Transactions of the Royal Society of Tropical Medicine and Hygiene 104 (2010) 524–528

Contents lists available at ScienceDirect

Transactions of the Royal Society ofTropical Medicine and Hygiene

journa l homepage: ht tp : / /www.e lsev ier .com/ locate / t rs tmh

Filarial selenium glutathione peroxidase: a probableimmunodiagnostic marker for lymphatic filariasis

Anchal Singha,1, Shaukat Kamalb, Sushma Rathaura,∗

a Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi, UP 221005, Indiab Regional Filarial Training and Research Centre, Vijaya Nagaram Palace, Varanasi, UP 221010, India

a r t i c l e i n f o

Article history:Received 11 March 2009Received in revised form 15 February 2010Accepted 15 February 2010Available online 15 March 2010

Keywords:

a b s t r a c t

Lymphatic filariasis (LF) caused by Wuchereria bancrofti is widely prevalent in tropical andsubtropical countries. Night blood film examination is most commonly used for diagnosisof filariasis but is cumbersome and labour intensive. In order to develop an indirect ELISA-based immunodiagnostic test, the importance of antifilarial IgG subclasses was evaluatedin bancroftian filariasis patients. Blood samples from healthy individuals and different cate-gories of LF patients were used to estimate the diagnostic potential of selenium glutathioneperoxidase antigen purified from the bovine filarial parasite Setaria cervi. This antigen

Lymphatic filariasisImmunodiagnosisSelenium glutathione peroxidaseELISASensitivity and specificityIndia

reacted with both IgG1 and IgG4; however, the IgG1 response was greater in microfilaraemicpatients and the IgG4 response was higher in chronic filarial patients. The diagnostic sensi-tivity of IgG1 and IgG4 was 97% and 96% whereas specificity was determined to be 95% and98% respectively. Our observations suggest that SeGSHPx could be an alternative diagnosticmarker for the detection of bancroftian filariasis in an endemic area.

Society

© 2010 Royal

1. Introduction

Lymphatic filariasis (LF) is a complex human infectionwith a wide spectrum of clinical manifestations. World-wide, more than 1.2 billion people are at risk of filariasis,and 120 million are already known to be infected1. It hasbeen estimated recently that there are more than 27 mil-lion microfilaraemic carriers, around 20.8 million cases ofsymptomatic filariasis and about 429 million individuals

at risk of filarial infections in India.2 The World HealthAssembly has inaugurated the Global Program to EliminateLymphatic Filariasis, and India is one of its signatories.3

Since 2006, a two-drug regimen using diethylcarbamazine

∗ Corresponding author. Tel.: +91 0542 2317231;fax: +91 0542 2368174.

E-mail address: sushmarathaur@yahoo.com (S. Rathaur).1 Present address: Amity Institute of Biotechnology, Amity University,

Uttar Pradesh, Sector-125, Noida, UP 201303, India.

0035-9203/$ – see front matter © 2010 Royal Society of Tropical Medicine and Hdoi:10.1016/j.trstmh.2010.02.007

of Tropical Medicine and Hygiene. Published by Elsevier Ltd. Allrights reserved.

(DEC) and albendazole has been adopted in India as astrategy to eliminate and eradicate LF; however, obtain-ing adequate supplies of both drugs is a major challengefor India.4

The decisions that affect the outcome of the massdrug administration (MDA) programme depend upon mon-itoring its effects, and defining target populations andendpoints for termination of MDA. Consequently there isa need for a reliable and accurate filarial diagnostic test.5

Until recently, diagnosis of filarial infection has dependedupon night blood film examination to detect circulatingmicrofilariae in the blood of infected individuals. This testhas major drawbacks, as it is neither convenient nor reli-able.

Active filarial infections are characterized by elevated

levels of antifilarial IgG4, which decline to normal levelsafter treatment.6 Within endemic populations different IgGsubclasses have been used for filarial diagnosis. An elevatedlevel of filarial specific IgG4 in urine has also been usedas a diagnostic tool for screening among young children

ygiene. Published by Elsevier Ltd. All rights reserved.

of Trop

igis

bamhcSwpoSabf(owTie

2

2

aIts2tcrpd

2d

affifwPdabdttceb

night blood films are referred to as true negatives. False

A. Singh et al. / Transactions of the Royal Society

nfected with filariasis.7 Currently, the immunochromato-raphic card test is used for filariasis detection, althought is facing challenges over its sensitivity, production andtorage, as well as cost-effectiveness for field trials.8

