Experimental Evidence That Granulocyte Transfusions Are Efficacious in Treatment of Neutropenic Hosts with Pulmonary Aspergillosis

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Experimental Evidence That Granulocyte Transfusions Are Efficacious in Treatment of Neutropenic Hosts with Pulmonary Aspergillosis
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  Experimental Evidence That Granulocyte Transfusions Are Efficaciousin Treatment of Neutropenic Hosts with Pulmonary Aspergillosis Marife Martinez, a Vicky Chen, a Ann-Jay Tong, a Kelsey Hamilton, a Karl V. Clemons, a,b,c David A. Stevens a,b,c California Institute for Medical Research, San Jose, California, USA a ; Division of Infectious Diseases, Santa Clara Valley Medical Center, San Jose, California, USA b ; Division of Infectious Diseases and Geographic Medicine, Stanford University, Stanford, California, USA c Although polymorphonuclear leukocytes (PMNs) are powerfully anti-  Aspergillus , transfusion therapy remains controversial, with conflicting results, and experimental support has been lacking. We devised a pulmonary infection model in neutropenicBALB/c mice, used an antibacterial regimen to prevent confounding sepsis, and optimized PMN induction, purifications, anddose. Mice were given 150 mg/kg cyclophosphamide every 4 days and a gentamicin-vancomycin-clindamycin-imipenem regimendaily beginning 4 days before intranasal challenge with 5  10 5  Aspergillus  conidia. This regimen produced leukopenia (  10% of normal white blood cell [WBC] count; < 10% PMNs) for 10 days, without bacterial superinfection. PMN donors given 100  g/kg recombinant murine granulocyte colony-stimulating factor (G-CSF) for 10 days yielded 11  10 7 to 13.6  10 7 WBC/ml (81 to87% PMNs). Infected mice were given PMN transfusions intravenously. In 2 experiments with up to 70% mortality of neutro-penic controls, transfusion of 10 7 PMNs 1 and 4 days after challenge had negligible effects on peripheral WBC counts but im-proved survival ( P   0.007, 0.02), decreased lung CFU ( P   0.03, 0.005), and cleared infection in 28 to 50% of survivors. Transfu-sion of 5  10 6 PMNs showed partial protection. Transfusions given every other day did not improve protection. Our presentresults provide an experimental basis for enthusiasm for PMN transfusions in the therapy of aspergillosis in humans. P rolonged neutropenia is a risk factor for the acquisition of invasive pulmonary aspergillosis, particularly in transplantandcancerpatients(1),andtheprognosisismuchworseifthereisnot autologous recovery of neutrophils during aspergillosis (2). Although the introduction of new therapeutics has improved theresponse to treatment, failures, breakthrough infections, and ul-timately mortality remain unacceptably high. One therapeuticmodalitythathasbeenusedperiodicallyforover40yearsisthatof white blood cell (WBC) or granulocyte transfusion (3–6). Differ- encesinthenumberofcellsthatcouldbecollectedandtransfused,as well as the cell collection techniques and donor matching, allhave contributed to the difficulty in assessing the efficacy of aWBC transfusion. Improvements in techniques used in this mo-dality were made with the use of granulocyte colony-stimulatingfactor (G-CSF) and steroids to improve granulocyte yield, andavailable data are suggestive that higher numbers of transfusedgranulocytes lead to improved outcome (5–9). In spite of these improvements, WBC transfusions remain acontroversial treatment, as their utility is difficult to discern sinceconflicting efficacy results have been reported (10, 11). A consen- sus on the value of this model of treatment has not been reached(12).Thespectrumofinfectionsinneutropeniafollowingtherapy  of hematologic cancers or stem cell transplantation has shiftedtoward those caused by molds (3). It has been proposed that thenumbers of patients needed in clinical trials to evaluate antifun-galssuggestthatthetrialsizemaybeprohibitiveforatrialofWBCtransfusions in mycoses (13). If WBC transfusions could beproven beneficial in experimental models, this, then, would sup-port the pursuit of WBC transfusions as a treatment option forhumans.Our goal in these studies was to create a satisfactory murinemodel for study of the treatment of invasivepulmonaryaspergillo-sis by granulocyte transfusion. Several challenges arose in determin-ingthecombinationoffungalinoculumandimmunosuppressiontoproducelethalityduetoinfectionandnotimmunosuppressionorsecondary bacterial infection; this required that a safe antibioticregimen be developed. Lastly, we needed to address the issues of polymorphonuclear leukocyte (PMN) induction in donor ani-mals, an optimal PMN separation method for mouse cells, and atransfusion schedule. Our results indicate that therapeutic trans-fusions of PMNs were beneficial in treating mice with invasivepulmonary aspergillosis.