Journal of Medical Microbiology (2014), 63, 393–398
DOI 10.1099/jmm.0.068726-0
Oropharyngeal and nasopharyngeal sampling for the detection of adolescent Streptococcus pneumoniae carriers Nicola Principi, Leonardo Terranova, Alberto Zampiero, Francesca Manzoni, Laura Senatore, Walter Peves Rios and Susanna Esposito Correspondence
Pediatric Highly Intensive Care Unit, Department of Pathophysiology and Transplantation, Universita` degli Studi di Milano, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy
Susanna Esposito [email protected]
Received 22 September 2013 Accepted 26 November 2013
Monitoring the dynamics of pneumococcal carriage makes it possible to evaluate the epidemiological characteristics of Streptococcus pneumoniae disease and the theoretical coverage offered by pneumococcal vaccines. It has been demonstrated that the nasopharyngeal (NP) sampling of respiratory secretions is superior to oropharyngeal (OP) sampling for identifying pneumococci carried by younger children, but adult data are conflicting and there are no published studies of adolescents. In order to compare the efficiency of OP and NP sampling in identifying and quantifying S. pneumoniae carriage in healthy adolescents, 2 swab samples were obtained from 530 adolescents aged 15–19 years, the first taken from the posterior pharyngeal wall through the mouth (OP) and the second through the nose (NP). Bacterial genomic DNA was tested for the autolysin-A-encoding gene (lytA) and wzg (cpsA) gene of S. pneumoniae in order to evaluate pneumococcal carrier status. All of the positive cases were serotyped. S. pneumoniae was identified in 35.8 % of the OP swabs and 3.5 % of the NP swabs (P,0.0001). The serotypes included in the 13-valent pneumococcal conjugate vaccine (PCV13) were found in all but two OP samples (98.9 %) and only 64.7 % of the NP samples (P,0.0001). The most frequently identified PCV13 serotype in both groups was 19F, followed by serotypes 5 and 9V. In conclusion, OP sampling appeared significantly more effective than NP sampling in identifying and characterizing pneumococcal carrier status in adolescents. This suggests that OP sampling should be used when evaluating the dynamics of pneumococcal carriage among adolescents and the theoretical coverage offered by PCV13.
INTRODUCTION Streptococcus pneumoniae is the leading bacterial cause of infection worldwide, causing mild diseases such as acute otitis media and rhinosinusitis or severe and sometimes fatal infections such as pneumonia, sepsis and meningitis (Esposito & Principi, 2013). S. pneumoniae colonizes the upper respiratory tract from the first months of life, and studies have clearly shown that pneumococcal carriage at the individual level is an immediate and necessary precursor to pneumococcal disease, and the most important cause of the spread of the pathogen in families and the community (Bogaert et al., 2004; Simell et al., 2012). Consequently, any changes in carriage characteristics may have a significant impact on disease epidemiology and vaccine efficacy, which explains the growing interest in studies of the dynamics of pneumococcal carriage. Abbreviations: NP, nasopharyngeal; OP, oropharyngeal.
068726 G 2014 SGM
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Pneumococcal colonization is usually evaluated in children because they are the subjects in whom the highest colonization rates have been repeatedly demonstrated, particularly in the youngest children (O’Brien et al., 2003). Colonization rates decline with age, probably because of shorter colonization episodes and less exposure (Ekdahl et al., 1997; Ho¨gberg et al., 2007; Hoti et al., 2009; Hill et al. 2010), but it could be important to evaluate S. pneumoniae colonization in adolescents and adults, particularly those with an underlying severe chronic disease who are at increased risk of severe pneumococcal infection and for whom pneumococcal vaccination is recommended (CDC, 2013). Knowing the characteristics of pneumococcal colonization in these subjects could also be useful for evaluating the theoretical coverage offered by current vaccines or the efficacy of any other preventive strategy. However, it is not clear which sampling site is best for collecting respiratory secretions and identifying the pneumococci carried by adolescents and adults. In the case of 393
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young children, a World Health Organization working group dedicated to defining core, standardized methods for studying pneumococcal colonization has established on the basis of various studies that nasopharyngeal (NP) sampling can be considered the best solution (O’Brien et al., 2003), but no studies comparing sampling methods in adolescents have yet been published and the few adult data are controversial: the findings of some studies support the use of NP sampling (Gladstone et al., 2012; Watt et al., 2004), but there are studies showing that oropharyngeal (OP) collection could lead to higher isolation rates (Boersma et al., 1993; Gladstone et al., 2012; Hendley et al., 1975; Trzcin´ski et al., 2013). The aim of this study was to compare the efficiency of OP and NP sampling in identifying and quantifying S. pneumoniae carriage in healthy adolescents.
METHODS
an RNase P-encoding gene in order to exclude PCR inhibition and DNA extraction failure. All of the positive cases were serotyped using primers and probes designed on the basis of the GenBank database sequences (www.ncbi.nlm.nih.gov) of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F [i.e. those in the 13-valent pneumococcal conjugate vaccine (PCV13) ], and synthesized by TIB Molbiol, as previously described (Esposito et al., 2012). Their analytical specificity was pre-evaluated by means of computer-aided analyses using Primer-BLAST (www.ncbi.nlm.nih.gov/tools/primerblast) and BLAST (www.blast.ncbi.nlm.nih.gov/Blast.cgi) software to compare the sequences with all of the sequences under ‘bacteria’ and ‘homo sapiens’. Statistical analysis. Continuous variables are expressed as mean
values±SD, and were analysed using Student’s t-test if the data were normally distributed (based on the Shapiro–Wilk statistic) or Wilcoxon’s rank sum test if the data were not. The categorical variables are expressed as numbers and percentages, and were analysed using contingency table analysis and the chi-squared or Fisher’s exact test as appropriate. All of the tests were two-sided, and a P value of ,0.05 was considered statistically significant. The data were analysed using SAS version 9.1 statistical software (SAS Institute).
