D M Whiley

Publications (22)52.59 Total impact

• Article: Comparison of a multiplexed MassARRAY system with real-time allele-specific PCR technology for genotyping of methicillin-resistant Staphylococcus aureus.

  M W Syrmis, R J Moser, D M Whiley, V Vaska, G W Coombs, M D Nissen, T P Sloots, G R Nimmo
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  ABSTRACT: The Sequenom MassARRAY iPLEX single-nucleotide polymorphism (SNP) typing platform uses matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) coupled with single-base extension PCR for high-throughput multiplex SNP detection. In this study, we investigated the use of iPLEX MassARRAY technology for methicillin-resistant Staphylococcus aureus (MRSA) genotyping. A 16-plex MassARRAY iPLEX GOLD assay (MRSA-iPLEX) was developed that targets a set of informative SNPs and binary genes for MRSA characterization. The method was evaluated with 147 MRSA isolates, and the results were compared with those of an established SYBR Green-based real-time PCR system utilizing the same SNP-binary markers. A total of 2352 markers belonging to 44 SNP-binary profiles were analysed by both real-time PCR and MRSA-iPLEX. With real-time PCR as the reference standard, MRSA-iPLEX correctly assigned 2298 of the 2352 (97.7%) markers. Sequence variation in the MRSA-iPLEX primer targets accounted for the majority of MRSA-iPLEX erroneous results, highlighting the importance of primer target selection. MRSA-iPLEX provided optimal throughput for MRSA genotyping, and was, on a reagent basis, more cost-effective than the real-time PCR methods. The 16-plex MRSA-iPLEX is a suitable alternative to SYBR Green-based real-time PCR typing of major sequence types and clonal complexes of MRSA.
  Clinical Microbiology and Infection 03/2011; 17(12):1804-10. · 4.54 Impact Factor
• Article: Rapid single-nucleotide polymorphism-based identification of clonal Pseudomonas aeruginosa isolates from patients with cystic fibrosis by the use of real-time PCR and high-resolution melting curve analysis.

  S N Anuj, D M Whiley, T J Kidd, K A Ramsay, S C Bell, M W Syrmis, K Grimwood, C E Wainwright, M D Nissen, T P Sloots
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  ABSTRACT: Pseudomonas aeruginosa genotyping relies mainly upon DNA fingerprinting methods, which can be subjective, expensive and time-consuming. The detection of at least three different clonal P. aeruginosa strains in patients attending two cystic fibrosis (CF) centres in a single Australian city prompted the design of a non-gel-based PCR method to enable clinical microbiology laboratories to readily identify these clonal strains. We designed a detection method utilizing heat-denatured P. aeruginosa isolates and a ten-single-nucleotide polymorphism (SNP) profile. Strain differences were detected by SYBR Green-based real-time PCR and high-resolution melting curve analysis (HRM10SNP assay). Overall, 106 P. aeruginosa sputum isolates collected from 74 patients with CF, as well as five reference strains, were analysed with the HRM10SNP assay, and the results were compared with those obtained by pulsed-field gel electrophoresis (PFGE). The HRM10SNP assay accurately identified all 45 isolates as members of one of the three major clonal strains characterized by PFGE in two Brisbane CF centres (Australian epidemic strain-1, Australian epidemic strain-2 and P42) from 61 other P. aeruginosa strains from Australian CF patients and two representative overseas epidemic strain isolates. The HRM10SNP method is simple, is relatively inexpensive and can be completed in <3 h. In our setting, it could be made easily available for clinical microbiology laboratories to screen for local P. aeruginosa strains and to guide infection control policies. Further studies are needed to determine whether the HRM10SNP assay can also be modified to detect additional clonal strains that are prevalent in other CF centres.
  Clinical Microbiology and Infection 12/2010; 17(9):1403-8. · 4.54 Impact Factor
• Article: Emerging respiratory agents: new viruses for old diseases?