(Thesestudieshavebeenpresentedinpartatthe3rdAdvancesagainst Aspergillosis meeting, Miami Beach, FL, Jan. 2008, ab-stract 90, p. 182, and the 51st Interscience Conference on Antimi-crobial Agents and Chemotherapy, Chicago, IL, September 2011,abstract M-300.) MATERIALS AND METHODS Infection model.  A series of preliminary studies were done to develop auseful model of pulmonary aspergillosis in neutropenic mice (14). In the course of these studies we examined different regimens and schedules of immunosuppression, different antibiotic dosages, and several inoculumnumbers. We found that the regimens of immunosuppression and anti-biotics and the inoculum numbers of conidia that are detailed below re-sulted in a murine model of progressive pulmonary aspergillosis in ani-mals that were neutropenic for the duration of the infection study, 10days. Immunosuppression.  Male BALB/c mice (six weeks old; CharlesRiver Laboratories, Hollister, CA) were used in these studies, in groups of 10. Neutropenia was induced by intraperitoneal injection of 150 mg/kgcyclophosphamide (Cytoxan; Baxter Health Care Corp., Deerfield, IL), Received  14 December 2012  Returned for modification  20 January 2013 Accepted  30 January 2013 Published ahead of print  4 February 2013Address correspondence to Karl V. Clemons, Clemons@cimr.org.Copyright © 2013, American Society for Microbiology. All Rights Reserved.doi:10.1128/AAC.02533-12 1882  aac.asm.org Antimicrobial Agents and Chemotherapy p. 1882–1887 April 2013 Volume 57 Number 4   on O c  t   o b  er  3  ,2  0 1 7  b  y  g u e s  t  h  t   t   p:  /   /   a a c . a s m. or  g /  D  ownl   o a d  e d f  r  om   andthemicewerehousedwithinmicroisolatorcages.Allmiceweregiventhe initial injection on day   4 prior to infection, and subsequent injec-tions were given every 4 days thereafter. Antibioticsregimen. Miceweregivendrinkingwaterwith33.3mg/kggentamicin (APP Pharmaceuticals, Schaumburg, IL), 167 mg/kg clinda-mycin (Patheon, Inc., Ontario, Canada), and 167 mg/kg vancomycin(NOVAPLUS, Hospira, Inc., Lake Forest, IL) beginning 4 days prior today 0 and every day thereafter. Fresh drinking water was prepared daily;mice were assumed to drink 5 ml of water per day. Mice were also given167 mg/kg imipenem/cilastin (Primaxin; Merck & Co., Inc., WhitehouseStation, NJ) subcutaneously beginning 4 days before infection and every day thereafter. Mice were weighed every 4 days, and doses of antibioticswere adjusted based on body weight. Organism.  Aspergillus fumigatus  10AF was thawed from  80°C stor-age and grown for 4 days at 37°C on potato dextrose agar (Difco PDA;Becton, Dickinson and Co., Sparks, MD) plates (15–18). Conidia were harvested, suspended in 0.05% Tween 80 saline, and washed three timesby centrifugation. Conidial viability was determined by plating on Sab-ouraud dextrose agar (BBL SDA; Becton, Dickinson), and numbers of conidia were also determined by the use of the hemacytometer. Infection.  Similar to a pulmonary model described previously (15), micewereinfectedbyintranasalchallengeof   A.fumigatus conidiagivenin30  l. The mice were anesthetized with isofluorane and conidia instilled.The inocula were 5.0  10 5 viable conidia in one study and 5.76  10 5 viable conidia, per mouse, in the second study described.Mortality was tallied daily through 10 days of infection. All survivingmice were euthanatized by CO 2  anoxia and the lungs removed for deter-mination of fungal burden by quantitative plating for CFU as describedpreviously (15, 19). Collection of granulocytes.  