1. Introduction
Even in the current era of novel drugs and targeted therapeutic approaches, autologous hematopoietic stem cell transplant (ASCT) remains a backbone in the therapeutic route of most patients with lymphoma and multiple myeloma. ASCT has always been considered a procedure at low risk of cytomegalovirus (CMV) infection and invasive fungal disease (IFD). However, in a near future these opportunistic infections might become a significant challenge for clinicians dealing with lymphoproliferative malignancies as a consequence of the introduction of novel therapeutic agents (i.e.,idelalisib, bortezomib, fludarabine, bendamustine) with a powerful immunosuppressive impact [
1,
2]. Many studies confirmed that CMV reactivation represents a relevant complication also in settings other than allogeneic stem cell transplant [
3,
4,
5,
6,
7,
8,
9,
10,
11,
12,
13]. As for IFD, most previously published studies in patients auto-grafted for the treatment of lymphoproliferative malignancies reported a low incidence ranging from 0 to 8%, though with a relevant mortality rate [
14,
15,
16,
17,
18,
19]. The putative relationship between CMV and IFD is still far from being clarified and the most relevant published studies have been performed in patients undergoing allogeneic stem cell transplant. Some of these studies showed that a diagnosis of CMV symptomatic reactivation/end-organ disease might be associated with a significant increase of IFD risk [
20,
21,
22]. To the best of our knowledge, studies aimed at investigating the relationship between CMV and IFD after ASCT are still lacking. In this article we present data on the incidence and risk factors for such infectious complications in a cohort of 347 ASCT performed in myeloma and lymphoma patients between May 2004 and April 2018 in our Institute, focusing on the relationship between IFD and CMV symptomatic reactivation/end-organ disease.
2. Results
Patient characteristics at transplant are summarized in
Table 1. About one third of transplants were performed in patients aged 60 years or older and 10% in heavily pre-treated subjects (≥3 chemotherapeutic lines). In case of tandem ASCT, the second transplant was excluded from this analysis. Almost all myeloma patients had undergone a bortezomib-based treatment before ASCT since 2011, whereas before this date they received a standard anthracyclines-based chemotherapy. As detailed in
Table 2, a comparison between lymphoma and myeloma patients showed a significantly higher rate of bacterial (42.5% vs. 28%,
p = 0.005) and fungal (5.3% vs 1.2%,
p = 0.035) infections in the former, whereas the rate of CMV symptomatic infection/end-organ disease was not significantly different (16% vs. 15%). Bacterial infections occurred earlier, as compared with IFD and CMV symptomatic reactivation/end-organ disease (7 vs. 14 vs. 34 days, respectively;
p < 0.001), involving either Gram-positive skin commensals (i.e.,
Staphylococcus aureus) or, more frequently, Gram-negative bacteria (i.e.,
Escherichia coli). A total of 12 cases of IFD were diagnosed with a crude overall incidence of 3.5%. Eight cases were classified as probable pulmonary aspergillosis and four were diagnosed as candidemia. As for CMV symptomatic infection/end-organ disease, a total of 54 cases were observed (15.5%). Bacterial infections were the most frequent cause of death (six cases), followed by both CMV (two cases) and fungal infections (two cases). Overall, only one patient died from a non-infectious cause. The overall case fatality rate was of 4%, 3.7% and 16.7% for bacterial infections, symptomatic CMV reactivation and IFD, respectively. Only a trend toward a higher transplant-related mortality (TRM) was observed in lymphoma as compared to myeloma patients (
p = 0.066).
Clinical and laboratory parameters and outcomes of 54 cases of CMV reactivation requiring specific antiviral treatment are detailed in
Table 3. No significant differences among lymphoma and myeloma patients were found. All symptomatic reactivation events were observed in patients presenting with CMV IgG before transplantation, with 34 days median time from transplant to first detection. However, only 5 cases of end-organ disease were diagnosed. Of them, three were classified as interstitial pneumonia and two as hemorrhagic colitis (the overall incidence of end-organ disease was 1.4%; the incidence of end-organ disease among cases of clinically relevant CMV reactivation was 9.2%).
