Human cytomegalovirus (HCMV) reactivation and disease remain relatively common after lung transplantation despite the use of specific prophylactic strategies that have been designed to minimize their impact (1–3)
Current routine diagnostic strategies for HCMV detection based on viral culture and shell vial assays are generally inefficient and poorly sensitive for the detection of HCMV (4–7) (8–10) (5,7) (11–13)
During active HCMV infection, viremia and DNA in the blood have both been recognized as major virologic risk factors for the progression to clinical disease (14,15) (5,16) (17–22)
Increasingly, improved quantitation assays measuring HCMV DNA viral load in at-risk solid organ and bone marrow transplant recipients as well as HIV/AIDS patients have been used to resolve significant increases in HCMV DNA levels (23–28) (25)
We have previously applied a highly sensitive, quantitative PCR assay for HCMV DNA that incorporates a competitive internal standard, fluorescently labeled primers, and fluoroimager detection of amplified product (29)
MATERIALS AND METHODS
Subjects
The Alfred Hospital is a Monash University–affiliated teaching hospital in Melbourne, Victoria, with a national Lung-Heart transplant service that averages 35–40 lung transplants per year. Between June 1997 and August 1998, 46 lung transplants were performed. Seventeen LTR were not considered for this study as their ongoing follow-up was to occur in other distant centers. Of the remaining 29 LTR, we recruited 26 who were able to be followed up with repeated blood tests and bronchoscopic sampling at the Alfred Hospital in Melbourne. Three patients were not recruited because of prolonged complications in the intensive care unit immediately postoperatively.
All patients were treated with a standard immunosuppressive regimen immediately after transplantation, comprising cyclosporine (CsA), azathioprine, and prednisolone adjusted according to a protocol to initially maintain CsA levels between 300 and 450 μg/L (SYVA, EMIT kit, monoclonal, Dade Behring), white blood cell levels between 5 and 10×109 /L, and diminishing corticosteroid doses from 1–2 mg/kg per day down to 15 mg/d by 6 months. Serologic testing was performed using an ELISA kit for HCMV IgG (Gull, Salt Lake City, UT). Ganciclovir prophylaxis was given to at-risk patients (donor or recipient seropositive for HCMV) at a dose of 5 mg/kg i.v. twice a day for 2 weeks followed by 5 mg/kg three times a week for a total of at least 8 weeks after transplantation, and this was increased to 12 weeks if there was a primary HCMV mismatch (D+/R−). The latter patient group also received HCMV hyper-Ig on days 1, 2, 3, 7, 14, 21, 28, and 35 after lung transplantation. HCMV pneumonitis was defined as a clinical HCMV syndrome in association with histopathologic evidence of HCMV inclusions in transbronchial biopsies. Bronchoscopy, bronchoalveolar lavage sampling, and transbronchial biopsies were performed as part of routine surveillance after lung transplantation at 2, 4, 8, 12, and 26 weeks and when clinically indicated. HCMV pneumonitis episodes were treated with a repeat course of full-dose ganciclovir (5 mg/kg twice daily, for 2 weeks).
Ganciclovir status was defined according to ganciclovir usage at the time HCMV DNA load was assessed relative to the previous sampling time point (e.g., Ganciclovir Added=ganciclovir had been commenced or augmented before the sampling time point; Ganciclovir Withdrawn=ganciclovir had been ceased before the sampling time point; Ganciclovir On=ganciclovir at prophylaxis doses was still being used before the sampling time point; No Ganciclovir=no ganciclovir in-between sampling time points).
