Evaluation of the filter paper blood collection method for detecting Og4C3 circulating antigen in bancroftian filariasis

TRANSACTIONS OFTHEROYALSOCIETYOFTROPICALMEDICINEANDHYGIENE(1998)92,407-410

Evaluation circulating

of the filter paper blood collection antigen in bancroftian filariasis

method

407

for detecting

Og4C3

John 0. Gyapong1y3, Kwabena Omane-Badu2 and Roger H. Webber3 ‘Health Research Unit, Ministry oj Health, I?O. Box 184, Accra, Ghana; 2Department of Community Health, School of Medical Sciences, University of Science and Technology, Kumasi, Ghana; 3Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London, WClE 7Hz UK Abstract Serological diagnosis of filariasis is generally known to be more reliable than detection of microfilariae. The recently developed Og4C3 enzyme-linked immunosorbent assay (ELISA) for detecting Wuchereria bancrofti circulating antigen has been shown to be very sensitive in diagnosing filiariasis using serum samples. The commercially available form of this ELISA, using whole blood collected on filter paper, has not been validated independently. We evaluated the sensitivity of this new method against standard 20 FL night blood films in 1808 paired samples from 18 communities in different endemic areas of Ghana. The diagnostic performance of the method was consistently low in all but 2 communities (sensitivity=50.3%). This method of diagnosing filariasis is not suitable for field use in its present form. Keywords: filariasis, linked immunosorbent

Wuchereria bancrofti, diagnosis, assay, Ghana

Og4C3,

Introduction Accurate diagnosis of lymphatic tilariasis still remains a problem for clinicians and epidemiologists working in control programmes. It is usually based on detection of microfilariae or clinical disease (WHO, 1992). Some of the practical limitations to the use of diagnostic methods based on detection of microfilaraemia are the nocturnal periodicity of the parasite (Wuchereria bancroft) and the dependence of the sensitivity of these methods on the blood volume examined (SOUTHGATE, 1974; DENNIS et al., 1976; DREYER et al., 1996). The development of diagnostic methods based on the detection of circulating antigens, which are not dependent on volume of blood or time of the collection of blood, were therefore a great advance in filariasis diagnosis oJ(IEIL et al., 1986; LIM, 1993; WAMAE, 1994). These features make the use of antigen assays desirable for rapid communitv diannosis of filariasis (RAMZI et aZ.. 199 1: FARIS \ et al., i9935. A monoclonal immunoglobulin M antibody-based sandwich enzyme-linked immunosorbent assay (ELISA) which detects a circulating antigen (Og4C3) specific for W. bancrofti in human sera has been developed using an antibody raised against Onchocerca gibsoni, which i&cts cattle (MORE & COPEMAN. 1990). Subseauent field evaluation on serum samples has shown the assay to be highly sensitive and specific for diagnosing W. bancrofii infections (TURNER et al., 1993; CHANTEAU et al., 1994; LAMMIE et al., 1994). A recent study in Brazil also showed the Og4C3 ELISA to be very sensitive in detecting infection in humans with undetectable or ultralow microfilaraemia (ROCHA et al., 1996). In order to make this technique applicable under field conditions, a filter paper method for collecting whole blood specimens has been developed and made commercially available by Tropical Biotechnology Pty Ltd, James Cook University, Townsville, Queensland, Australia (TROPBIO, 1996). The reliability of this method in the community diagnosis of lymphatic filariasis was evaluated in Ghana as part of a study initiated by the World Health Organization. This paper presents the findings of the study. Methods Eighteen communities in in the coastal and northern were identified, based on filariasis survey (GYAPONG

different endemicity zones Savannah areas of Ghana the findings of a national et al., 1996). Ethical ap-

