IN ERNAIIONAI. JOURNAI, O1 , LEPROSY^ Volume 67, Number 3

Printed in the U.S.A.(ISSN 014$-916X)

Seroprevalence Rates of Antibodies to PhenolicGlycolipid-I Among School Children as an Indicator of

Leprosy Endemicity 1

Stella van Beers, Mohammed Hatta, and Paul R. Klatser 2

Mycohacteriwn leprae is the causative or-ganism of leprosy, a disease which at presenthas a registered prevalence worldwide ofabout 830,000 people on antileprosy che-motherapy, although the estimated numberof cases in the world reaches about 1.2 mil-lion ( 15 ). The disease can cause permanentand extensive deformities of the skin andthe peripheral nerves. Because of the severeand often irreversible handicaps and, as aconsequence, social stigma and economicloss, the problem of leprosy is even greaterthan the number of cases would suggest.

The goal of the World Health Organiza-tion (WHO) is to eliminate leprosy as apublic health problem by the year 2000, de-fined as a global prevalence of below 1 per10,000. This program has resulted in inten-sified control activities, reducing the num-ber of leprosy patients ("), but the end ofthe problem is not yet in sight. The effec-tiveness of the current strategy for routineleprosy control, based on case detection andtreatment with multidrug therapy (MDT),has been questioned (`' 10). With the presentnumbers of unevenly distributed leprosycases and an incubation time of severalyears, many more leprosy patients can beexpected to emerge in several leprosy-en-demic countries beyond the year 2000 ( 2 ).

Information on the magnitude of the lep-rosy problem in any one arca is importantfor the health services for their planning,

' Received for publication on 30 March 1999. Ac-cepted for publication on 12 May 1999.

2 S. M. van Beers, Ph.D.; P. R. Klatser, Ph.D., De-partment of Biomedical Research, Royal Tropical In-stitute, Meibergdreef 39, 1105 ZA Amsterdam, TheNetherlands. M. Hatta, M.D., Ph.D., Department ofMicrobiology, Hasanuddin University, Ujung Pan-dang, South Sulawesi, Indonesia.

Reprint requests to Dr. Klatser at the above addressor e-mail: P.Klatser@kit.nl

monitoring and evaluation of leprosy con-trol activities. The epidemiological datagenerated routinely by the health serviceswill be greatly influenced by the policy andthe activities of the services. These data donot necessarily reflect the true situation.Until now, the most reliable assessment ofthe leprosy problem in an arca has been byrandom sample prevalence surveys. Butsuch surveys are costly and cannot be car-ried out routinely.

Previous studies have suggested that thepresence of IgM antibodies to phenolic gly-colipid-I (PGL-I) of M. leprae may be in-dicative of (past or present) infection (`)). Inaddition, it has been suggested that a highprevalence of anti-PGL-I IgM antibodiesamong children may be indicative of activetransmission of M. leprae in their surround-ings (L 4 ).

The aim of this study is to evaluate thevalidity of extrapolation of the seropreva-lence in school children 10 to 12 years oldas a proxy indicator for the levei of leprosyendemicity. School surveys were chosen asthe study design because they are relativelyeasy to perform and have the advantage oftargeting an easily accessible, stable andstandardized population.

MATERIALS AND METHODSStudy arcas. With the objective of com-

paring seroprevalence rates with leprosyendemicity, we selected three provinces inIndonesia, each with different leprosyprevalences. They were South, North andSouth-East Sulawesi, with registered preva-lences of 7.4, 3.5 and 2.1 per 10,000, andcase detection rates of 3.9, 1.8 and0.99/10,000, respectively.

Next, districts were selected. They hadto be part of a well-established leprosycontrol program, with at least 5 years of

243

244^ International Journal of Leprosy^ 1999

TAlil_E 1. Leprosy detection rates and characteristics of study population by arca.

Province

NorthSulawesi

SouthSulawesi

EastSulawesi

EastSulawesi

EastSulawesi

District Minahassa Maros Gowa Muna MunaLeprosy detection

rate /10,000 , 2.7 2.1 5.1 1.4 1.0Subdistrict Kauditan Bantimurung Bontomarannu Lawa TongkunoLeprosy detection

rate /10,000' 4.3 4.1 3.4 0.6 0.9No. schools

Registered 40 48 34 37 20Examined 31 39 34 37 20

No. pupilsRegistered in sample 539 663 784 644 423Clinical examination 500 650 734 579 381

Age (yrs.)Average 11.3 11.7 11.8 11.7 12.4S.D. 1.0 0.9 1.0 0.9 1.3

% BCG vaccinated 42.8 28.9 12.7 17.1 8.1

Calculated as average of the 3 years preceding the year of school survey.''Calculated from local registers, average of the 3 years preceding the year of school survey.

