melioidosis , an infectious disease caused by the tier  1 select agent burkholderia pseudomallei , is notoriously difficult to cure 1 .
north - eastern thailand is a hot spot for this infection , with an annual incidence of 21.0 per 100  000 population and a case - fatality rate of 40% 2 . for patients who survive their first episode of infection ,
this occurs in approximately 13% of patients followed for 10  years , and half of the recurrences occur within 12  months of the primary episode 3 .
approximately one - quarter of those with relapse will die as a direct result 4,5 .
biofilm formation has been described as an important factor associated with persistent infections in a number of infectious diseases , including burkholderia cepacia infection in cystic fibrosis patients 68 , but this has not been formally evaluated in relation to relapse of melioidosis . a previous study of a small number of isolates associated with relapse suggested that b.  pseudomallei with uncommon lipopolysaccharide ( lps ) types ( smooth type  b and rough type ) might be associated with relapse 9 . here , we evaluated biofilm formation and lps type of b.  pseudomallei isolates from patients with primary melioidosis drawn from a cohort described previously 5 , and determined their associations with relapse .
control study in which both cases and controls were drawn from a cohort of patients with primary melioidosis identified between 1986 and 2004 who survived to receive oral antimicrobial therapy and were observed until july 2005 5 .
cases were all patients who developed at least one episode of relapse during the study period , with relapse being verified by genotyping of the primary and relapse isolates 5 .
controls were randomly selected from those patients in the cohort who had not developed relapse by the time relapse was identified in cases .
cases and controls were matched for known risk factors for relapse , including choice and duration of oral antimicrobial therapy , positive blood culture , and multifocal distribution of infection at first presentation .
primary isolates from cases and controls and relapse isolates from cases were evaluated for quantitative biofilm formation and lps .
all isolates were stored at 80c prior to the evaluation . the first isolate cultured and saved from each episode
quantitative estimation of biofilm formation was performed with a modified microtitre plate test , as described previously 1012 .
all experiments were independently conducted twice , and the results reported were the average from those two experiments .
lps was extracted , and the type was defined as smooth type  a , smooth type  b , or rough type , as described previously 9 .
a conditional logistic regression model was used to evaluate the relationships between independent factors and the relapse outcome .
selection of controls and statistical analyses were conducted with stata , version  12.0 ( statacorp , college station , tx , usa ) .
the study was approved by the ethical and scientific review subcommittee of the thai ministry of public health 5 . of 86 and 202
primary isolates from cases and matched controls , 80 ( 93% ) and 184 ( 91% ) , respectively , were available for study and included in the analysis .
patients in the case and control groups had comparable characteristics for the matched variables ( table1 ) .
characteristics of cases and controls na , not applicable , as choice and duration of oral antimicrobial therapy , positive blood culture result and multifocal distribution of first presentation were matched variables ; od , optical density .
melioidosis was classified as localized ( single focus of infection and a negative blood culture result ) , multifocal ( one or more non - contiguous foci of infection and a negative blood culture result ) , bacteraemic ( a positive blood culture result plus a single or no identifiable focus of infection ) , and disseminated ( a positive blood culture result plus one or more non - contiguous foci of infection ) .
first , we determined whether the quantitative production of biofilm by the primary isolate influenced the likelihood that relapse would occur .
biofilm production by primary isolates from patients with relapse was higher than that for primary isolates from matched patients without relapse ( median corrected optical density ( od)630  nm of 0.95 ( interquartile range  0.751.28 ) vs. 0.79 ( interquartile range  0.631.06 ) ) .
this was independently associated with the relapse outcome ( conditional or  2.03 ; 95%  ci  1.273.25 ; p  0.003 ) .
overall , 99% of primary isolates from cases and 98% from controls had lps smooth type  a. lps smooth type  b was found in one ( 1% ) case and in two ( 1% ) controls , and rough - type lps was found in one ( 1% ) control .
an association between lps type of the primary isolate and relapse was not found ( p  0.74 ) .
next , we determined whether there was any difference in biofilm formation and in lps type between primary and relapse isolates from the same relapse cases . from 80 relapse cases ,
biofilm formation of the primary isolate and that of the relapse isolate were not different ( mean difference for corrected od630  nm of 0.002 ; 95%  ci  0.16 to 0.16 ; p  0.98 ) .
the lps type of the primary  relapse pair was the same for all 71 isolates ( lps smooth type  a , n  =  70 ; lps smooth type  b , n  =  1 ) . in this study , we have shown that a quantitative measure of biofilm formation by the primary isolate is associated with relapse in patients with melioidosis .
this was independent of known clinical risk factors for relapse , including choice and duration of oral antimicrobial therapy , positive blood culture , and multifocal distribution of infection at first presentation , factors that were matched by the nested case 
this provides the first evidence to suggest that biofilm formation of b.  pseudomallei in  vitro is associated with relapse in human melioidosis , and is consistent with findings reported for escherichia coli 
 6 and other biofilm - producing bacteria 7,8 .
we also observed that quantitative biofilm formation did not differ between paired primary and relapse isolates .
this lack of detectable change between isolates of the same lineage that are separated by the period spanning human infection , quiescence and re - emergence argues against the notion that increased biofilm formation occurs in  vivo through positive selection
. quantitative biofilm formation by isolates in this study was lower overall than that reported previously for 34 clinical b.  pseudomallei isolates ( mean corrected od630  nm of 1.98    0.32 ) 12 .
possible explanations are that the means used in the previous study were skewed by isolates with exceptionally high biofilm formation . in addition , the isolates in the study described here were only from patients who survived the first episode of acute infection , whereas the isolates in the previous study included those who died during the acute infection .
the majority of b.  pseudomallei isolates from the primary episode of melioidosis in this study expressed lps smooth type  a , and no association between lps type and relapse was found .
this contrasts with the findings of a previous study , in which three of 11 ( 27% ) patients with recurrent melioidosis had different lps types in the primary and relapse isolates 9 .
furthermore , isolates in this previous study were not genotyped , and the possibility that recurrence was attributable to re - infection with a different isolate rather than persistence and relapse with the primary isolate was not excluded 9 .
the proportions of uncommon , non - type  a lps in the primary episode of melioidosis in this study ( 1% in cases and 2% in controls ) were similar to those observed in the previous study ( 3% ) 9 .
our findings , based on a much larger number of bacterial isolates supported by a robust study design , do not provide evidence for a link between uncommon , non - type  a lps and relapse .
this study was supported by the thailand research fund ( mrg5480172 ) and the melioidosis research centre , khon kaen university , thailand .
s. j. peacock was supported by the national institute for health research cambridge biomedical research centre .
 we examined whether quantitative biofilm formation and/or lipopolysaccharide type of burkholderia pseudomallei was associated with relapsing melioidosis . 
 we devised a 1  :  4 nested case  
 control study in which both cases and controls were drawn from a cohort of patients with primary melioidosis . paired isolates from 80 patients with relapse and single isolates from 184 patients without relapse were tested . 
 relapse was associated with biofilm formation of the primary infecting isolate ( conditional or  2.03 ; 95%  ci  1.273.25 ; p  0.003 ) , but not with lipopolysaccharide type ( p  0.74 ) . 
 this finding highlights the importance of biofilm formation in relapsing melioidosis . 
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