Risk factors associated with Streptococcus pneumonia carriage in children under five years old with acute respiratory infection in Niger
Ibrahim Dan Dano1,&, Sani Ousmane1, Kamaye Moumouni2, Adamou Lagare1, Idi Issa1, Jean Testa1
1Centre de Recherche Médicale et Sanitaire (CERMES), 634 Boulevard de la Nation, Niamey, Niger, 2Hôpital National de Niamey (HNN), Service de Pédiatrie A, Niamey, Niger
Ibrahim Dan Dano, Centre de Recherche Médicale et Sanitaire (CERMES), 634 Boulevard de la Nation, Niamey, Niger
streptococcus pneumonia is a leading cause of bacterial pneumonia, meningitis and sepsis in children, and pneumococcal carriage is an important source of horizontal spread of these pathogens within the community.
a questionnaire was addressed to parents for the collection of sociodemographic and medical information. Nasopharyngeal swabbing was processed using a molecular method. We used logistic regression models to examine independent associations between pneumococcal carriage and potential risk factors. All associations with a p-value of < 0.25 in the bivariate regression analyses were subsequently entered in the multivariate regression model.
a total of 637 children aged 1 to 59 months admitted for acute respiratory infection were included. The rate of respiratory virus carriage was 76%, whereas that of bacteria was 47% and that of bacteria-virus co-colonization was 42%. A bivariate analysis showed that carriage was not related to gender, father's or mother's education level, father's occupation, type of housing or lighting, or passive exposure to cigarette smoking in the house. It was also not linked to complete vaccination with PCV-13 or PPSV-23 and antibiotic treatment prior to hospitalization. A multivariate analysis showed that carriage was related to age greater than 3 months, maternal occupation, house flooring type, and co-colonization of another bacterium and virus.
these results can be helpful to understand the dynamics of pneumococcal nasopharyngeal colonization; they confirm the interest of vaccinating infants before the age of 3 months with appropriate vaccine to prevent spread nasopharyngeal colonization and pneumococcal diseases in children.
streptococcus pneumonia is a significant human pathogen and a leading cause of bacterial pneumonia, meningitis, and sepsis in children . S. pneumonia causes an estimated 11% (8-12%) of all deaths in children aged 1-59 months . S. pneumonia is a commensal of the human respiratory tract that is carried by many people without becoming ill. An important feature is that pneumococcal diseases will not occur without preceding nasopharyngeal (NP) colonization with a homologous strain . Cross-sectional studies have shown that, at any given time, approximately 20 - 50% of healthy children harbour at least one serotype of this bacterium in the rhinopharynx, whereas longitudinal studies have shown that almost all children can carry this bacterial species [3, 4]. Pneumococcal carriage is an important source of horizontal spread of these pathogens within the community . Such pneumococcal NP carriage studies are important because carriage has a key role in pneumococcal disease and pneumococcal spread. We assessed risk factors associated with the carriage of S. pneumonia in children under five with signs of respiratory infection.
Study design: this cross-sectional study was conducted in the paediatric departments of the National Hospital of Niamey and that of Lamordé (Niger). Children aged 1 to 59 months with signs of respiratory infection (severe cough or tachypnea with axillary temperature above 38°C) who were hospitalized in one of the paediatric departments of the two hospitals were enrolled from January 2015 to June 2016. Children under the age of one month or over the age of five years and children suffering from heart disease or chronic respiratory infection were excluded. NP samples were obtained for molecular identification. Data on potential risk factors were gathered by confidential interview based on a questionnaire.
this study was approved by the National Consultative Ethics Committee of Niger by decision No 0016/2013/CCNE on 30 October 2013; approved consent from the parents of the children was requested before inclusion.
nasopharyngeal swabbing was performed with the nasopharyngeal flocked swabs kit, UTMTM
350C, Copan Diagnostics. The swabs were inserted along the nasal septum just above the floor of the passage to the nasopharynx until resistance was met; the swab was rotated gently against the NP mucosa for 10 - 15 seconds and then gently removed. After the swab removed from the patient, it was placed into the tube of UTMTM
transport medium all the way to the bottom of the tube.
Serum procalcitonin (PCT) level dosage:
the serum PCT level was determined for the diagnosis of bacterial infection with the BRAHMS PCT kit (reference 30450) on a VIDAS machine (BioMérieux SA, France). A PCT level below 0.1 µg/L was considered normal, 0.1 to 2 µg/L was considered moderate and above 2 µg/L was considered high.
