Over the last 25 years the Queensland Paediatric Infectious Diseases (QPID) laboratory has primarily focused on improving the health of Queensland children through the promotion of child health research. Particularly, by developing clinical research capacity in paediatric infectious diseases, and the participation in programmes that provided tangible improvements in medical service delivery to Queensland children, young people, and the community as a whole.
An important function of the QPID Laboratory has been the development of new rapid molecular assays for the detection and characterisation of microorganisms. These have included over 60 new tests that have been developed, validated and implemented for routine use by the Molecular Diagnostic Unit of the Microbiology Division of Pathology Queensland Central Laboratory, Health Services Support Agency.
These research and development initiatives have been generously supported by the Children’s Health Foundation through the Woolworths fundraising initiative, the National Health and Medical Research Council, the Australian Research Council and other external granting bodies.
At the QPID laboratory research activity occurs in six major programmes which address the three interlinking principles in the management of infectious diseases. These are:
- The Diagnosis and Characterisation of Infectious Diseases
- Improving Our Understanding of Respiratory Infections in Children
- Examining the Biology and Pathogenesis of Human Polyomaviruses
- The Epidemiology and Biology of Pseudomonas aeruginosa in Patients with Cystic Fibrosis
- Prevention of Infection
- Collaboration in Intra-Institute Studies
THE DIAGNOSIS AND CHARACTERISATION OF INFECTIOUS DISEASES
A/Professor David Whiley
Molecular diagnostics - another year, another influenza threat.
Our laboratory continues to be at the forefront of improving molecular diagnostics services, particularly as they relate to new or remerging infectious diseases. This year we witnessed yet another threat from the formidable influenza A virus, this time in the form of H10N8. While the alarm bells are not yet sounding for H10N8, this new avian influenza virus subtype follows on the heels of both H7N9 and H1N1 (swine flu), and once again creates new challenges for clinical laboratories. Early detection is pivotal to containing a pandemic virus yet the clinical presentation (ie that seen by a doctor) for influenza virus infection is typically indistinguishable from that due to seasonal influenza virus and other respiratory viruses. Timely and specific diagnosis is therefore a key aspect of the laboratory’s role. However, an increasing problem for laboratories is that with each new threatening strain of influenza virus that is discovered, considerable time and effort must be put into developing, implementing and maintaining new assays. Our research is now endeavouring to overcome this complexity by developing assays that give broad coverage for influenza A types of concern. For example, we have now developed a ‘matrix’-based assay that can detect the H7N9 and H10N8 subtypes but does not cross-react with current seasonal influenza subtypes. In short, approach simplifies testing algorithms and reduces costs, while also providing better diagnostic support to public health and clinical decision making. Ultimately, such approaches will increase the likelihood of early detection of potential pandemic strains.
Proactive surveillance of other global threats.
When developing an assay for a new emerging disease it is at times difficult for laboratories to obtain control material for testing due to importation restrictions. This has certainly been the case for the Middle East respiratory syndrome coronavirus (MERS-COV) which was first reported by the WHO in September of 2012. As of July 2014, WHO global case count was at 836 confirmed laboratory cases with 288 deaths. Whilst MERS-COV has not yet been reported in Australia, the potential significant public health implications have prompted QPID to have a MERS-COV assay on standby. We have previously designed a universal protocol that generates PCR positive reaction control material (called a Uni-control) without the need for the actual virus. The uni-controls are designed using two synthetic control oligonucleotides and irrelevant viral nucleic acid as an initiator template, was originally implemented to for swine flu. For MERS-COV, we have now developed PCR control material for two MERS-COV specific assays, targeting the ORF1b and the upE genes.
Fixing diagnostic problems - rotavirus detection and surveillance.
Rotavirus is a significant cause of disease with an estimated 450,000 children in developing countries dying of severe rotavirus-related gastroenteritis each year. While disease is typically less severe in developed counties such as Australia, it is still a significant cause of hospitalisations, emergency department visits, and general practice consultations. For these reasons accurate diagnosis is pivotal to ensure appropriate patient management and to ensure that vaccinations programs are performing as expected. As part of her PhD studies, Ms Sophia Ye recently investigated an unexplained increase in rotavirus positive results in a highly vaccinated population in Brisbane, where it was generally thought that rotavirus vaccination had been very successful in reducing rotavirus cases and hospitalizations. What Ms Ye found was that there was no data suggest a problem with vaccination, but rather there was a problem with the test use to diagnose rotavirus infection. She found that only 28-37% of positive results from this particular assay could be not be confirmed as positive using other testing. The results were highly suggestive of an unacceptably low specificity of this widely-used rotavirus immuno-chromatographic assay. Ms Ye has since published these findings in Eurosurveillance, and has more recently helped identify more optimal diagnostic methods that offer highly sensitive and specific detection of rotavirus detection in our population.
