Woon Bo Heo; Dong Il Won; Yoo Li Kim; Myeong Hee Kim; Heung Bum Oh; Jang Soo Suh

doi:10.3343/kjlm.2007.27.4.298

Journal List > Korean J Lab Med > v.27(4) > 1011411

Go to TopGo to Top Go to BottomGo to Bottom

TOOLS

Heo, Won, Kim, Kim, Oh, and Suh: Evaluation of Biosewoom™ Real-Q Cytomegalovirus Quantification kit for Cytomegalovirus Viral Load Measure

Original Article

Korean J Leg Med 2007;27(4):298-304.

Published online: 10 February 2007

DOI: https://doi.org/10.3343/kjlm.2007.27.4.298

Evaluation of Biosewoom™ Real-Q Cytomegalovirus Quantification kit for Cytomegalovirus Viral Load Measure

Woon Bo Heo, M.D.¹, Dong Il Won, M.D.¹, Yoo Li Kim, Ph.D.², Myeong Hee Kim, M.D.³, Heung Bum Oh, M.D.³, Jang Soo Suh, M.D.¹

¹Department of Laboratory Medicine, Kyung Pook National University Hospital, Daegu, Korea

²Biosewoom Institute of Bioscience & Biotechnology, Seoul, Korea

³Department of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, Seoul, Korea

Received 2 May 2007 Revised 20 June 2007 Accepted 25 June 2007

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Rapid and accurate laboratory tests are essential to detect cytomegalovirus (CMV) infections in solid organs and haematopoietic stem cell transplant recipients. We assessed the realtime quantitative PCR (RQ-PCR) technology for its usefulness in detecting CMV DNA.

Methods

We evaluated the analytical performance of CMV RQ-PCR using Real-Q Cytomegalovirus Quantification kit (BioSewoom Inc., Korea). To evaluate its clinical utility, we also compared it to pp65 antigenemia test, an immunostaining method, on 343 samples of total 84 patients, including 63 transplant recipients.

Results

The detection limit of RQ-PCR was 63 copies/mL and none of hepatitis B virus, hepatitis C virus, or human immunodeficiency virus showed a cross-reactivity with CMV. Total coefficient of variation (CV) was 10.4–19.5%. It detected CMV DNA in a linear range from 1 × 10² to 5 × 10¹¹ copies/mL (P<10^-13, R²=0.9994). The qualitative positive rates of pp65 antigenemia test and RQ-PCR were 4.7%, 16.3%, respectively and concordance rate between the two tests was 84.8% (K =0.221, P<10^-6). In comparison of quantitative results, the correlation between two tests was significant (r= 0.45, P<10^-17). In comparison among three groups by pp65 antigen level, CMV DNA level obtained with RQ-PCR increased significantly (P<10^-3 and P<10^-7, respectively).

Conclusions

The RQ-PCR is easier to perform than the immunostaining method, has good analytical performance and reflects the blood level of viral DNA well. It may be a new method substituting the pp65 antigenemia test. Further studies determining RQ-PCR value starting pre-emptive therapy will be required.

Keywords: RQ-PCR, pp65 antigenemia test, CMV DNA

References

1. Paya CV. Prevention of cytomegalovirus disease in recipients of solid-organ transplants. Clin Infect Dis. 2001; 32:596–603.

2. Boeckh M, Boivin G. Quantitation of cytomegalovirus: methodologic aspects and clinical applications. Clin Microbiol Rev. 1998; 11:533–54.

3. Camargo LF, Uip DE, Simpson AA, Caballero O, Stolf NA, Vilas-Boas LS, et al. Comparison between antigenemia and a quantitative-competitive polymerase chain reaction for the diagnosis of cytomegalovirus infection after heart transplantation. Transplantation. 2001; 71:412–7.

4. Guiver M, Fox AJ, Mutton K, Mogulkoc N, Egan J. Evaluation of CMV viral load using TaqMan CMV quantitative PCR and comparison with CMV antigenemia in heart and lung transplant recipients. Transplantation. 2001; 71:1609–15.

5. Monte PD, Lazzarotto T, Ripalti A, Landini MP. Human cytomegalovirus infection: a complex diagnostic problem in which molecular biology has induced a rapid evolution. Intervirology. 1996; 39:193–203.

