Two consecutive randomized controlled pertussis booster trials in children initially vaccinated in infancy with an acellular vaccine: The first with a five-component Tdap vaccine to 5-year olds and the second with five- or monocomponent Tdap vaccines at age 14-15 years.

Prior study children from a DTaP efficacy trial were recruited at ages 5 and 15 years to randomized booster trials addressing immunogenicity and reactogenicity; 475 preschool children received mixed or separate injections of a reduced antigen vaccine (Tdap5, Sanofi Pasteur MSD) and an inactivated polio vaccine, and 230 adolescents received the same or another booster vaccine (Tdap1, SSI, Denmark). Pre-vaccination antibody concentrations against pertussis antigens were significantly higher at 15 than 5 years of age, probably due to natural boosting between the studies. Tdap5 induced comparable anti-PT concentrations at both ages, but antibody responses were significantly higher to filamentous haemagglutinin, pertactin and fimbriae 2/3 in adolescents. As expected, a higher amount of PT (Tdap1, 20µg) induced a stronger anti-PT response than a lower amount (Tdap5, 2.5µg). The frequency of adverse events was low and there were no serious adverse reactions. All local reactions had an early onset and a short duration. A large swelling or redness of more than half of the upper arm circumference was reported in 8/475 5-year-olds and in 6/230 15-year-olds. Children vaccinated with Tdap5 reported more moderate pain in adolescence than at preschool age, whereas itching was only reported in preschool children. Sweden introduced DTaP vaccines in 1996 after a 17-year hiatus with no general pertussis vaccination and pertussis was still endemic at the time of the studies. The frequency of adverse events was nevertheless low in both preschool children and adolescents and antibody responses were adequate. These studies document immunogenicity and reactogenicity in a trial cohort consecutively vaccinated with acellular pertussis vaccines from infancy to adolescence. The adolescent study was registered at ClinicalTrials.gov on 26 March 2009 (NCT00870350).

Analysis of Bordetella pertussis clinical isolates circulating in European countries during the period 1998-2012.

Despite more than 50 years of vaccination, pertussis is still an endemic disease, with regular epidemic outbreaks. With the exception of Poland, European countries have replaced whole-cell vaccines (WCVs) by acellular vaccines (ACVs) in the 1990s. Worldwide, antigenic divergence in vaccine antigens has been found between vaccine strains and circulating strains. In this work, 466 Bordetella pertussis isolates collected in the period 1998-2012 from 13 European countries were characterised by multi-locus antigen sequence typing (MAST) of the pertussis toxin promoter (ptxP) and of the genes coding for proteins used in the ACVs: pertussis toxin (Ptx), pertactin (Prn), type 2 fimbriae (Fim2) and type 3 fimbriae (Fim3). Isolates were further characterised by fimbrial serotyping, multi-locus variable-number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE). The results showed a very similar B. pertussis population for 12 countries using ACVs, while Poland, which uses a WCV, was quite distinct, suggesting that ACVs and WCVs select for different B. pertussis populations. This study forms a baseline for future studies on the effect of vaccination programmes on B. pertussis populations.

Investigations into the emergence of pertactin-deficient Bordetella pertussis isolates in six European countries, 1996 to 2012.

Pathogen adaptation has been proposed to contribute to the resurgence of pertussis. A striking recent example is the emergence of isolates deficient in the vaccine component pertactin (Prn). This study explores the emergence of such Prn-deficient isolates in six European countries. During 2007 to 2009, 0/83 isolates from the Netherlands, 0/18 from the United Kingdom, 0/17 Finland, 0/23 Denmark, 4/99 Sweden and 5/20 from Norway of the isolates collected were Prn-deficient. In the Netherlands and Sweden, respectively 4/146 and 1/8 were observed in a later period (2010­12). The Prn-deficient isolates were genetically diverse and different mutations were found to inactivate the prn gene. These are indications that Prn-deficiency is subject to positive selective pressure. We hypothesise that the switch from whole cell to acellular pertussis vaccines has affected the balance between 'costs and benefits' of Prn production by Bordetella pertussis to the extent that isolates that do not produce Prn are able to expand. The absence of Prn-deficient isolates in some countries may point to ways to prevent or delay the spread of Prn-deficient strains. In order to substantiate this hypothesis, trends in the European B. pertussis population should be monitored continuously.

