ABSTRACT Here, we developed and evaluated a filter-paper urine collection method for chlamydia and gonorrhea testing. Dried urine strips (DUS) were developed to fit into the sample collection tube used for high throughput Hologic Aptima Combo 2 assay, minimizing processing post-sample collection. Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) bacterial stock dilutions were used to assess DUS performance and long-term stability at different temperature conditions. DUS diagnostic performance was evaluated against urine samples with Aptima Combo 2 Assay and an in-house RT-PCR method for CT and NG detection utilizing residual diagnostic urine specimens (n = 60 CT and NG negative, n = 50 CT-positive and n = 55 NG-positive, including 5 CT/NG dual-positive). Replacing the overnight strip drying step with use of additional desiccants was evaluated in order to reduce barriers to self-sample collection. We observed a high degree of diagnostic accuracy (>91% sensitivity and >98% specificity), compared to traditional urine samples across all evaluated DUS preparation and storage methods. DUS samples demonstrated high stability for prolonged periods at tested temperature storage conditions with some loss in sensitivity at lower bacterial loads. While drying of the sample was crucial to preserve sample integrity and improve recovery post-elution, placing the DUS directly into the sample storage bag with additional desiccants did not negatively impact diagnostic performance. DUS specimens were shown suitable for downstream CT genotyping and NG sequence typing. The developed DUS represents an accurate and stable sample collection method for CT and NG diagnostic testing and downstream pathogen characterization.IMPORTANCEInnovative sampling methods that are convenient, accurate, and user-friendly are crucial to increase sexually transmitted infection (STI) testing uptake. This study establishes a novel dried urine strip sample collection method for chlamydia and gonorrhea testing and downstream molecular characterization. Results show no inferiority of dried urine strips compared to urine, with regard to diagnostic performance, when analyzed using molecular in-house and commercial assays for chlamydia and gonorrhea detection. A dried urine strip sample collection method for chlamydia and gonorrhea testing offers significant advantages in resource-limited or stigmatized settings; it enables self-collection, mail-in return, and long-term storage at ambient temperatures, making it ideal for reaching underserved populations. The broader public health impact is the expansion of STI screening access and reduction in STI transmission by offering a discreet, scalable alternative to traditional specimen collection.

OBJECTIVES:Measles is a highly infectious virus with potentially serious complications. Infants too young to be vaccinated depend on maternally-derived, transplacentally-transferred antibodies for early protection. We aimed to identify factors influencing maternal measles immunity, and determine predictors of maternal susceptibility and infant immunity during the first year of life. METHODS:Using a prospective cross-sectional study design, we collected data and samples from infant-mother pairs admitted to the Hospital for Sick Children between 2018 and 2020. Measles antibody titres were measured using the gold-standard plaque reduction neutralisation test. RESULTS:We recruited 258 mothers aged 16-45 years, of whom 80.6% (95% CI: 75.8,85.4) had protective antibody levels. The proportion with protective antibodies was lowest among mothers <25 years at 67.9% (95% CI: 50.6,85.2), increasing to 100% (95% CI: 100.0,100.0) in those >40 years. Among the 233 individuals reporting vaccination status, 97.0% received at least one measles-containing vaccine. CONCLUSIONS:Nearly one-fifth of mothers in our study were seronegative to measles, increasing to nearly one-third of mothers under 30 years. The higher susceptibility observed in younger individuals within a highly-vaccinated cohort may suggest waning humoral immunity. Further research is needed to understand the significance of the high proportion of seronegative individuals in younger age groups.

INTRODUCTION:We reviewed evidence on whether vaccinated individuals can transmit measles and conducted a meta-analysis to understand transmission characteristics. METHODS:We searched and extracted data from peer-reviewed and gray literature and included studies reporting measles transmission events from vaccinated individuals. We meta-analyzed the data to calculate the median number of transmissions from vaccinated cases by measles burden, number of doses, and time since first and last dose. RESULTS:We identified 11,911 peer-reviewed and 22 gray literature records and included 33 articles in our review. Seventy individuals who had received 1 or more doses of measles-containing vaccine transmitted measles virus, resulting in 237 secondary cases. Vaccinated transmitters in eliminated areas were older (median age of 19 years (IQR: 3, 21)) than those in non-eliminated areas (median age 16 years; IQR 13, 18) (p = 0.12). Additionally, 91% (30/33) of studies provided data on subsequent transmission generations, leading to 812 measles cases traced back to vaccinated individuals, with a median of 4 (IQR: 1, 10) cases per vaccinated transmitter. CONCLUSION:Measles transmissions from vaccinated cases, although relatively uncommon, must be considered in public health investigations, as such transmissions can contribute to outbreaks.

