CYTOMEGALOVIRUS (CMV) GENOTYPING
Label Name: CMV GENO
Lab Discipline: Molecular Diagnostics
Institution:  Duke University Health System 
EAP ID:  LAB6247 
Last Review:  3/17/2017 10:11:22 AM
Specimen Type
  Plasma
Container & Volume
  Age Group   Container   Volume  
  0  - 3 Years LAVENDER TOP TUBE 2  ML
  3 Years - 18 Years LAVENDER TOP TUBE 3.5  ML
Label Reminders
  Must include patient name, MRN, date/time of collection and collector's initals.


Collection Notes
  Adult:
  • Collect 1 Lavender (dehydrated EDTA) tube (3.5 mL)


 
  All:
  • Use EDTA anticoagulant collection tubes. Dehydrated EDTA tubes are preferred.

    Specimens collected in liquid EDTA anticoagulant will yield results that are approximately 15% lower than those obtained from specimens collected in dehydrated EDTA due to the dilution.

    Never use heparin anticoagulants. Heparin inhibits PCR.

    Avoid freeze/thaw cycles, obvious microbial contamination, prolonged ambient temperature exposure (>24 hours).

    Prevent specimen-to-specimen contamination.

    Plasma MUST be separated from cells within 24 hours of collection or if refrigerated, within 72 hours.

    Test performance characteristics have not been established using neonatal specimens.



 
Storage
  DELIVER IMMEDIATELY TO MICROBIOLOGY IN A TIGHTLY SEALED CONTAINER WITH NO EXTERNAL SPILLAGE.

Transport at ambient temperature when delivering within 24 hours of collection.

Transport at 2-8°C when delivering within 72 hours of collection.

Separate plasma into a sterile plastic screw-top tube and deliver frozen if transport will be delayed beyond 72 hours of collection.

Transport
  DELIVER IMMEDIATELY TO MICROBIOLOGY IN A TIGHTLY SEALED CONTAINER WITH NO EXTERNAL SPILLAGE.

Transport at ambient temperature when delivering within 24 hours of collection.

Transport at 2-8°C when delivering within 72 hours of collection.

Separate plasma into a sterile plastic screw-top tube and deliver frozen if transport will be delayed beyond 72 hours of collection.

Turn Around Time -  Routine: 14 days   Stat: N/A
Reference Values


CMV GENO



No Reference Values
Methodology
  This assay uses PCR mediated amplification of the UL54 and UL97 genes followed by Sanger DNA sequencing to detect mutations in the CMV genome that confer anti-retroviral resistance. The CMV DNA sequences encoding amino acids 200-1015 of the UL54 gene and amino acids 430-708 of the UL97 gene are amplified from CMV genomic DNA using oligonucleotide PCR primers that contain M13 universal primer "tails" at their 5' ends, and have 3' ends that are homologous to their CMV genomic target sequence. The resulting PCR products are treated with an exonuclease/ phosphatase mixture (ExoSAP-IT) to remove excess PCR primers and nucleotides. These purified DNA amplicons are then sequenced using universal M13 forward and reverse primers and the Big Dye Terminator v3.1 Cycle Sequencing Kit (ABI). Sequencing products are purified with the Big Dye XTerminator Purification Kit (ABI) and resolved using the ABI3130xl Genetic Analyzer. Data is analyzed using the ABI SeqScape software v2.5. Sequences are compared to the GenBank reference DNA sequence of the human herpes virus 5 (strain AD169): UL54 gene (ID: HHV5gp060) and UL97gene (ID: HHV5gp091).

This test was developed and its performance characteristics determined by the DUHS Clinical Molecular Diagnostics Laboratory. It has not been cleared or approved by the U.S. Food and Drug Administration. This test is used for clinical purposes. It should not be regarded as investigational or for research. This laboratory is certified under the Clinical Laboratory Improvement Amendments of 1988 ("CLIA") as qualified to perform high complexity clinical testing.



   
   
Clinical Significance and Interpretive Data
    BACKGROUND:

CMV belongs to the beta class of herpes viruses, also designated as human herpes virus 5 (HHV5). The CMV linear double stranded DNA molecule encodes a large, complex 230-240 kb genome. The prevalence of CMV antibodies ranges from 50-80%, with higher incidences seen in developing countries and lower socioeconomic classes(1). In healthy individuals, infection is asymptomatic and becomes latent; however, for immunosuppressed patients, infection can result in a variety of clinical manifestations with sometimes life-threatening CMV disease. At risk individuals for active CMV infection or disease include: solid organ transplant recipients (SOTR), hematopoietic stem cell transplant recipients (HSCTR), AIDS patients, and neonates contracting congenital CMV from mothers with primary CMV infection.

