Blood Bank/Pheresis Chemistry Frisbee Laboratory Hematology		Immunology Microbiology Molecular Diagnostics Virology

THE CLINICAL VIROLOGY LABORATORY AT YALE-NEW HAVEN HOSPITAL

Phone: 203 688-3524

General description and hours of operation
The Clinical Virology Laboratory, located on the 5th floor of the Clinic Building in room CB521, is a full service virology laboratory that operates Monday through Friday from 6:30 am to 8:30 pm, and on Saturdays and Sundays from 8:00 am to 4:30 pm. In winter respiratory season, hours are extended to 7 days a week and up to 18 hours a day. Virology is a rapidly changing field and the laboratory is continuously modifying its test menu and test algorithms in order to keep abreast of current developments and provide the best services to our patients. The Laboratory Director and Laboratory Manager are available by phone or by beeper for consultations and questions. Clinical Virology Teaching Rounds are conducted biweekly for the infectious disease team and laboratory medicine residents. In addition, a Clinical Virology Newsletter is distributed 2 to 4 times per year and a pocket Guide to Viral Diagnosis, updated annually, is provided to clinical staff on request.

Services provided
Services provided include rapid detection of viral antigens in clinical samples, an ever-increasing menu of molecular tests, conventional and rapid virus isolation techniques, and determination of viral antibody response. In addition, a table of seasonal viruses, such as RSV and influenza, identified by the laboratory is updated weekly and provided below as a service to physicians in the community.

Direct detection of virus in clinical samples by immunofluorescence
Direct detection of viral antigens by cytospin-enhanced immunofluorescence (DFA) is employed for VZV and HSV in skin lesions (figure 1); HSV, VZV and adenovirus in eye swabs; RSV, influenza A and B, parainfluenza types 1-3 and adenovirus in respiratory samples. CMV antigenemia allows rapid quantification of CMV in peripheral blood cells (figure 2). Hepatitis B surface antigen is detected in serum and rotavirus in stool by ELISA.

Molecular methods ( see table 1)
Commercial Roche assays are used to detect and quantify HIV-1 RNA, HCV RNA and HBV DNA in plasma or serum, and to detect HIV-1 provirus in peripheral blood mononuclear cells. Real-time TaqMan PCR assays are used to detect HSV, VZV, CMV, EBV, enteroviruses, parvovirus B19, human metapneumovirus, influenza A and B, JC virus, BK virus, adenovirus, and norovirus. HCV genotyping and HIV-1 drug resistance genotyping are also available.

Culture methods

Rapid shell vial centrifugation cultures are routinely performed for CMV (figure 4). Samples are centrifuged onto cell cultures, incubated for 1 to 2 days, then fixed and stained with monoclonal antibody to the virus sought. This method detects viral proteins in infected cells prior to visible cytopathology and thus provides a more rapid result.

Antiviral susceptibility testing for HSV is performed by plaque reduction. Both a rapid assessment of sensitivity and a precise determination of 50% inhibitory concentration (IC 50 ) are available.

Antibody response for immune status and diagnosis of active infection
Antibody tests are available for hepatitis A, B, C, HIV, HSV, CMV, VZV, rubella, measles, mumps, parvovirus B19, and West Nile virus (ELISA), and for EBV (IF).

Clostridium difficile cytotoxin assay
Clostridium difficile diagnosis is a two-step process. First the bacteria are detected using an ELISA and negatives are reported the same day. Next, the bacteria-positive samples are assayed using the "gold standard" test of neutralization of cytotoxicity in cell culture. Cytotoxin results are reported at 4, 24 and 48 hours. Treatment is based on presence of cytotoxin.

Clinical Virology Laboratory Staff
Marie Louise Landry, M.D.
Director
Phone: 203 688-3475
Beeper: 1-888-631-0112

David Ferguson, M.T., A.S.C.P.
Laboratory Manager
Phone: 203 688-1102
Beeper: 412-0981

Sandra Cohen, B.S.
Section Coordinator
Phone: 203 688-3524

Robin Garner, M.T., A.S.C.P.
Coordinator, Molecular Virology

Clinical Virology Laboratory Staff: (Left to Right) Jocelyn Sadala, Sandy Cohen, Heather Wilson, Gerri Russo, Cheryl Tow-Keogh, Alicia Cimino, Dr. Marie Landry, Ruth Ferro, Robin Garner, Dave Ferguson, Jody Crisculo, Maureen Owen, Terri Constantinidi (Not pictured Lisa Voglesong, Clare Blake, Nicole Dubreiul)

Table 1. Molecular tests performed in Clinical Virology

• Most molecular tests are done Monday-Friday, and are not done on weekends or holidays, when staffing is minimal. The exceptions are HSV PCR and EV RT-PCR on CSF, which are done on Saturdays. Other CSF analytes or high priority samples will also be done when possible.
  
