Introduction
Cytomegalovirus (CMV) pneumonia is a serious and increasingly prevalent complication in patients with hematologic malignancies. Immunocompromised individuals, particularly those with cell-mediated immunodeficiencies, are at significant risk for CMV infection, which can manifest as a spectrum of syndromes from asymptomatic viremia to severe end-organ diseases. CMV pneumonia carries high morbidity and mortality, with reported incidence rates in hematologic malignancy patients ranging from 2.3% to 16% and overall mortality reaching 57%. Certain conditions and treatments, such as chronic lymphocytic leukemia, lymphoma, rituximab use, systemic steroid therapy, alemtuzumab, and busulfan, further elevate the risk of CMV pneumonia in non-transplant patients. The development of potent immunosuppressive agents and the enhanced sensitivity of CMV polymerase chain reaction (PCR) testing in bronchoalveolar lavage (BAL) fluid or serum have likely contributed to the rising incidence of CMV pneumonia in non-transplant hematologic malignancy patients. However, advancements in diagnostic methods for CMV pneumonia have lagged behind these developments.
The definitive diagnosis of CMV pneumonia relies on a combination of clinical presentation, including pulmonary symptoms and signs, and the detection of CMV in BAL fluid or lung tissue. Traditional diagnostic methods encompass virus isolation, histopathologic examination, immunohistochemical analysis, and in situ hybridization. PCR, especially quantitative real-time PCR (qRT-PCR), has emerged as a highly sensitive tool for CMV detection since the early 1990s. qRT-PCR enables the quantification of viral load in blood and BAL fluid. Blood viral load is a valuable predictor of CMV disease and guides pre-emptive therapy post-bone marrow transplantation (BMT). However, despite its high sensitivity and negative predictive value, BAL fluid viral load determined by qRT-PCR exhibits lower specificity and positive predictive value in the context of Cmv Pneumonia Diagnosis.
A significant challenge in cmv pneumonia diagnosis is differentiating between CMV infection and CMV end-organ disease, particularly in the lung. Asymptomatic pulmonary CMV infections have been documented in both immunocompromised and immunocompetent individuals. The crucial question remains whether viral titers are genuinely higher in patients with CMV pneumonia compared to those with asymptomatic viral shedding in the lungs. Standardization of CMV DNA viral load measurement methods is lacking, and critically, no established viral cut-off values exist to reliably distinguish CMV infection from cmv pneumonia diagnosis. This lack of clear diagnostic thresholds complicates clinical decision-making.
Currently, defined viral cut-off values for diagnosing CMV disease in hematological malignancy patients are absent for sample types other than whole blood, where CMV DNAemia can be assessed by real-time PCR. In lung transplant recipients, CMV culture of BAL fluid has shown sensitivity but is less informative than histological assessment of transbronchial biopsies. A 2005 study on the clinical utility of CMV load in BAL fluid in lung transplant recipients suggested a cut-off of >500,000 copies/mL (mean 1,638,450 copies/mL) with 100% sensitivity and specificity compared to positive lung biopsies using a quantitative hybrid capture assay. However, research specifically focusing on CMV qRT-PCR cut-off values in BAL fluid for cmv pneumonia diagnosis in hematologic malignancy patients remains scarce.
This study was designed to address this critical gap by evaluating the role of CMV qRT-PCR in the cmv pneumonia diagnosis in patients with hematologic malignancies. The primary aim was to establish viral load cut-off values in bronchial washing fluid that can effectively differentiate CMV pneumonia from CMV infection in this vulnerable patient population. Bronchial washing fluid, a less invasive alternative to BAL, was utilized to assess its diagnostic potential.
Materials and Methods
Data Collection and Patients
This retrospective study analyzed data from 565 adult patients (over 15 years old) with hematologic malignancies who underwent bronchoscopy at Seoul St. Mary’s Hospital (Seoul, Republic of Korea) between March 2008 and June 2014. This center performs over 450 BMTs annually, providing a substantial cohort for CMV pneumonia investigation. Patients were included if they underwent bronchoscopy to identify the causative pathogen of pneumonia, defined by new pulmonary infiltrates on chest radiograph, clinical signs of lower respiratory tract infection, and/or fever. Microbiological findings from bronchial washing fluids, along with blood CMV qRT-PCR and lung biopsy results, were retrospectively reviewed. Clinical data collected included age, sex, hematologic diagnosis, prior hematologic treatments, and transplantation details.