Peripheral Blood Samples and DNA Extraction
Peripheral blood samples were obtained within 4 weeks after transplantation and then at routine monthly intervals thereafter up to 6 months, which included sampling at the time of bronchoscopic and transbronchial biopsy investigation. Blood samples were analyzed for HCMV DNA in a blinded manner. HCMV PCR results were not used as a basis for clinical decision-making. Each sample consisted of 9 ml of blood in EDTA vacuum tubes, of which 3 ml was sent for HCMV culture. PBL were isolated from 6 ml of blood (3-ml duplicates) as previously described (29)
Competitive PCR Amplification
HCMV-specific fluorescently labeled primers directed at a conserved region within the DNA polymerase gene (HCMV strain AD169) were used to amplify a 314-base pair (bp) fragment as previously described by Greenfield et al. (30) (29)
A quantitative competitive PCR assay was performed using known amounts of competitor molecule as previously described (29) 2 , 200 μM nucleotides, 1 unit Taq Gold DNA polymerase, and 200 μM oligonucleotide primers. Master mixes were used whenever possible, and positive and negative controls were used. PCR amplification was conducted for 45 cycles with a preactivation step (94°C, 8 min), denaturation (94°C, 1 min), annealing/extension (68°C, 1 min), and a final extension (72°C, 7 min) in a thermal cycler fitted with a heated lid (Perkin-Elmer). Patient DNA samples were screened in duplicate with 1 μg of DNA and 100 copies of competitor. HCMV-positive DNA samples were run with increasing copies of competitor molecule. When both native and competitor molecules were quantifiable, the ratio between them could be measured, and thereby the input number of native DNA could be determined. The detection threshold of the PCR assay was 10 copies/μg of total DNA. Amplified PCR products were detected by electrophoresis in 3% agarose gels and quantitated as previously described (29)
Statistical Analysis
The chi-square test was used to determine the significance of differences in proportions between groups. To determine variables that predicted HCMV DNA levels, a generalized linear regression model was used incorporating categorical and continuous variables. All analyses were performed using SAS software package (v6.12, SAS Institute Inc., Cary, NC).
RESULTS
Clinical
The clinical characteristics of the cohort are shown in Table 1 . Fifteen bilateral sequential lung transplants and 11 single lung transplants were performed predominately for end-stage bronchiectasis (eight because of cystic fibrosis) and emphysema, respectively. One death in the cohort occurring in the first month after transplantation was related to airway anastomosis and sepsis complications.
Table 1: Patient cohort demographics
HCMV Pneumonitis, HCMV Serostatus, and HCMV Detection
During the first 6 months after lung transplantation, 13 episodes of HCMV pneumonitis (eight episodes in seven patients were diagnosed during routine surveillance bronchoscopy) were observed in nine subjects, with four patients experiencing two episodes. HCMV disease did not develop in the two D−/R− HCMV seromatches. There was no significant difference among the HCMV at-risk groups based on HCMV serostatus (i.e., of the nine patients in whom HCMV pneumonitis was observed, there were four HCMV D+/R+ matches, three D+/R−, and two D−/R+ mismatches from totals of 10, 8, and 6, respectively, in these serologic groups). However, there was a significant difference between HCMV detection, absolute levels, and relative change from baseline between the LTR who developed HCMV pneumonitis and those who did not (Fig. 1 ).
Figure 1: (A) HCMV viral loads from nine patients with histopathologically proven pneumonitis. PBL HCMV DNA load is reflective of HCMV pneumonitis in the lung allograft at the disease time points. There were 13 episodes of HCMV pneumonitis (8 episodes in 7 patients were diagnosed during routine surveillance bronchoscopy), and 6 of the 7 HCMV DNA levels above 2000 HCMV copies were associated with HCMV pneumonitis. HCMV DNA levels were relatively suppressed in the first 60 days after transplantation, which was related to routine ganciclovir prophylaxis, and were also quickly reduced after HCMV pneumonitis was diagnosed and full-dose ganciclovir treatment commenced. (B) HCMV viral loads from 16 patients without HCMV pneumonitis. Only two patients had HCMV levels above 2000 copies that were not associated with HCMV pneumonitis. Both patients had undiagnosed gastrointestinal and liver abnormalities during the study period. (n=16 because of early death of one patient).
Interestingly, there was no difference in the rate of HCMV detection in the circulating PBL among the HCMV at-risk serologic groups. Of the eight patients who were primary HCMV mismatches (D+/R−), there were three who had no HCMV DNA detected in any sample and five who had at least one positive sample, whereas of the 10 D+/R+ HCMV-matched patients, five had no HCMV DNA detected in any sample and five had at least one positive sample. Of the six patients who were reverse HCMV mismatches (D−/R+), all had at least one episode of detectable HCMV DNA in the PBL in the first 6 months after lung transplantation. Neither of the two HCMV D−/R− matches had detectable HCMV DNA levels in the PBL at any time.
HCMV Culture Results and HCMV DNA Detection
Of the 145 blood samples, HCMV culture was performed in 130 and was positive in six cases in six different individuals (Table 2 ).
