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Molecular and Culture-Based Bronchoalveolar Lavage Fluid Testing for the Diagnosis of Cytomegalovirus Pneumonitis

Molecular and Culture-Based Bronchoalveolar Lavage Fluid Testing for the Diagnosis of... Open Forum Infectious Diseases MAJOR A RTICLE Molecular and Culture-Based Bronchoalveolar Lavage Fluid Testing for the Diagnosis of Cytomegalovirus Pneumonitis 1,a 3,a 1 1 4 2 1,4 Susanna K. Tan, Elizabeth B. Burgener, Jesse J. Waggoner, Kiran Gajurel, Sarah Gonzalez, Sharon F. Chen, and Benjamin A. Pinsky 1 2 3 Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Department of Pediatrics, Division of Infectious Diseases, Department of Pediatrics, Division of Pulmonary Medicine, and Department of Pathology, Stanford University School of Medicine, Stanford, California Background. Cytomegalovirus (CMV) is a major cause of morbidity and mortality in immunocompromised patients, with CMV pneumonitis among the most severe manifestations of infection. Although bronchoalveolar lavage (BAL) samples are fre- quently tested for CMV, the clinical utility of such testing remains uncertain. Methods. Retrospective analysis of adult patients undergoing BAL testing via CMV polymerase chain reaction (PCR), shell vial culture, and conventional viral culture between August 2008 and May 2011 was performed. Cytomegalovirus diagnostic methods were compared with a comprehensive definition of CMV pneumonitis that takes into account signs and symptoms, underlying host immunodeficiency, radiographic findings, and laboratory results. Results. Seven hundred five patients underwent 1077 bronchoscopy episodes with 1090 BAL specimens sent for CMV testing. Cytomegalovirus-positive patients were more likely to be hematopoietic cell transplant recipients (26% vs 8%, P < .0001) and less likely to have an underlying condition not typically associated with lung disease (3% vs 20%, P < .0001). Histopathology was per- formed in only 17.3% of CMV-positive bronchoscopy episodes. When CMV diagnostic methods were evaluated against the com- prehensive definition, the sensitivity and specificity of PCR, shell vial culture, and conventional culture were 91.3% and 94.6%, 54.4% and 97.4%, and 28.3% and 96.5%, respectively. Compared with culture, PCR provided significantly higher sensitivity and negative predictive value (P ≤ .001), without significantly lower positive predictive value. Cytomegalovirus quantitation did not improve test performance, resulting in a receiver operating characteristic curve with an area under the curve of 0.53. Conclusions. Cytomegalovirus PCR combined with a comprehensive clinical definition provides a pragmatic approach for the diagnosis of CMV pneumonitis. Keywords. bronchoalveolar lavage; cytomegalovirus; diagnostics; pneumonitis. Cytomegalovirus (CMV) is a common human viral pathogen to the widespread use of CMV prevention strategies, the mor- with 45%–100% of adults demonstrating serologic evidence of tality associated with CMV pneumonitis remains high at 30%– CMV exposure [1]. Both primary infection and reactivation of 50% [4]. In SOT, the incidence of CMV pneumonitis varies latent virus may result in significant CMV-related disease in based on the type of organ transplanted [5]. Lung transplant re- immunocompromised patients. In particular, CMV is an im- cipients are at particularly high risk and have an overall CMV portant cause of morbidity and mortality after hematopoietic mortality estimated to be 2%–12% [6]. cell transplantation (HCT) and solid organ transplantation The definitive diagnosis of CMV pneumonitis is made via the (SOT) [2, 3]. detection of characteristic intranuclear inclusions or positive Pneumonitis is among the most serious manifestations of immunohistochemical staining in biopsied lung tissue [3]. CMV infection. Although the incidence of CMV pneumonitis This is a challenging diagnosis, however, because tissue for in HCT recipients has decreased to approximately 1% to 3% due histopathologic evaluation is rarely obtained. Many of these pa- tients have risk factors that preclude biopsy, such as significant thrombocytopenia or coagulopathy, leading physicians to forgo this invasive diagnostic procedure [7, 8]. Received 5 August 2015; accepted 24 December 2015. S. K. T. and E. B. B. contributed equally to this work. Bronchoalveolar lavage (BAL) is frequently used as a less in- Correspondence: S. K. Tan, 300 Pasteur Drive, Lane Building, Room L134, Stanford, CA 94305 vasive means by which to access lung pathology and aid in the (susietan@stanford.edu). ® diagnosis of CMV pneumonitis. Compared with lung biopsy, Open Forum Infectious Diseases © The Author 2016. Published by Oxford University Press on behalf of the Infectious Diseases BAL is more easily performed and results in fewer complica- Society of America. This is an Open Access article distributed under the terms of the Creative tions [8]. The lavage fluid can be sent for a variety of tests to Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/ detect CMV, including culture-based and polymerase chain re- by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work action (PCR) methods. Several studies have investigated these is properly cited. For commercial re-use, please contact journals.permissions@oup.com. diagnostic modalities in a variety of patient populations. Early DOI: 10.1093/ofid/ofv212 Diagnosis of Cytomegalovirus Pneumonitis � OFID � 1 studies evaluated the performance of conventional viral culture vortexed for 30 s in the original container, then vortexed and rapid shell vial culture of BAL specimens from patients in again for 30 s after the addition of 7–10 sterile glass beads. which CMV pneumonitis was diagnosed based on histopathol- For CMV PCR, 100 µL of each specimen was extracted on the ogy [9–12]. Although shell vial and traditional viral BAL cul- MagNA Pure LC (Roche, Indianapolis, IN) using the MagNA tures demonstrated similar sensitivity and specificity, the Pure LC DNA isolation kit with the DNA I Blood Cell High number of histologically confirmed cases was low, and, overall, Performance protocol. Amplification and detection were per- the sensitivity and specificity of shell vial and traditional culture formed using the Cobas Amplicor CMV Monitor and was compared with histopathology varied widely between studies. setup manually using 50 µL of the 100 µL elution [26]. Subsequent work evaluated CMV DNA detection in the BAL For culture methods, specimens were mixed 1:1 with antimi- fluid of HCT, SOT, and acquired immune deficiency syndrome crobial mix containing gentamicin (150 µg/mL), vancomycin patients using conventional PCR strategies [13–15]. In addition, (1000 µg/mL), and amphotericin B (35 µg/mL). Of the pro- numerous studies investigated the performance of CMV PCR cessed specimen, 0.25 mL was inoculated onto each conven- methods on BAL fluid specimens from lung transplant recipi- tional tube culture monolayer including human foreskin (HF) ents [16–24]. Although definitive diagnosis of CMV pneumoni- fibroblasts (Quidel/Diagnostic Hybrids) and human embryonic tis consistently relied upon review of biopsied lung tissue, the lung MRC-5 fibroblasts (Viromed) containing 1.0–1.5 mL of definition of CMV lung disease in the absence of histological the manufacturer’s culture medium. Conventional viral cultures assessment differed substantially between studies. For example, were incubated for 21 days at 35°C. Cell monolayers with de- some studies provided specific diagnostic criteria [12], whereas monstrable cytopathic effect (CPE) were scraped, washed with others used general clinical features without specific criteria phosphate-buffered saline (PBS) (Sigma-Aldrich) spotted to [25]. As a consequence, CMV PCR, even with quantitative Cel-Line Supercured HTC slides (Thermo Fisher Scientific), thresholds, and culture-based diagnostics demonstrated dis- and fixed for 10 minutes in acetone. The cells were then stained cordant sensitivities and specificities. for the presence of CMV by indirect immunofluorescence using To address these limitations, we developed a comprehensive anti-CMV antibodies targeting the immediate early antigen definition of CMV pneumonitis that takes into account clinical (Millipore) according to the manufacturer’s specifications. features, underlying host immunodeficiency, radiographic find- For shell vial cultures, 2 human fibroblast vials (1 HF and 1 ings, and laboratory results. In this context, we evaluated the MRC-5 from the vendors described above) were each inoculated performance of CMV diagnostic methods in BAL specimens, with 0.25 mL processed BAL after aspiration of the culture me- including conventional viral culture and shell vial culture as dium. The vials were centrifuged at 3000 rpm for 30 minutes, well as qualitative and quantitative CMV PCR. the monolayers were refed, and the vials were incubated at 35°C. At 24 hours, the cell monolayer from 1 vial (HF) was METHODS washed with PBS, fixed with acetone, and stained as described above with the same