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Global distribution of Streptococcus pneumoniae serotypes isolated from paediatric patients during 1999–2000 and the in vitro efficacy of telithromycin and comparators

¹Medical and Molecular Microbiology, GR Micro Limited, 7–9 William Road, London NW1 3ER, UK ²Carolinas Medical Center, Charlotte, NC, USA ³Institute of Medical Microbiology, National Reference Center for Streptococci, Aachen, Germany

Correspondence D. J. Farrell D.Farrell{at}grmicro.co.uk

Received February 20, 2004
Accepted July 6, 2004

Few data exist on the distribution of Streptococcus pneumoniae serotypes in many countries and in non-invasive disease overall. Here, data are presented from 772 paediatric isolates from children with community-acquired respiratory tract infections isolated from the PROTEKT global surveillance study during 1999–2000. Overall, 60.0 % of isolates were covered by the 7-valent pneumococcal vaccine formulation (PCV7), with greater coverage in the USA compared with Europe (69.6 vs 55.5 %, P = 0.014). Geographically dispersed clones of serogroups 3, 11 and 15 accounted for most of the isolates outside PCV7 coverage. Overall, macrolide, penicillin and cotrimoxazole non-susceptibility rates were high; however, all isolates were susceptible to telithromycin. Although only 7.4 % of isolates were resistant to amoxycillin/clavulanate, a higher prevalence of resistance was found in isolates from the USA and South Korea. This study shows the feasibility and importance of serotyping antibiotic surveillance study isolates and the potential of telithromycin as an important option for empiric therapy.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Streptococcus pneumoniae is the major causative pathogen of many childhood community-acquired respiratory tract infections (RTIs), including community-acquired pneumonia, acute otitis media and acute maxillary sinusitis. The 7-valent formulation of the pneumococcal conjugate vaccine (PCV7) was licensed by the US Food and Drug Administration and introduced in the USA in February 2000. In the USA, there is a general recommendation for the use of PCV7 in all children < 2 years old and the vaccine is licensed for children up to 5 years of age. In contrast, in Europe the vaccine is licensed for children up to 2 years of age. Most European countries do not have a general recommendation for use (the exceptions are Austria and France, although no government funding is provided to support the recommendations) and hence the vaccine is mainly used for high-risk children. When given in a four-dose regimen to infants, controlled clinical trials have demonstrated high efficacy in reducing invasive disease, but less efficacy in reducing non-invasive diseases such as acute otitis media (Black et al., 2002; Whitney et al., 2003).

Serotypes (STs) represented in PCV7 are 4, 6B, 9V, 14, 18C, 19F and 23F. Two other pneumococcal conjugate vaccines are currently under clinical investigation: the 9-valent (PCV9) vaccine provides PCV7 cover plus coverage of STs 1 and 5, and the 11-valent (PCV11) vaccine provides PCV9 cover plus coverage of STs 3 and 7F. Cross-coverage is thought to extend to other STs within the same serogroup (SG). For STs 6B and 19F, several studies have shown that significant cross-reactivity occurs with STs 6A and 19A, respectively (Nahm et al., 1997; Vakevainen et al., 2001; Yu et al., 1999). However, different amounts of cross-reacting opsonizing antibodies have been noted among different vaccine formulations and different methodologies used to determine antibodies may be unreliable for the assessment of cross-reaction. In addition, no data exist on the cross-reactivity of other SGs associated with STs in the vaccine (Yu et al., 1999). A study performed in South Africa found that ST 6A was still carried after vaccination, at a higher rate than expected (Mbelle et al., 1999). Although evidence is conflicting, some suggests that the carriage of non-vaccine STs may increase following vaccination (Obaro et al., 1996). The uncertainty regarding cross-protection and the potential for ST replacement underscore the need for ST surveillance.

