
 p. brasiliensis isolates and patient sera - five human 
 p. brasiliensis isolates were included in this study , one 
 belonging to the s1 species from botucatu , state of so paulo ( sp ) , brazil ( pb265 ) , 
 one of the ps3 species from colombia ( epm83 ) and the other three belonging to 
 p. lutzii from goinia , state of gois , brazil ( pb01 , pb8334 
 and pb66 ) .
the isolates were maintained on bbl mycosel 
 agar ( bd biosciences , sparks , md , usa ) and transformed into the yeast phase on 4% 
 glucose , 1% peptone , 0.5% yeast extract and 1% agar medium .
furthermore , the antigen 
 obtained via filtration of a b-339 culture , considered the standard antigen for 
 immunodiffusion assays , was added to the analyses ; this isolate was maintained on 
 potato agar medium and transformed into yeast on fava - netto agar .
regarding the sera from pcm patients , 71 samples were evaluated and 
 divided regionally , as follows : 20 serum samples from botucatu , 20 from jundia and 
 31 from the central - west region of brazil .
the sera from the botucatu region were 
 stored in the serum bank at the laboratory of tropical diseases - mycology area of 
 botucatu medical school of so paulo state university , whereas the samples from the 
 jundia and central - west regions were stored in the adolfo lutz institute / sp 
 collection . 

preparation of culture - filtered antigen - the yeast 
 cells from each fungal isolate were transferred to 10 slant cultures containing 
 fava - netto agar .
after five days of growth at 36c , the contents of the tubes were 
 inoculated into 500 ml 2% dextrose , 1% neopeptone , 0.018% thiamine and 0.036% 
 asparagine medium and incubated at 35c for 15 days with agitation at 100 rpm .
g / l final 
 concentration ) for four days under the same conditions and then filtered through 
 filter paper .
the preparations were concentrated using polyethylene glycol until 10% 
 of the initial volume was reached .
the preparations were then dialysed against 
 phosphate buffered saline ( pbs ) at ph 7.2 for three days at 4c . after this period , 

1.0-ml aliquots of the samples were separated , lyophilised and , at the moment of 
 use , resuspended in 20 l of pbs .
other methodologies were also tested to produce such antigens as the 
 somatic antigen ( franco et al .
1996 ) and 
 the  cell - free  antigen ( camargo et al .
1991 
 ) , which , in our experience , proved to be less sensitive than the culture - filtered 
 antigen in immunodiffusion assays ( data not shown ) . 

did assays - carefully cleaned slides were pre - coated 
 with 1.0 ml 1% agar solution , allowed to dry at 60c for 24 h and then coated with 
 3.0 ml citrate agar solution ( 1% purified agar , 0.9% sodium chloride , 0.4% sodium 
 citrate , 7.5% glycine or amino acetic acid and 0.01% thimerosal ) .
the citrate agar 
 was solidified at room temperature ( rt ) and the slides were stored in a humidified 
 chamber .
the medium on the slides was bored with the aid of a rosette - shaped mould 
 containing one central and six peripheral holes , each equidistant from the central 
 hole .
the slides received 10 l antigen ( central well ) , 10 l polyclonal anti- 
 p. brasiliensis antibody , the positive reaction control and 10 
 l test serum ( peripheral wells ) , and were incubated in a humidified chamber for 48 
 h at rt . after this period , the slides were incubated in 0.5% sodium citrate 
 solution for 45 min and in saline solution for 18 h. the wells were then covered 
 with 1% citrate agar and the slides were wrapped in humid filter paper and allowed 
 to dry for 8 h at 60c .
staining was performed for 10 min with 0.4% schwartz starch 
 solution and 10% glacial acetic acid or coomassie blue .
destaining was performed in 
 5% glacial acetic acid solution , twice for 10 min each .
the presence of a 
 precipitation line corresponded to an antigen - antibody pair and indicated a positive 
 reaction .
the most dubious results were subjected to one or , if necessary , two 
 replicates .
sodium dodecyl sulphate - polyacrylamide gel electrophoresis 
 ( sds - page ) - the gels and reagents for sds - page were prepared as 
 previously described ( laemmli 1970 ) ; a 10% 
 resolving gel and 3% acrylamide stacking gel were used .
after electrophoresis , the 
 gels were stained with coomassie blue solution ( 0.1% coomassie blue , 45% methanol 
 and 10% glacial acetic acid ) and the excess stain was removed with destaining 
 solution ( 10% glacial acetic acid and 10% methanol ) . 