antibody reagent used for conventional Ethics This study was reviewed and approved by the institutional viral culture. The other vial was processed (MRC-5) at 48 review board of Stanford University. hours. Results were reported only if the monolayers were >60% confluent, the negative control showed no specific fluo- Study Population rescence, and the positive control, cultured CMV AD169 refer- We retrospectively reviewed all BAL specimens sent for CMV ence strain (American Type Culture Collection), exhibited the PCR, shell vial culture, and conventional viral culture in patients expected apple green nuclear fluorescence. A positive result >18 years old who underwent bronchoscopy at our institution be- on a patient sample was reported if at least 1 vial had 2 or tween August 1, 2008 and May 31, 2011. Inclusion in the study more cells that demonstrated this characteristic staining. Shell required interpretable results for all 3 methods. Data were ana- vials that demonstrated intense nonspecific fluorescence or ex- lyzed for individual specimens, bronchoscopy episodes, and pa- cessive destruction of the monolayer by specimen toxicity or tients in the cohort. Patients were categorized as CMV positive contamination were reported as unsatisfactory. or CMV negative. Cytomegalovirus-positive patients were re- Histopathologic evaluation of formalin-fixed paraffin- quired to have at least 1 BAL specimen from 1 bronchoscopy ep- embedded tissues included the assessment of hematoxylin isode that was positive for CMV by PCR, shell vial culture, or and eosin (H&E)-stained slides for the degree and acuity of conventional viral culture. Cytomegalovirus-negative patients inflammation and the presence of viral-type inclusion bodies. had negative results for all 3 methods in BAL fluid for each bron- Cytomegalovirus immunohistochemical staining was per- choscopy episode, if more than 1 bronchoscopy was performed. formed at the discretion of the attending pathologist, using an anti-CMV antibody blend consisting of 2 mouse monoclonal Laboratory Methods for the Detection of Cytomegalovirus Bronchoalveolar lavage specimens were collected in sterile con- antibodies, CCH2 and DDG9 (Dako), that recognize immediate tainers and stored at 2–8°C before testing. Specimens were early and early antigens, respectively. Automated staining was 2 � OFID � Tan et al Table 1. Clinical Diagnostic Criteria for CMV Pneumonitis 1. At least 2 of the following (a, b, c): a. Signs/symptoms of pneumonia or evidence of organ dysfunction as manifested by 2 or more of the following: i. Fever ii. Cough iii. Dyspnea or tachypnea (RR > 20) iv. FEV ≤ 80% of baseline v. New or increasing oxygen requirement b. Host factors/predisposing conditions: SOT, HSCT, chemotherapy, rituximab, leukemia/lymphoma c. Radiographic changes consistent with CMV pneumonia i. CXR: reticulonodular or interstitial infiltrates ii. CT: ground glass opacities (GGOs), small nodules (<1 cm), absence of large nodules, air space consolidation AND 2. If a lung biopsy or autopsy was performed, this showed no evidence of a more likely explanation (acute rejection, GVHD, BCNU toxicity). AND 3. Requires that no other pathogen is isolated in culture or identified by DFA/staining that represents a more likely cause of the patient’s presentation and findings . Abbreviations: BCNU, bis-chloroethylnitrosourea; CMV, cytomegalovirus; CT, computed tomography; CXR, chest x-ray; DFA, direct fluorescent antibody; FEV , forced expiratory volume in 1 second; GVHD, graft-versus-host disease; HSCT, hematopoietic stem cell transplantation; HSV, herpes simplex virus; RR, respiratory rate; SOT, solid organ transplant. Although 20 breaths/minute is not a normal respiratory rate, this value was selected because it is frequently documented when the patient is breathing comfortably on room air. Coagulase-negative Staphylococcus, oropharyngeal flora, Candida spp, Mycobacterium gordonae, nontyped mycobacterial species, diphtheroids, HSV-1, HSV-2, Enterococcus,and Penicillium would not be considered pathogens. performed using the BOND-MAX instrument (Leica Biosys- 1, 2, and 3, herpes simplex virus (HSV)-1 and -2, and varicella- tems), at a 1:100 dilution, with citrate-induced antigen retrieval, zoster virus. A laboratory-developed real-time, reverse- and the Bond Polymer Refine Detection system. Staining was transcriptase PCR for influenza A was performed as described visualized using diaminobenzidine as the chromogen. previously [27], and it was available from November 2009 through the end of the study period. Other Viral Diagnostic Methods Respiratory viruses were evaluated by direct fluorescent anti- Criteria for the Diagnosis of Cytomegalovirus Pneumonitis body testing [27] at the discretion of the ordering clinician. In Based on review of the literature, we identified signs and symp- brief, 1 mL BAL fluid was transferred to a 15 mL conical tube, toms, predisposing host conditions, and radiographic findings diluted with 9 mL PBS, and centrifuged at 2500 rpm to generate consistent with CMV pneumonitis (Table 1)[4, 25, 28–30]. a cell pellet. After a second PBS wash, the well mixed sediment The diagnosis of CMV pneumonitis required a clinical syn- was spotted onto an 8-well Teflon masked slide. The cells were drome compatible with CMV pneumonitis, including the ab- then fixed with acetone and stained with a respiratory virus di- sence of a more likely cause for the patient’s presentation, and rect fluorescent antibody panel (Quidel/Diagnostic Hybrids) a positive result for CMV by at least 1 test on BAL fluid (PCR, that includes antibodies for the detection of influenza A, influ- shell vial, or conventional culture). enza B, respiratory syncytial virus (RSV), human metapneumo- Patients were considered to have a clinical syndrome compat- virus, adenovirus, and parainfluenza virus 1, 2, and 3. Samples ible with CMV pneumonitis if they met at least 2 of 3 criteria: were considered positive if 1 or more intact cells exhibited spe- signs and symptoms, predisposing host condition, or radio- cific fluorescence. A minimum sampling of 15 ciliated columnar graphic findings. Signs and symptoms were defined as having epithelial cells or alveolar macrophages per well was required 2 or more of the following: fever (temperature ≥38.5°C), for a negative result. All direct fluorescent antibody slides cough, dyspnea or tachypnea (respiratory rate >20/minute), were read by at least 2 clinical laboratory scientists, which was forced expiratory volume in 1 second (FEV ) ≤ 80% of baseline, standard protocol. and new or increasing oxygen requirement. Predisposing Bronchoalveolar lavage fluid for conventional viral culture underlying host conditions included HCT, SOT, leukemia or was processed as described above. In addition to the fibroblast lymphoma, exposure to chemotherapy, and rituximab use. Ra- cell lines used for CMV identification, 0.25 mL of the processed diographic findings for CMV pneumonitis were defined as the specimen was inoculated onto human lung carcinoma A549 presence of reticulonodular or interstitial infiltrates on chest cells and Rhesus Monkey Kidney cells (both obtained from Qui- x-ray or the presence of ground glass opacities, small nodules del/Diagnostic Hybrids). Cell monolayers were monitored for <1 cm, absence of large nodules >1 cm, and air space consolida- CPE, and the presence of virus was confirmed by direct or in- tion on computed tomography chest imaging. Microbiologic or direct immunofluorescence, depending on the target virus (re- histopathologic findings that were considered a more likely agents from Quidel/Diagnostic or Merck/Millipore/Light causefor thepatient’s presentation included acute rejection, Diagnostics; details available upon request). In addition to graft-versus-host disease, bis-chloroethylnitrosourea toxicity, CMV, these cell lines support the isolation of a number of virus- or identification of other pathogens in bronchoscopy speci- es that may be found in the lower respiratory tract, including mens, as described for viruses above and via routine microbio- influenza A, influenza B, RSV, adenovirus, parainfluenza virus logical cultures. Diagnosis of Cytomegalovirus Pneumonitis � OFID � 3 Statistical Analysis Table 2. Characteristics of Patients Who Underwent BAL With Specimens Sent for CMV Testing Demographics, underlying host conditions, bronchoscopy epi- sodes, and bronchoscopy indications in CMV-positive and CMV Positive CMV Negative CMV-negative patients were analyzed using univariate statistics Characteristic (n = 90) (n = 609) P Value as appropriate (Pearson χ or Fisher exact test for categorical Age, y, median (range) 52.9 (18.4–86.2) 55.7 (18.0–92.6) ns variables and Student’s t test for continuous variables). Differ- HCT (%) 24 (26) 47 (8) <.0001 ences in quantitative CMV PCR viral loads in BAL fluid in those Allogeneic-MRD 14 (58) 14 (30) .038 Allogeneic-MUD 7 (29) 21 (45) ns who did and did not meet histopathologic or clinical criteria for Allogeneic-Haploidentical 0 (0) 1 (2) ns CMV pneumonitis