Test Catalog

Test Id : CMVNG

Cytomegalovirus (CMV) Drug Resistance, Next-Generation Sequencing, Plasma

Useful For
Suggests clinical disorders or settings where the test may be helpful

Detecting mutations (variants) in the cytomegalovirus genes, UL97 and UL54, which are associated with antiviral resistance

 

This test is not useful for the initial diagnosis of CMV infection

Genetics Test Information
Provides information that may help with selection of the correct genetic test or proper submission of the test request

This test uses next-generation sequencing to assess whether variants (mutations) that are associated with antiviral resistance are present in genes UL97 and UL54.

Testing Algorithm
Delineates situations when tests are added to the initial order. This includes reflex and additional tests.

This test may be performed following quantitative nucleic acid amplification test results that suggest an increase, or stabilization, of cytomegalovirus viral load while the patient is on appropriate antiviral therapy.

Method Name
A short description of the method used to perform the test

Next-Generation Sequencing (NGS)

NY State Available
Indicates the status of NY State approval and if the test is orderable for NY State clients.

Yes

Reporting Name
Lists a shorter or abbreviated version of the Published Name for a test

CMV Antiviral Resistance by NGS, P

Aliases
Lists additional common names for a test, as an aid in searching

CMV by NGS

CMV Drug resistance

CMV sequencing

Cytomegalovirus antiviral resistance

UL97

UL54

Next Gen Sequencing Test

Ganciclovir

Cidofovir

Foscarnet

Testing Algorithm
Delineates situations when tests are added to the initial order. This includes reflex and additional tests.

This test may be performed following quantitative nucleic acid amplification test results that suggest an increase, or stabilization, of cytomegalovirus viral load while the patient is on appropriate antiviral therapy.

Specimen Type
Describes the specimen type validated for testing

Plasma EDTA

Ordering Guidance

This test is not intended for the initial diagnosis of cytomegalovirus (CMV) infection. If a diagnostic test is needed, order CMVQN / Cytomegalovirus (CMV) DNA Detection and Quantification by Real-Time PCR, Plasma.

 

Next-generation sequencing testing may be indicated if a patient with known CMV infection has a rising, or stabilizing, viral load while on antiviral therapy. This test can assist in determining whether CMV is present that has acquired mutations (variants) associated with antiviral resistance and should only be performed on patients who have had a recent (ie, within the previous 7 days) CMV quantitative plasma viral load of 500 IU/mL or greater.

 

Testing for CMV resistance should be performed no more frequently than once every 4 weeks.

 

Changes in CMV viral load 0.5 log or greater are considered significant and may prompt testing for antiviral resistance if the patient is on appropriate therapy.

Additional Testing Requirements

Plasma submitted for next-generation sequencing (NGS) testing must have been collected within 7 days of a viral load assay (CMVQN / Cytomegalovirus [CMV] DNA Detection and Quantification by Real-Time PCR, Plasma or similar test) with a result of 500 IU/mL or greater.

Shipping Instructions

1. Freeze plasma specimen immediately, and ship specimen frozen on dry ice.

2. If shipment will be delayed for more than 14 days, freeze plasma specimen at -20 to -80 degrees C (up to 30 days) until shipment on dry ice.

ORDER QUESTIONS AND ANSWERS

Question ID Description Answers
CMVQ1 CMV Quant >= 500 IU/mL last 7 days? Yes
No

Specimen Required
Defines the optimal specimen required to perform the test and the preferred volume to complete testing

Supplies: Sarstedt Aliquot Tube, 5 mL (T914)

Collection Container/Tube: Lavender top (EDTA)

Submission Container/Tube: Plastic vial

Specimen Volume: 1.2 mL

Collection Instructions: Centrifuge and aliquot plasma into a plastic vial.

Additional Information: Plasma for next-generation sequencing must be collected within 7 days of a viral load assay (ie, CMVQN) with a result of 500 IU/mL or greater.

Forms

If not ordering electronically, complete, print, and send a Microbiology Test Request (T244) with the specimen.

