urinary tract infections ( utis ) 
 affect millions of women each year , with an annual societal cost of billions of 
 dollars .
importantly , more than one quarter of women with a uti will have a 
 recurrent infection within six months . 

there are few established options for prevention of uti other than the 
 use of prophylactic antibiotics . 

however , resistance to commonly used antibiotics is increasing among 
 bacterial cystitis isolates . 

a growing body of evidence suggests that vaginal h202-producing 
 lactobacilli may have a protective effect against urogenital infections , 
 including uti [ 511 ] .
it is hypothesized 
 that lactobacilli prevent uropathogen colonization of the vagina , a necessary 
 step in ascending infection of the bladder . 

several clinical trials have demonstrated that certain lactobacillus species , mainly l. rhamnosus and   l. fermentum , can be given orally or vaginally with resulting 
 colonization of the vagina , reduction in vaginal coliform counts , and even 
 reduction in uti recurrence 
 [ 1218 ] .
specific 
 strains were chosen for their abilities to produce h202 , adhere to uroepithelial cells , interfere with uropathogen attachment and growth , and persist in the vagina [ 18 , 19 ] . 

the most frequently existing vaginal lactobacillus species , l. 
 crispatus [ 20 , 21 ] , also produces h202 and possesses the adherence and inhibitory qualities essential for a good probiotic 
 candidate [ 22 , 23 ] .
l. crispatus strain ctv-05 has been 
 developed as a vaginal suppository ( lactin - v ) for the prevention of recurrent 
 uti in women .
l. crispatus ctv-05 is 
 highly adherent to vaginal epithelial cells , and when given as a suppository 
 can colonize the vagina . 
 furthermore , l. crispatus ctv-05 has been reported to have a specific dna fingerprint that distinguishes 
 it from endogenous vaginal lactobacilli . 
 to further evaluate the safety of this new formulation of l. crispatus ctv-05 and its effect on 
 the vaginal microbial flora
, we conducted a phase i , randomized , double - blind , 
 placebo - controlled trial of vaginal l. 
 crispatus ctv-05 in premenopausal women with a history of recurrent uti .
we recruited premenopausal women aged 1835 
 years with a history of three or more uncomplicated utis diagnosed in the past 
 year , or two uncomplicated 
 utis diagnosed in the past six months , from ( student health center , university 
 of washington , wash , usa . ) additional eligibility requirements 
 included regular menstrual cycles or amenorrhea for at least six months 
 secondary to use of a hormonal contraceptive , a normal pap smear documented in 
 the last year or at the baseline clinic visit , abstinence 
 from sexual activity or participation in 
 a mutually monogamous sexual relationship , use of birth control , agreement not 
 to use other intravaginal products , agreement not to have sexual intercourse or 
 use tampons between the baseline and first follow - up visit , and capability to 
 understand english and provide informed consent .
exclusion criteria included history of 
 urologic abnormality , recent urologic surgery or urinary catheterization , 
 history of complicated pyelonephritis or renal calculi , hysterectomy , recent sexually 
 transmitted infection ( sti ) or bacterial vaginosis , risk factors for sti and 
 hiv , history of recurrent genital herpes , menses anticipated within ten days , pregnancy , 
 lactation , recent antibiotic or antifungal use , diabetes or other immunocompromised state , drug 
 or alcohol abuse , use of the ( nuvaring ) , prior use of the study drug or allergy 
 to any of its components , and abnormal initial pelvic examination .
the study was approved by ( institutional 
 review board ( irb ) , university of washington , wash , 
 usa . ) subjects were randomized in a double - blinded 
 fashion to l. crispatus ctv-05 at a 
 dose of 5  10 colony forming units 
 ( cfu ) or placebo vaginal suppository to be inserted daily for five days .
l. crispatus ctv-05 and placebo 
 suppositories were similar in appearance and consisted of a preservation matrix 
 and maltodextran .
, palo alto , 
 calif , usa ) according to a randomization 
 schedule and supplied to the study site sequentially labeled with a subject 
 number .
subjects 
 were seen at three clinic visits over a period of one month . at the first ( baseline )
visit , eligible subjects 
 provided informed consent before undergoing a structured medical and 
 gynecologic history and physical examination . 

