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serogroups of Streptococcus pneumoniae in nasopharyngeal carrier children younger than 2 years in Lima, Peru
Diagnostic Microbiology and Infectious Disease 52 (2005) 59 – 64 www.elsevier.com/locate/diagmicrobio
Penicillin resistance and serotypes/serogroups of Streptococcus pneumoniae in nasopharyngeal carrier children younger than 2 years in Lima, Peru Theresa J. Ochoaa,T, Rocio Rupab, Humberto Guerrac, Herminio Hernandezd, Eduardo Chaparrod, Jesus Tamarizb, Audrey Wangere, Edward O. Mason Jr.f a
Division of Pediatric Infectious Diseases, University of Texas Health Science Center at Houston, Houston, TX 77030, USA b Escuela de Tecnologı´a Me´dica, Universidad Peruana Cayetano Heredia, Lima 31, Peru c Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima 31, Peru d Departamento de Pediatrı´a, Universidad Peruana Cayetano Heredia, Lima 31, Peru e Department of Pathology, University of Texas Health Science Center at Houston, Houston, TX 77030, USA f Division of Pediatric Infectious Diseases, Baylor College of Medicine, Houston, TX 77030, USA Received 14 October 2004; accepted 21 December 2004
Abstract The purpose of this study was to determine the carriage rate, susceptibility pattern, and serotype distribution of Streptococcus pneumoniae in the nasopharynx of children younger than 2 years old in Lima, Peru. A total of 666 children were evaluated during 3 periods, 1997, 2001, and 2003. The overall pneumococcal carrier rate was 41%. Reduced susceptibility to penicillin was found in 5% (4/75) of isolates in 1997, 20% (15/75) in 2001, and 37% (40/109) in 2003. Reduced susceptibility to ceftriaxone was found in 12% of isolates in 2003. Serogroups 6, 19, 23, 15, and 14 accounted for 68% of all the isolates and for 81% of the penicillin-nonsusceptible strains. Only 65% of the isolated strains had serogroups found in the 7-valent conjugate pneumococcal vaccine. This highlights the importance of regional surveillance studies for effective vaccine strategies and treatment protocols. D 2005 Elsevier Inc. All rights reserved. Keywords: Penicillin resistance; Streptococcus pneumoniae; Lima, Peru
1. Introduction Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide, particularly in young children, patients with chronic diseases, the elderly, and immunocompromised individuals of all ages. Globally, approximately 1.2 million deaths due to pneumococcal pneumonia and meningitis are believed to occur among young children every year, mostly in developing countries (WHO, 1996). Antibiotic-resistant S. pneumoniae has become an increasing clinical and public health problem around the world. Levels of penicillin resistance vary geographically, as well as the serotypes. There are more than 90 pneumococcal serotypes, but less than a dozen are responsible for most of infections in children. Regional and local knowledge of the distribution of capsular serotypes and antibiotics susceptibility of pneumococci is essential for the developT Corresponding author. Tel.: +1-713-500-5714; fax: +1-713-500-5688. E-mail address: [email protected] (T.J. Ochoa). 0732-8893/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2004.12.014
ment of effective vaccine strategies and treatment protocols. In Peru, there are few studies on the susceptibility and serotype distribution of pneumococci and there are no prospective studies from a single location over time (Fukuda et al., 1996; Morales et al., 2001; Cullotta et al., 2002). The purpose of this study was to determine the carriage rate, susceptibility pattern, and serotype distribution of S. pneumoniae in nasopharyngeal carrier children younger than 2 years old in the Pediatric Outpatient Clinic at Hospital Nacional Cayetano Heredia in Lima, Peru, at 3 time intervals (1997, 2001, and 2003), to compare the susceptibility over time, and to determine associated factors for carriage of penicillin-nonsusceptible strains. 2. Methods 2.1. Study population Nasopharyngeal cultures were obtained from children at the Pediatric Outpatient Clinic of Hospital Nacional
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Cayetano Heredia during 3 periods: September 1996 to March 1997, March 2001 to September 2001, and June 2003 to December 2003. This hospital is the major medical facility serving the low socioeconomic communities of the northern part of Lima city, a population of almost 3 million inhabitants. Children 6 to 24 months of age who attended the clinic for a routine well-child visit or sick visit were consecutively sampled after informed consent was obtained from the parent or guardian. At the time that nasopharyngeal samples were obtained, demographic and clinical data were collected on each patient. The study protocol was reviewed and approved by the Ethical Committees of the Universidad Peruana Cayetano Heredia and Hospital Nacional Cayetano Heredia. 2.2. Laboratory analysis Nasopharyngeal samples were obtained using a calcium alginate swab and transported to the laboratory in sterile tubes containing trypticase soy broth. They were cultured on chocolate and blood agar (TSA with 5% sheep blood). S. pneumoniae was identified based on colony morphology, a-hemolysis, Gram stain, bile solubility, and optochin susceptibility. E-test (AB Biodisk, Solna, Sweden) was used to determine penicillin and ceftriaxone resistance as per the manufacturer’s instructions, using cation supplemented Mueller-Hinton agar with 5% sheep blood. Disk diffusion was performed on the same medium to determine resistance to chloramphenicol, clindamycin, erythromycin, and trimethoprim/sulfamethoxazole (TMP/SMX) (National Committee for Clinical Laboratory Standards [NCCLS], 1997). NCCLS standards were used for interpretation of disk diffusion zone diameters and the minimal inhibitory concentrations (MIC) for penicillin (susceptible V 0.06, intermediate 0.12 –1, resistant z 2 Ag/mL) and ceftriaxone for noncerebrospinal fluid (CSF) isolates (susceptible V 1, intermediate 2, resistant z 4 Ag/mL) (NCCLS, 2002). Isolates in the intermediate or resistant categories were considered nonsusceptible. S. pneumoniae were serotyped by capsular swelling observed microscopically (Quellung reaction) after suspension in type-specific antisera (Sattens Seruminstitute, Copenhagen, Denmark) (Sorensen, 1993).
