Michael A. Drebot

Evaluation of Commercially Available Anti–Dengue Virus Immunoglobulin M Tests

Anti–dengue virus immunoglobulin M kits were evaluated. Test sensitivities were 21%–99% and specificities were 77%–98% compared with reference ELISAs. False-positive results were found for patients with malaria or past dengue infections. Three ELISAs showing strong agreement with reference ELISAs will be included in the World Health Organization Bulk Procurement Scheme.

First Case of Zika Virus Infection in a Returning Canadian Traveler

A woman who recently traveled to Thailand came to a local emergency department with a fever and papular rash. She was tested for measles, malaria, and dengue. Positive finding for IgM antibody against dengue and a failure to seroconvert for IgG against dengue for multiple blood samples suggested an alternate flavivirus etiology. Amplification of a conserved region of the non-structural protein 5 gene of the genus Flavivirus yielded a polymerase chain reaction product with a matching sequence of 99% identity with Zika virus. A urine sample and a nasopharygeal swab specimen obtained for the measles investigation were also positive for this virus by reverse transcription polymerase chain reaction. Subsequently, the urine sample yielded a Zika virus isolate in cell culture. This case report describes a number of novel clinical and laboratory findings, the first documentation of this virus in Canada, and the second documentation from this region in Thailand.

Ixodes scapularis Ticks Collected by Passive Surveillance in Canada: Analysis of Geographic Distribution and Infection with Lyme Borreliosis Agent Borrelia burgdorferi

Abstract Passive surveillance for the occurrence of the tick Ixodes scapularis Say (1821) and their infection with the Lyme borreliosis spirochaetes Borrelia burgdorferi s.l. has taken place in Canada since early 1990. Ticks have been submitted from members of the public, veterinarians, and medical practitioners to provincial, federal, and university laboratories for identification, and the data have been collated and B. burgdorferi detected at the National Microbiology Laboratory. The locations of collection of 2,319 submitted I. scapularis were mapped, and we investigated potential risk factors for I. scapularis occurrence (in Québec as a case study) by using regression analysis and spatial statistics. Ticks were submitted from all provinces east of Alberta, most from areas where resident I. scapularis populations are unknown. Most were adult ticks and were collected in spring and autumn. In southern Québec, risk factors for tick occurrence were lower latitude and remote-sensed indices for land cover with woodland. B. burgdorferi infection, identified by conventional and molecular methods, was detected in 12.5% of 1,816 ticks, including 10.1% of the 256 ticks that were collected from humans and tested. Our study suggests that B. burgdorferi-infected I. scapularis can be found over a wide geographic range in Canada, although most may be adventitious ticks carried from endemic areas in the United States and Canada by migrating birds. The risk of Lyme borreliosis in Canada may therefore be mostly low but more geographically widespread than previously suspected.

Investigation of Genotypes of Borrelia burgdorferi in Ixodes scapularis Ticks Collected during Surveillance in Canada

ABSTRACT The genetic diversity of Borrelia burgdorferi sensu stricto, the agent of Lyme disease in North America, has consequences for the performance of serological diagnostic tests and disease severity. To investigate B. burgdorferi diversity in Canada, where Lyme disease is emerging, bacterial DNA in 309 infected adult Ixodes scapularis ticks collected in surveillance was characterized by multilocus sequence typing (MLST) and analysis of outer surface protein C gene (ospC) alleles. Six ticks carried Borrelia miyamotoi, and one tick carried the novel species Borrelia kurtenbachii. 142 ticks carried B. burgdorferi sequence types (STs) previously described from the United States. Fifty-eight ticks carried B. burgdorferi of 1 of 19 novel or undescribed STs, which were single-, double-, or triple-locus variants of STs first described in the United States. Clonal complexes with founder STs from the United States were identified. Seventeen ospC alleles were identified in 309 B. burgdorferi-infected ticks. Positive and negative associations in the occurrence of different alleles in the same tick supported a hypothesis of multiple-niche polymorphism for B. burgdorferi in North America. Geographic analysis of STs and ospC alleles were consistent with south-to-north dispersion of infected ticks from U.S. sources on migratory birds. These observations suggest that the genetic diversity of B. burgdorferi in eastern and central Canada corresponds to that in the United States, but there was evidence for founder events skewing the diversity in emerging tick populations. Further studies are needed to investigate the significance of these observations for the performance of diagnostic tests and clinical presentation of Lyme disease in Canada.