PRINCIPLE:

CMV infection is a serious complication for immunocompromised individuals, with an especially negative impact for SOTR. CMV-related symptoms affect up to 75% of organ recipients during the first year following transplantation (2). Primary CMV infection, resulting from a CMV-seropositive donor (D+) to CMV-seronegative recipient (R-), is the biggest risk factor for further development to CMV disease. Secondary CMV infection, caused by intrinsic reactivation or introduction of a new strain from the donor, occurs infrequently and shows a less severe response. Progression to CMV disease in D+/R- patients is associated with increased incidence of allograft rejection, infection of other opportunistic pathogens, tissue-invasive disease, and the long-term effect of decreased patient survival (3).

All D+/R- patients are recommended to receive antiviral prophylaxis for three months following transplant; however, approximately 30% still develop CMV disease after this treatment (4). From those developing delayed-onset primary CMV infection, a small number go on to harbor drug resistant strains, encouraged by suboptimal prophylactic antiviral doses and extended antiviral exposure (5). CMV drug resistant strains emerge almost exclusively from D+/R- transplants, with 94% of resistance detected from this population (6). Multiple studies have shown varied ranges for CMV drug resistance among SOTR, from very rare, 0.1%, up to 10% of all CMV-positive SOTR (6-8). Variation in resistance rates can be attributed to the specific organ transplanted, with lung showing highest incidences followed by combined kidney-pancreas, then heart, kidney, liver, and pancreas (6). Resistance variation rates can also be affected by the aggressiveness of preemptive treatment, potency of immunosuppressive drugs and use of prophylactics (6).

ANTIVIRAL AGENTS:

1. Ganciclovir (GCV) is the most widely used drug in the treatment of CMV. This nucleoside analog requires the UL97-encoded viral kinase to phosphorylate GCV. The active form, GCV-triphosphate, is then incorporated into the viral DNA by the UL54-encoded DNA polymerase and inhibits viral replication. Mutations in UL97 prevent the initial phosphorylation and activating step, while UL54 mutations result in the polymerase failing to incorporate GCV-nucleotides, with both mechanisms conferring GCV resistance (GCVr).
A UL54 GCVr mutation is almost always preceded by a UL97 mutation, with some UL54 GCVr mutations conferring cross resistance to PFA and CDV. Harboring a mutation in UL97 only is associated with low-level GCVr and shorter GCV treatment, while high-level GCVr is associated with mutations in both UL97 and UL54 and extended GCV therapy (9).

2. Foscarnet (PFA) requires no activation, and is a pyrophosphate analogue that inhibits the UL54-encoded DNA polymerase. It reversibly blocks the pyrophosphate binding site of the polymerase as a noncompetitive inhibitor, preventing cleavage of pyrophosphate from deoxynucleoside triphosphates. Mutations in UL54 are the only mechanism for PFA resistance (PFAr) and occur across several UL54 domains. Although most PFAr mutations confer singular drug resistance, some are also cross resistant to GCVr and CDVr.

3. Cidofovir (CDV) is a nucleoside phosphonate activated by host cellular enzyme phosphorylation. CDV-diphosphate acts as a competitive inhibitor of the CMV polymerase with incorporation into viral DNA inhibiting viral replication. Mutations in UL54 are the only mechanism for CDV resistance (CDVr) and occur across several UL54 domains. Generally CDVr mutations also confer GCVr, with a few conferring PFAr as well.

4. Maribavir (MBV) is currently undergoing Phase 3 clinical trials and has thus far shown to be a less toxic alternative. This benzimidazole riboside inhibits the UL97-encoded kinase and impairs viral DNA assembly. A few mutations have been reported conferring MBV resistance (MBVr) occurring in UL97; however no mutation have been described with cross resistance (10).

CMV MUTATIONS:

• UL97 mutations have been identified in over 90% of GCVr CMV isolates. Mutations are restricted to the ATP-binding site (codons 460-520) and substrate recognition site (codons 590-607), with rare mutations reported outside these regions (11). The most common UL97 GCVr mutations include A594V (~30%), L595S (~20%), M460V (~12%) and H520Q (~5%)(11).

• UL54 mutations occur in the highly conserved DNA polymerase regions distributed between codons 300-1000. UL54 has a diverse mutation spectrum with variability of GCVr, CDVr, and PFAr among the gene regions (12). The most common UL54 mutations include PFAr V715M, V781I, and L802M; GCVr and CDVr F412C, L501I/F, and P522S; and GCVr and PFAr A809V (11).

TEST INTERPRETATION:

• A sample showing a clear secondary base or homozygous change is evidence of a variant and indicates either a mutation or a polymorphism.

• A sequence change is also reported for samples showing clear evidence of a deletion, insertion, or duplication.

• Sequence variants will be compared to the MDL CMV Variant database. Individual known polymorphisms may not be reported.

• Alterations will be reported as: (1) Sequence variation is previously reported and has a known associated drug resistance. (2) Sequence variation is previously unreported and has an unknown associated drug resistance. (3) Sequence variation is previously unreported and is probably not associated with drug resistance (i.e. silent), or (4) Sequence variation is previously reported as a known polymorphism and is not associated with drug resistance. Sequence variants that are categorized as type (1) above will be referred to as “mutations.”

