Interlaboratory Comparison of the Pneumococcal Multiplex Opsonophagocytic Assays and Their Level of Agreement for Determination of Antibody Function in Pediatric Sera.

Opsonophagocytic assays are used to measure functional antibodies important in protection against pneumococcal capsular antigens. There have been efforts to standardize these methods, as the assays are commonly used to measure vaccine immunogenicity. We report here the results from three international laboratories using their own methods, based on the recommended WHO standard method. We tested 30 pediatric sera, before and after administration of a 13-valent conjugate pneumococcal vaccine, against all 13 serotypes. The three laboratories demonstrated good agreement using their own standardized multiplex opsonophagocytosis assay protocols, particularly postimmunization for those serotypes in the vaccine. While serotype-specific IgG methods have already been internationally standardized and are currently used as a measure of vaccine immunogenicity, this report demonstrates that despite minor differences in methods and a minor variation in response to nonvaccine serotypes, the results from opsonophagocytic assays across the three laboratories may be compared with confidence.IMPORTANCE When measuring a functional antibody response to pneumococcal immunization, it is imperative that a specific, reproducible, accurate, and standardized assay with acceptable inter- and intra-assay variation be advocated internationally to allow for meaningful comparison of results between laboratories. We report here the results of a collaboration between 3 international laboratories testing 30 pediatric samples against the 13 serotypes in Prevenar13.

Characterization of 19A-like 19F pneumococcal isolates from Papua New Guinea and Fiji.

Molecular identification of Streptococcus pneumoniae serotype 19F is routinely performed by PCR targeting the wzy gene of the capsular biosynthetic locus. However, 19F isolates with genetic similarity to 19A have been reported in the United States and Brazil. We screened 78 pneumococcal carriage isolates and found six 19F wzy variants that originated from children in Papua New Guinea and Fiji. Isolates were characterized using multilocus sequence typing and opsonophagocytic assays. The 19F wzy variants displayed similar susceptibility to anti-19F IgG antibodies compared to standard 19F isolates. Our findings indicate that these 19F variants may be more common than previously believed.

Pets at birth do not increase allergic disease in at-risk children.

BACKGROUND: The literature is contradictory concerning pet exposure and risk of allergic disease in childhood especially among those with a family history of allergy. OBJECTIVE: To investigate the relationship between cat and dog exposure at birth and allergic outcomes over the first 12 years in a birth cohort selected for familial allergy. METHODS: A prospective birth cohort of 620 infants with a family history of allergic diseases was recruited. Data on pet keeping, family demographics and cord blood samples were collected at birth. Information on childhood wheeze, eczema and hay fever was collected 18 times in the first 2 years, at 7 years and at 12 years. Skin prick tests were conducted at 2, 7 and 12 years, and in parents. Regression analyses were used to investigate the relevant associations while adjusting for potential confounders. RESULTS: Exposure to cats or dogs at birth showed a moderate reduction in risk of wheeze (aOR = 0.76; 95% CI 0.53, 1.09) and hay fever (aOR = 0.71; 0.49, 1.02) after 7 years of age. Protective effects were stronger in children of non-sensitized fathers (aOR wheeze 0.55; 0.31, 0.98; aOR hay fever 0.33; 0.15, 0.77 on exposure to cats alone, or cats or dogs at birth). Pet keeping was not related to cord blood IgE or sensitization from 2 to 12 years. CONCLUSIONS AND CLINICAL RELEVANCE: Pets at birth either decreased or had no effect on allergic disease up to age 12. We found no evidence that exposure to cats or dogs at birth increases the risk of allergic disease in high-risk children.

Serotype-specific avidity is achieved following a single dose of the 7-valent pneumococcal conjugate vaccine, and is enhanced by 23-valent pneumococcal polysaccharide booster at 12 months.

