key: cord-0942669-qh4adad5 authors: Schnell, David; Legoff, Jérôme; Mariotte, Eric; Seguin, Amélie; Canet, Emmanuel; Lemiale, Virginie; Darmon, Michael; Schlemmer, Benoît; Simon, François; Azoulay, Élie title: Molecular detection of respiratory viruses in immunocopromised ICU patients: Incidence and meaning date: 2012-05-29 journal: Respir Med DOI: 10.1016/j.rmed.2012.05.001 sha: 4d2e83e8e3bc27a0f3fd437ba8ad049aaeee3d99 doc_id: 942669 cord_uid: qh4adad5 PURPOSE: Prospective single-center study to assess the sensitivity and clinical relevance of molecular testing for respiratory viruses in critically ill immunocompromised patients with acute respiratory failure (ARF). METHODS: 100 consecutive critically ill immunocompromised patients with ARF in 2007–2009. Among them, 65 had hematologic malignancies (including 14 hematopoietic stem cell transplant recipients), 22 had iatrogenic immunosuppression, and 13 had solid malignancies. A multiplex molecular assay (MMA) was added to the usual battery of tests performed to look for causes of ARF. RESULTS: Nasopharyngeal aspirates and/or bronchoalveolar lavage fluid were tested for respiratory viruses using both the MMA and immunofluorescence. A virus was detected in 47 (47%) patients using the MMA and 8 (8%) patients using immunofluorescence (P = 0.006). MMA-positive and MMA-negative patients had similar clinical and radiographic presentations and were not significantly different for the use of ventilatory support (58% vs. 76%, P = 0.09), occurrence of shock (43% vs. 53%, P = 0.41), use of renal replacement therapy (26% vs. 23%, P = 0.92), SAPS II (35 [26–44] vs. 38 [27–50], P = 0.36), time spent in the ICU (6 vs. 7 days, P = 0.35), or ICU mortality (17% vs. 28%, P = 0.27). Using MMA, a virus was found in 6 of the 12 patients with no diagnosis at the end of the etiologic investigations. CONCLUSIONS: In critically ill immunocompromised patients, an MMA was far more sensitive than immunofluorescence for respiratory virus detection. Patients with RVs detected in the respiratory tract had the same clinical characteristics and outcomes as other patients. Molecular detection of respiratory viruses in immunocopromised ICU patients: Incidence and meaning Introduction Acute respiratory failure (ARF) occurs in up to 50% of patients with malignancies, who are then at high risk for death, particularly if they require mechanical ventilation. 1e4 Respiratory viruses (RVs) are detected in 10%e 20% of these ARF episodes. 5, 6 Together with the bacterial and fungal infections often seen in this setting, RV infections are potentially life-threatening in immunocompromised patients. 7e11 Viral culture has long been considered the reference standard for diagnosing RV infection but usually takes several days to yield results. Antigen detection is faster but less sensitive. 12 Molecular screening based on the polymerase chain reaction (PCR) is fast, more sensitive than earlier test methods, highly specific, and capable of detecting rhinoviruses and coronaviruses, which are missed by other tests. 13 Molecular assays have been reported to improve the diagnostic yield compared with conventional methods during acute respiratory illnesses in patients with hematological malignancies. 14e17 However, they detect only nucleic acids, as opposed to live organisms, and their clinical relevance is therefore unclear. A positive molecular assay on a respiratory sample may indicate viral infection, colonization, or contamination. The purpose of this study was to evaluate a multiplex molecular assay (MMA) comparatively with immunofluorescence for RV detection in immunocompromised patients admitted to intensive care unit (ICU) with ARF and to assess the clinical relevance of the MMA results. Immunocompromised patients with hypoxemic ARF admitted to our closed ICU in a teaching hospital from January 2007 to July 2009 were included prospectively in this cohort study. The MMA was the intervention. The institutional review board of the Clermont Ferrand teaching hospital approved this study and waived the need for informed consent. Immunocompromised status was defined as presence of a disease or treatment known to impair the immune system, such as a hematological or metastatic solid malignancy or long-term corticosteroid therapy, immunosuppressive therapy, cytotoxic chemotherapy, and/or bone marrow or hematopoietic stem cell transplantation (HSCT). Patients infected with the human immunodeficiency virus were not included. ARF was defined as a respiratory rate greater than 30 breaths per minute or respiratory distress symptoms or PaO 2 on room air lower than 8 kPa or a need for ventilatory support. The data reported in Tables 1e3 were collected for each study patient. Simplified acute physiology score II (SAPS II), time in the ICU and vital status at ICU discharge were also collected. 18 All patients were investigated using a previously described diagnostic strategy that relies heavily on noninvasive tests. 