• At the discretion of the director, the significance of a sequence change may be speculated upon based on the type of variant, position in the gene, and effect on the protein.

• All mutations will be reported with associated drug resistances for Ganciclovir, Foscarnet, and Cidofovir as available from the literature.

REFERENCES:

1. Centers for Disease Control and Prevention. http://www.cdc.gov/cmv

2. Pereyra F, Rubin RH. Prevention and treatment of cytomegalovirus infection in solid organ transplant recipients. Curr Opin Infect Dis. 2004:17; 357–361.

3. Razonable RR. Epidemiology of cytomegalovirus disease in solid organ and hematopoietic stem cell transplant recipients. Am J Health-Syst Pharm. 2005: 62(Supplement 1); S7-13.

4. Paya C, et. al. Efficacy and safety of valganciclovir vs. oral ganciclovir for prevention of cytomegalovirus disease in solid organ transplant recipients. Am J Transplant. 2004: 4; 611-620.

5. Drew WL, Paya CV, and Emery V. Cytomegalovirus (CMV) resistance to antivirals. American J Transplant.2001: 1; 307-312.

6. Baldanti F, Lurain N, Gerna G. Clinical and biologic aspects of human cytomegalovirus resistance to antiviral drugs. Human Immunology. 2004: 65; 403-409.

7. Limaye AP, et. al. Emergence of ganciclovir disease among recipients of solid-organ transplants. Lancet. 2000: 356; 645-649.

8. Lurain NS, et. al. Analysis and characterization of anti-viral drug-resistant cytomegalovirus isolates from solid organ transplant recipients. JID. 2002: 186; 760-8.

9. Smith IL, Cherrington JM, Jiles RE, et. al. High-level resistance of cytomegalovirus to ganciclovir is associated with alterations in both the UL97 and DNA polymerase genes. JID. 1997: 176; 69-77.

10. Chou S, Van Wechel LC, Marousek GI. Cytomegalovirus UL97 kinase mutations that confer maribavir resistance. JID. 2007:196; 91-94.

11. Gilbert C and Boivin G. Human cytomegalovirus resistance to antiviral drugs. Antimicrobial Agents and Chemotherapy. 2005: 49; 873-883.

12. Chou S, et. al. Viral DNA polymerase mutations associated with drug resistance in human cytomegalovirus. JID. 2003: 188; 32-9.

13. Castor J, et. al. Rapid detection directly from patient serum samples of human cytomegalovirus UL97 mutations conferring ganciclovir resistance. J Clin Microbiol. 2007: 45; 2681-3.

   
Indications
    In immunocompromised individuals with active CMV infection or disease, prolonged antiviral therapy is often required, increasing the patient’s risk for developing antiviral resistance. An increasing or high CMV viral load indicates a significant risk factor for resistant CMV infection, especially in conjunction with D+/R- SOT recipients (lung or kidney-pancreas transplants). Detection of a drug resistant mutation in CMV can confirm patient resistance and aid clinicians in antiviral therapy treatment options.

   
Limitations
    The specificity of DNA sequencing is high for the detection of nucleotide base changes and small deletions and insertions in the region analyzed.

• This assay may not detect an acquired mutation which is present below the 15% detection limit (i.e., mutant cell population of < 15%).

• Only amino acids of UL54 p.200-1015 (c.598-3045), and UL97 p.430-708 (c.1288-2124) of the CMV genome are examined. Changes outside of these region will not be detected.

• The presence of a mutant population containing a large deletion, duplication, insertion, or sequence alteration adversely affecting primer binding may not be identified using these methods.

• Up to 15% of CMV positive patients suspected of drug resistance show co-infection with more than one strain. Preferential amplification may occur in these cases resulting in failure to analyze an underrepresented strain present in the sample.

• The minimum viral load requirement for this assay is >1000 copies/mL; however, lower viral loads can be amplified and higher viral loads have shown insufficient.


   
Related Tests
    CYTOMEGALOVIRUS (CMV), PCR, QUANTITATIVE, PLASMA
   
Molecular Diagnostics Laboratory
(MDX)

Medical Director:
 Michael Datto, M.D., Ph.D.
 Phone: 919-684-6965
 Email: michael.datto@duke.edu
Lab Director:
 Catherine Rehder Ph.D, FACMG
 Phone: 919-613-8434
 Email: catherine.rehder@duke.edu
Lab Director:
 Siby Sebastian Ph.D., DABMG
 Phone: 919-613-8432
 Email: siby.s@duke.edu

Address: 
 Wadsworth Bldg, Cytogenetics, Rm 0220
 2351 Erwin Rd
 Durham,  NC  27705
 Phone: 919-684-2698
 FAX: 919-668-5424

Performing Times: