- Research article
- Open Access
- Open Peer Review
Oropharyngeal and nasal Staphylococcus aureus carriage by healthy children
BMC Infectious Diseases volume 14, Article number: 723 (2014)
As healthy children are the main reservoir of respiratory pathogens and the main cause of bacterial diffusion in the community, it could be interesting to investigate the type of screening that should be used during the early years of life in order to obtain a more precise estimate of Staphylococcus aureus circulation. The aim of this study was to evaluate oropharyngeal and nasal S. aureus carriage in otherwise healthy children and adolescents aged 6–17 years.
The oropharyngeal and nasal samples were collected in December 2013 from 497 healthy students attending five randomly selected schools in Milan, Italy, using an ESwab kit, and S. aureus was identified using the RIDA®GENE methicillin-resistant S. aureus (MRSA) system.
Two hundred and sixty-four subjects (53.1%) were identified as S. aureus carriers: 129 (25.9%) oropharyngeal carriers and 195 (39.2%) nasal carriers, of whom 60 (12.1%) were both oropharyngeal and nasal carriers. Oropharyngeal carriage increased with age (p < 0.001), whereas nasal carriage decreased. There was little or no agreement between oropharyngeal and nasal carriage in any of the age groups. MRSA was identified in only three cases (0.6%), always in nasal samples. There were no differences between the carriers and non-carriers in terms of the distribution of age, gender, ethnicity, the number of siblings in the household, exposure to passive smoking, previous clinical history, allergic sensitisation, or previous influenza, pneumococcal and meningococcal vaccinations. The frequency of male children was higher among the subjects with positive nasal and oropharyngeal swabs (66.7%) than among those with positive oropharyngeal swabs alone (46.4%; p = 0.02).
The oropharyngeal carriage of mainly methicillin-sensitive S. aureus is frequent in otherwise healthy children, including a relatively high proportion of those without nasal colonisation. These findings highlight the importance of adding throat to nasal screening when monitoring the circulation of S. aureus in the community.
Staphylococcus aureus carriers are at increased risk of developing S. aureus infections after invasive medical or surgical procedures, and more frequently develop S. aureus bacteremia when admitted to hospital . Consequently, routine admission screening is strongly recommended in high-risk units, and the general screening of all hospitalised patients is debated . The emergence of methicillin-resistant S. aureus (MRSA), and evidence that difficult to treat S. aureus-related diseases can also occur in the community has increased the importance of general screening in order to monitor S. aureus circulation and its susceptibility to antibiotics , and also explains why S. aureus carriage in the community has recently been periodically evaluated ,.
S. aureus screening is usually carried out using nasal swabs because the anterior nares are considered the primary site of S. aureus colonisation, which can be found in the nares of up to 40% of healthy subjects . Additional screening is currently considered unnecessary , but the findings of recent studies indicate that the throat can also be an important site of S. aureus colonisation, and that a relatively large number of subjects may be colonised exclusively in the throat -, and would be missed by nasal screening. This has led some authors to conclude that S. aureus screening should include both nose and throat swabs in order to allow a complete evaluation . Most studies of throat carriage have involved hospitalised adults, and there are very few data concerning children. However, as healthy children are the main reservoir of respiratory pathogens and the main cause of bacterial diffusion in the community , it could be interesting to investigate the type of screening that should be used during the early years of life in order to obtain a more precise estimate of S. aureus circulation.
The aim of this study was to evaluate oropharyngeal and nasal S. aureus carriage in otherwise healthy children and adolescents aged 6–17 years.
Oropharyngeal and nasal swabs were collected during the second and third week of December 2013 from children and adolescents attending five schools in Milan, Italy (two primary schools, two middle schools, and one high school), randomly selected with a computer-based list from the public schools considered representative of the middle class population living in Milan. Participation was completely voluntary, but was solicited by means of a letter and brochure distributed during lessons that described the characteristics of the study in detail and requested the written informed consent of both parents and the signed assent of the study subjects. Moreover, in order to maximise participation, all of the teachers reinforced the message in the week preceding the swabbing by giving detailed explanations concerning S. aureus carriage and its related diseases.
The study was approved by the Ethics Committees of the participating schools and the Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy. The children were enrolled after parental consent and subject assent had been obtained, and a brief demographic and clinical questionnaire had been completed. Children with a known, chronic underlying disease and those who had been treated with antibiotics in the previous three weeks were excluded from the study.
The swabbing was carried out in the medical room of each school at the end of the lessons on two consecutive days by a group of specifically trained pediatric residents supervised by a pediatrician (NP) using an ESwab kit containing a polypropylene screw-cap tube with an internal conical shape filled with 1 mL liquid Amies medium (cat. Number 480CE, Brescia, Copan, Italy). The oropharyngeal sampling involved pressing the tongue downward to the floor of the mouth by means of a spatula, and swabbing both tonsillar arches and the posterior nasopharynx without touching the sides of the mouth; the nasal sampling involved inserting the swab tip into one nostril and rotating it for three seconds. All the swabs were immediately transported to a central laboratory and processed within two hours.