Setaria cervi, a bovine filarial parasite, has a resem-lance to Wuchereria bancrofti in its nocturnal periodicitynd antigenic pattern and various biochemical,9 MALDIass sequencing10 and immunoprophylactic studies11

ave unequivocally established its closeness to W. ban-rofti. Previously we reported that different antigens of. cervi cross-react with sera of human patients infectedith filaria.12,13 We observed that selenium glutathioneeroxidase (SeGSHPx) was present in different life stagesf S. cervi, including its excretory-secretory products.14

ince the antigen is present in all stages of the wormnd is cross-reactive to W. bancrofti, SeGSHPx appears toe a very good candidate for use as a diagnostic antigenor detection of bancroftian filariasis in an endemic areaS. Rathaur, unpublished data). In this study, we reportn SeGSHPx reactivity with sera from humans infectedith filaria and from healthy controls in an indirect ELISA.

he efficacy of this test for the detection of an antifilar-al IgG subclass specific for the filarial parasite was alsovaluated.

. Materials and methods

.1. Study site and population

The study was carried out in Narottampur, a suburbanrea of Varanasi district during July 2006 and October 2006.nhabitants of this area vary in their socioeconomic sta-us, from very low to lower-middle class. Participants wereelected without any bias for age, sex and caste, until nearly0% of the total population of this area had been inves-igated. For each individual age, sex, duration within theommunity, and any previous treatment with DEC wereecorded. Oral informed consent was obtained from all thearticipants. None of them had received any antifilarialrugs in any form during the previous 5 years.

.2. Parasitological examination and collection ofifferent categories of sera

Finger-prick samples of night blood were taken fromll those participants who had been examined clinicallyor LF. Thick smears of night blood were prepared fromnger-prick samples and stained with Leishman’s stain

or identification of microfilariae to species. Microfilariaeere identified based on morphological features and size.

eripheral blood samples were collected using sterile nee-les and syringes after cleaning the volar surface of therm with cotton wool moistened with methylated spirit,etween 21:00 and 01:00 h. A 100-�l blood sample wasrawn into a heparinized capillary tube and immediately

ransferred to 900 ml of 3% acetic acid. The microfilariae inhe specimen were quantified later in laboratory, using theounting-chamber technique15 and microfilaraemia wasxpressed as mf/ml. On the basis of examination of nightlood films and clinical examination, cases were divided

ical Medicine and Hygiene 104 (2010) 524–528 525

into three groups: (i) microfilaraemic, (ii) asymptomaticmicrofilaraemic and (iii) symptomatic amicrofilaraemic.The average (mean ± SD) ages in these groups were 33 ± 4years, 35 ± 3 years and 38 ± 4 years respectively. The ratioof males to females in all groups was approximately 1:1.Twenty-five healthy individuals without any evidence ofpast filarial infection from the endemic area were selectedas endemic normal. The average age of the endemic normalgroup was 32 ± 4 years.

Sera from 15 normal individuals from a non-filariasisendemic area were used as controls to establish the cut-offfor the indirect ELISA (see below). For cross-reactivity stud-ies, well-defined ascariasis and hookworm sera obtainedfrom patients living outside the endemic area were used.These sera were generously provided by Prof. F.M. Tripathi,Institute of Medical Sciences, Banaras Hindu University,Varanasi, India.

2.3. Parasite collection and SeGSHPx purification fromadult female Setaria cervi

Setaria cervi female worms were collected from a localabattoir and somatic extract was prepared as describedpreviously.14 SeGSHPx was purified to homogeneity usinga combination of glutathione agarose affinity and SephadexG-200 column chromatography.14

2.4. Indirect ELISA for filarial specific IgG subclassantibodies against SeGSHPx antigen

ELISA plates were coated with 2 �g/ml of purifiedSeGSHPx and incubated overnight at 4 ◦C. Antigen-coatedplates were incubated with sera at 1:100 dilution from dif-ferent categories of participants: (i) normal, (ii) endemicnormal, (iii) symptomatic amicrofilaremic, (iv) asymp-tomatic microfilaremic, (v) ascariasis and (vi) hookwormsera. Plates were washed and anti-human IgG1-biotin orIgG4-biotin (Sigma-Aldrich, Bangalore, Karnataka, India) at1:1000 dilution was added. After completion of incubation,ELISA plates were washed three times with washing buffer.Finally colour was developed using streptavidin peroxi-dase (Sigma) (1:500) and orthophenylenediamine (Sigma)chromogen at 37 ◦C and optical density (OD) was takenat 595 nm in a BioRad ELISA reader (BioRad Laboratories,Hercules, CA, USA). The mean + 5 SD of the negative con-trol was used as the cut-off value for further analysis ofdata.

