sample collection : 20 ml of fresh urine was collected 
 from individual cattle in lopburi , chainat and nakhon nayok provinces in the central region 
 of thailand from october 2012 to february 2014 for the propose of disease surveillance . 

after collection , the samples were kept in cool storage ( 4c ) and transported to the 
 national institute of animal health .
all samples were subsequently examined with three 
 methods including bacterial culture , real - time pcr and lamp assay .
culture : one ml of urine samples was 
 added to 9 ml of 1% bovine serum albumin diluent with 5 fluro 
the 
 samples were cultured in emjh ( johnson and harris modification of the ellinghausen and 
 mccullough medium ) semi  solid media with 5 fluro 
uracil ( becton - dickinson biosciences , 
 detroit , mi , u.s.a . ) at 30c for 1216 weeks . leptospira spp . were 
 considered positive by the characteristic of thin helical structures with prominent hooked 
 ends and motility under a dark field microscope . 

the suspected samples were further subcultured in the emjh liquid media and purified with 
 0.2 m  pore 
dna extraction : dna in 10 ml of urine sample was 
 extracted using dynabeads dna direct universal kit ( invitrogen , oslo , norway ) 
 according to the manufacturer s instructions ; dna concentration was measured by 
 qubit fluorometer ( invitrogen ) . loop 
mediated isothermal amplification method ( lamp ) : the lamp was carried 
 out using primer and condition previously described in .
briefly , a total of 
 25 l reaction mixture contained 1.0 m outer primers f3 
 ( 5-gaaccctgaagcagcgac3 ) and b3 ( 5-gacttacatgtccgcctacg3 ) , 1.6 m 
 inner primers fip ( 5-cacatcgctgcttatttttccctgcgcgtgaacgatgaaggtct-3 ) and bip 
 ( 5-cctgcctaaagcaccggctaacgcccaatgattccgaacaa-3 ) , 0.8 m loop primer lb 
 ( 5agccgcggtaatacgtatggtg3 ) , 1x thermopol reaction buffer ( new england 
 biolabs , hitchin , u.k . ) , 4 mm mgso4 ( new england biolabs ) , 1 m betaine 
 ( sigma - aldrich , st .
louis , mo , u.s.a . ) , 0.4 mm dntps ( new england biolabs ) , 0.5 mm mncl , 25 
 m calcein and 8 units bst dna polymerase ( new england 
 biolabs ) and adjusted to a total volume of 25 l with sterile water ( new 
 england biolabs ) .
the reaction mixture was incubated at 61c for 90120 min in a heating 
 block and then was heated at 80c for 2 min to terminate the reaction .
the presence of 
 product was defined on the basis of green color detected with naked eyes .
real - time pcr : real - time pcr was conducted according to   by targeting the 241 bp region of 
 lipl32 gene which encoded the major outer membrane of pathogenic 
 leptospira spp .
the light cycler nanoinstrument ( roche 
 diagnostic , mannheim , germany ) was used for hydrolysis probe assay .
the dna product was 
 amplified using faststarttaq dna polymerase ( roche diagnostic ) with the primers lipl32 - 45f 
 ( 5-aagcattaccgcttgtggtg-3 ) , lipl32 - 286r ( 5-gaactcccatttcagcgatt-3 ) and probe189p ( [ fam ] 
 5-aaagccaggacaagcgccg-3[bhq1 ] .
the reaction mixture consisted of 10 ng 
 of extracted dna , 0.25 m each primers and probe and 1x faststart essential 
 dna probe master and was adjusted to a total volume of 20 l with sterile 
 water ( roche diagnostic ) . the thermal condition of a holding stage was performed at 95c for 
 10 min and followed by 45 cycles of two  step amplification from 95c to 55c for 30 sec .
were 
 calculated following ; accuracy of these tests was 
 calculated according to the formulas proposed in . 