Pneumococcal resistance to antimicrobials, including the commonly used penicillins, macrolides and cotrimoxazole, has been increasing in most countries over the last decade, with many isolates now resistant to multiple antibiotics (Felmingham & Washington, 1999; Felmingham & Gruneberg, 2000; Hoban et al., 2001; Sahm et al., 2000). Resistance to erythromycin and other macrolides among S. pneumoniae is increasing rapidly and is now more prevalent than ß-lactam resistance in many areas of the world (Felmingham et al., 2002). It has been recognized that information on the impact of vaccination on diseases caused by antibiotic-resistant pneumococci is of utmost importance (Hausdorff et al., 2000a). Serotyping isolates from a large global antimicrobial surveillance study would be a valuable and cost-effective method for obtaining such important information. In addition, although data exist on circulating STs in invasive pneumococcal disease (IPD), there is a paucity of data for non-invasive disease.

The PROTEKT (Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin) study is an international, longitudinal, antimicrobial-resistance surveillance study which was established with an educational grant from Aventis Pharma in 1999 to monitor the spread of resistance among bacterial pathogens isolated from community-acquired RTIs worldwide. The rationale for undertaking this investigation was that, at the time that this surveillance study was being carried out, various formulations of pneumococcal conjugate vaccines were under consideration or being introduced in several countries, providing the opportunity to obtain baseline data on the distribution of STs in many centres from countries across the world.

Telithromycin is a new ketolide antimicrobial with favourable pharmacokinetic and safety profiles (Drusano, 2001; Van Rensburg et al., 2002), a low capacity for induction of resistance (Davies et al., 2000; Stratton, 2003) and a favourable ecological profile in terms of resistance development in the normal microbiota (Edlund et al., 2000). It demonstrates a targeted spectrum of activity against the most common bacterial respiratory pathogens, including penicillin-, macrolide-, fluoroquinolone- and multi-resistant strains of S. pneumoniae (Farrell et al., 2002, 2003; Felmingham et al., 2002; Morrissey et al., 2003), paediatric isolates (Bozdogan et al., 2003) and atypical organisms such as Legionella pneumophila, Chlamydophila pneumoniae and Mycoplasma pneumoniae (Hammerschlag et al., 2001). Telithromycin, therefore, is a promising new alternative for the treatment of community-acquired RTIs.

The aims of this study were to determine the global distribution of S. pneumoniae STs in a large population of phenotypically and genotypically defined paediatric isolates from the first year of the PROTEKT study (1999/2000) and to determine the in vitro efficacy of the ketolide telithromycin and comparators against these isolates. The intention is to use these data as a baseline for comparison of ST and antimicrobial activity distribution changes in future years of the PROTEKT study.

	METHODS

TOP INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
During the 1999–2000 winter season, a total of 69 centres in 25 countries participated in the PROTEKT surveillance study. S. pneumoniae isolates were collected from children 14 years of age with community-acquired RTIs, including community-acquired pneumonia, sinusitis and acute otitis media. Sources of clinical isolates deemed acceptable included blood, sputum, bronchoalveolar lavage fluid, ear/tympanocentesis samples, nasopharyngeal swabs or aspirates, and sinus aspirates. Patients with nosocomial RTIs and those with cystic fibrosis were excluded from the study, as were duplicate strains or strains originating from existing collections. Isolates were determined to be pathogens by the attending physician based on local microbiological assessment and clinical judgement. The PROTEKT study objectives, design, methodology and data analysis have been previously described (Felmingham, 2002).

In total, 806 S. pneumoniae isolates were obtained from paediatric patients (14 years of age); of these, 772 were viable after storage and hence were available for serotyping. The geographical distribution of these isolates was as follows [country (number)]: Australia (59), Belgium (5), Brazil (84), Canada (71), France (27), Germany (90), Hong Kong (4), Hungary (23), Indonesia (1), Italy (23), Japan (108), Mexico (43), The Netherlands (6), Poland (9), Portugal (33), South Korea (28), Sweden (17), Switzerland (8), UK (31), USA (102).

Serotyping was performed by Neufeld Quelling reaction at GR Micro Ltd using Statens Serum Institut (Denmark) antisera. Statens Serum Institut was used as the reference laboratory for quality assurance and rare STs. Multilocus sequence typing (MLST) was carried out as described previously (Enright & Spratt, 1998).

Susceptibilities to new and existing antibacterials were determined at GR Micro Ltd using National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution methodology and NCCLS cut-off points (National Committee for Clinical Laboratory Standards, 2003). Tentative NCCLS cut-off points for telithromycin against S. pneumoniae are as follows: 1 µg ml^–1 is susceptible, 2 µg ml^–1 is intermediate and 4 µg ml^–1 is resistant (National Committee for Clinical Laboratory Standards, 2004). Non-susceptibility was defined as isolates resistant or intermediate to a particular antibiotic.

