Amelieke J. H. Cremers

The adult nasopharyngeal microbiome as a determinant of pneumococcal acquisition

BackgroundSeveral cohort studies have indicated associations between S. pneumoniae and other microbes in the nasopharynx. To study causal relationships between the nasopharyngeal microbiome and pneumococcal carriage, we employed an experimental human pneumococcal carriage model. Healthy adult volunteers were assessed for pneumococcal carriage by culture of nasal wash samples (NWS). Those without natural pneumococcal carriage received an intranasal pneumococcal inoculation with serotype 6B or 23F. The composition of the nasopharyngeal microbiome was longitudinally studied by 16S rDNA pyrosequencing on NWS collected before and after challenge.ResultsAmong 40 selected volunteers, 10 were natural carriers and 30 were experimentally challenged. At baseline, five distinct nasopharyngeal microbiome profiles were identified. The phylogenetic distance between microbiomes of natural pneumococcal carriers was particularly large compared to non-carriers. A more diverse microbiome prior to inoculation was associated with the establishment of pneumococcal carriage. Perturbation of microbiome diversity upon pneumococcal challenge was strain specific. Shifts in microbiome profile occurred after pneumococcal exposure, and those volunteers who acquired carriage more often diverted from their original profile. S. pneumoniae was little prominent in the microbiome of pneumococcal carriers.ConclusionPneumococcal acquisition in healthy adults is more likely to occur in a diverse microbiome and appears to promote microbial heterogeneity.

The post-vaccine microevolution of invasive Streptococcus pneumoniae

The 7-valent pneumococcal conjugated vaccine (PCV7) has affected the genetic population of Streptococcus pneumoniae in pediatric carriage. Little is known however about pneumococcal population genomics in adult invasive pneumococcal disease (IPD) under vaccine pressure. We sequenced and serotyped 349 strains of S. pneumoniae isolated from IPD patients in Nijmegen between 2001 and 2011. Introduction of PCV7 in the Dutch National Immunization Program in 2006 preluded substantial alterations in the IPD population structure caused by serotype replacement. No evidence could be found for vaccine induced capsular switches. We observed that after a temporary bottleneck in gene diversity after the introduction of PCV7, the accessory gene pool re-expanded mainly by genes already circulating pre-PCV7. In the post-vaccine genomic population a number of genes changed frequency, certain genes became overrepresented in vaccine serotypes, while others shifted towards non-vaccine serotypes. Whether these dynamics in the invasive pneumococcal population have truly contributed to invasiveness and manifestations of disease remains to be further elucidated. We suggest the use of whole genome sequencing for surveillance of pneumococcal population dynamics that could give a prospect on the course of disease, facilitating effective prevention and management of IPD.

Density and duration of experimental human pneumococcal carriage

The density and duration of pneumococcal carriage are considered to affect the likelihood of transmission and invasive disease. Because of its importance in both spreading and causing disease, carriage has been suggested as an endpoint in future vaccine studies. Culture is the current gold standard for detection, but may not be sensitive enough to detect changes at low density. Healthy adult volunteers received an intranasal inoculation of Streptococcus pneumoniae serotype 6B. Pneumococcal density in nasal washes collected at six time-points post-inoculation was determined by culture and quantitative PCR (qPCR). Natural pneumococcal carriers detected at initial screening were followed in parallel. In 331 nasal washes from 79 volunteers, the sensitivity and specificity of pneumococcal detection by qPCR, as compared with culture, were 92.3% and 75.9%. The estimation of pneumococcal density by culture and qPCR was highly correlated (rs = 0.73, p <0.0001), although qPCR had a lower detection limit. Pneumococcal density fluctuated within a carriage episode, and occasionally fell below the detection limit of both methods. The duration of carriage episodes was underestimated when only one method was used. Similar fluctuations in density were observed in natural carriers. Pneumococcal carriage is a dynamic event. Culture and qPCR are complementary for surveying the density and duration of pneumococcal carriage episodes.

