Cytomegalovirus

Context.-Human cytomegalovirus (CMV) infection is a progressive and life-threatening complication in immunocompromised patients even now. Therefore, early and accurate treatment based on rapid and certain detection is needed to prevent fatal CMV infection diseases.

Objective.-To study a quicker, simpler, and less expensive method of quantitative analysis using real-time polymerase chain reaction based on the SYBR Green I method of CMV detection for appropriate treatment of CMV infection in immunocompromised patients.

Design.-We quantified 50 samples tested by direct immunoperoxidase staining of leukocytes with peroxidase-labeled monoclonal antibody (C7-HRP test), 30 samples from healthy persons, and 47 samples from 7 patients suspected of having CMV infection diseases. We used the primer set in the pp65 gene of CMV and whole blood without a preparatory process. The setting for the study was the First Department of Pathology, Kurume University School of Medicine, St Mary's Hospital, and the Gene Section of the Clinical Laboratory at St Mary's Hospital, Fukuoka, Japan.

Results.-The results obtained with this method corresponded well with conventional C7-HRP tests and demonstrated excellent reproduction. Additionally, the results were better correlated with the clinical course than were C7-HRP tests.

Conclusions.-This method was more useful than the C7-HRP test as a rapid diagnostic test for early treatment of CMV infection. This test also demonstrated its usefulness for monitoring CMV infection during treatment using ganciclovir. Moreover, it was quicker, simpler, and cheaper than other real-time polymerase chain reaction methods.

(Arch Pathol Lab Med. 2005;129:200-204)

Cytomegalovirus (CMV) infection in immunocompromised patients has increased in incidence with advances in transplantation and chemotherapy.1,2 An early diagnosis is especially necessary for the accurate treatment of CMV pneumonia, because the symptoms are so serious and the death rate remains high.34

The C7-HRP test is the most-used method for diagnosing CMV infection, and it correlates well with the clinical course in pneumonia.25 However, it is a complicated procedure with a false-negative rate problem.6

On the other hand, polymerase chain reaction (PCR) is becoming a common method for detecting CMV infection, because it is both quick and sensitive. Although it is currently thought that real-time PCR using probes is the best method7 and many studies have been carried out, this method seems to be too expensive for many laboratories.8 Moreover, the diagnostic usefulness of PCR has not yet been accepted, because it is necessary to distinguish between CMV infection disease and abortive infection.9,10

Therefore, we studied a quicker, simpler, and more cost-saving method of quantitative analysis using real-time PCR based on the SYBR Green I method for appropriate treatment of CMV infection. Additionally, we investigated its usefulness as a marker in treatment with ganciclovir.

MATERIALS AND METHODS

Specimens

We tested whole blood categorized into the following 3 groups.

1. The first group included 50 samples of patients tested with the C7-HRP test from August 28 to September 26, 2001. Of these 50 patients, 32 were men and 18 were women, with an average age of 64 years.

2. The second group included 30 samples from healthy persons who underwent medical examination at our hospital. We selected 5 persons in each decade of life from the 20s through the 70s. Of these, 16 persons were men and 14 were women.

3. The third group included 47 samples from 7 patients with suspected CMV infection diseases after November 2001 that were tested with the C7-HRP test. Of these patients, 3 were men and 4 were women. The average age was 59 years. Their clinical diagnoses were adult T-cell leukemia (3 cases), aplastic anemia (2 cases), acute myelogenous leukemia (1 case), and chronic myelogenous leukemia (1 case).

Controls

Genomic DNA from CMV AD-169 was isolated using the QIAmp blood mini kit (QIAGEN, Hilden, Germany), and the pp65 gene of CMV AD-169 was amplified by the usual PCR method using the primer sets designed by Shibata et al.11 Their sequences were as follows: forward primer: 5'-AgACTATCAAC TTAATTTCTgATCA-3', reverse primer: 5'-CCTTCCTTCTTTTT gATTTTgTTT-3'.11 Polymerase chain reaction products were in 139 bp. This PCR was carried out in a total volume of 50 µL, containing 1.5mM MgCl^sub 2^, 0.2mM deoxynucleoside triphosphates, 1.25 U Taq DNA polymerase, 5 µL 10× buffer, 0.5 µM of each primer, and 0.5 µg genomic DNA of CMV AD-169.

The 139-bp PCR products of CMV AD-169 were purified using the Wizard PCR Preps DNA purification system (Promega, Madison, Wis). The concentration of purified products was measured. The positive controls were 10-fold-diluted solutions of purified products; the negative control was distilled water.

