David M. Whiley

Molecular Assays for Detection of Human Metapneumovirus

ABSTRACT The recent description of the respiratory pathogen human metapneumovirus (hMPV) has highlighted a deficiency in current diagnostic techniques for viral agents associated with acute lower respiratory tract infections. We describe two novel approaches to the detection of viral RNA by use of reverse transcriptase PCR (RT-PCR). The PCR products were identified after capture onto a solid-phase medium by hybridization with a sequence-specific, biotinylated oligonucleotide probe. The assay was applied to the screening of 329 nasopharyngeal aspirates sampled from patients suffering from respiratory tract disease. These samples were negative for other common microbial causes of respiratory tract disease. We were able to detect hMPV sequences in 32 (9.7%) samples collected from Australian patients during 2001. To further reduce result turnaround times we designed a fluorogenic TaqMan oligoprobe and combined it with the existing primers for use on the LightCycler platform. The real-time RT-PCR proved to be highly reproducible and detected hMPV in an additional 6 out of 62 samples (9.6%) tested during the comparison of the two diagnostic approaches. We found the real-time RT-PCR to be the test of choice for future investigation of samples for hMPV due to its speed, reproducibility, specificity, and sensitivity.

A Sensitive, Specific, and Cost-Effective Multiplex Reverse Transcriptase-PCR Assay for the Detection of Seven Common Respiratory Viruses in Respiratory Samples

Cell culture and direct fluorescent antibody (DFA) assays have been traditionally used for the laboratory diagnosis of respiratory viral infections. Multiplex reverse transcriptase polymerase chain reaction (m-RT-PCR) is a sensitive, specific, and rapid method for detecting several DNA and RNA viruses in a single specimen. We developed a m-RT-PCR assay that utilizes multiple virus-specific primer pairs in a single reaction mix combined with an enzyme-linked amplicon hybridization assay (ELAHA) using virus-specific probes targeting unique gene sequences for each virus. Using this m-RT-PCR-ELAHA, we examined the presence of seven respiratory viruses in 598 nasopharyngeal aspirate (NPA) samples from patients with suspected respiratory infection. The specificity of each assay was 100%. The sensitivity of the DFA was 79.7% and the combined DFA/culture amplified-DFA (CA-DFA) was 88.6% when compared to the m-RT-PCR-ELAHA. Of the 598 NPA specimens screened by m-RT-PCR-ELAHA, 3% were positive for adenovirus (ADV), 2% for influenza A (Flu A) virus, 0.3% for influenza B (Flu B) virus, 1% for parainfluenza type 1 virus (PIV1), 1% for parainfluenza type 2 virus (PIV2), 5.5% for parainfluenza type 3 virus (PIV3), and 21% for respiratory syncytial virus (RSV). The enhanced sensitivity, specificity, rapid result turnaround time and reduced expense of the m-RT-PCR-ELAHA compared to DFA and CA-DFA, suggests that this assay would be a significant improvement over traditional assays for the detection of respiratory viruses in a clinical laboratory.

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.

Two cases of failed ceftriaxone treatment in pharyngeal gonorrhoea verified by molecular microbiological methods.

Diagnostic, genotypic and antibiotic-resistance determinants of Neisseria gonorrhoeae were analysed by molecular methods to verify the failure of ceftriaxone treatment in two cases of pharyngeal gonorrhoea. Monoplex assays were needed to define competitive inhibition of a positive Chlamydia PCR in a duplex assay. Different penA changes were detected in the N. gonorrhoeae isolated from the two cases. These were associated with raised ceftriaxone MICs of 0.03 and 0.016 mg l(-1), which may have contributed to the treatment failures in these cases.

Diversity of penA Alterations and Subtypes in Neisseria gonorrhoeae Strains from Sydney, Australia, That Are Less Susceptible to Ceftriaxone

ABSTRACT Increasing numbers of Neisseria gonorrhoeae strains with decreased susceptibilities to ceftriaxone and other oral cephalosporins widely used for the treatment of gonorrhea have been isolated in Sydney, Australia, over several years. In this study, we examined the complete penicillin-binding protein 2 (PBP 2) amino acid sequences of 109 gonococci, selected on the basis of their diverse temporal and geographic origins and because they exhibited a range of ceftriaxone MICs: ≤0.03 μg/ml (n = 59), 0.06 μg/ml (n = 43), and 0.125 μg/ml (n = 7). Auxotyping, serotyping, and genotyping by N. gonorrhoeae multiantigen sequence typing sequence-based analysis was also performed. In total, 20 different amino acid sequence patterns were identified, indicating considerable variation in the PBP 2 sequences in this study sample. Only some of the N. gonorrhoeae isolates with significantly higher ceftriaxone MICs contained a mosaic PBP 2 pattern, while more isolates exhibited a nonmosaic PBP 2 pattern containing an A501V substitution. Although particular N. gonorrhoeae genotypes in our sample were shown to be less susceptible to ceftriaxone, the reduced susceptibility to ceftriaxone was not specific to any particular genotype and was observed in a broad range of auxotypes, serotypes, and genotypes. Overall, the results of our study show that N. gonorrhoeae strains exhibiting reduced sensitivity to ceftriaxone are not of a particular subtype and that a number of different mutations in PBP 2 may contribute to this phenomenon.

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.

Detection and Differentiation of Human Polyomaviruses JC and BK by LightCycler PCR

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.

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.

Emerging respiratory agents: New viruses for old diseases?

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 Kochs 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.

Merkel Cell Polyomavirus DNA in Respiratory Specimens from Children and Adults

Merkel cell polyomavirus (MCPyV) DNA was detected in 7 (1.3%) of 526 respiratory tract samples from patients in Australia with upper or lower respiratory tract symptoms. Partial T antigen and major capsid protein sequences of MCPyV identified in respiratory secretions showed high homology (99%–100%) to those found in Merkel cell carcinoma.