94^ International Journal of Leprosy^ 1992

controls and from BT patients (Fig. 1). Innon reactional LL patients there was no che-miluminescence with PMA stimulation. Incontrast, monocytes from LL patients withENL showed activation with PMA. In pa-tients with ENL reactions being treated withthalidomide, chemiluminescence inducedby PMA was lower than that seen in patientswith untreated ENL.

As expected, lymphocyte proliferation invitro in response to M. leprae was positivein some of the healthy controls and BT pa-tients, but was essentially negative in BLand LL patients (Fig. 2).

Although the number of patients studiedin each group is small, the results suggestthat during ENL the monocytes of LL pa-tients can respond to PMA although theirlymphocytes remain unresponsive to Al.leprae.

—Dilvani 0. Santos, B.Sc., M.Sc.Department of Cellular and

Molecular BiologyFederal Fluminense UniversityAv. Barros-Terra s/nValonguinhoNiteroi, R. J., Brazil 20400

—Jorge L. Salgado, B.Sc.Andre L. Moreira, M.D.

Leprosy UnitOswald() Cruz Foundation

—Euzenir N. Sarno, M.D., Ph.D.ChairmanLeprosy UtiitOswald() Cruz FoundationAv. Brasil 4365ManguinhosRio de Janeiro, R..1., Brazil 21040

Acknowledgment. This investigation received fi-nancial support from the UNDP/World Bank/WHOSpecial Program for Research and Training in TropicalDiseases (TDR) and from CNN (Brazil).

REFERENCES1. HOLZER, T. J., NELSON, K. E., SCHAUF, V., CR1SI'EN,

R. G. and ANDERSEN, B. R. Mycobacterium lepraefails to stimulate phagocytic cell superoxide aniongeneration. Infect. Immun. 51 (1986) 514-520.

2. HoRwiTz, M. H., LEVIS, W. K. and CHIN, Z. De-fective production of monocyte-activating cyto-kines in Lepromatous leprosy. J. Exp. Med. 159(1984) 666-672.

3. LAUNOIS, P., MAILLERE, B., DIEYE, A., SARTHOU, J.L. and BACH, M.-A. Human phagocyte oxidativeburst activation by BCG, M. leprae, and atypicalmycobacteria: defective activation by M. kora(' isnot reversed by interferon-y. Cell. Immunol. 124(1989) 168-174.

4. NATHAN, C. F., KAPLAN, G. and LEVIS, W. R. Localand systemic effects of intradermal recombinant in-terferon-y in patients with lepromatous leprosy. N.Engl. J. Med. 3 (1986) 6-15.

5. RIDLEY, M. J. and RIDLEY, D. S. The immuno-pathology of erythema nodosum leprosum: the roleof extravascular complexes. Lepr. Rev. 54 (1983)95-107.

Can Sandflies Be the Vector for Leprosy?

To THE EDITOR:There are several biting arthropods ex-

isting in leprosy-endemic areas of which anyone could theoretically act as a vector forthe transmission of leprosy. Arthropods suchas mosquitoes, bed bugs and a few otherstake up Mycobacterium leprae while feedingon lepromatous leprosy patients ( 1.3. 7 ). Ba-cilli remain viable for many days in the bodyof these arthropods (5.6 • 8 ). In tropical andsubtropical countries where leprosy is en-demic, sandflies are also commonly found.However, their role as a vector in the trans-

mission of leprosy has so far not been in-vestigated.

Acid-fast bacilli in field-collected sand-flies. As a preliminary approach to screenfor acid-fast bacilli (AFB), sandflies wereperiodically collected from the mud housesof known leprosy patients as well as fromhouses where there were no cases of leprosy.The sandflies were identified and classified( 2 . '). There are eight species of sandflies be-longing to the genera Phlebotomus and Ser-gentomyia existing in these areas of Agra,India. P. papatasi is found to be the most

60, 1^ Correspondence^ 95

predominant species of this region and ac-counted for 75% of the collection. Forty-seven percent of the sandflies caught fromthe houses of leprosy patients were foundto harbor AFB in their gut meal. The num-ber of AFB in the gut of these wild-caughtsandflies varied from 1 to 195. This findingprompted us to carry this study further todetermine the role of sandflies in the trans-mission of leprosy.

