INI1 RNAIIONAL J(H'RNAI, cgr Lkt'ROSY^

Volume 67. Numher 2P inted in lhe U.S.A.

(ISSN ()I.48-916X)

CORRESPONDENCE

Thi.s department is for the puhlication ofinfimnal conummications that are o/ alterembecause thev are in/nrnratit'e and stimulating, and /r the discussion of controrersialenatters. The mandate o/ this JOURN.-it. is to dissentinate in%ormation relating to leprosv inparticular and also other mvcohacterial diseases. Dissident cominem or inter pretation onpublished research is of cota -se valid, but personality attacks on individuais x'ould seemtcnnecessarv. Political comments, valid or not, also are unlrelconre. Tlrev ntight result ininterference mth the distributi on of the JOUXNAL and lhos interfere mth its prime purpose.

Infection by M. leprae is Governed by the Temperatureat the Entry Point: a Preliminary Note

To l i n: EDITOR:

The portal of entry of Mvcobacteriuntleprae, the mode of transmission of leprosyin the community, and the evolution of thedisease in an individual is still not clear.Previous studies conducted in nude micehave shown that M. leprae can be transmit-ted through unbroken nasal mucosa (I•')and abraded skin ( 2 ). It is well known thatthe nasal mucosa and feet are relativelycooler sites of the body. Infecting the nudemouse through intact and abraded skin atthe flank was not successful ('). In this pre-liminary experiment an attempt is made tofind out whether infection can take place iforganisms are injected into the flanks ofT900r mice which are almost similar tonude mice in their immunological status.

A group of 10 CBA mice were made im-munodeficient using standard methodologyexcept that the bone marrow transplantationwas performed by injecting the harvestedbone marrow into the peritoneal cavity ( 4 ).The M. leprae suspensions were obtainedfrom harvesting the lepromatous nodulesfrom the foot pads of a T900r mouse inocu-lated 15 months earlier with M. leprae. M.leprae suspensions were diluted so that 0.1ml of a sample contained 10' M. leprae. In10 T900r mice, hair was removed fromboth flanks and 0.1 ml of a suspension con-taining 10' bacilli was injected intrader-mally on both sides. Also, 0.03 ml of thebacillary suspension containing 10 1 bacilli

was injected into the hind foot pads of fivenormal CBA mice to confirm the viabilityof the bacilli. The animais were fed withstandard food pellets and unlimited water.At 6, 12, and IS months one animal fromeach group and two animais at 18 and 21months were sacrificed. Three animais dieddue to unknown causes. The skin alongwith the subcutaneous tissue and muscle atthe injected area on both sides were dis-sected out. The tissue from the left flankswas harvested and the acid-fast bacilli(AFB) counted. The tissue from the rightflanks was immediately fixed in 10%buffered formalin and used for histopatho-logical studies. The animais were autopsiedand the organs were studied grossly andhistopathologically. The tissues obtained atautopsy were also fixed in 10% bufferedformalin and processed for 4-1.tm-thickparaffin sections, and stained with hema-toxylin and eosin (H&E) and a modifiedFite-Faraco stain.

At 6, 9, 12, 18, and 21 months' growthup to l0' AFB was obtained from the footpads of normal mice, confirming that the M.leprae used in this experiment were viable.The results of the bacterial count obtainedfrom the T900r animais are given in TheTable, showing a steady decrease in thenumber of organisms inoculated into theflanks.

Sections of skin from the flanks showeda diffuse granuloma occupying the derruisand the subcutaneous tissue including bun-

162

67, 2^ Correspondence^ 163

Titi TAitl i . Bacterial counts from theflanks o! T900r mire tone mouse tias taken atmonths 6,12, and 15 and nro erice were taken

at months 18 and 21).

Growth in skinof the flank

Months^(Group 1)Inoculum = 10'

^

6^

1 x 10'

^

12^

8.9 x 10'

^

15^

3.4 x 10'

^

18^

3.1 x 10'2.6 x 10'

^

21^

5.3 x 10'3.9 x 10`

dles of striated muscles. The granulomaconsisted of macrophages, some withgranular and some with vacuolated cyto-plasm, lymphocytes, plasma cells and afew polymorphs (Fig. 1). They were mostlyarranged in scattered clumps. Acid-faststaining showed macrophages fìlled withAFB. Almost alI AFB were fragmented andgranular (Fig. 2). AFB were not found inany other areis. The above changes were

seen consistently at 6 and 15 months. Nohistopathological changes were observed inthe biopsy specimens obtained at month 12,15 or 21. The hind foot, ears, liver, spleen,lungs, heart and kidneys obtained at au-topsy from animais inoculated at the flanksshowed no signilicant lesion except for asmall, focal, nonspecific, mononuclear cellinflanunatory infiltration of the tiver andlungs. No acid-fast organisms were seen.

