Incidence and carrier frequency of Sandhoff disease in Saskatchewan determined using a novel substrate with detection by tandem mass spectrometry and molecular genetic analysis.

Sandhoff disease is a rare progressive neurodegenerative genetic disorder with a high incidence among certain isolated communities and ethnic groups around the world. Previous reports have shown a high occurrence of Sandhoff disease in northern Saskatchewan. Newborn screening cards from northern Saskatchewan were retrospectively screened in order to investigate the incidence and determine the carrier frequency of Sandhoff disease in these communities.

Detection of influenza H7N9 virus: all molecular tests are not equal.

The recent emergence of influenza A virus (H7N9) emphasizes the need for its rapid detection. While commercial nucleic acid amplification tests (NAATs) are commonly used to detect seasonal influenza virus, this study demonstrated that the analytical sensitivity of commercial assays is highly variable compared to that of CDC-based in-house NAATs for the detection of H7N9.

A polymerase chain reaction-based genotyping assay for detecting a novel Sandhoff disease-causing mutation.

Sandhoff disease is a rare genetic disorder, however, some northern Saskatchewan communities have a high incidence of the disease (for which the causative mutation has not been described). We discovered a novel mutation causing Sandhoff disease in this community and validated a molecular assay to detect the mutant allele. DNA sequencing was used to search for mutations in the HEXB gene from the most recently affected patient.

Molecular diagnostic assays for detection of viral respiratory pathogens in institutional outbreaks.

Outbreaks of viral respiratory disease in institutions may be associated with high morbidity and mortality, depending upon the viral etiology and the age and immune status of the affected patients. Control of outbreaks may include isolation and/or cohorting, and prophylaxis or treatment with specific antiviral agents may be indicated, all dependent upon the specific cause of the outbreak. Conventional methods of viral diagnosis detect only a limited number of the viruses that are known to cause outbreaks.

Elevated neonatal 3-OH isovalerylcarnitine due to breast milk sources in maternal 3-MCC deficiency.

We report a positive newborn screen for 3-hydroxyisovalerylcarnitine (C(5)OH) with an absence of 3-methylcrotonyl-coenzyme A carboxylase deficiency in the neonate. Subsequent blood tests demonstrated persistently elevated C(5)OH. Serial testing of the mother identified markedly elevated C(5)OH in both maternal blood and breast milk.

Diagnosis and high incidence of hyperornithinemia-hyperammonemia-homocitrullinemia (HHH) syndrome in northern Saskatchewan.

Mutations in the SLC25A15 gene, encoding the human inner mitochondrial membrane ornithine transporter, are thought to be responsible for hyperornithinemia-hyperammonemia-homocitrullinemia (HHH) syndrome, a rare autosomal recessive condition. HHH syndrome has been detected in several small, isolated communities in northern Saskatchewan (SK). To determine the incidence of HHH syndrome in these communities, a PCR method was set up to detect F188Δ, the common French-Canadian mutation.

Comparison of Shiga toxin-producing Escherichia coli detection methods using clinical stool samples.

Molecular diagnostic tools capable of identifying Shiga toxin-specific genetic determinants in stool specimens permit an unbiased approach to detect Shiga toxin-producing Escherichia coli (STEC) in clinical samples and can indicate when culture-based isolation methods are required. It is increasingly recognized that clinically relevant STEC are not limited to the singular O157 serotypes, and therefore diagnostic assays targeting toxin-encoding determinants must be able to account for any genetic variation that exists between serotypes. In this study conventional PCR and four real-time PCR assays (HybProbe, TaqMan, SYBR Green, and LUX) targeting the stx1 and stx2 Shiga toxin coding sequences were used to identify STEC in enriched stool samples (n = 36) and a panel of O157 and non-O157 strains (n = 64).

Human infection with a triple-reassortant swine influenza A(H1N1) virus containing the hemagglutinin and neuraminidase genes of seasonal influenza virus.

A reassortant influenza A(H1N1) virus of swine origin distinct from the pandemic H1N1 2009 strain was isolated from 3 patients, all of whom worked at the same large hog operation in Saskatchewan, Canada. The genomic composition of the isolates has not been previously reported, to our knowledge, and was the product of a genetic reassortment between seasonal H1N1 and triple-reassortant influenza virus that emerged in North American swine during the late 1990s. The neuraminidase and hemagglutinin genes of A/Saskatchewan/5350/2009, A/Saskatchewan/5351/2009, and A/Saskatchewan/5131/2009 were derived from human H1N1 virus and were closely related to those of A/Brisbane/59/2007.

