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The use of Streptococcus pneumoniae nasopharyngeal isolates from healthy children to predict features of invasive disease

KELLNER, JAMES D. MD; MCGEER, ALLISON MD; CETRON, MARTIN S. MD; LOW, DONALD E. MD; BUTLER, JAY C. MD; MATLOW, ANNE MD; TALBOT, JAMES MD; FORD-JONES, E. LEE MD

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The Pediatric Infectious Disease Journal: April 1998 - Volume 17 - Issue 4 - p 279-286
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Abstract

INTRODUCTION

As penicillin-nonsusceptible Streptococcus pneumoniae (PNSP) infections become more prevalent worldwide, it is of particular interest to determine whether studies of nasopharyngeal carriage isolates accurately represent the prevalence of PNSP found in invasive disease. The incidence of disease is related to the prevalence of asymptomatic carriage, but in certain developing countries nearly 100% of infants and children <5 years of age have continuous asymptomatic carriage.1, 2 The incidence of invasive disease in these populations is much higher than in children from developed countries where carriage prevalence is typically 25 to 50% in children <10 years of age.3-7 The prevalence of carriage also increases in children who are sick with respiratory infections or otitis media.8-11 Most individuals with invasive S. pneumoniae disease carry the same strain in their nasopharynx at the time of invasive infection.11, 12

The 10 most frequent S. pneumoniae serotypes found in collections of invasive or middle ear isolates are similar to those found in related collections of upper respiratory isolates; however, there are differences in the proportions of certain serotypes. In studies of children from developed countries, serotype 14 is found more commonly with invasive disease, whereas serogroup 3, 6, 19 or 23 may be found more commonly in upper respiratory and middle ear specimens.12-16

It is unclear whether antibiotic susceptibility patterns of isolates from healthy carriers accurately reflect the susceptibility patterns found among invasive isolates. Two studies that compared sterile site isolates with nasopharyngeal (NP) carriage isolates from healthy children found no difference in the rate of PNSP (MIC ≥ 0.1 μg/ml) between the groups.17, 18 Other studies compared sterile site isolates with upper respiratory isolates from children with respiratory infections or nasopharyngeal isolates from children hospitalized without infections and found either a higher or a lower rate of PNSP in sterile site isolates.19-23

The Centers for Disease Control and Prevention recently convened a working group to develop a strategy for minimizing the impact of PNSP. One objective (Objective C) is to "Investigate the transmission of antimicrobial-resistant pneumococci through studies of nasopharyngeal colonization" and to determine "if isolates identified through nasopharyngeal cultures are representative of isolates that cause invasive disease."24 We have addressed this question by comparing the results of two simultaneous community-based studies of nasopharyngeal carriage among child-care center attendees and invasive disease caused by S. pneumoniae.

METHODS

Patient population. Two simultaneous studies took place in Metropolitan Toronto and the surrounding urban area, known as Peel Region (population, 3.1 million) during 1995. The first (NP carriage study) was a prospective survey of children attending licensed child-care centers within Metropolitan Toronto. All 94 centers attended predominantly by children <4 years of age were considered eligible for participation. All children attending each participating center were eligible for enrollment unless they had an underlying illness that contraindicated an NP swab. A parent questionnaire was also obtained. The swabs were transported directly to the Hospital for Sick Children Microbiology Laboratory. This study was conducted from March, 1995, to February, 1996. All isolates obtained during 1995 were included in the present study.

The second study (invasive disease study) was a prospective surveillance for all cases of invasive S. pneumoniae disease (positive culture from a normally sterile site) in Metropolitan Toronto and Peel Region during 1995. Cases were identified through a network of 27 laboratories, serving all emergency and inpatient facilities in the area. An audit was performed to ensure that all cases were found. Clinical data were obtained from all cases.

Both studies received approval from the Research Ethics Boards of the Hospital for Sick Children (NP carriage study) and University of Toronto (invasive disease study).

Nasopharyngeal swab (child-care center study). A single NP swab was obtained from each participating child using a Dacron®-tipped flexible wire swab inserted into the posterior nares. The swabs were transported at ambient temperature to the Hospital for Sick Children and plated within 6 h.

Laboratory investigations. At the Hospital for Sick Children, each NP swab was inoculated onto two Columbia blood agar base plates (Quelab Labs, Inc., Montreal, Quebec, Canada) with 5% horse blood. Each plate was incubated overnight at 35°C, one in 5% CO2 and one anaerobically. Plates were read at 24 and 48 h. When there was growth up to five alpha-hemolytic colonies characteristic of S. pneumoniae were subcultured to another 5% horse blood agar plate with an optochin disk and incubated for 18 to 24 h. All optochin-susceptible isolates (≥15 mm inhibition) were confirmed as S. pneumoniae by bile solubility.

