BACTERIAL EPIDEMIOLOGY

Bacterial Epidemiology, Surveillance & Diagnostics

Personnel – Group leaders Professor Lyn Gilbert & Dr Vitali Sintchenko: Dr Fanrong Kong (CIDM staff); Dr Qinning Wang & Dr Matthew O’Sullivan (CIDM-PH, fellows); Danny Ko (NHMRC grant; research assistant); Dr Frank Lin (UNSW PhD student; NHMRC grant, research assistant), Michelle McKechnie (CIDM-PH PhD students); Mitchell Brown & Shahin Oftadeh (NSW Pneumococcal Reference); Heather Gidding (CIDM-PH epidemiologist); visiting scholars from various parts of  China (including Beijing, Wuhan, Tianjin, Shenzen, Shanghai and Hangzhou) - Hui Wang, Ying Sun, Lin Ma, Xianyu Zeng, Zouotao Zhau; Yajaun Wang; Yongwei Cai; Likuan Xiong; Fei Zhou; Zhonsheng Tong; Xiaoyou Chen; Xiaoyong Zhou)   .

 

Development of multiplex molecular diagnostic and typing systems

The study of sequence-based molecular epidemiology of pathogens of public health importance is a major focus of our research. We have developed a series of multiplex PCR-based reverse line blot (mPCR/RLB) assays to facilitate these investigations. They assays allow detection of up to 40 targets, simultaneously, and have been developed– for: a) genotyping and/or antibiotic resistance profiling of group B streptococcus (GBS), Streptococcus pneumoniae (23 polysaccharide vaccine serotypes; and less common serotypes; antibiotic resistance profile), Staphylococcus aureus, Chlamydia trachomatis, Bordetella pertussis, Salmonella Typhimurium, human papilloma virus; b) for identification of antibiotic gene target mutations that predict resistance in M. tunerculosis; c) for species identification of Mycoplasma and Mycobacterium spp.; and d) for simultaneous detection, in clinical specimens, of multiple causes of bacteraemia, urethritis and pneumonia.

Many of these assays were developed with the assistance of visiting Chinese scholars, who are now using them in their own laboratories in China; two are being used by scientists from the major New Zealand public health laboratory, who collaborated in their development. In future, they will be adaptable to microarrays; our GBS and S.pneumoniae serotype identification systems have used to develop prototype microarrays, by our collaborators at the Tianjin Biochip Corporation (TBC), with whom we share a patent. Other assays for diagnostic use will be adapted to faster, real-time PCR platforms. In 2006 we were invited to submit the protocol for development of mPCR/RLB assays to Nature Protocols (Kong & Gilbert, 2006).

Other studies in progress by this group include – molecular epidemiology and identification of Nocardia species; molecular epidemiology of the S. pneumoniae penicillin binding protein gene pbp2b. We have recently collaborated with others to identify an new serotype of group B streptococcus and to genotype strains of group B streptococcus causing disease in crocodiles and fish. We have independently identified a putative new S. pneumoniae serotype.

 

Development of informatics tools to interrogate and analyse data.

In collaboration with the Centre for Health Informatics (CHI), University of NSW, we are developing a program in health informatics to enhance the value of laboratory-generated data. Machine-learning algorithms are used to interrogate complex molecular data generated by genotyping systems and integrate them with clinical and epidemiological data, to develop risk assessment and decision support tools to improve clinical management and epidemiological investigations.

 

Major specific research projects and competitive research grants.

Translating bacterial molecular epidemiology into information to improve infectious disease risk assessment and control (NHMRC grant #358351; 2005-7) This project involves collaboration between CIDM-PH (Gilbert, Sintchenko, Kong), CHI and the CRC for Diagnostics, Queensland University of Technology, to study the molecular and clinical speidemiology of group B streptococcal and pneumococcal infections. It involves the development of novel molecular typing methods for group B streptococcus and Streptococcus pneumoniae and machine learning algorithms that integrate molecular, clinical and epidemiological data to develop decision support systems for clinicians and public health practitioners. Research assistants on the grant are Dr Frank Lin and Danny Ko with assistance from Mitchell Brown and Shahin Oftadeh. 

 

Optimisation of Salmonella genotyping and epidemiological data analysis for detection and investigation of outbreaks (NHMRC project grant # 457472; 2007-9). This project involves collaboration between CIDM-PH (Gilbert, Sintchenko, Kong) and microbiologists from Universities of Sydney and NSW and the Institute of Medical and Veterinary Science (IMVS), Adelaide. It involves the development of systems for rapid molecular typing of Salmonella Typhimurium and other serovars, for surveillance and outbreak identification. It will improve our understanding of the evolution, distribution and virulence of pathogenic salmonellae. We will also develop a spatiotemporal cluster identification system to provide early warning of outbreaks and more timely recognition and source identification of foodborne outbreaks. Dr Qinning Wang has been responsible for much of the early development of these methods. Rearch assistants and a PhD student (based at IMVS) will also contribute to this project. A prototype mPCR/RLB phage type-identification system has been developed and has already been used, successfully, in combination with multilocus variable number tandem repeat analysis (MLVA), to investigate several outbreaks of salmonellosis in NSW over the past 6 months.