Teasing out the challenges for children with cystic fibrosis.
Cystic Fibrosis (CF) is the most common lethal heritable disease in the Australian Caucasian population. The Australian CF Registry (2009) estimates around 3200 Australians are living with CF. Chronic airway infection is the major complication, which causes greater than 95% of mortality in CF patients. P. aeruginosa is the most significant pathogen for these patients and is associated with recurrent pulmonary exacerbations, accelerated lung function decline, increased hospitalization, reduced quality of life and the development of respiratory failure. Although aggressive P. aeruginosa eradication therapy is successful in delaying the onset of chronic P. aeruginosa infection in children with CF, prevalence of P. aeruginosa infection increases with age. It is now also recognised that certain P. aeruginosa strains may be shared between patients with CF, including the so-called ‘AUST-01’ and ‘AUST-02’ strains, however the clinical significance of shared strains has been a matter of hot debate both nationally and internationally. As part of her PhD studies, Dr Anna Tai has used novel molecular methods to show that that the presence of shared strains per se is not a problem, but that such strains can potentially evolve into a problem. Specifically, Dr Tai has shown evidence of a highly successful multi-drug-resistant sub-clonal variant of AUST-02. Studies are ongoing but are nevertheless likely to have considerable benefit for management of patients both within the clinic and the community.
Another problem for kids with CF, has been the increase in infections due to non-tuberculosis mycobacteria (NTMs) specifically Mycobacterium abscessus complex (MABSC). The MABSC consists of M. abscessus, M. bolletii and M. massiliense. All of the MABSC are naturally resistant to most standard antibiotic regimens and may also have antibiotic resistance gene mutations that allow them to be extremely difficult to treat especially in children with CF. M. massiliense has been implicated in patient to patient transmission in people with CF and MABSC has been shown to be the dominant NTM implicated in serious clinical decline in children with CF, therefore early detection is vital. Current laboratory protocols are time consuming and expensive and QPID’s Dr Melanie Syrmis has been working alongside staff from Queensland Mycobacterium Reference Laboratory to enable faster and more accurate molecular detection methods for this complex as well as specifically for M. massiliense. Earlier detection and treatment and/or isolation of patients infected with MABSC will allow for better clinical outcomes and health management.
Direct detection of antimicrobial resistance
Effective antimicrobial treatment is an essential element in the management and control of bacterial and viral infections, yet bacteria and viruses continue to mutate and evade therapy. Antimicrobial resistance is now a global problem affecting a range of clinically important agents, and is a problem that is expected to dramatically escalate unless measures are put in place to contain it. In particular, the emergence and spread of carbapenemases such as the New Delhi metallo-β-lactamase (NDM) and the Klebsiella pneumoniae carbapenemase (KPC) represents the single biggest threat to current patient management practices globally. If left unchecked, these resistance enzymes will potentially lead the world into a post-antibiotic era. The QPID laboratory continues to be at the forefront of dealing with this emerging problem and, as recommended by the World Health Organisation, is ensuring that laboratory-based surveillance practices for antimicrobial resistance are optimised. QPID’s Dr Syrmis is also collaborating with Dr David Paterson of UQCCR (one of Australia’s leading experts in antimicrobial resistance) to develop molecular protocols to rapidly detect these important resistance mechanisms. Such technologies will provide new important tools in our ongoing battle against resistant organisms and will ultimately improve outcomes for patients.
IMPROVING OUR UNDERSTANDING OF RESPIRATORY INFECTIONS IN CHILDREN
A/Professor Ian Mackay
Respiratory viruses in exacerbations of non-cystic fibrosis bronchiectasis in children.
Respiratory viruses are frequent triggers of exacerbation in a range of respiratory diseases among children. While this is well-established in the field of cystic fibrosis, the range and role of viruses in non-CF bronchiectasis is not well known. We sought 16 different viruses and virus groups among a cohort of 69 children, who were followed for 900 child-months using PCR-based diagnostic methods. This was the first such prospective cohort study of this kind.
A virus was associated with 48% of exacerbations, with more than one virus co-occurring in only 8% of exacerbations. These children were more likely to be hospitalized and have hypoxia and fever than those with virus-negative exacerbations. The most frequently identified viral pathogen was a rhinovirus (26% of 77 exacerbations). When we genotyped the rhinoviruses, we found most were of the species Rhinovirus A; members of Rhinovirus C were least often identified. Interestingly, viruses were not as frequently identified in non-CF bronchiectasis as has been found in cohorts of children with asthma. We described these findings in more detail in Kapur and colleagues article in Archives of Diseases in Childhood.
