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Nasopharyngeal pneumococcal colonization among Kenyan children: antibiotic resistance, strain types and associations with human immunodeficiency virus type 1 infection

RUSEN, IRA D. MD; FRASER-ROBERTS, LEIGH MD; SLANEY, LESLIE ART; OMBETTE, JOHN RT; LOVGREN, MARGUERITE ART; DATTA, PRATIBHA MD; NDINYA-ACHOLA, JACKONIAH MBCHB; TALBOT, JAMES A. MD, PHD; NAGELKERKE, NICOLAAS PHD; PLUMMER, FRANCIS A. MD; EMBREE, JOANNE E. MD

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The Pediatric Infectious Disease Journal: July 1997 - Volume 16 - Issue 7 - p 656-662
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Abstract

INTRODUCTION

Individuals with HIV-1 infection have an increased risk of severe illness caused by Streptococcus pneumoniae.1-3 The mechanics of this higher pneumococcal disease burden have not been completely delineated but may reflect decreased mucosal immunosurveillance allowing persistent colonization, or patients may be less able to eliminate the pathogen once bacteremia has occurred. Regardless the frequency of severe pneumococcal disease in children is an increasing public health concern because the HIV-1 epidemic coincides with the emergence and spread of pneumococcal strains resistant to penicillins and multiple other antibiotics.4-13 This is particularly true in sub-Saharan Africa where the prevalence of HIV-1 is high, the burden of disease caused by pneumococci is significant even among HIV-1-uninfected individuals and alternatives to penicillin are scarce.14, 15 In this situation vaccination to prevent pneumococcal disease is an attractive solution to this problem. The present 23-valent pneumococcal polysaccharide vaccine is inadequate for this purpose because of its poor immunogenicity in young children and HIV-1-immunosuppressed individuals as well as differences in serotype prevalences among populations.16-22 However, with the advent of improved technology related to the development of conjugated Haemophilus influenzae type b vaccines, effective pneumococcal vaccines have become a possibility.23-27 Therefore the objectives for this study were to: (1) compare pneumococcal nasopharyngeal colonization rates of HIV-1-infected children with those of HIV-1-exposed but -negative children born to HIV-1-positive mothers and those of seronegative controls to determine whether there is an association between HIV-1 infection and increased pneumococcal respiratory tract colonization; (2) document the prevalence of antibiotic resistant strains in this East African population; and (3) determine the distribution of pneumococcal serotypes in relation to the currently available 23-valent vaccine.

MATERIALS AND METHODS

Study population. Participants were drawn from a cohort of children born to both seropositive and seronegative mothers recruited into a perinatal HIV-1 transmission study which has been ongoing in Nairobi, Kenya since January, 1986.28 The study was approved by both the Ethics Committee of the University of Manitoba and the University of Nairobi. Briefly women admitted to the labor and delivery ward of a large maternity hospital in Nairobi, serving both rural and urban (often from overcrowded slums) women, were screened for HIV-1 antibody after obtaining informed consent for HIV-1 testing. Women and children were then followed to study mother-to-child HIV-1 transmission and the natural history of HIV-1 infection in women and children.

HIV-1 serologic status for both mothers and infants was established by an enzyme-linked immunosorbent assay (DuPont, HTLV-III ELISA from 1986 to 1988; Organon Technika, Vironostika from 1988 onward) and confirmed by Western blot (DuPont DeNemours, Geneva, Switzerland). Enzyme immunoassays and immunoblots were performed according to manufacturer's instructions. A Western blot was considered positive if antibodies were demonstrated against at least one HIV-1 core protein and one HIV-1 surface protein.29 Children born to seropositive mothers were considered to be infected when Western blot positive at or beyond 12 months of age. Children who became and remained seronegative were considered to be uninfected. Seropositive children in whom sera was unavailable at or beyond 12 months of life were classified as indeterminate30; they were excluded from the comparative analysis of colonization rates between the groups of children but were included in the examination of strain types and antibiotic resistance.

