Streptococcus pneumoniae Nasopharyngeal Carriage Prevalence, Serotype Distribution, and Resistance Patterns among Children on Lombok Island, Indonesia

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1 MAJOR ARTICLE Streptococcus pneumoniae Nasopharyngeal Carriage Prevalence, Serotype Distribution, and Resistance Patterns among Children on Lombok Island, Indonesia S. Soewignjo, 1 Bradford D. Gessner, 4 Agustinus Sutanto, 2 Mark Steinhoff, 5 Mulyati Prijanto, 3 Carib Nelson, 6 Anton Widjaya, 6 and Soemarjati Arjoso 3 1 Biomedical Research Unit, Mataram General Hospital, Mataram, and 2 Disease Prevention and Health Promotion Division, West Nusa Tenggara Provincial Health Office, Lombok, and 3 Center for Infectious Disease Research and Development, Ministry of Health, Jakarta, Indonesia; 4 Association Pour l Aide à lamédecine Préventive, Institut Pasteur, Paris; 5 Departments of International Health and Pediatrics and Schools of Medicine and Public Health, Johns Hopkins University, Baltimore; 6 Program for Appropriate Technology in Health, Seattle Few data exist on childhood pneumococcal carriage prevalence, serotype distribution, and resistance patterns for Indonesia, the world s fourth most populous country. During August 1997, nasopharyngeal samples were collected from a population-based, island-wide sample of 484 healthy children (age, 0 25 months) from Lombok Island, Indonesia. Two hundred twenty-one pneumococcal isolates were identified, for a carriage prevalence of 48%; 66% of isolates were of serogroup or serotype 6, 23, 15, 33, or 12. All isolates were susceptible to penicillin and cefotaxime. Twelve percent of the isolates were nonsusceptible to sulfamethoxazole or chloramphenicol and 4% were nonsusceptible to both of these drugs. Nonsusceptible organisms were most frequently serogroup or serotype 6, 12, and 33. Lombok has a moderate pneumococcal carriage prevalence and a relatively low proportion of resistant isolates. At least 3 of the 5 most common serogroups and serotypes and 2 of the 3 most common nonsusceptible serogroups and serotypes are not included in the current 7-valent pneumococcal conjugate vaccine. Streptococcus pneumoniae is one of the most common Received 30 May 2000; revised 18 August 2000; electronically published 20 March Informed consent was obtained from the parents of all study participants, and the study was conducted in conformance with guidelines for human experimentation specified by Johns Hopkins University and the US Department of Health and Human Services. The Program for Appropriate Technology in Health Institutional Review Board and the Indonesian Ministry of Health approved this study. Financial support: Office of Health and Nutrition, US Agency for International Development (cooperative agreement HRN-A with PATH); AventisPasteur (formerly Pasteur Mérieux Connaught) and the Mérieux Foundation (to A.M.P.). Aventis Pasteur is currently developing a pneumococcal conjugate vaccine. The opinions expressed herein are those of the authors and do not necessarily reflect the views of USAID, Aventis Pasteur, or the Mérieux Foundation. Reprints or correspondence: Dr. Bradford D. Gessner, Association Pour l Aide à la Médecine Préventive, Institut Pasteur, 28 rue du Docteur Roux, 75015, Paris, France ( @compuserve.com). Clinical Infectious Diseases 2001; 32: by the Infectious Diseases Society of America. All rights reserved /2001/ $03.00 causes of childhood morbidity and mortality worldwide, yet almost no data exist with regard to this organism in Indonesia, the world s fourth most populous country. In part because of this lack of data, the serotype composition of current and planned pneumococcal conjugate vaccines may not reflect the serotype distribution of invasive isolates in Indonesia. This situation will be difficult to remedy because of the logistical difficulties, expenses, and the time needed to acquire enough invasive isolates to make meaningful conclusions on serotype distribution, particularly in relatively poor rural areas where a substantial proportion of the Indonesia s children live. Several previous studies have found that carriage serotypes roughly parallel invasive serotypes (although the rank ordering differs) [1 4]. Moreover, carriage studies have been recognized as an appropriate method of conducting antibiotic resistance surveillance [5, 6]. Pneumococcal Carriage in Lombok Island, Indonesia CID 2001:32 (1 April) 1039

