Molecular Diagnosis of Vaginitis: Comparing Quantitative PCR and Microbiome Profiling Approaches to Current Microscopy Scoring.

Vaginitis is often diagnosed by microscopy and limited to testing for bacterial vaginosis (BV), vulvovaginal candidiasis, and trichomoniasis. Approximately 10% of vaginal swabs are negative but designated "altered flora" by BV Nugent score, leaving clinicians unsure how to treat patients. Accurate and comprehensive vaginitis diagnostics are needed to direct treatment and reduce risks of recurrent or more severe infections. Vaginal swabs were collected from 93 women (mean age, 23.53 years; range, 18 to 42 years) in a cross-sectional study. Microscopy results for BV and Candida were compared to those from two molecular approaches: (i) a comprehensive quantitative PCR (qPCR) assay, including testing for aerobic vaginitis (AV), Candida, sexually transmitted infections (STI), and BV (Applied Biosystems) with an accompanying BV interpretive algorithm (Coriell Life Sciences), and (ii) microbiome profiling of the 16S rRNA gene (Illumina). Microscopy plus BV Nugent score had 76% overall agreement with the qPCR plus BV interpretive algorithm method (24 positive, 47 negative). OF the nine samples designated altered flora by Nugent, five were categorized BV positive and four were BV negative by the qPCR method. Although BV negative, 3/4 of the latter samples had positive AV targets with one also was STI positive. Microscopic identification of Candida versus that by qPCR had 94% agreement (9 positive, 78 negative). The comprehensive qPCR assay revealed alternative etiologies summarized as 38% BV, 10% AV, 5% Candida, 2% STI, 10% mixed infection (positive targets in multiple panels), and 35% negative for all targets. 16S microbiome analysis confirmed the bacterial qPCR results and identified differentiating patterns between AV, BV, and Lactobacillus-dominated vaginal microbiomes.

Comparative diversity of microbiomes and Resistomes in beef feedlots, downstream environments and urban sewage influent.

BACKGROUND: Comparative knowledge of microbiomes and resistomes across environmental interfaces between animal production systems and urban settings is lacking. In this study, we executed a comparative analysis of the microbiota and resistomes of metagenomes from cattle feces, catch basin water, manured agricultural soil and urban sewage. RESULTS: Metagenomic DNA from composite fecal samples (FC; n = 12) collected from penned cattle at four feedlots in Alberta, Canada, along with water from adjacent catchment basins (CB; n = 13), soil (n = 4) from fields in the vicinity of one of the feedlots and urban sewage influent (SI; n = 6) from two municipalities were subjected to Illumina HiSeq2000 sequencing. Firmicutes exhibited the highest prevalence (40%) in FC, whereas Proteobacteria were most abundant in CB (64%), soil (60%) and SI (83%). Among sample types, SI had the highest diversity of antimicrobial resistance (AMR), and metal and biocide resistance (MBR) classes (13 & 15) followed by FC (10 & 8), CB (8 & 4), and soil (6 & 1). The highest antimicrobial resistant (AMR) gene (ARG) abundance was harboured by FC, whereas soil samples had a very small, but unique resistome which did not overlap with FC & CB resistomes. In the beef production system, tetracycline resistance predominated followed by macrolide resistance. The SI resistome harboured ß-lactam, macrolide, tetracycline, aminoglycoside, fluoroquinolone and fosfomycin resistance determinants. Metal and biocide resistance accounted for 26% of the SI resistome with a predominance of mercury resistance. CONCLUSIONS: This study demonstrates an increasing divergence in the nature of the microbiome and resistome as the distance from the feedlot increases. Consistent with antimicrobial use, tetracycline and macrolide resistance genes were predominant in the beef production system. One of the feedlots contributed both conventional (raised with antibiotics) and natural (raised without antibiotics) pens samples. Although natural pen samples exhibited a microbiota composition that was similar to samples from conventional pens, their resistome was less complex. Similarly, the SI resistome was indicative of drug classes used in humans and the greater abundance of mercury resistance may be associated with contamination of municipal water with household and industrial products.