 

Molecular typing and epidemiology of Bordetella pertussis in Australia (NHMRC project grant # 455308). This project also involves collaboration between CIDM-PH (Sintchenko, Gilbert) and colleagues at Universities of Sydney and NSW. We will develop a new culture-independent typing method for rapid molecular typing of Bordetella pertussis in respiratory samples and detailed investigation of the molecular epidemiology and evolution of B. pertussis, worldwide. This study will assist in maintaining a continuous short-term surveillance of circulating B.pertussis strains and improve its public health control in Australia.

 

Informatics approaches to improving risk assessment and response to outbreaks of communicable diseases ARC Linkage Grant #LP0667531. This project involves CIDM-PH Chief (Sintchenko) & Partner (Gilbert, Dwyer, Gidding) Investigators and colleagues from CHI and NSW Department of Health.. This project will develop and test a new model of outbreak detection and risk assessment based on coordinated syndromic and laboratory diagnostic surveillance.

 

Other projects, which have been completed in the past 3 years or are underway (supervised by Professo Gilbert and/or Dr Sintchenko):

• Surveillance of invasive pneumococcal disease, which involves serotyping of all invasive S. pneumoniae isolates from NSW (funded by Commonwealth Department of Health and Aging), is complemented by our pneumococcal genotyping project (see above). Antibiotic susceptibility testing against ~15 agents, of more than 2000 invasive pneumococci over a period of 5 years (funded by Wyeth (Australia).
• Development of mPCR/RLB assays to identify antibiotic resistance genes in Gram positive cocci (groups B streptococcus and S. pneumoniae – including common mutations of penicillin binding protein genes – pbp2b- and S. aureus) and Mycobacterium tuberculosis.   
• Prospective surveillance of S. aureus bacteraemia (community and healthcare associated, methicillin resistant and susceptible) using a novel molecular typing system, (BSc Honors project, Kathryn Shaw). Development of a rapid MRSA typing systems (mPCR/RLB assays to identify SCCmec  types/subtypes, toxin genes and antibiotic resistance genes; spa typing and PFGE) for use in hospital infection control (Supervisors: Gilbert & Sintchenko; researchers Qinning Wang & Matthew O’Sullivan). The use of “real-time” molecular typing of MRSA in infection control is the subject of a NHMRC project grant application for funding in 2008-10.
• Epidemiology and molecular typing of M.tuberculosis in NSW. Linh Nguyen (PhD student; supervisor Gilbert;); Peter Jelfs (CIDM) and Dr Vitali Sintchenko

Prospective study of causes of urethritis. Michelle McKechnie (PhD student, supervisors, Hillman, Gilbert) - in collaboration with Parramatta and Sydney Sexual Health Clinics and Sexually Transmisssible Infections Research Centre (STIRC).

MYCOLOGY RAPID DIAGNOSTICS

Rapid Diagnostics and Epidemiology of Nosocomial and Opportunistic Fungal infections

Personnel: Group Leader – Professor Tania Sorrell; Dr Geoffrey Playford (PhD student, Princess Alexandra Hospital, Brisbane); Dr Sharon Chen (CIDM); Dr Catriona Halliday (CIDM); Dr Anna Lau (PhD Student), Ms Namfon Pantarat, Dr Nicole Gilroy (CCRE IBHM), Professor Philip Kuchel (University of Sydney), Dr Jennifer Bodkin (June 05-Jun 06) & Dr Ronda Plummer (Mar 06-May 07), Molecular and Microbial Biosciences, University of Sydney) and visiting Chinese scholars (see 2.1 above).

 

Novel fungal diagnostics

a. Reverse line blot assays

Research group: Sorrell, Kong, Halliday, Playford, Sun, Wang.

Funding: NHMRC CCRE Program grant #264625

An RLB assay for simultaneous identification of medically important fungal species Candida, Aspergillus and Cryptococcus has been developed. This method is relatively inexpensive, sensitive and specific and has the advantage that it can identify mixed cultures. In general RLB is best suited to epidemiological studies (see Bacteriology section) but provides more rapid fungal species identification than conventional culture and phenotypic methods. Our visiting Chinese scholars plan to introduce our syndrome-or pathogen group-specific RLB assays into laboratories in China. We are currently adapting some primers developed for RLB to faster real time PCR or micro-array platforms (see below).

 

b. Multiplex tandem PCR (MT-PCR)

Research group : Sorrell, Lau (PhD project), Halliday, Chen, in collaboration with Iredell, Corbett Diagnostics, Sydney

Funding : NHMRC post-graduate student scholarship (Lau), WMI stipend top-up grant (Lau), NHMRC CCRE #264625, Corbett Diagnostics (in-kind support)

This project is initially targeted to rapid identification of fungi in cultured blood and in blood samples of patients with suspected bacteremia/fungemia. In practical terms, a platform for screening blood cultures which flag positive in laboratory automated systems for bacteria or fungi will be tested. Multiple specific primers will be used to simultaneously identify broad groups (eg fungi or bacteria) and up to 70 different pathogenic species. Blood collected up to 7 days before a positive sample is obtained will also be tested. The MT-PCR platform will subsequently be developed for fungal species that cause tissue/biofluid infection (a different spectrum from those which cause positive blood cultures). Once piloted and validated our intention is to add primers for detection of antifungal resistance determinants.