2 To provide estimates of pneumococcal serotype distribution, as well as to estimate carriage prevalence and antibiotic resistance patterns, we conducted a pneumococcal carriage study in Lombok, Indonesia. PATIENTS AND METHODS Study population. Lombok Island is located in the center of the Indonesian archipelago just east of Bali. The island has an area of 5435 km 2, a volcano rising 3726 m in height, and a tropical equatorial climate with a wet season from November through April. The population of 2.6 million is predominantly Muslim and of the Sasak ethnic group. Mataram is the largest city, with a population of 250,000. The current prospective, population-based study was conducted as part of a Haemophilus influenzae type b (Hib) carriage study whose methodology and results have been described elsewhere [7]. In brief, we stratified sampling by the 4 administrative districts of Lombok and then randomly selected 5 hamlets in each region for a total of 20 hamlets. In each hamlet, we obtained nasopharyngeal samples for culture from 8 children in each of 3 age groups (0 to!6 months, 6 to!12 months, and 12 to!25 months). We selected the first 8 children from each age group to come to a well-child clinic known locally as the outreach integrated health post or posyandu. Children with illness were excluded. Because extra children occasionally arrived for evaluation, we evaluated 484 study participants rather than the planned 480. Risk factor evaluation. A field-tested questionnaire was administered to collect information on the following risk factors: sex; current consumption of breast milk; current consumption of foods other than breast milk; consumption of premasticated food; use of wood for heat; use of kerosene for heat; the presence of someone who smoked cigarettes in the household; a previous history of at least 1 child death in the family; a history of sickness, diarrhea, or cough during the 30 days prior to the study period; the presence of 11 other child in the household, and a household size of 5 persons. Specimen collection. Field workers placed calcium alginate tipped swabs (Calgiswab; Spectrum Laboratories) into the nasopharynges of study subjects. The depth of swab insertion was estimated to be half the distance between the tip of the child s nose and the anterior portion of the ear. The swab was immediately placed in Amies transport medium for shipment to the central laboratory (Biomedical Research Unit, Mataram Hospital, Lombok). Laboratory methodology. Swabs were streaked onto trypticase soy agar plus 5% sheep blood plates the same day as collection. After overnight incubation, colonies that were morphologically consistent with S. pneumoniae (small, gray, and showing mucoid surrounded by a greenish zone of hemolysis) were subcultured onto the same media. Before incubation, a 6-mm 5-mg optochin disk was placed aseptically on the center of the streaking where the inoculum was densest. The plates then were incubated overnight in a CO 2 incubator. a-hemolytic strains with a growth inhibition zone of 14 mm in diameter were considered pneumococci. One or 2 colonies of presumptive pneumococci were selected and frozen at 70 C while we awaited the results of antibiotic susceptibility screening and serotype determination. Antibiotic susceptibility screening was done by use of the Kirby-Bauer disk diffusion method. This method involves an estimate of antimicrobial resistance appropriate for surveillance purposes, particularly in developing countries [8]. We used antibiotic disks that included oxacillin (1 mg), cefotaxime (30 mg), chloramphenicol (30 mg), and sulfamethoxazole (24 mg; Becton Dickinson Microbiology Systems). The oxacillin disk was used to screen for penicillin resistance [9]. Definitions of nonsusceptibility were based on the size of the zone of inhibition as follows: penicillin,!20 mm; cefotaxime,!15 mm (resistant) and mm (intermediate); chloramphenicol,!21 mm; and sulfamethoxazole,!15 mm (resistant) and mm (intermediate). Determination of MICs represents the reference standard for evaluating antimicrobial resistance. Consequently, isolates that were resistant to oxacillin and cefotaxime, as determined by use of disk diffusion, were submitted to Johns Hopkins Hospital for MIC determination by use of the Etest (AB Biodisk). Table 1. Prevalence of Streptococcus pneumoniae nasopharyngeal carriage according to age group, adjusted for design effect and weighted for age and region of sampling, Lombok Island, Indonesia, Age group, mo No. of specimens collected (% with pneumococcus identified) Weighted and adjusted prevalence of pneumococcal carriage, % (95% CI) RR of pneumococcal carriage (95% CI)!6 159 (38) 41 (32 49) Referent 6to! (48) 47 (40 54) 1.2 ( ) 12 to! (51) 52 (43 61) 1.3 ( ) Total 484 (46) 48 (42 54) 1040 CID 2001:32 (1 April) Soewignjo et al.