subjects inserted the first dose of study 
 drug in the clinic and were instructed to record any symptoms occurring during 
 the first week of study on a prepared diary card .
subjects were seen in follow - up 68 days ( 1-week ) 
 visit and 2634 days ( 4-week ) visit after enrollment . during these visits , new symptoms , new 
 urogenital infections , and any changes in clinical history were recorded .
finally , subjects were 
 contacted by telephone six months after enrollment to assess symptoms , 
 pregnancy , new diagnoses , or major medical events .
laboratory 
 testing included urine dipstick testing , urinalysis , urine culture , vaginal 
 fluid wet preparation slides , gram stains , and culture for facultative isolates 
 and lactobacillus species at all 
 visits .
repetitive element 
 sequence - based polymerase chain reaction ( rep - pcr ) for l. crispatus ctv-05 was performed on three lactobacillus colonies 
 from culture at each visit .
testing for neisseria gonorrhoeae and chlamydia 
 trachomatis by dna amplification was done at the baseline visit .
a 
 sample size of 30 was chosen to evaluate the primary outcome of safety as 
 assessed through self - reported symptoms , physical exam findings , and laboratory 
 studies .
secondary outcomes included 
 shifts in the vaginal flora assessed by vaginal culture and vaginal 
 colonization with l. crispatus ctv-05 
 assessed by rep - pcr .
two women randomized to l. 
 crispatus ctv-05 completed treatment over six days , and one woman 
 randomized to placebo used six suppositories over six days . all women remained on study through the 
 4-week visit ( figure 1 ) .
baseline 
 characteristics of subjects were similar in the two treatment groups ( table 1 ) .
the majority of women were young , white , 
 healthy , university students in their 20s . 

mild to moderate adverse events were 
 relatively common , however . of those 
 felt by the investigators to be related to study drug use ,
abnormal vaginal 
 discharge was the most frequently occurring , followed by external genital 
 irritation and vaginal candidiasis ( table 2 ) .
these adverse events occurred with 
 similar or greater frequency in women randomized to placebo as compared to women 
 randomized to l. crispatus ctv-05 .
three women reporting vaginal candidiasis also 
 reported multiple prior episodes in the preceding 12 months .
two women randomized to l. crispatus ctv-05 reported episodes of cystitis during the 
 study .
all subjects completed a diary card of symptoms that occurred during the period of 
 study drug insertion .
recorded symptoms generally 
 confirmed the reported adverse events and were similar to symptoms reported at the 
 1-week clinic visit ( below ) .
abnormal 
 vaginal discharge was the symptom most frequently reported by subjects at 
 clinic visits following study drug use ( figure 2 ) .
while no women reported abnormal 
 vaginal discharge at baseline , six randomized to l. crispatus ctv-05 and five randomized to placebo reported abnormal 
 vaginal discharge at the 1-week visit .
external genital or vaginal irritation was 
 also reported by six women in each treatment arm at follow - up visits .
vaginal odor was reported at the 1-week visit 
 by one woman randomized to l. crispatus ctv-05 , 
 and at the 4-week visit by two women randomized to placebo .
dysuria was reported by one woman randomized 
 to l. crispatus ctv-05 at the 1-week visit 
 and by one woman randomized to placebo at the 1- and 4-week visits .
headache , abdominal or pelvic cramps / abdominal 
 pain , and low back pain were frequently reported at baseline as well as at 
 follow - up visits .
there were no 
 significant differences in reported symptoms at any visit between women in the 
 two treatment arms .
external genital erythema was found at the 1- 
 and 4-week visits in more women randomized to l. crispatus ctv-05 than in women randomized to placebo ( 4 versus 1 , p=.33 ) .
moderate - to - profuse vaginal 
 discharge was frequently found in women from both treatment arms during 
 follow - up examinations 
 ( 12 versus 8 , p = 1.00 ) , though this finding was also often apparent at the 
 baseline visit ( 6 women in each arm ) . 

