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THE EMERGING VIRUSES GROUP
CHARACTERISING THE EXTENT AND SCOPE OF PAEDIATRIC RESPIRATORY VIRUS INFECTION.
Ian Mackay, Katherine Arden, Peter McErlean, Michael Nissen, Theo Sloots
Background:
This project aimed to increase the detection of viral agents responsible for acute respiratory tract infections among children visiting the Royal Children’s Hospital (RCH) by employing an intensive retrospective investigation of clinical specimens for the presence of viruses using molecular means.
Existing testing protocols had failed to detect a microbe in as much as 80% of specimens collected from patients with suspected respiratory infection. This project sought to identify what new assays should be developed for future deployment and whether some viruses have been under-characterised by previous studies.
Significance:
This project examined specimens from all four seasons in an attempt to determine what viruses were circulating that could not be detected by current laboratory assays or for which assays were not available. A preliminary study of the signs and symptoms upon presentation was conducted to determine the role of newly identified viruses in child health.
Several viruses were detected in Australia for the first time as a result of this study. We reported that human coronavirus (HCoV) NL63 was present in paediatric and adult patients with lower respiratory tract infections. More recently, HCoV-HKU1 and human bocavirus were found among specimens from the RCH. All three findings have initiated studies to clarify the role of these viruses in children with respiratory infection. We improved virus detection rates in our study population by more than 4-fold and proposed that similar improvements could be seen if some of these assays were employed prospectively.
This study was essential to characterise previously undetected viruses circulating among and possibly causing illness in young patients visiting or admitted to the RCH. Additionally, these data have formed the foundation for a vigorous virus characterisation program within the QPID laboratory.
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IMPROVING OUR UNDERSTANDING OF HUMAN CORONAVIRUSES IN A HOSPITAL-BASED COMMUNITY.
Ian Mackay, David Speicher, Peter McErlean, Michael Nissen, Theo Sloots
Background:
This project aimed to characterise in more detail the HCoVs we had identified during the previous project and build upon those studies employing new, more specific molecular assays for the four non-SASR HCoVs. Until the identification of the SARS-CoV, this family of viruses has not been extensively studied and were traditionally discounted as agents of the common cold. No previous molecular testing protocols existed, and this was the first study to characterise the HCoV strains among our hospital’s patients.
Significance:
Firstly we investigated local HCoV strains by sequencing complete and partial genes with a view to understanding what viruses were circulating at the RCH in the winter of 2004 and how these compared to viruses around the world. Secondly we improved our diagnostic capabilities by developing preliminary real-time PCR assays for all four human coronaviruses.
We were the first laboratory in Australia to report the existence of HCoV-NL63 and HCoV-HKU1. We have since determined that HCoVs contribute not simply to colds but to illness among young children visiting or admitted to the RCH with acute respiratory tract infections that includes bronchiolitis and pneumonia.
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STUDYING THE SCOPE OF VIRUS VARIATION: PICORNAVIRUS QUASISPECIES.
Ian Mackay, Peter McErlean, Michael Nissen and Theo Sloots
Background:
RNA viruses are considered to have an error-prone replication mechanism that can lead to many subtle and possibly more pathogenic, virus variants. However, the impact of such variation on modern testing methods, viral fitness, and patient outcomes as well as potential antiviral and vaccine candidates has not been a topic of intensive study. We have chosen the largest group of human respiratory pathogens, the picornaviruses, to begin addressing these issues.
Significance:
A preliminary proof-of-concept project began by looking at specimens employed in our earlier investigations that were positive for a picornavirus. This study has identified some subtle nucleotide changes among a sampling of cloned amplicons derived from a single specimen. It also identified that a similar but different co-infecting virus was present in this patient which could not have been identified in any other way. The implications of this are important since it suggests that a single patient may be able to harbour two separate picornavirus infections at the same time and the extent of possible co-infection is therefore unknown. We are now asking the question of whether the number of concurrent infections has an effect on the level of clinical illness, rather than the simple presence or absence of the one virus.
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PRELIMINARY IDENTIFICATION OF A NOVEL RESPIRATORY VIRUS IN CHILDREN.
Ian Mackay, Peter McErlean, Michael Nissen and Theo Sloots
Background:
Based on the results of our earlier investigations, we believe that many, as yet unidentified, respiratory viruses remain to be discovered, and their role(s) in acute respiratory tract infections needs to be studied. One such potentially new virus has been identified in our laboratory, and further more comprehensive investigations are being conducted to characterize this virus.
Significance:
In a preliminary study we have sequenced almost half of the new virus’ genome, and the sequenced regions bear striking resemblance to other members of the family Picornaviridae, while still being distinctive. Preliminary diagnostic studies indicate that this picornavirus-like virus (PLV) virus occurs in approximately 2% of children who present to Queensland Health clinics or hospitals with suspected acute respiratory tract infections. In many of these cases, no other virus was detected. Less than a quarter of PLV positives occurred in specimens positive for another microbe while more than 80% of positives occurred in children under the age of 5 years, most frequently in specimens collected during the winter months. We are developing methods to isolate PLV from clinical specimens using in vitro cell culture, and to identify and express viral proteins for antibody studies so that the scope of clinical illness among infected patients may be fully assessed.
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DIAGNOSTIC DEVELOPMENT GROUP
DEVELOPMENT OF NEW AND IMPROVED DIAGNOSTIC ASSAYS.