Swab collection. The study was carried out during the second and
third week of September 2012, and involved adolescents attending two high schools in Milan, Italy, several kilometres away from each other. Participation was completely voluntary, but was solicited by means of a brochure distributed during lessons; moreover, the science teachers reinforced the message in the week preceding the swabbing by giving detailed explanations of S. pneumoniae and its related diseases. The study was approved by the Ethics Committees of the participating schools and the University of Milan. All of the subjects gave their written informed consent, as did the parents of subjects aged ,18 years. The enrolled adolescents completed a brief questionnaire concerning their demographic and clinical data, and vaccination history. The swabbing was carried out by a group of paediatric nurses who were trained specifically for the task, supervised by a paediatrician (NP) in the medical room of each of the two schools at the end of lessons on 2 consecutive days. The same nurse obtained two consecutive pharyngeal samples from each subject (the first through the mouth and the second through the nose) using an ESwab kit containing a polypropylene screw-cap tube with an internal conical shape filled with 1 ml liquid Amies medium and one pernasal applicator swab with a flocked nylon fibre tip (code 480 or 482CE according to the size of the subject; Copan Italia). The NP sample was collected as follows. The distance between the patient’s nares and earlobe was measured to estimate the length of insertion. The swab was gently inserted up the nostril towards the pharynx for that distance until resistance was felt and was then rotated 3 times. The swab was then withdrawn and put in the transport medium. The OF sampling was carried out using a tongue spatula to press the tongue downward to floor of the mouth and swabbing both the tonsillar arches and the posterior nasopharynx, without touching the sides of the mouth. The completed swabs were immediately transported to a central laboratory and processed within 2 h. Identification of S. pneumoniae. Bacterial genomic DNA was
extracted by means of a NucliSENS easyMAG automated extraction system (BioMerie´ux) using a generic protocol and a 250 ml sample input. The extracted genomic DNA was tested for the autolysin-Aencoding gene (lytA) and wzg (cpsA) gene of S. pneumoniae by means of real-time PCR, as previously described (Marchese et al., 2011). Each sample was tested in triplicate, and was considered positive if at least two of the three tests revealed the presence of both the genes. In order to maximize sensitivity, no internal amplification control was used in the reaction, but there was an external control. The specimens that were real-time PCR negative were also tested for the presence of 394
RESULTS More than 95 % of the adolescents attending the high schools agreed to take part in the study. A total of 294 and 308 subjects aged 15–19 years were potentially enrolled in the two schools. However, because of mild upper respiratory tract infection, 34 (11.6 %) and 38 (12.3 %) students were discarded, respectively. Consequently, a total of 530 healthy adolescents entered the study. The analysis of the demographic characteristics of the enrolled subjects, the incidence of carrier state and the distribution of the studied serotypes revealed no differences between groups. Consequently, all the data concerning subjects and carrier state were grouped and analysed together. Among the 530 adolescents (101 males, 19.1 %; mean age±SD, 16.4±1.3 years), 190 (35.8 %) were S. pneumoniae carriers. Table 1 shows the general characteristics of the study population by S. pneumoniae carrier status. The two groups were similar in terms of the distribution of gender, age, race, active and passive smoking, the number of siblings in the household, the presence of a chronic underlying disease, and infections and antibiotic therapy in the 3 months before enrolment. Interestingly, a significantly higher proportion of S. pneumoniae-positive adolescents had travelled to a foreign country in the 3 months preceding enrolment (P,0.0001). These adolescents did not travel together and there was no similarity in serotype distribution in students who had travelled similarly to each other. None of the enrolled subjects had received any dose of pneumococcal vaccines. Table 2 compares OP and NP sampling in terms of the detection of S. pneumoniae carriage. The detection of carrier status was significantly more frequent using OP sampling (35.8 vs 3.5 %; P,0.0001). As all of the subjects with positive NP samples also had positive OP samples, OP sampling detected 100 % of the identified S. pneumoniae carriers, whereas NP sampling detected only 8.9 %. The higher positivity of OP swabbing led to a better definition Journal of Medical Microbiology 63
Streptococcus pneumoniae carrier status in adolescents
Table 1. General characteristics of the study population by S. pneumoniae carrier status Characteristic No. of males (%) Mean age in years±SD No. of white subjects (%) No. of smoking fathers (%) No. of smoking mothers (%) Mean no. of siblings±SD No. of subjects with chronic underlying disease (%) No. of infections in previous 3 months (%) No. of subjects receiving antibiotic therapy in previous 3 months (%) No. of travellers to a foreign country in previous 3 months (%) No. of smokers (%) No. of subjects who had received pneumococcal vaccine (%)
OP and/or NP swab positive for S. pneumoniae (n5190)
OP and/or NP swab negative for S. pneumoniae (n5340)
P value*
42 (22.1) 16.0±1.1 184 (96.8) 26 (13.7) 30 (15.8) 1.2±0.8 9 (4.7) 15 (7.9) 20 (10.5)
59 (17.4) 16.7±1.6 315 (92.6) 62 (18.2) 58 (17.1) 1.1±0.8 22 (6.5) 26 (7.6) 38 (11.2)
0.22 0.96 0.07 0.21 0.79 0.25 0.53 0.94 0.93
133 (70.0)
176 (51.8)
,0.0001
36 (18.9) 0 (0)
64 (18.8) 0 (0)
0.93 –
*P values for between-group comparison using the chi-squared or Fisher’s exact test for categorical data, and Student’s t-test or Wilcoxon’s rank sum test for continuous variables, as appropriate.
