INFO LINK. Your Future, Your Pathology. Inside this Issue. Human Papilloma Virus Expanding Molecular Biology

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1 INFO LINK Autumn 2008 Issue Twenty Inside this Issue Human Papilloma Virus Expanding Molecular Biology New Technology Your Future, Your Pathology Delivering quality comprehensive clinical and laboratory services for all your pathology needs

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3 Welcome to the winter 2008 edition of PaLMS Infolink. This edition introduces the PaLMS Northern Sydney Microbiology Department. Microbiology has incorporated the latest technology to set new standards for processing sample accuracy, precision, speed, quality, instrument reliability and efficiency. I would like to thank Mark Van Asten, Managing Director of Diagnostic Technology for his donation through the estate of his late aunt, Maria Binkhorst for the GeneXpert Real Time PCR Testing System. The donation was in gratitude for the care shown to his aunt whilst at Royal North Shore Hospital. PaLMS is extremely grateful for such a generous donation. The Microbiology Laboratory has also updated its automated technology procedures for performing routine molecular recovery and detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) in clinical specimens by introducing the BD Viper System. The formation of Pathology North provides enormous opportunities for public pathology over the coming years. The following are some of the strengths to be achieved by the cluster: Improved access for more remote laboratories to professional development and training opportunities. Improved quality of service for clinicians and patients Opportunities to increase efficiency and quality by upgrading and streamlining laboratory equipment and processes where appropriate. Opportunities to form new professional links with colleagues in cluster partner services. We are looking forward to an exciting future for Pathology North and to participation in an expanding, responsive and innovative pathology service. A highlight for PaLMS was welcoming the Mid North Coast on board in May PaLMS now extends from Northern Sydney to the Queensland border taking in the regions of Northern Sydney, Central Coast, the Mid North Coast and Northern Rivers. As of the 1st of July PaLMS will join with Hunter Area Pathology Service (HAPS) and Pathology New England to form the Pathology North Cluster. Dr. Campbell Tiley accepted the position of Acting Director PaLMS Pathology when Professor Leslie Burnett (PaLMS Director) commenced the role of Interim Director Pathology North, in Dr. Campbell Tiley is a clinical and laboratory haematologist at Gosford with strong interests in the use of information systems to support clinical practice. Dr. Campbell Tiley Acting Director PaLMS Pathology Info Link, PaLMS 1

4 Expanding Molecular Microbiology The following tests are now available on site at Northern Sydney PaLMS. This means faster turnaround times for results and you can discuss cases directly with the appropriate pathologists and scientists responsible for performing the tests for your patient. 1. Neisseria gonorrhoeae and Chlamydia trachomatis PCR: A qualitative detection of Neisseria gonorrhoeae and Chlamydia trachomatis DNA in endocervical swabs, male urethral swabs and in female and male urine specimens as evidence of infection with N.gonorrhoeae and/or C.trachomatis. Specimens may be from symptomatic or asymptomatic females and males. 2. Herpes Simplex Virus type 1 and 2 PCR: A qualitative detection of Herpes Simplex Virus DNA types 1 and 2 in cerebral spinal fluid, neonate swabs, eye swabs, vitreous fluid, skin swabs and genital swabs. 3. Hepatitis C Virus PCR (HCV): A qualitative assay for the detection of HCV RNA in human serum and plasma. The presence of HCV RNA is evidence of current HCV infection in patients presenting with clinical evidence of liver disease. 4. Hepatitis C Viral Load PCR: Assay for the quantitation of HCV RNA in human plasma or serum. The test is intended for use in conjunction with clinical presentation and other laboratory markers of HCV infection for the clinical management of patients with chronic HCV. The test can be used to assess the probability of a sustained viral response early in a course of antiviral treatment as measured by changes in serum or plasma HCV RNA levels. 