2.5. Diagnostic evaluation

Sensitivity and specificity of assay were calculated asdescribed by Galan and Gambino.16 Individuals who hadmicrofilariae in night blood films are referred to as truepositives. Individuals who did not have microfilariae in

negative refers to those individuals who had microfilariaein night blood films but who failed to be identified aspositive for microfilarial infection by ELISA. False positiverefers to those individuals who were negative for microfi-lariae in night blood films but positive by ELISA test.

526 A. Singh et al. / Transactions of the Royal Society of Tropical Medicine and Hygiene 104 (2010) 524–528

roxidasR (chrovel for I

Figure 1. IgG subclass reactivity of Setaria cervi selenium glutathione penormal, n = 15), EN (endemic normal, n = 25), MF (microfilariae, n = 50), Cexpressed as absorbance at 595 nm. Cut-off level for IgG4: −··−; cut-off le

Sensitivity of assay = (true positives × 100)/(true positives+ false negatives).

Specificity of assay = (true negatives×100)/(true negatives+ false positives).

3. Results

3.1. Antibody reactivity to Setaria cervi SeGSHPx by IgG1ELISA

In total, serum samples from 40 healthy individuals (15

non-endemic and 25 endemic controls) and 115 infectedindividuals were screened by ELISA (Figure 1). The cut-offOD for IgG1 ELISA was determined to be 0.439. Our resultsfor asymptomatic microfilaraemic sera turned out to be 2.8times higher than the cut-off value. Three ascariasis and

Table 1IgG subclass reactivity of selenium glutathione peroxidase in ELISA

Serum type Sample(no.)

IgG1a

ODb ± SD

Non-endemic control (normal) 15 0.31 ± 0.25

Endemic control (normal) 25 0.36 ± 0.02Asymptomatic microfilaraemic (range of mf density)

20–150 15 0.69 ± 0.19150–500 15 0.96 ± 0.28500–1300 20 1.20 ± 0.09

Symptomatic amicrofilaremic 25 0.62 ± 0.41Ascariasis 20 0.39 ± 0.45Hookworm 20 0.39 ± 0.28

a Antibody dilution 1:100.b OD at 595 nm; OD values are mean ± standard deviation.

e in indirect ELISA with different categories of sera: NEN (non-endemicnic, n = 25), ASC (ascariasis, n = 20), HW (hookworm, n = 20). Results aregG1: —–.

one hookworm patient who were microfilariae negativein parasitological examination (1.5%) showed positivity byIgG1-ELISA.

3.2. Antibody reactivity to Setaria cervi SeGSHPx by IgG4ELISA

The cut-off OD for IgG4 ELISA was determined as0.392. The IgG4-ELISA of asymptomatic serum sam-ples had mean OD (0.496), which was double that ofnon-endemic normal values (0.242). Only one of theascariasis-infected sera turned out to be ELISA positive.

However, three microfilaria positive cases were negativefor ELISA. The differences in mean OD values for otherhelminthic infections in comparison with that of endemicnormals (Table 1) for both antibody isotypes was insignif-icant.

IgG4a

Seropositivec

(no.)ODb ± SD Seropositivec

(no.)

0/15 0.24 ± 0.30 0/15

0/25 0.31 ± 0.32 0/25

13/15 0.41 ± 0.38 12/1515/15 0.49 ± 0.51 15/1520/20 0.58 ± 0.04 20/2025/25 0.99 ± 0.59 25/254/20 0.21 ± 0.19 1/201/20 0.28 ± 0.42 0/20

of Trop

3E

fid99wi

4

scIt

hsvuabbrsicupsotcgomrtiiioirsema

cwsicwmth

A. Singh et al. / Transactions of the Royal Society

.3. Diagnostic specificity and sensitivity of SeGSHPxLISA

Using data from all our samples and taking night bloodlm examination as the gold standard, the sensitivity ofetection of antifilarial IgG1 by the SeGSHPx ELISA was7% and specificity was 95%; for IgG4 the sensitivity was6% and specificity 98%. However, both IgG1 and IgG4 ELISAere able to detect all the chronic cases, thus the sensitivity

n this context can be regarded as absolute.

. Discussion

The SeGSHPx ELISA-based immunodiagnostic testcreened serum samples from healthy individuals and ban-roftian filariasis cases and a correlation with anti-SeGSHPxgG1 and IgG4 subclasses against SeGSHPx was observed inhe different categories of infected human sera.