  T P Sloots, D M Whiley, S B Lambert, M D Nissen
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  ABSTRACT: The recent advances in molecular technology have enabled the detection of several new viral agents in specimens collected from the human respiratory tract. Human metapneumovirus was first described in 2001, and is a significant respiratory pathogen, particularly of children. Following the identification of severe acute respiratory syndrome (SARS) associated coronavirus, two other newly detected coronaviruses, NL63 and HKU1, have been linked to respiratory disease in humans. However, identifying a new virus as the causative agent of a specific disease is difficult, and ideally would involve satisfying Koch's postulates. The recently described human bocavirus and polyomaviruses KI and WU have been detected in samples collected from humans with acute respiratory infection, but as yet, have not been conclusively proven to be agents of human disease. We review the new viral agents that have been detected in respiratory samples since 2001, and examine their contribution as agents of human disease.
  Journal of Clinical Virology 08/2008; 42(3):233-43. · 3.97 Impact Factor
• Article: Exploring 'best practice' for nucleic acid detection of Neisseria gonorrhoeae

  D. M. Whiley, S. M. Garland, G. Harnett, G. Lum, D. W. Smith, S. N. Tabrizi, T. P. Sloots, J. W. Tapsall
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  ABSTRACT: Nucleic acid detection tests (NADT) have considerable benefits for the detection of Neisseria gonorrhoeae (GC), including high sensitivity across a range of specimen types and use under widely differing settings and conditions. However, sexual health practitioners and others who use data generated by NADT for GC should be aware of some important limitations of these tests. False-positive results caused by cross reaction with commensal Neisseria species have been observed in many assays, and have lead to unacceptably low positive-predictive values in some patient populations. Further, false-negative results can be caused by GC sequence variation, with some gonococci lacking certain NADT target sequences. This review examines the issues associated with gonococcal NADT and considers best practice for use of these assays based on current knowledge. We emphasise the need for supplementary testing and extensive assay validation, and suggest appropriate strategies for these requirements irrespective of the setting in which they are used. Further, we highlight the need to maintain culture-based testing for certain specimen sites as well as for antimicrobial resistance surveillance.
  Sexual Health 04/2008; 5(1):17-23. · 1.45 Impact Factor
• Source

  Available from: Seweryn Bialasiewicz

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

  S Bialasiewicz, D M Whiley, S B Lambert, K Jacob, C Bletchly, D Wang, M D Nissen, T P Sloots
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  ABSTRACT: Currently, the role of the novel human polyomaviruses, KI (KIV) and WU (WUV) as agents of human disease remains uncertain. We sought to determine the prevalence of these viruses and their rate of co-detection with other viral respiratory pathogens, in an Australian population. 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. 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. 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.
  Journal of Clinical Virology 03/2008; 41(2):63-8. · 3.97 Impact Factor
• Source

  Available from: Seweryn Bialasiewicz

  Article: False-negative results in nucleic acid amplification tests-do we need to routinely use two genetic targets in all assays to overcome problems caused by sequence variation?

  D M Whiley, S B Lambert, S Bialasiewicz, N Goire, M D Nissen, T P Sloots
  [show abstract] [hide abstract]
  ABSTRACT: Nucleic acid amplification tests (NAATs) have numerous advantages over traditional diagnostic techniques and so are now widely used by diagnostic laboratories for routine detection of infectious agents. However, there is some concern over the increasing numbers of reports of NAAT false-negative results caused by sequence variation. Highly conserved NAAT target sequences have been reported for many organisms, yet sequence-related problems continue to be observed in commercial and in-house assays targeting a broad range of microbial pathogens. In light of these ongoing problems, it may be time to consider the use of two genetic targets in NAAT methods to reduce the potential for sequence-related false-negative results.
  Critical Reviews in Microbiology 02/2008; 34(2):71-6. · 6.27 Impact Factor
• Source

  Available from: Seweryn Bialasiewicz

  Article: Development and evaluation of real-time PCR assays for the detection of the newly identified KI and WU polyomaviruses.