David Whiley, Michael Nissen and Theo Sloots
Background:
The QPID Laboratory has continued to be instrumental in bringing about a radical change in the approach taken to the diagnosis of infectious disease in Queensland Health. We have developed a large number of rapid diagnostic tests (more than 70 to date) for a range of medically important viruses, bacteria, fungi and parasites using the latest in molecular technology. These assays have been made available to the RCH, QHPS, and other laboratories both locally and nationally. The tests are now in use for the routine diagnosis of infectious disease not only in children but also in adults. This has enhanced the management of patients with infectious diseases and has resulted in a significant improvement in patient outcome.
The development of these novel assays has resulted in the publication of the methods in scientific journals (see Publications) and book chapters. As a result, QPID is now recognised as a centre of excellence in molecular diagnostics, both nationally and internationally, and has exported the new assay methodology to other diagnostic centres through the transfer of protocols and the training of their staff.
New Diagnostic Tests:
Through close consultation with clinical staff of the RCH & HSD, we identified significant shortcomings in the diagnosis of infectious disease. These are being addressed through a test development programme commenced in 2000. This programme initially saw a rapid influx of molecular assays into QHPS aimed at detecting known viruses and bacteria of major clinical importance to paediatric patients (such as Herpes simplex virus and Neisseria meningitidis).
Advances in infectious disease research continue to create new needs and challenges for the QPID Diagnostic Development Group. New rapid diagnostic tests are still needed in virology, bacteriology, mycology and parasitology to address emerging diagnostic issues. For instance, the need for new diagnostic assays is created by the discovery of novel human viruses (eg. human metapneumovirus) or where animal viruses are now found to affect humans (such as bird flu). New tests are also needed in situations where commercially available tests fail to achieve desired performance characteristics in our local population (eg. eg. Neisseria gonorrhoeae) or where assays may not provide enough information to make help clinical decision making (eg. BK virus). Consequently, the QPID Diagnostic Development Group is continually working and responding to changing clinical needs and awareness. New assays developed have included molecular tests for the following organisms:
Herpes simplex virus – Herpes virus infections of the central nervous system resulting in encephalitis may present a considerable diagnostic dilemma for the clinician. Serological diagnosis is inadequate, and isolation of the virus from CSF is often unsuccessful. Other methods of diagnosis may involve invasive procedures like needle biopsy of infected brain tissue. This method is traumatic for the patient, and is only marginally more successful in providing a conclusive diagnosis. QPID have developed a rapid real-time PCR assay using the LightCycler instrument that can detect and characterise herpes simplex virus in the CSF of children with herpes virus encephalitis. This assay uses primers and probes directed to the HSV DNA polymerase gene. Clinical evaluation has shown this test to be highly sensitive (99%) and specific (100%) confirming that this assay offers a rapid and comprehensive alternative to existing methods for the rapid detection of herpes virus in CSF, thus presenting a much improved method for the diagnosis of herpes virus encephalitis.
Neisseria meningitidis – Acute meningitis is one of the most serious infections of children. It is an infection of the lining of the brain or meninges characterised by rapid onset, with a high risk of death or complications if not diagnosed early in its course. The major cause of bacterial meningitis is Neisseria meningitidis (the “meningococcus”), and is potentially the most damaging. Laboratory detection is achieved by isolation of the bacteria using standard bacteriological methods. Rapid treatment with antibiotics is an essential element of successful patient care, but results in inability to isolate the organism from these patients, thus leading to an inconclusive laboratory diagnosis. QPID has developed and published a PCR test simultaneously targeting the ctrA and porA genes present in the Neisseria meningitidis bacterial genome.
Until recently, traditional methods of diagnosis took up to 3 days for a result. The real-time PCR test we have developed produces a test result within 1 hour, and has made an enormous difference in patient prognosis.
This study addressed;
The development of a diagnostic test detecting meningococcus in spinal fluid or blood sample taken from a child with meningitidis.
The assay was adapted to real-time PCR on a LightCycler, thereby giving a result in 1 hour, instead of 2-3 days taken for bacterial culture which at best ahs a sensitivity of <50%.
Human metapneumovirus – Human metapneumovirus (hMPV) was first described in 2001 and has since been found to be a major cause of respiratory disease in children. In fact, QPID’s Emerging Virus Group recently completed a four year study and found that hMPV annually accounts for 7% of infections in paediatric patients admitted with respiratory tract disease. Publication of this research quickly generated interest in the virus in the local clinical community, driving the need for a new assay. QPID responded with the development of a real-time PCR assay for hMPV. This assay is now performed as part of the routine respiratory virus screen at QHPS.
Influenza A (H5N1) – Influenza type A has always been considered one of the major viruses causing respiratory disease in humans. It affects both children and adults and can be particularly dangerous in the very young and elderly. However, there are now renewed concerns over Influenza A as a result of the H5N1 Influenza A subtype. This H5N1 subtype or “bird flu” could potentially kill millions of people worldwide if a pandemic were to occur. Hence, it has received considerable media attention and has government agencies throughout the world busily developing strategies to combat this potential threat.
One area requiring attention has been diagnostics. Following the Hong Kong pandemic of 1968, the influenza A types infecting humans have remained fairly stable with the common subtypes causing disease including H1N1, H1N2, H2N2 and H3N2. As a consequence, molecular assays have traditionally been developed to ensure the detection of these subtypes only. Given established assays were unable to detect H5N1, there was a growing need to review diagnostic capabilities for influenza A.