5. Hepatitis B Viral Load PCR (HBV DNA): Assay for the quantitation of HBV DNA in human plasma. The test is intended for use in conjunction with clinical presentation and other laboratory markers as an aid in assessing viral response to antiviral treatment as measured by changes in plasma HBV DNA levels. 6. Mycobacterium tuberculosis complex, Mycobacterium avium and Mycobacterium intracellulare DNA PCR: A qualitative assay for the detection of M. tuberculosis complex, M. avium and M. intracellulare DNA in all clinical specimens. The isolation of an organism from the M. tuberculosis complex is required for the definitive diagnosis of tuberculosis. 7. Cytomegalovirus (CMV) DNA PCR: Assay for the quantitation of CMV DNA in human plasma. The test is intended for use in conjunction with clinical presentation and other laboratory markers as an aid in assessing viral response to antiviral treatment as measured by changes in plasma CMV DNA levels. 8. Human Papilloma virus (HPV) DNA Test: A qualitative assay for the detection of thirteen high-risk types of HPV DNA in cervical specimens. 9. Hepatitis C Virus (HCV) Genotyping: The HCV Genotype assay is designed to identify HCV genotypes 1 to 6. Substantial evidence has emerged indicating that typing and sub typing for HCV is clinically important in understanding HCV disease and therapy options. 10. Enterovirus PCR: A qualitative diagnostic test designed to detect Enterovirus RNA in cerebrospinal fluid specimens. Results from this test are intended for use as an aid in the diagnosis of Enterovirus associated meningitis. Enterovirus is taxonomically classified as those viruses consisting of Polioviruses, Coxsackieviruses, Echoviruses and Enteroviruses. 2 Info Link, PaLMS

5 Tom Karagiannis Senior Hospital Scientist, Northern Sydney PALMS - Microbiology Department Tom Karagiannis has worked in Pathology for the past 21 years with 18 of these years being in Microbiology with extensive experience in the clinical medical Microbiology Department e.g. Bacteriology, Molecular Biology, Mycology, Mycobacteria, Parasitology and Serology. He has worked at Royal North Shore Hospital (RNSH) since 2006, with previous experience at O Brien Pathology Randwick, Medical Pathology Services and St Vincent s Hospital Sydney. Prior to commencing at RNSH he was the Business Manager and Technical Support Specialist, Infectious Diseases Molecular and Serology, at Bayer Healthcare Australia Diagnostics Division. Tom s qualifications include a Bachelor of Science in Biomedical Science majoring in Microbiology from the University of Technology Sydney, 1997 and a Master of Science research thesis on Molecular typing of Shigella sonnei to investigate an ongoing epidemic in Eastern Sydney from the University of New South Wales, Prior to this Tom was working in pathology as a technician after gaining a Certificate in Pathology Technicians and an Associate Diploma of Health Science Pathology Techniques from the Institute of Technology, Sydney. Tom Karagiannis Telephone: (02) tkaragia@nsccahs.health.nsw.gov.au Tom s interests include Molecular Microbiology Infectious Diseases and Mycobacteria. Working with PaLMS has given him the opportunity to implement the latest technology in order to provide a superior pathology service. He continued his association until 2004 with the University of New South Wales, Sydney as a Clinical Microbiology Demonstrator and Tutor for the Student Diagnostic Unit for the School of Biotechnology and Biomolecular Sciences and the University of Technology, Sydney as a Clinical Microbiology Demonstrator and Tutor for the School of Biomedical Sciences. Tom has held the position as a National Association of Testing Authorities Australia (NATA) Assessor since February Info Link, PaLMS 3

