Effect of presumptive co-trimoxazole prophylaxis on pneumococcal colonization rates, seroepidemiology and antibiotic resistance in Zambian infants: a longitudinal cohort study

929Bulletin of the World Health Organization | December 2008, 86 (12) Effect of presumptive co-trimoxazole prophylaxis on pneumococcal colonization rates, seroepidemiology and antibiotic resistance in Zambian infants: a longitudinal cohort study CJ Gill,a V Mwanakasale,b MP Fox,a R Chilengi,c M Tembo,b M Nsofwa,b V Chalwe,b L Mwananyanda,d D Mukwamataba,b B Malilwe,b D Champo,b WB Macleod,a DM Thea a & DH Hamer a Objective To ascertain the microbiological consequences of WHO’s recommendation for presumptive co-trimoxazole prophylaxis for infants with perinatal HIV exposure. Methods Using a longitudinal cohort design, we followed HIV-exposed and HIV-unexposed infants trimonthly for up to 18 months per infant. HIV-exposed infants received daily co-trimoxazole prophylaxis from 6 weeks to ³ 12 months of age. Using Streptococcus pneumoniae as our sentinel pathogen, we measured how co-trimoxazole altered nasopharyngeal colonization, pneumococcal resistance to antibiotics and serotype distribution as a function of co-trimoxazole exposure. Findings From 260 infants followed for 3096 patient-months, we detected pneumococci in 360/1394 (25.8%) samples. HIV-exposed infants were colonized more frequently than HIV-unexposed infants (risk ratio, RR: 1.4; 95% confidence interval, CI: 1.0–1.9, P = 0.04). Co-trimoxazole prophylaxis reduced colonization by ca 7% but increased the risk of colonization with co-trimoxazole- resistant pneumococci within 6 weeks of starting prophylaxis (RR: 3.2; 95% CI: 1.3–7.8, P = 0.04). Prophylaxis with co-trimoxazole led to a small but statistically significant increase of nasopharyngeal colonization with pneumococci not susceptible to clindamycin (RR: 1.6; 95% CI: 1.0–2.6, P = 0.04) but did not increase the risk of non-susceptibility to penicillin (RR: 1.1; 95% CI: 0.7–1.7), erythromycin (RR: 1.0; 95% CI: 0.6–1.7), tetracycline (RR: 0.9; 95% CI: 0.6–1.5) or chloramphenicol (RR: 0.8; 95% CI: 0.3–2.3). Co-trimoxazole prophylaxis did not cause the prevailing pneumococcal serotypes to differ from those that are targeted by the 7-valent conjugate pneumococcal vaccine (RR: 1.0; 95% CI: 0.7–1.6). Conclusion Co-trimoxazole prophylaxis modestly suppresses pneumococcal colonization but accelerates infant acquisition of co-trimoxazole- and clindamycin-resistant pneumococci. Co-trimoxazole prophylaxis appears unlikely to compromise the future efficacy of conjugate vaccines. Bulletin of the World Health Organization 2008;86:929–938. Une traduction en français de ce résumé figure à la fin de l’article. Al final del artículo se facilita una traducción al español. .ةلاقلما هذهل لماكلا صنلا ةياهن في ةصلاخلا هذهل ةيبرعلا ةمجترلا a Department of International Health, Boston University School of Public Health, 710 Albany Street, Boston, MA, United States of America. b Tropical Diseases Research Centre (TDRC), Ndola, Zambia. c Africa Malaria Network Trust (AMANET), Dar es Salaam, United Republic of Tanzania. d Zambia-Emory HIV Research Project, Ndola, Zambia. Correspondence to Christopher J Gill (e-mail: cgill@bu.edu). doi:10.2471/BLT.07.049668 (Submitted: 16 November 2007 – Revised version received: 18 March 2008 – Accepted: 10 April 2008 – Published online: 5 August 2008 ) Introduction In 2000, the WHO and Joint United Nations Programme on HIV/AIDS (UNAIDS) secretariats recommended that infants in resource-poor settings with perinatal HIV exposure from an infected mother should receive co- trimoxazole (trimethoprim-sulfame- thoxazole) prophylaxis presumptively.1,2 Co-trimoxazole prophylaxis would con- tinue until two conditions are satisfied: (i) the child has been fully weaned and is no longer being exposed to maternal HIV; and (ii) the child can be proven uninfected with HIV. In resource-poor settings, presumptive prophylaxis would be necessary for at least a year in most cases. While WHO’s policy is intended to protect the subset of HIV-exposed infants who are (or become) HIV- infected in their first year, the majority of infants so targeted will escape HIV infection.3 In an earlier commentary on this policy,4 we drew attention to multiple potential adverse consequences of pre- sumptive co-trimoxazole prophylaxis, with a particular focus on antimicrobial resistance. To better understand how co-trimoxazole prophylaxis affects mi- crobial colonization and resistance rates, we implemented the co-Trimoxazole in Zambian Infants (TZI) project, a longitudinal cohort study designed to measure selected microbiological consequences of WHO’s policy. We selected Streptococcus pneumoniae as our sentinel pathogen for several reasons. First, the pneumococcus is a leading cause of morbidity/mortality among infants worldwide. Second, drug- resistant pneumococci are increasingly common and of particular public health concern. Third, multiple aspects of pneumococcal epidemiology can be assessed conveniently via nasopharyn- geal colonization surveys. Lastly, the 930 Bulletin of the World Health Organization | December 2008, 86 (12) Research Co-trimoxazole in Zambian infants study CJ Gill et al. effectiveness of antibiotics and vaccines, our two main tools for combating pneumococcal disease, could both be degraded by widespread presumptive co-trimoxazole prophylaxis. Exposure to sulfonamides is presumably the domi- nant force behind the emergence of co-trimoxazole-resistant pneumococci and might induce cross-resistance to other antibiotics.5–8 Moreover, because antibiotic resistance is often linked with specific serotypes,9–12 exposure to co-trimoxazole might shift the prevail- ing pneumococcal serotypes away from those represented by the 7-valent conju- gate pneumococcal vaccine. Hence, this analysis addressed the following questions: Does co-trimoxazole prophylaxis al-• ter nasopharyngeal pneumococcal colonization rates? Does co-trimoxazole prophylaxis in-• duce co-trimoxazole-resistant pneu- mococci and, if so, how quickly? Does co-trimoxazole prophylaxis in-• duce cross-resistance to other antibi- otic classes? Does co-trimoxazole exposure alter • the distribution of pneumococcal se- rotypes away from 7-valent vaccine strains? Methods Study overview The TZI project was a two-arm longi- tudinal cohort study whose principal objective was to measure the microbio- logical consequences of implementing WHO guidelines2 for presumptive