 

c. Phenotype arrays: biochemical profiling by nuclear magnetic resonance (NMR) spectroscopy for rapid identification of pathogens in cultured blood

Research group: Sorrell, Himmelreich, Kuchel, Sintchenko, Panatarat, Bodkin, Plummer

Funding: Merck, Sharpe and Dohme, US (Medical School grant).

We are evaluating NMR spectroscopy as a tool for rapid detection of organisms growing in blood cultures. Sample collection is ongoing and NMR spectra will be analysed using pattern recognition and multivariate statistical techniques (Sintchenko). It is anticipated that different bacteria and fungi will be distinguished by unique biochemical profiles on NMR spectroscopy. No processing of samples is required for NMR analysis (except addition to PBS-deuterated water in an NMR tube) and the technique can be automated, hence it is potentially extremely fast. Results will be compared with those obtained by MT-PCR.

 

d. Antifungal resistance testing by nuclear magnetic resonance (NMR) spectroscopy

Research group: Sorrell, Himmelreich, Kuchel, Pantarat, Bodkin, Plummer

Funding: NHMRC CCRE #264625; Merck, Sharpe and Dohme, US (untied Medical School grant).

We have established and published (Coen et al, 2006) the potential of NMR spectroscopy for rapid susceptibility testing of antifungal drugs (also presented at International Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, Dec 2005 and Ibid, San Francisco, Sep 2006). This works builds on our earlier feasibility study of the use of NMR spectroscopy in the diagnosis of meningitis (published 2005) .

 

Fungal disease epidemiology and prediction of risk – hospital epidemiology             

a. Candida infections in the Intensive Care Unit

Research group: Playford (PhD student), Sorrell, Lau (PhD student), Halliday, Chen, Iredell, ICU      clinicians, national collaborators

Funding: NHMRC post-graduate scholarship (Lau), Pfizer US, (NHMRC project application pending)

The National Candidemia study (funded by Pfizer US & Australia) [Chen, 2006] revealed that about 25% of all cases of candidemia arise in critically ill patients in intensive care units. Better strategies for prevention and early diagnosis and treatment are needed to improve outcomes in this group. Molecular studies have shown that most cases are endogenous though some are caused by in-hospital transmission. Clinical and Candida colonisation data will be collected to derive and validate a simple, robust and prospective risk predictive model for invasive candidiasis, which will incorporate serial determination of clinical risk factors, and quantitation and speciation of fungal colonising flora. Commencement is planned in May 2007.

 

b. Epidemiology of invasive fungal infections (IFIs) in patients with haematological malignancies and recipients of haematopoietic stem cell transplants

Research group:  Sorrell, Gilroy, Kabir, Gidding, Hale (PhD student), Isaacs, MacIntyre

Funding: NHMRC post-graduate scholarship and WMI stipend top-up (Hale); Pfizer Australia, NHMRC CCRE #264625, Gilead Sciences

In collaboration with the CCRE in Infections Bioethics in Haematological Malignancies (IBHM), a cohort study of risk factors for invasive fungal infections in adult and paediatric haematology patients is underway. We have added fungal infections to the National Bone Marrow Transplant Register to measure the incidence of fungal infections and inform studies to improve patient management and outcomes. Key collaborations with colleagues in Melbourne, Brisbane and New Zealand have been established to facilitate epidemiological studies of fungal infections and through the CCRE-IBHM to develop evidence based guidelines for prevention and management of nosocomial and invasive fungal infections.

ANTIBIOTIC RESISTANCE

Antibiotic Resistance, Rapid Diagnostics and Nosocomial Infections

Personnel – Group Leader Associate Professor Jonathan Iredell; Dr Sally Partridge (senior research fellow); Ahmad Khawaldeh (clinical research fellow); Damla Power/ Belinda Dillon (research assistants); Kiran Thapa (research assistant); Lee Thomas (former CIDM-PH scholar; CIDM scientific officer); Bjorn Espedido (PhD student); Jubelle Valenzuela (PhD student); Andrew Ginn (PhD student); Zong Zhidong (PhD student); Heather Gidding (CIDM-PH epidemiologist)

 

Screening for antibiotic resistance markers.

A longstanding interest in the molecular mechanisms of antibiotic resistance in Gram negative bacteria and their implications for clinical management has led to the identification of a novel (for Australia) carbapenemase resistance mechanism in hospital patients in western Sydney and analysis of the genetic basis of the two most important resistant Gram-negative pathogens in Australia, resulting in several publications and presentations.

            This basic research led to development of molecular screening methods for identification of resistance markers in Gram negative bacteria (independent of species) in the normal flora of hospitalised patients. This is being used for long term surveillance and epidemiological studies of the effects of various interventions on the emergence and prevalence of antibiotic resistance especially in ICU. It has allowed earlier recognition of multiresistant bacteria and implementation of control measures sooner than would otherwise have been possible. In western Sydney, where screening has been in place for the past 24 months, multiresistant Acinetobacter and Enterobacteriaceae were eradicated from the hospital microflora. By contrast, in Melbourne, where genetic screening was not introduced these organisms continue to cause infections and directly attributable deaths.

