Household Transmission of Carbapenemase-producing Enterobacterales in Ontario, Canada.

BACKGROUND: Data on household transmission of carbapenemase-producing Enterobacterales (CPE) remain limited. We studied risk of CPE household co-colonization and transmission in Ontario, Canada. METHODS: We enrolled CPE index cases (identified via population-based surveillance from January 2015 to October 2018) and their household contacts. At months 0, 3, 6, 9, and 12, participants provided rectal and groin swabs. Swabs were cultured for CPE until September 2017, when direct polymerase chain reaction (PCR; with culture of specimens if a carbapenemase gene was detected) replaced culture. CPE risk factor data were collected by interview and combined with isolate whole-genome sequencing to determine likelihood of household transmission. Risk factors for household contact colonization were explored using a multivariable logistic regression model with generalized estimating equations. RESULTS: Ninety-five households with 177 household contacts participated. Sixteen (9%) household contacts in 16 (17%) households were CPE-colonized. Household transmission was confirmed in 3/177 (2%) cases, probable in 2/177 (1%), possible in 9/177 (5%), and unlikely in 2/177 (1%). Household contacts were more likely to be colonized if they were the index case's spouse (odds ratio [OR], 6.17; 95% confidence interval [CI], 1.05-36.35), if their index case remained CPE-colonized at household enrollment (OR, 7.00; 95% CI, 1.92-25.49), or if they had at least 1 set of specimens processed after direct PCR was introduced (OR, 6.46; 95% CI, 1.52-27.40). CONCLUSIONS: Nine percent of household contacts were CPE-colonized; 3% were a result of household transmission. Hospitals may consider admission screening for patients known to have CPE-colonized household contacts.

Emergence of Carbapenemase-Producing Enterobacteriaceae, South-Central Ontario, Canada1.

We analyzed population-based surveillance data from the Toronto Invasive Bacterial Diseases Network to describe carbapenemase-producing Enterobacteriaceae (CPE) infections during 2007-2015 in south-central Ontario, Canada. We reviewed patients' medical records and travel histories, analyzed microbiologic and clinical characteristics of CPE infections, and calculated incidence. Among 291 cases identified, New Delhi metallo-ß-lactamase was the predominant carbapenemase (51%). The proportion of CPE-positive patients with prior admission to a hospital in Canada who had not received healthcare abroad or traveled to high-risk areas was 13% for patients with oxacillinase-48, 24% for patients with New Delhi metallo-ß-lactamase, 55% for patients with Klebsiella pneumoniae carbapenemase, and 67% for patients with Verona integron-encoded metallo-ß-lactamase. Incidence of CPE infection increased, reaching 0.33 cases/100,000 population in 2015. For a substantial proportion of patients, no healthcare abroad or high-risk travel could be established, suggesting CPE acquisition in Canada. Policy and practice changes are needed to mitigate nosocomial CPE transmission in hospitals in Canada.

Mycoplasma genitalium Antibiotic Resistance-Mediating Mutations in Canadian Women With or Without Chlamydia Trachomatis Infection.

Testing remnant Aptima specimens from women infected with Chlamydia trachomatis detected 13.4% (53/396) with Mycoplasma genitalium compared with 5.4% (22/406) in matched C. trachomatis-negative women. Overall, 9.4% (provincial ranges of 3-20%) were infected with M. genitalium and resistance mediating mutations were found in 47.3% (26/55) to macrolides and 1.9% (1/53) to fluoroquinolones by sequencing.

Impact of asymptomatic herpes simplex virus type 2 infection on mucosal homing and immune cell subsets in the blood and female genital tract.

