Kanti Pabbaraju

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.

Comparison of the Luminex xTAG Respiratory Viral Panel with In-House Nucleic Acid Amplification Tests for Diagnosis of Respiratory Virus Infections

ABSTRACT Detection of respiratory viruses using sensitive real-time nucleic acid amplification tests (NATs) is invaluable for patient and outbreak management. However, the wide range of potential respiratory virus pathogens makes testing using individual real-time NATs expensive and laborious. The objective of this study was to compare the detection of respiratory virus targets using the Luminex xTAG respiratory viral panel (RVP) assay with individual real-time NATs used at the Provincial Laboratory of Public Health, Calgary, Alberta, Canada. The study included 1,530 specimens submitted for diagnosis of respiratory infections from December 2006 to May 2007. Direct-fluorescent-antigen-positive nasopharyngeal samples were excluded from this study. A total of 690 and 643 positives were detected by RVP and in-house NATs, respectively. Kappa correlation between in-house NATs and RVP for all targets ranged from 0.721 to 1.000. The majority of specimens missed by in-house NATs (96.7%) were positive for picornaviruses. Samples missed by RVP were mainly positive for adenovirus (51.7%) or respiratory syncytial virus (27.5%) by in-house NATs and in general had low viral loads. RVP allows for multiplex detection of 20 (and differentiation between 19) respiratory virus targets with considerable time and cost savings compared with alternative NATs. Although this first version of the RVP assay has lower sensitivity than in-house NATs for detection of adenovirus, it has good sensitivity for other targets. The identification of picornaviruses and coronaviruses and concurrent typing of influenza A virus by RVP, which are not currently included in our diagnostic testing algorithm, will improve our diagnosis of respiratory tract infections.

Comparison of the Luminex xTAG™ Respiratory Viral Panel with xTAG® Respiratory Viral Panel Fast for diagnosis of respiratory virus infections

ABSTRACT Nucleic acid tests are sensitive and specific and provide a rapid diagnosis, making them invaluable for patient and outbreak management. Multiplex PCR assays have additional advantages in providing an economical and comprehensive panel for many common respiratory viruses. Previous reports have shown the utility of the xTAG respiratory viral panel (RVP) assay manufactured by Luminex Molecular Diagnostics for this purpose. A newer generation of this kit, released in Canada in early 2010, is designed to simplify the procedure and reduce the turnaround time by about 24 h. The assay methodology and targets included in this version of the kit are different; consequently, the objective of this study was to compare the detection of a panel of respiratory viral targets using the older Luminex xTAG RVP (RVP Classic) assay with that using the newer xTAG RVP Fast assay. This study included 334 respiratory specimens that had been characterized for a variety of respiratory viral targets; all samples were tested by both versions of the RVP assay in parallel. Overall, the RVP Classic assay was more sensitive than the RVP Fast assay (88.6% and 77.5% sensitivities, respectively) for all the viral targets combined. Targets not detected by the RVP Fast assay included primarily influenza B virus, parainfluenza virus type 2, and human coronavirus 229E. A small number of samples positive for influenza A virus, respiratory syncytial virus B, human metapneumovirus, and parainfluenza virus type 1 were not detected by the RVP Classic assay and in general had low viral loads.

Design and Validation of Real-Time Reverse Transcription-PCR Assays for Detection of Pandemic (H1N1) 2009 Virus

ABSTRACT Tracking novel influenza viruses which have the potential to cause pandemics, such as the pandemic (H1N1) 2009 virus, is a public health priority. Pandemic (H1N1) 2009 virus was first identified in Mexico in April 2009 and spread worldwide over a short period of time. Well-validated diagnostic tools that are rapid, sensitive, and specific for the detection and tracking of this virus are needed. Three real-time reverse transcription PCR (RT-PCR) assays for the amplification and detection of pandemic (H1N1) 2009 virus were developed, and their performance characteristics were compared with those of other published diagnostic assays. Thirty-nine samples confirmed to be positive for pandemic (H1N1) 2009 virus from Alberta, Canada, and six additional samples that were positive for influenza A virus but that were not typeable by using published seasonal influenza H1/H3 virus assays were available for this validation. Amplification and direct sequencing of the products was considered the “gold standard” for case identification. The new assays were sensitive and able to reproducibly detect virus in a 10−6 dilution of 4 × 106 50% tissue culture infective doses/ml when 5 μl was used as the template. They showed 100% specificity and did not cross-react with other respiratory viruses or seasonal influenza A virus subtypes. The coefficient of variation in crossing cycle threshold values for the detection of different template concentrations of pandemic (H1N1) 2009 virus was ≤3.13%, showing good reproducibility. The assays had a wide dynamic range for the detection of pandemic (H1N1) 2009 virus and utilized testing platforms appropriate for high diagnostic throughput with rapid turnaround times. We developed and validated these real-time PCR procedures with the goal that they will be useful for diagnosis and surveillance of pandemic (H1N1) 2009 virus. These findings will contribute to the informed management of this novel virus.