Real-Time PCR

Genomic DNA from whole blood was also isolated using the QIAmp blood mini kit (QIAGEN). The pp65 gene of CMV was amplified from genomic DNA by the real-time PCR method using LightCycler (Roche, Indianapolis, Ind).

Polymerase chain reaction was carried out in a total volume of 20 µL, containing 2 µL genomic DNA, 0.5 µM of each primer (the same primer set used for making the control), 4.0mM MgCl^sub 2^, and 2 µL LightCycler FastStart DNA Master SYBR Green I (Roche). The PCR was performed as follows: there was an activation step of Taq polymerase at 95°C for 4 minutes, followed by 35 cycles of denaturation at 95°C for 0 second, annealing at 65°C for 5 seconds, and extension at 72°C for 10 seconds and at 87°C for 3 seconds. We created the final step (87°C for 3 seconds) to measure the fluorescence signal. After the PCR reaction, the temperature was decreased to 65°C and then gradually raised to 95°C at a rate of 0.2°C/s. The fluorescence signal was continuously monitored during this process for melting curve analysis.

The results were judged positive or negative by the presence or absence of the melting temperature peak of 88°C and by the effectiveness of the quantitative score.

Additionally, 10 µL of PCR products were visualized by 2% agarose gel electrophoresis and ethidium bromide staining.

RESULTS

Correlation and Reproducibility

We tested between 1 and 108 copies/µL of the positive control. The amplification plots are shown (Figure 1). Primer dimers were not obtained during 35 cycles. The target peak of 88°C was obtained between 10^sup 2^ and 10^sup 8^ copies/µL (Figure 2), and a positive band on the electrophoresis occurred at the same point (photo not shown). The correlation of cycle numbers (cycle thresholds) and log concentration was excellent (y = -3.321x + 38.88, R^sup 2^ = 0.998), as shown in Figure 3.

On the other hand, the 3 sample concentrations we chose were different, and these were quantified 3 times each. The average coefficient of variation in simultaneous reproducibility was 0.92% (range, 0.44%-1.50%), and the average coefficient of variation in reproducible day difference during 5 days was 2.17% (range, 1.74%-2.99%).

Comparison With the C7-HRPTest

Fourteen of 50 samples were positive, and 36 samples were negative. Their quantitative scores were as high as 2.089 × 10^sup 3^ copies/µL, and as low as 4.428 × 10 copies/µL, and a positive band was obtained in all 14 cases for electrophoresis. The matching rate with C7-HRP tests was 82%, but in the 4 samples for which only real-time PCR was positive, the quantitative scores were low (5.860 × 10 copies/µL, 4.428 × 10 copies/µL, 5.370 × 10 copies/µL, and 5.325 × 10 copies/µL). On the other hand, positive cells of C7-HRP tests were 1 cell in 5 samples, and only the C7-HRP test was positive.

Healthy Persons

Two of 30 cases were positive, but their quantitative scores were low (5.040 × 10 copies/µL and 9.370 × 10 copies/µL).

Case Reports

In our cases, the quantitative scores were matched with the results of the C7-HRP tests and decreased after treatment with ganciclovir. The range of their quantitative scores was from 5.400 × 10 copies/µL to 1.129 × 10^sup 4^ copies/µL. We showed a case in which real-time PCR more closely matched the clinical course than the C7-HRP test did. The patient was 36 years old and had been diagnosed with chronic myelogenous leukemia. After the allo-bone marrow transcription, the patient developed fever, and the C7-HRP test was positive. He was diagnosed with CMV infection disease, and treatment with ganciclovir was started. The positive cells of the C7-HRP test and the quantitative score of real-time PCR decreased gradually. The C7-HRP test was negative on day 14, and treatment was stopped. However, the quantitative score was still high (1.435 × 10^sup 3^ copies/µL). The C7-HRP test done 2 days later was positive again, and treatment was restarted (Figure 4).

COMMENT

Cytomegalovirus is a common opportunistic infection, but CMV infections in immunocompromised patients are progressive and remain fatal.1-4 For this reason, preventive treatment using antiviral drugs may be used to protect against occurrences even without confirmation of CMV presence in patients who have undergone organ transplantation.12 However, antiviral drugs must be used initially for infectious diseases, and they also must be used very carefully to avoid side effects and an outbreak of a resistance virus.

Additionally, in the hematologic case, after bone marrow transplantation, for example, preventive treatment is impossible because of the "bone marrow inhibitory" side effect. Therefore, a rapid and certain method is needed to diagnose CMV infections for early and accurate treatment.3,9

In our hospital, the C7-HRP test is used for diagnosis and monitoring of treatment using ganciclovir for CMV pneumonia, because the test is covered by insurance and the results became positive even before the occurrence of symptoms, correlating with the clinical course.2,5 But the C7-HRP test procedure is complicated as a routine test at the laboratory in our hospital. Therefore, we entrust another institution with the test, which takes at least 2 days to be reported.

On the other hand, quantitative PCR is becoming a common method for detecting CMV infection because it is quick and sensitive and has good singularity. Additionally, real-time PCR is faster, as indicated by many reports.7,10 Furthermore, the LightCycler system (Roche) we used in this study is very simple to use and can save time in the PCR process, observing the real-time amplifying circumstances of PCR products. By analysis of the melting curve of PCR products, we can confirm the target products without electrophoresing on agarose gels.13,14 However, although many reports about real-time PCR of CMV have been published, there is no consensus regarding material or the gene amplified. In previous studies, some have used whole blood as material,15,16 but most have used plasma10,13,14,15 or leukocytes.4,10,12 At present, the most commonly used real-time method uses a specific fluorescent probe and can detect CMV in a few hours.7,16 Although it is reported to provide better results,4,16,17 this method has not become the norm in laboratory practice because the probes are expensive.8 Although the SYBR Green I method we used measures dsDNA and is thought to allow many nonspecific reactions, it is simpler, quicker, and less expensive than the probe method.8

Additionally, in this study we used whole blood without a sample preparation process.15,16 Therefore, the time to reporting from specimen introduction was less than 1 hour.

We had believed that we could quantify only the target PCR products because using hot-start PCR inhibited the nonspecific product and added the fluorescent measuring step after the extension. In fact, primer dimers were not obtained, and quantitative analysis using the real-time PCR method in this study showed excellent correlation and reproducibility.

We also examined 2 other primer sets in the MIE and IE genes besides the pp65 gene in the initial stage of the study. However, a good result was not obtained with these primers. Besides, the primer set we used in the pp65 gene did not cross-react with other microbes (Table).

Some previous studies have compared real-time PCR assay with the C7-HRP test and have reported that the result of PCR was better than that of the C7-HRP test.10,14,15 We also acquired a result almost equal to that of the C7-HRP test, and the result was obtained very rapidly compared with the C7-HRP test.

Moreover, it is well known that the C7-HRP test returns a false-negative result in cases with a marked decrease of white blood cells.6 Also in this study, there were some cases suspected of being falsely negative for the C7-HRP test in patients with decreased white blood cell counts, because the result did not correlate with the clinical course. However, in our method using LightCycler (Roche), it was more possible to achieve a result that correlated well with the clinical course than with the C7-HRP test in patients with a marked decrease of white blood cells.

We obtained positive results from healthy persons in some cases, but the copy numbers were very low, and there seemed to be no problem in diagnosis after careful consideration of the clinical course. Although the PCR method is a good method with very high sensitivity, usefulness should be confirmed by carrying out comparative examinations, not only in patients but also in healthy people, in order to distinguish simple infections from CMV infection diseases.18,19 In our study, a cutoff value of 10^sup 2^ copies/µL was considered appropriate.

Therefore, it is suggested that this method is quicker, simpler, and more cost-efficient than the C7-HRP test or real-time PCR using probes. Furthermore, it is useful not only for rapid diagnosis and early treatment but also as an exact monitor during treatment using ganciclovir in CMV infection diseases. Additionally, it was considered that it could be easily performed in any laboratory as a routine test.

We thank Professor Kouhei Harada, PhD, of the Department of Economics, Kurume University, for statistical advice.

References

1. Minamijima Y. Cytomegalovirus. Saishinn-naika-taikei. 1994;26:147-154.

2. Gondo H, Minematsu T, Harada M, et al. Cytomegalovirus (CMV) antigenaemia for rapid diagnosis and monitoring of CMV-associated disease after bone marrow transplantation. Br J Haematol. 1994;86:130-137.

3. Boriskin YS, Fuller K, Powles RL, et al. Early detection of cytomcgalovirus (CMV) infection in bone marrow transplant patients by reverse transcription-PCR for CMV spliced late gene UL 21.5: a 2 site evaluation. J Clin Virol. 2002;24:13-23.

4. Yun Z, Lewensohn-Fuches I, Ljungman P, Vahlne A. Real-time monitoring of cytomegalovirus infections after stem cell transplantation using TaqMan polymerase chain reaction assays. Transplantation. 2000;69:1733-1736.