HSV-2 infection is common and generally asymptomatic, but it is associated with increased HIV susceptibility and disease progression. This may relate to herpes-mediated changes in genital and systemic immunology. Cervical cytobrushes and blood were collected from HIV-uninfected African/Caribbean women in Toronto, and immune cell subsets were enumerated blindly by flow cytometry. Immune differences between groups were assessed by univariate analysis and confirmed using a multivariate model. Study participants consisted of 46 women, of whom 54% were infected with HSV-2. T cell activation and expression of the mucosal homing integrin α4ß7 (19.60 versus 8.76%; p < 0.001) were increased in the blood of HSV-2-infected women. Furthermore, expression of α4ß7 on blood T cells correlated with increased numbers of activated (coexpressing CD38/HLA-DR; p = 0.004) and CCR5(+) (p = 0.005) cervical CD4(+) T cells. HSV-2-infected women exhibited an increase in the number of cervical CD4(+) T cells (715 versus 262 cells/cytobrush; p = 0.016), as well as an increase in the number and proportion of cervical CD4(+) T cells that expressed CCR5(+) (406 versus 131 cells, p = 0.001; and 50.70 versus 34.90%, p = 0.004) and were activated (112 versus 13 cells, p < 0.001; and 9.84 versus 4.86%, p = 0.009). Mannose receptor expression also was increased on cervical dendritic cell subsets. In conclusion, asymptomatic HSV-2 infection was associated with significant systemic and genital immune changes, including increased immune activation and systemic α4ß7 expression; correlation of the latter with highly HIV-susceptible CD4(+) T cell subsets in the cervix may provide a mechanism for the increased HIV susceptibility observed in asymptomatic HSV-2-infected women.

Performance of the Cue COVID-19 point-of-care molecular test: insights from a multi-site clinic service model.

The COVID-19 pandemic highlighted the critical need for rapid and accurate molecular diagnostic testing. The Cue COVID-19 Point-of-Care Test (Cue POCT) is a nucleic acid amplification test (NAAT), authorized by Health Canada and FDA as a POCT for SARS-CoV-2 detection. Cue POCT was deployed at a network of clinics in Ontario, Canada with n = 13,848 patrons tested between 17 July 2021 and 31 January 2022. The clinical performance and operational experience with Cue POCT were examined for this testing population composed mostly of asymptomatic individuals (93.7%). A head-to-head prospective clinical verification was performed between 17 July and 4 October for all POCT service clients (n = 3,037) with paired COVID-19 testing by Cue and RT-PCR. Prospective verification demonstrated a clinical sensitivity of 100% and clinical specificity of 99.4% for Cue COVID-19 POCT. The lack of false negatives and low false positive rate (0.64%), underscores the high accuracy (99.4%) of Cue POCT to provide rapid PCR quality results. Low error rates (cancellation rate of 0% and invalid rate of 0.63%) with the current software version were additionally noted. Taken together, these findings highlight the value of accurate molecular COVID-19 POCT in a distributed service delivery model to rapidly detect cases in the community with the potential to curb transmission in high-exposure settings (i.e., in-flight, congregate workplace, and social events). The insights gleaned from this operational implementation are readily transferable to future POCT diagnostic services. IMPORTANCE This manuscript reports on the findings of a large asymptomatic population who underwent surveillance COVID testing on the Cue COVID-19 Point-of-Care Test (POCT). Review of test performance of this rapid molecular POCT, as compared to gold standard RT-PCR, is valuable to many audiences, including public health, emergency testing services, employers, and the general population of consumers who are seeking a user-friendly, accurate, cost-effective, and sustainable testing model for COVID screening. The findings from this operational experience also inform future models of POCT services beyond COVID.

Ano-genital human papillomavirus type 97 infection is detected in Canadian men but not women at risk or infected with the human immunodeficiency virus.

BACKGROUND: Human papillomavirus type 97 (HPV97) DNA was detected in nearly 5% of anal samples collected from HIV-seropositive men living in Montreal, Canada. The rate of detection of HPV97 in the genital tract of Canadian women is unknown. Whether HPV97 is a local epidemic in HIV-seropositive men living in Montreal is also unknown. The prevalence of human papillomavirus type 97 (HPV97) was assessed in cervicovaginal cells from women living in Canada and in anal samples from HIV-seropositive men living in Toronto. FINDINGS: Cervicovaginal lavages collected from 904 women (678 HIV-seropositive, 226 HIV-seronegative) women living in Canada and anal cells collected from 123 HIV-seropositive men living in Toronto were tested for the presence of HPV97 with PCR. HPV97-positive samples were further tested by PCR-sequencing for molecular variant analysis to assess if all HPV97-positive men were infected with the same strain. All cervicovaginal samples were negative for HPV97. HPV97 was detected in anal samples from 6 HIV-seropositive men (4.9%, 95% confidence interval 2.0-10.5%), of whom five had high-grade and one had low-grade anal intraepithelial neoplasia, in addition to 2 to 8 HPV genital genotypes per sample. Four HPV97 variants were defined by four variation sites in the viral control region. CONCLUSION: These findings indicate that HPV97 infects in the anal canal of HIV-seropositive men but is not detected in the genital tract of women.

