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High prevalence of carriage of antibiotic-resistant Streptococcus pneumoniae in children in Kampala Uganda
International Journal of Antimicrobial Agents 17 (2001) 395– 400 www.ischemo.org
Original article
High prevalence of carriage of antibiotic-resistant Streptococcus pneumoniae in children in Kampala Uganda M.L. Joloba a,b,c,*, S. Bajaksouzian b, E. Palavecino b, C. Whalen c, M.R. Jacobs b,d a Department of Microbiology, Makerere Uni6ersity Medical School, Kampala, Uganda Department of Pathology, School of Medicine, Case Western Reser6e Uni6ersity, 10900 Euclid A6enue, Cle6eland, OH, 44106 -4995, USA c Department of Epidemiology and Biostatistics, School of Medicine, Case Western Reser6e Uni6ersity, 10900 Euclid A6enue, Cle6eland, OH, 44106 -4995, USA d Department of Pathology, Uni6ersity Hospitals of Cle6eland, Cle6eland, OH, USA b
Received 20 June 2000; accepted 22 September 2000
Abstract There are few data on antibiotic-resistant Streptococcus pneumoniae in Uganda. A total of 191 healthy children in Kampala, Uganda were screened for nasopharyngeal carriage of S. pneumoniae; 118 (62%) of the children were carriers. Antimicrobial susceptibility and serotype of 115 strains was determined. Ninety-six (83.5%) of the isolates were of intermediate resistance to penicillin and 19 (16.5%) were susceptible. All strains were susceptible to cefotaxime. The rates of resistance to other drugs were trimethoprim–sulphamethoxazole (83.5%), tetracycline (28.7%) and chloramphenicol (10.4%). All strains were susceptible to rifampicin, erythromycin and clindamycin. Serogroups 6, 9, 14, 19 and 23 accounted for 80% of the isolates. These data show that the rate of carriage of antibiotic-resistant pneumococci by children is high in Kampala, Uganda. © 2001 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. Keywords: Streptococcus pneumoniae; Antibiotic resistance; Kampala, Uganda
1. Introduction Streptococcus pneumoniae remains a major cause of mortality and morbidity worldwide. It is the commonest cause of community-acquired bacterial respiratory tract infections [1–3]. It is also a major cause of meningitis and bacteraemia with incidence rates of 1.5 and 5 – 10 cases per 100 000 individuals per year respectively [4 – 6]. At least 1 million children die of pneumococcal disease every year [7]. The prevalence of antibiotic-resistant S. pneumoniae, documented in many parts of the world since the 1960s, is on the increase [2,8 –13]. Failures in therapy due to antibiotic resistant pneumococci have been reported in various places [14,15]. Since most antibiotic therapy in outpatients as well as initial * Corresponding author. Tel.: + 1-216-3684192; fax: +1-2163683970. E-mail address: [email protected] (M.L. Joloba).
treatment of inpatients is empiric, knowledge of local susceptibility patterns is important to guide such therapy. Uganda, with virtually uncontrolled antibiotic prescription, ready access to antibiotics without prescription, limited laboratory facilities and high prevalence of HIV infection, may well have significant morbidity and mortality due to antibiotic-resistant pneumococci. The characteristics, serotypes and susceptibility patterns of nasopharyngeal isolates have been shown to be similar to those of invasive strains [16 –19], findings consistent with the fact that pneumococcal disease follows nasopharyngeal carriage. Nasopharyngeal carriage of S. pneumoniae without disease occurs in all age groups. However, carriage rates vary with age, being greater in children in whom more than 95% have had an episode of nasopharyngeal colonization by the age of 2 years [2]. A colonizing strain is carried for an average of 6 weeks, but carriage can persist for up to 12
0924-8579/01/$20 © 2001 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. PII: S 0 9 2 4 - 8 5 7 9 ( 0 0 ) 0 0 3 4 5 - 9
M.L. Joloba et al. / International Journal of Antimicrobial Agents 17 (2001) 395–400
396
months [16]. As the prevalence of antibiotic-resistant pneumococci was not known in Uganda, a survey was undertaken to determine the nasopharyngeal carriage rate of S. pneumoniae in children to determine the prevalence of carriage, distribution of serotypes and antimicrobial susceptibility profiles of strains.
placed in the first streak area and plates were incubated overnight in candle extinction jars at 37°C. Pneumococci were indentified by a zone of inhibition around the optochin disk and the presence of smooth, grey, a-haemolytic colonies with depressed centres. Typical colonies were then subcultured on fresh TSA blood plates and incubated as described previously for purification.
2. Materials and methods
2.3. Storage and transportation of strains
2.1. Subjects The study was conducted in the paediatric outpatient department of Mulago Hospital, a national referral and teaching center in Kampala, the capital city of Uganda covering an area of 169 km2, with a population of 774 241 with 17.4% below 4 years of age [20]. All healthy children 3 years and below attending the outpatient department for routine checkups or immunizations were eligible. Written informed consent and ethical approval were obtained. A standardized questionnaire seeking data on demographics, recent antibiotic treatment and prophylaxis, and hospitalization was used. During the months of June and July of 1995, a total of 191 children were entered into the study.
2.2. Specimen collection and processing Pre-packed sterile calcium alginate swabs on flexible aluminum shafts (Calgiswab type 3, Fisher Scientific Co., Pittsburgh, PA) were used to collect nasopharyngeal secretions pernasally from the children. Each swab was immediately inoculated onto trypticase soy agar (TSA) plates supplemented with 5% whole sheep blood (Becton Dickson Microbiology Systems, Cockeysville, MD). The plates were kept at room temperature and transported to the laboratories of Makerere Medical School on the same day the swab was collected. The plates were then streaked, an optochin disk (Becton Dickson Microbiology Systems, Cockeysville, MD) was
Strains were stored by suspending fresh overnight growth in serum-glycerol freezing media and immediately placing in a − 70°C freezer. From the freezer, vials were immediately transferred into liquid nitrogen carriers and transported by airfreight to the laboratories of University Hospitals of Cleveland in accordance with local and international shipment regulations. From the carriers, the vials were kept frozen at −70°C until further analysis.