analysis of pbgp43 exon 2 selection pattern - the 
 sequences of pb gp43 exon 2 available in genbank were employed : 10 
 haplotypes of p. lutzii ( accessions : eu870212-eu870214 , eu870217 , 
 eu870218 , eu870220 , eu870221 and eu870226-eu870228 ) ( teixeira et al . 2009 ) and 11 from the other cryptic species 
 ( accessions : dq003724 , dq003741 , dq003744 , dq003746 , dq003748-dq003750 , 
 dq003771-dq003773 and dq003781 ) ( matute et al . 
 2006 ) were used .
the sequences were aligned using the software mega4 and 
 the synonymous and nonsynonymous substitution rates ( ds / dn ) were calculated pairwise 
 on the syn - scan website ( gonzales et al . 

2002 ) according to the method of nei - gojobori ( nei & gojobori 1986 ) , yielding a mean ds / dn value .

 double agar gel immunodiffusion testthe results of did 
 assays performed using 71 serum samples from the botucatu , jundia and central - west 
 regions are shown in table i . only 10 sera 
 from the botucatu region reacted against the standard antigen agb-339 at different 
 titrations .
the remaining 10 sera corresponded to patients with active disease or 
 confirmed pcm , but with no reactivity according to did .
of the five antigen 
 preparations evaluated , the one obtained from the ps3 isolate , epm83 ( agepm83 ) , 
 showed the highest reactivity index for the samples from the botucatu region . in 
 addition to reacting with three sera that were non - reactive to the standard antigen , 

this antigen was capable of detecting the presence of antibodies in all samples 
 reactive to b-339 . agepm83 and agb-339 presented a substantial strength of agreement 
 in the botucatu region , as represented by a kappa coefficient ( k ) of 0.7 ( table ii ) . in contrast , agpb265 ( s1 ) and 
 agpb01 ( p. lutzii ) did not react with any of the 20 serum samples 
 from the botucatu patients .
the other two antigens produced from the p. 
 lutzii isolates agpb8334 and agpb66 showed identical results ( k = 1.0 ) , 
 reacting with the same eight sera among the 10 positive for agb-339 and with none of 
 the 10 sera non - reactive to agb-339 .
the strength of agreement between these 
 antigens ( agpb8334 and agpb66 ) and agb-339 was also substantial ( k = 0.8 ) . 

table ireactivity profile shown by culture - filtered antigens in 
 immunodiffusion assays against sera from patients from botucatu , jundia 
 and central - west regions of brazil , reactive or not to the standard 
 antigen agb-339reactivity n ( % ) 
 agepm83agpb265agpb01agpb8334agpb66botucatureactive sera agb-339 ( n = 10)10 ( 100)0 ( 0)0 ( 0)8 ( 80)8 ( 80)non - reactive sera agb-339 ( n = 10)3 ( 30)0 ( 0)0 ( 0)0 ( 0)0 ( 0)jundiareactive sera agb-339 ( n = 10)7 ( 70)2 ( 20)2 ( 20)2 ( 20)3 ( 30)non - reactive sera agb-339 ( n = 10)2 ( 20)0 ( 0)3 ( 30)3 ( 30)3 ( 30)central - west regionreactive sera agb-339 ( n = 11)4 ( 36)1 ( 9)0 ( 0)3 ( 27)4 ( 36)non - reactive sera agb-339 ( n = 20)0 ( 0)0 ( 0)0 ( 0)0 ( 0)0 ( 0 ) 
 