90%-100% implementation of MDT, toprovide as reliable as possible estimates ofthe leprosy incidence. On the instigation ofthe leprosy control program, the followingdistricts with different leveis of endèmicitywere selected: in North Sulawesi, Minahasadistrict, with a case detection rate of2.7/10,000; in South Sulawesi, Maros andGowa districts with case detection rates of2.1/10,000 and 5.1/10,000 respectively; inSouth-East Sulawesi, Muna district, with aprevalence of 1.4 per 10,000. The case-de-tection rates cited are the averaged rates forthe 3 years prior to the year in which theschool survey was conducted to reduce theeffect of year-to-year variations.

In each district, a subdistrict was chosenas the reference arca for the school surveys.The selection of the subdistrict was not ran-dom but, again, based on the recommenda-tions of the control program for the most re-liable leprosy estimates. The subdistrictswere: Kauditan in Minahasa, Bantimurungin Maros, Bontomarannu in Gowa, andLawa and Tongkuno in Muna.

All patients from the local registers wereentered in a computcrized database usingthe Epi-Info software. Total detection rateswere calculated per subdistrict. Patientswho were actively detected in mass and fo-cal surveys were excluded to minimize theinfluence of different control activities onthe number of patients registered. The lep-

rosy case-detection rates of the preceding 3years were averaged and the result was usedas a proxy indicator of the leprosy endemic-ity for that arca.

School surveys. The school surveyswere carried out between 1994 and 1998. Ineach subdistrict, ali primary schools with agrade 5 were listed. In the subdistricts Kau-ditan and Bantimurung, the schools wereselected using single-stage cluster samplingwith a probability proportionate to size allo-cation scheme ( 5). Sample size calculationswere based on an expected prevalence of30% with an error of 5% and a design effectof 1.5. Expectations were based on previouscommunity-based studies. In the other dis-tricts, ali listed schools were enrolled in thestudy, since the required sample size wasGlose to the total number of students regis-tered.

All grade 5 pupils at the listed schoolswere included in the study. Each childpresent at the school during the survey wasexamined clinically by experienced localleprosy workers for signs of leprosy. Infor-mation on BCG vaccination status wasobtained by visual inspection for the pres-ence of a BCG scar. Household leprosycontact status was asked for and verifiedwith the local records. Finger-prick bloodwas collccted and serum separated from iton the same day. The serum was frozen at—20°C until testing. In Bantimurung subdis-

67, 3^ran Beers, et al.: Seropreralence Among School Children^245

TABU, 2. Number of newly found leprosy paticnts and seropositit'ity rates among schoolchildren in arcas of different endemicity.

Province

North Sulawesi South Sulawesi East Sulawesi

3 High-endemic arcasCase detection >3 /10,000

2 Low-endemic arcasCase detection 1.0/10,000

Subdistrict Kauditan Bantimurung Bontomarannu Lawa TongkunoLeprosy patients 2 O 3 O 0Household contact 10 4 6 O 0MLPA tested 498 649 729 579 380

Positive 26 28 26 7 795% CI 21.4-31.2 24.5-31.4 22.6-28.9 5.2-9.3 4.6-9.7Males % positive 23.4 25.1 20.5 7.5 7.5Fentales % positive 29.6 30.5 30.9 6.6 6.3

p''=0.14 p=0.15 p=0.002 p=0.8 p=0.8

" Number of children with household contact with a leprosy patient.Prohability of difference between males and females.

trict, a second school sero-survey was car-ried out 3 years aí -ter the first.

Serology. Antibodies to PGL-I of M.leprae were dctected in a simple gelatinparticle agglutination test (MLPA Serodia-leprae kit; Fujirebio, Japan) ( 5). This testwill be referred to as MLPA. Sera with atiter of >_1:32 were considered positive.

Data analysis. All data were entered ona personal computer and analyzed usingEpi-Info version 6.04. Analyses of variancewere applied as indicated in the text. Allprobabilities presented are two-tailed, un-less otherwise statcd. The heterogeneity ofseroprevalence among the different schoolclusters was examined with a goodness-of-fit test (E[(O - E)'/E]).