co-trimoxazole prophylaxis on pneu- mococcal colonization, drug resistance and seroepidemiology. The project was conducted at three antenatal clinics in Ndola, Zambia. Ndola is Zambia’s third- largest city, with most of its inhabitants living below the poverty level in periur- ban compounds. All mother/infant pairs were enrolled at the study clinics. Eligi- bility criteria were: (i) residence within the catchment zone of our study clinics; (ii) signed maternal informed consent; and (iii) maternal willingness to un- dergo HIV testing. “Case” infants were those born to HIV-positive women and thus requiring prophylaxis per WHO guidelines. “Comparison” infants were infants born to HIV-negative women, unexposed to HIV and hence not requir- ing prophylaxis per WHO guidelines. Comparison infants were recruited con- temporaneously 1:1 with case infants, and were age- and clinic-matched. Our sample size of 260 mother/infant pairs was powered to detect a 30% reduction in colonization and a twofold increase in co-trimoxazole resistance as a func- tion of co-trimoxazole exposure, while accommodating up to 30% attrition. As per WHO guidelines, case in- fants received daily prophylactic oral co-trimoxazole from 6 weeks until ³ 12 months of age, dosed at 10 mg/kg daily for trimethoprim and 50 mg/kg daily for sulfamethoxazole. Case infants still on study at 12 months were tested for HIV infection. If positive at 12 months and again at 15 months, they were considered true positives and offered co-trimoxazole prophylaxis indefinitely. Case infants who tested negative at 12 or 15 months and had fully weaned were considered HIV-negative and co- trimoxazole was stopped. All infants were enrolled at 6 weeks of age and followed according to a seven-visit, well-child care schedule at prespecified intervals (Fig. 1). At visit 1, no infants had started co-trimoxazole prophylaxis. From visits 2 to 5, all case infants received co-trimoxazole. Fig. 1. Design of longitudinal cohort study of co-trimoxazole prophylaxis among Zambian infants Birth 6 weeks AgeVisit number Period 1 – Pre-CTM Group: Case Comparison 1 3 months2 6 months3 9 months4 12 months5 15 months6 18 months7 Receiving CTM prophylaxis Not receiving CTM prophylaxis Period 2 – On CTM Period 3 – Post-CTM CTM, co-trimoxazole; TZI, co-trimoxazole in Zambian Infants. After visit 5, most case infants tested negative for HIV and stopped co- trimoxazole. This schedule creates three distinct periods for comparison (Fig. 1): pre-co-trimoxazole (period 1), on co-trimoxazole (period 2), and post- co-trimoxazole (period 3). The TZI project was jointly ap- proved by the ethical review boards at Boston University and the Tropical Diseases Research Centre (TDRC) in Ndola. All mothers provided written informed consent. Laboratory procedures Mothers attending antenatal clinics underwent HIV voluntary counselling and testing according to local standards. HIV screening used the Determine® 1 + 2 test (Abbott Laboratories, Abbott Park, IL, United States of America) and confirmed using the Capillus® test (Cambridge Biotech Ltd, Galway, Ireland),13 with the Bioline® test (Bionor AS, Skien, Norway) to resolve indeter- minate/discrepant results. The sensitiv- ity/specificity of this protocol exceeds 99%. This same protocol was used for case infants at 12 and 15 months.14 Research Co-trimoxazole in Zambian infants study 931Bulletin of the World Health Organization | December 2008, 86 (12) CJ Gill et al. HIV-positive mothers and their infants received peripartum nevirapine prophy- laxis according to the HIVNET 012 protocol.3 Antiretroviral drugs were virtually unavailable in Ndola during TZI. At every visit, we screened for S. pneumoniae colonization using pos- terior nasopharyngeal samples obtained with sterile calcium-alginate-tipped aluminium swabs advanced into both nostrils until meeting resistance and then rotated 180°.15 To maximize yields, swabs were plated immedi- ately on to room temperature soy- trypticase agar plates with 5% sheep’s blood/5% gentamicin (gent/BAP) and streaked later for optimal colony separation at the TDRC microbiology laboratory.15–17 Gentamicin increases the yield for S. pneumoniae by approxi- mately 40%.16,17 Screening plates were incubated at 37 °C under 5% CO2 at- mosphere for 48 hours. Colonies were presumptively identified as S. pneumo- niae by colony morphology (small grey “draftsman” mucoid α-haemolytic colonies), and typical diplococcoid morphology on Gram stain.18 For con- firmation, colonies from the screening gent/BAP plates were subcultured onto gentamicin-free BAP, and defined as S. pneumoniae on the basis of ³ 14 mm optochin (ethylhydrocupreine, Difco, Detroit,. MI, USA) inhibition zones, or bile-solubility for isolates with < 14 mm optochin inhibition.19 Drug resistance was determined using the elipsometer method (Etest®, AB Biodisk, Solna, Sweden).20,21 We measured the minimum inhibitory concentration (MIC) for each isolate against co-trimoxazole, penicillin, tetracycline, erythromycin, chloram- phenicol and clindamycin. Subcultures of pure pneumococcal isolates were used to create a bacterial lawn on 150 mm Mueller–Hinton broth agar plates, each plate accommodating all Etests® simul- taneously. Diameters of inhibition were measured at 24 hours of incubation. The point on the Etest® strip where inhibi- tion was first noted indicated the MIC for that isolate and was read directly off the calibration guide printed on each strip. Classification of inhibition zones for isolates into sensitive, intermediately and highly resistant pneumococci was per the National Committee for Clini- cal Laboratory Standards guidelines.18 Subcultured isolates were charac- terized to the serogroup and serotype level using the Statens Serum Institute (Copenhagen, Denmark) latex slide agglutination system, with subsequent factor typing of the dominant sero- types. Statistical analyses Data were dual entered at TDRC and cleaned/reconciled at Boston University. We conducted univariate estimates of risk ratios (RRs) with 95% confidence intervals (CI), t-tests, and/or tests of pro- portions (χ2 or Fisher’s exact). Because MICs for antibiotic resistance operate on a logarithmic scale, we log trans- formed MICs before conducting t-tests on the means and back-transformed the result using the exponent of the difference in the log means. Note that this procedure yields the ratio of the two medians of the back-transformed MICs, not their means [exp(Ln mean1 - Ln