of which pneumococcal serotypes were carried by the studied adolescents. At least one serotype included in PCV13 was identified in 188 of the 190 positive OP swabs (98.9 %), but in only 11 of the 17 NP samples (64.7 %) (P,0.0001). Moreover, OP sampling identified two or more of the serotypes included in PCV13 in 147 (77.4 %) positive cases, whereas NP sampling identified two or more serotypes in only 3 cases (17.6 %) (P,0.0001). The most frequently identified PCV13 serotype was 19F (45 % of all positive samples), followed by serotypes 5 and 9V.
DISCUSSION Reliably detecting S. pneumoniae in the upper respiratory tract is essential when studying pneumococcal colonization. Usually culture is used, although the differentiation of S. pneumoniae from other closely related viridans streptococci is not easy. The two classic phenotypic tests, bile solubility and optochin susceptibility, have been shown to fail to provide correct identifications on some occasions. This explains why, when molecular methods became available, they were largely used to evaluate pneumococcal carrier state and frequently reported, even if with some exceptions (Carvalho et al., 2013), more reliable than traditional culture in the identification of the pathogen in routine practice. Recently, Trzcin´ski et al. (2013) assessed with traditional culture and PCR paired trans-orally and trans-nasally obtained pharyngeal samples from 268 adults, and identified 52 (19 %) carriers by traditional culture and 105 (39 %) by PCR. Only when enriched cultures were used was the presence of S. pneumoniae found in a higher number of subjects than detected by PCR. Moreover, several studies have clearly evidenced the superior sensitivity of PCR to http://jmm.sgmjournals.org
non-enriched culture in the identification of S. pneumoniae in respiratory secretions or blood of subjects with pneumococcal diseases (Carvalho et al., 2013; Cvitkovic Spik et al., 2013). Differently from Carvalho et al. (2013), who for identification of pneumococcal carrier state used a molecular method based only on the amplification of the lytA gene, we performed this study, as did Trzcin´ski et al. (2013), amplifying both lytA and cpsA, a conserved gene in the capsular polysaccharide biosynthesis locus. Moreover, we considered positive only the samples in which both the genes were found in two out of three consecutive tests performed on the same OP and NP sample, so including only the encapsulated strains, those with the highest virulence. With this combined evaluation we are confident to have significantly reduced the risk of false-positive results because streptococci other than S. pneumoniae, such as other viridans streptococci and the recently described species Streptococcus pseudopneumoniae, that have the lytA gene are expected to be uncapsulated, and therefore escape from identification because they do not have the cpsA gene. Consequently, although positive molecular results may sometimes reflect only the transient presence of S. pneumoniae rather than true colonization, we believe that our data are sufficient to evaluate the efficiency of the two sampling methods in detecting real S. pneumoniae carriage. This conclusion is further supported by the characteristics of the tip used for sampling. Dube et al. (2013), who have evaluated the rate of detection of S. pneumoniae from different types of NP swabs in children, reported that the flocked swabs similar to those used in this study released more S. pneumoniae compared to both Dacron and rayon swabs from mock specimens. Similarly, higher bacterial loads were detected by quantitative PCR from flocked swabs compared with Dacron swabs from healthy children. 395
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Table 2. Comparison of OP and NP sampling for evaluating S. pneumoniae carrier status OP sampling (n5530) No. positive for S. pneumoniae (%) No. positive for one serotype included in PCV13 (%) No. positive for two or more serotypes included in PCV13 (%) No. positive for serotype 1 (%) No. positive for serotype 3 (%) No. positive for serotype 4 (%) No. positive for serotype 5 (%) No. positive for serotype 6A (%) No. positive for serotype 6B (%) No. positive for serotype 7F (%) No. positive for serotype 9V (%) No. positive for serotype 14 (%) No. positive for serotype 18C (%) No. positive for serotype 19A (%) No. positive for serotype 19F (%) No. positive for serotype 23F (%) No. positive only for serotypes not included in PCV13 (%)
190 (35.8) 41/190 (21.6) 147/190 (77.4) 1 (0.2) 4 (1.0) 40 (9.7) 110 (26.7) 6 (1.4) 2 (0.5) 3 (0.7) 44 (10.7) 2 (0.5) 1 (0.2) 14 (3.4) 184 (44.8) 1 (0.2) 2/190 (1.1)
NP sampling (n5530) 17 (3.5) 3/17 (17.6) 3/17 (17.6) – 1 (9.1) – 1 (9.1) – – 1 (9.1) 2 (18.2) – – 1 (9.1) 5 (45.5) – 11/17 (64.7)
P value* ,0.0001 0.76 ,0.0001
,0.0001
*P values for between-group comparison using the chi-squared or Fisher’s exact test for categorical data, and Student’s t-test or Wilcoxon’s rank sum test for continuous variables, as appropriate.