Specimen Minimum Volume
Defines the amount of sample necessary to provide a clinically relevant result as determined by the Testing Laboratory

1 mL

Reject Due To
Identifies specimen types and conditions that may cause the specimen to be rejected

Gross hemolysis OK
Gross lipemia OK

Specimen Stability Information
Provides a description of the temperatures required to transport a specimen to the performing laboratory, alternate acceptable temperatures are also included

Specimen Type Temperature Time Special Container
Plasma EDTA Frozen (preferred) 30 days
Refrigerated 14 days

Useful For
Suggests clinical disorders or settings where the test may be helpful

Detecting mutations (variants) in the cytomegalovirus genes, UL97 and UL54, which are associated with antiviral resistance

 

This test is not useful for the initial diagnosis of CMV infection

Genetics Test Information
Provides information that may help with selection of the correct genetic test or proper submission of the test request

This test uses next-generation sequencing to assess whether variants (mutations) that are associated with antiviral resistance are present in genes UL97 and UL54.

Testing Algorithm
Delineates situations when tests are added to the initial order. This includes reflex and additional tests.

This test may be performed following quantitative nucleic acid amplification test results that suggest an increase, or stabilization, of cytomegalovirus viral load while the patient is on appropriate antiviral therapy.

Clinical Information
Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test

Cytomegalovirus (CMV) is a DNA virus with a seroprevalence of approximately 50% in the United States. Acute infection can be asymptomatic or cause a mononucleosis-like illness in immunocompetent individuals. After acute infection, the virus enters a latent state. Reactivation of the virus can occur, particularly if a patient becomes immunosuppressed. Immunosuppressed patients are also at higher risk of severe acute infection. CMV disease may range from congenital disease, retinitis, inflammation of the gastrointestinal tract, encephalitis, and pneumonia.

 

Treatment for CMV typically involves antiviral drugs as well as decreasing the degree of immunosuppression (if applicable and medically advisable). Currently, anti-CMV drugs bind and inhibit either a viral kinase (UL97 gene product) or a viral DNA polymerase (UL54 gene product).

 

There are a number of assays available to test for the presence of CMV. Quantitative assays report the amount of CMV DNA present and can be used to monitor the viral load in patients who are at risk of CMV disease as well as assess response to antiviral therapy. A rising CMV viral load (ie, increase of > or =0.5 log between samples) correlates with an increased risk of CMV disease and may indicate treatment failure (ie, due to antiviral resistance) if the patient is on appropriate therapy.

 

Certain mutations (variants) in UL97 and UL54 have been associated with antiviral resistance. This test uses next-generation sequencing to analyze the sequence of UL97 and UL54. Identified mutations are reported if they have been previously associated with CMV antiviral resistance and are present in at least 15% of the sequences analyzed. This assay uses a database of known resistance-associated mutations that is periodically updated with new mutations reported in the scientific literature.

Reference Values
Describes reference intervals and additional information for interpretation of test results. May include intervals based on age and sex when appropriate. Intervals are Mayo-derived, unless otherwise designated. If an interpretive report is provided, the reference value field will state this.

None Detected/Not Predicted

Interpretation
Provides information to assist in interpretation of the test results

If a resistance-associated mutation (variant) is detected, a patient's antiviral regimen may need to be adjusted for optimal response.

 

If no resistance-associated mutations are detected, it is still important to assess the patient's clinical response and quantitative viral load before determining that the infecting virus is susceptible to a treatment regimen (see Cautions).

 

Predicted drug resistance is reported separately for each antiviral drug.

 

Predicted resistance to one antiviral may or may not be associated with predicted cross-resistance to other drugs.

Cautions
Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances

This assay is designed to detect resistance-associated mutations (variants) in the genes UL97 and UL54. Only mutations (variants) that have been reported in the literature to confer antiviral resistance and included in the sequence analysis will be reported. Unrecognized resistance mutations may exist, which are not reported in this assay. Furthermore, previously reported resistance-associated mutations may not result in a resistant phenotype in all cases.

Supportive Data

Accuracy:

The accuracy of the cytomegalovirus (CMV) drug resistance by next-generation sequencing (NGS) assay was determined by testing a combination of clinical and spiked plasma samples. Clinical samples were compared to the results of Sanger sequencing performed at another large reference laboratory. Spiked samples were compared to the expected results. Among 28 clinical plasma samples tested by Sanger sequencing and the NGS assay, an overall agreement of 96.4% (27/28) was observed at the level of determining overall drug resistance (Table 1).

 

Table 1. Comparison of NGS and Sanger sequencing for determination of antiviral drug resistance in clinical plasma samples (n=28).