West Nile Virus Surveillance and Diagnostic: A Canadian Perspective

A surveillance program has been in place since 2000 to detect the presence of West Nile virus (WNV) in Canada. Serological assays are most appropriate when monitoring for human disease and undertaking case investigations. Genomic amplification procedures are more commonly used for testing animal and mosquito specimens collected as part of ongoing surveillance efforts. The incursion of WNV into this country was documented for the first time in 2001 when WNV was demonstrated in 12 Ontario health units during the late summer and fall. In 2002 WNV activity was documented by avian surveillance in Ontario by mid-May with subsequent expansion of the virus throughout Ontario and into Quebec, Manitoba, Saskatchewan and Nova Scotia. Human cases were recorded in both Ontario and Quebec in 2002 with approximately 800 to 1000 probable, confirmed and suspect cases detected. The possible recurrence and further spread of WNV to other parts of Canada in 2003 must be anticipated with potential risk to public health. The continued surveillance and monitoring for WNV-associated human illness is necessary and appropriate disease prevention measures need to be in place in 2003.

Prognosis after West Nile Virus Infection

Context The long-term prognosis of West Nile virus infection is not well understood. Contribution In this longitudinal study of 156 patients with West Nile virus infection, physical and cognitive function seemed to return to population norms within about 1 year. Caution Patients who died were excluded from the analysis, and the analyses depended on statistical assumptions that the data did not always meet. Implication People infected with West Nile virus seem to recover physical and mental function within about 1 year. The Editors West Nile virus, endemic to Africa, Europe, the Middle East, and Asia, has caused recurrent outbreaks in the United States and Canada since 1999 (1, 2). Approximately 20% of infected persons develop a clinical presentation that can range from a mild influenza-like illness to neuroinvasive diseases, such as meningitis, encephalitis, and acute flaccid paralysis (3). Recent studies of persons infected with West Nile virus report that symptoms and signs, such as fatigue, cognitive dysfunction, and motor abnormalities, can persist for months after symptom onset (414). Little is known, however, about how physical and mental functioning changes over time or about long-term recovery among infected persons. Understanding such change patterns is essential to provide accurate prognostic information to patients and their families, as well as to help in the planning of care and evaluation of future interventions. Existing reports provide valuable information on self-reported outcomes (413) but have limitations, including single follow-up assessments (4, 69, 1113), follow-up until 12 months after symptom onset (4, 68, 1014), and lack of validated instruments to measure physical and mental functioning (48, 1113). Moreover, factors associated with slower recovery are unknown. The primary objectives of this study were to describe patterns of physical and mental outcomes after infection with West Nile virus by using longitudinal observations and to assess long-term outcomes. We hypothesized that such long-term outcomes would be worse in patients with neuroinvasive disease than in those with nonneuroinvasive disease. We conducted a longitudinal cohort study to develop prognostic curves for patient-relevant outcomes, such as physical and mental functioning, fatigue, and depression. We also assessed factors associated with delayed recovery of physical and mental outcomes. Methods Study Participants and Protocol Patients with positive West Nile virus IgM antibody-capture enzyme-linked immunosorbent assay (15) from serum or cerebrospinal fluid samples that was subsequently confirmed by plaque reduction neutralization assay (16) were eligible. We enrolled patients with neuroinvasive disease (meningitis, encephalitis, or acute flaccid paralysis) and nonneuroinvasive disease. We aimed to enroll participants within 4 weeks of symptom onset. To allow for feasibility of follow-up, we limited enrollment to geographic regions that had 4 or more infected individuals within an approximate radius of 200 km. Because complications of West Nile virus infection in children are generally less frequent, we excluded persons younger than age 18 years (17). Because the objective of the study was to assess prognosis attributable to West Nile virus, we excluded patients receiving experimental therapy for West Nile virus, as well as those being treated for an illness unrelated to West Nile virus that could interfere with interpretation of the outcome measures. Only 1 patient, who was receiving chemotherapy for a malignant solid organ condition, met the latter criterion. We classified participants with neuroinvasive disease by using previously published criteria for meningitis, encephalitis, and acute flaccid paralysis (4) (Table 1). We classified participants who met criteria for both meningitis and encephalitis as having meningoencephalitis. Only 3 patients had meningitis alone, so we combined them with the meningoencephalitis group for analysis. Table 1. Diagnostic Criteria for Meningitis, Encephalitis, and Acute Flaccid Paralysis We classified participants who were symptomatic but did not meet any of the case definitions for neuroinvasive disease as having nonneuroinvasive disease. We reviewed laboratory, hospital, and clinic medical records to confirm the case definition. Radiologic and