Multiplex real-time PCR assays for the prediction of cephalosporin, ciprofloxacin and azithromycin antimicrobial susceptibility of positive Neisseria gonorrhoeae nucleic acid amplification test samples.

BACKGROUND: The incidence of antimicrobial-resistant Neisseria gonorrhoeae (GC) is rising in Canada; however, antimicrobial resistance (AMR) surveillance data are unavailable for infections diagnosed directly from clinical specimens by nucleic acid amplification tests (NAATs), representing over 80% of diagnoses. We developed a set of 10 improved molecular assays for surveillance of GC-AMR and prediction of susceptibilities in NAAT specimens. METHODS: Multiplex real-time PCR (RT-PCR) assays were developed to detect SNPs associated with cephalosporin (ponA, porB, mtrR -35delA, penA A311V, penA A501, N513Y, G545S), ciprofloxacin (gyrA S91, parC D86/S87/S88) and azithromycin [23S (A2059G, C2611T), mtrR meningitidis-like promoter] resistance. The assays were validated on 127 gonococcal isolates, 51 non-gonococcal isolates and 50 NAATs with matched culture isolates. SNPs determined from the assay were compared with SNPs determined from in silico analysis of WGS data. MICs were determined for culture isolates using the agar dilution method. RESULTS: SNP analysis of the 50 NAAT specimens had 96% agreement with the matched culture RT-PCR analysis. When compared with MICs, presence of penA A311V or penA A501 and two or more other SNPs correlated with decreased susceptibility and presence of three or more other SNPs correlated with intermediate susceptibility to cephalosporins; presence of any associated SNP correlated with ciprofloxacin or azithromycin resistance. NAAT-AMR predictions correlated with matched-culture cephalosporin, ciprofloxacin and azithromycin MICs at 94%, 100% and 98%, respectively. CONCLUSIONS: We expanded molecular tests for N. gonorrhoeae AMR prediction by adding new loci and multiplexing reactions to improve surveillance where culture isolates are unavailable.

Validation of the Seegene RV15 multiplex PCR for the detection of influenza A subtypes and influenza B lineages during national influenza surveillance in hospitalized adults.

Background. The Serious Outcomes Surveillance Network of the Canadian Immunization Research Network (CIRN SOS) has been performing active influenza surveillance since 2009 (ClinicalTrials.gov identifier: NCT01517191). Influenza A and B viruses are identified and characterized using real-time reverse-transcriptase polymerase chain reaction (RT-PCR), and multiplex testing has been performed on a subset of patients to identify other respiratory virus aetiologies. Since both methods can identify influenza A and B, a direct comparison was performed.Methods. Validated real-time RT-PCRs from the World Health Organization (WHO) to identify influenza A and B viruses, characterize influenza A viruses into the H1N1 or H3N2 subtypes and describe influenza B viruses belonging to the Yamagata or Victoria lineages. In a subset of patients, the Seeplex RV15 One-Step ACE Detection assay (RV15) kit was also used for the detection of other respiratory viruses.Results. In total, 1111 nasopharyngeal swabs were tested by RV15 and real-time RT-PCRs for influenza A and B identification and characterization. For influenza A, RV15 showed 98.0 % sensitivity, 100 % specificity and 99.7 % accuracy. The performance characteristics of RV15 were similar for influenza A subtypes H1N1 and H3N2. For influenza B, RV15 had 99.2 % sensitivity, 100 % specificity and 99.8 % accuracy, with similar assay performance being shown for both the Yamagata and Victoria lineages.Conclusions. Overall, the detection of circulating subtypes of influenza A and lineages of influenza B by RV15 was similar to detection by real-time RT-PCR. Multiplex testing with RV15 allows for a more comprehensive respiratory virus surveillance in hospitalized adults, without significantly compromising the reliability of influenza A or B virus detection.

Comparison of two automated methods for detection and differentiation of herpes simplex virus in clinical specimens.