 table iidistribution of patients with active paracoccidioidomycosis from 
 different regions as to the hierarchical weights of agreement in 
 serological measuresorigen of the patientscomparisonsagreements ( + + ) ( - -)disagreements ( + - ) ( - + ) kappa coefficientconfidence intervalstrength of agreementmc nemar s testbotucatub339vsepm83107030.700.40 - 1.00substantial0.08  b339vspb2650101000.000.00 - 0.44slight < 0.01  b339vspb010101000.000.00 - 0.44slight < 0.01  b339vspb8334810200.800.54 - 1.00substantial0.16  b339vspb66810200.800.54 - 1.00substantial0.16pb01vspb66012800.000.00 - 0.54slight < 0.01pb01vspb8334012800.000.00 - 0.54slight < 0.01pb66vspb8334812001.001.00 - 1.00perfect0.32pb01vsepm83070130.000.00 - 0.32slight < 0.01pb66vsepm8387050.530.17 - 0.89moderate0.0254pb8334vsepm8387050.530.17 - 0.89moderate0.0254jundiab339vsepm8378320.500.12 - 0.88moderate0.65 
b339vspb265210800.200.00 - 0.46slight < 0.01  b339vspb0127830.000.00 - 0.28slight0.13  b339vspb833427830.000.00 - 0.28slight0.13  b339vspb6637730.000.00 - 0.40slight0.21pb01vspb66413120.620.23 - 1.00substantial0.5637pb01vspb8334515001.001.00 - 1.00perfect0.32pb66vspb8334413210.620.23 - 1.00substantial0.5637pb01vsepm83410150.370.00 - 0.79fair0.1025pb66vsepm8349250.270.00 - 0.71fair0.2568pb8334vsepm83410150.370.00 - 0.79fair0.1025central - west regionb339vsepm83420700.420.12 - 0.73moderate < 0.01  b339vspb2651201000.110.00 - 0.32slight < 0.01  b339vspb010201100.000.00 - 0.26slight0.01  b339vspb8334320800.330.03 - 0.62fair < 0.01  b339vspb66420700.420.12 - 0.73moderate < 0.01pb01vspb66027040.000.00 - 0.91slight0.0455pb01vspb8334028030.000.00 - 1.00slight0.0833pb66vspb8334327100.840.53 - 1.00high0.3173pb01vsepm83027040.000.00 - 0.90slight0.0455pb66vsepm83427001.001.00 - 1.00perfect0.32pb8334vsepm83327010.840.53 - 1.00high0.3173sodium dodecyl sulphate - polyacrylamide gel electrophoresis in 10% 
 acrylamide with antigens filtered from culture of paracoccidioides 
 brasiliensis stained with coomassie blue .
lines 1 , 7 : pageruler 
  plus prestained protein ladder ( fermentas ) ; 2 : 
 epm83 ; 3 : pb265 ; 4 : pb01 ; 5 : pb8334 ; 6 : pb66 . 
 
sodium dodecyl sulphate - polyacrylamide gel electrophoresis in 10% 
 acrylamide with antigens filtered from culture of paracoccidioides 
 brasiliensis stained with coomassie blue .
lines 1 , 7 : pageruler 
  plus prestained protein ladder ( fermentas ) ; 2 : 
 epm83 ; 3 : pb265 ; 4 : pb01 ; 5 : pb8334 ; 6 : pb66 . regarding the samples from the jundia region ( 10 sera reactive and 10 
 sera non - reactive to agb-339 ) , again , agepm83 presented the highest reactivity rate 
 among the five antigen preparations .
this antigen reacted with seven of the 10 
 samples reactive to b-339 and with two of the 10 non - reactive samples .
however , the 
 k of these antigens was 0.5 ( moderate ) , demonstrating a weaker agreement in 
 comparison to their performance in the botucatu region .
agpb265 displayed an 
 unsatisfactory performance , reacting with only two serum samples ( 20% ) that were 
 reactive to the standard antigen .
a similar reactivity pattern was noted for the 
 three antigens obtained from the p. lutzii isolates in sera 
 reactive to the standard antigen : agpb01 ( 20% ) , agpb8334 ( 20% ) and agpb66 ( 30% ) . 

