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Early Declines in Vaccine Type Pneumococcal Carriage in Children Less Than 5 Years Old After Introduction of 10-valent Pneumococcal Conjugate Vaccine in Mozambique

Sigaúque, Betuel PhD*,†,‡; Moiane, Benild BSc*; Massora, Sergio MSc*; Pimenta, Fabiana PhD§; Verani, Jennifer R. MD§; Mucavele, Helio MD*; Chaúque, Alberto BSc*; Quintó, Llorenç MSc; dos Santos, Rita T. MD; Carvalho, Maria da Gloria PhD§; Whitney, Cynthia G. MD§; Lessa, Fernanda C. MD§

Author Information
The Pediatric Infectious Disease Journal: October 2018 - Volume 37 - Issue 10 - p 1054-1060
doi: 10.1097/INF.0000000000002134


Streptococcus pneumoniae (pneumococcus) causes disease ranging from mild to severe and can lead to death.1 Pneumonia is the leading infectious cause of death in children <5 years of age globally, and 30%–50% of pneumonia episodes in children <5 years of age are estimated to be caused by pneumococcus, especially in countries where pneumococcal vaccine has not been introduced yet.2,3 According to the World Health Organization, 541,000 deaths caused by pneumococcus were estimated to occur in children <5 years of age in 2008. Of those, 12% were in HIV-infected children.4

Humans are the main reservoir of pneumococcus and the prevalence of pneumococcal nasopharyngeal (NP) carriage in healthy children in low-income countries can range from 49.8% to 90.0%.5,6 Although most children are asymptomatic carriers, in some instances, they may develop disease because of invasion of the pathogen into adjacent tissues or blood stream leading to otitis media, pneumonia, bacteremia or meningitis.7 HIV is an important risk factor for both pneumococcal colonization and disease.8,9 Pneumococcal colonization decreases with age, and children are the main drivers for transmission and spread of the pathogen in the community.10,11 Some studies have suggested that not only the presence of pneumococcus but also its colonization density in the upper respiratory tract can influence transmission, susceptibility to pneumonia, and disease severity.12–14

Pneumococcal conjugate vaccines (PCVs) prevent pneumococcal disease in large part by preventing acquisition of carriage of the serotypes included in the vaccine.15,16 The impact of PCV on NP carriage is considered a predictor of vaccine effectiveness,7 and carriage studies can be used to measure the impact of different formulations and schedules of PCV among vaccinated children (direct effect) and unvaccinated children (indirect effect by reducing transmission) in a variety of settings.17 Vaccination also has the potential to decrease pneumococcal colonization density. In a community randomized trial done in the United States,18 PCV was associated with significant decreases in pneumococcal density among vaccinated children who were colonized with vaccine-type (VT) pneumococcus.

Mozambique is a low-income country that has high incidence of invasive pneumococcal disease (IPD) in children under 5 years of age (416 cases per 100,000 children-year at risk)19 and a HIV prevalence of 16% among pregnant women.20 In April 2013, with the support of Gavi, the Vaccine Alliance, Mozambique introduced 10-valent PCV (PCV10) in the national immunization program using 3 primary doses (3 + 0 schedule) at 8, 12 and 16 weeks of age without a booster or any catch-up doses for older children. We evaluated the early impact of a 3 + 0 PCV10 dose schedule introduction on pneumococcal NP colonization and density in this previously naïve PCV population in a low-income setting with high HIV prevalence. We assessed differences in pneumococcal colonization and bacterial density between pre- and post-PCV introduction by vaccination and HIV status among children under 5 years of age.


