Of 104 HCT recipients, 87 had 100 stored BAL samples available for testing (). Thirty-eight patients had 45 stored BAL samples without sera available, and 17 patients had 37 stored sera without BALs. Forty-nine patients had 55 concomitant BAL and 95 concomitant serum samples. Sera had been collected a median of one day after BAL (interquartile range, 3 days before to 4 days after).
Specimens available for testing from 104 hematopoietic stem cell transplantation recipients.
Study cohort characteristics, grouped by type of samples available, are shown in . Most patients were lymphopenic in the week before pneumonia diagnosis. The groups differed with respect to proportion of pneumonia episodes accompanied by co-pathogens. Of 27 patients with pulmonary co-pathogens, 11 had more than one type. Aspergillus was most common, with 9 infections confirmed as Aspergillus fumigatus. Twelve patients had CMV pneumonia, and 10 had co-existing bacterial pathogens (5 Pseudomonas aeruginosa, 3 Streptococus pneumoniae, 2 Staphylococcus aureus, 3 coliforms). Other co-pathogens included 2 Candida species, 1 Mycobacterium fortuitum, and 1 rhizopus species.
Characteristics of 104 HCT recipients with virologically-confirmed respiratory virus pneumonia, grouped by stored samples available for respiratory virus testing by quantitative RT-PCR.
Quantitative respiratory virus detection in BAL fluid
Five BAL samples from five HCT recipients that had previously tested positive were negative upon retesting (2 RSV, 2 influenza A, one MPV); that is, viral RNA was below the limit of detection by quantitative RT-PCR. These samples were collected in 1990, 1999, and 2006 (3 samples) and had previously been positive by culture (RSV ×2), DFA (influenza A ×2, RSV), and RT-PCR (MPV). We analyzed quantitative viral load versus sample collection date and found no correlation between viral load and time over the study period to suggest that sample degradation may have consistently contributed to lack of RNA amplification (Pearson coefficient 0.08). Further analyses were performed using the 95 amplifiable BAL samples from 82 HCT recipients.
Three patients had two separate pneumonia episodes, and nine patients had more than one BAL sample per infection. Using the BAL with maximum quantitative viral load for each respiratory virus per pneumonia episode (85 BAL samples), median (range) respiratory virus copy number for each was: RSV (n=35) 2.6×106 (1.5×103 – 1.0×109) copies/ml, PIV (n=35) 4.9×107 (2.7×103 – 1.1×109) copies/ml, influenza (n=9) 6.8×105 (7.4×103 – 8.3×108) copies/ml, MPV (n=7) 3.9×107 (2.9×104 – 2.8×108) copies/ml, and CoV (n=4) 1.8×105 (2.5×103 – 2.0×107) copies/ml (.).
Figure 2 Respiratory virus–specific quantitative viral loads in BAL fluid. The BAL with the maximum viral load per pneumonia episode is shown and used for calculation of the median value per virus. A) Eighty-five BAL samples from 82 HCT recipients. (Total (more ...)
Quantitative respiratory virus detection in BAL samples and clinical outcomes
For 77 patients, we examined the association of quantitative viral load in BAL samples from first episodes of pneumonia (33 RSV, 30 PIV, five MPV, eight influenza, and two CoV) with the presence of lymphopenia, co-pathogens, and need for mechanical ventilation. Three patients with multiple respiratory viruses co-detected in BAL samples, and two patients for whom the first positive BAL was not available, were excluded. No statistically significant difference was found for median quantitative viral load between patients with and without lymphopenia (≤100 lymphocytes/μl ≤7 days before BAL) or co-pathogens; there was a trend toward higher viral load in patients who required mechanical ventilation (p=0.06; ). There was no association between quantitative viral load measured in first BAL and overall survival at one or 6 months (data not shown).