Gonococcal Conjunctivitis in Adults: Case Report and Retrospective Review of Cases in Alberta, Canada, 2000-2016.

BACKGROUND: A case of gonococcal conjunctivitis (GC) prompted us to review the reported cases and treatment regimens of GC in Alberta, Canada. METHODS: Gonococcal cases reported from 2000 to 2016 were extracted from the provincial sexually transmitted infection reporting database. The diagnosis of GC was based on a positive culture and/or nucleic acid amplification test from the eye. RESULTS: A total of 45 cases of GC were reported in adults. Three quarters (75.6%; n = 34) of the cases were diagnosed using culture, 57.8% (n = 26) of cases were among men, and 55.5% (n = 25) were diagnosed since 2014. Very few (13.3%; n = 6) of the cases were treated according to current Canadian Guidelines on Sexually Transmitted Infections, using 2 g of ceftriaxone in combination with azithromycin or doxycycline. Results of test of cures were available for 15.6% (n = 7) of the cases and occurred within 10 to 79 days (median = 26 days) after treatment; all were negative. CONCLUSIONS: Gonococcal conjunctivitis was relatively uncommon in our region, but given its potential for severe manifestations and sequelae coupled with the rising rates of gonorrhea; it remains important to consider this diagnosis in sexually active individuals presenting with purulent conjunctivitis. Additional studies are needed to inform treatment recommendations and to evaluate outcomes of infection.

Plasmid Distribution among Escherichia coli from Livestock and Associated Wastewater: Unraveling Factors That Shape the Presence of Genes Conferring Third-Generation Cephalosporin Resistance.

A key concern with agricultural wastewater storage ponds is that they may provide an environment conducive for horizontal exchange of antibiotic resistance genes (ARGs), thereby facilitating the emergence of antibiotic resistant pathogens. Central to this exchange are mobile genetic elements like plasmids; yet, the factors shaping their presence in agricultural environments remain poorly understood. Here, using Escherichia coli as a model bacterium, we examined genetic backgrounds and plasmid profiles of generic fecal and wastewater isolates and those possessing blaCTX-M and blaCMY-2 genes (which confer resistance to third-generation cephalosporins) to delineate factors shaping the environmental persistence of plasmid-associated ARGs in beef cattle feedlots. The wastewater environment exerted minimal influence on plasmid repertoires, as the number of plasmids and distribution of different incompatibility groups did not differ between generic fecal and wastewater isolates. The blaCTX-M and blaCMY-2 genes were associated with IncF and IncA/C plasmids, respectively, and host isolates possessing these ARGs had fewer plasmids than generic isolates, suggesting ARG-bearing plasmids may associate predominantly with such hosts to compensate for the metabolic burden imposed by these plasmids. Phylogeny also appeared to be a factor for blaCTX-M genes, as their bacterial hosts were restricted to particular genetic lineages, including the environmentally adapted ET-1 clade, as noted previously for these genes. Ultimately, these findings have important implications for evaluating human health risks of agricultural wastewater with respect to environmental persistence of ARGs and may help identify options for improving wastewater treatment.

Gonococcal and Chlamydial Cases of Pelvic Inflammatory Disease at 2 Canadian Sexually Transmitted Infection Clinics, 2004 to 2014: A Retrospective Cross-sectional Review.

One hundred thirteen patients with gonococcal and chlamydial pelvic inflammatory disease were reviewed at 2 Canadian sexually transmitted infection clinics. Most patients (81%) with pelvic inflammatory disease were diagnosed as having chlamydia alone. Three treatment failures were seen in patients treated with ofloxacin.

Increasing incidence of syphilis among patients engaged in HIV care in Alberta, Canada: a retrospective clinic-based cohort study.

