Switching gears for an influenza pandemic: validation of a duplex reverse transcriptase PCR assay for simultaneous detection and confirmatory identification of pandemic (H1N1) 2009 influenza virus.

Rapid methods for the detection and confirmatory identification of pandemic influenza A virus (also known as pandemic [H1N1] 2009) are of utmost importance. In this study, a conventional reverse transcriptase PCR (RT-PCR) assay for the detection of influenza A virus and the hemagglutinin of swine lineage H1 (swH1) was designed, optimized, and validated. Nucleic acids were extracted from 198 consecutive nasopharyngeal, nasal, or throat swab specimens collected early in the outbreak (127 negative specimens, 66 specimens with pandemic [H1N1] 2009 influenza virus, 3 specimens with seasonal [H1N1] influenza A virus, and 2 specimens with seasonal [H3N2] influenza A virus). The performance characteristics of the duplex RT-PCR assay were assessed and compared to those of various detection methods: a monoplex RT-PCR assay at the National Microbiology Laboratory, a real-time RT-PCR assay using a Centers for Disease Control and Prevention protocol, an in-house multiplex RT-PCR assay (targeting influenza A virus, influenza B virus, and respiratory syncytial virus), and a rapid antigen test (the Binax Now Influenza A & B assay). The sensitivity of the duplex RT-PCR assay for influenza A virus detection was 97.2%, whereas the sensitivities were 74.6%, 71.8%, 47.8%, and 12.7% for the other four assays, respectively. The duplex RT-PCR assay was also able to identify swH1 in 94% of the cases, thereby reducing the number of specimens forwarded to reference laboratories for confirmatory identification. Only a limited number of specimens that contained influenza A virus had amounts of virus that fell below the limit of detection of the assay with the swH1 primers. Overall, the duplex RT-PCR assay is a reliable method for the simultaneous detection and confirmatory identification of pandemic (H1N1) 2009 influenza virus and would be particularly attractive to laboratories without real-time RT-PCR capabilities.

Antimicrobial resistance among invasive isolates of Streptococcus pneumoniae collected across Canada.

Between 2002 and 2003, 736 nonduplicate Streptococcus pneumoniae isolated from blood cultures were collected from 7 of 10 Canadian provinces (10 tertiary care centers). Microdilution broth susceptibility testing was performed using the method prescribed by the Clinical Laboratory Standards Institute. Of the isolates, 16.85% were nonsusceptible to penicillin and 5.4% were highly resistant. Of the S.pneumoniae, 14.1% had reduced susceptibility to erythromycin and 47% had been accounted for by the M phenotype. No isolates were recovered that were resistant to telithromycin. Only 6 isolates were resistant to levofloxacin and gatifloxacin. Of these, 5 strains had intermediate susceptibility to moxifloxacin and 1 was considered susceptible. The rates observed in this study are in keeping with previous surveillance studies among noninvasive isolates.

Cephalosporin resistance in Klebsiella pneumoniae from Nova Scotia, Canada.

From 2116 Klebsiella pneumoniae strains isolated between January 2001 and December 2002 in Nova Scotia, Canada, 25 (1.18%) showed a reduced susceptibility to cefoxitin or extended-spectrum cephalosporins. Narrow-spectrum beta-lactamase genes (bla(SHV-11), bla(SHV-1), bla(SHV-26), bla(SHV-32), bla(SHV-36), and bla(SHV-40)) were the most prevalent. Four new variants were identified (bla(LEN-17), bla(OKP-B-13), bla(OKP-B-14), and bla(OKP-A-11)), representing the 1st description of bla(OKP) in the Americas. Among the extended-spectrum beta-lactamase (ESBL) genes, bla(SHV-2), bla(SHV2a), bla(SHV-12), and bla(CTX-M-15) were detected (ESBL prevalence of 0.14%). Nineteen strains were resistant to cefoxitin (MIC, 32 to >256 microg/mL). Nevertheless, an AmpC-like activity was detected in only 1 strain, which expressed CMY-2. The combined effects of narrow-spectrum beta-lactamase production and decreased or nonexpression of OmpK35/36 porins did not account for the cefoxitin resistance observed in some of these strains.

An evaluation of extended incubation time with blind subculture of blood cultures in patients with suspected endocarditis.