Detection and serotyping of pneumococci in community acquired pneumonia patients without culture using blood and urine samples

BackgroundTreatment of community acquired pneumonia (CAP) patients with antibiotics before laboratory-confirmed diagnosis leads to loss of knowledge on the causative bacterial pathogen. Therefore, an increasing number of pneumococcal infections is identified using non-culture based techniques. However, methods for serotyping directly on the clinical specimen remain scarce. Here we present three approaches for detection and serotyping of pneumococci using samples from patients with CAP.MethodsThe first approach is quantitative PCR (qPCR) analysis on blood samples (n = 211) followed by capsular sequence typing (CST) to identify the serotype. The second approach, a urinary antigen assay (n = 223), designated as inhibition multiplex immunoassay (IMIA), is based on Luminex technology targeting 14 serotypes. The third approach is a multiplex immunoassay (MIA) (n = 171) also based on Luminex technology which detects serologic antibody responses against 14 serotypes. The three alternative assays were performed on samples obtained from 309 adult hospitalized CAP patients in 2007–2010 and the results were compared with those obtained from conventional laboratory methods to detect pneumococcal CAP, i.e. blood cultures, sputum cultures and BinaxNOW® urinary antigen tests.ResultsUsing qPCR, MIA and IMIA, we were able to detect the pneumococcus in samples of 56% more patients compared to conventional methods. Furthermore, we were able to assign a serotype to the infecting pneumococcus from samples of 25% of all CAP patients, using any of the three serotyping methods (CST, IMIA and MIA).ConclusionThis study indicates the usefulness of additional molecular methods to conventional laboratory methods for the detection of pneumococcal pneumonia. Direct detection and subsequent serotyping on clinical samples will improve the accuracy of pneumococcal surveillance to monitor vaccine effectiveness.

Effect of antibiotic streamlining on patient outcome in pneumococcal bacteraemia

OBJECTIVES In blood culture-proven pneumococcal infections, streamlining empirical therapy to monotherapy with a penicillin is preferred in order to reduce the use of broad-spectrum antibiotics. However, adherence to this international recommendation is poor, and curiously it is unclear whether antibiotic streamlining may be harmful to individual patients. We investigated whether streamlining in bacteraemic pneumococcal infections is associated with mortality. METHODS Adults admitted to two Dutch hospitals between 2001 and 2011 with bacteraemic pneumococcal infections were retrospectively included. Detailed clinical data on patient characteristics, comorbidities and severity and outcome of disease were obtained in addition to data on antibiotic treatment. Those eligible for streamlining were selected for further analyses. RESULTS In the 45.8% of cases (126 of 275) where antibiotic treatment was streamlined, a lower mortality rate was observed (6.3% versus 15.4%, P = 0.021). The decision to streamline was only marginally explained by the 38 determinants accounted for. After correction for potential confounders, the OR for death while streamlining was 0.45 (95% CI: 0.18-1.11, P = 0.082) in all cases and 0.35 (95% CI: 0.12-0.99, P = 0.048) specifically in pneumonia cases. CONCLUSIONS Our results suggest that streamlining in eligible pneumococcal bacteraemia cases is safe, irrespective of patient characteristics, severity of disease or empirical treatment regimen.

Impact of Experimental Human Pneumococcal Carriage on Nasopharyngeal Bacterial Densities in Healthy Adults

Colonization of the nasopharynx by Streptococcus pneumoniae is a necessary precursor to pneumococcal diseases that result in morbidity and mortality worldwide. The nasopharynx is also host to other bacterial species, including the common pathogens Staphylococcus aureus, Haemophilus influenzae, and Moraxella catarrhalis. To better understand how these bacteria change in relation to pneumococcal colonization, we used species-specific quantitative PCR to examine bacterial densities in 52 subjects 7 days before, and 2, 7, and 14 days after controlled inoculation of healthy human adults with S. pneumoniae serotype 6B. Overall, 33 (63%) of subjects carried S. pneumoniae post-inoculation. The baseline presence and density of S. aureus, H. influenzae, and M. catarrhalis were not statistically associated with likelihood of successful pneumococcal colonization at this study’s sample size, although a lower rate of pneumococcal colonization in the presence of S. aureus (7/14) was seen compared to that in the presence of H. influenzae (12/16). Among subjects colonized with pneumococci, the number also carrying either H. influenzae or S. aureus fell during the study and at 14 days post-inoculation, the proportion carrying S. aureus was significantly lower among those who were colonized with S. pneumoniae (p = 0.008) compared to non-colonized subjects. These data on bacterial associations are the first to be reported surrounding experimental human pneumococcal colonization and show that co-colonizing effects are likely subtle rather than absolute.

