Quantitation of Cytomegalovirus DNA in Lung Tissue of Bone Marrow Transplant Recipients MOTOHIRO SHIBATA, MD, PHD, MAKOTO TERASHIMA, MD, HIROSHI KIMURA, MD, KIYOTAKA KUZUSHIMA, MD, PHD, JUN YOSHIDA, MD, PHD, KEIZO HORIBE, MD, PHD, AND TSUNEO MORISHIMA, MD, PHD Five bone marrow transplant recipients who died of respiratory failure were retrospectively analyzed with polymerase chain reaction (PCR) assay for pulmonary cytomegalovirus (CMV) infection. Two patients had CMV interstitial pneumonitis according to the virus isolation and the histologic and immunofluorescent examinations of the lungs, while the other three patients had non-CMV diseases (ie, idiopathic interstitial pneumonitis, pulmonary aspergillosis, or Streptococcus mitis septicemia). Cytomegalovirus DNA was amplified from the postmortem lung tissue with PCR. The PCR assay showed apparent PCR signals specific to CMV DNA in the two patients with CMV pneumonitis. In contrast, CMV DNA was hardly detectable or undetectable in the three patients without CMV disease. With quantitative PCR assay the initial CMV copy number in the lung tissue of the two patients with CMV pneumonitis was more than lo4 copies/fig DNA and was over l,OOO-fold more than that of the three patients without CMV pneumonitis. These results show that quantitative PCR assay could be useful as a diagnostic measure for pulmonary CMV infection. HUMPATHOL 23:911-915. Copyright 0 1992 by W.B. Saunders Company
Cytomegalovirus (CMV) is one of major etiologic agents of severe illness and death in immunocompromised hosts. Interstitial pneumonitis is a significant illness caused by CMV due to its high mortality rate. Combination therapy of ganciclovir with intravenous high-dose immunoglobulin has improved the prognosis of CMV interstitial pneumonitis.‘** Accurate diagnosis and early treatment are essential for the management of immunocompromised patients. Clinical diagnosis of CMV pneumonitis has been made based on (1) isolation of CMV from lung tissue or bronchoalveolar lavage (BAL) and (2) demonstration of interstitial infiltrates on chest radiograph. However, small amounts of CMV in samples may result in falsenegative results with virus culture. Furthermore, prophylactic administration of antiviral agents, such as ganciclovir, acyclovir, and immunoglobulin,“-6 may decrease the efficiency of virus isolation. Methods to detect viral DNA are therefore useful in determining the presence of CMV in samples. Polymerase chain reaction (PCR),
originally described by Saiki et ak7 is an extremely sensitive method to detect specific DNA and has been applied to CMV infections.“‘* Previous reports have shown that lung tissue of patients with CMV pneumonitis was positive for CMV DNA with PCR.‘3214 However, because immunocompromised patients might excrete CMV without any clinical disease, lung tissue from such immunocompromised patients could be positive for CMV DNA due to the high sensitivity of the PCR assay. It is noteworthy that many children in Japan acquire antibody to CMV by 5 years of age. Quantitative evaluation of CMV infection may be helpful in the diagnosis of CMV pneumonitis in that situation. In this study we performed quantitative PCR as a first step to show that quantitative PCR for CMV with sputum, BAL, or lung biopsy is potentially useful in differentiating CMV pneumonitis from other causes of pneumonitis in CMVpositive patients. The study was therefore designed to retrospectively analyze postmortem lung tissue from bone marrow transplant (BMT) recipients who had been examined for CMV infection with virus isolation, immunofluorescence, and histology. MATERIALS
AND METHODS
Virus Isolation For virus isolation, lung tissue or other samples (such as blood, liver, or kidney) obtained at autopsy were immediately inoculated into human embryonic lung cells and observed for cytopathic effect of CMV. Cytomegalovirus was identified by fluorescence microscopy using a murine monoclonal antibody to the immediate early antigen of CMV, which was kindly provided by Dr T. Furukawa, Kanazawa Medical University.
Histopathologic and lmmunofluorescence Examinations of Lung Tissue Lung tissue sections were fixed and stained by standard histologic techniques and viewed microscopically. A histologic diagnosis of CMV interstitial pneumonitis was made if large cells containing basophilic nuclear inclusions (Cowdry type A) and multiple smaller cytoplasmic inclusions were demonstrated.r5 For immunofluorescence, sections from frozen lung tissue were fixed in cold methanol and stained with the murine monoclonal antibody to the immediate early antigen of CMV. The slides were rinsed with phosphate-buffered saline and reacted with fluorescein-conjugated goat anti-mouse immunoglobulin (Organon Teknika-Cappel, West Chester, PA). The slides were then rinsed and observed under an ultraviolet microscope.
From the Department of Pediatrics, Nagoya University School of Medicine, Nagoya, Japan; and Department of Pediatrics, Nagoya First Red Cross Hospital, Nagoya, Japan. Accepted for publication October 8, 199 1. Key wordrc polymerase chain reaction, cytomegalovirus, quantitation, interstitial pneumonitis, bone marrow transplantation, Address correspondence and reprint requests to Motohiro Shibata, MD, Department of Pediatrics, Nagoya University School of Medicine, Tsuruma-cho 65, Nagoya 466, Japan. Copyright 0 1992 by W.B. Saunders Company 0046-8177/92/2308-0013$5.00/O
911
HUMAN PATHOLOGY
TABLE
1.
Volume 23, No. 8 (August
1992)
Histologic and Virologic Examinations of Five ImmunocomDromised Patients CMV Assay on Lung Tissue”
Case No. (Age lvrl /sex)
1 (10/F) 2 (g/M) 3 (14/M) 4 (18/M) 5 (G/F) Ahhreviations:
Chest X-ray
Inclusions
IP IP IP Pneumonia IP IF, immunofluorescence;
+ + _ _ _ IP, interstitial
* Lung tissue was examined by histology for inclusion isolation of CMV. t Serum CMV antibody was measured by complement
Preparation
IF + + ND ND ND
fixation or by enzyme immunoassay
of DNA From Lung Tissue
Othe1Pathoyen
+ ND + + +
_ Aspergillus Streptococcus
early antigen
milzs
of CMV, and by
for IgG antibody.
in the Southern blots was analyzed with the Fujix BAS 2000 Image Analyzing System (Fuji Photo Film, Tokyo, Japan)” and a standard curve was obtained at each experiment.