  S Bialasiewicz, D M Whiley, S B Lambert, A Gould, M D Nissen, T P Sloots
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  ABSTRACT: Recently, novel human polyomaviruses, KI (KIV) and WU (WUV) were described. Their role in human disease has not yet been determined. The aim of this study was to develop sensitive and specific assays for the detection of KIV and WUV. Two KIV (KI-A and KI-B) and three WUV (WU-A, WU-B and WU-C) real-time polymerase chain reaction (rtPCR) assays were developed and evaluated. Clinical sensitivities and specificities were determined by testing 200 respiratory specimens and the results compared to those for previously described conventional PCR assays. Limits of detection were determined, and the analytical specificities of the assays were investigated. No cross-reactivity was observed between the rtPCR methods and unrelated organisms. All five rtPCR assays could reliably detect 10 copies of genomic DNA equivalents per reaction, which was more sensitive than conventional methods. Compared to the conventional PCR assays, the sensitivity of the KI-A, KI-B, WU-A, WU-B and WU-C assays was 100%, 86.7% 95.5%, 100% and 100%, respectively. Specificity was 94.6%, 97.3%, 96.6%, 97.7% and 97.2%, respectively. The KI-A, WU-B and WU-C assays provide the most sensitive detection of KIV and WUV in clinical specimens and may be used for further research into these viruses.
  Journal of Clinical Virology 10/2007; 40(1):9-14. · 3.97 Impact Factor
• Article: Supplemental testing is still required in australia for samples positive for Neisseria gonorrhoeae by nucleic acid detection tests.

  G Lum, S M Garland, S Tabrizi, G Harnett, D W Smith, T P Sloots, D M Whiley, J W Tapsall
  Journal of Clinical Microbiology 12/2006; 44(11):4292-4; author reply 4293-4. · 4.15 Impact Factor
• Source

  Available from: Sepehr Tabrizi

  Article: A cluster of culture positive gonococcal infections but with false negative cppB gene based PCR.

  G Lum, K Freeman, N L Nguyen, E A Limnios, S N Tabrizi, I Carter, I W Chambers, D M Whiley, T P Sloots, S M Garland, J W Tapsall
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  ABSTRACT: To describe the prevalence and characteristics of isolates of Neisseria gonorrhoeae grown from urine samples that produced negative results with nucleic acid amplification assays (NAA) targeting the cppB gene. An initial cluster of culture positive, but cppB gene based NAA negative, gonococcal infections was recognised. Urine samples and suspensions of gonococci isolated over 9 months in the Northern Territory of Australia were examined using cppB gene based and other non-cppB gene based NAA. The gonococcal isolates were phenotyped by determining the auxotype/serovar (A/S) class and genotyped by pulsed field gel electrophoresis (PFGE). 14 (9.8%) of 143 gonococci isolated were of A/S class Pro(-/)Brpyut, indistinguishable on PFGE and negative in cppB gene based, but not other, NAA. This cluster represents a temporal and geographic expansion of a gonococcal subtype lacking the cppB gene with consequent loss of sensitivity of NAA dependent on amplification of this target. Gonococci lacking the cppB gene have in the past been more commonly associated with the PAU-/PCU- auxotype, a gonococcal subtype hitherto infrequently encountered in Australia. NAA based on the cppB gene as a target may produce false positive as well as false negative NAA. This suggests that unless there is continuing comparison with culture to show their utility, cppB gene based NAA should be regarded as suboptimal for use either as a diagnostic or supplemental assay for diagnosis of gonorrhoea, and NAA with alternative amplification targets should be substituted.
  Sexually Transmitted Infections 11/2005; 81(5):400-2. · 2.85 Impact Factor
• Article: Cryptic-plasmid-free gonococci may contribute to failure of cppB gene-based assays to confirm results of BD ProbeTEC PCR for identification of Neisseria gonorrhoeae.

