Ron Dagan

Evaluation and improvement of real-time PCR assays targeting lytA, ply, and psaA genes for detection of pneumococcal DNA.

ABSTRACT The accurate diagnosis of pneumococcal disease has frequently been hampered not only by the difficulties in obtaining isolates of the organism from patient specimens but also by the misidentification of pneumococcus-like viridans group streptococci (P-LVS) as Streptococcus pneumoniae. This is especially critical when the specimen comes from the respiratory tract. In this study, three novel real-time PCR assays designed for the detection of specific sequence regions of the lytA, ply, and psaA genes were developed (lytA-CDC, ply-CDC, and psaA, respectively). These assays showed high sensitivity (<10 copies for lytA-CDC and ply-CDC and an approximately twofold less sensitivity for psaA). Two additional real-time PCR assays for lytA and ply described previously for pneumococcal DNA detection were also evaluated. A panel of isolates consisting of 67 S. pneumoniae isolates (44 different serotypes and 3 nonencapsulated S. pneumoniae isolates from conjunctivitis outbreaks) and 104 nonpneumococcal isolates was used. The 67 S. pneumoniae isolates were reactive in all five assays. The new real-time detection assays targeting the lytA and psaA genes were the most specific for the detection of isolates confirmed to be S. pneumoniae, with lytA-CDC showing the greatest specificity. Both ply PCRs were positive for all isolates of S. pseudopneumoniae, along with 13 other isolates of other P-LVS isolates confirmed to be non-S. pneumoniae by DNA-DNA reassociation. Thus, the use of the ply gene for the detection of pneumococci can lead to false-positive reactions in the presence of P-LVS. The five assays were applied to 15 culture-positive cerebrospinal fluid specimens with 100% sensitivity; and serum and ear fluid specimens were also evaluated. Both the lytA-CDC and psaA assays, particularly the lytA-CDC assay, have improved specificities compared with those of currently available assays and should therefore be considered the assays of choice for the detection of pneumococcal DNA, particularly when upper respiratory P-LVS might be present in the clinical specimen.

Reduction of Nasopharyngeal Carriage of Streptococcus pneumoniae after Administration of a 9-Valent Pneumococcal Conjugate Vaccine to Toddlers Attending Day Care Centers

A double-blind, randomized study involving 264 toddlers attending day care centers was conducted to document the effect of a 9-valent pneumococcal conjugate vaccine on the carriage rate of pneumococci. Of 3750 cultures done on nasopharyngeal samples obtained from subjects during a 2-year follow-up period after vaccination, 65% were positive for Streptococcus pneumoniae. In all age windows, the rate of carriage of vaccine-type pneumococci was lower among subjects who received the pneumococcal vaccine than among control subjects, because the acquisition rate was lower in the former group. The effect was most pronounced among subjects aged < or =36 months. The sample size enabled us to study protection against carriage of S. pneumoniae serotypes 6B, 9V, 14, 19F, and 23F; significant protection against all serotypes except 19F was seen in the pneumococcal-vaccine group. The rate of carriage of serotype 6A (not included in the vaccine) was also reduced significantly, but the rate of carriage of serotype 19A (not included in the vaccine) was not. The rate of carriage of non-vaccine-type pneumococci (excluding serotype 6A) was higher in the pneumococcal-vaccine group than in the control group.

Serological criteria for evaluation and licensure of new pneumococcal conjugate vaccine formulations for use in infants

The World Health Organization (WHO) is undertaking a series of consultations on serological criteria for the evaluation and licensure of new formulations/combinations or different vaccination schedules of pneumococcal conjugate vaccines. The lack of a definitive serological correlate of protection and the multiplicity of antigens involved, especially since the clinical efficacy of most of the individual serotypes represented in the only licensed vaccine has not been established, are hindering the formulation of criteria for licensure of new formulations or combinations of the vaccine. This report analyses the various options with their relative merits and drawbacks and provides preliminary recommendations as guidance to regulatory agencies in evaluating these vaccines for the purposes of licensure. More detailed recommendations for production and control of pneumococcal conjugate vaccines, including criteria for evaluation for licensure, are currently being drafted.