Prospective characterization of protracted bacterial bronchitis (PBB) in children.
Protracted bacterial bronchitis, despite its name, is a disease that is also affected by the presence or absence of respiratory viruses. PBB affects young children, more boys than girls and can coincide with other structural anomalies of the central airway.
PBB has some clinical overlap with asthma however PBB responds to antibiotics therapy.
All children with PBB had bacterial infection but also two thirds had a virus detected compared to only one third of non-PBB control children. Adenoviruses featured significantly among children with PBB and they were more likely to be co-infected by Haemophilus influenzae than were adenovirus-positive control children. We employed an extended viral panel to seek other viruses from the lower airway samples of a small subset 27 children with PBB and 13 controls. Rhinoviruses were most often found in this subset (41% of cases versus 15% of controls). These findings were recently described in our Chest article by Wurzel and colleagues.
Adenovirus species C is associated with chronic suppurative lung diseases in children.
To expand our studies of human adenoviruses and PBB, we undertook genotyping studies to better understand the adenoviruses detected from among lower respiratory tract samples of children with chronic endobronchial suppuration (PBB and bronchiectasis).
We sequenced a variable and highly discriminatory portion of the adenovirus hexon gene and conducted a phylogenetic analysis to characterise the viral species and genotypes present. Of the 24 adenovirus genotyped, 96% were members of species C, specifically HAdV-1 and HAdV-2. This was not associated with a seasonal distribution. Further, specimens containing an adenovirus were more likely to be contain two or more bacterial species than specimens without an adenovirus. This was with the exception of Staphylococcus aureus which was negatively associated with adenovirus detection. Interestingly, in a subset of 52 children with extended viral testing, rhinoviruses were associated with adenovirus-positive children rather than adenovirus-negative children’s lower respiratory tract samples. These findings were recently published in Clinical infectious diseases by Wurzel and colleagues.
Respiratory virus detection in nasopharyngeal aspirate versus bronchoalveolar lavage is dependent on virus type.
Being able to determine clinical significance from the detection of a virus at a site distant from the disease is an important requirement. Most often an upper respiratory tract (URT) sample is collected and tested even when disease includes lower respiratory tract (LRT) features, because the URT is easier to sample and does not require anaesthesia or specialist input.
In this prospectively designed study we tested 150 paired nasopharyngeal aspirate (URT) and bronchoalveolar lavage (BAL) specimens. 35% of paired specimens yielded discordant results; they were positive at one site, but not the other. Rhinoviruses were not often found at both sites (mainly positive in the URT) while concurrent adenovirus detection in both URT and LRT samples was more common and, in contrast to rhinovirus detection, occurred in the presence of a low percentage of lymphocytes but an elevated BAL neutrophil numbers. The cellular infiltrate is suggestive of response to active adenovirus infection. We described these findings in more detail within Wurzel and colleagues’ article in the Journal of Clinical Virology.
Respiratory syncytial virus infection is associated with increased bacterial load in the upper respiratory tract in young children.
The study of viruses or bacteria in isolation is becoming more obviously an investment with diminishing returns. These potential pathogens do not occur this way in humans and so efforts are growing to study the interaction between them and to better capture the diversity. An analysis of 201 nasopharyngeal aspirates was undertaken to examine presence and nature of viral and bacterial co-infections.
Bacteria were significantly more often identified from the nasopharynx of ill children who were also infected with respiratory syncytial virus (RSV) than those who were not. Interestingly, RSV was significantly associated with detection of Streptococcus pneumoniae, while a rhinovirus infection was not. This study occurred during a peak RSV season, and as we know virus also associate both positively and negatively, future studies will require a longer sampling period to capture other interactions. These findings were published by Chappell and colleague in the Journal of Medical Microbiology and Diagnosis.
Deep sequencing to characterize viral genomes.
The process of determining the presence and gene or genome sequences of new, known or variant viruses has traditionally been laborious, technically demanding and time consuming.
To date we have used deep sequencing methods to characterize a human parainfluenza 4 virus variant, a divergent rhinovirus, a human parechovirus and two human enteroviruses; each from an ill child or adult. These analyses are ongoing and hope to improve our understanding of the viruses we know little about, find viruses we knew nothing about, quantify the genetic diversity among key human pathogen groups and, by observing the changes to viral genetic sequences, ensure our molecular diagnostics continue to perform with optimal efficiency.
