invasive mycoses represent a major cause of morbidity and mortality in patients with malignancy or undergoing hematopoietic stem cell transplantation ( hsct ) ( 1 , 2 ) . patients with hematologic malignancies are currently at higher risk of invasive fungal infections ( ifi ) caused by molds than yeasts , and the incidence of ifi is the highest among patients with acute myeloid leukemia . invasive aspergillosis ( ia ) occurs more often in patients with acute leukemia than in patients with chronic leukemia , lymphomas or multiple myeloma ( 4 ) . in most reports , prognosis of the established aspergillosis is dismal ; therefore , its prevention is of utmost importance . a meta - analysis of 50 studies on aspergillosis revealed mortality rates of approximately 60% in patients with acute leukemia and lymphoma and up to 90% in allogeneic stem cell recipients ( 5 ) . definitive diagnosis of ia requires histopathological evidences of deep - tissue invasion or a positive culture from normally sterile sites ( 6 ) . to diagnose the invasive pulmonary aspergillosis ( ipa ) in high - risk patients , bronchoalveolar lavage ( bal ) specimen early and precise diagnosis of ipa is important to start antifungal treatment in time and reduce the unnecessary use of toxic antifungal agents . although traditional approaches such as direct microscopic examination , histopathological evaluation and cultivation are still the gold standard , the diagnosis of ipa is generally difficult because of their inadequate sensitivity and specificity ( 7 ) . the current study aimed to evaluate the incidence of ipa in hsct and hematological malignancies patients by using bal specimens , and also the diagnostic potential of nested polymerase chain reaction ( pcr ) and real - time pcr were compared with conventional methods . during a 16 month period ( june 2009 to october 2010 ) , 46 consecutive bal fluid specimens were obtained from patients with hematological malignancies and hsct , at shariati hospital and medical mycology laboratory in the school of public health , tehran , iran . a portion of bal specimens ( 4 - 7 ml ) were obtained by specialist physicians under standardized techniques ( 8) , and then collected in sterile vessels free of preservatives , and transferred to the laboratory within one hour . the pellet was vortexed vigorously and resuspended in a small volume of supernatant , with the final volume of 400 to 600 microlitter . seventy - five - microliter aliquots of the specimens were planted on sabouraud glucose agar ( 4% ) ( difco , usa ) and brain heart infusion agar ( difco , usa ) plates equally ( total 150 l ) ; then were incubated for three to seven days at 30c . a wet mount was prepared with 150 l of the sediment with a drop of 20% potassium hydroxide ( koh ) , and examined by 100 and 400 magnification of microscope . for cell lysis of bal fluid , each specimen was subjected to four times freezing in liquid nitrogen and thawing in boiling water , and then was crushed with a conical grinder for one minute . for sticky and viscous specimens , 2% dithiothreitol ( wako , japan ) was added , then sonicated for two to five minutes ( 9 ) . finally , dna was extracted from each bal specimen by phenol - chloroform method as described by makimura et al . with a little modification ( dna of four specimens the homogenized bal sediment was added to an equal volume of the lysis buffer ( 100 mm tris - hcl , ph = 8 , 10 mm edta , 0.1% w / v sds , 100 mm nacl , 2% v / v triton 100x ) and an equal volume of phenol chloroform isoamyl alcohol ( 25 : 24 : 1 ) , and was centrifuged at 5'000 g for 10 minutes . chloroform was added to the supernatant and was centrifuged at 5'000 g for five minutes . sodium acetate ( 3 m ; 0.1 volume ) and 2-propanol ( equal volume ) were added to the supernatant and kept at -20c for 10 minutes , and then was centrifuged at 12'000 g for 12 minutes . the precipitated dna was washed with 300 l of 70% ethanol and re - suspended in 30 l of ultrapure water and was kept at -20c . furthermore , the analytical specificity of the pcr was assessed by testing the following fourteen genomic dna of standard fungal strains , which were provided by teikyo university institute of medical mycology ( timm ) , tokyo , japan : a. flavus ( jcm 2061 ) , a. fumigatus ( atcc 26430 , timm 3968 ) , a. niger ( timm 0113 ) , candida tropicalis ( atcc 750 ) , c. albicans ( timm 3313 ) , c. glabrata ( atcc 90030 ) , c. parapsilosis ( atcc 22019 ) , penicillium expansum ( timm 1293 ) , p. notatum ( timm 