Measurement of Antibodies Cytomegalovirus
to
Antibody to CMV was determined by microtiter complement fixation and by an enzyme-linked immunosorbent assay for IgM and IgG antibodies (Behring Werke, Marburg, Germany). A complement fixation titer of 1:s was considered positive. Enzyme-linked immunosorbent assay was carried out according to the manufacturer’s instructions, and the cut-off index was determined.
Chain Reaction
Polymerase chain reaction for CMV was performed as described previously. ‘* Briefly , a primer set was synthesized to amplify a 685 base pair sequence (828-15 12) from the immediate early gene 1 of CMV.” The sense primer CVl was 5’-GGCCATGGCGGCATTGCAGAACTTG-3’. The anti-sense primer CV2 was 5’-CTCTATCTCAGACACTGGCTCAGAC-3’. One microgram of DNA purified from lung tissue was applied to 35 cycles of PCR with Taq polymerase in a DNA thermal cycler (Perkin Elmer Cetus, Norwalk, CT). The amplified DNA products were subjected to agarose gel electrophoresis and transferred to a nylon membrane (Nytran, Schleicher & Schuell, Dassel, Germany). Southern blot hybridization was then performed with the 32P-labeled DNA probe derived from plasmid pIE14 that contains nucleotide sequences corresponding to 879-1267 nucleotides of the immediate early gene 1 of CMV.” As an internal control of tissue DNA, 25 cycles of PCR were also performed to amplify a 371 base pair sequence from @-actin DNA.17 The sense primer was 5’GCCATGTACGTTGCTATCCA-3’. The anti-sense primer was 5’-CGCTCATTGCCAATGGTGATGACCT-3’. Polymerase chain reaction for the actin DNA with 1 pg of the tissue DNA resulted in a similar intensity of DNA bands at the expected size after agarose gel electrophoresis and ethidium bromide staining. To avoid false-positive results with PCR we applied the contamination prevention measures of Kwok and Higuchi.‘s All PCR and Southern blots were run and analyzed in a blinded fashion.
Determination of Cytomegalovirus Number in DNA From Lung Tissue
+ + _ _ _
Serum CMV Antihodyt
pneumonitis; ND, not determined. bodies, by immunofluorescence for the immediate
Lung tissues obtained at autopsy were frozen at -70°C until the time of DNA preparation. Approximately 50 mg of frozen tissue was pulverized to a fine powder in liquid nitrogen and incubated at 56°C for 1 hour in TNE buffer (20 mmol/ L Tris-HCl [pH 7.51, 10 mmol/L NaCl, 20 mmol/L EDTA) containing 0.1% sodium dodecyl sulfate and 200 pg/mL proteinase K. DNA was extracted with equal volumes of phenol: chloroform and chloroform. DNA was then precipitated with ethanol and resuspended in TE buffer (10 mmol/L Tris-HCl [pH 8.01, 0.05 mmol/L EDTA). DNA concentration was determined by absorbance at 260 nm.
Polymerase
Isolation
Copy
For determining the initial CMV copy number in DNA samples, plasmid “E” DNA (kindly provided by D. Stinski’“) containing the immediate early gene 1 of CMV, was serially diluted with TE buffer and similarly amplified with PCR as external standards. The radioactivity of each CMV DNA band
912
Bone Marrow Transplantation Bone scribed.2’
marrow
transplantation
was performed
as de-
RESULTS We analyzed five immunocompromised patients who had received BMT for acute lymphoblastic leukemia (ALL) or acute myeloblastic leukemia and who died of respiratory failure. The results of virologic and pathologic examinations at autopsy are summarized in Table 1. Cytomegalovirus lung culture was positive in two patients and serum anti-CMV antibody was positive in four patients. DNA was purified from postmortem lung tissue and PCR was performed to amplify a 685 base pair sequence from the immediate early gene 1 of CMV, and was followed by Southern blot hybridization (Fig 1). CASE REPORTS Case No. 1 A lo-year-old girl with T-cell ALL was admitted to the Nagoya First Red Cross Hospital for BMT. Allogeneic BMT was performed from a partially HLA-matched cousin. On day 6 herpes zoster was observed on the right chest and successfully treated with acyclovir. On day 12 hemorrhagic cystitis caused by adenovirus type 11 developed and persisted until the patient’s death. Acute graft-versus-host disease was observed in skin and liver, and liver function gradually deteriorated. On day 59 chest roentgenogram showed interstitial pneumonitis; on the following day the patient died of respiratory failure. At autopsy, lung tissue showed CMV-specific histology and
CMV DNA IN LUNG TISSUE OF BMT RECIPIENTS
FIGURE I. Polymerase chain reaction and Southern blot hybridization for detecting CMV DNA in lung tissue. Lanes 1 to 5, cases no. 1 to 5; C, buffer control; P, plasmid “E” DNA (10 to IO4 copies). It should be noted that PCR signals in the 10 copies of plasmid DNA and in case no. 3 are visible in the original autoradiograph and may not be visible in the figure due to a loss of sensitivity during photographic reproduction.
(Shibata et al)
P 1
2
3
685bp+
4
5
c zaps
,‘
CMV antigen by microscopy and immunofluorescence. Cytomegalovirus was isolated from lung and kidney. The results of PCR assay revealed an apparent DNA band specific to CMV (Fig 1).
Adriamycin (Kyowd Hakko, Tokyo, Japan), methotrexate, and prednisolone, was performed without benefit. Pneumonia due to aspergillus developed 5 months after admission and the patient died of respiratory failure. At autopsy, pathologic examinations showed pulmonary aspergillosis. No histologic change for CMV infection was observed in the lung and no CMV was isolated from lung. Polymerase chain reaction assay of lung tissue showed no band for CMV DNA.
