Antimicrobial susceptibility and serotype distribution of Streptococcus pneumoniae causing meningitis in Egypt, 1998–2003

Momtaz O. Wasfy1,*, Guillermo Pimentel1, Mohammed Abdel-Maksoud1, Kevin L. Russell2, Christopher P. Barrozo2, John D. Klena1, Kenneth Earhart1 and Rana Hajjeh1,3

1 U.S. Naval Medical Research Unit No. 3, Cairo, Egypt; 2 Naval Health Research Center, San Diego, CA, USA; 3 Centers for Disease Control and Prevention, Atlanta, GA, USA


* Corresponding author. Tel: +20-2-348-0312; Fax: +20-2-342-7121; Email: wasfym{at}namru3.med.navy.mil

Received 30 January 2005; returned 14 February 2005; revised 17 February 2005; accepted 22 February 2005


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: To determine the antimicrobial susceptibility and serotype distribution of 205 isolates of Streptococcus pneumoniae, collected from the CSF of meningitis patients identified between 1998–2003, during sentinel meningitis surveillance in Egypt.

Methods: Antimicrobial susceptibility was evaluated against six antibiotics using disc diffusion and Etest methods. Serotyping was performed by latex agglutination and the Quellung test.

Results: Forty-nine percent of all isolates were found to be non-susceptible to penicillin (46% intermediate, MIC range 0.12–1.0 mg/L; 3% resistant, MIC = 2.0 mg/L), and 6% of the isolates were non-susceptible to ceftriaxone (5% intermediate, MIC = 1.0 mg/L; 1.3% resistant, MIC ≥ 2 mg/L). Resistance rates for tetracycline and trimethoprim/sulfamethoxazole were high (52 and 59.7%, respectively), but those for erythromycin and chloramphenicol were lower (11 and 9%, respectively). Five serotypes (6B, 1, 19A, 23F and 6A) accounted for 37% of the total isolates. Ten isolates (5%) were non-typeable. Overall, 29 and 42% of serotypes were represented in the 7- and 11-valent conjugate vaccines, respectively. However, vaccine coverage for children <2 years was 38 and 56% for the 7- and 11-valent, respectively.

Conclusions: Resistance to penicillin may be increasing among S. pneumoniae strains causing meningitis in Egypt, and a moderate proportion of these strains are not covered by current pneumococcal conjugate vaccines. In addition to intensifying education efforts about judicious use of antibiotics, laboratory-based surveillance for other forms of invasive pneumococcal disease, especially pneumonia, is needed before decisions can be made regarding the most effective vaccines for control of this disease in Egypt.

Keywords: pneumococci , serotypes , surveillance , vaccines


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide, due to a number of severe infections, including sepsis, pneumonia, meningitis and otitis media.1 Pneumococcal infections account for >1 million deaths each year among children <5 years of age in developing countries.2,3 Virulence of the organism is mainly associated with the presence of capsular polysaccharides that usually exhibit differences in size, composition, antiphagocytic properties and serotype-specific immunogenicity.4 So far, 90 S. pneumoniae serotypes are recognized.5

To reduce the burden of pneumococcal disease, polysaccharide vaccines were introduced first in the USA in 1946. The current 23-valent pneumococcal polysaccharide vaccine was licensed in the USA in 1983,6 and contains capsular polysaccharides from serotypes responsible for 88% of bacteraemic infections in the USA and 96% of those in the UK.7,8 This vaccine is now used in many countries to prevent invasive pneumococcal disease in adults. However, this vaccine has poor immunogenicity in infants and is not recommended for use in this age group.4,6 To increase immunogenicity, conjugate pneumococcal vaccines containing fewer capsular polysaccharides (7, 9 or 11 serotypes) were recently developed.9 Controlled clinical trials in the USA have shown that the 7-valent vaccine (Prevnar, Wyeth Lederle Vaccines) is highly efficacious against invasive pneumococcal disease.10 Following its introduction in the routine immunization schedule of infants and young children in the USA, the rate of invasive pneumococcal disease in children has been significantly reduced.11

Serotypes included in the 7-valent vaccine (4, 6B, 9V, 14, 18C, 19F and 23F) were responsible for 69–79% of reported pneumococcal disease in children aged <5 years in England and Wales,8,12 and ~82% of infants in the USA.13 The 11-valent (which includes four additional serotypes—1, 5, 3 and 7F) conjugate vaccine covered serotypes from 82–91% of pneumococcal infections in the respective areas.8,12,14 Whereas coverage provided by the conjugate vaccines against invasive S. pneumoniae disease was shown to be >75% in the USA and other developed countries,11,15 50% coverage or less was reported in other countries, whose prevailing S. pneumoniae serotypes were different from the vaccine serotypes.7,12,16 Therefore, it is critical to determine the local distribution of various S. pneumoniae serotypes in order to guide decisions for the most effective vaccine formulations in specific countries or regions.