BACKGROUND:Prior to receiving their first dose of measles-containing vaccine, infants may be protected against infection either via antibodies obtained transplacentally from their mothers or by herd immunity. However, there is evidence that transplacental protection may wane well-before the age of first measles vaccination. METHODS:We conducted a cross-sectional study of infants <12 months of age at The Hospital for Sick Children (SickKids) in Toronto, Ontario, and their mothers. We calculated the probability of measles susceptibility in infants and predicted the mean antibody titre by age. RESULTS:Measles seroprevalence decreased with age, starting at 70.4 % (38/54) in the first month of life and declining to 0.0 % (0/18) by four months of age. Regression models adjusted for maternal place of birth and measles immunity status indicated that the odds of susceptibility approximately tripled with every increasing month of age. INTERPRETATION:Infants in our study are likely susceptible to measles for the majority of their first year of life. All measles-exposed infants should be presumed susceptible, regardless of age. High population-level measles vaccine coverage is essential to protect this susceptible population.

ABSTRACT We report the development of a bench protocol and evaluation of bioinformatics pipelines for the whole genome sequence (WGS) of measles virus (MeV) genotype D8. We established a bench protocol using 1 kb amplicons tiling the MeV WGS. Four different pipeline parameters were assessed based on two basecallers and two quality thresholds: Guppy simplex with Q-score thresholds of 20 and 25 (G20 and G25), and Dorado duplex with Q-score thresholds of 20 and 25 (D20 and D25). Using a reference genome, we determined that complete genomes were obtained down to 10 copies/µL with all four parameters; however, errors began to be detected in the consensus sequence at 100 copies/µL. A panel of specimens from 32 measles cases, for which measles WGS had been obtained by other methods (reference sequences), was used to assess the utility and accuracy of the Oxford Nanopore Technologies (ONT) for the purposes of measles surveillance. We found that a crossing point (Cp) value of 31 (corresponding to approximately 100 copies/µL) or less could be considered a predictor for the generation of accurate and complete WGS. The GQ20 parameter achieved the most complete genomes (75%) and had the most identical sequences (84.4%). Error rates compared with the reference sequences for all parameters were below one nucleotide per whole genome. After assessing the reproducibility, GQ20 had the most identical sequences (97.4%). Finally, we inserted ONT-generated WGS and reference sequences into outbreaks with known epidemiological links, and our results show that the ONT WGS matches the epidemiological data. This evaluation establishes that NGS generated by ONT produces accurate and reliable MeV WGS. IMPORTANCE The use of ONT-sequencing platforms has the potential to expand the availability of measles sequencing as a result of its relatively lower cost and portability. This study establishes that measles sequences generated by ONT are accurate and reliable. This will enable sequencing in global regions where there is a lack of sequence data (which also tend to be the measles exporting regions) and more timely sequencing in low incidence settings, due also to the lower number of samples needed for the ONT platform. More timely generation of these data enables better investigation of cases, which informs public health response and outbreak management in measles-eliminated countries.

