Conceived and designed the experiments: IMOA MA CANO VI RAA. Performed the experiments: MA CE DN AB RAA. Analyzed the data: IMOA MA CE RAA. Contributed reagents/materials/analysis tools: MA CANO CE VI DN AB RAA. Wrote the paper: IMOA MA CANO RAA.
Current address: Infectious Diseases Development, Global Health Program, Bill & Melinda Gates Foundation, Seattle, Washington, United States of America
The authors have declared that no competing interests exist.
Introduction of pneumococcal vaccines in Nigeria is a priority as part of the Accelerated Vaccine Introduction Initiative (AVI) of the Global Alliance for Vaccines and Immunisation (GAVI). However, country data on the burden of pneumococcal disease (IPD) is limited and coverage by available conjugate vaccines is unknown. This study was carried out to describe the pre vaccination epidemiology and population biology of pneumococcal carriage in Nigeria.
This was a cross sectional survey. Nasopharyngeal swabs (NPS) were obtained from a population sample in 14 contiguous peri-urban Nigerian communities. Data on demographic characteristics and risk factor for carriage were obtained from all study participants. Pneumococci isolated from NPS were characterised by serotyping, antimicrobial susceptibility and Multi Locus Sequencing Typing (MLST).
The prevalence of pneumococcal carriage was 52.5%. Carriage was higher in children compared to adults (67.4% vs. 26%), highest (≈90%) in infants aged <9 months and reduced significantly with increasing age (P<0.001). Serotypes 19F (18.6%) and 6A (14.4%) were most predominant. Potential vaccine coverage was 43.8%, 45.0% and 62% for PCV-7, PCV-10 and PCV-13 respectively. There were 16 novel alleles, 72 different sequence types (STs) from the isolates and 3 Sequence Types (280, 310 and 5543) were associated with isolates of more than one serotype indicative of serotype switching. Antimicrobial resistance was high for cotrimoxazole (93%) and tetracycline (84%), a third of isolates had intermediate resistance to penicillin. Young age was the only risk factor significantly associated with carriage.
Pneumococcal carriage and serotype diversity is highly prevalent in Nigeria especially in infants. Based on the coverage of serotypes in this study, PCV-13 is the obvious choice to reduce disease burden and prevalence of drug resistant pneumococci. However, its use will require careful monitoring. Our findings provide sound baseline data for impact assessment following vaccine introduction in Nigeria.
Pneumonia is one of the leading causes of mortality in children <5 years old. It is responsible for 1.6 million of 8.8 million annual deaths in this age group
There are over 91 serotypes of pneumococci and worldwide, 6–11 serotypes are responsible for ≥70% of invasive disease (IPD) in children <5 years old
Since vaccination is the most effective public health tool to prevent IPD, pneumococcal conjugate vaccines (PCVs) containing 7, 10 and 13 serotypes have been developed although PCV-7 is being replaced by PCV-10 and -13 because it contains limited serotypes. Unlike the 23 serotype-containing polysaccharide vaccine, PCVs are more immunogenic in young children, induce immunological memory and provide serotype-specific protection against pneumococcal carriage. Although they contain serotypes most associated with disease and antimicrobial resistance worldwide, variation of serotypes geographically and antimicrobial resistant strains prevent universal protection. In addition, replacement in carriage and invasive disease by non-vaccine containing and drug resistant serotypes of pneumococci occurs with use of PCVs
In many countries needing PCV the most, data on the burden of pneumococcal carriage and disease; as well as the prevalent serotypes and coverage by PCVs is limited or unknown. Nigeria is one of the 10 countries where two-thirds of all deaths due to pneumonia in children <5 years occur
We investigated pneumococcal carriage in a Nigerian population to determine carriage rates, prevalent serotypes; theoretical coverage of pneumococcal conjugate vaccines and molecular epidemiology of pneumococci in this population.
