subcutaneous vit with a standard dose of 100  g is highly effective treatment [ 1 , 2 ] . the indications for vit are a history of an sr , positive venom skin or serum - specific ige antibodies [ 1 , 3 ] , the knowledge of the natural history and risk factors for severe outcome , and impaired quality of life . vit can however have side effects , the most serious being anaphylaxis [ 1 , 6 ] . hb venom and the build - up phase are well - known risk factors for side effects during vit . although there are no definite conclusions regarding the role of the dose increase schedule [ 6 , 7 ] , in a recent european multicentre study of 680 vit patients , the rapid dose increase ( ultrarush > rush > conventional phase ) appears to be a risk factor for side effects during the build - up phase . moreover , mast cell diseases and also an elevated bstc represent other risk factors [ 8 , 9 ] . in europe , vit may be performed using pa and npa venom extracts and paha preparations ( depot preparations ) . the latter of the three is used in the conventional build - up and maintenance phases , while the aqueous preparations are used in ultra - rush , rush , clustered , and maintenance phases . many european specialists switch from the aqueous extracts to paha preparations after updosing , whether or not they cause side effects . the efficacy of the pa and paha extracts is supported by studies using both sting challenge and in - field stings and is comparable to that of nonpurified preparations . in comparative trials , both pa and paha extracts appear to be better tolerated than npa extracts , especially in the prevention of severe large local reactions ( llrs ) [ 11 , 12 ] . as pa extracts and the corresponding npa extracts in hbv - allergic patients have not been compared so far , this study aims to prospectively compare treatment safety in terms of srs by administering pa and the corresponding npa bee - venom extracts during the same rush phase in patients with a normal bstc and evaluate the safety of depot versus npa extracts during a one - year maintenance phase . consecutive hbv - allergic patients with a history of a sr  mueller grade ii   were enrolled for vit with a pa or the corresponding npa extract . a diagnosis of hbv allergy was made if the patient had a history of an sr to a honeybee sting , was positive to skin testing , and/or had serum specific ige to apis mellifera venom . pregnancy and beta - blocker treatment were considered as standard exclusion criteria , as well as mastcell diseases and also a bstc  11.5  g / l ( immunofluorimetric cap assay , unicap 100 ; phadia , uppsala , sweden ) . the local ethics committee approved the study protocol and patients gave their written informed consent . the following extracts were used for treatment : ( 1 ) the traditional npa hbv preparations ( pharmalgen alk - abell aqueous extracts , hrsholm , denmark ) , reconstituted in albumin - containing saline diluent ; ( 2 ) a pa hbv preparation ( aquagen sq , alk - abell ) ; ( 3 ) a purified aluminium - adsorbed hbv depot preparation ( alutard sq , alk - abell ) . pa venom extracts are products with no low - molecular components present in the native venom extract ( cutoff : 1000  d ) . purification is mainly obtained through a sephadex - gel filtration process , which allows separation of the protein fractions by means of their molecular weight . pa venom extracts do not contain vasoactive amines like dopamine , histamine , and serotonin . in addition , a gel - filtration procedure reduces the presence of small peptides like apamin , kinins , and mast cell degranulating peptides in the final product . in the paha extract , the raw venom underwent the same purification procedure with recovery of the allergen - containing fraction only and subsequently adsorbed onto aluminium hydroxide . the patients underwent a 5-day rush vit regimen ( table 1 ) with the aqueous preparations and were split into groups a ( 40 patients ) and b ( 40 patients ) , which were administered npa ( pharmalgen alk - abell ) and pa extracts ( aquagen sq , alk - abell ) , respectively . patients experiencing srs during the build - up phase from both groups were switched from the aqueous to the paha extract and received weekly interval injections of up to 100  g of venom . during the maintenance phase , group a patients who developed no rush - phase srs went on to receive maintenance therapy with the non - purified aqueous extract while group b patients who tolerated the build - up phase were also treated with the paha preparation . only those patients with a history of anaphylaxis undergoing rush vit were hospitalised in our clinic . development of an sr during the rush - phase necessitated suspending treatment until the patient made a complete recovery , after which a depot extract was used . in the event of a large local or a mild sr , patients were instructed to immediately report any suspected delayed vit - related reactions experienced at home . the use of hospital sting challenges remains an ethical issue in italy and is generally avoided for testing vit efficacy . this being the case , patients were instructed to report details of any reaction to a field sting and any pharmacological treatment . to assess treatment efficacy in non beekeepers , only those stings typically attributable to bees , recognisable by the embedded stinger at the sting site , differences between the two groups in outcome variables ( i.e. , occurrence of srs ) were assessed using fisher 's exact test ( whenever an expected cell value was < 5 ) and the -square test . the number needed to harm ( nnh ) was calculated according to mcquay and moore . a multiple - logistic regression analysis was performed to identify clinical and demographic variables ( age , gender , reaction severity , baseline stc , type of vit , purified versus non - purified ) associated with the occurrence of srs during vit . the unpaired t - test and mann - whitney test , when appropriate , were used to compare quantitative variables . a two - sided p value < 0.05 was considered significant for all analyses . the power calculation assumed a difference between pa and npa treated subjects in the incidence of srs of at least 65% less ( relative risk reduction ) . this assumption provided 80% power at an alpha level of 0.05 for a sample size of at least 40 evaluable patients per group . ninety - seven consecutive patients with a history of an sr to hb stings were evaluated for the study , of whom 17 ( 17.5% ) were excluded on the grounds of a bstc  11.5  g / l ( figure 1 ) , leaving 80 to be allocated to two treatment groups : 40 in group a and 40 in group b. prior to vit , 16 patients ( 20% ) had experienced a grade ii sr , 28 patients ( 35% ) a grade iii sr , and 36 patients ( 45% ) a grade iv sr . the two groups of patients ( cases and controls ) were comparable with respect to age , sex , bstc and sr severity ( table 2 and figure 2 ) . the cumulative doses during the tolerated 5-day rush - phase was 223.11  g of hbv . eleven ( 27.5% ) ( 95%ci : from 13 to 41% ) group a patients developed an immediate sr : 6 were grade i reactions and 5 grade ii ( table 3 ) . treatment was suspended in these patients until they fully recovered , and in almost without exception vit was resumed with the equivalent dosage of the paha extract . a maintenance dose of 100  g was reached by weekly interval injections , with no side effects . thirty - nine group b patients tolerated the rush - phase and then switched to the paha extract for the hbv 100  g maintenance therapy with no side effects ( figure 3 ) . one ( 2.5% ) ( 95% ci : from 2 to 7% ) group b patients developed a grade i sr ( table 3 ) and switched from the aqueous purified extract to the paha extract ( figure 3 ) , with no sr , successfully reaching the maintenance dose of 100  g , with the result that patients treated with the npa extract during the rush phase experienced significant more frequent srs than those who underwent vit with the pa extract ( p = 0.0017 fisher 's exact test ) . the absolute risk reduction was 25% , and the relative risk ( purified versus nonpurified extracts ) was 91% ( 95% ci from 100 to 53 ) with a nnh of 4.2 . in the rush induction phase , the incidence of srs per total number of injections ( i.e 634 ) was 0.1%/doses in the purified - extract - treated patients ( group a ) compared to 1.9%/doses in the nonpurified - extract - treated group of patients ( total number of injections 555 ) ( p = 0.0017 chi - squared test ) . no patient in group a or b experienced late srs . maintenance dose was 100  g in all patients . twenty - nine group a patients underwent one - year maintenance treatment with the npa extract , while 51 ( 11 group a and 40 group b patients ) were given the paha extract ( figure 3 ) . the mean ( sd ) value of bstc was 5 ( 2.3 ) g / l ( median 4.5 ) and 5.3 ( 2 ) g / l ( median 4.7 ) in group a and b patients , respectively ( p =   n.s . ) ( figure 2).the mean value of bstc was 5,6  g / l ( median 5.5 ) in patients who developed srs and 5.3  g / l ( median 5.4 ) in those who did not ( p =   n.s . ) multiple logistic regression analysis did not reveal a significant correlation between the occurrence of an sr during vit with age , gender , reaction severity , or basal stcs , while , in contrast , the type of extract ( npa ) correlated significantly ( p = 0.0001 ) . thirteen out of the 29 ( 44.8% ) npa extract treated patients were re - stung during the one year maintenance phase without developing a reaction . fifteen out of the 51 patients ( 13.6% ) treated with the paha extract during the maintenance phase were re - stung without side effects , of whom one developed a mild sr during vit . while probably being the most effective form of allergen immunotherapy currently available to physicians [ 1 , 2 , 10 ] , subcutaneous vit is at the same time able to improve health - related quality of life . however , the treatment can cause side effects ranging from an llr to a severe sr . a recent paper comparing purified preparations with non - purified extracts reviewed the literature on the respective safety and effectiveness of the two . the authors found that in comparative trials purified extracts appear to be better tolerated than non - purified extracts , while paha extracts seem to be safer than the corresponding pa preparation , especially in the prevention of severe llrs . also , they concluded that further prospective - controlled studies are needed in order to evaluate the ability of purified extracts to reduce the frequency of srs over the corresponding non - purified preparation . this is the first study comparing the safety of purified and the corresponding non - purified aqueous venom preparations in terms of srs in hbv - allergic patients with a normal bstc under the same build - up protocol . we studied hv - venom - allergic patients as their risk of developing an sr during vit is greater than that of vespid - allergic patients . the scope of our paper is confined to the study of vit - induced srs . llrs , though frequent and bothersome , do not usually necessitate a dose reduction and do not prevent from reaching the full maintenance dose . patients with a bstc  11.5  g / l were excluded as this factor itself is a potential risk for side effects during vit . indeed , a recent paper reports that a rise in the bstc from 4.21 g / l to 20  g / l only in vespid allergic patients is accompanied by a simultaneous increase in requirement for emergency intervention during the build - up phase by a factor of approximately 3.75 . in this study , we compared the same build - up protocol in two homogeneous groups of patients who were treated with two different extracts from the same company . we have demonstrated that patients treated with the non - purified hbv experienced significantly more srs than those treated with the purified hbv preparation , due to a higher