            Our group has now identified the genetic basis for trasmissible resistance to carbapenems, fourth-generation cephalosporins, and the principal aminoglycosides in Gram-negative bacteria in two major Australian ICUs and incorporated these into diagnostic assays for clinical trialling in 2007-8. In addition, we have discovered and characterised the genetic basis of an epidemic of the highly transmissible CTX-M-15 ESBL enzyme, as well as a novel CTX-M variant. Data from these projects have been presented at international conferences (ICAAC) and are being prepared for publication.

            A large prospective collection of specimens (and relevant clinical data) from patients in several ICUs (Westmead, Royal Brisbane Hospital and Alfred Hospital, Melbourne) during periods of antibiotic cycling, is complete. This work is supported by a grant from Australia & New Zealand Intensive Care Society Foundation (ANZICS grant 04-5 Schedules Antibiotic Cycling in ICU) and seeding funds from the US National Institutes of Health (APUA/ROAR2 2005), with follow-up grant from ANZICS (grant 05-6 “RAPID”), in which newly developed rapid diagnostic techniques will be used and their impact on critical care evaluated. Preliminary analyses have shown an association with increased infection by antibiotic-resistant pathogens in ICU by the widely used drug, cefepime, when compared with the antipseudomonal PNC/ β-lactamase inhibitor combinations.

This work involes collaboration with Peking Union Medical College and the University of Western China,.

  

Rapid high throughput diagnostics

Rapid, simple methods for identification of blood borne pathogens have been developed and will soon be introduced into the routine diagnostic laboratory. The effect on antibiotic prescribing practice, of earlier diagnosis of the cause and antibiotic susceptibility of blood stream infections, will be evaluated prospectively (with funding from ANZICS Foundation – see above).

A novel method developed by colleagues at Corbett Research Pty Ltd – multiplex tandem PCR - will be adapted for rapid, simultaneous identification of large numbers of blood-borne and respiratory pathogens, including all avian and human subtypes and major antiviral resistance genes of influenza A virus as well as other major viral pathogens.  This project is funded by grants from AusIndustry (to Corbett Research Pty Ltd – 2004 START grant), ANZICS Foundation (RAPID grant, Iredell CI) and NHMRC project grant # 402870 “Novel, high-throughput platform for rapid identification, quantitation, differential diagnosis and resistance testing of influenza”. Working assays have been generated for respiratory diagnosis (influenza A-H1, -H3, and -H5, influenza B, and RSV) as well as for detection of neuraminidase-inhibitor resistance in influenza A, and these are now available (http://www.ausdiagnostics.com/qilan/Products). A recent survey uncovered an unrecognised resistant mutant strain in untreated clinical cases in Australia (unpubl. data), and clinical trials have begun at Westmead Hospital.

 

Other projects

• BioSignal Australia, in collaboration with the Iredell group, won a $1.5 million AusIndustry ICIP grant (Antimicrobial coatings and materials for biomedical devices http://www.ausindustry.gov.au) to study the efficacy of novel furanone compounds delivered on plastics (e.g. endovascular catheters) to prevent nosocomial infections. JI is a member of a 6-person international scientific advisory board which met for the first time in February 2007.
• Novel antibacterial activities: recent work has shown the value of certain ion-channel blocking agents in the treatment and eradication of key infections in cystic fibrosis patients. This work is being done in collaboration with Dr Peter Middleton (Westmead Hospital Respiratory Medicine), Antony George (UTS Molecular and Cell Biology) and Fred Widmer (CIDM) and is the subject of an NHMRC grant application for funding for 2008-10.
• An ARC Linkage grant (~$800,000) was awarded (JI is one of 5 CIs) in 2007 to fund a three-year program investigating the role of laser optics and novel immunofluorescence methods in microbial diagnostics.

A collaboration has been established with Special Phage Services to investigate bacteriophage therapeutics and is the subject of funding proposals in late 2007.

MYCOLOGY

 
Molecular Mycology

Personnel; Group leader: Associate Professor Wieland Meyer; Dr. Clement Tsui (Senior Post-doctorial Research Fellow), Dr. Alejandro Jover-Botella (Post-doctorial Research Fellow), Krystyna Maszewska (Technical Officer), Natalie van de Wiele (Visiting Research Assistant), Popchai Ngamskulrungroj (PhD student), Luciana Trilles (PhD student), Sirada Kaocharoen (PhD student)

 

Molecular typing of fungal pathogens

Research group : Meyer, Tsui, Maszewska, Wiele   

Funding : NHMRC Project grant # 352303

A sequenced-based molecular typing project focuses on identification of pathogenic fungi, especially the yeast genera Candida and Cryptococcus (NHMRC project grant #352303 Phylogeny as a basis for molecular identification of pathogenic fungi) is in its third year. This project involves the establishment of an accurate phylogenetic classification system for ascomycetous yeasts as a basis for the development of fast and reliable molecular identification methods from clinical specimens and pure cultures. Specific software modules within the BioloMICS program have been developed, to analyse multilocus gel patterns (such as PCR-fingerprinting or AFLP data). During the last year the phylogenetic relationships between the sexual and asexual Candida specie s have been improved by sequencing three additional genes RPB1 and RPB2 as well as the COX2 gene in addition the ACT1 and LSU gene and ITS1 and 2 region of the ribosomal rDNA gene cluster. The molecular species borders in yeast have been studied by sequencing of multiple clinical strains of one species. This will provide better cut off values for sequence based molecular identification. 

