Association of Neisseria gonorrhoeae genogroups and specific PBP2/MtrR/PorB mutation patterns with susceptibility to penicillin in a susceptible gonococcal population.

Objectives: To ascertain whether the antimicrobial susceptibility of Neisseria gonorrhoeae isolates with differing susceptibilities to penicillin is associated with genogroups (GGs) and combined mutation patterns in PBP2 (penA), the multiple transfer resistance repressor (MtrR; mtrR) and porin B (PorB; porB). Methods: The susceptibility of 146 clinical N. gonorrhoeae isolates to penicillin was determined using the agar dilution method and the interpretation criteria of CLSI. The DNA sequences of penA, mtrR and porB in isolates were compared with WT sequences and mutation patterns were determined. Isolates were typed by N. gonorrhoeae multi-antigen sequence typing (NG-MAST) and STs were grouped into specific GGs. Results: The isolates tested carried 9 mutation patterns in PBP2 and 12 mutation patterns in each of MtrR and PorB. Of the 146 isolates, 121 (82.9%) were grouped into 13 different GGs. Isolates with penicillin MICs of 0.03-0.06 mg/L were significantly associated with GG25 (P < 0.05) and PBP2/MtrR/PorB mutation pattern I/WT/WT (P < 0.01). Isolates with a penicillin MIC of 1.0 mg/L were associated (P < 0.05) with: (i) GG3655 and mutation pattern XXII/A-;G45D/G120K;A121N; (ii) GG921 and mutation pattern IX/G45D/G120D;A121N; and (iii) GG1109 and mutation pattern IX/G45D/WT. Sixty percent (9/15) of penicillin-resistant isolates (MIC ≥2 mg/L) were GG3654 (P < 0.0001) and carried mutation pattern IX/G45D/G120K;A121D or IX/G45D/G120D;A121D (P < 0.05). Conclusions: Specific mutation patterns in PBP2/MtrR/PorB were associated with specific GGs and penicillin susceptibility. This approach of typing strains and resistance patterns is ideal for predicting antimicrobial resistance and should be used in instances in which gonococcal culture is not available but DNA can be obtained from clinical specimens.

High levels of susceptibility to new and older antibiotics in Neisseria gonorrhoeae isolates from Saskatchewan (2003-15): time to consider point-of-care or molecular testing for precision treatment?

OBJECTIVES: The antimicrobial susceptibility of Neisseria gonorrhoeae isolates from Saskatchewan was determined retrospectively (2003-15) to ascertain temporal trends to both current and older antimicrobials used for treatment. METHOD: The agar dilution method was used to test the antimicrobial susceptibilities of 685 isolates to seven antibiotics. RESULTS: Over the period, only three (0.4%) gonococcal isolates had reduced susceptibility to cefixime and/or ceftriaxone. All isolates were susceptible to spectinomycin. Over 95% of the isolates tested were susceptible to azithromycin except in 2010 and 2013 (27.6% and 7.2% resistant, respectively). One isolate was resistant to both azithromycin and cefixime. Ciprofloxacin resistance was seen in < 5% of isolates prior to 2010, but in > 5% thereafter. From 2006 to 2012, and in 2015, penicillin resistance was detected in < 5% (0%-4.0%) of isolates, but in > 5% for the rest of the study period. Tetracycline resistance remained >5% (11.8%-89.1%) throughout the study. Plasmid-mediated resistance to tetracycline fluctuated between 0% and 17.5% of isolates tested. Four isolates were MDR and two isolates were XDR. CONCLUSIONS: N. gonorrhoeae isolates were largely susceptible (∼85%) to antibiotics no longer recommended for treatment, such as penicillin and ciprofloxacin. Gonorrhoea in Saskatchewan is primarily (>95%) diagnosed by nucleic acid amplification testing, which does not permit antimicrobial susceptibility testing. The development of molecular testing, or point-of-care tests, to evaluate antimicrobial susceptibility, would enhance knowledge of true levels of resistance and allow discretion as to whether older but still effective antibiotics could be used in individual patient care.

