Laura Selva

Emergence of invasive pneumococcal disease caused by multidrug-resistant serotype 19A among children in Barcelona☆

OBJECTIVE To describe the epidemiology of invasive pneumococcal disease (IPD) caused by Streptococcus pneumoniae serotype 19A. METHODS We studied all children and adolescents with IPD caused by serotype 19A who were admitted to a Childrens Hospital in Barcelona (1997-2007). Serotyping, antibiotic susceptibility and clonal analysis were performed. RESULTS Comparing the pre-vaccine period (1997-2001) with the early vaccine period (2002-2004) and the late vaccine period (2005-2007) there was an increase of IPD caused by serotype 19A: 1 of 58 episodes (1.7%) vs. 8 of 54 episodes (14.8%) vs. 27 of 123 episodes (21.9%), respectively (P = 0.002). All S. pneumoniae serotype 19A isolated in the pre-vaccine and early vaccine periods (n = 9) were penicillin susceptible, while in the late vaccine period, 12 of 27 (44%) were penicillin nonsusceptible (P = 0.01). A clonal analysis revealed 15 different sequence types (STs) expressing serotype 19A. 10 of them were preexisting STs associated with serotype 19A including the multidrug-resistant ST320 and ST276. CONCLUSION There was an increase of IPD caused by S. pneumoniae serotype 19A which was mainly related with the emergence of preexisting clones several of them closely related with international multidrug-resistant clones. These results should be considered when selecting the new conjugate pneumococcal vaccines.

Serotypes and clones causing invasive pneumococcal disease before the use of new conjugate vaccines in Catalonia, Spain

OBJECTIVES The objective of this study was to learn the serotype distribution and clonal composition of pneumococci causing invasive pneumococcal disease (IPD) in children and adults in Spain before the introduction of new 10-valent (PCV10) and 13-valent (PCV13) conjugate vaccines. METHODS This is a 1-year prospective study including all patients with culture-proved IPD admitted to 30 medical centers in Catalonia, Spain, during the year 2009. RESULTS A total of 614 episodes of IPD occurred in 612 patients. The rates of IPD were highest in children aged <24 months and adults >64 years (64.5 and 44.7 per 100,000 population). The burden of disease was mainly due to pneumonia in all age ranges. 609 of 614 strains were serotyped and 47 different serotypes were found. Among the 609 IPD cases with known serotype, 12.2% were caused by PCV7 serotypes, 51% by PCV10 serotypes, and 71.7% by PCV13 serotypes. 608 of 614 isolates were characterized by MLST. The main clonal types detected were ST306, CC191 and CC230. CONCLUSIONS PCV13 conjugate vaccine offers good coverage against IPD in Catalonia, Spain. However, the high genetic diversity of pneumococci highlights the importance of molecular surveillance systems for monitoring IPD during the vaccination period. SUMMARY This study shows that 13-valent conjugate vaccine offers good coverage against invasive pneumococcal disease in children and adults in Spain. However, the high genetic diversity of pneumococci highlights the importance of molecular surveillance systems for monitoring IPD during the vaccination period.

Rapid and Easy Identification of Capsular Serotypes of Streptococcus pneumoniae by Use of Fragment Analysis by Automated Fluorescence-Based Capillary Electrophoresis

ABSTRACT The purpose of this study was to develop a high-throughput method for the identification of pneumococcal capsular types. Multiplex PCR combined with fragment analysis and automated fluorescent capillary electrophoresis (FAF-mPCR) was utilized. FAF-mPCR was composed of only 3 PCRs for the specific detection of serotypes 1, 2, 3, 4, 5, 6A/6B, 6C, 7F/7A, 7C/(7B/40), 8, 9V/9A, 9N/9L, 10A, 10F/(10C/33C), 11A/11D/11F, 12F/(12A/44/46), 13, 14, 15A/15F, 15B/15C, 16F, 17F, 18/(18A/18B/18C/18F), 19A, 19F, 20, 21, 22F/22A, 23A, 23B, 23F, 24/(24A/24B/24F), 31, 33F/(33A/37), 34, 35A/(35C/42), 35B, 35F/47F, 38/25F, and 39. In order to evaluate the assay, all invasive pneumococcal isolates (n = 394) characterized at Hospital Sant Joan de Déu, Barcelona, Spain, from July 2010 to July 2011 were included in this study. The Wallace coefficient was used to evaluate the overall agreement between two typing methods (Quellung reaction versus FAF-mPCR). A high concordance with Quellung was found: 97.2% (383/394) of samples. The Wallace coefficient was 0.981 (range, 0.965 to 0.997). Only 11 results were discordant with the Quellung reaction. However, latex reaction and Quellung results of the second reference laboratory agreed with FAF-mPCR for 9 of these 11 strains (82%). Therefore, we considered that only 2 of 394 strains (0.5%) were not properly characterized by the new assay. The automation of the process allowed the typing of 30 isolates in a few hours with a lower cost than that of the Quellung reaction. These results indicate that FAF-mPCR is a good method to determine the capsular serotype of Streptococcus pneumoniae.