BACKGROUND: Syphilis is a global health concern disproportionately affecting HIV-infected populations. In Alberta, Canada, the incidence of syphilis in the general population has recently doubled with 25% of these infections occurring in HIV-infected patients. The Southern Alberta HIV Clinic (SAC) and Calgary STI Program (CSTI) analyzed the epidemiologic characteristics of incident syphilis infections in our well-defined, HIV-infected population over 11 years. METHODS: Since 2006, as routine practice of both the Southern Alberta Clinic (SAC) and Calgary STI Programs (CSTI), syphilis screening has accompanied HIV viral load measures every four months. All records of patients who, while in HIV care, either converted from being syphilis seronegative to a confirmed seropositive or were re-infected as evidenced by a four-fold increase in rapid plasma reagin (RPR) after past successful treatment, were reviewed. RESULTS: Incident syphilis was identified 249 times in 194 HIV-infected individuals. There were 36 individuals with repeated infections (28.5% of episodes). Following a prior decline in annual incident syphilis rates, the rates have tripled from 8.08/1000 patient-years (95% confidence interval (CI): 4.14-14.75) in 2011, to 27.04 per 1000 person-years (95% CI: 19.45-36.76) in 2016. Half of the syphilis episodes were asymptomatic. Patients diagnosed with syphilis were twice as likely not to be taking ART and had a higher likelihood of having plasma HIV RNA viral loads > 1000 copies/mL (19%). Incident syphilis was seen predominantly in Caucasians (72%, P < 0.001), males (94%, P < 0.001) and men who have sex with men (MSM) as their HIV risk activity (75%, P < 0.001). CONCLUSIONS: We have highlighted the importance of a regular syphilis screening program in HIV-infected individuals demonstrated by increasing rates of incident syphilis in our region. Targeted preventative strategies should be directed towards HIV-infected populations identified at highest risk, including; MSM, prior alcohol abuse, prior recreational drug use and those with prior syphilis diagnoses.

Retrospective Review of Gonococcal and Chlamydial Cases of Epididymitis at 2 Canadian Sexually Transmitted Infection Clinics, 2004-2014.

Fifty-seven cases of gonococcal and chlamydial infections complicated by acute epididymitis seen at 2 Alberta STI clinics from 2004 to 2014 were reviewed. The majority responded to treatment recommended by national guidelines. Three of 6 treatment failures were not treated according to guidelines.

Trichomonas vaginalis Prevalence and Correlates in Women and Men Attending STI Clinics in Western Canada.

Trichomonas vaginalis prevalence (2.8%) in female sexually transmitted infection clinic attendees was within the prevalence of chlamydia (5.8%) and gonorrhea (1.8%), while being very low for male attendees (0.2%). Correlates among women were indigenous ethnicity, other ethnicity, and being symptomatic.

Use of the APTIMA Combo 2 Assay and a Secondary Algorithm to Detect and Confirm Chlamydia trachomatis in Rectal-Only Infections.

We sought to confirm the results of 81 rectal specimens positive for Chlamydia trachomatis by the APTIMA Combo 2 assay among patients with concurrently collected negative genitourinary specimens. A total of 79 (97.5%) samples were confirmed by the APTIMA single target assay and/or sequencing of the C. trachomatis ompA gene.

Environmental Growth of Enterococci and Escherichia coli in Feedlot Catch Basins and a Constructed Wetland in the Absence of Fecal Input.

Population structures of fecal indicator bacteria (FIB) isolated from catch basins, a constructed wetland, and feces from a beef cattle feedlot were compared over a two-year period. Enterococcus hirae accounted for 92% of the fecal isolates, whereas secondary environments were characterized by greater relative abundance of environmentally adapted species including Enterococcus casseliflavus. While enterococci densities in the catch basins and wetland were similar under wet and drought conditions, E. hirae predominated during rainy periods, while E. casseliflavus predominated during drought conditions. Environmentally adapted species accounted for almost half of the erythromycin resistant enterococci isolated from the wetland. Densities of Escherichia coli were also comparable during wet versus drought conditions, and the relative abundance of strains from environmentally adapted clades was greater in secondary environments compared to feces. Unlike enterococci, fewer environmentally adapted E. coli strains were isolated on selective media containing ceftriaxone from the wetland compared to feces, suggesting resistance to this antibiotic may not be well maintained in the absence of selective pressure. Overall, these findings suggest that secondary environments select for environmentally adapted FIB. While these species and clades tend to be of limited clinical relevance, they could potentially serve as reservoirs of antimicrobial resistance.

