Molecular Diagnostic (PCR) Testing
Additional Specimen Information for Molecular Testing:
A variety of specimens can be tested for the presence of viruses when molecular amplification methods are used to detect viral DNA or RNA. Serum, plasma, and non-cellular fluids can all be tested with a quantitative assay to determine the viral concentration (copies/mL or International Units/mL). For cellular samples, biopsies, swabs, bone marrows, etc., samples are tested with a qualitative assay to detect the presence or absence of virus (present or not detected). Paraffin embedded tissues can also be tested for the presence of virus after deparaffinization. The best sample type for viral detection depends on the virus and associated clinical disease suspected. Specific tissue test codes exist for tissues routinely tested in the lab. Refer to the following information for the most common sample types tested for each virus.
Sample collections for PCR must be done in a sterile manner to avoid contamination of any kind from the environment and samples should be placed in sterile tubes for transport. After collection, all fluid specimens [plasma, serum, CSF, non-cellular fluids] should be frozen and stored at -20oC. Plasma should be separated from cells within 2 hours of collection if possible. Shipment should be on dry ice if possible, or wet ice if necessary. Whole blood should be collected in EDTA or CPD-A, stored at room temperature, and shipped immediately to arrive within 24 hours. Whole blood or plasma collected with heparin anticoagulant are NOT acceptable as heparin will inhibit PCR amplification reactions. BONE MARROW – it is also very important for bone marrow samples that they are not collected in sodium heparin anticoagulant, or that the syringe used to collect the specimen not be rinsed with heparin as this will inhibit the PCR reaction. Please collect the bone marrow into an unrinsed syringe and then rapidly place into an EDTA or CPD-A and mix rapidly to prevent clot formation. Swabs of lesion areas should be obtained with a Culturette swab, and stored frozen. Fresh tissue specimens should be placed in a small amount of PBS or culture fluid and transported to the lab as quickly as possible on ice. Frozen or fixed tissue should be shipped as is either frozen or at room temperature. Please consult the Molecular Virology, (206) 685-6656 for all questions concerning handling of other specimen types.
Adenovirus by PCR, Quant. - CSF, Plasma or Urine
Adenovirus infections are emerging as life-threatening complications in immunocompromised patients. In transplant recipients, adenoviruses can cause hemorrhagic cystitis, renal nephritis, enteritis, hepatitis, encephalitis, pneumonitis and multiple-organ failure. Disseminated infections frequently result in death. In patients with AIDS, adenoviruses can cause localized or disseminated disease. Adenoviruses can be isolated from CSF and brain from patients with meningoencephalitis. PCR assays allow sensitive and rapid diagnosis. Positive serum adenovirus PCR result can be used as an indicator of severe disseminated adenovirus infection. High adenoviral DNA levels in serum have been correlated with severe disseminated adenovirus infection in children with allogeneic stem-cell transplantation.
Adeno/Metapneumo/Rhino Virus PCR Panel - BAL, NP Swab
Search FUSAMR in the online Laboratory Test Guide.
BK Virus PCR assay - Plasma, Serum, Tissue or Urine
BK virus PCR detects the presence of BK virus DNA in a variety of clinical specimens. Most adults have childhood exposure to BK virus as indicated by the 60 to 80% of adults in the United States with positive tests for BK virus antibody. Some adults have low levels of BK virus in their urine but have no evidence of BK-related disease. Positive tests for BK virus are very rare in serum or plasma of normal adults. Conditions which lead to immunosuppression such as chemotherapy, bone marrow transplantation, high dose steroid therapy, leukemia, and lymphoma, can lead to “reactivation” of the virus and increased replication of BK virus in the kidney and/or bladder. Increasing levels of BK virus and very high levels of BK virus have been associated with late-onset hemorrhagic cystitis after bone marrow transplantation and nephritis after kidney transplantation. High levels of BK replication in either the kidney or bladder can be done by testing urine, serum, or plasma for BK virus by PCR Extremely high levels of BK virus, greater than 1 x 106 copies/ml, are often seen in the urine of infected patients.
See BKVQL in the online Laboratory Test Guide.
See UBKQN in the online Laboratory Test Guide.
Search BKVQN in the online Laboratory Test Guide.
Coronavirus (non SARS-CoV-2) PCR Panel
Search FUSCOV in the online Laboratory Test Guide.
