Potential animal and environmental sources of Q fever infection for humans in Queensland.

Q fever is a vaccine-preventable disease; despite this, high annual notification numbers are still recorded in Australia. We have previously shown seroprevalence in Queensland metropolitan regions is approaching that of rural areas. This study investigated the presence of nucleic acid from Coxiella burnetii, the agent responsible for Q fever, in a number of animal and environmental samples collected throughout Queensland, to identify potential sources of human infection. Samples were collected from 129 geographical locations and included urine, faeces and whole blood from 22 different animal species; 45 ticks were removed from two species, canines and possums; 151 soil samples; 72 atmospheric dust samples collected from two locations and 50 dust swabs collected from domestic vacuum cleaners. PCR testing was performed targeting the IS1111 and COM1 genes for the specific detection of C. burnetii DNA. There were 85 detections from 1318 animal samples, giving a detection rate for each sample type ranging from 2.1 to 6.8%. Equine samples produced a detection rate of 11.9%, whilst feline and canine samples showed detection rates of 7.8% and 5.2%, respectively. Native animals had varying detection rates: pooled urines from flying foxes had 7.8%, whilst koalas had 5.1%, and 6.7% of ticks screened were positive. The soil and dust samples showed the presence of C. burnetii DNA ranging from 2.0 to 6.9%, respectively. These data show that specimens from a variety of animal species and the general environment provide a number of potential sources for C. burnetii infections of humans living in Queensland. These previously unrecognized sources may account for the high seroprevalence rates seen in putative low-risk communities, including Q fever patients with no direct animal contact and those subjects living in a low-risk urban environment.

Polymerase chain reaction-based screening for the ceftriaxone-resistant Neisseria gonorrhoeae F89 strain.

Emergence and spread of Neisseria gonorrhoeae resistant to extended spectrum cephalosporins is a major problem threatening treatment of gonorrhoea and is further highlighted by the recent report of a second ceftriaxone-resistant N. gonorrhoeae strain (F89) in Europe, initially observed in France and subsequently identified in Spain. N. gonorrhoeae antimicrobial resistance (AMR) surveillance has acquired new importance and molecular tools have the potential to enhance bacterial culture-based methods. In this study, we established a polymerase chain reaction (PCR) protocol for direct detection of the F89 strain. A key component of this screening protocol was the development of a hybridisation probe-based melting curve analysis assay (mosaic501-hybPCR) to detect the presence of an A501P substitution on the N. gonorrhoeae mosaic penicillin binding protein 2 (PBP2) sequence, an important characteristic of the F89 strain. The mosaic501-hybPCR was evaluated using plasmid-derived positive controls (n=3) and characterised gonococcal (n=33) and non-gonococcal (n=58) isolates. The protocol was then applied to 159 clinical specimens from Sydney, Australia, collected during the first half of the year 2012 that were N. gonorrhoeae PCR-positive. Overall, the results indicate that the PCR-based protocol is suitable for direct detection of the N. gonorrhoeae F89 strain in non-cultured clinical samples. It therefore provides an additional tool to aid investigations into the potential spread of F89 strain throughout Europe and elsewhere.

A bivalent Neisseria meningitidis recombinant lipidated factor H binding protein vaccine in young adults: results of a randomised, controlled, dose-escalation phase 1 trial.

Neisseria meningitidis is a leading cause of meningitis and septicaemia, but a broadly-protective vaccine against endemic serogroup B disease is not licensed and available. The conserved, outer-membrane lipoprotein factor H binding protein (fHBP, also known as LP2086) is expressed as one of two subfamily variants in virtually all meningococci. This study investigated the safety, tolerability, and immunogenicity of a recombinant-expressed bivalent fHBP (r-fHBP) vaccine in healthy adults. Participants (N=103) aged 18-25 years were recruited into three ascending dose level cohorts of 20, 60, and 200µg of a bivalent r-fHBP vaccine formulation and randomised to receive vaccine or placebo at 0, 1, and 6 months. The vaccine was well tolerated. Geometric mean titres (GMTs) for r-fHBP subfamily-specific IgG antibodies increased 19-168-fold from pre-vaccination to post-dose 2 in a dose level-dependent manner. In addition, robust serum bactericidal assay using human complement (hSBA) responses for strains expressing both homologous and heterologous fHBP variants were observed. After three vaccinations, 16-52% of the placebo group and 47-90%, 75-100%, and 88-100%, of the 20, 60, and 200µg dose levels, respectively, had seroprotective (≥ 1:4) hSBA titres against six serogroup B strains. The bivalent r-fHBP vaccine was well tolerated and induced robust bactericidal activity against six diverse serogroup B strains in young adults at the 60 and 200µg dose levels.