The need for effective vaccines has also become more urgent with the emergence of penicillin-resistant S. pneumoniae isolates.17 Over the last 40 years, penicillin resistance among S. pneumoniae isolates has emerged and spread globally at an alarming rate.18 In a study from metropolitan Atlanta during 1994,19 invasive S. pneumoniae from children and adults displayed significant levels of resistance to both penicillin (25% overall, 7% with high level; MIC ≥ 2.0 mg/L) and cefotaxime (9% overall, 4% with high level; MIC ≥ 2 mg/L). A more recent analysis of 109 isolates from children and adults with pneumococcal meningitis in Atlanta, Baltimore and San Antonio, between 1994–1996, identified even higher levels of resistance to these two antibiotics (35% overall, 19% high-level for penicillin and 20% overall, 9% high-level for cefotaxime).20 A recent African multi-country study revealed that penicillin-non-susceptibility levels among S. pneumoniae isolates from different clinical specimens were in the range 9–61%.21 A study conducted in Cairo, Egypt, in the late 1970s showed that the percentage of penicillin-non-susceptible S. pneumoniae from patients with meningitis was 22.4%.22

Recent data from Egypt and nearby countries on the serotype distribution and antimicrobial resistance patterns of invasive S. pneumoniae isolates is minimal. We therefore conducted this study to obtain a better understanding of these issues among patients hospitalized with bacterial meningitis in Egypt during 1998–2003.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Collection of isolates

S. pneumoniae isolates were collected from CSF specimens of patients with meningitis, who were enrolled in a sentinel surveillance system for meningitis, conducted in a network of 13 infectious disease hospitals across Egypt, during a 5 year period (1998–2003). All specimens and/or isolates were transferred in Trans-Isolate Medium23 to the US Naval Medical Research Unit-3 (NAMRU-3), Cairo, for identification based on colony morphology, Gram stain reaction, susceptibility to optochin and bile solubility. All isolates were preserved in brain–heart infusion broth with 15% glycerol at –70°C.

Serotyping

Pneumococcal isolates were serotyped at the US Naval Health Research Center, San Diego, CA, using latex agglutination and the Quellung reaction for confirmation (Statens Serum Institute, Copenhagen, Denmark). Vaccine-related strains were defined as pneumococci with serotypes within the same serogroup as the vaccine serotypes,11 except for serotype 19A.7,24,25

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was performed and interpreted by the disc diffusion and Etest methods as recommended by the NCCLS.26,27 Quality control was carried out according to the recommendations of the NCCLS26,27 using S. pneumoniae ATCC 49619 as the control strain. Six antibiotics were tested: chloramphenicol (30 µg), tetracycline (30 µg), erythromycin (15 µg), trimethoprim/sulfamethoxazole (5 µg), penicillin (Etest) and ceftriaxone (Etest). The Etest (AB Biodisk, Solna, Sweden) was performed according to the manufacturer's instructions.28 The inoculum was prepared in 0.85% saline and turbidity adjusted to the equivalent of a 0.5 McFarland Standard. Pneumococcal isolates with resistance to at least three different classes of antibiotics were defined as multidrug-resistant (MDR).29 For comparative purposes, patient age groups were assigned as recommended by Hausdorff et al.12


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Isolates and serotype distribution

Two-hundred and five S. pneumoniae isolates were recovered from patients with acute bacterial meningitis. Forty-six serotypes were identified, with 10 isolates (5%) being non-typeable. Of the serotyped isolates, 13% were only characterized as reactive with serum pools C–I. Thirty-two percent of S. pneumoniae isolates were collected from adults 18–49 years old (predominantly serotypes 23F, 19A and 20), 29% from children 6–17 years old (mainly serotypes 1, 6A, 6B and 19A), 7% from 2–5-year-olds (6A, 6B and 19F) and 25% from infants <2 years (6B, 6A, 14, 5 and 23F; Figure 1). However, only 7% of the isolates were recovered from patients >49 years (mainly 16F and 20). Serotypes 14 and 5 were only recovered from infants <2 years. Isolates in serogroup 6 (serotypes 6A and 6B) were the most frequent in children <2 years (22%) and 2–5 years (31%) old.



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Figure 1. Distribution of S. pneumoniae isolated from bacterial meningitis patients per age group (n=205) in Egypt 1998–2003. Predominant serotypes per age group were 6B, 6A, 14 and 5 for infants < 2 years; 6A, 6B and 19F for children between 2–5 years old; 1, 6A, 6B and 19A for children 6–17 years; 23F, 19A and 20 for adults (18–49 years); and 16F and 20 for patients > 49 years.