All isolates resistant to erythromycin (MIC 1 µg ml^–1) were analysed by PCR for the presence of erm(A), erm(A) subclass erm(TR), erm(B), erm(C) and mef(A) sequences (Farrell et al., 2001). Detection of the transposon Tn1545 and the tetracycline-resistance gene tet(M) were performed as described previously (Farrell et al., 2004).

Statistical analysis was performed using a S² test.

	RESULTS

TOP INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Overall, 60.0, 61.4 and 67.4 % of S. pneumoniae isolates were STs represented in the PCV7, PCV9 and PCV11 vaccine formulations, respectively (Table 1 and Fig. 1). There was a significant increase in coverage between PCV11 and PCV7 (7.4 %, P = 0.003), while only a 1.4 % increase was found between PCV9 and PCV7 (1.4 %, P = 0.57).

View larger version (28K): [in this window] [in a new window]   Fig. 1. Serotype distribution of 772 S. pneumoniae isolates obtained from paediatric patients in PROTEKT Global 1999–2000 grouped by vaccine coverage. (a) Distribution by number of isolates; (b) cumulative percentage distribution.

Although the small number of isolates in some countries did not allow meaningful interpretation, there were sufficient isolate numbers to demonstrate a significantly higher coverage in the USA than in Europe for PCV7 (P = 0.014) and PCV11 (P = 0.003). Coverage was higher for the 2-year-old age group than for the 3–14-year-old age group for PCV7 (P = < 0.001). In the target age group (5 years), the coverage was significantly higher for PCV7 in the USA compared with Europe (72.3 vs 57.1 %; P = 0.016).

Blood-culture isolates were better covered by all formulations than non-blood-culture isolates (P = 0.003 for PCV7; Table 1). SG 14 was most often isolated from blood, SG 23 from bronchoalveolar lavage, SG 19 from the ear (including the small number of middle-ear fluids collected) and SGs 19 and 6 from sputum and nasopharyngeal specimens.

Isolates with STs represented in the PCV7, PCV9 and PCV11 formulations were less susceptible to the macrolides and penicillin than those with STs not represented (PCV7: P < 0.001 for erythromycin non-susceptibility; P < 0.001 for penicillin non-susceptibility; Table 2). Combined macrolide (MIC 1 µg ml^–1) and penicillin (MIC 2 µg ml^–1) resistance was also more prevalent in the STs represented in PCV7 [124/153 (81.0 %) of penicillin-resistant isolates co-resistant to macrolides] than in those not [21/41 (51.2 %)].

A total of 309/772 (40.0 %) isolates were not within the SG coverage of PCV7. Apart from ST 19A (38 isolates) and 6A (43 isolates), the most common SGs among these isolates were SG 3 (41 isolates), SG 11 (24 isolates) and SG 15 (24 isolates). Of the 309 isolates not represented by SG in PCV7, 60 were macrolide non-susceptible (59 resistant), 86 were intermediate (45) or resistant (41) to penicillin G and 40 were intermediate or resistant to both macrolides and penicillin G. All 106 isolates with reduced susceptibility to penicillin G or erythromycin A were susceptible to telithromycin (MIC < 1 µg ml^–1): MIC₅₀ 0.03 µg ml^–1, MIC₉₀ 0.5 µg ml^–1, range 0.008–1 µg ml^–1.

Among macrolide-resistant isolates (n = 274, 35.5 %), the distribution of resistance mechanisms was variable between different STs (Table 3). Macrolide resistance in ST 6B isolates was predominantly by erm(B)-mediated methylase, while the mechanism in ST 14 and ST 19F isolates was predominantly mef(A)-mediated efflux. Of the 30 isolates with combined methylase- and efflux-mediated macrolide resistance, 25 were SG 19: 16 from South Korea, seven from the USA and one each from Turkey and Hungary.

