To elucidate features of disease caused by S. pneumoniae serotype 5, we reviewed all cases of invasive pneumococcal disease in the northern Alberta area reported from 2005 through 2009. During the study period, Alberta was subdivided into 9 health regions. For cases originating in health regions 4 through 9 (located in northern Alberta), an extensive medical chart review was conducted. The total population for these health regions in 2008 was 1,888,881 (www.health.alberta.ca/documents/Population-Projections-2006.pdf). The clinical data collected were patient age, sex, aboriginal status (i.e., First Nations heritage), homelessness, substance abuse, type of invasive pneumococcal disease, outcome, and concurrent conditions (Table 1).

As part of its serotyping program, the National Centre for Streptococcus pneumococcal surveillance confirmed isolates as S. pneumoniae according to morphologic appearance and optochin susceptibility (9). All pneumococcal isolates that exhibited a positive quellung reaction when commercial type-specific antiserum (Statens Serum Institute, Copenhagen, Denmark) was used were assigned a serotype (10). Strains that were susceptible to optochin but for which no serotype was assigned were further tested by using the AccuProbe Streptococcus pneumoniae Culture Identification Test (Gen-Probe, San Diego, CA, USA) to confirm species identification.

Drug susceptibility was determined by using the reference broth microdilution method described by the Clinical and Laboratory Standards Institute (11). The following antimicrobial drugs were tested: penicillin, cefotaxime, ceftriaxone, chloramphenicol, erythromycin, clindamycin, tetracycline, trimethoprim/sulfamethoxazole, levofloxacin, and vancomycin. All antimicrobial agents were purchased from Sigma-Aldrich Canada Ltd, Oakville, Ontario, Canada. Interpretation of MICs was based on Clinical and Laboratory Standards Institute performance standards that were current at the time of testing (M100-S15 through M100-S17) (12,13).

S. pneumoniae chromosomal DNA was prepared as described (14). Chromosomal DNA was restricted with 20 U of SmaI (New England Biolabs, Beverly, MA, USA), and pulsed-field gel electrophoresis (PFGE) was performed by using a CHEF DR-III apparatus (Bio-Rad Laboratories [Canada] Limited, Mississauga, ON, Canada) for 23 h. The parameters used were as follows: initial pulse 3.5 s, final pulse 23.5 s, voltage 6 V/cm, and temperature 13°C. Salmonella Braenderup U9812 was used as a molecular size marker. The macrorestriction pattern was analyzed by using Bionumerics version 5 (Saint-Martens-Latem, Belgium).

Multilocus sequence typing was performed as described (15). The multilocus sequence type (MLST) was searched against the online pneumococcal database (http://spneumoniae.mlst.net).

LOGIT P (Serotype 5) = B₀ + B₁X₁ + B₂X₂ + … + B_pX_p …… (1)

In model 1, the significance of variables was assessed by using the Wald statistic. The variables that were significant at p<0.05 were retained in the model. All others were removed from the model unless they were possible confounders. In the final model, we tested βs, the effect of each variable on log odds of serotype 5 after adjustment for other associated variables. To calculate rates, we used the populations that were current for each province in January 1, 2009 (16).

From January 1, 2000, through December 31, 2004, the National Centre for Streptococcus serotyped 5,509 S. pneumoniae isolates from patients with invasive pneumococcal disease (1 isolate counted per case) from across Canada and identified 7 as serotype 5: one each in 2001, 2002, and 2003 and 4 in 2004. Since then, the Centre identified 52 isolates from patients with invasive pneumococcal disease as serotype 5 in 2005, 393 in 2006, 457 in 2007, 104 in 2008, and 42 in 2009, for a total of 1,048 cases (Figure 1). The number of cases caused by serotype 5 peaked in December 2006 and then slowly declined in 2007, 2008, and 2009 (Figure 1). Patients with invasive pneumococcal disease caused by S. pneumoniae serotype 5 were from British Columbia (343 [32.7%], 7.8 cases/100,000 population) Alberta (523 [49.9%], 14.4/100,000), Saskatchewan (85 [8.1%], 8.3/100,000), and Manitoba (92 [8.8%], 7.6/100,000) (Figure 1). During this 5-year period, only 5 isolates of serotype 5 were detected from elsewhere in Canada: 1 from Ontario in March 2007; 1 from Quebec in June 2009; and 3 from Northwest Territories in April, July, and December 2007.

In western Canada during 2000–2009, the numbers of serotype 5 and other serotype isolates identified increased (Figure 2). The increased number of isolates submitted for typing after the onset of the epidemic indicates greater interest on the part of public health officials in western Canada in identifying circulating serotypes from patients with invasive pneumococcal disease in their provinces.

The epidemic primarily affected young adults (median age 41 years) (Figure 3). Only a small subset of cases occurred among patients <5 years of age and even fewer in those >65 years of age. Most patients were male (637 male, 395 female, and 16 unknown).

The sources of specimens for the serotype 5 isolates from across Canada were as follows: 988 isolates from blood, 33 from lung/pleural fluid, 9 from cerebrospinal fluid, 7 from synovial fluid, 7 from chest/hip/leg fluid, 3 from pericardial fluid, and 1 from peritoneal fluid. For the univariable and multivariable analyses, isolates from patients with serotype 5 and nonserotype 5 invasive S. pneumoniae were collected from northern Alberta only (1,112 cases).