seven 
 ( 47% ) women randomized to l. crispatus ctv-05 had nine episodes of pyuria with a urine white blood cell count 8 per mm   ( range 8203 ) during a follow - up clinic visit ( table 3 ) .
in contrast , none 
 of the women randomized to placebo had pyuria at 1 or 4 weeks ( p = .01 ) .
pyuria was positively correlated with urine 
 leukocyte - esterase test results ( spearman r = 0.57 , p<.01 ) .
there 
 were no significant associations with vaginal or urinary symptoms or exam 
 findings .
microbes isolated from the 
 urine of women in the l. crispatus ctv-05 arm with pyuria included 
 mixed gram - positive organisms ( 6 ) , lactobacillus ( 3 ) , e. coli ( 1 ) , enterococcus ( 1 ) , group b streptococcus ( 1 ) , and yeast 
 ( 1 ) .
one subject had a negative urine 
 culture , and none had symptomatic infection . 

lactobacillus and mixed gram - positive rods were isolated at 10 - 10 cfu / ml from more women with pyuria ( 50% and 75% ) than without ( 19% and 46% , 
 resp . ) at the 1-week visit ( p=.22 for lactobacillus , p=.60 for 
 mixed gram - positive rods ) but not at the 4-week visit .
none of the other isolated organisms was found 
 at a significantly increased frequency in women randomized to l. crispatus ctv-05 versus placebo or 
 in women with pyuria compared to those without pyuria at any visit .
two women randomized to l. crispatus ctv-05 and one randomized to placebo had hematuria at 
 the 4-week visit ( 30 red blood cells per mm ) .
nine women in the l. crispatus ctv-05 arm and ten in the 
 placebo arm completed the six - month follow - up telephone call .
four women in the l. crispatus ctv-05 arm and one in the 
 placebo arm reported one or more episodes of cystitis .
28 of 30 women had vaginal 
 colonization with h202-producing 
 lactobacilli at baseline and all follow - up visits ( table 4 ) .
two women 
 randomized to placebo had no vaginal h202-producing 
 lactobacilli detected at any clinic visit
. 
 one of these women had large quantities of gardnerella and bacteroides species morphotypes on gram stain .
cultures 
 from women with h202-producing 
 lactobacilli yielded heavy ( 4 + ) growth .
vaginal 
 cultures from five women in each treatment arm yielded e. coli from one or more clinic visits .
e. coli was isolated from vaginal cultures taken at the 1- and 4-week visits from 4 
 women randomized to l. crispatus ctv-05 and from one women randomized to placebo ( p=.33 ) .
no inverse association between vaginal 
 colonization with h202-producing 
 lactobacilli and vaginal colonization with e. 
 coli was detected .
vaginal cultures 
 frequently yielded candida at baseline 
 as well as follow - up visits . using rep - pcr ,
l. crispatus ctv-05 was detected in the vaginas of four women 
 randomized to l. crispatus ctv-05 
 ( three women at each clinic visit ) .
three 
 women had a positive assay for l. 