Six (14.6 %) of the ST 3 isolates (four from Japan, one from South Korea and one from Hong Kong) were macrolide-resistant [erm(B)-mediated, erythromycin MIC >64 µg ml^–1], tetracycline-resistant, positive for the transposon Tn1545 and penicillin-susceptible. Thirteen (54.2 %) of the SG 15 isolates (global distribution) were macrolide-resistant [11 erm(B), one mef(A) and one ribosomal mutation-mediated).

The majority of the ST 3 isolates, including the six isolates positive for the erm(B) gene, represented a single clone (MLST 180), with a wide geographical distribution (Table 4). Interestingly, a macrolide-resistant isolate has been described from Taiwan with a macrolide–lincosamide–streptogramin B (MLS_B) phenotype (Table 4). The SG 11 and SG 15 isolates were also widely distributed with evidence of clonal spread.

The prevalence of cotrimoxazole, penicillin and macrolide resistance was high. Telithromycin demonstrated high in vitro potency and activity (100 % of isolates susceptible) against all 772 isolates, regardless of ST, genotype, geographical location, source of isolate or age group (Table 5). Amoxycillin/clavulanate was the most active comparator compound with 92.6 % of isolates susceptible. However, the majority of non-susceptible isolates were found in two countries, the USA (29/102, 28.4 %) and South Korea (15/28, 53.6 %). They appeared to be clonally related and were predominantly from ear specimens obtained from infants 2 years of age. The South Korean SG 19 clone, with combined mef(A)- and erm(B)-mediated macrolide resistance, was also seen among isolates from the USA.

	DISCUSSION

TOP INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
The ST distribution reported here differs considerably from that in recent reports (Hausdorff et al., 2000a; Whitney et al., 2003). STs 1 and 14 have been the STs most frequently found in blood cultures (Hausdorff et al., 2000b). Although ST 14 was the most prevalent found in our study, ST 1 was not prevalent. As previously reported (Hausdorff et al., 2000b; Joloba et al., 2001), ST 3 isolates were most frequently found in ear isolates. The data presented here suggest that current vaccine coverage may not be optimal in several countries. The PCV11 formulation may be advantageous, mainly due to its coverage of SG 3.

A recent study in Germany demonstrated 14 % lower potential coverage of STs prevalent in IPD than demonstrated in the large USA Kaiser Permanente Trial (Black et al., 2002; Von Kries et al., 2002). Even though coverage was lower than in the USA, the German study showed that the use of PCV7 in Germany predicted high efficacy for the reduction of IPD. The data in the present study show that the lower coverage rate seen in Germany may be a European trend in general. The potential increase in coverage by using PCV11 is greater in Europe than in the USA in the target population (children 5 years old). In general, the PCV9 formulation may not provide any additional benefits (more data would be needed to draw such a conclusion for any particular country). On the other hand, a recent South African study demonstrated efficacy with PCV9, emphasizing the need for local ST prevalence and efficacy studies when deciding upon vaccine introduction (Klugman et al., 2003).

Other parameters may define the impact of vaccine introduction and there is concern that vaccination may not eliminate the carrier state of PCV7 STs. In a recent study, vaccinated children in the 2–5-year-old age group were shown to be recolonized with STs present in the vaccine, suggesting that immunity may not be as long-lasting as was hoped (Lakshman et al., 2003). There have also been problems with vaccine uptake in the USA, with some physicians unwilling to prescribe and some parents unwilling to accept any risk, however small, associated with vaccination (Davis et al., 2001). Cost of the vaccine and the insurance status of the patient and family may also be a factor (Davis et al., 2003). In the Kaiser Permanente large, randomized, double-blind trial vaccination study, a reduction in community-acquired pneumonia by 32.2 % in the first year of life, 23.4 % in the first 2 years and 9.1 % in children >2 years was found (Black et al., 2002). Although these values represent a significant decrease in disease burden, it is far from elimination, and perhaps some of the concerns outlined above may be playing a role in the efficacy of vaccination.