QPID has now developed and published a real-time PCR assay capable of detecting all the common human influenza A subtypes as well as the avian subtypes now known to infect humans, including H5N1. The assay is now used routinely in QHPS and is forms part of the H5N1 preparedness strategy of Queensland Health.
Neisseria gonorrhoeae – Neisseria gonorrhoeae (the “gonococcus”) is very closely related to the bacterium Neisseria meningitidis (described above), but instead of causing bacterial meningitis, the gonococcus causes the very different disease gonorrhoea. Gonorrhoea is, of course, a well characterised sexually transmitted disease and falls outside of the paediatric disease umbrella. Nonetheless, gonorrhoea continues to be a problem for laboratory diagnosis. The reason for this is that the N. gonorrhoeae species comprises numerous subtypes which can vary considerably in their genetic make-up. Also, gonococcal strains often “share” their DNA with other bacterial species. The end result of all of this is that molecular assays for gonorrhoea are prone to both false-positive and false-negative results. This makes control of the disease even more difficult.
It is for these reasons that QPID’s skills in molecular diagnostics were sought out by the Communicable Diseases Unit of Queensland Health to development a reliable assay for detecting N. gonorrhoeae. QPID has since identified a target region on the porA pseudo gene of N. gonorrhoeae that is conserved across gonococcal subtypes and that has not been “shared” with other bacteria. A real-time PCR assay based on this target has been developed by QPID and is now been used in laboratories throughout Australia and the world. Queensland Health has also taken out an international patent application on the technology and is looking to commercialise the assay.
BK virus – Human polyoma virus BK (BKV) is carried by most people and is usually harmless. However, in transplant patients BKV can cause severe diseases including hemorrhagic cystitis and allograft nephropathy as a result of the patient’s immune system having been suppressed. Traditional methods for detecting BKV include serology and virus isolation, but are very insensitive. In 2001, QPID developed a real-time PCR assay for the qualitative detection (Yes or No) of BKV in the urine and serum of transplant patients. This proved to be reliable in excluding the possibility of BKV related disease for patients whose samples provide negative results in the assay. However, positive results were still left open to clinical interpretation. The reason for this is that BKV can be detected even in healthy individuals and so establishing a casual relationship between the detection of BKV and disease is difficult. Quite simply, the qualitative BKV PCR assay was not providing enough information for positive samples.
QPID has since developed a quantitative BKV real-time PCR that is now used routinely by QHPS to further investigate positive samples. The major benefit of this quantitative assay is that it can be used to determine the concentration of BK virus in a sample, rather than just determining its presence. Hence, high BKV loads indicating active infection can be distinguished from low loads of virus that are not clinically significant. Further, the assay can be used to monitor BKV viral load when adjusting a patient’s level of immunosuppression. Thus, the new quantitative BKV PCR assay plays a key role in risk management of BKV-related diseases in these patients.
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FUTURE DIAGNOSTIC ASSAY DEVELOPMENT:
Much of the work conducted by the Diagnostic Development Group to date has focused purely on developing assays for detecting the presence, absence or concentration of the target organism. Though dramatically enhancing the capabilities of diagnostic laboratories, the assays do not necessarily help in deciding treatment regimes. A notable limitation is the inability to provide antimicrobial resistance data, which is an emerging health concern given the increasing reports antimicrobial resistance in a broad range of pathogens. QPID has embarked on a broader assay development programme with the aim of developing assays for the detection of genetic resistance elements. In this way, clinicians will not only be provided with the identification of the organism, but also be provided with information helpful in deciding the appropriate antimicrobial treatment.
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ADDITIONAL PROJECTS:
Streamlining the QHPS molecular diagnostic service:
Although the creation of all these new molecular assays is a bonus for both clinicians and patients alike, it does come with its own problems. The increasing number of tests being developed at QPID has dramatically increased the molecular diagnostic workload within QHPS. Quite simply, more assays require more hands; therefore, having enough staff to run the assays has been a continuing challenge. The limited number of PCR instruments in the microbiology department of QHPS has also created a bottleneck. QHPS has in part resolved some of these problems by reorganising staff, hiring new staff and by purchasing new equipment. However, there has been a heavy impetus on QPID to find technological solutions to these problems.
Real-time PCR - One of the major ways in which QPID has helped solve the workload problems of QHPS is by the introduction of real-time technology. This is the latest technology in molecular diagnostics and considerably reduces the hands on time required to perform PCR. Briefly, conventional PCR requires two steps; an amplification step used to amplify the target DNA of interest, and a detection step used to detect this amplified DNA. Notably, this second step alone can take up to two hours. Real-time PCR combines both of these steps, enabling PCR amplification and detection to occur at the same time. Real-time PCR instruments utilise fluorogenic probe technology that enables “real-time” detection of PCR amplification products. The application of real-time PCR technology also has the added benefit of reducing the turnaround-time for results. The minimal time taken to obtain a result using conventional PCR was usually 5-6 hours. Real-time PCR technology enables the reduction of this time to about 1 hour for some assays (with a maximum of 2.5 hours).
In the past year, QPID has completed conversion of all PCR assays in the Microbiology Department of QHPS to real-time PCR, making it one of the most advanced laboratories of its kind in the southern hemisphere.