2.4. Identification, serotyping and antimicrobial susceptibility testing Optochin susceptibility and bile solubility were used for identification of the isolates as S. pneumoniae at University Hospitals of Cleveland [21]. The capsular reaction test using antisera to serogroups 6, 9, 14, 19 and 23 (Statens Seruminstitut, Copenhagen, Denmark) [21], was used to determine the serogroup. Other serotypes were not determined. Antimicrobial susceptibility was determined by disk diffusion as follows [21–23]. Colonies from an 18–24 h old subculture were suspended in saline and adjusted to a density equal to a 0.5 McFarland standard. Plates of Mueller–Hinton agar supplemented with 5% sheep blood (Becton Dickson Microbiology Systems, Cockeysville, MD) were inoculated by using cotton-tipped swabs (Hardwood Products Company, Guilford, ME), and antibiotic disks (Becton Dickson Microbiology Sys-
Table 1 Breakpoints for interpretation of disk diffusion testing Agent
Penicillin Erythromycin Chloramphenicol Tetracycline Trimethoprim–sulphamethoxazole Rifampicin Clindamycin a b
MIC determination required. NA, not applicable.
Zone diameter interpretation (mm) Disk content
Susceptible
Intermediate
Resistant
1 mg oxacillin 15 mg 30 mg 30 mg 25 mg 5 mg 2 mg
]20 ]21 ]21 ]23 ]19 ]19 ]19
519a 16–20 NAb 19–22 16–18 17–18 16–18
519a 515 520 518 515 516 515
M.L. Joloba et al. / International Journal of Antimicrobial Agents 17 (2001) 395–400 Table 2 Breakpoints used for interpretation of MIC testing Agent
Penicillin G Cefotaxime
397
3.2. Antibiotic use in the pre6ious 3 months
MIC interpretation breakpoints (mg/l) Susceptible
Intermediate
Resistant
50.06 50.5
0.1–1.0 1.0
]2.0 ]2.0
tems, Cockeysville, MD), were dispensed onto each plate and interpreted as shown in Table 1. In addition, minimum inhibitory concentrations (MICs) of penicillin and cefotaxime were determined by agar dilution as follows [24]. Mueller– Hinton agar plates supplemented with 5% sheep blood and containing doubling dilutions of penicillin (Squibb Institute for Medical Research, Princeton, NJ) or cefotaxime (Hoechst-Roussel Pharmaceuticals Inc., Somerville, NJ) were prepared. From a 0.5 McFarland turbidity saline suspension of fresh overnight growth, a 1:10 dilution was made in normal saline. With a Steers replicator, the diluted suspensions were inoculated onto Mueller– Hinton sheep blood agar plates containing antibiotics. Plates were incubated for 24 h at 35°C in 5–10% CO2. Zone diameters and MICs were read and interpreted using National Committee for Clinical Laboratory Standards criteria. The breakpoints are shown in Table 1 and Table 2 [22– 24].
3. Results A total of 191 children under 3 years of age were sampled. They were from 138 different suburbs of Kampala and surrounding villages. The ages of the subjects ranged from one week to 36 months with a mean of 11 months and standard deviation of 10.3 months. One hundred and forty-two (74.3%) of the children were below 12 months, 22 (11.5%) were 12–23 months and 27 (14.2%) 24– 35 months of age. There were 106 (55.5%) boys and 85 (45.5%) girls. There was no significant statistical difference in the age distribution by gender (P= 0.71).
3.1. Pre6alence of nasopharyngeal pneumococcal carriage Pneumococci were recovered from 118 (62%) of the 191 subjects. Carriage was present in 66 (62.3%) of the 106 boys and 52 (61.2%) of the 85 girls sampled. There was no statistically significant difference in carriage between boys and girls [OR=1.05; 95% CI 0.44– 1.89, P = 0.88]. Ninety-nine carriers (83%) were below 2 years of age, but there was no statistically significant difference in carriage between children above and below two years [OR= 0.64, 95% CI 0.24– 1.66, P = 0.32].
Seventy-eight (40.8%) of the children sampled had received at least one antibiotic in the preceding 3 months. Penicillins (ampicillin orally 16.8%, penicillin G parenterally 8.9%, cloxacillin orally 1.6%) and trimethoprim-sulphamethoxazole orally (25.7%) were the most frequently administered antimicrobial agents. Four agents, chloramphenicol, erythromycin, tetracycline and rifampin, had rates of use of 0.5–1.1% each. None of the children had received clindamycin or cefotaxime. Forty-six (39%) of the 118 pneumococcal carriers and 32 (43.8%) of the 73 non-carriers had received at least one antibiotic. This difference was not statistically significant [OR=0.82, 95% CI 0.43–1.54, P= 0.51.]
3.3. Hospitalization and attendance of day care centres History of hospitalization in the most recent 6 weeks was present in only three (1.6%) of the children. None of the children had attended day care centres. Therefore, no meaningful statistical computations could be done for these risk factors.