mean2) = median1/median2].22 In univariate analyses, we as- sumed that case infants received co- trimoxazole during period 2 but not in periods 1 or 3 (adjusting for the handful who tested positive for HIV and remained on co-trimoxazole), and no comparison infants received co- trimoxazole. However, we also tested the effect on colonization and drug resistance of intercurrent short-term sulfonamide treatment among the comparisons, such as brief courses of co-trimoxazole for acute infections or malaria treatment with sulfadoxine- pyrimethamine, a pharmacologically similar drug to co-trimoxazole that has been linked to increased colonization with co-trimoxazole-resistant pneu- mococci.23 Lacking a priori knowledge about what exposure level would be sufficient to induce resistance, we categorized any level of sulfonamide exposure during the preceding interval as ‘exposed to co-trimoxazole’ under this expanded definition. Conversely, Table 1. Baseline demographic and clinical characteristics of infants and mothers in longitudinal cohort study of the effect of co- trimoxazole prophylaxis on pneumococcal colonization rates, seroepidemiology and antibiotic resistance in Zambian infants Characteristics Case group Comparison group P-value Mothers No. single, of total (%) 13/129 (10.1) 14/128 (10.9) NS No. married, of total (%) 110/129 (85.3) 114/128 (89.1) NS No. divorced, of total (%) 2/129 (1.6) 0/128 (0.0) NS No. widowed, of total (%) 4/129 (3.1) 0/128 (0.0) 0.04 Mean age in years (SD) 25.9 (6.5) 24.2 (6.2) NS Mean weight in kg (SD) 56.2 (8.4) 56.5 (7.9) NS No. with primary language Bemba, of total (%) 111/132 (84.1) 109/128 (85.1) NS No. using insecticide-treated bednet, of total (%) 52/132 (39.4) 55/130 (42.3) NS Infants Mean birth weight in g (SD) 3059 (440) 3194 (622) 0.05 No. treated with co-trimoxazole since birth, of total (%) 6/132 (4.5) 5/128 (3.9) NS No. with infant health issues since birth No. having chronic cough, of total (%) 1/131 (0.8) 0/128 (0.0) NS No. having recurrent fevers, of total (%) 1/131 (0.8) 1/128 (0.8) NS NS, non-significant; SD, standard deviation. 932 Bulletin of the World Health Organization | December 2008, 86 (12) Research Co-trimoxazole in Zambian infants study CJ Gill et al. Fig. 2. Nasopharyngeal pneumococcal colonization among Zambian infants given co-trimoxazole prophylaxis and comparison group in longitudinal cohort study, by age Pn eu m oc oc ca l c ol on iz at io n (% ) 0 45 Case group Comparison group Baseline Age of infant (months) 40 35 30 25 20 15 10 5 3 6 9 12 15 18 TZI, co-Trimoxazole in Zambian Infants. only those infants with no reported exposure to sulfonamides were catego- rized as unexposed to co-trimoxazole. Owing to the longitudinal structure of the data set with multiple observations on individuals, we recalculated the RRs using a log-linear model with robust standard errors to adjust for the cluster- ing effect of repeated measures to see if our results changed significantly.22 Our sample size was based on a projected 30% reduction in colonization between the two arms from a typical baseline of 60% colonized, while adjusting for predicted rates of attrition of up to 50% by study end. Results Between December 2003 and Sep- tember 2004, we enrolled 132 HIV- exposed (case) and 128 HIV-unexposed (comparison) infants (260 total). We followed the mother/infant pairs for a total of 3096 person-months with the last patient visit in November 2005. Baseline characteristics were similar between the two groups (Table 1); 25 case versus 44 comparison infants were lost to follow-up (P < 0.01); 1 case versus 6 comparison infants withdrew (P = 0.05); 10 case versus 0 comparison infants died by study end (P = 0.001). Of these 10 case infants, only 2 had reached the 12-month visit and had undergone HIV testing; neither was HIV-positive. Fifteen of 105 HIV- exposed infants still on study by 12 months were HIV seropositive (14.3%, standard error: 3.0%). One tested posi- tive at 12 months but negative at 15 months, presumably a false positive due to residual maternal antibody. Although 48 versus 42 unscheduled clinical ill- ness visits occurred in the case and comparison arms respectively, the rates did not differ statistically after adjust- ing for person-time at risk. Intercurrent sulfonamide use occurred frequently among the comparison infants. By study end, 56.6% of the comparison infants had been treated at least once with a sulfonamide, occurring at 137 of 648 visits (21.1%). From 1394 nasopharyngeal swabs, 360 tested positive for S. pneumo- niae (25.8%). Of the 360 isolates, 45 (12.5%) were from period 1; 231 (64.2%) from period 2; and 84 (23.3%) from period 3. The sample positivity rate did not vary by the time of year of sampling (data not shown). By con- trast, nasopharyngeal colonization was strongly associated with the infants’ age (P < 0.001), peaking in both groups at 12 months (Fig. 2). Co-trimoxazole exposure modestly suppressed colonization among case infants. Colonization rates were 6.8% higher for cases than comparisons dur- ing period 1 (20.9% versus 14.1%, RR: 1.5; 95% CI: 0.9–2.6) and 7.1% higher in period 3 (28.7% versus 21.6%, RR: 1.3; 95% CI: 0.9–1.9). After combining the non-exposure periods (periods 1 and 3), case infants were significantly more likely to be colonized than comparisons (25.3% versus 18.1%, RR: 1.4; 95% CI: 1.0–1.9, P = 0.04). Adjusting for intercurrent sulfonamide treatments among the controls did not change this risk substantially (RR: 1.5; 95% CI: 1.2–1.9). By contrast, during co- trimoxazole exposure (period 2), colo- nization rates were similar between the two groups (29.8% case versus 27.2% comparison infants, RR: 1.1; 95% CI: 0.9–1.4, P = 0.41; Fig. 2). Table 2. Case:comparison ratios of median antibiotic MICs in pneumococci isolated from nasopharyngeal cultures taken from Zambian infants in longitudinal cohort study of co-trimoxazole prophylaxis, by study period a Antibiotic Case:comparison ratio (95% CI) Period 1 Period 2 Period 3 Co-trimoxazole 0.90 1.42* 1.30 (0.30–2.70) (0.98–2.08) (0.58–2.85) Penicillin 1.72 1.07 1.09 (0.86–3.47) (0.76–1.51) (0.66–1.81) Chloramphenicol 0.95 0.94 1.11 (0.78–1.15) (0.80–1.11) (0.84–1.44) Tetracycline 0.83 0.97 1.31 (0.23–3.00) (0.52–1.83) (0.41–4.17) Erythromycin 1.17 0.92 1.01 (0.76–1.80) (0.72–1.19) (0.80–1.28) Clindamycin 1.20 1.04 0.93 (0.77–1.87) (0.84–1.28) (0.73–1.19) CI, confidence interval; MIC, minimum inhibitory concentration. *P = 0.07. a Period 1: pre-co-trimoxazole; period 