Jf Clin Pathol 1994;47:547-551

Liver morphology and function in visceralleishmaniasis (Kala-azar)

I A El Hag, F A Hashim, I A El Toum, M Homeida, M El Kalifa, A M El Hassan

AbstractAim-To study the morphology and func-tion of the liver in visceral leishmaniasis(Kala-azar).Methods-Percutaneous liver biopsyspecimens from 18 patients with con-firmed visceral leishmaniasis were exam-ined under light and electron microscopybefore and after treatment with pentova-lent antimony. The tissue was also exam-ined for hepatitis B surface and coreantigens using immunoperoxidase stain-ing. Liver function was investigated innine patients before and after treatment.Results-Specimens before treatmentshowed Kupffer cells and macrophagescolonised by leishmania parasites in 40%of cases. A chronic mononuclear cellinfiltrate had affected the portal tractsand lobules. Ballooning degeneration ofthe hepatocytes, fibrosis of the terminalhepatic venules, and pericellular fibrosiswere common findings. The fibrosis wasrelated to Ito cells transforming to fibro-blast-like cells. None of the patients hadhepatitis B infection. All patients hadbiochemical evidence of liver dysfunctionbefore treatment. Liver function im-proved after treatment.Conclusion-Visceral leishmaniasis causesmorphological and functional distur-bance in the liver. Focal fibrosis ratherthan cirrhosis occurs. The exact aetiologyof hepatic damage is unclear but mayhave an immunological basis.

(i Clin Pathol 1994;47:547-551)

Department ofPathology, Faculty ofMedicine, UniversityofKhartoum, PO Box102, Khartoum, SudanI A El HagI A El ToumA M El HassanDepartment ofPhysiologyF A HashimDepartment ofInternal MedicineM HomeidaDepartment ofPathology, JohnsHopkins University,Baltimore, USAM El KalifaCorrespondence to:Professor Ahmed MohamedEl Hassan.

Accepted for publication14 December 1993

Visceral leishmaniasis is a major health prob-lem in the Sudan.' The disease is endemic inthe eastern and southern part of the countryand sometimes reaches epidemic propor-tions.2' In other parts of the world liver dis-ease and various histopathological changeswith some geographical variation have beendescribed. These changes include diffuseKupffer cells, hyperplasia with a heavyparasitic infiltrate, portal and intralobulargranulomas, diffuse fibrosis45 and fibrin ringgranuloma.6 Severe fibrogenic changes, theso-called Rogers' "cirrhosis", which is associ-ated with liver dysfunction and portal hyper-tension, is more common in India than otherendemic areas.5 In a recent study from India,however, no evidence of liver cirrhosis or por-tal hypertension in visceral leishmaniasis was

found.7 Cases of severe hepatitis with cytoly-

sis, cholestasis, and hepatic failure have beenreported from Tunisia.8 The presence of theparasites inside the hepatocytes has been doc-umented at the ultrastructural level and theirrole in relapse of the disease discussed.9 Thepathogenesis of liver dysfunction and fibrosisand the role of Ito cells in aetiology of the lat-ter are not well understood. We thereforedecided to study liver pathology in visceralleishmaniasis and its evolution in relation tohepatic function and response to treatment.Furthermore, hepatic disease in SudaneseKala-azar has not been reported before.

MethodsAfter informed consent 18 consecutivepatients with confirmed visceral leishmaniasiswere enrolled in the study. All patients werefrom the Bentiue area of southern Sudan orthe Masairya tribe of the west. The parasitesin both areas are of the same isoenzyme pat-tern.'0 The male:female ratio was 5:1, agesranged from 15 to 60 years. None of thepatients had a history of alcoholism, jaundice,hepatosplenic schistosomiasis, or bleedingtendency. None was taking medication. Therewas no clinical evidence of liver failure in anyof the patients. All had varying degrees ofhepatosplenomegaly. Patients were treatedwith intravenous Pentostam 10 mg/kg bodyweight daily for 30 days.

Percutaneous needle liver biopsy specimenswere taken from all patients before the start oftreatment. A second liver biopsy specimenwas obtained from only 13 patients within aweek of completion of treatment. Biopsy spec-imens were fixed in formalin-mercuric chlo-ride-acetic acid mixture for one hour,transferred to 70% ethanol, and processed forlight microscopy. Paraffin wax sections (2-3,um) were stained with haematoxylin andeosin; collagen, reticulin, and iron weredemonstrated by van Gieson stain, silverimpregnation and Perls's Prussian blue reac-tion, respectively. Immunohistochemistry forhepatitis B virus was performed using theDako PAP Kit system (Santa Barbara,California, USA) in which primary polyclonalantibodies against surface (HBsAg) and core(HBcAg) antigens of hepatitis B virus wereused. The manufacturer's instructions werefollowed. With each batch of test a knownhepatitis B positive slide was included.