Study design and Population

We conducted two cross-sectional surveys of pneumococcal NP carriage among children 6 weeks of age through 59 months of age in 3 geographic locations in Mozambique: Maputo (urban area in the south), Nampula (urban are in the north) and Manhiça (rural area in the south). The first survey was conducted from October 2012 to March 2013 (pre PCV 10), and the second was conducted from October 2014 to April 2015 (post PCV10). For both surveys, we enrolled HIV-infected children presenting for routine care at outpatient clinics in Maputo, Nampula and Manhiça and HIV-uninfected children from an existing Demographic Surveillance System (DSS) in Manhiça district.21 Children were considered HIV infected if HIV DNA polymerase chain reaction (PCR) was positive at any point in life or if HIV rapid test (Uni-Gold HIV, Trinity Biotech, Ireland) was positive in a child >18 months of age.22 Children were considered HIV uninfected if HIV PCR or rapid test (for children >18 months of age) was negative or if they were born from a mother with a documented negative HIV test during pregnancy. We performed an age-stratified (<12, 12–23, 24–59 mo) random sample of HIV-uninfected children using the DSS in Manhiça district. The DSS has been described elsewhere.21 Briefly, it covers a population of 92,000, and each person who is a resident of the DSS area is issued a permanent identification number to enable adequate follow-up. A resident is defined as a person living in the DSS area who is expected to stay for at least the following 3 months.

This study was approved by the institutional review boards of the Mozambican Ministry of Health and of the Centers for Disease Control and Prevention. Written informed consent was obtained from parents or guardians before study enrollment.

Data and Sample Collection

Trained study staff collected demographic information, vaccination history and a NP specimen for each eligible child whose parent or guardian provided informed consent for study participation. NP specimens were collected using flexible and sterile calcium alginate swab (Puritan, catalog number 22-029-501 and 22-029-500; Fisher Scientific, Pittsburg, PA). Each child was swabbed only once. All NP specimens were placed in 1.0 mL skim milk-tryptone-glucose-glycerol (STGG) medium, vortexed for 10–20 seconds to disperse the organisms from the swab and frozen at −70°C within 4 hours after collection. Study staff actively sought written evidence of immunization through the child immunization card or through vaccination records at immunization clinics if parents reported vaccine receipt and the immunization card was not available. We excluded from analysis any child without written documentation of vaccine receipt unless the parent or guardian reported that their child had not received any vaccinations beyond doses given at birth (ie, such children were included and considered to have received zero doses of PCV10).

Laboratory Methods

Pneumococcal isolation and identification was done by transferring 200 µL of the STGG-NP inoculated medium onto 5.0 mL Todd Hewitt broth containing 0.5% yeast extract and 1 mL of rabbit serum and incubating the inoculated broth tubes at 37°C in a carbon dioxide incubator for 6 hours. After incubation, 10 µL loop of cultured broth were transferred on tryptone soy agar plates with 5% sheep blood agar plate and incubated at 37°C in a carbon dioxide incubator and examined after 18–24 hours.23 All alpha-hemolytic, pneumococcal suspected colonies were tested for susceptibility to optochin and bile solubility. Pneumococcal isolates were serotyped by Quellung reaction. For nontypeable pneumococcal isolates, quantitative PCR (qPCR) was performed for pneumococcal lytA gene detection followed by conventional multiplex PCR for pneumococcal serotyping deduction.23 NP specimens that yielded serotypes 11A (non-PCV10 type), 19A (non-PCV10 but included in 13-valent PCV [PCV13]) and 19F (PCV10 type) were selected and processed for density using serotype specific monoplex wzy gene qPCR assays.24 DNA extracts were obtained from 200 µL of STGG-NP inoculated medium, transferred into 1.5 mL cryotubes containing 300 µL of buffer number 4 (MagNa Pure LC DNA Isolation Kit III, Roche Diagnostics, USA) and then placed into MagNA Pure Compact (Roche Diagnostics, USA) instrument using the external lysis protocol according to manufacturer instructions. Each DNA extraction batch also included a freshly prepared serial dilution of the 3 serotypes control cells in saline starting with 0.5 McFarland scale. A standard curve was prepared to correlate colony-forming units (CFUs) per mL and cycle threshold value. DNA extracts were stored at −20°C until qPCR were performed using PerfeCTa® qPCR ToughMix®, Low ROX™, Quanta BioSciences, USA.

Statistical Analysis

Sample size calculation to detect significant differences (α = 0.05) in the prevalence of VT pneumococcal carriage between pre- and post-PCV10 introduction periods was performed using the following assumptions: 50% VT pneumococcal carriage prevalence in the pre-PCV10 period among children <5 years of age, 80% PCV10 coverage, and an estimated PCV impact on VT-type carriage of 25% for HIV uninfected and 20% for HIV infected. Using these assumptions, we aimed to enroll in each survey round 300 HIV-uninfected and 400 HIV-infected children <5 years old.