2, on co-trimoxazole; period 3, post-co-trimoxazole. Research Co-trimoxazole in Zambian infants study 933Bulletin of the World Health Organization | December 2008, 86 (12) CJ Gill et al. Fig. 3. Proportion of nasopharyngeal pneumococcal isolates classified as sensitive, intermediately resistant or resistant to co-trimoxazole, over time, in longitudinal cohort study of co-trimoxazole prophylaxis among Zambian infants Pr op or tio n 0 1.0 Case group Comparison group Se ns iti ve Period 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 Visit 2 Period 2 Visit 3 Visit 4 Visit 5 Visit 6 Visit 7 Period 3 In te rm ed ia te Re si st an t Se ns iti ve In te rm ed ia te Re si st an t Se ns iti ve In te rm ed ia te Re si st an t Se ns iti ve In te rm ed ia te Re si st an t Se ns iti ve In te rm ed ia te Re si st an t Se ns iti ve In te rm ed ia te Re si st an t Se ns iti ve In te rm ed ia te Re si st an t Visit 1 The onset of co-trimoxazole pro- phylaxis led to a rapid increase in co-trimoxazole-resistant pneumococci from the case infants. During period 1, the mean Ln MICs for co-trimoxazole were comparable between the two groups (difference in means: −0.11; 95% CI: −1.2 to 1.0, P = 0.12). In period 2, the mean Ln MICs for co- trimoxazole increased in the case arm but stayed constant in the comparison arm (difference: +0.35; 95% CI: −0.03 to 0.73, P = 0.07). During period 3, the difference in the mean Ln MICs for co-trimoxazole declined among case infants, though it remained elevated relative to the comparison infants (difference: +0.26; 95% CI: −0.54 to 1.05, P = 0.52). During period 2, the median co-trimoxazole MICs were 1.4 times higher for case than comparison infants (P = 0.08) but were similar dur- ing periods 1 or 3. The median MICs did not differ significantly for any of the other antibiotics tested during the three periods (Table 2). When categorizing the MICs as sensitive (S), intermediately resistant (I) or resistant (R), the distributions were similar between cases and com- parisons during period 1 but diverged during period 2 (Table 3). This was largely explained by rapid increases in co-trimoxazole resistance between visits 1 and 2 (P = 0.04). At baseline (visit 1), the distribution was virtually identical between the two arms (14.8% Table 3. Degree of sensitivity to co-trimoxazole among pneumococci isolated from nasopharyngeal cultures taken from Zambian infants (n = 359)a given co- trimoxazole prophylaxis and comparison group, by study periodb Period/ interpretation of MICs Case group Comparison group Total Period 1 Sensitive 4 (14.8)c 3 (16.7) 7 (15.6) Intermediate 7 (25.9) 4 (22.2) 11 (24.4) Resistant 16 (59.3) 11 (61.1) 27 (60.0) Subtotal 27 18 45 (P = 0.96)d Period 2 Sensitive 6 (4.6) 9 (9.0) 15 (6.5) Intermediate 25 (19.1) 28 (28.0) 53 (22.9) Resistant 100 (76.3) 63 (63.0) 163 (70.6) Subtotal 131 100 231 (P = 0.08)d Period 3 Sensitive 7 (14.0) 7 (21.2) 14 (16.7) Intermediate 15 (30.0) 5 (15.2) 20 (24.1) Resistant 28 (56.0) 21 (63.6) 49 (59.3) Subtotal 50 33 83 (P = 0.27)d MIC, minimum inhibitory concentration. a Antibiotic sensitivities were not measured for one isolate. b Period 1: pre-co-trimoxazole; period 2, on co-trimoxazole; period 3, post-co-trimoxazole. c Figures in parentheses are percentages. d χ² of proportions within each exposure period. S, 25.9% I, 59.3% R in cases, versus 16.7% S, 22.2% I, 61.1% R in com- parisons; P = 0.96). By visit 2, 6 weeks later, all isolates from case infants were intermediately or highly resistant to co-trimoxazole (case versus comparison infants, RR: 2.2; 95% CI: 1.6–2.9), whereas the comparisons showed only a modest shift towards more highly resistant pneumococci (0.0% S, 7.4% I, 93.6% R in cases, versus 25.0% S, 16.7% I, 76.5% R in comparisons; P < 0.01). Thereafter, resistance rates increased in both groups so that after visit 3, intermediately or highly resistant isolates occurred in similar proportions between the two groups (Fig. 3). When combining both I and R categories together as non-susceptible, the relationship between co-trimoxazole prophylaxis and colonization with co-trimoxazole-resistant pneumococci became even stronger (RR: 3.2; 95% CI: 1.3–7.8, P = 0.01). Overall, ap- proximately 10% of all co-trimoxazole non-susceptibility in this population was accounted for by co-trimoxazole prophylaxis (attributable risk: 12%; 95% CI: 6– 18). When also consid- ering intercurrent non-prophylaxis exposure to sulfonamides among the controls, the risk of colonization with a co-trimoxazole non-susceptible pneu- mococcus increased further (RR: 4.4; 95% CI: 1.9–10.4). Repeating these analyses for the other antibiotics, co-trimoxazole pro- phylaxis led to a small but statistically significant increase of colonization with clindamycin non-susceptible pneu- mococci (RR: 1.6; 95% CI: 1.0–2.6, P = 0.04). Co-trimoxazole use did not increase the risk of non-susceptibility to 934 Bulletin of the World Health Organization | December 2008, 86 (12) Research Co-trimoxazole in Zambian infants study CJ Gill et al. penicillin (RR: 1.1; 95% CI: 0.7–1.7), erythromycin (RR: 1.0; 95% CI: 0.6–1.7), tetracycline (RR: 0.9; 95% CI: 0.6–1.5) or chloramphenicol (RR: 0.8; 95% CI: 0.3–2.3). In each of these analyses, the adjusted values after controlling for repeated measures and for baseline demographic variables were virtually identical to the results presented above (data not shown). We had serotype data for 354/360 samples (98%), of which 44% were covered by the 7-valent conjugate vac- cine. The probability that a given iso- late would be a 7-valent vaccine strain was not altered by whether the infant was exposed or not to co-trimoxazole (RR: 1.0; 95% CI: 0.7–1.6). However, 7-valent vaccine strain isolates were more likely to be non-susceptible to co-trimoxazole than non-vaccine iso- lates (RR: 2.2; 95% CI: 1.0–4.8). The distribution of serotypes between the two groups across the three exposure periods is summarized in Fig. 4, Fig. 5 and Fig. 6. The five most common se- rotypes were: 19f (16.0%), 6b (9.9%), 23f (7.5%), 15 (7.0%) and 14 (6.4%), with 5.9% untypable. The most strik- ing differences were a predominance of serotype 6 among the case infants (6b = 5, 6a = 3, 6 unfactorable = 3) during period 1 (Fig. 4), and an appar- ent loss of serotype diversity over time (Fig. 6): serotypes 2, 3, 5, 8, 12, 17, 18 and 33 were all found in period 1 and/ or period 2, but were absent from both groups in period 3. Discussion In this cohort of mostly HIV-negative Zambian