The ORChID birth cohort Keith Grimwood, Stephen Lambert, Robert Ware, Theo Sloots, Michael Nissen and David Wang (The ORChID Study Group).
It is important to identify the health and social impacts of respiratory infections in young children, yet surprisingly little is known about the scope and acquisition of respiratory pathogens and their role in clinical disease in this patient group. To gain a better understanding of these issues, we commenced a birth cohort study to examine the acquisition of respiratory viruses and their clinical impact in children in the first two years of life (The ORChID Study). This community-based study is unique in its prospective and comprehensive approach to documenting pathogens associated with respiratory disease or use of the respiratory tract as their route of entry.
The ORChID study commenced in 2010, and to date has reached the recruitment target of 165 subjects, of which 53 have withdrawn during the course of the study leaving a retention rate of 68%. More than 14,324 respiratory swabs have been received, of which 11224 have been analysed. The testing of the nose swabs for respiratory pathogens has revealed some significant results. For example, (i) evidence shows that protection against infection is afforded by maternal antibodies in the early months of life, (ii) several infants have had positive nose swabs for various viruses in the early months but remain asymptomatic, and (iii) rhinovirus, or the “common cold” virus, is the most frequently found virus in our sample population with 19.95% of swabs positive, followed by the recently discovered MW polyomavirus with 2.98%.
Of the bacterial pathogens, Streptococcus pneumonia was most frequently present (37.9%) with Moraxella catarrhalis (21.9%) and Haemophilus influenza (10.8%) the next most frequently detected. The ORChID study is set to complete at the end of 2014.
A second focus of the ORChid study is to examine the acquisition of gastrointestinal viruses during the first 2 years of life. For this purpose, swabs of stool samples were collected at the same time as the respiratory swabs, and were stored for appropriate analysis of 7 viral pathogens. The analysis of these samples has recently commenced.
EXAMINING THE BIOLOGY AND PATHOGENESIS OF HUMAN POLYOMAVIRUSES
Dr Seweryn Bialasiewicz
Seven years ago, the QPID Laboratory was involved in discovering a novel human polyomavirus, only the fourth known up until that point in time. Since then, an additional 9 human polyomaviruses have been discovered, including MCPyV, which has been strongly implicated in the development of an aggressive form of skin cancer. Little is known about the clinical impact of most of these new polyomaviruses, which has led us to expand our research into this rapidly growing family of viruses.
The epidemiology and seroprevalence of the novel human polyomaviruses
Previously we have shown that many of these viruses can be detected in the respiratory tract of children and adults. Recent work has focussed on trying to understand the early stages of polyomavirus infection, including analysing the antibody responses of the local South-East Queensland population to these viruses in collaboration with researchers at Leiden University, The Netherlands. In this study, we found that the majority of the local population was exposed to a wide range of different polyomaviruses from a very young age, including the cancer-associated MCPyV.
Figure: Seropositivity of MCPyV, HPyV6, HPyV7, TSPyV, HPyV9 and BKPyV in the SE Queensland population. (PLOS One)
Rebecca Rockett, a PhD candidate working on the polyomavirus family, conducted a complementary project looking at the presence of polyomaviruses in the ORChiD study, which consisted of testing the nose and nappy swabs collected from children at time of birth all the way through to the age of two. Rebecca found that not only did the exposure to the novel polyomaviruses start after the first 6 months of life in line with the previous serology data, but also that in a subset of these children the polyomavirus infections were correlated with respiratory symptoms.
Polyomaviruses as agents of disease
Traditionally, polyomaviruses have been known to cause disease only in patients with suppressed immune systems, such as transplant and AIDS patients. For this reason, we specifically focussed our work on these vulnerable populations to gain a better understanding of the new human polyomaviruses’ disease burdens. In collaboration with the kidney transplant physicians at the Princess Alexandra Hospital (PAH) and the paediatric oncologists at the Royal Children’s Hospital, we have been conducting two prospective longitudinal studies involving kidney transplant and paediatric Haematopoietic Stem Cell Transplant (HSCT) patients. The aims of both of these studies is to better understand the distribution of the new viruses in these populations in various bodily sites, and to associate their detection with disease or decreased clinical outcomes. In addition to our longitudinal studies, we have also been working with the physicians from both hospitals on active cases of patient infections, which allow for real-time translation of our lab work directly into the clinic and help inform the physicians on managing these difficult patient cases. Most recently, our work with the PAH physicians on a case of a rapidly deteriorating kidney transplant patient led to a radical therapy modification which potentially saved his life, as well as to us describing a new disease associated with JC polyomavirus.