Address for correspondence: Dr John 0. Gyapong, Health Research Unit, Ministry of Health, P.O. Box 184, Accra, Ghana; fax +233 21 226739, e-mail [email protected]

antigenaemia,

microfilaraemia,

filter

paper,

enzyme-

proval was obtained from the Ministry of Health, Ghana, and informed consent of all participants was also obtained. Using a modified expanded programme on immunization cluster survey sampling method (BENNETT et al., 1991), approximately 100 people of all ages were examined in each village. A 20 lrL sample of night blood was taken from each person and examined for microfilariae using the thick blood film method. At the same time, blood was collected on all 6 disks of the special filter paper used in the Og4C3 assay. Each disk when fully saturated contains about 5 FL of blood (TROPBIO, 1996). All blood samples were labelled clearly with unique identification numbers to facilitate data processing. All study communities were treated with ivermectin after the study. The thick blood films were stained with Giemsa’s stain at pH 7.2 using standard methods; the entire field was examined and all microtilariae counted (DENHAM, 1978). As a quality control measure, 10% of all slides were randomly re-examined ‘blindly’ by the laboratory technician and also by the principal investigator. The agreement between the different readings was assessed using the K statistic, a measure of inter-observer variation (FLEISS, 198 1). The filter paper samples were dried for about 12 h and stored in small self-sealing polythene bags in a freezer at -4°C for up to about one week in district hospitals. They were later transported in cold boxes and stored at -20°C until they were ready to be sent by courier to the reference laboratory* for analysis for circulating filarial antigens. The examination for antigenaemia was conductedbriefly as follows. Three filter paper disks saturated with blood were boiled with 200 l.tL of diluent to elute the parasite antigen. A standard ELISA was performed on each sample in duplicate and the results expressed as the mean optical density (OD) of both assays. The results were categorized into 8 titre groups depending on their OD. Groups 1 and 2 were classified as non-reactors (negative), group 3 as equivocal, and groups 4-8 as reactors or positive (TROPBIO, 1996). The remaining 3 filter paper disks saturated with blood were stored for future use in case the assay had to be repeated. The results of both the blood film examination and the ELISA were compared directly on an individual basis to assess the sensitivity, specificity and predictive value of the antigen assay, using the blood film examination as the reference standard. In addition, the geometric mean intensity of microfilaraemia in the community was calculated as antilog[Zlog(x+ 1)/n], where x was the *James Cook University, North Queensland,Townsville,

Tropical Biotechnology Australia.

Pty

Ltd,

408

JOHN

Results Both blood films and filter paper samples were processed horn 1808 individuals. The community prevalence and intensity of infection as recorded by the 2 methods is shown in Table 1. The prevalence of micro1. Community

prevalence

No. examined Females Males

Community

Total

Asemko Asemasa Mpatano Kanfakrom Kwamafokrom Katakor Atinchin Nsuekyir Gyanjinadzi Ateitu Gyahadzi Atechedo Osubonpayin Okyereko Azama Denugu Duri Nakom Total

92 106 117 106 100 85 157 109 131 123 101 80 120 54 74 102 86 1860;

Table

56 (60.9%) 56 (52.8%) 65 (55.6%) 63 (59.4%) 68 (68.0%) 52 (61.2%) 96(61.1%) 67(61.5%) 73 (55.7%) 63 (5 1.2%) 53(52.5%) 57 (71.3%) 68 (56.7%) 23 (42.6%) 36 (48.6%) 64 (62.7%) 40 (46.5%) 33 (50.8%) 1033 (57.1%)

2. Sex differences

in prevalence No.

examined Females Males Total

and intensity

1033 775 1808

of infection

with Wuchereria

Percentage microfilaraemic

36 (39.1%) 50(47.2%) 52(44*4%) 43 (40.6%) 32(32.0%) 33 (38.8%) 61(38.9%) 42(38.5%) 58 (44.3%) 60 (48.8%) 48 (47.5%) 23 (28.8%) 52 (43.3%) 3 1(57.4%) 38 (37.3%) 38 (37.3%) 46 (53.5%) 32 (49.2%) 775 (42.9%) and intensity

28.5 39.7 28.0 25.8 30.6 1.9 15.7 9.7 15.1 9.9 9.6 5.8 8.8 15.8 17.2 4.9 28.8 11.5 17.9 of infection

Prevalence (%) Microfilaraemia Antigenaemia 14.7 20.5 17.9

filaraemia was higher than the prevalence of antigenaemia in all but 2 communities. The sex differences in prevalence and intensity of infection are shown in Table 2. The prevalence of infection was significantly higher in males than in females (microfilaraemia: x2=1 1.61, RO.001; antigenaemia: x2=8.80, PO.003). This difference remained, even after controlling for differences in age group and transmission zone using logistic regression analysis (microtilaraemia: coefficient of regression @0.525, SE=O. 124, P
ETAL.

tive predictive values of 751% and 89.9%, respectively). The correlation between blood slide examination and the antigen assay at the community level is shown in Fig. 1; the coefficient of linear regression, r, was 0.75. The correlation between the measures of intensity of infection was equally good at community level (Fig. 2; r=O.69). The quality control checks on the 10% random sample of blood slides were excellent. The K score between the 2 different readings by the laboratory technician was 0.92, and that between the first reading of the laboratory technician and the principal investigator was 0.89. The few samples for which the readings were different

number of microfilariae per millilitre of blood in microfilaraemic individuals and n was the number of people examined. A similarly calculated geometric mean OD value was calculated for the antigen assay at the community level. Pearson’s correlation coefficient was used to assess the closeness of association between the 2 measures of prevalence and intensity of infection.

Table

0. GYAPONG

9.6 14.0 12.0

bancrofi

Geometric mean microfilaraemia (per mL) 457 792 782 841 1807 629 448 925 348 551 470 500 500 718 568 251 226 646 570

Percentage antigenaemic 15.5 37.9 21.6 21.0 24.0 5.0 8.1 8.7 6.5 ;:; 4.3 5.2 11.0 1.8 10.8 3.6 9.4 12.0

with Wuchereria Microfilaraemia 541 600 570

Geometric mean optical density 0.81 0.52 0.83 0.74 0.82 0.51 0.42 0.64 0.35 0.51 0.57 0.45 0.54 0.84 0.35 0.30 0.39 0.32 0.58

bancrofti

Geometric mean (per mL) Optical density 0.52 0.64 0.58

had very low microfilaraemia. The 2 15 blood slides giving discordant results in the 2 tests (Table 4; 54+ 16 1) were re-examined ‘blindly’ by the reference laboratory at James Cook University. The results were consistent with the original findings. The unused filter paper disks (3 per sample) were subsequently examined and the findings were not significantly different from the initial results. Discussion The 3 main findings of this study were the sex difference in prevalence and intensity of infection, the marked age-dependent pattern of infection, and the low sensitivity of the filter paper method for assessing Og4C3 antigenaemia (Tables 2-4). Sex differences in prevalence of infection with W. bancroft have been documented in several previous studies in different parts of the world (MCMAHON ec al., 1981; BRABIN, 1990; 1991; GYAPONG ef al., 1994, 1996; PANI et al., MICHAEL et al., 1996). All except one of the studies from northern Ghana reported a significantly higher prevalence of infection in males than in females. Sex differences in infection rate, and in overt clinical filariasis in general, are still not very clear-cut. There is no clear biologically plausible reason to explain the difference in terms of exposure to the vector or with regard to biolog-

FILTER

PAPER

Table

BLOOD

COLLECTION

3. Age differences

Age group (years)

FOR

in prevalence

No. examined

o-9 10-19 20-29 30-39 40-49 50-59 60-69

449 334 304

189 119

and intensity

of infection

with Wuchereria

409

bancrofti

Geometric mean (per mL) Optical

Microfilaraemia

density

7.9

6.1

522

9.3

594

11.5 13.8 15.1 17.5 12.8 13.3 13.0a

557 568 638 518 622 530

0.62

NSb

NSb

21.3 30.8 68.3a

-

BANCROFTIANTIGEN

8.8 16.3 25.2 23.1

29.4

86 73

x2 (trend)

OF WUCHERERIA

Prevalence (%) Microfilaraemia Antigenaemia

254

270

DETERMINATION

0.63 0.58 0.56 0.57 0.64

0.49 0.69

~f<0~001. bNo significant trend. Table

4.

bancrofti

Comparison microfilaraemia

between Wuchereria and antigenaemia

Antigenaemia Yes No Microfilaraemia Yes No Total

163

Total

161

54

217

1430

324 1484

1591

1808

40 35 30 25 20 15 10 5 0

6

0

10

Community

20 microfilaraemia

30

40 prevalence

Fig. 1. Correlation betweeen community prevalences (%) of microfilaraemia and antigenaemia. 0.9 0.6 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

I

! 0

500

Geometric

1000

mean

1500

2000

microfilaraemia

Fig. 2. Correlation between the community geometric mean intensity of microfilaraemia (per mL) and optical density. icaliphysiological differences between males and females. Most of the studies mentioned above also reported an age-dependent pattern of prevalence and intensity of infection; however, in the present study, no such trend was found with the intensity of infection. The filter paper antigenaemia assay recorded a lower prevalence of infection than the blood films in all but 2

communities (Table 1). Since the guidelines for collection and storage of the samples were strictly adhered to, it is unlikely that there was a problem with the blood collection procedure or storage and transportation of the samples, and the reference laboratory at James Cook University confirmed that the samples arrived in excellent condition. Thus we concluded that this method of collecting whole blood for the Og4C3 antigen assay was not sensitive enough adequately to detect infection status. Similar studies using the same filter paper blood collection method in other countries (India, Philippines, Tanzania) have found similarly low sensitivities (Dr I’. K. Das, Dr V. Bellizario and Dr I’. E. Simonsen, personal communications). A number of possible explanations can be suggested. The test was initially developed using serum samples from endemic and non-endemic areas in Papua New Guinea and Australia. It is therefore possible that we were dealing with a different subspecies of W. bancrofti in Ghana, with slightly different surface antigens. However, there is no strong evidence to support this speculation. Secondly, it is also possible that the cut-off point between positive and negative has been set too high and may have to be redefined after further tests with serum samples from different parts of the world. Thirdly, since this is the first time the filter paper method for detecting Og4C3 antigenaemia has been used under field conditions, some technical aspects may need to be looked at. For example, are 15 PL of whole blood collected on 3 disks of filter paper adequate to detect circulating tilarial antigens? Is the filter paper adequate for retaining Og4C3? The reliability of the blood film results should be considered. The 215 discordant readings were re-examined, and the findings were consistent. The 54 samples in which no microfilaria was seen but which contained antigen are understandable, as the serological test was expected to be more sensitive than the parasitological test, but the 16 1 samples classified as positive by parasitological examination but negative by serology cannot be explained. This finding is particularly worrying because the microscopical examination of a 20 PL blood slide is not a very sensitive test for the detection of microfilariae in peripheral blood; quite a number of microfilariae are lost during the staining process, especially during dehaemoglobinization (DENHAM et al., 197 1; SOUTHGATE, MAHON

1974;

ABARU

&

DENHAM,

1976;

Mc-

et al., 1979). It is also less sensitive than exam-

ining 100 uL of blood in a counting chamber and the filtration method. Even in the 2 communities where the prevalence of microfilaraemia was higher than that of antigenaemia, there were still some people who were microtilaraemic but negative for the Og4C3 antigen. WEIL

et al. (1987)

evaluated

a monoclonal

antibody-

based enzyme immunoassay for detecting soluble parasite antigen in sera collected in an area in South India endemic for W. bancrofti, and found it to be highly sensitive. In a follow-up study to compare the results obtained with whole blood, blood dried on filter paper,

JOHNO.

410

and serum, the filter paper blood specimens were to be less sensitive and less specific (SANTJXANAM

found et al.,

1989). Many more studies have since shown the superiority of immunodiagnosis of W’. bancrofti infection (RAMZY et al., 1991; FARIS 1993; WEIL et al., 1996),

et al.,

1993;

TURNER

et al.,

but all these studies used se-

rum samples. Until antigen assays for detecting filariasis infection are further developed under field conditions (e.g., by use of whole blood), they may not have a role in routine large-scale epidemiological surveys. A test such as the Og4C3 surface antigen assay using blood collected on filter paper, which has a sensitivity of only 50% compared to one of the least sensitive methods available (20 FL blood slides), requires further development if it is to have a role in the epidemiology and control of filariasis. The main conclusion that can be drawn from this study is that, even though the Og4C3 antigen assay of serum samples in the laboratory has a sensitivity of about 99% and a similar specificity, it was not possible to achieve these levels under field conditions, using whole blood collected on filter paper. Acknowledgements

We are grateful for the institutional support of the Health Research Unit, Ministry of Health, Accra, Ghana, especially to the director, Dr Sam Adjei, and Mrs Margaret Gyapong, a social scientist, for their valuable help during the data collection phase. The district health management teams of the study districts played a key role in data collection and community mobilization This investigation received financial support from the UNDPWorld BankWHO Special Programme for Research and Training in Tropical Diseases. The study was conducted while Dr John Gyapong was in receipt of a WHO Research Training Grant. Dr Roger Webber is a member of the malaria programme funded by the Department for International Development of the UK. References

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