From January, 1990, to November, 1990, all children <5 years of age attending the follow-up clinic established for the perinatal HIV-1 transmission study were enrolled in this nested study. Consent was obtained from all mothers to obtain nasopharyngeal specimens from their children. Additional information concerning symptoms and signs of respiratory tract illness were obtained. Recent antibiotic and medication use for treatment of coughing, breast-feeding, age, sex, height and weight of the child was recorded. A physical examination, which included an examination of the respiratory system, was performed by one of the clinic physicians (IDR or PD). Following the physical examination nasopharyngeal specimens were collected. Children with cough, coryza, respiratory distress and/or signs of pneumonia on physical examination were considered to have a respiratory illness at the time of the examination. Children without these signs were considered to be asymptomatic for respiratory disease. Failure to thrive was defined as weight for age falling below the fifth percentile on the National Center for Health Statistics (USA) growth charts.31

Specimen collection. Calcium alginate swabs (Calgi Swab type 1®) were used to collect nasopharyngeal specimens. Swabs were passed gently back from one nostril along the floor of the nasal cavity until it touched the posterior wall of the nasopharynx. It was withdrawn after remaining in place for a few seconds. Swabs were immediately inoculated onto plain chocolate blood agar plates.

Microbiologic methods. Plates were transported to the laboratory within 2 h of collection and incubated for 48 h in a candle jar (∼5% CO2 environment). Initial identification of pneumococci was by typical colonial appearance. Ethylhydrocopreine (optochin) sensitivity was performed for confirmation. Isolates (from one to two colonies per plate) were preserved frozen in skim milk for further testing. Serotyping was performed at the National Centre for Streptococcus, Edmonton, Canada. Assignment of serotype, based on the Danish nomenclature system, was determined by the Quelling reaction using pool, group and type antisera obtained from Statens Seruminstitut of Copenhagen, Denmark.32

Each isolate was screened for penicillin susceptibility using 1-μg oxacillin discs.33 Susceptibility testing by agar dilution was performed in the Department of Medical Microbiology at the University of Manitoba, Winnipeg, Canada. The organisms were suspended to 107 colony-forming units/ml and then 1-μl volumes were inoculated onto Mueller-Hinton agar plates, supplemented with 5% sheep blood containing the antibiotic. For each isolate MICs were performed against these antibiotics and were considered susceptible at the following concentrations: tetracycline <4 μg/ml; clindamycin <0.05 μg/ml; chloramphenicol <4 μg/ml; erythromycin <0.05 μg/ml; rifampin <1 μg/ml; and penicillin ≤0.06 μg/ml. Penicillin resistance was defined as intermediate in the range of ≥0.12 to ≤1.0 μg/ml and fully resistant when >1.0 μg/ml.

Statistical methods. Data were recorded on precoded case report forms and information entered onto a personal computer. Chi square testing were used for analysis of ordinal data and Student's t test was used for analysis of numerate data with SPSS/PC+.

RESULTS

Two hundred seven children born to 200 mothers with known HIV-1 serologic status at delivery took part in this study. No mother refused permission for her child to participate. Of the children born to seropositive mothers 26 were HIV-1-seropositive, 68 were seronegative and 28 were of indeterminate status. There were 85 children born to seronegative mothers. One hundred twenty-six children were cultured once during the study period; 54 children were cultured on 2 occasions; and 27 children were cultured multiple times (maximum, 5). As shown in Table 1 there was no difference in the number of clinic visits and frequency of nasopharyngeal sampling between the study groups. Ages and weights of children at the time of the clinic visits were similar. There were no differences in sex of the infants, percentages who were breast-feeding, had failure to thrive, fever or respiratory tract symptoms at the time of the visit, or medication use for coughs or antibiotics within 1 month of the clinic visit. All children had received their immunizations as appropriate for their age. No HIV-1-infected child had moderate or severe AIDS at the visits when the samples were obtained.

TABLE 1
TABLE 1:
Comparisons of characteristics of children in the study groups

Nasopharyngeal colonization rates are displayed in Figure 1. There were no statistically significant differences in colonization rates between HIV-1-infected and the seronegative groups of children when considering either first visits only or cumulative rates during the study interval. Colonization was higher among HIV-1-infected and uninfected children born to seropositive mothers compared with controls only when associated with respiratory illnesses (86% of 7 and 60% of 20 vs. 29% of 31, respectively, P = 0.004). No differences were observed when children were asymptomatic (20% of 35, 35% of 94 and 22% of 101, respectively). Colonization was not associated with fever, failure to thrive, or breast-feeding at the time of the visit, or with previous cough medication or antibiotic use during the month before the clinic visit.

