Rachel R. Higgins

Evaluation and Verification of the Seeplex Diarrhea-V ACE Assay for Simultaneous Detection of Adenovirus, Rotavirus, and Norovirus Genogroups I and II in Clinical Stool Specimens

ABSTRACT Acute viral gastroenteritis is an intestinal infection that can be caused by several different viruses. Here we describe the evaluation and verification of Seeplex Diarrhea-V ACE (Seeplex DV), a novel commercial multiplex reverse transcription-PCR (RT-PCR) assay that detects 5 diarrheal pathogens, including adenovirus, rotavirus, norovirus genogroup I (GI) and GII, and astrovirus. We describe a retrospective study of 200 clinical specimens of which 177 were stool specimens previously tested for the presence of gastrointestinal viruses by electron microscopy (EM) and/or real-time RT-PCR (rRT-PCR). The remaining 23 specimens comprised other human pathogens of viral or bacterial origin. Discordant norovirus GI and GII results were resolved using a commercial kit; discordant adenovirus and rotavirus results were resolved using a home brew multiplex rRT-PCR assay. Diagnostic sensitivities and specificities were calculated before and after discordant analysis. After discordant analysis, estimated diagnostic sensitivities were 100% for adenovirus, rotavirus, and norovirus GI and 97% for norovirus GII. Diagnostic specificities after discordant analysis were 100% for adenovirus, rotavirus, and norovirus GI and 99.4% for norovirus GII. The 95% limits of detection were 31, 10, 2, and 1 genome equivalent per reaction for adenovirus, rotavirus, and norovirus GI and GII, respectively. The results demonstrate that the Seeplex DV assay is sensitive, specific, convenient, and reliable for the simultaneous detection of several viral pathogens directly in specimens from patients with gastroenteritis. Importantly, this novel multiplex PCR assay enabled the identification of viral coinfections in 12 (6.8%) stool specimens.

Multidrug-Resistant Pandemic (H1N1) 2009 Infection in Immunocompetent Child

Recent case reports describe multidrug-resistant influenza A pandemic (H1N1) 2009 virus infection in immunocompromised patients exposed to neuraminidase inhibitors because of an I223R neuraminidase mutation. We report a case of multidrug-resistant pandemic (H1N1) 2009 bearing the I223R mutation in an ambulatory child with no previous exposure to neuraminidase inhibitors.

Multiple Influenza A (H3N2) Mutations Conferring Resistance to Neuraminidase Inhibitors in a Bone Marrow Transplant Recipient

ABSTRACT Immunocompromised patients are predisposed to infections caused by influenza virus. Influenza virus may produce considerable morbidity, including protracted illness and prolonged viral shedding in these patients, thus prompting higher doses and prolonged courses of antiviral therapy. This approach may promote the emergence of resistant strains. Characterization of neuraminidase (NA) inhibitor (NAI)-resistant strains of influenza A virus is essential for documenting causes of resistance. In this study, using quantitative real-time PCR along with conventional Sanger sequencing, we identified an NAI-resistant strain of influenza A (H3N2) virus in an immunocompromised patient. In-depth analysis by deep gene sequencing revealed that various known markers of antiviral resistance, including transient R292K and Q136K substitutions and a sustained E119K (N2 numbering) substitution in the NA protein emerged during prolonged antiviral therapy. In addition, a combination of a 4-amino-acid deletion at residues 245 to 248 (Δ245-248) accompanied by the E119V substitution occurred, causing resistance to or reduced inhibition by NAIs (oseltamivir, zanamivir, and peramivir). Resistant variants within a pool of viral quasispecies arose during combined antiviral treatment. More research is needed to understand the interplay of drug resistance mutations, viral fitness, and transmission.

