Risk factors for nasopharyngeal carriage of drug-resistant Streptococcus pneumoniae: data from a nation-wide surveillance study in Greece

A study group of 12 hospitals was organized under the coordination of the 4^th Department of Internal Medicine and the Infectious Diseases Laboratory of Athens University Medical School (see Acknowledgements section). The research protocol was approved by the Ministries of Health and Education and the Ethics Committees of all cooperating hospitals. A network was formed consisting of local teams with the responsibility to inform parents and collect appropriately filled questionnaires, collect and culture nasopharyngeal samples from the children, isolate presumed pneumococcal strains, store and ship isolated strains to the central laboratory (Laboratory for Infectious Diseases and Antimicrobial Chemotherapy, 4^th Dept of Internal Medicine, Athens University School of Medicine, University General Hospital ATTIKON). The single central laboratory undertook the microbiological procedures and the input of data from the questionnaires in a purpose-structured database.

Questionnaires were distributed to the parents by the local teams of doctors visiting the day-care centres 1–2 days before the sampling visit. On the sampling day, questionnaires were reviewed by the doctors' team. In the section of recent antibiotic use, parents were asked to provide generic or commercial names of all medications received in the last month or trimester and the appropriate classification was performed by the responsible doctors in the sampling site. Questionnaires were signed by the parents, were anonymous but labeled to match the respective sample per child, and consisted of three parts: a) demographic data, i.e. age, gender, name of daycare center, area and prefecture; b) data on antibiotic consumption in the last month and in the last trimester preceding the sampling, including: number of antibiotic courses, name of antimicrobial and reason for its prescription, mode of supply (over-the-counter or by prescription), c) family and household data, i.e. parents working in health care, number of siblings <8 years of age, and d) co morbidities and past medical history data: admission to hospital in the last year, history of « respiratory tract infections », otitis media history in the previous year and steroid use. Children without signed questionnaires by the parents were excluded from sampling, as well as those receiving antibiotics for active infection. Samples without appropriately filled questionnaires were microbiologically processed but were excluded from risk factor analysis.

The period of sampling started February 2004 and lasted until the end of May 2004. Carriage samples were collected per nasally with sterile swabs on flexible aluminium wire (Medical Wire & Equipment, Corsham, UK). Swabs were immediately plated on Columbia agar plates (Becton Dickinson, Sparks, MD) supplemented with 5% defibrinated horse blood and packed in 5% CO2 Gas Pak Pouches (Becton Dickinson).

Streptococcus pneumoniae isolates were identified by standard microbiological procedures. Susceptibility testing was performed with the E-test methodology (AB Biodisk, Solna, Sweden) for penicillin(PG), cefuroxime (CXM), ceftriaxone(CTX), erythromycin(ERY), trimethoprim/sulfamethoxazole (COT), tetracycline (TET), moxifloxacin, levofloxacin and ciprofloxacin (CIP). Strains displaying resistance were serotyped by the Quellung reaction using the 12 pooled antisera Pneumotest panel and selected factor sera (Statens Serum Institut, Copenhagen, Denmark). Methodology of sampling, isolation, identification and serotyping of pneumococcal strains, as well as susceptibility testing is mentioned elsewhere in detail [9].

Clinical and Laboratory Standards Institute methodology and breakpoints for non-susceptibility and resistance were used for all antibiotics [10], except for ciprofloxacin. For ciprofloxacin, the epidemiological cut-off of 2 mg/L was used as an indicator of resistance, according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2006 recommendations [11]. Strains with an MIC value higher than the susceptibility breakpoint were characterized as "non-susceptible", i.e. intermediate and fully resistant strains. Combined resistance was defined as resistance to two different classes of antibiotics (e.g. penicillin and erythromycin) irrespective of other classes. Multiple resistance (or multi-resistance) was defined as resistance to 3 or more antibiotics of different classes [2].

Between February and May 2004, consecutive nasopharyngeal swabs and questionnaires were collected from 2595 healthy children (median age ± S.D. 4.3 ± 1.1 years, male: female ratio 1:1), of which, after careful evaluation of the accompanying questionnaires, 2536 were included in the study analysis (2536/2595, i.e. 97.7% of original population). In Table 1, distribution of nasopharyngeal sampling specimens and isolated strains per geographic region is shown. Carriage rates differed significantly among differed regions. In Figure 1, a map of Greece is provided indicating the geographical location of the sampling sites, with the respective sampling population, carriage rates and penicillin resistance rates.