Fig. 1
Fig. 1:
Nasopharyngeal colonization rates with S. pneumoniae. P < 0.01 comparing cumulative colonization rates among all the groups. P = NS (not significant) comparing colonization rates at the first visit among all the groups. P = 0.003 comparing colonization rates between HIV-1-infected infants with and without respiratory illness. P = 0.04 comparing colonization rates between HIV-1-negative infants with and without respiratory illness. P = NS comparing colonization rates between control infants with and without respiratory illness. P = 0.004 comparing colonization rates between infants with respiratory illness born to seropositive mothers and controls.

Antibiotic susceptibility testing was available for 92 isolates. Fifty-four isolates were oxacillin-resistant; 51 also demonstrated MICs ≥0.12 μg/ml. Four oxacillinsensitive isolates had MICs to penicillin of ≥0.12 but ≤0.25 μg/ml. The one isolate not tested by oxacillin disc had an MIC to penicillin of ≥0.12 but ≤0.25 μg/ml. For data analysis resistance to penicillin was based on the MIC value; thus 56 (60.8%) of 92 isolates tested demonstrated resistance to penicillin. The MICs of resistant isolates ranged from ≥0.12 to 0.5 μg/ml. High level resistance (MIC >1 μg/ml) was not observed. There was no increased incidence of resistant organisms in the HIV-1-infected group as compared with the HIV-1-negative and control groups. Twenty-six (28.2%) isolates tested were resistant to tetracycline (MIC >16 μg/ml); 12 (46%) of these isolates were also penicillin-resistant. All strains were susceptible to the other antibiotics tested. Table 2 shows the resistance to penicillin among serotypes. Antibiotic use during the month before the nasopharyngeal swab was not associated with an increased detection of resistant organisms.

TABLE 2
TABLE 2:
Penicillin resistance of isolates classified by serotype

Ninety-four isolates from 73 children were available for serologic testing. In 5 cases 1 serotype was recovered on 2 consecutive visits from the same child. These instances were included only once when analyzing the differences in serotypes among the groups of children. Fifteen children were colonized with 2 or more strains. There was no significant difference in serotypes on the basis of HIV-1 status. Overall the isolates belonged to 16 different serotypes. The most common (in order of decreasing frequency) were 13, 15, 14, 6B and 19F. These 5 serotypes accounted for 73% of all isolates. Nonvaccine strains were isolated from 30 (41%) of the 73 colonized children studied.

DISCUSSION

In this study of children in Nairobi nasopharyngeal colonization rates for S. pneumoniae among children without respiratory illness varied from 20 to 35% and were similar to the colonization rates in children of 25.0% in Johannesburg, South Africa; 29.0% in Adelaide, Australia; and 31.9% in Lund, Sweden.6, 34, 35 This is despite the use of nonselective chocolate agar for the initial cultures. Colonization rates were significantly higher among children with respiratory illness born to seropositive mothers, but this was not seen among control children. Farley et al.36 found a relative risk of 12.6 (95% confidence interval, 5.4 to 28.8) for invasive pneumococcal infections in perinatally HIV-1-infected children during the first 3 years of life as compared with HIV-1-negative children born to HIV-1-positive mothers and control children; however, nasopharyngeal colonization rates were not studied. In our study no significant increase in colonization among HIV-1-infected children without respiratory illness was observed. Janoff et al.37 reported a similar finding among adult males infected with HIV-1 compared with uninfected men attending a sexually transmitted disease clinic in Denver. Thus the increased burden of pneumococcal disease in HIV-1-infected individuals is not likely the result of increased colonization and therefore is probably related to systemic immune dysfunction.