 

MLST and MLMT typing of the Crytococcus species complex

Research group : Meyer, Jover-Botella, Tsui, Maszewska, Ngamskulrungroj (PhD project), Trilles (PhD project), Kaocharoen (PhD project) in collaboration with Prof. E. Castaneda, Bogoda, Colombia, Dr. B. Wanke FIOCRUZ, Rio de Janeiro, Brazil   

Funding : University of Sydney Bridging grant, WMI stipend top-up grant (Ngamskulrungroj). Project GrantColciencias Bogota, Colombia, NH&MRC project grant application pending

A new multi-locus sequence typing (MLST) system, based on 6 genes (CAP59, LAC1, PLB1, URA5, IDE, IGS1) has been established for the Cryptococcus species complex. In addition a multilocus microsattelite typing (MLMT) system including 12 polymorphic loci was established for the Cryptococous species complex, this will allow for a quick and international standardised identification of those recently in the UK’s biological agents list newly listed species, which list Cryptococcus neoformans as one of only eight fungi which could potentially be used as a bioterrorist agent.

 

Quality controlled ITS sequencing database

Research group: Meyer, Tsui, Maszewska, Wiele in collaboration with Kong (CIDM), Chen (CIDM) & Halliday (CIDM).

Funding : NH&MRC project grant # 352303

A quality controlled sequence database has been established to store and share data on internal transcribed spacer (ITS) region sequences of more that 200 fungal type strains and clinical isolates. This database is available at the Molecular Mycology Research Laboratory (Sydney/Australia) and at the Centraalbureau voor Schimmelcultures (Utrecht/The Netherlands) webpages.

 

PCR-fingerprinting database-BioloMICS

Research group: Meyer, Maszewska, Wiele in collaboration with Dr. V. Robert (CBS, Utrecht, The

Netherlands)

Funding: NH&MRC project grant # 352303, CBS funding

A PCR-fingerprinting database of pathogenic yeasts has also been established as part of a new online fungal identification database using the BiloMICS program. These online databases are intended to provide remote access to reference sequences and profiles, to assist identification of fungal pathogens, by clinical diagnostic and research laboratories, without the need for reference tools.

 

Molecular epidemiology of Scedosporium

Research group: Meyer, Maszewska, Harun (PhD student), Chen (CIDM) and the Australian Mycology Interest Group and Dr. L. Delhaes (Pasteur Institute, Lille, France)

Funding : CIDM-PH one year seed funding, Merck, Sharp & Dohme, PhD scholarship from the Universiti Sains Malaysia, Malaysia (Harun)

Based on the Australian Scedosporium Study, we investigating the molecular epidemiology of Scedosporium species in Australia, using PCR-fingerprinting, AFLP and RFLP analysis and MLST.

 

Rolling circle amplification for the differentiation of species of the Crytococcus species complex

Research group : Meyer, Kaocharoen (PhD project), Tsui; Kong (CIDM), Bin Wang (WMI)

Funding: University of Sydney Bridging Grant

A new diagnostic tool, Rolling Circle Amplification (RCA), for the identification of the species and varieties of the Cryptococous species complex has been developed.

 

Other projects:

• Molecular phylogeny of pathogenic fungi and participation of the international research project AFTOL (Assembling the Fungal Tree of Life)
• Molecular Techniques for the Identification of Pathogenic Fungi
• Histone locus provides a target for rapid identification of Candida species in collaboration with an industry partner (MicroBioGen, Sydney, Australia)
• Development of a multilocus sequence typing (MLST) Scedosporium spp.
• Survey of simple sequence repeats in completed fungal genomes
• Ongoing speciation within Cryptococcus species complex
• Functional diversity within the human pathogen Cryptococcus neoformans
• Global molecular epidemiology of the Cryptococcus neoformans species complex in collaboration with colleagues in Brazil, Colombia, Greece, Thailand, The Netherlands and Japan; the aim is to predict potential outbreak hot spots and establish an early warning system to prevent or contain them
• Determination of fertility in global isolates of Cryptococcus gattii
• Has Cryptococcus gattii evolved into a more virulent genotype? Cryptococcus gattii molecular type VGII - a new emerging pathogen in temperate climates

Molecular investigation of virulence factors of pathogenic fungi using Cryptococcus gattii as model system, including gene knockout and complementation experiments and animal virulence studies

VIROLOGY

Rapid Diagnosis and Molecular Epidemiology of Viral Infections

Personnel: Group Leader: Dr Dominic Dwyer, Mala Ratnamoham (CIDM), Ken McPhie (CIDM), Jim Chew (CIDM), Linda Hueston (CIDM); Dr Belinda Herring (former CIDM-PH fellow); Dr Cheryl Toi (CIDM-PH fellow); Fei Zhou (CIDM-PH PhD scholar)

 

Rapid diagnosis of influenza and respiratory viral infections.