The fundamental link between pneumococcal carriage and disease

Streptococcus pneumoniae (pneumococcus) is a major cause of worldwide mortality and morbidity, and to a large extent is vaccine-preventable. Nasopharyngeal carriage of pneumococcus precedes disease and is the source of pneumococcal spread between people. The use of vaccine effect on carriage as part of the vaccine licensure and post-vaccine introduction evaluation could facilitate and expand the licensure of new, life-saving pneumococcal vaccines and enable a comprehensive estimate of population effects after vaccine introduction. The authors provide a review of the evidence supporting pneumococcal carriage at the individual level as an immediate and necessary precursor to pneumococcal disease. Based on such a causal link between carriage and disease, the authors emphasize the role of information on pneumococcal carriage in vaccine trials and in public health decision-making.

Potential interventions for the prevention of childhood pneumonia: geographic and temporal differences in serotype and serogroup distribution of sterile site pneumococcal isolates from children—implications for vaccine strategies

Streptococcus pneumoniae is a leading cause of fatal bacterial pneumonia in young children. Pneumococcal polysaccharide vaccines have not been promoted for use in young children because many constituent serotypes are not immunogenic in children < 2 years old. Conjugating pneumococcal polysaccharide epitopes to a protein carrier would likely increase vaccine immunogenicity in children. We reviewed published and unpublished pneumococcal serotype and serogroup data from 16 countries on 6 continents to determine geographic and temporal differences in serotype and serogroup distribution of sterile site pneumococcal isolates among children and to estimate coverage of proposed and potential pneumococcal conjugate vaccine formulas. The most common pneumococcal serotypes or groups from developed countries were, in descending order, 14, 6, 19, 18, 9, 23, 7, 4, 1 and 15. In developing countries the order was 6, 14, 8, 5, 1, 19, 9, 23, 18, 15 and 7. Development of customized heptavalent vaccine formulas, one for use in all developed countries and one for use in all developing countries, would not provide substantially better coverage against invasive pneumococcal disease than two currently proposed heptavalent formulas. An optimal nanovalent vaccine for global use would include serotypes 1, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F. Geographic and temporal variation in pneumococcal serotypes demonstrates the need for a species-wide pneumococcal vaccine.

Association of serotype with risk of death due to pneumococcal pneumonia: a meta-analysis.

BACKGROUND The 92 capsular serotypes of Streptococcus pneumoniae differ greatly in nasopharyngeal carriage prevalence, invasiveness, and disease incidence. There has been some debate, though, regarding whether serotype independently affects the outcome of invasive pneumococcal disease (IPD). Published studies have shown variable results with regard to case-fatality ratios for specific serotypes and the role of host factors in affecting these relationships. We evaluated whether risk of death due to IPD is a stable serotype-associated property across studies and then compared the pooled effect estimates with epidemiologic and biological correlates. METHODS We performed a systematic review and meta-analysis of serotype-specific disease outcomes for patients with pneumonia and meningitis. Study-specific estimates of risk of death (risk ratio [RR]) were pooled from 9 studies that provided serotype-specific data on pneumonia and meningitis using a random-effects method with serotype 14 as the reference. Pooled RRs were compared with RRs from adults with low comorbidity scores to evaluate potential confounding by host factors. RESULTS Significant differences were found in the RR estimates among serotypes in patients with bacteremic pneumonia. Overall, serotypes 1, 7F, and 8 were associated with decreased RRs, and serotypes 3, 6A, 6B, 9N, and 19F were associated with increased RRs. Outcomes among meningitis patients did not differ significantly among serotypes. Serotypes with increased RRs had a high carriage prevalence, had low invasiveness, and were more heavily encapsulated in vitro. CONCLUSIONS These results suggest that IPD outcome, like other epidemiologic measures, is a stable serotype-associated property.

Reduction of pneumococcal nasopharyngeal carriage in early infancy after immunization with tetravalent pneumococcal vaccines conjugated to either tetanus toxoid or diphtheria toxoid