• IVD = in vitro diagnostic; RUO= research use only; ASR = analyte specific reagent
  

Table 2: Weekly Report of Seasonal or Epidemic Viruses Detected by the Clinical Virology Laboratory *

* Samples tested may be obtained from patients at Yale New Haven Hospital, from other hospitals in the region, from community doctors’ offices or clinics
¹ Direct immunofluoresence
² PCR
³ ELISA

RSV, respiratory syncytial virus
HMPV, human metapneumovirus

Selected References:

1. Landry, M.L. and Hsiung, G.D. Primary isolation of viruses. In Clinical Virology Manual, Specter SS, Hodinka R and Young S (Eds), 3 rd edition, American Society for Microbiology Press, Washington, D.C. 2000.
2. Landry ML, Stanat S, Biron K, et al. Standardized plaque reduction assay for determination of drug susceptibilities of cytomegalovirus clinical isolates. Antimicrob Agents Chemother 44:688-692, 2000.
3. Landry ML, Ferguson D. SimulFluor Respiratory Screen for rapid detection of multiple respiratory viruses in clinical specimens by immunofluorescence staining. J Clin Microbiol 38:708-711, 2000.
4. Landry ML, Ferguson D. Reduced ability to culture cytomegalovirus from peripheral blood leukocytes isolated by direct erythrocyte lysis. J Clin Microbiol 38:3906, 2000.
5. Landry ML, Garner R, Ferguson D. Use of plastic vacutainer tubes for quantification of human immunodeficiency virus type 1 in blood specimens J Clin Microbiol 39:354-356, 2001.
6. Landry ML, Topal J , Ferguson D, Giudetti D, Tang Y. Evaluation of Biosite Triage Clostridium difficile Panel for the rapid detection of Clostridium difficile in stool. J Clin Microbiol 39: 1855-1858, 2001.
7. Landry ML. Rapid Viral Diagnosis. In the Manual of Clinical Laboratory Immunology, 6th Edition, American Society of Microbiology Press, Washington DC, 2002.
8. Landry ML, Garner R, Fergsuon D. Rapid enterovirus RNA detection in clinical specimens using nucleic acid sequence based amplification (NASBA). J Clin Microbiol 41:346-350, 2003.
9. Landry, ML, Ferguson D. Suboptimal detection of influenza in adults by Directigen Flu A + B and correlation with number of antigen-positive cells detected by cytospin immunofluorescence. J Clin Microbiol 41:3407-3409; 2003.
10. Habib-Bein NF , Beckwith WH, Mayo D, and Landry ML. SmartCycler Real-Time RT-PCR diagnosis of influenza A virus in a public health laboratory compared with direct immunofluorescence and cell culture in a medical center. J Clin Microbiol 41: 3597-3601, 2003.
11. Landry ML. Viral infections. In Burrows GN, Duffy TP and Copel J, Eds, Medical complications during pregnancy, 6th Edition, Elsevier, 2004.
12. Landry ML, Ferguson D, Cohen S, Peret TCT, Erdman DD. Detection of human metapneumovirus in clinical samples by immunofluorescence and shell vial centrifugation culture in three different cell lines. J Clin Microbiol 43:1950-1952, 2005.
13. Landry ML, Garner R, Ferguson D. Real-time nucleic acid sequence based amplification using molecular beacons for detection of enterovirus in clinical specimens. J Clin Microbiol 43:3136-3139, 2005.
14. Landry ML. Rapid Viral Diagnosis. In the Manual of Molecular and Clinical Laboratory Immunology, 7th Edition, American Society of Microbiology Press, Washington DC, 2006.
15. Campbell S and Landry ML. Rapid Antigen Tests. In Advanced Techniques in Diagnostic Microbiology, Kluwer Academic Publishers, 2006.
16. Kesebir D, Vazquez M, Weibel C, Shapiro E, Ferguson D, Landry ML, Kahn JS. Human bocavirus infection in young children in the United States: molecular epidemiology and clinical features associated with a newly emerging respiratory virus. J Infect Dis 194:1276-82, 2006.
17. Murray PR, Baron EJ, Jorgensen JH, Landry ML, and Pfaller MA. Eds, Manual of Clinical Microbiology, 9th Edition, American Society of Microbiology Press, Washington DC, 2007.