Five colonies of each confirmed S. pneumoniae isolate were then inoculated into trypticase soy broth (0.5 McFarland standard) and plated on Mueller-Hinton blood agar plates with a 1-μg oxacillin disc. The zone of inhibition was measured after 18 to 24 h of incubation at 37°C; all isolates with <20 mm inhibition were considered potentially penicillin-nonsusceptible. Invasive isolates were sent from the participating laboratories to the Mount Sinai Hospital where they were confirmed as S. pneumoniae and screened for penicillin resistance, as described above.

Nasopharyngeal and invasive isolates were stored frozen in glycerol citrate at ≤−70°C and tested for antibiotic susceptibility at the Mount Sinai Hospital. The minimum inhibitory concentrations of penicillin and other antibiotics were determined with a broth microdilution assay, using Mueller-Hinton broth with 3% laked horse blood. Isolates were classified as susceptible, intermediate or resistant according to National Committee for Clinical Laboratory Standards guidelines.25 Penicillin resistance was further defined to be penicillin-nonsusceptible S. pneumoniae (PNSP; MIC ≥ 0.1 μg/ml) or penicillin-resistant S. pneumoniae (PRP; MIC ≥ 2 μg/ml).26 Multidrug nonsusceptibility was defined by intermediate susceptibility or resistance to two or more drug classes of drugs including beta-lactam antibiotics and carbapenems, macrolides, trimethoprim/sulfamethoxazole (TMP/SMX), tetracycline, ofloxacin and chloramphenicol.27

The serotype of each NP isolate and invasive pediatric isolate was determined by the National Centre for Streptococcus in Edmonton, using the quellung reaction.28

When two or more colonies of S. pneumoniae with different morphologic appearances were identified from the NP swab of a single child, each morphologic type (morphotype) underwent oxacillin disc testing, broth microdilution antibiotic susceptibility testing and serotyping. It was presumed that different morphotypes from a single child were different S. pneumoniae strains if they had different serotypes or markedly different antibiotic susceptibility patterns, suggesting phenotypic or genetic differences. Thus in cases where each morphotype from a child had a different serotype, the morphotypes were considered to be different strains. If each morphotype had the same serotype, the morphotypes were considered to be different strains only if there was a 4-fold or greater difference in MIC for two or more of penicillin, cefotaxime or ceftriaxone, TMP/SMX and erythromycin.

Analysis. All data were recorded in FoxPro® (Version 2.6; Microsoft, Redmond, WA) and analyzed with SPSS® (Version 6.1.1; SPSS, Chicago, IL), SUDAAN (Version 7.0; Research Triangle Institute, Research Triangle Park, NC) and DIFFER® (Makapuu Medical Press, Honolulu, HI). Descriptive data were noted as simple frequencies. Associations between variables were measured using the chi square test or Fisher's exact test for categoric data. The unpaired Student t test was used to compare group means. To address the effect of clustering of particular strains of S. pneumoniae within child-care center(s) clustered data analysis of antibiotic susceptibility and serotype proportions from the NP carriage study was performed. A finite population-sampling approach was taken, using the overall population in eligible child-care centers in the area as the target population. Statistical estimation was based on the assumption that the participating centers represented a random sample from the overall pool and that children were sampled randomly within each center. Appropriate weights based on sampling probabilities were used to allow for the varying sampling fractions across centers. Variance calculations taking into account the two-stage sampling were computed by a Taylor series approach. The adjusted proportions derived from clustered data analysis were compared with the corresponding proportions from the invasive study using a two-tailed z test. The power to detect a difference between observed proportions in the case (invasive disease study) and control (NP carriage study) groups, assuming a significance level of 0.05, was calculated using the method of Schlesselman.29

RESULTS

Clinical data and culture results. The NP carriage study was conducted from March, 1995, to February, 1996. During 1995, 53 of 75 child-care centers that were approached agreed to participate (72%). From these centers 1139 of 2633 eligible children participated (43%). Another 6 centers participated in the study in 1996 and 13 centers that were eligible for participation were not approached (study sample size was achieved and thus enrollment ceased). S. pneumoniae was obtained from the NP swabs of 532 children (46% positive). A single S. pneumoniae morphotype was identified in 506 children, and 2 or more morphotypes were identified in 26 children (2 morphotypes in 25 children and 3 morphotypes in 1 child). In 13 cases the 2 morphotypes were distinct strains; thus a total of 545 distinct strains were obtained from the 532 children. The questionnaire was completed for 1048 children (92%). No child who participated in the carriage study was reported to have invasive S. pneumoniae disease during 1995.