were evaluated by Mann–Whitney U test. Autologous 3 (13) 11 (22.4) ns Receiver operating characteristic (ROC) curves were generated SOT (%) 28 (31) 207 (33) ns using XLSTAT 2014 (Microsoft, Redmond, WA). Sensitivity, Heart 2 (7) 12 (6) ns specificity, positive predictive value (PPV), and negative predic- Lung 18 (65) 135 (65) ns tive value (NPV) were calculated with comparisons between Heart-lung 3 (10) 28 (14) ns Other SOT 5 (17) 32 (15) ns tests made with Fisher exact testing. Statistical analysis was per- Hematologic malignancy (%) 9 (10) 75 (12) ns formed using XLSTAT 2014 (Microsoft). Acute leukemia 2 (22) 45 (60) .031 Other hematologic 7 (78) 30 (40) .031 RESULTS malignancy Solid malignancy (%) 8 (9) 69 (11) ns Patient Characteristics Connective tissue disease (%) 6 (6) 17 (3) ns During the study period, 705 patients underwent 1077 broncho- Other immune deficiency (%) 2 (2) 5 (1) ns scopy episodes and had 1090 specimens sent for CMV PCR, Chronic lung disease (%) 12 (13) 52 (8) ns shell vial culture, and conventional viral culture. Sixteen bron- Liver disease (%) 0 (0) 17 (3) ns choscopy specimens (1.5%) from 6 patients were excluded due k Other (%) 3 (3) 122 (20) <.0001 to uninterpretable shell vial cultures (n = 12), failed PCR Abbreviations: BAL, bronchoalveolar lavage; CMV, cytomegalovirus; HCT, hematopoietic cell (n = 2), and contaminated conventional viral culture (n = 2). transplant; SOT, solid organ transplant; MRD, matched related donor; MUD, matched unrelated donor; PCR, polymerase chain reaction. Of the excluded specimens, 93.8% (15 of 16) were negative by Conventional culture, shell vial culture, and CMV PCR in BAL fluid. the other 2 CMV BAL testing methods, whereas 1 of the con- b Defined as patients with at least 1 specimen positive for CMV by PCR, shell vial culture, or conventional culture from BAL fluid. ventional culture specimens excluded for contamination was Other SOT includes kidney, liver, heart-kidney, heart-liver-kidney, heart-lung-kidney, kidney- positive for CMV by both PCR and shell vial culture. Of the pancreas, liver-kidney, and lung-kidney. Acute leukemia includes acute myelogenous leukemia, acute lymphocytic leukemia, acute 699 patients included in the analysis, the median age was 53.2 promyelocytic leukemia, and acute myelomonocytic leukemia. years old. Two hundred thirty-five (33%) patients were SOT re- Other hematologic malignancy includes lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, multiple myeloma, Waldenstrom’s macroglobulinemia, myelofibrosis, and cipients, 71 (10%) were HCT recipients, 85 (12%) had hemato- myelodysplasia. logic malignancy, and 77 (11%) had solid malignancy. Solid malignancy includes lung, breast, esophageal, gastric, colon, pancreas, prostate, cervical, bladder, endometrial, kidney, melanoma, glioblastoma, thymoma, cholangio- Ninety patients tested positive for CMV in BAL fluid by PCR, carcinoma, hepatocellular carcinoma, Wilm’s tumor, and sarcoma. shell vial culture, or conventional viral culture. One hundred Connective tissue disease includes lupus, scleroderma, sarcoidosis, rheumatoid arthritis, Wegner’s granulomatosis, Still’s disease, giant cell arteritis, Addison’s disease, and seven positive specimens were obtained from these patients dur- myasthenia gravis. ing 103 bronchoscopy episodes. Cytomegalovirus-positive pa- Other immune deficiency includes human immunodeficiency virus and common variable immunodeficiency. tients were more likely to be HCT recipients (26% vs 8%, Chronic lung disease includes interstitial lung disease, chronic obstructive pulmonary P < .0001), less likely to have an underlying condition not typical- disease, bronchiectasis, pulmonary hypertension, allergic bronchopulmonary aspergillosis, and bronchiolitis obliterans. ly associated with lung disease (3% vs 20%, P < .0001), and had Liver disease includes cirrhosis, chronic liver disease, and fulminant hepatic failure. more bronchoscopy episodes per patient (1.81 vs 1.49, P = .019) Other includes none or unrelated conditions. compared with CMV-negative patients. The most frequent bron- choscopy indication was for respiratory compromise, which was also significantly higher in CMV-positive patients (69% vs 59%, logical changes, 3 demonstrated malignancies, 2 showed mild P = .031). Table 2 outlines patient characteristics, and Table 3 acute rejection, and 4 showed bronchiolitis. Only 1 specimen summarizes bronchoscopy episodes, specimens, and indications. was tested for CMV by immunochemistry (IHC). Neither CMV antigen nor histopathological changes were identified. Histopathologic Evaluation Of the patients with positive CMV tests on BAL who died Biopsy was performed in 20 of the 104 (19.2%) bronchoscopy episodes that were positive for CMV by PCR, shell vial culture, during the study period, 6 had at least 1 BAL collected within or conventional viral culture. In 18 episodes from 16 patients, 30 days of death (all without concurrent biopsy) and underwent autopsy that included evaluation of the lungs. Four autopsies sufficient tissue was obtained for histopathology. One histopa- thology specimen demonstrated inclusions consistent with showed no histopathologic evidence of CMV infection, whereas CMV. Eight of the specimens did not reveal any histopatho- 2 autopsies revealed CMV pneumonitis demonstrated by 4 � OFID � Tan et al Table 3. Summary of BAL Episodes, Specimens, and Indications in Thus, of the 24 bronchoscopy episodes from 22 patients with Patients Who Underwent CMV Testing at least 1 positive CMV test and available histopathology, a pa- thology-confirmed diagnosis of CMV pneumonitis was obtained CMV Positive CMV Negative P for 3 episodes from 3 patients. All 3 episodes also met the study Variable (n = 90) (n = 609) Value criteria for CMV pneumonitis. Of the remaining 21 episodes Episodes per patient 1.81 (1–8) 1.47 (1–10) .019 (range) from 19 patients without histopathologic evidence for CMV Positive episodes (%) 103 of 163 (63) 0 of 898 (0) end-organ disease, 7 episodes from 7 patients met clinical and Positive specimens (%) 107 of 168 (64) 0 of 906 (0) laboratory diagnostic criteria for CMV pneumonitis. Indication (% of total episodes) Respiratory 112 (69) 532 (59) .031 c Cytomegalovirus Detection in Bronchoalveolar Lavage Fluid symptoms To better understand the role of CMV PCR, shell vial culture, Transplant-related 32 (19) 221 (25) ns Imaging abnormality 16 (10) 99 (11) ns and conventional viral culture of BAL fluid for the diagnosis of Other 3 (2) 46 (5) ns CMV pneumonitis, we calculated the characteristics for each Abbreviations: BAL, bronchoalveolar lavage; CMV, cytomegalovirus; ns, not significant; PCR, test compared with a composite reference that included the cri- polymerase chain reaction. teria outlined in Table 1 and a positive result for CMV by at Conventional culture, shell vial culture, and CMV PCR in BAL fluid. b least 1 test on BAL fluid (Table 4). The sensitivity of PCR was Defined as patients having at least 1 specimen positive for CMV on bronchoalveolar lavage fluid by PCR, shell vial culture, or conventional culture. significantly higher than both shell vial culture (91.3% vs 54.4%, Includes hemoptysis, diffuse alveolar hemorrhage, atelectasis, airway obstruction, P < .001) and conventional viral culture (91.3% vs 28.3%, decreased pulmonary function with or without imaging abnormalities. Surveillance for rejection with or without respiratory symptoms or imaging abnormalities. P < .001). The sensitivity of shell vial culture was also signifi- Imaging abnormalities alone. cantly higher compared with conventional culture (54.4% vs Undefined indications or unrelated conditions such as for pulmonary toilet, lympha- 28.3%, P = .012). In addition, the specificity of PCR was signifi- denopathy, and pretransplant evaluation. cantly lower than both shell vial culture (94.6% vs 97.4%, P < .001) and conventional viral culture (94.6% vs 96.5%, characteristic viral inclusions. One was CMV IHC positive, and P < .001), whereas no significant difference in specificity was although CMV IHC was not performed in the other, PCR test- observed between the culture methods (97.4% vs 96.5%, ing of the autopsy lung tissue detected CMV DNA. P = .085). No significant differences in PPV were found between Table 4. Test Characteristics of CMV Detection Methods in BAL Specimens CMV Pneumonitis Test (+) (−) Sensitivity (%) Specificity (%) PPV (%) NPV (%) PCR (+) 42 56 91.3 94.6 42.9 99.6 PCR (−) 4 972 Shell vial culture (+) 25 27 54.4 97.4 48.1 97.9 Shell vial culture (−) 21 1001 Conventional culture (+) 13 15 28.3 96.5 46.4 96.9 Conventional culture (−) 33 1013 PCR and shell vial (+) 21 23 45.7 97.8 47.4 97.6 PCR and/or shell vial (−) 25 1005 PCR and culture (+) 12 13 26.1 98.7 48.0 96.8 PCR and/or culture (−) 34 1015 Shell vial and culture (+) 10 13 21.7 98.7 43.5 96.6 Shell vial and/or culture (−) 36 1015 PCR, shell vial, and culture (+) 9 12 19.6 98.8 42.6 96.6 PCR, shell vial, and/or culture (−) 37 1016 PCR and/or shell vial (+) 46 60 100.0 94.2 43.4 100.0 PCR