0883 ) , p. marneffei ( cbs 334.59 ) , fusarium solani ( tsy 0403 ) , trichosporon asahii ( cbs 2479 ) and sporobolomyces koalae ( jcm 18063 ) . all the primers used for the nested pcr had been designed by sugita et al . ( 11 ) . the pcr mixture for contained 12.5 l 2 premix ( ampliqon , denmark ) , 0.5 l ( 30 pmol ) of each primer , and 2 l of the dna template solution , and enough distilled water up to 25 l . the mixture was heated to 95c for five minutes followed by 30 cycles of 94c for 45 seconds ; 60c for one minute ; and 72c for one minute and a final step at 72c for seven minutes . for the species specific primer sets of a. flavus and a. fumigatus , 1 l of the first pcr product ( diluted 1/100 ) was used as a template . each mixture was heated to 95c for five minutes and pcr was performed in cycles of 94c for 50 seconds ; 58c for 40 seconds and 72c for 45 seconds for 25 cycles and a final extention at 72c for eight minutes . the reactions were run with a thermal cycler ( takara pcr thermal cycler dice mini , tp100 . real - time pcr was performed with a 7500 fast real - time pcr system ( applied biosystems ) based on taqman chemistry . the real - time pcr primers and probes were designed based on multiple alignments of various sequences of partial -tubulin gene by zarrinfar et al . as described previously all of the real - time pcr primers and probes were obtained from applied biosystems ( usa ) . reactions were performed using eagle taq mastermix with rox ( roche , basel , switzerland ) according to the manufacturer s recommended protocol . each reaction mixture contained 2 l of template dna solution , 0.2 l ( 10.4 m ) of probes a. fumi - p , 0.16 l ( 12.9 m ) of probe a. flavus - p , 0.4 l ( 10 m ) of primer a. fumi - f , 0.4 l ( 10 m ) of primer a. fumi - r , 0.4 l ( 10 m ) of primer a. flavus - f , 0.4 l ( 10 m ) of primer a. flavus - r , 10 l of master mix and enough distilled water up to the final volume of 20 l . following an initial denaturation step at 95c for 10 minutes , pcr amplification was performed for 45 cycles consisting of 95c for three seconds and 60c for 30 seconds dna extracted from a. fumigatus ( timm 3968 ) and a. flavus ( timm 2912 ) were used as positive and three tubes containing water instead of dna were used as negative controls in each run . a. fumigatus and a. flavus pcr products were cloned using a ta cloning kit and pcr 2.1 vector ( invitrogen corp ) . the pcr 2.1 plasmid containing the dna target was sequenced to confirm the insertion of a single copy of amplicon . the quantitation , accuracy , and precision of the real - time pcr assay were validated via preparation of serial dilutions of the plasmid containing the cloned target . the dna concentration was determined according to the absorbance at 260 nm and molecular weight of the plasmid . in addition , the plasmid was used as the positive control in all reaction runs . quantitative results were expressed by determination of the threshold cycle ( ct ) of detection , or crossing point ( cp ) , which marked the cycle at which fluorescence of the specimen became significantly different from the baseline signal . the analysis of the results for a positive specimen was designated when at least one replicate had a ct value of  38 cycles . the efficacy of the dna isolation system in releasing aspergillus dna from the fungal cells for pcr were evaluated by analyzing serial dilutions of a. fumigatus conidia in seven concentrations ( 10 , 10 , 10 , 10 , 10 , 10 and 10 conidia / ml saline ) . seventy - five - microliter aliquots of the specimens were planted on sabouraud glucose agar ( 4% ) ( difco , usa ) and brain heart infusion agar ( difco , usa ) plates equally ( total 150 l ) ; then were incubated for three to seven days at 30c . a wet mount was prepared with 150 l of the sediment with a drop of 20% potassium hydroxide ( koh ) , and examined by 100 and 400 magnification of microscope . for cell lysis of bal fluid , each specimen was subjected to four times freezing in liquid nitrogen and thawing in boiling water , and then was crushed with a conical grinder for one minute . for sticky and viscous specimens , 2% dithiothreitol ( wako , japan ) was added , then sonicated for two to five minutes ( 9 ) . finally , dna was extracted from each bal specimen by phenol - chloroform method as described by makimura et al . with a little modification ( dna of four specimens the homogenized bal sediment was added to an equal volume of the lysis buffer ( 100 mm tris - hcl , ph = 8 , 10 mm edta , 0.1% w / v sds , 100 mm nacl , 2% v / v triton 100x ) and an equal volume of phenol chloroform isoamyl alcohol ( 25 : 24 : 1 ) , and was centrifuged at 5'000 g for 10 minutes . chloroform was added to the supernatant and was centrifuged at 5'000 g for five minutes . sodium acetate ( 3 m ; 0.1 volume ) and 2-propanol ( equal volume ) were added to the supernatant and kept at -20c for 10 minutes , and then was centrifuged at 12'000 g for 12 minutes . the precipitated dna was washed with 300 l of 70% ethanol and re - suspended in 30 l of ultrapure water and was kept at -20c . furthermore , the analytical specificity of the pcr was assessed by testing the following fourteen genomic dna of standard fungal strains , which were provided by teikyo university institute of medical mycology ( timm ) , tokyo , japan : a. flavus ( jcm 2061 ) , a. fumigatus ( atcc 26430 , timm 3968 ) , a. niger ( timm 0113 ) , candida tropicalis ( atcc 750 ) , c. albicans ( timm 3313 ) , c. glabrata ( atcc 90030 ) , c. parapsilosis ( atcc 22019 ) , penicillium expansum ( timm 1293 ) , p. notatum ( timm 0883 ) , p. marneffei ( cbs 334.59 ) , fusarium solani ( tsy 0403 ) , trichosporon asahii ( cbs 2479 ) and sporobolomyces koalae ( jcm 18063 ) . all the primers used for the nested pcr had been designed by sugita et al . ( 11 ) . the pcr mixture for contained 12.5 l 2 premix ( ampliqon , denmark ) , 0.5 l ( 30 pmol ) of each primer , and 2 l of the dna template solution , and enough distilled water up to 25 l . the mixture was heated to 95c for five minutes followed by 30 cycles of 94c for 45 seconds ; 60c for one minute ; and 72c for one minute and a final step at 72c for seven minutes . for the species specific primer sets of a. flavus and a. fumigatus , 1 l of the first pcr product ( diluted 1/100 ) was used as a template . each mixture was heated to 95c for five minutes and pcr was performed in cycles of 94c for 50 seconds ; 58c for 40 seconds and 72c for 45 seconds for 25 cycles and a final extention at 72c for eight minutes . the reactions were run with a thermal cycler ( takara pcr thermal cycler dice mini , tp100 . real - time pcr was performed with a 7500 fast real - time pcr system ( applied biosystems ) based on taqman chemistry . the real - time pcr primers and probes were designed based on multiple alignments of various sequences of partial -tubulin gene by zarrinfar et al . as described previously ( 12 ) . all of the real - time pcr primers and probes were obtained from applied biosystems ( usa ) . reactions were performed using eagle taq mastermix with rox ( roche , basel , switzerland ) according to the manufacturer s recommended protocol . each reaction mixture contained 2 l of template dna solution , 0.2 l ( 10.4 m ) of probes a. fumi - p , 0.16 l ( 12.9 m ) of probe a. flavus - p , 0.4 l ( 10 m ) of primer a. fumi - f , 0.4 l ( 10 m ) of primer a. fumi - r , 0.4 l ( 10 m ) of primer a. flavus - f , 0.4 l ( 10 m ) of primer a. flavus - r , 10 l of master mix and enough distilled water up to the final volume of 20 l . following an initial denaturation step at 95c for 10 minutes , pcr amplification was performed for 45 cycles consisting of 95c for three seconds and 60c for 30 seconds dna extracted from a. fumigatus ( timm 3968 ) and a. flavus ( timm 2912 ) were used as positive and three tubes containing water instead of dna were used as negative controls in each run . a. fumigatus and a. flavus pcr products were cloned using a ta cloning kit and pcr 2.1 vector ( invitrogen corp ) . the pcr 2.1 plasmid containing the dna target was sequenced to confirm the insertion of a single copy of amplicon . the quantitation , accuracy , and precision of the real - time pcr assay were validated via preparation of serial dilutions of the plasmid containing the cloned target . the dna concentration was determined according to the absorbance at 260 nm and molecular weight of the plasmid . in addition , the plasmid was used as the positive control in all reaction runs . quantitative results were expressed by determination of the threshold cycle ( ct ) of detection , or crossing point ( cp ) , which marked the cycle at which fluorescence of the specimen became significantly different from the baseline signal . the analysis of the results for a positive specimen was designated when at least one replicate had a ct value of  38 cycles . the efficacy of the dna isolation system in releasing aspergillus dna from the fungal cells for pcr were evaluated by analyzing serial dilutions of a. fumigatus