Multiplex PCR – A recent initiative in our laboratory is the development of multiplex real-time PCR assays, which utilise multiple PCR tests in a single tube. This allows for the simultaneous detection of multiple pathogens using a single PCR reaction mix. For the diagnostic laboratory these multiplex assays have other significant benefits. Firstly, they reduce the detection workload and help free up valuable space on the real-time PCR instruments. Just as importantly, they can more than halve the costs of performing PCR reactions.
Our respiratory virus assays have been the flag-ship of our multiplex real-time PCR assay role-out. Following conversion to real-time PCR, our respiratory assays comprised seven separate PCR reactions, being one for each of the common respiratory viruses. We have now reduced this to three separate reactions by introducing two duplex assays and one triplex assay; the first duplex reaction combines tests for Influenza A and B, the second for RSV and Adenovirus, whereas the triplex combines PCR tests for Parainfluenza types 1, 2 and 3.
A centre of excellence in molecular diagnostics:
As a result of QPID’s considerable experience in molecular diagnostics, our expertise is now sought by laboratories all over Australia and the world. We now routinely provide advice and training to fellow scientists, run workshops at national conferences, and have taken part in establishing laboratory guidelines for nucleic acid testing. It has been our continuing goal to share our extensive knowledge so as to raise the standards of microbial molecular diagnostics.
Staff training - One of the few downsides to using molecular diagnostic techniques is that staff with a high degree of expertise are required to perform and interpret the assays. One way QPID has addressed this issue is by running both informal and formal training sessions. The formal training workshops at QHPS sessions are ran in conjunction with senior staff of the Molecular Diagnostic Unit. It is envisaged that these will be expanded in the future so as to provide to training to scientists from laboratories Australia-wide.
Conference workshops – QPID first started running molecular diagnostic workshops at the Australian Society for Microbiology annual meeting at Perth, in 2001. Since that time, QPID has maintained a strong presence at this national annual meeting, which is attended by molecular diagnostic staff from throughout Australasia. The molecular diagnostic workshops are designed to raise, identify and help resolve technical issues and problems associated with the use of the technology. Hence, they are pivotal in distributing our “hands-on” knowledge at a local level. At the upcoming Gold Coast ASM meeting, we will be running a workshop focusing on quality control in molecular diagnostics.
Nucleic acid testing guidelines - As already discussed above, molecular tests for N. gonorrhoeae have been fraught with problems. In response, the Public Health Laboratory Network (PHLN) recently convened a workshop in Melbourne of Australian experts in gonococcal nucleic acid testing. Both David Whiley and Theo Sloots from QPID attended. The aim of this workshop was to try to address the above problems by creating guidelines for using molecular diagnostic assays for diagnosis of gonorrhoea in Australia. The guidelines created by this workshop were endorsed by the members of the PHLN and the Communicable Diseases Network of Australia and have now been published in Communicable Diseases Intelligence.
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CYSTIC FIBROSIS RESEARCH GROUP
Background:
The overall focus of the Cystic Fibrosis Research Group (CFRG) is to assist in improving the clinical management of patients with cystic fibrosis (CF). The CFRG includes QPID heads Dr. Michael Nissen and Dr. Theo Sloots with Dr. Melanie Syrmis (Group Leader), Snehal Chandan (PhD student) and Seweryn Bialasiewicz (Research Assistant). The CFRG is part of the Brisbane Pseudomonas Research Group which also includes Dr. Scott Bell, Director adult CF clinic The Prince Charles Hospital (TPCH), Dr. Chris Coulter, Microbiologist and Director Infectious Diseases, (TPCH), Tim Kidd, Scientist, (QHPS), Penny Mitchell, CF liaison nurse and Dr. Claire Wainwright, Paediatric Respiratory Physician, (RCH). This group has an extensive track record in molecular epidemiology of Pseudomonas aeruginosa. The group has developed collaborative links with Professor Peter Bye and Dr. Barbara Rose’s group in Sydney and Dr. David Armstrong’s group in Melbourne and made a joint application to NIH and also to NH&MRC for a grant to investigate the molecular epidemiology, longitudinal clinical outcomes and pathogenicity of clonal strains of P. aeruginosa from six clinics across Queensland, NSW and Victoria. This study will expand on our previous studies of Ps aeruginosa in patients with CF and significantly add to understanding concerning the role of P. aeruginosa in CF. We believe this will form a sound basis for new research examining the pathogenesis of P. aeruginosa and identifying mechanisms for the treatment and prevention of bacterial biofilms, thereby improving patient outcome.
Specific areas that are being examined fall into three main categories (a) detection and transmission of P. aeruginosa, (b) antibiotic resistance of P. aeruginosa and (c) detection and characterisation of P. aeruginosa biofilm.
Significance:
Lung infection caused by P. aeruginosa is a major determinant of morbidity and mortality in cystic fibrosis (CF). The recent emergence of multi-drug resistant P. aeruginosa in CF patients with the capacity for person-to-person transmission has serious implications for patients and health providers. We have recently found that two hyper-transmissible P. aeruginosa strains (designated Pulsotypes 1 and 2) are widely spread within CF communities in eastern Australia. Insight into the factors that determine the transmissibility and pathogenicity of these epidemic strains is urgently needed so that more appropriate and improved clinical management of CF patients can be identified.
In our previous work, we described a clonal P. aeruginosa strain (Pulsotype 2 or P2) in many paediatric and adult patients attending CF centres in Brisbane, Australia. The investigation involved the use of the gold standard for genotyping P. aeruginosa, Pulsed-Field Gel Electrophoresis (PFGE), as well as the development of PCR genotyping techniques that could be used as more cost effective screening tools, e.g., ERIC-PCR, for epidemic strains for future studies. The following are three such studies that commenced from mid 2005.