Pieces of liver tissue (1-2 mm long) werefixed in 2-5% glutaraldehyde at 4°C overnightand then transferred to cacodylate buffer (pH7 2). The tissue was post-fixed in 1% osmium

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tetroxide and embedded in Epon. Ultrathinsections were stained with uranyl acetate andlead citrate and examined in a Phillips 300transmission electron microscope.

Sera from nine patients before and aftertreatment as well as from 15 healthy subjectswere tested for total protein, albumin, y gluta-myltransferase (GGT), alkaline phosphatase,aspartate aminotransaminase (AST), alanineaminotransaminase (ALT), lactate dehydro-genase, total bilirubin and direct bilirubin.The paired and unpaired two tailed Student's

t test were used.

ResultsThe main morphological changes seen underlight microscopy were inflammation, parasiteinvasion of Kupffer cells, fibrosis, and hepato-cyte changes in the form of swelling, dyspla-sia, and fatty change. Thrombosis was alsoseen.

INFLAMMATORY CHANGESInflammatory changes were seen in all liverbiopsy specimens before treatment, both inthe portal tracts (18 patients) and intralobu-larly (14 patients). Before treatment the portaltracts were enlarged and contained largenumbers of macrophages, lymphocytes, andoccasional plasma cells. There were no com-pact epithelioid granulomas nor Langhans'giant cells. In addition to this inflammatoryreaction, mild to moderate fibrosis was seen in28% of these specimens before treatment. Inanother 28% of cases limiting plates wereirregular and there was evidence of piecemealnecrosis (fig 1).

After treatment the inflammation in theportal areas disappeared in 23%, remainedthe same in 46%, and increased in 31% ofpatients. Intralobular inflammation was seenin 14 (78%) patients before treatment. It wasdiffuse in nature in 12 (86%) of them. Theinflammation consisted of hyperplastic andhypertrophic Kupffer cells and sinusoidal

inflammatory mononuclear cells. In the othertwo patients nodular collections of mono-nuclear cells were seen mainly around thecentral vein. After treatment intralobularinflammation was still present in seven out of13 (54%) patients.

Parasites were only identified in eight out of18 (44%) patients. The parasites were withinthe Kupffer cells and in histocytes in the portaltracts. Parasitised cells were usually associatedwith an inflammatory cellular reaction con-sisting of lymphocytes and plasma cells. Evenbefore treatment in 56% of patients theinflammatory reaction contained no para-sitised cells. No parasites were detected in thesamples after treatment despite the presenceof the inflammatory reaction.

FIBROSISFibrosis of the central vein was seen in 10 outof 18 (56%) patients before treatment and in12 out of 13 (92%) patients after treatment(fig 2). It was absent or seen in a milder formbefore treatment in patients with a nodularinflammatory reaction around central veins. Itappeared in these areas or increased in severityafter treatment.

Portal fibrosis was seen in 11 out of 13(85%) patients after treatment compared withfive out of 18 (28%) patients before treat-ment. Portal inflammatory reaction withoutfibrosis was seen in three cases before treat-ment. In these cases liver biopsy specimenstaken after treatment showed only fibrosiswithout inflammation.

Pericellular fibrosis was seen after treat-ment in eight out of 13 (62%) patients com-pared with five out of 18 (28%) patientsbefore treatment. It started around centralveins and portal tracts and spread to encirclecells in the vicinity of these structures first(fig 2).

Bridges between different liver compart-ments were seen in five out of 18 (28%)patients after treatment compared with twoout of 18 (11%) patients before treatment,with bridging seen before and after treatmentin one case.* : ... i ^ ........ . *. .,. . SS;:1::: Mw ES' E --i: ,l$ . S ?S" Sb. ' ;t: >' ws e c ... . s R :.:: ... ii .. ...... X1 .:: . :w5 ............ S .: ... ' : w.Bag03 ^ ,.6! .9.:Kai.::;°: : . :v . ::.":B ... :t" :. '.,'.'. '4 ............ :.|. ......... &g :.w .. ...... > .. . . &.&.Fi . ....... -ffii>. .::>! .t.ie8W. aF 5 ;

.:: :. oxi^'9 8 ' '' ? ; ... r

Figure 1 Edge ofa portal tract showing an irregular limiting plate with moderately heavymononuclear cell reaction infiltrating the hepatic parenchyma. A bile duct is at the top ofthe photograph (haematoxylin and eosin).

*.

Figure 2 Fibrosis of the wall ofa central vein. Note thefibrosis is extending into the surrounding liverparenchyma.Hepatocytes show ballooning degeneration (haematoxylinand eosin).

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Liver morphology andfunction in visceral leishmaniasis (Kala-azar)

Figure 3 Leishmaniaparasite inside twoadjacent Kupffer cells.There is collagen betweenthe two cells.