A Retrospective Review of Treatment Failures Using Azithromycin and Doxycycline in the Treatment of Rectal Chlamydia Infections in Women and Men Who Have Sex With Men.

We examined the prevalence of rectal chlamydia treatment failures in men who have sex with men and women attending Alberta sexually transmitted infection clinics. Among those completing a test of cure, there was no significant difference among patients treated initially with azithromycin (treatment failure, 39/460 [8.5%]; 95% confidence interval, 5.9%-11.0%) compared with patients treated with doxycycline (0/16; 95% confidence interval, 0%-0.2%; P = 0.63).

Evidence for increased Chlamydia case finding after the introduction of rectal screening among women attending 2 Canadian sexually transmitted infection clinics.

BACKGROUND: Chlamydia trachomatis is the most common notifiable disease in Canada, and extragenital sites are believed to serve as hidden reservoirs for ongoing transmission of infection. There are no specific Canadian screening guidelines for asymptomatic individuals from extragenital sites. We sought to determine the prevalence and factors associated with rectal C. trachomatis among female sexually transmitted infection (STI) clinic attendees in Alberta, Canada. METHODS: Between 20 July and 31 December 2012, all female attendees at 2 Provincial STI clinics receiving a pelvic examination, regardless of a history of anal intercourse, were screened for rectal C. trachomatis using the Gen-Probe Aptima COMBO 2 Assay. Demographic and behavior variables were compared between rectal-only chlamydia cases and genitourinary cases using χ(2) or Fisher exact test, Mann-Whitney test, and logistic regression. RESULTS: A total of 3055 women were screened for rectal chlamydia. The prevalence of rectal chlamydia ranged from 11.7% to 13.5%. There were 133 rectal-only cases, increasing case detection by 44.3% from 300 genitourinary cases to 433 total cases, ranging from 21.7% to 88.2% by clinic. Women who were a contact to an STI were less likely to have rectal-only chlamydia for both clinics (P ≤ .001). CONCLUSIONS: Our findings add to the growing body of evidence supporting universal rectal screening in high-risk women such as those undergoing pelvic exams at STI clinics.

Gonorrhea Treatment Failures With Oral and Injectable Expanded Spectrum Cephalosporin Monotherapy vs Dual Therapy at 4 Canadian Sexually Transmitted Infection Clinics, 2010-2013.

BACKGROUND: Antimicrobial resistance has developed to all antibiotics used to treat gonorrhea (GC), and trends in GC antimicrobial resistance have prompted changes in treatment guidelines. We examined treatment failures in sexually transmitted infection clinics. METHODS: Four Canadian sexually transmitted infection clinics reviewed treatment regimens, minimum inhibitory concentrations for cephalosporins and azithromycin, anatomical infection sites, and treatment outcomes for GC infections between January 2010 and September 2013, in individuals who returned for test of cure within 30 days of treatment. Treatment failure was defined as the absence of reported sexual contact during the posttreatment period and (i) positive for Neisseria gonorrhoeae on culture of specimens taken at least 72 hours after treatment or (ii) positive nucleic acid amplification test specimens taken at least 2 to 3 weeks after treatment, and matching sequence type pretreatment and posttreatment. χ Test and Fisher exact test were used to assess association of categorical variables. RESULTS: Of 389 specimens reviewed, GC treatment failures occurred in 13 specimens treated with cefixime 400-mg single dose (17.8% treatment failure rate regardless of anatomical site) and in 1 oropharyngeal specimen treated with cefixime 800-mg single dose. No treatment failures occurred using either ceftriaxone monotherapy or cefixime/ceftriaxone combined with azithromycin or doxycycline. CONCLUSIONS: In contrast to oral cefixime monotherapy, no treatment failures were identified with injectable ceftriaxone monotherapy or combination GC treatment. Our data support the use of combination treatment of GC with an extended spectrum cephalosporin (including oral cefixime) with azithromycin or doxycycline as well as ceftriaxone monotherapy.

