An easy method for detection of nasopharyngeal carriage of multiple Streptococcus pneumoniae serotypes

Journal of Microbiological Methods 75 (2008) 540–544

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Journal of Microbiological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h

An easy method for detection of nasopharyngeal carriage of multiple Streptococcus pneumoniae serotypes☆ Margit S. Kaltoft a, Uffe B. Skov Sørensen b, Hans-Christian Slotved a,⁎, Helle Bossen Konradsen a a b

Neisseria and Streptococcus Reference Laboratory, Department of Bacteriology, Mycology and Parasitology, Statens Serum Institut, Copenhagen, Denmark Institute of Medical Microbiology and Immunology, University of Aarhus, Aarhus, Denmark

a r t i c l e

i n f o

Article history: Received 14 May 2008 Received in revised form 12 August 2008 Accepted 29 August 2008 Available online 5 September 2008 Keywords: Streptococcus pneumoniae Pneumococcus Colonization Carriage Serotyping Diagnosis

a b s t r a c t In this paper, a simplified method for detection of pneumococcal carriage and for revealing the presence of several serotypes in a nasopharyngeal sample is evaluated. Enrichment broth was used for transportation and for the initial culturing of samples. All specimens were examined directly by the capsular reaction test for the presence of any of the 91 known pneumococcal serotypes. Sub-culturing on blood agar was used for isolation of the pneumococcal strains detected in the primary broth culture. A total of 693 nasopharyngeal swabs were obtained among children, their parents and employees in day care centres. Pneumococci were observed in 363 samples and 36 of these (9.9%) contained more than one serotype (multiple carriages). Two persons carried 3 different serotypes simultaneously. A significant increase in the positive sampling rate (5.8%) was achieved by using the simplified method compared to conventional streaking of the swabs directly on blood agar (p b 0.0001). © 2008 Elsevier B.V. All rights reserved.

1. Introduction Streptococcus pneumoniae (pneumococcus) is a major human pathogen causing otitis media, sinusitis, pneumonia, bacteremia and meningitis especially among young children and the elderly (Cherian, 2007; Konradsen and Kaltoft, 2002). By vaccination with 7-valent pneumococcal conjugate vaccine it is possible to prevent most cases of invasive and to some extent respiratory pneumococcal infections in young children (Black et al., 2000). The optimal formulation of pneumococcal vaccines is complicated by the fact that the prevalence of the serotypes causing disease changes over time both naturally and as a consequence of health interventions, including vaccination (Sandgren et al., 2004). Continuous surveillance of pneumococcal serotype distribution is, therefore, still needed (Berg et al., 2006; Beall et al., 2006). Colonization with more than one pneumococcal serotype has been observed to be as high as 29% among carriers in some populations (Gratten et al., 1989; Sá-Leão et al., 2002). Thus, it is of utmost importance to be able to detect carriers of multiple serotypes during

☆ The study was approved by The Region Zealand Committee on Biomedical Research Ethics and the Department on Children and Culture in Roskilde Municipality, Denmark. ⁎ Corresponding author. Department for Bacteriology, Mycology and Parasitology, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen, Denmark. Tel.: +45 32688422; fax: +45 32683865. E-mail address: [email protected] (H.-C. Slotved). 0167-7012/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2008.08.010

pneumococcal vaccine efficacy trials and during evaluation of the impact that vaccination may have on the dynamics of nasopharyngeal colonization (O'Brien and Nohynek, 2003). For studying genetic events such as capsular gene exchanges among pneumococci, a sensitive method for detection of all the pneumococcal populations simultaneously present in the nasopharynx is also needed (Sá-Leão et al., 2002; Claverys et al., 2000). Mouse intraperitoneal challenge assays were used in the past for evaluation of pneumococcal colonization (Austrian, 1986; Gundel and Okura, 1933). For ethical reasons, this technique has now been abandoned. The mouse inoculation assays were sensitive but timeconsuming and cost-ineffective (Huebner et al., 2000). In addition, mice have variable susceptibility to infections caused by different pneumococcal serotypes, which may bias the results (Briles et al., 1992). Converse and Dillon (1977) found that direct streaking of nasopharyngeal swabs onto blood agar plates containing gentamycin (5 μg/ml) was superior to the mouse inoculation assay for detection of pneumococci from nasopharyngeal specimens. It is possible to detect simultaneous carriage by consistent serotyping of a large number of suspected colonies obtained from primary agar plates. This is, however, a laborious procedure which is cost- and effort-ineffective (Huebner et al., 2000). Lankinen et al. (1997) showed that analysis by counter immune electrophoresis for the presence of pneumococcal capsular polysaccharides is a sensitive method for detection of multiple serotypes in a single sample. The present paper evaluates a method developed for easy examination of a large number of

