Ernesto Lombos

Development of a novel real-time reverse-transcriptase PCR method for the detection of H275Y positive influenza A H1N1 isolates.

Abstract During the 2007–2008 influenza season global strain surveillance for antiviral resistance revealed the sudden emergence of oseltamivir resistance in influenza A H1N1 isolates. Although oseltamivir resistance rates vary from region to region, 16% of isolates tested globally were found to be oseltamivir resistant by a histidine to tyrosine mutation of residue 275 of the neuraminidase gene of influenza A. In order to implement effective resistance testing locally a novel real-time reverse-transcriptase PCR (RT-PCR) assay was developed for the detection of the H275Y mutation. To evaluate this method, 40 oseltamivir resistant and 61 oseltamivir sensitive H1N1 influenza isolates were tested using Sanger sequencing, which is the reference method for detection of resistance, pyrosequencing and the novel H275Y RT-PCR assay. In comparison to Sanger sequencing, the sensitivity and specificity of the H275Y RT-PCR assay were 100% (40/40) and 100% (61/61) respectively, while the sensitivity and specificity of pyrosequencing were 100% (40/40) and 97.5% (60/61) respectively. Although all three methods were effective in detecting the H275Y mutation associated with oseltamivir resistance, the H275Y RT-PCR assay was the most rapid and could easily be incorporated into an influenza subtyping protocol.

Characterization of culture-positive adenovirus serotypes from respiratory specimens in Toronto, Ontario, Canada: September 2007–June 2008

This study describes the prevalence of culture-positive adenovirus serotypes in culture-positive respiratory specimens sent to the Central Public Health Laboratory, Toronto, Ontario, Canada for the period September 2007–June 2008. Total nucleic acid was extracted from virus cultures using an automated extraction method followed by polymerase chain reaction and Sanger sequencing of the adenovirus hexon gene hypervariable region 7. 73% of specimens (n = 70) were from patients ≤ 4 years of age. Of the 96 adenovirus isolates, the most common identified serotypes were serotype 3 (n = 44, 46%), serotype 2 (n = 25, 26%), serotype 1 (n = 17, 18%), and serotype 21 (n = 5, 5%). Adenovirus serotype 14 was not found in this study group. The leading serotype, Ad3, was identified throughout the duration of the study period. Molecular methods allow for the determination of circulating adenovirus serotypes and be used to document the spread of highly virulent adenoviral serotypes into a region.

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.

Evaluation of Altona Diagnostics RealStar Zika Virus Reverse Transcription-PCR Test Kit for Zika Virus PCR Testing

ABSTRACT With the emerging Zika virus (ZIKV) epidemic, accessible real-time reverse transcription-PCR (rRT-PCR) assays are needed to streamline testing. The commercial Altona Diagnostics RealStar ZIKV rRT-PCR test kit (Altona PCR) has been approved for emergency use authorization by the U.S. FDA. Our aim was to verify the Altona PCR by comparing it to the CDC-designed dual-target ZIKV rRT-PCR reference assay (reference PCR) and describe the demographics of patients tested for ZIKV by rRT-PCR in Ontario, Canada. A large set of clinical specimens was tested for ZIKV by the Altona PCR and the reference PCR. Positive or equivocal specimens underwent PCR and Sanger sequencing targeting the ZIKV NS5 gene. A total of 671 serum specimens were tested by the reference PCR: 58 (8.6%) were positive, 193 (28.8%) were equivocal, and 420 (62.6%) were negative. Ninety percent of the reference PCR-positive patients were tested in the first 5 days after symptom onset. The Altona PCR was performed on 284/671 specimens tested by the reference PCR. The Altona PCR was positive for 53/58 (91%) reference PCR-positive specimens and 16/193 (8%) reference PCR-equivocal specimens; the ZIKV NS5 PCR was positive for all 68 Altona PCR-positive specimens and negative for all 181 Altona PCR-negative specimens that underwent the NS5 PCR. The Altona PCR has very good sensitivity (91%) and specificity (97%) compared to the reference PCR. The Altona PCR can be used for ZIKV diagnostic testing and has less extensive verification requirements than a laboratory-developed test.

Respiratory infection in institutions during early stages of pandemic (H1N1) 2009, Canada.

Outbreaks of respiratory infection in institutions in Ontario, Canada were studied from April 20 to June 12, 2009, during the early stages of the emergence of influenza A pandemic (H1N1) 2009. Despite widespread presence of influenza in the general population, only 2 of 83 outbreaks evaluated by molecular methods were associated with pandemic (H1N1) 2009.

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.

A paucity of co-infecting respiratory viral pathogens in nasopharyngeal specimens from patients infected with H274Y-positive influenza A (H1N1) strains

The H274Ymutation in the neuraminidase gene of influenza A (H1N1) has been associated with oseltamivir resistance. Initial evaluations of this mutation in animal models suggested that this mutation had reduced fitness and reduced pathogenicity when compared to corresponding wild-type viruses. Earlier reports of clinical infections supported this view and H274Y was rarely reported. However, in the last year, there have been reports of dramatic increases in the proportion of H274Y mutations identified in clinical isolates throughout the world. If pathogenicity was indeed compromised in these most recent mutants, then it should be determined whether patients infected with oseltamivirresistant strains are presenting with illness associated with another co-infecting respiratory viral pathogen. The purpose of this brief investigation was to determine whether nasopharyngeal specimens from patients infected with strains of influenza A (H1N1) carrying the H274Y mutation were more likely than not to contain other commonly circulating respiratory viral pathogens. Nasopharyngeal specimens from patients with influenzalike illness were sent to the Ontario Public Health Laboratories. Isolates of influenza A collected from Toronto, Ontario, Canada (estimated population 2.7 million) during the period November 14, 2007 to February 14, 2008 were screened by reverse transcriptase (RT)-PCR for the H1N1 subtype using primers described previously. Strains of isolates were confirmed by Sanger sequencing and sequence alignment. Neuraminidase gene sequencing was undertaken and sequences were aligned using CLUSTALX. Isolate sequences were compared to sequenceswith describedH274Ymutations: GenBank accession No. EU516123 influenza A virus (A/Hawaii/28/ 2007(H1N1)); GenBank accession No. CY027037 influenza A virus (A/Kansas/UR06-0104/2007(H1N1)); GenBank accession No. EU516027 influenza A virus (A/Texas/31/2007(H1N1)). Specimens corresponding toH274Ymutant andH274wild-type isolateswere blindedand chosen at random for a retrospective investigation for other respiratory viral pathogens (adenovirus, coronavirus 229E/NL63, coronavirus OC43, influenza A/B, parainfluenza virus 1/2/3, respiratory syncytial virus A/B, rhinovirus A) using the SeeplexRVdetectionkit protocol (Seegene, Inc., Rockville, MD). Data analysis was carried out