BACKGROUND Pneumococcal nasopharyngeal colonization is important for transmission of the organisms. We assessed the ability of two tetravalent conjugate vaccines administered in early infancy to prevent carriage of vaccine-related pneumococci. METHODS A vaccine containing pneumococcal type 6B, 14, 19F and 23F polysaccharide conjugated to tetanus toxoid (Pnc-T) and a vaccine containing the same four polysaccharides conjugated to diphtheria toxoid (Pnc-D) were compared with placebo, in a double blinded study (25 infants per group). Vaccines (or placebo) were injected at 2, 4 and 6 months of age. At 12 months of age a native (nonconjugate) polysaccharide vaccine was administered as a booster. Serum type-specific anticapsular antibody concentrations were measured and nasopharyngeal cultures were obtained at 2, 4, 6, 7, 12 and 13 months of age. RESULTS In general carriage of all pneumococci (vaccine- and non-vaccine-related) was low at age 2 months and increased with age. However, for the vaccine-related serotypes (6A, 6B, 14, 19F and 23F) carriage was not increased with age in Pnc-D or Pnc-T recipients. Of all cultures obtained after the full primary series, 7 of 72 (10%), 3 of 62 (5%) and 19 or 70 (27%) were positive for the vaccine-related pneumococcal serotypes among the Pnc-D, Pnc-T and placebo recipients, respectively (P = 0.001 for Pnc-D vs. placebo; P = 0.014 for Pnc-T vs. placebo). Most of the antibiotic-resistant isolates belonged to the vaccine-related serotypes. CONCLUSIONS A significant reduction in the carriage of vaccine-related strains after administration of conjugate vaccines was observed. These preliminary results suggest that transmission of specific pneumococcal serotypes most often associated with disease and antibiotic resistance may at least partially be controlled by immunization.

Combined vaccination of Haemophilus influenzae type b conjugate and diphtheria-tetanus-pertussis containing acellular pertussis

After the introduction of effective Haemophilus influenzae type b (Hib) conjugate vaccines, clinical practice has driven the development of combination vaccines comprising Hib conjugates with the infant diphtheria-tetanus-pertussis (DTP) vaccines. However, when such combinations contain an acellular pertussis component (Pa), the antibody response to Hib is lower than that with separate injections and doubts have been raised about their efficacy. We believe that such concerns are unwarranted, since the serological correlates of efficacy previously applied for Hib polysaccharide vaccines seem inappropriate for Hib conjugates. Furthermore, our own studies have shown that the lower antibody responses are not associated with impaired function of the antibodies induced, nor, and possibly more importantly, with the induction of immune memory against Hib. Therefore, with the proviso that careful clinical surveillance of Hib disease is maintained, we encourage the introduction of DTPa-Hib combinations to facilitate the inclusion of Hib into the already crowded childhood immunisation schedule.

Nasopharyngeal Carriage of Streptococcus pneumoniae by Adults and Children in Community and Family Settings

The rate of Streptococcus pneumoniae carriage among adults was compared with that among children (age, < or =6 years) in the same population. Nasopharyngeal culture results for 1300 adults and 404 children were analyzed. S. pneumoniae was carried by only 4% of the adults, compared with 53% of children in the same community. Young age, day care center attendance, having young siblings, and no antibiotic use during the month before screening were associated with the high carriage rate among children, whereas the only risk factor associated with carriage among adults was the presence of a respiratory infection on the screening day. S. pneumoniae serotype distribution and antibiotic resistance patterns differed between adults and children. Isolates of the same serotype--even of the same clone--differed in their antibiotic susceptibility patterns between children and adults. In a subanalysis of 151 pairs of children and their parents and of 32 pairs of siblings, intrafamilial transmission of S. pneumoniae could not be demonstrated.

Identification and Characterization of a Novel Family of Pneumococcal Proteins That Are Protective against Sepsis

ABSTRACT Four pneumococcal genes (phtA, phtB, phtD, andphtE) encoding a novel family of homologous proteins (32 to 87% identity) were identified from the Streptococcus pneumoniae genomic sequence. These open reading frames were selected as potential vaccine candidates based upon their possession of hydrophobic leader sequences which presumably target these proteins to the bacterial cell surface. Analysis of the deduced amino acid sequences of these gene products revealed the presence of a histidine triad motif (HxxHxH), termed Pht (pneumococcal histidine triad) that is conserved and repeated several times in each of the four proteins. The four pht genes (phtA, phtB, phtD, and a truncated version of phtE) were expressed inEscherichia coli. A flow cytometry-based assay confirmed that PhtA, PhtB, PhtD and, to a lesser extent, PhtE were detectable on the surface of intact bacteria. Recombinant PhtA, PhtB, and PhtD elicited protection against certain pneumococcal capsular types in a mouse model of systemic disease. These novel pneumococcal antigens may serve as effective vaccines against the most prevalent pneumococcal serotypes.