Effects of 7-valent pneumococcal conjugate 1 vaccine on the severity of adult 2 bacteremic pneumococcal pneumonia

PURPOSE The introduction of a 7-valent conjugate pneumococcal vaccine (PCV7) in children largely affected the prevalence of adult pneumococcal pneumonia. In this study we investigated whether the clinical severity of adult bacteremic pneumococcal pneumonia has also altered following the introduction of pediatric PCV7 vaccination. METHODS Adults hospitalized with bacteremic pneumococcal pneumonia between 2001 and June 2011 at two Dutch hospitals were included retrospectively. Clinical data on patient characteristics, comorbidities and severity of disease were obtained and pneumococcal serotypes were determined. RESULTS Among 343 patients investigated, those infected with PCV7 serotypes had a higher PSI score (p=0.0072) and mortality rate (p=0.0083) compared with the remainder of the cohort. Since the introduction of PCV7 the proportion of pneumococcal pneumonias caused by serotypes 1 and 7F (p-values 0.037 and 0.025) increased, as well as the rate of pleural effusion and empyema (p-values 0.011 and 0.049). Whilst de proportion of adults infected with PCV7 serotypes decreased after the introduction of PCV7 (p=0.015), PSI scores in these patients remained higher (p=0.030), although mortality rates between PCV7 and non PCV7 types equalized. After the introduction of PCV7 a marked shortening in hospital stay was observed only among patients infected with non PCV7 serotypes (p=0.019). CONCLUSIONS The introduction of pediatric PCV7 vaccination was accompanied by subtle changes in clinical severity of adult bacteremic pneumococcal pneumonia. Expansion of serotypes covered by pneumococcal vaccination may again influence the clinical presentation of disease.

High pneumococcal density correlates with more mucosal inflammation and reduced respiratory syncytial virus disease severity in infants.

BackgroundRespiratory syncytial virus (RSV) is an important cause of lower respiratory tract infections in infants. A small percentage of the infected infants develops a severe infection, while most of these severely ill patients were previously healthy. It remains unclear why these children develop severe RSV infections. In this study, we investigate whether pneumococcal nasopharyngeal carriage patterns correlate with mucosal inflammation and severity of disease.MethodsIn total, 105 infants hospitalized with RSV infection were included and recovery samples were taken from 42 patients. The presence and density of Streptococcus pneumoniae was determined by RT qPCR to study its relation to viral load, inflammation (MMP-9 and IL-6) and severity of RSV disease.ResultsWe show that pneumococcal presence or absence in the nasopharynx does not correlate with viral load, inflammation or severity of disease. However, when pneumococcus is present in patients, a higher nasopharyngeal pneumococcal density was correlated with a higher RSV load, higher MMP-9 levels and a less severe course of disease.ConclusionsOur results show correlations between S. pneumoniae density and viral load, inflammation and disease severity, suggesting that pneumococcal density may be an indicator for severity in paediatric RSV disease.

The role of ZmpC in the clinical manifestation of invasive pneumococcal disease

INTRODUCTION The clinical severity and course of invasive pneumococcal disease (IPD) differs substantially between patients. Streptococcus pneumoniae harbors large genetic variability. Zinc metalloproteinase C (ZmpC), a secreted pneumococcal protein involved in neutrophil extravasation, inflammation and tissue remodeling, is present in a minority of IPD isolates. We investigated whether the presence of zmpC was associated with the clinical manifestation of IPD. MATERIAL AND METHODS IPD patients admitted to two Dutch hospitals between 2000 and 2013 were included in the study. Detailed clinical data were collected and the serotype and presence of zmpC were determined in the corresponding blood culture isolates. RESULTS ZmpC was present in 21% of the 542 included IPD cases and was mainly associated with serotypes 8, 4, 33A/F and 11A/D. Infection with S. pneumoniae positive for zmpC was more frequently observed in females (p=0.048) and patients with a history of smoking (p=0.033). Although no relation to clinical syndrome was observed, zmpC positive cases more often presented with cough, dyspnea and sepsis (p-values 0.026, 0.001 and 0.018), and more frequently required ICU admission (p=0.011) compared to zmpC negative cases. CONCLUSION The presence of zmpC was associated with a more severe clinical manifestation of IPD. This study demonstrates that information on pneumococcal genetic background may be useful to identify vulnerable individuals, to monitor clinical presentation and to predict the course of IPD.

Avidity of antibodies against infecting pneumococcal serotypes increases with age and severity of disease

ABSTRACT The incidence of invasive pneumococcal disease (IPD) rises with age. Among adult IPD patients, the avidity of antipneumococcal polysaccharide antibodies against the infecting serotype increased with age and severity of disease, indicating that susceptibility to IPD in the elderly may rather be due to flaws in other aspects of opsonophagocytosis.