Although not a strong component of our initial CBIG plans, studies on respiratory viruses have become a major focus in the last three years, driven by the emergence of SARS, potential pandemic influenza and other respiratory pathogens. The development of rapid and specific tests for these new viruses has enhanced our ability to provide rapid high-throughput laboratory testing to support public health interventions. This includes the use of rapid point of care tests and antivirals in nursing home influenza outbreaks (funded by ARC Linkage Grant LP0668279 2006-2008 Economic and social benefits of treating and preventing influenza in aged care facilities. CIs include Dwyer & MacIntyre). There are also studies guiding patient management and hospital/community infection control decisions in cases of respiratory virus infections (funded by Commonwealth Department of Health and Ageing 2006-2007 A cluster-randomised household study of mask use in control of respiratory virus transmission – a pilot study; CIs include Dwyer & MacIntyre). NHMRC and NIH applications on mask use in the community and in healthcare workers have been submitted for the 2007 funding round.

The Virology group is a key partner in a number of NHMRC urgent grants for Research into Potential Avian Influenza-Induced Pandemics, in collaboration with other groups in Australia and internationally. These include

• #402870 Novel, high-throughput platform for rapid identification, quantitation, differential diagnosis and resistance testing of influenza;
• #382917 Development of national protocols for the detection of influenza A H5N1;
• #408114 Use of rolling circle amplification, ligase chain reaction and real-time PCR to detect neuraminidase inhibitor resistance;
• #401175 A prospective study to examine the effectiveness and safety of antivirals against pandemic influenza, and
• #419224 Assessment of interventions for controlling pandemic influenza and determining data needs to inform these assessments.

These grants have allowed the development and evaluation of novel molecular assays for rapid detection of influenza, including potential pandemic strains and outbreaks, and assessment of neuraminidase inhibitor drug resistance.

 

Molecular typing of arboviruses

The virology group collaborates with the CIDM-PH Medical Entomology group, in studies on the molecular epidemiology of arboviruses. A sequence-based genotyping method for Ross River virus (RRV) identified varations in distibution of RRV genotypes in NSW. This study will be extended to other States. It has also been applied to Barmah Forest virus, which is responsible for large outbreaks in coastal NSW in recent years. This work is being undertaken by Belinda Herring, a former CIDM-PH postdoctoral fellow, who has recently taken up a Lecturer position in the School of Infectious Diseases and Immunology, University of Sydney, but is continuing her arbovirus research in collaboration with CIDM-PH.

 

Molecular typing of varicella-zoster virus

Cheryl Toi has joined CIDM-PH to provide expert molecular expertise in virology. She is developing a PCR-based method to separate vaccine strains from wild-type varicella zoster virus. This is an important capability, in the context of increasing use of live attenuatted varicella vaccination in children and the planned introduction of zoster vaccination in the elderly to prevent herpes zoster (shingles). Determining the viral cause of varicella-like rashes in both immunocompetent and immunosuppressed individuals will be especially important as part of these vaccine strategies.

 

Molecular typing of enteroviruses

Outbreaks of enteroviral diseases are common, both in Australia and in the region. More recently, poliviruses have reemerged. As current methods for typing enteroviruses from clinically significant disease outbreaks are done serologically (CIDM is the only laboratory in NSW that performs enterovirus typing) or with complicated molecular assays, there is a strong need to develop rapid and reliable typing methods that are readily transferable to other laboratories. Fei Zhou, a medical graduate from Shanghai, China, has commenced a PhD that will develop enterovirus typing methods using molecular methods, some of which are based on techniques used for bacterial typing mentioned above.

 

Molecular typing of noroviruses

We and others have demonstrated that the large outbreaks of norovirus gastroenteritis in 2004 were due to a particular new genotype, which raises the possibility of either common sources, with environmental contamination, or emergence of a more virulent, highly transmissible strain. The evaluation of laboratory tests for noroviruses has led to improved diagnostic capacity, particularly in the context of gastroenteritis outbreaks in closed environments.

 

HIV epidemiology and antiretroviral resistance

CIDM-PH has a longstanding collaboration with the Centre for Virus Research, Westmead Millennium Institute, in studies on HIV epidemiology and antiretroviral resistance. Monitoring of HIV drug resistance and subtyping has shown drug resistance in untreated HIV-infected individuals and the increasing rate of detection of novel imported HIV strains. This has led to changes in clinical practice so that HIV drug resistance testing is now performed before the commencement of antiretroviral therapy, so as to optimise clinical responses. A national HIV subtyping program has commenced; it is planned that results will be linked with State Departments of Health HIV databases to identify (and thus assist) community groups at risk of infection with imported or antiretroviral resistant strains.