Analysis of Swedish Bordetella pertussis isolates with three typing methods: characterization of an epidemic lineage.

Three Bordetella pertussis typing methods, pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), and multi-locus variable number tandem repeat analysis (MLVA) were compared using a collection of Swedish strains. Of the three typing methods used, PFGE was found to be the most discriminatory. MLVA and MLST were less discriminatory, but may be valuable for strain discrimination when culture is not possible as they are based on PCR. The combination of MLVA/MLST was found to be equally discriminatory as PFGE and should therefore also be considered. The relationship between predominant lineages in Sweden and The Netherlands, characterized by the PFGE type BpSR11 and the allele for the pertussis toxin promoter ptxP3, respectively, was investigated. Linkage was found between the PFGE type BpSR11 and ptxP3 in that all BpSR11 strains carried ptxP3. On the other hand ptxP3 was found in several other PFGE-types. The presence of the ptxP3 allele in different genetic backgrounds may indicate horizontal gene transfer within B. pertussis or homoplasy. Alternatively, this observation may be due to convergence of PFGE types.

Analysis of Bordetella pertussis populations in European countries with different vaccination policies.

Despite the widespread use of pertussis vaccines during the last decades, pertussis has remained an endemic disease with frequent epidemic outbreaks. Currently two types of vaccines are used: whole-cell vaccines (WCVs) and recently developed acellular vaccines (ACVs). The long-term aim of our studies is to assess the effect of different vaccination policies on the population structure of Bordetella pertussis and ultimately on the disease burden in Europe. In the present study, a total of 102 B. pertussis isolates from the period 1998 to 2001 from five European countries (Finland, Sweden, Germany, The Netherlands, and France) were characterized. The isolates were analyzed by typing based on variable number of tandem repeats (VNTR); by sequencing of polymorphic genes encoding the surface proteins pertussis toxin S1 and S3 subunits (ptxA and ptxC), pertactin (prn), and tracheal colonization factor (tcfA); and by fimbrial serotyping. The results reveal a relationship between geographic location and VNTR types, the frequency of the ptxC alleles, and serotypes. We have not observed a relationship between the strain characteristics we studied and vaccination programs. Our results provide a baseline which can be used to reveal changes in the B. pertussis population in Europe in the coming years.

Calibrated serological techniques demonstrate significant different serum response rates to an oral killed cholera vaccine between Swedish and Nicaraguan children.

Serum responses to oral cholera vaccines were assessed in three paediatric vaccine trials, two in León, Nicaragua and one in Stockholm, Sweden. A calibrated anti-cholera toxin B subunit (CTB) IgA ELISA was used together with an assay for vibriocidal antibodies. Swedish children had lower pre-vaccination levels of antibody, but serum responses were more pronounced in Swedish children than in Nicaraguan children. Post-vaccination levels of anti-toxin antibody were generally above those found after natural infections with enterotoxigenic Escherichia coli, that cross-reacts serologically with Vibrio cholerae. Adverse events seen after vaccination were generally mild and of little clinical significance.

How to make sense of pertussis immunogenicity data.

Studies on serologic correlates to protection in pertussis were reviewed. Trials in the 1950s showed that agglutinogen titers correlated to protection of whole-cell vaccines, but postvaccination antibodies against pertussis toxin (PT) and against filamentous hemagglutinin did not in a later trial of acellular vaccines. However, in household studies nested in 2 recent trials, preexposure antibody levels against pertactin and against fimbriae correlated with protection against typical and mild pertussis, and anti-PT correlated only with protection against typical pertussis. These findings could be used by regulatory agencies to license pertussis vaccines. A reference laboratory for pertussis should distribute panels to control interlaboratory variation in recommended assays, and a minimal response should be set for each pertussis antigen. We conclude that 2 studies have shown correlates between measurable anti-pertactin, anti-fimbriae, and anti-PT antibody levels at exposure and individual protection against pertussis. We suggest that postvaccination response rates may be used as surrogate markers of protection.

Community spread of Legionella pneumophila serogroup 1 in temporal relation to a nosocomial outbreak.