infants followed from age 6 weeks through 18 months, pneu- mococcal colonization was common, peaked in incidence during the first year of life, and was dominated by serotypes represented by the conjugate 7-valent pneumococcal vaccine. These findings are all consistent with what is generally understood to be typical for coloniza- tion patterns of pneumococci in young infants24 and thus increase our level of confidence in interpreting our subse- quent findings. Co-trimoxazole prophylaxis had the following effects on pneumo- coccal colonization dynamics. First, co-trimoxazole exposure significantly reduced colonization rates, though not Fig. 4. Frequency of nasopharyngeal pneumococcal serotypes in period 1 (pre- cotrimoxazole) of longitudinal cohort study of co-trimoxazole prophylaxis among Zambian infants (n = 45) Pe rc en t 0 40 Case group Comparison group 0 Serotype 20 10 30 1 42 5 6 7 8 9 10 11 15 18 19 20 23 33 below the rate in the comparison in- fants. While the magnitude of this effect was modest (ca 7% on an absolute scale), it may still be relevant at a population level, insofar as nasopharyngeal coloni- zation is considered a precondition to invasive pneumococcal disease.25,26 Second, co-trimoxazole prophylaxis induced a rapid rise in intermediate and particularly high-level co-trimoxazole resistance. This was our most strik- ing finding. On the one hand, co- trimoxazole-resistant pneumococci were exceedingly common in this population, cases and comparisons alike. While co- trimoxazole resistance in the colonizing pneumococci occurred rapidly with the onset of prophylaxis, resistance to co- trimoxazole increased over time in the comparison infants also, albeit some- what later. Thus, in a setting of wide- spread sulfonamide exposure, one could argue that co-trimoxazole prophylaxis merely accelerated a process that was already under way. On the other hand, the induction of co-trimoxazole resis- tance clearly validates concerns about rising drug resistance from presumptive co-trimoxazole prophylaxis. Moreover, co-trimoxazole prophylaxis accounted for slightly over 10% of the total in- crease in co-trimoxazole resistance in this study, a surprisingly large fraction given the already high background prevalence of resistance and frequency of sulfonamide exposure. This neces- sarily prompts the question of whether co-trimoxazole prophylaxis might have a more profound effect in settings where co-trimoxazole resistance is uncommon and/or where other sulfonamide use is less widespread. Third, co-trimoxazole exposure marginally increased the odds of non- susceptibility to clindamycin but did not appear to induce cross-resistance to other classes of antibiotics. Given that co-trimoxazole and clindamycin come from unrelated drug classes, this is unlikely to be explained by pharma- cological cross-tolerance. An alternative explanation is co-selection of linked antibiotic-resistance genes. Supporting this hypothesis is the fact that mul- tiple antibiotic-resistance genes among pneumococci have been demonstrated in clusters grouped together on trans- posons and that such transposons are linked with specific strains or clones of S. pneumoniae.27–29 Our results contrast with those obtained by Abdel-Haq et al., who noted that co-trimoxazole pro- phylaxis selected for colonization with multidrug resistant pneumococci.30 However, such selection presumably depends on whether such strains are Research Co-trimoxazole in Zambian infants study 935Bulletin of the World Health Organization | December 2008, 86 (12) CJ Gill et al. Fig. 5. Frequency of nasopharyngeal pneumococcal serotypes in period 2 (on co-trimoxazole) of longitudinal cohort study of co-trimoxazole prophylaxis among Zambian infants (n = 228) Pe rc en t 0 40 Case group Comparison group 0 Serotype 20 10 30 1 43 6 7 9 10 11 12 14 15 17 18 19 23 33 already circulating in a given commu- nity, so we do not feel that our results necessarily conflict with their findings. For the same reason, we would be cau- tious in inferring whether the pattern of cross-induction of clindamycin resistance observed in the context of this study and population should be extrapolated externally. In our view, a more generalizable interpretation is that our data provide additional evi- dence that co-trimoxazole exposure has consequences that may extend beyond induction of co-trimoxazole resistance alone but may include unrelated drug classes in patterns that are difficult to predict a priori. Fourth, co-trimoxazole exposure was not associated with any notable shifts in the distribution of pneumo- coccal serotypes. Thus, our data pro- vide no support for concerns that co- trimoxazole prophylaxis might reduce the future effectiveness of pneumococ- cal vaccines by shifting the distribution of prevailing serotypes away from vac- cine strains. The loss of serotype diversity that occurred in both study arms during period 3 was curious. This is unlikely to be explained by sample size, since there was a far greater diversity of serotypes during period 1, despite there being roughly half as many isolates as in pe- riod 3. More plausibly, this narrowed spectrum reflects the maturation of the infants’ mucosal immunity, with infants becoming refractory to colonization by certain serotypes over time. In partial support of this theory, Simell et al. noted that nasopharyngeal pneumococ- cal colonization in infants was inversely correlated with development of strain- specific immunoglobulin A.31 Of concern, co-trimoxazole resis- tance was extremely common even at baseline, indicating a high community background prevalence of co-trimox- azole-resistant pneumococci – perhaps unsurprising given how frequent sul- fonamide exposure was in this popula- tion. Though most of our infants were HIV negative, colonization rates were significantly higher among case infants even at baseline. Because this occurred so early in life and because so few of the infants proved to be infected with HIV, HIV-induced immunodeficiency is un- likely to be the explanation. More plau- sibly, it might reflect other aspects of these infants’ home/environments that increase their pneumococcal exposure risk or susceptibility to colonization, or qualitative/quantitative differences in passive immunity from residual mater- nal antibody. The TZI study was conducted to generate empirical population-level evidence about the possible microbio- logical effects of presumptive co-tri- moxazole prophylaxis among HIV- exposed