This is to the best of our knowledge the first study designed to evaluate methods of sampling respiratory secretions in order to identify adolescent S. pneumoniae carriers. The data indicate that OP sampling is significantly superior to NP sampling in detecting S. pneumoniae carriage in subjects of this age, and strongly support the use of trans-oral collection when studying adolescent carriage. The same conclusions were reached by Trzcin´ski et al. (2013), who found that trans-oral sampling was significantly superior in young adults regardless of the use of culture or molecular methods of S. pneumoniae detection. In younger children, NP swabbing usually detects more S. pneumoniae carriers than OP swabbing (Roca et al., 2012), but this may change with age. The nasopharynx is relatively easy to access via the trans-nasal route in younger children, but seems to be less accessible in adolescents and adults mainly because the NP approach is less tolerated (Trzcin´ski et al., 2013) and particularly in young people can be difficult to perform as requested. The head can be moved and the persistence of the tip in the right position for the required time can be lower than optimal. Moreover, an adequate sampling depth cannot be reached despite experienced nurses trying to obtain the sample (Trzcin´ski et al., 2013). The depth of swab insertion seems to be particularly important in defining S. pneumoniae identification rates as it has been found that OP swabbing is significantly more effective than NP swabbing when a rigid swab is used that limits sampling depth (Hendley et al., 1975). However, nasal specimens collected by otolaryngologists by means of anterior rhinoscopy yielded significantly fewer pneumococcal isolates than NP sampling (Ylikoski et al., 1989). In this study, starting from a greater number of positive samples, OP swabbing was also superior to NP 396
swabbing in characterizing the colonizing pneumococcal serotypes, thus providing a better evaluation of the theoretical coverage offered by PCV13. OP swabbing showed that all but two of the adolescents were colonized by at least one of the pneumococcal serotypes included in PCV13. Although some of these subjects may have also had nonvaccine serotypes, this suggests that PCV13 could have significantly reduced the carrier state of these serotypes that presently cause the greatest number of invasive pneumococcal disease. This information could not have been obtained using NP swabs because the detection of pneumococcal carriage was lower and serotype identification was significantly poorer. Although the proportions were significantly different, all of the PCV13 serotypes were found in the pharynges of the adolescents enrolled in this study. Because no difference in the distribution of the different serotypes was found between schools and these were sited very far from each other even if in the same town, it could be concluded that what is reported is representative of the serotypes that were circulating in Milan during the study period. However, more than 55 % of those identified using an OP swab were 19F and 9V, two of the serotypes included in the first conjugate pneumococcal vaccine [heptavalent pneumococcal conjugate vaccine (PCV7)] that has been licensed in Italy for use in children aged ,5 years for more than 10 years. This finding is different from that recently reported in an Italian study of pharyngeal pneumococcal colonization in adults aged ¢60 years living in Liguria (Italy): i.e. that pneumoccoccal carriage was quite similar to that of children living in the same geographical area and mainly based on serotypes not included in PCV7 (Ansaldi et al., 2013). This difference may be explained by the different Journal of Medical Microbiology 63
Streptococcus pneumoniae carrier status in adolescents
pneumococcal vaccination coverage of children aged ,5 years in Liguria and Lombardy (Italy) (where the present study was conducted): PCV7 vaccination coverage in Liguria had been .90 % for a number of years before the start of the colonization study by Ansaldi et al. (2013), whereas ,50 % of the children in Lombardy had received PCV7 in the years before this study. As carrier status in non-vaccinated subjects living in a given area is strictly related to the extent of childhood vaccination coverage (Davis et al., 2013), this could explain why so many of our adolescents were still colonized by the serotypes included in PCV7. However, a different mixing pattern between young vaccinated children and adolescents in Lombardy to the mixing pattern of vaccinated children and older adults in Liguria could, at least in part, explain this difference.
of pneumococcal pneumonia diagnostics by the use of rt-PCR on plasma and respiratory samples. Scand J Infect Dis 45, 731–737.
In conclusion, OP sampling is significantly more effective than NP sampling in evaluating pneumococcal carriage in adolescents. The use of PCV13 has recently been extended to these subjects because a relatively large number suffer from underlying conditions that are associated with an increased risk of S. pneumoniae disease. The evaluation of carriage is important when evaluating the potential impact of a vaccine and monitoring pneumococcal dynamics in vaccinated subjects, and OP sampling seems to lead to the best results.
Esposito, S., Marchese, A., Tozzi, A. E., Rossi, G. A., Da Dalt, L., Bona, G., Pelucchi, C., Schito, G. C., Principi, N. & Italian Pneumococcal CAP Group (2012). Bacteremic pneumococcal
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Impact of pneumococcal conjugate vaccines on nasopharyngeal carriage and invasive disease among unvaccinated people: Review of evidence on indirect effects. Vaccine. Dube, F. S., Kaba, M., Whittaker, E., Zar, H. J. & Nicol, M. P. (2013).
Detection of Streptococcus pneumoniae from different types of nasopharyngeal swabs in children. PLoS ONE 8, e68097. Ekdahl, K., Ahlinder, I., Hansson, H. B., Melander, E., Mo¨lstad, S., So¨derstro¨m, M. & Persson, K. (1997). Duration of nasopharyngeal
carriage of penicillin-resistant Streptococcus pneumoniae: experiences from the South Swedish Pneumococcal Intervention Project. Clin Infect Dis 25, 1113–1117. Esposito, S. & Principi, N. (2013). Pharmacotherapy for pneumo-
coccal infections: an update. Expert Opin Pharmacother 14, 65–77.
community-acquired pneumonia in children less than 5 years of age in Italy. Pediatr Infect Dis J 31, 705–710. Gladstone, R. A., Jefferies, J. M., Faust, S. N. & Clarke, S. C. (2012).