To clarify whether a nosocomial outbreak of legionnaires' disease in the Värnamo hospital in Sweden was part of a wider outbreak in the Värnamo community a number of investigations were performed. First, the proportion of cases of legionnaires' disease in a group with nosocomially acquired pneumonia (11%) was compared to the proportion within a group with community-acquired pneumonia (14%) and the difference was found not to be significant (p > 0.05). Second, the proportion of the nursing staff at the Värnamo hospital with an elevated antibody titre (> or = 16) to Legionella pneumophila serogroup (sg) 1 (33%, 84/258) was compared to the proportion in a group of local residents of Värnamo community (26%, 25/96) and found not to be significant; in contrast, comparison with the proportion in a group from the assistant nursing staff at another hospital 60 km away (5%, 4/80) was highly significant (p < 0.001). Furthermore, Legionella species were cultured from samples drawn from the hospital water supply as well from the water supply from municipal buildings. In 1996 a follow-up study was conducted, which showed that < 1% of the assistant nurses and local residents had an elevated titre to L. pneumophila sg 1. These results indicate that there was a temporary spread of L. pneumophila sg 1 in the Värnamo community at the beginning of 1991, both in the local hospital and the surrounding community. This implies that physicians should be aware of community-acquired cases of legionnaires' disease when a nosocomial outbreak is detected.

The sero-epidemiology of diphtheria in Western Europe. ESEN Project. European Sero-Epidemiology Network.

Seven countries in Western Europe collected large, representative serum banks across the entire age range and tested them for diphtheria anti-toxin (sample size ranged from 2991 to 7715). Although a variety of assays were used, the results were all standardized to those of a reference laboratory and expressed in international units. The standardization process, and the availability of similar, large data sets allowed comparative analyses to be performed in which a high degree of confidence could be ascribed to observed epidemiological differences. The results showed that there were large differences in the proportion of adults with insufficient levels of protection amongst different countries. For instance, roughly 35% of 50- to 60-year-olds were found to be seronegative (titre < or = 0.01 IU/ml) in Finland compared with 70-75% in the United Kingdom. Furthermore, the proportion of seronegative adults would be expected to increase in some countries, notably Italy and the western part of Germany. In those countries with vaccination of military recruits there was a marked sex-related difference in the proportion of seropositive individuals. All countries have high levels of infant vaccine coverage (> 90%) but the accelerated schedule in the United Kingdom appears to result in lower anti-toxin titres than elsewhere. In Sweden, booster doses are not offered until 10 years of age which results in large numbers of children with inadequate levels of protection. Although the United Kingdom and Sweden both have higher proportions of seronegative children than elsewhere the likelihood of a resurgence of diphtheria in these countries seems remote.

Performance of Uricult Trio assessed by a comparison method and external control panels in primary healthcare.

Using the comparison method, we have evaluated the technical performance of Uricult Trio by culturing on Uricult Trio and agar plates. Urine samples (477) from patients in primary healthcare were cultured in parallel in a microbiology laboratory. The result for Uricult Trio evaluated using the comparison method was incorrect in 32% of the cultures. We also studied the performance of Uricult Trio when used in primary healthcare by using external control panels. External control panels consisting of Uricult Trio, inoculated with known concentrations of certain bacterial strains, were used to assess the performance of Uricult Trio in primary healthcare during the period 1993-7. Aberrations in reports of concentration have ranged from 10% to 33%, failure in reporting of mixed culture from 0% to 91% and reporting of E. coli from 15% to 86%. There has been no sign of improvement over the years. The results indicate that Uricult Trio is unsuitable for indications other than exclusion of urinary tract infection or diagnosis of urinary tract infection caused by E. coli. Further, there is need for quality assurance and training activities at primary healthcare laboratories, probably best carried out in collaboration with local clinical microbiology laboratories.

Evaluation of two methods for improving quality of diagnosis of bacteriuria by culture in primary healthcare.