Live Attenuated Versus Inactivated Influenza Vaccine in Hutterite Children: A Cluster Randomized Blinded Trial.

BACKGROUND: Whether vaccinating children with intranasal live attenuated influenza vaccine (LAIV) is more effective than inactivated influenza vaccine (IIV) in providing both direct protection in vaccinated persons and herd protection in unvaccinated persons is uncertain. Hutterite colonies, where members live in close-knit, small rural communities in which influenza virus infection regularly occurs, offer an opportunity to address this question. OBJECTIVE: To determine whether vaccinating children and adolescents with LAIV provides better community protection than IIV. DESIGN: A cluster randomized blinded trial conducted between October 2012 and May 2015 over 3 influenza seasons. (ClinicalTrials.gov: NCT01653015). SETTING: 52 Hutterite colonies in Alberta and Saskatchewan, Canada. PARTICIPANTS: 1186 Canadian children and adolescents aged 36 months to 15 years who received the study vaccine and 3425 community members who did not. INTERVENTION: Children were randomly assigned according to community in a blinded manner to receive standard dosing of either trivalent LAIV or trivalent IIV. MEASUREMENTS: The primary outcome was reverse transcriptase polymerase chain reaction-confirmed influenza A or B virus in all participants (vaccinated children and persons who did not receive the study vaccine). RESULTS: Mean vaccine coverage among children in the LAIV group was 76.9% versus 72.3% in the IIV group. Influenza virus infection occurred at a rate of 5.3% (295 of 5560 person-years) in the LAIV group versus 5.2% (304 of 5810 person-years) in the IIV group. The hazard ratio comparing LAIV with IIV for influenza A or B virus was 1.03 (95% CI, 0.85 to 1.24). LIMITATION: The study was conducted in Hutterite communities, which may limit generalizability. CONCLUSION: Immunizing children with LAIV does not provide better community protection against influenza than IIV. PRIMARY FUNDING SOURCE: The Canadian Institutes for Health Research.

Molecular epidemiology of Neisseria gonorrhoeae isolates from Saskatchewan, Canada: utility of NG-MAST in predicting antimicrobial susceptibility regionally.

OBJECTIVES: To investigate the molecular epidemiology of isolates of Neisseria gonorrhoeae from Saskatchewan, Canada, using Neisseria gonorrhoeae multi antigen sequence typing (NG-MAST), and to assess associations between antimicrobial susceptibility (AMS) and specific strain types (STs). METHODS: 320 consecutive gonococcal isolates, collected between 2003 and 2008, were typed by NG-MAST. STs were grouped if one of their alleles was common and the other differed by ≤1% in DNA sequence. AMS was determined by agar dilution (CLSI) to seven antibiotics. RESULTS: N gonorrhoeae isolates were resolved into 82 individual NG-MAST STs and 18 NG-MAST ST groups with groups 25, 3655, 921, 3654, 3657 and 3656 comprising 53.4% (171/320) of the isolates. N gonorrhoeae isolates susceptible to all the tested antimicrobials were significantly (p<0.05) associated with ST 25 (87%). Other significant associations between ST and AMS included: ST 3654 and isolates with minimum inhibitory concentrations of ≥0.03 mg/L to third generation cephalosporins; ST 3711 (100%) and TRNG; and ST/group 3654 (43%) and chromosomal resistance to penicillin and tetracycline. Several NG-MAST STs/groups were significantly associated with isolates with chromosomal resistance to tetracycline. Isolates resistant to ciprofloxacin (n=5) and azithromycin (n=2) appeared as individual STs. Significant associations were observed among individual STs, sex and age of the patient, and regional and temporal distributions. CONCLUSIONS: Associations between N gonorrhoeae AMS and NG-MAST STs were identified and may be useful in predicting AMS regionally. Because STs in different countries vary considerably, the use of NG-MAST for the prediction of AMS globally requires further study.