BACKGROUND: The Aptima Herpes Simplex Virus (HSV) 1&2 Assay recently received Health Canada approved for detection and differentiation of HSV-1 and HSV-2 from anogenital sites. This assay uses target capture, transcription mediated amplification, and real-time detection of messenger RNA (mRNA) produced in host cells during active HSV infection. To evaluate its performance, the Aptima assay was compared to another Health Canada approved assay, the BD ProbeTec Herpes Simplex Viruses HSV 1&2 Qx Amplified DNA Assay, which uses strand displacement amplification technology. METHODS: As recommended by the manufacturers, the Aptima and ProbeTec assays were performed on the Panther and Viper instruments, respectively. Analytical sensitivity and specificity were assessed using 10-fold serial dilution of viruses in viral universal transport media (UTM), and nucleic acids extracted and concentrated from other viruses including all members of the Herpesviridae family. The clinical sensitivity and specificity were assessed retrospectively using 60 archived specimens, and prospectively using 158 swabs in UTM. Discrepant results were resolved with real-time PCR using the Altona Diagnostics RealStar alpha Herpes assay. RESULTS: Both the Aptima and ProbeTec assays showed excellent analytical and clinical specificity. However, the Aptima HSV assay failed to detect HSV in specimens with low viral loads, resulting in reduced sensitivity for HSV-2 during the retrospective evaluation at 85.0%, and for HSV-1 at 85.0% during the prospective evaluation. CONCLUSIONS: This study compared the Aptima and ProbeTec HSV assays and demonstrated that detection of HSV mRNA using the Aptima HSV assay was less sensitive in both retrospective and prospective analyses.

What is the real number of Lyme disease cases in Canada?

BACKGROUND: Lyme disease is emerging in Canada due to expansion of the range of the tick vector Ixodes scapularis from the United States. National surveillance for human Lyme disease cases began in Canada in 2009. Reported numbers of cases increased from 144 cases in 2009 to 2025 in 2017. It has been claimed that few (< 10%) Lyme disease cases are reported associated with i) supposed under-diagnosis resulting from perceived inadequacies of serological testing for Lyme disease, ii) expectation that incidence in Canadian provinces and neighbouring US states should be similar, and iii) analysis of serological responses of dogs to the agent of Lyme disease, Borrelia burgdorferi. We argue that performance of serological testing for Lyme disease is well studied, and variations in test performance at different disease stages are accounted for in clinical diagnosis of Lyme disease, and in surveillance case definitions. Extensive surveillance for tick vectors has taken place in Canada providing a clear picture of the emergence of risk in the Canadian environment. This surveillance shows that the geographic scope of I. scapularis populations and Lyme disease risk is limited but increasing in Canada. The reported incidence of Lyme disease in Canada is consistent with this pattern of environmental risk, and the differences in Lyme disease incidence between US states and neighbouring Canadian provinces are consistent with geographic differences in environmental risk. Data on serological responses in dogs from Canada and the US are consistent with known differences in environmental risk, and in numbers of reported Lyme disease cases, between the US and Canada. CONCLUSION: The high level of consistency in data from human case and tick surveillance, and data on serological responses in dogs, suggests that a high degree of under-reporting in Canada is unlikely. We speculate that approximately one third of cases are reported in regions of emergence of Lyme disease, although prospective studies are needed to fully quantify under-reporting. In the meantime, surveillance continues to identify and track the ongoing emergence of Lyme disease, and the risk to the public, in Canada.

Bacterial contamination of surgical loupes and headlights.

BACKGROUND: Medical equipment can transmit pathogenic bacteria to patients. This single-institution point prevalence study aimed to characterise the types and relative amount of bacteria found on surgical loupes, headlights and their battery packs. METHOD: Surgical loupes, headlights and battery packs of 16 otolaryngology staff and residents were sampled, cultured and quantified. Plate scores were summed for each equipment type, and the total was divided by the number of users to generate mean bacterial burden scores. Residents completed a questionnaire regarding their equipment cleaning practices. RESULTS: The contamination rates of loupes, headlights and battery packs were 68.75 per cent, 100 per cent and 75 per cent, respectively. Battery packs cultured more bacteria (1.58 per swab ± 1.00) than loupes (0.75 per swab ± 0.66; p = 0.024). Headlights had non-significantly greater growth (1.50 per swab ± 0.71) than loupes (p = 0.052). Bacterial growth was significantly higher from inner surfaces of loupes (p = 0.035) and headlights (p = 0.037). Potentially pathogenic bacteria were cultured from the equipment of five participants, including: Pantoea agglomerans, Acinetobacter radioresistens, Staphylococcus aureus, Acinetobacter calcoaceticus baumannii complex and Moraxella osloensis. CONCLUSION: This study demonstrates that surgical loupes and headlights used in otolaryngology harbour non-pathogenic skin flora and potentially pathogenic bacteria.