laboratory assessments were performed at the discretion of the attending physician. Supportive care, such as physiotherapy or psychotherapy, was under the discretion of the attending physician. We enrolled the first participant on August 2003 and the last on November 2006, with the final follow-up visit on May 2007. Provincial laboratories in Canada conducted all West Nile virus testing and forwarded the names of the physicians whose patients tested IgM-positive for West Nile virus to the study office. We asked these physicians to approach the patients (whose identity remained unknown to the research team, thereby maintaining confidentiality) or their families to see whether they were willing to be contacted about the study. A research nurse then assessed patient eligibility and obtained informed consent. We obtained ethics approval for the study protocol from the relevant review committees at McMaster University, University of Manitoba, University of Saskatchewan, and University of Alberta. All patients or their designated surrogate decision makers who agreed to participate in the study gave informed consent. Outcomes A trained research nurse assessed outcomes on enrollment into the study (baseline visit); on days 10, 20, and 30; and then every month for 12 months. In our original protocol, we planned to obtain repeated measurements at 24 and 36 months for participants enrolled in the first year of the study and to measure outcomes at 24 months (third year of the study) for those participants enrolled in the second year. Because participants were being visited more frequently to obtain blood work for an unrelated study from 15 to 36 months from their enrollment in this study, we obtained additional measurements at 3-month intervals from 15 to 36 months in these participants. A trained research nurse made most assessments during home visits, minimizing missing visits or selection bias based on participants ability to travel (although participants in Saskatchewan and Manitoba were seen in an ambulatory care setting at a tertiary care hospital for their convenience). We recorded age, sex, medical history, and premorbid chronic illnesses. Categories of comorbid conditions included cardiac disease (coronary artery disease and congestive heart failure), peripheral vascular disease, chronic obstructive pulmonary disease, diabetes, renal failure, peptic ulcer disease, cancer, and rheumatologic disease. We assessed all these conditions through interviews with participants and review of medical records. To assess physical functioning, we used the Physical Component Summary (PCS) of the Short Form-36 (18, 19). The Short Form-36 measures 8 health constructs by using 8 scales with 2 to 10 items per scale (total of 36 questions); raw scores range from 0 to 100 but are adjusted for population norms by using a linear transformation (18). For the PCS subscale, scores are standardized to the general U.S. population (mean score, 50 [SD, 10]). Very high scores indicate no physical limitations, disabilities, or decrements in well-being, as well as a high energy level. Very low scores indicate substantial limitations in self-care and physical, social, and role activities; severe bodily pain; or frequent tiredness. To assess mental functioning, we used the Mental Component Summary (MCS) of the Short Form-36. These scores are also standardized to the general U.S. population (mean score, 50 [SD, 10]). Very high scores indicate frequent positive affect and absence of psychological distress and limitations in usual social and role activities due to emotional problems. Very low scores indicate frequent psychological distress and substantial social and role disability due to emotional problems (18). We used the Depression Anxiety Stress Scale (DASS) (20, 21) and the Fatigue Severity Scale (FSS) (22) beginning in 2004 to capture depressive symptoms and persistent fatigue noted among participants after the study had begun. The DASS is a 14-item scale that assesses dysphoria and lack of interest or involvement. Scores range from 0 (no symptoms) to 42. Among a general adult population, 80% of people have a score of 9 or less and 70% have a score of 6 or less (23). The FSS measures the perceived level of fatigue by using a Likert scale, in which the score ranges from 1 (low fatigue level) to 7 (high fatigue level). Two thirds of the general population have a score between 2.7 and 5.3 (24). Statistical Analysis Prognosis We used data from 57 of 64 participants with neuroinvasive disease (meningoencephalitis [n= 32], encephalitis [n= 22], and meningitis [n= 3]) and 92 with nonneuroinvasive disease to estimate prognosis. We excluded 7 patients with acute flaccid paralysis from the analysis because this number was too small to estimate prognostic curves, and we grouped 3 participants with meningitis with the 32 participants in the meningoencephalitis group. We used nonlinear mixed-effects modeling to estimate the parameters of nonlinear models for PCS, MCS, DASS, and FSS scores (25). We compared participants with neuroinvasive disease with those with nonneuroinvasive disease. Of participants with neuroinvasive disease, we compared those with meningoencephalitis with those with encephalitis. The fixed effects in the nonlinear mixed-effects analysis describe the average pattern of change over time, and they indicate the typical course of recovery in this population. The random effects in the nonlinear mixed-effects analysis allow for orderly variations in the pattern of change among patients. We estimated predicted change curves for each patient, and we estimated the degree of i