RT- PCR for the detection of viral and bacterial NP colonization:
NP carriage of S. pneumonia
was determined by a multiplex real-time PCR with the FTD Respiratory pathogens 21 plus (Ref: FTD-2+.1-32. Fast-Track Diagnostics Luxembourg Sarl). This test enables the detection of the following respiratory pathogens: influenza A (Flu A), influenza A (H1N1) swl, influenza B (Flu B); coronaviruses NL63 (Cor63), 229E (Cor229), OC43 (Cor43) and HKU1 (CorHKU1); parainfluenza 1, 2, 3 and 4 (Para1, Para2, Para3, Para4); human metapneumovirus A and B (HMPVA and B); rhinovirus (Rhino); respiratory syncytial viruses A and B (RSV A and B); adenovirus (AV); enterovirus (EV); parechovirus (PV); bocavirus (HboV); Mycoplasma pneumonia
(Mpneu); Chlamydia pneumonia
(Cpneu); Streptococcus pneumonia
(Spneu); Haemophilus influenza
B (HIB); and Staphylococcus aureus
statistical analyses were performed with Epi info 7. We used logistic regression models to examine independent associations between pneumococcal carriage and potential risk factors. All associations with a p-value of < 0.25 in the bivariate regression analyses were subsequently entered in the multivariate regression model. Odd ratios and adjusted odd ratios with their 95% confidence intervals were computed. P-values ≤ 0.05 were considered statistically significant.
A total of 637 children aged 1 to 59 months admitted for acute respiratory infection were included in this study. The mean age of the children was 13.2 ± 12.65 months, and the female to male ratio was 0.8. The rate of respiratory virus carriage was 76%, that of bacteria carriage was 47% and that of bacteria-virus co-colonization was 42%. The most detected bacteria was S. pneumonia (39.6%), and the most detected virus was respiratory syncytial viruses A and B (23.7%), followed by rhinovirus (9.9%) and influenza A (7.1%). Details of the NP carriage rate of the respiratory pathogens detected are summarized in Figure 1, and the distribution according to age of the main pathogens detected is shown in Figure 2.
In the bivariate analysis, age older than 3 months (except for 24-36 months), house flooring material of tile, mother's profession of civil servant, non-attendance at a day care centre and co-carriage with another bacteria or virus were risk factors for S. pneumonia carriage (Table 1,Table 2). No association was found with regard to sex, duration of breast feeding, educational level, father's profession, type of house and electric lightning type of house, number of children less than 15 years old in the family, vaccination status, infection and prior antibiotic treatment.
Risk factors for S. pneumonia carriage in the multivariate logistic analysis were age older than 3 months (except for 24-36 months), house flooring material of tile, mother's profession of civil servant and co-carriage with another bacteria or virus (Table 3). In the simple logistic regression analysis, attendance at a day care centre was inversely related to S. pneumoniacarriage, but the association was not significant in the multivariate model.
The nasopharynx is known to be the main ecological reservoir of S. pneumonia and is where the bacteria give rise to disease after extending to other areas of the respiratory tract or penetrating normally sterile body fluids . Although NP isolates are not useful for predicting the causative agent of invasive disease in individuals, they reflect epidemiological aspects of pneumococcal disease in the community . In developing countries, particularly in Niger, there are few studies describing the epidemiology of NP carriage of S. pneumonia. This study focused on children under five years old with signs of respiratory infection. Most NP pneumococcal carriage studies enrolled children  or younger children [9-12]. Several studies have included healthy children  whereas others included children with concomitant respiratory infection . This study described the epidemiology of NP carriage of S. pneumonia. The bacterial carriage rate obtained in this study was comparable to those in previous studies in younger children [3, 4]; likewise, the viral carriage rate was also comparable [14-16]. Carriage of S. pneumonia was associated with age; the peak incidence of carriage in children is observed during the first 3 years of life , and the lowest carriage rate is observed in infants younger than 2 months of age . The mean age of first acquisition of S. pneumonia was reported to be 6 months with a range of 1-30 months [18-20]. Therefore, age is an important risk factor for pneumococcal colonization, and its influence varies with the phase of growth . A positive association between the carriage of other pathogens and the NP carriage of S. pneumonia was found in this study. Bacterial carriage is known to be influenced by bacterial and viral intra-and inter-species interactions (commensal and pathogenic) . These interactions can be synergistic or antagonistic. In general, the risk of S. pneumonia seemed to increase in the presence of particular respiratory viruses . The carriage of S. aureus has been reported to be inversely related to S. pneumonia carriage  likely due to bacterial interference ; however, this study failed to show such a negative association. It is unknown whether this association is direct or dependent on other determinants  such as age  and season . Environmental features are risks factors for S. pneumonia carriage . Low socio-economic status was also reported as a risk factor for the colonization and carriage of respiratory pathogens, likely due to cramped and poor housing .