3 Table 2. Prevalence of Streptococcus pneumoniae nasopharyngeal carriage according to region, adjusted for design effect and weighted for age and region of sampling, Lombok Island, Indonesia, Region No. of specimens collected (% with pneumococcus identified) Weighted and adjusted pneumococcal carriage prevalence, % (95% CI) Relative risk of pneumococcal carriage (95% CI) Mataram 122 (48) 49 (41 57) Referent West 120 (48) 49 (32 66) 1.0 ( ) Central 120 (34) 35 (24 45) 0.71 ( ) East 122 (53) 55 (49 61) 1.1 ( ) Total (42 54) According to methods described elsewhere [10], serogroups and serotypes (hereafter, serogroups/types ) were determined by means of the latex agglutination method with pooled antisera for 12 serogroups/types (Pneumotest; Statens Seruminstitut). This technique, which provides information on 90% 95% of the common serogroups/types, was chosen for several reasons. It is simple, it is consistent with the methodology used in other developing world surveillance studies, and full serotype determination of carriage specimens is of limited use because carriage serotypes are not adequate for designing vaccine formulations. Because of the expense involved, Statens Seruminstitut agreed to perform specific serotype determination for a limited number of specimens (not pure isolates). We selected 22 specimens (not pure isolates) for transport and testing. Statistical analysis. All analyses were done by use of the CSample function within Epi Info, version 6.01 (Centers for Disease Control and Prevention). To obtain prevalence estimates that were more representative of the entire population of Lombok, we adjusted for the effect of the district and hamlet sampling scheme and weighted for age and for the population!2 years of age within districts. RESULTS Prevalence. We identified an age-weighted and population-weighted pneumococcal carriage prevalence, adjusted for design effect, of 48%; there was little difference between age groups (table 1). The carriage prevalence did not vary between the 4 administrative districts on Lombok (table 2), despite the existence of substantial socioeconomic differences between the relatively urban Mataram region and the remaining 3 regions, which are rural. None of the evaluated risk factors were associated with pneumococcal carriage at the 95% CI; RRs varied between 0.92 and 1.1 for all evaluated risk factors with the exception of exclusive consumption of breast milk (RR, 1.4). Antibiotic susceptibility. All 221 specimens from which pneumococci were isolated that were identified at the laboratory in Lombok were tested for antibiotic susceptibility. Four (2.2%) were nonsusceptible to penicillin, none were nonsusceptible to cefotaxime, and 27 (13%) were nonsusceptible to at least 1 of the 4 antibiotics tested (table 3). Of the 4 isolates identified as nonsusceptible to penicillin by use of disk diffusion, the Johns Hopkins reference laboratory reported that all had MICs of penicillin of 0.06 mg/l (susceptible) and MICs of cefotaxime of mg/l (susceptible), for an overall penicillin nonsusceptibility prevalence of 0. Serotypes. The Lombok laboratory identified 232 individual pneumococcal isolates from the 221 pneumococcal specimens (11 specimens provided 2 serogroups/types). The most common serogroups/types were 6 and 23; serogroup/type 15 had the highest percentage of resistant organisms (table 4). The percentage of all isolates that would be included in the current 7-valent pneumococcal conjugate vaccine was 61%. The Statens Seruminstitut reference laboratory identified 24 specific sero- Table 3. Antibiotic resistance among 221 specimens of Streptococcus pneumoniae from children 0 25 months of age, adjusted for design effect and weighted for age and region of sampling, Lombok Island, Indonesia, Antibiotic, resistance category No. (%) of specimens Penicillin, nonsusceptible Chloramphenicol, nonsusceptible 13 (6.0) Cefotaxime Resistant 0 Intermediately resistant 0 Sulfamethoxazole Resistant 7 (3.5) Intermediately resistant 13 (5.4) Nonsusceptible to any antibiotic 25 (12) Nonsusceptible to any 2 antibiotics 8 (3.3) NOTE. Resistance was determined by means of disk diffusion (with confirmatory testing by use of Etest for penicillin-nonsusceptible specimens). a Four were found nonsusceptible by use of disk diffusion but susceptible by use of Etest. 0 a Pneumococcal Carriage in Lombok Island, Indonesia CID 2001:32 (1 April) 1041