Case-control study of household contacts to examine immunological protection from Bordetella pertussis transmission - study protocol.

BACKGROUND: There is mounting evidence that the recent resurgence of pertussis in many countries is in part related to the acellular vaccine, which has been administered in Canada since 1997. This vaccine elicits a different cell-mediated immune response than the previously used whole-cell vaccine, and its effectiveness wanes over time. The aim of this study is to understand the immunological, demographic and clinical factors that mediate protection from pertussis on exposure. METHODS: This is a household case-control study protocol. Following notification of an index case in a household, a study team will conduct a home visit to collect data and biological specimens. The study team will return to the household 8 weeks from the onset of illness in the index case. The Th1, Th2 and Th17 responses, cytokine expression, IgG subclass, blood cell counts and presence of Bordetella pertussis will be determined. We will use laboratory and statistical analyses to determine immunological differences between contacts who are infected with B. pertussis and contacts who remain healthy, and to determine which clinical and demographic covariates are associated with a reduced risk of infection. INTERPRETATION: The results of this study will be essential for understanding the immune response required for protection from infection with B. pertussis and will contribute to our understanding of the shortcomings of the current vaccine.

Randomized Intervention of Self-Collected Sampling for Human Papillomavirus Testing in Under-Screened Rural Women: Uptake of Screening and Acceptability.

BACKGROUND: Our aim was to determine if cervical cancer screening uptake would increase among under-screened women living in rural Ontario, Canada, if at-home self-collected sampling for human papillomavirus (HPV) testing was offered as a primary cervical cancer screening modality, compared to invited papanicolaou (Pap) testing or routine opportunistic screening. METHODS: Women 30-70 years of age who were overdue for cervical cancer screening were randomized to receive (1) an at-home self-collected HPV kit, (2) a reminder invitation for Pap testing, or (3) standard of care opportunistic screening. The first two arms were also asked demographic and screening history questions. Women randomized to arm 1 were asked about acceptability. RESULTS: In total, 818 eligible women were identified in a small rural community in Southwestern Ontario: 335 received a self-collected HPV testing kit, 331 received a reminder letter, and 152 received standard of care. In the HPV self-collection arm, 21% (70/335) returned the sample and questionnaire and 11% (37/335) opted to undergo Pap testing. In total, 32% from the HPV self-collection arm, 15% (51/331) from the Pap invitation arm, and 8.5% (13/152) with standard of care were screened. Women receiving the self-collected HPV kit were 3.7 (95% confidence interval 2.2-6.4) times more likely to undergo screening compared to the standard of care arm. In the HPV self-sampling arm, 80% (56/70) said they would be very likely to choose self-collected sampling in the future. CONCLUSIONS: Providing self-collected sampling for HPV testing was more effective than sending reminder letters to increase screening coverage in under-screened women.

A versatile plasmonic thermogel for disinfection of antimicrobial resistant bacteria.

The increasing occurrence of antimicrobial resistance among bacteria is a global problem that requires the development of alternative techniques to eradicate these superbugs. Herein, we used a combination of thermosensitive biocompatible polymer and gold nanorods to specifically deliver, preserve and confine heat to the area of interest. Our data demonstrates that this technique can be used to kill both Gram positive and Gram negative antimicrobial resistant bacteria in vitro. Our approach significantly reduces the antimicrobial resistant bacteria load in experimentally infected wounds by 98% without harming the surrounding tissues. More importantly, this polymer-nanocomposite can be prepared easily and applied to the wounds, can generate heat using a hand-held laser device, is safe for the operator, and does not have any adverse effects on the wound tissue and healing process.