Data were analyzed using STATA v.13.0 software program (StataCorp, College Station, TX) and SAS v 9.3 (Cary, NC). VT was defined as pneumococcal serotypes included in PCV10 (1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, 23F), PCV13 unique serotypes were defined as pneumococcal serotypes 3, 6A and 19A, and nonvaccine type were all other serotypes not included in the previous categories. A valid PCV10 dose was defined as a dose given at ≥6 weeks of age, ≥21 days after a previous PCV10 dose and ≥14 days before NP specimen collection. Children were considered fully vaccinated if they had received 3 valid PCV10 doses and unvaccinated if they had not received any PCV10 valid doses. Children with 1 or 2 valid doses as well as those <6 weeks old were excluded from the PCV10 impact analyses because the numbers were small and they were not age-eligible to have received any PCV dose, respectively. We compared baseline characteristics between children enrolled in the pre-PCV10 period and those enrolled in the post-PCV10 period using χ2 test for categorical variables and Wilcoxon rank-sum test for continuous variables. We used log binomial regression to calculate the prevalence ratio and 95% confidence interval for overall pneumococcal colonization, VT pneumococcal carriage and PCV13 unique serotypes by age group, vaccination and HIV status between pre- and post-PCV10 periods. The effect of PCV10 for each outcome of interest was calculated as (1−prevalence ratio) × 100%. We transformed qPCR results to Log10 CFU/mL for each study sample processed for bacterial density and compared the serotype-specific colonization density between the pre- and post-PCV10 periods by HIV status using Wilcoxon rank-sum test.


A total of 720 and 911 children were enrolled in the pre- and post-PCV10 period, respectively. Among the 911 children enrolled in the post-PCV10 period, 311 (34.1%) had 3 PCV10 valid doses, 92 (10.1%) had 1 or 2 valid doses and 508 (55.8%) had no valid doses. Among the 443 children age-eligible to have received at least 1 dose of PCV10 (children born on or after December 10, 2012), 309 (69.8%) received 3 PCV10 valid doses before NP swab collection. Children enrolled in the pre-PCV10 period were similar to those enrolled post-PCV10 with regard to age, gender, geographic location and HIV status. Children enrolled pre PCV10 were less likely to be well nourished compared with those enrolled post PCV (P = 0.02) (Table, Supplementary Digital Content 1, Pneumococcal carriage prevalence within each survey period did not differ by age group, gender or HIV status (Table, Supplementary Digital Content 2, However, among HIV-infected children, those enrolled from Maputo had lower pneumococcal carriage prevalence compared with children enrolled from Nampula or Manhiça. This difference was especially notable in the post-PCV10 period, where pneumococcal colonization prevalence was 73.4% for children from Maputo compared with 90.7% for children from Nampula and 89.1% (171/192 HIV infected) for children from Manhiça.

Changes in Pneumococcal Carriage Prevalence in Vaccinated HIV-Uninfected Children

Overall pneumococcal carriage prevalence remained stable after PCV10 introduction regardless of vaccination status, with the exception of children 6 weeks–11 months of age, in whom pneumococcal carriage prevalence increased from 81.7% in the pre-PCV10 to 96.8% in fully vaccinated children post-PCV10 period (Table 1). VT pneumococcal carriage declined by 42% (95% CI: 20%–58%; P < 0.001) among children <5 years old, from 35.9% (110/306) pre PCV10 to 20.7% (36/174 fully vaccinated) post PCV10. This decline was driven by both 6 weeks to 11 months and 12–23 months age groups, in whom VT pneumococcal carriage declined by 44% (95% CI: 1%–68%; P = 0.04) and by 41% (95% CI: 3%–63%; P = 0.03), respectively (Table 1). The prevalence of PCV13 unique serotypes pneumococcal carriage increased by 67% (95% CI: 10%–153%; P = 0.01) from 12.4% (38/306) to 20.7% (36/174 fully vaccinated) among children <5 years. This increase was mainly driven by children 6 weeks to 11 months old, who experienced an increase of 138% (95% CI: 16%–387%; P = 0.01) in the prevalence of PCV13 unique serotypes pneumococcal carriage in the post-PCV10. (Table 1).

