TKANSACT~NS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE (1989) 83, 189-192 189

Delayed-type hypersensitivity and lymphocyte proliferation in response to Leishmsnia major infection in a group of children in Jericho

Ricardo Alvatado’, Claes Enk2, Kamal Jabeg, Lionel Schnur’ and Shoshana Frankenburg’*2 ‘The Kuvin Centre for the Stua’y of Infectious and Tropical Diseases, 2Department of Dermatology, Hadassah University Hospital, Jerusalem, and the 3Public Health Department, Jericho, Israel

Abstract The cellular response to Leishnkznia major was

evaluated in v&o with a lymphocyte proliferation microtest, performed on 100 d of whole blood obtained -by finger prick. The &urn time and optimum conditions for storage of fresh blood before testing were determined, and the ability of the assay to evaluate cellular immunity to Leiskmania was compared to that of the classical Montenegro skin test. A positive correlation bet&en the diameter of the skin induration and the stimulation index was demonstrated. Defining a positive skin test by indura- tion 35 mm, and a positive proliferation assay by a stimulation index >2*6 and a response ~3000 ct/min. we found a significant correlation between the 2 tests: The proliferation assay was less sensitive than the skin test, but somewhat more specific. Diagnostic speci- ficities and sensitivities did not differ for the 2 tests.

Introduction The Jordan Valley is an area highly endemic for

cutaneous leishmaniasis. Jericho, the main city of the area, has been for many years an important focus of cutaneous leishmaniasis, giving the disease its local name, ‘Rose of Jericho’. During the last 2 decades there have been marked ecological changes in some of the areas endemic for cutaneous leishmaniasis in Israel, mainly in the Jordan Valley. These changes, largely the result of building civilian settlements, have led to a far greater exposure of the inhabitants to infected sandflies and contact with reservoir animals (GREENBLATT et al., 1985). Although cutaneous leishmaniasis is reDortable bv law in Israel. the disease is clearly under&ported. fro studies on’the human prevalence have so far been performed.

The chief indicator of the prevalence of cutaneous leishmaniasis in human populations is usually the Montenegro test, a dehyed-type hypersensitivity (DTHI skin test reaction which uses leishmanin (L&&u& promastigotes killed with phenol) as antigen (MANSON-BAHR, 1987). The main disadvan- tages of the Montenegro test are the difficulties of obtaining an objective measurement of induration (WHO, 1984) and the fact that varying proportions of the population in hyperendemic areas give positive leishmanial reactions in the absence of a historv of previous infection (MANSON-BAHR, 1987). -

Proliferation of human nerinheral blood lvmoho- cytes in titro in respon& t0 specific leishm&ial antigen has been shown to reflect previous contact

Author for correspondence: Dr Shoshana Frankenburg, The Kuvin Centre for the Studg of Infectious and Tropical Diseases, The Hebrew University Hadassah Medical School, Jerusalem 91010, Israel.

with the parasite ( WITZTUM et al., 1978; GREEN~Z al., 1983; SELLS & GOLDRING, 1987: FRANKENBURG. 1988j. The main disadvant&es of the conventiod lymfihocyte proliferation assay are the need for at least 5 ml of blood. which can be obtained onlv bv vein puncture, thus often arousing ethical atZ c;ltural resistance, and the relatively cumbersome procedure of separating mononuclear cells from the total blood by density gradient centrifugation. We have recently described a microtechnique for measuring specific lymphocyte proliferation in response to L. major antigen. This technique requires only 100 pl of blood, which can be obtained by finger prick, and no separation of mononuclear cells is needed (FRANKEN- BURG, 1988). Comparing the microtechnique with the conventional lymphocyte proliferation assay, we found that blood specimens from donors cured of a previous L. mujor infection responded to the L. major antigen in both assays, whereas blood from non- exposed controls did not respond in either assay. Furthermore, we investigated various aspects of the microtechnique, such as inter-donor variability, ways of defibrination of the blood, and differences between capillary and venous blood as a source of cells for the assay.

We now report a comparison between the classical Montenegro test and the micro-lymphocyte prolifera- tion test in a survey performed among seventh grade school children in the city of Jericho.