Integrated quantum dot barcode smartphone optical device for wireless multiplexed diagnosis of infected patients.

Inorganic nanoparticles are ideal precursors for engineering barcodes for rapidly detecting diseases. Despite advances in the chemical design of these barcodes, they have not advanced to clinical use because they lack sensitivity and are not cost-effective due to requirement of a large read-out system. Here we combined recent advances in quantum dot barcode technology with smartphones and isothermal amplification to engineer a simple and low-cost chip-based wireless multiplex diagnostic device. We characterized the analytical performance of this device and demonstrated that the device is capable of detecting down to 1000 viral genetic copies per milliliter, and this enabled the diagnosis of patients infected with HIV or hepatitis B. More importantly, the barcoding enabled us to detect multiple infectious pathogens simultaneously, in a single test, in less than 1 h. This multiplexing capability of the device enables the diagnosis of infections that are difficult to differentiate clinically due to common symptoms such as a fever or rash. The integration of quantum dot barcoding technology with a smartphone reader provides a capacity for global surveillance of infectious diseases and the potential to accelerate knowledge exchange transfer of emerging or exigent disease threats with healthcare and military organizations in real time.

Community-acquired respiratory viruses and co-infection among patients of Ontario sentinel practices, April 2009 to February 2010.

BACKGROUND: Respiratory viruses are known to cocirculate but this has not been described in detail during an influenza pandemic. OBJECTIVES: To describe respiratory viruses, including co-infection and associated attributes such as age, sex or comorbidity, in patients presenting with influenza-like illness to a community sentinel network, during the pandemic A(H1N1)pdm09 in Ontario, Canada. METHODS: Respiratory samples and epidemiologic details were collected from 1018 patients with influenza-like illness as part of respiratory virus surveillance and a multiprovincial case-control study of influenza vaccine effectiveness. RESULTS: At least one virus was detected in 668 (65·6%) of 1018 samples; 512 (50·3%) had single infections and 156 (15·3%) co-infections. Of single infections, the most common viruses were influenza A in 304 (59·4%) samples of which 275 (90·5%) were influenza A(H1N1)pdm09, and enterovirus/rhinovirus in 149 (29·1%) samples. The most common co-infections were influenza A and respiratory syncytial virus B, and influenza A and enterovirus/rhinovirus. In multinomial logistic regression analyses adjusted for age, sex, comorbidity, and timeliness of sample collection, single infection was less often detected in the elderly and co-infection more often in patients <30 years of age. Co-infection, but not single infection, was more likely detected in patients who had a sample collected within 2 days of symptom onset as compared to 3-7 days. CONCLUSIONS: Respiratory viral co-infections are commonly detected when using molecular techniques. Early sample collection increases likelihood of detection of co-infection. Further studies are needed to better understand the clinical significance of viral co-infection.

Analytical and clinical validation of novel real-time reverse transcriptase-polymerase chain reaction assays for the clinical detection of swine-origin H1N1 influenza viruses.

During the early stages of the 2009/2010 swine-origin H1N1 influenza A (S-OIV H1N1 FluA) outbreak, the development and validation of sensitive and specific detection methods were a priority for rapid and accurate diagnosis. Between May and June 2009, 2 real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) assays targeting the hemagglutinin and neuraminidase genes of the S-OIV H1N1 FluA virus were developed. These assays are highly specific, showing no cross-reactivity against a panel of respiratory viruses and can differentiate S-OIV H1N1 from seasonal FluA viruses. Analytical sensitivities of the 2 assays were found to be 10(-1) tissue culture infectious dose, 50%/ml. Clinical testing showed 99.2% sensitivity and 94.6-98.1% specificity. A large prospective analysis showed that 94.8-95.5% of S-OIV positive specimens were negative by seasonal H1/H3 subtyping. The large-scale validation data presented in this report indicate that these novel assays provide an accurate and efficient method for the rapid detection of S-OIV H1N1 FluA viruses.