This study evaluates the effect of training on the results from Uricult Trio and an established urine culture when used at primary healthcare laboratories in two Swedish counties, Uppsala and Värmland. Urine cultures and dipslides, Uricult Trio, performed at these laboratories were interpreted a second time at central laboratories. Interpretation errors at the primary healthcare laboratories were calculated. Primary healthcare laboratories also received external control panels with urine cultures and dipslides. There was one study period each year for 3 years in Uppsala and for 2 years in Värmland. A training programme was completed between study periods in Värmland. In Uppsala, primary healthcare laboratory results could be reviewed, as interpretations by the central laboratory were returned to them. The main outcome measures were the percentage of interpretation errors which, in the first study period, was 33-39%. This dropped to 15-19% in the second study period. In the results from the external control panels there were no striking differences between the studied areas and Sweden as a whole, except that Uppsala showed a better result in reporting E. coli and failed in 10% compared to Sweden 46%. A method for both quality assessment and education is to ask the primary healthcare laboratories to send cultures to the central laboratory for interpretation requesting their return to the primary healthcare laboratory with the interpretation from the central laboratory attached.

Towards European urinalysis guidelines. Introduction of a project under European Confederation of Laboratory Medicine.

Improved standardized performance is needed because urinalysis continues to be one of the most frequently requested laboratory tests. Since 1997, the European Confederation of Laboratory Medicine (ECLM) has been supporting an interdisciplinary project aiming to produce European urinalysis guidelines. More than seventy clinical chemists, microbiologists and ward-based clinicians, as well as representatives of manufacturers are taking part. These guidelines aim to improve the quality and consistency of chemical urinalysis, particle counting and bacterial culture by suggesting optimal investigative processes that could be applied in Europe. The approach is based on medical needs for urinalysis. The importance of the pre-analytical stage for total quality is stressed by detailed illustrative advice for specimen collection. Attention is also given to emerging automated technology. For cost containment reasons, both optimum (ideal) procedures and minimum analytical approaches are suggested. Since urinalysis mostly lacks genuine reference methods (primary reference measurement procedures; Level 4), a novel classification of the methods is proposed: comparison measurement procedures (Level 3), quantitative routine procedures (Level 2), and ordinal scale examinations (Level 1). Stepwise strategies are suggested to save costs, applying different rules for general and specific patient populations. New analytical quality specifications have been created. After a consultation period, the final written text will be published in full as a separate document.

Epidemiological typing of Bordetella pertussis isolates: recommendations for a standard methodology.

Pertussis is re-emerging in vaccinated populations, and to gain insight into the reasons for this development population-based studies are necessary. Unfortunately, various techniques are used to study Bordetella pertussis populations, hampering comparison between studies. A standard methodology for epidemiological typing of Bordetella pertussis isolates is proposed which is based on serotyping, pulsed-field gel electrophoresis and gene typing. Such a standard approach will allow comparisons between studies performed in different laboratories. Comparisons may reveal whether the epidemiological differences observed between countries are due for instance to different Bordetella pertussis populations or different vaccines used.

Diphtheria antitoxin response to DTP vaccines used in Swedish pertussis vaccine trials, persistence and projection for timing of booster.

Data from two Swedish pertussis vaccine trials with various combination vaccines were used to compare anti-diphtheria antitoxin concentrations over time between different vaccines, vaccine lots and vaccine schedules. The immune responses were measured with a validated ELISA method.Results are given for 1326 children, born 1992, that were recruited to the placebo (DT)-controlled Trial I which used a 2, 4, 6 month schedule. Two DTP acellular and one DTP whole cell vaccine were used. No DT boosters were given until 5 years of age. Trial II recruited children born 1993-94 and compared three DTP acellular vaccines with one DTP whole cell vaccine. Results are given for 306 children in a 2, 4, 6 month schedule and for 531 children in a 3, 5, 12 month schedule. The latter schedule gave significantly higher diphtheria antitoxin concentrations post third dose. The various DTP acellular vaccines and an inefficacious DTP whole cell vaccine gave lower antitoxin concentrations than both an efficacious DTP whole cell vaccine and the DT vaccine. The larger differences in antigen response between vaccines was reduced in the course of time. Generally, an initial rapid decline of antitoxin concentration was followed by a slower decline; the change typically occurring when the antitoxin concentration reached 0.13-0.16 EU/ml. The time needed to reach this level was between 6 and 10 months based on the initial vaccine response.A "best-fit" combined exponential regression model was used to predict the optimal timing for booster vaccinations against diphtheria.Our data support a 3, 5, 12 month schedule followed by a fourth dose 4-5 years after the third dose, depending upon the vaccine used.