The study protocol was approved by the Institutional Review Board of Seoul St. Mary’s Hospital (No. KC15RISI0153), with waived informed consent due to the retrospective nature of the analysis.
Fiber-optic Bronchoscopy Procedure
Bronchoscopy was performed following the identification of pulmonary infiltration on chest computed tomography (CT) scans, typically 2.54 ± 2.9 days after infiltrate detection. All procedures were conducted by experienced bronchoscopists using a flexible bronchoscope (BF-1T60t, Olympus, Tokyo, Japan). Bronchial washing was performed by wedging the bronchoscope into a segmental bronchus corresponding to the new infiltrate, followed by repeated instillation of 10 mL normal saline until at least 20 mL of aspirate was collected. This targeted approach aimed to sample the affected lung area effectively.
Microbiologic Assays
Bronchoscopic washing fluid specimens underwent comprehensive microbiologic analysis. Direct examination and culture were performed for bacteria, fungi, and viruses. Gram, Ziehl–Neelsen, Periodic-acid Schiff, and Gomori methenamine silver staining were used for bacterial, mycobacterial, and fungal detection, followed by culture. Respiratory virus detection utilized a multiplex qPCR assay (AdvanSure RV Real-time PCR kit, LG Life Sciences, Seoul, Korea) for a broad panel of common respiratory viruses. PCR assays were also employed for Pneumocystis jirovecii and mycobacteria detection.
For CMV detection, bronchial washing fluid was analyzed by qRT-PCR for CMV DNA, shell vial culture, and immunohistochemical (IHC) staining. Transbronchial lung biopsies, when available, were reviewed with hematoxylin-eosin and IHC staining for CMV by an experienced lung pathologist. DNA extraction for CMV qRT-PCR was performed from 200 μL of whole blood or bronchial washing fluid using the QIAamp DNA Blood Kit (Qiagen, Valencia, CA, USA). qRT-PCR was carried out using the ExiCycler™ 96 instrument (Bioneer Corporation, Daejeon, Korea) and AccuPower® CMV Quantitative PCR Kit (Bioneer). The limit of detection (LoD) was determined to be 380 copies/mL through probit analysis of control samples. Standardization was achieved using the WHO International Standard for human CMV (NIBSC code: 09/162), establishing a conversion factor of 7.3 International Units (IU) per copy of CMV DNA for the AccuPower® assay.
Definition of CMV Pneumonia
CMV pneumonia diagnosis was considered in patients presenting with pneumonia signs and symptoms and chest CT findings consistent with viral pneumonia. Established criteria were used to define CMV pneumonia, categorized as proven, probable, possible, or indeterminate based on a retrospective review of individual charts and chest CT findings by an infectious disease specialist (Lee DG). Patients with co-pathogens, particularly Aspergillus spp., and radiologic signs of invasive pulmonary aspergillosis (IPA) were excluded to ensure diagnostic specificity for CMV pneumonia.
Table 1. Definition of CMV pneumonia.
| Classification | Criteria |
|---|---|
| Proven | Positive CMV virus culture in bronchial washing fluid or the presence of intranuclear inclusion bodies or CMV detection by immunohistochemical staining in a lung biopsy specimen |
| Probable | Presence of intranuclear inclusion bodies or CMV detection by immunohistochemical staining in a cytologic specimen of bronchial washing fluid |
| Possible | Not classified as proven or probable |
| Indeterminate | Not classified as proven or probable and common respiratory virus other than CMV was isolated |
Statistical Analyses
Statistical analyses were performed using SPSS software (ver. 18.0.0 for Windows; SPSS, Inc., Chicago, IL, USA). Continuous variables are presented as means ± SEM, and categorical variables as proportions. CMV qRT-PCR results are expressed as means, medians, and ranges. The Mann-Whitney U-test was used to analyze differences in CMV PCR titers between patients with and without cmv pneumonia diagnosis. All tests were two-sided, with a P value < 0.05 considered statistically significant. Receiver operating characteristics (ROC) curves were generated to determine optimal CMV load cut-off values in bronchial washing fluid for cmv pneumonia diagnosis.