The Lagos University Teaching Hospital Primary Health Care and Rural Medicine Centre (LUTH PHC), Pakoto village is approximately 70 kilometres from Lagos, Nigeria. This health centre serves all communities within a 10 km radius covering two local government areas (LGAs) and it was the base for this study. Pakoto is one of 250 communities in Ifo LGA, Ogun State, Nigeria. The total population is 350,000 (2006 National Population Census, Nigeria). Majority of the inhabitants are Yoruba.
This was a cross sectional survey. The target sample was a range of 384–518 children aged <5 years required to estimate the prevalence of pneumococcal carriage with a precision of 5% (with 95% confidence) and 80–90% power. Since prevalence of carriage in the whole community has an impact on prevalence of carriage in children aged <5 years and vice versa, a sample including all age groups was preferred. To obtain a representative sample in an area with unknown age distribution, all consenting children aged <5 years; 1 in 3 persons aged 5–49 years and all those aged >50 years up to 1000 subjects were swabbed. Even if the proportion of children aged <5 years in the study communities was less than 25%, the sampling was designed to give an expected ratio of subjects of 25∶21∶11 (i.e. at least 440 <5 years). Communities selected for the study were within a 5 km radius of the health centre.
All consenting adults and children ordinarily resident in the study area were eligible for enrolment.
The Ethics Committees of the Lagos University Teaching Hospital, Lagos, Nigeria, the London School of Hygiene and Tropical Medicine, UK approved this study and acknowledgement received from the Gambian Government and MRC joint ethics committee. Parents/guardians of minors and every adult participant gave written informed consent before participation in study. Those 15–17 years old gave assent was in addition to written consent given by parents/guardians.
A questionnaire was administered to each subject (adolescent with their parents/guardians and adults) or to their parent or guardian (for minors) by a trained field worker to obtain demographic, personal, clinical, general and age-specific risk factors data. The risk factors for which data was obtained via questionnaire were household smoking, number of <5-year old residents in household, household cooking fuel, breastfeeding in children aged <6 months, antibiotic use, gender, age, ethnicity, location, overcrowding, and socioeconomic status.
All procedures (swabbing, storage and culture) were carried out using standard methods recommended by the World Health Organization (WHO)
Randomly selected isolates (201) had to microbial susceptibility tests to Penicillin G (PG), Cefotaxime (CT), Tetracycline (TC), Trimethoprim/sulfamethoxazole (TS), Erythromycin (E) and Chloramphenicol (CL) using E-tests (AB Biodisk, Solna, Sweden). Minimum inhibitory concentrations (MICs) with resistance and sensitivity according to the interpretive criteria provided in the product insert were recorded. Resistant Isolates were those with MICs above the threshold for sensitivity.
The MRC Unit, submits to the external quality assurance programme of the United Kingdom National External Quality Assessment Service
MLST was performed as previously described
All data were double entered into an Access relational database. Statistical analyses were performed using Stata 11.1 (Stata Corp LP, College Station, Texas). The main outcome variable was prevalence of pneumococcal carriage. The secondary outcomes were genetic structure of and serotypes found in carriage, their antibiotic susceptibility and risk factors (both general and specific) associated with carriage. Analysis of carriage was done with ‘person’ as unit of analysis while analysis of serotypes and antibiotic resistance was done with ‘isolate’ as unit of analysis.
The exposures considered were household smoking, number of <5-year old residents in household, household cooking fuel, breastfeeding in children aged <6 months, antibiotic use, gender, age, ethnicity, location, overcrowding, and socioeconomic status. Socioeconomic status was assessed using a modification of the socio-economic index score validated for Nigeria
Random effects logistic regression models were used to adjust for the effect of clustering on outcomes by location.
Out of 1025 consenting participants, 98.0% (1005) had useable NPS that were cultured. Their median age was 4.4 (interquartile range [IQR] 1.3–30) years. They were predominantly female (652, 63.8%) and Yoruba (952, 92.8%). As planned, there were more children (654, 63.8%) than adults were. A history of antibiotic use in sampled children was obtained in 100 (15.6%) of 641 children and from 76 out of 361 (21.1%) adults. Overall, 176 (17.5%) of total study population had taken a course of antibiotics at least 1 month prior to sampling. Antibiotics were obtained mostly by self-prescription and purchased from drug stores (66.7% [95% CI 54.8–77.1%], 50 of 75).