6. Delgado R, Lumbreras C, Alba C, Pedraza MA, Otero JR, Gomez R, et al. Low predictive value of polymerase chain reaction for diagnosis of cytomegalovirus disease in liver transplant recipients. J Clin Microbiol. 1992; 30:1876–8.

7. Schaade L, Kockelkorn P, Ritter K, Kleines M. Detection of cytomegalovirus DNA in human specimens by LightCycler PCR. J Clin Microbiol. 2000; 38:4006–9.

8. Ljungman P, Griffiths P, Paya C. Definitions of cytomegalovirus infection and disease in transplant recipients. Clin Infect Dis. 2002; 34:1094–7.

9. Patel R, Snydman DR, Rubin RH, Ho M, Pescovitz M, Martin M, et al. Cytomegalovirus prophylaxis in solid organ transplant recipients. Transplantation. 1996; 61:1279–89.

10. Kearns AM, Guiver M, James V, King J. Development and evaluation of a real-time quantitative PCR for the detection of human cytomegalovirus. J Virol Methods. 2001; 95:121–31.

11. Mandell GL, Bennett JE, editors. Principles and practices of infectious diseases. 6th ed.Philadelphia: Elsevier Churchill Livingstone;2005. p. 1786–7.

12. Meyers JD, Flournoy N, Thomas ED. Risk factors for cytomegalovirus infection after human marrow transplantation. J Infect Dis. 1986; 153:478–88.

13. Masur H, Whitcup SM, Cartwright C, Polis M, Nussenblatt R. Advances in the management of AIDS-related cytomegalovirus retinitis. Ann Intern Med. 1996; 125:126–36.

14. Nokta MA, Holland F, De Gruttola V, Emery VC, Jacobson MA, Griffiths P, et al. Cytomegalovirus (CMV) polymerase chain reaction profiles in individuals with advanced human immunodeficiency virus infection: relationship to CMV disease. J Infect Dis. 2002; 185:1717–22.

15. Emery VC, Sabin CA, Cope AV, Gor D, Hassan-Walker AF, Griffiths PD. Application of viral-load kinetics to identify patients who develop cytomegalovirus disease after transplantation. Lancet. 2000; 355:2032–6.

16. Sia IG, Wilson JA, Espy MJ, Paya CV, Smith TF. Evaluation of the COBAS AMPLICOR CMV MONITOR test for detection of viral DNA in specimens taken from patients after liver transplantation. J Clin Microbiol. 2000; 38:600–6.

17. Allice T, Enrietto M, Pittaluga F, Varetto S, Franchello A, Marchiaro G, et al. Quantitation of cytomegalovirus DNA by real-time polymerase chain reaction in peripheral blood specimens of patients with solid organ transplants: comparison with end-point PCR and pp65 antigen test. J Med Virol. 2006; 78:915–22.

18. Piiparinen H, Lautenschlager I. Quantitative TaqMan assay for the detection and monitoring of cytomegalovirus infection in organ transplant patients. Methods Mol Biol. 2006; 335:147–56.

19. Martin-Davila P, Fortun J, Gutierrez C, Marti-Belda P, Candelas A, Honrubia A, et al. Analysis of a quantitative PCR assay for CMV infection in liver transplant recipients: an intent to find the optimal cut-off value. J Clin Virol. 2005; 33:138–44.

20. Hernando S, Folgueira L, Lumbreras C, San Juan R, Maldonado S, Prieto C, et al. Comparison of cytomegalovirus viral load measure by real-time PCR with pp65 antigenemia for the diagnosis of cytomegalovirus disease in solid organ transplant patients. Transplant Proc. 2005; 37:4094–6.

21. Fan J, Ma WH, Yang MF, Xue H, Gao HN, Li LJ. Real-time fluorescent quantitative PCR assay for measuring cytomegalovirus DNA load in patients after haematopoietic stem cell transplantation. Chin Med J. 2006; 119:871–4.

22. Cha TA, Tom E, Kemble GW, Duke GM, Mocarski ES, Spaete RR. Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains. J Virol. 1996; 70:78–83.