Cytomegalovirus (HHV-5) DNA by PCR - Blood Spot, CSF, Plasma, Serum or Tissue
The cytomegalovirus (CMV) quantitative PCR detects the presence of CMV DNA in a variety of clinical specimens. CMV is an important pathogen in transplant recipients, HIV-infected individuals, and in other types of immunosuppression conditions. The assay is best suited for evaluation of plasma viremia and CMV CNS infection. In general, a high systemic CMV load is associated with CMV disease. In addition, quantitative CMV testing is useful in monitoring response to therapy, successful therapy being associated with a decrease in viral load.
Cytomegalovirus Drug Resist., Rapid UL97 and/or Rapid UL54 - Plasma, Serum or CSF
Studies done with CMV-infected patients who are non-responsive to anti-viral drugs have shown that sequence mutations within the CMV genome can lead to drug resistance. Mutations have been described in the UL97 gene, a phosphotransferase encoding region and the UL54 gene, the DNA polymerase gene. A number of mutations in both regions have been described and many of them have been shown in phenotype testing to correspond to drug resistance. In addition, some of them have been shown in gene transfer experiments where CMV sequence is transferred into a vaccinia virus vector system to result in changes in the viral replication rate. The most commonly used anti-viral for CMV therapy, Ganciclovir, has been shown to have resistance mutations localized to both the UL97 and UL54 regions, while the other two less used drugs, Cidofivir and Foxcarnet, have shown resistance mutations only in the UL54 region. For the UL97 gene, a number of mutations have been described which span about 650 bp of the 3′ end of the gene. For the UL54 gene, the mutations have been seen in over 1 kB of the gene.
Enterovirus by PCR
Enterovirus is the most common cause of aseptic meningitis. PCR detection of enterovirus RNA in CSF is the most rapid way to diagnose enteroviral meningitis. The assay detects Polio, Coxsackie A and B, Echoviruses, and Enteroviruses 68, 69, 70, and 71. During enterovirus season from July – October, the test is run each afternoon, M-F. During the off-season, the test is run 2 or 3 times a week, depending on volume. Sample types other than CSF which may contain Rhinoviruses may give positive results with this assay.
Search EPVQLT code in the online Laboratory Test Catalog.
Epstein-Barr virus (HHV-4) DNA by PCR - CSF, Plasma, Serum or Tissue
Epstein-Barr virus (EBV) quantitative PCR detects the presence of EBV DNA in clinical specimens, most commonly plasma or serum. EBV is the cause of infectious mononucleosis usually seen in children and young adults, which is typically a self-limiting disease. The EBV DNA test is useful for the diagnosis of infectious mononucleosis very early in the disease or when serological testing for EBV is non-diagnostic. Normal adults will have no detectable EBV DNA in their plasma/serum, although normal adults previously infected with EBV will have low levels of EBV DNA in their lymphocytes. For this reason, our test is performed on plasma or serum and not on whole blood or cells. A second and more common use of the EBV DNA test is the detection of “reactivation” of EBV virus in patients who have undergone immunosuppression secondary to chemotherapy or after organ or bone marrow transplantation. Patients with reactivation may have low levels of EBV in their plasma and circulating lymphocytes. Occasionally, immunosuppressed patients may develop a rapidly proliferating B cell lymphoproliferative disease, PTLD. PTLD is an EBV driven-B cell malignancy that can be rapidly fatal if not treated early. PTLD can also be seen in AIDS and HIV-1 infected individuals, and also may be seen as a form of newborn primary immunodeficiency. Development of these rapidly replicating EBV-associated lymphomas can be detected by observing rapidly increasing and very high levels of EBV in the serum or plasma of affected individuals.
EBV PCR can also be performed on tissue specimens; however, all individuals with prior exposure to EBV will have small numbers of lymphocytes containing the latent EBV genome in essentially all tissues and peripheral blood lymphocytes. Thus, the simple detection of EBV in a tissue sample is not sufficient to make the diagnosis of PTLD.
Please consult the laboratory (206) 685-6656 with requests for testing of tissue specimens.
Search EBVQ in the online Laboratory Test Catalog.
Search EBVQLT in the online Laboratory Test Guide.