Results
Patient Characteristics
Bronchoscopy was performed on average 2.54 ± 2.9 days after new pulmonary infiltrates suggestive of pneumonia were identified. Out of the initial 565 patients, 464 (82.1%) were CMV negative or had CMV levels below the limit of detection (380 copies/mL). Based on consensus criteria for cmv pneumonia diagnosis, 24 patients (23.8%) were diagnosed with CMV pneumonia, 70 (69.3%) had findings inconsistent with CMV pneumonia (CMV infection), and 7 (6.9%) were excluded due to suspected proven IPA. Among the 24 CMV pneumonia cases, 14 (58.3%) were classified as proven, 5 (20.8%) as possible, 3 (12.5%) as probable, and 2 (8.3%) as indeterminate (Figure 1, Supplementary Figure 1). Proven cases were based on positive CMV culture in bronchial washing fluid (9 cases) and lung biopsy specimens (5 cases). The two indeterminate cases were attributed to co-infections with other respiratory viruses (coronavirus and rhinovirus).
Table 2 summarizes the baseline clinical characteristics and prior treatments of the 24 patients diagnosed with CMV pneumonia. The mean age was 48 ± 3.0 years, and 75% were male. The most frequent underlying hematologic malignancies were acute myeloid leukemia (25.0%), hemophagocytic lymphohistiocytosis (16.7%), non-Hodgkin’s lymphoma (12.5%), and aplastic anemia (12.5%). A significant proportion (66.7%) were using immunosuppressants at the time of cmv pneumonia diagnosis, and 46.2% had received systemic chemotherapy within the preceding 30 days, including alemtuzumab (2 patients) and steroid pulse therapy (3 patients). All CMV pneumonia patients received antiviral treatment, with 58.3% experiencing pneumonia aggravation and 41.7% showing improvement. During a mean follow-up of 122 days, mortality was high (62.5%), with a 28-day mortality rate of 45.8%. CMV pneumonia was the direct cause of death in 58.3% of fatalities. Co-infections were common, observed in 62.5% of CMV pneumonia patients, involving bacteria, viruses, fungi, and Mycobacterium species.
Table 2. Baseline clinical characteristics of the patients (n = 24).
| Characteristic | Value |
|---|---|
| Age (yr), Mean ± SEM | 48 ± 3.0 |
| Male, n (%) | 18 (75.0) |
| Underlying hematologic disease, n (%) | |
| Acute myeloid leukemia | 6 (25.0) |
| Hemophagocytic lymphohistiocytosis | 4 (16.7) |
| Non-Hodgkin’s lymphoma | 3 (12.5) |
| Aplastic anemia | 3 (12.5) |
| Acute lymphoblastic leukemia | 2 (8.3) |
| Myelodysplastic syndrome | 2 (8.3) |
| Multiple myeloma | 1 (4.2) |
| Chronic myelogenous leukemia | 1 (4.2) |
| Chronic lymphocytic leukemia | 1 (4.2) |
| Primary myelofibrosis | 1 (4.2) |
| Prior treatment, n (%) | |
| Bone Marrow transplantation | 16 (66.7) |
| Use of immunosuppressant agent | 16 (66.7) |
| Systemic chemotherapy in last 30 days | 12 (46.2) |
| Alemtuzumab chemotherapy | 2 (8.3) |
| Steroid pulse therapy in last 30 days | 3 (12.5) |
| History of preemptive CMV therapy, n (%) | 4 (16.7) |
| Prognosis of pneumonia, n (%) | |
| Improved | 10 (41.7) |
| Aggravated | 14 (58.3) |
| Follow up periods (days), Mean ± SEM | 122.0 ± 65.0 |
| Death, n (%) | |
| Survived | 9 (37.5) |
| Before 28 days of diagnosis | 11 (45.8) |
| After 28 days of diagnosis | 4 (16.7) |
| Death due to the pneumonia, n (%) | 14 (58.3) |
Table 3 details transplantation characteristics for the 16 CMV pneumonia patients who had undergone BMT. The mean time to cmv pneumonia diagnosis post-BMT was 167.7 ± 60.9 days, with most cases (68.8%) diagnosed beyond 100 days post-transplant.
