Cheryl Bletchly

Comparing Nose-Throat Swabs and Nasopharyngeal Aspirates Collected From Children With Symptoms for Respiratory Virus Identification Using Real-Time Polymerase Chain Reaction

OBJECTIVES. The objective of this study was to calculate sensitivity values for the detection of major respiratory viruses of childhood by using combined nose-throat swabs and nasopharyngeal aspirates. METHODS. Children who had symptoms and presented to a pediatric teaching hospital and had a diagnostic respiratory specimen collected were enrolled, and paired nose-throat swab and nasopharyngeal aspirate specimens were collected. Parents were asked to collect the nose-throat swab specimen in the first instance but could defer to a health care worker if unwilling. Nose-throat swab collectors were asked to rate perceived quality of collection. All nasopharyngeal aspirates were collected by a health care worker by using a standard protocol. Real-time polymerase chain reaction for 8 respiratory viruses was performed in our hospitals diagnostic laboratory. RESULTS. Paired nose-throat swab/nasopharyngeal aspirate specimens were collected during 303 illnesses, with at least 1 respiratory virus identified in 186 (61%). For the major pathogens of childhood, influenza A virus and respiratory syncytial virus, collection by using the nose-throat swab had a sensitivity of 91.9% and 93.1%, respectively. A health care worker collected 219 (72%) of the nose-throat swab specimens; concordance with the nasopharyngeal aspirate was not related to health care worker collection or perceived quality of collection. CONCLUSIONS. Nose-throat swab specimens, in combination with sensitive molecular testing, are a less invasive diagnostic respiratory specimen with adequate sensitivity for use in the clinic and hospital outpatient settings and large-scale community studies through parent collection. For children who present to a hospital in which an avian or pandemic strain of influenza virus is reasonably part of the differential diagnosis, nasopharyngeal aspirates or a similar collection technique (eg, nasal washes) should continue to be used.

Detection of novel influenza A(H1N1) virus by real-time RT-PCR

Accurate and rapid diagnosis of novel influenza A(H1N1) infection is critical for minimising further spread through timely implementation of antiviral treatment and other public health based measures. In this study we developed two TaqMan-based reverse transcription PCR (RT-PCR) methods for the detection of novel influenza A(H1N1) virus targeting the haemagglutinin and neuraminidase genes. The assays were validated using 152 clinical respiratory samples, including 61 Influenza A positive samples, collected in Queenland, Australia during the years 2008 to 2009 and a further 12 seasonal H1N1 and H3N2 influenza A isolates collected from years 2000 to 2002. A wildtype swine H1N1 isolate was also tested. RNA from an influenza A(H1N1) virus isolate (Auckland, 2009) was used as a positive control. Overall, the results showed that the RT-PCR methods were suitable for sensitive and specific detection of novel influenza A(H1N1) RNA in human samples.

Presence of the newly discovered human polyomaviruses KI and WU in Australian patients with acute respiratory tract infection

Abstract Background Currently, the role of the novel human polyomaviruses, KI (KIV) and WU (WUV) as agents of human disease remains uncertain. Objectives We sought to determine the prevalence of these viruses and their rate of co-detection with other viral respiratory pathogens, in an Australian population. Study design Polymerase chain reaction assays previously described were used to examine the presence of KIV and WUV in 2866 respiratory specimens collected from January to December 2003 from Australian patients with acute respiratory infections. Results KIV and WUV were present in our population with an annual prevalence of 2.6% and 4.5%, respectively. There was no apparent seasonal variation for KIV, but a predominance of infection was detected during late winter to early summer for WUV. The level of co-infection of KIV or WUV with other respiratory viruses was 74.7% and 79.7%, respectively. Both viruses were absent from urine and blood specimens collected from a variety of patient sources. Conclusions KIV and WUV circulate annually in the Australian population. Although there is a strong association with the respiratory tract, more comprehensive studies are required to prove these viruses are agents causing respiratory disease.

Evaluation of the cobas 4800 CT/NG test for detecting Chlamydia trachomatis and Neisseria gonorrhoeae

Objectives To investigate the performance of the fully automated cobas 4800 CT/NG test for detection of Chlamydia trachomatis and Neisseria gonorrhoeae. Methods The study was conducted using 900 clinical specimens (496 urine and 404 swab specimens) for C trachomatis testing, of which 498 specimens (318 urine and 180 swab specimens) were also tested for N gonorrhoeae. The results of the cobas 4800 CT/NG test were compared with those obtained from the Roche COBAS AMPLICOR CT/NG and COBAS TaqMan CT assays. N gonorrhoeae-positive specimens were further tested using in-house, real-time PCR assays. A panel of 223 Neisseria isolates was used to further investigate the performance of the cobas 4800 N gonorrhoeae assay. Results For urine specimens, the sensitivity, specificity and negative and positive predictive values of the cobas 4800 CT/NG test were 94.5%, 99.5%, 98.8% and 97.7%, respectively, for C trachomatis, and 92.9%, 100%, 99.7% and 100%, respectively, for N gonorrhoeae. For swab specimens, the sensitivity, specificity and negative and positive predictive values were 92.0%, 100%, 99.5% and 100%, respectively, for C trachomatis, and 100%, 99.4%, 100% and 90.0%, respectively, for N gonorrhoeae. All N gonorrhoeae isolates were positive and all non-gonococcal Neisseria strains were negative by the cobas 4800 N gonorrhoeae assay. Conclusions The cobas 4800 CT/NG test is suitable for high through-put identification of C trachomatis and N gonorrhoeae infections.