Hepatitis B Quantitation by PCR - Plasma or Serum
The most sensitive marker of HBV replication is the PCR assay. Most acute and chronic HBV infections have evidence of 1 or more viral proteins, HBV surface, core or e antigen, as well as have HBV DNA in the blood. The HBV DNA test is more useful than the antigen detection tests when the virus concentration is low (<5,000 IU/mL) or when HBV mutants are present which do not produce normal protein antigens. The HBV DNA test is most often used to confirm HBV Ag positive tests, to help clarify confusing HBV serological tests, to screen transplant candidates and donors very sensitively, and to follow levels of HBV DNA during anti-viral therapy. Response to therapy has been associated with significant decreases in the HBV DNA quantity in the blood. HBV DNA has also been used to test chronic hepatitis patients and determine the presence or absence of low level HBV replication. The HBV DNA test may also be useful in testing early in the acute infection prior to the development of the antibody response.
Search HBVQNT in the online Laboratory Test Guide.
Hepatitis B Genotype and Drug Resistance - Plasma or Serum
HBV chronic infection can result in cirrhosis of the liver, liver cancer, liver failure and death. A variety of HBV types and subtypes (A-H genotypes, each genotype having 2-5 subtypes) have been identified and some are associated with either disease severity or risk for development of liver carcinoma. Current treatment options for chronically infected HBV patients include interferon, peg interferon and antiviral drugs such as Lamivudine, Adefovir, Entecavir, Telbivudine and Tenofovir targeted against the polymerase (Pol) region of HBV.
Hepatitis C RNA - Plasma or Serum
The HCV RNA test is used to confirm positive HCV serological tests and to demonstrate the presence of HCV RNA in the blood. Most individuals who have an HCV infection become life-long carriers of the virus. A positive test for HCV in the serum indicates active replication of the virus in the liver and possible liver damage. Once liver damage is confirmed and therapy initiated the HCV RNA test is useful for monitoring of response to therapy. Measurement of HCV RNA levels during antiviral therapy provides a direct measurement of the amount of ongoing viral replication. Drugs that inhibit HCV replication dramatically reduce the absolute levels of viral nucleic acid in serum. Likewise, patients who fail to respond to therapy show no decrease in viral nucleic acid level. Therefore, quantitative nucleic acid assays can be used to monitor direct antiviral response after therapy. At the end of therapy, the test is then used to periodically determine the continued lack of or the resumption of HCV replication.
See HCVQNT in the online Laboratory Test Guide.
Hepatitis C RNA Genotype - Plasma or Serum
The virus genotype present in an HCV-infected patient is a major predictor of the likelihood of clinical response to therapy and therefore the genotype test result is used to tailor the patient treatment protocol. The test should be done prior to the initiation of anti-viral therapy when the virus titer is highest to ensure that enough virus is present in the sample for the test to be accurate. Six major genotypes of HCV and numerous subtypes have been described.
Numerous studies have documented differences in response to therapy based on the HCV genotype present. HCV genotype 1 infections are less responsive to therapy than are Genotypes 2 and 3 and may require 12 months of combination therapy (interferon-a plus ribavirin) to obtain a significant response. However, with genotypes 2 or 3, optimal therapeutic responses usually require only 6 months of combination therapy.
See HCPCGT in the online Laboratory Test Guide.
Herpes Simplex Virus (HSV-1, HSV-2) DNA by PCR - CSF, Plasma, Serum or Tissue
Primary infection in children and adults is usually a localized skin or mucous membrane lesion. HSV DNA can usually be detected on swabs taken from localized lesions or from saliva or genital fluids. The virus then almost always becomes latent in the ganglia innervating the affected area. Recurrent skin lesions can then later be seen in the same localized area caused by reactivation of the virus. Occasionally, the virus can spread more systemically and cause more widespread skin lesions and organ and/or neurological disease. Disseminated HSV infections are an important cause of morbidity and mortality in neonates.
The PCR test is the most sensitive way to detect the presence of HSV DNA in CSF and is thus the most sensitive way to confirm suspected HSV encephalitis in adults, children, and neonates. The HSV PCR test is positive in up to 95% of CSF specimens from HSV encephalitis patients. Because HSV is not normally found in CSF, even low viral levels are associated with HSV encephalitis. HSV DNA can be detected immediately after the onset of neurologic symptoms, after the initiation of acyclovir therapy, and up to 3 weeks after the onset of symptoms.
Significant and life threatening infections with HSV can be seen in immunosuppressed patients who have “reactivation” of previously acquired HSV. HSV can cause a variety of severe infections in transplant cases with organ, bone marrow, and neurologic involvement, and often causes a severe interstitial pneumonia that is usually fatal. Detection of HSV DNA in these more systemic illnesses can be done with plasma or serum specimens from the patient.