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nasopharyngeal samples where a broth medium is used for transportation of swabs. The inoculated broths are initially incubated in order to promote growth of pneumococci and thereby increasing the sensitivity of the assay. Pneumococci are then detected directly in the broth cultures by the capsular reaction test using a panel of diagnostic antisera. By this procedure, it is possible to reveal the presence of different serotypes in one sample. Finally, the individual pneumococcal strains were isolated by qualified search for colonies of the recognized serotypes on the plates made from the primary cultures. 2. Materials and methods

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Table 2 Carriage rates of pneumococcus and the proportion of carriers colonized simultaneously with multiple serotypes

Number of participants Mean age at sampling Carriage rate, % (N)a Proportion carriers with N 1 serotype, % (N)a Proportion carriers with N 2 serotypes, % (N)a a b

Infants

Children

Parents

Employees Total

123 23 months 71b (87) 12 (10)

461 52.4 months 55 (253) 8 (20)

72 34 years 10 (7) 0

37 43 years 43 (16) 38 (6)

52 (363) 10 (36)

0

0

0

12.5 (2)

0.6 (2)

693

Strains of non-capsulated S. pneumoniae are included in the figures. Figures are rounded off.

2.1. Study design A prospective one-year-study of nasopharyngeal carriage was conducted in 27 day care centres located in the municipality of Roskilde, Denmark. Nasopharyngeal samples were obtained from 461 children (mean age 52.4 months) attending 23 kindergartens, and from 123 children (mean age 23 months) attending 4 nurseries. Nasopharyngeal samples from 72 parents to the children in the nurseries and from 37 staff members were also included. Written informed consent was obtained from the parents of each child and from the staff members. The local ethical committee and the Department on Children and Culture in Roskilde Municipality approved the study. 2.2. Media Serum broth made from beef infusion enriched with 5% horse serum and with 0.33% defibrinated horse blood (SSI Diagnostica, Hillerød, Denmark) was used both for transportation and for initial culturing of samples. Blood agar plates with gentamycin (G-HB agar, 5 μg gentamycin per ml, 5% horse blood, SSI Diagnostica) were used as a selective medium for isolation of pneumococci (O'Brien and Nohynek, 2003). Blood agar plates (10% horse blood, SSI Diagnostica) were used for sub-culturing. 2.3. Sampling procedure A deep nasopharyngeal sample was obtained from each participant as described by O'Brien and Nohynek (2003) by using a calcium alginate-tipped aluminium wire swab (Calgi swab type 1, Puritan, Hardwood Products Company, Guildford, MN, USA). The samples were transported after inserting the tip of the swab in a tube containing 1.5 ml serum broth and kept at 5 °C until it was processed in the laboratory within 3–5 h. 2.4. Microbiological techniques Specimens were processed as follows: i) Conventional culturing and serotyping was performed as described by O'Brien and Nohynek (2003) and Konradsen and Kaltoft (2002). Briefly, at least two preferably