The mother's occupation appeared to affect S. pneumonia
carriage more than the father's profession in this study. Pneumococcal carriage was reported to be associated with exposure to other children [19
]. Frequent and close person-to-person contact favours the development and transmission of pneumococci. Attendance at a day care centre was reported to be a risk factor for pneumococcal carriage [22
], particularly in developed countries [25
]. In this study, attendance at a day care centre was surprisingly inversely associated with pneumococcal carriage in the bivariate analysis, but it was not significant in the multivariate analysis. This could be a consequence of a confounding effect. The high prevalence of carriage in developing countries is a possible explanation of the lack of association between pneumococcal carriage and attendance at a day care centre [26
]. Close contact and poor hygienic conditions cannot explain increased pneumococcal carriage in day care centres. Additional factors are apparently associated, such as age, number of children attending the day care centre and pathogen interference. No association between pneumococcal carriage and the number of children less than 15 years old in the family was found. A study in Gambia [12
] also failed to identify an association with the number of children in the house or children sleeping in the same room. The relationship between the carriage of S. pneumonia
and educational level is not clear. Although some studies showed a close relationship between low educational level of the parents and higher nasopharyngeal colonization [12
], other studies [13
] such this study failed to show this association. Although reported in some studies [12
], this study did not identify any associations between the carriage of S. pneumonia
, smoking habits, breast feeding and recent antibiotic use. However, the results obtained showed that when the house floor material was tile, the prevalence of carriage increased. A study performed in Gambia [26
] did not identify this association, but they did not consider tile in their design. Several studies from different parts of the world [31-33
] have demonstrated the ability of different pneumococcal conjugate vaccines to reduce the rate of pneumococcal carriage. It has been reported [34
] that after vaccination with a conjugate vaccine, rapid replacement of pneumococci by new serotypes not included in the vaccine occurs; thus, cross protection can also occur. This might explain the lack of association between pneumococcal carriage and vaccination with PCV-13 observed in this study, which did not identify the different pneumococcal serotypes. Unlike other studies [14
], no association between NP carriage of S. pneumonia
and antibiotic pre-treatment was found, as reported by another study [37
The differences with other studies suggest that the factors that influence colonization are multiple and not entirely clear [22
]. This study included children with signs of respiratory infection who were visiting a paediatric care centre; thus, these results may not represent a healthy paediatric population.
These results can be helpful to understand the dynamics of pneumococcal nasopharyngeal colonization; they confirm the interest of vaccinating infants before the age of 3 months with appropriate vaccine to prevent nasopharyngeal colonization and pneumococcal diseases in children.
What is known about this topic
- Pneumococcal nasopharyngeal colonization is generally high in children;
- The rate of pneumococcal nasopharyngeal colonization vary by region;
- Pneumococcal nasopharyngeal colonization were affected by many factors.
What this study adds
- Description of risks factors associated to pneumococcal nasopharyngeal colonization in Niger;
- Risks factors associated to pneumococcal nasopharyngeal colonization in children observed in Niger differ from those observed in developed countries;
- Age appear as the most common risk factor observed and is important for preventive measures.
The authors declare no competing interest.
Study design: Ibrahim Dan Dano, Sani Ousmane, Kamaye Moumouni. Laboratory analysis: Ibrahim Dan Dano, Sani Ousmane, Adamou Lagare. Data analysis and interpretation: Ibrahim Dan Dano, Jean Testa. Wrote the manuscript: Ibrahim Dan Dano. Critical review and approval of manuscript: Sani Ousmane, Kamaye Moumouni Adamou Lagare Idi Issa Jean Testa. All the authors have read and agreed to the final manuscript.