Viral coinfection in children less than five years old with invasive pneumococcal disease.

Seventy-one patients <5 years of age who were hospitalized with invasive pneumococcal disease were studied in the period between August 2008 and December 2009. The purpose was to determine the proportion of episodes that were coinfected with respiratory virus. Viral coinfection was common (44/71; 62%), with rhinovirus and influenza virus being the most frequently detected. Highly invasive serotypes (1, 5, 7F, 14, 19A) were found in 31 of 71 patients, of whom 15 had viral coinfection (15/31; 48%). Viral detection occurred significantly more often in those episodes caused by nonhighly invasive serotypes (29/40; 72%), suggesting that a viral synergism could help those serotypes to make invasiveness more likely.

Effectiveness of 7-valent pneumococcal conjugate vaccine in the prevention of invasive pneumococcal disease in children aged 7-59 months. A matched case-control study.

The aim of this study was to evaluate the effectiveness of the administration of the 7-valent pneumococcal conjugate vaccine in a region with an intermediate vaccination coverage. A matched case-control study was carried out in children aged 7-59 months with invasive pneumococcal disease (IPD) admitted to two university hospitals in Catalonia. Three controls matched for hospital, age, sex, date of hospitalization and underlying disease were selected for each case. Information on the vaccination status of cases and controls was obtained from the vaccination card, the childs health card, the hospital medical record or the vaccination register of the primary healthcare center where the child was attended for non-severe conditions. A conditional logistic regression analysis was made to control for the effect of possible confounding variables. The adjusted vaccination effectiveness of the complete vaccination schedule (3 doses at 2, 4 and 6 months and a fourth dose at 15 months, 2 doses at least two months apart in children aged 12-23 months or a single dose in children aged >24 months) in preventing IPD caused by vaccine serotypes was 93.7% (95% CI 51.8-99.2). It was not effective in preventing cases caused by non-vaccine serotypes. The results of this study carried out in a population with intermediate vaccination coverage confirm those of other observational studies showing high levels of effectiveness of routine 7-valent pneumococcal conjugate vaccination.

PsrP, a protective pneumococcal antigen, is highly prevalent in children with pneumonia and is strongly associated with clonal type.

ABSTRACT Invasive pneumococcal disease (IPD) is a major health problem worldwide. Due to ongoing serotype replacement, current efforts are focused in an attempt to identify the pneumococcal antigens that could be used in a next-generation multivalent protein vaccine. The objective of our study was to use real-time PCR to determine the distribution and clonal type variability of PsrP, a protective pneumococcal antigen, among pneumococcal isolates from children with IPD or healthy nasopharyngeal carriers. psrP was detected in 52.4% of the 441 strains tested. While no differences were determined when the prevalence of psrP in colonizing strains (n = 89) versus that in all invasive strains (n = 352) was compared, a strong trend was observed when the prevalence of psrP in all pneumonia isolates (n = 209) and colonizing isolates (P = 0.067) was compared, and a significant difference was observed when the prevalence in all pneumonia isolates and those causing bacteremia (n = 76) was compared (P = 0.001). An age-dependent distribution of psrP was also observed, with the incidence of psrP being the greatest in strains isolated from children >2 years of age (P = 0.02). Strikingly, the presence of psrP within a serotype was highly dependent on the clonotype, with all isolates of invasive clones such as clonal complex 306 carrying psrP (n = 88), whereas for sequence type 304, only 1 of 19 isolates carried psrP; moreover, this was inversely correlated with antibiotic susceptibility. This finding suggests that inclusion of psrP in a vaccine formulation would not target resistant strains. We conclude that psrP is highly prevalent in strains that cause IPD but is most prevalent in strains isolated from older children with pneumonia. These data support the potential use of PsrP as one component in a multivalent protein-based vaccine.

Streptococcus pneumoniae serotype 1 causing invasive disease among children in Barcelona over a 20‐year period (1989–2008)

Fifty-six isolates of serotype 1 were identified during a 20-year prospective study (1989-2008), including all children with culture-proven invasive pneumococcal disease (IPD) admitted to a childrens hospital in Barcelona. Forty-eight of them (85.7%) were in children aged >2 years. Complicated pneumonia (n = 28) and non-complicated pneumonia (n = 20) were the main clinical presentations. The frequency of serotype 1 IPD increased from 1999-2003 to 2004-2008: 1.2 to 4.4 episodes/100 000 children (p <0.001). The ST306 clone were identified in 70.4% of isolates. As IPD caused by serotype 1 is mainly detected in older children, a vaccination programme for children >2 years should be considered.