the formulas described in   were used in positive 
 and negative predictive values calculation .
equations 17 were used in these analyses ; percent positivity ; pp=(tp+fp)/(tp + fp + tn + fn ) ( 1 ) sensitivity ; se = tp/(tp + fn ) ( 2 ) specificity ; sp = tn/(tn + fp ) ( 3 ) gold standard prevalence ; gsp=(tp+fn)/(tp + fp + tn + fn ) ( 4 ) positive predictive value ; ppv = ( se  gsp)/((se  gsp ) + ( ( 1-sp ) (1-gsp ) ) ) ( 5 ) negative predictive value ; npv = ( sp  ( 1-gsp))/(((1-se )  gsp ) + ( ( sp ) (1-gsp ) ) ) ( 6 ) accuracy=(tp + tn)/(tp + fp + tn + fn ) ( 7 ) , where tp , fp , tn and fn refer to true positive , false positive , true negative and false 
 negative , respectively .
cohen s kappa statistics for agreement of all the tests were analyzed , and the strength of 
 agreement was interpreted as proposed in .
k=(po - pe)/(1- pe ) ( 8) , where k , po and pe represent cohen s kappa coefficient , relative 
 observed agreement of the tests and hypothetical probability of agreement , respectively .
all statistical analyses were carried out with program r version 3.2.2 ( r development core 
 team , vienna , austria ) .
caret were used in the calculation of se , 
 sp , ppv and npv , whereas cohen s kappa 
 statistics were performed within the package  fmsb .
prevalence : a total of 533 urine samples were collected from 266 beef and 
 267 dairy cattle .
it was found that 31 , 45 and 58 animals were positive from leptospiral 
 culture , lamp and real - time pcr , respectively .
detection from these three assays was 5.8% ( confidence 
 interval ( ci ) 4.08.2 ) , 8.4% ( 95% ci 6.211.1 ) and 10.9% ( 95% ci 8.413.8 ) , 
 respectively .
accuracy : lamp and real - time pcr were evaluated using bacterial culture as 
 a gold standard .
the examination results comparing bacterial culture with two diagnostic 
 assays are illustrated in table 1table 1 .
comparison of examination results from lamp and real - time pcr with 
 culture method in the detection of leptospira spp . in urine samples 
 collected from cattle in central region of thailandculture resultlampreal - time pcrno .
lamp was more sensitive and more specific than real - time pcr as could be obviously seen 
 that the sensitivity of lamp and real - time pcr was 96.8% ( 95% ci 81.599.8 ) and 74.2% ( 95% 
 ci 55.187.5 ) , while the specificity of lamp and real - time pcr was 97.0% ( 95% ci 94.998.2 ) 
 and 93.0% ( 95% ci 90.395.0 ) , respectively .
95.298.3 ) was significantly higher than that of real - time pcr ( 91.9% ; 89.394.1 ) .
both 
 assays had higher negative predictive values ( lamp : 99.8% ; 95% ci 98.7100.0 and real - time 
 pcr : 98.3% ; 95% ci 96.699.2 ) than the positive ones ( lamp : 66.7% ; 95% ci 50.979.6 and 
 real - time pcr : 39.7% ; 95% ci 27.353.4 ) . comparing these two assays , higher positive and 
 negative predictive values
agreement of the tests : to further assess the strength of agreement among 
 all studied assays , cohen s kappa statistics were then analyzed .
culture method and lamp 
 substantially agreed with each other ( 77.4% ; 95% ci 66.588.3 ) , whereas real - time pcr only 
 moderately agreed with culture ( 47.7% ; 95% ci 32.762.7 ) and lamp ( 45.3% ; 95% ci 
 31.059.6 ) .
the present study evaluated the diagnostic accuracy of two rapid molecular methods 
 including lamp and real - time pcr , in the detection of leptospira genetic 
 materials in urine samples from cattle in central region of thailand . in this study , 
 bacterial culture
true prevalence. comparing the 
 true prevalence with the prevalence derived from lamp and real - time pcr ( table 1 . )
, overestimated prevalence was found in both assays , 
 especially from real - time pcr ( 10.9% ; 95% ci 8.413.8 ) ; it was almost two times higher than 
 the true prevalence ( 5.8% ; 95% ci 4.08.2 ) , whereas that of lamp fell in the middle at 8.4% 
 ( 95% ci 6.211.1 ) .
our results corresponded with the previous study , suggesting that positivity in molecular techniques could be obtained 
 with no positive culture results . regarding diagnostic accuracy of lamp and real - time pcr , lamp was more accurate method in 
 the detection of leptospira spp . with higher sensitivity and specificity 
 than real - time pcr .
the lamp assay targeting 16s rdna gene used in the present study had a 
 low detection limit at 10100 copies .
therefore , 
 its sensitivity was high as it could detect the pathogen even with low amount of genetic 
 materials .
in contrast , it was found that 
 real - time pcr targeting lipl32 gene was a low sensitive method with the 
 sensitivity of 43% ( 95% ci 3452% ) , and the 
 detection limit of real - time pcr was found at 1001,000 copies per reaction under the 
 author s conditions .