3.4. Antimicrobial susceptibility Of the 118 strains isolated, 115 were available for susceptibility testing. High rates of resistance to penicillin (83.5%), trimethoprim–sulphamethoxazole (83.5%) and tetracycline (28.7%) were found (Table 3). The frequency of resistance to chloramphenicol was lower (10.4%), whereas all strains were susceptible to cefotaxime, erythromycin, rifampicin and clindamycin. Resistance to penicillin was of intermediate level, with MICs ranging from 0.125 to 1.0 mg/l, and MIC50 and Table 3 Antimicrobial susceptibility of Streptococcus pneumoniae to penicillin and cefotaxime by MIC determination and other agents by disk diffusion testing (N= 115) Agent
Susceptible
Intermediate
Resistant
Penicillina Cefotaxime Trimethoprim –sulphamethoxazole Tetracycline Chloramphenicol Erythromycin Clindamycin Rifampicin
19 (16.5)b 115 (100) 17 (14.8)
96 (83.5) 0 2 (1.7)
0 0 96 (83.5)
78 (67.8) 103 (89.6) 115 (100) 115 (100) 115 (100)
4 (3.5) 0 0 0 0
33 (28.7) 12 (10.4) 0 0 0
a
Seven strains with oxacillin zones of 11–19 were susceptible (MICs 0.03–0.06 mg/l). b No. (percentage) of strains.
M.L. Joloba et al. / International Journal of Antimicrobial Agents 17 (2001) 395–400
398
Table 4 Susceptibility patterns and seroroups of Streptococcus pneumoniae. Strains with intermediate resistance to tetracycline and trimethoprim– sulphamethoxazole are included with resistant strains in this analysis Resistance patterna
Serogroup 6
None P S T PS PT TS PTS PCS PCTS TOTALS
1c 4
9
Total 14
2 3
19
23
1 1
2
14
4
4
20 1
6 1
4
2
4
5
3 26
11
8
3 1 3 30
2 16
cillin, chloramphenicol, tetracycline and trimethoprimsulphamethoxazole). Of the penicillin resistant strains, 90% were resistant to at least one of the non-b-lactam drugs tested, with trimethoprim-sulphamethoxazole accounting for the majority of these.
NDb 3 2 2 2 8 2 2 1 2 24
4 11 6 2 56 2 2 20 2 10 115
a P, penicillin G; S, trimethoprim–sulphamethoxazole; T, tetracycline; C, chloramphenicol. b ND, not determined. c Number of strains with indicated serogroup and resistance pattern.
MIC90 values of 0.25 and 0.50 mg/l respectively. Only 19 (16.5%) strains were susceptible (MICs 0.008– 0.06 mg/l) to penicillin, with MIC50 and MIC90 values of 0.3 and 0.06 mg/l, respectively. The overall MIC50 and MIC90 values were 0.250 and 0.50 mg/l (range 0.008– 1.0 mg/l). Although all pneumococcal isolates were susceptible to cefotaxime (MICsB0.50 mg/l) with an MIC range of 0.015 to 0.25 mg/l, and MIC50 and MIC90 values of 0.125 and 0.25 mg/l, respectively, the penicillin intermediate strains were less susceptible to cefotaxime (MICs 0.12– 0.25 mg/l) than the penicillin susceptible strains (MICs 0.015– 0.06 mg/l).
3.5. Serotype and resistance pattern distribution Ninety-one (79.1%) of the isolates belonged to one of the five serogroups tested, with 30 (26.1%) belonging to serogroup 19, 26 (22.6%) to serogroup 6, 16 (13.9%) to serogroup 23, 11 (9.6%) to serogroup 9 and 8 (7.0%) to serogroup 14. Twenty-four (20.9%) isolates were not reactive to types 6, 9, 14, 19 or 23 anticapsular sera and their serogroups were not further determined. All strains of serogroups 9, 14, 19, 23 and all but one of serogroup 6 strains were resistant to at least one of the antibiotics tested. One hundred and eleven (96.5%) of the 115 strains were resistant to at least one agent and 92 (80%) to two or more agents. Serogroup and susceptibility pattern analyses are shown in Table 4. The most common resistance pattern was resistance to penicillin and trimethoprim–sulphamethoxazole, in 56 strains (48.7%), whereas 20 strains were also resistant to tetracycline. Ten strains were resistant to four agents (peni-
4. Discussion Pneumococcal disease usually follows nasopharyngeal colonization [16,18]. As such, the characteristics of the pathogenic isolates have been found to be similar to the colonizing strains [18,19]. In both developed and developing countries pneumococcal colonization in young children has been detected in 30–60% of individuals [2,25,26]. This study has shown that the nasopharyngeal carriage rate of pneumococci in children under 3 years of age in Kampala was 61.8%. Similar studies conducted in South Africa [18] and Pakistan [19] showed colonization rates of 53% (53 of 100) and 61.9% (631 of 1019) respectively. In this study, gender, age and previous antibiotic use were not associated with significant differences in carriage. Most strains were resistant to one or more antibiotics, but lack of prior use of antibiotics did not predispose to colonization with susceptible strains. This is presumably due to the fact that most strains colonizing children (96.5%) are resistant, with few susceptible strains present in this age group. High rates of resistance to agents prescribed to the study population were found in the S. pneumoniae strains isolated, whereas no resistance was found to agents rarely (B 1.1%) prescribed (erythromycin, clindamycin and rifampicin). However, resistance to tetracycline (28.7%) was found despite usage of 0.5%, whereas resistance to chloramphenicol (10.4%) was found with 1.1% usage. The reasons for these differences are unknown, but may be related to use of tetracycline and chloramphenicol in other age groups, or to strains acquiring transposons coding for multiple resistance factors [2]. One hundred and eleven (96.5%) of the 115 isolates were resistant to at least one of the antibiotics used. Trimethoprim–sulphamethoxazole (cotrimoxazole) and the penicillins are the cheapest drugs available in Uganda, and as a result are the most frequently prescribed. The widespread use of these inexpensive agents may have created selective pressure for such strains and account for the high prevalence of resistance to these agents. Once resistant strains develop, they are readily transmitted to new carriers and maintained in the population, especially in children. All strains tested were susceptible to erythromycin and clindamycin. The same findings were reported in Kenya [26,27] and Rwanda [28], both neighbouring countries, although some of these studies included clin-