Case No. 2 A g-year-old boy with pre-T-cell A1.L was admitted to the Nagoya University Hospital for BMT. Allogeneic BMT was performed from a partially HLA-matched cousin. Severe acute graft-versus-host disease was observed in skin and digestive tracts 3 weeks after BMT. Dyspnea suddenly developed on day 32 and chest roentgenogram revealed interstitial pneumonitis. On the following day the patient died of respiratory failure. At autopsy, CMV was isolated from the lung. Histologic examinations of lung tissue showed inclusion bodies specific to CMV, and CMV antigen was also detectable by immunofluorescence. No causative agents other than CMV were isolated from the lung. The results of PCR assay showed a strong DNA band specific to CMV.
Case No. 5
Case No. 3 A 14-year-old boy with T-cell AIdI, received BMT from a partially HLA-matched aunt at the Nagoya University Hospital. On day 24 acute graft-versus-host disease was observed in the skin and was successfully treated with methylprednisolone. Four months after BMT, chronic graft-versus-host disease of the skin and liver and interstitial pneumonitis developed. Because IgM antibody to CMV by enzyme-linked immunosorbent assay became positive, CMV was clinically considered as a cause of pneumonitis. Corticosteroids and azathioprine were administered with some benefit, and interstitial pneumonitis subsequently was resolved. Eight months after BMT the patient developed bronchopneumonia in the right middle lung field that did not respond to antibiotics, antifungal agents, or sulfamethoxazole/trimethoprim. Subsequently, interstitial shadow was observed in a chest roentgenogram. Intravenous imnnmoglobulin and ganciclovir were administered for possible CMV pneumonitis. However, the patient gradually deteriorated and died of multiple organ failure. At autopsy, lung tissue showed no pathologic changes of CMV infection and CMV was not isolated from the lung, pleural fluid, liver, or kidney. Interstitial pneumonitis was diagnosed as idiopathic. Polymerase chain reaction assay of lung tissue showed a weak band for CMV DNA.
Case No. 4 An 1B-year-old man with acute myeloblastic leukemia who had received a BMT from his brother 2 years before was admitted to the Nagoya University Hospital for a relapse of leukemia. Multiple drug therapy, including cytosine arabinoside,
913
A G-year-old girl with Philadelphia chromosome-positive ALL who had received autologous BMT 4 months previously was admitted to the Nagoya University Hospital for a relapse of leukemia. Multiple drug therapy, including vincristine, cyclophosphamide, methotrexate, r.-asparaginase, and cytosine arabinoside, was administered without benefit. Three months after admission the patient developed dyspnea and chest xray revealed interstitial shadow in both lung fields. Streptococrzu mitis was isolated from blood. The patient’s condition progressively deteriorated and she died of respiratory failure. At autopsy, lung pathology revealed pulmonary edema and mild interstitial change due to sepsis. No CMV was isolated from the lung, liver, or peripheral blood. Polymerase chain reaction assay of lung tissue showed no DNA band for CMV.
Determination of Cytomegalovirus Number in the Lungs
Copy
Quantitative PCR was performed to estimate CMV copy number in the lungs. Figure 2 shows an example of a standard curve to determine the initial copy number of CMV in DNA samples. Cases no. 1 and 2 had over lo4 copies of CMV per microgram DNA and case no. 3 had approximately 10 copies per microgram. In cases no. 4 and 5 CMV copy number was below the sensitivity of the quantitative PCR assay (less than 10 copies per microgram DNA). Repeated experiments showed similar results.
DISCUSSION The manifestations of CMV infections vary from asymptomatic to fatal illnesses. Interstitial pneumonitis due to CMV is a major cause of morbidity and mortality in immunocompromised patients. The diagnosis of CMV pneumonitis is, however, quite complex because CMV is isolated from various sites of immunocompromised patients without any manifestations.22 Lung tissue is the most reliable sample to diagnose CMV pneumonitis. In this study we have applied the PCR assay to amplify CMV DNA from lung tissue of patients who died of
HUMAN PATHOLOGY
Volume 23, No. 8 (August
cause small variations in reaction conditions influence the efficiency of PCR. However, recent studies have shown that quantitation with PCR assay is possible by comparing each measurement of PCR signals with a known standard that has similarly been amplified by PCR.2”.‘4 In this study we used plasmid DNA containing the immediate early gene 1 of CMV as an external PCR standard. The quantitation of the PCR products by comparison with the external standard showed that the copy number of the CMV genome in the lung tissue of CMV pneumonitis patients is over 1,OOO-fold more than that of the patients without CMV disease. The CMV copy number in case no. 3 was approximately 10 copies/ pg DNA, which corresponds to 1 CMV genome per 3 X 10” cells. It appears that the copy number of CMV genome in case no. 3 is too few to cause CMV disease. Recently, BAL specimens have been used for CMV isolation to diagnose CMV pneumonitis. However, falsepositive results are a problem since CMV has been isolated from BAL of immunocompromised patients without any manifestations.22 Quantitative PCR with BAL might be helpful in differentiating CMV pneumonitis from other causes of pneumonitis in CMV-positive patients. In conclusion, we used the PCR assay for quantitation of CMV DNA in lung tissue from BMT recipients and found that stronger PCR signals were correlated with the presence of CMV pneumonitis, which was determined with virus isolation and CMV-specific histology. These results are in agreement with those of a previous report’” that used the conventional DNA hybridization technique to analyze human CMV DNA in lungs from BMT recipients and to show that the quantitative PCR assay could be useful as one of the diagnostic procedures for CMV pneumonitis.
m 0 -
x -
ZE e 0
j
1
2
COPY
3
4
1992)
LOG
NUMBER
FIGURE 2. A standard curve for the quantitative PCR assay. Plot of counts per minute (cpm) versus the logarithm of the initial plasmid DNA copy number.