During 1995 there were 96 invasive pediatric cases (age <18 years) and 374 invasive adult cases. Data reported here are for the pediatric cases only. There were 3 deaths (2 in children with chronic underlying diseases). The sources of isolates were blood (92%), cerebrospinal fluid (5%), joint fluid (2%), sinus aspirates (2%), nasal septal hematoma (1%) and brain abscess (1%). There were positive cultures from more than 1 sterile site in 3 cases. The primary clinical diagnoses included bacteremia without focus (49%), pneumonia (17%), otitis media (17%), meningitis (6%), cellulitis (6%), septic arthritis (2%) and other (5%).

The mean age of participants in the NP carriage study was 30 months (range, 1 to 78 months) and 33 months (range, newborn to 17 years) for the invasive disease study (P = 0.51). In the NP carriage study 46% of participants were female, compared with 40% in the invasive disease study (P = 0.32).

Questionnaire data from the NP carriage study revealed that 27% of children had received antibiotics within 1 month of participating in the study. Seven percent had been hospitalized in the previous 6 months and 60% had at least 1 episode of otitis media in the previous 12 months. Fifty-two percent of children with invasive disease had received antibiotics in the preceding 3 months.

Serotypes. Serotyping was performed on all viable NP carriage isolates (536 of 545, 98%) and all viable invasive isolates (89 of 96, 93%). Of these, 18 carriage isolates (3%) and 1 invasive isolate (1%) were non-typable. There were 26 serotypes from 19 serogroups in the NP carriage study and 15 serotypes from 11 serogroups in the invasive disease study. The distribution of the most common serotypes from each study and comparison between studies are summarized in Table 1. Seventy-four (83%) invasive isolates and 322 (60%) NP carriage isolates were from serotypes included in experimental conjugate vaccines (serotypes 4, 6B, 9V, 14, 18C, 19F and 23F).30 After cluster analysis the adjusted proportions of each serotype within the NP carriage study differed little from the unadjusted proportions within the NP carriage study. In the NP carriage study 5% of isolates were serotype 11A and 2% were serotype 15A. Apart from these 2 serotypes, the 13 most frequent serotypes in each study were the same, although the rank order for some serotypes was different. Specifically serotype 14 was more prevalent among invasive isolates (41% of total, rank first) than among the NP carriage isolates (6% of total, rank fifth). In addition serotypes 6B and 6A were more common among NP carriage isolates (23 and 11%, ranks first and fourth, respectively) than among the invasive isolates (12 and 2%, ranks second and ninth, respectively).

TABLE 1
TABLE 1:
Distribution of Streptococcus pneumoniae serotypes from 536 nasopharyngeal carriage isolates and 89 invasive isolates

Antibiotic susceptibility. Susceptibilities were performed on all viable isolates (545 carriage and 91 invasive isolates). The proportions of resistant isolates are summarized in Table 2. All isolates were fully susceptible to vancomycin, ofloxacin and rifampin. All invasive isolates were fully susceptible to tetracycline. After cluster analysis the adjusted proportions of each antibiotic from the NP carriage study differed little from the unadjusted proportions within the same study. The adjusted proportions were compared with the invasive study proportions. In all instances when there was a difference in the groups, the resistance rate was higher in the NP carriage isolates. The proportion of isolates that were multidrug-nonsusceptible was similar in both studies (16% of carriage isolates, 12% of invasive isolates; P = 0.34). The powers of these observations to detect a difference among the proportions resistant to penicillin, TMP/SMX and erythromycin in each study were 23, 79 and 29%, respectively.

TABLE 2
TABLE 2:
Streptococcus pneumoniae antibiotic susceptibility and comparison of resistance from 545 nasopharyngeal carriage isolates and 91 invasive isolates

Antibiotic resistance and serotype distribution. Eighty-eight NP carriage isolates and 10 invasive isolates were PNSP. One PNSP carriage isolate was not serotyped. Four serotypes (6B, 19A, 19F and 23F) accounted for 87% of all PNSP NP carriage isolates, compared with 47% of penicillin-susceptible S. pneumoniae (PSSP isolates) [odds ratio (OR) 7.7; 95% confidence interval (95% CI), 4.0, 14.8; P < 0.0001]. These same 4 serotypes accounted for 70% of all PNSP invasive isolates, compared with 30% of PSSP isolates (OR 5.4; 95% CI 1.3; 22.5; P = 0.01).