Age-matched male BALB/c mice wereplaced into groups of 20 and given 100   g/kg of recombinant murineG-CSF(donatedbyAmgen,ThousandOaks,CA)viadailyintraperitonealinjection for 12 days prior to the day of PMN collection. This regimeninduces a maximal response of the donor mice (J. Andresen, Amgen,personalcommunication;datanotshown)toproducesufficientnumbersofPMNsforcollectionfromperipheralbloodandsubsequenttransfusion(Table 1). Donor mice were bled via brachial artery incision, and blood was placed in tubes containing heparin; approximately 1 ml of blood wasobtained from each donor mouse. The heparinized whole blood was di-luted 1:2 with 0.9% sodium chloride and then mixed with an equal vol-ume of 6% dextran (molecular weight [MW], 200,000 to 250,000; SigmaD-7265).Thissuspensionwasincubatedfor1hatambienttemperaturetosediment red blood cells (RBC). The upper portion (ca. three-fourths of the total volume) of the suspension was collected and centrifuged. Theremaining RBC were lysed with 0.85% ammonium chloride for 10 to 15min on ice and the cells pelleted by centrifugation. Cell pellets were sus-pended in saline. PMNs were separated from mononuclear cells using aFicoll-Hypaque method, with 6 ml of cell suspension layered over 6 ml of Ficoll-Hypaque(Histopaque1077;Sigma).Gradientswerecentrifugedat365   g   for 30 min at 4°C. Pelleted PMNs were recovered and suspendedin RPMI 1640 medium with  L -glutamine and NaHCO 3  (Sigma). Thesecells were washed once by centrifugation and suspended in 2 ml of RPMI1640.Thismethodproducedapurifiedpreparationof   99%PMNs,withviability of 93 to 99% and with  29% PMN recovery from donor blood.Cellnumbersweredeterminedbyhemacytometercount.Cellviability was determined by dye exclusion using 0.2% Trypan blue in phosphate-buffered saline (PBS) (pH 7.2). Dilutions to reach the final desired cellnumbersweredoneinRPMI1640.PMNswereheldat4°Cuntiltheyweretransfused into the infected mice on the same day as they were collected. Transfusion.  Neutropenic, infected recipient mice were given PMNtransfusions via injection into a lateral tail vein. Transfusions were doneon days 1 and 4 postinfection. Preliminary studies had shown this sched-uletobeoptimal.Micereceived5  10 6 or1  10 7 PMNspermouse.OnegroupofinfectedmicedidnotundergoPMNtransfusionandwasusedasan untreated control group. Statistics.  Analyses of survival were done using a log rank test. Com-parisons of CFU were done using a Mann-Whitney U test. Data pointsmissingduetodeathfrominfectionofthemicewereassignedanarbitrary value of log 10  5 CFU, a value close to that found just prior to death, andincluded in the statistical analyses (20, 21). Assignment of this value en- sures that death is considered a worse outcome than is survival with any burden (20, 21). RESULTS Leukopenia and WBC counts for donor and infected recipientmice.  Through empirical investigation, we devised a satisfactory system of producing and maintaining leukopenia in mice, withminimal numbers of deaths due to immunosuppression, second-ary infection, or antibiotic toxicity. Using the dose of cyclophos-phamideandantibioticregimendescribedinMaterialsandMeth-ods, we induced a steady leukopenia of approximately 10 6 WBC/mlfor10dayswithoutbacterialsuperinfection.Thisis10%of the normal numbers of murine WBC and  10% PMNs. Thebaseline total WBC count of the mice 1 day prior to the first doseofcyclophosphamideaveraged1.28  10 7 WBC/ml.Onday0,justprior to infection, the average WBC count was 2.17    10 6 WBC/ml (Fig. 1). These kinetics, and the lack of intercurrent in-fection,aresimilartowhatisseeninmicegiventhisantibioticandimmunosuppressive regimen but not challenged with  Aspergillus (data not shown).Total WBC and differential counts were performed on days of  FIG 1  WBC counts of mice throughout the duration of the experiment. Ar-rows indicate days when cyclophosphamide was given.  