Sampling methods for the study of pneumococcal carriage: a systematic review. Vaccine 30, 6738–6744. Hendley, J. O., Sande, M. A., Stewart, P. M. & Gwaltney, J. M., Jr (1975). Spread of Streptococcus pneumoniae in families. I. Carriage
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ACKNOWLEDGEMENTS
Hill, P. C., Townend, J., Antonio, M., Akisanya, B., Ebruke, C., Lahai, G., Greenwood, B. M. & Adegbola, R. A. (2010). Transmission of
This study was supported by a grant from the Italian Ministry of Health (Bando Giovani Ricercatori 2009). The authors have no potential conflict of interest to declare.
Ho¨gberg, L., Geli, P., Ringberg, H., Melander, E., Lipsitch, M. & Ekdahl, K. (2007). Age- and serogroup-related differences in observed
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of Streptococcus pneumoniae and identification of pneumococcal serotypes by real-time polymerase chain reaction using blood samples from Italian children ¡ 5 years of age with community-acquired pneumonia. Microb Drug Resist 17, 419–424. O’Brien, K. L., Nohynek, H. & World Health Organization Pneumococcal Vaccine Trials Carriage Working Group (2003).
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Journal of Medical Microbiology 63
DOI 10.1099/jmm.0.068726-0
Oropharyngeal and nasopharyngeal sampling for the detection of adolescent Streptococcus pneumoniae carriers Nicola Principi, Leonardo Terranova, Alberto Zampiero, Francesca Manzoni, Laura Senatore, Walter Peves Rios and Susanna Esposito Correspondence
Pediatric Highly Intensive Care Unit, Department of Pathophysiology and Transplantation, Universita` degli Studi di Milano, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy
Susanna Esposito [email protected]
Received 22 September 2013 Accepted 26 November 2013
Monitoring the dynamics of pneumococcal carriage makes it possible to evaluate the epidemiological characteristics of Streptococcus pneumoniae disease and the theoretical coverage offered by pneumococcal vaccines. It has been demonstrated that the nasopharyngeal (NP) sampling of respiratory secretions is superior to oropharyngeal (OP) sampling for identifying pneumococci carried by younger children, but adult data are conflicting and there are no published studies of adolescents. In order to compare the efficiency of OP and NP sampling in identifying and quantifying S. pneumoniae carriage in healthy adolescents, 2 swab samples were obtained from 530 adolescents aged 15–19 years, the first taken from the posterior pharyngeal wall through the mouth (OP) and the second through the nose (NP). Bacterial genomic DNA was tested for the autolysin-A-encoding gene (lytA) and wzg (cpsA) gene of S. pneumoniae in order to evaluate pneumococcal carrier status. All of the positive cases were serotyped. S. pneumoniae was identified in 35.8 % of the OP swabs and 3.5 % of the NP swabs (P,0.0001). The serotypes included in the 13-valent pneumococcal conjugate vaccine (PCV13) were found in all but two OP samples (98.9 %) and only 64.7 % of the NP samples (P,0.0001). The most frequently identified PCV13 serotype in both groups was 19F, followed by serotypes 5 and 9V. In conclusion, OP sampling appeared significantly more effective than NP sampling in identifying and characterizing pneumococcal carrier status in adolescents. This suggests that OP sampling should be used when evaluating the dynamics of pneumococcal carriage among adolescents and the theoretical coverage offered by PCV13.
INTRODUCTION Streptococcus pneumoniae is the leading bacterial cause of infection worldwide, causing mild diseases such as acute otitis media and rhinosinusitis or severe and sometimes fatal infections such as pneumonia, sepsis and meningitis (Esposito & Principi, 2013). S. pneumoniae colonizes the upper respiratory tract from the first months of life, and studies have clearly shown that pneumococcal carriage at the individual level is an immediate and necessary precursor to pneumococcal disease, and the most important cause of the spread of the pathogen in families and the community (Bogaert et al., 2004; Simell et al., 2012). Consequently, any changes in carriage characteristics may have a significant impact on disease epidemiology and vaccine efficacy, which explains the growing interest in studies of the dynamics of pneumococcal carriage. Abbreviations: NP, nasopharyngeal; OP, oropharyngeal.
068726 G 2014 SGM
Printed in Great Britain
Pneumococcal colonization is usually evaluated in children because they are the subjects in whom the highest colonization rates have been repeatedly demonstrated, particularly in the youngest children (O’Brien et al., 2003). Colonization rates decline with age, probably because of shorter colonization episodes and less exposure (Ekdahl et al., 1997; Ho¨gberg et al., 2007; Hoti et al., 2009; Hill et al. 2010), but it could be important to evaluate S. pneumoniae colonization in adolescents and adults, particularly those with an underlying severe chronic disease who are at increased risk of severe pneumococcal infection and for whom pneumococcal vaccination is recommended (CDC, 2013). Knowing the characteristics of pneumococcal colonization in these subjects could also be useful for evaluating the theoretical coverage offered by current vaccines or the efficacy of any other preventive strategy. However, it is not clear which sampling site is best for collecting respiratory secretions and identifying the pneumococci carried by adolescents and adults. In the case of 393
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young children, a World Health Organization working group dedicated to defining core, standardized methods for studying pneumococcal colonization has established on the basis of various studies that nasopharyngeal (NP) sampling can be considered the best solution (O’Brien et al., 2003), but no studies comparing sampling methods in adolescents have yet been published and the few adult data are controversial: the findings of some studies support the use of NP sampling (Gladstone et al., 2012; Watt et al., 2004), but there are studies showing that oropharyngeal (OP) collection could lead to higher isolation rates (Boersma et al., 1993; Gladstone et al., 2012; Hendley et al., 1975; Trzcin´ski et al., 2013). The aim of this study was to compare the efficiency of OP and NP sampling in identifying and quantifying S. pneumoniae carriage in healthy adolescents.