Evaluation of single- and dual antigen delayed fluorescence immunoassay in comparison to an ELISA and the in vivo toxin neutralisation test for detection of diphtheria toxin antibodies.

An evaluation of the delayed fluorescence immunoassay (Delfia) against an ELISA method for determination of diphtheria antitoxin levels in serum was performed. The Delfia was also validated in the in vivo toxin neutralisation test (Txn) in rabbits. Two variants of the Delfia were studied, a single-antigen Delfia (sDelfia) with only the diphtheria toxin included and a dual-antigen Delfia (dDelfia) with tetanus toxoid included for simultaneous detection of antibodies against two antigens. The diphtheria antitoxin cut-off levels in the sDelfia and the dDelfia were 0.004 and 0.002 AU/ml, respectively, which is lower than the internationally accepted level showing any protection against diphtheria (0.01 IU/ml). Both Delfia variants showed good correlation with the ELISA procedure above the ELISA cut-off level of 0.02 AU/ml. Results from samples assayed in the in vivo Txn assay indicated that the low antitoxin levels detected by the Delfia were valid. These results show that the Delfia could be considered as an in vitro reference method for detection of diphtheria antitoxin in seroepidemiological surveys and vaccine studies.

Bordetella pertussis, Bordetella parapertussis, Mycoplasma pneumoniae, Chlamydia pneumoniae and persistent cough in children.

Material collected during a prospective pertussis vaccine trial in 1992-95 was examined for Bordetella pertussis (culture and serology), Bordetella parapertussis (culture), Mycoplasma pneumoniae and Chlamydia pneumoniae (PCR). From 64% (99/155) of episodes with cough for less than 100 d, 115 aetiological agents were identified in one southern and one northern subset of DT-recipients. The most common single agent was B. pertussis, representing 56%(64/115), with a median cough period of 51 d, followed by M. pneumoniae 26%(30/115), 23 d, C. pneumoniae 17% (19/115), 26 d, and B. parapertussis 2% (2/115). For co-infections, the median duration of cough was about 60 d. Spasmodic cough for 21 d or more (clinical WHO criteria for pertussis) was present in 82% (41/50) of infections with B. pertussis as single agent, 38% (17/45) with B. parapertussis, 38% (5/13) with C. pneumoniae, 26% (5/19) with M. pneumoniae and 30%(17/56) in cases where no aetiology was found. In children with cough for more than 100 d (n = 78) using all vaccine arms, B. pertussis was responsible in 83% (65/78), in 21%(16/78) together with other agents. Acellular vaccines were more efficient against serious disease than whole cell vaccine. Antibiotic treatment was more common at the southern (34%) study site than at the northern one (12%). The findings indicate that diagnosis should rely on laboratory confirmation, both for rational treatment of an individual case and for monitoring outbreaks.

Microbiological and serological diagnosis of pertussis.

Swedish vaccine trials have been used to examine sensitivity and specificity of diagnostic procedures for Bordetella pertussis infection. The proportions of cases diagnosed by culture and serology were 55% and 45%, respectively, when both methods were optimized. The culture method included nasopharyngeal aspiration, direct inoculation on plates, enrichment, and repeated collection of samples. An enzyme-linked immunosorbent assay for IgG antibodies to pertussis toxin (PT) and to filamentous hemagglutinin, with paired sera, was used for serology. Preexposure sera other than the acute serum increased the sensitivity of serology by 10%. A serology quality-assurance program to control imprecision and allow comparability over time and between laboratories is described. The direct fluorescent antibody technique had a sensitivity of 38% and a specificity of 99.6% in comparison with culture. A nested polymerase chain reaction (PCR) with the PT promoter region as target was 95% sensitive in comparison with culture if a cation-exchange resin was used to reduce inhibition. PCR enabled us to identify 83 positive samples in addition to 215 culture-positive ones-an increase of 38%--all with other indicators of pertussis infection.

Marked decline in pertussis followed reintroduction of pertussis vaccination in Sweden.

Immunisation against pertussis with an acellular pertussis vaccine for children at 3, 5, and 12 months was included in the Swedish vaccination programme in January 1996, 17 years after the withdrawal of whole cell vaccine in 1979. Within months coverage r