different looking colonies were selected from the primary G-HB agar plate and sub-cultured on 10% blood agar plates. The strains were examined for optochin sensitivity (Optochin Diagnostic Tablets 10 μg, Rosco, Taastrup, Denmark), bile solubility and were serotyped as described below. ii) Direct inspection of overnight serum broth cultures was used as an easy, sensitive and fast method for revealing the presence of one or more of the 91 known pneumococcal serotypes. The tests were carried out as follows. Tubes containing swabs placed in serum broth [see i)] were incubated overnight at 35 °C. The next morning, a drop of the serum broth from each tube was streaked on G-HB agar (secondary plate), and an optochin tablet was placed on each plate. The secondary plates were used for comparison with the primary plates, for control of results and for isolation of pneumococcal strains. The direct inspections of the serum broth cultures were performed by the capsular reaction tests by using pool, group and type-specific diagnostic pneumococcal antisera according to the procedure given by the manufacturer (SSI Diagnostica, Hillerød, Denmark). If a single common serotype was observed both by direct examination of the broth culture and on the primary plate, the results were confirmed by serotyping of an additional colony obtained from the secondary plate. If more than one serotype were detected by either of the two former tests, then several colonies obtained from the secondary plate were serotyped in order to find and isolate the pneumococcal strains in question. Pneumococci were identified and characterized by the following criteria (O'Brien and Nohynek, 2003): alpha-hemolysis, optochin susceptibility (Gardam and Miller, 1998), bile solubility (Facklam and Washington, 1991) and serotyping (Konradsen and Kaltoft 2002). All cultures were incubated overnight in a 5% CO2 incubator at 35 °C. 2.5. Statistics For comparison of two paired binomial samples, McNemar's test was conducted, and furthermore 95% confidence limits for the difference in probability of a positive sampling with or without direct serotyping of a serum broth culture, respectively, were calculated (Altman, 1991). p b 0.05 was chosen as the significance level. 3. Results

Table 1 Comparison of two techniques for detection of pneumococci in nasopharyngeal samples Primary G-HBa Positive Negative Total

Secondary G-HBb Positive

Negative

Total

319 42 361c

2 330 332

321 372 693

Nasopharyngeal swabs were kept in serum broth and plated on 5% horse blood agar plates containing 5.0 μg gentamycin per ml (G-HB). See Materials and methods for details. a The primary plates were prepared from the samples without preincubation. b Samples were incubated for 18 h before the secondary plates were prepared. c The total number of positive samples was 363.

A total of 693 nasopharyngeal swabs were collected; of these, 46% (321) were positive for pneumococci on the primary G-HB agar plate, while 52% (361) were positive on the secondary G-HB plate (Table 1). Only two of the samples were found to be positive on the primary plates but negative on the secondary plates. Both of these samples contained type 18C pneumococci. In contrast, 40 samples out of the 363 positive samples (11%) showed growth of pneumococci only on the secondary plates. The sensitivities of the two techniques were 99% (361/363) and 88% (321/363), respectively. Thus, the incubation step significantly increased the positive sampling rate by 5.8% (95% CI: 4.0%–7.6%, p b 0.0001).

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By using the improved method including the direct examination of the serum broth culture by the capsular reaction test 36 of the 363 positive samples (10%) were found to contain mixed populations of pneumococci. Among them, 34 samples contained two different pneumococcal serotypes and two samples contained 3 serotypes simultaneously. In the majority of the samples containing mixed populations of pneumococci, i.e. 28, including the two samples containing 3 different serotypes simultaneously, all serotypes were detected by both methods. However, in four samples only one serotype were found on the primary G-HB plate while two different serotypes were detected in the serum broths and on the secondary G-HB plate. Four other samples showed no growth of pneumococci on the primary G-HB plates while pneumococci of two different serotypes were found by the direct method. Thus, the conventional plating technique missed more than one fifth (8/36) of these double positive samples. All serum broth cultures, which were found to contain capsulated pneumococci by direct capsular reaction test, exhibited growth of pneumococci of the same serotype on the secondary control plates. This confirmed that direct serotyping on broth cultures is a reliable method, although the technique does not allow for detection of rough pneumococci. Carriage of pneumococci was examined among children attending 27 day care centres, employees and parents. Table 2 shows the total carriage rates and the rates for carriage of mixed populations of pneumococci for each group of participants. The lowest carriage rates were found among the parents (10%) and none of them carried more