Changes in Pneumococcal Carriage Prevalence in Vaccinated HIV-Infected Children

Overall pneumococcal carriage prevalence remained stable at around 80% among HIV-infected children <5 years old, while VT pneumococcal carriage declined by 43% (95% CI: 12%–61%; P = 0.002) from 34.8% (144/414) to 19.7% (27/137 fully vaccinated). The decline in VT pneumococcal carriage prevalence among HIV-infected children was mainly driven by changes among the age group 6 weeks to 11 months, in whom VT pneumococcal carriage prevalence declined by 64% (95% CI: 18%–84%; P = 0.01) from 34.9% pre PCV10 to 12.8% in fully vaccinated children during post-PCV10 (Table 2). The prevalence of PCV13-unique serotypes carriage remained stable in HIV-infected children across all age groups regardless of vaccination status post PCV10 (Table 2).

Changes in Pneumococcal Carriage Prevalence Among Unvaccinated Children

Because of the high vaccination coverage for at least 1 PCV10 dose, 94.6% (211/223) for HIV-uninfected and 78% (172 /220) for HIV-infected children age-eligible for vaccination (born on or after December 10, 2012), the highest number of unvaccinated children was in the oldest age group of 24–59 months. Among unvaccinated HIV-uninfected children 24–59 months old, VT pneumococcal carriage went from 36.0% pre PCV10 to 28.1% post PCV10, but the decline was not statistically significant (Table 1). Among HIV-infected children in this same age group, VT pneumococcal carriage went from 39.1% pre PCV10 to 27.3% post PCV10 with a statically significant decline of 30% (95% CI: 10%–46%; P = 0.005) (Table 2).

Impact of PCV10 Introduction on Carriage Rates Among HIV-Uninfected Children by Age and Vaccination Status, Mozambique, 2012–2015
Impact of PCV10 Introduction on Carriage Rates Among HIV-Infected Children by Age and Vaccination Status, Mozambique, 2012–2015

Serotype-Specific Changes

Among 242 and 335 HIV-uninfected children colonized with pneumococcus pre- and post-PCV period, 251 and 361 pneumococcal isolates were recovered, respectively. After excluding nontypeable isolates, 45.8% (110/240) pre PCV10 and 27.5% (92/335) post PCV10 were a PCV10 type. PCV13 unique serotypes (19A, 6A, and 3) increased from 15.8% (38/240) to 20.3% (68/335), with the largest increase in serotype 19A, which increased from 3.3% (8/240) pre PCV10 to 8.1% (27/335) post PCV10 among HIV-uninfected children (Fig. 1A). Among 337 and 431 HIV-infected children colonized with pneumococcus pre and post PCV period, 343 and 456 pneumococcal isolates were recovered, respectively. After excluding nontypeable isolates, 45.5% (145/319) pre PCV10 and 30.8% (132/429) post PCV10 were a PCV10 type. The prevalence of PCV13 unique serotypes was 19.8% (63/319) pre PCV10 and 18.9% (81/429) post PCV among HIV-infected children (Fig. 1B).

Figure 1.
Figure 1.:
Serotype distribution by HIV status pre and post introduction of 10-valent pneumococcal conjugate vaccine in children < 5 years of age in Mozambique. A: HIV-uninfected children. *Not including nontypeable isolates (n = 11 pre PCV10 and n = 26 post PCV10). B: HIV-infected children. *Not including nontypeable isolates (n = 24 pre PCV10 and n = 27 post PCV10).

Compared with HIV-uninfected children, those who were HIV infected had a higher median bacterial colonization density in both the pre- and post-PCV10 introduction; however, this difference was only significant for the 11A serotype in the post-PCV period (P = 0.009). No changes in bacterial density for serotypes 11A, 19A and 19F were observed comparing levels pre-PCV10 introduction to post-PCV10 introduction (Fig., Supplemental Digital Content 3,


We observed a substantial reduction of VT pneumococcal NP carriage in HIV-infected and HIV-uninfected children in Mozambique within 2 years after PCV10 introduction in the national immunization program. However, VT pneumococcal carriage prevalence remained high despite observed reductions in the post-PCV period. Declines in VT pneumococcal colonization occurred in both vaccinated and unvaccinated children. Children 6 weeks to 11 months old had the largest declines in VT pneumococcal carriage rate, 64% and 44% in HIV-infected and HIV-uninfected children, respectively. This youngest age group is at the highest risk of pneumococcal disease, with incidence at least 3-fold higher than among older children.25 Therefore, early declines in VT pneumococcal carriage prevalence in this age group are encouraging.