these antigens also showed reactivity to three of the 10 samples negative for the 
 standard antigen .
the assessment of agepm83 performance against the 40 serum samples from 
 the botucatu and jundia regions , considered to be endemic for p. 
 brasiliensis , indicated that this antigen preparation was capable of 
 discriminating among 22 samples , whereas agb-339 reacted with 20 .
in addition , 
 agepm83 reacted with five of the 20 ( 25% ) sera that were non - reactive to the 
 standard antigen .
interestingly , the p. lutzii antigens reacted 
 with five of the serum samples from the jundia region , three of which had not shown 
 reactivity against the antigen agb-339 . considering the 31 serum samples from the central - west region of brazil , 
 11 showed serological reactivity against the b-339 antigen and none of the evaluated 
 antigen preparations was capable of reacting with more than four of these 11 serum 
 samples . similarly , none of the 20 sera non - reactive to the standard antigen was 
 reactive to any of these antigens .
all of the antigens showed low k values when 
 compared to the standard antigen .
all the antigen preparations were also evaluated for specificity by a did 
 assay against nine serum samples from patients with confirmed histoplasmosis and no 
 cross - reaction was noted against the heterologous sera . 

sds - page - among the antigen preparations , agepm83 had 
 the most evident gp43 level by sds - page ( figure ) and also exhibited an intense band 
 near 90 kda .
the antigens obtained from the p. lutzii isolates 
 showed several common bands between 100 - 60 kda . agpb01 and agpb8334 showed 
 additional bands at 43 kda , but of lower intensity than the signal for agepm83 . 

analysis of ds / dn rate of pbgp43 exon 2 - exon 2 of the 
 pb gp43 gene from the p. lutzii and p. 
 brasiliensis groups had a ds / dn mean rate higher than 1.0 
 ( supplementary data ) , indicating that the isolates in this region have undergone 
 purifying selection and amino acid sequence conservation ( nei & kumar 2000 ) .
however , the mean ds / dn ratio for the 
 p. lutzii group ( 1.13 ) was lower than for the p. 
 brasiliensis group ( 1.52 ) , suggesting that the gp43 amino acid sequence 
 is more variable among the p. lutzii isolates .
comparing the five antigen preparations , the antigen obtained from the 
 epm83 isolate had a reactivity profile similar to the profile observed for the 
 standard antigen ( agb-339 ) against the serum samples from botucatu .
an analysis of 
 the antigenic profile of culture filtrates by sds - page indicated that agepm83 had a 
 more evident band at 43 kda compared to the other antigens .
this finding confirms 
 the importance of the 43-kda glycoprotein in pcm immunodiagnosis ( puccia et al .
1986 ) , at least for regions in 
 which the s1 and ps2 species occur ( batista jr et al .
it must be highlighted 
 that the epm83 isolate belongs to the species ps3 and not to the species occurring 
 in the botucatu and jundia regions .
regardless , ps3 isolates are known to be 
 phylogenetically close to s1 ( matute et al . 
 2006 , theodoro et al .
the reaction of agepm83 , associated with high gp43 secretion , with sera 
 non - reactive to the standard antigen in the botucatu and jundia regions suggests 
 that this specific antigen may serve as an alternative when the standard antigen 
 ( agb-339 ) is not capable of detecting circulating anti- p. 
 brasiliensis antibodies in the residents of regions in which the 
 particular species s1 and ps2 occur .
however , certain samples reactive to agb-339 
 from the jundia region did not show reactivity against agepm83 , resulting in a 
 moderate strength of agreement between these antigens in this region .
this finding , 
 associated with the low positivity of agepm83 reactivity with serum samples from the 
 central - west region of brazil , indicates that it is not possible to use only a 
 single antigen preparation to diagnose a disease caused by highly diverse agents , 
 particularly in such a large country as brazil .
additionally , other authors have 
 noted that an antigen produced from an isolate from the central - west region of 
 brazil was capable of reacting with a larger number of sera from that region in 
 comparison to the standard antigen from an isolate from sp ( b-339 ) .
nevertheless , 
 when tested with sera from sp , the antigen from central - west of brazil did not 
 produce satisfactory results ( batista jr et al .
these findings confirm the 
 necessity of using different strains to produce antigens and indicate the epm83 
 isolate as a potential alternative that should be further studied .
the present study confirmed the practical difficulties in producing 
 antigens reactive to sera from patients from the central - west region of brazil , even 
 against three isolates of p. lutzii , a genotype that appears to be 
 the most prevalent in this region .
this difficulty must be related to the greater 
 diversity of gp43 in this species . given its higher nonsynonymous mutation rate 
 compared to the isolates of the p. brasiliensis group , the amino 
 acid sequence of this glycoprotein must be more variable in p. 
 lutzii isolates .
thus , patients from central - west of brazil would be 
 sensitised by various gp43 forms , which could explain the difficulty in producing a 
 single antigen reactive in all patients from this region .
these facts cast doubt on 
 the potential of gp43 as a universal antigen for all species in immunodiffusion 
 assays , thus requiring a search for new alternatives .
in addition to constituting a 
 problem in serological diagnosis , the variability of this antigenic portion could 
 represent an escape mechanism for avoiding host immune responses ( matute et al .
1995 ) , p. brasiliensis strains 
 were observed to express variable quantities of gp43 ( berzaghi et al . 2005 ) .
this result corroborates the difficulty 
 in using this molecule for the universal serological diagnosis of pcm because a 
 patient infected with an isolate that produces gp43 at low rates will not show 
 antibodies specific for this antigen at detectable concentrations . indeed , the 
 determination of variations in the expression of genes encoding gp43 in p. 
 brasiliensis is long - standing , but still unresolved problem .
although a 
 large number of isolates from this group remain to be evaluated , our results suggest 
 that isolates of the p. lutzii group generally have low gp43 
 production .
speciation events , however , are not sufficient to fully explain the 
 variation in gp43 rates because not all the isolates of the s1 , ps2 and ps3 species 
 produce high quantities of this molecule .
eventual variations in the promoter region 
 of the gp43 gene among the isolates and interference in posttranscriptional 
 processes , such as alternative splicing and silencing by rna interference , can 
 influence this variability . in summary , considering speciation within p. 
 brasi - liensis is important for the serological diagnosis of the 
 disease , particularly for the more distant genotypes represented by the p. 
 lutzii group . regarding the group represented by the s1 and ps3 
 genotypes , the most relevant factor in serological diagnosis is the expression of 
 gp43 , which is highly variable among isolates . 