Prevalence and characteristics of rectal chlamydia and gonorrhea cases among men who have sex with men after the introduction of nucleic acid amplification test screening at 2 Canadian sexually transmitted infection clinics.

We sought to determine the prevalence of rectal chlamydia and gonorrhea after the introduction of nucleic acid amplification tests for screening in men reporting receptive anal intercourse. The rectal chlamydia prevalence was 14.1% (95% confidence interval, 11.9-16.3), and the gonorrhea prevalence was 5.9% (95% confidence interval, 4.4-7.3). Most cases were positive only from the rectum.

Utility of specimens positive for Neisseria gonorrhoeae by the Aptima Combo 2 assay for assessment of strain diversity and antibiotic resistance.

In our jurisdiction, the Aptima Combo 2 assay (Gen-Probe, Inc.) is used to detect Neisseria gonorrhoeae from specimens collected at clinics for sexually transmitted infections (STI) and from select community patients. In addition, swabs are also collected for N. gonorrhoeae culture, susceptibility testing, and sequence typing (ST). Since only a small proportion of samples from provincial cases undergo culture, the available trends in antimicrobial susceptibility and predominant strain types may not be representative of all N. gonorrhoeae infections. Due to the limitations facing the use of N. gonorrhoeae culture to understand these trends in the general community, we performed a molecular analysis for markers of cephalosporin resistance and ST determination by using nucleic acid extracts of specimens sent for Aptima testing. Thirty-four samples submitted for both Aptima testing and N. gonorrhoeae culture from the same anatomic location (within 24 h) were included in the study. Sequence type was determined based on the sequence of the por and tbpB genes, and amino acid changes in the PBP 2 protein, encoded by the penA gene, were considered representative for the assessment of antimicrobial susceptibility. Sequence identity of 100% was observed between the sequences obtained from Aptima-analyzed samples and culture samples. Sequencing results showed an association between decreased susceptibility to extended-spectrum cephalosporins (ESC(ds)), tbp allele 110, ST 1407, and amino acid changes (G545S, I312M, and V316T) in the PBP 2 protein. Our data, generated based on a few representative genes, suggest that gonococcal samples positive by Aptima testing can be used to determine single nucleotide polymorphisms associated with ESC(ds) and the sequence type based on molecular strain typing. Confirmation of these findings may obviate the need for gonorrhea culture in the future.

Retrospective review of pharyngeal gonorrhea treatment failures in Alberta, Canada.

Our review of Neisseria gonorrhoeae pharyngeal treatment failures from sexually transmitted infection clinics in Alberta suggests that treatment failures with oral cefixime monotherapy were not related to elevated cefixime minimum inhibitory concentrations. Dual therapy with oral cefixime and azithromycin may be a suitable alternate for the treatment of pharyngeal gonorrhea.

Neisseria gonorrhoeae multiantigen sequence typing is beneficial in further characterizing gonococcal populations in Alberta, Canada.

