Test Id : CMVNG

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.

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.

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.

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.

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.

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

1 mL

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

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

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.

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.

None Detected/Not Predicted

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.

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.

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).

(*) 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.

(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.

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

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)

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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.

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