An example of DNA banding profiles of the three most prominent pulsotypes, P1, P2 and P42 by gel electrophoresis following PFGE and ERIC-PCR. Banding patterns showed clear discrimination between pulsotypes for each method.
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MOLECULAR IDENTIFICATION OF CLONAL AND TRANSMISSIBLE STRAINS OF PSEUDOMONAS AERUGINOSA BACTERIAL PATHOGENS IN CYSTIC FIBROSIS.
Melanie Syrmis, Seweryn Bialasiewicz, Snehal Chandan, Theo Sloots, Claire Wainwright, Chris Coulter, Tim Kidd, Scott Bell, and Michael Nissen
Our previous work indicated the presence of 6 clonal strain types from 100 patients attending the RCH and TPCH CF Clinics. This study was developed to screen P. aeruginosa isolates from all patients with CF attending RCH and TPCH clinics in order to determine the overall prevalence of epidemic P. aeruginosa strains in our Brisbane clinics. To date, more than 800 P. aeruginosa isolates have been collected and stored. Genotyping of these P. aeruginosa isolates has also commenced with 97 already tested using ERIC-PCR. This work will not only provide clinicians with data regarding the spread of strain types through clinics, but establish a large bank of characterised P. aeruginosa isolates from patients with CF.
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AIR PARTICLE TRANSMISSION OF PSEUDOMONAS AERUGINOSA.
Claire Wainwright, Snehal Chandan, Tim Kidd, Melanie Syrmis, Megan France, Zoran Ristovski, Karen Furlong, Joyce Cheney, Megan Hargreaves, Cheryl Swanson, Kevin Fennelly, Chris Coulter, Michael Nissen, Theo Sloots, and Scott Bell.
The transmission of respiratory agents such as influenza, RSV, adenovirus, and Mycobacterium tuberculosis (M. tuberculosis), is well-supported by epidemiological studies. However, to date little data exists for the transmission of P. aeruginosa in the context of CF. A method has recently been reported to assess cough-generated aerosols in patients with M. tuberculosis. We sought to adapt the method to assess whether cough-generated infective aerosols were evident in patients with CF during voluntary and induced cough manoeuvres. We aimed to assess the particle size distribution of infectious aerosols, and determine if the concentration of cough aerosols were related to disease severity, nature of cough (spontaneous or induced) and the presence of clonal P. aeruginosa in children and adults with CF.
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ENVIRONMENTAL INITIAL ACQUISITION STUDY.
Snehal Chandan, Tim Kidd, Melanie Syrmis, Joyce Cheney, Theo Sloots, Claire Wainwright and Michael Nissen
This pilot study focuses on identifying P. aeruginosa strains in the home environment and whether these strains are also present in the children who live in these homes. The young children, taking part in the NH&MRC funded ACFBAL study, have standardised clinical management including an eradication protocol when P. aeruginosa infection is detected. All isolates of P. aeruginosa from oropharyngeal, sputum or broncho-alveolar lavage specimens will be stored. Environmental testing will be performed in patients clear of infection with P. aeruginosa and at the time of confirmation of P. aeruginosa infection. Testing will include air sampling following toilet flush, use of sink and showering from the homes of 20 families and will focus on areas of moisture around each household including taps, pipes, air vents, damp walls, soap dishes, toothbrushes, wet bathroom containers and toys. Any P. aeruginosa isolates will be stored and genotyped using ERIC-PCR as the initial screening tool combined with PFGE as the confirmation assay. Environmental isolates will be compared with past and future clinical isolates from children with CF who have not yet acquired chronic P. aeruginosa infection and also with the predominant clonal strains of P. aeruginosa isolated from patients with CF as part of a previous study.
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THE USE OF MULTILOCUS RESTRICTION TYPING (MLRT) AS AN EPIDEMIOLOGICAL TOOL FOR PSEUDOMONAS AERUGINOSA IN CYSTIC FIBROSIS.
Snehal Chandan, Melanie Syrmis, Theo Sloots, Claire Wainwright, Scott Bell, Chris Coulter and Michael Nissen.
The utility of PFGE and PCR-based typing techniques has not been validated when comparing geographically and/or spatially distinct isolates of P. aeruginosa. Global epidemiological tools include multi-locus sequence typing, which is an expensive typing technique. One cheaper alternative is multi-locus restriction typing (MLRT). The MLRT has recently been successfully applied to Burkholderia cepacia isolates, another important infectious agent for people with CF. MLRT involves the use of several housekeeping genes that are assessed for allelic variation via PCR combined with enzyme restriction. For each housekeeping gene, the different alleles are assigned a numerical number so that the final result will be a numerical code.
Results of TaqI digestion of aprA gene PCR products. In A, each isolate differs from the others by at least two bands. Legend: M: DNA Marker, P1: Pulsotype 1, Man: Manchester epidemic strain, P7: Pulsotype 7 (unique strain type).
This study will examine the validity of MLRT as a method to assess relatedness of strains of P. aeruginosa in patients with cystic fibrosis.
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SPECIFIC DETECTION OF THE MAJOR CLONAL PSEUDOMONAS AERUGINOSA STRAIN IN BRISBANE CF CLINICS.