Table 2: HCMV culture versus HCMV PCR detection in PBL
HCMV DNA Detection and HCMV Pneumonitis
HCMV DNA was detected at some point in the first 6 months in all LTR who exhibited histopathologically proven HCMV pneumonitis in that time period (Table 3 ). HCMV DNA detection in the PBL had a sensitivity of 92% for HCMV pneumonitis and a specificity of 76% (Table 3 ). Negative and positive likelihood ratios from this dichotomous data were 9.5 and 4, respectively. Unadjusted odds ratio for HCMV pneumonitis was 37.5. When the odds ratio was adjusted for ganciclovir status (withdrawing ganciclovir), age (older), and sex (female), it further increased to 107 (95% confidence interval, 14–821;P <0.005).
Table 3: HCMV PCR detection in PBL and HCMV pneumonitis
Absolute HCMV DNA Detectable Levels and HCMV Pneumonitis
The distribution of the patient samples with detectable HCMV DNA levels was normally distributed after a log. transformation. In those with detectable HCMV DNA in PBL (n=44), a generalized, multivariate linear regression model identified HCMV pneumonitis as the major determining factor (P =0.03), with HCMV DNA levels being 4.4 (95% confidence interval, 1.2–16.8) times higher in those with HCMV pneumonitis than in those without HCMV pneumonitis [adjusted geometric mean of 1151 copies/μg of total DNA with disease (range, 311–4188) and 260 copies/μg of total DNA without disease (range, 503–2592)]. Other variables such as age, sex, D/R HCMV seromatch, and ganciclovir status did not significantly influence the model.
Relative Changes in HCMV DNA Levels and HCMV Pneumonitis
Tenfold changes in HCMV DNA levels predicted for HCMV pneumonitis episodes are shown in Table 4 . A greater than 10-fold increase in HCMV DNA levels in any individual sample in the first 6 months after lung transplantation had a specificity of 93% and a sensitivity of 67% for HCMV pneumonitis (positive likelihood ratio, 11; negative likelihood ratio, 3), whereas HCMV DNA levels tended not to change or to fall when HCMV pneumonitis did not develop. A representative example of HCMV viral load in a patient with HCMV pneumonitis is illustrated in Figure 2 .
Table 4: Relative changes in HCMV DNA levels and HCMV pneumonitis
Figure 2: (A) Representative results of HCMV DNA viral load in a patient with HCMV pneumonitis. The asterisk indicates episodes of histopathologically proven HCMV pneumonitis, and arrows indicate the time points when the patient was receiving ganciclovir. Episodes of HCMV pneumonitis were associated with HCMV viral loads that increased by greater than 10-fold compared with baseline levels. In addition HCMV DNA levels tended to decrease with ganciclovir treatment. (B) Agarose gel fluorometrically detected competitive PCR results taken from the same patient (samples 1–7 screened with 100 copies of competitor and equal amounts of sample DNA). Lane 1, molecular weight markers; lane 2, PCR-negative control; lane 3, HCMV competitor alone; lane 4, HCMV-positive control; lanes 6–12, samples 1–7 of HCMV pneumonitis-positive patient.
Ganciclovir Status Versus HCMV Pneumonitis and HCMV DNA Levels
The effect of ganciclovir prophylaxis on HCMV DNA levels and on the development of HCMV pneumonitis is shown in Figure 1 . Although HCMV DNA levels were generally suppressed during routine thrice weekly ganciclovir prophylaxis, there were still four episodes of HCMV pneumonitis in three patients during this period (three episodes were related to a period of augmented immunosuppression and one to proven ganciclovir resistance). HCMV pneumonitis episodes occurred throughout the study period with some clustering during the period early after ganciclovir prophylaxis. Ganciclovir in treatment doses was very effective in controlling HCMV pneumonitis and HCMV DNA levels in the PBL (Fig. 1 ). Indeed, in the latter situation HCMV viral loads decreased by at least 10-fold in 7 of 12 cases (58%). This compares with HCMV DNA levels increasing by greater than 10-fold in 11 of 27 (41%) cases when ganciclovir was withdrawn.