and shell vial (−) 0 968 PCR and/or culture (+) 43 58 93.4 94.4 42.6 99.7 PCR and culture (−) 3 970 Shell vial and/or culture (+) 28 29 60.8 97.2 49.1 98.2 Shell vial and culture (−) 18 999 PCR, shell vial, and/or culture (+) 46 61 100.0 94.1 42.3 100.0 PCR, shell vial, and culture (−) 0 967 Abbreviations: BAL, bronchoalveolar lavage; CMV, cytomegalovirus; NPV, negative predictive value; PCR, polymerase chain reaction; PPV, positive predictive value. Criteria for CMV pneumonitis as defined in Table 1 and Methods. Diagnosis of Cytomegalovirus Pneumonitis � OFID � 5 any of the methods. However, the NPV of PCR was significantly symptoms, predisposing host conditions, radiographic findings, higher compared with both shell vial (99.6% vs 97.9%, P = .001) laboratory results, and an assessment of the most likely cause in and routine culture (99.6% vs 96.9%, P < .001). The NPV of a differential diagnosis. The intention for creating this defini- shell vial and routine culture was not statistically different tion was to provide a framework for the diagnosis of CMV (P = .130). pneumonitis that is systematic and reflective of clinical practice. Requiring positive results for PCR and 1 or both of the cul- Utilizing this comprehensive definition as reference, compar- ture methods resulted in significantly lower sensitivity and sig- ison of BAL CMV diagnostic methods revealed that PCR pro- nificantly higher specificity compared with PCR testing alone vided significantly higher sensitivity and NPV compared with (P < .001). Although there were no significant differences in both shell vial and conventional cultures. These results suggest PPV, the NPV of PCR testing alone was significantly higher that CMV PCR of BAL may be most effectively used to rule out compared with combinations requiring positive results from CMV pneumonitis. The high sensitivity of PCR was achieved both PCR and culture (P < .001). Alternatively, if positive labo- with a moderate loss of specificity, which was significantly ratory testing was defined as a positive result in at least 1 of 2 lower than the culture methods, although all CMV tests and methods including PCR, or 1 of the 3 methods, no significant combinations of tests demonstrated specificities ≥94.1%. differences in sensitivity, specificity, PPV, or NPV were ob- Although clinicians frequently interpret culture as providing served compared with PCR alone. In sum, PCR testing alone an improved PPV, we found that shell vial culture and conven- offers optimal NPV and similar PPV when compared with tional culture individually and in combination with PCR dem- the various combinations of tests. onstrated consistently low PPV, ranging from 42.3% to 49.1%. When test characteristics in HCT recipients were compared Although these values, in part, reflect the relatively low preva- with test characteristics in patients with other underlying dis- lence of CMV pneumonitis in the overall study population, no eases, the sensitivity, specificity, PPV, and NPV of shell vial cul- significant differences in PPV were identified compared with tures and conventional cultures were not significantly different. PCR testing alone. Furthermore, requiring positive results Likewise, PCR demonstrated no significant differences in sensi- from either shell vial or conventional culture and PCR resulted tivity, specificity, and NPV. However, PCR had a significantly in high false-negative rates, suggesting that phasing out culture higher PPV in HCT recipients compared with patients with methodsinfavor of moleculardiagnostics, asisoccurring in other underlying diseases (76.9% vs 30.1%, P = .02). Further- many clinical microbiology and virology laboratories, is unlikely more, in HCT recipients, as described above, PCR testing to impact the diagnosis of CMV pneumonitis. It is notable that alone resulted in optimal test characteristics compared with a subset analysis of HCT recipients revealed a higher PPV of the various possible combinations of CMV tests. PCR in HCT recipients compared with the other groups, Cytomegalovirus quantitation in BAL fluid by PCR was also which was likely a reflection of the higher prevalence of patients evaluated to determine whether a threshold could be identified who met clinical criteria in HCT recipients in our cohort. This to aid in the diagnosis of CMV pneumonitis. A ROC curve was finding suggests improved utility of a positive PCR result for the generated, and the area under the curve (AUC) was 0.53. Fur- diagnosis of CMV pneumonitis in patients that had undergone thermore, quantitative CMV DNA levels in BAL fluid were not HCT. statistically different between those who did and did not meet We also investigated CMV quantitation in BAL fluid as a the study criteria for CMV pneumonitis (P = .635). We gener- means by which to further improve test performance. Results ated a ROC curve specifically for HCT recipients and found from previous studies in lung transplant recipients have been moderate improvement in the AUC of 0.70, with the optimal mixed, with 1 study identifying a threshold for CMV pneumo- viral load threshold of 791 copies/mL yielding a sensitivity of nitis of 500 000 copies/mL [22] and others showing no correla- 95.0%, specificity 50%, PPV 86.4%, and NPV 75.0%. In addi- tion of CMV levels with pneumonitis or bronchiolitis tion, no significant difference in DNA levels was identified be- obliterans, respectively [18, 24]. Similar to the latter studies, tween those who did and did not meet criteria for CMV we found CMV levels to be highly variable overall, as well as pneumonitis in HCT recipients (P = .142). in the subset of HCT recipients, providing limited improvement in discriminating between those cases that met the criteria for DISCUSSION CMV pneumonitis and those that did not. These observations In this report, we evaluated the performance of CMV diagnostic may be due, in part, to the variation in fluid volume and cells methods in the largest set of BAL specimens to date, collected collected from each lavage and the focal nature of CMV end- from patients with a wide variety of underlying diseases includ- organ disease. ing, but not limited to, HCT and SOT. We compared conven- Although the identification of characteristic inclusions by tional viral culture and shell vial culture, as well as qualitative H&E and CMV antigens by IHC remain the reference methods and quantitative CMV PCR, with a comprehensive definition for the diagnosis of CMV pneumonitis, lung biopsy tissue was of CMV pneumonitis that takes into account signs and infrequently obtained in this study during episodes in which 6 � OFID � Tan et al CMV was detected by any laboratory method in BAL speci- this diagnostic dilemma that combines the comprehensive clin- mens. Furthermore, although H&E staining has been shown ical definition with highly sensitive CMV nucleic acid amplifi- to be insensitive compared with IHC for the detection of cation testing. Future studies will be required to evaluate this CMV-infected cells in lung biopsy tissue [21, 31], CMV IHC approach and to identify additional biomarkers that will be was performed in only 5.6% (1 of 18) of total episodes and in able to improve the diagnosis of CMV pneumonitis. none of the subset of 7 episodes that met clinical and laboratory criteria for CMV pneumonitis. This limited and suboptimal use Acknowledgments of lung biopsy tissue brings into question the benefit of this in- We thank the hardworking, dedicated staff of the Stanford Clinical Virol- ogy Laboratory for commitment to quality and positive impact on patient vasive procedure, and it suggests that the comprehensive defini- care. tion developed in this manuscript may provide a more practical Financial support. This work was funded by National Institutes of approach to the diagnosis of CMV pneumonitis. Health (NIH) training grant 5T32AI007502-19 (to S. K. T.) and a generous gift from Beta Sigma Phi (to S. K. T.), an international women’s service or- Further limitations include the observational design of the ganization. Further salary support was provided by NIH award K08 study and the absence of data regarding the clinical progression AI110528-01 (to J. J. W.). of patients that tested positive for CMV in BAL fluid and met Potential conflicts of interest. All authors: No reported conflicts. All clinical criteria but did not receive anti-CMV therapy. These authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. patients comprise 34.8% (16 of 46) of the CMV-positive pa- tients that met our clinical definition of CMV pneumonitis; fu- References ture prospective studies will be required to assess disease 1. Bate SL, Dollard SC, Cannon MJ. Cytomegalovirus seroprevalence in the United progression in such patients. Although the clinical criteria cou- States: the national health and nutrition examination surveys, 1988-2004. 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Health Technol Assess 2007; 11: e334. ix–51. 8 � OFID � Tan et al http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Open Forum Infectious Diseases Oxford University Press