The Impact of Prior Season Vaccination on Subsequent Influenza Vaccine Effectiveness to Prevent Influenza-related Hospitalizations Over 4 Influenza Seasons in Canada.

BACKGROUND: Recent studies have demonstrated the possibility of negative associations between prior influenza vaccines and subsequent influenza vaccine effectiveness (VE), depending on season and strain. We investigated this association over 4 consecutive influenza seasons (2011-2012 through 2014-2015) in Canada. METHODS: Using a matched test-negative design, laboratory-confirmed influenza cases and matched test-negative controls admitted to hospitals were enrolled. Patients were stratified into 4 groups according to influenza vaccine history (not vaccinated current and prior season [referent], vaccinated prior season only, vaccinated current season only, and vaccinated both current and prior season). Conditional logistic regression was used to estimate VE; prior vaccine impact was assessed each season for overall effect and effect stratified by age (<65 years, ≥65 years) and type/subtype (A/H1N1, A/H3N2, influenza B). RESULTS: Overall, mainly nonsignificant associations were observed. Trends of nonsignificant decreased VE among patients repeatedly vaccinated in both prior and current season relative to the current season only were observed in the A/H3N2-dominant seasons of 2012-2013 and 2014-2015. Conversely, in 2011-2012, during which B viruses circulated, and in 2013-2014, when A/H1N1 circulated, being vaccinated in both seasons tended to result in a high VE in the current season against the dominant circulating subtype. CONCLUSIONS: Prior vaccine impact on subsequent VE among Canadian inpatients was mainly nonsignificant. Even in circumstances where we observed a trend of negative impact, being repeatedly vaccinated was still more effective than not receiving the current season's vaccine. These findings favor continuation of annual influenza vaccination recommendations, particularly in older adults. CLINICAL TRIALS REGISTRATION: NCT01517191.

Lyme disease: clinical diagnosis and treatment.

BACKGROUND: Lyme disease is an emerging zoonotic infection in Canada. As the Ixodes tick expands its range, more Canadians will be exposed to Borrelia burgdorferi, the bacterium that causes Lyme disease. OBJECTIVE: To review the clinical diagnosis and treatment of Lyme disease for front-line clinicians. METHODS: A literature search using PubMed and restricted to articles published in English between 1977 and 2014. RESULTS: Individuals in Lyme-endemic areas are at greatest risk, but not all tick bites transmit Lyme disease. The diagnosis is predominantly clinical. Patients with Lyme disease may present with early disease that is characterized by a "bull's eye rash", fever and myalgias or with early disseminated disease that can manifest with arthralgias, cardiac conduction abnormalities or neurologic symptoms. Late Lyme disease in North America typically manifests with oligoarticular arthritis but can present with a subacute encephalopathy. Antibiotic treatment is effective against Lyme disease and works best when given early in the infection. Prophylaxis with doxycyline may be indicated in certain circumstances. While a minority of patients may have persistent symptoms, evidence does not demonstrate that prolonged courses of antibiotics improve outcome. CONCLUSION: Clinicians need to be aware of the signs and symptoms of Lyme disease. Knowing the regions where Borrelia infection is endemic in North America is important for recognizing patients at risk and informing the need for treatment.

Comparative evaluation of the Bio-Rad Geenius HIV-1/2 Confirmatory Assay and the Bio-Rad Multispot HIV-1/2 Rapid Test as an alternative differentiation assay for CLSI M53 algorithm-I.

