[ASIAN PACIFIC JOURNAL °E A~f.ERGY AND IMMUNOLOGY (2003) 21: 253-257

Comparative Assessment of an Og4C3ELISA and an ICT Filariasis Test: AStudy of .Myanmar Migrants in Thailand

Surang Nuchprayoon1,3,Chantima Porksakorn\ Alisa Junpee\ Vivornpun Sanprasere andYong Poovorawan2

Lymphatic filariasis, mainlycaused by the parasitic filarialworms Wuchereria bancrofti andBrugia malayi, is still a major pub-lic health problem in the tropics.Over 90% of the disease burden isdue to W. bancrofti.1 The diseaseaffects over 120million people2andpersists as the second leading causeof clinical debility and disabilityworldwide.3The World Health As-sembly has set the goal to eliminatelymphaticfilariasisas a public healthproblem.2To achieve this goal, theelimination program requires thecollaboration of the public healthsystems in the affected countries.Furthermore, assessment and moni-toring of the control programs' byefficient surveillanceprocedures areneeded to determine where controlefforts should be initiated, how ef-fective they are, and when they maybe discontinued.3

Recently, it has been re-ported that Myanmar immigrants to

SUMMARY Detection of circulating filarial antigen has now emerged as analternative method for the diagnosis of bancroftian filariasis. We comparedtwo antigen detection assays, an Og4C3 ELlSAand an ICT(immunochroma-tography) Filariasis test, for the diagnosis of Wuchereria bancrofti infec-tions in migrant Myanmar workers in Tak province, Western Thailand. A to-tal of 337 Myanmars participated in this study. The microfilarial rate was 3.3%.The Og4C3 ELlSAcould detect 19.1% of bancroftian filariasis while the ICTtest detected 12.7%.Both antigen assays could detect all microfilaremics. TheOg4C3 ELlSAdetected 14.8% of amicrofilaremics while the ICTtest identified8.1%. Those who were positive for the ICT test were also positive by theOg4C3 ELlSA.Those Og4C3 positive cases, that were ICTnegative (ICT-velOg4C3+ve)had statistically significant (p < 0.05, unpaired i-test) lower Og4C3antigen levels (409.5 units, range 117-2,389) than those that were ICT posi-tive (ICT+ve/Og4C3+ve)(5,252.0 units, range 130-28,062).Our results empha-size the problem of bancroftian filariasis in Myanmar migrants working inThailand. Close. monitoring and control of this disease in Myanmar migrantsare of public health importance. Antigen detection systems are promisingtools for the surveillance of bancroftian filariasis.

Thailand carry W. bancrofti with aprevalence of 2%-8%,4-7while theprevalence in Thai people was only1.62 casesIlOO,OOO.8Conventionally,diagnosis of bancroftian filariasis isbased on the microscopic detectionof microfilariae in blood samples

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h 6-79-10 B ... . filta en at mg 1.' ancroltIan 1a-riasis is often underreported be-cause people with a low level ofmicrofilaremia or amicrofilaremia

can be missed by the conventionalmicroscopicexamination.11Detectionof circulatingfilarialantigenhas nowemerged as alternative pathway forthe diagnosis of W bancrofti infec-tions.7,1O,12Commercial kits utilizingspecific monoclonal antibodies toFrom the 10epartment of Parasitology, 20e-partment of Pediatrics and 3Chula MedicalResearch Center, Faculty of Medicine, Chu-lalongkorn University, Bangkok, ThailandCorrespondence: Surang Nuchprayoon

254 NUCHPRAYOON, ET AL.

detect W.bancrofti antigens are cur-rently available. An enzyme-linkedimmunosorbentassay (ELISA)basedon Og4C3monoclonalantibody13hasbeen used to diagnose microfi-laremic as well as amicrofilaremicindividuals with high specificityand sensitivity.9,13Recently,a wholeblood immunochromatographic(ICT)filariasis card test has been devel-oped using the AD 12.1 monoclonalantibody,14with high specificityandsensitivity to W.bancrofti antigen.15The card test is a promising field-ready diagnostictool due to its easeofuse.15

In the present study, weevaluated the burden of bancroftianfilariasis in Myanmar migrants inThailandby comparingthe ICT Fila-riasis test to the Og4C3 ELISA forantigen detection.

MATERIALS AND METHODS

Study population

The study subjects wereMyanmar migrants recruited fromtwo factoriesin Mae Sot District,TakProvince, Western Thailand. Verbalinformed consent was obtained ftomeach adult or child's parent or guard-ian in the presence of two witnesses.Individuals who were sick or had ahistory of receiving diethylcarbam-azine (DEC) treatment were excluded

from this study. Signs and symp-toms of lymphatic filariasis wereestablished through an interviewandphysical examination. A translatorwho spoke Thai and Myanmesehelped with the communication.Thisstudy was approved by the EthicsCommittee of the Faculty of Medi-cine, Chulalongkorn University,Bangkok, Thailand.