mutation pattern observed at pb gp43 exon 2 region in haplotypes of 
 the paracoccidioides lutzii group seq 1   seq 2   sd   nd   s   n   ps   pn   ds   dn   ds / dn   01   7455   0.00   2.00   96.50   326.50   0.00   0.01   -0.00   0.01   *   01   84   0.00   1.00   96.67   326.33   0.00   0.00   -0.00   0.00   *   01  
6810   0.00   3.00   96.50   326.50   0.00   0.01   -0.00   0.01   *   01   717   0.00   2.00   96.50   326.50   0.00   0.01   -0.00   0.01   *   01   3171   0.00   2.00   96.50   326.50   0.00   0.01  
-0.00   0.01   *   01   189   1.00   1.00   96.17   326.83   0.01   0.00   0.01   0.00   3.42   01   218   0.00   2.00   96.67   326.33   0.00   0.01   -0.00   0.01   *   01   133   12.75   37.25   96.67   326.33   0.13   0.11   0.15   0.12   1.17   01   769   9.62   39.38   97.17   325.83   0.10   0.12   0.11   0.13   0.81   7455   84   0.00   1.00   96.83   326.17   0.00   0.00   -0.00   0.00   *   7455   6810   0.00   1.00   96.67   326.33   0.00   0.00   -0.00   0.00   *   7455   717   0.00   0.00   96.67   326.33  
0.00   0.00   -0.00   -0.00   *   7455   3171   0.00   2.00   96.67   326.33   0.00   0.01   -0.00   0.01   *   7455   189   1.00   3.00   96.33   326.67   0.01   0.01   0.01   0.01   1.13   7455   218   0.00   2.00   96.83   326.17  
0.00   0.01   -0.00   0.01   *   7455   133   12.75   37.25   96.83   326.17   0.13   0.11   0.14   0.12   1.17   7455   769   9.62   39.38   97.33   325.67   0.10   0.12   0.11   0.13   0.80   84   6810   0.00   2.00   96.83   326.17   0.00   0.01   -0.00   0.01   *   84   717   0.00   1.00   96.83   326.17   0.00   0.00   -0.00   0.00   *   84   3171   0.00   1.00   96.83   326.17   0.00   0.00   -0.00   0.00   *   84   189   1.00   2.00   96.50   326.50   0.01   0.01   0.01   0.01   1.70   84   218   0.00   1.00   97.00   326.00   0.00   0.00   -0.00   0.00   *   84   133   12.75   36.25   97.00   326.00   0.13   0.11  
0.14   0.12   1.20   84   769   9.62   38.38   97.50   325.50   0.10   0.12   0.11   0.13   0.83   6810   717   0.00   1.00   96.67   326.33   0.00   0.00   -0.00   0.00   *   6810   3171   0.00   3.00   96.67   326.33   0.00   0.01   -0.00   0.01   *   6810   189   1.00   4.00   96.33   326.67   0.01   0.01   0.01   0.01   0.85   6810   218   0.00   3.00   96.83   326.17   0.00   0.01   -0.00   0.01   *   6810   133   12.75   38.25   96.83   326.17   0.13   0.12   0.14   0.13   1.14   6810   769   9.62   40.38   97.33   325.67   0.10   0.12   0.11   0.14   0.78   717   3171   0.00   2.00   96.67   326.33   0.00   0.01   -0.00   0.01   *   717   189   1.00   3.00   96.33   326.67   0.01  
0.01   0.01   0.01   1.13   717   218   0.00   2.00   96.83   326.17   0.00   0.01   -0.00   0.01   *   717   133   12.75   37.25   96.83   326.17   0.13   0.11   0.14   0.12   1.17   717   769   9.62   39.38   97.33   325.67   0.10   0.12   0.11   0.13   0.80   3171   189   1.00   3.00   96.33   326.67   0.01   0.01   0.01   0.01   1.13   3171   218   0.00   2.00   96.83   326.17   0.00   0.01   -0.00   0.01   *   3171   133   12.75   37.25   96.83   326.17   0.13   0.11   0.14   0.12   1.17   3171   769   9.62   39.38   97.33   325.67   0.10   0.12   0.11   0.13   0.80   189   218   1.00   3.00   96.50   326.50   0.01   0.01  
0.01   0.01   1.13   189   133   11.75   37.25   96.50   326.50   0.12   0.11   0.13   0.12   1.07   189   769   10.62   39.38   97.00   326.00   0.11   0.12   0.12   0.13   0.90   218   133   12.75   37.25   97.00   326.00   0.13   0.11   0.14   0.12   1.17   218   769   9.38   39.62   97.50   325.50   0.10   0.12   0.10   0.13   0.77   133   769   3.00   11.00   97.50   325.50   0.03   0.03   0.03   0.03  
0.91   mean   4.19   15.09   96.80   326.20   0.04   0.05   0.05   0.05   1.13 dn : jukes - cantor correction for multiple values of proportion of 
 observed nonsynonymous mutations ( pn ) ; ds : jukes - cantor correction 
 for multiple values of proportion of observed synonymous mutations 
 ( ps ) ; ds / dn : rate of synonymous mutations over nonsynonymous 
 mutations ; n : number of potential nonsynonymous substitutions ; nd : 
 number of observed nonsynonymous mutations ; s : number of potential 
 synonymous substitutions ; sd : number of observed synonymous 
 mutations .
asterisk means that ds is not significant due to the high 
 similarity among sequences . 
 