CMV or non-CMV respiratory diseases. The advantage of the PCR assay is that it can amplify and detect the noninfectious CMV genome in infected cells as well as the viral genome in free viral particles. The administration of antiviral agents, such as ganciclovir, may affect the efficiency of CMV isolation and, hence, methods to detect the viral genome could be useful in determining the presence of CMV in samples. However, the high sensitivity of the PCR assay may be disadvantageous because the prevalence of the anti-CMV antibody among Japanese children is so high that any lung tissue from immunocompromised patients could be found positive for CMV DNA with the PCR assay. To determine the validity of the PCR assay we retrospectively analyzed well-characterized lung samples from immunocompromised patients. In the two patients (cases no. 1 and 2) who were shown to have CMV interstitial pneumonitis by the pathologic and virologic examinations, the PCR assay apparently showed strong signals specific to CMV. In contrast, CMV DNA was undetectable or hardly detectable in the three patients (cases no. 3, 4, and 5) whose respiratory illness was considered irrelevant to CMV by virus isolation and histology. The weak PCR signal in case no. 3 was not considered to indicate that interstitial pneumonitis of this patient was caused by CMV, but rather to reflect the characteristic of CMV that infects humans persistently. To clarify the quantitative difference between the patients with and without CMV pneumonitis, the initial CMV copy number in the DNA samples was determined with the PCR assay. Exact quantitation of viral DNA by PCR assay is difficult be-
REFERENCES 1. Emanuel D, Cunningham I, Jules-Elysee K, et al: Cytomegalovirus pneumonia after bone marrow transplantation successfully treated with the combination of ganciclovir and high-dose intravenous immune globulin. Ann Intern Med 109:777-782, 1988 2. Keed EC, Bowden RA, Dandliker PS, et al: Treatment of cytomegalovirus pneumonia with ganciclovir and intravenous cytomegalovirus immunoglobulin in patients with bone marrow transplants. Ann Intern Med 109:783-788, 1988 3. Winston DJ, Ho WG, Lin C-H, et al: Intravenous immune globulin for prevention of cytomegalovirus infection and interstitial pneumonia after bone marrow transplantation. Ann Intern Med 106: 12-18, 1987 4. Sullivan KM, Kopecky KJ, Jocom J, et al: Imrnunomodulator); and antimicrobial efficacy of intravenous immunoglobulin in bone marrow transplantation. N Engl J Med 323:705-712, 1990 5. Meyers JD, Reed EC, Shepp DH, et al: Acyclovir for prevention of cytomegalovirus infection and disease after allogeneic marrow transplantation. N Engl J Med 3 18:70-75, 1988 6. Schmidt GM, Horak DA, Niland JC, et al: A randomized, controlled trial of prophylactic ganciclovir for cytomegalovirus pulmonary infection in recipients of allogeneic bone marrow transplants. N Engl J Med 324:1005-1011, 1991 7. Saiki RK, Scharf S, Faloona F, et al: Enzymatic amplification of fl-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230: 1350-l 354, I985 8. Demmler GJ, Buffone GJ, Schimbor CM, et al: Detection of cytomegalovirus in urine from newborns by using polymerase chain reaction DNA amplification. J Infect Dis 158: 1177-l 184, 1988
914
CMV DNA IN LUNG TISSUE OF BMT RECIPIENTS 9. Shibata D, Martin WJ, Appleman MD, et al: Detection of cytomegalovirus DNA in peripheral blood of patients infected with human immunodeficiency virus. J Infect Dis 158:1185-l 192, 1988 10. Hsia K, Spector DH, Lawrie J, et al: Enzymatic amplification of human cytomegalovirus sequences by polymerase chain reaction. J Clin Microbial 27: 1802-l 809, 1989 11. Jiwa NM, Van Gamert GW, Raap AK, et al: Rapid detection of human cytomegalovirus DNA in peripheral blood leukocytes of viremic transplant recipients by the polymerase chain reaction. Transplantation 48:72-76, 1989 12. Shibata M, Morishima T, Terashima M, et al: Human cytomegalovirus infection during childhood: Detection of viral DNA in peripheral blood by means of polymerase chain reaction. Med Microbiol Immunol 179:245-253, 1990 13. Cassol SA, Poon M-C, Pal R, et al: Primer-mediated enzymatic amplification of cytomegalovirus (CMV) DNA. Application to the early diagnosis of CMV infection in marrow transplant recipients, J Clin Invest 83:1109-1115, 1989 14. Jiwa M, Steenbergen RDM, Zwaan FE, et al: Three sensitive methods for the detection of cytomegalovirus in lung tissue of patients with interstitial pneumonitis. Am J Clin Path01 93:491-494, 1990 15. Hackman RC, Myerson D, Meyers JD, et al: Rapid diagnosis of cytomegaloviral pneumonia by tissue immunofluorescence with a murine monoclonal antibody. J Infect Dis 151:325-329, 1985 16. Stenberg RM, Thomsen DR, Stinski MF: Structural analysis of the major immediate early gene of human cytomegalovirus. J Virol 49:190-199, 1984 17. Ng S-Y, Gunning P, Eddy R, et al: Evolution of the functional human @-actin gene and its multi-pseudogene family: Conservation
915
(Shibata et al)
of noncoding regions and chromosomal dispersion of pseudogenes. Mol Cell Biol 5:2720-2732, 1985 18. Kwok S, Higuchi R: Avoiding false positives with PCR. Nature 339:237-238, 1989 19. Thomsen DR, Stinski MF: Cloning of the human cytomegalovirus genome as endonuclease XbaI fragments. Gene 16:207-216, 1981 20. OzawaT, Tanaka M, Ikebe S, et al: Quantitative determination of deleted mitochondrial DNA relative to normal DNA in parkinsonian striatum by a kinetic PCR analysis. Biochem Biophys Res Commun 172:483-489, 1990 21. Morishima Y, Morishita Y, Tanimoto M, et al: Low incidence of acute graft-versus-host disease by the administration of methotrexate and cyclosporine in Japanese leukemia patients after bone marrow transplantation from human leukocyte antigen compatible siblings: Possible role of genetic homogeneity. Blood 74:2252-2256, 1989 22. Ruutu P, Ruutu T, Volin L, et al: Cytomegalovirus is frequently isolated in bronchoalveolar lavage fluid of bone marrow transplant recipients without pneumonia. Ann Intern Med 112:913916, 1990 23. Dickover RE, Donovan RM, Goldstein E, et al: Quantitation of human immunodeficiency virus DNA by using the polymerase chain reaction. J Clin Microbial 28:2130-2133, 1990 24. Katz JP, Bodin ET, Coen DM: Quantitative polymerase chain reaction analysis of herpes simplex virus DNA in ganglia of mice infected with replication-incompetent mutants. J Virol 64:4288-4295, 1990 25. Churchill MA, Zaia JA, Forman SJ, et al: Quantitation of human cytomegalovirus DNA in lungs from bone marrow transplant recipients with interstitial pneumonia. J Infect Dis 155:501-509, 1987