BACKGROUND:Outbreaks of lymphogranuloma venereum (LGV) have been reported among gay, bisexual, and men who have sex with other men (gbMSM) in Europe and North America. In Alberta, Canada, a universal surveillance program was initiated to assess LGV rates among gbMSM seen at provincial sexually transmitted infection clinics. Our study examines the epidemiological characteristics and treatment of cases. METHODS:We describe a prospective, multicenter LGV surveillance program using chlamydia-positive nucleic acid amplification test specimens from gbMSM collected at 3 sexually transmitted infection clinics between April 2018 and July 2022. Chlamydia-positive specimens from rectal, pharyngeal, and urine specimens were sent for LGV typing. Treatment and test of cure for LGV-confirmed cases were followed. Demographic variables were compared between LGV-positive and LGV-negative specimens using χ2 tests for categorical variables and Mann-Whitney U tests for continuous variables. RESULTS:Eighty-five percent (n = 2333) of all chlamydia-positive specimens were tested for LGV, and 45 (1.9%; 95% confidence interval, 1.4%-2.5%) specimens were confirmed to be LGV. A majority (n = 1374) of specimens submitted for LGV testing were rectal swabs, of which 37 (2.7%) were positive. One-half (51.2% [22 of 43]) of cases were asymptomatic. Of the 22 cases with a negative test-of-cure result, 9 (40.9%) patients received doxycycline 100 mg twice a day for 21 days, 7 (31.8%) patients received azithromycin 1 g as a single dose, 5 (22.7%) patients received doxycycline 100 mg twice a day for 7 days, and 1 patient (4.6%) was treated with doxycycline 100 mg twice a day for 14 days. CONCLUSIONS:Universal testing increased the detection of LGV among asymptomatic patients. Future research should consider the impact of asymptomatic LGV in transmission networks and the role of alternate, shorter treatment regimens than the recommended 21 days of doxycycline.

Nucleic acid amplification tests (NAATs) are the method of choice for Chlamydia trachomatis diagnosis, but these strategies are susceptible to target site mutations. C. trachomatis variants escaping detection with the Aptima Combo 2 (AC2) assay on the Hologic Panther instrument from 23S rRNA mutations have been reported in Nordic countries, England, Japan, and the United States. Given the potential for false negative results, this study investigated whether strains of C. trachomatis with AC2 target site mutations were present in Canada. Surveillance was conducted in Canadian laboratories from 2019 to 2021. Specimens suspected of AC2 target site mutations included those with low-value detections on the AC2 assay, with subsequent high-value detections on the Aptima Chlamydia Trachomatis (ACT) assay used for confirmatory testing. Specimens with AC2/ACT discrepant results were subjected to sequencing of the AC2 target (i.e., 23S rRNA). Sequencing revealed 15 (4.8%) diagnostic escape variants which were carrying either C1514T, G1523A, or G1526A mutations. All specimens with a diagnostic escape mutation were detected with a reformulated version of the AC2 assay. Overall, while the prevalence of C. trachomatis variants was rare, their presence in the Canadian population supports the use of the new AC2 kit formulation and the need for ongoing genetic surveillance for NAAT-based assays. IMPORTANCE Molecular tests are commonly used for the detection of sexually transmitted infections (STIs) like Chlamydia trachomatis. Mutations impacting C. trachomatis molecular target detection on the Hologic Panther AC2 assay have been reported in several countries, raising concerns about potential false negative results. This study showed C. trachomatis target detection failures in specimens submitted for C. trachomatis testing in Canadian laboratories from 2019 to 2021. A reformulated version of the AC2 molecular test is now available that can identify C. trachomatis strains harboring target site mutations that were impacted by the previous test formulation. While target site mutations were rare in Canada, revealing their presence is important to ensure accurate molecular detection of C. trachomatis with existing testing methods. This study supports ongoing genetic monitoring of C. trachomatis molecular test target sites, as well as the use of the reformulated test to avoid false negative results and subsequent transmissions.

Objectives: To determine the prevalence of human papillomavirus (HPV) types by genotyping high-grade squamous intraepithelial lesion (HSIL), adenocarcinoma in situ (AIS), and early-stage invasive cervical cancer (ICC) in patients who have been exposed or are na & iuml;ve to the HPV vaccine. Methods: This was a cross-sectional study. All patients over the age of 18 years who presented to the colposcopy clinic with HSIL, AIS, or ICC who were expected to undergo a cervical biopsy, loop electrosurgical excisional procedure, or cone biopsy were eligible and approached for informed consent. HPV typing was performed to identify the causative HPV types. Results: Between November 2016 and May 2023, 113 patients (34 vaccinated with at least 1 dose, and 79 non-vaccinated) consented to this study. The median ages at coitarche and study entry were 18 (range 14-37) and 34 (range 24-66) years, respectively. Only 3 patients were vaccinated prior to coitarche. Histology was as follows: HSIL = 97, AIS = 9, HSIL and AIS = 2, squamous cell carcinoma = 4, and 1 patient with adenocarcinoma. The causative HPV type was 16 or 18 in 59% of the vaccinated group and in 66% of the non-vaccinated group. Most vaccinated patients (74%) reported receiving 2-3 doses of HPV vaccine. Conclusions: In our cohort, the distribution of causative HPV 16 and 18 in patients presenting with HSIL/AIS/ICC was similar between vaccine-na & iuml;ve and vaccinated patients. This data suggests cervical screening guidelines should not differentiate between "vaccinated" and "non-vaccinated" women without further details of their vaccination.