conidia in seven concentrations ( 10 , 10 , 10 , 10 , 10 , 10 and 10 conidia / ml saline ) . forty - six bal specimens were obtained from allogeneic hsct ( n = 18 ) and patients with hematological malignancies ( n = 28 ) , 70% being men ( n = 32 ) with an average age of 38 and 30% being women ( n = 14 ) with an average age of 35 years . among the patients with hematological malignancies , six were all ( acute lymphoblastic leukemia ) ; 11 aml ( acute myeloid leukemia ) ; five had cll ( chronic lymphocytic leukemia ) ; one had nhl ( non - hodgkin lymphoma ) and also five had lymphoma ( table 1 ) . according to the eortc / msg 2008 criteria ( 13 ) , a diagnosis of proven ia can be established by the culture from a sterile tissue biopsy specimen , or the needle aspiration , or the microscopic detection of branched septate hyphae in such specimens with histopathological evidence of the associated tissue damage . is regarded as evidence for probable infection in a high - risk patient with relevant clinical and radiological findings . the radiological findings include typical pulmonary high resolution computed tomographic findings , such as the halo sign , air - crescent sign , or a cavity with a consolidated area , also findings in other tissues suggestive of fungal infection . if the radiological and mycological evidence for ia are not obtained but include the host factor and clinical criteria consistent with the infection , the diagnosis can only be classified as possible . = 1 ) were classified as proven ipa , 21.7% ( n = 10 ) as probable ipa , 41.3% ( n = 19 ) as possible ipa , and 34.8% ( n = 16 ) as not ipa ( table 2 ) . abbreviations : bal , bronchoalveolar lavage fluid ; all , acute lymphoblastic leukemia ; aml , acute myeloid leukemia ; cll , chronic lymphocytic leukemia ; nhl , non - hodgkin lymphoma .   abbreviations : ipa , invasive pulmonary aspergillosis ; all , acute lymphoblastic leukemia ; aml , acute myeloid leukemia ; cll , chronic lymphocytic leukemia ; nhl , non - hodgkin lymphoma ; hsct , hematopoietic stem cell transplants . some of bal specimens had positive results in nested pcr for a. flavus and a. fumigatus , and in real - time pcr for a. flavus and a. fumigatus , together . branched septated hyphae were observed in wet mount preparitons of seven ( 15.2% ) bal specimens , and one tissue biopsy specimen in the direct microscopic examination . eleven ( 23.9% ) specimens were positive in the culture including eight ( 72.7% ) a. flavus and three ( 27.3% ) a. fumigatus . of the 46 specimens tested , 22 ( 47.8% ) were positive by the nested pcr including 13 a. flavus and one a. fumigates specimens , and eight specimens were positive for both a. flavus and a. fumigatus . among the 10 probable ipa specimens , two ( 20% ) showed positive results for a. flavus and three ( 30% ) for both a. flavus and a. fumigatus . among the 19 specimens with possible ipa , five ( 26% ) showed positive results of the nested pcr for a. flavus , 1 ( 5% ) for a. fumigatus and three ( 16% ) for both a. flavus and a. fumigatus . of the 16 specimens with not ipa , five ( 31% ) had positive nested pcr for a. flavus and two ( 12.5% ) were positive for both a. flavus and a. fumigatus . eight specimens showed positive results by real - time pcr , including five for a. flavus and three for a. fumigatus . one of the specimens showed positive results for both a. flavus and a. fumigatus . between the 10 specimens with probable ipa , two ( 20% ) of the , two ( 10.5% ) out of 19 specimens with possible ipa had positive real - time pcr results , which one of them was positive for a. flavus and the other one for both a. flavus and a. fumigatus . two ( 12.5% ) out of the 16 specimens without ipa showed positive real - time pcr results for a. flavus and one ( 6.25% ) showed positive real - time pcr result for a. fumigatus . in the current study , the lower limit of detection ( lod ) of used taqman real - time pcr method was 35 copies per assays for a. fumigatus and 40 copies per assays for a. flavus . specificity assays did not show cross - reaction with dna of the other fungal standard strains . in addition , the lowest detectable number of conidia in 1 ml saline was 10 , which would be detectable by the molecular methods . branched septated hyphae were observed in wet mount preparitons of seven ( 15.2% ) bal specimens , and one tissue biopsy specimen in the direct microscopic examination . eleven ( 23.9% ) specimens were positive in the culture including eight ( 72.7% ) a. flavus and three ( 