than a single serotype. In contrast, the carriage rate among staff members was 43% (16/37), and 38% (6/37) carried more than one pneumococcal serotype simultaneously. Infants were more often colonized (71%) than younger children (55%) and the rate of carrying mixed pneumococcal populations were 12% and 8%, respectively. The distribution of the serotypes detected in the nasopharyngeal samples is presented in Table 3. A total of 32 different serotypes and rough pneumococci were found. Except for the two 18C isolates mentioned above, all the serotype(s) detected on the primary plates were also found in the serum broth cultures by direct serotyping. There were only minor differences in the distribution among serotypes detected by the two techniques. Thus, none of them seemed to miss the detection of particular serotypes. Table 3 also shows that the total number of rough pneumococci found on the secondary plates were 30, while only 25 rough strains were detected by random picking of colonies from the primary G-HB plates. It was not possible to detect rough pneumococci directly in the serum broth, as they do not react with the diagnostic antisera. However, they were identified from the secondary G-HB as alpha-hemolytic colonies, optochin sensitive and positive in a bile solubility test. Overall, 7.5% of the pneumococcal isolates detected in this study were non-capsulated. The number of isolates expressing a serotype covered by the current 7-valent pneumococcal conjugate vaccine was 144 and 127, corresponding to a coverage rate of 35.9% and 31.7%, by the direct serotyping of the serum broth cultures and of colonies obtained from the primary plates, respectively. 4. Discussion

Table 3 Serotype distribution among 401 pneumococci isolated from nasopharyngeal samples Serotypea

Numberb

% of allc (N = 401)

6A 23F 19F Rough 11A 14 19A 6B 22F 18Ce 15B 16F 15C 4 20 35F 3 24F 9V 7F 38 37 33F 21 10A 8 7B 18F 18B 9N 35B 24A 17F

57/53 38/35 34/33 30d/25 27/26 26/19 22/17 18/13 17/15 13/13 14/11 12/0 11/8 10/9 9/8 8/0 8/7 6/5 5/0 5/4 5/0 3/0 3/0 3/2 3/0 2/1 2/0 2/0 2/1 1/0 1/0 1/0 1/0

14.2 9.5 8.5 7.5 6.7 6.5 5.5 4.5 4.2 3.7 3.5 3.0 2.7 2.5 2.2 2.0 2.0 1.5 1.2 1.2 1.2 0.7 0.7 0.7 0.7 0.5 0.5 0.5 0.5 0.2 0.2 0.2 0.2

a Pneumococcal serotypes and non-typeable “rough” pneumococci listed according to decreasing order of frequency. Types covered by the 7-valent pneumococcal conjugate vaccine are shown in boldface. b Number of isolates of the given serotype detected by: direct serotyping of broth cultures after incubation/serotyping of randomly selected colonies obtained from primary plates prepared from swabs before incubation of the transport media. c Distribution of serotypes found by direct serotyping. d Rough pneumococci were detected on blood agar plates only. e The total number of serotype 18C isolates was 15.

In 2003, the WHO Pneumococcal Vaccine Working Group recommended a method for detection of upper respiratory carriage of S. pneumoniae (O'Brien and Nohynek, 2003). This core consensus method is useful and allows comparisons of data from different carriage studies. This method is, however, not very sensitive in detecting of carriers colonized with multiple pneumococcal serotypes and an optimized method is, therefore, still needed (O'Brien and Nohynek, 2003). The purpose of our study was to evaluate an easy and sensitive method for detection of pneumococci in nasopharyngeal specimens and for detection of simultaneous carriage of multiple pneumococcal serotypes. Compared to other media, serum broth enhances the growth of pneumococci (Slotved and Kerrn, 2005) and the present study demonstrated that incubation of the cultures before microscopic examination increases the sensitivity of the assay significantly. Lankinen et al. (1997) included gentamycin in the enrichment broth, but they did not gain any increase in the yield of positive samples. Antibiotics were, therefore, omitted from the serum broth used in our study. The WHO Pneumococcal Vaccine Working Group has evaluated and recommended the STGG transport medium based on skimmed milk for transportation and storage of the nasopharyngeal swabs during field trails (O'Brien et al., 2001). We used a commercially available enhancement medium for both transportation and for optimal culturing of the specimens and initially kept the samples at 5 °C in order to compare techniques. However, our observations show that there is no need for cooling since preincubation enhances the recovery of pneumococci when samples are examined within 18 h. The capsular reaction test was chosen for simultaneous detection and typing of pneumococci in the overnight broth culture (Austrian, 1976). In addition, all serum broth cultures were sub-cultured on selective G-HB plates. This step is included when strains are needed for testing of antibiotic resistance, for detection of non-capsulated pneumococci or for other purposes. The frequency of non-capsulated pneumococci in this study (7.5%) is similar to the rates found in other carriage studies (Sá-Leão et al., 2008). In a study from 2004, a typespecific enzyme immunoassay covering seven frequent serotypes was used for detection of pneumococcal capsular polysaccharide antigen