The DNA PCR test detects either HSV-1 or HSV-2 (HHV-1, HHV-2). If distinction between HSV-1 and HSV-2 in a positive specimen is clinically indicated, type-specific PCR can be performed from the extracted sample. Please call the lab to add on this test if desired (206) 685-6656.
Also available: HSV Type 1 and HSV Type 2 Drug Resistance to Acyclovir in the Thymidine Kinase (UL23 only) gene detected by Sanger Sequencing. See HSVDR in the online Laboratory Test Guide.
Search HS12QN in the online Laboratory Test Guide.
The DiaSorin Simplexa™ HSV 1 & 2 Direct FDA approved assay is a rapid real-time PCR that enables the direct amplification, detection and differentiation of HSV-1 and/or HSV-2 DNA from both cutaneous and mucocutaneous swab specimens and CSF specimens. Well conserved regions of the HSV-1 and HSV-2 DNA polymerase genes are targeted to identify HSV-1 and HSV-2 DNA respectively in the specimen. Results are reported as “detected” or “not detected” and a semi-qualitative result is provided by reporting the PCR Cycle threshold (Ct) value for positive specimens.
Search RPDHSV in the online Laboratory Test Guide.
HHV-6 DNA by PCR - CSF, Plasma, Serum or Tissue
Most adults have been previously exposed to HHV-6 during childhood. Rarely, HHV-6 causes high fevers and sepsis in infants 3-24 months old and it also appears to be a common cause of febrile seizures. HHV-6 can be present in normal adult, previously exposed individuals in saliva, PBMC’s and other tissues while the virus is almost always absent in plasma, serum and CSF from the same individuals. Occasionally, overwhelming HHV-6 infections can be seen in neonates and children and can be confirmed by HHV-6 DNA detection in either CSF or plasma. Immunocompromised individuals who have been previously exposed to HHV-6 may “reactivate” the virus and have a more systemic viral illness due to HHV-6 infection. Detection of these infections is also best done with plasma or CSF specimens.
Search HH6QN in the online Laboratory Test Guide.
Search HHV6QLT in the online Laboratory Test Guide.
HHV-6 Chromosomal Integration - Whole Blood
Studies have estimated that about 1-2% of the population may have HHV-6 integrated into their DNA. The presence of integrated HHV-6 in all cells of an individual can cause a major complication for the interpretation of HHV-6 PCR testing for children and transplant and other immunosuppressed patients, because the normal PCR assays used to detect HHV-6 infections also detect the integrated DNA. Recently, multiple case reports have identified uninfected individuals with integrated HHV-6 who have been falsely diagnosed with HHV-6 infections due to very high levels of HHV-6 in their blood (>1 x 10e6 copies/mL). HHV-6 PCR quantities present depend upon the sample type used for testing; whole blood assays detect HHV-6 at levels equivalent to the WBC, while serum, plasma, and CSF samples may contain lower levels of virus due to smaller quantities of cellular DNA contaminants. The HHV-6 Chromosomal Integration assay can be used to identify individuals who have suspected “false positive” HHV-6 PCR assays due to viral integration. The test is done on whole blood and samples that contain cells with integrated DNA should give a ratio of approximately 1 copy of HHV-6 per cell.
Search HH6ABC in the online Laboratory Test Catalog.
HHV-8 DNA by PCR (KS or Kaposi's Sarcoma Virus) - Plasma or Tissue
Human Herpes Virus 8, also known as Kaposi’s sarcoma associated herpes virus (HHV-8, KSHV), has been found in the cells of Kaposi’s sarcoma, body cavity lymphomas (primary effusion lymphoma), and Castleman’s disease. The viral genome has also been identified in B-Cell lymphomas occurring in HIV-positive subjects. HHV-8 genomes can be identified by in-situ hybridization to be present in endothelial and spindle cells of Kaposi’s sarcoma tissues. However, a large number of HIV-1+ individuals have been shown to have exposure to HHV-8 virus, so the presence of virus is not diagnostic for the KS-related lymphomas. Rising levels of HHV-8 in plasma or serum is correlated with increasing infected-tissue burden and is the best indication of advancement of the sarcoma or lymphoma. A low level of HHV-8 DNA in peripheral blood has been described for both healthy and HIV negative individuals and is an indication of either low viral replication or the presence of latent virus. The best specimens for testing are biopsies (usually skin biopsies or lymph node biopsies) or plasma.
Search HH8QN in the online Laboratory Test Catalog.
Search HHV8QLT in the online Laboratory Test Catalog .