Comparison of the BD Viper System with XTR Technology to the Gen-Probe APTIMA COMBO 2 Assay using the TIGRIS DTS system for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine specimens.

BACKGROUND: Performances of the BD ProbeTec Chlamydia trachomatis (CT)/Neisseria Gonorrhoeae (GC) Q(x) Amplified DNA Assay reagents on a BD Viper System with XTR Technology and APTIMA COMBO 2 Assay reagents on a TIGRIS DTS platform, for detection of both CT and GC were compared. METHODS: A total of 1018 first-void urine specimens were tested for the presence of CT and GC DNA using the 2 assays. RESULTS: CT was detected in 143 specimens (14%). Eight specimens exhibited discordant results, and they were divided equally between the 2 assays. Based on the original results, the overall agreement for CT was 99.2%, with 97.1% and 99.5% in agreement with positive and negative specimens, respectively. Cohen's Kappa was 0.967. GC was detected in 27 specimens (2.6%). Two specimens exhibited discordant results, and they were divided equally between the 2 assays. Based on the original results, the overall agreement was 99.8%, with 96.2% and 99.9% in agreement for positive and negative specimens, respectively. Cohen's Kappa was 0.961. CONCLUSIONS: There was a high level of agreement between the systems for both CT and GC detection.

Effect of influenza vaccination of children on infection rates in Hutterite communities: a randomized trial.

CONTEXT: Children and adolescents appear to play an important role in the transmission of influenza. Selectively vaccinating youngsters against influenza may interrupt virus transmission and protect those not immunized. OBJECTIVE: To assess whether vaccinating children and adolescents with inactivated influenza vaccine could prevent influenza in other community members. DESIGN, SETTING, AND PARTICIPANTS: A cluster randomized trial involving 947 Canadian children and adolescents aged 36 months to 15 years who received study vaccine and 2326 community members who did not receive the study vaccine in 49 Hutterite colonies in Alberta, Saskatchewan, and Manitoba. Follow-up began December 28, 2008, and ended June 23, 2009. INTERVENTION: Children were randomly assigned according to community and in a blinded manner to receive standard dosing of either inactivated trivalent influenza vaccine or hepatitis A vaccine, which was used as a control. MAIN OUTCOME MEASURES: Confirmed influenza A and B infection using a real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay and by measuring serum hemagglutination inhibition titers. RESULTS: The mean rate of study vaccine coverage among eligible participants was 83% (range, 53%-100%) for the influenza vaccine colonies and 79% (range, 50%-100%) for the hepatitis A vaccine colonies. Among nonrecipients, 39 of 1271 (3.1%) in the influenza vaccine colonies and 80 of 1055 (7.6%) in the hepatitis A vaccine colonies had influenza illness confirmed by RT-PCR, for a protective effectiveness of 61% (95% confidence interval [CI], 8%-83%; P = .03). Among all study participants (those who were and those who were not vaccinated), 80 of 1773 (4.5%) in the influenza vaccine colonies and 159 of 1500 (10.6%) in the hepatitis A vaccine colonies had influenza illness confirmed by RT-PCR for an overall protective effectiveness of 59% (95% CI, 5%-82%; P = .04). No serious vaccine adverse events were observed. CONCLUSION: Immunizing children and adolescents with inactivated influenza vaccine significantly protected unimmunized residents of rural communities against influenza. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00877396.

Predictors of influenza a molecular viral shedding in Hutterite communities.

BACKGROUND: Patterns of influenza molecular viral shedding following influenza infection have been well established; predictors of viral shedding however remain uncertain. OBJECTIVES: We sought to determine factors associated with peak molecular viral load, duration of shedding, and viral area under the curve (AUC) in children and adult Hutterite colony members with laboratory-confirmed influenza. METHODS: A cohort study was conducted in Hutterite colonies in Alberta, Canada. Flocked nasal swabs were collected during three influenza seasons (2007-2008 to 2009-2010) from both symptomatic and asymptomatic individuals infected with influenza. Samples were tested by real-time reverse-transcription polymerase chain reaction for influenza A and influenza B, and the viral load was determined for influenza A-positive samples. RESULTS: For seasonal H1N1, younger age was associated with a larger AUC, female sex was associated with decreased peak viral load and reduced viral shedding duration, while the presence of comorbidity was associated with increased peak viral load. For H3N2, younger age was associated with increased peak viral load and increased AUC. For pandemic H1N1, younger age was associated with increased peak viral load and increased viral AUC, female sex was associated with reduced peak viral load, while inapparent infection was associated with reduced peak viral load, reduced viral shedding duration, and reduced viral AUC. CONCLUSIONS: Patterns of molecular viral shedding vary by age, sex, comorbidity, and the presence of symptoms. Predictor variables vary by influenza A subtype.

Effect of Influenza Vaccination of Children on Infection Rate in Hutterite Communities: Follow-Up Study of a Randomized Trial.

BACKGROUND: An earlier cluster randomized controlled trial (RCT) of Hutterite colonies had shown that if more than 80% of children and adolescents were immunized with influenza vaccine there was a statistically significant reduction in laboratory-confirmed influenza among all unimmunized community members. We assessed the impact of this intervention for two additional influenza seasonal periods. METHODS: Follow-up data for two influenza seasonal periods of a cluster randomized trial involving 1053 Canadian children and adolescents aged 36 months to 15 years in Season 2 and 1014 in Season 3 who received the study vaccine, and 2805 community members in Season 2 and 2840 in Season 3 who did not receive the study vaccine. Follow-up for Season 2 began November 18, 2009 and ended April 25, 2010 while Season 3 extended from December 6, 2010 and ended May 27, 2011. Children were randomly assigned in a blinded manner according to community membership to receive either inactivated trivalent influenza vaccine or hepatitis A. The primary outcome was confirmed influenza A and B infection using RT-PCR assay. Due to the outbreak of 2009 H1N1 pandemic, data in Season 2 were excluded for analysis. RESULTS: For an analysis of the combined Season 1 and Season 3 data, among non-recipients (i.e., participants who did not receive study vaccines), 66 of the 2794 (2.4%) participants in the influenza vaccine colonies and 121 of the 2301 (5.3%) participants in the hepatitis A colonies had influenza confirmed by RT-PCR, for a protective effectiveness of 60% (95% CI, 6% to 83%; P = 0.04); among all study participants (i.e., including both those who received study vaccine and those who did not), 125 of the 3806 (3.3%) in the influenza vaccine colonies and 239 of the 3243 (7.4%) in the hepatitis A colonies had influenza confirmed by RT-PCR, for a protective effectiveness of 63% (95% CI, 5% to 85%; P = 0.04). CONCLUSION: Immunizing children and adolescents with inactivated influenza vaccine can offer a protective effect among unimmunized community members for influenza A and B together when considered over multiple years of seasonal influenza. TRIAL REGISTRATION: Clinicaltrials.gov NCT00877396.

Characteristics of respiratory viral infections during influenza season in Canadian Hutterite Communities.

OBJECTIVES: To determined the pathogen-specific incidence of respiratory virus infection in Hutterite communities occurring over the 2008-2009 influenza season and assess temporal characteristics of respiratory illness related to infection. METHODS: 3273 participants community members enrolled in a cluster randomized trial of influenza vaccine were studied. RESULTS: One hundred forty-nine participants had laboratory-confirmed influenza, and 595 had at least one episode of laboratory-confirmed respiratory viral infection other than influenza. Entero/rhinovirus had the highest incidence among children<5 years. CONCLUSIONS: A decline in the incidence of infections with age was observed for influenza as well as for most other respiratory viruses.