270 in the third period). Children were between 6 and 24 months of age (median 10 months); 50% were girls. More children from the third period attended day care and were from households with z 3 children. The use of antibiotics was more common in the second period (data not available for the third period) (Table 1). The most commonly used antibiotics were amoxicillin (50%) and TMP/SMX (20%). All children were healthy; approximately 15, 20, and 15%, respectively, of the children from each period had a mild common cold. None of the children have had received the conjugated pneumococcal vaccine. 3.2. Pneumococcal nasopharyngeal carriers Of 666 children, 272 had a positive nasopharyngeal culture for S. pneumoniae. The overall pneumococcal carriage rate was 41%. Of 170 children, 75 (44%) were found to be carriers in the first period, 75 of 226 (33%) in the second period, and 122 of 270 (45%) in the third period. There were no significant differences in carriage rates among the different age groups. Carrier rates were significantly higher in children from households with more than 3 children (36%) compared with children from households with 2 or less children (26%) ( P b 0.01). 3.3. Penicillin resistance Reduced susceptibility to penicillin increased over the study period from 5% to 37%. In the first period, 4 of 75 strains were penicillin-nonsusceptible (5.3%), including 1 strain with high-level resistance (MIC = 1.5 Ag/mL). In the second period, 15 of 75 strains were penicillinnonsusceptible (20%), including 6 strains (8%) with high resistance (MIC = 1.5 and 2.0 Ag/mL). In the third period, 109 of 122 strains were available for MIC testing (the remaining strains were lost in storage). Of those, 40 strains were penicillin-nonsusceptible (36.7%), including 26 strains (23.8%) with high resistance (MICs as high as 8 and Table 1 Demographics of 666 children from the pediatric outpatient clinic at Cayetano Heredia hospital in Lima, Peru
2.3. Statistical analysis Comparison of the characteristics of the study population was done using ANOVA for continuous variables and m2 for trend for categorical variables. Comparison between percentage of penicillin and ceftriaxone resistance among the periods was done using m2 for trend. Some factors associated with nasopharyngeal carriage of penicillin-nonsusceptible strains were evaluated by m2 test with Yates correction. 3. Results 3.1. Study population characteristics Nasopharyngeal cultures were obtained from 666 children (170 in the first period, 226 in the second period, and
Number of patients Age (months), median (25%, 75%) Girls Day-care attendance Households with z 3 children Prior antibiotic useb
Period 1 (1997) n (%)
Period 2 (2001) n (%)
Period 3 (2003) n (%)
P
170
226
270
–
9 (6, 14)
10 (7, 15)
12 (9, 19)
b 0.001a
80 (47.0) 1 (0.6)
114 (50.0) 9 (3.9)
142 (52.6) 22 (8.1)
0.302 b 0.001
37 (21.8)
62 (27.4)
103 (38.2)
b 0.01
53 (31.2)
107 (47.3)
–
b 0.01
a Children from period 3 were significantly older than children from periods 1 and 2, which do not differ (Tukey-Kramer post hoc comparisons). b At least once in the prior 3 months. Data are available only for the first and second periods (396 children).
T.J. Ochoa et al. / Diagnostic Microbiology and Infectious Disease 52 (2005) 59–64
% of isolates
A
Table 3 S. pneumoniae serotypes/serogroups in nasopharyngeal carrier children in Lima, Peru (211 strains)
1997 40 30
10
0. 0 0. 12 0 0. 16 02 0. 3 0 0. 32 04 0. 7 0 0. 64 09 0. 4 12 0. 5 1 0. 9 25 0. 38 0. 0. 5 75 1 1. 5 2 3 4 6 8 12
MIC (ug/mL)
% of isolates
Serotype/serogroup
Period 1 (1997), n
Period 2 (2001), n
Period 3 (2003), n
All 3 periods, total n (%)
19 6 23 15 14 9 33 Other serogroupsa Cross-reactionb Nontypeable Total strains
14 17 8 5 2 3 3 9 2 3 66
14 7 13 3 1 2 3 16 1 5 65
14 16 11 10 8 3 0 15 3 0 80
42 40 32 18 11 8 6 40 6 8 211
20
0
B
2001
40
61
30 20 10
0. 0 0. 12 01 0. 6 0 0. 23 0 0. 32 0 0. 47 0 0. 64 0 0. 94 12 0. 5 1 0. 9 25 0. 38 0. 0. 5 75 1 1. 5 2 3 4 6 8 12
0
(19.9) (19.0) (15.2) (8.5) (5.2) (3.8) (2.8) (19.0) (2.8) (3.7) (100)
a Other serogroups: 34 (five strains), 10, 11, 18 (4 strains each), 24 (3 strains), 13, 16, 17, 21, 22 (2 strains each), 3, 4, 12, 20, 38, 39 (1 strain each). b Cross reaction: 35– 42 (3 strains) 29 –35, 34 – 47, 35 – 42 – 47 (1 strain each).
MIC (ug/mL)
% of isolates
C
common in children who attended day care ( P b 0.05) or had prior antibiotic exposure ( P b 0.001).
2003
40 30
3.4. Resistance to other antibiotics
20
Resistance to other antibiotics increased during the study periods. Considering the new ceftriaxone MIC breakpoints for non-CSF isolates, there were no high or intermediate resistant strains in the second period. In the third period, 13 of 109 strains (12%) had intermediate resistance to ceftriaxone, with no high level resistance. Based on disk diffusion, TMP/SMX had the highest resistance rate (56% in 2003) and clindamycin had the lowest (7% in 2003) (Table 2). Six strains (8%) in the first period, 17 (23%) in the second period, and 10 (8%) in the third period were multiresistant (resistant to 3 or more antibiotics).
10
0. 0 0. 12 01 0. 6 0 0. 23 0 0. 32 0 0. 47 0 0. 64 0 0. 94 12 0. 5 1 0. 9 25 0. 38 0. 0. 5 75 1 1. 5 2 3 4 6 8 12
0
MIC (ug/mL)
Fig. 1. Penicillin MIC distribution for S. pneumoniae isolates from nasopharyngeal carrier children in Lima, Peru, during 3 periods, 1997 (75 isolates, A), 2001 (75 isolates, B), and 2003 (109 isolates, C).