6 Human Papilloma Virus by Tom Karagiannis, Senior Hospital Scientist Human Papilloma Viruses (HPV) are composed of an icosahedral viral particle (virion) containing an 8000 base pair double-stranded circular DNA molecule surrounded by a protein capsid. Following infection of epithelial cells, the viral DNA becomes established throughout the entire thickness of the epithelium, but intact virions are found only in the upper layers of the tissue. Thus, viral DNA can be found either in virions or as episomal or integrated HPV sequences, depending upon the type and grade of lesion. The presence of certain HPV types in the female genital tract is associated with a number of diseases, including Condyloma, Bowenoid papulosis, cervical, vaginal and vulvar intraepithelial neoplasia and carcinoma (1). Cancer of the cervix is the third most common form of cancer among women worldwide, with ,000 new cases each year and 230,000 women die annually because of this disease (2,3). In developing countries, cancer of the cervix is the most frequent female malignancy and is responsible for about 24% of all cancers in women. In developed countries, cervical cancer accounts for 7% of all female cancers (2). The principle cause of invasive cervical cancer and its precursor lesions is infection with oncogenic types of HPV which is found in close to 100% of cancers (1). In young women, the incidence of HPV infection is high, but the infection is often of short duration. The prevalence of high-risk (HR) HPV DNA is highest among sexually active teenagers, declining in women 20 to 30 years of age, and is still lower in women over the age of 30 (Figure 1). Persistent HR HPV infections, which are more common in older women, greatly increase the risk of cervical intraepithelial neoplasia (CIN) or cervical cancer, especially if the viral load is high (1). The HPV family consists of many different types; more than 100 types have been identified to date, of which 40 types have been detected in the anogenital mucosa. Several HPV types, such as types 16, 18, 31, 33 and 35 have been implicated as sexually transmitted agents with an aetiological role in cervical carcinogenesis, whereas other types, such as 6 and 11 are frequently detected in benign lesions such as condylomata acuminata. Therefore, HPV types 6 and 11 are termed low-risk (LR) HPV types and HPV types involved in carcinogenesis (such as types 16 and 18) are termed HR HPV types (2). It is generally accepted that women infected with an HR HPV type are thus at higher risk of developing cervical cancer than those who are not infected with HPV or who are infected with one of the LR HPV types. The strong association between infection with certain HPV types and cervical cancer that has emerged has brought new urgency to the need for establishing accurate methods to diagnose HPV infection. Although cytological screening by the Papanicolaou test, or Pap smear, continues to play a major role in the early diagnosis of cervical neoplastic lesions, assays that detect HPV DNA in cervical smears may represent an important source of adjunct information for the purpose of counselling, selection of therapy, and follow-up (3). Several HPV DNA detection methods have been described in the literature, each of which allows the detection of a wide spectrum of HPV types. Amplification based methods, mainly PCR, are currently the most sensitive methods for detection of HPV DNA. They are ideal for research and epidemiological purposes since they allow the detection of low-viral load infection. However, due to frequent contamination problems and consequent false-positive results and the high cost of amplification technology they are not widely used in the clinical diagnostic laboratory for the routine detection of HPV infection (3). Greater sensitivity might be undesirable for some clinical applications, because of the high prevalence of self limited HPV infection among young sexually active females. 4 Info Link, PaLMS

7 Human Papilloma Virus (Continued) The Microbiology Department at PaLMS Royal North Shore Hospital has recently introduced the Digene Hybrid Capture 2 Test for the detection of 13 oncogenic HPV types for the detection of CIN 2-3 and cancer (CIN 3+) in cervical specimens. The Hybrid Capture 2 assay relies on the formation of target HPV DNA-RNA complexes during the hybridisation step in specimens containing sufficient HPV DNA and the chemiluminescent detection of these hybrids by an alkaline phosphatase-conjugated monoclonal antibody specific to DNA-RNA complexes with substrate in a enzyme-linked immunosorbent assay (ELISA) format (4). The Hybrid Capture 2 assay has been shown to have similar analytic sensitivity to some PCR methods for HPV DNA detection and is a sensitive test for the detection of CIN 2-3 and cervical cancer (CIN 3+) and has received FDA approval for guiding the management of women with equivocal cytology (atypical squamous cells) (5). Sexually transmitted HPVs also cause a major fraction of anal cancers and approximately 25% of cancers of the mouth and upper throat (known as the oropharynx) (figure 2). The latter commonly present in the tonsil area and HPV is linked to the increase in oral cancers in non-smokers. Engaging in anal sex or oral sex with an HPV-infected partner may increase the risk of developing these types of cancers (6). Testing for HPV has not yet been validated in clinical studies for these diagnosis. Figure 2: Cervix Anus Vagina/Vulva Penis Mouth Throat References HPV-Induced Total 0 100, , , , ,000 Annual number of cases worldwide 1. The Digene Hybrid Capture 2 High-Risk HPV DNA Test. An In Vitro nucleic acid hybridization assay with signal amplification using microplate chemiluminescence for the qualitative detection of 13 high-risk types of human papillomavirus (HPV) DNA in cervical specimens. 2. Soderlund-Strand, A., P. Rymark, P. Andersson, J. Dillner, and L. Dillner Comparison between the Hybrid Capture II Test and a PCR-Based Human Papillomavirus Detection Method for Diagnosis and Post treatment Follow-Up of Cervical Intraepithelial Neoplasia. J.Clin.Microbiol. 43: van Ham, M.A.P.C., J. M.J.E. Bakkers, G.K. Harbers, W.G.V. Quint, L.F.A.G. Massuger and W.J.G. Melchers Comparison of Two Commercial Assays for Detection of Human Papillomavirus (HPV) in Cervical Scrape Specimens: Validation of the Roche Amplicor HPV Test as a Means To Screen for HPV Genotypes Associated with a Higher Risk of Cervical Disorders. J.Clin.Microbiol. 43: Incidence Pre-Cancer Genital HPV Infection Cancer 4. Poljak, M., A. Brencic, K. Seme, A. Vince and I.J. Marin Comparative Evaluation of First- and Second- Generation Digene Hybrid Capture Assays for Detection of Human Papillomaviruses Associated with High or Intermediate Risk for Cervical Cancer. J.Clin.Microbiol. 37: Castle, P.E., A.T. Lorincz, D.R. Scott, M.E. Sherman, A.G. Glass, B.B. Rush, S. Wacholder, R.D. Burk, M.M. Manos, J.E. Schussler, P. Macomber and M. Schiffman Comparison between Prototype Hybrid Capture 3 and Hybrid Capture 2 Human Papillomavirus DNA Assays for Detection of High-Grade Cervical Intraepithelial Neoplasia and Cancer. J.Clin.Microbiol. 41: Figure 1: Age (Years) Baseman JG, Koutsky LA (2005). The epidemiology of human papillomavirus infections. J. Clin. Virol. 32 Suppl 1: S Gillison, M.L., W.M. Koch, R.B. Capone, M. Spafford, W.H. Westra, L. Wu, M.L. Zahurak, R.W. Daniel, M. Viglione, D.E. Symer, K.V. Shah and D. Sidransky Evidence for a Causal Association Between Human Papillomavirus and a Subset of Head and Neck Cancers. J. Natl. Cancer Inst. 92: Info Link, PaLMS 5

8 Northern Sydney PaLMS Microbiology Incorporates Latest Novel Robotic Technology for Molecular Testing and Diagnosis of Sexually Transmitted Diseases. Royal North Shore Hospital PaLMS Microbiology Laboratory has updated its procedures for performing routine molecular recovery and detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) in clinical specimens by introducing the BD Viper System. The BD Viper System is an automated, high throughput platform that can perform nucleic acid purification, real-time amplification and detection in a closed system format. It is designed for use with a multitude of specimen types, including male and female urine, male urethral swabs and female endocervical and vaginal swabs. The system uses Strand Displacement Amplification (SDA) based technology for CT and NG that each incorporate an internal control to monitor for sample inhibition and verify that proper amplification conditions exists in each specimen. BD Viper automation uses Selective Compliance Assembly Robotic Arm (SCARA) technology incorporated in the automotive, pharmaceutical and microprocessor industries. The key features of the BD Viper instrumentation is designed for high throughput, minimal labour, real-time isothermal amplification technology equates to fast time to results, no reagent preparation, a closed sealed solid-barrier amplification system reducing the opportunity of contamination due to no pipetting of amplified product. The novel engineering contains no vacuum pumps, syringes, valves or reagent tubing. Pipette tips do not cross over another sample, tube or micro well. The system requires no maintenance by the scientist, no fluidic system to maintain, only an end of day clean. By automating the most labour intensive steps for amplified tests sample handling and amplification/ detection, the BD Viper system allows CT and NG testing in Microbiology to set new standards for processing accuracy, precision, speed, quality, instrument reliability and efficiency. 6 Info Link, PaLMS

9 Donation to Northern Sydney PaLMS Microbiology Department of a GeneXpert PCR System for testing Enterovirus in Cerebrospinal Fluid Mark Van Asten, Managing Director of Diagnostic Technology recently donated through the estate of his late aunt, Maria Binkhorst a single module GeneXpert Real Time PCR Testing System in gratitude for the care shown to his aunt whilst at RNSH. PaLMS is extremely grateful for such a generous donation. The technology allows PaLMS Microbiology Department to offer a rapid, sensitive molecular Enterovirus (EV) test that can help physicians quickly identify patients with EV meningitis, thereby preventing unnecessary use of antibiotics and the possibility of a repeat spinal tap. The test takes approximately 2.5 hours. The GeneXpert System automates and integrates sample purification, nucleic acid amplification, and detection of the target sequence in samples using real-time PCR and reverse transcription PCR. The system consists of an instrument, personal computer, and preloaded software for running tests on collected samples and viewing the results. The system requires the use of single-use disposable GeneXpert cartridges that hold the PCR reagents and host the PCR process. Because the cartridges are self-contained, cross-contamination between samples is eliminated. The Cepheid Xpert Enterovirus assay performed on the GeneXpert System is a qualitative in vitro diagnostic test designed to detect EV RNA in cerebrospinal fluid (CSF) specimens. Results from this test are intended for use as an aid in the diagnosis of EV-associated meningitis. Enterovirus is taxonomically classified as those viruses consisting of Polioviruses, Coxsackieviruses, Echoviruses and Enteroviruses. About 90% of viral meningitis cases are caused by EV, usually self-resolves within 7-10 days. Info Link, PaLMS 7

10 Past Issues Issue Nineteen Issue Eighteen Issue Seventeen, Issue Sixteen, Issue Fifteen, Issue Fourteen, B-type Natriuretic Peptide (BNP) Down Syndrome - Population Screening Anti-Neutrophil Cytoplasmic Antibodies - ANCA Changes in the use of RH D immunoglobin products in Australia: Introduction of Antenatal Prophylaxis Laboratory Diagnosis for Infection with Human Immunodeficieny Virus (HIV) Hereditary Haemochromatosis Infectious Mononucleosis Issue Thirteen, Issue Twelve, Issue Eleven, Issue Ten, Issue Nine, Issue Eight, Issue Seven, Issue Six, Issue Five, Issue Four, Issue Three, Issue Two, Issue One, Thrombophilia Faecal Occult Blood Testing Acute Phase Response New Targeted Treatment for CML Laboratory Markers of Bone Metabolism Thyroid Function Tests - Dead Easy or Impossible Synovial Fluid Analysis in the Investigation of Arthritis The Biopsy Diagnosis of Gastritis Diabetes Mellitus New Markers for Coronary Artery Diseases Laboratory Investigation of the Small, Pale Red Cell: Microcytic, Hypochromic Anaemia Auto-Antibodies in Connective Tissue Diseases Laboratory Tests to Detect Allergiv Reactions to Drugs Investigation of Hypertension Important Concepts in Laboratory Utilisation Visit the PaLMS Web site at to view past issues or contact PaLMS Marketing on (02) for a copy to be sent to you.

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12 PaLMS COLLECTION FACILITIES Northern Sydney Enquiries or PaLMS Chatswood Katherine Street Medical Centre, Unit 235/1 Katherine Street, Ph/Fax: (02) Monday to Friday: 8.00am to 4.30pm Dee Why (enter via Fisher Rd) 1ST Floor, 671 Pittwater Rd Ph/Fax: Saturday 8.30 am to midday Eastwood Ryde Hospital Denistone Road, Eastwood Ph: (02) Monday to Friday: 8.00 am to 4.30 pm Saturday 9.00 am to12.00 midday Hornsby Hospital Adj. Main Entrance Palmerston Rd Ph: Fax: Saturday 8.30 am to midday Manly Hospital West Wing, Darley Road Ph: (02) Monday to Friday: 8.00 am to 4.30 pm Saturday 9.00 am to12.00 midday Mona Vale Hospital Level 2, Coronation Street Ph: (02) Monday to Friday: 8.00 am to 4.30 pm Saturday 9.00 am to midday Neutral Bay Suite 3, 5 Waters Road Ph: Fax: St Leonards North Shore Private Hospital PaLMS Collection Suite, Ground Floor, Westbourne St Ph: Fax: Monday to Friday: 8.00 am to 6.00 pm Saturday 9.00 to 1.00 pm St Leonards Royal North Shore Hospital Clinic 13, Outpatient Department, Level 3, Pacific Highway Phone: (02) Willoughby 130 Mowbray Rd Ph: (02) Monday to Friday: 8.00 am to 2.00 pm Saturday CLOSED Central Coast Enquiries (02) Gosford Hospital Pathology Collection Centre (near Outpatients Clinic) Ph: (02) Monday to Friday: 8.00 am to 4.00 pm Wyong Hospital Pathology Collection Centre (near Outpatients Clinic) Ph: (02) Monday to Friday: 8.00 am to 2.00 pm Mid North Coast Enquiries: Please contact your local hospital collection centre telephone number Coffs Harbour Hospital 345 Pacific Highway COFFS HARBOUR SOUTH 2450 Ph: (02) Monday to Friday: 8.00 am to 5.00 pm Forster South Street FORSTER NSW 2428 Ph: (02) Monday to Friday: 8.00 am to 4.30 pm Kempsey 3/24 Clyde Street KEMPSEY NSW 2440 Ph: (02) Monday to Friday: 8.00 am to 4.30 pm Kempsey Hospital 119 River Street KEMPSEY NSW 2440 Ph: (02) Monday to Friday: 8.00 am to 5.00 pm Macksville Hospital Boundary Street MACKSVILLE NSW 2447 Ph: (02) Monday to Friday: 8.00 am to 4.30 pm Manning Base Hospital York Street Ph: (02) TAREE NSW 2430 Monday to Friday: 8.00 am to 4.00 pm Taree 17 York Street TAREE NSW 2430 Ph: (02) Monday to Friday: 8.00 am to 5.00 pm Wauchope Hospital 69 High Street WAUCHOPE NSW 2446 Ph: (02) Monday to Friday: 8.30 am to pm Northern Rivers Enquiries: Please contact your local hospital collection centre telephone number Ballina District Hospital Fox Street Ballina 2478 Phone: (02) Monday to Friday: 9.00 am pm Grafton Base Hospital Arthur Street Grafton 2460 Phone: (02) Lismore Base Hospital 76 Uralba Street Lismore 2480 Phone: (02) Maclean Hospital 21 Union Street Maclean Monday to Friday: 9.00 am pm Murwillumbah District Hospital Ewing Street Murwillumbah Phone: (02) Monday to Friday 7.30 am pm St Vincents Hospital 20 Dalley Street Lismore 2480 Phone: (02) The Tweed Hospital Florence Street Tweed Heads 2485 Ph: (07) Monday to Friday: 8.00 am to 5.00 pm For further information visit palms@nsccahs.health.nsw.gov.au CATALOGUE NO: 09875