Snehal Chandan, Melanie Syrmis, Chris Coulter, Scott Bell, Claire Wainwright, Theo Sloots, and Michael Nissen
Identification of genetically related or identical strains of P. aeruginosa may be performed using PFGE or repetitive element based PCR. However these methods can be time consuming and expensive and they require testing of each individual strain that is isolated on culture. The P2 P. aeruginosa was identified initially in nearly 40% of the 100 adult and paediatric patients with CF attending either the RCH or TPCH clinics. Thus, screening sputum samples rapidly for the P2 Brisbane clonal strain using a PCR assay would be clinically very useful and in addition, a PCR marker would be very helpful for screening large sample numbers of environmental isolates to enable rapid detection of the clonal strain. Results from previous work i.e. rapid PCR typing (ERIC & BOX PCR) and multi-locus restriction typing methods will assist in identifying possible molecular targets/primers for these specific PCR assays. Confirmation of identification would be required using standard methods.
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ANTIBIOTIC RESISTANCE OF PSEUDOMONAS AERUGINOSA.
Background:
Antimicrobial resistance in P. aeruginosa is of major clinical importance given it is a significant pathogen of patients with cystic fibrosis. There have been renewed concerns over resistance in P. aeruginosa with reports of potential patient-to-patient transmission of antibiotic resistant clonal P. aeruginosa strains. The P2 P. aeruginosa identified in our initial study had higher resistance to a variety of antibiotics including aminoglycoside (gentamicin, tobramycin, and amikacin) and -lactam (meropenem, ceftazidime, piperacillin, and ticarcillin/clavulanic acid) drug classes when compared to non-clonal strains. Horizontal gene transfer mechanisms such as plasmids, transposons and integrons containing resistance genes can enhance the spread and development of antibiotic resistance in bacteria.
Distribution of the aadB gene in clonal strain types and non-clonal Pseudomonas aeruginosa isolates. The P2 P. aeruginosa were more likely to carry the aadB gene than the non-clonal strains
Significance:
Initial work performed within our laboratory indicated a high prevalence of an integron associated-aminoglycoside resistance gene, the aadB, in many P. aeruginosa isolates from patients with CF. The aadB gene encodes the nucleotidyltransferase ANT(2”)-I, that imparts resistance to the aminoglycosides, gentamicin and tobramycin. The integron was detected in plasmid extracts from P. aeruginosa isolates from patients with CF. Plasmids are also an example of an acquired resistance mechanism.
The P2 P. aeruginosa were found to carry this resistance mechanism more than non-clonal strains which may explain part of the increased resistance observed. The findings are of clinical importance, as tobramycin one of the most commonly used antibiotics for the treatment of P. aeruginosa infection in patients with CF. Therefore, long term treatment may select for strains with this resistance mechanism. In addition to tobramycin, resistance to colistin, another important antibiotic for treating P. aeruginosa infection was observed in the P2 strains. As both of these antibiotics are very important in the management of P. aeruginosa infection in patients with CF, a second study was developed to validate the findings with gold standard antibiotic susceptibility techniques not commonly available in routine clinical laboratories.
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ASSESSMENT OF METHODS OF ANTIBIOTIC SUSCEPTIBILITY FOR TOBRAMYCIN AND COLISTIN FOR CLINICAL PSEUDOMONAS AERUGINOSA CYSTIC FIBROSIS ISOLATES.
Tim Kidd, C Strahan, Melanie Syrmis, Claire Wainwright, Theo Sloots, Michael Nissen, Chris Coulter, Scott Bell.
In vitro susceptibility testing is used as a guide for antibiotic choice in the treatment of patients with CF. Utility of such testing and the optimal methods for its assessment are controversial. The gold standard method for susceptibility testing is broth dilution minimal inhibitory concentration (MIC) however, is rarely performed in clinical microbiology laboratories due to labor and time intensity. Most CF microbiology laboratories utilise disc diffusion (DD) testing as it is relatively inexpensive and easy to use. MIC testing using the AB BIODISK Etest® has recently become available; however its routine use on clinical CF isolates is somewhat limited. Whilst guidelines are well established for standardisation of susceptibility testing for beta-lactam, quinolone and aminoglycoside antibiotics, controversy exists for colistin sulphate. The aim of this study was to compare the results of in vitro susceptibility testing of P. aeruginosa isolates for tobramycin and colistin using NCCLS DD, Etest® and broth microdilution (BM).
BM and Etest® provided similar results for tobramycin and colistin. Material and labor costs will remain significant limitations in the widespread utilisation of BM and Etest® (~Aus$8/antibiotic/isolate). Divergence of results with DD is of concern, though very major error rates were relatively low. Most clinical P. aeruginosa isolates remain susceptible to colistin despite its frequent use in Australian patients.
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DETECTION AND CHARACTERISATION OF BIOFILMS.
Background:
Over 60% of P. aeruginosa infections in CF patients result in the establishment of a biofilm, with P. aeruginosa of a mucoid phenotype most closely associated with their establishment. Biofilms act as a major virulence factor by providing a defence against local humeral and cellular responses by the host. The presence of biofilms in patients with CF is a major problem, as once established, they are difficult, sometimes impossible, to eradicate and require aggressive antibiotic therapy. The best clinical outcome results from early detection of biofilm formation in children, followed by appropriate, effective, antibiotic treatment.
Mucoid P. aeruginosa from a Horse Blood Agar culture plate.