12 Ag/mL) (Fig. 1). Although this study does not represent active surveillance over 6 years, there has been a 4-fold increase in the percentage of resistant strains between the first 2 periods of the study and 7-fold increase between the first and third period. The trend is statistically significant ( P b 0.001). To determine associated factors for carriage of penicillinnonsusceptible pneumococci, we analyzed together patients from all the 3 periods. We compared the associated conditions in 59 patients that carried a nonsusceptible isolate to 200 patients that carried a penicillin-susceptible isolate. We found that carriage of nonsusceptible pneumococci was more
3.5. Pneumococcus serotypes/serogroups Of 272 strains, 211 (77.6%) were available for serotyping (66, 65, and 80 strains from the first, second, and third periods, respectively). Twenty-six pneumococcus serogroups were found. The serogroup distribution was not similar in the 3 periods. For example, there were more strains with serotypes 14 and 15 in period 3 than periods 1 and 2, but this difference was not statistically significant (Table 3). Serogroups 6, 19, 23, 15, and serotype 14 accounted for 68% of all the isolates and for 81% of the
Table 2 Susceptibility pattern of S. pneumoniae in nasopharyngeal carrier children in Lima, Peru, based on disk diffusion (272 strainsa) Susceptible (%)
Clindamycin Chloramphenicol Erythromycin TMP/SMX a
Intermediate resistant (%)
1997
2001
2003
Total
1997
2001
2003
88 77 81 48
88 73 84 27
93 89 86 39
90 82 84 38
11 – 17 19
7 – 8 23
0 – 2 5
75 isolates in 1997, 75 in 2001, and 122 in 2003.
High resistant (%) Total 5 – 8 14
1997
2001
2003
Total
1 23 1 33
5 27 8 50
7 11 11 56
5 18 7 48
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Percentage of strains
25 20 15 10
23
20
19
21
19 15
16
5
9
2
5
0 19
6
23
15
14
Serogroup All strains
Penicillin non-susceptible strains
Fig. 2. S. pneumoniae serotypes/serogroups in nasophayngeal carrier children in Lima, Peru. Serogroups 19, 6, 23, 15, and 14 accounted for 68% of all 211 isolated strains (white bars) and for 81% of the 43 penicillinnonsusceptible strains (black bars).
penicillin-nonsusceptible strains. Serotype 14 accounted only for 5% of the strains but accounted for 21% of the penicillin-nonsusceptible strains ( P b 0.01) (Fig. 2). Serotype 14 accounted for 31% (4/13) of the ceftriaxonenonsusceptible strains. Six strains had cross-reaction between 2 and 3 serogroups. 4. Discussion The most important finding of this study is the marked increase in penicillin resistance over the studied period (5% to 37%). In part, this can be explained by the small but higher proportion of patients that attended day care and higher proportion of patients from households with larger number of children, during the third period of the study. This probably reflects the socioeconomic trend in this population (more mothers working, more day-care attendance, and more crowded living conditions). Overall, carriage of a resistant pneumococcal strain was associated with day-care attendance and prior antibiotic use, which are well-known risk factors (Levine et al., 1999; Finkelstein et al., 2003; Regev-Yochay et al., 2003). It should be noted that the samples where collected during different seasons of the year for each time interval; this could influence the carrier rate and susceptibility profile. The increase in penicillin resistance found in this study reflects what is happening in other parts of the world. In a multicenter surveillance of invasive pneumococcal infections in children from the United States from 1993 to 1999, penicillin resistance showed a 4-fold increase (from 14% to 37%) (Kaplan et al., 2002). A surveillance study in 6 Latin American countries between 1993 and 1999 found that 29% of invasive strains isolated from children were penicillin resistant. Resistance varied by country and increased during this period (Di Fabio et al., 2001). In Peru, in 1994, Fukuda et al. found 3.3% penicillin resistance in 61 adult invasive isolates; in 2000, Cullotta et al. found 15% reduced susceptibility in 146 pediatric nasopharyngeal isolates; and in 2000, Morales et al. found 27% penicillin reduced
susceptibility in 52 pediatric invasive isolates from around the country. The present study, done in the same population using the same methodology, showed that between 1997 and 2003, there was a significant increase in penicillin resistance from 5% to 37%. Using the new MIC breakpoint for non-CSF isolates, the rates of high level resistance to third-generation cephalosporins has not increased during this study. It is important to point out that previous data from Peru (27% ceftriaxone resistance) (Morales et al., 2001) and other Latin American countries (26%, 22%, and 20% third-generation cephalosporins resistance in Uruguay, Mexico, and Argentina, respectively) (Hortal et al., 2001) were published using the old breakpoints, explaining the discrepancies observed among these studies (Daily et al., 2004). The serotype distribution of nasopharyngeal isolates is usually predictive of invasive isolates in a given population, although the rank order of specific serotypes may be different (Ghaffar et al., 1999; Kellner et al., 1998; Lehmann et al., 1997). In all age groups, serogroups 1 and 14 are more often isolated from blood; serogroups 6, 10, and 23, from CSF. In small children, serogroups 3, 19, and 23 are more often isolated from middle ear fluid (Hausdorff et al., 2000). In the present study, the most commonly isolated serogroups were 6, 19, 23, 15, and 14. These data are similar to previous studies in Peru in which serogroups 23, 6, 15, and 19 were the most common among nasopharyngeal isolates from children (Cullotta et al., 2002), and serotype 14 and serogroups 18, 1, 6, and 23 were the most common among pediatric invasive isolates (Diaz et al., 2001). The increase in the penicillin resistance rate in this study was not associated with an increase in the isolation of resistant serotypes, such as serotype 14. Geographic and temporal variation in pneumococcal serotypes demonstrates the need for a better worldwide pneumococcal vaccine. The serogroups of 65% of the isolates in the present study