dn : jukes - cantor correction for multiple values of proportion of 
 observed nonsynonymous mutations ( pn ) ; ds : jukes - cantor correction 
 for multiple values of proportion of observed synonymous mutations 
 ( ps ) ; ds / dn : rate of synonymous mutations over nonsynonymous 
 mutations ; n : number of potential nonsynonymous substitutions ; nd : 
 number of observed nonsynonymous mutations ; s : number of potential 
 synonymous substitutions ; sd : number of observed synonymous 
 mutations .
asterisk means that ds is not significant due to the high 
 similarity among sequences . 

mutation pattern observed at pb gp43 exon 2 region in haplotypes of 
 the paracoccidioides brasiliensis group seq 1   seq 2   sd   nd   s   n   ps   pn   ds   dn   ds / dn   a4   v3   1.00   5.00   97.83   325.17   0.01   0.02   0.01   0.02   0.66   a4   v2   2.00   11.00   97.83   325.17   0.02   0.03   0.02   0.03   0.60   a4   v1   2.00   5.00   97.83   325.17   0.02   0.02   0.02   0.02   1.33   a4   c1   0.00   4.00   97.83   325.17   0.00   0.01   -0.00   0.01   *   a4   b26   1.00   10.00   97.83   325.17   0.01   0.03   0.01   0.03   0.33   a4   b25   0.00   1.00   98.00   325.00   0.00   0.00   -0.00   0.00   *   a4   b23   2.00   11.00   97.50   325.50   0.02   0.03   0.02   0.03   0.60   a4   b21   0.00   3.00   97.83   325.17   0.00   0.01   -0.00   0.01   *   a4   b18   0.00   5.00   97.83   325.17   0.00   0.02   -0.00   0.02   *   a4   b1   1.00   1.00   98.00   325.00   0.01   0.00   0.01   0.00   3.33   v3   v2   3.00   8.00   97.33   325.67   0.03   0.02   0.03   0.02   1.26   v3   v1   1.00   0.00   97.33   325.67   0.01   0.00   0.01   -0.00   *   v3   c1   1.00   1.00   97.33   325.67   0.01   0.00   0.01   0.00   3.36   v3   b26   2.00   7.00   97.33   325.67   0.02   0.02   0.02   0.02   0.96   v3   b25   1.00   4.00   97.50   325.50   0.01   0.01   0.01   0.01   0.83   v3   b23   3.00   8.00   97.00   326.00   0.03   0.02   0.03   0.02   1.27   v3   b21   1.00   2.00   97.33   325.67   0.01   0.01   0.01   0.01   1.68   v3   b18   1.00   2.00   97.33   325.67   0.01   0.01   0.01   0.01   1.68   v3   b1   2.00   4.00   97.50   325.50   0.02   0.01   0.02   0.01   1.68   v2   v1   4.00   8.00   97.33   325.67   0.04   0.02   0.04   0.02   1.69   v2   c1   2.00   7.00   97.33   325.67   0.02   0.02   0.02   0.02   0.96   v2   b26   1.00   1.00   97.33   325.67   0.01   0.00   0.01   0.00   3.36   v2   b25   2.00   10.00   97.50   325.50   0.02   0.03   0.02   0.03   0.66   v2   b23   0.00   1.00   97.00   326.00   0.00   0.00   -0.00  