M.L. Joloba et al. / International Journal of Antimicrobial Agents 17 (2001) 395–400
ical isolates from all age groups. There was no resistance to cefotaxime or rifampicin, agents that are rarely prescribed. However, 80% of the strains showed reduced susceptibility to cefotaxime (MICs 0.125–0.25 mg/l), a phenomenon explained by cross-resistance between b-lactams. As previously found worldwide, serogroups 6, 9, 14, 19 and 23, the most common serogroups in children [2,3,16,29–31], accounted for 80% of isolates. They are also the types most often associated with resistance. Although the 23-valent vaccine contains these serogroups, it is ineffective in children below two years of age [7,16]. The data from this study show that the rates of pneumococcal carriage and antimicrobial resistance are high in Kampala, Uganda, with strains being predominantly resistant to penicillin and trimethoprim–sulphamethoxazole. These high rates of resistance call for documentation of resistance rates in pneumococcal disease, evaluation of outcomes with agents in current use and changes in practice. Treatment of pneumococcal infections should depend on the site of infection and underlying conditions [32,33]. Trimethoprim–sulphamethoxazole, with a resistance rate of 83.5%, is not suitable as an empiric agent for infections where pneumococci are major pathogens. The high rate of intermediate penicillin resistance (83.5%), raises concerns about the usefulness of penicillin G in the empiric treatment of pneumococcal meningitis [32]. Chloramphenicol, with reduced bactericidal effect in penicillin-resistant strains [32– 35], is also not indicated as a single agent or in combination therapy of childhood pneumococcal meningitis before susceptibility results are available. Cefotaxime or ceftriaxone are suitable agents for empiric management of pneumococcal meningitis in Uganda [32,33]. In pneumococcal pneumonia and bacteraemia parenterally administered penicillin and other b-lactams can still be used as levels of the drugs many times higher than the MIC can be achieved in these sites [32]. However, higher doses may be required for patients with underlying conditions such as overt HIV infections. In otitis media and sinusitis, oral b-lactams with good bioavailability such as amoxycillin and cefuroxime axetil, are active against pneumococcal strains with intermediate levels of penicillin-resistance and are therefore useful agents for management of these infections [32,33]. Erythromycin is also a suitable agent for management of outpatient pneumococcal infections in this area as no erythromycin resistance was detected. However, these are considerably more expensive than agents in current use in Uganda, and such recommendations may be difficult to implement. The high rates of antimicrobial resistance in S. pneumoniae found in this study are presumably a conse-
399
quence of antimicrobial abuse. Since this phenomenon is not limited to S. pneumoniae, there is need to re-evaluate indications for prophylactic and therapeutic use of antibiotics as well as monitoring the availability of prescribed and self-administered antibiotics. Continued surveillance of antimicrobial susceptibility profiles of S. pneumoniae in all age groups and in other parts of the country is needed in Uganda to guide empiric antimicrobial therapy.
Acknowledgements The study was supported in part by a grant from the AIDS International Training and Research Program of the Fogarty International Center (grant no. TW00011), and a grant from Merck & Co., Cincinnati, OH. We thank Dr L. Marum, Dr C. Ndugwa, Dr P.M. Mudido, Dr Baingana Baingi, Dr I. Kalyesubula, Dr J. Johnson, Ms K. Edmonds, Dr P. Waibale, Mr A. Kaddu and Mr S. Kabengera for assistance rendered. We also thank Dr G. Bukenya and Dr J. Ellner for their support and encouragement for this project.
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[15] Muhe L, Klugman KP. Pneumococcal and Haemophilus influenzae meningitis in a children’s hospital in Ethiopia: serotypes and susceptibility patterns. Trop Med Int Health 1999;4:421 –7. [16] Fedson DS, Musher DM. Pneumococcal vaccine. In: Plotkin SA, Mortimer EA Jr., editors. Vaccines. Philadelphia, PA: Saunders, 1994:517 – 64. [17] Friedland IR, Klugman KP. Antibiotic-resistant pneumococcal disease in South African children. Am J Dis Child 1992;146:920 – 3. [18] Koornhof HJ, Wasas A, Klugman KP. Antimicrobial resistance in Streptococcus pneumoniae: a South African perspective. Am J Dis Child 1992;15:84 –94. [19] Mastro TD, Nomani NK, Ishaq Z, et al. Use of nasopharyngeal isolates of Streptococcus pneumoniae and Haemophilus influenzae from children in Pakistan for surveillance of antimicrobial resistance. Pediatr Infect Dis J 1993;12:824 –30. [20] United Nations Children’s Fund, Kampala, Uganda. Children and Women in Uganda. A situation analysis. 1989. [21] Jacobs MR, Appelbaum PC. Antibiotic-resistant pneumococci. Rev Med Microbiol 1995;6:77 –93. [22] National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial susceptibility testing. Sixth informational supplement M 100-S7. National Committee for Clinical Laboratory Standards, Villanova, PA, 1997. [23] National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial disk susceptibility tests, 6th ed. Approved standard M2-A6. National Committee for Clinical Laboratory Standards, Villanova, PA, 1997. [24] National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M7-A4. National Committee for Clinical Laboratory Standards, Villanova, PA, 1997. [25] Klugman KP, Friedland IR. Antibiotic resistant pneumococci in pediatric disease. Microb Drug Resist 1995;1:5 – 8.