27.3% ) a. fumigatus . of the 46 specimens tested , 22 ( 47.8% ) were positive by the nested pcr including 13 a. flavus and one a. fumigates specimens , and eight specimens were positive for both a. flavus and a. fumigatus . among the 10 probable ipa specimens , two ( 20% ) showed positive results for a. flavus and three ( 30% ) for both a. flavus and a. fumigatus . among the 19 specimens with possible ipa , five ( 26% ) showed positive results of the nested pcr for a. flavus , 1 ( 5% ) for a. fumigatus and three ( 16% ) for both a. flavus and a. fumigatus . of the 16 specimens with not ipa , five ( 31% ) had positive nested pcr for a. flavus and two ( 12.5% ) were positive for both a. flavus and a. fumigatus . eight specimens showed positive results by real - time pcr , including five for a. flavus and three for a. fumigatus . one of the specimens showed positive results for both a. flavus and a. fumigatus . between the 10 specimens with probable ipa , two ( 20% ) were positive for a. fumigatus . of the , two ( 10.5% ) out of 19 specimens with possible ipa had positive real - time pcr results , which one of them was positive for a. flavus and the other one for both a. flavus and a. fumigatus . two ( 12.5% ) out of the 16 specimens without ipa showed positive real - time pcr results for a. flavus and one ( 6.25% ) showed positive real - time pcr result for a. fumigatus . in the current study , the lower limit of detection ( lod ) of used taqman real - time pcr method was 35 copies per assays for a. fumigatus and 40 copies per assays for a. flavus . specificity assays did not show cross - reaction with dna of the other fungal standard strains . in addition , the lowest detectable number of conidia in 1 ml saline was 10 , which would be detectable by the molecular methods . as a reliable and rapid diagnosis would improve the survival rate in high - risk patients especially among hsct and patients with hematological malignancies , the current study evaluated routine laboratory methods , nested pcr and real - time pcr to diagnose pa due to a. fumigatus and a. flavus . the assay was carefully validated and applied to an analysis of bal specimens . to define ipa , the study used the diagnostic criteria described by the european organization for research and treatment of cancer / invasive fungal infections cooperative group and the national institute of allergy and infectious diseases , mycoses study group ( eortc / msg ) ( 13 ) . infection is one of the main causes of death in hsct ( 14 ) and patients with hematological malignancies ( 15 ) . on time although conventional mycological and histopathological methods are still useful for a definite diagnosis of ipa , new non - invasive diagnostic methods including molecular biomarkers are now available ( 16 ) . these new diagnostic methods facilitate an early diagnosis of invasive fungal disease and allow for utilization of a pre - emptive treatment approach , which may ultimately lead to improved treatment outcomes in hsct and patients with hematological malignancies . although microscopic examination and cultivation of clinical specimens for pa diagnosis are still gold - standard methods ; in general , they do not have enough sensitivity and specificity . furthermore these methods show positive results only in the end stages of infection where an increased fungal burden exists . on the other hand , furthermore , fungal culture is often confounded by antifungal treatment , since the initiation of empirical treatment is a common practice in hsct and patients with hematological malignancies . although there are many published articles on the application of pcr to detect aspergillus dna , to date , a standard commercially developed molecular diagnostic test is not available . real - time pcr assay is widely used to detect fungal pathogens in the molecular studies , and considerably rapid and highly sensitive results are obtained in pediatric patients ( 7 ) . according to the definition provided by eortc / msg ( 13 ) , the ipa incidence of patients examined in the current study was 28% and 21% in hsct and patients with hematological malignancies , respectively . while in the other reported studies ( 3 , 7 , 17 ) , the incidence of ipa was lower than that of the current study . in the current study , 7 ( 15.2% ) specimens were positive in direct examination while the positive results of culture ( 23.9% ) , real - time pcr ( 17.4% ) and nested pcr ( 47.8% ) were higher than it . only two specimens had positive results by all the