Polyomaviruses have been long suspected to be involved in human cancer, as they are able to cause tumours in laboratory animals and transform human cells in culture; yet no conclusive evidence of oncogenesis was reported until the discovery of the link of Merkel Cell polyomavirus with Merkel Cell Carcinoma. To examine the oncogenic potential of polyomaviruses generally, the QPID Laboratory has embarked on a series of investigations into the potential role of the other novel human polyomaviruses in cancer. Previously we have described the involvement and integration of BK polyomavirus into aggressive bladder cancer in kidney transplant patients. These continuing investigations have led us to establish a collaboration with the head of the Tumor Virus Molecular Biology Section, Dr. Chris Buck, at the NIH (USA) Center for Cancer Research, and Mr Clay Winterford and Dr Nigel Waterhouse at QIMR Berghofer. With their assistance, we have expanded our research into identifying the oncogenic potential of the novel polyomaviruses in breast and lung cancer.
Figure: Histochemical staining with monoclonal antibody to the polyomavirus LT antigen in adenocarcinoma tissue from a lung cancer patient
THE EPIDEMIOLOGY AND BIOLOGY OF PSEUDOMONAS AERUGINOSA IN PATIENTS WITH CYSTIC FIBROSIS
Dr Tim Kidd
Viability of Pseudomonas aeruginosa within cough aerosols generated by persons with cystic fibrosis
Person-to-person transmission of respiratory pathogens, including Pseudomonas aeruginosa, is a challenge facing many cystic fibrosis (CF) centres, but how this occurs and the transmission pathways involved remain uncertain. During our earlier work we determined that P. aeruginosa is capable of surviving in aerosols produced during coughing (Wainwright et al, 2009). These data raise the possibility of airborne transmission, however it was unclear how far cough generated aerosols travel or how long they remain viable in the airborne phase. In this study we used purpose-built equipment to measure viable P aeruginosa in cough aerosols at 1, 2 and 4-metres from human subjects and after allowing aerosols to age for 5, 15 and 45 min in a slowly rotating drum to minimise gravitational settling and inertial impaction. Nineteen patients with CF chronically infected with P. aeruginosa, and 10 age-matched healthy controls participated in the study. Viable P. aeruginosa were detected in cough aerosols from all patients with CF, but not from controls; travelling 4-metres in 17/18 (94%) and persisting for 45-mininutes in 14/18 (78%) of the CF group. Marked inter-subject heterogeneity of aerosol production was observed, suggesting that some patients may be ‘super-spreaders’ of P. aeruginosa. Sputum P. aeruginosa concentration was also a strong predictor of viable cough aerosol concentrations. In summary, this study indicates that when patients with CF harbouring P. aeruginosa cough, they produce an aerosol containing viable organisms capable of travelling 4-metres and persisting in the air for 45-minutes. These data challenge current CF infection control guidelines that remain in place in many parts of the world, including the separation of patients by 1–2 metres to prevent cross-infection by respiratory bacteria.
Epidemiology, acquisition and treatment efficacy of Pseudomonas aeruginosa among young children with cystic fibrosis
In CF, antibiotic-susceptible Pseudomonas aeruginosa isolates are acquired from an early age. Eventually, a single strain dominates and establishes a chronic infection. Chronic infections are difficult to eradicate and patients have increased morbidity and mortality. Fortunately, early diagnosis and treatment can prevent or delay the onset of chronic infection. However, uncertainties exist over the origins of initial infecting P. aeruginosa strains, especially in settings where some strains are shared by several patients, suggesting person-to-person transmission. There are also limited and conflicting data on whether re-isolated strains differ following eradication treatment, and if paired upper and lower airway isolates are genotypically indistinguishable. This information is crucial for planning eradication, surveillance and infection control strategies for CF clinics and the CF community. In this study we investigated the epidemiology of early P. aeruginosa infection in children with CF, eradication treatment efficacy, and genotypic relationships between upper and lower airway isolates among 155 patients aged ≤5-years recruited to the Australasian Cystic Fibrosis BronchoAlveolar (BAL) Lavage trial. Overall, 201/271 (74%) P. aeruginosa isolates from BAL and oropharyngeal cultures were available for genotyping, including those collected before and after eradication therapy. Eighty-two (53%) subjects acquired P. aeruginosa with most strains representing unique genotypes indicating limited cross-infection in this age group. Initial eradication success rate was 87%, but 36 (44%) reacquired P. aeruginosa. An important observation was the significant differences in upper and lower airway genotypes following eradication treatments. While detecting P. aeruginosa in BAL cultures following eradication therapy usually revealed different genotypes to the original strains, upper airway isolates detected months after treatment had generally unaltered genotypes. Overall, these data show that CF children frequently acquire environmental P. aeruginosa strains. Discordance between BAL and oropharyngeal strains also raises important questions over the possible presence upper airway infection reservoirs and how to best determine successful eradication in children with CF.