METHODS
an RNase P-encoding gene in order to exclude PCR inhibition and DNA extraction failure. All of the positive cases were serotyped using primers and probes designed on the basis of the GenBank database sequences (www.ncbi.nlm.nih.gov) of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F [i.e. those in the 13-valent pneumococcal conjugate vaccine (PCV13) ], and synthesized by TIB Molbiol, as previously described (Esposito et al., 2012). Their analytical specificity was pre-evaluated by means of computer-aided analyses using Primer-BLAST (www.ncbi.nlm.nih.gov/tools/primerblast) and BLAST (www.blast.ncbi.nlm.nih.gov/Blast.cgi) software to compare the sequences with all of the sequences under ‘bacteria’ and ‘homo sapiens’. Statistical analysis. Continuous variables are expressed as mean
values±SD, and were analysed using Student’s t-test if the data were normally distributed (based on the Shapiro–Wilk statistic) or Wilcoxon’s rank sum test if the data were not. The categorical variables are expressed as numbers and percentages, and were analysed using contingency table analysis and the chi-squared or Fisher’s exact test as appropriate. All of the tests were two-sided, and a P value of ,0.05 was considered statistically significant. The data were analysed using SAS version 9.1 statistical software (SAS Institute).
Swab collection. The study was carried out during the second and
third week of September 2012, and involved adolescents attending two high schools in Milan, Italy, several kilometres away from each other. Participation was completely voluntary, but was solicited by means of a brochure distributed during lessons; moreover, the science teachers reinforced the message in the week preceding the swabbing by giving detailed explanations of S. pneumoniae and its related diseases. The study was approved by the Ethics Committees of the participating schools and the University of Milan. All of the subjects gave their written informed consent, as did the parents of subjects aged ,18 years. The enrolled adolescents completed a brief questionnaire concerning their demographic and clinical data, and vaccination history. The swabbing was carried out by a group of paediatric nurses who were trained specifically for the task, supervised by a paediatrician (NP) in the medical room of each of the two schools at the end of lessons on 2 consecutive days. The same nurse obtained two consecutive pharyngeal samples from each subject (the first through the mouth and the second through the nose) using an ESwab kit containing a polypropylene screw-cap tube with an internal conical shape filled with 1 ml liquid Amies medium and one pernasal applicator swab with a flocked nylon fibre tip (code 480 or 482CE according to the size of the subject; Copan Italia). The NP sample was collected as follows. The distance between the patient’s nares and earlobe was measured to estimate the length of insertion. The swab was gently inserted up the nostril towards the pharynx for that distance until resistance was felt and was then rotated 3 times. The swab was then withdrawn and put in the transport medium. The OF sampling was carried out using a tongue spatula to press the tongue downward to floor of the mouth and swabbing both the tonsillar arches and the posterior nasopharynx, without touching the sides of the mouth. The completed swabs were immediately transported to a central laboratory and processed within 2 h. Identification of S. pneumoniae. Bacterial genomic DNA was
extracted by means of a NucliSENS easyMAG automated extraction system (BioMerie´ux) using a generic protocol and a 250 ml sample input. The extracted genomic DNA was tested for the autolysin-Aencoding gene (lytA) and wzg (cpsA) gene of S. pneumoniae by means of real-time PCR, as previously described (Marchese et al., 2011). Each sample was tested in triplicate, and was considered positive if at least two of the three tests revealed the presence of both the genes. In order to maximize sensitivity, no internal amplification control was used in the reaction, but there was an external control. The specimens that were real-time PCR negative were also tested for the presence of 394
RESULTS More than 95 % of the adolescents attending the high schools agreed to take part in the study. A total of 294 and 308 subjects aged 15–19 years were potentially enrolled in the two schools. However, because of mild upper respiratory tract infection, 34 (11.6 %) and 38 (12.3 %) students were discarded, respectively. Consequently, a total of 530 healthy adolescents entered the study. The analysis of the demographic characteristics of the enrolled subjects, the incidence of carrier state and the distribution of the studied serotypes revealed no differences between groups. Consequently, all the data concerning subjects and carrier state were grouped and analysed together. Among the 530 adolescents (101 males, 19.1 %; mean age±SD, 16.4±1.3 years), 190 (35.8 %) were S. pneumoniae carriers. Table 1 shows the general characteristics of the study population by S. pneumoniae carrier status. The two groups were similar in terms of the distribution of gender, age, race, active and passive smoking, the number of siblings in the household, the presence of a chronic underlying disease, and infections and antibiotic therapy in the 3 months before enrolment. Interestingly, a significantly higher proportion of S. pneumoniae-positive adolescents had travelled to a foreign country in the 3 months preceding enrolment (P,0.0001). These adolescents did not travel together and there was no similarity in serotype distribution in students who had travelled similarly to each other. None of the enrolled subjects had received any dose of pneumococcal vaccines. Table 2 compares OP and NP sampling in terms of the detection of S. pneumoniae carriage. The detection of carrier status was significantly more frequent using OP sampling (35.8 vs 3.5 %; P,0.0001). As all of the subjects with positive NP samples also had positive OP samples, OP sampling detected 100 % of the identified S. pneumoniae carriers, whereas NP sampling detected only 8.9 %. The higher positivity of OP swabbing led to a better definition Journal of Medical Microbiology 63
Streptococcus pneumoniae carrier status in adolescents
Table 1. General characteristics of the study population by S. pneumoniae carrier status Characteristic No. of males (%) Mean age in years±SD No. of white subjects (%) No. of smoking fathers (%) No. of smoking mothers (%) Mean no. of siblings±SD No. of subjects with chronic underlying disease (%) No. of infections in previous 3 months (%) No. of subjects receiving antibiotic therapy in previous 3 months (%) No. of travellers to a foreign country in previous 3 months (%) No. of smokers (%) No. of subjects who had received pneumococcal vaccine (%)
OP and/or NP swab positive for S. pneumoniae (n5190)
OP and/or NP swab negative for S. pneumoniae (n5340)
P value*
42 (22.1) 16.0±1.1 184 (96.8) 26 (13.7) 30 (15.8) 1.2±0.8 9 (4.7) 15 (7.9) 20 (10.5)
59 (17.4) 16.7±1.6 315 (92.6) 62 (18.2) 58 (17.1) 1.1±0.8 22 (6.5) 26 (7.6) 38 (11.2)
0.22 0.96 0.07 0.21 0.79 0.25 0.53 0.94 0.93
133 (70.0)
176 (51.8)
,0.0001
36 (18.9) 0 (0)
64 (18.8) 0 (0)
0.93 –
*P values for between-group comparison using the chi-squared or Fisher’s exact test for categorical data, and Student’s t-test or Wilcoxon’s rank sum test for continuous variables, as appropriate.