 crispatus ctv-05 detected at the baseline visit before administration of the 
 study drug .
no women randomized to 
 placebo had a positive assay at any visit ( p = .22 each visit ) .
we 
 compared our rep - pcr results ( figure 3 ) 
 to previously published rep - pcr patterns   in order to identify the 
 bacterial species . at baseline
, 80% of 
 subjects had l. crispatus , 37% had l. jensenii , 17% had l. gasseri , and 27% had other lactobacillus species detected in 
 vaginal culture .
the following 
 combinations of lactobacillus species 
 were found : l. crispatus ( 27% ) , l. crispatus and l. jensenii ( 20% ) , l. 
 crispatus and l. gasseri ( 7% ) , l. crispatus and other lactobacillus species ( 13% ) , l. crispatus , l. jensenii , and l. gasseri ( 3% ) , l. crispatus , l. jensenii , and other lactobacillus species ( 7% ) , l. crispatus , l. gasseri , and other lactobacillus species ( 3% ) , l. jensenii ( 7% ) , l. gasseri ( 3% ) , other lactobacillus species ( 3% ) , and no lactobacillus ( 7% ) .
the prevalences of individual species were 
 relatively stable over time ( figure 4 ) .
there were no significant differences in lactobacillus species prevalence between 
 subjects in the two treatment arms with the exception of l. gasseri at the 1-week visit ( p = .02 ) .
in phase i , placebo - controlled trial , use of a l. crispatus ctv-05 vaginal suppository , 
 for the prevention of recurrent uti was well tolerated with minimal side effects .
there were no serious adverse events , and 
 despite the occurrence of abnormal vaginal discharge and external genital or 
 vaginal irritation in several women , compliance was high .
these mild to moderate symptoms appear to be 
 secondary to the act of suppository use or a reaction to the preservation 
 matrix rather than a consequence of l. 
 crispatus ctv-05 as they occurred with similar frequency among women in 
 each treatment arm .
however , 
 given the frequency of recurrent vaginal candidiasis experienced by subjects 
 prior to enrollment and the prevalence of candida in baseline vaginal cultures , the relationship of this condition to study drug 
 use is not compelling .  
l. crispatus ctv-05 use was associated 
 with pyuria ( detected by microscopy and urine leukocyte - esterase ) in seven women 
 at either the 1- or 4-week visits ( p = .04 at 4 weeks ) .
pyuria was not associated with urogenital 
 symptoms , exam findings , or symptomatic uti . 
 while lactobacillus was not isolated from the urine during every episode 
 of pyuria , it is possible that some lactobacilli were misclassified as mixed gram - positive 
 rods , as both were more frequently found in women with pyuria than in women 
 without .
our data suggest that vaginal 
 instillation of l. crispatus ctv-05 induces 
 a mild inflammatory response in the bladder or vaginal mucosa of some subjects 
 without causing prolonged urogenital infection . 