Cytomegalovirus (CMV) is one of major etiologic agents of severe illness and death in immunocompromised hosts. Interstitial pneumonitis is a significant illness caused by CMV due to its high mortality rate. Combination therapy of ganciclovir with intravenous high-dose immunoglobulin has improved the prognosis of CMV interstitial pneumonitis.‘** Accurate diagnosis and early treatment are essential for the management of immunocompromised patients. Clinical diagnosis of CMV pneumonitis has been made based on (1) isolation of CMV from lung tissue or bronchoalveolar lavage (BAL) and (2) demonstration of interstitial infiltrates on chest radiograph. However, small amounts of CMV in samples may result in falsenegative results with virus culture. Furthermore, prophylactic administration of antiviral agents, such as ganciclovir, acyclovir, and immunoglobulin,“-6 may decrease the efficiency of virus isolation. Methods to detect viral DNA are therefore useful in determining the presence of CMV in samples. Polymerase chain reaction (PCR),
originally described by Saiki et ak7 is an extremely sensitive method to detect specific DNA and has been applied to CMV infections.“‘* Previous reports have shown that lung tissue of patients with CMV pneumonitis was positive for CMV DNA with PCR.‘3214 However, because immunocompromised patients might excrete CMV without any clinical disease, lung tissue from such immunocompromised patients could be positive for CMV DNA due to the high sensitivity of the PCR assay. It is noteworthy that many children in Japan acquire antibody to CMV by 5 years of age. Quantitative evaluation of CMV infection may be helpful in the diagnosis of CMV pneumonitis in that situation. In this study we performed quantitative PCR as a first step to show that quantitative PCR for CMV with sputum, BAL, or lung biopsy is potentially useful in differentiating CMV pneumonitis from other causes of pneumonitis in CMVpositive patients. The study was therefore designed to retrospectively analyze postmortem lung tissue from bone marrow transplant (BMT) recipients who had been examined for CMV infection with virus isolation, immunofluorescence, and histology. MATERIALS
AND METHODS
Virus Isolation For virus isolation, lung tissue or other samples (such as blood, liver, or kidney) obtained at autopsy were immediately inoculated into human embryonic lung cells and observed for cytopathic effect of CMV. Cytomegalovirus was identified by fluorescence microscopy using a murine monoclonal antibody to the immediate early antigen of CMV, which was kindly provided by Dr T. Furukawa, Kanazawa Medical University.
Histopathologic and lmmunofluorescence Examinations of Lung Tissue Lung tissue sections were fixed and stained by standard histologic techniques and viewed microscopically. A histologic diagnosis of CMV interstitial pneumonitis was made if large cells containing basophilic nuclear inclusions (Cowdry type A) and multiple smaller cytoplasmic inclusions were demonstrated.r5 For immunofluorescence, sections from frozen lung tissue were fixed in cold methanol and stained with the murine monoclonal antibody to the immediate early antigen of CMV. The slides were rinsed with phosphate-buffered saline and reacted with fluorescein-conjugated goat anti-mouse immunoglobulin (Organon Teknika-Cappel, West Chester, PA). The slides were then rinsed and observed under an ultraviolet microscope.
From the Department of Pediatrics, Nagoya University School of Medicine, Nagoya, Japan; and Department of Pediatrics, Nagoya First Red Cross Hospital, Nagoya, Japan. Accepted for publication October 8, 199 1. Key wordrc polymerase chain reaction, cytomegalovirus, quantitation, interstitial pneumonitis, bone marrow transplantation, Address correspondence and reprint requests to Motohiro Shibata, MD, Department of Pediatrics, Nagoya University School of Medicine, Tsuruma-cho 65, Nagoya 466, Japan. Copyright 0 1992 by W.B. Saunders Company 0046-8177/92/2308-0013$5.00/O
911
HUMAN PATHOLOGY
TABLE
1.
Volume 23, No. 8 (August
1992)
Histologic and Virologic Examinations of Five ImmunocomDromised Patients CMV Assay on Lung Tissue”
Case No. (Age lvrl /sex)
1 (10/F) 2 (g/M) 3 (14/M) 4 (18/M) 5 (G/F) Ahhreviations:
Chest X-ray
Inclusions
IP IP IP Pneumonia IP IF, immunofluorescence;
+ + _ _ _ IP, interstitial
* Lung tissue was examined by histology for inclusion isolation of CMV. t Serum CMV antibody was measured by complement
Preparation
IF + + ND ND ND
fixation or by enzyme immunoassay
of DNA From Lung Tissue
Othe1Pathoyen
+ ND + + +
_ Aspergillus Streptococcus
early antigen
milzs
of CMV, and by
for IgG antibody.
in the Southern blots was analyzed with the Fujix BAS 2000 Image Analyzing System (Fuji Photo Film, Tokyo, Japan)” and a standard curve was obtained at each experiment.
Measurement of Antibodies Cytomegalovirus
to
Antibody to CMV was determined by microtiter complement fixation and by an enzyme-linked immunosorbent assay for IgM and IgG antibodies (Behring Werke, Marburg, Germany). A complement fixation titer of 1:s was considered positive. Enzyme-linked immunosorbent assay was carried out according to the manufacturer’s instructions, and the cut-off index was determined.