Humoral and cell-mediated immunity to pandemic H1N1 influenza in a Canadian cohort one year post-pandemic: implications for vaccination.

We evaluated a cohort of Canadian donors for T cell and antibody responses against influenza A/California/7/2009 (pH1N1) at 8-10 months after the 2nd pandemic wave by flow cytometry and microneutralization assays. Memory CD8 T cell responses to pH1N1 were detectable in 58% (61/105) of donors. These responses were largely due to cross-reactive CD8 T cell epitopes as, for those donors tested, similar recall responses were obtained to A/California 2009 and A/PR8 1934 H1N1 Hviruses. Longitudinal analysis of a single infected individual showed only a small and transient increase in neutralizing antibody levels, but a robust CD8 T cell response that rose rapidly post symptom onset, peaking at 3 weeks, followed by a gradual decline to the baseline levels seen in a seroprevalence cohort post-pandemic. The magnitude of the influenza-specific CD8 T cell memory response at one year post-pandemic was similar in cases and controls as well as in vaccinated and unvaccinated donors, suggesting that any T cell boosting from infection was transient. Pandemic H1-specific antibodies were only detectable in approximately half of vaccinated donors. However, those who were vaccinated within a few months following infection had the highest persisting antibody titers, suggesting that vaccination shortly after influenza infection can boost or sustain antibody levels. For the most part the circulating influenza-specific T cell and serum antibody levels in the population at one year post-pandemic were not different between cases and controls, suggesting that natural infection does not lead to higher long term T cell and antibody responses in donors with pre-existing immunity to influenza. However, based on the responses of one longitudinal donor, it is possible for a small population of pre-existing cross-reactive memory CD8 T cells to expand rapidly following infection and this response may aid in viral clearance and contribute to a lessening of disease severity.

Seroprevalence of pandemic influenza H1N1 in Ontario from January 2009-May 2010.

BACKGROUND: We designed a seroprevalence study using multiple testing assays and population sources to estimate the community seroprevalence of pH1N1/09 and risk factors for infection before the outbreak was recognized and throughout the pandemic to the end of 2009/10 influenza season. METHODS: Residual serum specimens from five time points (between 01/2009 and 05/2010) and samples from two time points from a prospectively recruited cohort were included. The distribution of risk factors was explored in multivariate adjusted analyses using logistic regression among the cohort. Antibody levels were measured by hemagglutination inhibition (HAI) and microneutralization (MN) assays. RESULTS: Residual sera from 3375 patients and 1024 prospectively recruited cohort participants were analyzed. Pre-pandemic seroprevalence ranged from 2%-12% across age groups. Overall seropositivity ranged from 10%-19% post-first wave and 32%-41% by the end of the 2009/10 influenza season. Seroprevalence and risk factors differed between MN and HAI assays, particularly in older age groups and between waves. Following the H1N1 vaccination program, higher GMT were noted among vaccinated individuals. Overall, 20-30% of the population was estimated to be infected. CONCLUSIONS: Combining population sources of sera across five time points with prospectively collected epidemiological information yielded a complete description of the evolution of pH1N1 infection.

Increased levels of immune activation in the genital tract of healthy young women from sub-Saharan Africa.