Measles virus genotype B3 coding-complete genome sequence from a 2019 case showed a novel mutation in the phosphoprotein (P) gene that abrogates the established stop codon. A downstream stop codon has been identified, resulting in a putative P that would be 19 amino acids longer than wild type.

Recent mumps outbreaks have been observed in vaccinated young adults due to the mumps virus (MuV) of genotype G, whereas the current vaccine is a mixture of two genotype A strains. These outbreaks could be attributed to waning vaccine immunity or the antigenic differences between the HN and F glycoproteins in the vaccine and circulating MuV. These glycoproteins are essential targets for the immune system, and antigenic variations may reduce the recognition of mumps antibodies, rendering the population susceptible to the MuV. We established stable cell lines expressing the MuV glycoproteins to study cross-reactivity between genotype A and genotype G. Cross-reactivity between the genotypes was evaluated via immunofluorescence using patient sera from vaccinated individuals, infected individuals, and vaccinated individuals infected with genotype G. Titer ratios showed that the vaccinated individuals exhibited a titer 3.68 times higher for the HN protein and 2.3 times higher for the F protein when comparing genotype A with genotype G. In contrast, the infected individuals showed a lower titer for genotype A compared with genotype G, at 0.43 and 0.33 for the HN and F proteins, respectively. No difference in titer ratio was observed for individuals vaccinated and subsequently infected with mumps. These findings suggest that antigenic variations between the two genotypes may potentially result in immune escape of the circulating strain, resulting in individuals susceptible to the MuV.

Measles and rubella serological diagnoses are done by IgM detection. The World Health Organization Global Measles and Rubella Laboratory Network previously endorsed Siemens Enzygnost enzyme-linked immunosorbant assay kits, which have been discontinued. A recommended replacement has not been determined. We aimed to search for suitable replacements by conducting a systematic review and meta-analysis of IgM detection methods that are currently available for measles and rubella. A systematic literature search was performed in Medline, Embase, Global Health, Cochrane Central, and Scopus on March 22 and on 27 September 2023. Studies reporting measles and/or rubella IgM detection with terms around diagnostic accuracy were included. Risk of bias was assessed using QUADAS tools. Meta-DiSc and R were used for statistical analysis. Clinical samples totalling 5,579 from 28 index tests were included in the measles meta-analysis. Sensitivity and specificity of the individual measles studies ranged from 0.50 to 1.00 and 0.53 to 1.00, respectively. Pooled sensitivity and specificity of all measles IgM detection methods were 0.94 (CI: 0.90-0.97) and 0.94 (CI: 0.91-0.97), respectively. Clinical samples totalling 4,983 from 15 index tests were included in the rubella meta-analysis. Sensitivity and specificity of the individual rubella studies ranged from 0.78 to 1.00 and 0.52 to 1.00, respectively. Pooled sensitivity and specificity of all rubella IgM detection methods were 0.97 (CI: 0.93-0.98) and 0.96 (CI: 0.93-0.98), respectively. Although more studies would be ideal, our results may provide valuable information when selecting IgM detection methods for measles and/or rubella.