AIM: To evaluate whether the avidity of serotype-specific IgG to pneumococcal serotypes is enhanced by an increased number of doses of the 7-valent pneumococcal conjugate vaccine (PCV) in infancy or by a 12 month 23-valent pneumococcal polysaccharide vaccine (23vPPS) booster, and/or subsequent re-exposure to a small dose of pneumococcal polysaccharide antigens (mPPS) at 17 months. METHODS: Fijian infants aged 6 weeks were recruited, stratified by ethnicity and randomized to 8 groups to receive 0, 1, 2, or 3 doses of PCV, with or without 23vPPS at 12 months. All children received mPPS at 17 months of age. Avidity of serotype-specific IgG for PCV serotypes in the first 12 months and for all 23vPPS serotypes thereafter was assessed by EIA after sodium thiocyanate elution. RESULTS: At one month post primary series, the 2 and 3 PCV dose groups demonstrated similar avidity, with the single dose group tending to have lower avidity. However, by age 9 months, the single dose group had similar avidity to the 2 and 3 PCV groups for most serotypes. The 23vPPS booster enhanced affinity maturation for most serotypes and this was most marked in those groups that received a single PCV dose. There was little further increase following the mPPS. CONCLUSIONS: By 9 months of age, similar avidity can be induced following one, 2 or 3 doses of PCV. A 23vPPS booster at 12 months enhanced affinity maturation with an increase in antibody avidity for most serotypes. Subsequent re-challenge with mPPS at 17 months did not further enhance the avidity of serotype-specific response in the 12 month 23vPPS groups.

Opsonophagocytic activity following a reduced dose 7-valent pneumococcal conjugate vaccine infant primary series and 23-valent pneumococcal polysaccharide vaccine at 12 months of age.

Opsonophagocytic activity (OPA) was measured following reduced infant doses of 7-valent pneumococcal conjugate vaccine (PCV-7) with or without 23-valent pneumococcal polysaccharide vaccine (PPV-23) at 12 months, and subsequent re-exposure to a small dose of pneumococcal polysaccharide antigens (mPPS) at 17 months. Fijian infants were randomized to receive 0, 1, 2, or 3 PCV-7 doses. Half received PPV-23 at 12 months and all received mPPS at 17 months. OPA was performed on up to 14 serotypes. Three and 2 PCV-7 doses resulted in similar OPA for most PCV-7 serotypes up to 9 months and for half of the serotypes at 12 months. A single dose improved OPA compared with the unvaccinated group. PPV-23 significantly improved OPA for all serotypes tested but in general, was associated with diminished responses following re-challenge.

Antibodies to serotype 9V exhibit novel serogroup cross-reactivity following infant pneumococcal immunization.

Cross-reactivity within the pneumococcal immune response was examined in this study. Significant cross-reactivity between serotypes 9V, 15B and 19A was identified in infant post-immunization serum that could not be effectively titrated during specific IgG measurements. Pre-absorption using serotype 9V inhibited this cross-reactivity and normalized titratability in the WHO ELISA for serotypes 15B and 19A. However, this did not affect functional avid IgG and was associated with fewer pneumococcal conjugate vaccine (PCV) doses, suggesting that cross-reactive antibodies were of low avidity. The results in this study have important implications for assessment of vaccine immunogenicity.

Safety and immunogenicity of the 23-valent pneumococcal polysaccharide vaccine at 12 months of age, following one, two, or three doses of the 7-valent pneumococcal conjugate vaccine in infancy.

Fijian infants aged 6 weeks were stratified by ethnicity and randomized to receive 0, 1, 2, or 3 PCV-7 doses with or without the 23-valent pneumococcal polysaccharide vaccine (PPV-23) at 12 months. Strong booster effects for all 7 PCV-7 serotypes were elicited, and for 4/7 serotypes these responses were highest in the single PCV-7 group. There were fourfold rises in GMC for all non-PCV-7 serotypes. By 17 months the PPV-23 group still had significantly higher GMC (each p<0.001) for all serotypes. The PPV-23 was well tolerated and induced excellent responses for all serotypes which were greatest in the single PCV-7 group.

Hyporesponsiveness to re-challenge dose following pneumococcal polysaccharide vaccine at 12 months of age, a randomized controlled trial.

BACKGROUND: To evaluate the immunological impact of the 23-valent pneumococcal polysaccharide vaccine (23vPPS) at 12 months, for children who have received zero to three infant doses of seven-valent pneumococcal conjugate vaccine (PCV), on responses to a subsequent exposure to a small dose of 23vPPS (mPPS). METHODS: Five hundred and fifty-two Fijian infants were stratified by ethnicity and randomized into eight groups to receive zero, one, two, or three PCV doses at 14 weeks, six and 14 weeks, or six, ten, and 14 weeks. Within each group, half received 23vPPS at 12 months and all received mPPS at 17 months. Sera were taken prior and one month post-mPPS. FINDINGS: By 17 months, geometric mean antibody concentrations (GMC) to all 23 serotypes in 23vPPS were significantly higher in children who had received 23vPPS at 12 months compared to those who had not. Post-mPPS, children who had not received the 12 month 23vPPS had a significantly higher GMC for all PCV serotypes compared with those who had (each p<0.02). For the non-PCV serotypes, children who had not received the 12 month 23vPPS had significantly higher GMC for six of 16 non-PCV serotypes (7F, 9N, 12F, 19A, 22F, 33F) than those who did (each p<0.02). After adjusting for the pre-mPPS level, exposure to 23vPPS was associated with a lower response to mPPS for all serotypes (each p<0.001). INTERPRETATION: Despite higher antibody concentrations at 17 months in children who had received 23vPPS at 12 months, the response to a re-challenge was poor for all 23 serotypes compared to children who had not received the 12 month 23vPPS.