AFB in experimentally fed sandflies.Four-day-old laboratory-reared 1'. papatasi(4), about 20 in number, were made to feedon untreated, active lepromatous leprosypatients. The patients' bacterial index (BI)ranged from 4+ to 5+ with the morpho-logical index (MI) between 1.2% and 3%.All were bacteremic with a load of 1 to 6 x10' bacilli per ml. After allowing the sand-flies to have a full bloodmeal, groups of 10sandflics were sacrificed immediately bychilling. The remaining sandflies were sac-rificed periodically thereafter at daily inter-vals up to 10 days. The sandflies were dis-sected, and a uniform smear was made ofall the teased-out components of the pro-boscis, gut, and fecal deposits on the slideand stained for AFB. The results of periodicscreening of the proboscis, gut, and fecaldeposits showed that the number of AFBpresent in these sites declined over time.Occasional AFB were still present in theproboscis on the 8th day; the gut contentand fecal deposit did not show any AFB bythe 8th day. The maximum number of AFBfound was 290 in the proboscis. 595 in thegut meal, and 437 in the fecal deposits.

Inoculating mice with gut meals of sand-flies. Sandflies fed on lepromatous leprosypatients were placed in groups of 10 and 20for each experiment. Each day, from day 0to day 10, sandflies were sacrificed by chill-ing, dissected, and the gut contents werepooled. It was observed that a small pro-portion of AFB were present in these pooledgut suspensions only up to day 7. Thesesuspensions were inoculated into the footpads of mice, and foot pad harvests at 12months postinoculation revealed the pres-ence of an insignificant number of bacilli inonly one mouse each in two groups of sand-flies. In both of these groups mice were in-oculated with day 0 suspensions. In all othermice, including the controls, the harvestswere negative for AFB.

Feeding half-fed sandflies on foot pads ofmice. Sandflies half fed on lepromatousleprosy patients were allowed to refeed ona mouse to determine whether M. lepraecould be carried mechanically through theircontaminated proboscis. The sandflies weremade to refeed on the hind foot pads of micefrom day 0 to day 8 in batches of 1, 6, and10. The mouse foot pads were harvestedimmediately and also at the end of the 12-month post-refeeding. Only a single mousein each group, harvested immediately afterrefeeding, showed a few AFB.

From these experiments it is evident thatwhile taking a bloodmeal sandflies pick upAFB through their proboscis. Some of theseorganisms are ingested and subsequentlyexcreted. However, very few bacilli werecarried mechanically by the contaminatedproboscis. The decline in the number of AFBin the proboscis, gut and fecal deposits withthe passage of time and the failure to mul-tiply significantly in the foot pads of miceindicate that the organisms are nonviable.The mere presence of AFB in the proboscis,gut, and fecal deposits may not make thesandfly a vector for the transmission of lep-rosy.

In conclusion, the present study indicatesthat sandflies do not seem to have any ep-idemiological significance in the transmis-sion of leprosy.

—Sreevatsa, Ph.D.Bhavaneshwar K. Girdhar, M.D.

Central JALMA Institute for LeprosyAgra 282001, U.P., India

—Ipe M. Ipe, Ph.D.St. John's CollegeAgra 282001, U.P., India

—Kothapalli V. Desikan, M.D.Leprosy Histology Center, MGIMSSevagram 442102Wardha, Maharashtra, India

Present address for Dr. Sreevatsa: CJILField Unit, Avadi, Madras 600054, TamilNadu, India.

Ackncmledgment. The authors are grateful to Dr.NI. D. Gupta, Officer-in-Charge, CJIL Field Unit, forthe permission to publish this correspondence. Thetechnical assistance of Mr. Rajendra, F. Lal, and thesecretarial assistance of Mr. A. S. Kumar is gratefullyacknowledged.

96^ International Journal of Leprosy^ 1992

REFERENCES^6.