4 Table 4. Serogroup/type distribution of 232 pneumococcal isolates from 221 children who were 0 25 months of age, adjusted for design effect and weighted for age and region of sampling, Lombok Island, Indonesia, Serogroup/type No. of isolates % of total isolates (95% CI) No. nonsusceptible to 1 antibiotic (%) 6 a (21 30) 7 (9.9) 23 a (13 29) 5 (9.8) (3.6 12) 2 (10) (2.1 14) 1 (7.2) ( ) 5 (53) 19 a ( ) 1 (8.0) ( ) 0 14 a ( ) 1 (15) 9 a ( ) 2 (38) 18 a (0 3.7) (0 2.5) (0 2.8) (0 0.68) (0 1.5) (0 1.5) (0 2.0) 0 Nontypeable (7.3 14) 1 (2.9) a Included in or cross-reacting with licensed 7-valent (serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F) or proposed 9-valent (7-valent serotypes plus serotypes 1 and 5) pneumococcal conjugate vaccine. Proportion of all isolates, 61%. types in 22 pneumococcal specimens. Serotypes included 6B (4), 19F (3), 10A, 15C, 18C, 23B, and 31 (2 each), and 6A, 20, 23A, 23F, 32F, 35B, and 35F (1 each); 10 of these isolates (42%) would be included in the current 7-valent pneumococcal conjugate vaccine. Agreement between the Lombok and reference laboratories could not be determined because neither the specimens tested in Lombok nor those sent to the reference laboratory were pure cultures. DISCUSSION To our knowledge, this is the first published report of a study that has examined childhood pneumococcal carriage in Indonesia and one of the few population-based carriage studies conducted anywhere. Other studies in Asia, Europe, and North America have found carriage prevalences of 11% 100% [1, 2, 11 14]. Some of the differences among populations may relate to sampling or laboratory methodology. Some studies, though, have identified specific risk factors for carriage, such as the presence of younger or older children in the household, day care attendance, parental smoking, overcrowded living conditions, and rural residence [1, 11, 14]. Antibiotic resistance patterns found among carriage specimens provide a rough estimate of those found among invasive isolates [1, 5, 6], which indicates that pneumococcal carriage studies may be useful for monitoring resistance patterns. In Lombok, all tested pneumococci were susceptible to penicillin, a finding consistent with reports from relatively rural and underdeveloped areas of Malaysia and Lesotho (Africa) [15, 16]. This contrasts with studies in urban areas of Southeast Asia that have found a much higher proportion of penicillin nonsusceptibility, sometimes in excess of 60% [17 21]. Similar to isolates from other areas of the world, many of the nonsusceptible isolates in Lombok were serogroups/types 6 or 23 [17, 22 24]. The primary risk factor for development of resistant pneumococci is antibiotic use [25 28], particularly use of low levels of antibiotics for prolonged periods [29]. Other identified risk factors include day care attendance and urban residence [1, 3, 16, 25, 30]. Because of the absence of nonsusceptible pneumococcal specimens in our study, we could not appropriately evaluate risk factors. In Lombok, however, few children attend day care centers, most live in a relatively rural setting (even in Mataram region), and use of antibiotics is believed to be low because of a combination of a highly structured medical care system, training of village-level health care providers on appropriate antibiotic use, the poverty of the general population, and a lack of street pharmacies. Published serotype distribution data for invasive isolates from Indonesia do not exist, and because of the expense involved, it is unlikely that Indonesia will collect this information before the introduction of pneumococcal conjugate vaccines. Some studies have found that carriage serotypes are similar invasive serotypes, although the rank ordering differs and a few serotypes are almost always invasive [1 4]; consequently, our data may provide an approximation of clinically significant serotypes in Lombok. We found that fewer than two-thirds of carriage isolates belonged to serogroups/types included in the current 7-valent or proposed 9-valent pneumococcal conjugate vaccines [31, 32]. This is consistent with reports from Asia (excluding Indonesia) that have documented that fewer than two-thirds of invasive isolates belonged to serotypes included in either the 7-valent or 9-valent vaccines [13]. By contrast, serotypes in the 9-valent vaccine caused 80% 90% of invasive pneumococcal disease in areas of the world outside of Asia. Our findings on antimicrobial resistance and serogroup/type distribution patterns lead to 2 summary conclusions. First, because antimicrobial resistance in Lombok remains low, trimethoprim-sulfamethoxazole or amoxicillin may continue to be used as a first-line outpatient treatment for childhood pneumonia. Future studies will need to monitor trends in antimicrobial resistance patterns to document the continuing appropriateness of this recommendation. Second, our data on serogroup or serotype distribution suggest that although still substantial the proportion of pneumococcal disease preventable with currently formulated conjugate vaccines may be less in Lombok, and perhaps other areas of Indonesia, than in other 1042 CID 2001:32 (1 April) Soewignjo et al.

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