Although VT pneumococcal carriage was 41% lower in HIV-uninfected children 12–23 months old who received 3 PCV10 valid doses compared with baseline, no significant decline was observed among HIV-infected children in this age group. HIV-infected children in this age group had a lower vaccination coverage with 3 PCV10 doses compared with HIV-uninfected children (64.9% vs. 88.9%). In addition, the prevalence of VT pneumococcal carriage pre PCV10 in this patient population was lower compared with HIV-uninfected children (26.5% vs. 35.1%), possibly explained in part by the fact that 90% of HIV-infected children 12–23 months old were receiving trimethoprim-sulfamethoxazole prophylaxis at the time of enrollment. Failure to detect a significant decrease in carriage among HIV-infected children might therefore have resulted from low vaccination coverage and low baseline prevalence, which decreased statistical power, as opposed to a diminished immune response to the vaccine in these children.

Among HIV-uninfected children <5 years of age, the prevalence of PCV13 unique serotypes (3, 6A, 19A) increased from 12.4% (38/306) to 20.7% (36/174 fully vaccinated) during post-PCV10 period. This increase was mainly driven by increases that occurred among children 6 weeks to 11 months old who received 3 valid doses of PCV10 and by serotype 19A. In comparison, data on PCV10 effect on NP pneumococcal carriage from Kilifi county in Kenya showed an increase in the 19A and 6A carriage prevalence post PCV in children < 5 years of age; however, the increase was not statistically significant. Although Kenya has a PCV schedule similar to Mozambique, Kilifi county did a catch-up campaign for children <5 years of age when the vaccine was introduced in 2011. Furthermore, the Kilifi county analysis differed from ours in that PCV13 unique serotype carriage post PCV10 introduction was not assessed among children who had received 3 valid PCV10 doses.26 The significant increase in PCV13 serotypes we observed was mainly among those who received 3 PCV10 valid doses. In contrast, in Brazil, which introduced a PCV10 3 + 1 dose schedule with catch-up for children <23 months old, no increases in PCV13 unique serotype carriage were observed. This is consistent with the results of a clinical trial from Finland, which showed reduction of serotype 19A carriage only in children 18–22 months who had received a 3 + 1 schedule.27,28 Although we observed an increase in PCV13 unique serotype carriage prevalence in HIV-uninfected children, no increases were observed in HIV-infected children who were fully vaccinated. Therefore, continued monitoring of the changes in the pneumococcal carriage prevalence for the three PCV13 unique serotypes is needed to confirm if observed increases will hold as new birth cohorts are vaccinated.

Despite observed early declines in VT pneumococcal carriage prevalence among fully vaccinated children in both HIV-infected and HIV-uninfected groups, the prevalence of VT carriage 2 years after PCV10 introduction remains high, at 20.4% and 20.7%, respectively. In other countries that have introduced PCV, VT pneumococcal carriage dropped dramatically within 3 years after introduction. In Brazil, VT pneumococcal carriage prevalence dropped from 19.8% to 1.8% 3 years after PCV10 introduction.28 In the United States, VT pneumococcal carriage dropped from 29% to 3% also within 3 years after PCV13 introduction.16 In both of these countries, a booster dose at 12 months of age was given, which may have led to a more rapid decline. In the Gambia, where a 3 + 0 PCV13 dose schedule is used, VT pneumococcal carriage decreased from 23.7% to 13.7% within 1 year after PCV13 replaced 7-valent PCV (PCV7).17 Data on the impact of PCV10 introduction on VT pneumococcal carriage from African countries are limited. Multiple factors might contribute to a persistently high VT pneumococcal carriage prevalence among vaccinated children in Mozambique. These include the following: 1) high prevalence of VT pneumococcal carriage pre PCV10 introduction, 2) a previously PCV-naïve population and 3) lack of a booster dose at 12 months of age. In addition, the lack of a catch-up campaign with vaccination for older infants and toddlers might result in a need for more time to realize the full effects of the PCV10 program on carriage in Mozambique. A recent U.S. study among the Navajo Nation and the White Mountain Apache American Indian tribes, toddlers and older children were more involved in the pneumococcal transmission, so it is possible that VT carriage rates will continue to decline as vaccinated children age and additional birth cohorts are vaccinated.29 However, any decision to reduce the PCV 3-dose schedule to a 2-dose schedule in countries with high PCV coverage in Africa, as it was recently done in the United Kingdom,30 requires more compelling reductions in VT colonization than what we observed.