RESULTSLeprosy indicators. Examination of the

local records revealed that thc incidencerates at the levei of the subdistricts showeda different pattern in comparison with thedistricts and the provinces (Table 1). Basedon the local data, we divided the subdis-tricts involved into regions of different en-demicity: threc arcas, Kauditan, Maros andGowa, with a case detection rate of at lcast3.0/10,000, will be referred to as the high-endemic regions. Two arcas, Lawa andTongkuno, with a case-detection rate of lessthan 1/10,000, were considered thc low-en-demic regions.

School surveys. The characteristics ofthe schools included in the surveys are

shown in Table I. For the first survcys,from the total of 3203 childrcn registered atthe different schools 2844 (89%) were in-cluded in the sample and examined. Serawcre collected and tested from 2835 chil-dren (89% of the number registered).

The male-to-female ratio was not signif-icantly different among the subdistricts(chi-squared, p = 0.21). The mcan age ofthe school childrcn was also similar in thethree subdistricts.

In the high-endemic arcas, tive childrenwere newly diagnoscd with leprosy duringthe survey, and 20 children reported havinga member of the household with clinicaileprosy (Table 2).

The BCG vaccination coverage washighest in the high-cndemic arca; it rangedfrom 42.8% there to 8.1% in one of thelow-endemic arcas.

Serology. Table 2 shows the results ofthe scrological examinations. The seroposi-tivity rates in the threc high-endemic arcaswere similar, ranging from 26% to 28%.Thcy were notably higher than the 7%found in the low-endemic arcas. The corre-lation coefficient between incidence andseroprevalence was 0.98.

A signifìcant difference in scropreva-ience was found between male and femalesubjects in the high-endemic arcas, but notin the low-endemic arcas (Table 2).

To look for a possible age effect, theschool childrcn wcre divided into twogroups: undcr 12 years of age and 12 yearsor older. No difference was found in the

246^ International Journal of Lcpros.v^ 1999

seropositivity rates between these two agegroups (results not shown).

No relation was found between seropos-itivity and BCG status.

To investigate the hetcrogcneity of sem-prevalence in the different school clusters,the expected prevalence rate in each clusterwas calculated and compared with the oh-served prevalence rate. Seropositivity wasclearly uniformly distributed in all schoolclusters in four of the tive arcas (p >0.3).However, in Bantimurung subdistrict, ahigh-endemic arca, we observed a higherseropositivity rate than expected in two ofthe 39 schools (p <0.005 and p = 0.04, re-spectively), which brought the assumptionof hetcrogcneity of the clusters to a proba-bility of p = 0.07.

In the second survey in Bantimurung, 3years after the initial survey, 905 childrenfrom grade 5 were examined. The sero-prevalence of 28% was exactly the same asthat found in the first survey. The leprosycase detection rate was also similar 3 yearslater.

DISCUSSIONA prerequisite for any disease control

program is to be able to measure, orai leastto estimate, the prevalence of the disease.Such information is essential not only forthe allocation of resources, bui also forevaluating the effect of the control men-sures. At present, leprosy control programsface difficulties in estimating the burden ofdisease. Because leprosy prevalence is gen-erally low, accurate assessment of the preva-lence of the disease can only be obtainedthrough laborious and expensive mass sur-veys. Official data from registers are oftennot reliable, since they are greatly influ-enced by the policies and the quality of thelocal or national leprosy control program.We have looked at an alternative approachto assess the magnitude of the leprosy prob-1em, extrapolation of the seropositivity ratesfor the species-specific antigen PGL-I inschool children. The results suggest thatthis seropositivity is related to the leprosyincidence in that population. This approachmay thus provido a rapid, reliable and rela-tively inexpensive method to estimate theleprosy burden in a selected arca.

We have determined the seroprevalencerates in school children in tive selected ar-

cas in Sulawesi, indonésia. Threc had simi-lar high leprosy case-detection rates of 3.4to 4.3/10,000 and seroprevalence rates of26% to 28%. The other two regions hadmuch lower incidencc rates of fess than1.0/10,000 and also lower seropositivityrates (7.1 %). It should be noted that thesecase-detection rates are based on passivecase finding only.