Five serotypes (6A, 6B, 19A, 19F and 23F) accounted for 89% of all TMP/SMX-resistant NP carriage isolates, compared with 53% of TMP/SMX-susceptible S. pneumoniae (OR 7.4; 95% CI 4.5, 12.2; P < 0.0001). These same five serotypes accounted for 67% of all TMP/SMX-resistant invasive isolates, compared with 28% of TMP/SMX-susceptible isolates (OR 5.2; 95% CI 1.8, 14.7; P = 0.001).

Risk factors for PNSP. Data regarding age, sex and antibiotic use were available from each study. An age of <24 months, female sex and recent antibiotic use (use at time of study or in previous 1 month) were associated with carriage of PNSP (Table 3). These same factors (with recent antibiotic used defined as use in the previous 3 months) were not associated with invasive disease caused by PNSP. The power of these observations to detect a difference between the proportions of NP PNSP and PSSP cases was 98, 46 and 79% for age <24 months, female sex and recent antibiotic use, respectively. However, the power to detect a difference between the proportions of invasive PNSP and PSSP cases was 25% or less for each factor.

TABLE 3
TABLE 3:
Factors associated with PNSP compared with PSSP in children with nasopharyngeal carriage or invasive disease

DISCUSSION

This study shows that within a community a survey of NP carriage isolates of S. pneumoniae reflects the antibiotic susceptibility rates of invasive isolates found in the same time period for most antibiotics. However, even a large study like this may have limited power to detect a difference between groups. The prevalence of isolates with reduced susceptibility to penicillin, erythromycin, clindamycin and chloramphenicol were somewhat higher, but not significantly different, in the NP carriage study than in the invasive isolate study, whereas reduced susceptibility to TMP/SMX was definitely more prevalent in the NP carriage study. Also the NP carriage isolates and invasive isolates share the 13 most common serotypes (apart from serotypes 11A and 15A, which were not found among the invasive isolates), but the relative proportions of each and rank order are variable.

A strength of this study was the use of clustered data analysis that adjusted for the effect of clustering of features at one center or another. However, there was little difference in the estimated proportions for antibiotic resistance rates and serotypes using clustered data analysis or simple proportions calculated.

There are limitations to our findings. First, the power of our observations to detect a difference between the two study groups (or to confirm that no difference between groups exists) is limited by the relatively small number and low prevalence of invasive PNSP cases. This limitation could potentially be overcome by obtaining a larger number of invasive PNSP cases, either by extending the study period or by performing a similar study in an area with a higher prevalence of PNSP.

It is possible that antibiotic-resistant strains of S. pneumoniae are inherently more likely to be carried in the nasopharynx and less likely to be invasive. It is more likely, however, that most of the difference in resistance may be explained by the difference (or trends in difference) in proportions of specific serotypes found within the NP carriage and invasive isolates. As has been found by others, only a few serotypes were predominant among PNSP strains.31 In this study the predominant PNSP serotypes were 6B (more common in NP carriage isolates), 19A, 19F and 23F, but not serotype 14 (the predominant invasive serotype). There were too few invasive PNSP isolates to do serotype by serotype comparisons with the NP carriage isolates; however, such an adjustment would be necessary more accurately to compare groups of isolates. Some serotypes are more prevalent in the nasopharynx than with invasive disease in children, i.e., serogroups 6 and 23, whereas the converse is true for serotype 14.13, 32

Before 1993 the reported prevalence of PNSP isolates among S. pneumoniae infections in Canada was 3% or less.33-36 However, in Toronto during 1993 and 1994, a survey found 7% of 274 S. pneumoniae infections were PNSP (invasive and noninvasive isolates, all ages).37 In 1994 and 1995, 12% of 1089 S. pneumoniae infections from Toronto and other Canadian centers were PNSP (invasive and noninvasive isolates, all ages).38 Thus our study was performed at a time when the prevalence of PNSP was rapidly increasing in Toronto. It is possible that at such a time the difference in PNSP prevalence between NP carriage and invasive isolates will be exaggerated by the predominance of PNSP prevalence in specific serotypes more commonly found in the nasopharynx, such as 6B.12-14 Later, when the prevalence of PNSP is higher and at a plateau, the difference between NP and invasive isolates may lessen as resistance becomes more common in important invasive serotypes, such a serotype 14.12-14

Another limitation is that data obtained from children attending child-care centers may not be completely representative of the general population of children. Children in child-care centers have more frequent illness than in other care settings and may have more frequent exposure to antibiotics.39, 40 Transmission of PNSP has been demonstrated in a child-care center.7 Child-care center attendance has also been shown to be a risk factor for carriage of PNSP by one study,41 but not by another.42 Thus a higher rate of PNSP might be expected from children attending childcare centers.