Detection of a broad range of human adenoviruses in respiratory tract samples using a sensitive multiplex real‐time PCR assay

Human adenoviruses (hAdVs) are associated with acute respiratory tract infections in pediatric populations and have been identified as a cause of outbreaks in institutional settings. Rapid diagnosis of hAdV infection is critical for appropriate and timely management. This study reports the design and validation of a sensitive and specific multiplex real‐time PCR for the detection of a broad range of hAdV serotypes in respiratory samples. The assay targets the conserved region of the hAdV hexon gene and utilizes hydrolysis probes for the detection of amplified products. The assay was evaluated using retrospectively (n = 864) and prospectively (n = 11,451) collected samples from November 2005 to July 2006. Seasonality studies and analysis of outbreaks was conducted over a 2‐year period from January 2005 to December 2006 (n = 33,067 samples). The assay gave a hAdV positive rate of 7.1% (n = 811) for specimens tested prospectively and was able to detect a broad range of hAdV serotypes with good sensitivity and specificity. A high rate of co‐infection was noted (21.7%). Adenovirus infections were more prevalent in the young with a median age of 24 months for positive patients. Sequence analysis of hAdV positives showed that serotype 7 was the most prevalent followed by serotypes 2 and 3. Association of hAdVs with respiratory outbreaks was low at 2.3% (6 of 266 outbreaks tested) and no seasonal variation was observed for hAdV infections during the 2‐year study period. This assay can improve the detection of hAdVs in respiratory samples and can be used to provide valuable epidemiological information. J. Med. Virol. 80:856–865, 2008.

Longitudinal Study of Influenza Molecular Viral Shedding in Hutterite Communities

BACKGROUND The nature of influenza viral shedding during naturally acquired infection is not well understood. METHODS A cohort study was conducted in Hutterite colonies in Alberta, Canada. Flocked nasal swabs were collected during 3 influenza seasons (2007-2008 to 2009-2010) from both symptomatic and asymptomatic individuals infected with influenza. Samples were tested by real-time reverse-transcription polymerase chain reaction for influenza A and influenza B, and the viral load (VL) was determined for influenza A positive samples. RESULTS Eight hundred thirty-nine participants were included in the cohort; 25% (208) tested positive for influenza viruses. They experienced 238 episodes of viral shedding, of which 23 (10%) were not accompanied by symptoms. For seasonal and pandemic H1N1, VL peaked at or before onset of acute respiratory infection. For H3N2, VL peaked 2 days after the onset of acute respiratory infection, which corresponded to peaks in systemic and respiratory symptom scores. Although the duration of shedding was shorter for asymptomatic participants, the peak level of VL shedding was similar to that of symptomatic participants. Viral loads for children and adults revealed similar patterns. CONCLUSIONS Molecular viral shedding values follow symptom scores, but timing of peak VL varies by subtype. Asymptomatic infections are infrequent.

Full-genome analysis of avian influenza A(H5N1) virus from a human, North America, 2013.

Full-genome analysis was conducted on the first isolate of a highly pathogenic avian influenza A(H5N1) virus from a human in North America. The virus has a hemagglutinin gene of clade 2.3.2.1c and is a reassortant with an H9N2 subtype lineage polymerase basic 2 gene. No mutations conferring resistance to adamantanes or neuraminidase inhibitors were found.

Diagnosis and epidemiological studies of human metapneumovirus using real-time PCR

Abstract Background Human metapneumovirus (hMPV) is prevalent in children, the elderly and immunocompromised individuals, but available epidemiological data is limited. Objectives (1) To develop and validate a real-time PCR method for hMPV diagnosis. (2) To determine the percentage of hMPV in respiratory specimens from the community and its association with outbreaks in our geographic area. (3) To provide epidemiological data in terms of age distribution, seasonality and co-infections. Study design A real-time PCR assay was designed for detection of hMPV lineages A and B. Prospective testing for hMPV over a 22-month period was then undertaken. Results The real-time PCR was sensitive and specific for detection of both lineages of hMPV. hMPV was detected in 9.5% (n =8239) of the specimens and 25% of the outbreaks (n =100) tested. The hMPV-positive patients ranged in age from 18 days to 99 years with a median age of 24 months. The number of positive samples peaked during the winter months of December, January and February. A high rate of co-infections was noted in the samples tested. Conclusions hMPV is common in the community and is associated with outbreaks. Including hMPV in routine testing improves etiological diagnosis of acute respiratory infections.

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.

Comparison of the Hybrid Capture 2 and cobas 4800 Tests for Detection of High-Risk Human Papillomavirus in Specimens Collected in PreservCyt Medium

ABSTRACT Clinical cervical cytology specimens (n = 466) collected in PreservCyt (Hologic Inc.) were used to evaluate the agreement between Hybrid Capture 2 (hc2; Qiagen) and cobas 4800 (c4800; Roche Molecular Diagnostics) for the detection of high-risk human papillomavirus (HR HPV) genotype infections. The agreement between the two assays was 93.8% (kappa = 0.87; 95% confidence interval, 0.828 to 0.918), with 186 and 251 concordant positive and negative results, respectively. All 186 concordant positives were confirmed using the Linear Array (LA; Roche Molecular Diagnostics) genotyping test. Of the 29 samples with discordant results (6.2%), 18 were hc2 positive and LA verified 17 as positive for HR HPV. Eleven discordant specimens were c4800 positive, and LA confirmed 5 as positive for HR HPV. As of October 2009, practice guidelines in Alberta, Canada, recommend reflex HPV testing for women over 30 years old with atypical squamous cells of undetermined significance (ASCUS) and for women over 50 years old with low-grade squamous intraepithelial lesions (LSIL) to help prioritize those who should undergo further evaluation. In this study, agreement between hc2 and c4800 results for samples from women over 30 years old with ASCUS cytology was 92.3% (n = 13), while no samples from women over 50 years old with LSIL cytology were identified for analysis.