are found in the 7-valent conjugate pneumococcal vaccine (serogroups 6, 9, 18, 19, 23, and serotypes 4 and 14). In other Latin American countries, serotypes 14, 6A/6B, 5, 1, and 23F ranked as the most common serotypes. The expected coverage for the conjugated vaccines in this region for the prevention of pneumonia and meningitis was 58% for the 7-valent vaccine, 76% for the 9-valent (7-valent + serotypes 1 and 5), and 81% for the 11-valent (9-valent + serotypes 3 and 7F) (Di Fabio et al., 2001). In areas where a conjugated vaccine is being introduced (Peru), key research areas include establishing long-term surveillance for pneumococcal nasopharyngeal carriage and invasive and noninvasive disease. S. pneumoniae is the major pathogen causing meningitis, bacteremia, pneumonia, and empyema in pediatric patients. Pneumococcal disease is considered to occur subsequent to nasopharyngeal colonization which is a highly dynamic process (Gray et al., 1980; Meats et al., 2003). Although nasopharyngeal isolates are not useful for predicting the cause of invasive disease in an individual patient, they are
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useful in predicting resistance rates and serotype distribution of invasive isolates in a given population. Resistance rates among invasive strains may be less than those isolates obtained from nonsterile sites (Ghaffar et al., 1999; Kellner et al., 1998; Lehmann et al., 1997). Knowledge of the local susceptibility patterns is of great importance for the physician making decisions on initial empirical treatment of infections. Several studies have demonstrated that the clinical characteristics and the course and outcome do not differ between patients with infections due to susceptible and nonsusceptible pneumococci, in cases of pneumonia, bacteremia, and nonmeningeal invasive disease (Kaplan et al., 2001; Silverstein et al., 1999; Tan et al., 1998; Deeks et al., 1999). One limitation of this study is that the results cannot be extrapolated to the general population. Samples were collected from children visiting a hospital that serves several low socioeconomic communities of Lima. Although, these communities may be representative of most of the population of Lima, the results cannot be reliably generalized. In summary, the overall pneumococcal carriage rate was 41%. The percentage of penicillin-nonsusceptible pneumococci has increased 7-fold (5% to 37%) from 1997 to 2003 in the Pediatric Outpatient Clinic of Hospital Nacional Cayetano Heredia in Lima, Peru. Serogroups 6, 19, 23, 15, and serotype 14 accounted for 68% of all the isolates and for 81% of the penicillin-nonsusceptible strains. Acknowledgments This work was funded in part by a grant from Consejo Nacional de Ciencia y Tecnologı´a (CONCYTEC, Peru) and by an award from Fondo de Apoyo al Investigador Farmaindustria (FAIF 2000, Peru) and supported by the following Peruvian laboratories: Roche, Hoechst Marion Roussel, Eli Lilly, and Becton Dickinson. The authors thank Dr Thomas Cleary for his helpful suggestions and review of the manuscript, Dr Kathleen Kennedy for her help with the data analysis, and Susan Kirby for her secretarial support. References Cullotta AR, Kalter HD, Delgado J, Gilman RH, Facklam RR, Velapatino B, Coronel J, Cabrera L, Urbina M (2002) Antimicrobial susceptibilities and serotype distribution of Streptococcus pneumoniae isolates from a low socioeconomic area in Lima, Peru. Clin Diagn Lab Immunol 9:1328 – 1331. Daily P, Farley M, Jorgensen JH, Barret N, Sanza LT, Glennen A, Dumas N, Hatch J, Craig A, Whitney CG, Greene CM (2004) Effect of new susceptibility breakpoints on reporting of resistance in Streptococcus pneumoniae—United States, 2003. MMWR Morb Mortal Wkly Rep 53:152 – 154. Deeks SL, Palacio R, Ruvinsky R, Kertesz DA, Hortal M, Rossi A, Spika JS, Di Fabio JL (1999) Risk factors and course of illness among children with invasive penicillin-resistant Streptococcus pneumoniae. The Streptococcus pneumoniae Working Group. Pediatrics 103:409 – 413. Di Fabio JL, Castaneda E, Agudelo CI, De La Hoz F, Hortal M, Camou T, Echaniz-Aviles G, Noemi M, Barajas C, Heitmann I, Hormazabal JC, Brandileone MC, Dias Vieira VS, Regueira M, Ruvinski R, Corso A, Lovgren M, Talbot JA, De Quadros C (2001) Evolution of
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Streptococcus pneumoniae serotypes and penicillin susceptibility in Latin America, Sireva-Vigia Group, 1993 to 1999. PAHO SirevaVigia study group. Pan American Health Organization. Pediatr Infect Dis J 20:959 – 967. Diaz S, Morales S, Gonzalez D, Salazar E (2001) Serotipos de Streptococcus pneumoniae aislados de procesos invasivos, 1999 – Abril 2000. Proceedings of the VII Congreso Peruano de Enfermedades Infecciosas y Tropicales; 2001 Aug 30 – Sep 2; Lima, Peru. p. 30. Finkelstein JA, Huang SS, Daniel J, Rifas-Shiman SL, Kleinman K, Goldmann D, Pelton SI, DeMaria A, Platt R (2003) Antibiotic-resistant Streptococcus pneumoniae in the heptavalent pneumococcal conjugate vaccine era: Predictors of carriage in a multicommunity sample. Pediatrics 112:862 – 869. Fukuda J, Echevarria J, Llanos F, Yichu A, Palomino S, Gotuzzo E, Carrillo C (1996) Streptococcus pneumoniae resistentes a penicilina en Lima, Peru´. Rev Med Hered 7:11 – 16. Ghaffar F, Friedland IR, McCracken Jr GH (1999) Dynamics of nasopharyngeal colonization by Streptococcus pneumoniae. Pediatr Infect Dis J 9:638 – 646. Gray BM, Converse III GM, Dillon Jr HC (1980) Epidemiologic studies of Streptococcus pneumoniae in infants: Acquisition, carriage, and infection during the first 24 months of life. J Infect Dis 142:923 – 933. Hausdorff WP, Bryant J, Kloek C, Paradiso PR, Siber GR (2000) The contribution of specific pneumococcal serogroups to different disease manifestations: Implications for conjugate vaccine formulation and use, part II. Clin Infect Dis 30:122 – 140. Hortal M, Lovgren M, de la Hoz F, Agudelo CI, Brandileone MC, Camou T, Casagrande S, Castaneda E, Corso A, Echaniz G, Hormazabal JC, Pace J, Palacio R, Perez-Giffoni G, Ruvinsky R, Di Fabio JL, PAHO ˜ a Study Group. (2001) Antibiotic resistance in StreptoSIREVA-VigA coccus pneumoniae in six Latin American countries: 