates were tested for 6C using monoclonal antibodies. The opsonophagocytic and antibiotic susceptibilities of the isolates were determined.
The prevalence of 6A was 9.6% (33/343) before 2001, 8.0% (18/226) in 2004, and 2.9% (12/416) in 2007. In contrast, the prevalence of 6C was 0.6% (2/343) before 2001, 2.2% (5/226) in 2004, and 8.7% (36/416) in 2007 (P < .001 for 2/343 vs. 36/416). 6C isolates from 2007 were more susceptible to antibiotics than were 6A isolates. PCV7 induced a low ability to opsonize different isolates of 6C.
Among NP isolates, the prevalence of 6C isolates has increased and the prevalence of 6A isolates has decreased since the introduction of PCV7 in Massachusetts in 2000. The observed increase in serotype 6C prevalence may be explained by the induction by PCV7 of low amounts of functional anti-6C antibody, compared with anti-6A and anti-6B antibodies.
Strategies to limit complement deposition on Streptococcus pneumoniae are established as virulence features for invasive disease, but their role in respiratory tract infection requires further analysis. We evaluated complement C3 protein deposition on discordant S. pneumoniae isolates of the same serotype (6A) and their capacity to cause nasopharyngeal (NP) colonization and experimental otitis media (EOM) in an animal model. We compared C3 binding to five 6A isolates from asymptomatic NP carriers with five 6A strains that caused invasive disease, and we observed less C3 (∼10-fold less fluorescence) binding to invasive isolates. We selected two high-level C3-binding carriage and two low-level C3-binding invasive 6A isolates for further study. In the EOM model, 11/12 (92%) ears challenged with a low-level C3-binding 6A strain became infected. Only 2/8 (25%) ears challenged with the discordant high-level C3-binding 6A isolate developed disease (P = 0.005). Results with the second discordant 6A isolate pair were comparable. Cobra venom factor (CoVF) treatment, which depletes C3 and consumes complement, restored virulence of the high-level C3-binding strain; 8/8 (100%) ears in CoVF-treated animals developed EOM compared to only 25% of ears in naïve animals (P = 0.007). These studies demonstrate the critical role for complement evasion in pneumococcal EOM. Colonization with carriage isolates that bound high levels of C3 caused EOM in fewer animals compared to low-level C3-binding invasive strains. Thus, limiting C3 deposition on the surface of S. pneumoniae correlates with increased incidence of EOM following NP colonization and barotrauma in the animal model.
The incidence of community-associated methicillin-resistant Staphylococcus aureus (MRSA) has risen dramatically in the U.S., particularly among children. Although Streptococcus pneumoniae colonization has been inversely associated with S. aureus colonization in unvaccinated children, this and other risk factors for S. aureus carriage have not been assessed following widespread use of the heptavalent pneumococcal conjugate vaccine (PCV7). Our objectives were to (1) determine the prevalence of S. aureus and MRSA colonization in young children in the context of widespread use of PCV7; and (2) examine risk factors for S. aureus colonization in the post-PCV7 era, including the absence of vaccine-type S. pneumoniae colonization.
Swabs of the anterior nares (S. aureus) were obtained from children enrolled in an ongoing study of nasopharyngeal pneumococcal colonization of healthy children in 8 Massachusetts communities. Children 3 months to <7 years of age seen for well child or sick visits in primary care offices from 11/03–4/04 and 10/06–4/07 were enrolled. S. aureus was identified and antibiotic susceptibility testing was performed. Epidemiologic risk factors for S. aureus colonization were collected from parent surveys and chart reviews, along with data on pneumococcal colonization. Multivariate mixed model analyses were performed to identify factors associated with S. aureus colonization.
Among 1,968 children, the mean age (SD) was 2.7 (1.8) years, 32% received an antibiotic in the past 2 months, 2% were colonized with PCV7 strains and 24% were colonized with non-PCV7 strains. The prevalence of S. aureus colonization remained stable between 2003–04 and 2006–07 (14.6% vs. 14.1%), while MRSA colonization remained low (0.2% vs. 0.9%, p = 0.09). Although absence of pneumococcal colonization was not significantly associated with S. aureus colonization, age (6–11 mo vs. ≥5 yrs, OR 0.39 [95% CI 0.24–0.64]; 1–1.99 yrs vs. ≥5 yrs, OR 0.35 [0.23–0.54]; 2–2.99 yrs vs. ≥5 yrs, OR 0.45 [0.28–0.73]; 3–3.99 yrs vs. ≥5 yrs, OR 0.53 [0.33–0.86]) and recent antibiotic use were significant predictors in multivariate models.