Identification of S. aureus
S. aureus was identified using the RIDAGENE methicillin-resistant S. aureus (MRSA) system (R-Biopharm AG, Darmstadt, Germany) , a multiplex real-time PCR for the direct, qualitative detection of MRSA and its differentiation from methicillin-sensitive S. aureus (MSSA). It has been validated for use with human nasal specimens and cultures and has a detection limit of ≤5 DNA copies per reaction .
DNA was extracted from the nasal and oral specimens using 200 μL of lysis buffer (provided with the kit) mixed with 100 μL of Copan swab transport media. The preparation tubes were vortexed for 60 seconds, and incubated in a heating block at 95°C for 10 minutes. The samples were then centrifuged for one minute at 12,000 rpm and supernatant was collected. After DNA isolation, the mecA/mecC gene, the SCCmec/orfX junction (type I, II, III, IV, V, VI, VII, IX and XI), and the orfX gene specific for MRSA were amplified by means of TaqMan technology-based assays in accordance with the manufacturer’s instructions. Each assay contains internal control DNA, which is added to the samples in the extraction step in order to detect possible PCR inhibition or DNA extraction failure. The samples were classified as negative if there was no amplification signal but the internal control DNA was positive; MRSA if they were positive for the mecA/mecC, the SCCmec/orfX junction and the orfX gene; MSSA if they were positive for both the SCCmec/orfX junction and the orfX gene, or only for the orfX gene.
The groups were compared using contingency table analysis with the chi-squared or Fisher’s exact test, as appropriate. A Cochran-Armitage test for trend was used to compare the ordered categorical data between groups. Subgroup analyses were made on the basis of age (<10, 10–14, and ≥15 years). Agreement between the oropharyngeal and nasal swab results was examined by computing weighted kappa coefficients, using Fleiss-Cohen weights. All of the tests were two-sided, and a p value of <0.05 was considered statistically significant. The data were analysed using SAS version 9.2 statistical software (SAS Institute, Cary, NC, USA).
Results and discussion
The parents of more than 70 % of the children attending the selected schools agreed to allow their children to take part in the study, but 35 (6.6%) of the 532 potential participants were excluded because of mild upper respiratory tract infections. Of the 497 remaining children, 264 (53.1%) were identified as S. aureus carriers: 129 (25.9%) oropharyngeal carriers and 195 (39.2%) nasal carriers, of whom 60 (12.1%) were both oropharyngeal and nasal carriers. This indicates that the use of oropharyngeal swabs increased the identification of S. aureus carriage by about 13.8%. Table 1 shows the oropharyngeal and/or nasal carriage of S. aureus in the study population by age group and methicillin susceptibility. Oropharyngeal carriage increased with age (p < 0.001), whereas nasal carriage decreased. However, the differences between the age groups were statistically significant only in the case of oropharyngeal carriage. There was little or no agreement between oropharyngeal and nasal carriage in any of the age groups. MRSA was identified in only three cases (0.6%), always in nasal samples.
Table 2 shows the general characteristics of the study population by S. aureus carrier status. There were no differences between the carriers and non-carriers in terms of the distribution of age, gender, ethnicity, the number of siblings in the household, exposure to passive smoking, previous clinical history, allergic sensitisation, or previous influenza, pneumococcal and meningococcal vaccinations.
Table 3 shows the main characteristics of the children carrying S. aureus by site of positivity. The children with positive nasal swabs alone were younger than those with positive oropharyngeal swabs alone (p < 0.001) or positive nasal and oropharyngeal swabs (p = 0.01). The frequency of male children was higher among the subjects with positive oropharyngeal and nasal swabs (66.7%) than among those with positive oropharyngeal swabs alone (46.4%; p = 0.02). There were no statistically significant differences in any of the other considered characteristics.