DISCUSSION
In this study we have used quantitative competitive PCR to monitor HCMV DNA load and its dynamics in the PBL during the first 6 months after lung transplantation in a cohort of 26 patients. Our results indicate that the detection of HCMV DNA in the PBL, its absolute levels, and its change relative to prior baseline levels are all significant predictors of histopathologically proven HCMV pneumonitis in the lung allograft. In addition, we have shown that although ganciclovir is very effective in the treatment of HCMV pneumonitis and suppressing HCMV DNA load in the PBL, thrice weekly ganciclovir prophylaxis only partially controlled HCMV DNA levels and did not eliminate HCMV pneumonitis risk, particularly in the setting of augmented immunosuppression. This finding, which is in keeping with the primarily virostatic rather than virucidal effect of ganciclovir, was further supported by the tendency of HCMV DNA levels to increase when ganciclovir was withdrawn. Positive HCMV D/R serostatus compared with a negative HCMV D/R match crudely predicted for whether or not there were any HCMV DNA-positive samples in the PBL in the first 6 months after lung transplantation and HCMV pneumonitis risk. Hence, none of the negative HCMV D/R matches had any positive HCMV DNA samples detected or any episodes of HCMV pneumonitis, whereas 16 of the remaining 24 patients had at least one episode of HCMV DNA being detected in the circulating PBL, and nine of these patients had HCMV pneumonitis. However, there were no significant differences among HCMV D+/R−, D+/R+, and D−/R+ serogroups with respect to the number of episodes or absolute levels of HCMV DNA detection after lung transplantation, nor indeed were there any significant differences in the proportion of patients in each group that developed HCMV pneumonitis. Although the relatively low rate of HCMV pneumonitis and HCMV DNA detection in the primary HCMV-mismatched group may be partly explained by the extra ganciclovir prophylaxis that these patients received according to our protocols, other factors such as virus-host immunity dynamic interactions may also be important, inasmuch as the lack of difference in the rate of HCMV pneumonitis among the three HCMV at-risk serologic groups in LTR was well described in the era before ganciclovir prophylaxis (1)
There was a wide intrasubject and intersubject variation in the HCMV DNA PBL levels throughout the first 6 months after transplantation, which is in keeping with previous reports of PBL HCMV DNA levels in immunosuppressed subjects (25,29,31)
There were four HCMV pneumonitis episodes in which HCMV viral load in the PBL was relatively stable (<10-fold rise), and one episode in which it was not detected. The major reason for this discrepancy is likely to be that the PBL compartment only partially reflects what is occurring in the lung allograft. Other possibilities include the presence of an HCMV variant strain or even another herpesvirus (e.g., HHV6, which is a related β-herpesvirus with many similar characteristics to HCMV) causing pneumonitis with “typical” cytoplasmic and intranuclear inclusions but unable to be detected by our PCR assay (32,33)
Although we have shown that HCMV DNA load in the PBL is dynamically reflective of biologically significant influences (HCMV pneumonitis in the allograft and ganciclovir use), and that quantitation of HCMV DNA significantly improves on the predictive power of qualitative HCMV DNA detection for HCMV pneumonitis, our results also clearly indicate that other factors must also be important contributors to the development of HCMV disease in the allograft and the response to ganciclovir. In particular, local immunopathologic processes in the lung allograft may play an important role in the development of HCMV pneumonitis. In addition, thrice weekly ganciclovir prophylaxis is clearly variable in being able to completely suppress HCMV DNA loads in the PBL, and it is likely that ganciclovir pharmacodynamic effects are even more variable in the lung allograft.
The use of histopathologically proven HCMV pneumonitis as the diagnostic “gold standard” in this study may have increased the possibility of false-positive HCMV DNA detection in the PBL. There were seven cases in which 10-fold increases in HCMV DNA levels were not associated with biopsy-proven HCMV pneumonitis, and it is still possible that these may have represented true HCMV-related disease as transbronchial biopsy sampling may have missed HCMV pneumonitis in the lung or HCMV disease may have been present elsewhere in the body (e.g., gastrointestinal tract, reticuloendothelial system). Interestingly, in both cases in which HCMV DNA levels were unexplainably very high (Fig. 1 B), there were undiagnosed gastrointestinal and liver abnormalities that may well have been HCMV related.
There was a large discrepancy between HCMV DNA load in the PBL and detectable HCMV viremia in our immunosuppressed cohort, which is consistent with previous reports (34–38) (39–42) (1,42,43)
In conclusion, our study has provided further evidence to show that HCMV viral load in the PBL is an important and clinically useful correlate of HCMV pneumonitis in LTR. Although HCMV DNA quantitation in the blood cannot be the sole basis for therapeutic decisions in patients with suspected HCMV infection, quantitative virology for HCMV is likely to prove to be a powerful diagnostic tool that can be used to aid diagnosis and monitoring of HCMV pneumonitis and other HCMV disease-related syndromes after lung transplantation. A randomized control trial comparing improved HCMV antiviral strategies with standard strategies is now required to assess whether improvements in HCMV control after lung transplantation is associated with further reductions in HCMV pneumonitis and potentially other transplantation-related complications such as acute and chronic rejection and opportunistic infection rates that may be influenced by HCMV reactivation.
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