JC Virus PCR - Brain Biopsy Tissue, CSF or Urine
JC virus is a frequent cause of self-limiting infection in children and by the time of adulthood, a significant number of individuals will have been exposed to the virus. Rarely, the virus infects the myelin-producing cells and causes progressive multifocal leukoencephalopathy (PML). Greater than 90% of affected individuals will have JC virus DNA in their CSF. The affected brain tissue will also contain high levels of the virus. JC virus has also been reported in urine of patients who are immunosuppressed. About 1/3 of kidney transplant patients have “reactivated” JC virus DNA in their urine, and a significant subset of patients have both JC and BK virus present. Unlike BK virus, JC has not been reported to cause clinical disease in these patients.
Search JCVQN. in the online Laboratory Test Guide.
Search UJCVQN for urine sample in the online Laboratory Test Guide.
Norovirus 1 & 2, Rotaviruses and Adenoviruses for Stool samples
The Enteric stool panel PCR assay targets the orf1/orf2 junction of Norovirus types 1 and 2, the NSP3 gene of Rotavirus type A, and the hexon/penton genes of Adenoviruses. Results are reported as “Positive” or “None detected”.
Search SVIPCR in the online Laboratory Test Guide.
Parainfluenza PCR Panel (PIV1, 2, 3, 4)
The UW Clinical Virology Laboratory in the Department of Laboratory Medicine and Pathology incorporates the modified FDA approved Panther Fusion® assay for the detection and differentiation of Human parainfluenza viruses type 1, type 2 , type 3 and type 4 using real-time PCR.
The assays targets the hemagglutinin neuraminidase gene for each virus. Results are reported as Detected or NOT detected and a semi-qualitative result is provided by reporting the PCR Cycle threshold (Ct) value for positive specimens.
Search FUSPIV in the online Laboratory Test Guide.
Human Parvovirus B19 PCR Assay - Bone Marrow, Plasma or Serum
Parvovirus B19 causes a wide spectrum of disease including erythema infectiosum, aplastic crisis in patients with hemolytic anemias, hydrops fetalis, acute arthritis, persistent anemias and neutropenia in immunocompromised patients. PCR amplification of Parvovirus B19 DNA can be used for early diagnosis of B19 infection, as B19 DNA may precede the appearance of IgM antibody.
Search B19PCQ in the online Laboratory Test Guide.
Search B19QLT in the online Laboratory Test Guide.
Sensitive detection of respiratory viruses is important for early diagnosis of infection, especially in immunocompromised patients. This panel of qualitative real-time PCR assays tests for the presence of any of 12 respiratory viruses including RSV, influenza types A and B, human metapneumovirus, parainfluenza types 1, 2, 3, and 4, adenoviruses, coronaviruses, rhinoviruses, and human bocavirus. The PCR assay is both more sensitive and detects a broader range of viruses than does detection by fluorescent antibody staining and is more rapid than viral culture.
Varicella Zoster Virus (HHV-3) DNA by PCR - CSF or Plasma
VZV infections cause chicken pox in children or zoster/shingles in adults. Primary infections with VZV are becoming less common due to vaccination of children with VZV vaccine. Occasionally, systemic infections can be seen in neonates and children which can be very severe and involve the skin, internal organs, and the brain. Neonatal infections are particularly severe in infants born to VZV seronegative mothers. VZV can also cause the skin disease shingles in adults many years after the primary infection and is particularly severe when it occurs in immunocompromised patients including HIV-1 positive individuals and in bone marrow transplant patients. Systemic infections can also often cause significant brain disease including aseptic meningitis and encephalitis that can be detected by the presence of VZV in the CSF. Plasma samples can be used to document systemic infections while skin biopsy and swab material can detect skin and organ disease due to the virus.
Search VZVQN in the online Laboratory Test Guide.
Search VZVQLT in the online Laboratory Test Guide.
Varicella Zoster Virus (Rapid)
The DiaSorin Simplexa™ Varicella Zoster Virus (VZV) Direct FDA approved assay is a rapid real-time PCR that enables the direct amplification, detection of VZV from both cutaneous and mucocutaneous swab specimens and CSF specimens. A well conserved region of the VZV DNA polymerase gene is targeted to identify VZV in the specimen. Results are reported as Detected or NOT detected and a semi-qualitative result is provided by reporting the PCR Cycle threshold (Ct) value for positive specimens.
Search VZVRPD in the online Laboratory Test Guide.