Rapid Antigen-Capture Assay To Detect West Nile Virus in Dead Corvids

The utility of the VecTest antigen-capture assay to detect West Nile virus (WNV) in field-collected dead corvids was evaluated in Manitoba and Ontario, Canada, in 2001 and 2002. Swabs were taken from the oropharynx, cloaca, or both of 109 American Crows, 31 Blue Jays, 6 Common Ravens, and 4 Black-billed Magpies from Manitoba, and 255 American Crows and 28 Blue Jays from Ontario. The sensitivity and specificity of the antigen-capture assay were greatest for samples from American Crows; oropharyngeal swabs were more sensitive than cloacal swabs, and interlaboratory variation in the results was minimal. The sensitivity and specificity of the VecTest using oropharyngeal swabs from crows were 83.9% and 93.6%, respectively, for Manitoba samples and 83.3% and 95.8%, respectively, for Ontario birds. The VecTest antigen-capture assay on oropharyngeal secretions from crows is a reliable and rapid diagnostic test that appears suitable for incorporation into a WNV surveillance program.

Ross River virus disease reemergence, Fiji, 2003-2004.

We report 2 clinically characteristic and serologically positive cases of Ross River virus infection in Canadian tourists who visited Fiji in late 2003 and early 2004. This report suggests that Ross River virus is once again circulating in Fiji, where it apparently disappeared after causing an epidemic in 1979 to 1980.

Susceptibility of Pigs and Chickens to SARS Coronavirus

An outbreak of severe acute respiratory syndrome (SARS) in humans, associated with a new coronavirus, was reported in Southeast Asia, Europe, and North America in early 2003. To address speculations that the virus originated in domesticated animals, or that domestic species were susceptible to the virus, we inoculated 6-week-old pigs and chickens intravenously, intranasally, ocularly, and orally with 106 PFU of SARS-associated coronavirus (SARS-CoV). Clinical signs did not develop in any animal, nor were gross pathologic changes evident on postmortem examinations. Attempts at virus isolation were unsuccessful; however, viral RNA was detected by reverse transcriptase-polymerase chain reaction in blood of both species during the first week after inoculation, and in chicken organs at 2 weeks after inoculation. Virus-neutralizing antibodies developed in the pigs. Our results indicate that these animals do not play a role as amplifying hosts for SARS-CoV.

Distribution and Phylogenetic Comparisons of a Novel Mosquito Flavivirus Sequence Present in Culex tarsalis Mosquitoes from Western Canada with Viruses Isolated in California and Colorado

In a previous study, a new flavivirus genome sequence was identified in Culex tarsalis mosquitoes obtained in Alberta, Canada and was shown to be genetically related to but distinct from members of the insect-specific flaviviruses. Nonstructural protein 5-encoding sequences amplified from Cx. tarsalis pools from western Canada have shown a high similarity to genome sequences of novel flaviviruses isolated from mosquitoes in California and Colorado. Despite wide distribution of this virus, designated Calbertado virus, strains demonstrate a high degree of nonstructural protein 5 nucleotide (> 90%) and amino acid (> 97%) identity. The ecology and geographic range of Calbertado virus warrants further study because it may potentially influence transmission of mosquito-borne flaviviruses, including important human pathogens such as West Nile and Saint Louis encephalitis viruses.