Chain Reaction
Polymerase chain reaction for CMV was performed as described previously. ‘* Briefly , a primer set was synthesized to amplify a 685 base pair sequence (828-15 12) from the immediate early gene 1 of CMV.” The sense primer CVl was 5’-GGCCATGGCGGCATTGCAGAACTTG-3’. The anti-sense primer CV2 was 5’-CTCTATCTCAGACACTGGCTCAGAC-3’. One microgram of DNA purified from lung tissue was applied to 35 cycles of PCR with Taq polymerase in a DNA thermal cycler (Perkin Elmer Cetus, Norwalk, CT). The amplified DNA products were subjected to agarose gel electrophoresis and transferred to a nylon membrane (Nytran, Schleicher & Schuell, Dassel, Germany). Southern blot hybridization was then performed with the 32P-labeled DNA probe derived from plasmid pIE14 that contains nucleotide sequences corresponding to 879-1267 nucleotides of the immediate early gene 1 of CMV.” As an internal control of tissue DNA, 25 cycles of PCR were also performed to amplify a 371 base pair sequence from @-actin DNA.17 The sense primer was 5’GCCATGTACGTTGCTATCCA-3’. The anti-sense primer was 5’-CGCTCATTGCCAATGGTGATGACCT-3’. Polymerase chain reaction for the actin DNA with 1 pg of the tissue DNA resulted in a similar intensity of DNA bands at the expected size after agarose gel electrophoresis and ethidium bromide staining. To avoid false-positive results with PCR we applied the contamination prevention measures of Kwok and Higuchi.‘s All PCR and Southern blots were run and analyzed in a blinded fashion.
Determination of Cytomegalovirus Number in DNA From Lung Tissue
+ + _ _ _
Serum CMV Antihodyt
pneumonitis; ND, not determined. bodies, by immunofluorescence for the immediate
Lung tissues obtained at autopsy were frozen at -70°C until the time of DNA preparation. Approximately 50 mg of frozen tissue was pulverized to a fine powder in liquid nitrogen and incubated at 56°C for 1 hour in TNE buffer (20 mmol/ L Tris-HCl [pH 7.51, 10 mmol/L NaCl, 20 mmol/L EDTA) containing 0.1% sodium dodecyl sulfate and 200 pg/mL proteinase K. DNA was extracted with equal volumes of phenol: chloroform and chloroform. DNA was then precipitated with ethanol and resuspended in TE buffer (10 mmol/L Tris-HCl [pH 8.01, 0.05 mmol/L EDTA). DNA concentration was determined by absorbance at 260 nm.
Polymerase
Isolation
Copy
For determining the initial CMV copy number in DNA samples, plasmid “E” DNA (kindly provided by D. Stinski’“) containing the immediate early gene 1 of CMV, was serially diluted with TE buffer and similarly amplified with PCR as external standards. The radioactivity of each CMV DNA band
912
Bone Marrow Transplantation Bone scribed.2’
marrow
transplantation
was performed
as de-
RESULTS We analyzed five immunocompromised patients who had received BMT for acute lymphoblastic leukemia (ALL) or acute myeloblastic leukemia and who died of respiratory failure. The results of virologic and pathologic examinations at autopsy are summarized in Table 1. Cytomegalovirus lung culture was positive in two patients and serum anti-CMV antibody was positive in four patients. DNA was purified from postmortem lung tissue and PCR was performed to amplify a 685 base pair sequence from the immediate early gene 1 of CMV, and was followed by Southern blot hybridization (Fig 1). CASE REPORTS Case No. 1 A lo-year-old girl with T-cell ALL was admitted to the Nagoya First Red Cross Hospital for BMT. Allogeneic BMT was performed from a partially HLA-matched cousin. On day 6 herpes zoster was observed on the right chest and successfully treated with acyclovir. On day 12 hemorrhagic cystitis caused by adenovirus type 11 developed and persisted until the patient’s death. Acute graft-versus-host disease was observed in skin and liver, and liver function gradually deteriorated. On day 59 chest roentgenogram showed interstitial pneumonitis; on the following day the patient died of respiratory failure. At autopsy, lung tissue showed CMV-specific histology and
CMV DNA IN LUNG TISSUE OF BMT RECIPIENTS
FIGURE I. Polymerase chain reaction and Southern blot hybridization for detecting CMV DNA in lung tissue. Lanes 1 to 5, cases no. 1 to 5; C, buffer control; P, plasmid “E” DNA (10 to IO4 copies). It should be noted that PCR signals in the 10 copies of plasmid DNA and in case no. 3 are visible in the original autoradiograph and may not be visible in the figure due to a loss of sensitivity during photographic reproduction.
(Shibata et al)
P 1
2
3
685bp+
4
5
c zaps
,‘
CMV antigen by microscopy and immunofluorescence. Cytomegalovirus was isolated from lung and kidney. The results of PCR assay revealed an apparent DNA band specific to CMV (Fig 1).
Adriamycin (Kyowd Hakko, Tokyo, Japan), methotrexate, and prednisolone, was performed without benefit. Pneumonia due to aspergillus developed 5 months after admission and the patient died of respiratory failure. At autopsy, pathologic examinations showed pulmonary aspergillosis. No histologic change for CMV infection was observed in the lung and no CMV was isolated from lung. Polymerase chain reaction assay of lung tissue showed no band for CMV DNA.
Case No. 2 A g-year-old boy with pre-T-cell A1.L was admitted to the Nagoya University Hospital for BMT. Allogeneic BMT was performed from a partially HLA-matched cousin. Severe acute graft-versus-host disease was observed in skin and digestive tracts 3 weeks after BMT. Dyspnea suddenly developed on day 32 and chest roentgenogram revealed interstitial pneumonitis. On the following day the patient died of respiratory failure. At autopsy, CMV was isolated from the lung. Histologic examinations of lung tissue showed inclusion bodies specific to CMV, and CMV antigen was also detectable by immunofluorescence. No causative agents other than CMV were isolated from the lung. The results of PCR assay showed a strong DNA band specific to CMV.
