Or the number of people divided by the number of beds in the house. Household contact with children less than two years old was defined as contact of at least 4 hours per day. Isolation of pneumococci Between January 2008 and January 2009, nasopharyngeal swabs were collected from each child at four times, at enrollment and then again at three month intervals. Samples were collected with calcium alginate swabs (Calgiswab type 1, Spectrum USA) and inoculated into modified Stuart transport medium and sent to the Clinical Microbiology Laboratory at the Gon lo Moniz Research Institute. All swabs were plated within 4 hours onto agar plates with 5 sheep blood and 5.0 / mL of gentamicin. Plates were incubated at 35 in 5 CO2-enriched atmosphere for up to 48 hours. Three -hemolytic colonies exhibiting morphologic characteristics suggestive of S. pneumoniae were isolated. Identification of these isolates as S. pneumoniae was confirmed by optochin disc susceptibility (BBL Microbiology Systems, Cockeysville, USA) and the bile solubility test. One S. pneumoniae colony per plate was then sub-cultured, harvested, and kept frozen at -70 for further testing. When S. pneumoniae isolates from the same primary plate exhibited a clearly different colony morphology, dissimilar colonies were frozen separately.Author PF-04418948 site manuscript Author Manuscript Author Manuscript Author ManuscriptVaccine. Author manuscript; available in PMC 2017 February 03.Menezes et al.PageSerotypingAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptThe isolates were serotyped by multiplex-PCR as described elsewhere [12]. DNA extraction and PCR conditions were performed as described by the US Centers for Disease Control and Prevention (CDC) [12]. Isolates with negative multiplex PCR results were subjected to single-plex-PCR with primer 19F variation [13] and Quellung reaction testing for capsular type definition. Antimicrobial susceptibility testing The broth microdilution method was performed according to Clinical and Laboratory Standard Institute recommendations [14] to determine susceptibility of isolates to penicillin, cefotaxime, tetracycline, erythromycin, trimethoprim/sulfamethoxazole (TMP/SMX) and levofloxacin (Sigma ldrich, Germany). Quality control was performed by testing S. pneumoniae ATCC 49619. Isolates with a penicillin MIC value 0.12 /mL were defined as penicillin non-susceptible. Genotyping Pulse field gel electrophoresis (PFGE) analysis was performed to define the molecular profile of the isolates. Chromosomal digests generated by SmaI were prepared and analyzed as described elsewhere [15]. A CHEF DRII apparatus (Bio-Rad, Hercules, CA) was used for running the gels. The bacterial strains were also analyzed by multilocus sequence typing (MLST), as described elsewhere [16]. Data management and statistical analysis Data were order PD0325901 entered and managed by Epi Info version 3.5.1 (CDC, Atlanta, GA, USA). Statistical analyses were performed in SAS v9.3. Univariate and multivariate logistic regression models were constructed to identify risk factors for colonization (PROC GLIMMIX). To construct confidence intervals that accounted for the non-independence of samples from the same individual, we created 1000 bootstrap samples, where all observations from an individual were grouped together and sampled with replacement. Household crowding was analyzed as continuous variables. A variable was considered to be significantly associated with colonization (p<0.05) if the.Or the number of people divided by the number of beds in the house. Household contact with children less than two years old was defined as contact of at least 4 hours per day. Isolation of pneumococci Between January 2008 and January 2009, nasopharyngeal swabs were collected from each child at four times, at enrollment and then again at three month intervals. Samples were collected with calcium alginate swabs (Calgiswab type 1, Spectrum USA) and inoculated into modified Stuart transport medium and sent to the Clinical Microbiology Laboratory at the Gon lo Moniz Research Institute. All swabs were plated within 4 hours onto agar plates with 5 sheep blood and 5.0 / mL of gentamicin. Plates were incubated at 35 in 5 CO2-enriched atmosphere for up to 48 hours. Three -hemolytic colonies exhibiting morphologic characteristics suggestive of S. pneumoniae were isolated. Identification of these isolates as S. pneumoniae was confirmed by optochin disc susceptibility (BBL Microbiology Systems, Cockeysville, USA) and the bile solubility test. One S. pneumoniae colony per plate was then sub-cultured, harvested, and kept frozen at -70 for further testing. When S. pneumoniae isolates from the same primary plate exhibited a clearly different colony morphology, dissimilar colonies were frozen separately.Author Manuscript Author Manuscript Author Manuscript Author ManuscriptVaccine. Author manuscript; available in PMC 2017 February 03.Menezes et al.PageSerotypingAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptThe isolates were serotyped by multiplex-PCR as described elsewhere [12]. DNA extraction and PCR conditions were performed as described by the US Centers for Disease Control and Prevention (CDC) [12]. Isolates with negative multiplex PCR results were subjected to single-plex-PCR with primer 19F variation [13] and Quellung reaction testing for capsular type definition. Antimicrobial susceptibility testing The broth microdilution method was performed according to Clinical and Laboratory Standard Institute recommendations [14] to determine susceptibility of isolates to penicillin, cefotaxime, tetracycline, erythromycin, trimethoprim/sulfamethoxazole (TMP/SMX) and levofloxacin (Sigma ldrich, Germany). Quality control was performed by testing S. pneumoniae ATCC 49619. Isolates with a penicillin MIC value 0.12 /mL were defined as penicillin non-susceptible. Genotyping Pulse field gel electrophoresis (PFGE) analysis was performed to define the molecular profile of the isolates. Chromosomal digests generated by SmaI were prepared and analyzed as described elsewhere [15]. A CHEF DRII apparatus (Bio-Rad, Hercules, CA) was used for running the gels. The bacterial strains were also analyzed by multilocus sequence typing (MLST), as described elsewhere [16]. Data management and statistical analysis Data were entered and managed by Epi Info version 3.5.1 (CDC, Atlanta, GA, USA). Statistical analyses were performed in SAS v9.3. Univariate and multivariate logistic regression models were constructed to identify risk factors for colonization (PROC GLIMMIX). To construct confidence intervals that accounted for the non-independence of samples from the same individual, we created 1000 bootstrap samples, where all observations from an individual were grouped together and sampled with replacement. Household crowding was analyzed as continuous variables. A variable was considered to be significantly associated with colonization (p<0.05) if the.