1993–1999 surveillance. Microb Drug Resist 7:391 – 401. Kaplan SL, Mason Jr EO, Barson WJ, Tan TQ, Schutze GE, Bradley JS, Givner LB, Kim KS, Yogev R, Wald ER (2001) Outcome of invasive infections outside the central nervous system caused by Streptococcus pneumoniae isolates nonsusceptible to ceftriazone in children treated with beta-lactam antibiotics. Pediatr Infect Dis J 20:392 – 396. Kaplan SL, Mason Jr EO, Wald E, Tan TQ, Schutze GE, Bradley JS, Givner LB, Kim KS, Yogev R, Barson WJ (2002) Six year multicenter surveillance of invasive pneumococcal infections in children. Pediatr Infect Dis J 21:141 – 147. Kellner JD, McGeer A, Cetron MS, Low DE, Butler JC, Matlow A, Talbot J, Ford-Jones EL (1998) The use of Streptococcus pneumoniae nasopharyngeal isolates from healthy children to predict features of invasive disease. Pediatr Infect Dis J 17:279 – 286. Lehmann D, Gratten M, Montgomery J (1997) Susceptibility of pneumococcal carriage isolates to penicillin provides a conservative estimate of susceptibility of invasive pneumococci. Pediatr Infect Dis J 16:297 – 305. Levine OS, Farley M, Harrison LH, Lefkowitz L, McGeer A, Schwartz B (1999) Risk factors for invasive pneumococcal disease in children: A population-based case – control study in North America. Pediatrics 103:E28. Meats E, Brueggemann AB, Enright MC, Sleeman K, Griffiths DT, Crook DW, Spratt BG (2003) Stability of serotypes during nasopharyngeal carriage of Streptococcus pneumoniae. J Clin Microbiol 41:386 – 392. Morales S, Diaz S, Gonzalez D, Huapaya B, Sua´rez V, Salazar E (2001) Susceptibilidad antimicrobiana de Streptococcus pneumoniae utilizando la prueba de concentracio´n inhibitoria mı´nima (CIM). An˜os 1990 – 2000. Proceedings of the VII Congreso Peruano de Enfermedades Infecciosas y Tropicales; 2001 Aug 30 – Sep 2; Lima, Peru. p. 40. National Committee for Clinical Laboratory Standards. (1997) Performance standards for antimicrobial disk susceptibility tests. Approved standards [Document M2-A6]. Wayne (Pa)7 NCCLS.
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Penicillin resistance and serotypes/serogroups of Streptococcus pneumoniae in nasopharyngeal carrier children younger than 2 years in Lima, Peru Theresa J. Ochoaa,T, Rocio Rupab, Humberto Guerrac, Herminio Hernandezd, Eduardo Chaparrod, Jesus Tamarizb, Audrey Wangere, Edward O. Mason Jr.f a
Division of Pediatric Infectious Diseases, University of Texas Health Science Center at Houston, Houston, TX 77030, USA b Escuela de Tecnologı´a Me´dica, Universidad Peruana Cayetano Heredia, Lima 31, Peru c Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima 31, Peru d Departamento de Pediatrı´a, Universidad Peruana Cayetano Heredia, Lima 31, Peru e Department of Pathology, University of Texas Health Science Center at Houston, Houston, TX 77030, USA f Division of Pediatric Infectious Diseases, Baylor College of Medicine, Houston, TX 77030, USA Received 14 October 2004; accepted 21 December 2004
Abstract The purpose of this study was to determine the carriage rate, susceptibility pattern, and serotype distribution of Streptococcus pneumoniae in the nasopharynx of children younger than 2 years old in Lima, Peru. A total of 666 children were evaluated during 3 periods, 1997, 2001, and 2003. The overall pneumococcal carrier rate was 41%. Reduced susceptibility to penicillin was found in 5% (4/75) of isolates in 1997, 20% (15/75) in 2001, and 37% (40/109) in 2003. Reduced susceptibility to ceftriaxone was found in 12% of isolates in 2003. Serogroups 6, 19, 23, 15, and 14 accounted for 68% of all the isolates and for 81% of the penicillin-nonsusceptible strains. Only 65% of the isolated strains had serogroups found in the 7-valent conjugate pneumococcal vaccine. This highlights the importance of regional surveillance studies for effective vaccine strategies and treatment protocols. D 2005 Elsevier Inc. All rights reserved. Keywords: Penicillin resistance; Streptococcus pneumoniae; Lima, Peru
1. Introduction Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide, particularly in young children, patients with chronic diseases, the elderly, and immunocompromised individuals of all ages. Globally, approximately 1.2 million deaths due to pneumococcal pneumonia and meningitis are believed to occur among young children every year, mostly in developing countries (WHO, 1996). Antibiotic-resistant S. pneumoniae has become an increasing clinical and public health problem around the world. Levels of penicillin resistance vary geographically, as well as the serotypes. There are more than 90 pneumococcal serotypes, but less than a dozen are responsible for most of infections in children. Regional and local knowledge of the distribution of capsular serotypes and antibiotics susceptibility of pneumococci is essential for the developT Corresponding author. Tel.: +1-713-500-5714; fax: +1-713-500-5688. E-mail address: [email protected] (T.J. Ochoa). 0732-8893/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2004.12.014
ment of effective vaccine strategies and treatment protocols. In Peru, there are few studies on the susceptibility and serotype distribution of pneumococci and there are no prospective studies from a single location over time (Fukuda et al., 1996; Morales et al., 2001; Cullotta et al., 2002). The purpose of this study was to determine the carriage rate, susceptibility pattern, and serotype distribution of S. pneumoniae in nasopharyngeal carrier children younger than 2 years old in the Pediatric Outpatient Clinic at Hospital Nacional Cayetano Heredia in Lima, Peru, at 3 time intervals (1997, 2001, and 2003), to compare the susceptibility over time, and to determine associated factors for carriage of penicillin-nonsusceptible strains. 2. Methods 2.1. Study population Nasopharyngeal cultures were obtained from children at the Pediatric Outpatient Clinic of Hospital Nacional