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in nasopharyngeal specimens (Lankinen et al., 2004). A similar assay for detection of multiple serotypes has been developed (Bronsdon et al., 2004), but none of these methods are recommended for routine diagnostic purposes. The mouse inoculation assay was previously used for detection of multiple pneumococcal carriages (Gundel and Okura, 1933), but has been abandoned for ethical reasons. Picking and serotyping randomly selected colonies from blood agar plate has been used in most studies of carriage of multiple serotypes (Gratten et al., 1989); this procedure is, however, labor-intensive and may underestimate the number of serotypes present (Charalambous et al., 2008). The rates of carriers colonized with a single or with multiple pneumococcal strains are at the same level in this study or higher than rates reported in other recent studies from western countries (Garciade-Lomas et al., 1997; Kellner and Ford-Jones, 1999; St Sauver et al., 2000; Syrjanen et al., 2001). A carriage rate of 42% was found among 2912 children attending day care centres in Malmö, Sweden (Nilsson and Laurell, 2001). In a similar Finnish study, the carriage rate among children aged 2 to 24 months was only 21%, and the multiple carriage rate was 3.7% (Syrjanen et al., 2001). In the present study, the carriage rate was found to vary from 54% to 70% dependent on the age of the children, and the rate of carrying multiple pneumococcal strains varied from 8% to 12% (Table 2). An explanation of the observed discrepancies among the studies might be that the children in our study were all day care attendees, while most of the children in the Finnish study were taken care of at home. The pneumococcal carriage rate and the rate of carrying multiple strains were high among staff members in day care centres. We were not able to find other recent studies reporting similar carriage rates among adults. However, similar observations were reported among military recruits in the past (Hodges et al., 1946). The carriage rates found among healthy children and infants by using our improved method were at the same level as the rates detected among children suffering from respiratory infections by using latex agglutination and counter immune electrophoresis (Lankinen et al., 1997; Lloyd-Evans et al., 1996). After receiving the conjugate vaccine children are less colonized with the vaccine types than unvaccinated children, and massvaccination seems to lead to herd immunity (Jacobs et al., 2008; Dagan et al., 2002). However, vaccination may lead to replacement with other serotypes. Furthermore, capsular switching is known to occur naturally among pneumococci during carriage (Sandgren et al., 2004; Barnes et al., 1995), a phenomenon also observed among the resistant pneumococci (Garcia-Suarez Mdel et al., 2006; Gonzales et al., 2006). Thus, it is important to continue the global surveillance of the serotype distribution among pneumococci isolated from patients and from healthy carriers in order to ensure the optimal formulation of the pneumococcal vaccines in use. In this paper, we have suggested a simplified method which will be useful for the future surveillance of pneumococcal serotype distributions. Acknowledgements We are grateful to technicians Susanne Sauer and Kirsten Burmeister from the former Streptococcus Unit at SSI for their excellent technical support, biostatistician Jesper Madsen, Department of Biostatistics, SSI for the statistical support, and to the children, parents and staff members of the 27 day care centres in Roskilde Municipality for participating in the study. Financial support: This study was partially supported by a grant from Leo Pharma Nordic. References Altman, D.G.,1991. Practical Statistics for Medical Research. Chapman and Hall, pp. 237–238. Austrian, R., 1976. The quellung reaction, a neglected microbiologic technique. Mt. Sinai. J. Med. 43, 699–709.

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