Results from an inter-laboratory comparison of pneumococcal serotype-specific IgG measurement and critical parameters that affect assay performance.

Quantitation of specific IgG to polysaccharides (serotypes) of Streptococcus pneumoniae provides the basis for evaluating vaccine efficacy. Different enzyme-linked immunosorbent assay (ELISA) methods are used internationally, making comparisons between laboratories difficult. We undertook an inter-laboratory comparison between two international laboratories performing serotype-specific IgG ELISAs using a panel of well-characterized serum samples: the Murdoch Childrens Research Institute Pneumococcal Laboratory (Melbourne, Australia) and the Vaccine Immunology Laboratory, National Public Health Institute (Helsinki, Finland). While good agreement was found for the inter-laboratory comparison for most serotypes, differences in ELISA methodology influenced specific IgG measurement. Therefore, use of the World Health Organization (WHO)-based ELISA methods for measurement of serotype-specific IgG is reliable, accurate and provides consistent results between international laboratories.

Immunogenicity following one, two, or three doses of the 7-valent pneumococcal conjugate vaccine.

The aim was to identify an appropriate infant pneumococcal vaccination strategy for resource poor countries. Fijian infants received zero, one, two, or three doses of 7-valent pneumococcal conjugate vaccine (PCV) in early infancy. Following three PCV doses, geometric mean concentration (GMC) to all seven serotypes were > or = 1.0 microg/mL, and >85% of children achieved antibody levels > or = 0.35 microg/mL at 18 weeks. Following two doses, GMC were lower for 6B, 14, and 23F, but higher for 19F compared with three doses. Following a single dose, significant responses were seen for all serotypes post-primary series compared with the unvaccinated. By 12 months, differences between two and three doses persisted for serotype 14 only. Although GMC following three doses are higher than after two doses, the differences were small. A single dose may offer some protection for most serotypes.

The clinical syndrome of specific antibody deficiency in children.

Specific antibody deficiency (SAD) is an immune deficiency which has been reported in adults and children with recurrent respiratory tract infections; however, the clinical features of SAD are not well described. This study evaluated formally the clinical syndrome of SAD, by comparing the clinical features of children with SAD and those of children with recurrent infection but normal immune function tests. SAD was defined as an adequate IgG antibody response to less than 50% of 12 pneumococcal serotypes tested following 23-valent unconjugated pneumococcal immunization. An adequate IgG antibody response was defined as a post-immunization titre of >or= 1.3 microg/ml or >or= four times the preimmunization value. Seventy-four children with recurrent infection were evaluated where immune deficiencies other than SAD had been excluded. Eleven (14.9%) of these children had SAD. Clinical features differed between the group with SAD and the group with normal antibody responses. A history of otitis media, particularly in association with chronic otorrhoea was associated with SAD [relative risk (RR) of SAD in those with chronic otorrhoea 4.64 (P = 0.02)]. SAD was associated with allergic disease, particularly allergic rhinitis [RR of SAD in those with allergic rhinitis 3.77 (P = 0.04)]. These two clinical associations of SAD were independent in this study [RR of chronic otorrhoea in those with allergic rhinitis 0.85 (P = 0.28)]. SAD was not an age-related phenomenon in this population. SAD has a distinct clinical phenotype, presenting as recurrent infection associated with chronic otorrhoea and/or allergic disease, and the condition should be sought in children with these features.

Screening for consumptive coagulopathy in preeclampsia.