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Cayetano Heredia during 3 periods: September 1996 to March 1997, March 2001 to September 2001, and June 2003 to December 2003. This hospital is the major medical facility serving the low socioeconomic communities of the northern part of Lima city, a population of almost 3 million inhabitants. Children 6 to 24 months of age who attended the clinic for a routine well-child visit or sick visit were consecutively sampled after informed consent was obtained from the parent or guardian. At the time that nasopharyngeal samples were obtained, demographic and clinical data were collected on each patient. The study protocol was reviewed and approved by the Ethical Committees of the Universidad Peruana Cayetano Heredia and Hospital Nacional Cayetano Heredia. 2.2. Laboratory analysis Nasopharyngeal samples were obtained using a calcium alginate swab and transported to the laboratory in sterile tubes containing trypticase soy broth. They were cultured on chocolate and blood agar (TSA with 5% sheep blood). S. pneumoniae was identified based on colony morphology, a-hemolysis, Gram stain, bile solubility, and optochin susceptibility. E-test (AB Biodisk, Solna, Sweden) was used to determine penicillin and ceftriaxone resistance as per the manufacturer’s instructions, using cation supplemented Mueller-Hinton agar with 5% sheep blood. Disk diffusion was performed on the same medium to determine resistance to chloramphenicol, clindamycin, erythromycin, and trimethoprim/sulfamethoxazole (TMP/SMX) (National Committee for Clinical Laboratory Standards [NCCLS], 1997). NCCLS standards were used for interpretation of disk diffusion zone diameters and the minimal inhibitory concentrations (MIC) for penicillin (susceptible V 0.06, intermediate 0.12 –1, resistant z 2 Ag/mL) and ceftriaxone for noncerebrospinal fluid (CSF) isolates (susceptible V 1, intermediate 2, resistant z 4 Ag/mL) (NCCLS, 2002). Isolates in the intermediate or resistant categories were considered nonsusceptible. S. pneumoniae were serotyped by capsular swelling observed microscopically (Quellung reaction) after suspension in type-specific antisera (Sattens Seruminstitute, Copenhagen, Denmark) (Sorensen, 1993).
270 in the third period). Children were between 6 and 24 months of age (median 10 months); 50% were girls. More children from the third period attended day care and were from households with z 3 children. The use of antibiotics was more common in the second period (data not available for the third period) (Table 1). The most commonly used antibiotics were amoxicillin (50%) and TMP/SMX (20%). All children were healthy; approximately 15, 20, and 15%, respectively, of the children from each period had a mild common cold. None of the children have had received the conjugated pneumococcal vaccine. 3.2. Pneumococcal nasopharyngeal carriers Of 666 children, 272 had a positive nasopharyngeal culture for S. pneumoniae. The overall pneumococcal carriage rate was 41%. Of 170 children, 75 (44%) were found to be carriers in the first period, 75 of 226 (33%) in the second period, and 122 of 270 (45%) in the third period. There were no significant differences in carriage rates among the different age groups. Carrier rates were significantly higher in children from households with more than 3 children (36%) compared with children from households with 2 or less children (26%) ( P b 0.01). 3.3. Penicillin resistance Reduced susceptibility to penicillin increased over the study period from 5% to 37%. In the first period, 4 of 75 strains were penicillin-nonsusceptible (5.3%), including 1 strain with high-level resistance (MIC = 1.5 Ag/mL). In the second period, 15 of 75 strains were penicillinnonsusceptible (20%), including 6 strains (8%) with high resistance (MIC = 1.5 and 2.0 Ag/mL). In the third period, 109 of 122 strains were available for MIC testing (the remaining strains were lost in storage). Of those, 40 strains were penicillin-nonsusceptible (36.7%), including 26 strains (23.8%) with high resistance (MICs as high as 8 and Table 1 Demographics of 666 children from the pediatric outpatient clinic at Cayetano Heredia hospital in Lima, Peru
2.3. Statistical analysis Comparison of the characteristics of the study population was done using ANOVA for continuous variables and m2 for trend for categorical variables. Comparison between percentage of penicillin and ceftriaxone resistance among the periods was done using m2 for trend. Some factors associated with nasopharyngeal carriage of penicillin-nonsusceptible strains were evaluated by m2 test with Yates correction. 3. Results 3.1. Study population characteristics Nasopharyngeal cultures were obtained from 666 children (170 in the first period, 226 in the second period, and
Number of patients Age (months), median (25%, 75%) Girls Day-care attendance Households with z 3 children Prior antibiotic useb
Period 1 (1997) n (%)
Period 2 (2001) n (%)
Period 3 (2003) n (%)
P
170
226
270
–
9 (6, 14)
10 (7, 15)
12 (9, 19)
b 0.001a
80 (47.0) 1 (0.6)
114 (50.0) 9 (3.9)
142 (52.6) 22 (8.1)
0.302 b 0.001
37 (21.8)
62 (27.4)
103 (38.2)
b 0.01
53 (31.2)
107 (47.3)
–
b 0.01
a Children from period 3 were significantly older than children from periods 1 and 2, which do not differ (Tukey-Kramer post hoc comparisons). b At least once in the prior 3 months. Data are available only for the first and second periods (396 children).
T.J. Ochoa et al. / Diagnostic Microbiology and Infectious Disease 52 (2005) 59–64
% of isolates
A
Table 3 S. pneumoniae serotypes/serogroups in nasopharyngeal carrier children in Lima, Peru (211 strains)
1997 40 30
10
0. 0 0. 12 0 0. 16 02 0. 3 0 0. 32 04 0. 7 0 0. 64 09 0. 4 12 0. 5 1 0. 9 25 0. 38 0. 0. 5 75 1 1. 5 2 3 4 6 8 12
MIC (ug/mL)
% of isolates
Serotype/serogroup
Period 1 (1997), n
Period 2 (2001), n
Period 3 (2003), n
All 3 periods, total n (%)
19 6 23 15 14 9 33 Other serogroupsa Cross-reactionb Nontypeable Total strains
14 17 8 5 2 3 3 9 2 3 66
14 7 13 3 1 2 3 16 1 5 65
14 16 11 10 8 3 0 15 3 0 80
42 40 32 18 11 8 6 40 6 8 211
20
0
B
2001
40
61
30 20 10
0. 0 0. 12 01 0. 6 0 0. 23 0 0. 32 0 0. 47 0 0. 64 0 0. 94 12 0. 5 1 0. 9 25 0. 38 0. 0. 5 75 1 1. 5 2 3 4 6 8 12
0
(19.9) (19.0) (15.2) (8.5) (5.2) (3.8) (2.8) (19.0) (2.8) (3.7) (100)
a Other serogroups: 34 (five strains), 10, 11, 18 (4 strains each), 24 (3 strains), 13, 16, 17, 21, 22 (2 strains each), 3, 4, 12, 20, 38, 39 (1 strain each). b Cross reaction: 35– 42 (3 strains) 29 –35, 34 – 47, 35 – 42 – 47 (1 strain each).