Data comparing antibiotic susceptibility between invasive and carriage S. pneumoniae isolates are limited and conflicting. Two studies compared sterile site isolates to NP carriage isolates from healthy children.11, 18 Henderson et al.18 compared data from a longitudinal survey of nasopharyngeal carriage in a child-care center with sterile site isolates collected from all patients in a nearby hospital. There was no difference in the rate of PNSP in both groups (8% PNSP (9 of 113) in sterile site isolates, 12% PNSP (25 of 210) in non-sterile site isolates, P = 0.10). The power to detect a difference was 20%. Mastro et al.11 studied urban and rural well children (n = 133 and 285, respectively) and urban children with acute lower respiratory tract infection (n = 601). Thirty-six percent of the ill children had positive blood cultures, one-half of which were S. pneumoniae. There was no significant difference in penicillin resistance when blood isolates (11% PNSP) were compared with carriage isolates from children with acute respiratory infection (12% PNSP), urban well children (14% PNSP) and rural well children (7% PNSP). The power to detect a difference in the groups was 26% or less for each comparison.

Few studies have directly compared invasive and noninvasive serotypes from similar populations. Some series have considered middle ear and invasive isolates together16 and noninvasive isolates have come from the upper respiratory tract of healthy,14, 16, 32 or sick children.13, 16 Two prospective cohort studies have demonstrated that there is nearly complete concordance between the most common serotypes or serogroups found in sterile sites and nasopharyngeal isolates obtained at the time of disease from individual children with invasive S. pneumoniae disease or otitis media.11, 12 Other studies have compared independent but contemporaneous collections of invasive or noninvasive isolates.13, 14, 16, 32 All studies of sterile site isolates found that serotype 14 was more prevalent among invasive isolates than among noninvasive isolates.13, 16, 32 Other frequent and predominant invasive isolates (compared with noninvasive) have included 5,32 616 and 18.13, 16 Although one study found serogroup 6 more commonly with invasive than with noninvasive isolates,16 serogroups 6 and 23 have usually been found to be more prevalent in noninvasive isolates.13, 32 In addition in one study it was found that serogroups 6, 19 and 23 were found with similar frequency in the middle ear fluid of a group of children with otitis media compared with the nasopharynx of another group of healthy children.14 Thus there are differences in the prevalence and rank order of serotypes between invasive and noninvasive collections and these differences vary depending on the population and geographic location. In addition because PNSP isolates are linked to a limited number of serotypes,31 any valid comparison of S. pneumoniae isolates from different sites must take serotypes into account.

Antibiotic resistance to S. pneumoniae is now common worldwide. The emergence of resistance in a community or region may be very rapid. Once significant resistance is present, antibiotic choices must be modified for empiric and definitive treatment of infections presumed or proved to be caused by S. pneumoniae.43, 44 It is possible that interventions to alter antibiotic use may result in decreased levels of resistance, also over a brief period of time.45 Thus it is important for communities to obtain accurate and current knowledge of local antibiotic resistance patterns. Although periodic surveys of nasopharyngeal carriage of S. pneumoniae in healthy children in childcare centers, schools or ambulatory health clinics could rapidly provide a large number of isolates, the cost of necessary serotyping and limited power of such surveys limits their feasibility for routine surveillance.

ACKNOWLEDGMENTS

We thank Heather Watson and Lisa Palermino (Hospital for Sick Children) for coordinating the NP carriage study; Irene Kyle (University of Guelph), Michael Bates and Drs. Howard Njoo and Barbara Yaffe (cities of Toronto and North York) for supporting the involvement of child care centers; Margaret Roscoe (Hospital for Sick Children Microbiology Laboratory) for coordinating the microbiology studies; Marguerite Lovgren (National Centre for Streptococcus, Edmonton) for serotyping; Dr. Rollin Brant and Victoria Stagg (University of Calgary) for performing the clustered data analysis; and Dr. Ronald Gold (Hospital for Sick Children) for helpful suggestions. Funding for the NP carriage study was provided by grants from Lederle Praxis Biologics Inc. and Pfizer Canada. Funding for the invasive disease study was provided by Centers for Disease Control RFP Grant 200-94-0806. JDK was supported by a Canadian Infectious Diseases Society Eli Lilly Research Fellowship.

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Figure

No caption available.
Keywords:

Streptococcus pneumoniae; carriage; serotypes; antibiotic resistance

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