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DETECTION OF ANTIBODIES TO PSEUDOMONAS AERUGINOSA BIOFILM.
Melanie Syrmis, Theo Sloots and Michael Nissen.
This project focuses on the development an improved method for the early detection of P. aeruginosa biofilm development in children with CF, so that early antibiotic intervention may be applied. Secondly, the most effective antibiotic treatment regimen will be determined for P. aeruginosa in biofilms, and compared to antibiograms that are determined routinely in the diagnostic microbiology laboratory for planktonic non-biofilm Pseudomonads. Together the findings of these studies will have a major impact on the clinical management and treatment of children with CF who are at the early stage of P. aeruginosa biofilm development. Effective treatment of biofilm during childhood will result in a reduced incidence of biofilm in adult patients with CF, thus providing a better prognostic outcome for these patients. In addition, during the course of this study we will identify the major proteins (and the genes coding for these) specific to mucoid P. aeruginosa in biofilm.
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THE CLINICAL STUDIES GROUP
Previous research at the Qpid Laboratory has enhanced our ability to initiate and continue significant new studies into the role of microbial pathogens in paediatric disease. Current research projects are focussed on the following:
Improvements in sampling methods for virus detection by PCR
Molecular characterisation of respiratory syncytial virus (RSV)
The immunological response to hMPV infection
COMPARING VIRUS DETECTION BY PCR IN SIMULTANEOUSLY COLLECTED ASOPHARYNGEAL ASPIRATES AND NOSE-THROAT SWABS IN CHILDREN.
Stephen Lambert, Sarah Tozer, Theo Sloots and Michael Nissen.
Nasopharyngeal aspirates (NPAs) are currently the only method and specimen type accepted by QHPS for the detection of respiratory viruses in the upper respiratory tract. NPAs are distressing, noisy, and invasive for children, parents, and the person collecting the specimen. This project aims to assess the possibility of using a combined nose-throat swab (NTS) as a replacement specimen for NPAs. This study will also allow for the validation of NTS specimens on the respiratory virus PCR platform currently used by QHPS – an important component of pandemic influenza planning.
The study commenced at the RCH in early July 2006, and every child having an NPA for a medical indication can be involved. To date we have collected over 40 paired NPA/NTS specimens and concordance has been high with 44 paired specimens showing the same result. Of the 4 discordant pairs: 3 had adenovirus found in the NPA but not the NTS, and one had hMPV identified in the NTS but not the NPA.
The aim is to continue this study over the winter/spring season this year, collecting between 100 and 200 paired specimens, with a review of the results early next year. At this stage a recommendation may be made to consider a move to NTS specimens as a replacement for NPAs.
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MOLECULAR CHARACTERISATION OF RESPIRATORY SYNCYTIAL VIRUS (RSV).
Sarah Tozer, Rashmi Dixit, Theo Sloots and Michael Nissen.
Background:
RSV infection is the major cause of acute lower respiratory illness in infants and young children throughout the world. Most infants become infected in their first year of life, with the majority of the remainder infected by their second year. Further RSV is a precipitant of asthma attacks and infection may predispose susceptible children to recurrent wheezing later in life. Re-infection is common and occurs despite the presence of antibody to the virus. This high frequency of re-infection declines only slightly in adulthood and suggests the development of only partial immunity. Genetic variation may play a critical role in the ability of RSV to escape the immune response and establish infections. This may also have implications for vaccine development against Australian RSV isolates, and future antiviral therapies.
Aims:
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To determine the genetic identity of respiratory syncytial virus (RSV) isolates circulating in South-east Queensland children.
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To analyse whether genotype correlates with the severity of RSV infection,
To determine RSV viral load has a bearing on clinical severity of acute RSV infection and bronchiolitis.
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To determine whether the endogenous cytokine, Interleukin-8, is a predictor of severe acute RSV infection and bronchiolitis.
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CHARACTERISATION OF THE CYTOKINE RESPONSE IN CHILDREN INFECTED WITH HUMAN METAPNEUMOVIRUS (HMPV).
Maxine Preston, Rashmi Dixit, Theo Sloots and Michael Nissen
Background:
Human metapneumovirus (hMPV) is a major cause of acute respiratory disease in infants and young children, and accounts for a significant number of hospitalisations. The virus can also cause considerable disease in the elderly and in those patients with a compromised immune system, for example, organ transplant patients. Very little is known about how the virus causes disease in the respiratory tract, and how the immune system either contributes to the disease process or helps the body to overcome the infection. There is a need to look at the role of the immune system in infection with hMPV. This research project aims to examine the expression of cytokines, soluble proteins that direct and control an immune response to viral infection, in infants and young children suffering acute infection with hMPV. The results of this study will greatly contribute to the future development of vaccines and treatments for hMPV infection in all patient groups.
Aims:
- To determine the hMPV-specific cytokine response in infants and young children infected with hMPV, with particular reference to Th1- and Th2-type cytokines.
- To enumerate the cellular subsets induced by hMPV in infants and young children following hMPV infection.
- To compare the above with the cellular and cytokine response in infants and young children not infected with hMPV.