OBJECTIVE: To identify a practical and cost-effective profile of tests to screen for consumptive coagulopathy in preeclampsia (PE). METHODS: Retrospective analysis of the results of measurements of platelet count, prothrombin time (PT), activated partial thromboplastin time (APTT), plasma fibrinogen and D-dimers in 100 patients presenting with PE uncomplicated by other disease or antepartum hemorrhage. Twenty-four patients had pregnancy-induced hypertension only, and 76 hypertension with proteinuria. RESULTS: The incidence of abnormal tests on presentation was raised D-dimers 34%, thrombocytopenia 14%, prolonged APTT 12%, prolonged PT 3%, and low fibrinogen 2%. Prolonged APTT without thrombocytopenia occurred in 8% of patients. In 19 patients with elevation of D-dimers alone, only one showed evidence of consumption of coagulation factors on subsequent testing. CONCLUSIONS: A combination of platelet count and APTT is probably a practical and cost-effective combination to screen for consumptive coagulopathy in PE.

Reference ranges for haematology parameters in pregnancy derived from patient populations.

Reference ranges for several haematology parameters in pregnancy were determined by the mathematical detection of Gaussian and Gamma distributions in partitioned but unselected patient data. For each trimester, red cell parameters were shown to be well described by Gaussian distributions. Platelet and white cell parameters were best described by Gamma distributions with the exception of eosinophil and basophil counts for which neither distribution was applicable. The reference ranges derived for each trimester are compared.

Maternofetal transfer of IgG anti-Escherichia coli antibodies with enhanced avidity and opsonic activity in very premature neonates.

Total IgG concentrations, IgG antibody concentrations to pooled Escherichia coli antigens, and IgG anti-E. coli antibody avidity were measured in cord and maternal serum samples collected from 52 mother-infant pairs after premature delivery (mean gestational age 28 wk, range 23-33 wk). The mean IgG anti-E. coli antibody concentration in cord serum (1.86 relative units/mL) was markedly lower than in maternal serum (5.42 relative units/mL) at this gestation (p less than 0.0001). Cord serum IgG anti-E. coli antibody concentrations correlated closely with maternal IgG anti-E. coli concentrations when controlled for the effect of gestational age (partial correlation coefficient 0.89; p less than 0.001) but only weakly with gestational age when controlled for maternal IgG antibody concentrations (partial correlation coefficient 0.23; p = 0.06). The mean ratio of cord to maternal IgG anti-E. coli antibody concentrations was considerably lower than the mean ratio for total IgG concentrations (0.34 versus 0.72; p less than 0.001). The mean avidity of IgG antibody for the pooled E. coli antigens was significantly greater in cord serum than in maternal serum (2.45 versus 1.99M; p less than 0.0001). There was a close correlation between cord and maternal antibody avidity (r = 0.70; p less than 0.001), but cord IgG antibody avidity did not correlate with gestational age (r = -0.07; p = 0.61), nor with cord IgG anti-E. coli antibody concentrations (r = 0.10; p = 0.50).(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoglobulin and anti-Escherichia coli antibody in lower respiratory tract secretions from infants weighing less than 1500 g at birth.

Concentrations of immunoglobulins and anti-Escherichia coli antibody were studied longitudinally in tracheobronchial aspirates from 33 premature intubated neonates, median gestational age 27 weeks. Aspirates collected at birth contained IgG, IgA, and IgM in 100%, 93%, and 79% of samples, respectively. The median IgA concentration at birth was 0.7 micrograms/mg total protein and increased to 5.8 micrograms/mg protein by the sixth week. IgG and IgM antibodies to E coli were present in 90% and 30%, respectively, of tracheobronchial aspirates collected at birth. Samples from three of 28 neonates (11%) contained IgA anti-E coli antibody at birth, and the proportion with IgA antibody rose to 50% during the sixth week. Secretory component associated IgA and IgM were detectable in samples tested at birth and at 4 weeks of age, and secretory component associated anti-E coli antibody was present in aspirates from three of nine neonates studied at 4 weeks of age, but had not been detectable at birth.

IgE responses to environmental antigens in atopic children.

The levels of IgE antibodies to inhaled and dietary antigens and total serum IgG, IgA, IgM and IgE were studied in two highly selected populations of children who had either severe asthma and no history of eczema, or severe asthma and generalized eczema. These populations were age-matched with a healthy control population. The results showed that the eczematous children had significantly higher levels of IgE antibodies to common environmental antigens than the non-eczematous patients with severe asthma. Both patient groups had significantly higher IgE antigen specific antibody levels than the control population to these antigens. Minor differences in total IgG, IgA and IgM levels were noted between the groups. The variations in total IgE levels between the groups showed the same pattern as for specific IgE antibodies.