            In the next year, drugs from two novel anti-HIVdrug classes will be available –integrase inhibitors and CCR5 antagonists. New resistance testing assays for these drugs are needed for both individual patient care, and for understanding the transmission of drug resistance mutations in the HIV-infected population. Studies of the prevalence of transmitted drug resistance in Australia are being developed.

 

Other projects:

• Comparison of rapid point-of-care tests with direct immunofluorescence (DFA) and PCR for detection of human and avian influenza, respiratory syncytial virus
• Herpes simplex virus IgG EIA Commercial Typing Kits compared with Western Immunoblot Typing.
• Investigation of new respiratory viruses in outbreaks of respiratory illness
• Are there cryptic foci of Murray Valley encephalitis virus (MVE) in NSW?

Barmah Forest virus in NSW (PhD project, Linda Hueston)

ENTOMOLOGY

Entomology

Personnel: Group Leader: Professor Richard Russell, Merilyn Geary (CIDM), Stephen Doggett (CIDM), John Clancy (CIDM), John Haniotis (CIDM), Dr Cameron Webb.

 

Molecular identification and typing of arboviruses in mosquitoes.

With Belinda Herring we have continued the analysis of strains of Ross River and Barmah Forest viruses. Ongoing studies on arboviruses associated with notifications in NSW in the last few years have generated data that will improve our understanding of the movements (in both mosquitoes and humans) of these viruses in coastal NSW. We have coordinated with public health unit officers to assist public health interventions in prevention of community disease. Associated with these studies is a program to detect previously uncharacterised arboviruses, with the eventual aim of determining whether these are of clinical significance.

 

Arboviral vector competence studies.

The vector competence of three coastal mosquito species of secondary pest importance, Verrallina carmenti, Verrallina lineata and Mansonia septempunctata, as vectors of Ross River virus has been assessed. All three mosquito species could be infected but transmission rates were relatively low, indicating that the three species may not be locally significant vectors of RRV (and therefore not require specific targeting by control programs), but have the potential to contribute to local transmission cycles.

 

Bed bug management

The urban pest control industry has been struggling in recent years with the increasing problem of commercial and domestic bed bug infestations; we have provided expert ‘remote’ and ‘on-site’ advice and assistance, and undertaken the preparation of an industry ‘code-of-conduct’ for Australia (that is also being accessed internationally).

 

Wetlands management

We have developed an outreach consultancy arm for government and private industry development projects that have mosquito-associated health risks; we are supplying expertise in assessing and managing mosquito threats for the burgeoning industry of remediated and constructed wetlands that are required to manage waste and stormwater in residential and industrial developments, and we have generated a paradigm shift in urban planning considerations for environmental health in NSW.

 

Medicinal maggots for wound therapy

A major achievement has been the establishment of the first service in Australia providing disinfected maggots for wound therapy; we provide maggot shipments on a weekly basis on average to clinical units throughout Australia (and overseas). We are now in the process of establishing a leech colony for provision to surgical units.

PARASITOLOGY

Parasitology

Personnel: Dr Rogan Lee (CIDM), Nina Santiago (CIDM)

 

Improved methods in malaria diagnosis

Dr Nick Adams (CIDM Microbiology, registrar, 2006) investigated the use of the Smart Cycler as a means to test for presence of malaria in blood specimens. His initial studies showed that comparison of annealing temperatures could not differentiate the 4 species of malaria and variation in some primer sites could have the potential of missing some strains of malaria.  Dr Rod James willl continue this project.

 

Improved serodiagnosis of strongyloidiasis

Western blot analysis of Strongyloides ratti antigen as a method of identifying low reacting serum antibodies in current ELISA assays. Stocks of antigen have been accumulated for extraction of stage 3 larvae.  The preparation of material for SDS PAGE and western blotting will proceed with the help of Nina Santigo in the laboratory.

 

Toxoplasma gondii in patients immunosuppressed by cancer chemotherapy.

The Parasitology Unit is collaboratiing with the University of Malaya.  Blood and survey interviews have been collected from Malayan cancer patients.  Parasitology carried out the initial serological testing of these patients by measuring anti-toxoplasma IgG levels. IgM antibody levels will also be measured to determine whether any of these patients show recent exposure to this parasite and whether these patients will develop a relapse of the infection after chemotherapy.

SEXUAL HEALTH

Sexually transmissible  infections

Personnel: Professor A Mindel, Dr R Hillman (STIRC), Dr S Sawleshwarkar (STIRC), C Chung (STIRC).

 

Human papilloma virus (HPV) research

a. HPV therapeutic vaccine trial

Evaluating the safety of a therapeutic vaccine targeting HPV type 16 in MSM; funded by CSL

 

b. HPV prophylactic vaccine trial

Evaluating safety & efficacy of prophylactic vaccines against HPV types 6, 11, 16 & 18 in men aged 18-27; funded by MSD. Collaborators: R Findlayson (Taylor Square Private Clinic), M Bloch (Holdsworth House).

 

c. HPV types in anal cancer specimens

100 archived anal cancer specimens from St Vincent’s Pathology will be tested for multiple types of HPV using in situ PCR. Collaborators: S Tabrizi (Melbourne), R Ward & A Meagher (St Vincent’s)

 

d. Anal cytology/histology correlation.