 
The distribution of S. pneumoniae serotypes recovered from Cairo was similar to that of isolates from other infectious disease hospitals in the sentinel surveillance network. Overall, the most common S. pneumoniae serotypes were 6B (10%), 1 (7%), 19A (7%), 23F (6%) and 6A (6%), constituting 37% of the total isolates (Table 1). Ten serotypes (5%) were isolated only once. The remaining serotypes ranged in frequency between 1–4%. Of the total typeable S. pneumoniae isolates, only 29 and 42% were represented in the current 7- and 11-valent conjugate vaccine formulas, respectively. Among isolates recovered from children <2 years old, 49 and 69% were covered in the 7- and 11-valent conjugate vaccines, respectively (Table 2). For children 2–5 years old, S. pneumoniae coverage was 61 and 69% in the 7- and 11-valent conjugate vaccines, respectively. Serotypes 19A (7%), 6A (6%) and 16F (3%) were the major non-vaccine types.


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Table 1. Serotype distribution and antibiotic resistance profiles of S. pneumoniae isolated from bacterial meningitis patients in Egypt, 1998–2003 (n=205)

 

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Table 2. S. pneumoniae serotype representation in the 7- and 11-valent conjugate vaccine formulations for isolates recovered from age groups ≤ 5 years and ≥ 50 years

 
Antimicrobial susceptibility

Nearly 50% of all S. pneumoniae isolates (100/205) were non-susceptible (intermediate and resistant) to penicillin. Of all penicillin-non-susceptible S. pneumoniae isolates 47 and 13% were vaccine- and vaccine-related strains, respectively. Penicillin-resistant isolates (3%) showed MIC values of 2.0 mg/L. Thirteen isolates (6%) were non-susceptible to ceftriaxone, with 5% intermediate and 1.3% resistant. Resistance to tetracycline, trimethoprim/sulfamethoxazole, erythromycin and chloramphenicol was 52, 59, 11 and 9%, respectively. The predominant serotypes (6B, 1, 19A, 23F and 6A) were significantly more likely, as a group, to be non-susceptible to penicillin compared with the other serotypes (85 versus 25%, P<0.001). All serotype 1 isolates were susceptible to penicillin. Only 4% (32/805) of all isolates were MDR. Of note, the majority of MDR isolates (50%) were within serotypes 23F, 6B and 6A (Table 1). Details of S. pneumoniae susceptibility profiles by serotype are summarized in Table 1. For children <2 years old, 51 and 9% of S. pneumoniae isolates were non-susceptible to penicillin and ceftriaxone, respectively.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although pneumococcal meningitis represents a relatively small proportion of invasive pneumococcal disease compared with pneumonia and bacteraemia,4,13 meningitis is the most severe form, resulting in death or disability in 40–75% of children in developing countries who contract the disease.30,31 Therefore, information on meningitis S. pneumoniae strain distribution is very useful in understanding the epidemiology of pneumococcal disease, and for implementing vaccination programmes.16 This study revealed an increase in penicillin resistance among S. pneumoniae strains from CSF in Egypt, an important finding both for management of patients with meningitis, as well as for prevention. In addition, our findings regarding the distribution of the various S. pneumoniae strains from meningitis patients can have important implications for decisions related to the use of pneumococcal vaccines in this country.

Characterization of S. pneumoniae isolates from meningitis patients in Egypt revealed a wide diversity of 47 serotypes, with serotypes 6B, 1, 19A, 23F and 6A being the most common. This is somewhat different from a previous report in 1990 showing that serotypes 1, 6A, 9L, 12A, 19A and 29 were the predominant serotypes.32 Temporal differences in the proportions and serotype distribution of S. pneumoniae have been reported previously,7,33 and could explain some of these variations. Furthermore, unlike the previous study where isolates were from one referral hospital, the isolates in our study were collected from multiple community-based hospitals throughout Egypt, and could be more representative of prevalent meningitis S. pneumoniae strains in the community. Our findings demonstrate a decline in the proportion of serotype 1 from 32–7.3%, with serotype 6B emerging as a more frequently isolated invasive strain. The decline in the distribution of serotype 1 is similar to a previous observation in the USA, where serogroups 1–3 and 5 decreased significantly from 71–7% in adults and from 18–2% in children during the period 1928–98.34 Similarly, serotypes 1 and 5 were important in Spain in the early 1980s, but in recent times are rarely isolated.33 Whereas these two serotypes have caused illness with much greater frequency in Latin America than elsewhere, they were especially uncommon in the USA and Canada.35 In our study, serotypes 5 and 3 were identified at relatively low rates (2.4 and 1.9%, respectively). Overall, our S. pneumoniae serotype distribution by patient age is more or less similar to that reported from the USA, Europe and Africa,16 although it is slightly different from those of neighbouring countries such as Israel36,37 and Saudi Arabia.38 It is important to note that isolates from these countries covered a wider spectrum of invasive pneumococcal disease, including pneumonia and bacteraemia, in addition to meningitis.