We thank the total Corporate Foundation who financed this work through the department of International Affairs, Institut Pasteur International Network. We warmly thank the personnel of the Bacteriology-Virology Unit and Epidemiology Unit of CERMES, the personnel of the Pediatric Department of National Reference Hospital Niamey and National Reference Hospital of Lamordé, Niamey for their contributions.
Tables and figures
Table 1: sociodemographic potential risk factors estimated by bivariate logistic regression for the carriage of S. pneumoniae in children under five years old with acute respiratory infection
Table 2: medical potential risk factors estimated by bivariate logistic regression for the carriage of S. pneumonia in children under five years old with acute respiratory infection
Table 3: adjusted ORs estimated by multivariate logistic regression for S. pneumoniae carriage, according to possible risk factors
Figure 1: NP bacterial and viral carriage legende: Flu A: influenza A; H1N1: influenza A swl; Flu B: influenza B; Cor63: coronaviruses NL63; Cor229: coronaviruses 229E; Cor43: coronaviruses OC43; CorHKU1: coronaviruses HKU; Para 1: parainfluenza 1; Para 2: parainfluenza 2; Para 3: parainfluenza 3; Para 4: parainfluenza 4; HMPVA and B: human metapneumovirus A and B; Rhino : rhinovirus; RSV A and B: respiratory syncytial viruses A and B; AV: adenovirus; EV: enterovirus; PV: parechovirus; HboV: bocavirus; Mpneu: Mycoplasma pneumoniae; Cpneu: Chlamydia pneumoniae; Spneu: Streptococcus pneumoniae; HIB: Haemophilus influenzae B; Saur: Staphylococcus aureus
Figure 2: NP carriage of main pathogens detected according to age
- Katherine L O'Brian, Lara JJ, James PW, Emily Henkle, Maria Deloria-Knoll, Nathalie McCall et al. Burden of diseases caused by Streptococcus pneumoniae in children younger than 5 years: global estimates. Lancet. 2009; 374(9693): 893-902. PubMed | Google Scholar
- Bogaert D, de Groot R, Hermans PWH. Streptococcus pneumoniae colonization: the key to pneumococcal disease. Lancet Infect Dis. 2004; 4(3): 144-154. PubMed | Google Scholar
- Cohen R, Levy C, Bingen E, Bechet S, Derkx V, Werner A, Koskas M, Varon E. Nasopharyngeal carriage of children 6 to 60 months during the implementation of the 13-valent pneumococcal conjugate vaccine. Arch Pediatr. 2012; 19(10): 1132-1139. PubMed | Google Scholar
- Sharma Dolly, Baughman Wendy, Holst Amy, Stephanie Thomas, Delois Jackson MS, Carvalho da Gloria Maria et al. Pneumococcal carriage and invasive disease in children before introduction of the 13-valent conjugate vaccine: comparison with the era before 7-valent conjugate vaccine. Pediatr Infect Dis J. 2013; 32(2): e45-e53. PubMed | Google Scholar
- Kazunori Oishi, Kazuyo Tamura, Yukihiro Akeda. Global Control of Pneumococcal Infections by Pneumococcal vaccines. Tropical Medicine and health. 2014; 42(2): s83-s86. PubMed | Google Scholar
- Robert Austrain. Some aspects of pneumococcal carrier state. J Antimicrob Chemother. 1986; 18(Suppl A): 35-45. PubMed | Google Scholar
- Brueggemann, AB, Griffiths DT, Emma Meats, Timothy Peto, Crook DW, and Spratt BG (2003). Clonal relationship between invasive and carriage Streptococcus pneumoniae and serotype and clone-specific differences in invasive disease potential. J Infect Dis. 2003; 187(9): 1424-1432. PubMed | Google Scholar
- Cardozo Déa M, Nascimento-Carvalho Cristiana MC, Souza Fabiane R, Silva Nivea MS. Nasopharyngeal colonization and penicillin resistance among pneumococcal strains: a world-wide 2004 update. Barz J Inf Dis. 2006; 10(4): 293-304. Google Scholar
