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Molecular Diagnostic (PCR) Testing

Note: For HIV PCR testing, see HIV 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 (which requires an additional billing charge). 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, HSV, CMV, EBV, HHV-6, BK, and VZV. If detection of a virus is needed for viruses other than these 6, request the "Virus Detection by PCR, Tissue" test and indicate on the requisition slip the virus to be tested. Refer to the following information for the most common sample types tested for each virus.

Two specimens must be collected if both culture and PCR testing are requested. 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) 667-6999 for all questions concerning handling of other specimen types.

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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.

Search "ADVQN" code in the online Laboratory Test Catalog for information on Adenoviruses, PCR Quantitative.

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.

Search "BKVQL" code in the online Laboratory Test Catalog for information on BK Virus PCR Qual. for tissue biopsy, bone marrow, PBMC, or swabs.

Search "UBKVQN" code in the online Laboratory Test Catalog for information on BK Virus PCR Quant. Urine for urine samples.

Search "BKVQN" code in the online Laboratory Test Catalog for information on BK Virus PCR Quant. for blood and CSF.

Cytomegalovirus (HHV-5) DNA by PCR - 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.

Search "CMVQN" code in the online Laboratory Test Catalog for information on CMV Quant. by PCR for non-cellular body fluid samples.

Search "CMVQLT" code in the online Laboratory Test Catalog for information on CMV Qual. by PCR for tissue biopsy, bone marrow, PBMC, or swabs.

Cytomegalovirus Drug Resist., Rapid UL97 - Plasma or Serum

The test amplifies and sequences two overlapping regions of the UL97 gene spanning amino acids 446-638 of the UL97 protein. Clinical resistance to Ganciclovir has been shown to map to multiple amino acid mutations within this region. Mutations within the UL97 protein, which functions to phosphorylate Ganciclovir within infected cells, have been shown to result in decreased activity of the drug. Studies of the variety of mutations in the UL97 gene are on-going to determine which mutations are most strongly associated with phosphorylase dysfunction and clinical drug resistance.

* Eight different amino acid substitutions and 2 different deletions in the UL97 account for more than 90% of the reported cases associated with drug resistance. CMV strains isolated from these patients have shown strong in-vitro drug resistance when cultured with Ganciclovir. In addition, viral constructs containing these 10 specific mutations have been tested with in-vitro marker experiments and shown to result in decreased Ganciclovir phosphorylation. Thus these 10 mutations have been strongly associated in a variety of ways with Ganciclovir drug resistance. More than 30 additional mutations at other locations within the UL97 gene have been reported in one or more patients with clinical resistance but have not been as carefully studied with all available techniques. These mutations are therefore less well characterized at this time. Results from our UL97 sequencing test will contain the name of any mutation found and an interpretation which describes whether the mutation has been strongly associated with resistance at this time or not.

The other two common anti-viral drugs, Foscarnet and Cidofivir, do not require activation by the UL97 gene product, so this screening test cannot be used to measure CMV drug resistance to these two drugs. Mutations of the UL54 gene, the DNA polymerase, have been shown to be associated with resistance to Ganciclovir as well as Foscarnet and Cidofivir.

Search "CMVQLT" code in the online Laboratory Test Catalog for information on CMV Drug Resist, Rapid UL97.

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 "ENTPCR" code in the online Laboratory Test Catalog for information on Enterovirus by PCR.

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) 667-6999 with requests for testing of tissue specimens.

Search "EBVQ" code in the online Laboratory Test Catalog for information on EBV Quant. by PCR for non-cellular body fluid samples.

Search "EBVQLT" code in the online Laboratory Test Catalog for information on EBV Qual. by PCR for tissue biopsy, bone marrow, PBMC, or swabs.

Hepatitis B 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 copies/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 "HBVPCQ" code in the online Laboratory Test Catalog for information on Hepatitis B by PCR Quantitative.

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.

The HCV assay is now performed by the RT-PCR method and sensitivity for the assay is <60 International Units/mL. The RT-PCR assay is also linear from 60 to 1 x 108 International Units/mL. Extensive in-house evaluations demonstrated excellent correlation of the quantitative results with those of the Bayer bDNA test.

Search "HCVQNT" code in the online Laboratory Test Catalog for information on Hepatitis C RNA, Quantitative information.

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.

Search "HCPCGT" code in the online Laboratory Test Catalog for information on Hepatitis C RNA Genotyping by PCR information.

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) 667-6999.

Search "HSVQN" code in the online Laboratory Test Catalog for information on HSV Quant. by PCR for blood and CSF information.

Search "HSVQLT" code in the online Laboratory Test Catalog for information on HSV Qual. by PCR for tissue biopsy, bone marrow, PBMC, swabs information.

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" code in the online Laboratory Test Catalog for information on HHV-6 Quant. by PCR for non-cellular body fluid samples information.

Search "HH6QLT" code in the online Laboratory Test Catalog for information on HHV-6 Qual. by PCR for tissue biopsy, bone marrow, PBMC, swabs information.

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" code in the online Laboratory Test Catalog for information on HHV-8 PCR, Quant. information.

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" code in the online Laboratory Test Catalog for information on Parvovirus B19 by PCR information.