INTRODUCTION: The CLSI-M53-A, Criteria for Laboratory Testing and Diagnosis of Human Immunodeficiency Virus (HIV) Infection; Approved Guideline includes an algorithm in which samples that are reactive on a 4th generation EIA screen proceed to a supplemental assay that is able to confirm and differentiate between antibodies to HIV-1 and HIV-2. The recently CE-marked Bio-Rad Geenius HIV-1/2 Confirmatory Assay was evaluated as an alternative to the FDA-approved Bio-Rad Multispot HIV-1/HIV-2 Rapid Test which has been previously validated for use in this new algorithm. METHODS: This study used reference samples submitted to the Canadian - NLHRS and samples from commercial sources. Data was tabulated in 2×2 tables for statistical analysis; sensitivity, specificity, predictive values, kappa and likelihood ratios. RESULTS: The overall performance of the Geenius and Multispot was very high; sensitivity (100%, 100%), specificity (96.3%, 99.1%), positive (45.3, 181) and negative (0, 0) likelihood ratios respectively, high kappa (0.96) and low bias index (0.0068). The ability to differentiate HIV-1 (99.2%, 100%) and HIV-2 (98.1%, 98.1%) Ab was also very high. CONCLUSION: The Bio-Rad Geenius HIV-1/2 Confirmatory Assay is a suitable alternative to the validated Multispot for use in the second stage of CLSI M53 algorithm-I. The Geenius has additional features including traceability and sample and cassette barcoding that improve the quality management/assurance of HIV testing. It is anticipated that the CLSI M53 guideline and assays such as the Geenius will reduce the number of indeterminate test results previously associated with the HIV-1 WB and improve the ability to differentiate HIV-2 infections.

Oseltamivir pharmacokinetics in morbid obesity (OPTIMO trial).

BACKGROUND: Detailed pharmacokinetics to guide oseltamivir (Tamiflu®) dosing in morbidly obese patients is lacking. METHODS: The OPTIMO trial was a single-centre, non-randomized, open-label pharmacokinetic study of single-dose and steady-state oral oseltamivir phosphate and its carboxylate metabolite in healthy, morbidly obese [body mass index (BMI) >  40)] and healthy, non-obese (BMI  <  30) subjects. RESULTS: In the morbidly obese versus control subjects, respectively, the single-dose median oseltamivir oral clearance (CL/F) [840 (range 720-1640) L/h versus 580 (470-1800) L/h] was higher, the area under the curve from time zero to infinity (AUC(0-∞)) [89 (46-104) ng·h/mL versus 132 (42-160) ng·h/mL] was lower and the volume of distribution (V/F) [2320 (900-8210) L versus 1670 (700-7290) L] was unchanged. In the morbidly obese versus control subjects, respectively, the single-dose median oseltamivir carboxylate CL/F [22 (17-40) L/h versus 23 (12-33) L/h], AUC(0-∞) [3100 (1700-4100) ng·h/mL versus 3000 (2100-5900) ng·h/mL] and V/F [200 (130-370) L versus 260 (150-430) L] were similar. Similar results for oseltamivir and oseltamivir carboxylate CL/F, AUC0₋12 and V/F values were observed in the multiple-dose study. CONCLUSIONS: With single and multiple dosing, the systemic exposure to oseltamivir is decreased but that of oseltamivir carboxylate is largely unchanged. Based on these pharmacokinetic data, an oseltamivir dose adjustment for body weight would not be needed in morbidly obese individuals.

Ovine-associated Q fever.

In Atlantic Canada, the traditional risk factor for acquisition of Q fever infection has been exposure to infected parturient cats or newborn kittens. In this study we describe the first case of Q fever in Nova Scotia acquired as a result of direct exposure to sheep. A serosurvey of the associated flock was undertaken using an indirect immunofluorescence assay (IFA) testing for antibodies to phase I and phase II Coxiella burnetii antigens. This serosurvey revealed that 23 of 46 sheep (50%) were seropositive for the phase II antibody. Four of these sheep had titres of 1:64 including three nursing ewes, one of which had delivered two lambs that died shortly after delivery. Only one ewe had phase I antibodies but had the study's highest phase II antibody titre (1:128). Molecular studies using polymerase chain reaction (PCR) failed to detect C. burnetii DNA in any of the milk specimens.

