the global program to eliminate lymphatic filariasis ( gpelf ) currently implements two diagnostic tools : detection of microfilariae ( mf ) and the detection of circulating filarial antigen ( cfa ) . both tools have proven to be extremely useful during the elimination program stages , where parasite prevalence is relatively high , but there is concern that they might not be sensitive enough to detect residual endemnicity or resurgence in the postprogram phase [ 1 , 2 ] . of particular concern is the slow - evolving nature of the disease since individuals may not become cfa or mf positive until up to 12 months post infection . filarial antibodies develop in response to exposure to the parasite and in endemic countries occur during the first few years of life . in the past , antibody assays in endemic areas were not useful , since the community was constantly exposed and thus antibody positive . with the approach of the end of the mass drug administrations ( mdas ) , the age prevalence should have shifted , with younger individuals born after the mda theoretically antibody negative if there has been a cessation of lf transmission . therefore , it has been suggested that antibody serology be included in the repertoire of lf diagnostics . the lf research community has witnessed the move from earlier antibody assays , reliant on crude parasite lysates , to the recent more specific and sensitive recombinant antigen - based antibody assays ( for review see ) . the earlier assays based on crude parasite lysate were limited in terms of specificity [ 6 , 7 ] , making their use in lf programs inadequate . the advent of recombinant antigen detection systems has increased the specificity of the antibody assays by reducing cross - reaction with other parasitic diseases . the recombinant antigens commercially available are the bm14 , wbsxp , and the bmr1 . bmr1 is a brugia malayi recombinant antigen , which has been shown to react specifically with sera from b. malayi - infected individuals [ 9 , 10 ] ; however its specificity for w. bancrofti has been reported as quite low . the use of the recombinant antigen bm14 in the commercially available filariasis celisa ( cellabs pty ltd . the bm14 assay is an igg4-specific elisa whereby the plates are coated with the recombinant bm14 antigen ( cellabs pty ltd , australia ) . the bm14 gene belongs to a family of genes encoding proteins that are strong immunogens   and was originally isolated from a cdna library in 1992 for its potential application in lf diagnostics . these initial studies demonstrated the affinity of antibodies isolated from microfilaraemic individuals for the expressed recombinant antigen [ 12 , 13 ] . the recombinant antigen has been demonstrated to react with sera from patients with brugian or bancroftian filariasis with reported sensitivities of 96% and 91% , respectively , and no cross reaction was reported with 19 serum samples from strongyloides patients . unfortunately the assay reacted with 72% of the loa loa and onchocerca volvulus positive sera limiting its usefulness in african regions . although the bm14 antibody assay has been commercially available from cellabs , pty ltd australia as a diagnostic tool since 2006 , application to large population sizes in field studies has not been thoroughly assessed . concerns about cross - reactivity in population 's endemic for other helminth parasites , similar to earlier antibody assays , as well as interlaboratory variation have been raised . however , there have been many studies utilising the bm14 , in a research laboratory - based elisa format , with favourable results [ 1417 ] . the commercially available filariasis celisa anti - bm14 igg4 assay differs slightly from the original prototype research - based elisa . however , a recent multicentre evaluation has shown promising results with serum ( weil et al . , in press ) but for any long - term survey work an easier sampling method would be required since venous collection , transportation , and storage of serum can be difficult in endemic areas . filter paper sampling is more cost - effective , easier , and there has been reports of limited sample variation due to fluctuations in temperature since specimens thoroughly dried can be stable at room temperature for up to a week . samples could be collected by filter paper method , which has been shown to be a suitable alternative for antifilarial igg4 antibody assays based on crude protein lysate . filter paper studies for the recombinant antigen bm14 in the filariasis celisa are yet to be ascertained , and the aim of this study was to assess the efficacy of filter paper sampling in a w. bancrofti endemic country . areas of low lf prevalence were chosen for comparison of sampling techniques and for the effect of storage temperature on filter paper samples . the research was conducted under the human ethics approval numbers h1423 and h2816 , as approved by the james cook university research human ethics committee . forty - five nonendemic volunteers , irrespective of age and gender , from townsville , australia were selected as negative controls . ninety four individuals in the south pacific country tuvalu were randomly selected irrespective of age , gender , or previous lf test results . the study was conducted with the assistance of the ministry of health , tuvalu . following verbal consent , 495 participants in the village of siufaga in samoa were screened for antifilarial igg4 antibodies using the filariasis celisa ( filariasis celisa , cellabs pty ltd . the 200 samples chosen were based on the initial optical density ( od ) value , whereby a sample was considered reactive