Pneumococcus was identified in swabs from 528 of 1005 subjects. The prevalence of carriage was 52.5% (95% Confidence Interval [CI]: 49.4–55.7%). As seen in
Pneumococcal carriage was higher in children (those <18 years), 434 of 644 (67.4% [95% CI 63.6–71%]) compared to adults, 94 of 361 (26% [95% 21.6–30.9%] and this difference was significant (p<0.001). Carriage was highest (244 of 328) in children aged <2 years i.e. 74.4% (69.3%–79.0%). As shown in
Forty-two (42) serotypes of pneumococcus were identified, while 17 isolates (3.2%) were non-typeable (NT). The distribution of serotypes also varied by age group with almost 80% of all serotypes carried by children <5 years diminishing to ≈50% in the older age groups as shown in
All | <5years | 5–14 years | >15–39years | ≥40 | |
|
528 | 375 | 63 | 57 | 33 |
|
52.5 | 71.0 | 11.9 | 10.8 | 6.3 |
|
42 | 33 | 21 | 21 | 19 |
|
|||||
|
19F (94) | 19F (71) | 19F (10) | 19F (11) | 11 (3) |
|
6A (76) | 6A (65) | 11 (7) | 6A (7) | 23F (3) |
|
6B (72) | 6B (60) | NT (6) | 23F (6) | 3 (3) |
|
23F (37) | 23F (27) | 18C (5) | 11 (4) | 6B (3) |
|
11 (27) | 15B (14) | 6B (5) | 18C (4) | 12 (2) |
|
15B (21) | 11 (13) | 3 (4) | 6B (4) | 13 (2) |
|
3 (19) | 14 (13) | 6A (4) | 15B (3) | 17 (2) |
|
NT (17) | 19A (10) | 15B (3) | 3 (2) | 19F (2) |
|
18C (15) | 3 (10) | 20 (3) | 4 (2) | 7C (2) |
|
9V (15) | 9V (10) | 21 (3) | 7F (2) | 9V (2) |
|
74.4% | 78.1% | 79.4% | 78.9% | 72.7% |
|
231 (43.8%) | 173 (46.1%) | 19 (30.2%) | 28 (49.1%) | 11 (33.3%) |
|
235 (44.5%) | 174 (46.4%) | 20 (31.8%) | 30 (52.6%) | 11 (33.3%) |
|
325 (61.6%) | 264 (70.4%) | 27 (42.9%) | 38 (66.7%) | 14 (42.4%) |
Serotypes9V, 14, 18C, 3 and 15C accounted for 2–4% of overall pneumococcal isolates, while other serotypes not previously mentioned made up less than 2% of all serotypes found.
The theoretical coverage for the earliest available conjugate vaccine-PCV7, was poor for all age categories ranging from 30.2%–49.1% and there was very little difference between the coverage offered by the newer PCV10 when compared to PCV7 (
MLST was performed on 99 pneumococcal isolates representing 29 different serotypes selected randomly from the 528 pneumococcal isolates. We found 16 novel alleles [(
Each circle represents an ST. The area of each circle corresponds to the number of isolates. Thick, short, solid lines connect single locus variants; thin longer solid lines connect double locus variants and broken lines connect three or more locus variants. Texts in red and inside the circles are the serotypes.