This study shows that when M. lepraewere injected intradermally into the skin ofthe flanks of T900r mice they were taken upby the macrophages which aggregated inthe injected sites. Clumps of macrophagescontaining AFB were seen at the injectedsite at 6 and IS months. Morphologically,the AFB were ali fragmented and granular.Their counts decreased from 10' to 10" at 6months, 8.9 x 10 5 at 12 months, 3.4 x 10' at15 months, 3.1 x 10' at 18 months and 3.9x 10' at 21 months, clearly showing thatthere was no multiplication at the injectedsites. There was also no evidence of dis-semination of the infection to other paris ofthe hody since autopsy of the animais did

Fui. I. Photomicrograph showing microgranulunta^Fio. 2. Photomicrograph showing clumps of hro-in the skin of the ilank of T900r coice 6 months after^ken and granular AFB in macrophages of the ,granu-inoculation with 10' viahle ;tl. livra(' (11&E x250).^loura ,hown in figure I (modified Fite x800).

164^ International Journal of Leprosv^ 1999

not show any organisms in organs such asthe lungs, liver, spleen or ears.

One possible reason for the lack of mul-tiplication may be that the skin at the flanksis warmer than that at the foot pads. It isreasonable, therefore, to suggest that al-though M. leprae may enter into the bodythrough any skin site it can multiply anddisseminate only in a suitable environmentwith a temperature less than normal corebody temperature. The other reason maybe that unlike the foot pad, the region ofthe flanks is a large arei and the injectedM. leprae may have fanned out without be-ing localized and are thus lost to furtherevaluation.

'The absence of granuloma or any histo-pathological changes at the right flanks ofthe animal examined at intervals of 12, 15,and 21 months, although AFB were har-vested from the left lide, is difficult to ex-plain. The fact that even minimal inflamma-tory changes were absent suggests that thebiopsy might have missed the site com-pletely.

Further experiments are being done toconfirm this study since the number of miceused here was small, and there were no con-trol animais infected in the foot pads usingthe same inoculum and the same dosage forcomparison.

—Gigi J. Ebenezer, M.D.Head, Department of

Histopathologv andExperimental Pathnlogy

Shantha ArumugamLaboralorV Tecla icianDepartment of Experimental Patltologv

—Charles K. Job, M.D., F.R.C.Path.,F.A.M.S.

Emeritus ScientistSchieffelin Leprosv Research

and Training CenterKarigiri, V llore DistrictTanril Nada, Ilidia 632 /06

REFERENCESCIII:IU., S., Jon, C. K. and HASTINGS, R. C. Trans-mission of leprosy ia nude coice. Am. J. Trop. Med.Hyg. 34 (1985) 1161-1166.lias, C. K., CnI.nL, S. and HASrINGS, R. C. Newfinding on the mode of entry of M. leprae in nudecoice. (Letter) Int. J. Lepr. 58 (1990) 726-729.MCDERNwrr-LANCASTER, R. D. and MCDOUGALL,A. C. Mode of transmission of M. leprae infectionin nade mice. Int. J. Exp. Pathol. 71 (1990) 689-700.SIIANTIIA, A., Jon , C. K. and EIENEZER, G. J.Preparation of thymectomizcd and irradiated T900rmice: a modification. Indian J. Lepr. 69 (1997)200-201.

"Large Numbers" of Mycobacterium leprae areDischarged from the Intact Skin of Lepromatous

Patients; a Preliminary Report

To THE EDITOR:

In 1970 Pedley in an innovative study ofthe skin of leprosy patients concluded thatthe number of Mycobacterium leprae dis-charged from the intact skin of lepromatouspatients was negligible and, therefore, nasalsecretions were the major source of infec-tion (`'). Although his findings seem to bewidely accepted and quoted, no attempt hasbeen made to confirm or disprove his hy-pothesis. In the method employed in hisstudy, called composite skin contact smears

(CSCS), a glass slide was pressed 10 timesrepeatedly on the skin lesions of leproma-tons patients with a high bacterial index(BI), heat fixed, stained for M. leprae, andexamined under a microscope with an oilimmersion lens. From 11 patients, 34 CSCSwere studied and only 20 acid-fast organ-isms (AFB) were detected. On the basis ofthis study he concluded "that very fewbacilli, if any, are discharged from the intactskin of a lepromatous patient."

In this study under report, biopsies from13 patients belonging to the lepromatous