This first study of oropharyngeal and nasal S. aureus colonisation rates in healthy school children shows that, like adults with a history of exposure to the healthcare system -, they frequently carry S. aureus in the throat and that this may occur even in the absence of nasal colonisation in a relatively high proportion of cases. Although oropharyngeal sampling led to the detection of S. aureus carriage in fewer children than nasal sampling, it did identify about 14% of children who would have been considered negative on the basis of a nasal swab alone. The global carriage rate of more than 53% was similar to that observed in adults who have provided both nasal and oropharyngeal samples -, but significantly higher than that reported in healthy children who have undergone nasal evaluation alone , thus highlighting the importance of throat sampling. In this study S. aureus was identified by PCR assay and not by culture. Consequently, genotyping or phenotyping data of the pathogen cannot be presented and it was not possible to establish whether more than one strain type of S. aureus could be identified in children carrying this pathogen. However, molecular techniques used in this study can be adequate for an easier and faster epidemiological evaluation of S. aureus carriage in the general pediatric population in order to have useful information concerning the circulation of MRSA in the community and the risk of the occurrence of community acquired (CA)-S. aureus illnesses that are potentially difficult to treat. The very limited circulation of MRSA found in this study could be, at least in part, due to the enrolment of children who did not receive any antibiotic treatment in the previous three weeks and for whom no antibiotic selection of oropharyngeal flora favouring MRSA emergence occurred. However, the finding suggests a marginal risk of CA disease due to MRSA in Milan: however, it is worth remembering that, although such diseases are relatively uncommon among the general population of Europe, they are considerably more frequent in intensive care units and hospital wards caring for patients with chronic diseases -.
The fact that the rates of exclusively oropharyngeal carriage were significantly higher among the older children suggests that age may play a role in conditioning the site of colonisation. The carriage of respiratory bacteria is strictly age dependent as it is significantly higher in younger children than in adolescents , and is modified by vaccine use . Moreover, there are data showing that the primary site of colonisation of bacteria such as Streptococcus pneumoniae can vary with age . As it has been demonstrated that significant interactions can occur between bacterial species and between bacteria and viruses, and that these can affect the carriage rates of a given infectious agent , it can be hypothesised that the age-related differences in pharyngeal S. aureus colonisation rates may be due to time-related variations in bacterial balance in different body sites.
Regardless of age, a minority of children showed both oropharyngeal and nasal colonisation. This is different from the findings of some adult studies indicating that bacterial colonisation in the nose is almost always accompanied by colonisation in the throat . However, the difference may be due to the different characteristics of the studied subjects: we only enrolled otherwise healthy children with little or no exposure to the healthcare system, whereas the adult studies mainly involved hospitalised patients. Furthermore, the adult studies also showed that dual colonisation is significantly more frequent in subjects in close contact with other S. aureus carriers, such as frequently hospitalised patients .
Although it has not been established what risk oropharyngeal carriers represent in terms of bacterial spread and infection, the findings of this study highlight the importance of adding throat to nasal screening when monitoring the circulation of S. aureus in the community. The additional cost of throat screening could be minimised by pooling samples in the laboratory and, as in this study, using molecular methods that are more cost-effective for screening S. aureus than traditional cultures because of the high cost of unnecessary isolation .
Methicillin-resistant S. aureus
Methicillin-sensitive S. aureus
Wertheim HF, Melles DC, Vos MC, van Leeuwen W, van Belkum A, Verbrugh HA, Nouwen JL: The role of nasal carriage in Staphylococcus aureus infections. Lancet Infect Dis. 2005, 5: 751-62. 10.1016/S1473-3099(05)70295-4.
Weber SG, Huang SS, Oriola S, Huskins WC, Noskin GA, Harriman K, Olmsted RN, Bonten M, Lundstrom T, Climo MW, Roghmann MC, Murphy CL, Karchmer TB, Society for Healthcare Epidemiology of America, Association of Professionals in Infection Control and Epidemiology: Legislative mandates for use of active surveillance cultures to screen for methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci: position statement from the Joint SHEA and APIC Task Force. Infect Control Hosp Epidemiol. 2007, 28: 249-60. 10.1086/512261.
Palavecino EL: Clinical, epidemiologic, and laboratory aspects of methicillin-resistant Staphylococcus aureus infections. Methods Mol Biol. 2014, 1085: 1-24. 10.1007/978-1-62703-664-1_1.
Dey S, Rosales-Klintz S, Shouche S, Pathak JP, Pathak A: Prevalence and risk factors for nasal carriage of Staphylococcus aureus in children attending anganwaries (preschools) in Ujjain, India. BMC Res Notes. 2013, 6: 265-10.1186/1756-0500-6-265.
Gardella N, Murzicato S, Di Gregorio S, Cuirolo A, Desse J, Crudo F, Gutkind G, Mollerach M: Prevalence and characterization of methicillin-resistant Staphylococcus aureus among healthy children in a city of Argentina. Infect Genet Evol. 2011, 11: 1066-71. 10.1016/j.meegid.2011.03.019.
Sivaraman K, Venkataraman N, Cole AM: Staphylococcus aureus nasal carriage and its contributing factors. Future Microbiol. 2009, 4: 999-1008. 10.2217/fmb.09.79.
Nilsson P, Ripa T: Staphylococcus aureus throat colonization is more frequent than colonization in the anterior nares. J Clin Microbiol. 2006, 44: 3334-9. 10.1128/JCM.00880-06.