Molecular and Culture-Based Bronchoalveolar Lavage Fluid Testing for the Diagnosis of Cytomegalovirus Pneumonitis

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Abstract

Open Forum Infectious Diseases MAJOR A RTICLE Molecular and Culture-Based Bronchoalveolar Lavage Fluid Testing for the Diagnosis of Cytomegalovirus Pneumonitis 1,a 3,a 1 1 4 2 1,4 Susanna K. Tan, Elizabeth B. Burgener, Jesse J. Waggoner, Kiran Gajurel, Sarah Gonzalez, Sharon F. Chen, and Benjamin A. Pinsky 1 2 3 Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Department of Pediatrics, Division of Infectious Diseases, Department of Pediatrics, Division of Pulmonary Medicine, and Department of Pathology, Stanford University School of Medicine, Stanford, California Background. Cytomegalovirus (CMV) is a major cause of morbidity and mortality in immunocompromised patients, with CMV pneumonitis among the most severe manifestations of infection. Although bronchoalveolar lavage (BAL) samples are fre- quently tested for CMV, the clinical utility of such testing remains uncertain. Methods. Retrospective analysis of adult patients undergoing BAL testing via CMV polymerase chain reaction (PCR), shell vial culture, and conventional viral culture between August 2008 and May 2011 was performed. Cytomegalovirus diagnostic methods were compared with a comprehensive definition of CMV pneumonitis that takes into account signs and symptoms, underlying host immunodeficiency, radiographic findings, and laboratory results. Results. Seven hundred five patients underwent 1077 bronchoscopy episodes with 1090 BAL specimens sent for CMV testing. Cytomegalovirus-positive patients were more likely to be hematopoietic cell transplant recipients (26% vs 8%, P < .0001) and less likely to have an underlying condition not typically associated with lung disease (3% vs 20%, P < .0001). Histopathology was per- formed in only 17.3% of CMV-positive bronchoscopy episodes. When CMV diagnostic methods were evaluated against the com- prehensive definition, the sensitivity and specificity of PCR, shell vial culture, and conventional culture were 91.3% and 94.6%, 54.4% and 97.4%, and 28.3% and 96.5%, respectively. Compared with culture, PCR provided significantly higher sensitivity and negative predictive value (P ≤ .001), without significantly lower positive predictive value. Cytomegalovirus quantitation did not improve test performance, resulting in a receiver operating characteristic curve with an area under the curve of 0.53. Conclusions. Cytomegalovirus PCR combined with a comprehensive clinical definition provides a pragmatic approach for the diagnosis of CMV pneumonitis. Keywords. bronchoalveolar lavage; cytomegalovirus; diagnostics; pneumonitis. Cytomegalovirus (CMV) is a common human viral pathogen to the widespread use of CMV prevention strategies, the mor- with 45%–100% of adults demonstrating serologic evidence of tality associated with CMV pneumonitis remains high at 30%– CMV exposure [1]. Both primary infection and reactivation of 50% [4]. In SOT, the incidence of CMV pneumonitis varies latent virus may result in significant CMV-related disease in based on the type of organ transplanted [5]. Lung transplant re- immunocompromised patients. In particular, CMV is an im- cipients are at particularly high risk and have an overall CMV portant cause of morbidity and mortality after hematopoietic mortality estimated to be 2%–12% [6]. cell transplantation (HCT) and solid organ transplantation The definitive diagnosis of CMV pneumonitis is made via the (SOT) [2, 3]. detection of characteristic intranuclear inclusions or positive Pneumonitis is among the most serious manifestations of immunohistochemical staining in biopsied lung tissue [3]. CMV infection. Although the incidence of CMV pneumonitis This is a challenging diagnosis, however, because tissue for in HCT recipients has decreased to approximately 1% to 3% due histopathologic evaluation is rarely obtained. Many of these pa- tients have risk factors that preclude biopsy, such as significant thrombocytopenia or coagulopathy, leading physicians to forgo this invasive diagnostic procedure [7, 8]. Received 5 August 2015; accepted 24 December 2015. S. K. T. and E. B. B. contributed equally to this work. Bronchoalveolar lavage (BAL) is frequently used as a less in- Correspondence: S. K. Tan, 300 Pasteur Drive, Lane Building, Room L134, Stanford, CA 94305 vasive means by which to access lung pathology and aid in the (susietan@stanford.edu). ® diagnosis of CMV pneumonitis. Compared with lung biopsy, Open Forum Infectious Diseases © The Author 2016. Published by Oxford University Press on behalf of the Infectious Diseases BAL is more easily performed and results in fewer complica- Society of America. This is an Open Access article distributed under the terms of the Creative tions [8]. The lavage fluid can be sent for a variety of tests to Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/ detect CMV, including culture-based and polymerase chain re- by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work action (PCR) methods. Several studies have investigated these is properly cited. For commercial re-use, please contact journals.permissions@oup.com. diagnostic modalities in a variety of patient populations. Early DOI: 10.1093/ofid/ofv212 Diagnosis of Cytomegalovirus Pneumonitis � OFID � 1 studies evaluated the performance of conventional viral culture vortexed for 30 s in the original container, then vortexed and rapid shell vial culture of BAL specimens from patients in again for 30 s after the addition of 7–10 sterile glass beads. which CMV pneumonitis was diagnosed based on histopathol- For CMV PCR, 100 µL of each specimen was extracted on the ogy [9–12]. Although shell vial and traditional viral BAL cul- MagNA Pure LC (Roche, Indianapolis, IN) using the MagNA tures demonstrated similar sensitivity and specificity, the Pure LC DNA isolation kit with the DNA I Blood Cell High number of histologically confirmed cases was low, and, overall, Performance protocol. Amplification and detection were per- the sensitivity and specificity of shell vial and traditional culture formed using the Cobas Amplicor CMV Monitor and was compared with histopathology varied widely between studies. setup manually using 50 µL of the 100 µL elution [26]. Subsequent work evaluated CMV DNA detection in the BAL For culture methods, specimens were mixed 1:1 with antimi- fluid of HCT, SOT, and acquired immune deficiency syndrome crobial mix containing gentamicin (150 µg/mL), vancomycin patients using conventional PCR strategies [13–15]. In addition, (1000 µg/mL), and amphotericin B (35 µg/mL). Of the pro- numerous studies investigated the performance of CMV PCR cessed specimen, 0.25 mL was inoculated onto each conven- methods on BAL fluid specimens from lung transplant recipi- tional tube culture monolayer including human foreskin (HF) ents [16–24]. Although definitive diagnosis of CMV pneumoni- fibroblasts (Quidel/Diagnostic Hybrids) and human embryonic tis consistently relied upon review of biopsied lung tissue, the lung MRC-5 fibroblasts (Viromed) containing 1.0–1.5 mL of definition of CMV lung disease in the absence of histological the manufacturer’s culture medium. Conventional viral cultures assessment differed substantially between studies. For example, were incubated for 21 days at 35°C. Cell monolayers with de- some studies provided specific diagnostic criteria [12], whereas monstrable cytopathic effect (CPE) were scraped, washed with others used general clinical features without specific criteria phosphate-buffered saline (PBS) (Sigma-Aldrich) spotted to [25]. As a consequence, CMV PCR, even with quantitative Cel-Line Supercured HTC slides (Thermo Fisher Scientific), thresholds, and culture-based diagnostics demonstrated dis- and fixed for 10 minutes in acetone. The cells were then stained cordant sensitivities and specificities. for the presence of CMV by indirect immunofluorescence using To address these limitations, we developed a comprehensive anti-CMV antibodies targeting the immediate early antigen definition of CMV pneumonitis that takes into account clinical (Millipore) according to the manufacturer’s specifications. features, underlying host immunodeficiency, radiographic find- For shell vial cultures, 2 human fibroblast vials (1 HF and 1 ings, and laboratory results. In this context, we evaluated the MRC-5 from the vendors described above) were each inoculated performance of CMV diagnostic methods in BAL specimens, with 0.25 mL processed BAL after aspiration of the culture me- including conventional viral culture and shell vial culture as dium. The vials were centrifuged at 3000 rpm for 30 minutes, well as qualitative and quantitative CMV PCR. the monolayers were refed, and the vials were incubated at 35°C. At 24 hours, the cell monolayer from 1 vial (HF) was METHODS washed with PBS, fixed with acetone, and stained as described above with the same antibody reagent used for conventional Ethics This