Introduction:Measles is caused by the highly infectious measles virus, MeV, for which there is an effective vaccine. Monitoring of progress of measles elimination requires enhanced surveillance and tracking of MeV strains, including documenting the absence of an endemically circulating strain. Due to a reduction in the number of circulating genotypes, additional sequence information, beyond the standardized 450 nucleotide window of the nucleoprotein (N450), is required to corroborate the information from epidemiological investigations and, ideally, fill in gaps in the surveillance data. Methods:This study applies MeV sequencing tools, namely the N450, the non-coding region between the matrix and fusion genes (MF-NCR), and the complete coding sequence of the genome (WGS-t), to clinical specimens obtained from cases occurring over a three-year time period in Canada. This data was systematically analyzed, including with Bayesian evolutionary analysis by sampling trees (BEAST) of the WGS-t. Results and discussion:Of the 143 reported cases, N450, MF-NCR, and WGS-t sequences were obtained from 101, 81, and 75 cases, respectively. The BEAST analysis confirmed that the two most frequently detected lineages (B3 named strain MVi/Marikina City.PHL/10.18 and D8 named strain MVs/Gir Somnath.IND/42.16) were the result of repeated importations. Of the 16 outbreaks occurring during the study period, the analysis conclusively corroborated the epidemiological information for 13. BEAST analysis of the WGS-t convincingly demonstrated the expansion of two outbreaks by the inclusion of additional contemporary cases for which the epidemiological investigation had been unable to identify links. Furthermore, the analysis revealed the existence of three additional unrecognized outbreaks among the cases categorized as unknown source. One outbreak was without WGS-t and could not be resolved. Conclusion:Measles WGS-t data corroborated and expanded upon the outbreak analysis from traditional epidemiological investigations of measles outbreaks. However, both are needed for fulsome investigations in elimination settings.

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INTRODUCTION:The incidence of varicella in Canada has decreased by almost 99% since vaccination was introduced. However, variation in the timing and eligibility of vaccination programs across the country has resulted in some cohorts being under-vaccinated and therefore potentially susceptible to infection. METHODS:We used nationally representative specimens from the Biobank of Statistics Canada's Canadian Health Measures Survey (CHMS) as well as residual specimens from Ontario collected between 2009-2014 to estimate population immunity across age-groups and geography, and identify any groups at increased risk of varicella infection. RESULTS:The weighted proportion of specimens with antibody levels above the threshold of protection was 93.6% (95% CI: 92.4, 95.0). Protection was lowest among those aged 3-5 years (54.3%; 95% CI: 47.3, 61.4), but increased with age. Individuals born outside Canada had more than twice the odds of varicella susceptibility than those born in Canada (aOR: 2.7; 95% CI: 1.4, 5.0; p = 0.004). There were no differences by sex or geography within Canada, and there were no statistically significant differences when Ontario CHMS sera were compared to Ontario residual sera, apart from in participants aged 12-19 year age-group, for whom the CHMS estimate (91.2%; 95% CI: 86.7, 95.7) was significantly higher (p = 0.03) than that from residual specimens (85.9%, 95% CI: 81.1, 90.8). DISCUSSION:Varicella immunity in Canada is changing. Children appear to have low population immunity, placing them at greater risk of infection and at increased risk of severe disease as they age. Our results underscore the importance of performing periodic serosurveys to monitor further population immunity changes as the proportion of vaccine-eligible birth-cohorts increases, and to continually assess the risk of outbreaks.

Despite the provision of a mumps vaccination program in Canada for over three decades, mumps has not reached elimination. Instead, a re-emergence has been observed in vaccinated populations, particularly in young adults. These outbreaks have been almost exclusively due to genotype G infections, a trend that has been seen in other countries with high mumps vaccination rates. To characterize mumps outbreaks in Canada, genomes from samples from Manitoba (n = 209), Newfoundland (n = 25), and Nova Scotia (n = 48) were sequenced and analysed by Bayesian inference. Whole genome sequencing was shown to be highly discriminatory for outbreak investigations compared to traditional Sanger sequencing. The results showed that mumps virus genotype G most likely circulated endemically in Canada and between Canada and the US. Overall, this Canadian outbreak data from different provinces and ancestral strains demonstrates the benefits of molecular genomic data to better characterize mumps outbreaks, but also suggests genomics could further our understanding of the reasons for potential immune escape of mumps genotype G and evolution in highly vaccinated populations. With a possible endemic circulation of mumps genotype G and the remaining risk of new imported cases, increased surveillance and alternative vaccination strategies may be required for Canada to reach the current target for mumps or a future elimination status.