Table 3. Transplantation characteristics of the patients diagnosed with CMV pneumonia (n = 16).
| Characteristic | Value |
|---|---|
| Donor type, n (%) | |
| Sibling | 5 (31.3) |
| Unrelated | 4 (25.0) |
| Familial mismatched transplantation | 4 (25.0) |
| Cord | 1 (6.3) |
| Autologous | 2 (12.5) |
| Source of graft, n (%) | |
| Bone marrow | 2 (12.5) |
| Peripheral blood stem cell | 13 (81.3) |
| Cord blood stem cell | 1 (6.3) |
| Risk of CMV disease, n (%) | |
| High risk† | 10 (62.5) |
| Low risk‡ | 6 (37.5) |
| Time since BMT (days), Mean ± SEM | 167.7 ± 60.9 |
| Before 100 days after BMT, n (%) | 11 (68.8) |
† High risk: Unrelated donors, mismatched related donors, and related donors with acute graft-versus-host disease (GVHD) of grades II–IV or severe chronic GVHD. ‡ Low risk: Related donors with acute GVHD of grade I or without acute/chronic GVHD.
qRT-PCR of Bronchial Washing Fluid
Figure 2 illustrates the distribution of CMV qRT-PCR viral load in bronchial washing fluid for patients with and without cmv pneumonia diagnosis. Patients diagnosed with CMV pneumonia exhibited significantly higher median log10 CMV qRT-PCR copies/mL (5.3, range 3.56–8.19) compared to those without CMV pneumonia (3.4, range 2.7-5.05) (P < 0.001).
Table 4 compares qRT-PCR results between CMV pneumonia and non-CMV pneumonia groups. Median CMV loads in bronchial washing fluid were significantly elevated in CMV pneumonia patients (1.8 × 105 copies/mL) compared to those with CMV infection (3.0 × 103 copies/mL) (P < 0.001). Similarly, blood CMV loads were also significantly higher in the CMV pneumonia group (3.4 × 104 copies/mL vs. 5.4 × 103 copies/mL, P = 0.006). Patients with cmv pneumonia diagnosis had approximately six-fold higher median blood CMV loads.
Table 4. Comparison of quantitative polymerase chain reaction results between patients diagnosed with CMV pneumonia or not.
| Measurement | CMV pneumonia (n = 24) | Not CMV pneumonia (n = 70) | P Value |
|---|---|---|---|
| Bronchial washing fluid (copies/ml) | < 0.001 | ||
| Mean | 7,378,508.6 | 10,899.2 | |
| Median | 187,224.5 | 3,055 | |
| Minimum – Maximum | 3,642 – 156,666,945 | 506 – 113,000 | |
| Blood (copies/ml) | 0.006 | ||
| Mean | 683,659.1 | 20,915.4 | |
| Median | 33,839.5 | 5,486.5 | |
| Minimum – Maximum | 882 – 5,570,000 | 689 – 280,870 |
Determination of CMV DNA Cut-off Values
ROC curve analysis was performed to determine optimal CMV load cut-off values in bronchial washing fluid for cmv pneumonia diagnosis. The analysis included 94 patients, categorized as post-BMT (n=59) and no-BMT (n=35). Among post-BMT patients, the optimal cut-off value was 18,900 copies/mL (137,970 IU/mL) (AUC 0.91 ± 0.041, P < 0.001) (Figure 3A). For no-BMT patients, the cut-off was considerably higher at 316,415 copies/mL (2,309,825 IU/mL) (AUC 0.93 ± 0.051, P < 0.001) (Figure 3B). Considering all patients, the optimal cut-off value for cmv pneumonia diagnosis was 28,774 copies/mL (210,054 IU/mL) (AUC 0.908 ± 0.033, 95% CI 0.843–0.973; P < 0.001) (Figure 3C), with a sensitivity of 75% and specificity of 88.6%.
Figure 3: ROC Curves for CMV Pneumonia Diagnosis based on Bronchial Washing Fluid Viral Load. These curves illustrate the diagnostic accuracy of different viral load cut-off values for (A) post-BMT patients, (B) no-BMT patients, and (C) all patients.
A. Post-bone marrow transplantation (BMT) patients; viral cut-off was 18,900 copies/mL B. no-BMT patients; viral cut-off was 316,415 copies/mL. C. All patients; viral cut-off was 28,774 copies/mL.
Discussion
This study provides compelling evidence that CMV loads in bronchial washing fluid, as determined by qRT-PCR, are indicative of lung involvement in CMV disease and are valuable for cmv pneumonia diagnosis. Patients definitively diagnosed with CMV pneumonia exhibited significantly higher qRT-PCR CMV titers in both bronchial washing fluid (61-fold higher) and whole blood (6-fold higher) compared to those with CMV infection without pneumonia. The ROC curve analysis identified a clinically relevant cut-off value of 28,774 copies/mL CMV DNA in bronchial washing fluid, which correlates with cmv pneumonia diagnosis with a sensitivity of 75% and specificity of 88.6%. Given the low prevalence of CMV pneumonia in the study center (0.85%-0.86%), this cut-off yields a positive predictive value of 5.3% and a high negative predictive value of 99.8%.