Human papillomavirus typing and viral gene expression analysis for the triage of women with abnormal results from papanicolaou test smears to colposcopy.

Context: A cascade of molecular tests for human papillomavirus (HPV), as a follow-up to Papanicolaou test screening, could eliminate unnecessary colposcopy. Tests based on detection of HPV E6 messenger RNA (mRNA) are already being used as screening tools, but there is a good biological rationale for expecting that an increase in the relative amounts of HPV E6 mRNA in cervical samples may better predict cancerous transformation.

Objective: To compare some of the available diagnostic methods and our novel method of relative quantification (RQ) of HPV gene expression for the effective triage of women with abnormal results from Papanicolaou tests to colposcopy.

The impact of the distribution of human papillomavirus types and associated high-risk lesions in a colposcopy population for monitoring vaccine efficacy.

Context: Impact studies of the new human papillomavirus (HPV) vaccines will be biased unless local baseline distribution studies are conducted. Vaccine cross protection for other important oncogenic HPV types and the emergence of potential genotype replacements require the knowledge of the prevaccine epidemiology of HPV.

Objective: To determine the prevaccine distribution of HPV types in Saskatchewan, using a subpopulation of women referred to a colposcopy clinic.

Rapid detection of Norovirus based on an automated extraction protocol and a real-time multiplexed single-step RT-PCR.

Background: Molecular diagnosis of Norovirus infection can be a complex multistep process, which requires significant user intervention and expertise, and is not amenable to automation without extensive validation and optimization.

Objectives: To develop a real-time multiplexed RT-PCR assay with automated sample preparation that requires only a single-step and a single-tube for reverse transcription, amplification, and detection while exceeding the sensitivity of conventional PCR for broad-spectrum Norovirus detection.

Study Design: Limit of detection was assessed against dilutions of clinical specimens.

Development of a triplex real-time PCR assay for detection of Panton-Valentine leukocidin toxin genes in clinical isolates of methicillin-resistant Staphylococcus aureus.

Community-associated methicillin-resistant Staphylococcus aureus harboring Panton-Valentine leukocidin (PVL) genes is an emerging pathogen. A novel real-time PCR assay for identification of MRSA isolates containing PVL was developed. The PVL assay was used in a triplex format allowing simultaneous amplification of mecA, nuc, and PVL genes in 614 clinical isolates.

Rapid genotyping of hepatitis C virus by primer-specific extension analysis.

Quick and accurate genotyping of hepatitis C virus (HCV) is becoming increasingly important for clinical management of chronic infection and as an epidemiological marker. Furthermore, the incidence of HCV infection with mixed genotypes has clinical significance that is not addressed by most genotyping methods. We have developed a fluorescence-based genotyping assay called primer-specific extension analysis (PSEA) for the most prevalent HCV genotypes and have demonstrated the capacity of PSEA-HCV for detecting mixed-genotype HCV infections.

High-throughput qualitative multiplex 5' nuclease assay using post-only PCR analysis.

Homogenous fluorescence PCR assays offer distinct advantages for qualitative testing and are gaining immense popularity in fields like diagnostic microbiology. To meet the demand of high-volume laboratories, we developed a protocol for qualitative multiplex 5' nuclease assays using post-only PCR analysis. This novel approach overcomes throughput problems encountered with the established methods for TaqMan detection on the ABI PRISM 7700 Sequence Detection system and permits off-site TaqMan PCR, which can be analyzed several days after the reactions are completed.

West Nile virus in Canadian blood donors.

Background: Blood donation screening for West Nile virus (WNV) RNA by nucleic acid testing (NAT) was implemented in Canada in July 2003, and 14 WNV RNA-positive donations were identified. Samples were screened in minipools of six donations with a WNV assay (TaqScreen, Roche). Two of the donors were identified by single-donor screening that was initiated in the province of Saskatchewan, which had the highest prevalence of WNV in the country, in early September 2003.

The use of TaqMan PCR assay for detection of Bordetella pertussis infection from clinical specimens.

Objective: The routine clinical laboratory detection of Bordetella pertussis is through culture, which can require 5 to 7 days for the bacteria to grow. Using a polymerase chain reaction (PCR) assay can shorten this detection time while increasing the sensitivity of detection with similar specificity. This study compared culture with TaqMan PCR for detection of B pertussis in clinical specimens and the turnaround time for each assay during the pertussis season.