The relative test performance characteristics of two commercial assays for the detection of Mycobacterium tuberculosis complex in paraffin-fixed human biopsy specimens

The Seeplex™ TB Detection-2 assay (Rockville, MD) is a nested endpoint PCR for the Mycobacterium tuberculosis complex (MTBC) targets IS6110 and MPB64 that utilizes dual priming oligonucleotide technology. When used to detect the presence of MTBC DNA in formalin-fixed paraffin-embedded tissue specimens, the sensitivity and specificity of this assay is equivalent to a labor-intensive traditional endpoint PCR assay and is more sensitive than a commercial real-time PCR assay.

Recovery of Influenza B Virus with the H273Y Point Mutation in the Neuraminidase Active Site from a Human Patient

ABSTRACT The H275Y oseltamivir resistance mutation confers high-level resistance to oseltamivir in isolates of human A(H1N1) influenza. We report the recovery and identification of an influenza B virus with the H273Y neuraminidase point mutation directly from a human patient. The H273Y influenza B isolate is resistant to oseltamivir and peramivir but sensitive to zanamivir.

Neuraminidase-inhibitor resistance testing for pandemic influenza A (H1N1) 2009 in Ontario, Canada

BACKGROUND Oseltamivir resistance-associated H275Y mutation in the neuraminidase (NA) gene of pandemic influenza A (H1N1) 2009 was occasionally reported worldwide during the 2009-2010 influenza season. A significant proportion of those were found in immunocompromised or severely ill persons. This phenomenon remains infrequent and clear recommendations for resistance testing are lacking. OBJECTIVES Present the suggested clinical selection criteria for antiviral susceptibility testing for influenza in Canada and to describe the Ontarian experience during the 2009-2010 influenza season. STUDY DESIGN Using a defined algorithm, we prospectively screened for OsR with pyrosequencing and phenotypic testing during the 2009-2010 influenza season. Zanamivir resistance was screened using phenotypic and sequencing technique on selected occasions. Clinical data was gathered for the resistant cases. RESULTS A total of 804 clinical H1N1 (2009) positive samples from Ontario were screened for oseltamivir resistance between June 2009 and March 2010. We identified oseltamivir resistance in 5 (0.6%) distinct patients aged 9-62 years. All the resistant strains bore the H275Y mutation. Susceptibility to zanamivir was maintained in all of them. Three patients harboring oseltamivir resistant strain were intensive care unit patients and four were immunocompromised. All were tested for susceptibility because of a repeat positive result for influenza A PCR. CONCLUSION Oseltamivir resistance was not frequent during the 2009-2010 influenza season but was identified with a systematic and prospective approach to resistance testing. In order to be as sensitive as possible in the detection of those few cases, we report the suggested indications for antiviral susceptibility testing in Canada.

Temporal changes in respiratory adenovirus serotypes circulating in the greater Toronto area, Ontario, during December 2008 to April 2010

BackgroundCertain adenovirus serotypes cause severe infections, especially in children. It is important to monitor temporal changes in serotypes causing clinical disease. The objective of this study was to document circulating respiratory adenovirus serotypes by sequencing adenovirus culture isolates from the Greater Toronto Area, Ontario, during December 2008 to April 2010.MethodsNucleic acid extraction was performed on 90 respiratory tract adenovirus culture isolates. PCR amplification was conducted with primers targeting the adenovirus hexon gene hypervariable region 7. Sanger sequencing and phylogenetic analyses were performed to determine serotype identities.ResultsAmong 90 clinical respiratory isolates sequenced, eight different serotypes were identified. Serotype 3 (34, 38%), serotype 2 (30, 30%), and serotype 1 (14, 16%) isolates were most common; serotypes 5, 6, 11, 17 and 21 were also observed. Seventeen (50%) of the 34 HAdV-3 isolates were identified between December 2008 and February 2009, while none were identified from December 2009 to February 2010. Between December 2008 and April 2009, the two most common serotypes were HAdV-3 and HAdV-2, detected in 18 (53%) and 8 (24%) of the 34 cultures isolates, respectively. However, from December 2009 to April 2010, there was an increase in HAdV-2, which became the most prevalent serotype, detected in 10 (50%) of the 20 isolates identified (p = 0.05).ConclusionsThere was a gradual shift in prevailing adenovirus serotypes during the 17 month study period, from predominantly HAdV-3 to HAdV-2. If an adenovirus vaccine were to be broadly implemented, multiple serotypes should be included.