Table 4 and
Table 5 show univariate and multivariate analysis, respectively, of putative risk factors for fungal, bacterial and CMV infections. A diagnosis of lymphoma, a refractory disease status at transplant and a longer duration of neutropenia (defined as ≥7 days with neutrophils count lower than 500/mm
3) were all significantly associated with the risk of post-transplant bacterial infections (
p = 0.005,
p = 0.04 and
p < 0.001, respectively). However, at multivariate analysis, only a neutropenia persistent for more than 7 days was significantly associated with the risk of bacterial infections (OR 2.16, 95%CI 1.29–3.74;
p = 0.006;
Table 5).
Regarding fungal infections, a diagnosis of lymphoma (
p = 0.035), three or more previous treatment lines (
p = 0.009) and an age over 60 years (
p = 0.001) were identified as potential risk factors at univariate analysis. Multivariate analysis confirmed an independent role of all these risk factors in favoring fungal infections (OR 4.09, 95%CI 1.2–16.23;
p = 0.039; OR 2.91, 95%CI 1.29–6.55;
p = 0.012; OR 10.34, 95%CI 2.55–40.11;
p = 0.001, respectively;
Table 5). Finally, a refractory disease status at transplant and a longer duration of neutropenia were the two variables significantly associated with the risk of post-transplant CMV symptomatic reactivation/end-organ disease (
p = 0.039 and
p = 0.004, respectively). However, at multivariate analysis, only a longer duration of neutropenia conserved its significant association with the occurrence of CMV symptomatic reactivation/end-organ disease (OR 2.4; 95%CI 1.2–4.9;
p = 0.009;
Table 5). Results of a sub-analysis including only lymphoma patients are summarized in
Table 6. The duration of neutropenia was found to be the only factor significantly associated with bacterial infections (
p = 0.022). Conversely, the diagnosis (T-cell lymphoma > B-cell lymphoma > Hodgkin’s lymphoma) was the only factor significantly associated with CMV infection (
p = 0.028), whereas more than three previous treatment lines and an age older than 60 years were the two factors significantly associated with the occurrence of fungal infections both at univariate (
p = 0.031 and
p = 0.002, respectively) and multivariate analysis (OR 4.53; 95%CI 1.1–18.53;
p = 0.036; OR 7.58; 95%CI 1.83–31.7;
p = 0.005, respectively;
Table 6). A strong correlation between IFD and CMV symptomatic infection/end-organ disease was observed. In particular, in 8 (66.7%) of the 12 cases of IFD a simultaneous or more often subsequent symptomatic CMV reactivation occurred (Pearson chi-square value: 24.9; continuity correction: 21.01;
p < 0.0001).
4. Materials and Methods
4.1. Patients
A single institution, retrospective, cohort study was conducted on a total of 347 non CD34+ selected autografts performed at the Hematology and Stem Cell Transplant Unit of Regina Elena National Cancer Institute of Rome (Italy) in the period comprised between May 2004 and April 2018. In case of tandem ASCT, the second transplant was excluded from the analysis. Over the 347 patients, 188 were affected by non-Hodgkin’s and Hodgkin’s lymphoma, whereas the remaining 159 by multiple myeloma. The patients who underwent an ASCT for an acute leukemia in the same period were excluded from this analysis. Patient characteristics at transplant are described in detail in
Table 1. All patients received the same anti-infectious prophylaxis. In particular, all patients received an antiviral prophylaxis with valaciclovir and anti-Pneumocystiis prophylaxis with cotrimoxazole given from the day of transplant until six months after intervention and an anti-bacterial prophylaxis with ciprofloxacin from the day of transplant until the resolution of severe neutropenia. No mold-active prophylaxis was routinely administered. All patients were observed for a period of six months from transplant, with the aim to properly evaluate the occurrence of bacterial, fungal and viral infectious complications, as well as TRM: all clinical events occurring during this period were promptly captured and registered into an electronic database. All patients had signed an informed consent for transplant, also granting the use of sensitive data for scientific purposes.