of which pneumococcal serotypes were carried by the studied adolescents. At least one serotype included in PCV13 was identified in 188 of the 190 positive OP swabs (98.9 %), but in only 11 of the 17 NP samples (64.7 %) (P,0.0001). Moreover, OP sampling identified two or more of the serotypes included in PCV13 in 147 (77.4 %) positive cases, whereas NP sampling identified two or more serotypes in only 3 cases (17.6 %) (P,0.0001). The most frequently identified PCV13 serotype was 19F (45 % of all positive samples), followed by serotypes 5 and 9V.
DISCUSSION Reliably detecting S. pneumoniae in the upper respiratory tract is essential when studying pneumococcal colonization. Usually culture is used, although the differentiation of S. pneumoniae from other closely related viridans streptococci is not easy. The two classic phenotypic tests, bile solubility and optochin susceptibility, have been shown to fail to provide correct identifications on some occasions. This explains why, when molecular methods became available, they were largely used to evaluate pneumococcal carrier state and frequently reported, even if with some exceptions (Carvalho et al., 2013), more reliable than traditional culture in the identification of the pathogen in routine practice. Recently, Trzcin´ski et al. (2013) assessed with traditional culture and PCR paired trans-orally and trans-nasally obtained pharyngeal samples from 268 adults, and identified 52 (19 %) carriers by traditional culture and 105 (39 %) by PCR. Only when enriched cultures were used was the presence of S. pneumoniae found in a higher number of subjects than detected by PCR. Moreover, several studies have clearly evidenced the superior sensitivity of PCR to http://jmm.sgmjournals.org
non-enriched culture in the identification of S. pneumoniae in respiratory secretions or blood of subjects with pneumococcal diseases (Carvalho et al., 2013; Cvitkovic Spik et al., 2013). Differently from Carvalho et al. (2013), who for identification of pneumococcal carrier state used a molecular method based only on the amplification of the lytA gene, we performed this study, as did Trzcin´ski et al. (2013), amplifying both lytA and cpsA, a conserved gene in the capsular polysaccharide biosynthesis locus. Moreover, we considered positive only the samples in which both the genes were found in two out of three consecutive tests performed on the same OP and NP sample, so including only the encapsulated strains, those with the highest virulence. With this combined evaluation we are confident to have significantly reduced the risk of false-positive results because streptococci other than S. pneumoniae, such as other viridans streptococci and the recently described species Streptococcus pseudopneumoniae, that have the lytA gene are expected to be uncapsulated, and therefore escape from identification because they do not have the cpsA gene. Consequently, although positive molecular results may sometimes reflect only the transient presence of S. pneumoniae rather than true colonization, we believe that our data are sufficient to evaluate the efficiency of the two sampling methods in detecting real S. pneumoniae carriage. This conclusion is further supported by the characteristics of the tip used for sampling. Dube et al. (2013), who have evaluated the rate of detection of S. pneumoniae from different types of NP swabs in children, reported that the flocked swabs similar to those used in this study released more S. pneumoniae compared to both Dacron and rayon swabs from mock specimens. Similarly, higher bacterial loads were detected by quantitative PCR from flocked swabs compared with Dacron swabs from healthy children. 395
N. Principi and others
Table 2. Comparison of OP and NP sampling for evaluating S. pneumoniae carrier status OP sampling (n5530) No. positive for S. pneumoniae (%) No. positive for one serotype included in PCV13 (%) No. positive for two or more serotypes included in PCV13 (%) No. positive for serotype 1 (%) No. positive for serotype 3 (%) No. positive for serotype 4 (%) No. positive for serotype 5 (%) No. positive for serotype 6A (%) No. positive for serotype 6B (%) No. positive for serotype 7F (%) No. positive for serotype 9V (%) No. positive for serotype 14 (%) No. positive for serotype 18C (%) No. positive for serotype 19A (%) No. positive for serotype 19F (%) No. positive for serotype 23F (%) No. positive only for serotypes not included in PCV13 (%)
190 (35.8) 41/190 (21.6) 147/190 (77.4) 1 (0.2) 4 (1.0) 40 (9.7) 110 (26.7) 6 (1.4) 2 (0.5) 3 (0.7) 44 (10.7) 2 (0.5) 1 (0.2) 14 (3.4) 184 (44.8) 1 (0.2) 2/190 (1.1)
NP sampling (n5530) 17 (3.5) 3/17 (17.6) 3/17 (17.6) – 1 (9.1) – 1 (9.1) – – 1 (9.1) 2 (18.2) – – 1 (9.1) 5 (45.5) – 11/17 (64.7)
P value* ,0.0001 0.76 ,0.0001
,0.0001
*P values for between-group comparison using the chi-squared or Fisher’s exact test for categorical data, and Student’s t-test or Wilcoxon’s rank sum test for continuous variables, as appropriate.