The high level of non-susceptibility to cotrimoxazole (48.4 %) in these isolates is of concern, particularly if these strains and clones are able to, or indeed have already, spread to developing countries. The World Health Organization protocol for case management in children over 2 months directs that, in patients with non-severe pneumonia, cotrimoxazole is the preferred drug in most settings because of its broad-spectrum efficacy, low cost, ease of administration and relatively low rate of adverse effects (World Health Organization, 1990). The high and increasing prevalence of macrolide and penicillin resistance is limiting the choice of empiric therapy in less-severe community-acquired RTIs, particularly in paediatric patients in which the use of fluoroquinolone therapy is currently not an option. Our data show that telithromycin and amoxycillin/clavulanate are the most active compounds against S. pneumoniae in this global paediatric population. As with telithromycin, amoxycillin/clavulanate has a favourable pharmacokinetic profile and high clinical efficacy in the treatment of RTIs, including drug-resistant strains (File et al., 2002). However, close monitoring of local resistance to amoxycillin/clavulanate, particularly in ear infections, is needed, as the data from the USA and South Korea demonstrate in this study.

Of the 41 SG 3 isolates, six (from Asia) were found to be resistant to macrolides, with erm(B) as the mechanism of resistance. These isolates were also resistant to tetracycline [tet(M)-mediated] and harboured the mobile genetic element Tn1545, which is known to carry both erm(B) and tet(M). The combination of a virulent SG 3 clone, with a mobile genetic element encoding multiple resistance genes, some evidence of geographical spread (genetically related isolates found in South Korea, Japan and Hong Kong, with a report of the same clone being previously isolated in Taiwan) and lack of vaccine coverage is of concern and needs close monitoring.

These data from the PROTEKT 1999–2000 global surveillance study demonstrate the potential for ST replacement to occur – penicillin- and macrolide-resistant S. pneumoniae in STs outside PCV7 coverage (SGs 3, 11 and 15 in particular) is prevalent. However, since the purpose of this study was to obtain baseline data, the results cannot be used to infer any impact of vaccine introduction. A statistically significant shift in ST distribution data in the same centres in later years (i.e. PROTEKT years 2, 3 and 4) could be used as scientific evidence to suggest that ST replacement is occurring due to vaccine introduction. Serotype replacement is a potential risk to the continued success of the vaccine and therefore needs to be monitored closely.

These data suggest that telithromycin, with its high activity against all STs in this paediatric population, may be more effective than macrolides or penicillins in potentially limiting ST replacement by antimicrobial-resistant isolates that are not covered by PCV7 (such as the SG 3 and SG 15 isolates described here).

The greatest burden of pneumococcal disease is non-serious illness, and current data analysing the impact of PCV7 and PCV9 conjugate formulations and the 23-valent polysaccharide formulation suggest but do not prove that vaccination has a significant impact on the reduction of non-serious illness (Jackson et al., 2003; Klugman et al., 2003; Whitney et al., 2003). Until alternative strategies prove to be effective in reducing this burden, antimicrobial chemotherapy in non-serious illness, particularly in non-bacteraemic pneumonia, is a valuable therapeutic option. As the choice of antimicrobial therapy for community-acquired pneumonia is usually empiric and increasingly limited due to the prevalence of antimicrobial resistance, telithromycin may be an option for the treatment of less-severe community-acquired RTIs.

This is the first global surveillance study to combine antimicrobial, genotype and ST surveillance and provides valuable baseline data on the global prevalence of STs causing community-acquired RTIs. We are in the process of serotyping >2000 isolates from paediatric patients from the first year of PROTEKT US (2000–2001), a surveillance study conducted with sites across the USA only, to obtain detailed baseline data focused on the USA. We are repeating these tests in both populations of isolates collected during the 2002–2003 respiratory season to monitor changes in ST distribution, antimicrobial resistance (including mechanisms of resistance) and the epidemiology of resistant strains. It is hoped that the data presented here will prove to be a useful adjunct to ST prevalence studies to understand better the epidemiology of S. pneumoniae.

	ACKNOWLEDGEMENTS

TOP INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
We are grateful to our colleagues worldwide for the supply of bacterial isolates as part of the PROTEKT study and the GR Micro PROTEKT team who performed the serotyping, genotyping and MIC determinations. Aventis is acknowledged for their financial support of the PROTEKT study. Preliminary data were presented at the 13th European Congress of Clinical Microbiology and Infectious Diseases, Glasgow, UK, 2003 (abstract).

	REFERENCES

TOP INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES