INFO LINK. Your local pathology provider. Issue Twenty FOUR. Inside this Issue. A division of Pathology North

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1 INFO LINK 24 SPRING 2011 Issue Twenty FOUR Inside this Issue Molecular Microbiology Detecting Respiratory Viruses by Multiplex PCR Your local pathology provider Delivering quality comprehensive clinical and laboratory services for all your pathology needs A division of Pathology North

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3 Infolink Introduction Spring 2011 Welcome to the spring 2011 edition of Infolink. The article in this addition is: Detecting Respiratory Viruses by Multiplex Polymerase Chain Reaction (PCR). Acute respiratory disease accounts for an estimated 75% of all acute morbidities in developed countries with approximately 80% of these infections being viral. Nucleic acid amplification procedures, including Multiplex PCR, have been developed for identifying most respiratory viruses. These are highly sensitive tests and are becoming the preferred routine tests in clinical laboratories for detecting respiratory viruses. Also included in this addition are the details of PaLMS Northern Sydney Microbiologists and a summary of the Molecular Microbiology tests available. Individual cases can be discussed directly with the appropriate pathologist and scientist responsible for performing and/or reporting on the tests for your patient. RNSH REGISTRAR RECEIVES RESEARCH AWARD In recognition of the service and expertise of A/Prof Jeanette Philips, the Australasian Division of the International Academy of Pathology has instituted the Jeanette Philips award. This award, sponsored by the PaLMS pathologists, recognises A/Prof Philips' contribution to education and research, particularly in the field of endocrine pathology. A/Prof Philips, who is now retired, was a long serving staff specialist at PaLMS Royal North Shore Hospital. The ward is to be given annually for the best original research presentation, preferably based on an article published in the peer reviewed literature in the field of endocrine pathology, by a pathologist in training. It will be administered through the newly formed Australasian Endocrine Pathology Society. The inaugural award was shared by Dr Daniel Wong from PathWest pathology in Perth and Dr Julie Paik, a registrar in Jeanette's former department of PaLMS Anatomical Pathology at Royal North Shore Hospital. Dr Paik's original research, published in the internationally recognised journal Human Pathology, demonstrated that Merkel Cell Carcinoma in Australia is only infrequently driven by the Merkel Cell polyoma virus. This finding is in striking contrast to previous studies which demonstrated a high level of virus driven neoplasia in Merkel Cell Carcinoma, but were performed in North American and Dr Julie Paik (registrar anatomical pathology, Royal North Shore Hospital) receiving the inaugural Jeanette Philips award This award was presented by Jeanette s husband, Dr Ron Joffe. European populations. Dr Paik has postulated that in Australia, with its high level of sun exposure and predominantly fair skinned population, a second sun-driven pathway, is primarily responsible for most cases of Merkel Cell Carcinoma. Dr. Tom Kennedy Assistant Director Pathology North Pacific Laboratory Medicine Services (PaLMS) Northern Sydney, Central Coast MBBS (Monash) MBA (Tas) MClinEpid (Newcastle) FRCPA Info Link, PaLMS 1

4 Molecular Microbiology The following tests are available on site at Northern Sydney PaLMS delivering you fast turnaround times for results. Individual cases can be discussed directly with the appropriate pathologist and scientist responsible for performing and/or reporting on the tests for your patient. Fees are based on the Medicare Benefit Schedule. Rebate only or bulk billing is available on request. 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, 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. 11. Bordetella pertussis PCR: A qualitative diagnostic assay designed to detect whooping cough in children and adults from nasopharyngeal aspirates and throat swabs. 12. Influenza A (Swine H1N1 09) PCR: A qualitative diagnostic test designed to detect Influenza A and the specific Swine H1N1 09 virus derived from Mexico in throat, nose and respiratory specimens. 13. Varicella Zoster PCR: A qualitative diagnostic test designed to detect Varicella-Zoster virus from clinical specimens e.g. cerebrospinal fluid and skin swabs. January Info Link, PaLMS

5 George Kotsiou MB BS FRACP FRCPA MCEpi DTM&H Dr Kotsiou graduated from the University of Adelaide in He has dual fellowships in Microbiology and Infectious Diseases. His major interests include nosocomial infections, infection control, outpatient antibiotic therapy and HIV medicine. He also holds the position of Director of HIV Medicine and is involved with outreach rural clinics in HIV & Infectious Diseases. Phone: Bernie Hudson MB BS DTPH FACTM FAFPHM FRACP FRCPA Dr Hudson is a Clinical Microbiologist and Infectious Diseases Physician. He holds specialist qualifications in pathology (microbiology), internal medicine (infectious diseases) and public health. He also holds positions of Clinical Senior Lecturer in Infectious Diseases at Sydney University and Associate Professor at James Cook University Townsville. Phone: BHudson@nsccahs.health.nsw.gov.au Dr Robyn Hardiman, FRACP FRCPA Dr Robyn Hardiman is a Clinical Microbiologist and Infectious Diseases Physician. Previously Dr Hardiman was Head of Department at POW Infectious Diseases and Microbiology Department. Dr Hardiman is involved in infection control; she assists with the management and treatment of patients with infection and HIV within the Area and community. She is responsible for overseeing of the Virology Laboratory. Phone: RPHardim@nsccahs.health.nsw.gov.au Archie Darbar, BSc MB BS FRACP FRCPA Dr Darbar is a clinical microbiologist and infectious diseases physician who has special interests in travel medicine, tuberculosis and streptococcal infections. His roles include directing management of patients with infections due to multi resistant organisms as well as overseeing the serology laboratory. Phone: ADarbar@nsccahs.health.nsw.gov.au Info Link, PaLMS 3

6 Tom Karagiannis Senior Hospital Scientist, Northern Sydney PaLMS - Microbiology Department Tom Karagiannis has worked in Pathology for the past 24 years with 21 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 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. Telephone: (02) tkaragia@nsccahs.health.nsw.gov.au 6 June 2011 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 was given the position as a National Association of Testing Authorities Australia (NATA) Assessor since February Info Link, PaLMS

7 Detecting Respiratory Viruses by Multiplex PCR By Tom Karagiannis Introduction Acute respiratory disease (ARD) accounts for an estimated 75% of all acute morbidities in developed countries and most of these infections (approximately 80%) are viral. Upper respiratory tract infections (URTI) such as rhinitis, pharyngitis and laryngitis are among the most common infections in children. The Centers for Disease Control and Prevention s national statistics report indicates that there are between 12 and 32 million episodes of URTI each year in children aged 1 to 2 years. URTI can lead to acute asthma exacerbations, acute otitis media and lower respiratory tract infection (LRTI) such as bronchitis, bronchiolitis and pneumonia. Acute viral respiratory tract infection is the leading cause of hospitalization for infants and young children in developed countries and is a major cause of death in developing countries. Studies in Britain have shown for example that of fifty cases of children with LRTI only one showed evidence of a bacterial pneumonia. By far the majority of cases appeared to be viral in origin. Bacterial infections are important because they can be life-threatening and are treatable with antibiotics whereas there are as yet no widely-used antiviral agents for respiratory viruses. One of the problems is that any respiratory infection requiring medical attention is almost invariably treated with antibiotics. Only the problem cases are admitted into hospital where viral diagnostic facilities exist. Even so, a high proportion of respiratory problems in small children do not seem to respond to antibiotics and are presumed to be viral. In clinical practice a specific virus is often difficult or not diagnosed because current tests lack of sensitivity. Viral Respiratory Pathogens The major causes of ARD in children and adults are influenza A and B viruses, parainfluenza virus types 1, 2, 3, respiratory syncytial virus (RSV), adenovirus and rhinovirus. Other viruses such as coronavirus, bocavirus, enterovirus, parainfluenza 4, parvovirus type 4 and 5 infect the respiratory tract at a much lower frequency. It is now clear that rhinoviruses and coronaviruses once thought to cause only a common cold can cause LRTI and ARD and can be fatal in some cases. The viruses mentioned have overlapping clinical presentations and cause both URTI and LRTI and attending physicians usually cannot distinguish the causative agent without a laboratory diagnosis. Since 2000, newly discovered respiratory viruses include new avian influenza viruses, human metapneumovirus, severe acute respiratory syndrome (SARS) and the 2009 H1N1 pandemic influenza strain. Traditional Diagnostic Methods Over the past two decades, virus isolation and serology have been the mainstay of the clinical laboratory for diagnosing respiratory virus infections. Virus isolation was performed using a number of cell lines and, together with embryonated hen eggs for influenza virus, provided the means for isolating respiratory viruses. A number of serological tests including haemagglutination inhibition (HAI) test, complement fixation and enzyme immunoassay (EIA) were used for testing paired acute and convalescentphase sera for diagnosing infections. In the early 1990s tube cultures were replaced by shell vial culture (SVC) and together with specific monoclonal antibodies could detect specific viral antigens in 1 to 2 days instead of 8 to 10 days for tube culture. Direct fluorescent antibody (DFA) staining of cells derived from nasopharyngeal swabs or nasopharyngeal aspirates became the mainstay for many laboratories and provided a rapid test result. EIAs were introduced in the 1980s and 1990s but these tests lacked sensitivity and were usually relegated to point-of-care testing in defined settings. Info Link, PaLMS 5

8 Multiplex Polymerase Chain Reaction (PCR) Nucleic acid amplification procedures including PCR have been developed for most respiratory viruses and today these highly sensitive tests are becoming routine, and the preferred tests in clinical laboratories for detecting respiratory viruses. Multiplex PCR represents the latest diagnostic approach for the clinical laboratory and completes the evolution of diagnostics for respiratory viruses. The introduction of multiplex PCR for respiratory virus detection at PaLMS Microbiology Department, Royal North Shore Hospital Sydney, to detect 15 respiratory viruses (Table 1) from clinical specimens such as nasopharyngeal aspirates, nasopharyngeal swabs broncho alveolar lavage has greatly increased detection sensitivity and specificity of a causative respiratory viral agent. Table 1. Viruses detected using Multiplex PCR Adenovirus A/B/C/D/E Coronovirus 229E/NL63 Parainfluenza virus 1 Parainfluenza virus 2 Parainfluenza virus 3 Coronavirus OC43 Rhinovirus A/B/C Influenza A virus Respiratory syncytial virus A Respiratory syncytial virus B Bocavirus 1/2/3/4 Influenza B virus Parainfluenza virus 4 Enterovirus Human metapneumovirus Dual respiratory virus infections are poorly detected using DFA and SVC methods because of subjective DFA results. Dual or even triple infections are routinely reported using multiplex PCR and have accounted for between 8 and 11% of positive specimens. The ability to easily detect dual infections provides the means and impetus for studies to examine the clinical importance of dual infections and in particular whether certain individuals are at greater risk for dual infections or whether they result in a poorer outcome for the patient. The overall sensitivity offered by multiplex PCR means that infected patients will be diagnosed more accurately, more often, especially at times during the course of their infection when they are shedding low levels of virus that would be missed by non-molecular tests. The benefits of a more accurate diagnosis are (a) benefits the patient in terms of receiving the appropriate antiviral drugs such as oseltamivir or zanamivir in the case of influenza virus (b) assists infection control practitioners in providing appropriate infection control measures to minimize the risk of nosocomial spread (c) assists the primary care practitioner by stopping the search for a diagnosis even if there is no beneficial antiviral agent for the respiratory virus that was found (d) minimizes unnecessary antibiotic use and (e) provides more accurate information to public health authorities regarding what viruses are circulating in the community so that they can adjust public health policy. The adoption of multiplex PCR tests for respiratory viruses will have a great benefit over the next few years since multiplex tests offer laboratories the ability to detect a wide range of viral infections. This was not previously possible. These tests will allow laboratories to report infections with conventional viruses such as rhinovirus as well as newly discovered viruses using a single test. Testing for multiple viruses in a single test may provide a saving of resources and cost less than the aggregate cost of performing multiple uniplex (single agent) PCR tests. References 1. Mahony, J.B. Detection of Respiratory Viruses by Molecular Methods. Clinical Microbiology Reviews. Oct.2008, p Seeplex Multi detection at Once Molecular Diagnostics Automatic Detection DPO-based Multiplex PCR. Respiratory Pathogen Detection. Seegene Inc. Korea. 3. Keen, A. Viral Respiratory Infections. 6 Info Link, PaLMS

9 Past Issues Issue Twenty Three Issue Twenty Two Issue Twenty One Issue Twenty Issue Nineteen Issue Eighteen Issue Seventeen, Issue Sixteen, Issue Fifteen, Issue Fourteen, 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, Haemoglobin A1c A New Era Lab Tests Online Trace Elements in Health and Disease PaLMS Toxicology Cytogenetic advances in the detection of prognostic factors in B cell malignancies, Trace Elements in Health and Disease Human Papilloma Virus, Expanding Molecular Biology 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 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. Info Link, PaLMS 7

10 Infolink Mailout and Change of Details Please select the relevant box and fill in the details below: I would like to: Be included in the Infolink mail out (please fill in details below) Change my current details (please fill in details below) Be removed from the Infolink mail out Name:... Telephone:... New Details Title:... Name:... Position:... Provider Number:... Speciality area:... Business Name:... Address:... Phone:... Fax: Please photocopy this page and fax to Vicki Johnston, PaLMS Marketing and Communication Manager (02) Or send an vjohnston@nsccahs.health.nsw.gov.au 8 Info Link, PaLMS

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12 PaLMS COLLECTION FACILITIES Northern Sydney Enquiries or PaLMS Chatswood Katherine Street Medical Centre Unit 235, 1 Katherine Street Chatswood NSW 2067 Ph: (02) Monday to Friday: 8.00am to 4.30pm Dee Why Unit 1 "Seascape" Fisher Road Dee Why NSW 2099 Ph: (02) Monday to Friday: 8.30am to 5.00pm Eastwood / Ryde Hospital Denistone Road, Eastwood Ryde NSW 2122 Ph: (02) Monday to Friday: 8.00am to 4.30pm Saturday: 9.00am to midday Frenchs Forest Building 1, Level 2 Frenchs Forest Road Frenchs Forest NSW 2086 Ph: (02) Monday to Friday: 8.00am to 4.30pm Hornsby Ku-ring-gai Hospital Adj. Main Entrance Palmerston Rd Hornsby NSW 2077 Ph: (02) Monday to Friday: 8.30am to 5.00pm Saturday: 8.30am to midday Manly Hospital West Wing, Darley Road Manly NSW 2095 Ph: (02) Monday to Friday: 8.00am to 4.30pm Saturday: 9.00am to12.00 midday Mona Vale Hospital Level 2, Coronation Street Mona Vale NSW 2103 Ph: (02) Monday to Friday: 8.00am to 4.30pm Saturday: 9.00am to midday St Leonards North Shore Private Hospital PaLMS Collection Suite, St Leonards NSW 2065 Ground Floor, Westbourne St Ph: (02) Monday to Friday: 8.00am to 6.00pm Saturday: 9.00 to 1.00pm St Leonards Royal North Shore Hospital Clinic 13, Outpatient Department, Level 3, Pacific Highway St Leonards NSW 2065 Ph: (02) Monday to Friday: 8.30am to 5.00pm Central Coast Enquiries (02) Gosford Hospital Central Coast Specialist Centre Holden Street Gosford NSW 2250 Ph: (02) Monday to Thursday: 9.00am to 3.00pm Outpatient Department Level 3, Holden Street Gosford Hospital Ph: (02) Monday to Friday: 8.00am to 4.00pm Wyong Hospital Pathology Collection Centre (near Outpatients Clinic) Pacific Highway Wyong NSW 2259 Ph: (02) Monday to Friday: 8.00am to 2.00pm For further information visit or Neutral Bay Suite 3, 5 Waters Road Neutral Bay NSW 2089 Ph: (02) Monday to Friday: 8.30am to 5.00pm Saturday: 9.00am to midday 12 palms@nsccahs.health.nsw.gov.au CATALOGUE NUMBER: NS09875