n  3 or 4 samples pertime point. Day 1 and 4 values are 3 h prior to transfusion with PMNs. Days 3and 5 to 9 represent values 24 h or more posttransfusion. Day   5 representsthe value for nonneutropenic mice, and day 0 represents the value at the timeof infection. TABLE 1  WBC counts and differentials of mice given recombinantmurine G-CSF a Donor group WBC count (cells/ml) % PMNs1 1.34  10 8 81%2 1.10  10 8 87% a Blood was obtained via brachial artery and collected in tubes containing sodiumheparin. WBC counts on donor blood were performed on the day of collection, whichwas 1 day after the cessation of treatment with recombinant murine G-CSF. WBCcounts prior to treatment with recombinant murine G-CSF fell within a range of 6.4  10 6 to 1.6  10 7 WBC/ml of blood (average, 1.14  10 7 WBC/ml; PMN average was10%). PMN Transfusion against AspergillosisApril 2013 Volume 57 Number 4 aac.asm.org  1883   on O c  t   o b  er  3  ,2  0 1 7  b  y  g u e s  t  h  t   t   p:  /   /   a a c . a s m. or  g /  D  ownl   o a d  e d f  r  om   transfusion on pooled blood from donor mice (Table 1). Recipi- ent mice were bled prior to transfusion and again 1 hour post-transfusion. Subsequent WBC counts were taken at 24 and 48hours after the first transfusion and 24 hours after the secondtransfusion (Fig. 2). Infected mice remained pancytopenicthroughout the experiment, with an average WBC range between6.0  10 5 WBC/ml and 2.75  10 6 WBC/ml on any day.Onehourafterthefirsttransfusion,thetotalWBCcountswereslightly lower than the pretransfusion counts. However, the per-centageofPMNsinthedifferentialcountsincreasedalmost3-foldfrom 6.3% to 17.7%. Counts at 24 and 48 h posttransfusion de-clined further, and the percentage of PMN decreased to   9%.AfterthesecondPMNtransfusion,therewasa  2-foldincreaseinthe total WBC count and a substantial increase in the percentageof PMNs to   39%. By 24 h posttransfusion, the WBC countsdeclined slightly and the percentage of PMNs had dropped toabout 20%, lower than at 1 h posttransfusion but higher than inthe pretransfusion samples (Fig. 2). EfficacyofWBCtransfusionagainstpulmonaryaspergillosisin neutropenic mice.  Data on survival and CFU were gatheredfrom two experiments. From the first experiment, one mousefrom the untreated control group died immediately after infec-tion. This mouse did not recover from anesthesia and was notincluded in the data on the assumption that the mouse died fromthe procedure and not from infection. There were no mice thatdied immediately after infection for the second experiment. Atday 10 postinfection, there were 3 survivors in the untreated con-trol group, 7 survivors in the group given 5  10 6 PMNs/mouse,and 9 survivors in the group given 1  10 7 PMNs/mouse. Simi-larly,atday10postinfectioninthesecondexperiment,therewere6 survivors in the untreated control group and 8 survivors in thegroup given 5  10 6 PMNs/mouse and all 10 mice survived in thegroup given 1  10 7 PMNs/mouse.Figure 3 shows the survival for the groups in the first experi-ment. There was a significant difference between the untreatedcontrol group and the group given 1  10 7 PMN/mouse for bothsurvival( P   0.007)andCFU( P   0.04).Thegroupgiven5  10 6 PMNs/mouse showed a trend toward prolonged survival, but thedifference was not significant compared to results for the un-treatedcontrolgroup( P   0.1),norwasthereanydifferencefromthegroupgiven1  10 7 PMNs/mouse( P   0.2).ThislowerPMNdose of 5    10 6 per mouse also was intermediate between theeffective1  10 7 doseandthecontrolsinloweringlungCFU,withno significant difference in lung CFU for the group given 5  10 6 PMNs/mouse and the untreated control group ( P   0.32) or thegroupgiven1  10 7 PMNs/mouse( P   0.17)(Fig.4).However,4 mice in the 1  10 7 PMNs/mouse group were free of detectablepulmonary infection with  A. fumigatus , compared to 2 given thelesser number of cells.Figure 5 shows the survival for the groups in the second exper-iment. Again, there was a significant difference between the un-treated control group and the group given 1  10 7 PMN/mousefor both survival ( P     0.03) and CFU ( P     0.005). The groupgiven 5  10 6 PMNs/mouse again showed a trend toward pro-longed survival, but the difference was not significant comparedto results for the untreated control group ( P   0.3), nor was there FIG2  WBCcountsbeforeandafterPMNtransfusion.Thefirsttransfusionisindicatedat0h,andthesecondtransfusionisshownat72h.ArrowsindicatePMNtransfusions.  