Materials and Methods stuay poplllatiofis

Two groups of subjects were studied. (i1 Thirtv school &l&en were randomly chosen fr’om 157 12-13 years old children in the 7th zrade 115 of 109 boys fr-om the Al-Buhtari Elemen& and Intermedi- ate School and 15 of 48 girls from the Jericho Elementary and Intermediate School for Girls). A questionnaire was completed by a physician in an interview with each child, and the physician checked each child for leishmanial scars. The operational definition of leishmanial antecedents consisted of a positive clinical history and the finding of susmted scars. None of the children had, in the past-week, fever, cough, rash or bleedina disorders. Onlv one child- was ;&eiving antibiotics against tonsil& (ii) Four subjects cured of L. mujor infection, verified by the demonstration of parasites in the lesion, and 2 subjects not previously exposed.

Blood from patients and non-exposed subjects was obtained on the same day.

Montenegro (leishnuanin) test One hundred ~1 of saline with 0.5% phenol

containing lo6 L. major promastigotes (LRC-L137)

190

were injected intradermally, and the induration evalu- ated 72 h later. An area of induration with a diameter 35 mm was considered positive.

1OOOOO

Micmlymphocyte prolifmation test The test was adapted from the technique described

by LEROUX et al. (1985). BriefIy, 100 4 of blood were taken by finger prick, using sterile 100 ~1 disposable micropipettes (Blaubrand, Intra Mark, Federal Re- public of Germany). The blood was transferred to a tube containing 1.5 ml RPMI-1640 (Ysum, Israel) supplemented with 2 mu glutamine, 100 &ml streptomycin, 100 u/ml penicillin (Teva, Israel) and 1 N of heparin (Leo, Ballerup, Denmark, without preservatives). In some of the experiments, blood was drawn into heparinixed capillaries. The diluted blood was thoroughly mixed and distributed in 100 fl aliquots into round bottomed 96-well plates (Cell- Cult, Sterilin, England). The mitogen used was phytohaemagglutinin-P (PHA, 0.3 l.tg/well, Serva, Federal Republic of Germany). The antigen used was a freeze-thawed and sonicated preparation of L. major promastigotes (FTS). The FTS was lyophilixed and stored at -80X, and diluted in RPMI-1640 and sterilized by filtration before use. One or 10 pg in 25 gl of medium were added to each well; every test was performed in triplicate. The plates with mitogen were incubated for 3 d and the plates with antigen were incubted for 6 d at 37°C in an atmosphere of air and 6% COr. Eighteen h before harvesting, 1 clcj of [3H]thymidine (2 Ci/mmol, Nuclear Research Centre, Israel) was added. The cells were harvested with an automatic cell harvester (Titertek) and the incorpo- rated radioactivity was measured by means of a liquid scintillation counter (Packard Tricarb). Results are presented as counts per minute (ct/min) or as stimula- tion indices (SI, ct/min of wells with antigen divided by ct/min of control wells with cells only). A response of at least 3000 ct/min and an SI of at least 2.6 (with either concentration of FTS) was considered positive.

Results Conditions for blood storage

A first series of experiments was performed in order to establish the optimum conditions for blood storage before assaying. Blood from immune donors was stored at 4”C, either diluted in RPMI-1640 (100 ~1 of blood in 1.5 ml of culture medium) or without previous dilution (100 u.l of blood in heparinixed capillaries) and tested for lymphocyte proliferation after various periods of time. The results (Fig. 1) showed no difference between the response to FTS of blood stored with and without dilution (P>O*O5, Willcoxon U test). It was also found that storage at 4°C was preferable to storage at room temperature (results not shown). Fig. 2 shows the decrease in lymphocyte proliferation with progressive storage: the blood could be stored for as long as 2 d without significant loss of activity, but after 3 d the activity decreased to 20% (P<O*O5, Friedmann two-way analysis of variance). Based on these results, the blood samples were collected during the morning and kept refrigerated until the next day, when they were distributed into wells and incubated.

Testing in vivo and in vitro Of the 30 children submitted to both tests, 5 did

0” -I

100

8 . .

t! . . . 0 0

:

0

H ; : .

0

.

i 0 : x . . : A 4 1 2 3

Days of storage Fig. 1. The effect on the proliferative response of storing blood diluted (0) and undilttted (m).

0

0 .

.

! : :

.