- Pablo YN, Porat Drora, Fraser Felicia, Prajgrod Marlene, MeriresLesley, McGee KP et al. Acquisition, carriage and transmission of pneumococci with decreased antibiotic susceptibility in young children attending a day care facility in southern Israel. J Infect Dis. 1998; 177(4): 1003-1012. PubMed | Google Scholar
- Henderson WF, Gilligan PH, Kimberly Wait, Goff DA. Nasopharyngeal carriage of antibiotic-resistant pneumococci by children in group day care. J Infect Dis. 1998; 157(2): 256-263. Google Scholar
- Ergin Çiftçi, Ülker Dogru DA, Erdal ince, Haluk Güriz. Investigation of risk factors for penicillin-resistant Streptococcus pneumoniae carriage in Turkish. Children Pediatr Int. 2001; 43(4): 385-390. PubMed | Google Scholar
- Coles CL, Kanungo Reba, Rahmathullah Lakshmi, Ramilla DT, Joanne SCD, Santosham Mathuram et al. Pneumococcal nasopharyngeal colonization in young South Indian infants. Pediatr Infect Dis J. 2001; 20(3): 289-295. PubMed | Google Scholar
- Boost MV, O'Donoghue MM, Dooley JS. Prevalence of carriage of antimicrobial resistant strains of Streptococcus pneumoniae in primary school children in Hong Kong. Epidemiol Infect. 2001; 127(1): 49-55. PubMed | Google Scholar
- van den Bergh MR, Giske Biesbroek, Rossen JWA, de Steenhuijsen Piters WAA, Astrid ATM Bosch, Elske JM van Gils et al. Associations between Pathogens in the Upper Respiratory Tract of Young Children: Interplay between Viruses and Bacteria. PLoS ONE. 2012; 7(10): e47711. PubMed | Google Scholar
- Rosalyn JS, Lisa RB, Karen Miernyk, Carolynn DeByle, Lori Pruitt, Kimberlee BH et al. Viral respiratory infections in hospitalized and community control children in Alaska. J Med Virol. 2010; 82(7): 1282-1290. PubMed | Google Scholar
- Selma PW, Glenys RC, Lea-Ann SK, Karli JC, Eva NM, Shyan Vijayasekaran et al. High detection rates of nucleic acids of a wide range of respiratory viruses in the nasopharynx and the middle ear of children with a history of recurrent acute otitis media. J Med Virol. 2011; 83(11): 2008-2017. PubMed | Google Scholar
- Ron Dagan, Ofra Shriker, Inbal Hazan, Eugene Leibovitz, David Greenberg, Francis Schlaeffer et al. Prospective study to determine clinical relevance of detection of pneumococcal DNA in sera of children by PCR. J Clin Microbiol. 1998; 36(3): 669-673. PubMed | Google Scholar
- Ghaffaar Faryal, Friedland IR and McCracken GH. Dynamics of nasopharyngeal colonization by Streptococcus pneumoniae. Pediatric Infectious Disease Journal. 1999; 18(7): 638-646. PubMed | Google Scholar
- Howard Faden, Linda Duffy, Raymond Wasielewski, Judy Wolf, Debra Krystofik, Ying Tung. Relationship between nasopharyngeal colonization and the development of otitis media in children. Journal of Infectious Diseases. 1997; 175(6): 1440-1445. PubMed | Google Scholar
- Raymond Josette, Armand-Lefevre Laurence, Moulin Florence, Dabernat Henri, Commeau Anne, Gendrel Dominique et al. Nasopharyngeal colonization by Haemophilus influenzae in children living in an orphanage. Pediatric Infectious Disease Journal. 2001; 20(8): 779-784. Google Scholar
- Cardozo DM, Nascimento-Carvalho CM, Anna Lucia SSA, Silva Neto AM, Daltro CHC, Maria-Angelica SB et al. Prevalence and risk factors for nasopharyngeal carriage of Streptococcus pneumoniae among adolescents. Journal of Medical Microbiology. 2008; 57(2): 185-189. PubMed | Google Scholar
- García-Rodríguez JA, Fresnadillo Martínez MJ. Dynamics of nasopharyngeal colonization by potential respiratory pathogens. J Antimicrob Chemother. 2002; 50(S2): 59-73. PubMed | Google Scholar
- Gili Regev-Yochay, Ron Dagan, Meir Raz, Yehuda Carmeli, Bracha Shainberg, Estela Derazne et al. Association Between Carriage of Streptococcus pneumoniae and Staphylococcus aureus in Children. JAMA. 2004; 292(16): 716-720. PubMed | Google Scholar