infants, but was not intended to study the efficacy of co-trimoxazole for reducing clinical disease. Clearly, we could have selected any number of pathogens to study, but we felt that the pneumococcus was a logical choice. Studying colonization dynamics in a relatively small cohort closely over an extended period of time reduced the risk that secular events (time of year, intercurrent outbreaks of pneumococ- cal disease in the community) would confound our results, and allowed us to evaluate the effect of starting and stop- ping co-trimoxazole prophylaxis, fur- ther strengthening inferences regarding cause and effect. However, an obvious limitation is that studying colonization dynamics is not equivalent to studying invasive pneumococcal disease. Another limitation is that the higher rates of study attrition in the comparison-arm infants could have introduced some degree of bias into our measurements. That said, given that pneumococcal colonization is generally asymptomatic, it seems unlikely that colonization and attrition would be confounded. Conclusion Co-trimoxazole prophylaxis modestly suppresses nasopharyngeal colonization with S. pneumoniae. Unfortunately, this comes at the price of accelerated acquisition of high-level resistance to co-trimoxazole and potentially other antibiotic classes as well (i.e. clin- damycin). The clinical significance of the co-trimoxazole resistance is un- certain, particularly in settings where co-trimoxazole-resistant pneumococci are highly prevalent and sulfonamide exposure extremely common, as in this African community. The lack of effect of co-trimoxazole exposure on the distribution of pneumococ- cal serotypes is reassuring from the perspective of the future effectiveness of conjugate pneumococcal vaccines. Taken in the context of recent reports of co-trimoxazole’s ancillary benefits in African populations,32,33 on balance, our findings support WHO’s current policy on presumptive co-trimoxazole pro- phylaxis. That said, our data reinforce the pressing need for a refined strategy 936 Bulletin of the World Health Organization | December 2008, 86 (12) Research Co-trimoxazole in Zambian infants study CJ Gill et al. Résumé Effet d’un traitement prophylactique présomptif par le cotrimoxazole sur les taux de colonisation pneumococcique, la séro-épidémiologie et la résistance aux antibiotiques chez les nourrissons zambiens : étude de cohorte longitudinale Objectif Evaluer les conséquences microbiologiques de la recommandation de l’OMS concernant le traitement présomptif par le cotrimoxazole des nourrissons exposés au VIH pendant la période périnatale. Méthodes Dans le cadre d’une étude longitudinale de cohorte, nous avons suivi trois fois par mois des nourrissons exposés et non exposés au VIH sur une durée allant jusqu’à 18 mois par enfant. Les nourrissons exposés au VIH ont reçu un traitement prophylactique quotidien par le cotrimoxazole de l’âge de 6 semaines à celui de 12 mois au moins. En utilisant Streptococcus pneumoniae comme agent pathogène sentinelle, nous avons mesuré l’effet obtenu sur la colonisation nasopharyngée, la résistance aux antibiotiques et la distribution par sérotypes des pneumocoques, en fonction de l’exposition au cotrimoxazole. Résultats Parmi 260 nourrissons suivis sur 3096 patients-mois, nous avons détecté des pneumocoques sur 360/1394 échantillons (25,8 %). Les nourrissons exposés au VIH étaient colonisés plus fréquemment que ceux non exposés à ce virus (rapport des risques, RR = 1,4 ; intervalle de confiance à 95 %, IC : 1,0-1,9, p = 0,04). La prophylaxie par le cotrimoxazole réduit la colonisation d’environ 7 %, mais accroît le risque de colonisation par des pneumocoques résistants à ce médicament dans les 6 semaines après le début du traitement (RR = 3,2 ; IC à 95 % = 1,3-7,8, p = 0,04). Ce traitement entraîne une augmentation faible, mais statistiquement significative de la colonisation nasopharyngée par des pneumocoques non sensibles à la clindamicyne (RR = 1,6 ; IC à 95 % = 1,0-2,6, p = 0,04), mais n’accroît pas le risque de non sensibilité à la pénicilline (RR = 1,1 ; IC à 95 % = 0,7-1,7), à l’érythromycine (RR = 1,0 , IC à 95 % = 0,6-1,7), à la tétracycline (RR = 0,9 ; IC à 95 % = 0,6-1,5) ou au chloramphénicol (RR = 0,8 ; IC à 95 % = 0,3-2,3). Le cotrimoxazole n’entraîne pas de divergence de la distribution sérotypique dominante des pneumocoques par rapport à celle visée par le vaccin antipneumococcique conjugé heptavalent (RR = 1,0 ; IC à 95 % = 0,7-1,6). Conclusion Le traitement prophylactique par le cotrimoxazole élimine modérément la colonisation pneumococcique et accélère l’acquisition par les nourrissons de pneumocoques résistants au cotrimoxazole et à la clindamycine. Il semble peu probable qu’il compromette l’efficacité future des vaccins antipneumococciques conjugués. Fig. 6. Frequency of nasopharyngeal pneumococcal serotypes in period 3 (post- cotrimoxazole) of longitudinal cohort study of co-trimoxazole prophylaxis among Zambian infants (n = 84) Pe rc en t 0 40 Case group Comparison group 0 Serotype 20 10 30 1 4 6 7 9 10 11 14 15 19 23 for early diagnosis of infant HIV infec- tion, both to minimize unnecessary drug exposure and to optimize the use of precious resources. ■ Acknowledgements We thank Ms Anne Bolmström, presi- dent of AB-Biodisk for donating a portion of the Etests® used in this study; Mr Theo Leuenberger of Roche Pharmaceuticals for donating co-tri- moxazole; also Dr Anne von Gottberg; Ms Christine Ayash, Ms Anna Knapp; Ms Sushma Hyoju and Dr Stephen Pelton. We also thank the study nurses Victoria Luo, Joyce M Mulenga, Joyce W Mulenga, Rosaline Kapupa, Edna Mungwa and Sister Kasongo. The nevirapine and Determine® tests were donated by Boehringer Ingelheim an®®®d Abbott Laboratories through the Axios Corporation. Some of these data were presented in abstract form at the 2004 American Society of Tropical Medicine and Hygiene conference in Miami, FL, USA. Funding: TZI was supported by NIH/ NIAID K23 AI 62208 and a coop- erative agreement between Boston University and the Office of Health and Nutrition of the United States Agency for International Development: GHS- A-00-03-00020-00. The funders and commercial donors listed above played no role in the design, implementation, analysis/interpretation of the data, or writing of the manuscript. Competing interests: None declared. hcraeseR yduts stnafni naibmaZ ni elozaxomirt-oC )21( 68 ,8002 rebmeceD | noitazinagrO htlaeH dlroW eht fo nitelluB739 .la te lliG