occurrence of srs during the build - up phase . in fact , during the same 5-day rush phase , 27.5% of the npa - extracts - treated patients developed an sr compared with 2.5% of the pa - extract - treated subjects ( p = 0.0017 ; nnh = 4.2 ) . reactions were nonsevere with six patients developing a grade i and five a grade ii reaction . the lower frequency of srs following injections of the pa preparations compared with the npa extracts , as demonstrated by our study , may be due to the former 's purification of low - molecular - weight irritants , with recovery of the allergen - containing fraction only . this finding can not be attributed to the cumulative dose applied over a given span of time as the two groups of patients underwent the same 5-day rush protocol . patients who had srs during the build - up phase were switched to the paha extract from the npa solution , as were those treated with the pa extract whether they had experienced an sr or not . during the one - year maintenance phase , no srs occurred in either group . though there was no control group , our data indirectly confirm the safer profile of the paha extracts . in fact , all those patients who experienced pa- or npa - induced srs during the rush phase could have benefited from being switched to the same dosage of a paha preparation and have subsequently achieved the 100  g maintenance dose with weekly interval injections . to date , the only study which compares pa and paha extracts with the corresponding npa preparations dates back to 1986 and was done in yellow - jacket - venom- ( yjv- ) allergic patients . the authors demonstrated that pa - induced srs were milder than those caused by npa preparations , but more frequent with the paha extracts . in hbv - allergic patients , a few studies compare the paha extract with the pa extract under different protocols [ 1618 ] , but none with the corresponding npa extract . in hbv - allergic patients , rueff demonstrated that treatment with the pa extract caused significantly more frequent llrs than the depot extract during the 5-day rush and maintenance phases . on the other hand , though not significant , aqueous preparations induced srs more frequently during the updosing phase . another study of hbv - allergic patients , in which the safety of the pa and paha extracts and of three different induction protocols were compared , demonstrated that depot cluster vit was better tolerated than pa rush vit , which in turn was less well tolerated than the pa cluster protocol . the efficacy of the pa and paha preparations is supported by studies implementing sting challenges in hbv- and yjv - allergic patients and is comparable to that of non - purified extracts . in - hospital this procedure , however , remains an ethical issue in italy , making in - field stings the only available feedback on the efficacy of the treatment . though the observational period was over one year and only a few patients had in - field restings , our experience shows that the efficacy of the non - purified and purified extracts is highly comparable . our study suffers from a few limitations which need to be taken into account from a results point of view . firstly , we conducted an unblinded open trial . despite this , the main outcome was the incidence of srs which should be considered as the  hard  end point . also , the clinical definition of sr is well defined and accepted with a low margin to subjectivity . secondly , the sample size required to verify the trial hypothesis was calculated in advance and the relative risk reduction ( rrr ) in the number of srs we observed ( 91% ) in the purified extract - treated patients was far greater than the rrr forecast in calculation of the sample size ( 65% ) . in conclusion , our study demonstrates the superiority of the pa extract in hbv - allergic subjects over the corresponding non - purified extracts under the same rush incremental phase . though similar in terms of their efficacy , hb pa extracts are safer option than npa preparations for specialists who perform rush vit , followed by maintenance treatment with the paha preparation . the use of the hb depot extracts for the conventional incremental and maintenance phases could be proposed as a workable solution for specialists with less experience in managing vit . moreover , in patients with srs caused by both pa and npa extracts , the switch to a paha extract could be safely made and allow the generally adequate maintenance dose of 100  g of venom to be reached .
in this study , we compared a purified aqueous extract and the corresponding nonpurified aqueous preparation under the same build - up protocol in bee venom allergic patients with a normal baseline mast cell tryptase concentration . 
 eighty patients with a history of a systemic reaction were enrolled for immunotherapy using a 5-day rush protocol . 
 patients treated with the purified extract and those treated with the non purified aqueous extract who developed a systemic reaction underwent maintenance therapy with the purified aluminium hydroxide adsorbed preparations . 
 patients treated with the nonpurified aqueous extract who did not experience a systemic reaction during the rush phase underwent the maintenance phase with that extract . 
 systemic reactions during the build - up phase occurred significantly more often in patients treated with nonpurified aqueous extract than in those treated with the corresponding purified aqueous preparations . during the one - year maintenance phase , no systemic reactions occurred in either of the groups . 
 neither age nor baseline mast cell tryptase concentration presented a significant correlation with the occurrence of a systemic reaction during the treatment , while the type of extract did . in conclusion , nonpurified aqueous extracts induced more frequent systemic reactions than the purified aqueous preparations , during the same rush protocol . 
 the efficacy seemed to be comparable .