if the od reading was 0.400 , as per manufacturer 's instructions . one hundred and one of the chosen samples were reactive , where 50 samples were high reactors with an od reading > 1.1 . the remaining 51 of the reactive samples had lower od readings ranging from 0.400 to 0.611 . fifty one of the nonreactive samples had initial od readings close to the positive cut - off value of 0.400 in order to determine if after storage these nonreactive samples became false positives . the study protocol was reviewed by the samoan ministry of health for approval prior to commencing the research and carried out with the assistance of world health organisation , samoa . filter paper used for collection was the tropbio filter paper disc ( tropbio pty ltd , qld , australia ) , with six protrusions that specifically soak 10  l of blood each . blood was collected by fingerprick method directly onto the six protrusions of the filter paper . blood was collected from the same individual for a paired plasma and filter paper sample . approximately 200  l of blood was collected using the fingerprick method by capillary action into edta vacutainers ( bd biosciences , becton , dickinson and company , north ryde , nsw , australia ) . following collection , six x 10 l blood was blotted onto filter paper , using a micropipette , and left to dry overnight . the remaining blood in the vacutainer was left overnight at 4c to allow red cell sedimentation . both filter papers and plasma samples were stored at 4c and transported back to australia at 4c at the conclusion of the study . upon arrival to the australian laboratory , both filter papers and plasma were stored at 20c until tested . blood was collected by fingerprick and soaked directly onto each of the 6 protrusions of the filter paper . following collection , filter papers were left to thoroughly dry , then placed in ziplock bags , and transported back to australia . sample diluent was prepared according to manufacturer 's instructions and 500  l was transferred into separate serum tubes using a micropipette . a single blood - soaked protrusion was excised into the serum tube , which was then vortexed to ensure complete saturation of the disc . as each protrusion soaks exactly 10  l , it was assumed that half of this volume constituted serum . diluting 5  l of serum in 500  l of sample diluent the following day the eluates were warmed to room temperature ( rt ) before commencing the assay . antifilarial igg4 antibodies were detected using the commercially available filariasis celisa kit ( cellabs pty ltd , manly , australia ) and samples were tested in duplicate . initial sample incubation was for 2 hours at 37c , incubation with secondary igg4 conjugate was for 45 minutes at 37c , and the final incubation with tetramethylbenzidine ( tmb ) substrate was in the dark at rt for 15 minutes . the washing steps between incubations were performed with an automated plate washer ( multidrop combi nl , pathtec , vic , australia ) using 200  l of washing buffer per well .       on completion the od of the samples was measured at a dual wavelength of 450  nm/650  nm with a multiskan ex type 355 primary v.2.1 - 0 ( pathtec , vic , australia ) using the software labsystems genesis version 3.00 ( pathtec , vic , australia ) . serum samples have been shown to have a lower cut - off value for positivity of od > 0.250 . all analyses were performed using the statistical software spss version 17.0 . the chi - squared test , with the kappa agreement statistic , was used to analyse the sensitivity , specificity , positive predictive value ( ppv ) , and the negative predictive value ( npv ) of the filter paper sample compared to the gold standard . differences in od readings , between sampling techniques and following storage , were analysed using the mann - whitney u test . the average od absorbance value obtained for the 45 nonendemic samples was 0.08 , and thus well below the positive cut - off value for reactivity of 0.400 ( figure 1 ) . there was a significant correlation between the two methods of sampling using the kappa agreement statistic ( r = 0.90 ; p < .01 ) , although there was a significantly higher average od absorbance value for filter paper samples ( 0.550 ) than serum ( 0.401 ) ( z = 6.273 ; p < .001 ) . in comparison to the gold standard , the sensitivity , specificity , ppv , and npv for filter paper sampling the filter paper technique reported a sensitivity of  92% ( 95%-ci 7599 ) , specificity of  77% ( 95%-ci 6586 ) , ppv of  60% ( 95%-ci 4375 ) , and an npv of  96% ( 95%-ci 87100 ) . the initial maximum and minimum od values of the filter paper samples , prior to storage , were 3.9 and 0.013 respectively , with a median of 0.415 . following 10 months of storage at 20c , the od maximum and minimum values decreased to 3.5 and 0 , respectively , with a median drop to 0.08 . this loss of reactivity was significant by mann - whitney u test ( z = 10.9 ; p < .001 ) . following 10-month storage , 67 of the original 101 reactive samples dropped below the reactivity cut - off value of 0.400 and were deemed nonreactive ( table 2 ) . to preserve integrity of samples for sero - epidemiological studies it is important to implement not only the correct collection method but also the correct storage prior to testing . thus serum collection in endemic countries is often infeasible , not only for difficulties concerning sample preservation but also for cumbersome large - scale sampling that is required . there are many advantages for collecting blood by fingerprick including : less invasive , less patient side effects such as reduced risk of haematoma , and reduced