JC nemuseR al y aígoloimedipeores al ,nóicazinoloc ed sasat sal ne lozaxomirtoc noc nóicnuserp ed sixalfiorp al ed otcefE setrohoc ed lanidutignol oidutse :aibmaZ ed setnatcal ne sacicócomuen aicnetsiserrocitoibitna ed sixalfiorp al ed socigóloiborcim sotcefe sol raulavE ovitejbO sol arap SMO al rop adadnemocer lozaxomirtoc noc nóicnuserp .HIV la latanirep nóicisopxe noc setnatcal óiugis es ,setrohoc ed lanidutignol oidutse nu etnaideM sodotéM HIV la sotseupxe on y HIV la sotseupxe setnatcal ed nóiculove al rop sesem 81 atsah ed oicapse rop lartsemirt dadicidoirep noc ocitcálfiorp lozaxomirtoc noreibicer suriv la sotseupxe soL .etnatcal .dade ed sesem 21 sol ominím omoc atsah sanames 6 sal edsed onegótap etnega omoc eainomuenp succocotpertS odnasU nóicazinoloc al ne lozaxomirtoc led sotcefe sol somidem ,alenitnec ed nóicubirtsid al y socitóibitna sol a aicnetsiser al ,aegnírafosan .lozaxomirtoc la nóicisopxe al ed nóicnuf ne sopitores sol otneimiuges a soditemos setnatcal 062 sol ertnE sodatluseR ne sococomuen somatceted ,etneicap-sesem 6903 etnarud HIV la sotseupxe setnatcal soL .sartseum )%8,52( 4931/063 la sotseupxe on sol euq odunem a sám nóicazinoloc noratneserp led )CI( aznafinoc ed olavretni ,4,1 :RR ,sogseir ed etneicnoc( suriv al ojuder lozaxomirtoc noc sixalfiorp aL .)40,0 = p ,9,1–0,1 :%59 ogseir le ótnemua orep etnemadamixorpa %7 nu ne nóicazinoloc ne lozaxomirtoc la setnetsiser sococomuen rop nóicazinoloc ed ,2,3 :RR( sixalfiorp al ed ozneimoc la setneiugis sanames 6 sal lozaxomirtnoc noc sixalfiorp aL .)40,0 = p ,8,7–3,1 :%59CI ovitacfiingis etnemacitsídatse orep oregil otnemua nu ócovorp a selbisnesni sococomuen rop aegnírafosan nóicazinoloc al ed on orep ,)40,0 = p ,6,2–0,1 :%59CI ,6,1 :RR( anicimadnilc al :%59CI ;1,1 :RR( anilicinep al a dadilibisnesni ed ogseir le ótnemua :RR( anilcicartet ,)7,1–6,0 :%59CI ;0,1 :RR( anicimortire ,)7,1–7,0 .)3,2–3,0 :%59CI ;8,0 :RR( locinefnarolc o )5,1–6,0 :%59CI ;9,0 sopitores ed lfirep le óretla on lozaxomirtoc noc sixalfiorp aL anaid omoc sodicelbatse sol noc nóicarapmoc ne socicócomuen ,0,1 :RR( etnelavatpeh adagujnoc acicócomuenitna anucav al ed .)6,1–7,0 :%59CI etnemadaredom emirpus lozaxomirtoc noc sixalfiorp aL nóisulcnoC ed nóicisiuqda al areleca y sococomuen rop nóicazinoloc al sol ne anicimadnilc al y lozaxomirtoc la setnetsiser sococomuen sal a aicacfie etser sixalfiorp ase euq elbaborpmi sE .setnatcal .orutuf le ne sadagujnoc sanucav ملخص تأثير الاتقاء التـرجيحي بالكوتـريموكسازول على معّدلات استعمار المكورات الرئوية، وعلى الوبائيات السيرولوجية، وعلى المقاومة للمضادات الحيوية لدى الرضَّ ع في زامبيا: دراسة أتـرابية طولانية الهدف: استهدفت هذه الدراسة الاستيقان من العواقب المكروبيولوجية لتوصية منظمة الصحة العالمية بإعطاء جرعة اتقائية تـرجيحية من الكوتـريموكسازول للرضَّ ع الذين تعرَّضوا لفيروس الإيدز في الفترة المحيطة بالولادة. الطريقة: استخدم الباحثون تصميًما أترابيا طولانيا ً لتتبع كل رضيع من الرضَّ ع المتعرّضين لفيروس الإيدز وغير المتعرّضين له، وذلك كل 3 أشهر ولمدة 81 شهرا.ً وتلقَّ ى الرضَّ ع المتعرّضون لفيروس الإيدز جرعات اتقائية يومية بالكوتريموكسازول بداية من عمر 6 أسابيع إلى 21 شهرا ً فأكثر. كما قام الباحثون، على اعتبار أن العقدية الرئوية هي العامل الخافر المسبب للمرض، بقياس مدى تغيير الكوتريموكسازول للاستعمار الأنفي البلعومي، ومقاومة المكورات الرئوية للمضادات الحيوية، وتوزع النمط المصلي، باعتبار ذلك كله دالة للتعرض للكوتريموكسازول. النتائج: بعد متابعة 062 رضيعا ًلمدة 6903 مريض – شهر، اكتشف الباحثون وجود المكورات الرئوية لدى 063 من 4931 عينة (8.52%). ولوحظ تكرار المستعمرات لدى الرضَّ ع المتعرضين لفيروس الإيدز أكثر مما كان لدى غير المتعرّضين للفيروس (نسبة الاختطار: 4.1، عند فاصلة ثقة 59%: إذ تراوحت من 1 إلى 9.1، عند نسبة احتمال 40.0). ولوحظ أن الجرعة الاتقائية بالكوتريموكسازول قللت الاستعمار بحوالي 7%، ولكنها رفعت خطر الاستعمار بالمكورات الرئوية المقاومة للكوتريموكسازول خلال 6 أسابيع من بدء إعطاء الجرعات الاتقائية (نسبة الاختطار 2.3، عند فاصلة ثقة 59%: إذ تراوحت من 3.1 إلى 8.7، عند نسبة احتمال 40.0). كما بيَّنت الدراسة أن الجرعة الاتقائية بالكوتريموكسازول أدت إلى زيادة بسيطة، ولكنها مهمة إحصائيا، في الاستعمار الأنفي البلعومي بالمكورات الرئوية العديمة التأثر بالكلينداميسين ( نسبة الاختطار: 6.1 ، عند فاصلة ثقة 59%: إذ تراوحت من 1 إلى 6.2، ونسبة احتمال 40.0 )، ولكن لم تؤد الجرعة الاتقائية إلى زيادة في خطر عدم التأثر بالبنسلين ( نسبة الاختطار: 1.1 ، عند فاصلة ثقة 59% : إذ تراوحت من 7.0 إلى 7.1 )، أو للإريثروميسين ( نسبة الاختطار: 1 ؛ عند فاصلة ثقة 59%: إذ تراوحت من 6.0 إلى 7.1) ، أو للتتراسيكلين (نسبة الاختطار: 9.0 عند فاصلة ثقة 59%: إذ تراوحت من 6.0 إلى 5.1)، أو للكلورامفينيكول ( نسبة الاختطار 8.0، عند فاصلة ثقة 59%: إذ تراوحت من 3.0 إلى 3.2). كما بينت الدراسة أن الجرعة الاتقائية بالكوتريموكسازول لم تؤد إلى اختلاف النمط المصلي السائد للمكورات الرئوية عن الأنماط المصلية المستهدفة بلقاح المكورات الرئوية المقترن السباعي التكافؤ ( نسبة الاختطار: 1؛ عند فاصلة ثقة 59%: إذ تراوحت من 7.0 إلى 6.1). الاستنتاج: تؤدي الجرعات الاتقائية من الكوتريموكسازول إلى كبت مستعمرات المكورات الرئوية بدرجة بسيطة، ولكنها أدت إلى تسريع إصابة الرضَّ ع بالمكورات الرئوية المقاومة للكوتريموكسازول والكلينداميسين. ويبدو من غير المرجح أن تؤدي الجرعات الاتقائية بالكوتريموكسازول إلى تقليل نجاعة اللقاحات المتقارنة في المستقبل. 938 Bulletin of the World Health Organization | December 2008, 86 (12) Research Co-trimoxazole in Zambian infants study CJ Gill et al. References Guidelines on co-trimoxazole prophylaxis for HIV-related infections 1. among children, adolescents and adults in resource-limited settings: recommendations for a public health approach. Geneva: WHO/UNAIDS; 2006. UNAIDS/WHO hail consensus on use of co-trimoxazole for prevention of 2. HIV-related infections in Africa, 2000 [Press release]. Geneva: WHO; 2000. Available from: http//who.int/inf-pr-2000/en/pr2000-23.html [accessed on 4 August 2008]. Guay LA, Musoke P, Fleming T, Bagenda D, Allen M, Nakabiito C, et al. 3. Intrapartum and neonatal single-dose nevirapine compared with zidovudine for prevention of mother-to-child transmission of HIV-1 in Kampala, Uganda: HIVNET 012 randomised trial. Lancet 1999;354:795-802. PMID:10485720 Gill4. CJ, Sabin LL, Tham J, Hamer DH. Reconsidering empirical co-trimoxazole prophylaxis for infants exposed to HIV infection. Bull World Health Organ 2004;82:290-7. PMID:15259258 Porat5. N, Leibovitz E, Dagan R, Coman G, Sfartz S, Peled N, et al. Molecular typing of Streptococcus pneumoniae in northeastern Romania: unique clones of S. pneumoniae isolated from children hospitalized for infections and from healthy and human immunodeficiency virus-infected children in the