False-negative results using Neisseria gonorrhoeae porA pseudogene PCR - a clinical gonococcal isolate with an N. meningitidis porA sequence, Australia, March 2011.

The gonococcal porA pseudogene is a popular target for in-house Neisseria gonorrhoeae PCR methods. With this study we present two novel findings: the first case of an N. gonorrhoeae porA pseudogene PCR false-negative result caused by sequence variation, and in the same organism, the first description of a clinical N. gonorrhoeae strain harbouring an N. meningitidis porA sequence.

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

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.

Evaluation of a clinical scoring system and directed laboratory testing for respiratory virus infection in hematopoietic stem cell transplant recipients.

A simple clinical screening (CS) tool for respiratory virus (RV) infection was introduced and evaluated in a single hematology ward, as part of a strategy to reduce nosocomial RV infection. Up to 6 clinical symptoms or signs were scored and a predefined threshold score of ≥ 2 prompted paired nose/throat swab (NTS) collection for RV testing. The criterion standard for RV infection was positive immunofluorescence (IF) or polymerase chain reaction (PCR) for 7 and 15 viruses, respectively. The tool was shown to be most beneficial at excluding infection at a threshold score of 1 (negative predictive value [NPV] 89%, [95% confidence interval 78-96%], sensitivity 85% [70-94%], specificity 35% [27-43%]), compared with a score of 2 (NPV 85% [76-91%], sensitivity 63% [46-77%], specificity 57% [48-65%]) at a prevalence of 22%. The tool's ability to diagnose infection was limited (positive predictive value 27% and 29% at thresholds 1 and 2). The sensitivity of IF compared with PCR was 45% for the 7 viruses common to both, and 23% for the extended virus panel detected by PCR. An algorithm incorporating CS, paired NTS collection at a threshold of 1 symptom or sign, and sensitive testing including PCR can guide infection control measures in hospitalized hematopoietic stem cell transplant recipients.

Q fever seroprevalence in metropolitan samples is similar to rural/remote samples in Queensland, Australia.

Q fever is a vaccine preventable disease; however, despite this, high notification numbers are still recorded annually in Australia. We investigated the seroprevalence of Coxiella burnetii, the Q fever agent, in a Queensland sample population. Notification data (N = 6425) from 1984-2008 were collated, identifying high risk areas of Q fever exposure. Of these 177 were recorded in children. Serum samples were collected from Queensland and screened using both an immunoflourescence assay at 1:10 dilution and a commercially available ELISA kit. Results were collated based on age, geographical location and sex. From 1988 Queensland samples screened, 103 were identified as Q fever IgG-positive, giving a seroprevalence of 5.2% (95% CI 4.3-6.2%). Seroprevalence in the rural/remote population was 5.3% (95% CI 4.6-6.6%), and the metropolitan Brisbane population, which is considered not at risk, was 5.0% (95% CI 3.7-6.7%). Sixty-three seropositive males and 40 females were identified, along with an increase in seropositivity with increasing age. The seropositivity of children was 1.3% (95% CI 0.7-2.3%) from 844 samples. We have shown that both metropolitan and paediatric populations which are considered low risk of Coxiella exposure have surprisingly high seropositivity. These emerging groups require further investigation and consideration for the introduction of preventive measures.

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.

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.

Molecular characterization and distinguishing features of a novel human rhinovirus (HRV) C, HRVC-QCE, detected in children with fever, cough and wheeze during 2003.

BACKGROUND: Human rhinoviruses (HRVs) are associated with more acute respiratory tract infections than any other viral group yet we know little about viral diversity, epidemiology or clinical outcome resulting from infection by strains, in particular the recently identified HRVs. OBJECTIVES: To determine whether HRVC-QCE was a distinct HRV-C strain, by determining its genome and prevalence, by cataloguing genomic features for strain discrimination and by observing clinical features in positive patients. STUDY DESIGN: Novel real-time RT-PCRs and retrospective chart reviews were used to investigate a well-defined population of 1247 specimen extracts to observe the prevalence and the clinical features of each HRV-QCE positive case from an in- and out-patient pediatric, hospital-based population during 2003. An objective illness severity score was determined for each HRVC-QCE positive patient. RESULTS: Differences in overall polyprotein and VP1 binding pocket residues and the predicted presence of a cis-acting replication element in 1B defined HRVC-QCE as a novel HRV-C strain. Twelve additional HRVC-QCE detections (1.0% prevalence) occurred among infants and toddlers (1-24 months) suffering mild to moderate illness, including fever and cough, who were often hospitalized. HRVC-QCE was frequently detected in the absence of another virus and was the only virus detected in three (23% of HRVC-QCE positives) children with asthma exacerbation and in two (15%) toddlers with febrile convulsion. CONCLUSIONS: HRVC-QCE is a newly identified, genetically distinct HRV strain detected in hospitalized children with a range of clinical features. HRV strains should be independently considered to ensure we do not overestimate the HRVs in asymptomatic illness.