  J W Tapsall, E A Limnios, N L Nguyen, I Carter, G Lum, K Freeman, S N Tabrizi, S M Garland, D M Whiley, T P Sloots, I W Chambers
  Journal of Clinical Microbiology 05/2005; 43(4):2036-7. · 4.15 Impact Factor
• Article: A new confirmatory Neisseria gonorrhoeae real-time PCR assay targeting the porA pseudogene.

  D M Whiley, P J Buda, J Bayliss, L Cover, J Bates, T P Sloots
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  ABSTRACT: The Roche Cobas Amplicor system is widely used for the detection of Neisseria gonorrhoeae but is known to cross react with some commensal Neisseria spp. Therefore, a confirmatory test is required. The most common target for confirmatory tests is the cppB gene of N. gonorrhoeae. However, the cppB gene is also present in other Neisseria spp. and is absent in some N. gonorrhoeae isolates. As a result, laboratories targeting this gene run the risk of obtaining both false-positive and false-negative results. In the study presented here, a newly developed N. gonorrhoeae LightCycler assay (NGpapLC) targeting the N. gonorrhoeae porA pseudogene was tested. The NGpapLC assay was used to test 282 clinical samples, and the results were compared to those obtained using a testing algorithm combining the Cobas Amplicor System (Roche Diagnostics, Sydney, Australia) and an in-house LightCycler assay targeting the cppB gene (cppB-LC). In addition, the specificity of the NGpapLC assay was investigated by testing a broad panel of bacteria including isolates of several Neisseria spp. The NGpapLC assay proved to have comparable clinical sensitivity to the cppB-LC assay. In addition, testing of the bacterial panel showed the NGpapLC assay to be highly specific for N. gonorrhoeae DNA. The results of this study show the NGpapLC assay is a suitable alternative to the cppB-LC assay for confirmation of N. gonorrhoeae-positive results obtained with Cobas Amplicor.
  European Journal of Clinical Microbiology 10/2004; 23(9):705-10. · 2.86 Impact Factor
• Article: Preliminary comparison of three LightCycler PCR assays for the detection of herpes simplex virus in swab specimens.

  D M Whiley, M W Syrmis, I M Mackay, T P Sloots
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  ABSTRACT: Three Herpes Simplex Virus LightCycler polymerase chain reaction assays were compared for the detection of herpes simplex virus in 48 swab specimens. The assays comprised of one in-house assay and two commercial kits: the Artus HSV LC RealArt PCR kit and the Roche LightCycler HSV 1/2 Detection kit. On the whole, the three assays had comparable sensitivities. However, differentiation of herpes simplex virus types 1 and 2 by melting curve analysis was problematic in all assays. Overall, the results highlight the limitations of typing herpes simplex virus by melting curve analysis.
  European Journal of Clinical Microbiology 01/2004; 22(12):764-7. · 2.86 Impact Factor
• Article: Preliminary Comparison of Three LightCycler PCR Assays for the Detection of Herpes Simplex Virus in Swab Specimens

  D.M. Whiley, M.W. Syrmis, I.M. Mackay, T.P. Sloots
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  ABSTRACT: Three Herpes Simplex Virus LightCycler polymerase chain reaction assays were compared for the detection of herpes simplex virus in 48 swab specimens. The assays comprised of one in-house assay and two commercial kits: the Artus HSV LC RealArt PCR kit and the Roche LightCycler HSV 1/2 Detection kit. On the whole, the three assays had comparable sensitivities. However, differentiation of herpes simplex virus types 1 and 2 by melting curve analysis was problematic in all assays. Overall, the results highlight the limitations of typing herpes simplex virus by melting curve analysis.
  European Journal of Clinical Microbiology 11/2003; 22(12):764-767. · 2.86 Impact Factor
• Source

  Available from: Theodorus P. Sloots

  Article: Detection and differentiation of human polyomaviruses JC and BK by LightCycler PCR.