\

O! 1 2 3 4

Days of storage Fig. 2. Effect of storage time on the proliferative response. Median values (0) and range (vertical bars) of ten observations.

not return for skin test reading and were excluded from the study. The results of the Montenegro skin tests and the FTS-induced lymphocyte proliferation tests are summarized in Table 1. All subjects reacted positively to PHA stimulation (results not shown).

When comparing the results of the skin test and of lymphocyte proliferation after antigen stimulation, it was found that 14 subjects were positive by both tests; 4 subjects were negative by both tests, and 7 subjects were positive by skin test but negative by lymphocyte proliferation (P<O*05, Fisher exact probability test). Thus, there was significant correlation between the outcome of the 2 tests in binominal terms of positive and negative test results. Furthermore, a positive correlation existed between the diameter of the skin

191

Table 1. Montenegro skin tests and FTS-induced proliferative responsesa

Subjects

1

Leishmunia Skin tests antecedents (mm induration)

ves 11

Lymphocyte proliferation Cthill indexb

5.590 19.2

Ii 24 25

;. no no no Yes yes Yes no yes yes yes no no no no no no no yes

g no yes

15 9

1: 14 8

1; 4 5 4 2

10 9 8

13370 13130 7294 6878 7028

18782 9826

18614 4430

18592 6874 5612

19824 1103 1628

2:: 354

1078 414 880 220

1%:

27.3 24.3 17.1 30.1 6.0

18.5 15.6 51.0 5.3

11.7

1z.i 18-7 2.6 1.5 0.4

A:; 0.7

;:;

;:z 1.6

Positive controlC yes 14 10489 7.6 Negative controld no 1 934 1.5

“FTS=freeze-thawed, sonicated promastigote preparation. bStimulation index calculated as (ct/min of antigen stimulated cells)+(ct/min of unstimulated cells). ‘Cells from a subject with culture-verified previous disease. “Cells form a non-exposed subject.

. ,

. . . . .

. . . . .

Fig. 3. Correlation between lymphocyte proliferation stimulation index and skin induration.

induration and the stimulation index (Spearman rank correlation coefficient, r,=O*63, P<O*O5) (Fig. 3).

Comparing the two tests by calculating nosographic sensitivity and specificity ratios (Table 2), it was found that the sensitivity of the proliferation assay was somewhat less than that of the skin test, whereas the proliferation assay had much greater specificity. The diagnostic specificities and sensitivities (i.e., predictive values of positive and negative results, respectively) of the 2 tests did not differ.

Discussion In this study a modified lymphocyte proliferation

assay was compared to the Montenegro skin test as a method of screening for previous contact with L. major. Whereas skin testing is traditionally used to survey the distribution of leishmanial infection, the lymphocyte proliferation assay has mainly been used to study the cell mediated character of the response to infection.

We studied a group of children living in a hyperendemic area, without clinical symptoms of present disease. Previous disease was defined by a positive clinical history and the presence of suspected scars. A positive correlation between the diameter of the skin induration and the stimulation index was demonstrated. Defining a positive skin test by indura- tion k5 mm @IANSON-BAHR, 1987)., and a positive proliferation assay by a stimulation index of at least

192

Table 2. Sensitivity and specihity of tests in vitro and in viva

Leishmunia antecedents Nosographic

Yes No sensitivity specificity

Skin test Positive 10 11

o-91 0.21 Negative 1 3

Proliferation” Positive 8 6

0.72 0.57 Negative 3 8

eLymphocyte proliferation after antigen stimulation.

Diagnostic specificity sensitivity

0.48 o-75

0.57 o-73

2.6 and a response of at least 3000 cr/ntin (FRANKEN- BURG, 1988), we found a signilicant correlation between the 2 tests. The nosographic sensitivity of the skin test was higher than that of the proliferation assay, whereas the specificity was higher for the latter test; the diagnostic specificities of the 2 tests did not differ.

It has been reported (MANSON-BAHR, 1987) that, in areas endemic for leishmaniasis, varying proportions of the population show positive L&km&a skin tests in the absence of a history of past infection. In our study, the proportion of skin test-positive cases without leishmaniasis antecedents was 11114=0-79, whereas the proportion of proliferation-positive cases without antecedents was 6/14=0~43. Whether these cases represent false positives or cases with subclinical infections remains to be established. Elucidation of this uncertainty is obviously important in comparing the 2 assays for epidemiological studies.