Cough-generated aerosols of Pseudomonas aeruginosa and other Gram-negative bacteria from patients with cystic fibrosis.

BACKGROUND: Pseudomonas aeruginosa is the most common bacterial pathogen in patients with cystic fibrosis (CF). Current infection control guidelines aim to prevent transmission via contact and respiratory droplet routes and do not consider the possibility of airborne transmission. It was hypothesised that subjects with CF produce viable respirable bacterial aerosols with coughing. METHODS: A cross-sectional study was undertaken of 15 children and 13 adults with CF, 26 chronically infected with P aeruginosa. A cough aerosol sampling system enabled fractioning of respiratory particles of different sizes and culture of viable Gram-negative non-fermentative bacteria. Cough aerosols were collected during 5 min of voluntary coughing and during a sputum induction procedure when tolerated. Standardised quantitative culture and genotyping techniques were used. RESULTS: P aeruginosa was isolated in cough aerosols of 25 subjects (89%), 22 of whom produced sputum samples. P aeruginosa from sputum and paired cough aerosols were indistinguishable by molecular typing. In four cases the same genotype was isolated from ambient room air. Approximately 70% of viable aerosols collected during voluntary coughing were of particles

Do rhinoviruses reduce the probability of viral co-detection during acute respiratory tract infections?

BACKGROUND: Human rhinoviruses (HRVs) are often concurrently detected with other viruses found in the respiratory tract because of the high total number of HRV infections occurring throughout the year. This feature has previously relegated HRVs to being considered passengers in acute respiratory infections. HRVs remain poorly characterized and are seldom included as a target in diagnostic panels despite their pathogenic potential, infection-associated healthcare expenditure and relatively unmoderated elicitation of an antiviral state. OBJECTIVES: To test the hypothesis that respiratory viruses are proportionately more or less likely to co-occur, particularly the HRVs. STUDY DESIGN: Retrospective PCR-based analyses of 1247 specimens for 17 viruses, including HRV strains, identified 131 specimens containing two or more targets. We investigated the proportions of co-detections and compared the proportion of upper vs. lower respiratory tract presentations in the HRV positive group. Both univariate contingency table and multivariate logistic regression analyses were conducted to identify trends of association among the viruses present in co-detections. RESULTS: Many of the co-detections occurred in patterns. In particular, HRV detection was associated with a reduced probability of detecting human adenoviruses, coronaviruses, bocavirus, metapneumovirus, respiratory syncytial virus, parainfluenza virus, influenza A virus, and the polyomaviruses KIPyV and WUPyV (p < or = 0.05). No single HRV species nor cluster of particular strains predominated. CONCLUSIONS: HRVs were proportionately under-represented among viral co-detections. For some period, HRVs may render the host less likely to be infected by other viruses.

A novel gel-based method for self-collection and ambient temperature postal transport of urine for PCR detection of Chlamydia trachomatis.

OBJECTIVES: The aim of this study was to develop a novel urine transport method to be used in self-collection-based screening for Chlamydia trachomatis. The method needed to be suitable for C trachomatis PCR detection, be economical and suitable for transport by standard envelope mailing. METHODS: An anhydrous gel composed of super-absorbent polymer and buffering agent was used to desiccate urine into a dry granulous state, which could subsequently be reconstituted upon arrival at a laboratory. DNA was then extracted from the reconstituted solution using the Roche MagNA Pure protocol for the detection of C trachomatis by PCR. Collections of urine specimens from three populations with widely differing chlamydia prevalence (100%,n = 56; 47%, n = 70; 3%, n = 97) were used. We determined the gel method's impact on C trachomatis PCR sensitivity and specificity using neat and gel-processed urine specimens. An equine herpes virus PCR was used to test for assay inhibition. RESULTS: Overall, the sensitivity of the gel-based method ranged from 94.6-100% compared with neat urine, with a specificity of 100%. No PCR inhibition or decrease in analytical sensitivity was observed using the gel-processed extracts. CONCLUSIONS: The gel-based method was found to be suitable for the detection of C trachomatis by PCR. In addition, its ease of use, effectiveness at ambient temperature and low cost makes it well-suited for self-collection kits used in population-based C trachomatis screening, particularly for geographically and socially isolated individuals.