In Massachusetts, S. aureus and MRSA colonization remained stable from 2003–04 to 2006–07 among children <7 years despite widespread use of pneumococcal conjugate vaccine. S. aureus nasal colonization varies by age and is inversely correlated with recent antibiotic use.
Human immunodeficiency virus (HIV)-infected children and adolescents who are failing antiretrovirals may have a better virologic response when drug exposures are increased, using higher protease inhibitor doses or ritonavir boosting. We studied the pharmacokinetics and safety of high-dose lopinavir-ritonavir (LPV/r) in treatment-experienced patients, using an LPV/r dose of 400/100 mg/m2 orally every 12 h (p.o. q12h) (without nonnucleoside reverse transcriptase inhibitor [NNRTI]), or 480/120 mg/m2 p.o. q12h (with NNRTI). We calculated the LPV inhibitory quotient (IQ), and when the IQ was <15, saquinavir (SQV) 750 mg/m2 p.o. q12h was added to the regimen. We studied 26 HIV-infected patients. The median age was 15 years (range, 7 to 17), with 11.5 prior antiretroviral medications, 197 CD4 cells/ml, viral load of 75,577 copies/ml, and a 133-fold change in LPV resistance. By treatment week 2, 14 patients had a viral-load decrease of >0.75 log10, with a median maximal decrease in viral load of −1.57 log10 copies/ml at week 8. At week 2, 19 subjects showed a median LPV area under the concentration-time curve (AUC) of 157.2 (range, 62.8 to 305.5) μg·h/ml and median LPV trough concentration (Ctrough) of 10.8 (range, 4.1 to 25.3) μg/ml. In 16 subjects with SQV added, the SQV median AUC was 33.7 (range, 4.4 to 76.5) μg·h/ml and the median SQV Ctrough was 2.1 (range, 0.2 to 4.1) μg/ml. At week 24, 18 of 26 (69%) subjects remained in the study. Between weeks 24 and 48, one subject withdrew for nonadherence and nine withdrew for persistently high virus load. In antiretroviral-experienced children and adolescents with HIV, high doses of LPV/r with or without SQV offer safe options for salvage therapy, but the modest virologic response and the challenge of adherence to a regimen with a high pill burden may limit the usefulness of this approach.
The typically recovered quantity of nontypeable Haemophilus influenzae (NTHi) bacteria in an ex vivo middle ear (ME) aspirate from the chinchilla model of experimental otitis media is insufficient for direct analysis of gene expression by microarray or of lipopolysaccharide glycoforms by mass spectrometry. This prompted us to investigate a strategy of multiple consecutive lavage samplings to increase ex vivo bacterial recovery. As multiple consecutive lavage samples significantly increased the total number of bacterial CFU collected during nasopharyngeal colonization or ME infection, this led us to evaluate whether bacteria sequentially acquired from consecutive lavages were similar. Comparative observation of complete ex vivo sample series by microscopy initially revealed ME inflammatory fluid consisting solely of planktonic-phase NTHi. In contrast, subsequent lavage samplings of the same infected ear revealed the existence of bacteria in two additional growth states, filamentous and biofilm encased. Gene expression analysis of such ex vivo samples was in accord with different bacterial growth phases in sequential lavage specimens. The existence of morphologically distinct NTHi subpopulations with varying levels of gene expression indicates that the pooling of specimens requires caution until methods for their separation are developed. This study based on multiple consecutive lavages is consistent with prior reports that NTHi forms a biofilm in vivo, describes the means to directly acquire ex vivo biofilm samples without sacrificing the animal, and has broad applicability for a study of mucosal infections. Moreover, this approach revealed that the actual burden of bacteria in experimental otitis media is significantly greater than was previously reported. Such findings may have direct implications for antibiotic treatment and vaccine development against NTHi.
Nontypeable (NT) Haemophilus influenzae is an important cause of otitis media in children. We have shown previously that NT H. influenzae mutants defective in their ability to sialylate lipopolysaccharide (LPS), called siaB mutants, show attenuated virulence in a chinchilla model of experimental otitis media (EOM). We show that complement is a key arm of host innate immunity against NT H. influenzae-induced EOM. Depleting complement in chinchillas by use of cobra venom factor (CoVF) rendered two otherwise avirulent siaB mutants fully virulent and able to cause EOM with severity similar to that of wild-type strains. Clearance of infection caused by siaB mutants in CoVF-treated animals coincided with reappearance of C3. Wild-type strains were more resistant to direct complement-mediated killing than their siaB mutants. The serum-resistant strain bound less C3 and C4 than the serum-sensitive strain. Neither NT H. influenzae strain tested bound factor H (alternative complement pathway regulator). Selective activation of the alternative pathway resulted in more C3 binding to siaB mutants. LPS sialylation had a more profound impact on the amount of alternative-pathway-mediated C3 binding (∼5-fold decrease in fluorescence) when LPS was the main C3 target, as occurred on the more serum-resistant strain. In contrast, only an ∼1.5-fold decrease in fluorescence intensity of C3 binding was seen with the serum-sensitive strain, where surface proteins predominantly bound C3. Differences in binding sites for C3 and C4 may account for variations in serum resistance between NT H. influenzae strains, which in turn may impact their virulence. These data demonstrate a central role for complement in innate immune defenses against NT H. influenzae infections and specifically EOM.
