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Need to evaluate the performance of real-time PCR assays for the quantitation of cytomegalovirus DNA load in lower respiratory tract specimens

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There is an increasing appreciation for the potential clinical value of the quantitation of cytomegalovirus (CMV) DNA in the lower respiratory tract in critically ill patients lacking canonical immunosuppression, in view of the possible pathogenic role of CMV in these patients [1]. No data have been published on the analytical performance of real-time PCR assays for this purpose.

We present our data on the performance of the COBAS® AmpliPrep/COBAS® TaqMan CMV PCR Assay (Roche Diagnostics, Mannheim, Germany) for the quantitation of CMV DNA in tracheal aspirates (TA). This CMV PCR assay has been approved recently by the US Food and Drug Administration for use with plasma specimens [2]. We chose TA for the analyses because of the simplicity of their collection in critically ill patients undergoing mechanical ventilation.

We pooled TA obtained from five ICU patients with undetectable CMV DNA levels in TA and in plasma specimens [3]. The pool was fluidized by treatment with N-acetyl cysteine (1 % in PBS) at a 1:1 volume ratio (vortexed for 30 seconds, incubated for 10 minutes at room temperature and centrifuged at 1,600 rpm for 10 minutes). The pellet was then resuspended with distilled water to obtain the initial volume. Four aliquot portions were spiked with different quantities (2.69, 3.69, 4.69, and 5.69 log10 IU/ml) of the First World Health Organization International Standard for CMV (National Institute for Biological Standards and Control, Hertfordshire, UK) [4]. The testing pools were assayed in heptuplicate on three consecutive days.

The fitted regression line between copies/ml and IU/ml for tracheal aspirates was y = 1.0013x – 0.0517 (R2 = 0.999). According to our calculations, 1 copy/ml was equated to 0.90 IU/ml (similar to that for plasma specimens [5]). The overall intra-assay and inter-assay coefficients of variation were 8.2 % (95 % confidence interval (CI), –0.6 to 17.0 %) and 10.77 % (95 % CI, 1.69 to 19.8 %), which are slightly higher than those for plasma specimens [5]. The analytical performance of the assay was analyzed with clinical samples containing different copy numbers of CMV DNA. The data are shown in Table 1. The overall intra-assay and inter-assay coefficients of variation were 14.0 % (95 % CI, 9.8 to 18.1 %) and 15.4 % (95 % CI, 12.1 to 18.8 %), respectively.

Table 1 Performance of the cytomegalovirus PCR assay for quantitation of cytomegalovirus DNA load in tracheal aspirates

Our data indicate that this CMV PCR assay performs well with fluidized TA containing low to intermediate CMV DNA loads (between 500 and 50,000 copies/ml), and may therefore be used for monitoring CMV replication in the lower respiratory tract in critically ill patients undergoing mechanical ventilation.

Abbreviations

CI:

Confidence interval

CMV:

Cytomegalovirus

PBS:

Phosphate-buffered saline

PCR:

Polymerase chain reaction

TA:

Tracheal aspirates.

References

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    Hirsch HH, Lautenschlager I, Pinsky BA, Cardeñoso L, Aslam S, Cobb B, Vilchez RA, Valsamakis A: An international multicenter performance analysis of CMV viral load tests. Clin Infect Dis 2013, 56: 367-373. 10.1093/cid/cis900

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    Chilet M, Aguilar G, Benet I, Belda J, Tormo N, Carbonell JA, Clari MA, Costa E, Navarro D: Virological and immunological features of active cytomegalovirus infection in nonimmunosuppressed patients in a surgical and trauma intensive care unit. J Med Virol 2010, 82: 1384-1391. 10.1002/jmv.21825

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    Fryer JF, Heath AB, Anderson R, Minor PD: Collaborative Study Group: Collaborative Study to Evaluate the Proposed 1stWHO International Standard for Human Cytomegalovirus (HCMV) for Nucleic Acid Amplification (NAT)-based Assays . In WHO ECBS Report 2010, WHO/BS/10.2138. Geneva: WHO Press; 2010.

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Acknowledgements

The authors are grateful to Roche Diagnostics for providing the reagents used in the current study.

Author information

Correspondence to David Navarro.

Additional information

Competing interests

DN has received honoraria from Roche Diagnostics for participating in several conferences. The remaining authors declare that they have no competing interests.

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Keywords

  • Lower Respiratory Tract
  • Organization International Standard
  • Tracheal Aspirate
  • Plasma Specimen
  • Fitted Regression Line