A total of 793 pneumococcal strains was isolated in the study from the original 2595 nasopharyngeal specimens (mean carriage rate within study period 793/2595 = 30.55%). Of those, 780 isolates survived shipment and storage and were microbiologically processed, and finally 746 strains were included in the present protocol analysis of the 2536 properly filled questionnaires (mean carriage in per protocol analysis 746/2536 = 29.41%). Carriage of pneumococcal strains was related to age, period and region of sampling, use of antibiotics in the last trimester (last month not included) and history of respiratory infections in the last year. Antibiotic use during the last month did not affect carriage rates of pneumococci. In Table 2, analysis of potential risk factors for nasopharyngeal carriage of pneumococci, as defined by the protocol questionnaire, is listed.

In Table 3, results for resistance rates and MIC distribution parameters for all antibiotics tested are shown per region of sampling. Resistance rates differed significantly among different geographic regions. Multiresistant strains formed 23.86% of the carriage isolates (178/746), while combined resistance to penicillin and erythromycin was documented for 75/746 of the total sample (10.05%). No resistance to moxifloxacin and levofloxacin was documented.

Recent antibiotic use in the last month-irrespective of antibiotic class, duration of administration and number of courses of antibiotics- was strongly associated with the isolation of resistant pneumococci to a single or multiple antibiotics. Moreover, increasing number of antibiotic courses in the last month increased the likelihood of isolating a resistant pneumococcal strain Odds Ratio (OR) per antibiotic course for penicillin resistance = 1.35, 95% Confidence Interval (CI) <0.99–1.83> P = 0.05, and OR for erythromycin resistance = 1.43, CI <1.11–1.83> P = 0.004). Combined resistance to penicillin and erythromycin and multiresistance (3 or more classes of antibiotics) were also strongly related to antibiotic use in the month preceding sampling (OR for combined resistance = 2.09, CI <1.24–3.52> P = 0.002, and OR for multiresistance = 2.02, CI <1.38–2.96> P < 0.001). In terms of non-susceptibility (i.e. intermediate and fully resistant strains together), antibiotic use both in the last month and in the preceding 3 months (last month excluded) held a statistically significant correlation with carriage of a penicillin and/or erythromycin non susceptible strain. For children that had received an antibiotic course in the last trimester, the possibility of carrying a resistant (or a non-susceptible) strain in their nasopharynx was even greater, if they had additionally received an antibiotic course in the last month (P < 0.001, OR 4.18 for resistance, 95%CI 2.06–8.54). In Table 4, risk factors for carriage of a resistant or non-susceptible strain are listed in full detail. Carriage of ciprofloxacin resistant strains was independent of antibiotic use.

A significant number of children from the total study population had at least one antibiotic course in the month preceding sampling (31.03%) or in the previous trimester (42.43%). Administration of antibiotics did not differ among geographic regions. The majority of antibiotic courses in the carriers' group were prescribed by a doctor either in the preceding month (92.5%) or trimester (87.8%). The rest of the consumed antibiotics were purchased over the counter by parents' initiative (5.55% and 9.8%) or by pharmacists' advice (1.85% and 2.3% of cases for the last month and trimester respectively).

The most common infections for prescribing an antibiotic were similar in rates in both groups (carriers and non-carriers). Rates for most common causes were as follows: acute otitis media 33.56 – 31%, tonsillitis 13.34 – 11%, lower respiratory tract infections 4.26 – 4%, and virus-related respiratory tract infections 28.2 – 28% (mostly rhinopharyngitis, tracheobronchitis and laryngitis), respectively for each group. Most commonly prescribed antibiotics were amoxicillin-clavulanate (35%) and oral 2^nd generation cephalosporins (31%), namely cefaclor, cefprozil and cefuroxime axetil.

Among the 380 pneumococcal strains displaying resistance to at least 1 antibiotic class, serotyping was available for 317 strains (83.5% of resistant strains). The distribution of serotypes per geographic region and the respective coverage of the heptavalent pneumococcal conjugate vaccine is presented in Table 5. Serotypes 19F, 14, 9V, 23F and 6B formed 70.6% of the total number of resistant strains serotyped.