A random selection of 201 pneumococcal isolates was tested for susceptibility to the following antibiotics; Erythromycin (E), Tetracycline (TC), Cefotaxime (CT), Trimethoprim-Sulphamethoxazole (TS), Penicillin G (PG) and Chloramphenicol (C). Isolates were only fully susceptible to erythromycin and 1 (0.5%) isolate had high-level resistance to CT otherwise others were fully susceptible (see
Penicillin | TMP-SMX | Tetracycline | Cefotaxime | Erythromycin | Chloramphenicol | |
|
62 (30.9) | 187 (93.0) | 169 (84.1) | 1 (0.5) | 0 (0.0) | 17 (8.5) |
|
||||||
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13 (39.4) | 30 (90.9) | 28 (84.8) | 1 (3.0) | 0 (0.0) | 2 (6.1) |
|
8 (25.8) | 31 (100) | 29 (93.6) | 0 (0.0) | 0 (0.0) | 3 (9.7) |
|
4 (15.4) | 24 (92.3) | 25 (96.2) | 0 (0.0) | 0 (0.0) | 5 (19.2) |
|
4 (25.0) | 16 (100.0) | 13 (81.3) | 0 (0.0) | 0 (0.0) | 0 (0.0) |
|
2 (12.5) | 13 (81.2) | 6 (37.5) | 0 (0.0) | 0 (0.0) | 2 (12.5) |
|
1 (14.3) | 7 (100.0) | 7 (100.0) | 0 (0.0) | 0 (0.0) | 1 (14.3) |
|
6 (85.7) | 7 (100.0) | 7 (100.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) |
|
6 (66.7) | 9 (100.0) | 8 (88.9) | 0 (0.0) | 0 (0.0) | 1 (11.1) |
|
0 (0.0) | 3 (100.0) | 3 (100.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) |
|
0 (0.0) | 4 (100.0) | 4 (100.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) |
|
26 (29.9) | 82 (94.3) | 77 (88.5) | 1 (1.2) | 0 (0.0) | 6 (6.9) |
|
26 (29.6) | 83 (94.3) | 78 (88.6) | 1 (1.1) | 0 (0.0) | 6 (6.8) |
|
40 (32.5) | 118 (95.9) | 111 (90.2) | 1 (0.8) | 0 (0.0) | 7 (5.7) |
Antibiotic use was associated with age (p<0.001) and history of prior ingestion was highest in those aged <5 years (93 of 523, 17.8%) and aged >40 years (40 of 139, 29%); and least in the 5–14 years age category (6 of 125, 4.8%). Overall, there were no differences in prevalence of carriage by history of antibiotic use (52.3% vs. 54.0%, p = 0.7). When children (<18 years) were compared to adults, no differences in carriage by antibiotic use were seen as well.
In multivariable analysis, pneumococcal carriage was adjusted for age, gender, overcrowding, predominant household cooking fuel, and total household residents aged <5 years. At the level of the individual, age and overcrowding were significantly associated with pneumococcal carriage. The odds of pneumococcal carriage in the <5 years age group was 7 times more than in those aged >40 years. They also had 2.5 times the odds of carriage found in the 5–14 years age group. As shown in
Risk factor | P- value | p-value | ||
Age group (years) | ||||
≥40 | 1.0 | 1.0 | ||
15–39 | 1.1 (0.64–1.81) | <0.001 | 1.1 (0.65–1.91) | 0.70 |
5–14 | 2.9 (1.69–5.03) | <0.001 | 2.9 (1.63–5.0) | <0.001 |
<5 | 7.1 (4.53–11.03) | <0.001 | 7.4 (4.60–11.85) | <0.001 |
Gender | ||||
Female | 1.0 | 1.0 | ||
Male | 1.1 (0.79–1.44) | 0.65 | 1.1 (0.81–1.48) | 0.57 |
Predominant cooking fuel | ||||
Kerosene | 1.0 | 1.0 | ||
Other | 0.4 (0.17–1.12) | 0.08 | 0.5 (0.18–1.16) | 0.10 |
Overcrowding index | ||||
≤3 | 1.0 | 1.0 | ||
≥4 | 1.3 (1.02–1.83) | 0.03 | 1.3 (0.97–1.78) | 0.07 |
Number of resident under fives | ||||
0 | 1.0 | 1.0 | ||
≥1 | 0.8 (0.60–1.01) | 0.14 | 0.8 (0.61–1.11) | 0.20 |
*OR-Odds Ratio from Logistic regression model.
**Adjusted for clustering.
On adjusting the data for clustering by location in a random effect logistic regression model, age remained the key risk factor for carriage at community level. The evidence for an association between overcrowding and pneumococcal carriage weakened when data was adjusted for clustering.