I. DtiNGAL, N. Is leprosy transmitted by arthropods?Lepr. Rev. 32 (1961) 28-35.LEWIS, D. J. The phlebotomine sanctifies of west

Pakistan. Bull. Br. Nlus. Hist. (Ent.) 19 (1967) I-57.

3. MANJA, K. S., NARAYANAN, E., KASTIJRI, G.,KIRCHHEIMER, W. F. and BALASUBRAMANYAN, M.Non-cultivable mycobacteria in some field collected

arthropods. Lepr. India 45 (1973) 231-234.4. Nlorm, G. B. and Trsii, R. 13. A simple technique

for mass rearing Luizomyia longipalpis and Phle-

botomu.s. papatasi (Diptera: Psychodidae) in the lab-

orators. J. Med. Entomol. 20 (1983) 568-569.5. NARAYANAN, E., SHANKAR MANIA, K., 13Ern. II. M.

S., RIRCHHEIMER, W. F. and BALASUBRANIANYAN,

M. Arthropods feeding experiments in leproma-

tous leprosy. Lepr. India 43 (1972) 188-193.

NARAYANAN, E., SREEVATSA, KIRCHHEIMER, W. F.and 13EDI, 13. M. S. Transfer of leprosy bacilli from

patients to mouse foot pads by . , lales aegipti. Lepr.

India 49 (1977) 181-186.NARAYANAN, E., SREEVATSA, DANIEL RAJ, A.,KIRCIIIILIMER, NV. F. and Brim, B. NI. S. Persistence

and distribution of M. leprae in Aeries ae,elpti and

Culex . fini,ecius experimentally fed on leprosy pa-tients. Lepr. India 50 (1978) 26-36.SAHA, K., JAIN, NI., NIIIKIIERJEE, M. K., CHAWLA,

and PRAKASII. N. Vi-

ability of Mycobacterium leprae within the gut ofAeries aegypt i after they feed on multibacillary lep-

romatous patients: a study by fluorescent and elec-

tron microscopes. Lepr. Rev. 56 (1985) 279-290.THEopoR, 0. Classification of the old world species

of the subfamily phlebotominae (Diptera: Psy-chodidae). Bull. Ent. Res. 39 (1948) 85-115.

7.

8.

9.

Comparison of PGL-I Level with AFB Numbers inFoot Pad Suspension

To THE EDITOR:

Since the time that phenolic glycolipid-I(PGL-I) was first isolated and characterizedas a Mycobacterium frprae-specific product( 4 ' 5 ), it has been widely used in serologicaltests for leprosy. Besides its use for purposesof detecting antibodies to the antigen, PGL-1has been found in various clinical speci-mens, such as serum ( 1-3 . '), urine (`'), andtissues (`. '), etc. However, PGL-I has neverbeen assayed in tissues of mouse foot padsinoculated with M. leprae where it couldprove to be a useful surrogate of acid-fastbacilli (AFB) numbers. Therefore, we at-tempted to measure the levels of PGL-I in

a mouse foot pad suspension using the dotenzyme-linked immunosorbent assay(ELISA) described previously ( 2 . 3 ). Briefly,foot pad suspensions (1.0-1.7 ml) were ly-ophilized, and the lipids were extracted withchloroform : methanol (2:1) solution. Afterapplication to a florisil column, the chlo-roform : methanol (19:1) elute was exam-ined for the presence of PGL-1 by dot-ELISAusing rabbit anti-M. leprae antiserum con-taining anti-PGL-I antibodies. A series ofnormal mouse foot pad suspensions con-taining different amounts of the standardPGL-I were processed using the same pro-cedures to determine the test parameters for

THE TABLE. Detection of PGL-1 in foot pad suvensions.

AFI3 numbers counted^No. assayedPGL-1-positive.'^PGL-I level (ng)

No. (%)^ Mean -±

<7.22 x 10'or <1.77 x 10 4 14 1^(7.1) 7.0

7.22 x^10'-9.4 x 10' 15 8 (53.3) 34.7 ± 39.6

1.0 x^10'-1.0 x^10" 15 15 (100) 98.0 ± 89.7

>1.0 x^10' 14 14 (100) 353.4^428.2

" Determined by dot-ELISA.Calculated based on the suspensions containing PGL-I.