This is to the best of our knowledge the first study designed to evaluate methods of sampling respiratory secretions in order to identify adolescent S. pneumoniae carriers. The data indicate that OP sampling is significantly superior to NP sampling in detecting S. pneumoniae carriage in subjects of this age, and strongly support the use of trans-oral collection when studying adolescent carriage. The same conclusions were reached by Trzcin´ski et al. (2013), who found that trans-oral sampling was significantly superior in young adults regardless of the use of culture or molecular methods of S. pneumoniae detection. In younger children, NP swabbing usually detects more S. pneumoniae carriers than OP swabbing (Roca et al., 2012), but this may change with age. The nasopharynx is relatively easy to access via the trans-nasal route in younger children, but seems to be less accessible in adolescents and adults mainly because the NP approach is less tolerated (Trzcin´ski et al., 2013) and particularly in young people can be difficult to perform as requested. The head can be moved and the persistence of the tip in the right position for the required time can be lower than optimal. Moreover, an adequate sampling depth cannot be reached despite experienced nurses trying to obtain the sample (Trzcin´ski et al., 2013). The depth of swab insertion seems to be particularly important in defining S. pneumoniae identification rates as it has been found that OP swabbing is significantly more effective than NP swabbing when a rigid swab is used that limits sampling depth (Hendley et al., 1975). However, nasal specimens collected by otolaryngologists by means of anterior rhinoscopy yielded significantly fewer pneumococcal isolates than NP sampling (Ylikoski et al., 1989). In this study, starting from a greater number of positive samples, OP swabbing was also superior to NP 396
swabbing in characterizing the colonizing pneumococcal serotypes, thus providing a better evaluation of the theoretical coverage offered by PCV13. OP swabbing showed that all but two of the adolescents were colonized by at least one of the pneumococcal serotypes included in PCV13. Although some of these subjects may have also had nonvaccine serotypes, this suggests that PCV13 could have significantly reduced the carrier state of these serotypes that presently cause the greatest number of invasive pneumococcal disease. This information could not have been obtained using NP swabs because the detection of pneumococcal carriage was lower and serotype identification was significantly poorer. Although the proportions were significantly different, all of the PCV13 serotypes were found in the pharynges of the adolescents enrolled in this study. Because no difference in the distribution of the different serotypes was found between schools and these were sited very far from each other even if in the same town, it could be concluded that what is reported is representative of the serotypes that were circulating in Milan during the study period. However, more than 55 % of those identified using an OP swab were 19F and 9V, two of the serotypes included in the first conjugate pneumococcal vaccine [heptavalent pneumococcal conjugate vaccine (PCV7)] that has been licensed in Italy for use in children aged ,5 years for more than 10 years. This finding is different from that recently reported in an Italian study of pharyngeal pneumococcal colonization in adults aged ¢60 years living in Liguria (Italy): i.e. that pneumoccoccal carriage was quite similar to that of children living in the same geographical area and mainly based on serotypes not included in PCV7 (Ansaldi et al., 2013). This difference may be explained by the different Journal of Medical Microbiology 63
Streptococcus pneumoniae carrier status in adolescents
pneumococcal vaccination coverage of children aged ,5 years in Liguria and Lombardy (Italy) (where the present study was conducted): PCV7 vaccination coverage in Liguria had been .90 % for a number of years before the start of the colonization study by Ansaldi et al. (2013), whereas ,50 % of the children in Lombardy had received PCV7 in the years before this study. As carrier status in non-vaccinated subjects living in a given area is strictly related to the extent of childhood vaccination coverage (Davis et al., 2013), this could explain why so many of our adolescents were still colonized by the serotypes included in PCV7. However, a different mixing pattern between young vaccinated children and adolescents in Lombardy to the mixing pattern of vaccinated children and older adults in Liguria could, at least in part, explain this difference.
of pneumococcal pneumonia diagnostics by the use of rt-PCR on plasma and respiratory samples. Scand J Infect Dis 45, 731–737.
In conclusion, OP sampling is significantly more effective than NP sampling in evaluating pneumococcal carriage in adolescents. The use of PCV13 has recently been extended to these subjects because a relatively large number suffer from underlying conditions that are associated with an increased risk of S. pneumoniae disease. The evaluation of carriage is important when evaluating the potential impact of a vaccine and monitoring pneumococcal dynamics in vaccinated subjects, and OP sampling seems to lead to the best results.
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ACKNOWLEDGEMENTS
Hill, P. C., Townend, J., Antonio, M., Akisanya, B., Ebruke, C., Lahai, G., Greenwood, B. M. & Adegbola, R. A. (2010). Transmission of
This study was supported by a grant from the Italian Ministry of Health (Bando Giovani Ricercatori 2009). The authors have no potential conflict of interest to declare.
Ho¨gberg, L., Geli, P., Ringberg, H., Melander, E., Lipsitch, M. & Ekdahl, K. (2007). Age- and serogroup-related differences in observed
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