The use of the simplified and miniaturized version of the lymphocyte proliferation test may have some technical advantages over skin testing. One of the major drawbacks of skin testing for epidemiological purposes is the fact that the same population has to be called upon twice-once to inject the sensitin and once to read the results. In contrast, the lymphocyte proliferation microassay requires seeing the patient only once; furthermore, blood can be obtained by lingerprick and can be stored up to 2 d before assaying without losing activity. -

DTH does not necessarilv correlate with resistance to reinfection. The reactionbecomes positive early in the course of the infection, whereas the lesion heals some months later. Reinoculation of an individual hearing an active lesion and displaying a positive skin test reaction leads to a typical second ulcer that heals together with the original lesion (DOSTROVSKY et al:, 1952). In experimental murine leishmaniasis there is also a clear dissociation between DTH and resistance to secondary challenge (LIEW er ul., 1984). We are now evaluating the correlation between the lympho- cyte proliferation test and immunity to disease.

Acknowledgementa This study was supported by research contract no.

l-Al-22668NIH-NIAID, Epidemiology and Control of Arthropod Borne Diseases, and grant no. 8600155 from the US-Israel Binational Science Foundation (BSF), Jerusalem,

Israel. C. Enk is a postdoctoral research fellow of the Heiscr Program for Research in Leprosy. We are endebted to Mr Musa Abu Hammad, Headmaster of the Al Bubtari School, and to Mrs Faixa Jaher, Headteacher of the Jericho Elementary and Intermediate School for Girls, for their help and cooperation, and to Smadar Uli and Maria Westerman for excellent technical assistance.

References Dostrovsky, A., Sagber, F. & Zuckerman, A. (1952).

Isopbas~c reaction following experimental superinfection of Leishmania nopiea. Archives of Dewnawlogy and Syphilology, 66, 665475.

Frankenburg, S. (1988). A simplified microtechnique for measuring bmnan lymphocyte proliferation after stim- ulation with mitogen. Journal of Zmmunological Methods, 112, 177-182.

Green, M. S., Kark, J. D., Greenblatt, C. L., Londner, M. V., Frankenbnrg, S. & Jacobson, R. L. (1983). Tbe cellular and bumoral response in subjects vaccinated against cutaneous leisbmaniasis using Leishmania fmpka major promasdgotes. Paratire Zmmunology, 5, 337-344.

Greenblatt. C. L.. Scblein. Y. & Scbnur. L. F. (19851. Leisbn&asis &I Israel -and vicinity. In: Leishm&ia&, Cbang, K. P. SC Bray, R. S. (editors). Amsterdam: Elsevier, pp. 415426.

Leroux, M., Scbindler~ L., Bratm, R., Doerr, H. W., Geisen. H. P. & Ktrchner, H. (1985). A whole blood lympb~proliferarion assay for measuring cellular immun- ity against herpes viruses. 3oumal of Zrwuuwlagical Methods, 79, 252-262.

Liew, F. Y.., Howard, J. G. & Hale, C. (1984). Prophylactic immumzation against experimental leisbmaniasis. III. Protection against fatal Leishmania tropica infection induced by irradiated promastigotes involves Lyt. 1 + 2 - T cells which do not mediate cutaneous DTH. Joumul of Zmmunolo~, 132, 456-461.

Manson-Babr, P. E. C. (1987). Diagnosis. In: The Leish- mantises in Biologv and Medicine, Peters, W. & Killick- Kendrick, R. (editors). London: Academic Press, pp. 703-729.

Sells, P. G. & Goldring, 0. L. (1987). Lymphocyte transformation in cutaneous leisbmaniasis patients. Annuls of Tropical Medicine and Parasitology, 81, 105- 116.

WHO, (1984). The leishmaniases. Report of a WHO expert committee. Geneva: World Health Organixation, Tech- nical Report Series No 701.

Witztum, E., Spira? D. T. & Zuckerman, A. (1978). Blast transformation m different stages of cutaneous leish- maniasis. Is~a~lJownal of Medical Sciences, 14,244-248.

Received 20 April 1988; revised 25 July 1988; accepted for publication I I October 1988