Geographical differences in first acquisition of Pseudomonas aeruginosa in cystic fibrosis
Newborn screening programs for CF enables early diagnosis and the opportunity to detect infection before the development of symptoms and significant structural changes. In this prospective study, we investigated whether geographical differences in the residence of patients with CF were associated with first acquisition of P. aeruginosa in children diagnosed after newborn screening. We performed bronchoalveolar lavage and upper airway cultures in children newly diagnosed with CF to identify infection with P. aeruginosa during infancy and early childhood. Children were assessed according to their residence in a regional or metropolitan area. Multilocus sequence typing was used to determine P. aeruginosa genotype and an environmental questionnaire was also administered. Our analysis revealed that children living in regional areas were more significantly likely than metropolitan dwellers to acquire infection with P. aeruginosa. Most strains were also associated with environmental genotypes suggesting that the increased risk of acquiring P. aeruginosa in regional areas may be due to exposure to a greater burden of environmental P. aeruginosa organisms. Overall, we showed clear geographical differences in the first acquisition of infection with P. aeruginosa in infants with CF. This study and other recent studies, one including two CF cohorts from Australia, suggest that the climate and local environment play a significant part in predicting early infection with this important organism that is known to be associated with significantly worse early lung disease in CF.
The social network in cystic fibrosis centre care and the risk of shared Pseudomonas aeruginosa strain infection
During a recent national prevalence study of 983 Australian patients with CF two predominant Pseudomonas aeruginosa genotypes, AUST-01 and AUST-02, were identified in 22% and 18% patients, respectively (Kidd et al, 2013). These findings indicated widespread person-to-person transmission between within and between Australian CF centres. However, it was unclear whether patient movement plays a role in the distribution and prevalence of these strains. In this study we used social network analysis to explore prior patient movement between Australian CF centres and other hospitals, and to describe and visualise the network characteristics of all patients, those with AUST-01 and AUST-02 infection, and adult versus paediatric patients. Overall, we observed a highly complex web of interactions between Australian CF centres and other healthcare facilities. The proportion of AUST-01 and AUST-02 infections in patients treated at multiple hospitals was significantly higher than patients treated at individual centres. Adult patients were also more likely to have had contact with other hospitals than paediatric patients. High level network connectivity between Australian CF centres suggests the potential for widespread P. aeruginosa cross infection. Capturing patient-hospital network connectivity improves our ability to explain the potential transmission of dominant shared P. aeruginosa stains. Our data highlights that social network analysis may be useful for the implementation of targeted infection control interventions and identifying previously unrecognised transmission pathways.
Motility and biofilm production of cystic fibrosis and environmental Pseudomonas aeruginosa
In the CF airway Pseudomonas aeruginosa uses motility mechanisms for adherence and biofilm formation. There are few studies which compare P. aeruginosa motility and biofilm formation among isolates from CF patients and other ecological niches. It is also unclear if shared (genotypically indistinguishable) and unique strains of P. aeruginosa from CF patients display similar motility and biofilm capabilities. In this study we sought to compare the motility characteristics and biofilm formation capabilities of shared and unique strains P. aeruginosa strains cultured from the CF airway and isolates collected from other ecological settings. Motility (swim, swarm and twitch) and short-term microtitre based biofilm formation assays performed under aerobic, microaerophilic and anaerobic conditions were determined for isolates collected from CF patients (n=60, including AUST-02 [n=26]), animal infections (n=14), non-CF clinical infections (n=20) and the environment (n=34). Environmental isolates displayed higher rates of motility and biofilm formation in all three atmospheric conditions when compared to all other isolates. In contrast, strains cultured from CF patients displayed the lowest rates of motility and biofilm production when compared with isolates from other ecological settings. In particular, the shared AUST-02 strains demonstrated significant reductions in motility and biofilm formation when compared to environmental isolates. These studies may provide important clues as to why some strains of P. aeruginosa are more transmissible than others. Further long-term biofilm and mechanistic studies are now being conducted to investigate the significance of these findings.