Figure 4 Hepatocytesshowing pronouncedoedema of the cytoplasm.

Figure 5 The bottom ofthe photograph shows partof the cytoplasm of an Itocell containing manyfilaments. At the top is ahepatocyte in intimatecontact with the Ito cell.

Dysplasia, defined by increased nuclearstaining and pleomorphism, was seen in 12out of 18 (67%) patients before treatment.Dysplasia remained after treatment.

Parasitised Kupffer cells were hypertro-phied and occasionally contained phagocy-tosed red cells.

Apoptotic Kupffer cells containing para-sites were seen in three patients.

Increased iron content in hepatocytes andKupffer cells was seen in all patients beforeand after treatment. It was more noticeable inKupffer cells.

THROMBOSISThrombosis was seen in six patients. In onepatient an organised thrombus was found in aportal vein. In the remaining five patientssinusoidal thrombosis was widespread.

In all cases biopsy specimens were negativefor HBsAG and HBcAG.

At electron microscopic examination, para-sites were seen inside the Kupffer cells (fig 3).They were enveloped in a unit host mem-brane. The pellicular membrane showed typi-cal microtubules. Internal structures includedthe nucleus, kinetoplasts, and the flagellarpocket. Erythrophagocytosis was observed inparasitised Kupffer cells.Some hepatocytes showed dilatation of the

endoplasmic reticulum and microvesicles sur-rounding the dilated endoplasmic reticulum.The latter showed disruption of the ribo-somes. Some hepatocytes, particularly aftertreatment, showed pronounced oedema of thecytosol in addition to dilatation and disrup-tion of the endoplasmic reticulum (fig 4).

Ito cells were identified by their typicallarge fat droplets. Some Ito cells showedextensive dilatation of their endoplasmic retic-ulum, a sign of increased secreting activity. Insome areas cells in the typical position of Itocells had no lipid droplets and showed intra-cytoplasmic filaments (fig 5). These were

CHANGES IN HEPATOCYTES AND KUPFFER CELLSSwollen pale-staining hepatocytes were com-mon and were seen in 11 out of 18 (61%)patients before treatment and in 11 out of 13(85%) patients afterwards (fig 2). The swollenhepatocytes formed rosettes similar to thoseseen in chronic active hepatitis. Focal andindividual hepatic cell necrosis was rarelyencountered. Severe fatty change was seen inonly one patient, who was very ill and whodied five days after the start of treatment.

Figure 6 At the top is a lymphocyte in intimate contactwith a parasitised Kupffer cell and an Ito cell. The lattercontains lipid droplets.

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considered to be Ito cells completely trans-formed into fibroblast-like cells. ParasitisedKupffer cells, Ito cells, and lymphocytes werecontiguous (fig 6).

Collagen fibres were seen around hepato-cytes and occasionally between adjacentKupffer cells (fig 3).

Before and after treatment alkaline phos-phatase and GGT values were significantlyhigher in patients than controls (table). Otherliver enzymes (ALT, AST, lactate dehydroge-nase) were significantly higher in patientsbefore treatment than in controls but theyreturned to normal after treatment.

DiscussionMorphological changes in the liver in visceralleishmaniasis involve Kupffer cells, hepato-cytes, Ito cells, portal tracts, sinusoids andhepatic veins. Hypertrophy and hyperplasia ofthe Kupffer cells occur, and in 44% of casesthese cells contain parasites. In about two-thirds of patients the hepatocytes showed bal-looning degeneration which worsened aftertreatment despite improved liver functionafter treatment. The hepatic changes were notrelated to the presence of parasites or to theinflammatory reaction in the biopsy speci-mens. In some samples that had been treated,in which parasites had been eliminated andthe inflammation had subsided, ballooningdegeneration was still evident. The pathogene-sis of this change is unknown. Concomitanthepatitis B infection has been excluded. Thefact that in some patients the hepatic changesworsen after treatment may be partly due tothe effect of the drug. Functionally, the GGTand alkaline phosphatase values did not dropto normal after treatment, although serumalbumin and other enzymes returned to nor-mal. The piecemeal necrosis, seen even in theabsence of demonstrable parasites, couldmean an immunological injury to the hepato-cytes, particularly at the periphery of the lob-ules. The inflammatory cellular reaction,which consists of parasitised macrophages,lymphocytes, and plasma cells, affects theportal tracts and is found inside the lobuleeither diffusely in the sinusoid or focally.Although parasites disappeared after treat-ment, the inflammatory reaction was still pre-sent or even increased after treatment in 77%

of the patients. However, the patients recov-ered clinically and most of the liver functiontests returned to normal. The only patientwho died had severe fatty changes. We havealready seen another patient who died of vis-ceral leishmaniasis and had a fatty liver atnecropsy. It seems that severe fatty change ofthe liver in visceral leishmaniasis is a sign ofpoor prognosis. Compact epithelioid granulo-mas and Langhans' giant cells do not occur.This contrasts with cutaneous leishmaniasiswhere well developed compact granulomasare associated with the development of adelayed hypersensitivity reaction, as indicatedby a positive leishmanin test." Patients withvisceral leishmaniasis are leishmanin negativeand their peripheral mononuclear cells do notreact to leishmania antigen. 12 Patients withasymptomatic (subclinical) visceral leishmani-asis are leishmanin positive and show welldeveloped epithelioid granuloma in the liver.'3