risk of needlestick injury to collector . the reduced risk of needlestick injury is particularly important in areas where night bleeding is still required , or areas where there is potential transmission of blood - borne infectious diseases such as human immunodeficiency virus ( hiv ) . it is an easy sample to collect and also can be easily obtained from individuals where venous collection is difficult such as children . another favourable aspect of filter paper sampling is that it has been found in other neglected tropical disease ( ntd ) programmes , such as the onchocerciasis programme , that introduction of filter paper techniques has increased the number of volunteers willing to participate in blood collection , again contributing to economical feasibility and ease of surveying . the present study clearly demonstrates the applicability of filter paper sampling for detection of antifilarial igg4 antibodies using the filariasis celisa . although filter paper sampling requires overnight elution , the sample preparation is quicker and easier . filter paper sampling requires submerging the protrusion in sample diluent prior to elution , whereas serum samples are diluted by pipetting . compared to the gold standard , filter paper sampling had excellent sensitivity of 92% , only missing 2 positive serum samples . the specificity was lower ( 77% ) as 16 samples tested positive by filter paper but negative by plasma . this dropped the ppv to be 60% ; so when testing a sample , it could possibly be a false positive 40% of the time . the npv was also excellent ( 96% ) making filter paper sampling quite robust for accurately testing for nonreactive status . the lower specificity , coupled with a low ppv , means that filter paper sampling may result in false positives at an approximate rate of 40% ( 95%-ci 25% to 57% of the time ) . controlled laboratory experiments , utilising blood spiked with a known amount of antibody onto filter paper , showed no significant differences between serum samples and filter paper samples . there could be a number of reasons for the higher od readings for filter paper samples in the field setting . however , addition of a blocking step for nonspecific proteins did not alter the results ( data not shown ) . it was assumed that 5  l of serum was eluted from 10  l of blood . if the individual was anaemic with a low haematocrit , which can be common in lf endemic countries , the serum to whole blood ratio would increase and potentially more than 5  l could be eluted . eluting a higher volume of serum in an antifilarial igg4 positive individual would alter the od readings of the assay . further studies would be required to ascertain the cause of the higher reactivity observed in the field setting . forty percent of samples potentially being false positive ( a low ppv ) could be disadvantageous for diagnostic testing . however , from a programmatic perspective , the low ppv should not impact greatly on survey work since antifilarial igg4 prevalence rates would be compared annually . any increase in antifilarial igg4 prevalence rates would flag a problem , rather than the individual 's results . if follow - up studies in problem areas required individual diagnostic results , serum samples could be used for confirmation . the high sensitivity and npv are the crucial aspect for sero - epidemiological studies since a high percentage of false negatives would be more detrimental to the lf program . therefore , the high npv and sensitivity observed in this study when using filter paper sampling are advantageous for the lf program . however , until the question of alleged false positivity has been resolved , filter paper test results should be regarded with caution . the decrease in sensitivity observed following 10 months of storage was in agreement with previous studies , which detected antibodies against onchocerca volvulus . in the previous study a significant decrease in antibody detection was observed following 7 months of filter paper storage at 70c , 20c , 4c , and rt . therefore , the results from the current study suggest that in order to detect individuals with low antifilarial igg4 titres , filter paper testing should occur within the first 10 months of storage . the filter papers utilised in the previous study were the whatman no.2 papers , which may be less robust than the tropbio filter paper discs . to ascertain the effect of storage of blood - soaked tropbio filter paper discs , further storage studies need to be conducted looking at several time points and storage conditions , including 70c , 20c , 4c , and rt .      in conclusion , the filter paper collection technique for the detection of antifilarial igg4 antibodies by the commercial kit filariasis celisa is a feasible option for future sero - epidemiological surveys . if serology is pursued as part of the lf diagnostic repertoire , filter paper sampling in endemic countries would be more cost - effective and less laborious than venepuncture techniques . for filter paper sampling , the high npv , coupled with high assay sensitivity , would be advantageous as lf prevalence drops in endemic countries , adding to the usefulness of the assay in post - mda surveys or future surveillance work . future work to ascertain the robustness of the assay in large - scale filter paper sampling in an endemic country would be required , including further evaluating the efficacy of storage of filter papers . overall , filter paper sampling would be more cost - effective and easier than venepuncture and is a favourable alternative method for detection of antifilarial igg4 during survey work in lf endemic areas .