Multidrug resistance and microevolution of Pseudomonas aeruginosa in cystic fibrosis
Pseudomonas aeruginosa is the most common pathogen causing chronic airway infection in CF. During the course of chronic airway infection, P. aeruginosa develops in vivo adaptive genetic mutations. AUST-02 is the predominant Queensland strain but its potential for intra-strain diversification is unknown. In this study we sought to assess intra-strain diversity, antimicrobial resistance of AUST-02 isolates collected from the Royal Children’s and The Prince Charles Hospital (TPCH) CF centers between 2001 and 2008. Each isolate was subjected to mexZ and lasR gene sequencing and broth microdilution susceptibility testing. Prospective molecular surveillance for AUST-02 variants was thereafter conducted among patients attending the TPCH in 2007 and 2012. Overall, we showed temporal and spatial diversification of the AUST-02 strain over time with isolates from 2007/08 showing more genetic mutations than those collected in 2001/02. Most patients harboured AUST-02 with unique mexZ/lasR sequence types. However, a new multidrug resistant AUST-02 variant associated with cross-transmission and poor clinical outcome was detected in some patients. These data provide evidence of ongoing cross-infection and the ongoing adaptation of highly successful P. aeruginosa strains in the CF airway.
Phenotypic screening and epidemiology of carbapenemase producing Pseudomonas aeruginosa in cystic fibrosis
Carbapenems are a powerful group of broad-spectrum antibiotics which are often used as a last resort treatment against multi-resistant Pseudomonas aeruginosa strains. However, over the past decade acquired resistance to these antibiotics (i.e. via the acquisition of genes encoding carbapenemase enzymes) has been increasingly reported. Despite this there are currently no carbapenemase screening guidelines that exist for P. aeruginosa. In this study we evaluated a range of carbapenemase screening tests among 80 P. aeruginosa isolates collected from persons with CF. Our analysis revealed high false positive rates among the most commonly used phenotypic screening methods. In contrast, real-time PCR and the Carba NP test demonstrated superior specificity compared to other phenotypic tests correctly identifying all true positive and negative isolates and controls. Information gleaned from these studies can be used for developing a cost effective and reliable approach to carbapenemase surveillance. In another investigation we sought to determine whether carbapenemase producing P. aeruginosa isolates were present among a large cohort of Australian patients with CF. The isolates were tested by real-time PCR targeting a range of common carbapenemase and acquired resistance genes. In summary, no carbapenemase genes were identified among any of the clinical isolates tested. These data provide the broader clinical community with a reassurance that to date there have be no widespread epidemics of carbapenemases among the Australian CF population.
Epidemiology of Burkholderia cepacia complex infection in cystic fibrosis
Burkholderia cepacia complex organisms are important transmissible pathogens found in patients with CF. In recent years, the rates of cross-infection of epidemic strains have declined due to effective infection control efforts. However, cases of sporadic B. cepacia complex infection continue to occur in some centers. The acquisition pathways and clinical outcomes of sporadic B. cepacia complex infection are unclear. In this study sought to determine the patient clinical characteristics, outcomes, incidence, and genotypic relatedness for all cases of B. cepacia complex infection at the Royal Children’s and The Prince Charles Hospital CF centers. We also sought to study the external conditions that influence the acquisition of infection. From 2001 to 2011, 67 individual organisms were cultured from the respiratory samples of 64 patients. Sixty-five percent of the patients were adults, in whom chronic infections were more common. The incidence of B. cepacia complex infection increased by a mean of 12% per year. The rates of transplantation and death were similar in the incident cases who developed chronic infection compared to those in patients with chronic P. aeruginosa infection. Multilocus sequence typing revealed 50 individual strains from 65 isolates. Overall, 85% of the patients were infected with unique strains, suggesting sporadic acquisition of infection. The yearly incidence of nonepidemic B. cepacia complex infection was positively correlated with the amount of rainfall. In summary, this study demonstrated that despite strict cohort segregation, new cases of unrelated B. cepacia complex infection continue to occur. These data also support an environmental origin of infection and suggest that climatic conditions may be associated with the acquisition of B. cepacia complex infections.
Microevolution of Burkholderia pseudomallei during chronic infection
Some bacterial pathogens establish long-term infections that are difficult or impossible to eradicate with current treatments. Rapid advances in genome sequencing technologies provide a powerful tool for understanding bacterial persistence within the human host. Burkholderia pseudomallei is considered a highly pathogenic bacterium because infection is commonly fatal. Here, we used whole-genome sequencing to investigate within-host evolution of B. pseudomallei in a unique case of human infection with ongoing chronic carriage. Genomic comparison of isolates obtained 139 months (11.5 years) apart showed a strong signal of adaptation within the human host, including inactivation of virulence and immunogenic factors, and deletion of pathways involved in environmental survival. Two global regulatory genes were mutated in the 139-month isolate, indicating extensive regulatory changes favoring bacterial persistence. This study provides important insights into B. pseudomallei pathogenesis and, more broadly, identifies parallel evolutionary mechanisms that underlie chronic persistence of all bacterial pathogens.