4.2. Criteria for Diagnosis of CMV Symptomatic Infection and End-Organ Disease
The criteria were based on published recommendations [
43,
44,
45,
46,
47]. According to local policy [
13] and published guidelines [
43,
47], CMV DNAemia was determined only upon clinical suspicion of post-transplant reactivation, therefore no routine monitoring CMV strategy was adopted. Criteria of clinical suspicion and assessment of CMV DNAemia, were defined as follows: presence of fever (temperature >38 °C) and overt clinical signs of bone marrow suppression in the absence of concomitant bacterial, viral (i.e., HHV-6, EBV, parvovirus B19) or fungal co-infections. Bone marrow suppression was defined as a delay of neutrophils and/or platelet recovery from ASCT (absence of complete neutrophils and platelets recovery after 14 and 21 days from transplantation, respectively) or a drop in neutrophils and/or platelet count after recovery (absolute count of neutrophils or platelets < 1.000/mm
3 or 100.000/mm
3, respectively, or a decrease of at least 30% of the counts in two consecutive determinations). CMV symptomatic infection was defined as a documented CMV DNAemia, confirmed by two consecutive determinations, in the presence of clinical suspicion criteria of reactivation. CMV end-organ disease was defined by the presence of signs consistent with CMV infection, as determined by a combination of imaging and clinical and histopathological/molecular evaluations. In particular, CMV gastrointestinal disease was defined by the presence of a combination of clinical symptoms from the upper or lower gastrointestinal tract, findings of macroscopic mucosal lesions on endoscopy, and demonstration of the presence of CMV inclusion bodies in the tissue biopsy, further confirmed by positive immunohistochemical staining of CMV antigens in tissue sections of the gastrointestinal tract. CMV pneumonia was defined by the presence of clinical (hypoxemia) and radiological signs of interstitial pulmonary disease, combined with the detection of high CMV viral loads by quantitative PCR in bronchoalveolar lavage fluid and confirmed by the detection of CMV by direct immunostaining of alveolar cells [
44,
46]. Lung tissue biopsies to demonstrate the presence of CMV inclusion bodies in the tissue biopsy were not performed, considering the high risk of complications derived from a pulmonary biopsy in patients with a severe respiratory distress and a great hemorrhagic risk. In the presence of signs and symptoms of CMV reactivation, as above specified, an antiviral treatment was administered. The choice of the antiviral agent to treat a symptomatic reactivationwas based on the clinical features of the patients at the time of reactivation.
4.3. Quantification of CMV DNA
Automated nucleic acid sample preparation systems NucliSENSeasyMAG® (BioMerieux, Durham, USA) has been used for DNA extraction from plasma, according to the manufacturer’s instructions. Amplification for detection and quantification of viral DNA has been performed using commercially available real-time PCR assays (Affigene® CMV Trender diagnostic assay, Cepheid AB, Bromma, Sweden), according to the manufacturer’s instructions (Cepheid AB, Bromma, Sweden) on a Mx3000P® System (Stratagene, La Jolla, CA, USA) until August 2013 then the analogous Geneproof CMV PCR kit (GeneProof, Brno, Czech Republic.) on SLAN® Real-Time PCR Detection System (Shanghai Hongshi Medical Technology Co., Ltd., Shanghai, China). The limit of detection was 88 copies/mL with both kits.