community. J Infect Dis 2000;181:966-74. PMID:10720519 doi:10.1086/315316 Leibovitz6. E, Dragomir C, Sfartz S, Porat N, Yagupsky P, Jica S, et al. Nasopharyngeal carriage of multidrug-resistant Streptococcus pneumoniae in institutionalized HIV-infected and HIV-negative children in northeastern Romania. Int J Infect Dis 1999;3:211-5. PMID:10575151 doi:10.1016/ S1201-9712(99)90027-9 Tarasi7. A, Chong Y, Lee K, Tomasz A. Spread of the serotype 23F multidrug- resistant Streptococcus pneumoniae clone to South Korea. Microb Drug Resist 1997;3:105-9. PMID:9109101 Barnes8. DM, Whittier S, Gilligan PH, Soares S, Henderson FW, Tomasz A, et al. Transmission of multidrug-resistant serotype 23F Streptococcus pneumoniae in group day care: evidence suggesting capsular transformation of the resistant strain in vivo. J Infect Dis 1995;171:890-6. PMID:7706816 Kellner9. JD, McGeer A, Cetron MS, Low DE, Butler JC, Matlow A, et al. The use of Streptococcus pneumoniae nasopharyngeal isolates from healthy children to predict features of invasive disease. Pediatr Infect Dis J 1998;17:279-86. PMID:9576381 doi:10.1097/00006454-199804000-00004 Scott10. JA, Hall AJ, Hannington A, Edwards R, Mwarumba S, Lowe B, et al. Serotype distribution and prevalence of resistance to benzylpenicillin in three representative populations of Streptococcus pneumoniae isolates from the coast of Kenya. Clin Infect Dis 1998;27:1442-50. PMID:9868658 doi:10.1086/515013 Rusen11. ID, Fraser-Roberts L, Slaney L, Ombette J, Lovgren M, Datta P, 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. PMID:9239769 doi:10.1097/00006454-199707000-00007 Gratten12. M, Nimmo G, Carlisle J, Schooneveldt J, Seneviratne E, Kelly R, et al. Emergence of further serotypes of multiple drug-resistant Streptococcus pneumoniae in Queensland. Commun Dis Intell 1997;21:133-6. PMID:9170700 McKenna13. SL, Muyinda GK, Roth D, Mwali M, Ng’andu N, Myrick A, et al. Rapid HIV testing and counseling for voluntary testing centers in Africa. AIDS 1997;11 Suppl 1;S103-10. PMID:9376093 Phili14. R, Vardas E. Evaluation of a rapid human immunodeficiency virus test at two community clinics in Kwazulu-Natal. S Afr Med J 2002;92:818-21. PMID:12432808 O15. ’Brien KL, Nohynek H. Report from a WHO Working Group: standard method for detecting upper respiratory carriage of Streptococcus pneumoniae. Pediatr Infect Dis J 2003;22:e1-11. PMID:12586987 doi:10.1097/00006454- 200302000-00010 Converse16. GM 3rd, Dillon HC Jr. Epidemiological studies of Streptococcus pneumoniae in infants: methods of isolating pneumococci. J Clin Microbiol 1977;5:293-6. PMID:16032 Sondag17. JE, Morgens RK, Hoppe JE, Marr JJ. Detection of pneumococci in respiratory secretions: clinical evaluation of gentamicin blood agar. J Clin Microbiol 1977;5:397-400. PMID:16034 Performance18. standards for antimicrobial susceptibility testing: 9th informational supplement [NCCLS document M100-S9]. Villanova, PA: National Committee for Clinical Laboratory Standards; 1991. Pikis A, Campos JM, Rodriguez WJ, Keith JM. Optochin resistance 19. in Streptococcus pneumoniae: mechanism, significance, and clinical implications. J Infect Dis 2001;184:582-90. PMID:11474432 doi:10.1086/322803 Kacou-N’Douba A, Bouzid SA, Guessennd KN, Kouassi-M’Bengue AA, 20. Faye-Kette AY, Dosso M. Antimicrobial resistance of nasopharyngeal isolates of Streptococcus pneumoniae in healthy carriers: report of a study in 5-year- olds in Marcory, Abidjan, Cote d’Ivoire. Ann Trop Paediatr 2001;21:149-54. PMID:11471259 Giger O, Mortensen JE, Clark RB, Evangelista A. Comparison of five different 21. susceptibility test methods for detecting antimicrobial agent resistance among Haemophilus influenzae isolates. Diagn Microbiol Infect Dis 1996;24:145-53. PMID:8724400 doi:10.1016/0732-8893(96)00026-0 Ramsey FL, Schafer DW. 22. The statistical sleuth – a course in methods of data analysis. Belmont, CA: Duxbury Press; 1997. Feikin DR, Dowell SF, Nwanyanwu OC, Klugman KP, Kazembe PN, Barat LM, 23. et al. Increased carriage of trimethoprim/sulfamethoxazole-resistant Streptococcus pneumoniae in Malawian children after treatment for malaria with sulfadoxine/pyrimethamine. J Infect Dis 2000;181:1501-5. PMID:10762585 doi:10.1086/315382 Yomo A, Subramanyam VR, Fudzulani R, Kamanga H, Graham SM, 24. Broadhead RL, et al. Carriage of penicillin-resistant pneumococci in Malawian children. Ann Trop Paediatr 1997;17:239-43. PMID:9425380 Gray BM, Converse GM 3rd, Dillon HC Jr. Epidemiologic studies of 25. Streptococcus pneumoniae in infants: acquisition, carriage, and infection during the first 24 months of life. J Infect Dis 1980;142:923-33. PMID:7462701 Gray BM, Dillon HC Jr. Clinical and epidemiologic studies of pneumococcal 26. infection in children. Pediatr Infect Dis 1986;5:201-7. PMID:3952010 doi:10.1097/00006454-198605010-00011 Courvalin P, Carlier C. Transposable multiple antibiotic resistance in 27. Streptococcus pneumoniae. Mol Gen Genet 1986;205:291-7. PMID:3027505 doi:10.1007/BF00430441 Puopolo KM, Klinzing DC, Lin MP, Yesucevitz DL, Cieslewicz MJ. A composite 28. transposon associated with erythromycin and clindamycin resistance in group B Streptococcus. J Med Microbiol 2007;56:947-55. PMID:17577061 doi:10.1099/jmm.0.47131-0 Montanari MP, Cochetti I, Mingoia M, Varaldo PE. Phenotypic and molecular 29. characterization of tetracycline- and erythromycin-resistant strains of Streptococcus pneumoniae. Antimicrob Agents Chemother 2003;47:2236-41. PMID:12821474 doi:10.1128/AAC.47.7.2236-2241.2003 Abdel-Haq N, Abuhammour W, Asmar B, Thomas R, Dabbagh S, Gonzalez R. 30. Nasopharyngeal colonization with Streptococcus pneumoniae in children receiving trimethoprim-sulfamethoxazole prophylaxis. Pediatr Infect Dis J 1999;18:647-9. PMID:10440445 doi:10.1097/00006454-199907000- 00017 Simell B, Kilpi TM, Käyhty H. Pneumococcal carriage and otitis media induce 31. salivary antibodies to pneumococcal capsular polysaccharides in children. J Infect Dis 2002;186:1106-14. PMID:12355361 doi:10.1086/344235 Zar HJ, Hanslo D, Tannenbaum E, Klein M, Argent A, Eley B, et al. Aetiology 32. and outcome of pneumonia in human immunodeficiency virus-infected children hospitalized in South Africa. Acta Paediatr 2001;90:119-25. PMID:11236037 doi:10.1080/080352501300049163 Thera MA, Sehdev PS, Coulibaly D, Traore K, Garba MN, Cissoko Y, et al. 33. Impact of trimethoprim-sulfamethoxazole prophylaxis on falciparum malaria infection and disease. J Infect Dis 2005;192:1823-9. PMID:16235184 doi:10.1086/498249