19 Most of the patients underwent noninvasive tests for infections, such as sputum examination for bacteria, mycobacteria, and fungi; induced sputum for Pneumocystis jirovecii pneumonia; serum and blood tests for circulating Cytomegalovirus and Aspergillus; blood cultures; specific PCR tests for herpes viruses on blood and respiratory samples; and urine tests for bacterial antigens. Bronchoscopy and bronchoalveolar lavage (BAL) were performed when deemed appropriate by the attending physician. BAL fluid was collected as previously described 19 and was used for bacterial, mycobacterial, and fungal cultures; RV antigen detection by immunofluorescence; and cytological examination. Echocardiography, chest computed tomography, and thoracocentesis were also performed when deemed appropriate by the attending physician. Clinically documented infection was defined as a strong clinical and radiographic suspicion of pneumonia without microbiological documentation but with either septic shock or complete resolution after antibacterial treatment. 5 Immunofluorescence (Argene, Verniolle, France) was performed routinely to test nasopharyngeal aspirates (NPA) and BAL fluid for influenza A and B viruses; respiratory syncytial virus (RSV); parainfluenza viruses (PIV) 1, 2, and 3; and adenoviruses. Human metapneumovirus (hMPV) was sought starting in October 2007. All respiratory specimens were also investigated for RVs using an MMA based on the Multiplex Ligation-dependent Probe-Amplification (MLPA) technology (RespiFinder19Ò, Pathofinder, Maastricht, The Netherlands) that allows the detection and differentiation of 14 respiratory viruses, including influenza viruses A and B; PIV-1 to PIV-4; RSV A and B; rhinovirus; human coronaviruses 229E, OC43 and NL63; hMPV; and adenovirus. 20 Additionally, this MMA detects influenza A H5N1 and four bacteria (Chlamydophila pneumoniae, Mycoplasma pneumoniae, Legionella pneumophila, and Bordetella pertussis). Samples were stored at À80 c. Before extraction, 5 ml of an internal amplification control which contained an encephalomyocarditis virus RNA transcript was added into the sample. Nucleic acids were purified from 400 ml of samples with the EasyMag system (Biom » rieux, Marcy l'Etoile, France) and eluted in 100 ml of elution buffer, of which 10 were used for amplification. The three steps of pre-amplification, hybridization and ligation-PCR were performed on a C1000 themocycler (Bio-Rad, Marnes la Coquette, France). The amplified MLPA products were analyzed on an ABI 3100 genetic analyzer (Applied Biosystems, Foster City, CA). Fragment sizing analysis was performed with the GeneMarker software (SoftGenetics, LLC, State College, PA). Two clinicians reviewed the medical charts of all study patients to determine the etiologies of ARF. This was made on Table 1 Baseline characteristics, symptoms, chest radiography patterns, need for life-supporting interventions, and outcomes of the 100 immunocompromised patients with acute respiratory failure and comparaison of patients with and without a positive MMA. the basis of clinical, radiographic, microbiological, and histological findings, according to predefined criteria. 1, 6, 21 The reviewers also aimed at evaluating the clinical significance of RV detection by MMA in these patients. In the absence of validated criteria, this was mainly based on the presence or absence of a definite etiology of ARF, and on the possible interplay between viral infection and this etiology. Quantitative parameters are reported as median and interquartile range (IQR, 25th À75th percentiles) and qualitative parameters as number and percentage. Categorical variables were compared using the c 2 test or Fisher's exact test, as appropriate. Continuous variables were compared using the ManneWhitney U test or the Wilcoxon test, as appropriate. Associations between patient characteristics and positive MMA results were assessed using a logistic regression model. Multivariable analysis was performed using stepwise forward selection to introduce variables yielding P values smaller than 0.20 by univariate analysis. We also introduced variables that seemed clinically relevant. Then, the absence of a significant increase in the likelihood value after omission of each of the remaining variables was checked. Odds ratios (ORs) and their 95% confidence intervals (95%CIs) were computed. P values less than 0.05 were considered significant. Statistical analyses were performed using Statview 5.0 (SAS Institute, Cary, NC). We included 100 immunocompromised patients admitted to our ICU with ARF during the study period. Their main characteristics are listed in Table 1 . Median symptom duration at ICU admission was 3 1e6 days. The admission leukocyte count was 7.7 (3.4e14.9) 10 9 L À1 , and 6 (6%) patients had neutropenia at admission. At admission, PaO 2 was 11.3 (8.7e14.6) kPa with a mean oxygen rate of 6 2e12 L$min À1 , PaCO 2 was 4.88 (4.00e5.9) kPa, and pH was 7.42 (7.37e7.46). The use of life-supporting interventions and outcomes over time are reported in Table 1 . The MMA was performed on NPA in 45 (45%) patients and BAL fluid in 55 (55%). Immunofluorescence was positive in 8 (8%) patients and the MMA in 47 (47%) patients (P Z 0.006) ( Table 2) . After excluding rhinoviruses and coronaviruses, the difference remained highly significant (31% versus 8%, P < 0.0001). Coinfection with two viruses was detected in 5 patients using the MMA; the combinations were influenza A and rhinovirus, influenza A and hMPV, influenza A and adenovirus, influenza B and adenovirus, and PIV 2 and coronavirus OC43. .0] 10 9 L À1 vs. 7.2 [3.6e15.6]10 9 L À1 , respectively; P Z 0.67). By