Case No. 5
Case No. 3 A 14-year-old boy with T-cell AIdI, received BMT from a partially HLA-matched aunt at the Nagoya University Hospital. On day 24 acute graft-versus-host disease was observed in the skin and was successfully treated with methylprednisolone. Four months after BMT, chronic graft-versus-host disease of the skin and liver and interstitial pneumonitis developed. Because IgM antibody to CMV by enzyme-linked immunosorbent assay became positive, CMV was clinically considered as a cause of pneumonitis. Corticosteroids and azathioprine were administered with some benefit, and interstitial pneumonitis subsequently was resolved. Eight months after BMT the patient developed bronchopneumonia in the right middle lung field that did not respond to antibiotics, antifungal agents, or sulfamethoxazole/trimethoprim. Subsequently, interstitial shadow was observed in a chest roentgenogram. Intravenous imnnmoglobulin and ganciclovir were administered for possible CMV pneumonitis. However, the patient gradually deteriorated and died of multiple organ failure. At autopsy, lung tissue showed no pathologic changes of CMV infection and CMV was not isolated from the lung, pleural fluid, liver, or kidney. Interstitial pneumonitis was diagnosed as idiopathic. Polymerase chain reaction assay of lung tissue showed a weak band for CMV DNA.
Case No. 4 An 1B-year-old man with acute myeloblastic leukemia who had received a BMT from his brother 2 years before was admitted to the Nagoya University Hospital for a relapse of leukemia. Multiple drug therapy, including cytosine arabinoside,
913
A G-year-old girl with Philadelphia chromosome-positive ALL who had received autologous BMT 4 months previously was admitted to the Nagoya University Hospital for a relapse of leukemia. Multiple drug therapy, including vincristine, cyclophosphamide, methotrexate, r.-asparaginase, and cytosine arabinoside, was administered without benefit. Three months after admission the patient developed dyspnea and chest xray revealed interstitial shadow in both lung fields. Streptococrzu mitis was isolated from blood. The patient’s condition progressively deteriorated and she died of respiratory failure. At autopsy, lung pathology revealed pulmonary edema and mild interstitial change due to sepsis. No CMV was isolated from the lung, liver, or peripheral blood. Polymerase chain reaction assay of lung tissue showed no DNA band for CMV.
Determination of Cytomegalovirus Number in the Lungs
Copy
Quantitative PCR was performed to estimate CMV copy number in the lungs. Figure 2 shows an example of a standard curve to determine the initial copy number of CMV in DNA samples. Cases no. 1 and 2 had over lo4 copies of CMV per microgram DNA and case no. 3 had approximately 10 copies per microgram. In cases no. 4 and 5 CMV copy number was below the sensitivity of the quantitative PCR assay (less than 10 copies per microgram DNA). Repeated experiments showed similar results.
DISCUSSION The manifestations of CMV infections vary from asymptomatic to fatal illnesses. Interstitial pneumonitis due to CMV is a major cause of morbidity and mortality in immunocompromised patients. The diagnosis of CMV pneumonitis is, however, quite complex because CMV is isolated from various sites of immunocompromised patients without any manifestations.22 Lung tissue is the most reliable sample to diagnose CMV pneumonitis. In this study we have applied the PCR assay to amplify CMV DNA from lung tissue of patients who died of
HUMAN PATHOLOGY
Volume 23, No. 8 (August
cause small variations in reaction conditions influence the efficiency of PCR. However, recent studies have shown that quantitation with PCR assay is possible by comparing each measurement of PCR signals with a known standard that has similarly been amplified by PCR.2”.‘4 In this study we used plasmid DNA containing the immediate early gene 1 of CMV as an external PCR standard. The quantitation of the PCR products by comparison with the external standard showed that the copy number of the CMV genome in the lung tissue of CMV pneumonitis patients is over 1,OOO-fold more than that of the patients without CMV disease. The CMV copy number in case no. 3 was approximately 10 copies/ pg DNA, which corresponds to 1 CMV genome per 3 X 10” cells. It appears that the copy number of CMV genome in case no. 3 is too few to cause CMV disease. Recently, BAL specimens have been used for CMV isolation to diagnose CMV pneumonitis. However, falsepositive results are a problem since CMV has been isolated from BAL of immunocompromised patients without any manifestations.22 Quantitative PCR with BAL might be helpful in differentiating CMV pneumonitis from other causes of pneumonitis in CMV-positive patients. In conclusion, we used the PCR assay for quantitation of CMV DNA in lung tissue from BMT recipients and found that stronger PCR signals were correlated with the presence of CMV pneumonitis, which was determined with virus isolation and CMV-specific histology. These results are in agreement with those of a previous report’” that used the conventional DNA hybridization technique to analyze human CMV DNA in lungs from BMT recipients and to show that the quantitative PCR assay could be useful as one of the diagnostic procedures for CMV pneumonitis.
m 0 -
x -
ZE e 0
j
1
2
COPY
3
4
1992)
LOG
NUMBER
FIGURE 2. A standard curve for the quantitative PCR assay. Plot of counts per minute (cpm) versus the logarithm of the initial plasmid DNA copy number.