This study investigated pneumococcal carriage in a Nigerian population to determine carriage rates, prevalent serotypes, theoretical coverage of available pneumococcal conjugate vaccines and population biology of isolated pneumococci. Since recently acquired pneumococci in carriage are usually the primary source of serotypes that cause invasive disease, carriage of pneumococcus can provide information on prevalence of antimicrobial resistance and some insight into need for vaccines and potential impact of selected vaccine on IPD.
Nigeria is one of the countries in the west sub-Saharan Africa region that currently plans to introduce one of the available PCVs. This may likely be PCV-13 if the example of other African countries (Mali, Rwanda, Gambia and Sierra Leone) is followed. If introduced, the likely benefit of this vaccine is currently unknown and baseline data to inform expected changes or trends in pneumococcal carriage and IPD are unavailable. We hope this study will address some of these knowledge gaps. To our knowledge, this is the first investigation into the risk factors and population structure of carried pneumococcal in Nigeria.
The prevalence of pneumococcal carriage varies within country and setting ranging from <10% in some developed countries like Italy and France
The 42 pneumococci serotypes found in this study population provide the first detailed description of the repertoire of carried pneumococcal serotypes in Nigeria. The most common serotypes were 19F, 6A, 6B, 23F, 11 and 15B. Interestingly, these are among the most common serotypes reported in carriage studies in similar settings
Although data on serotypes implicated in IPD in Nigeria is limited, serotypes 19F, 4 and 5 were found in a recent report on surveillance of IPD
The serotype coverage for PCV-7 (44%) is limited in Nigeria and similar settings because of the few serotypes it contains. In particular, it does not contain serotypes 1 and 5 that have been implicated 25–30% of IPD found in low resource settings
Although replacement by non-vaccine serotypes in both carriage and disease occurs following prolonged routine use of PCVs
MLST analysis shows not only a higher proportion of novel STs but also a highly diverse pneumococcal population structure in Nigeria which is consistent with other studies of carried pneumococcal population in similar settings like The Gambia and Ghana
We also found evidence of capsular switching in three STs (280, 310 and 5543). After the introduction of the pneumococcal conjugate vaccine, particular strains with genetic advantage may change their capsules from vaccine serotypes to non-vaccine serotype through capsular switching
The high frequency of resistance to commonly used antibiotics is a source of worry especially as TS, to which 93% of isolates were resistant, is one the WHO recommended first line antibiotic for treatment for respiratory tract infections in most developing countries
Age <5 years
The absence of community age-distribution data during determination of sample size did not affect our results. The study did not have sufficient power for serotype specific sub analysis despite achieving the target sample size. Our risk factor analyses did not include day care or school attendance so the contribution if any of this exposure to pneumococcal carriage among children in this population is still unknown. However, this risk factor was not associated with carriage in other studies conducted in settings like ours
In conclusion, carriage of pneumococcus is very high in this Nigerian community indicating a high burden of pneumococcal disease. Young age is a major risk factor for carriage.
The consequence of findings from this study, and essential to the introduction of routine immunization against pneumococcus is the need for an extensive regional/nationwide surveillance of pneumococcal carriage, IPD and antimicrobial resistance that are crucial for vaccination impact assessment. The global vaccine preventable invasive bacterial disease (VP-IBD's) 3-tiered approach to sentinel surveillance should be the model implemented in Nigeria alongside introduction of PCV-13. This should start with tier-1 i.e. surveillance for suspected meningitis in children under five years across the country with rapid expansion to tier-2 level surveillance that includes cases of suspected pneumonia and septicaemia. Revisions to local antibiotic policy and prescription practice are required in light of the significant resistance seen to commonly used antibiotics.
Pneumococcal serotypes and MLST distribution (new alleles are underlined).
(DOCX)
Dr. M.O. Ota for critical review and help with preparing this manuscript, Aminata Dumbuya, Fatou Bah Baldeh, Zainab Kalokoh all of the Research Microbiology Laboratory, MRC The Gambia Unit; Mrs. Afolabi, Coordinator; Mr.Fadeyi and the field team all of the LUTH Pakoto Health Centre; and Caroline Jackson (London School of Hygiene and Tropical Medicine).