univariate analysis, a positive MMA was associated with being a solid organ transplant recipient (P Z 0.01), receiving immunosuppressants in the past month (P Z 0.05), and being tested on NPA (60% vs. 32%, P Z 0.01). No variable was significantly associated with a positive MMA by multivariate analysis. Study patients were classified into diagnostic categories based on a medical chart review (Fig. 1) . The MMA was positive in 30 of the 48 patients with lung infections and 17 of the 52 patients with non-infectious lung diseases. Results of microbial investigations are given in Table 3 . Coinfections with an RV and bacteria or fungi were found in 11 patients with infectious lung disease. These coinfections are described in Table 4 . All patients were considered as having bacterial or fungal lung infection. A patient with non-infectious lung disease had a respiratory sample positive with coagulase-negative Staphylococci and coronavirus NL63. These pathogens were not considered significant. Also, Table 3 compares the MMA-positive and MMA-negative patients in the subgroups with and without lung infection. In the subgroup of patients with noninfectious lung diseases, 12 patients had no diagnosis found at the end of the etiologic investigations. Multiplex molecular assay was positive in 6 (50%) of these patients. Among immunocompromised patients admitted to the ICU with ARF, about half had an RV detected by MMA, which was more than 5-fold the rate of RV detection by immunofluorescence. The MMA still had a significantly higher sensitivity when we considered only the viruses included in the immunofluorescence panel. All viruses detected by immunofluorescence were also detected by the MMA. To our knowledge, this is the first MMA study in immunocompromised patients with ARF. About half these patients had at least one RV detected by the MMA. This high sensitivity is consistent with the results of previous studies in HSCT recipients with acute respiratory illnesses. 15, 16 Not surprisingly, rhinoviruses were the RVs most commonly detected by the MMA in our population. 15, 16, 22 Rhinoviruses and coronaviruses are not detected by immunofluorescence, a fact that may explain the higher sensitivity of the MMA. However, the MMA remained significantly more sensitive than immunofluorescence when we considered only the RVs detected by both methods. Whether the higher sensitivity of RV by molecular screening improves the etiologic diagnosis of ARF remains uncertain. The significance of a positive MMA in immunocompromised patients with ARF cannot be determined from our data. In our study cohort, the need for life-supporting interventions and the mortality rate were both low in MMApositive patients, but neither was significantly different from that in MMA-negative patients. The clinical and radiographic presentations were also similar. A positive MMA with negative results of other tests for viruses has been associated with lower viral loads and fewer respiratory symptoms compared to concomitant detection by MMA and other tests. Thus, PCR-based methods may help to detect asymptomatic or mildly symptomatic stages of RV infections. 14 Studies have established that RVs including PIV, hMPV, and rhinoviruses are sometimes detected by MMA in respiratory samples from symptom-free HSCT, indicating that asymptomatic RV shedding can occur. 16, 17, 23 Table 3 Microorganisms, use of life-supporting interventions, and outcomes in patients with and without documented lung infections and in patients with and without a positive MMA for respiratory viruses. RV shedding lasts longer in immunocompromised patients than in immunocompetent patients, 17,24e26 and a positive MMA may merely indicate a low level of shedding of limited clinical significance. Therefore, in some of our patients, a positive MMA may have indicated asymptomatic shedding unrelated to the cause of the ARF. Finally, MMAs detect only the nucleic acids of RVs, so that a positive MMA can be related to sample contamination. In the subgroup of patients without documented lung infections, a positive MMA was associated with significantly less use of mechanical ventilation, a significantly shorter duration of hemodynamic failure, and nonsignificantly lower values for use of renal replacement therapy, time in the ICU, and ICU mortality. Thus, in immunocompromised patients with ARF, the MMA may identify patients with probable viral pulmonary involvement, a diagnosis associated with better outcomes than those assumed to be present when all tests are inconclusive. Indeed, negative tests for the cause of ARF constitute a major diagnostic criterion for severe non-infectious conditions such as drugrelated pulmonary toxicity or pulmonary infiltration by the malignancy. Consequently, MMA may deserve to be added to the list of investigations performed routinely to detect the cause of ARF. Isn't so, every effort should be done to make the difference between a positive test and a diagnostic test. In contrast, in the subgroup of patients with lung infections, there were no significant differences between MMA-positive and MMA-negative patients. When there is a known bacterial or fungal lung infection, identifying an RV probably has no major impact on the management strategy or patient outcome. An RV may act merely as a risk factor