study was reviewed and approved by the institutional viral culture. The other vial was processed (MRC-5) at 48 review board of Stanford University. hours. Results were reported only if the monolayers were >60% confluent, the negative control showed no specific fluo- Study Population rescence, and the positive control, cultured CMV AD169 refer- We retrospectively reviewed all BAL specimens sent for CMV ence strain (American Type Culture Collection), exhibited the PCR, shell vial culture, and conventional viral culture in patients expected apple green nuclear fluorescence. A positive result >18 years old who underwent bronchoscopy at our institution be- on a patient sample was reported if at least 1 vial had 2 or tween August 1, 2008 and May 31, 2011. Inclusion in the study more cells that demonstrated this characteristic staining. Shell required interpretable results for all 3 methods. Data were ana- vials that demonstrated intense nonspecific fluorescence or ex- lyzed for individual specimens, bronchoscopy episodes, and pa- cessive destruction of the monolayer by specimen toxicity or tients in the cohort. Patients were categorized as CMV positive contamination were reported as unsatisfactory. or CMV negative. Cytomegalovirus-positive patients were re- Histopathologic evaluation of formalin-fixed paraffin- quired to have at least 1 BAL specimen from 1 bronchoscopy ep- embedded tissues included the assessment of hematoxylin isode that was positive for CMV by PCR, shell vial culture, or and eosin (H&E)-stained slides for the degree and acuity of conventional viral culture. Cytomegalovirus-negative patients inflammation and the presence of viral-type inclusion bodies. had negative results for all 3 methods in BAL fluid for each bron- Cytomegalovirus immunohistochemical staining was per- choscopy episode, if more than 1 bronchoscopy was performed. formed at the discretion of the attending pathologist, using an anti-CMV antibody blend consisting of 2 mouse monoclonal Laboratory Methods for the Detection of Cytomegalovirus Bronchoalveolar lavage specimens were collected in sterile con- antibodies, CCH2 and DDG9 (Dako), that recognize immediate tainers and stored at 2–8°C before testing. Specimens were early and early antigens, respectively. Automated staining was 2 � OFID � Tan et al Table 1. Clinical Diagnostic Criteria for CMV Pneumonitis 1. At least 2 of the following (a, b, c): a. Signs/symptoms of pneumonia or evidence of organ dysfunction as manifested by 2 or more of the following: i. Fever ii. Cough iii. Dyspnea or tachypnea (RR > 20) iv. FEV ≤ 80% of baseline v. New or increasing oxygen requirement b. Host factors/predisposing conditions: SOT, HSCT, chemotherapy, rituximab, leukemia/lymphoma c. Radiographic changes consistent with CMV pneumonia i. CXR: reticulonodular or interstitial infiltrates ii. CT: ground glass opacities (GGOs), small nodules (<1 cm), absence of large nodules, air space consolidation AND 2. If a lung biopsy or autopsy was performed, this showed no evidence of a more likely explanation (acute rejection, GVHD, BCNU toxicity). AND 3. Requires that no other pathogen is isolated in culture or identified by DFA/staining that represents a more likely cause of the patient’s presentation and findings . Abbreviations: BCNU, bis-chloroethylnitrosourea; CMV, cytomegalovirus; CT, computed tomography; CXR, chest x-ray; DFA, direct fluorescent antibody; FEV , forced expiratory volume in 1 second; GVHD, graft-versus-host disease; HSCT, hematopoietic stem cell transplantation; HSV, herpes simplex virus; RR, respiratory rate; SOT, solid organ transplant. Although 20 breaths/minute is not a normal respiratory rate, this value was selected because it is frequently documented when the patient is breathing comfortably on room air. Coagulase-negative Staphylococcus, oropharyngeal flora, Candida spp, Mycobacterium gordonae, nontyped mycobacterial species, diphtheroids, HSV-1, HSV-2, Enterococcus,and Penicillium would not be considered pathogens. performed using the BOND-MAX instrument (Leica Biosys- 1, 2, and 3, herpes simplex virus (HSV)-1 and -2, and varicella- tems), at a 1:100 dilution, with citrate-induced antigen retrieval, zoster virus. A laboratory-developed real-time, reverse- and the Bond Polymer Refine Detection system. Staining was transcriptase PCR for influenza A was performed as described visualized using diaminobenzidine as the chromogen. previously [27], and it was available from November 2009 through the end of the study period. Other Viral Diagnostic Methods Respiratory viruses were evaluated by direct fluorescent anti- Criteria for the Diagnosis of Cytomegalovirus Pneumonitis body testing [27] at the discretion of the ordering clinician. In Based on review of the literature, we identified signs and symp- brief, 1 mL BAL fluid was transferred to a 15 mL conical tube, toms, predisposing host conditions, and radiographic findings diluted with 9 mL PBS, and centrifuged at 2500 rpm to generate consistent with CMV pneumonitis (Table 1)[4, 25, 28–30]. a cell pellet. After a second PBS wash, the well mixed sediment The diagnosis of CMV pneumonitis required a clinical syn- was spotted onto an 8-well Teflon masked slide. The cells were drome compatible with CMV pneumonitis, including the ab- then fixed with acetone and stained with a respiratory virus di- sence of a more likely cause for the patient’s presentation, and rect fluorescent antibody panel (Quidel/Diagnostic Hybrids) a positive result for CMV by at least 1 test on BAL fluid (PCR, that includes antibodies for the detection of influenza A, influ- shell vial, or conventional culture). enza B, respiratory syncytial virus (RSV), human metapneumo- Patients were considered to have a clinical syndrome compat- virus, adenovirus, and parainfluenza virus 1, 2, and 3. Samples ible with CMV pneumonitis if they met at least 2 of 3 criteria: were considered positive if 1 or more intact cells exhibited spe- signs and symptoms, predisposing host condition, or radio- cific fluorescence. A minimum sampling of 15 ciliated columnar graphic findings. Signs and symptoms were defined as having epithelial cells or alveolar macrophages per well was required 2 or more of the following: fever (temperature ≥38.5°C), for a negative result. All direct fluorescent antibody slides cough, dyspnea or tachypnea (respiratory rate >20/minute), were read by at least 2 clinical laboratory scientists, which was forced expiratory volume in 1 second (FEV ) ≤ 80% of baseline, standard protocol. and new or increasing oxygen requirement. Predisposing Bronchoalveolar lavage fluid for conventional viral culture underlying host conditions included HCT, SOT, leukemia or was processed as described above. In addition to the fibroblast lymphoma, exposure to chemotherapy, and rituximab use. Ra- cell lines used for CMV identification, 0.25 mL of the processed diographic findings for CMV pneumonitis were defined as the specimen was inoculated onto human lung carcinoma A549 presence of reticulonodular or interstitial infiltrates on chest cells and Rhesus Monkey Kidney cells (both obtained from Qui- x-ray or the presence of ground glass opacities, small nodules del/Diagnostic Hybrids). Cell monolayers were monitored for <1 cm, absence of large nodules >1 cm, and air space consolida- CPE, and the presence of virus was confirmed by direct or in- tion on computed tomography chest imaging. Microbiologic or direct immunofluorescence, depending on the target virus (re- histopathologic findings that were considered a more likely agents from Quidel/Diagnostic or Merck/Millipore/Light causefor thepatient’s presentation included acute rejection, Diagnostics; details available upon request). In addition to graft-versus-host disease, bis-chloroethylnitrosourea toxicity, CMV, these cell lines support the isolation of a number of virus- or identification of other pathogens in bronchoscopy speci- es that may be found in the lower respiratory tract, including mens, as described for viruses above and via routine microbio- influenza A, influenza B, RSV, adenovirus, parainfluenza virus logical cultures. Diagnosis of Cytomegalovirus Pneumonitis � OFID � 3 Statistical Analysis Table 2. Characteristics of Patients Who Underwent BAL With Specimens Sent for CMV Testing Demographics, underlying host conditions, bronchoscopy epi- sodes, and bronchoscopy indications in CMV-positive and CMV Positive CMV Negative CMV-negative patients were analyzed using univariate statistics Characteristic (n = 90) (n = 609) P Value as appropriate (Pearson χ or Fisher exact test for categorical Age, y, median (range) 52.9 (18.4–86.2) 55.7 (18.0–92.6) ns variables and Student’s t test for continuous variables). Differ- HCT (%) 24 (26) 47 (8) <.0001 ences in quantitative CMV PCR viral loads in BAL fluid in those Allogeneic-MRD 14 (58) 14 (30) .038 Allogeneic-MUD 7 (29) 21 (45) ns who did and did not meet histopathologic or clinical criteria for Allogeneic-Haploidentical 0 (0) 1 (2) ns CMV pneumonitis were evaluated by Mann–Whitney U test. Autologous 