23. Zweygberg Wirgart B, Brytting M, Linde A, Wahren B, Grillner L. Sequence variation within three important cytomegalovirus gene regions in isolates from four different patient populations. J Clin Microbiol. 1998; 36:3662–9.

Fig. 1.

Linearity range of Real-Q CMV Quantification kit.

Fig. 2.

Quantitative comparison of pp65 antigen values and CMV DNA levels obtained by RQ-PCR (r=0.45, P<10^-17).

Table 1.

Classification of the patients in this study

Underlying disease	No	No of CMV infection
Underlying disease	No	I	II	III
Transplant recipients	63	16		1
Peripheral blood stem cell	49	12		1
Bone marrow	7
Kidney	6	3
Liver	1	1
HIV infection	3	1	1
Hepatitis	4
Gastroenteritis	2		1
Pneumonia/Pneumonitis	4
Pericarditis	1		1
Chronic renal failure	2
Leukemia/Lymphoma	2
Aplastic anemia	1
Systemic lupus erythematosus	1
Total	83	17	3	1

I, Asymptomatic infection; II, End organ disease; III, CMV syndrome.

Abbreviations: CMV, cytomegalovirus; HIV, human immunodeficiency virus.

Table 2.

Analytical sensitivity of Real-Q CMV Quantification kit

Input CMV (copies/mL)	Result
Input CMV (copies/mL)	Replicates	Positive reaction	%
10,000	150	150	100
5,000	150	150	100
1,000	150	150	100
500	150	150	100
100	150	150	100
75	150	145	97
50	150	130	87

Abbreviation: CMV, cytomegalovirus.

Table 3.

Analytical specificity of Real-Q CMV Quantification kit

	CMV (VIC)^*	Internal control (FAM)^*
Hepatitis B virus	−	+
Hepatitis C virus 1a	−	+
Hepatitis C virus 1b	−	+
Hepatitis C virus 2a	−	+
Hepatitis C virus 2b	−	+
Hepatitis C virus 3a	−	+
Hepatitis C virus 3b	−	+
Hepatitis C virus 4	−	+
Hepatitis C virus 4h	−	+
Hepatitis C virus 5a	−	+
Hepatitis C virus 6a	−	+
HIV A	−	+
HIV B	−	+
HIV C	−	+
HIV D	−	+
HIV E	−	+
HIV F	−	+
HIV G	−	+
HIV H	−	+

^* VIC and FAM are the reporter dyes.

Abbreviations: See Table 1.

Table 4.

Comparison between pp65 antigenemia and CMV DNA obtained with RQ-PCR among 343 samples

	pp65 antigenemia^*		Total
	Positive	Negative	Total
CMV DNA^*
Positive	10 (2.9%)	46 (13.4%)	56 (16.3%)
Negative	6 (1.7%)	281 (81.9%)	287 (83.7%)
Total	16 (4.7%)	327 (95.3%)	343 (100%)

^* Concordance rate 84.8%, Kappa coefficient 0.221 (P<10^-6).

Abbreviations: CMV, cytomegalovirus; RQ-PCR, real-time quantitative PCR.

Table 5.

CMV DNA levels according to the pp65 antigen levels

Patient group (pp65 antigen positive cells/200,000 PBLs, N)	CMV DNA levels^* (Log₁₀ copies/mL)
0 (N=327)	0.38±1.03
1–10 (N=12)	1.59^†±1.73
11–20 (N=4)	5.23^†±0.31

^* Mean±SD.

^† Scheffe's test, P<10³, and P<10^-7, respectively, compared with the group above.

Abbreviations: CMV, cytomegalovirus; PBLs, peripheral blood leukocytes.

Table 6.

pp65 antigenemia and CMV DNA levels according to the clinical status of CMV infection

	pp65 antigen (positive cells/200,000 PBLs)	CMV DNA levels (Log₁₀ copies/mL)
Asymptomatic CMV infection (N=17)	1.5±3.9^*	2.72±1.54^*
CMV end-organ disease (N=3)	0, 0, 0	0, 0, 5.06
CMV viral syndrome (N=1)	16	5.20

^* Mean±SD.

Abbreviations: See Table 5.

TOOLS

Similar articles