The present conjugate vaccines were reported to elicit cross-reactive antibodies to S. pneumoniae serotypes not included in the formulation,7,39 but some recent studies have shown limited efficacy for coverage against some serotypes from the same serogroup (i.e. serotype 19A).24,25 Assuming that protective immunity to related S. pneumoniae serotypes is induced along with the vaccine-specific strains, the 11-valent vaccine would cover up to 69% of S. pneumoniae meningitis strains among Egyptian children. Our isolates were only identified from the CSF and may not represent all serotypes causing different forms of pneumococcal diseases, especially given recent reports suggesting that certain serotypes may have a propensity to invade one clinical site rather than another.12,16

Although aggressive antibiotic therapy is lifesaving for many patients with pneumococcal disease, the emergence of drug-resistant pneumococci has compromised the effectiveness of antibiotics, and highlights the need for better preventive measures. Our data show that penicillin-non-susceptibility among S. pneumoniae isolates has significantly increased during the last few decades.22,32,40 Although the meningitis surveillance system through which we collected the S. pneumoniae isolates was not population-based, our results are representative of S. pneumoniae strains causing meningitis in Egypt, since the sentinel surveillance covered a large number of infectious disease hospitals throughout the country, and all meningitis patients have to be admitted to infectious disease hospitals by law. The percentage of penicillin-non-susceptible S. pneumoniae from Egyptian meningitis rose from 22% in 1978 to 37–52% in 2003.22,32,41 A similar increase in penicillin-non-susceptibility has been observed in Eastern, Western and Southern African countries,21 the USA42 and other parts of the world.43 In comparison with the antibiotic resistance profiles of neighbouring countries, a small proportion of S. pneumoniae isolates from Saudi Arabia collected from different clinical specimens were found to be penicillin-non-susceptible (18%, MIC = 0.1–1 mg/L and 1% showing MIC ≥ 1.0 mg/L), and a higher proportion were resistant to tetracycline, chloramphenicol and erythromycin.38 The Egyptian isolates showed a higher resistance to erythromycin than those from Saudi Arabia (11 versus 4%). In comparison with isolates from the USA,19,42,44 resistance profiles of isolates from Egypt for tetracycline and trimethoprim/sulfamethoxazole were 2–3 times higher and for ceftriaxone lower, but resistance to erythromycin and chloramphenicol was in the same range. The multiple drug resistance rates in S. pneumoniae serotypes from Egypt were higher than those in the USA.42 It is interesting to note, however, that all isolates belonging to serotype 1 were susceptible to penicillin and all isolates from serotypes 6B and 19A were non-susceptible to penicillin. Overall, the observed differences in antibiotic susceptibility profiles of S. pneumoniae in this and other studies is likely to reflect the pattern of treatment at the local hospitals (using inexpensive antibiotics as first choice of therapy).

The marked increase in penicillin-non-susceptibility among S. pneumoniae isolates in Egypt is of concern. Strategies to control the spread of resistant pneumococcal infections through programmes that encourage a more judicious use of antibiotics are recommended, as well as continued surveillance for antibiotic resistance. Future surveillance efforts in Egypt should include other forms of invasive disease, in particular pneumonia and bacteraemia, to obtain a more complete description of invasive S. pneumoniae strains.


    Acknowledgements
 
We express our sincere appreciation and thanks to the many physicians, nurses and clinical laboratory staff of the infectious disease hospitals involved in this study. Thanks are extended to the following infectious disease hospital directors: Dr Yehia Sultan, Dr Atef Sadaka, Dr Abdel Halim Abdel Hamid, Dr Bothaina Bakry, Dr Mohamed El Ragaby, Dr Ragaa Sakr, Dr Mohamed Abdel Hady, Dr Mofreh Ramzy, Dr Ahmed Ibrahim, Dr Said Ibrahim, Dr Mokhtar Wahba and Dr Abdel Fattah Ibrahim. We also thankfully acknowledge support from the staff of the Central Public Health Laboratory of the Ministry of Health and Population who assisted with training and monitoring the activities within the infectious disease hospitals. We are especially grateful to Drs W. Hausdorff, C. Whitney and S. Tai for their critical review of the manuscript.

Financial Support: The work was supported by a grant from the US Agency for International Development (USAID) and the Department of Defense Global Emerging Infectious Systems (DoD-GEIS) (work unit number E022). Disclaimers: The opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large, or the Egyptian Ministry of Health and Population. The study protocol was approved by the US Naval Medical Research Unit No.3 Institutional Review Board, Protocol #30968 in compliance with all Federal regulations governing the protection of human subjects.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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