BK virus nephropathy in a heart transplant recipient: case report and review of the literature.

The human polyomavirus BK virus (BKV) remains latent in the urinary tract and may be reactivated in immunocompromised states. BKV is noted to be the etiologic agent of polyomavirus-associated nephropathy (PVAN), which is a significant cause of allograft failure in renal transplant patients. Renal dysfunction following non-renal solid organ transplantation is common and is typically attributed to drug toxicity or patient comorbidities. In this article we describe a case of PVAN in the native kidneys of a heart transplant recipient and review the literature. Although this is only the fourth case reported, BKV nephropathy should be considered in the differential diagnosis of new renal failure following non-kidney solid organ transplantation, as early diagnosis of PVAN is necessary to prevent irreversible renal damage.

The effect of C. burnetii infection on the quality of life of patients following an outbreak of Q fever.

Sixty-six cases of Q fever were diagnosed in people affiliated with a goat-farming co-operative in rural Newfoundland in the spring of 1999. Follow-up studies which included administration of the Short Form 36 Health Survey (SF-36) were conducted 3 and 27 months after the initial outbreak to prospectively follow the effects of acute Q fever on the quality of life of the participants. Twenty-seven months after the outbreak 51% of those who had Q fever reported persistent symptoms including seven participants whose symptoms had initially resolved 3 months after the outbreak. Individuals with Q fever had significantly lower scores on five of the eight scales in the SF-36 and lower scores in the mental and physical summary scales compared to uninfected controls. Although this supports the hypothesis of a 'post Q fever fatigue syndrome' (QFFS), further study is warranted.

Atypical manifestations of chronic Q fever.

Chronic Q fever is uncommon, with the majority of cases manifesting as culture-negative endocarditis. In this report, we describe 3 patients who present with atypical manifestations of chronic Q fever. These were a 43-year-old man whose site of chronic Q fever was the central nervous system, a 53-year-old woman who underwent coronary angioplasty 6 days before the onset of symptoms of acute Q fever and within 4 months had serologic evidence consistent with chronic Q fever, and a 66-year-old man with fever of unknown origin, a pancreatic mass, and aorto-bifemoral grafts.

Goat-associated Q fever: a new disease in Newfoundland.

In the spring of 1999 in rural Newfoundland, abortions in goats were associated with illness in goat workers. An epidemiologic investigation and a serologic survey were conducted in April 1999 to determine the number of infections, nature of illness, and risk factors for infection. Thirty-seven percent of the outbreak cohort had antibody titers to phase II Coxiella burnetii antigen >1:64, suggesting recent infection. The predominant clinical manifestation of Q fever was an acute febrile illness. Independent risk factors for infection included contact with goat placenta, smoking tobacco, and eating cheese made from pasteurized goat milk. This outbreak raises questions about management of such outbreaks, interprovincial sale and movement of domestic ungulates, and the need for discussion between public health practitioners and the dairy industry on control of this highly infectious organism.

Pseudomonas aeruginosa community-acquired pneumonia in previously healthy adults: case report and review of the literature.

We report a case of rapidly fatal Pseudomonas aeruginosa community-acquired pneumonia (CAP) in a previously healthy 67-year-old woman. Eleven published case reports of P. aeruginosa CAP in previously healthy adults are reviewed. According to our review, the mean age of affected patients is 45.3 years. Five patients described in the literature were smokers with a mean smoking history of 40 pack-years. The clinical presentation is nonspecific, and although the pneumonia can be rapidly fatal, only 33% of the patients who were reported died. However, mortality may be independent of treatment within the first 36 hours of presentation. Exposure to aerosols of contaminated water is a risk factor for P. aeruginosa CAP in this population. Pseudomonas CAP should be considered in the differential diagnosis for anyone with a smoking history who presents with rapidly progressive pneumonia. We discuss treatment recommendations that are based on evidence in the currently available literature on the subject.