Anal cytology has been reported to be ~60% sensitive and specific for high grade dysplasia. This study investigates the relationship between anal cytology and histology, as influenced by timing of the sample and HIV status, using the AIN database at St Vincent’s Hospital.

 

e. Prevalence & risk factors for anal HPV infection in MSM with & without HIV infection

The HIM & PH studies are longstanding community-based cohorts of Australian men investigating sexually transmitted infections, HIV and risk behaviour. In 2005, anal specimens were taken from a subgroup & analysed for the presence of HPV.  Collaborators: A Grulich, C Vajdic, G Prestage (NCHER)

 

f. Prevalence & risk factors for ASIL in MSM with & without HIV infection

Anal cytological specimens were taken from a sample of the HIM & PH cohorts. Those with abnormalities were offered HRA follow up, with biopsy. These findings will be related to their demographic characteristics. Collaborators: A Grulich, C Vajdic, G Prestage (NCHER) & G Medley (Melbourne)

 

g. Anal cancer review.

A retrospective case note review of all cases of anal cancer presenting to St Vincent’s Hospital since 1994. Data will be related to demographic and outcome variables. Collaborators: R Ward & A Meagher (St Vincent’s)

 

h. High grade dysplasia review

Retrospective case review of all cases of high grade anal dysplasia presenting to St Vincent’s Hospital since 1994. Data will be related to demographic and outcome variables.A Meagher & M Whitfeld (St Vincent’s)

 

i. Twenty year review of anal dysplasia

Pioneering work was done in 1987 at the Albion Street Clinic characterizing anal HPV infection and dysplasia in men. This study will perform a retrospective case note review to see which men progressed to anal cancer. Collaborators: R Melville, J Saragapany & V Furner, Albion Street Clinic

 

j. Imiquimod & AIN

There are currently no well-validated treatment modalities for high grade anal dysplasia. Surgical treatment is the most widely practiced in Australia. Surgery may not be technically appropriate or desired by many with dysplasia. Imiquimod is known to be highly effective against non-anal dysplasia, such as Bowen’s disease & it is proposed to evaluate this as an option for anal problems. M Whitfeld (St Vincent’s)

 

k. HPV vaccine for prevention of cervical cancer

A phase III, double-blind, randomized, controlled, multi-center study to evaluate the efficacy of GlaxoSmithKline Biologicals’ HPV-16/18 VLP/AS04 vaccine compared to hepatitis A vaccine as control in prevention of persistent HPV-16 or HPV-18 cervical infection and cervical neoplasia, administered intramuscularly according to a 0, 1, 6 month schedule in healthy females 15-25 years of age. Funding:  GSK

 

Herpes simplex virus

A double-blind, randomized, controlled study to evaluate the immunogenicity and safety of GlaxoSmithKline Biologicals’ herpes simplex candidate vaccine (gD2‑AS04) in healthy HSV seronegative and seropositive female subjects aged 10 – 17 years. Funding:  GSK

 

Chlamydia trachomatis

Interactive, internet-based education and information for young people about Chlamydia trachomatis: a randomised controlled trial Collaborators: M Kang, R Skinner, C Sullivan, M Webb, J Burns, & T Usherwood. Funding Department of Health and Aged Care Australian Federal Government

 

Bettering the Evaluation and Care of Health (BEACH)

A collaboration between STIRC and General Practice Statistics and Classification Unit (GPSCU), using the national BEACH (Bettering the Evaluation and Care of Health) database to study patterns of diagnostic testing for STIs by GPs.

 

Helicobacter pylori as an STI

Pilot study to evaluate seroprevalence of H. pylori in patients attending Parramatta Sexual Health Clinic.

 

SMS to Improve patient Attendance in Sexual Health Clinics

Using short message service (SMS) software to improve patient attendance for regular STI screening in priority population groups.

 

Gambling and Sexual Health

This study has been carried out in conjunction with the department of psychology at Westmead Hospital. The aim is to assess links between risk taking gambling and sexual behaviours.

 

Anal STIs as risk factors for HIV seroconversion: data from the HIM cohort

Collaborators: F Jin , GP Prestage, J Imrie ; SC Kippax, CM Pell ; B Donovan, DJ Templeton ; PH Cunningham, AL Cunningham,  JM Kaldor, AE Grulich.

 

Non-occupational post-exposure prophylaxis (non-PEP) in two sexual health clinics: Risk assessment, treatment delivery, compliance, and STI investigations

 

The epidemiology of urogenital organisms in patients in selected populations.

Research group: Hillman (STIRC), Couldwell & Lagios (Parramatta Sexual Health Clinic); Freeman (Sydney Sexual Health Clinic); Gilbert, Sintchenko, Gidding (CIDM-PH) and McKechnie (CIDM-PH PhD student).  This project involves a proposective studiy of men with urethritis and controls to compare the incidence of a large range of putative urethritis pathogens, using mPCR/RLB in the two groups. In addition, Michelle’s PhD project includes a study of the prevalence of putative urethritis pathogens in women attending sexual health clinical and their correlation with demographic and social factors.