Prevention of Infection
The first step in ameliorating infectious disease is to limit or prevent the infectious process through the application of effective antimicrobial compounds to inhibit microbial replication, or ultimately to prevent the infectious process completely through vaccination of the population at risk. In this context, the QPID laboratory participates in preventative based research studies involving external collaborative organisations.
Evaluating anti-malarial drug efficacy
We have developed a range of novel quantitative PCR assays which increase the sensitivity of detection of blood-stage malarial parasites by at least a hundredfold compared with traditional microscopy. These assays have been used extensively in anti-malarial drug trials, and have been successfully applied to demonstrate quantitative changes in parasitaemia.
To date the QPID laboratory, in collaboration with The Clinical Tropical Medicine Laboratory, QIMR Berghofer, and Q-Pharm, has performed twenty clinical trials to monitor the anti-parasitic drug efficacy in healthy volunteers infected with P.falciparum. The result of these trials contributes significant information for the design, administration and introduction of new anti-malarial treatments globally.
In an extension of this work, the QPID laboratory is now engaged in monitoring anti-malaria drug efficacy in patients infected in the field, in South-East Asia, with wild type P.falciparum. This study is a collaboration between the QPID laboratory, QIMR Berghofer and Novartis Pharma AG. This study is ongoing.
Developing a Q fever vaccination strategy in children
Q fever is a vaccine-preventable zoonosis caused by Coxiella burnetii and is primarily considered as a disease of adults residing in rural Australia, particularly those in close contact with livestock. Recent research outcomes show that subjects from urban Southeast Queensland, including children, have evidence of previous exposure to the bacteria at a higher rate than previously assumed. Also, these results show that the likely source of exposure are domestic animals. However, knowledge of the disease in children is extremely limited, and a vaccination strategy for paediatric subjects does not exist. Based on the results generated from this study, an effective vaccine intervention strategy can now be developed thereby limiting the threat of more serious or chronic infections. Studies examining the genetic diversity of bacterial strains circulating in the environment, and the immune response to exposure to the bacteria are continuing as part of this research program, and will be paramount in designing vaccines that are effective in our local population.
Figure: Notification rates of Q-fever in Queensland since 1984
Assessing the population coverage of a new meningococcal B vaccine
A multicomponent vaccine (4CMenB) has been developed for the prevention of invasive serogroup B (MenB) meningococcal disease by Novartis. The vaccine is composed of three protein antigens (fHbp, NHBA and NadA) and Outer Membrane Vesicles (OMVs) of a New Zealand MenB strain. A Meningococcal Antigen Typing System (MATS), was applied in the QPID laboratory to evaluate strain coverage by the vaccine, using MenB isolates collected from January 2009 to June 2011 by five National Neisseria Network reference laboratories in Australia. The results to date show that the overall estimate of strain coverage is 76% (95%CI) and that the 4CMenB vaccine has the potential to protect against a majority of the Meningococcal B strains that have caused invasive disease in Australia. Recently additional isolates have become available from the Public Health laboratory in Victoria, and these are currently being tested. Further historical testing for non-Men B isolates is being completed.
COLLABORATION IN INTRA-INSTITUTE STUDIES
As part of a wider collaborative agreement with other research groups in the Queensland Children’s Medical Research Institute, the QPID laboratory has participated as investigators in clinical research studies of respiratory disease in children. These were:
Professor Anne Chang - Cough and Asthma Research Group
- Bronchiolitis (ABIS 1 & 2) study
- Central Australia pneumonia study
- Bronchiectasis BAL study.
Doctor Kerry-Ann O’Grady - Respiratory infection Outreach and Research Team
- ARI- Department of Emergency Medicine Cough study
- Central Australia swab study
Professor Peter Sly - Children's Lung, Environment and Asthma Research
- RELX study – a multicenter RCT testing the efficacy of omalizumab on asthma exacerbations
- AVB study – Examining the protective immunity in special populations
- COMBAT study – a RCT examining azithromycin in the prevention of bronchiectasis in infants with CF.
The operation of the QPID laboratory continues to be generously supported by the Children’s Health Foundation through the QCMRI Research Programme and the Woolworths “Fresh Food Kids” Hospital Appeal. QPID is also supported by external research grants from the NHMRC, ARC and NIH. Additional support is received from Pathology Queensland and Queensland Health.