In our study, pneumococcal colonization prevalence was lower in children from Maputo, which is the capital of Mozambique. This could be explained by differences in outpatient antibiotic consumption in Maputo compared with Manhiça and Nampula. In Maputo, antibiotics are widely available without prescription at private pharmacies. In contrast, in Manhiça, outpatient antibiotics are provided only at health centers with frequent out of stock, and in Nampula, antibiotic use is limited by the challenges in health care access.

No significant changes in bacterial density of serotypes 11A, 19A, 19F pneumococcal colonization were observed after PCV10 introduction. This finding is similar to what was observed in the Gambia among children <5 years of age for both VT and non-VT colonization after PCV7 introduction12 and in Peru for VT pneumococcal colonization after PCV7 introduction.31 The median colonization density for VT pneumococci of 5.5 log10 CFU/mL found in children <5 years of age in Peru was very similar to the median bacterial colonization density we found for 19F pneumococcal colonization pre and post PCV10 introduction. Although bacterial density did not change after PCV10 introduction, we observed a higher pneumococcal colonization density in HIV-infected children, which may put these children at a higher risk of pneumonia and other forms of severe pneumococcal disease.13,14

Our study has several limitations. We only enrolled HIV-uninfected children from Manhiça, where demographic disease surveillance was available and coverage of infant vaccines is usually high, limiting our ability to generalize the findings to the entire country. Second, for the sample size calculation, we estimated a higher VT pneumococcal prevalence in the pre-PCV10 era (50%) than what we observed (36.2% in HIV uninfected and 34.2% in HIV infected), and although we attempted to enroll more participants in the post-PCV10 survey, we may have lacked power to detect statistical differences in some of the age groups. Third, HIV-infected children were recruited from HIV outpatient clinics as a convenience sample, and the majority of them were on antiretroviral therapy. Therefore, the PCV impact we observed in this group may not be generalizable to those HIV-infected children who are not on antiretroviral therapy.

Despite these limitations, we were able to demonstrate an early impact of PCV10 introduction on VT pneumococcal carriage in both HIV-infected and HIV-uninfected children in a low-income setting. These findings represent the first evidence to suggest that PCV10 introduction in Mozambique was effective in reducing VT pneumococcal colonization among vaccinated and unvaccinated children. However, VT pneumococcal carriage prevalence remains high after PCV introduction, and increases in PCV13 unique serotype carriage is concerning. Continued monitoring of non-PCV10 type carriage and disease will be important to confirm observed increases in PCV13 unique serotype pneumococcal carriage, as well as to determine whether these serotypes are increasingly causing disease. Surveillance data and observational studies with an endpoint of IPD in Mozambique will be critical to inform PCV impact on VT and non-VT IPD.


The authors thank the children and their parents for participating in the study. The authors thank Jorge Uqueio for the clinical study coordination, Fortunato Romão, Berta Juga, Elsa Balze for the sample collection, all clinicians and Bacteriology laboratory technicians for their dedication to implementing this study. The authors also would like to thank the staff from Manhiça District Hospital, Mavalane Genaral Hospital and Nampula Central Hospital for their contributions towards this study. Finally, the authors thank the district health authorities from Manhiça District and the Ministry of Health for their support of this study.


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pneumococcus; carriage; pneumococcal conjugate vaccine; vaccine impact

Supplemental Digital Content

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