Predominance of different immunoglobulin subtypesas been reported to be dependent upon the clinicaltage of the filarial disease.17 Noordin et al.18 found ele-ated levels of IgG1 in onchocerciasis patients when theysed recombinant antigen (BmR1). Rathaur et al.19 havelso reported a strong IgG1 response against a recom-inant antigen of W. bancrofti (glutathione-S-tranferase)y ELISA. In contrast, Kurniawn-Atmadja et al.20 haveeported both IgG1 and IgG4 response against differenttages of Brugia malayi. On the basis of review literaturen the area of immunodiagnostics of filarial parasites itan be easily concluded that differences in immunoglob-lin isotype response are dependent upon the nature ofarasitic antigen rather than being specific to differenttages of parasite. Anti-SeGSHPx IgG1 was detected in 96%f W. bancrofti infected sera; a negligible cross-reactivityo other helminthic infections was also observed. Thisross-reactivity could be attributed either to heterolo-ous antigens or to cross-reacting filarial antigens.13 Webserved a strong IgG1 response against SeGSHPx amongicrofilaraemic patients while IgG4 was a predominant

esponse in chronic cases. These observations suggest thathe presence of SeGSHPx at different life stages of the filar-al parasite will be an added advantage for diagnosis ofnfection and perhaps for the classification of different clin-cal stages of the infection. These assumptions are basedn preliminary data which were used to develop a basicmmunodiagnostic test and a larger sample size is war-anted to establish the correlation of SeGSHPx with clinicaltages of filarial infection. The IgG1 response increased withlevated levels of worm burden, thus it might serve as aarker for worm exposure and ongoing transmission. This

spect requires further detailed investigations too.Elevated levels of antifilarial IgG4 in microfilaraemic

arriers have been observed in response to adult B. malayiorm extracts.6,21 However, in our study, chronic patients

howed highest levels of IgG4 followed by microfilar-ae infected carriers when compared with non-endemic

ontrol individuals. Elevated levels of anti-SeGSHPx IgG4ere found in all the chronic cases, whereas only 94%icrofilaraemics could be identified as IgG4 seroposi-

ive. Anti-SeGSHPx IgG4 isotype cross-reactivity to otherelminth infections was very low as indicated by a

ical Medicine and Hygiene 104 (2010) 524–528 527

specificity of 98.7% which is close to the specificity ofrecombinant filarial antigens such as Bm14 and BmR1.22,23

The main limitations of these commercially available testsare cost and inconsistent availability. Therefore screeningof large populations in an endemic developing country likeIndia by any of these tests is not feasible.

In the present study antibodies against the antigen havebeen detected in all stages of filarial disease. A successfulfilarial diagnostic test should be a sensitive quantitativetest that can also indicate which patients are at riskof developing disease symptoms. Our findings suggestthat the immunological sensitivity and specificity of anti-SeGSHPx IgG4 can be utilized for the diagnosis of LF inasymptomatic patients and also those patients at differentclinical stages of LF in endemic areas. Our data demonstratethat SeGSHPx has the potential to be a good alternativediagnostic tool to detect filarial infection. This preliminarystudy can form the basis for the development of an immun-odiagnostic test to screen large populations. Currently weare working towards improving the sensitivity and speci-ficity of this test.

Authors’ contributions: SR conceived the study; SR andAS designed the experiments; SK was responsible forcollecting blood samples; AS, SK and SR analysed and inter-preted the data; AS drafted the manuscript. All authorsrevised the article for intellectual content and read andapproved the final version. All authors are guarantors ofthe paper.

Acknowledgements: The authors are grateful to Prof. F.M.Tripathi for providing hookworm and ascariasis infectedsera. The authors wish to thanks Prof. A.S. Verma, AmityInstitute of Biotechnology, Amity University, Uttar Pradesh,Noida, India, for his critical suggestions and English usagecorrections.

Funding: AS acknowledges the University Grants Com-mission for providing financial support in the form of juniorand senior research fellowships.

Conflicts of interest: None declared.

Ethical approval: Banaras Hindu University, Varanasi Eth-ical Committee, India. Oral informed consent was obtainedfrom all study participants.

References

1. Ottesen EA. The global programme to eliminate lymphatic filariasis.Trop Med Int Health 2000;5:591–4.

2. Sabesan S, Palaniyandi M, Das PK, Michael E. Mapping of lymphaticfilariasis in India. Ann Trop Med Parasitol 2000;94:591–606.

3. WHO. Global Programme to Eliminate Lymphatic Filariasis Wkly Epi-demiol Rec 2006, 81: 221–32.

4. WHO. Global Programme to Eliminate Lymphatic Filariasis Wkly Epi-demiol Rec 2008, 37: 333–48.

5. Weil GJ, Ramzy RM. Diagnostic tools for filariasis elimination pro-grams. Trends Parasitol 2007;23:78–82.

6. Haarbrink M, Terhell AJ, Abadi K, Asri M, de Medeiros F, Yazdan-bakhsh M. Anti-filarial IgG4 in men and women living in Brugiamalayi-endemic areas. Trop Med Int Health 1999;4:93–7.