0.00   *   v2   b21   2.00   8.00   97.33   325.67   0.02   0.02   0.02   0.02   0.83   v2   b18   2.00   8.00   97.33   325.67   0.02   0.02   0.02   0.02   0.83   v2   b1   3.00   10.00   97.50   325.50   0.03   0.03   0.03   0.03   1.00   v1   c1   2.00   1.00   97.33   325.67   0.02   0.00   0.02   0.00   6.77   v1   b26   3.00   7.00   97.33   325.67   0.03   0.02   0.03   0.02   1.44   v1   b25   2.00   4.00   97.50   325.50   0.02   0.01   0.02   0.01   1.68   v1   b23   4.00   8.00   97.00   326.00   0.04   0.02   0.04   0.02   1.70   v1   b21   2.00   2.00   97.33   325.67   0.02   0.01   0.02   0.01   3.38   v1   b18   2.00   2.00   97.33   325.67   0.02   0.01   0.02   0.01   3.38   v1   b1   3.00   4.00   97.50   325.50   0.03   0.01   0.03   0.01   2.54   c1   b26   1.00   6.00   97.33   325.67   0.01   0.02   0.01   0.02   0.55   c1   b25   0.00   3.00   97.50   325.50   0.00   0.01   -0.00   0.01   *   c1   b23   2.00   7.00   97.00   326.00   0.02   0.02   0.02   0.02   0.96   c1   b21   0.00   1.00   97.33   325.67   0.00   0.00   -0.00   0.00   *   c1   b18   0.00   1.00   97.33   325.67   0.00   0.00   -0.00   0.00   *   c1   b1   1.00   3.00   97.50   325.50   0.01   0.01   0.01   0.01   1.11   b26   b25   1.00   9.00   97.50   325.50   0.01   0.03   0.01   0.03   0.37   b26   b23   1.00   1.00   97.00   326.00   0.01   0.00   0.01   0.00   3.38   b26   b21   1.00   7.00   97.33   325.67   0.01   0.02   0.01   0.02   0.47   b26   b18   1.00   7.00   97.33   325.67   0.01   0.02   0.01   0.02   0.47   b26   b1   2.00   9.00   97.50   325.50   0.02   0.03   0.02   0.03   0.74   b25   b23   2.00   10.00   97.17   325.83   0.02   0.03   0.02   0.03   0.67   b25   b21   0.00   2.00   97.50   325.50   0.00   0.01   -0.00   0.01   *   b25   b18   0.00   4.00   97.50   325.50   0.00   0.01  
-0.00   0.01   *   b25   b1   1.00   0.00   97.67   325.33   0.01   0.00   0.01   -0.00   *   b23   b21   2.00   8.00   97.00   326.00   0.02   0.02   0.02   0.02   0.84   b23   b18   2.00   8.00   97.00   326.00   0.02   0.02   0.02   0.02   0.84   b23   b1   3.00   10.00   97.17   325.83   0.03   0.03   0.03   0.03   1.01   b21   b18   0.00   2.00   97.33   325.67   0.00   0.01  
-0.00   0.01   *   b21   b1   1.00   2.00   97.50   325.50   0.01   0.01   0.01   0.01   1.67   b18   b1   1.00   4.00   97.50   325.50   0.01   0.01   0.01   0.01   0.83   mean   1.45   5.04   97.42   325.58   0.01   0.02   0.02   0.02   1.52 dn : jukes - cantor correction for multiple values of proportion of 
 observed nonsynonymous mutations ( pn ) ; ds : jukes - cantor correction 
 for multiple values of proportion of observed synonymous mutations 
 ( ps ) ; ds
/ dn : rate of synonymous mutations over nonsynonymous 
 mutations ; n : number of potential nonsynonymous substitutions ; nd : 
 number of observed nonsynonymous mutations ; s : number of potential 
 synonymous substitutions ; sd : number of observed synonymous 
 mutations .
asterisk means that ds is not significant due to the high 
 similarity among sequences . 
 