OBJECTIVES: To determine whether healthy, young women in sub-Saharan Africa have a more activated immune milieu in the genital tract (i.e. activated CD4 T cells) than a similar population in the United States. DESIGN: A cross-sectional study nested in a phase 1 microbicide trial. METHODS: Cervical cytobrushes were collected from 18 to 24-year-old women in San Francisco, California, USA (n = 18) and Kisumu, Kenya (n = 36) at enrollment into a phase 1 microbicide trial. All participants tested negative for HIV, herpes simplex virus 2, gonorrhea, chlamydia, and trichomonas, and had abstained from sex for at least 7 days prior to enrollment. Cryopreserved T-cell populations were assayed by flow cytometry in a central laboratory. Secretory leukocyte protease inhibitor levels were assayed in cervicovaginal lavage samples. The Wilcoxon rank-sum test was used to compare immune parameters between sites. RESULTS: The total number of endocervical CD4(+) T cells was slightly higher in participants from San Francisco, but participants from Kisumu had a substantially higher number and proportion of CD4(+) T cells expressing the early activation marker CD69, with and without the HIV coreceptor C-C chemokine receptor type 5, and a greater proportion of activated CD8(+) T cells. Median (interquartile range) genital levels of secretory leukocyte protease inhibitor were lower in participants from Kisumu compared with those from San Francisco [190 (96-519) vs. 474 (206 817) pg/ml, P < 0.03]. CONCLUSION: Activated mucosal T cells were increased in the genital tract of young, sexually transmitted infection/HIV-free Kenyan women, independent of common genital coinfections, and secretory leukocyte protease inhibitor levels were reduced. The cause of these mucosal immune differences is not known, but could partly explain the high HIV incidence in young women from sub-Saharan Africa.

Cervical HIV-specific IgA in a population of commercial sex workers correlates with repeated exposure but not resistance to HIV.

We conducted a comprehensive cross-sectional analysis of total and HIV-specific cervical antibody levels in HIV-1-resistant, uninfected, and infected women in order to examine the role of HIV-specific antibody responses in the female genital tract and examine the effect on antibody levels of various epidemiologic factors in this population. Cervical lavages were collected from 272 subjects of the Pumwani commercial sex worker cohort. Total and HIV-specific genital tract IgA and IgG levels were measured using an ELISA and correlated with behavioral and demographic factors. No significant difference was seen between cervical HIV-specific IgA levels in infected, uninfected, and resistant individuals, nor were any correlations between cervical HIV-specific IgA and neutralization capacity or viral shedding seen. We did, however, note increased HIV-specific IgA in HIV-negative women with four or more clients per day, and decreased HIV-specific IgA in both long-term nonprogressors and long-term survivors. These results show that there is not a strong cohort-wide correlation between HIV-specific cervical IgA levels and resistance to infection by HIV-1 as previously believed, but there is a correlation between exposure to HIV and HIV-specific cervical IgA. Our findings do not preclude the possibility that functional differences in the cervical IgA of HEPS women may play a role in resistance, but argue that HIV-specific responses may not be a universal protective factor. They also indicate that resistance to HIV is a complex condition related to more factors than exposure. Further studies of correlates of immune protection in these individuals would be beneficial to the field.

Mucosal Neisseria gonorrhoeae coinfection during HIV acquisition is associated with enhanced systemic HIV-specific CD8 T-cell responses.

BACKGROUND: The host immune response against mucosally acquired pathogens may be influenced by the mucosal immune milieu during acquisition. As Neisseria gonorrhoeae can impair dendritic cell and T-cell immune function, we hypothesized that coinfection during HIV acquisition would impair subsequent systemic T-cell responses. METHODS: Monthly screening for sexually transmitted infections was performed in high risk, HIV seronegative Kenyan female sex workers as part of an HIV prevention trial. Early HIV-specific CD8 T-cell responses and subsequent HIV viral load set point were assayed in participants acquiring HIV, and were correlated with the presence of prior genital infections during HIV acquisition. RESULTS: Thirty-five participants acquired HIV during follow-up, and 16 out of 35 (46%) had a classical sexually transmitted infection at the time of acquisition. N. gonorrhoeae coinfection was present during HIV acquisition in 6 out of 35 (17%), and was associated with an increased breadth and magnitude of systemic HIV-specific CD8 T-cell responses, using both interferon-gamma gamma and MIP-1 beta as an output. No other genital infections were associated with differences in HIV-specific CD8 T-cell response, and neither N. gonorrhoeae nor other genital infections were associated with differences in HIV plasma viral load at set point. CONCLUSION: Unexpectedly, genital N. gonorrhoeae infection during heterosexual HIV acquisition was associated with substantially enhanced HIV-specific CD8 T-cell responses, although not with differences in HIV viral load set point. This may have implications for the development of mucosal HIV vaccines and adjuvants.