Number of interpretations by Sanger sequencing predicting

Number of interpretations by NGS predicting

Susceptible

Ganciclovir resistance

Cidofovir resistance

Foscarnet resistance

Susceptible

11

0

0

0

Ganciclovir resistance

0

16

0

1(*)

Cidofovir resistance

0

0

1

0

Foscarnet resistance

0

0

0

2

(*) This sample showed an A692S allelic variant (which confers Foscarnet resistance) in UL54 by Sanger sequencing, but M460I and M460V allelic variants (which confer Ganciclovir resistance) in UL97 were detected by NGS.

 

The data were analyzed at the level of individual resistance-associated variants, and showed the following results:

Table 2.

Number of variants detected by Sanger Sequencing

UL97

UL54

 

 

DEL 598-603

A594V

C603W

C607Y

H520Q

L595S

L595W

M460V

M460I

A692S

A987G

L501F

V781I

Not detected

TNP

Number of variants detected by NGS

UL97

DEL 598-603

1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A594V

 

3

 

 

 

 

 

 

 

 

 

 

 

 

 

C603W

 

 

1

 

 

 

 

 

 

 

 

 

 

 

 

C607Y

 

 

 

0

 

 

 

 

 

 

 

 

 

1(a)

 

H520Q

 

 

 

 

5

 

 

 

 

 

 

 

 

 

 

L595S

 

 

 

 

 

5

 

 

 

 

 

 

 

 

 

L595W

 

 

 

 

 

 

1

 

 

 

 

 

 

 

 

M460V

 

 

 

 

 

 

 

3

 

 

 

 

 

1(b)

1(c)

M460I

 

 

 

 

 

 

 

 

0

 

 

 

 

 

1(c)

UL54

A692S

 

 

 

 

 

 

 

 

 

0

 

 

 

 

 

A987G

 

 

 

 

 

 

 

 

 

 

1

 

 

 

 

L501F

 

 

 

 

 

 

 

 

 

 

 

1

 

 

 

V781I

 

 

 

 

 

 

 

 

 

 

 

 

2

 

 

 

Not detected

 

 

 

1(d)

 

 

 

 

 

1(c)

 

 

 

11

 

 

TNP

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0

 

(a) This specimen showed a C607Y variant (15.7% prevalence) in UL97 by NGS; however, this variant was not detected during initial testing by Sanger sequencing. Sanger sequencing utilizes a prevalence threshold of 20%. In contrast, NGS analysis utilizes a lower prevalence threshold of 15%. The predicted resistance of this specimen was the same by both assays.

(b) This specimen showed a M460V variant (19.3% prevalence) in UL97 by NGS; however, this variant was not detected during initial testing by Sanger sequencing. Sanger sequencing utilizes a prevalence threshold of 20%. In contrast, NGS analysis utilizes a lower prevalence threshold of 15%. The predicted resistance of this specimen was the same by both assays.

(c) This specimen showed an A692S variant in UL54 (conferring resistance to Foscarnet) by Sanger; however, no UL54 variants were detected by NGS. The NGS assay detected two variants in UL97 (M460V, M460I), but Sanger did not generate a result for UL97 due to poor sequence. Quantity not sufficient for testing by a third method.

(d) This specimen showed a C607Y variant in UL97 by Sanger sequencing. This variant was detected by NGS, but at a prevalence of 9.02%. The predicted resistance of these specimens was the same by both assays.

 

To supplement the number of positive samples achieved by clinical plasma samples, spiking studies were performed. Sixteen analyte-negative plasma samples were spiked with a plasmid containing variants known to confer resistance and were then analyzed by the NGS assay. The NGS assay exhibited an overall percent agreement of 87.5% (14/16) with the expected result (at the variant level) upon initial testing. After discordant analysis, the NGS assay showed an overall agreement of 100% (16/16).

 

Limit of Detection:

The limit of detection for this assay was established as 500 IU/mL.

 

Analytical Specificity:

Specificity was determined using a panel of viruses commonly found in blood along with CMV-positive plasma samples that did not contain resistance variants. In addition, basic local alignment search tool (BLAST) analysis for the UL97 and UL54 primer sequences was performed. The UL97 and UL54 primers were specific for specimens that contained CMV nucleic acid. No variants were detected in wild-type CMV plasma samples. No cross-reacting organisms were identified by BLAST analysis of the primer sequences.