CMV or non-CMV respiratory diseases. The advantage of the PCR assay is that it can amplify and detect the noninfectious CMV genome in infected cells as well as the viral genome in free viral particles. The administration of antiviral agents, such as ganciclovir, may affect the efficiency of CMV isolation and, hence, methods to detect the viral genome could be useful in determining the presence of CMV in samples. However, the high sensitivity of the PCR assay may be disadvantageous because the prevalence of the anti-CMV antibody among Japanese children is so high that any lung tissue from immunocompromised patients could be found positive for CMV DNA with the PCR assay. To determine the validity of the PCR assay we retrospectively analyzed well-characterized lung samples from immunocompromised patients. In the two patients (cases no. 1 and 2) who were shown to have CMV interstitial pneumonitis by the pathologic and virologic examinations, the PCR assay apparently showed strong signals specific to CMV. In contrast, CMV DNA was undetectable or hardly detectable in the three patients (cases no. 3, 4, and 5) whose respiratory illness was considered irrelevant to CMV by virus isolation and histology. The weak PCR signal in case no. 3 was not considered to indicate that interstitial pneumonitis of this patient was caused by CMV, but rather to reflect the characteristic of CMV that infects humans persistently. To clarify the quantitative difference between the patients with and without CMV pneumonitis, the initial CMV copy number in the DNA samples was determined with the PCR assay. Exact quantitation of viral DNA by PCR assay is difficult be-
REFERENCES 1. Emanuel D, Cunningham I, Jules-Elysee K, et al: Cytomegalovirus pneumonia after bone marrow transplantation successfully treated with the combination of ganciclovir and high-dose intravenous immune globulin. Ann Intern Med 109:777-782, 1988 2. Keed EC, Bowden RA, Dandliker PS, et al: Treatment of cytomegalovirus pneumonia with ganciclovir and intravenous cytomegalovirus immunoglobulin in patients with bone marrow transplants. Ann Intern Med 109:783-788, 1988 3. Winston DJ, Ho WG, Lin C-H, et al: Intravenous immune globulin for prevention of cytomegalovirus infection and interstitial pneumonia after bone marrow transplantation. Ann Intern Med 106: 12-18, 1987 4. Sullivan KM, Kopecky KJ, Jocom J, et al: Imrnunomodulator); and antimicrobial efficacy of intravenous immunoglobulin in bone marrow transplantation. N Engl J Med 323:705-712, 1990 5. Meyers JD, Reed EC, Shepp DH, et al: Acyclovir for prevention of cytomegalovirus infection and disease after allogeneic marrow transplantation. N Engl J Med 3 18:70-75, 1988 6. Schmidt GM, Horak DA, Niland JC, et al: A randomized, controlled trial of prophylactic ganciclovir for cytomegalovirus pulmonary infection in recipients of allogeneic bone marrow transplants. N Engl J Med 324:1005-1011, 1991 7. Saiki RK, Scharf S, Faloona F, et al: Enzymatic amplification of fl-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230: 1350-l 354, I985 8. Demmler GJ, Buffone GJ, Schimbor CM, et al: Detection of cytomegalovirus in urine from newborns by using polymerase chain reaction DNA amplification. J Infect Dis 158: 1177-l 184, 1988
914
CMV DNA IN LUNG TISSUE OF BMT RECIPIENTS 9. Shibata D, Martin WJ, Appleman MD, et al: Detection of cytomegalovirus DNA in peripheral blood of patients infected with human immunodeficiency virus. J Infect Dis 158:1185-l 192, 1988 10. Hsia K, Spector DH, Lawrie J, et al: Enzymatic amplification of human cytomegalovirus sequences by polymerase chain reaction. J Clin Microbial 27: 1802-l 809, 1989 11. Jiwa NM, Van Gamert GW, Raap AK, et al: Rapid detection of human cytomegalovirus DNA in peripheral blood leukocytes of viremic transplant recipients by the polymerase chain reaction. Transplantation 48:72-76, 1989 12. Shibata M, Morishima T, Terashima M, et al: Human cytomegalovirus infection during childhood: Detection of viral DNA in peripheral blood by means of polymerase chain reaction. Med Microbiol Immunol 179:245-253, 1990 13. Cassol SA, Poon M-C, Pal R, et al: Primer-mediated enzymatic amplification of cytomegalovirus (CMV) DNA. Application to the early diagnosis of CMV infection in marrow transplant recipients, J Clin Invest 83:1109-1115, 1989 14. Jiwa M, Steenbergen RDM, Zwaan FE, et al: Three sensitive methods for the detection of cytomegalovirus in lung tissue of patients with interstitial pneumonitis. Am J Clin Path01 93:491-494, 1990 15. Hackman RC, Myerson D, Meyers JD, et al: Rapid diagnosis of cytomegaloviral pneumonia by tissue immunofluorescence with a murine monoclonal antibody. J Infect Dis 151:325-329, 1985 16. Stenberg RM, Thomsen DR, Stinski MF: Structural analysis of the major immediate early gene of human cytomegalovirus. J Virol 49:190-199, 1984 17. Ng S-Y, Gunning P, Eddy R, et al: Evolution of the functional human @-actin gene and its multi-pseudogene family: Conservation
915
(Shibata et al)
of noncoding regions and chromosomal dispersion of pseudogenes. Mol Cell Biol 5:2720-2732, 1985 18. Kwok S, Higuchi R: Avoiding false positives with PCR. Nature 339:237-238, 1989 19. Thomsen DR, Stinski MF: Cloning of the human cytomegalovirus genome as endonuclease XbaI fragments. Gene 16:207-216, 1981 20. OzawaT, Tanaka M, Ikebe S, et al: Quantitative determination of deleted mitochondrial DNA relative to normal DNA in parkinsonian striatum by a kinetic PCR analysis. Biochem Biophys Res Commun 172:483-489, 1990 21. Morishima Y, Morishita Y, Tanimoto M, et al: Low incidence of acute graft-versus-host disease by the administration of methotrexate and cyclosporine in Japanese leukemia patients after bone marrow transplantation from human leukocyte antigen compatible siblings: Possible role of genetic homogeneity. Blood 74:2252-2256, 1989 22. Ruutu P, Ruutu T, Volin L, et al: Cytomegalovirus is frequently isolated in bronchoalveolar lavage fluid of bone marrow transplant recipients without pneumonia. Ann Intern Med 112:913916, 1990 23. Dickover RE, Donovan RM, Goldstein E, et al: Quantitation of human immunodeficiency virus DNA by using the polymerase chain reaction. J Clin Microbial 28:2130-2133, 1990 24. Katz JP, Bodin ET, Coen DM: Quantitative polymerase chain reaction analysis of herpes simplex virus DNA in ganglia of mice infected with replication-incompetent mutants. J Virol 64:4288-4295, 1990 25. Churchill MA, Zaia JA, Forman SJ, et al: Quantitation of human cytomegalovirus DNA in lungs from bone marrow transplant recipients with interstitial pneumonia. J Infect Dis 155:501-509, 1987