Background: Before the early 2000s, the sexually transmitted infection lymphogranuloma venereum (LGV) was rare in high-income countries. Initially, most cases in these countries were among symptomatic men who have sex with men (MSM) living with HIV. In the context of widespread HIV preexposure prophylaxis (PrEP), LGV's epidemiology may be changing. We aimed to characterize the epidemiology and clinical presentation of LGV in the PrEP era. Methods: A retrospective chart review was performed on all LGV cases occurring between November 2004 to October 2022 in British Columbia (BC), Canada. Cases were stratified by having occurred before (2004-2017) or after widespread PrEP availability in BC (2018-2022). Annual rates and test positivity percentages were calculated. Bivariate logistic regression was performed to identify drivers of asymptomatic infection in the PrEP era. Results: Among 545 cases identified, 205 (37.6%) occurred pre-PrEP and 340 (62.4%) occurred during the PrEP era. Most cases were among MSM (97.2%). The estimated rate of LGV has doubled from 2018 to 2022, reaching 1535.2 cases per 100,000 PrEP users. Most PrEP-era cases were among HIV-negative individuals (65.3%), particularly those on PrEP (72.6%). Cases in the PrEP era were often asymptomatic compared with pre-PrEP (38.6% vs. 19.3%; P < 0.001). Users of PrEP were more likely to experience asymptomatic infection compared with HIV-negative PrEP nonusers (odds ratio, 2.07; 95% confidence interval, 1.07-3.99). Conclusions: In the context of increased asymptomatic testing, LGV may be increasing in BC. Most infections now occur among HIV-negative MSM. A high proportion of infections are asymptomatic.

BACKGROUND:Human papillomavirus (HPV) infection is associated with anal cancers and is more prevalent in gay, bisexual, and men who have sex with men (gbMSM), partly due to their vulnerability to HIV infection. Baseline HPV genotype distributions and risk factors can inform the design of next-generation HPV vaccines to prevent anal cancer. METHODS:A cross-sectional study was conducted among gbMSM receiving care at a HIV/STI clinic in Nairobi, Kenya. Anal swabs were genotyped using a Luminex microsphere array. Multiple logistic regression methods were used to identify risk factors for four HPV outcomes (any HPV, any HR-HPV, and 4- and 9-valent vaccine-preventable HPVs). RESULTS:Among 115 gbMSM, 51 (44.3%) were HIV-infected. Overall HPV prevalence was 51.3%; 84.3% among gbMSM living with HIV and 24.6% among gbMSM without HIV (p < 0.001). One-third (32.2%) had HR-HPV and the most prevalent vaccine-preventable HR-HPV genotypes were 16, 35, 45, and 58. HPV-18 was uncommon (n = 2). The 9-valent Gardasil vaccine would have prevented 61.0% of HPV types observed in this population. In multivariate analyses, HIV status was the only significant risk factor for any HPV (adjusted odds ratio [aOR]:23.0, 95% confidence interval [95% CI]: 7.3-86.0, p < 0.001) and for HR-HPV (aOR: 8.9, 95% CI: 2.8-36.0, p < 0.001). Similar findings were obtained for vaccine-preventable HPVs. Being married to a woman significantly increased the odds of having HR-HPV infections (aOR: 8.1, 95% CI: 1.6-52.0, p = 0.016). CONCLUSIONS:GbMSM living with HIV in Kenya are at higher risk of anal HPV infections including genotypes that are preventable with available vaccines. Our findings support the need for a targeted HPV vaccination campaign in this population.

On November 18-19, 2019, the Immunity of Canadians and Risk of Epidemics (iCARE) Network convened a workshop in Toronto, Ontario, Canada. The objectives of the workshop were to raise the profile of sero-epidemiology in Canada, discuss best practice and methodological innovations, and strategize on the future direction of sero-epidemiology work in Canada. In this conference report, we describe the presentations and discussions from the workshop, and comment on the impact of the COVID-19 pandemic on serosurveillance initiatives, both in Canada and abroad.