MIC (ug/mL)
% of isolates
C
common in children who attended day care ( P b 0.05) or had prior antibiotic exposure ( P b 0.001).
2003
40 30
3.4. Resistance to other antibiotics
20
Resistance to other antibiotics increased during the study periods. Considering the new ceftriaxone MIC breakpoints for non-CSF isolates, there were no high or intermediate resistant strains in the second period. In the third period, 13 of 109 strains (12%) had intermediate resistance to ceftriaxone, with no high level resistance. Based on disk diffusion, TMP/SMX had the highest resistance rate (56% in 2003) and clindamycin had the lowest (7% in 2003) (Table 2). Six strains (8%) in the first period, 17 (23%) in the second period, and 10 (8%) in the third period were multiresistant (resistant to 3 or more antibiotics).
10
0. 0 0. 12 01 0. 6 0 0. 23 0 0. 32 0 0. 47 0 0. 64 0 0. 94 12 0. 5 1 0. 9 25 0. 38 0. 0. 5 75 1 1. 5 2 3 4 6 8 12
0
MIC (ug/mL)
Fig. 1. Penicillin MIC distribution for S. pneumoniae isolates from nasopharyngeal carrier children in Lima, Peru, during 3 periods, 1997 (75 isolates, A), 2001 (75 isolates, B), and 2003 (109 isolates, C).
12 Ag/mL) (Fig. 1). Although this study does not represent active surveillance over 6 years, there has been a 4-fold increase in the percentage of resistant strains between the first 2 periods of the study and 7-fold increase between the first and third period. The trend is statistically significant ( P b 0.001). To determine associated factors for carriage of penicillinnonsusceptible pneumococci, we analyzed together patients from all the 3 periods. We compared the associated conditions in 59 patients that carried a nonsusceptible isolate to 200 patients that carried a penicillin-susceptible isolate. We found that carriage of nonsusceptible pneumococci was more
3.5. Pneumococcus serotypes/serogroups Of 272 strains, 211 (77.6%) were available for serotyping (66, 65, and 80 strains from the first, second, and third periods, respectively). Twenty-six pneumococcus serogroups were found. The serogroup distribution was not similar in the 3 periods. For example, there were more strains with serotypes 14 and 15 in period 3 than periods 1 and 2, but this difference was not statistically significant (Table 3). Serogroups 6, 19, 23, 15, and serotype 14 accounted for 68% of all the isolates and for 81% of the
Table 2 Susceptibility pattern of S. pneumoniae in nasopharyngeal carrier children in Lima, Peru, based on disk diffusion (272 strainsa) Susceptible (%)
Clindamycin Chloramphenicol Erythromycin TMP/SMX a
Intermediate resistant (%)
1997
2001
2003
Total
1997
2001
2003
88 77 81 48
88 73 84 27
93 89 86 39
90 82 84 38
11 – 17 19
7 – 8 23
0 – 2 5
75 isolates in 1997, 75 in 2001, and 122 in 2003.
High resistant (%) Total 5 – 8 14
1997
2001
2003
Total
1 23 1 33
5 27 8 50
7 11 11 56
5 18 7 48
62
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Percentage of strains
25 20 15 10
23
20
19
21
19 15
16
5
9
2
5
0 19
6
23
15
14
Serogroup All strains
Penicillin non-susceptible strains
Fig. 2. S. pneumoniae serotypes/serogroups in nasophayngeal carrier children in Lima, Peru. Serogroups 19, 6, 23, 15, and 14 accounted for 68% of all 211 isolated strains (white bars) and for 81% of the 43 penicillinnonsusceptible strains (black bars).