7. Weerasooriya MV, Itoh M, Islam MZ, Qiu XG, Fujimaki Y, Kimura E.Prevalence and levels of filaria-specific urinary IgG4 among chil-dren less than five years of age and the association of antibody

of Trop

528 A. Singh et al. / Transactions of the Royal Society

positivity between children and their mothers. Am J Trop Med Hyg2003;68:465–8.

8. Dreyer G, Noroes R, Lanfredi R, Lins R, Menzes Oliviera A, Figuerdo-Silva J. Preliminary observations on fluids incubated with W. bancroftiusing the imunochromatographic test. Rev Soc Bras Med Trop2008;41:209–11.

9. Pokharel DR, Rathaur S. Purification and characterization of a Leucineaminopeptidase from bovine filarial parasite Setaria cervi. Acta Trop2008;106:1–8.

10. Gupta S, Singh A, Yadav M, Singh K, Rathaur S. MALDI masssequencing and characterization of filarial glutathione-S-transferase.Biochem Biophys Res Commun 2007;356:381–5.

11. Pokharel DR, Rai R, Kodumudi KN, Reddy MVR, Rathaur S. Vacci-nation with Setaria cervi 175 kDa collagenase induces high levelof protection against Brugia malayi infection in jirds. Vaccine2006;24:6208–15.

12. Sharma S, Misra S, Rathaur S. Secretory acetylcholinesterase ofSetaria cervi microfilariae and its antigenic cross-reactivity withWuchereria bancrofti. Trop Med Int Health 1998;3:46–51.

13. Gupta S, Bhandari YP, Reddy MV, Harinath BC, Rathaur S. Setariacervi: immunoprophylactic potential of glutathione-S-transferaseagainst filarial parasite Brugia malayi. Exp Parasitol 2005;109:

252–5.

14. Singh A, Rathaur S. Identification and characterization of a selenium-dependent glutathione peroxidase in Setaria cervi. Biochem BiophysRes Commun 2005;331:1069–74.

15. McMahon JE, Marshall TF, Vaughan JP, Abaru DE. Bancroftian filar-iasis: a comparison of microfilariae counting techniques using

ical Medicine and Hygiene 104 (2010) 524–528

counting chamber, standard slide and membrane (nuclepore) filtra-tion. Ann Trop Med Parasitol 1979;73:457–64.

16. Galan RS, Gambino SR. Beyond normality—the predictive value andefficiency of medical diagnosis. New York: John Wiley and Sons; 1975.

17. Maizels RM, Kurniawan A, Sartono E, Partono F, Selkirk ME, Yazdan-bakhsh M. T cell activation and the balance of antibody isotypes inhuman lymphatic filariasis. Parasitol Today 1995;11:50–6.

18. Noordin R, Aziz RA, Ravindran B. Homologs of the Brugia malayi diag-nostic antigen BmR1 are present in other filarial parasites but inducedifferent humoral immune responses. Filaria J 2004;3:10.

19. Rathaur S, Fischer P, Domagalski M, Walter RD, Liebau E. Brugiamalayi and Wuchereria bancrofti: gene comparison and recombinantexpression of pi-class related glutathione S-transferases. Exp Para-sitol 2003;103:177–81.

20. Kurniawan-Atmadja A, Sartono E, Partono F, Yazdanbakhsh M,Maizels RM. Antibody responses to filarial infective larvae are notdominated by the IgG4 isotype. Parasite Immunol 1998;20:9–17.

21. Wongkamchai S, Rochjanawatsiriroj C, Monkong N, Nochot H,Loymek S, Jiraamornnimit C, et al. Diagnostic value of IgG isotyperesponses against Brugia malayi antifilarial antibodies in the clinicalspectrum of brugian filariasis. J Helminthol 2006;80:363–7.

22. Chandrashekar R, Curtis KC, Ramzy RM, Liftis F, Li BW, Weil GJ. Molec-

ular cloning of Brugia malayi antigens for diagnosis of lymphaticfilariasis. Mol Biochem Parasitol 1994;64:261–74.

23. Rahmah N, Taniawati S, Shenoy RK, Lim BH, Kumaraswami V, AnuarAK, et al. Specificity and sensitivity of a rapid dipstick test (BrugiaRapid) in the detection of Brugia malayi infection. Trans R Soc TropMed Hyg 2001;95:601–4.