Detection of Streptococcus pneumoniae and Haemophilus influenzae type B by real-time PCR from dried blood spot samples among children with pneumonia: a useful approach for developing countries.

Background Dried blood spot (DBS) is a reliable blood collection method for storing samples at room temperature and easily transporting them. We have previously validated a Real-Time PCR for detection of Streptococcus pneumoniae in DBS. The objective of this study was to apply this methodology for the diagnosis of S. pneumoniae and Haemophilus influenzae b (Hib) in DBS samples of children with pneumonia admitted to two hospitals in Mozambique and Morocco. Methods Ply and wzg genes of S. pneumoniae and bexA gene of Hib, were used as targets of Real-Time PCR. 329 DBS samples of children hospitalized with clinical diagnosis of pneumonia were tested. Results Real-Time PCR in DBS allowed for a significant increase in microbiological diagnosis of S. pneumoniae and Hib. When performing blood bacterial culture, only ten isolates of S. pneumoniae and none of Hib were detected (3·0% positivity rate, IC95% 1·4-5·5%). Real-Time PCR from DBS samples increased the detection yield by 4x fold, as 30 S. pneumoniae and 11 Hib cases were detected (12·4% positivity rate, IC95% 9·0-16·5%; P<0·001). Conclusion Real-Time PCR applied in DBS may be a valuable tool for improving diagnosis and surveillance of pneumonia caused by S. pneumoniae or Hib in developing countries.

Prevalence and Clonal Distribution of pcpA, psrP and Pilus-1 among Pediatric Isolates of Streptococcus pneumoniae

Streptococcus pneumoniae is the leading cause of vaccine-preventable deaths globally. The objective of this study was to determine the distribution and clonal type variability of three potential vaccine antigens: Pneumococcal serine-rich repeat protein (PsrP), Pilus-1, and Pneumococcal choline binding protein A (PcpA) among pneumococcal isolates from children with invasive pneumococcal disease and healthy nasopharyngeal carriers. We studied by Real-Time PCR a total of 458 invasive pneumococcal isolates and 89 nasopharyngeal pneumococcal isolates among children (total = 547 strains) collected in Barcelona, Spain, from January 2004 to July 2010. pcpA, psrP and pilus-1 were detected in 92.8%, 51.7% and 14.4% of invasive isolates and in 92.1%, 48.3% and 18% of carrier isolates, respectively. Within individual serotypes the prevalence of psrP and pilus-1 was highly dependent on the clonal type. pcpA was highly prevalent in all strains with the exception of those belonging to serotype 3 (33.3% in serotype 3 isolates vs. 95.1% in other serotypes; P<.001). psrP was significantly more frequent in those serotypes that are less apt to be detected in carriage than in disease; 58.7% vs. 39.1% P<.001. Antibiotic resistance was associated with the presence of pilus-1 and showed a negative correlation with psrP. These results indicate that PcpA, and subsequently Psrp and Pilus-1 together might be good candidates to be used in a next-generation of multivalent pneumococcal protein vaccine.

Detection of human papillomavirus infection in women attending a colposcopy clinic.

BACKGROUND Infection by human papillomavirus (HPV) is the main cause of and a necessary factor for cervical cancer. There are more than 100 known HPV genotypes, although HPV genotypes do not all have the same risk of inducing cervical cancer. The main aim of this study is to know the distribution of different types of HPV in women attending a colposcopy clinic for cervical dysplasia. METHODS We prospectively identified and enrolled a cohort of women followed for cervical dysplasia who were referred to the colposcopy clinic of University Hospital Sant Joan de Déu in Barcelona, Spain, from April 2003 to April 2007. Two cervical scrape samples from each patient were collected for routine cytology and for identification of HPV DNA. During the study period, 2 techniques (Line Probe assay and microarray assay) were used consecutively. FINDINGS HPV DNA was detected in 68% of patients (338 of 496 women) with statistically significant differences in positive results according to the cytologic category. Overall, the type distribution showed a predominance of genotype HPV-16 (27% of the total patients) followed by HPV-53 (9.4%), HPV-51 (8%), and HPV-51 (8%). Multiple genotype detection was observed in 35.5% of the patients with HPV infection. HPV-16 was mainly associated with the probable high-risk genotypes: HPV-53 and HPV-66. CONCLUSION The higher prevalence of high-risk nonvaccine genotypes should be considered to increase vaccine efficiency.