Laboratory testing and phylogenetic analysis during a mumps outbreak in Ontario, Canada

BackgroundIn September 2009, a mumps outbreak originated in New York and spread to Northeastern USA and Canada. This study compares the performance of different diagnostic testing methods used in Ontario and describes molecular characteristics of the outbreak strain.MethodsBetween September 2009 and February 2010, specimens from suspect cases were submitted to Public Health Ontario Laboratory for mumps serology, culture and/or real-time reverse-transcriptase PCR (rRT-PCR) testing. rRT-PCR-positive specimens underwent genotyping at Canada’s National Microbiology Laboratory. Whole genome sequencing was performed on four outbreak and three sporadic viral culture isolates.ResultsSix hundred ninety-eight patients had IgM serology testing, of which 255 (37%) had culture and rRT-PCR. Among those, 35/698 (5%) were IgM positive, 39/255 (15%) culture positive and 47/255 (18%) rRT-PCR-positive. Buccal swabs had the highest rRT-PCR positivity (21%). The outbreak isolates were identical to that in the New York outbreak occurring at the same time. Nucleotide and amino acid identity with the Jeryl Lynn vaccine strain ranged from 85.0-94.5% and 82.4-99.4%, depending on the gene and coding sequences. Homology of the HN protein, the main immunogenic mumps virus protein, was found to be 94.5 and 95.3%, when compared to Jeryl Lynn vaccine major and minor components, respectively.ConclusionsDespite higher sensitivity than serology, rRT-PCR testing is underutilized. Further work is needed to better understand the suboptimal match of the HN gene between the outbreak strain and the Jeryl Lynn vaccine strain.

Rotavirus genotypes circulating in Ontario, Canada, before and after implementation of the rotavirus immunization program

BACKGROUND AND OBJECTIVES Ontario introduced a universal publicly-funded group A rotavirus (RVA) immunization program in August 2011, using monovalent vaccine. RVA immunization programs have decreased the incidence of RVA acute gastroenteritis in many countries but it is unclear if it will contribute to the emergence of certain genotypes. We monitored RVA trends and genotypes in Ontario before and after implementation of the publicly-funded immunization program. METHODS RVA detection was conducted at Public Health Ontario Laboratories from January 2009 to December 2011 (pre-program period) and January 2012 to October 2015 (publicly-funded RVA immunization program period) and number of RVA-positive specimens and percent positivity were analysed. A convenience sample of RVA-positive stool specimens, from September 2010 to December 2011 (pre-program period) and January 2012 to June 2013 (program period), were genotyped using heminested PCR. A literature review on the burden of illness from emergent genotype was performed. RESULTS Stool specimens showed a significant decrease in RVA percent positivity from the 36 month pre-program period (14.4%; 1537/10700) to the 46 month program period (6.1%; 548/9019). An increase in the proportion of RVA G10 among genotyped specimens, associated with five different P genotypes, from the pre-program (6.3%; 13/205) to the program (31.5%; 40/127) period was observed. Our literature review identified approximately 200 G10-positive human stool specimens from 16 different countries. CONCLUSIONS This study documented a decrease in the number of RVA-positive specimens and percent positivity after implementation of the immunization program. An unexpected increase in the proportion of RVA G10 was detected following program introduction. Ongoing RVA surveillance is important in evaluating both the long-term impact of immunization and emergence of RVA genotypes.

Laboratory-confirmed influenza B infection in immunized long-term care facility residents receiving oseltamivir prophylaxis in Ontario.

We report on an influenza B outbreak in an Ontario long-term care facility in which 2 immunized residents receiving oseltamivir prophylaxis for at least 5 days developed laboratory-confirmed influenza B infection. All isolates were tested for the most common oseltamivir resistance, and none of them had resistance identified.