BACKGROUND: In a small proportion of patients, bacterial endocarditis is due to organisms that grow slowly and may not be recovered in conventional blood cultures incubated for five days. This has led to recommendations for prolonged incubation and routine subculture of negative cultures. OBJECTIVE: The above-mentioned approach is evaluated. METHOD: The microbiology of all blood cultures subjected to prolonged incubation and the charts of individuals who had organisms recovered after five days were evaluated to determine their clinical significance. RESULTS: In all, 507 blood cultures were handled using an extended incubation and blind subculture protocol. Fifty-three blood cultures in 27 patients were positive. Blood cultures were positive after five days in only five cases; patient outcomes were not affected by the results in any of these cases, although several fastidious organisms (ie, Haemophilus paraphrophilus and Haemophilus parainfluenzae) were recovered in the first five days of incubation. CONCLUSION: Prolonged incubation and blood subcultures in patients with suspected endocarditis or infections due to fastidious organisms do not represent a wise use of increasingly scarce resources.

A comparison between the Strep A Rapid Test Device and conventional culture for the diagnosis of streptococcal pharyngitis.

BACKGROUND: Rapid antigen detection tests are frequently used to diagnose pharyngitis due to Streptococcus pyogenes. Because a large number of kits are available commercially, performance characteristics may vary considerably. The present study evaluated one such kit currently in use in Canadian laboratories for which published evaluations are not available. OBJECTIVE: To evaluate the performance characteristics of the Strep A Rapid Test Device (SARTD) (Nova Century Scientific Inc, Canada). METHODS: Pharyngeal swabs from 818 patients with suspected streptococcal pharyngitis were tested. Swabs were initially inoculated onto the surface of a blood agar plate and then used to perform the rapid antigen test. The test was performed in accordance with the product monograph. Beta-hemolytic colonies were identified as S pyogenes using conventional means. RESULTS: Four hundred ninety specimens were obtained from children and 328 from adults. S pyogenes was recovered from 171 (21%) patients. The SARTD detected S pyogenes antigens in 123 of 171 specimens from which S pyogenes was isolated on culture; the screen was negative in 610 of 647 specimens from which cultures were negative. The positive and negative predictive values of the SARTD were 76.9% and 92.7%, respectively. CONCLUSIONS: The SARTD was much less sensitive (72%) than was suggested in the product monograph (90%). Laboratories should vigorously evaluate such products in-house, optimize specimen collection and transport, and choose more sensitive kits for use.

The impact of switching to polymerase chain reaction for the diagnosis of Chlamydia trachomatis infections in women.

BACKGROUND: We noted a marked increase in Chlamydia trachomatis (CT) infections in the Capital Health Region of NS coincident with substitution of a PCR for an enzyme immunoassay (EIA). We reviewed our experience to determine the cost of switching and the impact on the number of new infections diagnosed. METHODS: Information on the number of EIA and PCR tests performed on women was retrieved from an abstracted laboratory information database. We examined records of testing performed between April 1998 and December 2001. Prior to June 2001, all genital swabs were tested using the MicroTrak, II Chlamydia EIA and confirmed by direct fluorescence examination. After July 2001, genital swabs were tested using the COBAS AMPLICOR C. trachomatis test. RESULTS: During the study period, 62,288 EIA tests were performed on specimens submitted; 2,061 (3.33%) were positive. In the six months when testing was performed by the PCR method, 9,559 PCR tests were performed, 463 (4.84%) were positive; 46% increase. In the three years before PCR testing was implemented, an average of 1,626 specimens were submitted monthly. An average of 54 tests were positive (3.3%). The cost for each positive detected by PCR was 208 dollars Cdn and 226 dollars by EIA. CONCLUSIONS: The switch to PCR for the diagnosis of CT produced a marked increase in the number of chlamydia infections diagnosed. The recent increase in the number of reported CT cases in Canada may be due in large part to more sensitive tests. Surprisingly, the cost of each positive test by PCR was 18 dollars Cdn less than that of the EIA.

Predominance of herpes simplex virus type 1 from patients with genital herpes in Nova Scotia.

The epidemiology of genital herpes is changing with evidence to suggest an increasing incidence of herpes simplex virus type 1 (HSV-1) infections. The results of 6529 HSV genital cultures taken between April 1998 and December 2001 were reviewed. overall, HSV-1 was recovered more often than HSV-2; 1213 versus 1045. This trend was particularly striking in young women 30 years of age or less, in whom 70.8% of isolates were HSV-1. In men of the same age range, 45% of isolates were HSV-1. The proportion of women with HSV-1 declined from 73.7% in those younger than 31 years of age to 4.5% in those older than 60 years of age.These observations have important implications. The decline in the relative proportion of HSV-1 isolates from young adults may be the result of changing sexual practices, changing susceptibility or increased exposure to HSV-1 during vaginal intercourse. In this setting HSV-2 vaccines may be less likely to produce the desired reduction in the overall prevalence of genital herpes infections.