Widmer AF, Mertz D, Frei R: Necessity of screening of both the nose and the throat to detect methicillin-resistant Staphylococcus aureus colonization in patients upon admission to an intensive care unit. J Clin Microbiol. 2008, 46: 835-10.1128/JCM.02276-07.
Mertz D, Frei R, Periat N, Zimmerli M, Battegay M, Flückiger U, Widmer AF: Exclusive Staphylococcus aureus throat carriage: at-risk populations. Arch Intern Med. 2009, 169: 172-8. 10.1001/archinternmed.2008.536.
Lee CJ, Sankaran S, Mukherjee DV, Apa ZL, Hafer CA, Wright L, Larson EL, Lowy FD: Staphylococcus aureus oropharyngeal carriage in a prison population. Clin Infect Dis. 2011, 52: 775-8. 10.1093/cid/cir026.
Fall C, Richard V, Dufougeray A, Biron A, Seck A, Laurent F, Breurer S: Staphylococcus aureus nasal and pharyngeal carriage in Senegal. Clin Microbiol Infect. 2014, 20: O239-41. 10.1111/1469-0691.12385.
Mertz D, Frei R, Jaussi B, Tietz A, Stebler C, Flückiger U, Widmer AF: Throat swabs are necessary to reliably detect carriers of Staphylococcus aureus . Clin Infect Dis. 2007, 45: 475-7. 10.1086/520016.
RIDA®GENE MRSA real-time PCR. Available at: http://www.infung.com.tw/en/download/42.html Accessed on 22 November 2014.
Baud O, Giron S, Aumeran C, Mouly D, Bardon G, Besson M, Delmas J, Coignard B, Tristan A, Vandenesch F, Illes G, Lesens O: First outbreak of community-acquired MRSA USA300 in France: failure to suppress prolonged MRSA carriage despite decontamination procedures. Eur J Clin Microbiol Infect Dis 2014, Epub 11 May.,
Naidoo R, Nuttall J, Whitelaw A, Eley B: Epidemiology of Staphylococcus aureus bacteraemia at a tertiary children’s hospital in Cape Town, South Africa. PLoS One. 2013, 8: e78396-10.1371/journal.pone.0078396.
Rodriguez M, Hogan PG, Burnham CA, Fritz SA: Molecular epidemiology of Staphylococcus aureus in households of children with community-associated S. aureus skin and soft tissue infections. J Pediatr. 2014, 164: 105-11. 10.1016/j.jpeds.2013.08.072.
Esposito S, Zampiero A, Terranova L, Montinaro V, Peves Rios W, Scala A, Ansuini V, Galeone C, Principi N: Comparison of posterior pharyngeal wall and nasopharyngeal swabbing as a means of detecting the carriage of Neisseria meningitidis in adolescents. Eur J Clin Microbiol Infect Dis. 2013, 32: 1129-33. 10.1007/s10096-013-1856-2.
Dunne EM, Manning J, Russell FM, Robins-Browne RM, Mulholland EK, Satzke C: Effect of pneumococcal vaccination on nasopharyngeal carriage of Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Staphylococcus aureus in Fijian children. J Clin Microbiol. 2012, 50: 1034-8. 10.1128/JCM.06589-11.
Principi N, Terranova L, Zampiero A, Manzoni F, Senatore L, Rios WP, Esposito S: Oropharyngeal and nasopharyngeal sampling for the detection of adolescent Streptococcus pneumoniae carriers. J Med Microbiol. 2014, 63: 393-8. 10.1099/jmm.0.068726-0.
van den Bergh MR, Biesbroek G, Rossen JW, de Steenhuijsen Piters WA, Bosch AA, van Gils EJ, Wang X, Boonacker CW, Veenhoven RH, Bruin JP, Bogaert D, Sanders EA: Associations between pathogens in the upper respiratory tract of young children: interplay between viruses and bacteria. PLoS One. 2012, 7: e47711-10.1371/journal.pone.0047711.
Harbarth S, Masuet-Aumatell C, Schrenzel J, Francois P, Akakpo C, Renzi G, Pugin J, Ricou B, Pittet D: Evaluation of rapid screening and pre-emptive contact isolation for detecting and controlling methicillin-resistant Staphylococcus aureus in critical care: an interventional cohort study. Crit Care. 2006, 10: R25-10.1186/cc3982.
This study was supported by a grant from the Italian Ministry of Health (Bando Giovani Ricercatori 2009).
The authors declare that they have no competing interests.
SE designed the study, supervised the data entry and management and drafted the manuscript; LT and AZ performed the laboratory analyses; VI and WPR participated in the enrollment and data entry; CP performed the statistical analysis; NP critically revised the study design, supervised the enrollment and co-drafted the manuscript. All of the authors read and approved the final version of the manuscript.