A broad spectrum, one-step reverse-transcription PCR amplification of the neuraminidase gene from multiple subtypes of influenza A virus

BackgroundThe emergence of high pathogenicity strains of Influenza A virus in a variety of human and animal hosts, with wide geographic distribution, has highlighted the importance of rapid identification and subtyping of the virus for outbreak management and treatment. Type A virus can be classified into subtypes according to the viral envelope glycoproteins, hemagglutinin and neuraminidase. Here we review the existing specificity and amplification of published primers to subtype neuraminidase genes and describe a new broad spectrum primer pair that can detect all 9 neuraminidase subtypes.ResultsBioinformatic analysis of 3,337 full-length influenza A neuraminidase segments in the NCBI database revealed semi-conserved regions not previously targeted by primers. Two degenerate primers with M13 tags, NA8F-M13 and NA10R-M13 were designed from these regions and used to generate a 253 bp cDNA product. One-step RT-PCR testing was successful in 31/32 (97%) cases using a touchdown protocol with RNA from over 32 different cultured influenza A virus strains representing the 9 neuraminidase subtypes. Frozen blinded clinical nasopharyngeal aspirates were also assayed and were mostly of subtype N2. The region amplified was direct sequenced and then used in database searches to confirm the identity of the template RNA. The RT-PCR fragment generated includes one of the mutation sites related to oseltamivir resistance, H274Y.ConclusionOur one-step RT-PCR assay followed by sequencing is a rapid, accurate, and specific method for detection and subtyping of different neuraminidase subtypes from a range of host species and from different geographical locations.

Structure of the dengue virus glycoprotein non-structural protein 1 by electron microscopy and single-particle analysis

The flavivirus non-structural protein 1 (NS1) is a glycoprotein that is secreted as a soluble hexameric complex during the course of natural infection. Growing evidence indicates that this secreted form of NS1 (sNS1) plays a significant role in immune evasion and modulation during infection. Attempts to determine the crystal structure of NS1 have been unsuccessful to date and relatively little is known about the macromolecular organization of the sNS1 hexamer. Here, we have applied single-particle analysis to images of baculovirus-derived recombinant dengue 2 virus NS1 obtained by electron microscopy to determine its 3D structure to a resolution of 23 Å. This structure reveals a barrel-like organization of the three dimeric units that comprise the hexamer and provides further insights into the overall organization of oligomeric sNS1.

Production of the baculovirus-expressed dengue virus glycoprotein NS1 can be improved dramatically with optimised regimes for fed-batch cultures and the addition of the insect moulting hormone, 20-Hydroxyecdysone

A perennial problem in recombinant protein expression is low yield of the product of interest. A strategy which has been shown to increase the production of baculovirus-expressed proteins is to utilise fed-batch cultures. One disadvantage of this approach is the time-consuming task of optimising the feeding strategy. Previously, a statistical optimisation routine was applied to develop a feeding strategy that increased the yield of beta-Galactosidase (beta-Gal) by 2.4-fold (Biotechnol. Bioeng. 59 (1998) 178). This involves the single addition of nutrient concentrates (amino acids, lipids, glucose and yeastolate ultrafiltrate) into Sf 9 cell cultures grown in SF 900II medium. In this study, it is demonstrated that this optimised fed-batch strategy developed for a high-yielding intracellular product beta-Gal could be applied successfully to a relatively low-yielding glycosylated and secreted product such as the dengue virus glycoprotein NS1. Optimised batch infections yielded 4 microg/ml of NS1 at a peak cell density of 4.2 x 10 (6) cells/ml. In contrast, optimised fed-batch infections exhibited a 3-fold improvement in yield, with 12 microg/ml of NS1 produced at a peak cell density of 11.3 x 10 (6) cells/ml. No further improvements in yield were recorded when the feed volumes were doubled and the peak cell density was increased to 23 x 10 (6) cells/ml, unless the cultures were stimulated by the addition of 4 microg/ml of 20-Hydroxyecdysone (an insect moulting hormone). In this case, the NS1 yield was increased to 20 microg/ml, which was nearly 5-fold higher than optimised batch cultures.