In our opinion, it is valid to comparethese rates from the different arcas, sincethe control programs active in these arcasare under the same management policies.However, our data also illustrate that it is analmost impossible task to obtain true inci-dence rates in a leprosy-endemic arca. Atbest, the case detection rates can be used,which we have also done in our studies, butthese rates are highly dependem on thecolnmitment of the local leprosy workers.In addition, the figures reporte(' representaggregatcd data which may completely oh-scure pockets of high leprosy endemicitysuch as those we have encountered in NorthSulawesi. Although this province was elas-sified as a low-endemic arca and very fewcases had been reported in the past yearsfrom the selected subdistrict, the examina-tion of the local registers revealed the sub-district to be highly endemic. The low num-bers of patients reportcd may have beencaused by a change of the local leprosyhealth worker followed by a temporary ab-sence of one. Thc load of undetected casesand, therefore, the difficulty in obtaining atrue picture of the leprosy burden is furtherillustrated by the high number of leprosypatients who were detectcd actively in theoccasional mass surveys. Nevertheless, wefeel confident that the designation of thestudied arcas into high- and low-endemicarcas is justified. We have presented the av-erage case detection rates over the last 3years, yet inspection of the data of previousyears gave a similar ranking of the arcas.

A relation between the leprosy burdenand seroprevalence in different paris of theworld has been documented by severalother studies (4 . 11 .9. In addition, Baumgart,et al. (') found that, upon introduction ofMDT in a closed community in Papua NewGuinea, the decrease in leprosy prevalencewas accompanied by a reduction in theseropositivity rate among children. Unfor-tunately, a valid comparison of either sero-

67, 3^ran Becrs, et al.: Seropreralence Arnong School Children^247

positivity or leprosy prevalence rates be-tween these studies is not possible, lincedifferent methods were applied to obtainthe data. Wc have alrcady discussed lhe dif-ficulties in comparing the officia] data onleprosy prevalence. Comparison of serolog-ical results is only possible when thc samestandardized test has been used. Wc havethereforc used a commercially available,standardized assay in kit formal (S) whichguarantees comparable results.

Since seropositivity rates have previ-ously been shown to be age-dependent (' ' 1 ),we chose school children as the studypopulation. This approach offers the com-bined advantages of having an age-stan-dardized population and a rclatively easyaccess to the study population when con-ducting the surveys. Only in the high-en-demic arcas did we Gnd a higher seroposi-tivity rate among females, as has been re-ported for older age groups ( 7. "). This mayreflect a more avid response in femalesupon repeated exposure. The rate of posi-tive BCG vaccination status was highest inthe high-endemic arcas. Clearly, risk factorsother than BCG status are predominam indctcrmining the development of leprosy inthesc arcas.

One of the features of the epidemiologyof leprosy can be its clustered distribution("). However, in four of our arcas, theseropositivity rates were uniformly distrib-uted in ali clusters, suggesting that seropos-itivity reflected an endemic exposure to M.leprae on a population leve]. In only onearca did one particular school cluster showan unexpectedly high seroprevalence rate.From a total of 161 school clusters thenumber having only one homogeneouscluster is so low that it does not influencethe applicability of estimating seropreva-lence by using a single-stage cluster designwith school as sampling units. Heterogene-ity of seropositivity in populations at thevillage levei in Sulawesi has been reportedbefore (' 4). A second survey conductedmore than 3 years ]ater showed exactly thesame seroprevalence rates, revealing theconsistency of seropositivity which wasparalleled by a consistency in case-detec-tion rates.

Although the leprosy detection rate andseroprevalence showed a strong correlation,the number of arcas studied is too small for

fino numerical conclusions. We also lackedan arca with an intermediate endemicity. Itis, thereforc, not possible to predict whetherthe nature of the relationship will be linearor logarithmic in nature.

We propose to validate the approach toassessment of the leprosy problem throughextrapolation of seropositivity rates inschool children in other arcas, both in In-donesia and elsewhere in endemic coun-tries. In Chis study we used a standardizedparticle agglutination assay (MLPA) for thedetection of antibodies to PGL-I, a tech-nique which still requires a laboratory facil-ity. We have now developed a simple dip-stick assay to detect antibodies to PGL-I ofM. leprae which can be performed in thefield and does not depend on a cold chain(`). Using this method, togelher with a sim-plified sampling frame, may make rapidepidemiological mapping of leprosy a feasi-ble prospect in the near future.

SUMMARYIn order to study whether the seropreva-

lence of antibodies to phenolic glycolipid-I(PGL-I) among school children is a usefulindicator of the leprosy problcm in certainarcas, school surveys were carried out.These surveys have the advantage of target-ing an easily accessible, stable and stan-dardized population. Antibodies to the spe-cies-specifie PGL-I of Mvcobacteriu,n lep-ras were detectcd in a simple gelatinparticle agglutination test.