for other infections or play a role in generating ARF, either alone or in conjunction with a bacterial or fungal infection. Finally, the higher sensitivity of MMA compared to immunofluorescence seems to have only limited clinical consequences. The high frequency of RVs detection does not appear to have significant impact on patients' presentation and outcomes. This may be particularly true in our cohort of critically ill patients. To gain insight into the clinical significance of a positive MMA, routinely performing the MMA on both BAL fluid and NPA and/or obtaining lung biopsies might have been of interest. Histology can show a cytopathic effect but lacks sensitivity. Positive results are difficult to interpret, and no criteria are available for determining the causal role for the RV in the lung disease. High viral loads are more often accompanied with clinical disease, but low viral loads do not exclude clinical disease due to the virus. Moreover, the pathogenesis of viral infection depends not only on intrinsic viral pathogenicity, but also on genetic host factors such as those involved in the immune response. For rhinoviruses in particular, the clinical manifestations may reflect the immune response to the infection rather than the viral cytopathic effect. 27 In our prospective cohort of immunocompromised patients admitted to the ICU with ARF, an MMA was far more sensitive than immunofluorescence for RV detection. The clinical characteristics and outcomes were not significantly different between MMA-positive and MMA-negative patients. However, the MMA can suggest alternative diagnoses to noninfectious lung diseases. Studies evaluating how this diagnostic strategy translates into improved diagnostic efficacy with further increased survival are warranted. This study was supported by a grant from the Assistance-Publique Hôpitaux de Paris [AOM 04139], a nonprofit institution. The prognosis of acute respiratory failure in critically ill cancer patients Characteristics and outcomes of cancer patients requiring mechanical ventilatory support for >24 hrs Noninvasive ventilation in immunosuppressed patients with pulmonary infiltrates, fever, and acute respiratory failure Improved survival in cancer patients requiring mechanical ventilatory support: impact of noninvasive mechanical ventilatory support Diagnostic bronchoscopy in hematology and oncology patients with acute respiratory failure: prospective multicenter data Pulmonary infiltrates in non-HIV immunocompromised patients: a diagnostic approach using non-invasive and bronchoscopic procedures Clinical implications of respiratory virus infections in solid organ transplant recipients: a prospective study Respiratory virus infections in adults with hematologic malignancies: a prospective study Risk factors for pneumonia in immunocompromised patients with influenza Respiratory viral infections in adults with hematologic malignancies and human stem cell transplantation recipients: a retrospective study at a major cancer center Respiratory viral infections in immunocompetent and immunocompromised persons Performance of six influenza rapid tests in detecting human influenza in clinical specimens Comparison of real-time PCR assays with fluorescentantibody assays for diagnosis of respiratory virus infections in children Comparison of conventional and molecular detection of respiratory viruses in hematopoietic cell transplant recipients Polymerase chain reaction is more sensitive than viral culture and antigen testing for the detection of respiratory viruses in adults with hematological cancer and pneumonia Frequent detection of respiratory viruses in adult recipients of stem cell transplants with the use of real-time polymerase chain reaction, compared with viral culture Respiratory virus infection among hematopoietic cell transplant recipients: evidence for asymptomatic parainfluenza virus infection A new simplified acute physiology score (SAPS II) based on a European/North American multicenter study Diagnostic strategy for acute respiratory failure in patients with haematological malignancy RespiFinder: a new multiparameter test to differentially identify fifteen respiratory viruses Outcome of diffuse alveolar hemorrhage in hematopoietic stem cell transplant recipients Respiratory virus infections after marrow transplant: the Fred Hutchinson Cancer Research Center experience Persistent symptomless human metapneumovirus infection in hematopoietic stem cell transplant recipients Prolonged shedding of multidrug-resistant influenza A virus in an immunocompromised patient Prolonged excretion of amantadine-resistant influenza a virus quasi species after cessation of antiviral therapy in an immunocompromised patient Influenza infections after hematopoietic stem cell transplantation: risk factors, mortality, and the effect of antiviral therapy Relationship of upper and lower airway cytokines to outcome of experimental rhinovirus infection This study was supported by a grant from the Assistance-Publique Hôpitaux de Paris (AOM 04139), a nonprofit institution. Assistance-Publique Hôpitaux de Paris was not involved in the study design, data collection or analysis, writing of the manuscript, or decision to submit the manuscript for publication. No potential conflicts of interest occurred for any of the authors.