Clinical Reference
Recommendations for in-depth reading of a clinical nature

1. Chou S, Ercolani RJ, Sahoo MK, et al: Improved detection of emerging drug-resistant mutant cytomegalovirus subpopulations by deep sequencing. Antimicrob Agents Chemother. 2014 Aug;58(8):4697-4702

2. Garrigue I, Moulinas R, Recordon-Pinson P, et al: Contribution of next generation sequencing to early detection of cytomegalovirus UL97 emerging mutants and viral subpopulations analysis in kidney transplant recipients. J Clin Virol. 2016 Jul;80:74-81

3. Razonable RR: Drug-resistant cytomegalovirus: clinical implications of specific mutations. Curr Opin Organ Transplant. 2018 Aug;23(4):388-394

4. Houldcroft CJ, Bryant JM, Depledge DP, et al: Detection of low frequency multi-drug resistance and novel putative maribavir resistance in immunocompromised pediatric patients with cytomegalovirus. Front Microbiol. 2016 Sep 9;7:1317

Method Description
Describes how the test is performed and provides a method-specific reference

This test involves amplification of cytomegalovirus UL97 and UL54 genes by polymerase chain reaction (PCR), followed by next-generation sequencing (NGS) of the amplified PCR products using the MiSeq (Illumina). Sequencing results are compared to a reference database of gene mutations (variants) previously reported to be associated with antiviral drug resistance. Only the UL97 and UL54 genes are sequenced. The use of NGS allows this assay to detect resistance-associated mutations even if they are present in a subset of the sequences analyzed. Mutations are reported if they are present in 15% or greater of the sequence reads analyzed.(Unpublished Mayo method)

PDF Report
Indicates whether the report includes an additional document with charts, images or other enriched information

No

Day(s) Performed
Outlines the days the test is performed. This field reflects the day that the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time before the test is performed. Some tests are listed as continuously performed, which means that assays are performed multiple times during the day.

Monday through Friday

Report Available
The interval of time (receipt of sample at Mayo Clinic Laboratories to results available) taking into account standard setup days and weekends. The first day is the time that it typically takes for a result to be available. The last day is the time it might take, accounting for any necessary repeated testing.

10 to 14 days

Specimen Retention Time
Outlines the length of time after testing that a specimen is kept in the laboratory before it is discarded

14 days

Performing Laboratory Location
Indicates the location of the laboratory that performs the test

Rochester

Fees
Several factors determine the fee charged to perform a test. Contact your U.S. or International Regional Manager for information about establishing a fee schedule or to learn more about resources to optimize test selection.

  • Authorized users can sign in to Test Prices for detailed fee information.
  • Clients without access to Test Prices can contact Customer Service 24 hours a day, seven days a week.
  • Prospective clients should contact their Regional Manager. For assistance, contact Customer Service.

Test Classification
Provides information regarding the medical device classification for laboratory test kits and reagents. Tests may be classified as cleared or approved by the US Food and Drug Administration (FDA) and used per manufacturer instructions, or as products that do not undergo full FDA review and approval, and are then labeled as an Analyte Specific Reagent (ASR) product.

This test was developed, and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. This test has not been cleared or approved by the US Food and Drug Administration.

CPT Code Information
Provides guidance in determining the appropriate Current Procedural Terminology (CPT) code(s) information for each test or profile. The listed CPT codes reflect Mayo Clinic Laboratories interpretation of CPT coding requirements. It is the responsibility of each laboratory to determine correct CPT codes to use for billing.

CPT codes are provided by the performing laboratory.

87910

LOINC® Information
Provides guidance in determining the Logical Observation Identifiers Names and Codes (LOINC) values for the order and results codes of this test. LOINC values are provided by the performing laboratory.

Test Id Test Order Name Order LOINC Value
CMVNG CMV Antiviral Resistance by NGS, P 72852-7
Result Id Test Result Name Result LOINC Value
Applies only to results expressed in units of measure originally reported by the performing laboratory. These values do not apply to results that are converted to other units of measure.
604560 CMV UL97 mutations 72833-7
604561 CMV UL54 mutations 33631-3
604562 Ganciclovir Resistance 72854-3
604563 Cidofovir Resistance 72856-8
604564 Foscarnet Resistance 42317-8
CMVQ1 CMV Quant >= 500 IU/mL last 7 days? 86955-2

Test Setup Resources

Setup Files
Test setup information contains test file definition details to support order and result interfacing between Mayo Clinic Laboratories and your Laboratory Information System.

Excel | Pdf

Sample Reports
Normal and Abnormal sample reports are provided as references for report appearance.

Normal Reports | Abnormal Reports

SI Sample Reports
International System (SI) of Unit reports are provided for a limited number of tests. These reports are intended for international account use and are only available through MayoLINK accounts that have been defined to receive them.

SI Normal Reports | SI Abnormal Reports