Blood specimens and parasitologi-cal analysis

Two to five milliliters ofvenous blood were collected from337 individuals by sterile techniqueand universal precaution between8.00 p.m. and midnight. Thick-bloodfilms were prepared in duplicate asdescribed previously.6-7,16-18All of themicrofilaria-postive specimens wereW. bancrofti.

Due to difficulties in trans-portation, a total of 220 whole bloodsamples and 272 sera were availablefor the ICT Filariasistest and Og4C3ELISA, respectively.Sera from non-infectedhealthypeople living in non-endemic areas were used as nega-tive controls.

Detection of W. bancrofti specificantigens

The Og4C3 circulating anti-gen detection test was done by asandwich ELISA13according to the

manufacturer's recommendations(TropBio Pty Ltd, Townsville, Aus-tralia).

The whole blood version ofthe ICT test kit (AMRAD Opera-tions, New South Wales, Australia)was performed according to themanufacturer's instruction for thequalitative detection of W.bancroftiantigen.

Data analysis

The data were collected and

analyzed using Excel 6.0@softwareas well as the unpaired [-test andthe Chi-square test with the level ofsignificance set atp < 0.05.

RESULTS

Among the 337 Myanmarmigrant workers, 58 (17.2%) weremale and 279 (82.8%) were female(Table 1).The mean age of the studypopulation was 22.3 I 6.0 years(range 10-56 years). Clinical mani-festations of lymphatic obstructionwere observed in 13 individuals.Eight individuals had enlarged in-guinal lymph nodes. Five individu-als had enlarged scrotums. By usingblood smear examination under amicroscope, microfilariae were de-tected in 11 (3.3%) individuals, 2 ofwhom were male and 9 were female.

Table 1 Prevalence of bancroftian filariasis in Myanmar migrants classified by sex, and diagnosticmethods

Test No. examined (%) No. positive (%)

Total Male Female Total Male Female

Thick-blood film 337 58 (17.2) 279 (82.8) 11(3.3) 2 (3.4) 9 (3.2)ICTfilariasis test 220 47 (21.4) 173 (78.6) 28 (12.7) 4 (8.5) 24 (13.9)

Og4C3 ELlSA 272 47 (17.3) 225 (82.7) 53 (19.5) 6 (12.8) 47 (20.9)

Og4C3 AND ICT FILARIASIS TESTS

The prevalence of filarialantigenemiawas 12.7% (28 of 220)as assessed by the ICT Filariasistest, and 19.5% (53 of 272) as as-sessed by the Og4C3 ELlSA (Table1).The ICT Filariasis test identified4 antigen-positive males and 24females, resulting in prevalencerates of 8.5% and 13.9%, respec-tively. Out of the 53 Og4C3 anti-genemiccases, there were 6 (12.8%)antigen-positive males and 47(20.9%) females. There was no sta-tistical significance in gender dif-ferences in the prevalence as as-sessed by thick-blood films, theICT Filariasis test, and ELISA forOg4C3 antigen.

The data were further ana-lyzed to compare the prevalencerates for bancroftian filariasis as as-sessed by thick-blood film, the ICTFilariasis test and Og4C3 ELISA(N = 220). Forty-two (19.1%) indi-viduals were positive for at leastone test, all of which were positivefor Og4C3 antigen. Both antigendetection tests identified all micro-filaremic cases. All the 28 ICT-positive cases were also positivewith the Og4C3 ELlSA. In amicro-filaremic individuals, the positiverate of the ICT Filariasis test for

specific W. bancrofti circulatingantigen was 8.1% (17 of 209) whilethe ELISA for Og4C3 antigencould detect 14.8% (31 of 209) ofamicrofilaremics(data not shown).

The Og4C3 antigen levelsfor the ICT+ve/Og4C3+ve, ICT-velOg4C3+ve, and ICT-ve/Og4C3-vegroups were 5,252.0 (130-28,062,N = 28), 409.5 (117-2,389, N = 14),and 10.4(10-61, N = 178) units, re-spectively(data not shown). Amongthe Og4C3positivecases, those withICT negative (ICT-ve/Og4C3+ve)had statistically significant lower

Og4C3 antigen levels than the ICTpositive (ICT+ve/Og4C3+ve) indi-viduals (p < 0.05, unpaired I-test).

DISCUSSION

The antigenemic status ofW. bancrofti infection among mi-grant Myanmar workers was as-sessed using the ICT Filariasis testand an ELISA for Og4C3 antigendetection. Previous baseline data onthe prevalence of bancroftian fila-riasis among migrant Myanmarworkers in Thailand were based on

parasitological diagnosis usingthick film bloodsmears.s,8 Similarto previous studies which estimatedthat 2%-8% of these migrants carryW. bancrofti infection,4-6we foundthat the migrants had a microfilarialrate of 3.3%, while both antigentests iden-tified 2-5 times morecases (Table 1).

It is estimated that hundreds

of thousands of Myanmar migrantshave settled in the urban cities ofThailand. The infected immigrantscarry the nocturnal periodic form(urban type) of W. bancrofti whichhas Culex quinquefasciatus as themain vector species.7,19Cx. quin-quefasciatus is abundant in thebig cities of Thailand, and has thepotential to transmit bancroftianfilariasis19 thus putting the Thaipopulation at risk of acquiring thisinfection. Therefore there is a ma-jor concern that bancroftian filaria-sis may re-emerge as a major healthproblem for Thai citizens. It is nec-essary to set up well-plannedstrategies for the control of lym-phatic filariasis while the Myanmarmigrants are working in Thailand.Obviously, using improved diag-nostic methods is essential to fa-cilitate surveillance activities, andmonitor and evaluate control ef-

255

forts.

Both diagnostic kits used inthis study detected antigens secretedfr d 1 1420-21. d

. .om aut worms,' m lcatmg

active infection. As both antigendetection tests were capable of de-tecting W. bancrofti circulating an-tigens in microfilaremic as well asamicrofilaremic individuals, the useof thick-blood films would haveunderestimated the real prevalenceby 2-5 folds in this population (Ta-ble 1). The fact that the prevalencewould be higher when using anti-gen tests for screening instead ofmicroscopic examination for micro-filariae was reported previ-ously.7,10,ls,n-26Circulating antigensas markers for the diagnosis ofbancroftian filariasis can detect in-dividuals with low levels of or ab-

. fil . 2427F hsent lTIlCro1arelTIla.' urt ermore,large numbers blood specimens canbe obtained during daytime fast andeasily. As part of the global elimi-nation program, antigen testingwould therefore be a useful rapidscreening tool for determining theprevalence and distribution of W.bancrofti.1s,28

As reported previously/3,29-33the ICT Filariasis test and Og4C3ELISA could detect all microfi-laremic cases. The Og4C3 antigenlevels of our microfilaremic groupwere significantly higher than of theamicrofilaremic group (p < 0.05,unpaired I-test; data not shown).

We found that the ICTFilariasis test could detect less

patients with active W. bancroftiinfection than the Og4C3 ELISA(Table 1), as previously de-scribed.26,32This indicates that theOg4C3 ELISA is a more sensitivetest. ill supportof this hypothesis, allleT-positive (ICT +ve) individuals

256 NUCHPRAYOON, ET AL.

were also positive for the Og4C3antigen. Also those in the ICT-ve/Og4C3+ve group had statisti-cally significant lower antigen lev-els than the ICT+vel Og4C3+vegroup. The Og4C3 antigen is re-leased from adult worms and is

thought to correlate with the adultworm burden.2OIt is possible thatthe ICT-ve/Og4C3+ve group had alower adult worm burden than theICT+ve/Og4C3+ve group. Alter-natively, the Og4C3 monoclonalantibody (mAb) may recognizemore epitopes compared to theICT's AD 12.1 mAb. Furthermore,the test protocol of the Og4C3ELISA includes a boiling step torelease antigen possibly trapped in-side the immune complexes, whichincreases the chance of detection bythe Og4C3 mAb.

While the global elimina-tion program of lymphatic filariasisis ongoing, highly sensitive andspecific diagnostic assays are nec-essary to monitor and control theprogram. This study demonstratedthe value of antigen detection testsas a means of surv.eillancefor ban-croftian filariasis in a migrantMyanmar population. The Og4C3ELISA has a higher performance indetecting amicrofilaremics than theICT Filariasis test. However, theICT Filariasis test may be more ap-propriate in remote areas becauseof its ease of use, as finger-prickblood is sufficient and results areobtained within 15 minutes.

ACKNOWLEDGMENTS

We are thankful for thesupport from the Thailand ResearchFund (TRF) and the UNDPIWorldBank/WHO Special Program forResearch and Training in TropicalDiseases. CP and VS are Ph.D. stu-

dents in the Royal Golden Jubilee(RG]) Ph.D. Program supported byTRF. AJ is a graduate student sup-ported by National Center for Ge-netic Engineering and Biotechnol-ogy (BIOTEC). yP is a Senior Re-search Scholar of the Thailand Re-search Fund. We are thankful to Dr.Saravudh Suwanadabba (Ministryof Public Health) for his helpfuladvice, to Ms. Kobkam Kam-janopas for co-ordination in thefield, to Ms. Songpun Sangprakan,and Ms. Chantharat Krainara fortechnical assistance, and to Ms. Ji-rapom Songtrus and all the healthpersonnel from Vector-borne Dis-eases Center 18 and the FilariasisDivision, CDC Department, Minis-try of Public Health; and the staffsat the Department of Parasitology,Faculty of Medicine, Chulalong-kom University, for their technicalassistance in the field and laboratorywork.

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