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?

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.

Emerging respiratory agents: new viruses for old diseases?

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.

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

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.

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

BACKGROUND: Recently, novel human polyomaviruses, KI (KIV) and WU (WUV) were described. Their role in human disease has not yet been determined. OBJECTIVES: The aim of this study was to develop sensitive and specific assays for the detection of KIV and WUV. STUDY: 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. RESULTS: 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. CONCLUSIONS: 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.

Characterisation of a newly identified human rhinovirus, HRV-QPM, discovered in infants with bronchiolitis.

BACKGROUND: Human rhinoviruses (HRVs) are some of the earliest identified and most commonly detected viruses associated with acute respiratory tract infections (ARTIs) and yet the molecular epidemiology and genomic variation of individual serotypes remains undefined. OBJECTIVES: To molecularly characterise a novel HRV and determine its prevalence and clinical impact on a predominantly paediatric population. STUDY DESIGN: Nucleotide sequencing was employed to determine the complete HRV-QPM coding sequence. Two novel real-time RT-PCR diagnostic assays were designed and employed to retrospectively screen a well-defined population of 1244 specimen extracts to identify the prevalence of HRV-QPM during 2003. RESULTS: Phylogenetic studies of complete coding sequences defined HRV-QPM as a novel member the genus Rhinovirus residing within the previously described HRV-A2 sub-lineage. Investigation of the relatively short VP1 sequence suggest that the virus is resistant to Pleconaril, setting it apart from the HRV A species. Sixteen additional HRV-QPM strains were detected (1.4% of specimens) often as the sole micro-organism present among infants with suspected bronchiolitis. HRV-QPM was also detected in Europe during 2006, and a closely related virus circulated in the United States during 2004. CONCLUSIONS: We present the molecular characterisation and preliminary clinical impact of a newly identified HRV along with sequences representing additional new HRVs.

Method for detection of respiratory viruses in the sputa of patients with cystic fibrosis.

Since the role of respiratory viruses in lung exacerbations of patients with cystic fibrosis has been hampered by the difficulty of detecting viruses in viscous sputum specimens, a multiplex reverse transcriptase PCR (RT-PCR) assay combined with colorimetric amplicon detection was tested for the identification of seven common respiratory viruses in the sputa of cystic fibrosis patients. Of 52 sputa from 38 patients, 12 (23%) samples from 12 patients were positive for a respiratory virus (4 for influenza B, 3 for parainfluenza 1, 3 for influenza A and 2 for respiratory syncytial virus). These results suggest that the RT-PCR method carried out on sputum may provide a convenient means of investigating the role of virus infection in lung exacerbations of cystic fibrosis patients.

Clonal strains of Pseudomonas aeruginosa in paediatric and adult cystic fibrosis units.

Despite recent reports of clonal strains of Pseudomonas aeruginosa in cystic fibrosis (CF) units, the need for routine microbiological surveillance remains contentious. Sputum was collected prospectively from productive patients attending the regional paediatric and adult CF units in Brisbane, Australia. All P. aeruginosa isolates were typed using pulsed-field gel electrophoresis. Spirometry, anthropometrics, hospitalisations and antibiotic sensitivity data were recorded. The first 100 sputum samples (first 50 patients at each clinic) harboured 163 isolates of P. aeruginosa. A total of 39 patients shared a common strain (pulsotype 2), 20 patients shared a strain with at least one other patient and 41 patients harboured unique strains. Eight patients shared a strain identical to a previously reported Australian transmissible strain (pulsotype 1). Compared with the unique strain group, patients harbouring pulsotype 2 were younger and had poorer lung function. Treatment requirements were similar in these two groups, as were the rates of multiresistance. In conclusion, 59% of patients harboured a clonal strain, supporting the need for routine microbiological surveillance. In contrast to previously described clonal strains, the dominant pulsotype was indistinguishable from nonclonal strains with respect to both colonial morphology and multiresistance. The clinical significance of clonal strains remains uncertain and requires longitudinal study.