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[26] Rusen ID, Fraser-Roberts L, Slaney L, et al. Nasopharyngeal pneumococcal colonization among Kenyan children: antibiotic resistance, strain types and associations with human immunodeficiency virus type 1 infection. Pediatr Infect Dis J 1997;16:656 – 62. [27] Kell CM, Jordens ZJ, Daniels M, et al. Molecular epidemiology of penicillin-resistant pneumococci isolated in Nairobi, Kenya. Infect Immun 1993;61:4382 – 91. [28] Bogaerts J, Lepage P, Taelman H, et al. Antimicrobial susceptibility and serotype distribution of Streptococcus pneumoniae from Rwanda, 1984 – 1990. J Infect 1993;27:157 – 68. [29] Cheong YM, Jegathesan M, Henrichsen J, Wong YH, Ng AJ, Louis A. Antibiotic susceptibility and serotype distribution of Streptococcus pneumoniae in Malaysian children. J Trop Pediatr 1988;34:182 – 5. [30] Fenoll A, Martin-Bourgon C, Munoz R, Vicioso D, Casal J. Serotype distribution and antimicrobial resistance of Streptococcus pneumoniae isolates causing systemic infections in Spain, 1979 – 1989. Rev Infect Dis 1991;13:56 – 60. [31] Gray BM, Dillon HC. Clinical and epidemiological studies of pneumococcal infection in children. Pediatr Infect Dis 1986;5:201 – 7. [32] Jacobs M.R. Drug-resistant Streptococcus pneumoniae: rational antibiotic choices. Am J Med 1999;106:19S-25S; 48S-25S. [33] Friedland I.R., McCracken G.H. Jr.@ Management of infections caused by antibiotic-resistant Streptococcus pneumoniae, N. Engl. J. Med. 1994; 331:377-382. [34] Friedland IR, Klugman KP. Recurrent penicillin-resistant pneumococcal meningitis after chloramphenicol therapy. Pediatr Infect Dis J 1991;10:705 – 7. [35] Jacobs MR. Treatment and diagnosis of infections caused by drug resistant-Streptococcus pneumoniae. Clin Infect Dis 1992;15:119 – 27.
Original article
High prevalence of carriage of antibiotic-resistant Streptococcus pneumoniae in children in Kampala Uganda M.L. Joloba a,b,c,*, S. Bajaksouzian b, E. Palavecino b, C. Whalen c, M.R. Jacobs b,d a Department of Microbiology, Makerere Uni6ersity Medical School, Kampala, Uganda Department of Pathology, School of Medicine, Case Western Reser6e Uni6ersity, 10900 Euclid A6enue, Cle6eland, OH, 44106 -4995, USA c Department of Epidemiology and Biostatistics, School of Medicine, Case Western Reser6e Uni6ersity, 10900 Euclid A6enue, Cle6eland, OH, 44106 -4995, USA d Department of Pathology, Uni6ersity Hospitals of Cle6eland, Cle6eland, OH, USA b
Received 20 June 2000; accepted 22 September 2000
Abstract There are few data on antibiotic-resistant Streptococcus pneumoniae in Uganda. A total of 191 healthy children in Kampala, Uganda were screened for nasopharyngeal carriage of S. pneumoniae; 118 (62%) of the children were carriers. Antimicrobial susceptibility and serotype of 115 strains was determined. Ninety-six (83.5%) of the isolates were of intermediate resistance to penicillin and 19 (16.5%) were susceptible. All strains were susceptible to cefotaxime. The rates of resistance to other drugs were trimethoprim–sulphamethoxazole (83.5%), tetracycline (28.7%) and chloramphenicol (10.4%). All strains were susceptible to rifampicin, erythromycin and clindamycin. Serogroups 6, 9, 14, 19 and 23 accounted for 80% of the isolates. These data show that the rate of carriage of antibiotic-resistant pneumococci by children is high in Kampala, Uganda. © 2001 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. Keywords: Streptococcus pneumoniae; Antibiotic resistance; Kampala, Uganda
1. Introduction Streptococcus pneumoniae remains a major cause of mortality and morbidity worldwide. It is the commonest cause of community-acquired bacterial respiratory tract infections [1–3]. It is also a major cause of meningitis and bacteraemia with incidence rates of 1.5 and 5 – 10 cases per 100 000 individuals per year respectively [4 – 6]. At least 1 million children die of pneumococcal disease every year [7]. The prevalence of antibiotic-resistant S. pneumoniae, documented in many parts of the world since the 1960s, is on the increase [2,8 –13]. Failures in therapy due to antibiotic resistant pneumococci have been reported in various places [14,15]. Since most antibiotic therapy in outpatients as well as initial * Corresponding author. Tel.: + 1-216-3684192; fax: +1-2163683970. E-mail address: [email protected] (M.L. Joloba).
treatment of inpatients is empiric, knowledge of local susceptibility patterns is important to guide such therapy. Uganda, with virtually uncontrolled antibiotic prescription, ready access to antibiotics without prescription, limited laboratory facilities and high prevalence of HIV infection, may well have significant morbidity and mortality due to antibiotic-resistant pneumococci. The characteristics, serotypes and susceptibility patterns of nasopharyngeal isolates have been shown to be similar to those of invasive strains [16 –19], findings consistent with the fact that pneumococcal disease follows nasopharyngeal carriage. Nasopharyngeal carriage of S. pneumoniae without disease occurs in all age groups. However, carriage rates vary with age, being greater in children in whom more than 95% have had an episode of nasopharyngeal colonization by the age of 2 years [2]. A colonizing strain is carried for an average of 6 weeks, but carriage can persist for up to 12
0924-8579/01/$20 © 2001 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. PII: S 0 9 2 4 - 8 5 7 9 ( 0 0 ) 0 0 3 4 5 - 9
M.L. Joloba et al. / International Journal of Antimicrobial Agents 17 (2001) 395–400
396
months [16]. As the prevalence of antibiotic-resistant pneumococci was not known in Uganda, a survey was undertaken to determine the nasopharyngeal carriage rate of S. pneumoniae in children to determine the prevalence of carriage, distribution of serotypes and antimicrobial susceptibility profiles of strains.
placed in the first streak area and plates were incubated overnight in candle extinction jars at 37°C. Pneumococci were indentified by a zone of inhibition around the optochin disk and the presence of smooth, grey, a-haemolytic colonies with depressed centres. Typical colonies were then subcultured on fresh TSA blood plates and incubated as described previously for purification.