We have determined the seroprevalencerates in 2835 school children from tive dif-ferent arcas in ihree provinces of Sulawesi,Indonesia. Three arcas with a case-detec-tion rate of over 3.4/10,000 were desig-nated as high-endemic arcas. The other twowere designated as low-endemic arcas, hav-ing a case-detection rate of less than1/10,000. The seroprevalence rates in theihree high-endemic arcas ranged from 26%to 28% (95% CI 21%-31%). In both low-endemic arcas the seroprevalence rate was7% (95% CI 5%-10%). In a second surveyconducted in one high-endemic arca 3 yearsafter the first survey, the seroprevalence ratewas the same as in the first survey. Theseresults indicate that seropositivity ratesamong school children may reflect the lep-rosy incidence. They illustrate the potencialapplicability of seroprevalence as an indica-

248^ International Journal ofLeprosy^ 1999

tor of the magnitude of the leprosy problemin a selected arca.

RESUMENSe hizo un estudio para establecer si la seropreva-

lencia de anticuerpos contra el glicc)lipidó fenôlico-Ide Mycobacterium leprae (PGL-i) en los escolares, esun indicador útil de infección leprosa en determinadasáreas geográficas. El estudio tiene la ventaja depoderse enfocar a una población fácilmente accesible,estable y estandarizada. Los anticuerpos anti-PGL-I semidieron usando la prueba simple de aglutinación departículas de gelatina.

Determinamos Ias tases de prevalencia en 2835 es-colares de 5 diferentes áreas de 3 provincial de Su-lawesi, Indonesia. Tres áreas con una Casa de detecciónde casos de 3,4/10,000 se designaron como áreas dealta endemia. Las otras dos se designaron como áreasde haja endemia con una tasa de detección de casos demenos de 1/10,000. La seroprevalencia de anticuerposen las 3 áreas de alta endemia varió del 36% al 28%(95% CI 21%-3I%). En las dos áreas de haja endemiala tasa de prevalencia fue del 7% (95% CI 5%-10%).En un segundo estudio efectuado 3 anos después enuna de las áreas de alta endemia, la seroprevalencia fuela misma que la encontrada en el primer estudio. Losresultados indican que las talas de seroprevalencia en-tre los escolares pueden reflejar la incidencia de leprae ilustran la potencial aplicabilidad de la seroprevalen-cia como un indicador de la magnitud del problema dela lepra en un área seleccionada.

RÉSUMÉAfio de savoir si la séro-prévalence des anticorps

dirigés contre le glycolipide phénolique-1 (PGL-I)parmi les enfants scolarisés est un indicateur utile detendance vers un problème de lepre dans certaineszones, des enquêtes dans les écoles furent réalisées.Ces enquétes ont l'avantage de cibler une populationfacilement accessible, relativement stable et hien stan-dardisée. Les anticorps spécifiques d'espèce, dirigéscontre le PGL-I de Mycobacterium leprae, furent dé-tectés un utilisant un test simple d'agglutination surparticule de gélatine. Nous avons obtenu les taux deséro-prévalence chez 2835 enfants allant à l'écoledans 5 zones de 3 provinces du Sulawesi en Indonésie.Trois zones oú le taux de détection de cas est au-dessus de 3,4 pour 10 000 furent désignées zoneshautement endémiques. Les 2 autres furent qualifiéesde zones de faible endémicité, ayant un taux de détec-tion de cas de moins de 1 pour 10 000. Les taux deséro-prévalence dans les 3 zones variaient de 26% à28% [Intervalle de confiance à 95% (IC95%) 21%-31%]. Dans les 2 zones à faible endémicité, le taux deséro-prévalence était de 7% (IC95%: 5%-10%). Aucours d'une seconde enquète menée dans une zonehautement endémique 3 années après la premièrc, letaux de séro-prévalence était identique à la premièrc.Ces résultats suggèrent que le taux de séro-prévalenceparmi les enfants scolarisés reflete l'incidence de la

lèpre et illustrent l'application potenticlle de la séro-prévalence confine un indicateur de l'amplitude deproblème de la lcpre, au moios dans certaines zones.

Acknowledgment. We express our gratitude to theheads of the Oflices of the Ministry of 1lealth, Repub-lic of Indonesia, in Sulawesi and to the leprosy ofliccrsin the Ministry of Health and in the provinces of Su-lawesi for their cooperation. We are obliged to Dr. De-ter Lever and Dr. Frankie Lopreng tor their valuableadvice. We thank Mr. Romi and Mr. Marrwani for ex-cellent technical assistance. We are especially indebtedto all the school children and their parents who coop-erated voluntarily in this study. The financial supportby the Netherlands Leprosy Relief Association (NSL)is greatly appreciated.

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