  D M Whiley, I M Mackay, T P Sloots
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  ABSTRACT: Human polyomaviruses JC and BK may cause several clinical manifestations in immunocompromised hosts, including progressive multifocal leukoencephalopathy and hemorrhagic cystitis. Molecular detection by PCR is recognized as a sensitive and specific method for detecting human polyomaviruses in clinical samples. In this study, a real-time PCR assay using the LightCycler platform was evaluated and compared to an "in-house" PCR assay using a conventional detection method. A total of 122 urine specimens were tested, and human polyomavirus was detected in 49 specimens (40%) by both conventional PCR and LightCycler PCR. The remaining 73 specimens (60%) were found negative by both assays. For 46 of the 49 positive specimens, LightCycler PCR and conventional PCR identified the same polyomavirus type. These samples included 30 samples with JC virus (JCV), 14 samples with BK virus (BKV), and 2 samples in which both viruses were detected. In the remaining three samples, both JCV and BKV were detected by the conventional assay, but only JCV was detected by the LightCycler assay. The results of this study show that the LightCycler PCR assay displays sensitivity and specificity similar to those of a conventional PCR assay. These data, combined with its rapid turnaround time for results and decreased hands-on time, make the LightCycler PCR assay highly suitable for the rapid detection and differentiation of JCV and BKV in the clinical laboratory.
  Journal of Clinical Microbiology 01/2002; 39(12):4357-61. · 4.15 Impact Factor
• Article: Clinical Virology

  D M Whiley, T P Sloots
• Article: Pseudomonas

  T. J. Kidd, D M Whiley, S C Bell, K Grimwood
• Article: Detection of Neisseria Meningitidis in clinical samples by a duplex real-time PCR targeting the porA and ctrA genes

  D M Whiley, M. E. Crisante, M. E. Syrmis, I. M. Mackay, T P Sloots
• Article: Detection of Influenza type A virus by LightCycler RT-PCR

  D M Whiley, M W Syrmis, I. M. Mackay, T P Sloots
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  ABSTRACT: Using the Roche LightCycler we developed a real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay using the Influenza A LightCycler RT-PCR (FA-LC-RTPCR) for the rapid detection of Influenza A. The assay was used to examine 178 nasopharyngeal aspirate (NPA) samples, from patients with clinically recognised respiratory tract infection, for the presence of Influenza A RNA. The results were then compared to a testing algorithm combining direct immunofluorescent assy (DFA) and a culture augmented DFA (CA-DFA) assay. In total, 76 (43%) specimens were positive and 98 (55%) specimens were negative by both the FA-LC-RTPCR and the DFA and CA-DFA algorithm. In addition, the FA-LC-RTPCR detected a further 4 (2%) positive specimens, which were confirmed by a conventional RT-PCR method. The high level of sensitivity and specificity, combined with the rapid turnaround time for results, makes the LC-RT-PCR assay suitable for the detection of Influenza A in clinical specimens.
• Article: A cluster of culture positive gonococcal infections but with false negative cppB gene based PCR

  G Lum, K Freeman, N L Nguyen, E A Limnios, S. M. Tabrizi, I Carter, I W Chambers, T P Sloots, D M Whiley, S M Garland, J Tapsall
• Article: False-Negative results in nucleic acid amplification tests: do we need to routinely use two genetic targets in all assays to overcome problems caused by sequence variation?

  D M Whiley, S. Lambert, S Bialasiewicz, N. Goire, M D Nissen, T P Sloots
  [show abstract] [hide abstract]
  ABSTRACT: Nucleic acid amplification tests (NAATs) have numerous advantages over traditional diagnostic techniques and so are now widely used by diagnostic laboratories for routine detection of infectious agents. However, there is some concern over the increasing numbers of reports of NAAT false-negative results caused by sequence variation. Highly conserved NAAT target sequences have been reported for many organisms, yet sequence-related problems continue to be observed in commercial and in-house assays targeting a broad range of microbial pathogens. In light of these ongoing problems, it may be time to consider the use of two genetic targets in NAAT methods to reduce the potential for sequence-related false-negative results.