Median quantitative BAL viral load from first positive BAL samples associated with respiratory virus pneumonia, in the presence and absence of clinical characteristics (N=77 patients)
Quantitative respiratory virus detection in serum
One hundred thirty-two serum samples obtained from 66 HCT recipients near the date of diagnostic BAL were tested by quantitative RT-PCR, corresponding to 68 episodes of pneumonia: 41 RSV, 17 PIV, 5 influenza, three MPV, one PIV2/CoV co-infection, and one MPV/influenza A/CoV co-infection (). Forty-nine patients had concomitant BAL (n=55) and serum (n=95) samples. Respiratory viral RNA was detected in sera from six patients: four (10%) with RSV pneumonia, one with influenza B, and the patient with MPV/influenza A/CoV co-infection (influenza and MPV RNA detected). Median serum RNA detection in copies/ml for RSV was 5.3×102 (range 3.0×102 – 1.2×104) copies/ml, 3.3×102 copies/ml for influenza B, and 7.9×102 and 3.7×102 copies/ml for MPV and influenza A, respectively. The six positive serum samples were collected a median of one day after closest concomitant positive BAL sample (range, 11 days before to six days after), similar to the 126 negative serum samples (median one day after BAL, range 16 days before to 19 days after; p=0.78).
Quantitative RT-PCR results of serum samples in 66 HCT recipients
. provides quantitative viral loads for BAL samples corresponding to negative and positive serum samples. For two patients with RSV RNA detected in serum, corresponding BAL samples with maximum viral load had 1.7×108 – 1.0×109 copies/ml RNA detected. One patient with detection of influenza B RNA in serum had 6.7×104 copies/ml in BAL, and the patient with MPV and influenza A detected in serum had 7.8×107 and 7.4×103 copies/ml, respectively, in BAL. No viral RNA was detected in serum of 18 patients with PIV pneumonia, even among patients with highest BAL viral loads.
The association of quantitative RSV viral load in BAL samples and detection of RSV RNA in serum was analyzed for 23 HCT recipients with RSV pneumonia and quantitative viral load from concurrent BAL and serum samples. A higher median maximum RSV BAL viral load was present in two patients with detection of RSV RNA in serum (5.9 ×108 copies/ml) compared with patients without (3.2×106, copies/ml); p=0.05.
Detection of viral RNA in serum and clinical outcomes
All six patients with viral RNA detected in serum underwent allogeneic bone marrow transplantation with pneumonia and serum RNA detection in the first 120 days after HCT; additional characteristics are in . Serum viral RNA was detected in three patients after receiving at least a week of antiviral therapy, two with aerosolized ribavirin for RSV (patients #3, #4) and one with oseltamivir (patient #6). Positive serum samples were collected within 1–12 days of positive BAL. Five of the six patients died within one week of a positive serum or BAL sample and viral pneumonia was regarded as the final diagnosis and cause of death. Autopsies were performed for patients #5 and # 6; both had diffuse alveolar damage and negative viral cultures; focal bronchiolitis obliterans organizing pneumonia was also reported for patient #5. No patients with viral RNA detected in serum had bacterial, fungal, or viral co-pathogens in the BAL.
Characteristics of six HCT recipients with pneumonia and respiratory virus RNA detected in serum samples.
Characteristics from were similar between patients with and without viral RNA detection in serum (data not shown). When the entire population was analyzed, there was no difference in timing of pneumonia diagnosis after HCT. However, for the subset of 40 patients with RSV pneumonia after HCT, the 4 patients with RSV RNA detected in serum were diagnosed with pneumonia earlier after transplant than patients without RSV RNA in serum (median 10 days [range 4–27] versus 48 days [range 8–237], p=0.02).
For first episodes of respiratory virus-associated pneumonia, detection of viral RNA in serum was assessed as a risk factor for receipt of mechanical ventilation and death within 30 days after first positive BAL. This analysis was restricted to the 59 patients who underwent allogeneic transplantation. In univariate analysis, patients with detection of viral RNA in serum had increased risk of mechanical ventilation (RR 2.4, p=0.02) and death (RR 2.0, p=0.005) within 30 days of first positive BAL; the association with death persisted in an adjusted model (RR for death 1.8, p=0.02; ).
Table 4 Outcomes among allogeneic HCT recipients with post-transplantation pneumonia, with and without viral RNA detection in serum, shown separately for all patients with (A) pneumonia due to all virus infections and (B) pneumonia with RSV alone. Prevalence (more ...)