dn : jukes - cantor correction for multiple values of proportion of 
 observed nonsynonymous mutations ( pn ) ; ds : jukes - cantor correction 
 for multiple values of proportion of observed synonymous mutations 
 ( ps ) ; ds / dn : rate of synonymous mutations over nonsynonymous 
 mutations ; n : number of potential nonsynonymous substitutions ; nd : 
 number of observed nonsynonymous mutations ; s : number of potential 
 synonymous substitutions ; sd : number of observed synonymous 
 mutations .
asterisk means that ds is not significant due to the high 
 similarity among sequences .
 we aimed to evaluate whether the occurrence of cryptic species of 
 paracoccidioides brasiliensis , s1 , ps2 , ps3 and 
 paracoccidioides lutzii , has implications in the 
 immunodiagnosis of paracoccidioidomycosis ( pcm ) . 
 small quantities of the antigen 
 gp43 were found in culture filtrates of p. lutzii strains and 
 this molecule appeared to be more variable within p. lutzii 
 because the synonymous - nonsynonymous mutation rate was lower , indicating an 
 evolutionary process different from that of the remaining genotypes . 
 the 
 production of gp43 also varied between isolates belonging to the same species , 
 indicating that speciation events are important , but not sufficient to fully 
 explain the diversity in the production of this antigen . 
 the culture filtrate 
 antigen agepm83 , which was obtained from a ps3 isolate , showed large quantities 
 of gp43 and reactivity by immunodiffusion assays , similar to the standard 
 antigen ( agb-339 ) from an s1 isolate . 
 furthermore , agepm83 was capable of 
 serologically differentiating five serum samples from patients from the botucatu 
 and jundia regions . 
 these patients had confirmed pcm but , were non - reactive to 
 the standard antigen , thus demonstrating an alternative for serological 
 diagnosis in regions in which s1 and ps2 occur . 
 we also emphasise that it is not 
 advisable to use a single antigen preparation to diagnose pcm , a disease that is 
 caused by highly diverse pathogens . 