3 (13) 11 (22.4) ns Receiver operating characteristic (ROC) curves were generated SOT (%) 28 (31) 207 (33) ns using XLSTAT 2014 (Microsoft, Redmond, WA). Sensitivity, Heart 2 (7) 12 (6) ns specificity, positive predictive value (PPV), and negative predic- Lung 18 (65) 135 (65) ns tive value (NPV) were calculated with comparisons between Heart-lung 3 (10) 28 (14) ns Other SOT 5 (17) 32 (15) ns tests made with Fisher exact testing. Statistical analysis was per- Hematologic malignancy (%) 9 (10) 75 (12) ns formed using XLSTAT 2014 (Microsoft). Acute leukemia 2 (22) 45 (60) .031 Other hematologic 7 (78) 30 (40) .031 RESULTS malignancy Solid malignancy (%) 8 (9) 69 (11) ns Patient Characteristics Connective tissue disease (%) 6 (6) 17 (3) ns During the study period, 705 patients underwent 1077 broncho- Other immune deficiency (%) 2 (2) 5 (1) ns scopy episodes and had 1090 specimens sent for CMV PCR, Chronic lung disease (%) 12 (13) 52 (8) ns shell vial culture, and conventional viral culture. Sixteen bron- Liver disease (%) 0 (0) 17 (3) ns choscopy specimens (1.5%) from 6 patients were excluded due k Other (%) 3 (3) 122 (20) <.0001 to uninterpretable shell vial cultures (n = 12), failed PCR Abbreviations: BAL, bronchoalveolar lavage; CMV, cytomegalovirus; HCT, hematopoietic cell (n = 2), and contaminated conventional viral culture (n = 2). transplant; SOT, solid organ transplant; MRD, matched related donor; MUD, matched unrelated donor; PCR, polymerase chain reaction. Of the excluded specimens, 93.8% (15 of 16) were negative by Conventional culture, shell vial culture, and CMV PCR in BAL fluid. the other 2 CMV BAL testing methods, whereas 1 of the con- b Defined as patients with at least 1 specimen positive for CMV by PCR, shell vial culture, or conventional culture from BAL fluid. ventional culture specimens excluded for contamination was Other SOT includes kidney, liver, heart-kidney, heart-liver-kidney, heart-lung-kidney, kidney- positive for CMV by both PCR and shell vial culture. Of the pancreas, liver-kidney, and lung-kidney. Acute leukemia includes acute myelogenous leukemia, acute lymphocytic leukemia, acute 699 patients included in the analysis, the median age was 53.2 promyelocytic leukemia, and acute myelomonocytic leukemia. years old. Two hundred thirty-five (33%) patients were SOT re- Other hematologic malignancy includes lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, multiple myeloma, Waldenstrom’s macroglobulinemia, myelofibrosis, and cipients, 71 (10%) were HCT recipients, 85 (12%) had hemato- myelodysplasia. logic malignancy, and 77 (11%) had solid malignancy. Solid malignancy includes lung, breast, esophageal, gastric, colon, pancreas, prostate, cervical, bladder, endometrial, kidney, melanoma, glioblastoma, thymoma, cholangio- Ninety patients tested positive for CMV in BAL fluid by PCR, carcinoma, hepatocellular carcinoma, Wilm’s tumor, and sarcoma. shell vial culture, or conventional viral culture. One hundred Connective tissue disease includes lupus, scleroderma, sarcoidosis, rheumatoid arthritis, Wegner’s granulomatosis, Still’s disease, giant cell arteritis, Addison’s disease, and seven positive specimens were obtained from these patients dur- myasthenia gravis. ing 103 bronchoscopy episodes. Cytomegalovirus-positive pa- Other immune deficiency includes human immunodeficiency virus and common variable immunodeficiency. tients were more likely to be HCT recipients (26% vs 8%, Chronic lung disease includes interstitial lung disease, chronic obstructive pulmonary P < .0001), less likely to have an underlying condition not typical- disease, bronchiectasis, pulmonary hypertension, allergic bronchopulmonary aspergillosis, and bronchiolitis obliterans. ly associated with lung disease (3% vs 20%, P < .0001), and had Liver disease includes cirrhosis, chronic liver disease, and fulminant hepatic failure. more bronchoscopy episodes per patient (1.81 vs 1.49, P = .019) Other includes none or unrelated conditions. compared with CMV-negative patients. The most frequent bron- choscopy indication was for respiratory compromise, which was also significantly higher in CMV-positive patients (69% vs 59%, logical changes, 3 demonstrated malignancies, 2 showed mild P = .031). Table 2 outlines patient characteristics, and Table 3 acute rejection, and 4 showed bronchiolitis. Only 1 specimen summarizes bronchoscopy episodes, specimens, and indications. was tested for CMV by immunochemistry (IHC). Neither CMV antigen nor histopathological changes were identified. Histopathologic Evaluation Of the patients with positive CMV tests on BAL who died Biopsy was performed in 20 of the 104 (19.2%) bronchoscopy episodes that were positive for CMV by PCR, shell vial culture, during the study period, 6 had at least 1 BAL collected within or conventional viral culture. In 18 episodes from 16 patients, 30 days of death (all without concurrent biopsy) and underwent autopsy that included evaluation of the lungs. Four autopsies sufficient tissue was obtained for histopathology. One histopa- thology specimen demonstrated inclusions consistent with showed no histopathologic evidence of CMV infection, whereas CMV. Eight of the specimens did not reveal any histopatho- 2 autopsies revealed CMV pneumonitis demonstrated by 4 � OFID � Tan et al Table 3. Summary of BAL Episodes, Specimens, and Indications in Thus, of the 24 bronchoscopy episodes from 22 patients with Patients Who Underwent CMV Testing at least 1 positive CMV test and available histopathology, a pa- thology-confirmed diagnosis of CMV pneumonitis was obtained CMV Positive CMV Negative P for 3 episodes from 3 patients. All 3 episodes also met the study Variable (n = 90) (n = 609) Value criteria for CMV pneumonitis. Of the remaining 21 episodes Episodes per patient 1.81 (1–8) 1.47 (1–10) .019 (range) from 19 patients without histopathologic evidence for CMV Positive episodes (%) 103 of 163 (63) 0 of 898 (0) end-organ disease, 7 episodes from 7 patients met clinical and Positive specimens (%) 107 of 168 (64) 0 of 906 (0) laboratory diagnostic criteria for CMV pneumonitis. Indication (% of total episodes) Respiratory 112 (69) 532 (59) .031 c Cytomegalovirus Detection in Bronchoalveolar Lavage Fluid symptoms To better understand the role of CMV PCR, shell vial culture, Transplant-related 32 (19) 221 (25) ns Imaging abnormality 16 (10) 99 (11) ns and conventional viral culture of BAL fluid for the diagnosis of Other 3 (2) 46 (5) ns CMV pneumonitis, we calculated the characteristics for each Abbreviations: BAL, bronchoalveolar lavage; CMV, cytomegalovirus; ns, not significant; PCR, test compared with a composite reference that included the cri- polymerase chain reaction. teria outlined in Table 1 and a positive result for CMV by at Conventional culture, shell vial culture, and CMV PCR in BAL fluid. b least 1 test on BAL fluid (Table 4). The sensitivity of PCR was Defined as patients having at least 1 specimen positive for CMV on bronchoalveolar lavage fluid by PCR, shell vial culture, or conventional culture. significantly higher than both shell vial culture (91.3% vs 54.4%, Includes hemoptysis, diffuse alveolar hemorrhage, atelectasis, airway obstruction, P < .001) and conventional viral culture (91.3% vs 28.3%, decreased pulmonary function with or without imaging abnormalities. Surveillance for rejection with or without respiratory symptoms or imaging abnormalities. P < .001). The sensitivity of shell vial culture was also signifi- Imaging abnormalities alone. cantly higher compared with conventional culture (54.4% vs Undefined indications or unrelated conditions such as for pulmonary toilet, lympha- 28.3%, P = .012). In addition, the specificity of PCR was signifi- denopathy, and pretransplant evaluation. cantly lower than both shell vial culture (94.6% vs 97.4%, P < .001) and conventional viral culture (94.6% vs 96.5%, characteristic viral inclusions. One was CMV IHC positive, and P < .001), whereas no significant difference in specificity was although CMV IHC was not performed in the other, PCR test- observed between the culture methods (97.4% vs 96.5%, ing of the autopsy lung tissue detected CMV DNA. P = .085). No significant differences in PPV were found between Table 4. Test Characteristics of CMV Detection Methods in BAL Specimens CMV Pneumonitis Test (+) (−) Sensitivity (%) Specificity (%) PPV (%) NPV (%) PCR (+) 42 56 91.3 94.6 42.9 99.6 PCR (−) 4 972 Shell vial culture (+) 25 27 54.4 97.4 48.1 97.9 Shell vial culture (−) 21 1001 Conventional culture (+) 13 15 28.3 96.5 46.4 96.9 Conventional culture (−) 33 1013 PCR and shell vial (+) 21 23 45.7 97.8 47.4 97.6 PCR and/or shell vial (−) 25 1005 PCR and culture (+) 12 13 26.1 98.7 48.0 96.8 PCR and/or culture (−) 34 1015 Shell vial and culture (+) 10 13 21.7 98.7 43.5 96.6 Shell vial and/or culture (−) 36 1015 PCR, shell vial, and culture (+) 9 12 19.6 98.8 42.6 96.6 PCR, shell vial, and/or culture (−) 37 1016 PCR and/or shell vial (+) 46 60 100.0 94.2 43.4 100.0 PCR and shell vial (−) 0 968 PCR and/or culture (+) 