Recovery of cephalosporin-resistant Escherichia coli and Salmonella from pork, beef and chicken marketed in Nova Scotia.

BACKGROUND: Antimicrobial use in farm animals is a potentially important contributor to the emergence of antimicrobial resistance. Resistant Salmonella may lead to serious human infections and resistant Escherichia coli may transfer plasmid-encoded resistance genes to other pathogens. OBJECTIVE: To determine the prevalence of E coli and Salmonella species resistant to the third generation of cephalosporins in retail meat products in Halifax, Nova Scotia in 2002. METHODS: Ground beef, ground pork and chicken wings were tested for E coli and Salmonella. E coli were selected on ceftriaxonecontaining media. Beta-lactamases were characterised by isoelectric focusing, polymerase chain reaction and sequencing. Pulsed field gel electrophoresis was performed to determine the relationship of strains. The transferability of plasmids and location of resistance genes was also determined. RESULTS: Forty-three of 75 packages of chicken wings contained ceftriaxone-resistant E coli; 42 of these contained beta-lactamases with isoelectric points at approximately 8.7. Six of seven CMY primer amplicons that were sequenced contained plasmid-mediated Citrobacter freundii-derived blaCMY-2; the other contained a CMY-2- like beta-lactamase. Pulsed field gel electrophoresis patterns demonstrated that strains were not clonal in nature. Four chicken samples contained Salmonella, one of which contained bla CMY-2-mediated resistance and an E coli bearing the same gene, but on different plasmids. Four of 100 beef samples contained blaCMY-2-bearing E coli; none contained Salmonella. Two of 75 pork samples contained ceftriaxone resistant E coli, one of which encoded for CMY-2. One susceptible Salmonella strain was recovered from pork. CONCLUSIONS: Chicken from retail outlets located in Halifax, Nova Scotia, commonly contained blaCMY-2-bearing E coli. The relationship antibiotics used in food-producing animals and its effect on resistance of commensals and pathogens needs to be determined.

Analysis of 3789 in- and outpatient Escherichia coli isolates from across Canada--results of the CANWARD 2007-2009 study.

Escherichia coli was the most commonly isolated pathogen in the Canadian Ward Surveillance Study 2007-2009 (3789 isolates). Susceptibility to cefazolin (34.1%), trimethoprim-sulfamethoxazole (73.8%), ciprofloxacin (78.4%), and levofloxacin (78.8%) was lowest. Susceptibility was above 90% for meropenem (100%), tigecycline (99.9%), piperacillin-tazobactam (97.6%), nitrofurantoin (96.9%), ceftazidime (95.6%), amoxicillin-clavulanate (94.9%), ceftriaxone (94.1%), cefoxitin (92.3%), and gentamicin (90.8%). Over the study period, there was a significant reduction in susceptibility to amoxicillin-clavulanate and trimethoprim-sulfamethoxazole for urinary tract isolates. Inpatient status was associated with greater resistance to nearly all antimicrobials including greater multidrug resistance (MDR). Increasing age was associated with resistance to fluoroquinolones, ceftriaxone, piperacillin-tazobactam, and MDR. Female gender was associated with susceptibility to fluoroquinolones and nitrofurantoin. In conclusion, greater antimicrobial resistance and MDR in E. coli were observed in inpatients, males, and with increasing age. The deterioration of susceptibility to trimethoprim-sulfamethoxazole continues with the greatest reduction in urinary isolates. Significant regional differences in resistance rates were apparent.

The value of multiple surveillance cultures for methicillin-resistant Staphylococcus aureus.