BACKGROUND: Antimicrobial resistance testing and behavioral data combined with Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST) can help to define gonococcal populations and identify, characterize, and compare clusters of infection. METHODS: Antimicrobial resistance testing, using E test, was reviewed for gonococcal isolates in Alberta, Canada, from 2007 to 2011. Antimicrobial resistance testing was conducted on isolates demonstrating antimicrobial resistance and those with cefixime minimum inhibitory concentrations (MICs) of 0.06 µg/mL or greater. Demographic and behavioral information was obtained from provincial surveillance data. NG-MAST typing was conducted on a proportion of isolates. RESULTS: Gonococcal isolates were available for 2250 (26.4%) of 8535 cases of gonorrhea in Alberta from 2007 to 2011. The proportion of cases with decreased susceptibility to cefixime (≥0.06 µg/mL) increased from 0.7% to 2.4% between 2007 and 2009 to a high of 10.1% in 2010 and 8.9% in 2011. Six isolates with cefixime MIC of 0.25 µg/mL were noted: 5 were from men who have sex with men (MSM) and 1 was a pharyngeal isolate from a heterosexual female. Twenty-four (1.1%) isolates were azithromycin resistant (MIC ≥2.0 µg/mL); there were no significant differences between cases resistant or susceptible to azithromycin. NG-MAST of gonococcal isolates in Alberta suggests the entry of multiple strains into the province. Three clusters were identified: Cluster A predominantly in MSM, including sequence type 1407, a ST previously associated with decreased susceptibility to expanded spectrum cephalosporins; Cluster B, a predominantly heterosexual cluster with most cases in Edmonton; and Cluster C among MSM. CONCLUSIONS: Our data highlight the use of NG-MAST in further defining gonococcal populations.

Pregnancy and neonatal outcomes of women with reactive syphilis serology in Alberta, 2002 to 2006.

OBJECTIVES: To describe the maternal characteristics, diagnosis, and pregnancy, and the neonatal outcomes of pregnant women with reactive syphilis serology in a Canadian cohort. METHODS: We conducted a retrospective chart review of pregnant women in Alberta with reactive syphilis serology between 2002 and 2006. Clinical staging of syphilis in mothers and infants was determined using provincial and national surveillance criteria. RESULTS: Seventy-five pregnancies met the inclusion criteria. Thirty women were adequately treated pre-conception, 20 women had infectious syphilis (10 primary, 5 secondary, 5 early latent), 24 had late latent syphilis, and one had disease of unknown stage. Seven infants with congenital syphilis and one infant with presumed congenital syphilis were born to women with primary (n = 4), secondary (n = 2), early latent (n = 1), and unknown stage (n = 1) syphilis. Treatment was provided prior to delivery in one woman; five women did not access prenatal care. Four infants had long-term sequelae. CONCLUSION: All infants with congenital syphilis were born to women with infectious syphilis who had limited prenatal care. Initiatives to reach women at high risk are required to decrease the incidence of congenital syphilis.

Molecular typing of Treponema pallidum strains in western Canada: predominance of 14d subtypes.

BACKGROUND: Resurgence of syphilis in Canada and worldwide requires laboratories to update their methods for molecular epidemiology investigation and surveillance. This study utilizes polymerase chain reaction diagnostic tests for syphilis, identifies macrolide resistance, and uses a molecular typing system to characterize Treponema pallidum clinical strains causing syphilis in Alberta and Northwest Territories, Canada. METHODS: In total 449 specimens including genital swabs, whole blood, sera, and cerebrospinal fluid were obtained from 374 patients with suspect syphilis in Alberta and Northwest Territories. Molecular subtyping was based on genetic characterization of treponemal repeat genes, arp and tpr. Detection of macrolide resistance was accomplished by identification of the 23S rRNA gene mutation associated with the resistance pattern. RESULTS: Forty-nine specimens obtained from 43 patients were found to be positive for T. pallidum DNA using bmp, tpp47 and polA polymerase chain reaction assays. Four molecular subtypes were identified, with one type, 14d, accounting for 70% of all cases and 83% of typeable strains. Seven patients (16%) were found to be infected by macrolide-resistant strains, of which 6 were men who have sex with men and 1 whose infection was acquired in China. CONCLUSIONS: A single molecular type of T. pallidum, characterized as 14d, caused the majority of the syphilis cases identified in this study. A more discriminatory typing method would be required to determine if these strains are clonal. Treatment of infectious syphilis with macrolide antibiotics should be restricted to patient populations where resistance is rare and clinical and serological follow up of patients is possible.