the importance of this phenomenon with respect to safety or potential efficacy 
 is unclear .
it is possible that induction of an asymptomatic inflammatory 
 response by lactobacilli protects against uropathogen colonization of the 
 vagina or infection in the bladder .
limited 
 existing data from prior studies suggest that l. crispatus therapy is safe . 

women surveyed after participation in a clinical trial of a vaginal 
 capsule formulation of l. crispatus ctv-05 
 for treatment of bacterial vaginosis rarely reported adverse effects , and those 
 reported were largely related to a perceived difference in vaginal 
 discharge . satisfaction with the vaginal 
 capsule was high .
no adverse effects were reported in a pilot study of l. crispatus ctv-05 given 
 intravaginally to a small group of healthy women .
nine women with a history of recurrent uti 
 reported no adverse effects when given l. 
 crispatus strain ga198332 as a vaginal suppository every other day for one 
 year .
studies of other probiotic 
 strains of lactobacillus have 
 indicated that they can be given to women safely with minimal or no side 
 effects [ 1218 ] . to our knowledge , pyuria 
 resulting from vaginal instillation of lactobacillus has not been previously reported , and should be evaluated further in future 
 studies .
we 
 did not see an effect of l. crispatus ctv-05 use on vaginal lactobacillus growth . however , most study participants had heavy 
 vaginal growth of h2o2-producing 
 lactobacilli at baseline , making detection of subsequent changes difficult .
data from a pilot study indicated that l. crispatus ctv-05 did not displace other 
 endogenous vaginal lactobacilli .
vaginal 
 colonization was most successful in women lacking vaginal h2o2-producing 
 lactobacilli at the outset , suggesting that a lactobacillus probiotic may be 
 most effective at establishing vaginal colonization in women with abnormal 
 flora , such as those with bacterial vaginosis or recurrent uti .
to our knowledge , our study represents the first attempt to use rep - pcr to identify the genetic fingerprint of the probiotic strain l. crispatus ctv-05 in a larger - scale clinical study .
although none of the placebo recipients were 
 colonized with lactobacillus strains having the characteristic rep - pcr pattern of l. crispatus ctv-05 at any time , three 
 women randomized to l. crispatus ctv-05 had at least one isolate in their vaginal culture at baseline that bore 
 the characteristic fingerprint .
. as there are no 
 large - scale surveys of the prevalence and distribution of lactobacilli bearing this fingerprint , we do not know whether the rep - pcr technique failed to 
 distinguish l. crispatus ctv-05 from 
 other genetically closely related strains of l. crispatus naturally colonizing the vagina at baseline , or 
 whether the identical strain was prevalent in our study population . of note ,
subjects in our study had a much 
 higher prevalence of vaginal h2o2-producing 
 lactobacilli in baseline vaginal cultures than found in previous studies . by comparing our rep - pcr results
to previous 
 published rep - pcr patterns , we characterized lactobacillus isolates at the 
 species level and found l. crispatus , l. jensenii , and l. gasseri to be the most prevalent species , with relatively stable 
 prevalences over time .
our prevalence 
 data are consistent with what has been previously documented [ 20 , 21 ] .
additional tests are under way to compare 
 quantitative pcr results for l. crispatus in women before and after the introduction of active suppositories or placebo .
our study was not 
 designed nor statistically powered to evaluate the effect of l. crispatus ctv-05 on the rate of uti 
 recurrence .
therefore , it is difficult 
 to interpret the report of cystitis in two women randomized to l. crispatus ctv-05 .
other small studies have suggested that use of 
 vaginally administered l. crispatus may 
 be associated with a lower rate of uti recurrence [ 16 , 29 ] .
the major strengths 
 of our study include the placebo - controlled , randomized study design , excellent 
 compliance , and extensive and complete followup .
use of a study diary provided finer details 
 of our subjects experiences that confirmed adverse events and symptoms 
 reported from memory at clinic visits .
weaknesses 
 that should be mentioned include the inability to accurately document vaginal 
 colonization with the probiotic strain , and the inherent lack of statistical 
 power due to the small sample size typical of phase i studies .
we are currently conducting a phase ii trial 
 that will have more power to clarify some of the issues raised in this 
 study . in 
 conclusion ,
l. crispatus ctv-05 is 
 well tolerated when given as a vaginal suppository to healthy women with a 
 history of recurrent uti .
mild to 
 moderate side effects related to suppository use occur but do not affect 
 compliance .
timing of administration and efficacy in 
 preventing recurrent cystitis should be evaluated further in larger studies .
 objectives : we performed a phase i trial to assess the safety and tolerance of a lactobacillus vaginal suppository for prevention of recurrent uti . 
 methods : premenopausal women with a history of recurrent uti were randomized to use l. crispatus ctv-05 or placebo vaginal suppositories daily for five days . 
 results : 30 women were randomized ( 15 to l. crispatus ctv-05 ) . 
 no severe adverse events occurred . 
 mild to moderate vaginal discharge and genital irritation were reported by women in both study arms . 
 seven women randomized to l. crispatus ctv-05 developed pyuria without associated symptoms . 
 most women had high concentrations of vaginal 
 h202-producing lactobacilli before randomization 
 . l. crispatus , 
 l. jensenii , and l. gasseri were the most common 
 lactobacillus species identified , with stable prevalence over time . 
 
 conclusions : l. crispatus ctv-05 can be given as a vaginal suppository with minimal sideeffects to healthy women with a history of recurrent uti . 
 mild inflammation of the urinary tract was noted in some women . 