JC Virus PCR - Brain Biopsy Tissue or CSF

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 "JCVPCQ" code in the online Laboratory Test Catalog for information on JVC PCR Quant. information.

Respiratory Virus by PCR (REVQLT)

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.

Search "REVQLT" code in the online Laboratory Test Catalog for information on Respiratory Virus Qualitative PCR information.

SARS Coronavirus by RT-PCR - Fluids

This RNA virus is a previously unrecognized strain of coronavirus that causes severe atypical pneumonia. First recognized in November, 2002, it has infected more than 8000 people worldwide and resulted in more than 800 deaths. The primary means of transmission is by close person-to-person contact or from direct contact with infectious material from a person who has SARS.

The detection of the genomic RNA from SARS-CoV is based upon reverse transcription of specific genomic RNA sequences followed by real-time PCR amplification. The SARS-CoV RT-PCR test is intended to be used in combination with clinical symptoms and other diagnostic test results to help diagnosis patients suspected of have a SARS-CoV infection. All positive results are confirmed by performing a second RT-PCR assay that targets a different SARS-CoV gene.

Search "SARPCR" code in the online Laboratory Test Catalog for information on SARS Qualitative PCR information.

Vaccinia Virus by PCR - Skin or Mucous Membrane Lesion

Vaccinia virus is used to immunize high-risk persons against smallpox or to protect laboratory technologists who work with vaccinia virus in the research setting. Occasionally, vaccinia virus can be transferred from the active vaccination site to a site elsewhere on the skin through transfer of infection, usually by the fingers. A more generalized infection is sometimes seen in one of three forms: eczema vaccinatum as a complication in children or adults who suffer from chronic dermatitis; true generalized vaccinia, which is a limited, non-fatal infection that occurs in persons with normal skin and a delayed antibody response to vaccination; or progressive vaccinia (vaccinia gangrenosa), which is rare and represents a slow necrotizing progression from the initial vaccination site or elsewhere due to a continued vaccinia viremia. In addition, vaccinia virus may be transferred to a close contact, usually a household member or sexual partner. As such, vaccinia virus should be considered in the differential diagnosis of dermatitis and genital ulcer disease.

To identify vaccinia in skin lesions and distinguish this orthopoxvirus from variola virus (the etiologic agent of smallpox) and varicella-zoster virus (the herpes virus that causes chickenpox and shingles; see Varicella-zoster virus (VZV/HHV-3) DNA by PCR), the UW Clinical Retrovirology Laboratory uses electron microscopy (EM) in combination with real-time reverse polymerase chain reaction (PCR) amplification assays.

Specimen: Touch prep on microscope slide; swab or biopsy - pea-sized amount of material, in a dry, screw-cap tube.

Performed: On an as needed basis, initial EM results within one working day of specimen receipt; PCR results within two working days. Notification of Laboratory Medicine Resident recommended to prioritize and coordinate sample testing.

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" code in the online Laboratory Test Catalog for information on Varicella Zoster Virus by PCR for non-cellular body fluid samples information.

Search "VZVQLT" code in the online Laboratory Test Catalog for information on Varicella Zoster Qual. by PCR for tissue biopsy, bone marrow, PBMC, swabs information.

Virus Detection by PCR, Tissue

Fresh tissue biopsies, needle biopsies, bone marrow, whole blood or PBMC, or any other sample type with cellular material can be tested to detect either DNA or RNA viruses. Fresh biosies and other fluids containing cells should be kept cool and send to the laboratory as soon as possible. Bone marrow and blood specimens should be drawn in EDTA, CPD-A, or PPT tubes and must not be drawn into heparinized tubes or through heparin-wetted syringes as heparin inhibits the DNA amplification reaction. Paraffin imbedded samples can also be tested, but because of tissue fixation variation, false negative results are sometimes seen. If testing for HSV, EBV, CMV, HHV-6, BK, or VZV, please order the specific qualitative virus test. For HBV, HCV, JC, Parvo. B19, West Nile, HHV-8, please order the "Virus Detection by PCR, Tissue" test.

West Nile Virus by PCR - CSF or Serum

The West Nile Virus PCR assay can be used in conjunction with the IgM serology assay as an aid in the diagnosis of viral meningitis or systemic infections due to West Nile Virus. The assay is specific for West Nile virus (Lineage 1 or the New York 1999 strain) and does NOT detect the closely related West Nile Lineage 2 (Uganda) strain. The Lineage 1 (NY99) strain has been identified in the USA and Canada while the Ugandan strain has not yet been seen in North America and is not as strongly associated with the occurrence of meningitis. The PCR assay is also negative when tested with other mosquito-born Flaviviruses including St. Louis, Eastern and Western Equine, and Murray Valley encephalitis viruses. Sensitivity of the PCR assay is much lower (around 60% in early studies) than that of the IgM serology test but is significantly more specific because the serological test is cross-reactive with a number of other Flaviviruses. The low sensitivity of the PCR assay is due to the short period of viremia (a few days) and to low levels (1 x 103 to 1 x 105 copies/mL) of the virus seen in the blood or CSF.

Search "WNVQN" code in the online Laboratory Test Catalog for information on West Nile Virus PCR Quant. information.

Last updated: 11/27/12

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