43 58 93.4 94.4 42.6 99.7 PCR and culture (−) 3 970 Shell vial and/or culture (+) 28 29 60.8 97.2 49.1 98.2 Shell vial and culture (−) 18 999 PCR, shell vial, and/or culture (+) 46 61 100.0 94.1 42.3 100.0 PCR, shell vial, and culture (−) 0 967 Abbreviations: BAL, bronchoalveolar lavage; CMV, cytomegalovirus; NPV, negative predictive value; PCR, polymerase chain reaction; PPV, positive predictive value. Criteria for CMV pneumonitis as defined in Table 1 and Methods. Diagnosis of Cytomegalovirus Pneumonitis � OFID � 5 any of the methods. However, the NPV of PCR was significantly symptoms, predisposing host conditions, radiographic findings, higher compared with both shell vial (99.6% vs 97.9%, P = .001) laboratory results, and an assessment of the most likely cause in and routine culture (99.6% vs 96.9%, P < .001). The NPV of a differential diagnosis. The intention for creating this defini- shell vial and routine culture was not statistically different tion was to provide a framework for the diagnosis of CMV (P = .130). pneumonitis that is systematic and reflective of clinical practice. Requiring positive results for PCR and 1 or both of the cul- Utilizing this comprehensive definition as reference, compar- ture methods resulted in significantly lower sensitivity and sig- ison of BAL CMV diagnostic methods revealed that PCR pro- nificantly higher specificity compared with PCR testing alone vided significantly higher sensitivity and NPV compared with (P < .001). Although there were no significant differences in both shell vial and conventional cultures. These results suggest PPV, the NPV of PCR testing alone was significantly higher that CMV PCR of BAL may be most effectively used to rule out compared with combinations requiring positive results from CMV pneumonitis. The high sensitivity of PCR was achieved both PCR and culture (P < .001). Alternatively, if positive labo- with a moderate loss of specificity, which was significantly ratory testing was defined as a positive result in at least 1 of 2 lower than the culture methods, although all CMV tests and methods including PCR, or 1 of the 3 methods, no significant combinations of tests demonstrated specificities ≥94.1%. differences in sensitivity, specificity, PPV, or NPV were ob- Although clinicians frequently interpret culture as providing served compared with PCR alone. In sum, PCR testing alone an improved PPV, we found that shell vial culture and conven- offers optimal NPV and similar PPV when compared with tional culture individually and in combination with PCR dem- the various combinations of tests. onstrated consistently low PPV, ranging from 42.3% to 49.1%. When test characteristics in HCT recipients were compared Although these values, in part, reflect the relatively low preva- with test characteristics in patients with other underlying dis- lence of CMV pneumonitis in the overall study population, no eases, the sensitivity, specificity, PPV, and NPV of shell vial cul- significant differences in PPV were identified compared with tures and conventional cultures were not significantly different. PCR testing alone. Furthermore, requiring positive results Likewise, PCR demonstrated no significant differences in sensi- from either shell vial or conventional culture and PCR resulted tivity, specificity, and NPV. However, PCR had a significantly in high false-negative rates, suggesting that phasing out culture higher PPV in HCT recipients compared with patients with methodsinfavor of moleculardiagnostics, asisoccurring in other underlying diseases (76.9% vs 30.1%, P = .02). Further- many clinical microbiology and virology laboratories, is unlikely more, in HCT recipients, as described above, PCR testing to impact the diagnosis of CMV pneumonitis. It is notable that alone resulted in optimal test characteristics compared with a subset analysis of HCT recipients revealed a higher PPV of the various possible combinations of CMV tests. PCR in HCT recipients compared with the other groups, Cytomegalovirus quantitation in BAL fluid by PCR was also which was likely a reflection of the higher prevalence of patients evaluated to determine whether a threshold could be identified who met clinical criteria in HCT recipients in our cohort. This to aid in the diagnosis of CMV pneumonitis. A ROC curve was finding suggests improved utility of a positive PCR result for the generated, and the area under the curve (AUC) was 0.53. Fur- diagnosis of CMV pneumonitis in patients that had undergone thermore, quantitative CMV DNA levels in BAL fluid were not HCT. statistically different between those who did and did not meet We also investigated CMV quantitation in BAL fluid as a the study criteria for CMV pneumonitis (P = .635). We gener- means by which to further improve test performance. Results ated a ROC curve specifically for HCT recipients and found from previous studies in lung transplant recipients have been moderate improvement in the AUC of 0.70, with the optimal mixed, with 1 study identifying a threshold for CMV pneumo- viral load threshold of 791 copies/mL yielding a sensitivity of nitis of 500 000 copies/mL [22] and others showing no correla- 95.0%, specificity 50%, PPV 86.4%, and NPV 75.0%. In addi- tion of CMV levels with pneumonitis or bronchiolitis tion, no significant difference in DNA levels was identified be- obliterans, respectively [18, 24]. Similar to the latter studies, tween those who did and did not meet criteria for CMV we found CMV levels to be highly variable overall, as well as pneumonitis in HCT recipients (P = .142). in the subset of HCT recipients, providing limited improvement in discriminating between those cases that met the criteria for DISCUSSION CMV pneumonitis and those that did not. These observations In this report, we evaluated the performance of CMV diagnostic may be due, in part, to the variation in fluid volume and cells methods in the largest set of BAL specimens to date, collected collected from each lavage and the focal nature of CMV end- from patients with a wide variety of underlying diseases includ- organ disease. ing, but not limited to, HCT and SOT. We compared conven- Although the identification of characteristic inclusions by tional viral culture and shell vial culture, as well as qualitative H&E and CMV antigens by IHC remain the reference methods and quantitative CMV PCR, with a comprehensive definition for the diagnosis of CMV pneumonitis, lung biopsy tissue was of CMV pneumonitis that takes into account signs and infrequently obtained in this study during episodes in which 6 � OFID � Tan et al CMV was detected by any laboratory method in BAL speci- this diagnostic dilemma that combines the comprehensive clin- mens. Furthermore, although H&E staining has been shown ical definition with highly sensitive CMV nucleic acid amplifi- to be insensitive compared with IHC for the detection of cation testing. Future studies will be required to evaluate this CMV-infected cells in lung biopsy tissue [21, 31], CMV IHC approach and to identify additional biomarkers that will be was performed in only 5.6% (1 of 18) of total episodes and in able to improve the diagnosis of CMV pneumonitis. none of the subset of 7 episodes that met clinical and laboratory criteria for CMV pneumonitis. This limited and suboptimal use Acknowledgments of lung biopsy tissue brings into question the benefit of this in- We thank the hardworking, dedicated staff of the Stanford Clinical Virol- ogy Laboratory for commitment to quality and positive impact on patient vasive procedure, and it suggests that the comprehensive defini- care. tion developed in this manuscript may provide a more practical Financial support. This work was funded by National Institutes of approach to the diagnosis of CMV pneumonitis. Health (NIH) training grant 5T32AI007502-19 (to S. K. T.) and a generous gift from Beta Sigma Phi (to S. K. T.), an international women’s service or- Further limitations include the observational design of the ganization. Further salary support was provided by NIH award K08 study and the absence of data regarding the clinical progression AI110528-01 (to J. J. W.). of patients that tested positive for CMV in BAL fluid and met Potential conflicts of interest. All authors: No reported conflicts. All clinical criteria but did not receive anti-CMV therapy. These authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. patients comprise 34.8% (16 of 46) of the CMV-positive pa- tients that met our clinical definition of CMV pneumonitis; fu- References ture prospective studies will be required to assess disease 1. Bate SL, Dollard SC, Cannon MJ. Cytomegalovirus seroprevalence in the United progression in such patients. Although the clinical criteria cou- States: the national health and nutrition examination surveys, 1988-2004. 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Health Technol Assess 2007; 11: e334. ix–51. 8 � OFID � Tan et al

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Published: Feb 1, 2015

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