Significance:
Little is known about the immunobiology of human metapneumovirus (hMPV) infection in humans. The primary immune response to hMPV infection has been studied in an animal model, and, whilst these results are useful, it is imperative to collect comparable data for this very important human pathogen in it’s natural host. HMPV is a recognised cause of acute and frequently debilitating respiratory tract disease worldwide, with considerable impacts on health care in Queensland. Approximately 65% of children admitted to the RCH with infectious disease suffer from respiratory infection, and during the epidemic season (August to November), 28% of these are due to hMPV. It is likely that the results from our study will significantly contribute to the elucidation of the mechanisms of immunity associated with hMPV infection, which, in turn, will provide a basis for patient management and for the development of effective vaccines and virus-specific therapies.
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A VACCINE FOR HUMAN METAPNEUMOVIRUS ASSOCIATED RESPIRATORY DISEASE.
This is a collaborative project with Dr Robert Tindle and staff, SASVRC (see section: Viral Immunology Research Unit). Some aspects of this research were commenced in 2003 through funding from the RCH Foundation. This project proposes to examine the feasibility of developing a polyvalent vaccine to a number of different respiratory pathogens including hMPV. If successful this technology may be applied to other viral pathogens, and agents such as meningococcus and Pseudomonas aeruginosa.
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THE CLINICAL TRIALS GROUP
The Clinical Trials Group (CTG) began conducting industry sponsored clinical trials within the RCH & HSD during 2004. Since that time the group has grown in size and this has made it possible to conduct a number of studies simultaneously.
The conduct of clinical trials allows the RCH & HSD to participate in the development of improved, safer, and more effective treatment options for children. A particular interest of the CTG is infectious diseases, particularly vaccine preventable diseases, in children, but in 2006 we have participated in two studies involving adults, and we have also been involved in the planning for studies in children not related to infectious diseases.
At present the Clinical Trials Group consists of:
- Michael Nissen – infectious diseases paediatrician and medical microbiologist;
- Stephen Lambert – paediatric infectious diseases fellow;
- Aaron Buckner – clinical studies co-ordinator;
- Lisa Mulhearn – study co-ordinator;
- Jane Yunus – study co-ordinator.
The CTG is currently conducting 8 clinical trials. These are:
Combination HibMenC study:
This study is comparing a new vaccine for 12-month old children combining the vaccines for Hib (Haemophilus influenzae type B) and group C meningococcal disease. Both of these bacteria can cause meningitis and septicaemia (blood-poisoning) in childhood, and there are currently two separate vaccines given at 12-months of age against these infections.
Combination HibMenCY study:
This study is comparing a new vaccine for children given during the first year of life at 2, 4, and 6-months of age. The vaccine combines the vaccines for Hib (Haemophilus influenzae type B), group C meningococcal disease, and group Y meningococcal disease. Group Y meningococcal disease is a common cause of meningococcal disease in other parts of the world. This vaccine could replace separate injections given to infants and allow early protection against serious infections in childhood.
Group B Meningococcal vaccine studies:
The CTG is currently involved in a Phase I adult study using a protein-based meningococcal B vaccine. We have successfully enrolled 45 healthy adults aged between 18 and 25-years, and are currently following them during their three dose vaccine schedule over a seven month period. We are also involved in the planning stages for studies using the same vaccine in adolescent and toddler age groups.
Flu Questionnaire Development study:
In preparation for future trials using newly developed influenza vaccines, we are participating in a study aimed at developing a patient-generated questionnaire to capture the impact of influenza in adults. The study is being conducted over the flu season, and aims to get community-based people with laboratory confirmed influenza to assist in the development and validation of an influenza impact questionnaire.
RSV monoclonal antibody preparation for high risk children:
This study involves providing a new improved RSV monoclonal antibody to children at high risk of serious RSV disease and hospitalisation during the winter season. Such children include those who were born prematurely or born with underlying serious heart or lung conditions.
Singular (Montelukast) Bronchiolitis Study:
RSV Bronchiolitis and Asthma share many features. Both of these conditions trigger the release of substances called ‘CysLT’s’ in the body, which leads to inflammation of the airways. SINGULAIR® (generic name: Montelukast) is a proven asthma medication that works by blocking these substances, and therefore reducing the airway inflammation and narrowing. A recent study was conducted in Denmark, where they successfully trialled SINGULAIR on children with RSV Bronchiolitis. This study showed that SINGULAIR significantly reduced symptoms, in particular cough and wheezing. Children who are diagnosed with RSV Bronchiolitis were offered participation to potentially help reduce the severity of RSV Bronchiolitis symptoms.
Inhaled Aztreonam Clinical Trial:
This study is being done to test an antibiotic called Aztreonam Lysinate for Inhalation (AI). This drug is being tested in Cystic Fibrosis (CF) patients who have Pseudomonas Aeruginosa. The study drug is taken by inhalation via an experimental nebulizer device called an eFlow® Electronic Nebulizer, which is small, portable, and can deliver the medication in approx. 3 minutes. The CTG is also preparing to commence a Follow-On, Open Label Study, open to participants of this study.
MCID Study:
The MCID (Minimal Clinically Important Difference) Study was developed to gain understanding of the use of the CFQ-R (Cystic Fibrosis Questionnaire – Revised) as an adequate measurement tool of Quality of Life in patients with CF, to be used as a qualitative measure in Clinical Trials. Recruits were given a known and measurable treatment regime upon exacerbation of the disease, and were asked to complete the questionnaire pre- and post-treatment. Pulmonary function tests were also performed as a quantitative measure of treatment effect with which to compare the results in the questionnaire.
The CTG is in discussion about further clinical studies, including first-in-man studies for a potential Group A Strep vaccine and for a new IV iron preparation for children with kidney disease.
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