4.4. Criteria for Diagnosis of Invasive Fungal Infections
Invasive fungal infections were defined according to criteria of the EORTC/MSG [
48]. In particular, a probable diagnosis of aspergillosis required that a host factor (i.e., recent history of neutropenia), a clinical feature (i.e., typical radiological findings) and a mycological evidence (i.e., galactomannan antigen detected in plasma, serum, bronchoalveolar lavage fluid or cerebrum-spinal fluid) were present. On the other hand, a proven aspergillosis required microscopic or microbiological evidence from a sterile culture of
Aspergillus strains. A candidemia was diagnosed on the basis of at least one blood culture positive for a
Candida spp strain, associated to clinical symptoms consistent with fungal pathogens (i.e., fever, hypotension, respiratory distress, gastrointestinal symptoms, hepatic or spleen involvement). Candidemia was considered as CVC-related if the isolated strain grew from CVC drawn blood or from a removed CVC-tip [
49]. In the presence of signs and symptoms of invasive fungal infection, as above specified, an antifungal treatment was performed. As for therapeutic algorithm, we adopted a clinically-driven strategy in case of fever or lung infiltrates during neutropenic phase, according to published indications [
50], without any planned weekly surveillance of serological markers of IFD. The choice of the antifungal agent was based on local policy and international guidelines.
4.5. Galactomannan Detection
The diagnostic Galactomannan testing, in serum and bronchoalveolar lavage (BAL) fluid samples, was performed in a routine clinical setting by the Platelia Aspergillus enzyme immunoassay (Bio-Rad Laboratories, Marnes-la-Coquette, France), according to the manufacturer’s instructions. Galactomannan level was measured spectrophotometrically by using the automated PhD™ lx System (Bio-Rad Laboratories, Hercules, CA, USA). Serum and BAL samples with an optical density index ≤ 0.5, were considered negative for galactomannan antigen
4.6. Criteria for Diagnosis of Bacterial Infections
A BSI was defined by the isolation of a bacterium in at least one blood culture, in association with signs and symptoms of sepsis; two positive cultures were required for diagnosis of coagulase-negative staphylococci or Corynebacterium spp. BSI. Clinically documented infections were defined as infections clinically or radiologically documented in the absence of a microbiological determination (i.e., unspecified pneumonia, colitis, skin and soft tissues infections).
4.7. Statistical Analysis
Data were analyzed by Statistical Package of Social Sciences software (SPSS, version 20.0, Chicago, IL, USA). For statistical analysis we considered each transplant as a single event and patients undergoing a double transplant were considered as two different events. Univariate analysis was performed in order to identify risk factors for bacterial, CMV and fungal infections by using χ2 test (Fisher or Pearson) and analysis of variance for categorical and quantitative variables, respectively. Two-sided p-values below 0.05 were considered to be statistically significant for the multivariate analysis. In case of two or more significant variables with reciprocal competitive effect, only the variable statistically more significant or clinically more relevant was included in the final model. Binary logistic regression model was used to analyze the associations between significant risk factors and the occurrence of infections. Enter and remove limits were 0.05 and 0.1, respectively.
5. Conclusions
Our data, gathered from adult patients with lymphoproliferative malignancies undergoing ASCT indicate that (1) Myeloma patients are at lower risk of bacterial infections and IFD when compared with lymphoma patients, but are at equal risk of CMV symptomatic reactivation, likely as a consequence of a selective impact of bortezomib on Herpes Viruses infection. (2) a significant association exists in the autologous setting between CMV reactivation and IFD, though a cause-effect relationship between these post-transplant events remains to be determined; (3) Bacterial infections, in particular by gram negative microorganisms, are the most frequent infectious complications with an overall case fatality rate of 4%; (4) The overall incidence of post-transplant CMV symptomatic infection and end-organ disease is about 15% and 1% respectively, with a low fatality rate (3.7%). Among lymphoma patients a diagnosis of T-cell lymphoma is the only factor significantly associated with the risk of CMV reactivation; (5) The overall IFD incidence is of about 3.5% with a fatality rate of about 17%. Patients with lymphoma, age of 60 years or older and having received three or more previous treatment lines of chemotherapy are at higher risk of IFD at multivariate analysis. Although, these data need to be confirmed by further prospective studies, the results strongly indicate that clinicians should be aware that IFD is a rare complication after ASCT but burdened by a high mortality rate, frequently associated with a CMV reactivation, and more often observed in lymphoma patients with advanced age and a long previous treatment history.