Treatment of acute otitis media (AOM) with azithromycin results in apparent clinical success, but tympanocentesis performed 4 to 6 days after initiation of therapy in children with nontypeable Haemophilus influenzae (NTHI) recovered from initial middle ear cultures demonstrates persistence of infection in more than 50% of episodes. We sought to determine the effect of azithromycin at different doses on the density of middle ear infection due to NTHI to provide additional understanding of this dichotomy between clinical and microbiologic outcome measures in AOM. In a chinchilla model of experimental otitis media (EOM), animals treated with placebo were compared to animals receiving a single daily dose 30 or 120 mg of azithromycin per kg of body weight per day for 5 days. Microbiologic outcome was assessed by obtaining quantitative cultures from the middle ear during a 5-day course and for 1 week following therapy. Azithromycin concentrations were measured to ascertain whether a concentration-dependent effect was present. Azithromycin at 30 and 120 mg/kg/day demonstrated a dose-dependent effect on the quantitative assessment of middle ear infection due to NTHI. A 30-mg/kg dose of azithromycin daily resulted in levels in serum and areas under the serum concentration-time curve at 24 h comparable to published data obtained with children given azithromycin at 5 to 10 mg/kg in multiday regimens. Increased doses of azithromycin (120 mg/kg) achieved 2.5- to 4-fold-higher levels in serum and 3- to 6-fold-higher total levels and levels in extracellular middle ear fluid as well as more rapid reduction in bacterial density and a greater proportion of middle ears with complete sterilization than either placebo or the 30-mg/kg/day regimen.
Candidate vaccine antigens for preventing otitis media caused by nontypeable Haemophilus influenzae (NTHI) should possess one or more conserved epitopes. We sought to evaluate the candidacy of P1, a surface-expressed outer membrane protein knowing that this antigen is subject to diversifying selection. Therefore, we selected NTHI strains from among >500 phylogenically variant isolates representative of the diversity found in natural populations of H. influenzae. Twenty-three variants of P1 (≤95% similarity) were identified among 42 strains. When chinchillas were immunized with recombinant P1 (rP1) obtained from one of these isolates (BCH-3), all animals developed antibodies specific for rP1. Immunized animals were protected against disease when challenged with BCH-3, but not with an ompP1 mutant of BCH-3 or a strain (BCH-2) possessing a heterologous P1 (91% identity). We conclude that (i) while P1 induces protection against NTHI-mediated otitis media, development of a polyvalent vaccine reflecting the variability of P1 would be necessary to construct an efficacious vaccine and (ii) use of a phylogenically characterized collection of representative isolates in concert with gene sequencing, cloning, gene inactivation, and animal testing offers an efficient, rational, and rigorous strategy for evaluating the potential problems associated with variability of vaccine targets and specificity of related immune responses.
Viral peptides are recognized by cytotoxic T lymphocytes (CTL) as a complex with major histocompatibility complex (MHC) class I molecules, but the extent to which a single HLA allele can accommodate epitope peptides of different length and sequence is not well characterized. Here we report the identification of clonal CTL responses from the same donor that independently recognize one of two HLA-B57-restricted epitopes, KAFSPEVIPMF (KF11; p24Gag residues 30 to 40) and KAFSPEVI (KF8; p24Gag residues 30 to 37). Although lysis studies indicated that the KF11 peptide stabilized the HLA-B57-peptide complex more efficiently than the KI8 peptide, strong clonal responses were directed at each epitope. In samples from a second donor, the same phenomenon was observed, in which distinct CTL clones recognized peptide epitopes presented by the same HLA class I allele (in this case, HLA-A3) which were entirely overlapping. These data are relevant to the accurate characterization of CTL responses, which is fundamental to a detailed understanding of MHC class I-restricted immunity. In addition, these studies demonstrate marked differences in the length of peptides presented by HLA-B57, an allele which is associated with nonprogressive human immunodeficiency virus infection.