penicillin-nonsusceptible strains. Serotype 14 accounted only for 5% of the strains but accounted for 21% of the penicillin-nonsusceptible strains ( P b 0.01) (Fig. 2). Serotype 14 accounted for 31% (4/13) of the ceftriaxonenonsusceptible strains. Six strains had cross-reaction between 2 and 3 serogroups. 4. Discussion The most important finding of this study is the marked increase in penicillin resistance over the studied period (5% to 37%). In part, this can be explained by the small but higher proportion of patients that attended day care and higher proportion of patients from households with larger number of children, during the third period of the study. This probably reflects the socioeconomic trend in this population (more mothers working, more day-care attendance, and more crowded living conditions). Overall, carriage of a resistant pneumococcal strain was associated with day-care attendance and prior antibiotic use, which are well-known risk factors (Levine et al., 1999; Finkelstein et al., 2003; Regev-Yochay et al., 2003). It should be noted that the samples where collected during different seasons of the year for each time interval; this could influence the carrier rate and susceptibility profile. The increase in penicillin resistance found in this study reflects what is happening in other parts of the world. In a multicenter surveillance of invasive pneumococcal infections in children from the United States from 1993 to 1999, penicillin resistance showed a 4-fold increase (from 14% to 37%) (Kaplan et al., 2002). A surveillance study in 6 Latin American countries between 1993 and 1999 found that 29% of invasive strains isolated from children were penicillin resistant. Resistance varied by country and increased during this period (Di Fabio et al., 2001). In Peru, in 1994, Fukuda et al. found 3.3% penicillin resistance in 61 adult invasive isolates; in 2000, Cullotta et al. found 15% reduced susceptibility in 146 pediatric nasopharyngeal isolates; and in 2000, Morales et al. found 27% penicillin reduced
susceptibility in 52 pediatric invasive isolates from around the country. The present study, done in the same population using the same methodology, showed that between 1997 and 2003, there was a significant increase in penicillin resistance from 5% to 37%. Using the new MIC breakpoint for non-CSF isolates, the rates of high level resistance to third-generation cephalosporins has not increased during this study. It is important to point out that previous data from Peru (27% ceftriaxone resistance) (Morales et al., 2001) and other Latin American countries (26%, 22%, and 20% third-generation cephalosporins resistance in Uruguay, Mexico, and Argentina, respectively) (Hortal et al., 2001) were published using the old breakpoints, explaining the discrepancies observed among these studies (Daily et al., 2004). The serotype distribution of nasopharyngeal isolates is usually predictive of invasive isolates in a given population, although the rank order of specific serotypes may be different (Ghaffar et al., 1999; Kellner et al., 1998; Lehmann et al., 1997). In all age groups, serogroups 1 and 14 are more often isolated from blood; serogroups 6, 10, and 23, from CSF. In small children, serogroups 3, 19, and 23 are more often isolated from middle ear fluid (Hausdorff et al., 2000). In the present study, the most commonly isolated serogroups were 6, 19, 23, 15, and 14. These data are similar to previous studies in Peru in which serogroups 23, 6, 15, and 19 were the most common among nasopharyngeal isolates from children (Cullotta et al., 2002), and serotype 14 and serogroups 18, 1, 6, and 23 were the most common among pediatric invasive isolates (Diaz et al., 2001). The increase in the penicillin resistance rate in this study was not associated with an increase in the isolation of resistant serotypes, such as serotype 14. Geographic and temporal variation in pneumococcal serotypes demonstrates the need for a better worldwide pneumococcal vaccine. The serogroups of 65% of the isolates in the present study are found in the 7-valent conjugate pneumococcal vaccine (serogroups 6, 9, 18, 19, 23, and serotypes 4 and 14). In other Latin American countries, serotypes 14, 6A/6B, 5, 1, and 23F ranked as the most common serotypes. The expected coverage for the conjugated vaccines in this region for the prevention of pneumonia and meningitis was 58% for the 7-valent vaccine, 76% for the 9-valent (7-valent + serotypes 1 and 5), and 81% for the 11-valent (9-valent + serotypes 3 and 7F) (Di Fabio et al., 2001). In areas where a conjugated vaccine is being introduced (Peru), key research areas include establishing long-term surveillance for pneumococcal nasopharyngeal carriage and invasive and noninvasive disease. S. pneumoniae is the major pathogen causing meningitis, bacteremia, pneumonia, and empyema in pediatric patients. Pneumococcal disease is considered to occur subsequent to nasopharyngeal colonization which is a highly dynamic process (Gray et al., 1980; Meats et al., 2003). Although nasopharyngeal isolates are not useful for predicting the cause of invasive disease in an individual patient, they are
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useful in predicting resistance rates and serotype distribution of invasive isolates in a given population. Resistance rates among invasive strains may be less than those isolates obtained from nonsterile sites (Ghaffar et al., 1999; Kellner et al., 1998; Lehmann et al., 1997). Knowledge of the local susceptibility patterns is of great importance for the physician making decisions on initial empirical treatment of infections. Several studies have demonstrated that the clinical characteristics and the course and outcome do not differ between patients with infections due to susceptible and nonsusceptible pneumococci, in cases of pneumonia, bacteremia, and nonmeningeal invasive disease (Kaplan et al., 2001; Silverstein et al., 1999; Tan et al., 1998; Deeks et al., 1999). One limitation of this study is that the results cannot be extrapolated to the general population. Samples were collected from children visiting a hospital that serves several low socioeconomic communities of Lima. Although, these communities may be representative of most of the population of Lima, the results cannot be reliably generalized. In summary, the overall pneumococcal carriage rate was 41%. The percentage of penicillin-nonsusceptible pneumococci has increased 7-fold (5% to 37%) from 1997 to 2003 in the Pediatric Outpatient Clinic of Hospital Nacional Cayetano Heredia in Lima, Peru. Serogroups 6, 19, 23, 15, and serotype 14 accounted for 68% of all the isolates and for 81% of the penicillin-nonsusceptible strains. Acknowledgments This work was funded in part by a grant from Consejo Nacional de Ciencia y Tecnologı´a (CONCYTEC, Peru) and by an award from Fondo de Apoyo al Investigador Farmaindustria (FAIF 2000, Peru) and supported by the following Peruvian laboratories: Roche, Hoechst Marion Roussel, Eli Lilly, and Becton Dickinson. The authors thank Dr Thomas Cleary for his helpful suggestions and review of the manuscript, Dr Kathleen Kennedy for her help with the data analysis, and Susan Kirby for her secretarial support. References Cullotta AR, Kalter HD, Delgado J, Gilman RH, Facklam RR, Velapatino B, Coronel J, Cabrera L, Urbina M (2002) Antimicrobial susceptibilities and serotype distribution of Streptococcus pneumoniae isolates from a low socioeconomic area in Lima, Peru. Clin Diagn Lab Immunol 9:1328 – 1331. Daily P, Farley M, Jorgensen JH, Barret N, Sanza LT, Glennen A, Dumas N, Hatch J, Craig A, Whitney CG, Greene CM (2004) Effect of new susceptibility breakpoints on reporting of resistance in Streptococcus pneumoniae—United States, 2003. MMWR Morb Mortal Wkly Rep 53:152 – 154. Deeks SL, Palacio R, Ruvinsky R, Kertesz DA, Hortal M, Rossi A, Spika JS, Di Fabio JL (1999) Risk factors and course of illness among children with invasive penicillin-resistant Streptococcus pneumoniae. The Streptococcus pneumoniae Working Group. Pediatrics 103:409 – 413. Di Fabio JL, Castaneda E, Agudelo CI, De La Hoz F, Hortal M, Camou T, Echaniz-Aviles G, Noemi M, Barajas C, Heitmann I, Hormazabal JC, Brandileone MC, Dias Vieira VS, Regueira M, Ruvinski R, Corso A, Lovgren M, Talbot JA, De Quadros C (2001) Evolution of
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