Although penicillin-resistant S. pneumoniae is an emerging problem worldwide, there is marked geographic variability in levels of penicillin resistance.6, 7, 38, 39 In a review of 1627 isolates from 30 centers in the United States, a range of 4 to 48% of isolates were resistant to penicillin, with an overall rate of 16%.40 A previous study from Kenya reported an overall resistance rate to penicillin of 17% of pneumococcal isolates associated with community-acquired pneumonia, with resistant isolates significantly more common in HIV-1-positive adults.41 In this study no such association between resistance to penicillin and HIV-1 infection was seen. Also in our study, whereas 60% of the strains isolated exhibited intermediate resistance to penicillin, high level penicillin resistance was not seen; only 28% were resistant to tetracycline and multiple drug resistance was not observed. Standard beta-lactam therapy for organisms with intermediate resistance to penicillin is effective for nonmeningeal illness, but concentrations of penicillin in cerebrospinal fluid may not be adequate to eradicate pneumococci with intermediate penicillin resistance.38, 39, 42 Therefore the high prevalence of relative resistance to penicillin raises public health concerns regarding the availability and cost of alternative antibiotic therapies for pneumococcal meningitis in Kenya.

Strategies using prophylactic antibiotics and intravenous immunoglobulin have been proposed as a means to prevent invasive pneumococcal disease in HIV-1-seropositive individuals. Although potentially effective these would be impractical for countries such as Kenya.43, 44 Universal pneumococcal vaccination is a more promising method, but barriers to effective immunization for young children in developing nations are numerous. In this study population, 33 (36%) of 91 isolates belonged to serotypes not included in the current vaccine. This was primarily because of the high rate of recovery of serotype 13, accounting for 31% of all isolates. Serotype 13 is one of the serotypes that is rarely isolated. A multicenter study of pneumococcal serotypes in Africa45 identified type 13 in <1% of all invasive isolates and serotype 13 accounted for only 0.9% of isolates in a recent Canadian survey.8 The reason for the predominance of this serotype is unclear because it was isolated throughout the study period and no obvious clustering occurred other than that it was primarily isolated from seronegative and control children. Its isolation is also of clinical importance given that >90% of these isolates demonstrated intermediate resistance to penicillin. Although we did not examine the serotypes responsible for invasive pneumococcal disease, others have noted an association between strains colonizing the nasopharynx, and invasive disease and surveillance of colonizing strains has been suggested as a means of monitoring circulating strains in populations.46-49 Although the development of a protein-polysaccharide conjugate vaccine holds promise, because fewer strains can be included, those selected must be specific for the target population and, in Kenya, should likely include serotype 13.

A combined program of passive and active immunization for children in developing countries merits consideration. Shahid et al.50 prospectively assessed the passive transmission of specific IgG antibodies to serotypes 6B and 19F to infants of healthy women immunized during pregnancy. Maternal immunization was associated with significantly higher IgG antibody to both serotypes among infants of immunized mothers. The higher titers persisted in the majority of infants at 5 months of age. These results suggest that passive immunization strategies might protect infants from invasive pneumococcal disease during the first several months of life, when active immunization options are insufficiently immunogenic. Possible detrimental effects such as interference with the efficacy of active immunization require further exploration.

The final obstacle to the widespread use of pneumococcal vaccines is economic. Trials of the efficacy of new vaccines are large and expensive, and current pneumococcal vaccines are beyond the means of many countries in sub-Saharan Africa.51 However, development of an affordable vaccine, which is immunogenic in young infants and includes the strains circulating in the community, is essential to improve the health care for children in these regions.52

ACKNOWLEDGMENTS

These data were presented in part at the Fifth Annual Conference on HIV/AIDS Research, Winnipeg, Manitoba, Canada, June 8 to 11, 1955.53 FAP is the recipient of a Senior Scientist award from the Medical Research Council of Canada. JEE is the recipient of a Fellowship from the Medical Research Council of Canada. This work was supported in part by grants from the International Development Research Centre (Ottawa) and the National Health Research Development Program (Ottawa). This project would not have been possible without the contribution of the study nurses, Mrs. Agnes Wanjala, Mrs. Janet Muriithi, Ms. Janet Itweru and Mrs. Elizabeth Agoki, and we thank them for their dedication. We thank the Nairobi City Commission and the staff of Pumwani Maternity Hospital for their continued support and cooperation. We also acknowledge the assistance of the laboratory staff at the National Centre for Streptococcus, Provincial Laboratory for Public Health for Northern Alberta.

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Keywords:

Pneumococcal colonization; human immunodeficiency virus type 1; strains; antibiotic resistance; Kenya; children

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