5. Vlieger AM, Boland GJ, Jiwa NM, et al. Cytomegalovirus antigenemia assay or PCR can be used to monitor ganciclovir treatment in bone marrow transplant recipients. Bone Marrow Transplant. 1992;9:247-253.

6. Flexman J, Kay I, Fonte R, Herrmann R, Gabbay E, Palladino S. Differences between the quantitative antigenemia assay and the cobas amplicor monitor quantitative PCR assay for detecting CMV viraemia in bone marrow and solid organ transplant patients. J Med Virol. 2001;64:275-282.

7. Provenzano M, Rossi CR, Mocellin S. The usefulness of quantitative real-time PCR in immunogenetics. ASHI Q. 2001;25:89-91.

8. Ponchel F, Toomes C, Bransfield K, et al. Real-time PCR based on SYBR Green I fluorescence: an alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions. BMC Biotechnol. 2003;3:18.

9. Toyokawa M, Nishi I. Cytomegalovirus infection. Rinsho Byori. 2002;123: 71-78.

10. Tanaka N, Kimura H, Iida K, et al. Quantitative analysis of cytomegalovirus load using a real-time PCR assay. J Med Virol. 2000;60:455-462.

11. Shibata D, Martin WJ, Appleman MD, Causey DM, Leedom JM, Arnheim N. Detection of Cytomegalovirus DNA in peripheral blood of patients infected with human immunodeficiency virus. J Infect Dis. 1988;158:1185-1192.

12. Dykewicz CA, Kaplan JE, for the Guidelines Working Group of the Centers for Disease Control and Prevention, Infectious Disease Society of America, and American Society of Blood and Marrow Transplantation. Guidelines for preventing opportunistic infections among hematopoietic stem cell transplant recipients. MMWR Morb Mortal Wkly Rep Recommendation Rep. 2000;49(RR10):1-128.

13. Schaade L, Kockelkorn P, Ritter K, Kleines M. Detection of Cytomegalovirus DNA in human specimens by Lightcycler PCR. J Clin Microbiol. 2000;38:4006-4009.

14. Pang XL, Chui L, Fenton J, LeBlanc B, Preiksaitis JK. Comparison of LightCycler-based PCR, COBAS amplicor CMV monitor, and pp65 antigenemia assays for quantitative measurement of cytomegalovirus viral load in peripheral blood specimens from patients after solid organ transplantation. J Clin Microbiol. 2003;41:3167-3174.

15. Leruez-Ville M, Ouachee M, Delarue R, et al. Monitoring cytomegalovirus infection in adult and pediatric bone marrow transplant recipients by a real-time PCR assay performed with blood plasma. J Clin Microbiol. 2003;41:2040-2046.

16. Jebbink J, Bai X, Rogers BB, Dawson DB, Scheuermann RH, Domiati-Saad R. Development of real-time PCR assays for the quantitative detection of Epstein-Barr virus and cytomegalovirus, comparison of TaqMan probes, and molecular beacons. J Mol Diagn. 2003;5:15-20.

17. Griscelli F, Barrais M, Chauvin S, Lastere S, Bellet D, Bourhis JH. Quantification of human cytomegalovirus DNA in bone marrow transplant recipients by real-time PCR. J Clin Microbiol. 2001;39:4362-4369.

18. Machida U, Kami M, Fukui T, et al. Real-time automated PCR early diagnosis and monitoring of cytomegalovirus infection after bone marrow transplantation. J Clin Microbiol. 2000;38:2536-2542.

19. Funato T, Satou N, Abukawa D, et al. Quantitative evaluation of cytomegalovirus DNA in infantile hepatitis. J Viral Hepat. 2001;8:217-222.

Sanae Numata, MT; Yasuhiro Nakamura, MD; Yutaka Imamura, MD; Junichi Honda, MD; Seiya Momosaki, MD; Masamichi Kojiro, MD

Accepted for publication October 11, 2004.

From the First Department of Pathology (Ms Numata and Drs Momosaki and Kojiro) and the First Department of Internal Medicine (Dr Honda), Kurume University School of Medicine, Fukuoka, Japan; and the Departments of Gene Diagnostics (Ms Numata), Pathology (Dr Nakamura), and Hematology (Dr lmamura), St Mary's Hospital, Fukuoka, Japan.

The authors have no relevant financial interest in the products or companies described in this article.

Reprints: Sanae Numata, MT, Department of Gene Diagnostics, St Mary's Hospital, 422, Tsubukuhon-machi, Kurume-shi, Fukuoka, 830-8543, Japan (e-mail: s-numa@st-mary-med.or.jp).

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