Cytomegalovirus (CMV) DNA Quantitation in Bronchoalveolar Lavage Fluid From Hematopoietic Stem Cell Transplant Recipients With CMV Pneumonia

Abstract

Background: Quantitative cytomegalovirus (CMV) DNA-specific polymerase chain reaction (PCR) analysis is widely used as a surveillance method for hematopoietic stem cell transplant (HCT) recipients. However, no CMV DNA threshold exists in bronchoalveolar lavage (BAL) to differentiate pneumonia from pulmonary shedding.

Methods: We tested archived BAL fluid samples from 132 HCT recipients with CMV pneumonia and 139 controls (100 patients with non-CMV pneumonia, 18 with idiopathic pneumonia syndrome [IPS], and 21 who were asymptomatic) by quantitative CMV and β-globin DNA-specific PCR.

Results: Patients with CMV pneumonia had higher median viral loads (3.9 log10 IU/mL; interquartile range [IQR], 2.6-6.0 log10 IU/mL) than controls (0 log10 IU/mL [IQR, 0-1.6 log10 IU/mL] for patients with non-CMV pneumonia, 0 log10 IU/mL [IQR, 0-1.6 log10 IU/mL] for patients with IPS, and 1.63 log10 IU/mL [IQR, 0-2.5 log10 IU/mL] for patients who were asymptomatic; P < .001 for all comparisons to patients with CMV pneumonia). Receiver operating characteristic curve analyses and predictive models identified a cutoff CMV DNA level of 500 IU/mL to differentiate between CMV pneumonia and pulmonary shedding, using current CMV pneumonia prevalence figures. However, different levels may be appropriate in settings of very high or low CMV pneumonia prevalence. The presence of pulmonary copathogens, radiographic presentation, or pulmonary hemorrhage did not alter predictive values.

Conclusion: CMV DNA load in BAL can be used to differentiate CMV pneumonia from pulmonary shedding.

Keywords: cytomegalovirus; pneumonia.; viral load.

Figure 1.

Quantitative cytomegalovirus (CMV) load in bronchoalveolar lavage (BAL) fluid. A, Viral load in BAL fluid was significantly higher in CMV pneumonia cases than in any of the control subgroups. B, Viral load in BAL fluid from CMV pneumonia cases did not differ according to the absence (CMV only) or presence of a copathogen (BAL specimens were examined by Gram, fungal, and acid-fast bacilli staining; cytologic examination; cultures for bacteria, mycobacteria, fungi, and viruses; shell vial centrifugation culture for respiratory syncytial virus [RSV]; direct fluorescent antibody testing for Legionella, Pneumocystis jiroveci, RSV, influenza virus, parainfluenza virus types 1–3, and adenovirus; and Aspergillus galactomannan (GM) enzyme-linked immunosorbent assay [performed on all archived samples]). The viral loads of control groups (asymptomatic patients, patients with idiopathic pneumonia syndrome [IPS], and patients with non-CMV infectious pneumonia) are shown for comparison.

Figure 2.

Quantitative cytomegalovirus (CMV) load in bronchoalveolar lavage (BAL) fluid according to possible effect modifiers. A, Viral load in BAL fluid from CMV pneumonia cases did not differ with respect to the radiologic appearance of the lungs at the time of the BAL. Radiographic presentation was categorized by a pulmonologist blinded to the CMV results in the following categories: (1) focal nodule(s), (2) focal ground-glass opacities (GGOs), (3) diffuse nodules, (4) diffuse GGOs. Outcomes in subjects with non-CMV pneumonia with a high viral load were evaluated by chart review. If multiple locations were lavaged on the same day, each is represented separately. One CMV pneumonia case with multiple BALs on the same day was excluded because BAL polymerase chain reaction (PCR) results could not be assigned to a specific BAL location. A total of 147 BALs were performed on 131 patients. B, Quantitative CMV load in BAL fluid from CMV pneumonia cases did not differ according to the absence (CMV only) or presence of any specific copathogen subset. Detection of Aspergillus galactomannan in the BAL or blood counted as detection of a fungal copathogen. Co-occurrence of fungal copathogens with other copathogens is denoted by green markers. If bacteria were present in addition to a viral copathogen, the BAL finding is grouped with viral copathogens but is denoted with an open marker. A total of 68 subjects had CMV only detected, 33 had a fungal copathogen detected, 22 had a viral copathogen detected, and 9 had a bacterial copathogen detected. C, Viral loads in cases and controls were similar in the presence or absence of pulmonary hemorrhage in patients with CMV DNAemia within 7 days of BAL (data are for 92 patients with CMV pneumonia and 33 controls).

Figure 3.

Receiver operating characteristic (ROC) curves and predictive models. A, Patients with cytomegalovirus (CMV) pneumonia (n = 132) versus controls with idiopathic pneumonia syndrome (IPS) or non-CMV pneumonia (n = 118). The optimal cutoff was 99.7 IU/mL, with a sensitivity of 90.2% and specificity of 80.5%. B, Patients with CMV pneumonia (n = 132) versus asymptomatic controls (n = 21). The optimal cutoff was 203.3 IU/mL, with a sensitivity of 84.1% and a specificity of 76.2%.

Figure 4.

Predictive models. Positive predictive values with thresholds of 100, 500, 1000 IU/mL across a range of cytomegalovirus (CMV) pneumonia prevalences in patients who underwent a bronchoalveolar lavage for evaluation of pulmonary infiltrates. A, Data for all patients (132 patients with CMV pneumonia and 118 controls with non-CMV pneumonia). C, Patients without antiviral treatment (99 patients with CMV pneumonia and 78 controls with non-CMV pneumonia). Corresponding negative predictive values are shown in panels B and D.

Figure 5.

Clinical outcome in controls with cytomegalovirus (CMV)–specific polymerase chain reaction findings of >500 IU/mL in bronchoalveolar lavage (BAL) fluid (1 patient had idiopathic pneumonia syndrome [IPS], and 11 patients had non-CMV pneumonia). A, Viral loads of >500 IU/mL are shown by solid markers. The dotted line represents the threshold. B, The likelihood of subsequent development of CMV disease in subset. “Unlikely” denotes clinical presentation consistent with other causes, CMV-negative blood specimen; “possible” denotes CMV detected in blood specimen, treated with antiviral agents, other possible causes present; “probable” denotes CMV detected in blood specimen, no other causes of pneumonia in subsequent follow-up evaluations, clinical course consistent with viral pneumonia; and “proven” denotes subsequent CMV pneumonia diagnosed by standard criteria (eg, at autopsy).