Roger first described "cirrhotic" changes inpatients with visceral leishmaniasis, in India in1908.14 It was not a true cirrhosis but a diffusefibrosis, isolating small groups of hepatocytes,with evidence of regeneration. A better termfor the change is diffuse intralobular fibrosis.4Most of our patients showed fibrosis of thewall of the central veins and pericellular fibro-sis in its vicinity. This was confirmed by elec-tron microscopy. In some patients there wasbridging fibrosis connecting central veins toportal tracts or portal tracts to portal tracts inparts of the liver. Because of the focal natureof the fibrosis, it is doubtful if these patientswill ever progress to cirrhosis. Over the pastthree years, in a longitudinal study of visceralleishmaniasis in a village in an endemic area inthe Sudan, no patient developed cirrhosisafter being treated for visceral leishmaniasis(El Hassan, unpublished data). Long termfollow up, which was not possible in ourpatients, is needed to determine the final out-come of the hepatic changes.The source of fibrosis seems to be Ito cells.

Regardless of the cause of hepatic injury, heal-ing by fibrosis in the liver involves Ito cells.The fibrosis may follow physical injury,'5 alco-holic liver disease,'6 17 and fatty liver of obe-sity.'8 Transformation of Ito cells tofibroblast-like cells is believed to be the causeof fibrosis in these varied clinical situations.'9The most reliable indicator of transformation is

Mean (SD) Results of liverfunction tests in controls (n = 15) and patients (n = 9) before and after treatmentTotal Alkaline Lactate Total Directprotein Albumin phosphatase ALT dehydrogenase GGT AST bilirubin bilirubin(gldl) (mgldl) (U/I) (U/I) (U/i) (U/i) (UIl) (mgldl) (mgldl)

Control 8-0 (10) 4-3 (07) 74 (29) 12 (6) 210 (72) 20 (9) 34 (11) 0 7 (0 6) 01 (01)Pre-treatment 7-4 (2 9) 2-8 (0 7)a 234 (221)c 57 (50w) 352 (172)9 66 (54)1 159 (121)' 0 4 (01) 0-2 (0-1)Post-treatment 7 9 (1-4) 3-7 (0-4)b 190 (105)d 15 (5) 156 (29) 52 (47) 50 (16) 0-6 (0-2) 0 1 (0 1)

Significantly different from:a control, p < 0 001.b control and pre-treatment groups, p < 0-02 and p < 0 01.c control, p < 0-05.d control, p < 0 01.e control, p < 0-02.pre-treatment, p < 0 05.

9 control, p < 0-05.h control and pre-treatment, p < 0-02.control, p < 0-02.control and post-treatment, p < 0 01 and p < 0 05.

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dilatation and an increase in the rough endo-plasmic reticulum rather than a change in thelipid content of these cells.'9 We found dilatedendoplasmic reticulum in Ito cells and com-plete transformation of these cells into fibro-blast-like cells. The stimulus for Ito celltransformation is probably mediated by cyto-kines. Transforming growth factor B, pro-duced by Kupffer cells, endothelial cells, andIto cells induces synthesis of fibronectin,laminin, collagen and protoglycans and italso down-regulates collagenase expression.20Interleukin 1 stimulates DNA synthesis andproliferation of Ito cells in primary culture,and platelet derived growth factor activatesIto cells towards a myofibroblast appear-ance.20 We have shown that parasitisedKupffer cells, Ito cells, and lymphocytes arecontiguous. Activation of platelets was evi-denced by microthrombi in the sinusoides ofsome patients.

We are indebted to Miss Wafaa Salih and Mr Sayed OsmanYousif of the electron microscopy unit, University ofKhartoum, Susan Peters of the Department of Anatomy,University of Copenhagen, for technical assistance with elec-tron microscopy, and Mr El Tahir Fadel for bone marrowbiopsies. The hepatitis B kit was kindly donated by Dako A/SDenmark.

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11 Ridley DS, Ridley MS. Late stage cutaneous leishmaniasis:Immunopathology of tuberculoid lesions in skin andlymphnodes. BrJ Exp Pathol 1984;65:337-46.

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20 Clement B, Lorel 0, Lawarsur F, Guellouzo A. Newchallenges in hepatic fibrosis. J Hepatol 1993;18:1-4.

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