Molecular Typing of Pneumococci Causing Parapneumonic Empyema in Spanish Children Using Multilocus Sequence Typing Directly on Pleural Fluid Samples

To the Editors: Byington et al1 reported an increase in pediatric parapneumonic empyema (PPE) in children from Utah after the introduction of the PCV7 vaccine. Streptococcus pneumoniae serotype 1 was the most common cause of PPE with a higher prevalence in the PCV7 period compared with before PCV7 use. An emergence of cases resulting from serotypes 3 and 19A was observed in the later time period. The authors investigated only culture-positive cases of PPE and acknowledge that epidemiologic profile of empyema cases without confirmed bacterial etiology may differ. The rate of PPE among Spanish children younger than 14 years is also increasing in the past decade with reported incidence rates of 42.9 per 100,000 children in 2003 in Madrid area compared with 18.1 per 100,000 children in 1993.2 Etiologic investigation of this common disease has been hampered both by low sensitivity of conventional cultures and by failure to perform serotype analysis on a regular basis in confirmed pneumococcal cases. PCV-7 was introduced in Spain in July 2001 and is only available in the private market. Estimated coverage for the target population is presently 40% to 50%. We have retrospectively reviewed patients <14 years of age with a diagnosis of PPE from the 2 largest tertiary care hospitals in southern Spain. The mean number of PPE cases per year increased from 15 (range, 5–22) in 1998 to 2001 to 43 (range, 27–62) in the period 2002 to 2005 (P < 0.02). S. pneumoniae (SP) was the most common etiologic agent identified in both time periods but was only cultured in 16% of PPE cases. Molecular diagnosis of PPE was introduced in the second half of 2004 and since then, 39 samples (76%) that were culture-negative tested positive for pneumolysin (ply) by polymerase chain reaction. We aimed to determine whether direct molecular typing of ply-positive/culture-negative pleural fluid (PF) samples would be a feasible approach to define better the current epidemiology of PPE in southern Spain. DNA extracts from 36 pleural fluids that were culture-negative/ply-positive were sent to Oxford where MLST was performed using standard methods for aroE, gki, spi and ddl genes and substituting first-round cerebrospinal fluid primers for gdh, recP, xpt because of nonspecific amplification.3 Second-round polymerase chain reaction was carried out using conventional primers. Fifteen DNA extracts were fully and 3 partially genotyped (50%); 18 samples were MLST-negative in first-round PCR; these were associated with more prolonged receipt of antibiotic presampling. Preliminary results from the second round of nested PCR gave positive results in 13 of 18 DNA extracts that were initially MLST-negative (sequencing in progress). Available pneumococcal isolates from pediatric patients diagnosed with PPE during last 4 years were also genotyped. Table 1 shows the sequence types identified in the 27 samples completely genotyped along with the predicted (culture negative) or confirmed (culture positive) serotypes. The sequence type was designated according to the MLST web site (http://www.mlst.net). All clones found in the study have been well established in Spain for a number of years.4,5TABLE 1: Sequence Types (ST) and Predicted or Confirmed Serotypes in 27 Pleural Fluids Completely GenotypedIn conclusion, use of MLST to identify pneumococci directly from culture-negative PFs may be a useful adjunct for investigating the complex epidemiology of pediatric PPE. As seen by Byington et al1 in the United States, our preliminary results suggest a predominance of nonvaccine serotypes, particularly serotype 1, in the etiology of PPE in southern Spain both in culture-positive and -negative cases. David Sánchez-Tatay was supported by a grant from Consejería de Salud, Junta de Andalucía. Luis A. Arroyo was supported by a grant from Fundación El Monte. MLST was funded by GlaxoSmithKlineBiologicals. Ignacio Obando, MD Pediatric Infectious Diseases Unit Hospital Infantil Virgen del Rocío Sevilla, Spain Luis A. Arroyo, MD David Sánchez-Tatay, BSc Investigation Unit Hospital Infantil Virgen del Rocío Sevilla, Spain David Moreno, MD Pediatric Infectious Diseases Unit Hospital Carlos Haya Málaga, Spain William P. Hausdorff, PhD GlaxoSmith Kline Biologicals Rixensart, Belgium Angela B. Brueggemann, PhD Zoology Department University of Oxford Oxford, U.K.