2. Materials and methods
2.3. Storage and transportation of strains
2.1. Subjects The study was conducted in the paediatric outpatient department of Mulago Hospital, a national referral and teaching center in Kampala, the capital city of Uganda covering an area of 169 km2, with a population of 774 241 with 17.4% below 4 years of age [20]. All healthy children 3 years and below attending the outpatient department for routine checkups or immunizations were eligible. Written informed consent and ethical approval were obtained. A standardized questionnaire seeking data on demographics, recent antibiotic treatment and prophylaxis, and hospitalization was used. During the months of June and July of 1995, a total of 191 children were entered into the study.
2.2. Specimen collection and processing Pre-packed sterile calcium alginate swabs on flexible aluminum shafts (Calgiswab type 3, Fisher Scientific Co., Pittsburgh, PA) were used to collect nasopharyngeal secretions pernasally from the children. Each swab was immediately inoculated onto trypticase soy agar (TSA) plates supplemented with 5% whole sheep blood (Becton Dickson Microbiology Systems, Cockeysville, MD). The plates were kept at room temperature and transported to the laboratories of Makerere Medical School on the same day the swab was collected. The plates were then streaked, an optochin disk (Becton Dickson Microbiology Systems, Cockeysville, MD) was
Strains were stored by suspending fresh overnight growth in serum-glycerol freezing media and immediately placing in a − 70°C freezer. From the freezer, vials were immediately transferred into liquid nitrogen carriers and transported by airfreight to the laboratories of University Hospitals of Cleveland in accordance with local and international shipment regulations. From the carriers, the vials were kept frozen at −70°C until further analysis.
2.4. Identification, serotyping and antimicrobial susceptibility testing Optochin susceptibility and bile solubility were used for identification of the isolates as S. pneumoniae at University Hospitals of Cleveland [21]. The capsular reaction test using antisera to serogroups 6, 9, 14, 19 and 23 (Statens Seruminstitut, Copenhagen, Denmark) [21], was used to determine the serogroup. Other serotypes were not determined. Antimicrobial susceptibility was determined by disk diffusion as follows [21–23]. Colonies from an 18–24 h old subculture were suspended in saline and adjusted to a density equal to a 0.5 McFarland standard. Plates of Mueller–Hinton agar supplemented with 5% sheep blood (Becton Dickson Microbiology Systems, Cockeysville, MD) were inoculated by using cotton-tipped swabs (Hardwood Products Company, Guilford, ME), and antibiotic disks (Becton Dickson Microbiology Sys-
Table 1 Breakpoints for interpretation of disk diffusion testing Agent
Penicillin Erythromycin Chloramphenicol Tetracycline Trimethoprim–sulphamethoxazole Rifampicin Clindamycin a b
MIC determination required. NA, not applicable.
Zone diameter interpretation (mm) Disk content
Susceptible
Intermediate
Resistant
1 mg oxacillin 15 mg 30 mg 30 mg 25 mg 5 mg 2 mg
]20 ]21 ]21 ]23 ]19 ]19 ]19
519a 16–20 NAb 19–22 16–18 17–18 16–18
519a 515 520 518 515 516 515
M.L. Joloba et al. / International Journal of Antimicrobial Agents 17 (2001) 395–400 Table 2 Breakpoints used for interpretation of MIC testing Agent
Penicillin G Cefotaxime
397
3.2. Antibiotic use in the pre6ious 3 months
MIC interpretation breakpoints (mg/l) Susceptible
Intermediate
Resistant
50.06 50.5
0.1–1.0 1.0
]2.0 ]2.0
tems, Cockeysville, MD), were dispensed onto each plate and interpreted as shown in Table 1. In addition, minimum inhibitory concentrations (MICs) of penicillin and cefotaxime were determined by agar dilution as follows [24]. Mueller– Hinton agar plates supplemented with 5% sheep blood and containing doubling dilutions of penicillin (Squibb Institute for Medical Research, Princeton, NJ) or cefotaxime (Hoechst-Roussel Pharmaceuticals Inc., Somerville, NJ) were prepared. From a 0.5 McFarland turbidity saline suspension of fresh overnight growth, a 1:10 dilution was made in normal saline. With a Steers replicator, the diluted suspensions were inoculated onto Mueller– Hinton sheep blood agar plates containing antibiotics. Plates were incubated for 24 h at 35°C in 5–10% CO2. Zone diameters and MICs were read and interpreted using National Committee for Clinical Laboratory Standards criteria. The breakpoints are shown in Table 1 and Table 2 [22– 24].
3. Results A total of 191 children under 3 years of age were sampled. They were from 138 different suburbs of Kampala and surrounding villages. The ages of the subjects ranged from one week to 36 months with a mean of 11 months and standard deviation of 10.3 months. One hundred and forty-two (74.3%) of the children were below 12 months, 22 (11.5%) were 12–23 months and 27 (14.2%) 24– 35 months of age. There were 106 (55.5%) boys and 85 (45.5%) girls. There was no significant statistical difference in the age distribution by gender (P= 0.71).