four methods ; however , two other specimens showed positive results in direct examination , culture and nested pcr together . the reasons why there were mismatches between the various methods is not quite clear . potential explanation includes the possibility of the empirical therapies in these patients ( 18 ) , lower sensitivity of traditional methods and specimen contamination ( 7 ) . moreover , isolation of the fungus from non - sterile specimens such as bal specimen may also reflect colonization of the airway instead of invasive infection ( 19 ) . in the present study , unlike most reports that show a. fumigatus as a common cause of pa , in this study , a. flavus was the most frequent species isolated by culture and pcr while a. fumigatus was the second etiologic agent ( 20 ) . these findings may be consistent with the increase of non - fumigatus aspergillus spp . and or other studies conducted in iran have shown that a. flavus is the most frequent species isolated from patients and air ( 12 , 21 - 23 ) . in the 16 specimens with not ipa , none of them had a positive direct examination and culture but seven and three of the specimens had positive nested pcr and positive real - time pcr , respectively . this may be due contamination and or a hidden suppressed immune system in the patients ( 24 ) . however , in immunocompromised patients with characteristic clinical presentation , observation of aspergillus in culture or pcr assay , even if obtained from sputum or bal , has a high diagnostic value for ipa ( 25 ) . although this conclusion is based on a limited number of patients with hematological malignancies and hsct for whom pa were suspected , the performance was promising . despite this seemingly small number of subjects , the study reports a high number of proven / probable ipa cases in terms of evaluating diagnostic tests or surrogate parameter performance in bal specimens for pa . although there was a weak correlation between the results of several methods that may not increase the diagnosis power , it is useful to make diagnostic decisions especially in patients with hematological malignancies and hsct with clinical signs suspicion to pa . in conclusion , incidence of ipa in allogeneic hsct and patients with hematological malignancies was relatively high and a. flavus was the most common cause of pa . as molecular methods had higher sensitivity , it may be useful as the screening methods in hsct and patients with hematological malignancies , or to determine when empirical antifungal therapy can be withheld .
background : pulmonary aspergillosis ( pa ) is one of the most serious complications in immunocompromised patients , in particular among hematopoietic stem cell transplants ( hsct ) and patients with hematological malignancies.objectives:the current study aimed to evaluate the incidence of pa and utility of molecular methods in hsct and patients with hematological malignancies , four methods including direct examination , culture , nested polymerase chain reaction ( pcr ) and real - time pcr were performed on bronchoalveolar lavage ( bal ) specimens in tehran , iran.patients and methods : during 16 months , 46 bal specimens were obtained from individuals with allogeneic hsct ( n = 18 ) and patients with hematological malignancies ( n = 28 ) . 
 direct wet mounts with 20% potassium hydroxide ( koh ) and culture on mycological media were performed . 
 the molecular detection of aspergillus fumigatus and a. flavus was done by amplifying the conserved sequences of internal transcribed spacer 1 ( its1 ) ribosomal dna by nested - pcr and the -tubulin gene by taqman real - time pcr.results:seven ( 15.2% ) out of 46 specimens were positive in direct examination and showed branched septate hyphae ; 11 ( 23.9% ) had positive culture including eight ( 72.7% ) a. flavus and three ( 27.3% ) a. fumigatus ; 22 ( 47.8% ) had positive nested - pcr and eight ( 17.4% ) had positive real - time pcr . 
 the incidence of invasive pulmonary aspergillosis ( ipa ) in these patients included proven ipa in 1 ( 2.2% ) , probable ipa in 10 ( 21.7% ) , possible ipa in 19 ( 41.3% ) and not ipa in 16 cases ( 34.8%).conclusions : the incidence of ipa in allogeneic hsct and patients with hematological malignancies was relatively high and a. flavus was the most common cause of pa . 
 as molecular methods had higher sensitivity , it may be useful as screening methods in hsct and patients with hematological malignancies , or to determine when empirical antifungal therapy can be withheld .