BACKGROUND: We evaluated our experience in a low prevalence setting to determine the extent to which multiple swabs increased detection rates and the incremental costs of doing so. METHODS: Nasal and groin swabs submitted in pairs were cultured onto a single plate (Oxoid MRSA Denim Blue Agar; Oxoid Company, Napean, ON, Canada). We determined whether MRSA was detected when swabs submitted in the preceding 3 days were negative. We explored the costs associated with screening and of each additional colonized patient detected. RESULTS: In all, 60,049 paired nose and perineal swabs were submitted from 21,599 patients. In all, there were 12,750 duplicate, 1437 triplicate, and 112 instances when >4 swabs were processed within 3 days. The first culture was positive in 106 of 12,750 (0.83%%), 42 of 12,750 (0.33%) on the second when the first was negative, 7 of 1642 (0.43%) on the third or subsequent swab pair when the preceding 2 were negative. CONCLUSION: Overall, the sensitivity of the first of multiple cultures of a set was 74.3%. Had the 14,392 multiple samples not been submitted, 49 colonized patients would not have been identified. Additional laboratory costs associated with multiple samples equaled $2088 per patient identified.

Risk of coinfection with Chlamydia trachomatis and Neisseria gonorrhoeae in Nova Scotia.

BACKGROUND: The frequency of Chlamydia trachomatis and Neisseria gonorrhoeae coinfection can vary depending on their individual incidence and prevalence rates. OBJECTIVE: To determine the frequency of C trachomatis and N gonorrhoeae coinfections by evaluating the results of testing in 2007 and 2008 to better inform testing and treatment decisions. METHODS: Specimens from the same patient submitted on the same day served as the basis for the present study. The age, sex and the source of the specimen were also linked to the accession number. Infection and coinfection rates were analyzed in both males and females. RESULTS: Concurrent testing was performed on 41,567 female specimens and 1827 male specimens, of which, 1495 female samples (3.6%) tested positive for C trachomatis infection and 88 (0.2%) tested positive for N gonorrhoeae infections. Only 31 females were coinfected; however, for those between 11 and 25 years of age, 25 of 61 females (40.1%) with N gonorrhoeae infection also tested positive for C trachomatis infection; conversely, 25 of 1248 females (2.0%) with C trachomatis infection also tested positive for N gonorrhoeae infection. For males, 213 (11.7%) tested positive for C trachomatis infection, and 59 (3.2%) tested positive for N gonorrhoeae infection. In 30 males with N gonorrhoeae between 11 and 25 years of age, and 149 males with C trachomatis, eight coinfections were observed (26.7% and 5.3%, respectively). Of those older than 25 years of age, only five of 905 men and six of 19,465 women were coinfected. None of the 10,935 women who were 30 years of age or older had coinfections. CONCLUSION: The N gonorrhoeae coinfection rate in males with C trachomatis may justify empirical antimicrobials; however, in females, the proportion of coinfected may not justify empirical treatment for N gonorrhoeae infection when the C trachomatis test is positive and N gonorrhoeae testing has not been performed.

A study of the prevalence of cytotoxic and non-cytotoxic Klebsiella oxytoca fecal colonization in two patient populations.

BACKGROUND: Klebsiella oxytoca is a cause of antibiotic-associated hemorrhagic colitis. Few reports of the occurrence of K oxytoca within stool exist and there is no gold standard method for its isolation. METHODS: MacConkey agar was modified to culture K oxytoca. Ampicillin was added and adonitol was substituted for lactose. Rectal swabs from 200 patients being screened for vancomycin-resistant enterococci (VRE) and stool specimens from 429 patients who tested negative for Clostridium difficile cytotoxin were cultured. K oxytoca isolates were evaluated for cytotoxicity to HEp-2 cells. Available charts of K oxytoca-positive patients and a convenience sample of 93 K oxytoca-negative patients who underwent testing for C difficile cytotoxicity were reviewed retrospectively for documentation of bloody stool. RESULTS: K oxytoca was isolated from 14 of 200 patients (7.0%) being screened for VRE; only one of the 14 isolates (7.1%) was cytotoxic. The organism was isolated from 42 of 429 patients (9.8%) tested for C difficile cytotoxicity; 10 isolates (23.8%) were cytotoxic. Differences in isolation and cytotoxicity rates between groups were not statistically significant. Two of 13 (15.4%) K oxytoca-positive patients screened for VRE, three of 27 (11.1%) K oxytoca-positive patients tested for C difficile cytotoxicity, and 11 of 93 (11.8%) patients from the convenience sample had documented bloody stool. CONCLUSIONS: A medium that greatly facilitates isolation of K oxytoca was developed. Occurrence of K oxytoca colonization was similar in the two patient populations studied and isolation of cytotoxic K oxytoca was not usually associated with hematochezia. Current understanding of the occurrence and causality of antibiotic-associated hemorrhagic colitis is insufficient for clinical laboratories to begin culturing K oxytoca and testing for cytotoxicity.