3.1. Pre6alence of nasopharyngeal pneumococcal carriage Pneumococci were recovered from 118 (62%) of the 191 subjects. Carriage was present in 66 (62.3%) of the 106 boys and 52 (61.2%) of the 85 girls sampled. There was no statistically significant difference in carriage between boys and girls [OR=1.05; 95% CI 0.44– 1.89, P = 0.88]. Ninety-nine carriers (83%) were below 2 years of age, but there was no statistically significant difference in carriage between children above and below two years [OR= 0.64, 95% CI 0.24– 1.66, P = 0.32].
Seventy-eight (40.8%) of the children sampled had received at least one antibiotic in the preceding 3 months. Penicillins (ampicillin orally 16.8%, penicillin G parenterally 8.9%, cloxacillin orally 1.6%) and trimethoprim-sulphamethoxazole orally (25.7%) were the most frequently administered antimicrobial agents. Four agents, chloramphenicol, erythromycin, tetracycline and rifampin, had rates of use of 0.5–1.1% each. None of the children had received clindamycin or cefotaxime. Forty-six (39%) of the 118 pneumococcal carriers and 32 (43.8%) of the 73 non-carriers had received at least one antibiotic. This difference was not statistically significant [OR=0.82, 95% CI 0.43–1.54, P= 0.51.]
3.3. Hospitalization and attendance of day care centres History of hospitalization in the most recent 6 weeks was present in only three (1.6%) of the children. None of the children had attended day care centres. Therefore, no meaningful statistical computations could be done for these risk factors.
3.4. Antimicrobial susceptibility Of the 118 strains isolated, 115 were available for susceptibility testing. High rates of resistance to penicillin (83.5%), trimethoprim–sulphamethoxazole (83.5%) and tetracycline (28.7%) were found (Table 3). The frequency of resistance to chloramphenicol was lower (10.4%), whereas all strains were susceptible to cefotaxime, erythromycin, rifampicin and clindamycin. Resistance to penicillin was of intermediate level, with MICs ranging from 0.125 to 1.0 mg/l, and MIC50 and Table 3 Antimicrobial susceptibility of Streptococcus pneumoniae to penicillin and cefotaxime by MIC determination and other agents by disk diffusion testing (N= 115) Agent
Susceptible
Intermediate
Resistant
Penicillina Cefotaxime Trimethoprim –sulphamethoxazole Tetracycline Chloramphenicol Erythromycin Clindamycin Rifampicin
19 (16.5)b 115 (100) 17 (14.8)
96 (83.5) 0 2 (1.7)
0 0 96 (83.5)
78 (67.8) 103 (89.6) 115 (100) 115 (100) 115 (100)
4 (3.5) 0 0 0 0
33 (28.7) 12 (10.4) 0 0 0
a
Seven strains with oxacillin zones of 11–19 were susceptible (MICs 0.03–0.06 mg/l). b No. (percentage) of strains.
M.L. Joloba et al. / International Journal of Antimicrobial Agents 17 (2001) 395–400
398
Table 4 Susceptibility patterns and seroroups of Streptococcus pneumoniae. Strains with intermediate resistance to tetracycline and trimethoprim– sulphamethoxazole are included with resistant strains in this analysis Resistance patterna
Serogroup 6
None P S T PS PT TS PTS PCS PCTS TOTALS
1c 4
9
Total 14
2 3
19
23
1 1
2
14
4
4
20 1
6 1
4
2
4
5
3 26
11
8
3 1 3 30
2 16
cillin, chloramphenicol, tetracycline and trimethoprimsulphamethoxazole). Of the penicillin resistant strains, 90% were resistant to at least one of the non-b-lactam drugs tested, with trimethoprim-sulphamethoxazole accounting for the majority of these.
NDb 3 2 2 2 8 2 2 1 2 24
4 11 6 2 56 2 2 20 2 10 115
a P, penicillin G; S, trimethoprim–sulphamethoxazole; T, tetracycline; C, chloramphenicol. b ND, not determined. c Number of strains with indicated serogroup and resistance pattern.
MIC90 values of 0.25 and 0.50 mg/l respectively. Only 19 (16.5%) strains were susceptible (MICs 0.008– 0.06 mg/l) to penicillin, with MIC50 and MIC90 values of 0.3 and 0.06 mg/l, respectively. The overall MIC50 and MIC90 values were 0.250 and 0.50 mg/l (range 0.008– 1.0 mg/l). Although all pneumococcal isolates were susceptible to cefotaxime (MICsB0.50 mg/l) with an MIC range of 0.015 to 0.25 mg/l, and MIC50 and MIC90 values of 0.125 and 0.25 mg/l, respectively, the penicillin intermediate strains were less susceptible to cefotaxime (MICs 0.12– 0.25 mg/l) than the penicillin susceptible strains (MICs 0.015– 0.06 mg/l).