According to univariable analysis, serotype 5 was more prevalent than other serotypes among patients who were male (66.2% vs. 57.0%), of First Nations heritage (21.8% vs. 10.0%), or homeless (16.1% vs. 4.7%) (Table 1). Among the substance-abuse categories, associations with tobacco use, alcoholism, and illicit drug use were considered significant (p<0.001 for each; Table 1). With respect to concurrent conditions, cases of invasive pneumococcal disease caused by serotype 5 were significantly associated with cancer within 5 years before onset of invasive pneumococcal disease, cardiovascular disease, hematologic abnormalities, diabetes mellitus, cirrhosis, chronic renal failure, musculoskeletal impairment, and hepatitis C (Table 1). For patients with bacteremia and pneumonia, invasive pneumococcal disease caused by S. pneumoniae serotype 5 occurred significantly more often with pneumonia than did that caused by other serotypes (94.7% vs. 74.6%; Table 1). In addition, meningitis was more common for patients in the non–serotype 5 group than in the serotype 5 group (8.0% vs. 0.7%, respectively; p<0.001; Table 1). Death was less associated with infection caused by serotype 5 than by other serotypes (3.2% vs. 14.1%, respectively; Table 1).

The multivariable logistic regression model used to examine the associations of different factors with S. pneumoniae serotype 5 that were identified by univariable analyses found that First Nations heritage and homelessness were significantly associated with serotype 5 (adjusted odds ratio [aOR] 2.34; 95% CI 1.53–3.57 and aOR 1.83, 95% CI, 1.07–3.12, respectively) (Table 2). Tobacco use (aOR 1.90, 95% CI 1.29–2.81) and illicit drug use (aOR 1.89, 95% CI, 1.31–2.73) were also significantly associated, whereas alcoholism was not (Table 2). Among concurrent conditions, the following were significantly associated: cancer within 5 years before invasive pneumococcal disease (aOR 0.32, 95% CI 0.14–0.71), cardiovascular disease (aOR 0.51, 95% CI 0.32–0.82), hematologic abnormalities (aOR 0.19, 95% CI 0.06–0.55), and cirrhosis (aOR 0.18; 95% CI 0.06–0.50) (Table 2). Associations with musculoskeletal disease and hepatitis C infection, although significant according to univariable analysis, were not significant according to multivariable analysis.

Antimicrobial drug susceptibility testing of 1,009 isolates indicated that all S. pneumoniae serotype 5 isolates tested were susceptible to cefotaxime, ceftriaxone, tetracycline, levofloxacin, and vancomycin. A small percentage (5 [0.5%]) of the 1,009 were intermediately resistant to penicillin (MIC ≥0.125 mg/L), 2 (0.2%) were resistant to chloramphenicol, 4 (0.4%) were resistant to clindamycin, 2 (0.2%) were intermediately resistant to erythromycin, and 4 (0.4%) were fully resistant to erythromycin. For trimethoprim/sulfamethoxazole, 976 (96.7%) isolates showed intermediate resistance (MICs 1.0–2.0 mg/L) and 18 (1.8%) showed resistance (4.0 to >16.0 mg/L). The remaining 39 isolates were not available for testing.

During the epidemic, a subset of S. pneumoniae serotype 5 isolates (91 isolates), encompassing each year of the epidemic from the 4 affected western provinces were randomly selected and subjected to PFGE for restriction fragment-length polymorphism (RFLP) analysis (12 isolates in 2005, 26 in 2006, 13 in 2007, 20 in 2008, and 20 in 2009). All isolates typed by PFGE had either an identical RFLP pattern or differed by 1 band (Figure 4). Extending the RFLP analysis back to the 7 serotype 5 isolates from Canada from 2000 through 2004 showed that the first similar fingerprint detected was from a person who lived in a small town in rural southeastern Alberta in March 2004.

To determine whether this clone had been found in the United States, we compared it with 6 serotype 5 isolates from the US Centers for Disease Control and Prevention. Three isolates were from a small cluster of cases in San Francisco, California, in 2002 and 3 were from sporadic cases in the United States in 2006 (B. Beall, pers. comm.). Of these 6 isolates, the RFLP pattern for 5 isolates was identical to that of the epidemic clone and 1 isolate had a single band difference, suggesting that the serotype 5 clone in western Canada had been circulating in the United States in 2002 and 2006. This clone might have been imported into Canada from the United States; however, it might also have been imported from elsewhere in the world because sequence type (ST) 289 is the major circulating serotype 5 clone.

MLST analysis showed the allelic profile of the S. pneumoniae serotype 5 clone to be ST289 (aroE16, gdh12, gki9, recP1, spi41, xpt33, ddl33). ST289 has been listed in the MLST database (http://spneumoniae.mlst.net). The ST289 clone was originally reported from Colombia and is contained in the Pneumococcal Molecular Epidemiology Network list of worldwide antimicrobial drug–resistant clones (designation Colombia⁵-19) (www.sph.emory.edu/PMEN) (17).