These findings offer valuable insights into cmv pneumonia diagnosis by providing an optimal cut-off point for CMV DNA in bronchial washing fluid in hematologic malignancy patients. This is particularly significant as lung biopsy, the gold standard for diagnosis, carries a considerable risk of bleeding in this patient population. The study suggests that the less invasive bronchial washing procedure, combined with qRT-PCR and the established cut-off values, can serve as a robust alternative to invasive lung biopsy for cmv pneumonia diagnosis. The identified cut-off points can also serve as supplementary information for early detection of CMV in pulmonary infiltrates.
Previous studies in the 1990s evaluated PCR for CMV detection in BAL fluid in BMT recipients. Cathomas et al. reported high sensitivity (100%) but lower specificity (93.5%) for PCR in BAL fluid, which was improved by combining it with CMV immunostaining. Hohenthal et al. also found CMV PCR positivity in BAL fluid from hematologic malignancy patients with pneumonia, but the clinical significance was often uncertain. In lung transplant recipients, Chemaly et al. reported a much higher viral load cut-off of 500,000 copies/mL in BAL fluid. The discrepancy in cut-off values may be attributed to differences in host immune status and study populations. Notably, this study is unique in its use of qRT-PCR to quantitatively differentiate CMV pneumonia from CMV infection and its inclusion of a larger patient cohort compared to prior research.
Bronchial washing, as performed in this study, offers a simplified and less invasive approach compared to BAL while still effectively identifying pathogens in pulmonary infiltrates. The incidence of CMV pneumonia observed in this study aligns with previous reports, supporting the efficacy of bronchial washing in this context.
This study acknowledges several limitations. Firstly, the inclusion of clinically diagnosed CMV pneumonia cases without pathologic confirmation may introduce some diagnostic uncertainty. However, rigorous clinical review by an experienced specialist aimed to exclude other etiologies, and all patients responded to anti-CMV therapy. Secondly, the cohort included both BMT and non-BMT patients. Recognizing the potential influence of BMT status on CMV reactivation risk, separate ROC curve analyses were performed. The significantly higher cut-off value in no-BMT patients (316,415 copies/mL) compared to post-BMT patients (18,900 copies/mL) highlights the clinical relevance of considering BMT status in cmv pneumonia diagnosis. While excluding no-BMT patients slightly lowered the overall cut-off, the relatively small number of post-BMT CMV pneumonia cases (n=16) may limit the precision of the ROC curve for this subgroup. Larger studies are needed to validate these cut-off values prospectively and to further refine them based on BMT status and CMV disease risk. Finally, the potential influence of pulmonary hemorrhage on BAL viral load was considered. While a small number of patients with pulmonary hemorrhage were identified, excluding these cases did not substantially alter the overall viral load cut-off value.
Conclusions
This study demonstrates that CMV loads in bronchial washing fluid and whole blood, quantified by qRT-PCR, serve as valuable indicators of lung involvement in CMV disease and are crucial for cmv pneumonia diagnosis. A cut-off value of 28,774 copies/mL CMV DNA in bronchial washing fluid shows a strong correlation with cmv pneumonia diagnosis in patients with hematologic malignancies. These findings provide a less invasive and quantitative approach to aid in the timely and accurate cmv pneumonia diagnosis, potentially improving patient management and outcomes.
Supplementary Figure
oncotarget-08-39736-s001.pdf (522KB, pdf)
Abbreviations
BMT: Bone marrow transplantation
CMV: Cytomegalovirus
CT: Computed tomography
GVHD: Graft-versus-host disease
IHC: Immunohistochemical
PCR: Polymerase chain reaction
qRT-PCR: Quantitative real-time PCR
Footnotes
Author contributions
HY Lee and CK Rhee drafted the manuscript. CK Rhee designed the study, HY Lee and JY Choi collected the patients’ data. HY Lee helped performing statistical analysis. DG Lee reviewed and coordinated the data and drafted the manuscript. All authors approved the final version of the manuscript.
CONFLICTS OF INTEREST
The authors declare that they have no conflict of interests.
REFERENCES
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Associated Data
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Supplementary Materials
oncotarget-08-39736-s001.pdf (522KB, pdf)