3.5. Serotype and resistance pattern distribution Ninety-one (79.1%) of the isolates belonged to one of the five serogroups tested, with 30 (26.1%) belonging to serogroup 19, 26 (22.6%) to serogroup 6, 16 (13.9%) to serogroup 23, 11 (9.6%) to serogroup 9 and 8 (7.0%) to serogroup 14. Twenty-four (20.9%) isolates were not reactive to types 6, 9, 14, 19 or 23 anticapsular sera and their serogroups were not further determined. All strains of serogroups 9, 14, 19, 23 and all but one of serogroup 6 strains were resistant to at least one of the antibiotics tested. One hundred and eleven (96.5%) of the 115 strains were resistant to at least one agent and 92 (80%) to two or more agents. Serogroup and susceptibility pattern analyses are shown in Table 4. The most common resistance pattern was resistance to penicillin and trimethoprim–sulphamethoxazole, in 56 strains (48.7%), whereas 20 strains were also resistant to tetracycline. Ten strains were resistant to four agents (peni-
4. Discussion Pneumococcal disease usually follows nasopharyngeal colonization [16,18]. As such, the characteristics of the pathogenic isolates have been found to be similar to the colonizing strains [18,19]. In both developed and developing countries pneumococcal colonization in young children has been detected in 30–60% of individuals [2,25,26]. This study has shown that the nasopharyngeal carriage rate of pneumococci in children under 3 years of age in Kampala was 61.8%. Similar studies conducted in South Africa [18] and Pakistan [19] showed colonization rates of 53% (53 of 100) and 61.9% (631 of 1019) respectively. In this study, gender, age and previous antibiotic use were not associated with significant differences in carriage. Most strains were resistant to one or more antibiotics, but lack of prior use of antibiotics did not predispose to colonization with susceptible strains. This is presumably due to the fact that most strains colonizing children (96.5%) are resistant, with few susceptible strains present in this age group. High rates of resistance to agents prescribed to the study population were found in the S. pneumoniae strains isolated, whereas no resistance was found to agents rarely (B 1.1%) prescribed (erythromycin, clindamycin and rifampicin). However, resistance to tetracycline (28.7%) was found despite usage of 0.5%, whereas resistance to chloramphenicol (10.4%) was found with 1.1% usage. The reasons for these differences are unknown, but may be related to use of tetracycline and chloramphenicol in other age groups, or to strains acquiring transposons coding for multiple resistance factors [2]. One hundred and eleven (96.5%) of the 115 isolates were resistant to at least one of the antibiotics used. Trimethoprim–sulphamethoxazole (cotrimoxazole) and the penicillins are the cheapest drugs available in Uganda, and as a result are the most frequently prescribed. The widespread use of these inexpensive agents may have created selective pressure for such strains and account for the high prevalence of resistance to these agents. Once resistant strains develop, they are readily transmitted to new carriers and maintained in the population, especially in children. All strains tested were susceptible to erythromycin and clindamycin. The same findings were reported in Kenya [26,27] and Rwanda [28], both neighbouring countries, although some of these studies included clin-
M.L. Joloba et al. / International Journal of Antimicrobial Agents 17 (2001) 395–400
ical isolates from all age groups. There was no resistance to cefotaxime or rifampicin, agents that are rarely prescribed. However, 80% of the strains showed reduced susceptibility to cefotaxime (MICs 0.125–0.25 mg/l), a phenomenon explained by cross-resistance between b-lactams. As previously found worldwide, serogroups 6, 9, 14, 19 and 23, the most common serogroups in children [2,3,16,29–31], accounted for 80% of isolates. They are also the types most often associated with resistance. Although the 23-valent vaccine contains these serogroups, it is ineffective in children below two years of age [7,16]. The data from this study show that the rates of pneumococcal carriage and antimicrobial resistance are high in Kampala, Uganda, with strains being predominantly resistant to penicillin and trimethoprim–sulphamethoxazole. These high rates of resistance call for documentation of resistance rates in pneumococcal disease, evaluation of outcomes with agents in current use and changes in practice. Treatment of pneumococcal infections should depend on the site of infection and underlying conditions [32,33]. Trimethoprim–sulphamethoxazole, with a resistance rate of 83.5%, is not suitable as an empiric agent for infections where pneumococci are major pathogens. The high rate of intermediate penicillin resistance (83.5%), raises concerns about the usefulness of penicillin G in the empiric treatment of pneumococcal meningitis [32]. Chloramphenicol, with reduced bactericidal effect in penicillin-resistant strains [32– 35], is also not indicated as a single agent or in combination therapy of childhood pneumococcal meningitis before susceptibility results are available. Cefotaxime or ceftriaxone are suitable agents for empiric management of pneumococcal meningitis in Uganda [32,33]. In pneumococcal pneumonia and bacteraemia parenterally administered penicillin and other b-lactams can still be used as levels of the drugs many times higher than the MIC can be achieved in these sites [32]. However, higher doses may be required for patients with underlying conditions such as overt HIV infections. In otitis media and sinusitis, oral b-lactams with good bioavailability such as amoxycillin and cefuroxime axetil, are active against pneumococcal strains with intermediate levels of penicillin-resistance and are therefore useful agents for management of these infections [32,33]. Erythromycin is also a suitable agent for management of outpatient pneumococcal infections in this area as no erythromycin resistance was detected. However, these are considerably more expensive than agents in current use in Uganda, and such recommendations may be difficult to implement. The high rates of antimicrobial resistance in S. pneumoniae found in this study are presumably a conse-
399
quence of antimicrobial abuse. Since this phenomenon is not limited to S. pneumoniae, there is need to re-evaluate indications for prophylactic and therapeutic use of antibiotics as well as monitoring the availability of prescribed and self-administered antibiotics. Continued surveillance of antimicrobial susceptibility profiles of S. pneumoniae in all age groups and in other parts of the country is needed in Uganda to guide empiric antimicrobial therapy.
Acknowledgements The study was supported in part by a grant from the AIDS International Training and Research Program of the Fogarty International Center (grant no. TW00011), and a grant from Merck & Co., Cincinnati, OH. We thank Dr L. Marum, Dr C. Ndugwa, Dr P.M. Mudido, Dr Baingana Baingi, Dr I. Kalyesubula, Dr J. Johnson, Ms K. Edmonds, Dr P. Waibale, Mr A. Kaddu and Mr S. Kabengera for assistance rendered. We also thank Dr G. Bukenya and Dr J. Ellner for their support and encouragement for this project.
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