Screening for H7N9 influenza A by matrix gene-based real-time reverse-transcription PCR.

Rapid detection of novel influenza A strains, including H7N9, is pivotal to ensuring appropriate public health-based responses and real-time reverse-transcription polymerase chain reaction (RT-PCR) methods are used typically for this purpose. However, the utility of such methods can be undermined by ongoing sequence variations, particularly when targeting the variable influenza A haemagglutinin (HA) and neuraminidase (NA) genes. This may often be a source of frustration for clinical laboratories that are implementing methods in preparation for potential pandemics as primers and probe targets may need to be checked regularly and updated. In this study, screening methods were developed for H7N9 influenza A strains based on the highly-conserved influenza A matrix gene. Three assays were developed and evaluated in parallel, and included two methods which simply involved inclusion of a single H7N9 probe sequence into an established influenza A and B multiplex RT-PCR (FluAB-PCR). The detection limits of the methods were compared using ten-fold dilutions of H7N9 RNA, and the specificity of the methods were tested using 32 influenza A RT-PCR-positive samples and a panel of 18 influenza A isolates, including representives of seasonal H3N2, seasonal H1N1, pandemic H1N1, H5N1, H5N3, H9N2 and H7N7. The detection limits of the three methods were the same, and no cross-reactions were observed with sH3N2, sH1N1, pH1N1 or H5N1. However, cross-reactions were observed with H5N3, H9N2 and H7N7. Overall, the results show that the methods are useful for front-line screening for H7N9.

Respiratory syncytial virus infection is associated with increased bacterial load in the upper respiratory tract in young children

Background: Respiratory syncytial virus (RSV) is the leading cause of severe respiratory tract infection requiring hospitalization among infants and young children. Furthermore, RSV infection has been shown to stimulate increased bacterial load in the nasopharynx and to promote bacterial otitis media and/or pneumonia. Objective: To assess the diversity and prevalence of bacterial pathogens associated with viral infections of the respiratory tract among young children. Study design: During the 2012 seasonal RSV epidemic in South East Queensland, Australia, 201 nasopharyngeal aspirates (NPAs) collected from symptomatic children under five years of age and sent for pathology laboratory screening were examined. Samples were analysed for common viral and bacterial pathogens by quantitative PCR and culturing techniques. For a subset of 29 samples the complete microbial community was profiled using cultureindependent PCR and pyrosequencing of the 16S ribosomal RNA gene. Results: RSV infection was confirmed in 67 patients, of which 49 were positive for high bacterial load in the upper respiratory tract. Bacterial detection was significantly higher amongst RSV positive samples (73.1%) than amongst RSV negative samples (56.7%) (p=0.03) and was independent of other viral pathogens. The predominant bacterial species detected during RSV infections were Moraxella catarrhalis (22), Streptococcus pneumoniae (17), Haemophilus influenzae (5) and Staphylococcus aureus (6). Notably, microbial profiling analysis showed that during either RSV or rhinovirus infections a single bacterial species can constitute between 80 and 95% of the bacterial community present. Conclusions: In nasopharyngeal samples collected from symptomatic children, high levels of bacteria were found more commonly in the presence of RSV infections. Most significantly, RSV infection was associated with a 3-fold increase in S. pneumoniae detection.

Improved detection of genetic markers of antimicrobial resistance by hybridization probe-based melting curve analysis using primers to mask proximal mutations: Examples include the influenza H275Y substitution

OBJECTIVES Numerous real-time PCR assays have been described for detection of the influenza A H275Y alteration. However, the performance of these methods can be undermined by sequence variation in the regions flanking the codon of interest. This is a problem encountered more broadly in microbial diagnostics. METHODS In this study, we developed a modification of hybridization probe-based melting curve analysis, whereby primers are used to mask proximal mutations in the sequence targets of hybridization probes, so as to limit the potential for sequence variation to interfere with typing. The approach was applied to the H275Y alteration of the influenza A (H1N1) 2009 strain, as well as a Neisseria gonorrhoeae mutation associated with antimicrobial resistance. Assay performances were assessed using influenza A and N. gonorrhoeae strains characterized by DNA sequencing. RESULTS The modified hybridization probe-based approach proved successful in limiting the effects of proximal mutations, with the results of melting curve analyses being 100% consistent with the results of DNA sequencing for all influenza A and N. gonorrhoeae strains tested. Notably, these included influenza A and N. gonorrhoeae strains exhibiting additional mutations in hybridization probe targets. Of particular interest was that the H275Y assay correctly typed influenza A strains harbouring a T822C nucleotide substitution, previously shown to interfere with H275Y typing methods. CONCLUSIONS Overall our modified hybridization probe-based approach provides a simple means of circumventing problems caused by sequence variation, and offers improved detection of the influenza A H275Y alteration and potentially other resistance mechanisms.