- Joseph AL, Noga Givon-Lavi, Amit Huppert, Melinda MP, Regev-Yochay, Ron Dagan et al. Epidemiological Markers for Interactions Among Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus in Upper Respiratory Tract Carriage. JID. 2016; 213(10): 1585-1605. PubMed | Google Scholar
- Gili Regev-Yochay, Meir Raz, Ron Dagan, Nurith Porat, Bracha Shainberg, Erica PincoNathan et al. Nasopharyngeal carriage of Streptococcus pneumoniae by adults and children in community and family settings. Clin Infect Dis. 2004; 38(5): 632-639. PubMed | Google Scholar
- Philip CH, Abiodun Akisanya, Kawsu Sankareh, Yin Bun Cheung, Mark Saaka, George Lahai et al. Nasopharyngeal Carriage of Streptococcus pneumoniae in Gambian Villagers. Clinical Infectious Diseases. 2006; 43(6): 673-679. PubMed | Google Scholar
- Jennifer St Sauver, Marrs CF, Betsy Foxman, Patricia Somsel, Robbie Madera, Janet RG. Risk factors for otitis media and carriage of multiple strains of Haemophilus influenzae and Streptococcus pneumoniae. Emerging Infectious Diseases. 2000; 6(6): 622-630. PubMed | Google Scholar
- Fontanals D, Bou R, Pons I, Sanfeliu I, Domínguez A, Pineda V et al. Prevalence of Haemophilus influenzae carriers in the Catalan preschool population. European Journal of Clinical Microbiology and Infectious Diseases. 2000; 19(4): 301-304. PubMed | Google Scholar
- Principi Nicola, Marchisio Paola, Schito GC, Mannelli Stefania. Risk factors for carriage of respiratory pathogens in the nasopharynx of healthy children. Pediatr Infect Dis. 1999; 18(6): 517-523. PubMed | Google Scholar
- Petrosillo N, Pantosti A, Bordi E, SpanóM A, Del Grosso, Tallarida B et al. Prevalence, determinants and molecular epidemiology of Streptococcus pneumoniae isolates colonizing the nasopharynx of healthy children in Rome. Eur J Clin Microbiol Infect Dis. 2002; 21(3): 181-188. PubMed | Google Scholar
- Alberto Leiberman, Ron Dagan, Eugene Leibovitz, Pablo Yagupsky Fliss DM. The bacteriology of the nasopharynx in childhood. Int J Pediatr Otorhinolaryngol. 1999; 49(suppl 1): S151-S153. PubMed | Google Scholar
- Ron Dagan, Fraser Drora. Conjugate pneumococcal vaccine and antibiotic-resistant Streptococcus pneumoniae: herd immunity and reduction of otitis morbidity. Pediatr Infect Dis J. 2000; 19(Suppl 5): S79-S88. PubMed | Google Scholar
- Obaro SKF, Adegbola RAF, Chang IH. Safety and immunogenicity of a nonavalent pneumococcal vaccine conjugate to CRM197 administered simultaneously but in a separate syringe with diphtheria, tetanus and pertussis vaccines in Gambian infants. Pediatr Infect Dis J. 2000; 19(5): 463-469. PubMed | Google Scholar
- Ron Dagan, Noga Givon-Lavi, Orly Zamir, Merav Sikuler-Cohen, Lior Guy, Jacob Janco et al. Reduction of Nasopharyngeal Carriage of Streptococcus pneumoniae after Administration of a 9-Valent Pneumococcal Conjugate Vaccine to Toddlers Attending Day Care Centers. JID. 2002; 185(7): 927-936. PubMed | Google Scholar
- Shelley LD, Rosario Palacio, Raúl Ruvinsky, Daniel AK, Maria Hortal, Alicia Rossi et al. Risk factors and course of illness among children with invasive penicillin-resistant Streptococcus pneumoniae. Pediatrics. 1999; 103(2): 409-413. PubMed | Google Scholar
- Deeks SL, Palacio R, Ruvinsky R, Kertesz DA, Hortal M, Rossi A. Risk factors for invasive pneumococcal disease in children: a population-based case-control study in North America. Pediatrics. 1999; 103(3): E28. PubMed | Google Scholar
- Paola Marchisio, Susanna Esposito, Schito GC, Anna Marchese, Roberta Cavagna, Nicola Principi et al. Nasopharyngeal Carriage of Streptococcus pneumoniae in Healthy Children: Implications for the Use of Heptavalent Pneumococcal Conjugate Vaccine. Emerging Infectious Diseases. 2002; 8(5): 479-484. PubMed | Google Scholar