n  3 or 4 samples per time point. FIG 3  Survival curve of mice from experiment 1. Arrows indicate days of PMN transfusion of either 1  10 7 or 5  10 6 PMNs per mouse. Control micereceived no treatment. There was a significant difference in survival betweenthe control group and the group given 1  10 7 PMNs/mouse ( P   0.007). Martinez et al. 1884  aac.asm.org Antimicrobial Agents and Chemotherapy   on O c  t   o b  er  3  ,2  0 1 7  b  y  g u e s  t  h  t   t   p:  /   /   a a c . a s m. or  g /  D  ownl   o a d  e d f  r  om   adifferencefromthegroupgiven1  10 7 PMNs/mouse( P   0.2).There was no significant difference in lung CFU for the groupgiven5  10 6 PMNs/mouseandtheuntreatedcontrolgroup( P   0.2) or the group given 1  10 7 PMNs/mouse ( P   0.2) (Fig. 6). Similar to the first study, 5 mice given 1  10 7 PMNs/mouse werefree of detectable pulmonary infection.We also assessed the number of transfusions needed to obtainan optimal result. We found that giving 5  10 6 or 1  10 7 WBCevery other day (i.e., on days 1, 3, 5, and 7 postinfection) did notimprove protection. In other experiments with a more lethal out-come (80% lethal dose [LD 80 ] or LD 90  in controls), a trend forimproved survival and reduction of lung CFU with transfusion of 10 7 WBC on days 1 and 4 remained but did not reach statisticalsignificance (data not shown).In addition to the survival and CFU, at the time of organ re-moval,thelungsofthesurvivingcontrolmiceonaverageweighedmore than those from PMN-treated mice. The control mouselungslookedhemorrhagiccomparedtothoseofthePMN-treatedmice, which appeared more normal (pink). DISCUSSION Our aims in these studies were 2-fold and included the develop-ment of a persistently neutropenic model of pulmonary aspergil-losis and the assessment of the utility of PMN transfusion as atherapeuticintervention.Throughaseriesofempiricalstudies,wedeveloped a reproducible model of invasive pulmonary aspergil-losis in persistently neutropenic BALB/c mice. As a result, wefound that an inoculum of 5  10 5 conidia, which is an inoculumup to 10-fold or more lower than those used in other models of pulmonaryaspergillosisinitiatedbyintratrachealorintranasalin-oculation of conidia, induced lethal disease (22, 23). We found that the model required the administration of a multiple-drugantibiotic regimen to prevent secondary bacterial infection.Furthermore,thedoseandscheduleofcyclophosphamidewerecritical to induce neutropenia but not cause drug-related lethaltoxicity, with 150 mg/kg given every 4 days satisfying this re-quirement.Performance of granulocyte transfusions using cells isolatedfrom peripheral blood would not have been possible without theinduction of the donor mice with G-CSF; induction was requiredto obtain sufficient numbers of PMNs, with one donor mouseproviding enough cells for transfusion of two recipient mice. De-spite infusing up to 1  10 7 cells/mouse, the peripheral leukocytecount in recipient mice did not increase significantly after trans-fusion. However, most importantly, we demonstrated that thepercentage of PMNs in the blood of the recipient mice increasedsubstantially. We presume that the transfused WBC largely trafficto the site of infection rather than remaining in the peripheralcirculation, which would be consistent with what is known aboutthe physiology of WBC during infection. The neutrophil concen-tration determined in venous blood represents about half of in-travascular neutrophils, with the remainder adherent to vascularwalls (24). Studies in humans have shown that transfused granu- locytes go to areas of infection in neutropenic patients, includingsites of   Aspergillus  infection (25, 26). The homing of infused neu- trophils to infection is consistent with our efficacy results. Thus, atransfusion of 1  10 7 PMNs to each infected mouse resulted insignificant prolongations of survival and reduction of pulmonary burdenofaspergillosis.Also,wefoundthattwodosesgiven3daysapartweresufficienttoresultinsignificantsurvivaladvantageandreduction of fungal burden in the lungs.The utility of PMN transfusion as a treatment against infec-tious disease in the neutropenic patient has been a controversialtopic for many years. Although there is a lack of convincing con-trolled trial data (6, 12), reviews of case studies and small trials would speak to a role for granulocyte transfusions in addition toantifungal or antibacterial therapy, with positive results in up to FIG 6  CFU count for experiment 2. There was significant difference betweenthe control group and the group given 1  10 7 PMNs/mouse. ( P   0.005).Horizontal bars represent the median values. FIG 4  CFU count for experiment 1. There was significant difference betweenthe control group and the group given 1    10 7 PMNs/mouse ( P     0.03).Horizontal bars represent the median values. FIG 5  Survival curve of mice from experiment 2. Arrows indicate days of PMN transfusion. There was a significant difference in survival between thecontrol group and the group given 1  10 7 PMNs/mouse ( P   0.02). PMN Transfusion against AspergillosisApril 2013 Volume 57 Number 4 aac.asm.org  1885   on O c  t   o b  er  3  ,2  0 1 7  b  y  g u e s  t  h  t   t   p:  /   /   a a c . a s m. or  g /  D  ownl   o a d  e d f  r  om   70% of patients. Open trials in mycoses in neutropenia (2) and favorable results in fungal infections in chronic granulomatousdisease patients are particularly relevant (27–29). Treatment pro- tocols and recommendations vary among investigators (6, 12). Our study likely addresses the most difficult hurdle for WBCtransfusion, as most (4, 30–34) but not all (35–37) studies have suggested that fungal infections, particularly those caused by molds, are more refractory than bacterial infections to the poten-tial benefits of these transfusions.Our data, using a model of pulmonary aspergillosis in neutro-penicmice,indicatethatPMNtransfusionappearstobebeneficialand is dependent on the number of cells given. This is well illus-trated by our results showing that only the highest number of transfused cells resulted in significantly prolonged survival andreduction of fungal burden in the lungs; up to 50% of the treatedmice were free of detectable pulmonary infection. The relation of success to number of transfused cells is emphasized throughoutthe clinical literature.Our experimental model has several parallels to clinical expe-rience. We used G-CSF to stimulate our donors; G-CSF has beenshown to prime the oxidative burst in donor cells and to increasecell survival, phagocytosis, microbicidal activity, recruitment tositesofinfection,andproteinsinvolvedincelladhesion(3,38,39). Repeated G-CSF administrations have been shown to be superiortosingleadministrationsinimprovingthesefunctionalproperties(40). It is recommended that the granulocytes be transfused as soon as possible after they are obtained, without storage (41, 42), as we did, although it has been shown that neutrophil functionscan be preserved for 24 h  ex vivo  (43), and viability after G-CSF mobilization is preserved up to 72 h after collection, while inter-leukin 1 (IL-1) or IL-8, implicated in transfusion reactions, re-mains low (44). Between collection and transfusion the cells were refrigerated, as is optimal with human cells (41). Our WBC dose calculates as 4  10 8 granulocytes/kg, which is close to the dose of   3  10 8 granulocytes/kgrecommendedinhumans(34,45).Our recipient WBC kinetics after transfusion are similar to those de-scribed in humans (3, 5, 39, 42), and survival has not correlated with a measurable increase in neutrophils (34). Daily WBC trans-fusions do not appear to be necessary for success (40). Our model, however, may be favored over the situation in hu-mans, as our donors and recipients are isogenic, a situation rarely obtained in humans. The genetic identity removes the possiblecomplications of graft-versus-host disease (necessitating, in hu-mans, radiation of the WBC) and alloimmunization (44). We are less aware of infusion-related side effects (none were noted) andunaware of any as subjectively reported in humans (3, 27). It will be of interest in future studies to determine whetherPMN transfusion in combination with conventional antifungaltherapy may lessen the number of PMNs needed to induce a clin-ical benefit as well as determine whether regimens that will resultin cure of all mice can be found. Thus, our results provide anexperimental basis for continued interest in granulocyte transfu-sionasabeneficialtherapeuticoption,particularlyagainstpulmo-nary aspergillosis. ACKNOWLEDGMENTS These studies were funded by the Foundation for Research in InfectiousDiseases.We have no conflicts of interest to declare. REFERENCES 1.  Walsh TJ, Stevens DA.  2011. Aspergillosis, chapter 347.  In  GoldmanL, Schafer A (ed), Cecil textbook of medicine, 24th ed. Elsevier, Phila-delphia, PA.2.  Dignani MC, Anaissie EJ, Hester JP, O’Brien S, Vartivarian SE, Rex JH,Kantarjian H, Jendiroba DB, Lichtiger B, Andersson BS, Freireich EJ. 1997. 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