the lower sensitivity of real - time pcr , compared to lamp , was 
 attributed to the difference in lower detection limits of the two methods .
however , the low 
 sensitivity of real - time pcr in this study also resulted from the high number of false 
 negative samples ( 25.8% ; 8/31 ) which were negative with real - time pcr but positive with 
 culture .
the plausible explanation was that a very low 
 count in the original sample might stochastically present in the aliquot prepared for 
 culture but not in that for real - time pcr . in 
 terms of specificity ,
both lamp and real - time pcr were considered a highly specific assay 
 which was consistent with other previous publications [ 4 , 10 , 24 , 25 ] .
the primers used in the present 
 study were preliminarily tested with some intermediate species of leptospires and 
 demonstrated some mismatched characteristics .
however , the test did not guarantee that the 
 primer set never amplifies the target gene from any other intermediate species . to prove 
 this ,
the possible amplification of 
 target gene from intermediate species by the lamp primer set may contribute to higher 
 sensitivity of lamp than real - time pcr .
apart from sensitivity and specificity , predictive values changed proportional to the 
 prevalence changes .
the predictive values gave a 
 basic idea on how we can predict the results using a specific test with a specific 
 population in a specific time .
in this study , npv was high in both lamp ( 99.8% ; 95% ci 
 98.7100.0% ) and real - time pcr ( 98.3% ; 95% ci 96.699.2% ) .
in contrast , the ppv of these two tests was moderate to low 
 ( 66.7% and 39.7% for lamp and real - time pcr , respectively ) .
however , as the predictive values depended 
 largely on prevalence , the values were not transferrable from one to another study with 
 different spaces and times . based on the cohen s kappa statistics calculated in this study , real - time pcr
this result was in agreement with the 
 previous studies providing a notice on the possible disagreement of real - time pcr and 
 culture [ 7 , 25 ] . moreover , lamp was much more sensitive and slightly more specific than 
 real - time pcr . therefore , the agreement of these two tests was only at moderate level .
bacterial culture which was considered the gold standard in the present study was regarded 
 as a technique with low sensitivity , laborious , time consuming   and not practical in the early detection of leptospires . to confront with this problem ,
various molecular 
 techniques have been employed to rapidly identify the pathogens including real - time pcr and 
 lamp .
however , real - time pcr was challenging in term of finance , technical and instrumental 
 requirements and might not be applicable in developing countries where leptospirosis has 
 been endemic [ 1 , 26 ] .
therefore , the less expensive method with less expertise requirement , such as 
 lamp , has been developed and implemented for the early detection of 
 leptospira pathogen . as 
 demonstrated in our study , lamp was more effective than real - time pcr in the detection of 
 pathogenic leptospira spp . in the urine of cattle with substantially less 
 cost and more simplicity .
accordingly , lamp was an excellent alternative for routine 
 surveillance of leptospirosis in cattle .
 leptospirosis has been one of the worldwide zoonotic diseases caused by pathogenic 
 leptospira spp . 
 many molecular techniques have consecutively been 
 developed to detect such pathogen including loop  
 mediated isothermal amplification method 
 ( lamp ) . 
 the objectives of this study were to evaluate the diagnostic accuracy of lamp 
 assay and real - time pcr using bacterial culture as the gold standard and to assess the 
 agreement among these three tests using cohen s kappa statistics . in total , 533 urine 
 samples were collected from 266 beef and 267 dairy cattle reared in central region of 
 thailand . sensitivity and 
 specificity of lamp were 96.8% ( 95% ci 81.599.8 ) and 97.0% ( 95% 
 ci 94.998.2 ) , respectively . 
 the accuracy of lamp ( 97.0% ) was significantly higher than 
 that of real - time pcr ( 91.9% ) at 95% ci . 
 with cohen s kappa statistics , culture method and 
 lamp were substantially agreed with each other ( 77.4% ) , whereas real - time pcr only 
 moderately agreed with culture ( 47.7% ) and lamp ( 45.3% ) , respectively . 
 consequently , lamp 
 was more effective than real - time pcr in detecting leptospira spp . in the 
 urine of cattle . 
 besides , lamp had less cost and was simpler than real - time pcr . 
 thus , 
 lamp was an excellent alternative for routine surveillance of leptospirosis in cattle . 
MATERIALS AND METHODS
RESULTS
DISCUSSION